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Modulation of atrial fibrillation

Geuzebroek, G.S.C.

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Citation for published version (APA):Geuzebroek, G. S. C. (2013). Modulation of atrial fibrillation.

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Modulation of Atrial Fibrillation

ISBN: 978-94-6182-265-9

Cover: Sander Minnoye, DIDID.eu

Printed: Offpage

© 2013 Guillaume Geuzebroek

All rights reserved. No part of this thesis may be reproduced, stored in a retrievel system, or transmitted in any form or by any means without permission of the author. The copyright of the articles that are accepted for publication or have been published has been transferred to the respective journals.

This thesis has been made possible by collaboration between the department of Cardio-Thoracic Surgery, St. Antonius Hospital, Nieuwegein, The Netherlands and the department of Experimental Cardiology, Academic Medical Center, Amsterdam, The Netherlands.

I gratefully thank the following companies for their financial support:AtriCure Europe BV, AM-pharma BV, Maquet Netherlands BV, St. Jude Medical Nederland BV, Johson & Johnson Medical BV, Vascutek Nederland, Biosense Webster BV, Boehringer Ingelhem BV, Sorin Group Nederland NV, Krijnen Medical Innovations BV, MI Consultancy BV.

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctoraan de Universiteit van Amsterdamop gezag van de Rector Magnificus

prof. dr. D.C. van den Boomten overstaan van een door het college voor promoties ingestelde

commissie, in het openbaar te verdedigen in de Aula der Universiteitop woensdag 15 mei 2013, te 13:00 uur

doorGuillaume Stephan Caesar Geuzebroek

geboren te Baarn

Modulation of Atrial Fibrillation

Promotiecommissie

Promotor: Prof. dr. ir. J.M.T. de Bakker

Copromotor: Dr. R. Coronel Dr. M.W. Veldkamp

Overige leden: Prof. dr. N.M. van Hemel Prof. dr. P. Kirchhof Prof. dr. mr. B.A.J.M. de Mol Dr. T. op ‘t Hof Prof. dr. U. Schotten Prof. dr. A.A.M. Wilde

Faculteit der Geneeskunde

Content

Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

Chapter 10

General introduction and outline of thesis

Freedom from atrial arrhythmias after classic Maze III surgery: A 10-year experience

Medium-term outcome of different surgical methods to cure atrial fibrillation: Is less worse?

Completely thoracoscopic pulmonary vein isolation with ganglionic plexus ablation and left atrial appendage amputation for treatment of atrial fibrillation

Increased amount of atrial fibrosis in patients with atrial fibrillation secondary to mitral valve disease

Effects of acetylcholine and noradrenalin on action potentials of isolated rabbit sinoatrial and atrial myocytes

Neuropeptide substance-P causes action potential prolongation and resting membrane depolarization in rabbit atrial myocytes

Summary

Samenvatting

Dankwoord

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Epidemiology, risk factors and symptoms of atrial fibrillationAtrial fibrillation is the most common sustained arrhythmia with an overall prevalence of 5.5% above 55 years of age.1 In the USA alone it is estimated that a total of 2.3 million citizens suffer from atrial fibrillation and that this number will increase with time.2

The prevalence of atrial fibrillation is strongly correlated with age and has a prevalence of almost nil before 55 years, but of almost 10% above 80 years of age.1-3 Other risk factors for atrial fibrillation are diverse and include heart failure, male gender, diabetes, coronary artery disease, valvular heart disease and hypertension.3-8

The main symptoms of atrial fibrillation are palpitations, fatigue, vertigo, chest pain, dizziness, syncope and dyspnea. Not all patients do, however, experience symptoms.5,9 Even in patients who do experience symptoms of atrial fibrillation, episodes of atrial fibrillation are more often asymptomatic than symptomatic.10

Atrial fibrillation can not only cause discomfort to the patient, but also imposes great health risks. Common symptoms are caused by a combination of an (fast) irregular heartbeat and a decreased cardiac output. This can lead to heart failure via 2 mechanisms. One is a direct effect caused by a decreased ventricular filling due to a decreased atrial contractile function and a shortening of diastolic ventricular intervals.11 The second mechanism is an indirect effect caused by remodeling due to prolonged high ventricular rhythm, which reduces systolic function (tachycardiomyopathy).12 Indeed, patients with atrial fibrillation have a 3 times increased risk of heart failure.13 Furthermore, atrial fibrillation is associated with a 5 times increased risk of ischemic stroke due to functional stand-still of the atria.14 In addition, strokes caused by atrial fibrillation are more lethal than non-AF related strokes.15 Recent studies also show that atrial fibrillation is an independent risk factor for dementia.16,17

Overall, atrial fibrillation causes decreased quality of life, increased morbidity and is even an independent risk factor for death. It is responsible for a relative mortality risk of 1.5 in men and 1.9 in women from an age of 55 years.18

Historical perspective of the mechanisms of atrial fibrillation During the last few decades progress has been made in understanding the mechanism of initiation and maintenance of atrial fibrillation. This increased

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knowledge of the mechanisms behind atrial fibrillation in combination with the low efficacy of pharmacological treatment and the associated life threatening drug induced arrhythmias in the form of Torsades de Pointes, has led to enormous progress in the non-pharmacological treatment of atrial fibrillation.19-21 To get a clear understanding of this progress it is necessary to know the historical perspective of the mechanisms of atrial fibrillation. In the early 1900s, three main theories existed on the underlying mechanism of atrial fibrillation. These theories encompassed the presence of one or multiple rapid autonomic foci or reentry.22-24 In 1924 Garrey and co-workers recognized that a minimum amount of tissue (‘critical mass’) is necessary to maintain atrial fibrillation.25 This observation refuted both a single autonomic focus as well as multiple autonomic foci as a main cause of atrial fibrillation, and supported the concept of reentry being the underlying mechanism. Some years earlier, reentry had already been described by Mayer in jellyfish and later also in myocardium of tortoises and frog by Mines.26,27 The observed reentrant activations were always dependent on an anatomical obstacle and became known as circus movement reentry (figure 1, figure 2A).24

Figure 1. Circus movement reentry as it was originally describes by Lewis.At the bottom of the circle the tissue is stimulated (circle 1). At point A, a unidirectional block prevents clockwise conduction, but conduction is continued in counter-clockwise manner (circle 2). When the activation front has reached point A (circle 5), the tissue at this point has been repolarized and reentrant activation ensues. Reproduced with permission from the publisher24


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A new concept of atrial fibrillation was proposed in 1959 by Moe and Abildskov and is known as multiple wavelet theory (figure 2B).28 Moe and co-worker hypothesized that multiple activation fronts (which they called ‘wandering wavelets’) are present during atrial fibrillation and that they continuously change in direction, size and number.28 They also hypothesized that the stability of the arrhythmia increases with a larger number of wandering wavelets, which would decrease the chance of simultaneous termination of all wandering wavelets. The number of wandering wavelets would depend on the tissue mass, conduction velocity and refractory period.28 A larger tissue mass can potentially harbor more wavelets, while a slower conduction velocity and a shorter refractory period allow smaller reentry circuits and thus more wandering wavelets in the same volume of tissue. The smallest possible reentry circuit can be expressed as the wavelength, which is a multiplication of the conduction velocity and refractory period. In 1964, the feasibility of the multiple wavelet theory as an underlying mechanism of atrial fibrillation was confirmed by a computer model and the mechanism was later confirmed by detailed activation mapping of atrial fibrillation in a canine model.29,30

A B C D

Figure 2. Mechanisms of reentry and atrial fibrillation.Schematic drawing of different mechanisms of reentry. A: Circus movement reentry. This reentry occurs around an anatomical obstacle and because the length of the pathway is dependent on the circumference of the obstacle, which is larger than the wavelength, an excitable gap is present. Due to the excitable gap, this form of reentry can be entrained by pacing. B: Multiple wavelet theory. During atrial fibrillation multiple wandering activation wavelets are present. C: Leading circle reentry. The reentry is not dependent on an anatomical obstacle and because it will adapt to the smallest possible reentrant circuit (wavelength) there is no excitable gap and thus cannot be entrained. The center of reentry is kept continuous in a refractory state due to continuously invading activation fronts D: Spiral wave reentry. It strongly resembles the leading circle reentry, but the core (black dot) is not refractory like the leading circle reentry. The core is excitable, but not excited.

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When the activation pathway is the same in every cycle of the arrhythmia, sequential mapping can be applied to map the activation path. Sequential mapping requires a single roving probe (electrode) and is technically less challenging than simultaneous mapping with large electrode grids and multichannel data acquisition systems. Until the 1970s sequential mapping techniques only permitted the demonstration of reentry with a fixed activation pathway around an anatomical obstacle (circus movement reentry). In 1973, Allessie and co-workers were, however, able to provoke and map functional reentry in atria of rabbit hearts, which led to a flutter-like tachycardia.31,32 This reentry mechanism became known as the leading circle reentry (figure 2C). The reentrant activation pathway of a leading circle reentry forms a circle which not only activates the surrounding tissue, but keeps the core refractory. The latter is caused by a continuously invasion of the core by activation fronts from all directions. The leading circle will adopt the smallest possible activation pathway (wavelength) and, in contrast to circus movement, almost no excitable gap is present (figure 2A and 2C).31,32

Apart from circus movement reentry, multiple wandering wavelets and leading circle reentry, a fourth reentry mechanism has been suggested as a mechanism for atrial fibrillation. A spiral wave reentry (figure 2D) is a form of functional reentrant activation and strongly resembles the leading circle reentry. The main difference between a leading circle reentry and a spiral wave reentry is the state of the core. While the core of the leading circle is thought to be continuously refractory, the core of a spiral wave is excitable but is not excited.32-34

Until the late 1990s, non-pharmacological treatment for atrial fibrillation was mainly based on reentry as the prime mechanism of atrial fibrillation. This changed substantially when Haïssaguerre and co-workers revealed that atrial fibrillation was initiated by ectopic foci originating from the myocardial sheaths of the pulmonary veins, and that ablating these foci could terminate atrial fibrillation.35 From that time onward, non-pharmacological therapy for atrial fibrillation focused more on modification of the triggering mechanisms in the pulmonary veins.

Until today, the exact underlying mechanisms of atrial fibrillation remain unknown.36 Independent of the exact mechanism or mechanisms of atrial fibrillation, the arrhythmia can, however, be considered as a condition which is initiated by triggers, maintained or realized by the substrate and influenced by modulating


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factors.37-39

Trigger and substrate of atrial fibrillationThe trigger is the initiator of the arrhythmia, while the substrate harbors or maintains the arrhythmia.40 The mechanisms behind the triggers (or foci) that initiate atrial fibrillation are not known. Abnormal automaticity, triggered activity and micro-reentry are the three major possibilities that have been postulated as mechanisms for these focal activations.41,42 The possibility of automaticity as a mechanism for ectopic foci came from the observation of Brunton and Fayer in 1876.43 They saw that the pulmonary veins could contract independently from the rest of the heart. This observation suggested that pulmonary veins contain myocardial cells that harbor pacemaker properties. Later studies showed indeed that the pulmonary veins in both animals and humans are excitable and contain sleeves of atrial myocardial cells and that some cells strongly resemble sinus node cells.44-46 Furthermore, spontaneous diastolic depolarization, a pacemaker property, was recorded in pulmonary vein myocardium of dogs.47 Whether automaticity really leads to atrial fibrillation remains unclear. Another possible mechanism for ectopic foci is triggered activity. Triggered activity is a condition when a normal action potential is followed by depolarization of the membrane which reaches the activation threshold and leads to an action potential.42 Two different types of these membrane depolarizations exist, early after depolarizations (EADs) and delayed after depolarizations (DADs). An EAD is an abnormal transient depolarizion of the membrane (potential) during phase 2 or 3 of the action potential. The exact mechanism behind an EAD remains a topic of debate. Two main mechanisms seem to play a role. The first is a transient inward current due to re-activation of the L-type calcium channels. The second is spontaneous Ca2+ release from the sarcoplasmic reticulum under conditions of elevated diastolic intracellular Ca2+ concentrations.48 In the latter case, the excess Ca2+ is removed from the cell through the Na+/Ca2+-exchanger, generating a net inward current by exchanging 3 Na+-ions for 1 Ca2+-ion. The DAD is an abnormal transient depolarization of the membrane (potential) during phase 4, when the membrane is completely repolarized. In contrast to EADs, general consensus exists on the mechanism of DADs. This mechanism is similar to one of the proposed

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mechanism of an EAD: generation of a net inward current by the Na+/Ca2+-exchanger under conditions of elevated diastolic intracellular Ca2+.49,50

Micro-reentry has been suggested as a third mechanism of ectopic activation initiating atrial fibrillation because of the distinctive structure of the pulmonary veins. Sleeves of myocardium reach up to 2 cm into the pulmonary veins and have a criss-cross fiber direction.51,52 Additionally, an increased amount of fibrous tissue is found in the pulmonary veins of patients with atrial fibrillation.53 These two characteristics of the pulmonary veins can lead to severe conduction delays and block.54,55 The severe conduction delay can potentially result in an extremely short wavelength (micro-reentry), while the highly anisotropic tissue characteristics favor unidirectional conduction block and thus reentry.

Modulating factorsIn 1987, Coumel already stated that apart from the simultaneous presence of a trigger and substrate, a third factor, the modulating factor, is necessary in the genesis of an arrhythmia (figure 3).40 He proposed that the autonomic nervous system could act as an important modulating factor.40 This concept of a trigger, substrate and modulating factor is schematically depicted in figure 3 and is known as the Triangle of Coumel (figure 3). To date, this trinity of a trigger, a substrate and a modulating factor is the leading concept of atrial fibrillation.

Figure 3. Triangle of CoumelThe triangle of Coumel schematically depicts the trinity of a substrate, trigger and modulating factor. This trinity is responsible for atrial fibrillation (AF). Adapted with permission from the publisher40


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Other modulating factors, different from the autonomic nervous system, exist. Endothelial dysfunction, inflammation, hormonal factors, obesity and atrial stretch have all been suggested to modulate arrhythmogenesis.37 The main modulating factor is, however, probably atrial fibrillation itself. This phenomenon is generally described as ‘atrial fibrillation begets atrial fibrillation’ after the publication by Wijffels and co-workers of a goat model of AF.56 They demonstrated that when atrial fibrillation was continuously re-induced by atrial pacing (in healthy goats), the arrhythmia became self-sustainable due to shortening of the refractory period. Indeed, while the onset of atrial fibrillation in man is often paroxysmal in nature, it becomes eventually persistent or even permanent.57 In addition to electrical remodeling, atrial fibrillation is also associated with structural and contractile remodeling, which may add to the arrhythmogenic vulnerability of the substrate.58,59

Electrophysiological remodeling by atrial fibrillation in the form of a decreased refractory period has been a consistent finding.56,60 The decreased refractory period caused by atrial fibrillation is the result of shortening of the action potential mainly by a decreased L-type Ca2+ current and an increased inward rectifying K+ current (IK1).

61,62

Structural remodeling observed in subjects with AF is diverse and, in part, consists of atrial dilatation, increased cell-size, myolysis, perinuclear accumulation of glycogen, alterations in connexin expression, fragmentation of sarcoplasmic reticulum and changes in mitochondrial shape.58,60,63 The clinical relevance of these structural changes remains, however, unknown.58

Logan and co-workers were the first who recognized loss of contractile function.64 They observed that immediately following electrical cardioversion the left atrium lacked contraction despite activation. Subsequent echocardiographic studies confirmed that atrial contractility was diminished after termination of atrial fibrillation and that the time of complete recovery of contractility depended on the duration of atrial fibrillation before the electric cardioversion.65 The exact mechanism for this phenomenon is not fully understood. Various studies, however, suggest that a decreased intracellular calcium concentration resulting from a reduced L-type Ca2+ current is the culprit process.61,66,67

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Fibrous tissue and atrial fibrillationIt is generally believed that atrial fibrosis plays an important role in atrial fibrillation.68 While there is a clear relation between atrial fibrosis and atrial fibrillation, the causality between the two is less clear. Both experimental and clinical studies have shown a relation between atrial fibrillation and fibrosis.69-71 In man, an increased amount of fibrous tissue is found in atria of patients with atrial fibrillation compared to those of patients in sinus rhythm and of patients with mitral valve disease compared to those without mitral valve disease.53,69,70 Furthermore, mice with increased amounts of fibrous tissue, due to genetically overexpressing TGF-β1, are more vulnerable to atrial fibrillation.71

The increased amount of fibrous tissue observed in patients with atrial fibrillation could be due to structural remodeling.53,58,69 However, the increased amount of fibrous tissue could also have a different origin and be the cause instead of the result of atrial fibrillation. Fibrosis itself may provide the substrate for reentry and thus for atrial fibrillation in two manners. One is ‘current-to-load mismatch’ and the other anisotropic or ‘zigzag’ conduction.54,72,73

Fibrosis can create nonconductive barriers that can cause conduction delay and block via so-called current-to-load mismatch. Fibrotic barriers cause variation in the tissue architecture. At sites of tissue expansion more depolarizing current is required for successful propagation. At these sites conduction delay may arise and finally lead to conduction failure if the available depolarizing current is insufficient.74 This form of conduction block depends on the spatial variation within the myocardium and is therefore often unidirectional in nature and thus favors unidirectional block which is necessary for reentrant arrhythmias like atrial fibrillation.74 The second mechanism by which fibrosis makes the substrate more susceptible for atrial fibrillation is due to ‘zigzag’ conduction. The nonconductive barriers caused by fibrosis causes the activation front to travel around these barriers by creating a ‘zigzag’-like path, which results in prolongation of the activation time in mainly the transversal direction.72 This causes increased anisotropic conduction which favors reentry and thus atrial fibrillation.75

Autonomic nervous system and atrial fibrillationIn 1987, Coumel already recognized that the autonomic nervous system plays a key-role


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in atrial fibrillation.40 He recognized that especially paroxysmal atrial fibrillation is dependent on a dysbalance of the autonomic nervous system. He described both vagally and adrenergically induced atrial fibrillation.76 Vagally induced paroxysmal atrial fibrillation is often preceded by a bradycardia and occurs predominantly at night.76 This type of paroxysmal atrial fibrillation is mainly seen in patients without structural heart diseases.76 Adrenergically driven atrial fibrillation is less common and is often secondary to a cardiac disorder.76 This type of atrial fibrillation occurs typically at daytime during exercise or stress and often coexists with structural heart disease. A large observational study in Europe led to the identification of the onset of atrial fibrillation either as vagal or as adrenergic in 33%.77 Vagally-only induced atrial fibrillation was present in 6% of the patients, adrenergically-only in 15% and the remainder (12%) had a mixed pattern. Surprisingly, the prevalence of structural heart diseases did not differ between patients with vagal and adrenergic induced atrial fibrillation.77

The intrinsic nervous system of the heart is a complex system of ganglionic plexus, which at the atrial level are predominantly located in the fat pads around the pulmonary veins.78,79 Each ganglion can contain several hundreds of neurons.79,80 These neurons contain not only acetylcholine and noradrenalin, but many other neurotransmitters such as substance-P.80

It has become clear that the autonomic nervous system can exert a large effect on the electrophysiological properties of the atria. Both vagal and adrenergic stimulation shorten the atrial refractory period and thus allow shorter wavelengths favoring reentry.81 Many animal models of atrial fibrillation use some form of vagal stimulation to initiate and maintain atrial fibrillation. The susceptibility for atrial fibrillation during vagal stimulation is thought to be caused not only by the shorting of the refractory period, but also by an increased dispersion of refractoriness.82

Only in the last years cardiologists and cardiac surgeons have gained special interest in the autonomic nervous system in atrial fibrillation. This is for a large part caused by seminal work of the group from Oklahoma.83,84 They have shown which drastic effects the autonomic nervous system can have on both the substrate and the initiating factor.83 At present, some non-pharmacological therapies not only target the myocardial sleeves of the pulmonary veins, but also attempt to modify the atrial autonomic nervous system. Both surgical epicardial and percutaneous

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endocardial procedures are combining pulmonary vein isolation with ablation of ganglionic plexus.85-87

From experimental and clinical studies it appears that targeting the autonomic nervous system can be beneficial in the treatment of atrial fibrillation.83,86,88,89 However, no randomized trials which compare pulmonary vein isolation with or without ganglionic plexus ablation have been published.

History of surgery for atrial fibrillationDue to low efficacy of anti-arrhythmic medication in maintaining sinus rhythm and the side effects of both anti-arrhythmic and anti-coagulant medication, surgical procedures were developed for the treatment of atrial fibrillation in the late 1980s and early 1990s. Two of the first surgical treatments were the Corridor and the Maze procedures. Although the Maze procedure was very successful, many various surgical approaches were in use and developed over time and some are described below.

Left atrial isolation and Corridor procedureIn 1980, the ‘left atrial isolation’ was described and was the first surgical procedure for atrial fibrillation.90 It was originally designed to treat autonomic or ectopic supraventricular tachycardias originating from the left atrium (figure 4). The procedure electrically isolated the left atrium from the right atrium, thereby prevented tachycardias originating from the left atrium from conducting to the right atrium. This procedure appeared also to be beneficial for atrial fibrillation. After the procedure was performed for atrial fibrillation, the sino-ventricular co-ordination was restored, but the left atrium continued to fibrillate or became electrically silent. The ‘Corridor’ procedure was described by Guiraudon in 1985.91 It surgically created an electrical isolated corridor from the sinus node to the AV-node (figure 5). Like the left atrial isolation procedure, it restored the sino-ventricular co-ordination, but both the left and right atrium continued to fibrillate. This procedure was considered as an alternative for pacemaker implantation after His-ablation for drug-refractory atrial fibrillation.92 Its advantage over pacemaker implantation is that the ventricles are physiological paced by the sinus node instead of a pacemaker. Both the Corridor procedure and the left atrial isolation procedure


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have a success rate of approximately 70-80% in restoring the sino-ventricular co-ordination92-95 However, the risk of thrombo-embolism remains due to the loss of atrial systolic function and thus patients remain dependent on anti-coagulation therapy.

Figure 4. Left atrial isolation procedure.Drawing of the left atrial isolation procedure. This procedure electrically isolates the left atrium from the right atrium. (LAA= left atrial appendage, CS= coronary sinus, MV= mitral valve).Reproduced with permission from the publisher94

Figure 5. Corridor procedure.Schematic overview of the Corridor procedure. The procedure surgically creates a small path of tissue (‘Corridor’) from the sinus node (arrow) to the AV-node. This electrically isolated path prevents reentry and thus atrial fibrillation within the path. This restores sino-ventricular co-ordination, but the left and right atrium remain susceptible for atrial fibrillation. (PV= pulmonary vein, MI mitral valve circumference, TRI = tricuspid valve circumference)Reproduced with permission from the publisher95

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Maze procedureThe Maze operation was the first treatment of atrial fibrillation which could overcome all negative effects of atrial fibrillation and was designed by Cox and co-workers in the late 1980s.96 In contrast to the Corridor and left atrial isolation procedures, the Maze not only restored the sino-ventricular co-ordination, but also reduced the risk for thrombo-embolism and restored atrial contractility. The development of the Maze procedure was based on the multiple wavelet theory and reentry as the mechanism of atrial fibrillation. Cox and co-workers recognized that preventing or disrupting these reentrant activities could prevent atrial fibrillation. The objective of the Maze procedure was to create a labyrinth (hence Maze) of non-conductive lines in the atria, with the sinus node as entrance and the AV-node as exit.96 These non-conductive lines were created in a fixed pattern in both atria by cutting and sewing (figure 6). The initial Maze procedure which was performed on man (now called Maze I) led in many patients to sinus node dysfunction.97 It was therefore modified to the Maze II and eventually to the Maze III procedure (figure 6).98,99 The Maze III procedure became a well-accepted and widely used therapy for drug-refractory symptomatic atrial fibrillation and was long-time considered as the golden standard for non-pharmacological treatment of atrial fibrillation.

Modified Maze procedureDespite its high success rate, the Maze III operation was not widely adopted and the classical ‘cut-and-sew’ method is nowadays almost totally abandoned. The reason was that the procedure is time consuming, technically challenging, requires a sternotomy and involves the use of extra-corporeal circulation. Especially for lone atrial fibrillation, the morbidity and mortality caused by the operation is large in comparison to the gravity of the disease. Not only disadvantages inherent to the Maze operation, but also the development of ablation devices that were able to create lines of non-conductive tissue, changed surgery for atrial fibrillation. These ablation devices replaced the time consuming ‘cut-and-sew’ technique and led amongst others to the Maze IV procedure.100 This procedure strongly resembled the Maze III procedure, but uses mostly radiofrequency ablation to create the lines of non-conductive tissue.100


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At the top the incisions in the right atrium are depicted. First the right atrial appendage is amputated. From this a short perpendicular incision in the free wall is made and an incision to the tricuspid annulus which is completed by a cryo-lesion. This is followed by an atriotomy between the superior caval and inferior caval vein. From this incision a second incision to the tricuspid annulus is made and also completed by a cryo-lesion. Finally a septal incision is made to provide access to the left atrium.

At the bottom the incisions in the left atrium are depicted. First, the left atrial appendage is amputed. The septal incision is extended to form a button around the pulmonary veins and is connected to the incision of the amputation of the left atrial appendage. From the button an incision to the mitral valve annulus is made and completed with a cryo-lesion. Reproduced with permission from the publisher114

Picture 6. Drawing of the classical Maze III procedure. The Maze III procedure creates a labyrinth in the atria by a fixed pattern of lines which are created by cutting and sewing the myocardium.

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The last factor that led to further abolishment of the Maze III procedure is our understanding of atrial fibrillation. The Maze III operation was totally based on the idea that atrial fibrillation existed of multiple wandering reentry wavelets. In fact Cox and co-workers wrote: “Despite these observations, the erroneous concept that atrial fibrillation was caused by multiple autonomic foci persisted in the medical literature for the next 35 to 40 years”.101 While during atrial fibrillation multiple reentry wavelets are indeed presents, the importance of autonomic foci became clear in the late 1990s when Haïssaguerre and co-workers showed the importance of ectopic foci located in the pulmonary vein.35 From that time onward, both cardiologist and cardiac surgeons focused on the pulmonary veins in the treatment of, especially, lone paroxysmal atrial fibrillation. Many variations on the classical Maze procedure have been in use since the development of the Maze III procedure. These variations included both the type of ablation technique and the pattern of (ablation) lines.102 Different studies suggest that these modified procedures are as effective as the classical Maze III procedure.102-104

Pulmonary vein isolation A broad scala of different techniques of percutaneous catheter ablations have been reported. Some even mimic parts of the Maze procedure.105 While the long-term success of surgery for atrial fibrillation is well established, the long-term success of catheter ablations for persistent atrial fibrillation remains controversial.106,107

While percutaneous catheter techniques are often the first choice for drug-refractory symptomatic atrial fibrillation, new minimally invasive surgical techniques have been developed.108 Surgical isolation of the pulmonary veins has 3 major advantages compared to catheter based ablation. First, surgical epicardial ablation is performed under direct vision, while endocardial is dependent on navigation systems and fluoroscopy. Second, ganglion plexus can be detected and ablated. Third, the left atrial appendage can be amputated, which is thought to be an important source of thrombo-emboli.109 In 2005 Wolf and co-workers described a minimally invasive procedure during which they isolated the pulmonary veins by bipolar radiofrequency ablation, ablated the ganglionic plexus and amputated the left atrial appendage.108 This


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technique was later modified to a completely thoracoscopic procedure.110

Hybrid epicardial-endocardial approachThe thoracoscopic epicardial approach for atrial fibrillation has also some disadvantages. Confirmation of pulmonary vein isolation can be difficult, a ‘classical’ ablation line to the mitral valve annulus is not possible and confirmation of conduction block of additional lines is dependent on custom made catheters.87 Recently, some groups are performing simultaneous epicardial and endocardial approaches to treat atrial fibrillation.111-113 First results are promising, but a clear disadvantage is that it needs a hybrid operation room which also harbors the possibility of fluoroscopy.

Concluding remarksOver the last decades enormous progress has been made in understanding the pathophysiology and etiology of atrial fibrillation. Non-pharmacological therapy became successful after the development of the Maze operation in the 1990s. From then on an evolution of techniques and recognition of the importance of the pulmonary vein has led to a broad range of both surgical and catheter based therapies. Nowadays, both percutaneous endocardial and surgical epicardial ablation are highly successful for especially lone paroxysmal atrial fibrillation with limited peri-procedural morbidity.

Aim and outline of the thesisThis thesis is collaboration between the departments of cardiothoracic surgery of the Sint Antonius Hospital in Nieuwegein and Experimental Cardiology of the Academic Medical Center of Amsterdam. The aim of the thesis was to assess the success of different surgical techniques as treatment of atrial fibrillation and to obtain a better insight in the role of fibrosis and autonomic nervous in atrial fibrillation. In Chapter 2, 3 and 4 we will address the question of the success rates of the classical Maze III procedure, of modified Maze procedures and of a completely thoracoscopic pulmonary vein isolation, respectively. In Chapter 5 we will address the question whether more fibrosis is present in atria of patients with concomitant atrial fibrillation than in atria of

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patients with lone atrial fibrillation, and whether the left and right atria are equally affected. In Chapter 6 we will describe the effect of noradrenalin or acetylcholine on the action potential of atrial and automatic (sinoatrial nodal) cells. Finally, in Chapter 7 we will describe the electrophysiological effect and ionic mechanism of the neurotransmitter substance-P on single atrial myocytes.


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2. Go AS, Hylek EM, Phillips KA, Chang Y, Henault LE, Selby JV, Singer DE. Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) Study. JAMA 2001;285:2370-2375

3. Feinberg WM, Blackshear JL, Laupacis A, Kronmal R, Hart RG. Prevalence, age distribution, and gender of patients with atrial fibrillation. Analysis and implications. Arch Intern Med 1995;155:469-473

4. Benjamin EJ, Levy D, Vaziri SM, D’Agostino RB, Belanger AJ, Wolf PA. Independent risk factors for atrial fibrillation in a population-based cohort. The Framingham Heart Study. JAMA 1994;271:840-844

5. Psaty BM, Manolio TA, Kuller LH, Kronmal RA, Cushman M, Fried LP, White R, Furberg CD, Rautaharju PM. Incidence of and risk factors for atrial fibrillation in older adults. Circulation 1997;96:2455-21461

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55. Hocini M, Ho SY, Kawara T, Linnenbank AC, Potse M, Shah D, Jaïs P, Janse MJ, Haïssaguerre M, De Bakker JM. Electrical conduction in canine pulmonary veins: electrophysiological and anatomic correlation. Circulation 2002;105:2442-2448

56. Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation 1995;92:1954-1968

57. de Vos CB, Pisters R, Nieuwlaat R, Prins MH, Tieleman RG, Coelen RJ, van den Heijkant AC, Allessie MA, Crijns HJ. Progression from paroxysmal to persistent atrial fibrillation clinical correlates and prognosis. J Am Coll Cardiol 2010;55:725-731

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58. Allessie M, Ausma J, Schotten U. Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovasc Res 2002;54:230-246

59. Schotten U, Duytschaever M, Ausma J, Eijsbouts S, Neuberger HR, Allessie M. Electrical and contractile remodeling during the first days of atrial fibrillation go hand in hand. Circulation 2003;107:1433-1439

60. Morillo CA, Klein GJ, Jones DL, Guiraudon CM. Chronic rapid atrial pacing. Structural, functional, and electrophysiological characteristics of a new model of sustained atrial fibrillation. Circulation 1995;91:1588-1595

61. Yue L, Feng J, Gaspo R, Li GR, Wang Z, Nattel S. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ Res 1997;81:512-525

62. Dobrev D, Graf E, Wettwer E, Himmel HM, Hála O, Doerfel C, Christ T, Schüler S, Ravens U. Molecular basis of downregulation of G-protein-coupled inward rectifying K(+) current (I(K,ACh) in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced I(K,ACh) and muscarinic receptor-mediated shortening of action potentials. Circulation 2001;104:2551-2557

63. Ausma J, Wijffels M, Thoné F, Wouters L, Allessie M, Borgers M. Structural changes of atrial myocardium due to sustained atrial fibrillation in the goat. Circulation 1997;96:3157-3163

64. Logan WF, Rowlands DJ, Howitt G, Holmes AM. Left atrial activity following cardioversion. Lancet 1965;2:471-473

65. Manning WJ, Silverman DI, Katz SE, Riley MF, Come PC, Doherty RM, Munson JT, Douglas PS. Impaired left atrial mechanical function after cardioversion: relation to the duration of atrial fibrillation. J Am Coll Cardiol 1994;23:1535-1540

66. Leistad E, Aksnes G, Verburg E, Christensen G. Atrial contractile dysfunction after short-term atrial fibrillation is reduced by verapamil but increased by BAY K8644. Circulation 1996;93:1747-1754

67. Daoud EG, Marcovitz P, Knight BP, Goyal R, Man KC, Strickberger SA, Armstrong WF, Morady F. Short-term effect of atrial fibrillation on atrial contractile function in humans. Circulation 1999;99:3024-3027

68. Burstein B, Nattel S. Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation. J Am Coll Cardiol 2008;51:802-809

69. Frustaci A, Chimenti C, Bellocci F, Morgante E, Russo MA, Maseri A. Histological substrate of atrial biopsies in patients with lone atrial fibrillation. Circulation 1997;96:1180-1184

70. Boldt A, Wetzel U, Lauschke J, Weigl J, Gummert J, Hindricks G, Kottkamp H, Dhein S. Fibrosis in left atrial tissue of patients with atrial fibrillation with and without underlying mitral valve disease. Heart 2004;90:400-405


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71. Verheule S, Sato T, Everett T 4th, Engle SK, Otten D, Rubart-von der Lohe M, Nakajima HO, Nakajima H, Field LJ, Olgin JE. Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-beta1. Circ Res 2004;94:1458-1465

72. de Bakker JM, van Capelle FJ, Janse MJ, Tasseron S, Vermeulen JT, de Jonge N, Lahpor JR. Slow conduction in the infarcted human heart. ‘Zigzag’ course of activation. Circulation 1993;88:915-926

73. Hoogendijk MG, Potse M, Vinet A, de Bakker JM, Coronel R. ST segment elevation by current-to-load mismatch: an experimental and computational study. Heart Rhythm 2011;8:111-118

74. Rohr S, Salzberg BM. Characterization of impulse propagation at the microscopic level across geometrically defined expansions of excitable tissue: multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures. J Gen Physiol 1994;104:287-309

75. Spach MS, Josephson ME. Initiating reentry: the role of nonuniform anisotropy in small circuits. J Cardiovasc Electrophysiol 1994;5:182-209

76. Coumel P. Paroxysmal atrial fibrillation: a disorder of autonomic tone? Eur Heart J 1994;15 Suppl A:9-16

77. de Vos CB, Nieuwlaat R, Crijns HJ, Camm AJ, LeHeuzey JY, Kirchhof CJ, Capucci A, Breithardt G, Vardas PE, Pisters R, Tieleman RG. Autonomic trigger patterns and anti-arrhythmic treatment of paroxysmal atrial fibrillation: data from the Euro Heart Survey. Eur Heart J 2008;29:632-639

78. Armour JA, Murphy DA, Yuan BX, Macdonald S, Hopkins DA. Gross and microscopic anatomy of the human intrinsic cardiac nervous system. Anat Rec 1997;247:289-298

79. Pauza DH, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart. Anat Rec 2000;259:353-382

80. Hoover DB, Isaacs ER, Jacques F, Hoard JL, Pagé P, Armour JA. Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia. Neuroscience 2009;164:1170-1179

81. Zipes DP, Mihalick MJ, Robbins GT. Effects of selective vagal and stellate ganglion stimulation of atrial refractoriness. Cardiovasc Res 1974;8:647-655

82. Liu L, Nattel S. Differing sympathetic and vagal effects on atrial fibrillation in dogs: role of refractoriness heterogeneity. Am J Physiol 1997;273:H805-H816

83. Scherlag BJ, Patterson E, Po SS. The neural basis of atrial fibrillation. J Electrocardiol 2006;39:S180-S183

84. He B, Scherlag BJ, Nakagawa H, Lazzara R, Po SS. The intrinsic autonomic nervous system in atrial fibrillation: a review. ISRN Cardiol 2012;2012:490674

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85. Scherlag BJ, Nakagawa H, Jackman WM, Yamanashi WS, Patterson E, Po S, Lazzara R. Electrical stimulation to identify neural elements on the heart: their role in atrial fibrillation. J Interv Card Electrophysiol 2005;13:37-42

86. Pokushalov E, Romanov A, Artyomenko S, Turov A, Shirokova N, Katritsis DG. Left atrial ablation at the anatomic areas of ganglionated plexi for paroxysmal atrial fibrillation. Pacing Clin Electrophysiol 2010;33:1231-1238

87. Krul SP, Driessen AH, van Boven WJ, Linnenbank AC, Geuzebroek GS, Jackman WM, Wilde AA, de Bakker JM, de Groot JR. Thoracoscopic video-assisted pulmonary vein antrum isolation, ganglionated plexus ablation, and periprocedural confirmation of ablation lesions: first results of a hybrid surgical-electrophysiological approach for atrial fibrillation. Circ Arrhythm Electrophysiol 2011;4:262-270

88. Pappone C, Santinelli V, Manguso F, Vicedomini G, Gugliotta F, Augello G, Mazzone P, Tortoriello V, Landoni G, Zangrillo A, Lang C, Tomita T, Mesas C, Mastella E, Alfieri O. Pulmonary vein denervation enhances long-term benefit after circumferential ablation for paroxysmal atrial fibrillation. Circulation 2004;109:327-334

89. Scherlag BJ, Yamanashi W, Patel U, Lazzara R, Jackman WM. Autonomically induced conversion of pulmonary vein focal firing into atrial fibrillation. J Am Coll Cardiol 2005;45:1878-1886

90. Williams JM, Ungerleider RM, Lofland GK, Cox JL. Left atrial isolation: new technique for the treatment of supraventricular arrhythmias. J Thorac Cardiovasc Surg 1980;80:373-380

91. Guiraudon GM, Campbell CS, Jones DL, McLellan DG, MacDonald, JL. Combined sinoatrial node atrioventricular node isolation: a surgical alternative to His bundle ablation in patients with atrial fibrillation. Circulation 1985;72:220

92. Defauw JJ, Guiraudon GM, van Hemel NM, Vermeulen FE, Kingma JH, de Bakker JM. Surgical therapy of paroxysmal atrial fibrillation with the “corridor” operation. Ann Thorac Surg 1992;53:564-570

93. Leitch JW, Klein G, Yee R, Guiraudon G. Sinus node-atrioventricular node isolation: long-term results with the “corridor” operation for atrial fibrillation. J Am Coll Cardiol 1991;17:970-975.

94. Graffigna A, Pagani F, Minzioni G, Salerno J, Viganò M. Left atrial isolation associated with mitral valve operations. Ann Thorac Surg 199;54:1093-1097.

95. van Hemel NM, Defauw JJ, Kingma JH, Jaarsma W, Vermeulen FE, de Bakker JM, Guiraudon GM. Long-term results of the corridor operation for atrial fibrillation. Br Heart J. 1994;71:170-176.

96. Cox JL, Schuessler RB, D’Agostino HJ Jr, Stone CM, Chang BC, Cain ME, Corr PB, Boineau JP. The surgical treatment of atrial fibrillation. III. Development of a definitive surgical procedure. J Thorac Cardiovasc Surg 1991;101:569-583

97. Cox JL. The surgical treatment of atrial fibrillation. IV. Surgical technique. J Thorac Cardiovasc Surg 1991;101:584-592


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98. Cox JL, Boineau JP, Schuessler RB, Jaquiss RD, Lappas DG. Modification of the Maze procedure for atrial flutter and atrial fibrillation. I. Rationale and surgical results. J Thorac Cardiovasc Surg. 1995;110:473-484

99. Cox JL, Jaquiss RD, Schuessler RB, Boineau JP. Modification of the Maze procedure for atrial flutter and atrial fibrillation. II. Surgical technique of the Maze III procedure. J Thorac Cardiovasc Surg 1995;110:485-495

100. Gaynor SL, Diodato MD, Prasad SM, Ishii Y, Schuessler RB, Bailey MS, Damiano NR, Bloch JB, Moon MR, Damiano RJ Jr. A prospective, single-center clinical trial of a modified Cox Maze procedure with bipolar radiofrequency ablation. J Thorac Cardiovasc Surg 2004;128:535-542

101. Cox JL, Schuessler RB, Boineau JP. The surgical treatment of atrial fibrillation. I. Summary of the current concepts of the mechanisms of atrial flutter and atrial fibrillation. J Thorac Cardiovasc Surg 1991;101:402-405

102. Khargi K, Hutten BA, Lemke B, Deneke T. Surgical treatment of atrial fibrillation; a systematic review. Eur J Cardiothorac Surg 2005;27:258-265

103. Wang J, Meng X, Li H, Cui Y, Han J, Xu C. Prospective randomized comparison of left atrial and biatrial radiofrequency ablation in the treatment of atrial fibrillation. Eur J Cardiothorac Surg 2009;35:116-122

104. Albrecht A, Kalil RA, Schuch L, Abrahão R, Sant’Anna JR, de Lima G, Nesralla IA. Randomized study of surgical isolation of the pulmonary veins for correction of permanent atrial fibrillation associated with mitral valve disease. J Thorac Cardiovasc Surg 2009;138:454-459

105. Pappone C, Manguso F, Vicedomini G, Gugliotta F, Santinelli O, Ferro A, Gulletta S, Sala S, Sora N, Paglino G, Augello G, Agricola E, Zangrillo A, Alfieri O, Santinelli V. Prevention of iatrogenic atrial tachycardia after ablation of atrial fibrillation: a prospective randomized study comparing circumferential pulmonary vein ablation with a modified approach. Circulation 2004;110:3036-3042

106. Weerasooriya R, Khairy P, Litalien J, Macle L, Hocini M, Sacher F, Lellouche N, Knecht S, Wright M, Nault I, Miyazaki S, Scavee C, Clementy J, Haissaguerre M, Jais P. Catheter ablation for atrial fibrillation: are results maintained at 5 years of follow-up? J Am Coll Cardiol 2011;57:160-166

107. Tilz RR, Rillig A, Thum AM, Arya A, Wohlmuth P, Metzner A, Mathew S, Yoshiga Y, Wissner E, Kuck KH, Ouyang F. Catheter ablation of long-standing persistent atrial fibrillation: 5-year outcomes of the hamburg sequential ablation strategy. J Am Coll Cardiol 2012;60:1921-1929

108. Wolf RK, Schneeberger EW, Osterday R, Miller D, Merrill W, Flege JB, Gillinov AM. Video-assisted bilateral pulmonary vein isolation and left atrial appendage exclusion for atrial fibrillation. J Thorac Cardiovasc Surg 2005;130:797-802

109. Halperin JL, Hart RG. Atrial fibrillation and stroke: new ideas, persisting dilemmas. Stroke. 1988;19:937-941

110. Yilmaz A, Van Putte BP, Van Boven WJ. Completely thoracoscopic bilateral pulmonary vein isolation and left atrial appendage exclusion for atrial fibrillation. J Thorac Cardiovasc Surg 2008;136:521-522

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111. Mahapatra S, LaPar DJ, Kamath S, Payne J, Bilchick KC, Mangrum JM, Ailawadi G. Initial experience of sequential surgical epicardial-catheter endocardial ablation for persistent and long-standing persistent atrial fibrillation with long-term follow-up. Ann Thorac Surg 2011;91:1890-1898

112. Gehi AK, Paul Mounsey J, Pursell I, Landers M, Boyce K, Chung EH, Schwartz J, Jennifer Walker T, Guise K, Kiser AC. Hybrid epicardial-endocardial ablation using a pericardioscopic technique for the treatment of atrial fibrillation. Heart Rhythm 2013;10:22-28

113. La Meir M, Gelsomino S, Lucà F, Pison L, Colella A, Lorusso R, Crudeli E, Gensini GF, Crijns HG, Maessen J. Minimal invasive surgery for atrial fibrillation: an updated review. Europace 2013;15:170-182

114. Ballaux PK, Geuzebroek GS, van Hemel NM, Kelder JC, Dossche KM, Ernst JM, Boersma LV, Wever EF, Brutel de la Rivière A, Defauw JJ. Freedom from atrial arrhythmias after classic Maze III surgery: a 10-year experience. J Thorac Cardiovasc Surg 2006;132:1433-1440


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Freedom from atrial arrhythmias after classic Maze III surgery: A 10-year experience

2

Ph.K.E.W. BallauxG.S.C. Geuzebroek

N.M. van HemelJ.C. Kelder

K.M.E. DosscheJ.M.P.G. Ernst

L.V.A. BoersmaE.F.D. Wever

A. Brutel de la RevièreJ.J.A.M.T. Defauw

J Thorac Cardiovasc Surg 2006;132:1433-1440

Chapter 2

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AbstractObjectives: We studied the persistence of favorable outcome, the occurrence of new atrial arrhythmias, and sinus node dysfunction in patients who underwent the Maze III procedure.

Methods: Preoperative, in-hospital, and follow-up data of 203 patients who underwent the Maze III procedure between June 1993 and June 2003 were collected. A total of 139 patients underwent the Maze procedure for lone atrial fibrillation, and 64 patients underwent the Maze procedure and concomitant cardiac surgery.

Results: There was no 30-day postoperative mortality. During a mean follow-up of 4.0 ± 2.6 years, 12 patients (6%) died (2 cardiac related). At the end of follow-up, freedom from supraventricular arrhythmias was 80% for the lone atrial fibrillation group and 64% for the concomitant atrial fibrillation group. Freedom from stroke during follow-up was 100% in the lone atrial fibrillation group and 97% in the concomitant group. Multivariate analysis revealed that rhythm at 1-year follow-up (P <.001; odds ratio 9.56, 95% confidence limits 3.92-23.31) and preoperative left atrium dimension (P = .028; odds ratio 1.06 for every millimeter, 95% confidence limits 1.01-1.12) were predictors of success at the end of follow-up.

Conclusions: This study shows that the favorable results of the Maze III procedure in terms of freedom from supraventricular arrhythmias persist in most patients for at least 4 years.

Maze III surgery for atrial fibrillation

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IntroductionThe unmodified Maze III operation is considered the most successful design for exclusion, and channeling of left atrial (LA) and right atrial (RA) areas to eliminate atrial fibrillation (AF) resulting from random reentry or initiated by rapidly firing foci.1,2 The surgically created pathways preclude AF, preserve sinus node function, and maintain atrial contraction and filling in the majority of patients who undergo the procedure. The avoidance of intraoperative mapping of atrial arrhythmias is a clear advantage. Since 1990, many reports have addressed the favorable effects of Maze III surgery in patients undergoing concomitant cardiac surgery, predominantly mitral valve surgery.3-10 However, there are few reports focusing on Maze III surgery performed exclusively for symptomatic and drug-refractory AF without detectable cause (lone AF).4,6,11,12

The results of percutaneous methods to abolish AF with radiofrequency or ice-cooled catheter ablation techniques, of which the design and landmarks frequently mimic those of the Maze III surgery, are improving.2,13,14 Until now it is unclear how to identify patients with AF who most profit from the various nonpharmacologic AF therapies. Thus far, patients treated with catheter ablation have predominantly paroxysmal AF (PAF). Despite the absence of associated cardiac disease, smaller left atria, and shorter follow-up, the success rate cannot match that of Maze surgery.

Our 10-year experience with Maze III surgery enabled us to determine whether the initially favorable effects of this surgery continue in the long term. For this reason we performed a single-center outcome study to define the predictors of long-term AF suppression and to evaluate the maintenance of normal sinus node function and freedom from other supraventricular arrhythmias.

Material and MethodsInclusion for Maze SurgeryPatients with symptomatic PAF or chronic AF (CAF) were offered Maze III surgery. AF was classified as paroxysmal if spontaneous episodes of sinus rhythm intervened with bouts of AF. AF without documented sinus rhythm for more than 1 week

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was classified as CAF. The classification of persistent and permanent AF could not be applied because this surgery was first performed in 1993, whereas the AF classification was established later.15 In the lone AF group, failure of more than 3 anti-arrhythmic drugs and more than 1 attempted DC cardioversion were required before acceptance for surgery. In the concomitant AF group, these criteria were not a prerequisite for cardiac surgery. Reasons to exclude patients from Maze surgery were documented incompetent sinus node,12 LA diameter greater than 90 mm,16 impaired left and/or right ventricular function,17 and calcified LA wall. Previous failed radiofrequency catheter ablation of the atria to eradicate AF was no reason to exclude a patient from Maze surgery. Verbal consent for surgery was obtained after extensive oral information about the surgery and its alternatives was given.

Preoperative StudiesThe years of presence of PAF or CAF were determined. The number of failed antiarrhythmic drugs and DC cardioversions were noted. Standard electrocardiogram, 2-channel Holter recordings, and bicycle stress testing were used to document AF and to exclude sinus node disease and abnormal cardiac conduction. Two-dimensional and pulsed-wave Doppler echocardiographic studies were performed to measure cardiac chamber size and function and to diagnose structural cardiac disease. Left and right sided heart catheterization and coronary angiographies were performed to identify abnormalities.

Surgical ProcedureThe Maze III operation was carried out as described in the initial reports18 and as mentioned before by our group (Figure 1).12

Postoperative MethodsAntiarrhythmic drugs were administered if AF persisted more than 12 hours. Electrical cardioversion was carried out if antiarrhythmic drugs failed. The temporary epicardial pacemaker wires served to pace in case of bradycardia, to diagnose postoperative arrhythmias and, in subgroups, to attempt to induce AF. If sinus node function failed after more than 14 days postsurgery, a rate-responsive atrioventricular (AV) pacemaker was implanted.


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Figure 1. Unmodified Maze III procedure. A Incisions on the right atrium. Amputation of the right auricle and from this opening 2 perpendicular incisions, 1 in the free wall and 1 toward the tricuspid annulus, where a cryolesion is placed at the annulus. A longitudinal atriotomy lateral in the superior caval vein extends to the inferior caval vein. A perpendicular incision from the longitudinal incision to the tricuspid annulus, where a second cryolesion is placed. An incision is placed in the septum. B Incisions on the left atrium. Amputation of the left auricle. The pulmonary veins are separated from the remainder to the heart in a button. Two connecting lesions from the button are made: 1 to the left auricle and 1 to the mitral valve. One cryolesion is placed at the mitral annulus and one at the coronary sinus, which is exposed through this last incision.

A

B

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Follow-up StudiesThe majority of patients who underwent surgery for lone AF were followed at our own outpatient department at 6 weeks, 3 and 6 months, and then annually. Patients were asked about palpitations, symptoms of cerebrovascular accidents (CVAs), and daily lifestyle and exercise tolerance. Holter monitoring and bicycle stress testing19 were performed (systematically at our own institution for approximately half of the series and sporadically at other institutions) to detect atrial arrhythmias and sinus node incompetence. Echocardiographic studies were performed. Most of the patients who underwent surgery for concomitant AF were followed by the referring cardiologist, who was responsible for the timing and examinations of the follow-up.

Collection of DataThe inpatient and outpatient clinic files were searched for baseline, 1-year follow-up, and end of follow-up data. If data were incomplete, questions about follow-up were mailed to the referring cardiologist for the completion of latest follow-up data. If the investigators were not able to procure recent or complete information, the general practitioner was also consulted. As a last resort, the patients were contacted by phone. This was the case for only 5 patients. For the patients who most recently underwent operation, we only accepted a follow-up of at least 6 months. Follow-up was gathered from December 1, 2003, to March 31, 2004.

Variables Studied During Follow-upThe time and reason of death were noted. Rhythm was documented at every contact with the patient. Echocardiographic data were examined at 1 year and at the end of follow-up, including LA diameter (long axis) in the parasternal view, largest RA diameter, grade of mitral valve regurgitation, and left ventricular ejection fraction. The reason for pacemaker implantation and occurrence of CVA and transient ischemic attack were noted. The use of oral anticoagulation and antiarrhythmic drugs was studied. Antiarrhythmic drugs included all beta-blockers and calcium antagonists prescribed as antihypertensive treatment but with a possible heart rate-slowing effect. Postoperative occurrence of congestive heart failure, onset of valvular pathology, and DC cardioversion were noted.


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DefinitionsAll foramen ovale or atrial septal defect closures without a patch were considered to be found coincidentally and categorized as ‘lone AF surgery’. All atrial septal defects requiring a patch were considered to be a separate cardiac abnormality and qualified as ‘concomitant AF surgery’.

A regular atrial rhythm less than 100 beats/min at rest was labeled as normal sinus rhythm. Whenever AF occurred, the patient was categorized as having AF. All runs of 3 or more atrial beats were categorized as supraventricular tachycardia.

Success was labeled as the absence of symptomatic or asymptomatic AF, atrial flutter (AFL), and other atrial tachyarrhythmias more than 6 months after surgery. Implantation of a pacemaker for incompetent sinus node function or AV block did not rule out ‘success’.

Failure was defined as symptomatic or asymptomatic AF, AFL, and other atrial tachyarrhythmias occurring more than 6 months after surgery. Pacing after His bundle ablation for intractable atrial arrhythmias was also categorized as a failure.

Statistical AnalysisAll values are expressed as mean ± standard deviation. For hypothesis testing in categoric variables, the Fisher exact test was used. For normally distributed data, a paired or an unpaired t test was used. Non-normally distributed data were compared by the Mann-Whitney U test. Kaplan-Meier curves for freedom from AF could not be constructed because the precise time of AF recurrence after the operation could often not be determined. A multivariate analysis was carried out with the use of logistic regression to determine factors influencing surgical success (see definition) more than 1 year after surgery. The following factors were considered: age, type of preoperative AF, preoperative duration of AF longer or less than 5 years, preoperative CVA or transient ischemic attack, history of preoperative radiofrequency ablation, concomitant procedure or not, aortic crossclamp and cardiopulmonary bypass time, and highest degree of postoperative AV block. All calculations were performed with SAS v 8.2 (SAS Inc, Cary, NC).

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ResultsPreoperative DataBetween June 1993 and June 2003, 203 consecutive patients underwent the unmodified Maze III operation: 139 for lone AF and 64 for a concomitant procedure. AF caused a mean of 2.5 symptoms before surgery; the most frequent symptoms in the lone group were palpitations (83.2%), fatigue (46.0%), dyspnea on exertion (33.6%), vertigo (20.4%), chest pain (18.2%), syncope (8.0%), and decreased exertion capacity (8.0%). In the concomitant group, symptoms were hard to attribute specifically to AF because of the associated pathology. Five patients in the concomitant group experienced congestive heart failure preoperatively. In the lone AF group, male gender dominated, and patients were younger, had more PAF, and showed smaller atrial dimensions than the concomitant AF group (Table 1). The duration of AF could be determined more accurately in the lone group and is probably underestimated in the concomitant AF group. The high number of different antiarrhythmic drugs tried and the number of failed catheter ablations and DC cardioversions performed reflect the large effort to prevent and treat AF in the lone AF group (Table 1).

Perioperative DataAs expected, the duration of extracorporeal circulation and crossclamp time were considerably longer in patients with concomitant surgery (Table 2). In the lone AF group, 8 atrial septal defects/foramen ovale were closed, and 1 strumectomy and 1 open lung biopsy were performed. In 64 patients in whom surgery was combined with the Maze operation, 77 procedures were carried out, including 27 mitral valve replacements, 24 mitral valve repairs, 10 coronary artery bypass grafts, 6 tricuspid valve repairs, 8 atrial septal defect closures, 1 aortic valve replacement, and 1 transection of a Kent bundle for Wolff-Parkinson-White syndrome. There was no difference in postoperative AV block, grading of the AV block, and incidence of temporary pacing between the 2 groups (Table 2).


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There was no 30-day mortality. In 33 patients of the lone AF group, 46 in-hospital complications were identified: tamponade in 5%; fever of unknown origin, congestive heart failure, and pneumonia in 4.6% each; and pneumothorax, lung atelectasis, bleeding and rethoracotomy, urinary retention, and pleural effusion in 1.4% each. In the concomitant group 30 patients experienced 33 postoperative complications: tamponade in 7.7%; bronchitis/pneumonia in 7.7%; bleeding and rethoracotomy in 4.6%; transient peripheral neurologic deficit in 4.6%; and congestive heart failure, urinary tract infection, permanent neurologic deficit, and atelectasis in 3.1% of the patients. In 1 patient the right coronary artery located in the wall of the RA was damaged, which was repaired with a venous bypass graft.

Table 1. Baseline characteristics of 203 patients treated with Maze III surgery.

Lone AF 139 (68%)

Concomitant procedures

64 (32%)

P value

Male/female (%) 85.6/14.4 65.6/34.4 .003 Mean age (y) 50.7 ± 8.1 58.1 ± 10.9 <.0001 Duration of AF (No. of patients) Mean (months) Minimum Maximum

(139) 84 ± 60.7

8 360

(56) 51 ± 49.8

2 216

<.001

Type AF (No. of patients) Paroxysmal Chronic

(139) 108 (77.7%) 31 (22.3%)

(63) 34 (53.1%) 30 (46.9%)

<.001

LA diameter (No. of patients) Mean (mm) Minimum Maximum

(124) 41.9 ± 7.6

28 68

(57) 51.7 ± 9.8

35 74

<.001

RA diameter (No. of patients) mean (mm) Minimum Maximum

(90) 53.0 ± 6.1

38 70

(12) 57.0 ± 6.9

44 64

.04

Preoperative CVA Preoperative TIA

3 (2.2%) 7 (5.0%)

2 (3.1%) 3 (4.7%)

.65 1

Antiarrhythmic drugs (No. of patients) Drugs per patient

(132) 4.9 ± 1.7

(62) 2.5 ± 1.6

<0.001

No. of patients with previous 1 or more treatments for arrhythmia

22 (15.8%) 3 (4.6%) .023

Mean No. of preoperative electrical cardioversions 3.0 1.2 .002

CVA, cerebrovascular accident; TIA, transient ischemic attack; AF, atrial fibrillation; LA, left atrial; RA, right atrial.

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Follow-up ObservationsData were complete in 84.7% for the 1-year follow-up and in 97% for the latest follow-up. The total follow-up time was 796 patient years with a mean of 4.0 ± 2.6 years per patient.

Twelve patients (5.9%) died during follow-up, 1 because of colon carcinoma in the lone AF group (0.7%). Two of the other 11 deaths (17.2%) in the concomitant AF group were attributable to cardiac complications: asystole (1) and ventricular fibrillation (1). The remainder were attributable to carcinoma (4), cerebral sarcoidosis (1), CVA (1), dementia (1), and unknown cause (2).

Rhythm ResultsThe rhythms at the end of follow-up are depicted in Figure 2. Freedom from AF at the latest follow-up was 89.7% for the lone AF group and 69.4% for the concomitant group. By applying our definition of success, freedom from any atrial arrhythmia was 77.7% and 76.9% at 1 year and 80.1% and 64.5% at the latest follow-up for

Table 2. Perioperative data.

Lone AF 139 (68%)

Concomitant procedures

64 (32%)

P value

Duration of ECC (No. of patients) Mean (minutes) Minimum Maximum

(136) 114 ± 18.5

76 182

(63) 148 ± 30.6

84 259

<.0001

Duration of Aoclamp (No. of patients) Mean (minutes) Minimum Maximum

(136) 61 ± 18.1

37 149

(63) 90 ± 22.5

60 150

<.0001

No. of additional flutter treatment 3 (2.2%) 0 .553 Transient in-hospital AV block (no. of patients) 1 2 3 total

(135)

28 (20.5%) 11 (8.1%) 1 (0.7%)

16 (11.9%)

(64)

19 (29.2%) 8 (12.3) 3 (4.6)

8 (12.3)

.214 Patients needing pacing (no. of patients) Temporary Definitive Total

(139) 69 (49.6%)

2 (1.4%) 71 (51.1%)

(64) 31 (48.4%)

1 (1.6%) 32 (50%)

.763 Duration of hospital stay (days) Median

9.0 ± 4.0

10.0 ± 5.3

<.001

ECC, Extracorporeal circulation; AV, atrioventricular.


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the lone AF group and the concomitant AF group, respectively (Figure 3). In the lone AF group, 41.1% of the patients with supraventricular arrhythmias were free from palpitations; this was 30.0% in the concomitant AF group. During follow-up, transcutaneous catheter ablation was carried out in 3 patients for recurrent drug-refractory AF but was successful in only 1 patient. In the remaining 2 patients, drug treatment was continued with other combinations.

Figure 2. Rhythms at the end of follow-up. FU, Follow-up; PMAF, atrial fibrillation and pacemaker; chron AF, chronic atrial fibrillation; SVT, supraventricular tachycardia; AFL, atrial flutter; PAF, paroxysmal atrial fibrillation; PMfreeAF, freedom from atrial fibrillation with pacemaker implanted; SR, sinus rhythm; AF, atrial fibrillation.

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For the whole group, multivariate analysis revealed that (1) surgical success (see definition) at the end of follow-up was strongly related to the absence of AF at 1 year after surgery (P < .001; odds ratio [OR] 9.56, 95% confidence limit (CL) 3.92-23.31); (2) preoperative LA diameter correlated with the occurrence of AF at the end of follow-up (P = .028; OR 1.06 for every millimeter, CL 1.01-1.12); and (3) failure at 1-year follow-up was related to the preoperative duration of AF of more than 5 years (P = .066; OR 2.06, CL 0.95-4.46). No correlation was found between the preoperative duration of AF and the latest follow-up results.

We could not demonstrate a significant (P >.05) difference of success between the lone AF group and the concomitant AF group.

Of the patients with surgical success, freedom from antiarrhythmic medication at the latest follow-up was 75.9% in the lone group and 63.2% in the concomitant group. Freedom from oral anticoagulants was 90.7% in the lone AF group and 50.9% in the concomitant AF group. Eighty-eight percent of the patients of the concomitant AF group without mechanical valves did not use anticoagulation.

Figure 3. Surgical success and freedom from AF of the Maze III procedure at 1 year and end of follow-up. AF, Atrial fibrillation; FU, follow-up.


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Pacemaker ImplantationAt the latest follow-up, 14 patients (10.4%) in the lone AF group and 7 patients (11.1%) in the concomitant group had a pacemaker. Two of the pacemakers in patients of the lone AF group were already implanted before the Maze operation. In the lone AF group, pacemakers were implanted for sick sinus syndrome (6), for total AV block (2), for bradycardia (2), and after His ablation (4). In the concomitant group, pacemakers were implanted for sick sinus syndrome (3), for total AV block (1), and after His ablation (3).

StrokeIn the lone AF group, no CVA occurred during follow-up. In the concomitant AF group, 2 patients had a CVA. One patient had an intracerebral bleed attributable to an elevated international normalized ratio (5.7); he used oral anticoagulants for a mechanical valve in the mitral position. The other patient had an embolic stroke while using oral anticoagulants after mitral valve repair. Both patients were in sinus rhythm.

Sinus Node FunctionOne half of the patients in the lone AF group (63/139) underwent maximal heart rate exercise testing during follow-up in the appropriate condition: no heart-slowing medication and not in AF or other atrial tachyarrhythmias. The mean maximal heart rate was 90% ± 3% of the predicted value. In the concomitant AF group, 23 patients fulfilled these criteria, and the mean of the maximal heart rate was 91% ± 11% of predicted.

EchocardiographyEchocardiography showed a significant reduction of the LA dimensions in both groups at 1-year follow-up compared with baseline characteristics, but the dimensions increased again afterward (Table 3). The mitral regurgitation in the lone AF group increased significantly during follow-up and increased nonsignificantly in the concomitant AF group.

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DiscussionSuccess was defined as freedom from AF, AFL, and other supraventricular arrhythmias. According to our definition, the cumulative medium-term freedom from these arrhythmias can be determined by the preoperative LA diameter. The success at the end of follow-up can be predicted at 1 year after the operation. This observation indicates that success persists during follow-up and depends on the preoperative LA diameter. Other clinical risk factors could not be determined. The duration of preoperative AF significantly influenced only the 1-year results. The absence of a significant correlation with success at the end of follow-up may be attributable to a lack of power. Our definition of success, determined by the absence of all atrial arrhythmias, because they all affect the patient, deviates strongly from success criteria used by others. If we use freedom from AF, as many authors do, our success is 89.7% for lone AF and 69.4% for concomitant AF at the end of follow-up.

Baseline

1 year

End

n Average ± SD n Average ± SD n Average ± SD

Atrial diameters in lone AF LA (mm) 124 41.9 ± 7.5 73 40.7 ± 6.8 64 40.8 ± 8.8

RA (mm) 90 53.6 ± 6.1 73 50.6 ± 6.1 47 53.5 ± 6.5

Atrial diameters in concomitant AF LA (mm) 57 51.7 ± 9.8 43 44.3 ± 7.3 40 48.8±10.1

RA (mm) 15 56.7 ± 6.9 39 51.0 ± 8.8 28 54.0±9.3

Ejection Fraction In lone AF(%) 71 55.3 ± 11.0 60 57.0 ±9.6

In conc AF (%) 38 53.3 ± 8.4 33 50. 9± 10.9

Mitral incompetence In lone AF (n/4) 71 0.3 ± 0.5 66 0.5 ± 0.6

In conc AF (n/4) 41 0.4 ± 0.8 45 0.4 ± 0.6

SD, standard deviation; AF, atrial fibrillation; LA, left atrial; RA, right atrial; EF, ejection fraction; MI, mitral incompetence; conc, concomitant.

P < .0001

P = .0413

P = .0001

Table 3. Echocardiographic data.


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Differences in SuccessMultivariate regression analysis did not show a significant difference in success between the 2 groups. The differences of success at the end of follow-up were attributable to baseline differences, especially LA diameters (Table 1). Preoperative LA diameters were larger in the concomitant group, reflecting a more extensive cardiac disease process, in part because of the high incidence of mitral valve disease. A correlation between LA dimension and long-term results has clearly been identified by others.3 The absence of a significant difference between the lone and concomitant AF groups was also identified by others.4 Our data show a significant decrease in LA diameter as a result of the operation, partly because of the suture line and partly because of the treatment of mitral valve pathology. Although not significant, there is a larger increase in atrial diameters of the concomitant AF group during follow-up as a consequence of more degenerated substrate and more progressed disease. Recent evidence demonstrates that aggressively reducing the size of giant left atria can positively influence the success.20 No atrial size reduction was performed in our patients.

Previous PublicationsOur success is similar to that of other large series,3,5,7 but seems to be inferior to others.4 A reason could be the application of a cryolesion only at the inside of the coronary sinus instead of circumferentially.21 Five patients had a left-sided flutter during follow-up catheter mapping. However, the main reason for the difference in reported results is caused by our strict definition of success and the rigorous documentation of supraventricular arrhythmias.22,23 Whereas most authors define success as freedom from only AF, we used a more strict definition of success. After all, many patients free from AF still have tachycardia. In contrast with other authors,4,8 we did not rely on subjective patient information obtained by phone contact. Up to 41% of our patients with postoperative AF had no palpitations. The occurrence of asymptomatic episodes of supraventricular tachycardias, even in patients who have had symptomatic episodes before, was already proven.24

In-Hospital and Late SurvivalNo 30-day mortality was present in our series, and the cardiac death rate during

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follow-up was minimal (1%), which may be explained by the fact that the patients in the lone AF group were young and fit with little comorbidity. Despite the complexity of the operation, mortality and morbidity are minimal in most large series.

Antiarrhythmic DrugsThe freedom from antiarrhythmic drugs is low because many patients used beta-blocking agents, digoxin, or calcium antagonists for reasons other than suppression or prevention of AF and supraventricular arrhythmias. The percentage of patients receiving antiarrhythmic drugs is in line with other groups.5

PacemakerThe incidence of postoperative pacemaker implantation is in the range of what other authors found, notwithstanding the fact that normal sinus node function was requested for surgery. Incidences up to 16% can be found.8 Could extensive mobilization of the superior caval vein, and thus damage to the sinus node region, play a role or have we failed to document sick sinus syndrome preoperatively in a few patients? Abolition of AF, but with an implanted pacemaker, does not necessarily mean a failure because atrial transport can be maintained, and antiarrhythmic drugs and oral anticoagulants can be withdrawn.

StrokeIn recent years it has become clear that the Maze operation is also highly successful in the prevention of ischemic CVA, which was also the case in our series.5,25

Comparison With Modified Maze OperationsBecause of the complexity and duration of classic Maze III surgery, many attempted to simplify the design and application of the original concept.26-28 Until now it has been unclear whether these therapies are as efficacious as the full Maze procedure. To our knowledge, no randomized studies have been published comparing the full cut-and-sew Maze procedure with operations with a more simple lesion pattern or using other energy devices. A recent review found no difference.29


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Indications for Surgical Treatment of Atrial FibrillationOur current strategy is to offer Maze surgery to symptomatic patients with lone AF if antiarrhythmic drugs, cardioversions, and percutaneous catheter ablation failed or AF and AFL coexist; the latter because catheter ablation of both arrhythmias will require lengthy procedures with a long exposure to radiation.

For patients with concomitant AF, the threshold is lower for performing an additional Maze procedure to treat AF because of the severe consequences of AF. In the future, ablation devices will simplify the creation of lesions in atrial myocardium. Most patients undergoing a concomitant procedure can benefit from AF surgery, especially patients in whom oral anticoagulants can be withdrawn and the surgical risk is acceptable.

Study LimitationsThe definitions applied for surgical success are not widely used because of a lack of consensus.22,23 This complicates a fair comparison of the surgical success with that of other institutions. Lack of intensive and long duration electrocardiographic recordings of the cardiac rhythm, because these registrations could negatively influence the result of the study.

ConclusionsThis study shows that the favorable unmodified Maze III results of abolishment of AF and other atrial arrhythmias in patients with and without additional cardiac surgery persist for at least 4 years after surgery and can be predicted at the end of the first year after Maze surgery. This outcome can be used for preoperative patient information and postoperative strategy of patient care.

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References

1. Cox JL, Canavan TE, Schuessler RB, Cain ME, Lindsay BD, Stone C, Smith PK, Corr PB, Boineau JP. The surgical treatment of atrial fibrillation. II. Intraoperative electrophysiologic mapping and description of the electrophysiologic basis of atrial flutter and atrial fibrillation. J Thorac Cardiovasc Surg 1991;101:406-426


3. Gillinov AM, Sirak J, Blackstone EH, McCarthy PM, Rajeswaran J, Pettersson G, Sabik FJ 3rd, Svensson LG, Navia JL, Cosgrove DM, Marrouche N, Natale A. The Cox Maze procedure in mitral valve disease: predictors of recurrent atrial fibrillation. J Thorac Cardiovasc Surg 2005;130:1653-1660.

4. Prasad SM, Maniar HS, Camillo CJ, Schuessler RB, Boineau JP, Sundt TM 3rd, Cox JL, Damiano RJ Jr. The Cox Maze III procedure for atrial fibrillation: long-term efficacy in patients undergoing lone versus concomitant procedures. J Thorac Cardiovasc Surg 2003;126:1822-1828

5. Bando K, Kobayashi J, Kosakai Y, Hirata M, Sasako Y, Nakatani S, Yagihara T, Kitamura S. Impact of Cox Maze procedure on outcome in patients with atrial fibrillation and mitral valve disease. J Thorac Cardiovasc Surg 2002;124:575-583

6. Lönnerholm S, Blomström P, Nilsson L, Oxelbark S, Jideus L, Blomström-Lundqvist C. Effects of the Maze operation on health-related quality of life in patients with atrial fibrillation. Circulation 2000;101:2607-2611

7. Schaff HV, Dearani JA, Daly RC, Orszulak TA, Danielson GK. Cox-Maze procedure for atrial fibrillation: Mayo Clinic experience. Semin Thorac Cardiovasc Surg 2000;12:30-37

8. Ad N, Cox JL. Combined mitral valve surgery and the Maze III procedure. Semin Thorac Cardiovasc Surg 2002;14:206-209

9. McCarthy PM, Gillinov AM, Castle L, Chung M, Cosgrove D 3rd. The Cox-Maze procedure: the Cleveland Clinic experience. Semin Thorac Cardiovasc Surg 2000;12:25-29

10. Kim KB, Cho KR, Sohn DW, Ahn H, Rho JR. The Cox-Maze III procedure for atrial fibrillation associated with rheumatic mitral valve disease. Ann Thorac Surg. 1999;68:799-803

11. Albåge A, Lindblom D, Insulander P, Kennebäck G. Electrophysiological evaluation of the sinus node and the cardiac conduction system following the Maze procedure for atrial fibrillation. Pacing Clin Electrophysiol 2004;27:194-203

12. Jessurun ER, van Hemel NM, Defauw JA, Stofmeel MA, Kelder JC, de la Rivière AB, Ernst JM. Results of Maze surgery for lone paroxysmal atrial fibrillation. Circulation 2000;101:1559-1567

13. Haïssaguerre M, Jaïs P, Shah DC, Garrigue S, Takahashi A, Lavergne T, Hocini M, Peng JT, Roudaut R, Clémenty J. Electrophysiological end point for catheter ablation of atrial fibrillation initiated from multiple pulmonary venous foci. Circulation 2000 Mar;101:1409-1417


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14. Pappone C, Oreto G, Rosanio S, Vicedomini G, Tocchi M, Gugliotta F, Salvati A, Dicandia C, Calabrò MP, Mazzone P, Ficarra E, Di Gioia C, Gulletta S, Nardi S, Santinelli V, Benussi S, Alfieri O. Atrial electroanatomic remodeling after circumferential radiofrequency pulmonary vein ablation: efficacy of an anatomic approach in a large cohort of patients with atrial fibrillation. Circulation 2001;104:2539-2544

15. Gallagher MM, Camm AJ. Classification of atrial fibrillation. Pacing Clin Electrophysiol 1997;20:1603-1605

16. Kosakai Y. Treatment of atrial fibrillation using the Maze procedure: the Japanese experience. Semin Thorac Cardiovasc Surg 2000;12:44-52

17. Cox JL, Ad N, Palazzo T, Fitzpatrick S, Suyderhoud JP, DeGroot KW, Pirovic EA, Lou HC, Duvall WZ, Kim YD. Current status of the Maze procedure for the treatment of atrial fibrillation. Semin Thorac Cardiovasc Surg 2000;12:15-19

18. Cox JL, Jaquiss RD, Schuessler RB, Boineau JP. Modification of the Maze procedure for atrial flutter and atrial fibrillation. II. Surgical technique of the Maze III procedure. J Thorac Cardiovasc Surg 1995;110:485-495

19. Löllgen H, Ulmer HV, Crean P. Recommendations and standard guidelines for exercise testing. Eur Heart J 1998;9(Suppl K):3-37

20. Romano MA, Bach DS, Pagani FD, Prager RL, Deeb GM, Bolling SF. Atrial reduction plasty Cox Maze procedure: extended indications for atrial fibrillation surgery. Ann Thorac Surg 2004;77:1282-1287

21. Cox JL. The surgical treatment of atrial fibrillation. IV. Surgical technique. J Thorac Cardiovasc Surg 1991;101:584-592

22. Gillinov AM, Blackstone EH, McCarthy PM. Atrial fibrillation: current surgical options and their assessment. Ann Thorac Surg 2002;74:2210-2217

23. van Hemel NM, Defauw JJAMT. Maze procedure. Letter to the editor. Ann Thorac Surg 2004;77:83-84

24. Page RL, Tilsch TW, Connolly SJ, Schnell DJ, Marcello SR, Wilkinson WE, Pritchett EL; Azimilide Supraventricular Arrhythmia Program (ASAP) Investigators. Asymptomatic or “silent” atrial fibrillation: frequency in untreated patients and patients receiving azimilide. Circulation 2003;107:1141-1145

25. Cox JL, Ad N, Palazzo T. Impact of the Maze procedure on the stroke rate in patients with atrial fibrillation. J Thorac Cardiovasc Surg 1999;118:833-840

26. Mohr FW, Fabricius AM, Falk V, Autschbach R, Doll N, Von Oppell U, Diegeler A, Kottkamp H, Hindricks G. Curative treatment of atrial fibrillation with intraoperative radiofrequency ablation: short-term and midterm results. J Thorac Cardiovasc Sur. 2002;123:919-927

27. Knaut M, Tugtekin SM, Spitzer S, Gulielmos V. Combined atrial fibrillation and mitral valve surgery using microwave technology. Semin Thorac Cardiovasc Surg 2002;14:226-231

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28. Nakajima H, Kobayashi J, Bando K, Niwaya K, Tagusari O, Sasako Y, Nakatani T, Kitamura S. The effect of cryo-Maze procedure on early and intermediate term outcome in mitral valve disease: case matched study. Circulation 2002;106:I46-I50

29. Khargi K, Hutten BA, Lemke B, Deneke T. Surgical treatment of atrial fibrillation; a systematic review. Eur J Cardiothorac Surg 2005;27:258-265


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Medium-term outcome of different surgical methods to cure atrial fibrillation: is less worse?

3

G.S.C. GeuzebroekPh.K.E.W. Ballaux

N.M. van HemelJ.C. Kelder

J.J.A.M.T. Defauw

Interact Cardiovasc Thorac Surg 2008;72:201-206

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AbstractDifferent lesion sets and ablation techniques have been performed. We compared these outcomes in search of the best method. We performed a retrospective analysis of patients who have undergone AF surgery different from the Maze III. The surgical lesion sets were pulmonary vein isolation (PVI) alone, left atrial Maze (LAM) and bi-atrial Maze (BAM) and were made with different ablation techniques. During surgery one patient died due to bleeding of a pulmonary vein. The number of patients in the PVI-, LAM-, BAM-groups was 12, 28 and 26, respectively, with freedom from AF at latest follow-up [22.0 ± 15.6 (3.1–81.2) months] of 33%, 59% and 60%, respectively. Atrial flutter occurred less in the BAM-group (4%) than in the left-sided procedures (15.4%) (P=0.231). Multivariate analysis demonstrated a higher recurrence of AF for PVI alone (OR 4.42, CL 0.95–20.6, P=0.0583) and a lower recurrence for the ‘cut-and-sew’ technique (OR 0.13, CL 0.030–0.60, P=0.0084). Left- and bi-atrial Maze procedures are equally effective in the suppression of AF, whereas omission of right-sided lesions results in a higher prevalence of atrial flutter. The ‘cut-and-sew’ technique is superior in terms of freedom from AF compared to bipolar and unipolar radiofrequency.

Modified Maze surgery for atrial fibrillation

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IntroductionThe classical Maze III is highly successful in terminating AF, but it has some drawbacks. We still perform the classical Maze III procedure in our department and have reported the results recently1, but because of its drawbacks we also performed less extensive lesion sets with alternative ablation techniques.

Since the introduction of the Maze procedure our understanding of AF has grown enormously. Haïssaguerre et al. showed that lone paroxysmal AF is often initiated by ectopic foci originating from the pulmonary veins and that ablating or isolating these foci can cure AF.2 Recent evidence shows that focal activation in the left atrium close to the pulmonary veins plays also an important role in patients with chronic AF and mitral valve disease.3 The experience with the surgical left atrial isolation procedure also discloses the important role of the left atrium in the arrhythmogenesis of AF.4 Therefore, both cardiologists and surgeons are now more and more focusing on the left atrium with special interest in the pulmonary veins5 and one could expect that left-sided procedures are highly effective.

In search of the best alternative for the Maze III, we retrospectively compared three different lesion sets and three different ablation techniques.

Material and methodsStudy populationBetween March 1999 and June 2005, a total of 67 consecutive patients had undergone a surgical procedure for AF different from the classical Maze III procedure. Preoperative, perioperative and follow-up data of these patients have been collected from in-patient and out-patient clinic files and a questionnaire was mailed to the referring cardiologists. Two patients were lost to follow-up (97% completeness of follow-up).

Surgical proceduresThree different types of lesion sets have been applied to eliminate AF: pulmonary vein isolation (PVI) alone, left atrial Maze (LAM) and bi-atrial Maze (BAM). The LAM consists of a PVI and an ablation line through the left isthmus, but without right-sided lesions. The BAM mimics the classical Maze III procedure, but the ‘cut-and-sew’

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technique is replaced by ablation lines. All BAM procedures were made with bipolar RF ablation, except for three procedures which had been made with unipolar RF ablation. The BAM procedure resembles strongly the Maze IV operation described by Damiano and his group.6 Amputation of the left atrial appendage was carried out in all procedures. We did not perform intra-operative pacing to test transmurality or conduction block of the ablation lines.

Choice of surgical methodThe type of lesion set as well as the ablating technique have been an evolution of available technology and understanding of the arrhythmogenesis of AF. Although the classical Maze III was still the standard surgical treatment of AF at our department in this period, we performed surgery for AF other than the classical Maze III as a consequence of its drawback.

Anti-arrhythmic managementPatients did not receive standard amiodaron postoperatively for prevention or treatment of AF. Sotalol was first treatment of choice to prevent or treat AF postoperatively. If sotalol was inadequate or not tolerated, the preoperative anti-arrhythmic drugs were given for either rate or rhythm control. At least one electrical cardioversion was performed before hospital discharge if a patient had a relapse of AF. After hospital discharge the anti-arrhythmic treatment was left to the referring cardiologist.

DefinitionsWe defined ‘success’ as the freedom from AF after a minimal follow-up of three months after surgery. This time window was chosen because of the time dependent success of procedures for AF.7 The freedom from AF excludes AF regardless of its type or duration. Rhythm results were based on electrocardiograms and 24 h Holter recordings, but never on symptoms related to AF.

Statistical analysisAll values are expressed as mean ± S.D. or percentages.For formal hypothesis testing we computed t-tests, ANOVA and Fisher’s exact test


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where appropriate. Multivariate analysis by means of logistic regression was carried out to

determine baseline factors that influence the recurrence of AF at latest follow-up. For this analysis the determinants were age, sex, type and duration of AF, left and right atrial diameters, previous CVA or TIA, left ventricular ejection fraction, number of failed anti-arrhythmic drugs, concomitant surgery for cardiac valve disease and atherosclerotic heart disease, aortic cross-clamp time, cardiopulmonary bypass time and occurrence of in-hospital AF after surgery.

A second multivariate analysis was performed to determine the effect of the different surgical methods on the recurrence of AF at latest follow-up. The determinants were the type of lesion set (PVI, LAM and BAM) and the ablation technique (‘cut-and-sew’, unipolar radiofrequency and bipolar radiofrequency). All calculations have been performed with SAS v 8.2; P<0.05 was considered significant.

ResultsPatient demographics and baseline findingsThe baseline characteristics are described in Table 1.

The number of patients in the PVI, LAM and BAM-group was 12, 28 and 26, respectively. Figure 1 displays the distribution of the applied techniques: the ‘cut-and-sew’ and bipolar RF were the most frequently used techniques. The BAM-group did not consist of any ‘cut-and-sew’ technique, because this method would constitute the classical Maze III.

In-hospital resultsOne patient died shortly after surgery due to non-repairable damage of the right upper pulmonary vein caused by blunt trauma during positioning of the bipolar radiofrequency device.

Further perioperative results are presented in Table 2.

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Figure 1. Surgical methods.The figure displays the different techniques that have been used for the three different lesion sets. PVI, pulmonary vein isolation; LAM, left atrial Maze procedure; BAM, bi-atrial Maze procedure; RF, radiofrequency. *Remainder consist of cryothermy and diathermy ablation techniques for the PVI-group and unipolar radiofrequency ablation for the LAM- and BAM-group.

Total† n= 67

PVI

n= 12

LAM n= 28

BAM n= 26

P value

Male Age (years) Duration AF (months) Type of AF Paroxysmal

Persistent Permanent Number of failed AAD Number of previous ECV Preoperative TIA Preoperative CVA LA diameter (mm) RA diameter (mm) LVEF (%) Concomitant AF MV disease TV disease AoV disease CABG

46 (69%) 61.8 ± 10.0 76.7 ± 79.1

38 (58%) 19 (29%) 9 (14%) 2.9 ± 1.8 2.2 ± 3.0 2 (3.0%) 9 (14%)

51.5 ± 11.3 57.9 ± 7.4 50.1 ± 8.5 60 (90%) 45 (67%) 3 (4.5%) 8 (12%)

17 (25%)

7 (58%) 59.6 ± 13.5 118 ± 103

6 (50%) 3 (25%) 3 (25%) 3.3 ± 1.7 2.5 ± 3.5 1 (8.3%) 1 (8.3%)

54.3 ± 17.2 59.5 ± 7.7 48.3± 2.8 10 (83%) 10 (83%) 1 (8.3%) 2 (17%) 0 (0%)

10 (64%) 61.1 ± 10.3

74 ± 82

19 (68%) 6 (21%) 3 (11%) 3.2 ± 2.0 2.0 ± 2.7 2 (7.1%) 0 (0%)

50.4 ± 10.1 56.8 ± 6.9 48.4 ± 8.0 23 (82%) 17 (61%) 1 (3.6%) 4 (14%) 6 (21%)

*

21 (81%) 63.3 ± 7.9

60 ± 58

19 (52%) 9 (36%) 3 (12%) 2.3 ± 1.5 2.3 ± 3.3 6 ( 24%) 1 (4%)

51.1 ± 9.2 57.8 ± 8.4 52.8 ± 9.4 26 (100%) 17 (65%) 1 (3.8%) 2 (7.7%) 11 (42%)

N.S. N.S. N.S.

N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.

*0.008

Table 1 represents the baseline characteristics of all patients and its three subdivisions of lesion sets (exclusive pulmonary vein isolations (PVI), the left atrial maze procedures (LAM) and the bi-atrial maze procedures (BAM)). LVEF, left ventricular ejection fraction; CABG, coronary artery bypass grafting; AAD, anti-arrhythmic drugs; ECV, electric cardioversion; LA, left atrium; RA, right atrium; MV, mitral valve; TV, tricuspid valve; AoV, aortic valve; N.S., not significant. †The total group includes one patient who died shortly postoperative. This patient is excluded in the subgroups.

Table 1. Baseline characteristics.


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Mid-term rhythm resultsDuring a mean follow-up of 22.0 ± 15.6 (3.1–81.2) months, two patients died due to congestive heart failure. Time of death was in both cases more than four years after surgery. Although the difference is not significant, the success at latest follow-up of the PVI-group was lowest: 33% vs. 59% and 60% for the LAM- and BAM-group, respectively (P=0.176 for PVI vs. LAM, P=1.000 for LAM vs. BAM, P=0.170 for PVI vs. BAM).

The success of the three largest uniform subgroups are: 85% for the left atrial Maze made with the ‘cut-and-sew’ technique (n=13), 40% for the left atrial Maze made with ‘bipolar RF ablation’ (n=10) and 57% for the bi-atrial Maze made with ‘bipolar RF ablation’ (n=23).

Atrial flutter occurred in 4.0% (1/25) of the patients who had undergone a BAM procedure. Patients with only left-sided lesion (PVI- and LAM-group) experienced atrial flutter in 15.4% (6/39) (P=0.231).

Total n= 66

PVI

n= 12

LAM n= 28

BAM n= 26

P value

Duration of ECC (n) Mean (min)

61 130 ± 45

11 102 ± 28

27 126 ± 42

¶

23 147 ± 49

¶= 0.0092

Duration of Aoclamp (n) Mean (min)

61 84 ± 36

11 68 ± 28

27 83 ± 34

23 94 ± 41

N.S.

In hospital AF

58% 25% 74% 56% N.S.

Transient AV-block (n) 1 2 3 In total

64 9.4% 3.1% 14% 22%

12 8.3% 0%

8.3%

17%

28 7.1% 7.1% 7.1%*

21%

24 13% 0%

25% 28%

N.S. N.S. N.S. N.S.

Patients needing pacing (n) Temporary Definitive In total

64 39% 1.6% 41%

12 17% 0%

17%

28 25% 3.6% 29%

†

24 67% 0%

67%

†<0.02

Duration of hospital stay (n)** Mean (days)

48 11.7 ± 4.9

10 11.1 ± 5.6

20 10.7 ± 3.2

18 13.2 ± 6.0

N.S.

ECC, extra corporal circulation; Aoclamp, Aortic cross clamp; n, number of patients. *One patient had a permanent third degree AV-block and received a pacemaker. **Some patients were transferred back to the department of cardiology of the referring hospital.

Table 2. In-hospital results.

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Follow-up observationsFreedom from pacemaker at latest follow-up was 94% (61/65). One patient received a pacemaker for a persisting third degree AV-block shortly after his left atrial Maze (LAM) procedure with mitral valve replacement. During follow-up, another two patients received a pacemaker for a third degree AV-block and another one for symptomatic bradycardias.

At latest follow-up, 46% of the patients who were free from AF were free from anti-arrhythmic drugs. Freedom from oral anticoagulantia was 44% for patients who were free from AF and did not have a mechanical valve. Freedom from CVAs or TIAs was 100% (after hospital discharge). Echocardiographic follow-up showed a significant decrease of the right and left atrial diameter (Table 3).

Multivariate analysisMultivariate analysis of the baseline characteristics showed that the recurrence of AF at latest follow-up is higher for patients with mitral valve disease (OR 4.04, CL 1.08–15.1, P=0.038) and lower for patients of male gender (OR 0.16, CL 0.044–0.61, P=0.0069).

Multivariate analysis for the surgical methods revealed a higher recurrence of AF at latest follow-up after PVI (OR 4.42, CL 0.95–20.6, P=0.058) and a lower recurrence of AF for the ‘cut-and-sew’ technique (OR 0.13, CL 0.030–0.60, P=0.0084).

Baseline

End of follow-up

P value

LA diameter (mm) LA diameter (mm) With MVS Without MVS LA diameter (mm) With success Without success RA diameter (mm) LVEF (%)

51.5 ± 11.3

55.6 ± 12.0 44.2 ± 5.1

46.9 ± 7.3 56.9 ± 13.1

57.9 ± 7.4

50.1 ± 8.5

47.2 ± 6.9

47.3 ± 7.5 47.0 ± 4.5

47.3 ± 5.3 48.1 ± 7.7

53.3 ± 11.9

48.6 ± 10.8

0.0042

0.0017 0.5281

0.1337 0.0323

0.0092

0.0513

Different echocardiographic results before surgery (baseline) and at the end of follow-up. The left atrial diameter presents the long axis in the parasternal view and the right atrial diameter presents the largest available diameter. MVS, mitral valve surgery.

Table 3. Echocardiographic results.


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DiscussionKey resultsAlthough this study lacks power for any significant results between the different ablation sets, it shows that the PVI alone had the lowest success rate, whereas the LAM and BAM procedure scored equally. Secondly, the ‘cut-and-sew’ technique resulted in a significant higher success than the other techniques.

AF recurrenceMultivariate analysis of the baseline characteristics demonstrated that patients with mitral valve disease had a higher recurrence of AF. This finding can be ascribed to a particular substrate for AF and concurs with our previous results of the classical Maze III procedure.1 In contrast to AF without mitral valve disease, AF with mitral valve disease is often caused by a volume overload of the left atrium.8 Secondly multivariate analysis of the baseline characteristics also found that male patients had lower failure rate than female ones. The small sample size or a higher incidence of underlying structural heart disease, which could influence the substrate of AF in female patients, could contribute to this gender difference. It is noteworthy to mention that women underwent more frequently mitral valve surgery than men (90% vs. 57%, respectively, P=0.0059).

It is unclear whether the low success rate of the PVI has to be ascribed to an unsatisfying method as such or to the unfavourable arrhythmic profile of the patients who received this surgical method. These patients had longstanding and mostly permanent AF, more failed antiarrhythmic drugs, electrical cardioversions and the largest diameters of both atria, and were mostly treated in combination with mitral valve disease. Although the baseline characteristics do not differ statistically (Table1), PVI alone is probably not the ideal method to apply in patients with chronic and complex AF and a more extensive AF ablative method would be preferable.

Surgical method: ablation techniqueMultivariate analysis shows that the ‘cut-and-sew’ technique resulted in a significant higher freedom from AF. Whether this is caused by the obtained transmurality or by the configuration of pulmonary vein exclusion is unclear. The non ‘cut-and-sew’ techniques lack the production of consistent

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transmural ablation lines or conduction blocks. Even with the bipolar RF ablation device, which gives an indication when a lesion is transmural, about three separate ablations are needed for electrical isolation of the pulmonary veins.9

With the ‘cut-and-sew’ technique we excluded all pulmonary veins in one large button. This could cure AF by deminishing the total mass of atrium (‘critical mass theory’)10 and is supported by recent data showing that two connecting lesions improves success.11 Excluding a large patch of left atrium can also exclude a large area of important substrate, but can drastically effect the atrial filling fraction and be potentially thrombogenic. The superiority of the ‘cut-and-sew’ method has also been described by Stulak et al.12 They showed that patients were five times more likely to be in atrial fibrillation after a ‘RF ablation Maze III’ than after a ‘cut-and-sew’ Maze III.

Left vs. bi-atrial MazeFreedom from AF of the LAM-group and the BAM-group were comparable (59% vs. 60%, respectively). This outcome suggests that left-sided ‘Maze-like’ procedures have comparable success rates as bi-atrial ‘Maze-like’ procedures; this observation concurs with the report of Deneke et al.7 Although encouraging results of left-sided ‘Maze-like’ procedures have been reported by others. We found, however, a trend towards a higher prevalence of atrial flutter in the procedures when right-sided ablation lines were not performed. This outcome was also found by Usui et al.13

Mortality and safetyOne patient died shortly after surgery, which was related to the use of the bipolar radiofrequency device. The damage was not caused by the heating of tissue, but by trauma incurred during surrounding of the pulmonary veins. This complication occurred at the beginning of our experience with the bipolar RF device. We are not aware of any reports describing similar events with this device.

Implications: optimal surgical methodPVI alone can cure AF, but multivariate analysis of this study shows that this lesion set has a significant worse rhythm result. The most important advantage of PVI alone is that it can be performed off-pump, minimal invasive and that it is a short


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and relative easy procedure in combination with alternative ablation techniques. The specific patient profile which might be suitable for PVI alone waits further exploration. Because arrhythmogenesis of AF is predominantly restricted to the left atrium and because of the superiority of the ‘cut-and-sew’ technique, our data suggest a ‘cut-and-sew’ left-sided Maze procedure with a right atrial approach to prevent flutter or post-incisional macro-reentry. This is supported by the success of 85% of the subgroup ‘cut-andsew’ left atrial Maze which is even comparable with the classical Maze III.1 The left atrial procedure should include an ablation line from the pulmonary vein isolation to the mitral valve and amputation of the left auricle to prevent thrombo-embolic events.14,15

Another option would be a hybrid approach with percutaneous catheter techniques to cure or prevent atrial flutter by ablation of the cavotricuspid isthmus.

Study limitationsThe small number of patients in the three groups which we retrospectively compared, weakens the message of this study. Secondly, we used different ablation techniques without preoperative control of the electrical isolation of the pulmonary veins. This could have led to incomplete conduction blocks. Long rhythm registration during follow-up was not available in all patients.

ConclusionsLeft atrial Maze procedures seem to be effective in eliminating AF as are bi-atrial Maze procedures, but omission of right atrial ablation lines seems to result in a higher prevalence of atrial flutter. A right atrial ablation should therefore be considered to prevent atrial flutter. The exact patient profile for PVI alone has yet to be determined, but it seems it is less suitable for chronic and complex AF. This study showed that the ‘cut-and-sew’ technique is superior in curing AF in comparison with the bipolar and unipolar radiofrequency ablation.

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References

1. Ballaux PK, Geuzebroek GS, van Hemel NM, Kelder JC, Dossche KM, Ernst JM, Boersma LV, Wever EF, Brutel de la Rivière A, Defauw JJ. Freedom from atrial arrhythmias after classic Maze III surgery: A 10-year experience. J Thorac Cardiovasc Surg 2006;132:1433-1440

2. Haissaguerre M, Jais P, Shah DC, Takahashi A, Hocini M, Quiniou G, Garrigue S, Le Mouroux A, Le Metayer P, Clementy J. Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins. N Engl J Med 1998;339:659-666.

3. Nitta T, Ishii Y, Miyagi Y, Ohmori H, Sakamoto S, Tanaka S. Concurrent multiple left atrial focal activations with fibrillatory conduction and right atrial focal or reentrant activation as the mechanism in atrial fibrillation. J Thorac Cardiovasc Surg 200;127:770-778

4. Williams JM, Ungerleider RM, Lofland GK, Cox JL. Left atrial isolation: new technique for the treatment of supraventricular arrhythmias. J Thorac Cardiovasc Surg 1980;80:373-380

5. Heidbuchel H. Left atrial ablation pendulum swinging back towards the pulmonary veins. Eur Heart J 2005;26:2484-486

6. Gaynor SL, Schuessler RB, Bailey MS, Ishii Y, Boineau JP, Gleva MJ, Cox JL, Damiano RJ Jr. Surgical treatment of atrial fibrillation: predictors of late recurrence. J Thorac Cardiovasc Surg 2005;129:104-111

7. Deneke T, Khargi K, Grewe PH, von Dryander S, Kuschkowitz F, Lawo T, Muller KM, Laczkovics A, Lemke B. Left atrial versus bi-atrial Maze operation using intraoperatively cooled-tip radiofrequency ablation in patients undergoing open-heart surgery: safety and efficacy. J Am Coll Cardiol 2002;39:1644-1650

8. Allessie MA, Boyden PA, Camm AJ, Kleber AG, Lab MJ, Legato MJ, Rosen MR, Schwartz PJ, Spooner PM, Van Wagoner DR, Waldo AL. Pathophysiology and prevention of atrial fibrillation. Circulation 2001;103:769-777


10. Byrd GD, Prasad SM, Ripplinger CM, Cassilly TR, Schuessler RB, Boineau JP, Damiano RJ Jr. Importance of geometry and refractory period in sustaining atrial fibrillation: testing the critical mass hypothesis. Circulation 2005;112:I7-I13

11. Gillinov AM, McCarthy PM, Blackstone EH, Rajeswaran J, Pettersson G, Sabik JF, Svensson LG, Cosgrove DM, Hill KM, Gonzalez-Stawinski GV, Marrouche N, Natale A. Surgical ablation of atrial fibrillation with bipolar radiofrequency as the primary modality. J Thorac Cardiovasc Surg 2005;129:1322-1329.

12. Stulak JM, Dearani JA, Sundt TM 3rd, Daly RC, McGregor CG, Zehr KJ, Schaff HV. Superiority of cut-and-sew technique for the Cox Maze procedure: comparison with radiofrequency ablation. J Thorac Cardiovasc Surg 2007;133:1022-1027


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13. Usui A, Inden Y, Mizutani S, Takagi Y, Akita T, Ueda Y. Repetitive atrial flutter as a complication of the left-sided simple Maze procedure. Ann Thorac Surg 2002;73:1457-1459

14. Garcia-Fernandez MA, Perez-David E, Quiles J, Peralta J, Garcia-Rojas I, Bermejo J, Moreno M, Silva J. Role of left atrial appendage obliteration in stroke reduction in patients with mitral valve prosthesis: a transesophageal echocardiographic study. J Am Coll Cardiol 2003;42:1253-1258

15. Cox JL, Ad N, Palazzo T. Impact of the Maze procedure on the stroke rate in patients with atrial fibrillation. J Thorac Cardiovasc Surg 1999;118:833-840

Completely thoracoscopic pulmonary vein isolation with ganglionic plexus ablation and left atrial appendage amputation for treatment of atrial fibrillation

4

Eur J Cardiothorac Surg 2010;383:356-360

A. Yilmaz*

G.S.C. Geuzebroek*

B.P. van PutteL.V.A. Boersma

U. SonkerJ.M.T. de Bakker

W-J van Boven

*These authors have contributed equally to this study

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AbstractObjectives: Percutaneous catheter pulmonary vein isolation (PVI) has been the preferred choice for invasive treatment of symptomatic, drugrefractory lone atrial fibrillation (AF). Incomplete ablation lines, procedure-related morbidity and long-term success remain, however, a problem. A minimally invasive surgical approach can provide an attractive and secure alternative. Surgery offers an epicardial, bipolar approach under direct vision, but the invasiveness of surgery remains a problem. Therefore, we developed a completely thoracoscopic procedure. The objective of this study was to assess the feasibility, safety and effectiveness of a completely thoracoscopic surgical procedure to cure lone AF.

Methods: Bilateral ‘video-assisted thoracoscopy’ was performed to isolate the bilateral pairs of pulmonary veins using bipolar RF-energy, to ablate the ganglionic plexus (GP) and to amputate the left atrial appendage. Preoperative, in-hospital and follow-up data were collected for our first 30 patients.

Results: AF was paroxysmal in 63%, persistent in 27% and permanent in 10% of cases. The mean (±SD) left atrial diameter was 42.1 ± 7.4 mm and the mean duration of AF was 79.0 ± 63.9 months. Freedom from AF was obtained in 77% of the patients during a mean follow-up of 11.6 months. Forty-three percent of the patients had previously undergone a percutaneous PVI and were all free from AF during follow-up. Mean operation time was 137.4 ± 24.7 min. All patients were extubated in the operating room and left the recovery room within 12 h. The mean hospital stay was 5.1 ± 1.8 days. Two patients ultimately underwent a median sternotomy. No CVAs or pacemaker implantation were identified and none of the patients died.

Conclusions: We report our initial experience of a completely thoracoscopic PVI with GP-ablation and amputation of the left atrial appendage and demonstrate that the procedure is feasible, safe and effective for the treatment of lone AF.

Minimal invasive surgery for atrial fibrillation

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IntroductionThe classical Maze III procedure has been the gold standard for the treatment of symptomatic, drug-refractory atrial fibrillation (AF). Despite its high success rates, it has not been widely adopted due to many reasons. This is mainly because the procedure is technically challenging and performed through a median sternotomy with the use of extra-corporal circulation.1

Less invasive procedures have been developed due to increasing knowledge about the pathophysiology of AF and due to the development of ablation devices, which replace the original ‘cut-and-sew’ technique for scarification.

Ectopic foci from the pulmonary veins (PVs) play an important role in the pathophysiology, especially in lone paroxysmal AF.2 Therefore, both cardiologists and surgeons focus on these vessels for the cure of AF. Recent clinical and experimental studies also suggest an important role of the autonomic nervous system.3,4 Wolf et al. have developed an off-pump procedure in which the PVs are isolated, ganglionic plexus (GP) are ablated and the left atrial appendage is amputated through a bilateral thoracotomy.5 A completely thoracoscopic procedure is even less invasive and should benefit perioperative morbidity and patient discomfort.

We have recently described the operation technique for a completely thoracoscopic approach to isolate the pulmonary veins, to ablate the GP and to amputate the left atrial appendage for the treatment of AF.6 The present study evaluates our first 30 completely thoracoscopic procedures with a medium-term follow-up.

Material and methodsPatient selectionCompletely thoracoscopic procedures for patients suffering from symptomatic, drug-refractory, (lone) AF have been performed since January 2007. Patients with drug-refractory AF, between the ages of 18 and 80 years and at least a moderate heart function, were eligible for surgery. Previous cardiac or lung surgery, very longstanding AF (more than 10 years) and a left atrium larger than 70 mm were relative contraindications. A previous unsuccessful percutaneous catheter ablation was not a contraindication for surgery. Patients were included after informed

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consent.

Operation technique (Figure 1)A completely bilateral video-assisted thoracoscopy (VATS) was performed as previously described by our group.6 Briefly, the patient is placed in the supine position under general anaesthesia and double lumen tube intubation. The procedure is performed through three thoracoports on both sides. Thoracoports are inserted in the fourth and sixth intercostal space in the midaxillary line. A third port is placed about 5 cm anterior to the midaxillary line in the third or fourth intercostal space. The camera is inserted through the port which is positioned in the fourth intercostal space. The remaining two ports are used for two thoracoscopic instruments.

On the right side, the pericardium is opened a few centimetres anterior to the phrenic nerve, followed by exploration of Waterstone’s groove for optimal positioning of the ablation device later on. Before isolation of the pulmonary veins, the GP at the base of the PVs are located with the AtriCure Transpolar Pen (AtriCure, Inc., West Chester, Ohio, USA) by high-frequency pacing (1000 beats min-1 with a potential of 18 V). Plexus locations are identified by a highfrequency pacing-induced slowing of the heart rate, which is considered to be a vagal response. This is defined by a ventricular asystole of at least 3 s or ventricular rate slowing of more than 50% when AF is present. A positive GP location is ablated for about 20 s with the AtriCure Transpolar Pen (AtriCure, Inc., West Chester, Ohio, USA). High-frequency pacing is again performed at the same position to verify that the GP is successfully ablated. If necessary, ablation can be repeated.

Blunt dissection around the PVs is performed using the AtriCure Lumitip Dissector (AtriCure, Inc., West Chester, Ohio, USA) and isolation of the PVs is achieved by bipolar radiofrequency ablation with the AtriCure Isolator Synergy ablation clamp (AtriCure, Inc., West Chester, Ohio, USA). At least three overlapping ablation lesions are performed at the antrum of the left and right PVs.7 A conduction block is confirmed by the absence of PV potentials if AF is present. This is performed by bipolar mapping of the PVs just beside the ablation line. Pacing from the PVs is used to confirm conduction block when a sinus rhythm is present. An extra ablation lesion is made if necessary.

The procedure is repeated on the left side, except that the pericardium


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is opened posterior to the phrenic nerve. In addition, the ligament of Marshall is dissected by electrocautery. The left atrial appendage was initially excluded by a PDS endoloop (Ethicon, Amersfoort, The Netherlands), but in later procedures, it was amputated by using an Autosuture Endo Gia stapler (Tyco Healthcare Group, North Haven, Connecticut, USA). A stapling device was used later on, because this guarantees exclusion of the cavity of the appendage. Amputation or exclusion of the left atrial appendage was performed after a thrombus was excluded by transoesophageal echocardiograph.

On completion of the procedure, the patient is extubated in the operating room and transferred to the recovery room.

Figure 1. Completely thoracoscopic pulmonary antrum isolation with GP-ablation.Panel A: The whole procedure is performed thoracoscopically through 3 thoracoports bilaterally. The black lines with numbers indicate the intercostal spaces (ICS). The thick red line indicates the mid-axillary line. The thin red line shows the position of the thoracoport in the 6th ICS relative to the xyphoid. Panel B: This shows the bipolar RF-ablation clamp (RF-clamp) around the right set of pulmonary veins. RA, right atrium; SCV, superior cavalvein. Panel C: Controlling the electrical isolation with an EP-catheter. The arrow indicates the ablation line. Panel D: Amputation of the left atrial appendage (LAA) with a stapling device (stapler).

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In-hospital post-operative managementAll patient received oral anticoagulation for at least 3 months after surgery. Further, the preoperative antiarrhythmic drugs were usually continued. Sotalol was started if a patient was only on rate-control medication with a β-blocker such as verapamil or digoxin. Electrical cardioversion (ECV) was performed if a patient had symptomatic AF less than 48 h or was adequately anticoagulated (International Normalised Ratio >2.0). Patients with asymptomatic AF were often discharged without an attempt for an electrical cardioversion. In that case, the referring physician was advised to perform one later on.

Follow-upBaseline characteristics, in-hospital and follow-up data were collected from clinical and out-patient clinic files. The referring cardiologist was sent a questionnaire for follow-up. Heart rhythm documentation was based on electrocardiograms (ECGs) and 24-h Holter-monitoring. Patients had continuous rhythm monitoring in the hospital. ECGs after discharge were performed after 1 week, 3 weeks, 3 months, 6 months and then annually. Holter-monitoring was performed at 3, 6 and 12 months for patients from our centre. Patients who were followed by a referring cardiologist and did not receive a Holter-monitoring were offered one at our centre. The surgical procedure was considered to be unsuccessful if an episode of AF (of duration >30 s, independently of amount of episodes or symptoms) occurred after a blankingperiod of 3 months.

Changes in medication (withdrawal of anti-arrhythmic drugs and/or anticoagulation after a successful procedure) were an autonomous decision of the referring cardiologist.

Statistical analysisData are presented as mean ± standard deviation. The Fisher’s exact test was performed where appropriate.


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ResultsBaseline characteristicsThe mean age of the patients was 55.6 ± 8.6 (range 35.9-69.8) years. The mean duration of AF was 79.0 ± 63.9 (range 36-240) months and the mean left atrial diameter was 42.1 ± 7.4 (range 35-55) mm. None of the patients had a significant mitral or tricuspid valve insufficiency. Three patients had a diminished left ventricular function with an estimated left ventricular ejection fraction of 30-40%. Eighteen patients (60%) had previously undergone a percutaneous catheter ablation(s), including left-sided ablation for AF in 13 (43%). All of them, except one, had paroxysmal AF.

In total, 19 patients (63%) suffered from paroxysmal AF, eight (27%) from persistent AF and three (10%) from permanent AF. The baseline characteristics are presented in Table 1.

Results (n=30)

Age (yrs) 55.6 ± 8.6 Man 77% (23/30) AF duration (months) For paroxysmal AF For persistent AF Longstanding persistent For permanent AF

79.0 ± 63.9 97.3 ± 70.6 34.5 ± 7.7 40.0 ± 6.9 88.0 ± 59.2

Type AF Paroxysmal Persistent Longstanding persistent Permanent

63% (19/30) 27% (8/30) 10% (3/30) 10% (3/30)

Preoperative LA† ø (mm) 42.1 ± 7.4 Preoperative RAa ø (mm) 55.8 ± 5.3 Number of failed AAD Including amiodaron

4.1 ± 1.4 52%

Number of failed ECV 2.3 ± 1.9 Previous failed percutaneous catheter PVI For paroxysmal AF For persistent AF (none longstanding) For permanent AF

43% (13/30) 92% (12/13) 8% (1/13) 0% (0/13)

Previous percutaneous catheter ablation (other than PVI) 17% (5/30) Congestive heart failure 7% (2/30) Preoperative CVA 7% (2/30) Preoperative TIA 3% (1/30) Preoperative pacemaker implantation

0% (0/30)

LA, left atrium; RA, right atrium; Ø, diameter; AAD, anti-arrhythmic drugs; ECV, electrical cardioversion; PVI, pulmonary vein isolation; CVA, cerebrovascular accident; TIA, transient ischaemic attack. †long axis in parasternal view along axis in 4-chamber view

Table 1. Patient characteristics.

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In-hospital resultsThe procedure had to be converted to a median sternotomy in the third and sixth patients, one due to severe pleural adhesions and the other due to bleeding from the right lower PV. In the following 24 patients, there were no complications during surgery. In all cases, the PVs could be electrically isolated and the left atrial appendage amputated. The mean duration of surgery was just over 2 h (137.4 ± 24.7 (range 101-197) min).

All patients could leave the recovery room within 12 h and were discharged from the hospital after a mean hospitalization of 5.1 ± 1.8 (range 3-9) days. Two patients received a thoracostomy drain because of a residual pneumothorax. This was secondary to inadequate removal of the drains and not due to a pulmonary lesion. In-hospital results are presented in Table 2.

Follow-up resultsThere was no peri- or post-operative mortality. The mean follow-up duration was 11.6 ± 3.9 (5.3-19.0) months. Taking a blanking period of 3 months after surgery into account, 77% of the patients did not have a ‘single’ registration of AF during follow-up. Holter-monitoring was available in 91% of the patient who were free from AF. Two patients did not want to receive a Holter-monitoring at our own hospital, but were free from complaints and had multiple ECGs with sinus rhythm. All patients who had a previously failed percutaneous left-sided catheter ablation for AF were free from AF after a mean follow-up of 9.8 ± 4.1 months.

Freedom from AF was 84%, 75% and 33% for paroxysmal, persistent and permanent AF, respectively ( p > 0.05).

Freedom from AF of the patients who had an in-hospital relapse of AF was

Results (n=30)

Mortality 0% (0/30) Conversion to sternotomy 7% (2/30) Procedure time (minutes) 137.4 ± 24.7 (range 101 - 197) In-hospital AF 60% (18/30) Discharged with AF 47% (14/30) Duration of hospital stay (days) 5.1 ± 1.9 (range 3 - 9) Temporary pacemaker dependency 0% (0/30)

Table 2. In-hospital results.


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61% (11/18). This was 57% (8/14) for patients who were discharged while still being in AF. Six of the 23 (26%) patients, who were free from AF, had undergone an ECV during follow-up (by definition of ‘success’ within 3 months after surgery); four of them were discharged with AF.

For patient who had a successful procedure, the freedom from anti-arrhythmic drugs was 65%, freedom from coumarines 48% and freedom from both was 26%. Focussing on the first 15 patients with a longer mean follow-up of 12.8 ± 4.2 months, this was 73%, 55% and 36%, respectively.

During the follow-up period, none of the patients received a pacemaker or suffered a cerebrovascular accident (CVA) or transient ischaemic attack (TIA).

DiscussionThis study reports our experience of the first 30 completely thoracoscopic PV isolations with GP-ablation and amputation of the left atrial appendage with a mean follow-up of almost 1 year. It shows that a completely thoracoscopic approach is feasible and safe, although two patients needed a sternotomy because of bleeding. This complication occurred within the first six cases and the authors are convinced that this is a part of the learning curve. No complication during surgery occurred in the last 24 cases. Furthermore, the procedure is effective, even for patients with longstanding AF or who had a previously unsuccessful percutaneous catheter ablation.

Freedom from AF was 77% for the whole group after a mean follow-up of almost a year, but still, 35% were using anti-arrhythmic drugs. Comparing success rates with other groups is difficult and depends on patient selection, definition of success and duration of follow-up. The technique of isolating the PVs and ablation of the ganglionic plexus has been previously described by Wolf et al.5 Our results are in concordance with those of others who also adopted this technique.8,9 This procedure appears to be less successful than the more elaborate classical Maze III operation1; however, the advantages of a completely thoracoscopic procedure are clear. It is an off-pump, minimal invasive procedure and has a shorter operating time, intensive care and hospital stay.

The success in patients who had a previous percutaneous catheter PVI is of special mention. All of them were free from AF during follow-up. This could be

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either due to previous inadequate percutaneous endocardial ablation and now a successful addition of bipolar RF-ablation or due to the additional GP-ablation.

Autonomic nervous system and GP-ablationIn 1958, Alessi et al. already showed that vagal stimulation leads to non-uniform shortening of the atrial refractory period in dogs.10 Shortly thereafter, Moe et al. could sustain AF with vagal stimulation without a focal discharge.11 More recent experimental evidence comes from the group of Oklahoma. These investigators showed that conversion of a premature (pulmonary) depolarisation to AF depends on GP stimulation.12 They could even initiate ectopic foci from the PVs by GP stimulation alone.13 These experimental data question the classical ‘trigger-substrate-hypothesis’ and it is clear that the autonomic nervous system plays a key role in the initiation and maintenance of AF.

The first important clinical indication for the role of vagal denervation came from Pappone et al.3 They showed that patients with a vagal response during RF-ablation (hence, vagal ablation) had a higher success rate in comparison to patients without a vagal response. RF-ablation was, however, not directed against the GP. Scherlag et al. were the first to compare PVI with and without GP-ablation in a prospective study.14 The follow-up was short and the groups were small, but the data suggest better outcome with additional GP-ablation. Some groups have even performed only GP-ablation for AF. The reported success rates vary between 29% and 84% and the follow-up is short.15-17

GP-ablation is, therefore, expected to contribute to the success of procedures for AF. Whether the effect of GP-ablation is persistent is a matter of debate. In dogs, autonomic innervations restore within 4 weeks18 and even reinnervations occur in totally denervated hearts of patients, who have undergone heart transplantation.19

Detecting GP is another point of debate. From an anatomical point of view, the GP is quite large and complex.20 Fields of ganglia (GP) contain both sympathetic and parasympathetic neurons and form complicated networks. The clinical relevance of a vagal reaction during high-frequency stimulation of GPs is not completely understood. This reaction is not pathologic per se and due to a network of ganglia, it could well be that the site of stimulation is not the site of neurotransmitter release.

In summary, there is limited evidence that GP-ablation in combination with


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a PVI leads to a higher success rate than PVI alone. There is a need for a randomised, prospective trial.

Extended lesion set and alternative ablation devicesIn this study, we describe our first 30 completely thoracoscopic PVIs with GP-ablation. PV isolation is a well-accepted method to treat AF. However, AF was treated successfully in only one of the three patients with permanent AF. One may expect that pulmonary vein isolation alone is adequate to treat paroxysmal AF. Additional lines in the left atrium could however lead to better results and extension of the indications for surgery.21,22 With the recently available Coolrail (AtriCure, Inc, West Chester, Ohio, USA), totally left-sided Maze III lesion sets can be achieved relatively easy. In permanent AF and longstanding persistent AF, the efficacy of the procedure could benefit from this more extensive lesion set. The transmurality of these additional epicardial ablation lines however remains a problem. An epicardial ablation line from the pulmonary veins to the mitral valve annulus (‘left fibrous trigone line’) is especially difficult. Extensive pacing and mapping has to be performed to check whether there is a complete conduction block.

We perform a bilateral thoracoscopic procedure with the use of bipolar RF-energy for the treatment of lone AF. Other groups have performed a unilateral approach with the use of other energy sources, such as microwave, high-intensity focussed ultrasound and vacuum-assisted RF, to isolate the pulmonary veins.23-25 All these energy sources are capable of making transmural lesions. A unilateral approach is, however, not suitable for amputation of the left atrial appendage. Further, positioning of the ablation device is not totally performed under direct vision and could lead to loss of contact with the epicardium.

LimitationsWe could not arrange a Holter registration for two patients, who did not have any documented relapse of AF after 3 months. These patients had, however, multiple registrations of sinus rhythm during follow-up and did not have any complaints, which could indicate a relapse of AF. Some patients experienced the procedure as a success because they had fewer and shorter episodes of AF, but by definition this was seen as failure.

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The majority of our patients come from referring hospitals. There may have been differences between centres in stopping anti-arrhythmic drugs and/or anticoagulation when a patient is in sinus rhythm. This and the duration of follow-up may influence the freedom from anti-arrhythmic drugs and anticoagulation.

Verification of a conduction block requires extensive mapping and expertise. To maximise success after surgery for AF, especially in the case of additional ablation lines, we need to develop reliable and easy-to-use mapping devices. A hybrid approach with endocardial verification of electrical isolation could also be of great value.

We did not record the exact number of application of the ablation device necessary to isolate the pulmonary veins and the number of GPs and their exact location.

ConclusionThe present study indicates that a completely thoracoscopic procedure for drug-refractory AF is feasible, safe and effective. The advantage of the surgical procedure in comparison to a percutaneous catheter PVI is its epicardial approach under direct vision, possibility of GP-ablation and amputation of the left atrial appendage. The procedure could therefore offer an alternative for percutaneous catheter ablation and is even effective after unsuccessful ones.


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References

1. Ballaux PK, Geuzebroek GS, van Hemel NM, Kelder JC, Dossche KM, Ernst JM, Boersma LV, Wever EF, Brutel de la Rivière A, Defauw JJ. Freedom from atrial arrhythmias after classic Maze III surgery: A 10-year experience. J Thorac Cardiovasc Surg 2006;132:1433-1440

2. Haissaguerre M, Jais P, Shah DC, Takahashi A, Hocini M, Quiniou G, Garrigue S, Le Mouroux A, Le Metayer P, Clementy J. Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins. N Engl J Med 1998;339:659-666.

3. Pappone C, Santinelli V, Manguso F, Vicedomini G, Gugliotta F, Augello G, Mazzone P, Tortoriello V, Landoni G, Zangrillo A, Lang C, Tomita T, Mesas C, Mastella E, Alfieri O. Pulmonary vein denervation enhances long-term benefit after circumferential ablation for paroxysmal atrial fibrillation. Circulation 2004;109:327-334

4. Zhou J, Scherlag BJ, Edwards J, Jackman WM, Lazzara R, Po SS. Gradients of atrial refractoriness and inducibility of atrial fibrillation due to stimulation of ganglionated plexi. J Cardiovasc Electrophysiol 2007;18:83-90

5. Wolf RK, Schneeberger EW, Osterday R, Miller D, Merrill W, Flege JB Jr, Gillinov AM. Video-assisted bilateral pulmonary vein isolation and left atrial appendage exclusion for atrial fibrillation. J Thorac Cardiovasc Surg 2005;130:797-802

6. Yilmaz A, Van Putte BP, Van Boven WJ. Completely thoracoscopic bilateral pulmonary vein isolation and left atrial appendage exclusion for atrial fibrillation. J Thorac Cardiovasc Surg 2008;136:521-522


8. Edgerton JR, Edgerton ZJ, Weaver T, Reed K, Prince S, Herbert MA, Mack MJ. Minimally invasive pulmonary vein isolation and partial autonomic denervation for surgical treatment of atrial fibrillation. Ann Thorac Surg 2008;86:35-38

9. Wudel JH, Chaudhuri P, Hiller JJ. Video-assisted epicardial ablation and left atrial appendage exclusion for atrial fibrillation: extended follow-up. Ann Thorac Surg 2008;85:34-38

10. Alessi R, Nusynowitz M, Abildskov JA, Moe GK. Nonuniform distribution of vagal effects on the atrial refractory period. Am J Physiol 1958;194:406-410.

11. Moe GK, Abildskov JA. Atrial fibrillation as a self-sustaining arrhythmia independent of focal discharge. Am Heart J 1959;58:59-70

12. Scherlag BJ, Yamanashi W, Patel U, Lazzara R, Jackman WM. Autonomically induced conversion of pulmonary vein focal firing into atrial fibrillation. J Am Coll Cardiol 2005;45:1878-1886

13. Patterson E, Po SS, Scherlag BJ, Lazzara R. Triggered firing in pulmonary veins initiated by in vitro autonomic nerve stimulation. Heart Rhythm 2005;2:624-631


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15. Platt M, Mandapati R, Scherlag B J, Yamanashi WS, Nakagawa H, Lazzara R., Jackmann WM.. Limiting the number and extent of radiofrequency applications to terminate atrial fibrillation and subsequently prevent its inducibility. Heart Rhythm 2004,1, S-11

16. Lemery R, Birnie D, Tang AS, Green M, Gollob M. Feasibility study of endocardial mapping of ganglionated plexuses during catheter ablation of atrial fibrillation. Heart Rhythm 2006;3:387-396

17. Scanavacca M, Pisani CF, Hachul D, Lara S, Hardy C, Darrieux F, Trombetta I, Negrão CE, Sosa E. Selective atrial vagal denervation guided by evoked vagal reflex to treat patients with paroxysmal atrial fibrillation. Circulation 2006;114:876-885

18. Oh S, Zhang Y, Bibevski S, Marrouche NF, Natale A, Mazgalev TN. Vagal denervation and atrial fibrillation inducibility: epicardial fat pad ablation does not have long-term effects. Heart Rhythm 2006;3:701-708

19. Kaye DM, Esler M, Kingwell B, McPherson G, Esmore D, Jennings G. Functional and neurochemical evidence for partial cardiac sympathetic reinnervation after cardiac transplantation in humans. Circulation 1993;88:1110-1118

20. Pauza DH, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart. Anat Rec 2000;259:353-382.

21. Geuzebroek GS, Ballaux PK, van Hemel NM, Kelder JC, Defauw JJ. Medium-term outcome of different surgical methods to cure atrial fibrillation: is less worse? Interact Cardiovasc Thorac Surg 2008;7:201-206

22. Gillinov AM, Bhavani S, Blackstone EH, Rajeswaran J, Svensson LG, Navia JL, Pettersson BG, Sabik JF 3rd, Smedira NG, Mihaljevic T, McCarthy PM, Shewchik J, Natale A. Surgery for permanent atrial fibrillation: impact of patient factors and lesion set. Ann Thorac Surg 2006;82:502-513

23. La Meir M, De Roy L, Blommaert D, Buche M. Treatment of lone atrial fibrillation with a right thoracoscopic approach. Ann Thorac Surg 2007;83:2244-2245

24. Klinkenberg TJ, Ahmed S, Ten Hagen A, Wiesfeld AC, Tan ES, Zijlstra F, Van Gelder IC. Feasibility and outcome of epicardial pulmonary vein isolation for lone atrial fibrillation using minimal invasive surgery and high intensity focused ultrasound. Europace 2009;11:1624-1631

25. Bevilacqua S, Gasbarri T, Cerillo AG, Mariani M, Murzi M, Nannini T, Glauber M. A new vacuum-assisted probe for minimally invasive radiofrequency ablation. Ann Thorac Surg 2009;88:1317-1321

Inreased amount of atrial fibrosis in patients with atrial fibrillation secondary to mitral valve disease

5

G.S.C. GeuzebroekS.C.M. van Amersfoorth

M.G. HoogendijkJ.C. Kelder

N.M. van HemelJ.M.T. de Bakker

R. Coronel

J Thorac Cardiovasc Surg 2012;144:327-333

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AbstractObjectives: Atrial fibrosis is related to atrial fibrillation but may differ in patients with mitral valve disease or lone atrial fibrillation. Therefore, we studied atrial fibrosis in patients with atrial fibrillation + mitral valve disease or with lone atrial fibrillation and compared it with controls.

Methods: Left and right atrial appendages amputated during Maze III surgery for lone atrial fibrillation (n = 85) or atrial fibrillation + mitral valve disease (n = 26) were embedded in paraffin, sectioned, and stained with picrosirius red. Atria from 10 deceased patients without a cardiovascular history served as controls. A total of 1048 images (4-μm sections, 10-fold magnification, 4 images per appendage) were obtained and digitized. The percentage of fibrous tissue was calculated by quantitative morphometry.

Results: Irrespective of the presence or absence of atrial fibrillation or mitral valve disease, more fibrous tissue was present in right atrial appendages than in left atrial appendages (12.7% ± 5.7% vs 8.2% ± 3.9%; P < .0001). The mean amount of fibrous tissue in the atria was significantly larger in patients with atrial fibrillation + mitral valve disease than in patients with lone AF and controls (13.6% ± 5.8%, 9.7% ± 3.2%, and 8.8% ± 2.4%, respectively; P < .01). No significant differences existed between patients with lone atrial fibrillation and patients without a cardiovascular history (controls).

Conclusions: Atria of patients with atrial fibrillation and mitral valve disease have more fibrosis than atria of patients with lone atrial fibrillation. However, patients with lone atrial fibrillation have an equal amount of atrial fibrosis compared with controls. These findings support the notion that fibrosis plays a more important role in the pathogenesis of atrial fibrillation secondary to mitral valve disease than in lone atrial fibrillation and potentially explains the relatively poor success of antiarrhythmic surgery in patients with mitral valve disease.

Fibrosis and atrial fibrillation

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IntroductionAtrial fibrillation (AF) is the most common sustained arrhythmia in humans and has an increased prevalence in elderly patients and patients with mitral valve disease (MVD), heart failure, hypertension, or pulmonary disease.1

Both electrical and structural remodeling of the atria play an important role in the pathogenesis of AF.2 Experimental and clinical investigations have shown a relation between atrial interstitial fibrosis and AF with or without structural heart diseases.3-5 Fibrosis resulting from chronic atrial dilatation and stretch may cause AF6, whereas AF itself may cause atrial fibrosis because of structural remodeling.2 The amount of fibrous tissue probably affects the success of nonmedical therapy directed at AF suppression and prevention, such as Maze(-like) surgery. Although surgery directed against AF can be effective in the treatment of lone AF, it is less effective in the treatment of AF secondary to MVD.7

Organized lines of fibrotic scar tissue created by Maze(-like) surgery prevents AF by reducing the tissue mass that could sustain reentry. Diffuse interstitial fibrosis also reduces the electrical tissue mass but facilitates reentry arrhythmias via 2 mechanisms. First, it forms barriers of nonconductive tissue that can lead to longer activation paths (‘zigzag’ conduction) with mainly transversal conduction slowing and thus increased anisotropic conduction, which is associated with reentry.8,9 The ‘zigzag’ conduction locally prolongs atrial activation time and thereby shortens the dimension of a potential reentrant activation path. Second, these barriers create geometric variation within the myocardium and cause sites of sudden myocardial expansion that can lead to current-to-load mismatch.10 Both mechanisms promote the occurrence of unidirectional conduction block, which is a prerequisite for the initiation and persistence of reentry arrhythmias, such as AF.

Because AF is associated with MVD and more difficult to treat than lone AF1,7, we hypothesized that atria of patients with AF and MVD contain more diffuse fibrotic tissue than atria of patients with lone AF. Furthermore, comparison of the fibrous tissue content in atria of patients with lone AF with that of control patients without a history of AF may elucidate fibrosis secondary to AF. A secondary study objective was the assessment of predictors for the amount of fibrous tissue in the atria. We therefore analyzed left atrial appendages (LAAs) and right atrial appendages (RAAs) and collected clinical data of patients who had undergone

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surgery for AF with or without mitral valve surgery. Postmortem tissues for control were obtained from patients without a cardiovascular history.

Material and MethodsStudy PopulationAll tissue samples were obtained at the St Antonius Hospital, Nieuwegein, The Netherlands. The study was in accordance with the declaration of Helsinki and approved by the institutional review board. The study conformed to our local institutional protocol, which describes the use of human tissues for research. Data were anonymously and retrospectively collected. In accordance with our local protocol, no informed consent was necessary because the tissue samples were resected as part of standard therapy or diagnosis.

LAAs and RAAs were routinely removed during elective classic Maze III surgery without (lone AF) or with mitral valvuloplasty or mitral valve replacement (AF + MVD). The lone AF group consisted of 85 patients who had undergone a classic Maze III procedure only and included 6 patients with a small type II atrial septal defect (ASD). These ASDs were peroperatively detected by coincidence, could be closed without a patch, were not visible on echocardiography peroperatively, and were therefore considered to have no significant hemodynamic consequences. All other ASDs (type I or II) were excluded. The group with AF and mitral valve surgery comprised 26 patients. Inclusion criteria for Maze III surgery have been described in detail.11

Patients without a cardiac history served as controls. Tissue samples for the control group were obtained from hearts of 10 deceased patients after permission for autopsy from relatives. LAAs, RAAs, and samples of the left and right atrial free wall were gathered from this group. Causes of death were alcohol abuse (n = 2), cardiogenic shock after acute myocardial infarction (without a cardiac history, n = 1), respiratory failure due to chronic obstructive pulmonary disease (n = 1), hepatic coma (n = 1), metastasized small cell lung carcinoma (n = 1), metastasized rectum carcinoma (n = 1), sepsis with known non–Hodgkin lymphoma (n = 1), sepsis eci (n = 1), and cerebrovascular attack (n = 1). There was no documentation or suspicion of AF in the medical history of the control patients.


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Collected Patient DataPre-, peri-, and postoperative data were retrospectively collected from inpatient and outpatient hospital files of the patients who had undergone a Maze III procedure. These included patient demographics, medication, echocardiographic data, left- and right-sided pressure, and type of surgery.

Collected patient data entailed age, gender, length, weight, previous catheter ablations, type of AF, duration of AF, serum creatinine, history of diabetes mellitus, and blood pressure. The last 3 blood pressure recordings before surgery were noted. AF was classified as paroxysmal, persistent, or permanent. We deviated from the Heart Rhythm Society’s consensus article because this consensus was not available at the time of operation.12

The use of angiotensin inhibitors, statins, aldosterone antagonists, and antihypertensive drugs during the last 3 months before operation and amiodarone use anytime before operation were noted.

Echocardiographic data consisted of the left atrial diameter (long parasternal axis), right atrial diameter (long axis in 4-chamber view), left ventricular end-diastolic/systolic diameter, and left ventricular ejection fraction. From the catheterization records, we obtained wedge pressure; right atrial pressure; left ventricular systolic and diastolic pressures; right ventricular systolic pressure; and diastolic, systolic, and mean pulmonary artery pressures.

HistologyTissue samples were fixed in 4% buffered formalin immediately after excision. After fixation, tissues were embedded in paraffin, and 4-μm transversal sections at the base of the appendages were cut and stained with picrosirius red for visualization and quantification of collagen in the fibrous tissue.

Four digital pictures were taken with a 10 × objective at 3, 6, 9, and 12 o’clock of the tissue sections. Background, perivascular, endocardial fibrosis, and epicardial fibrosis were excluded by manually revising every picture with Adobe Photoshop 9.0 (Adobe Systems Inc, San Jose, Calif) (Figure 1). Thereafter, the amount of fibrous tissue was calculated by quantitative spectrometry with Image-pro 6.2 (Media Cybernetics Inc, Bethesda, Md). The percentage of fibrous tissue was expressed as the amount of red (collagen) relative to the total amount of tissue (perivascular,

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endocardial, and epicardial fibrous tissue excluded). A total of 1048 pictures were taken, revised, and analyzed without any knowledge of the patient characteristics.

StatisticsValues are presented as mean ± standard deviation, percentages ± standard deviation, or different when indicated. Continuous variables were compared with paired Student t, unpaired Student t test, Mann-Whitney test, or analysis of variance test with post hoc Tukey honestly significant difference comparison, where appropriate. Categoric variables were compared with the Pearson’s chi-square test with Yates’ continuity correction. For correlation, the squared Pearson correlation coefficient was computed along with a scatter plot for visual inspection.

The distribution of the percentage fibrosis in the atrial appendages was skewed to the right and near normal after log-transformation. For the univariate statistics, we used the nontransformed data to keep the original units. For the multivariate analysis, we used the log-transformed data. The multivariate analysis consisted of linear regression with log-transformed percentage fibrosis in the atrial appendage as the dependent variable. All variables with P < .1 in a bivariate analysis

Figure 1. Picrosirius red staining of a 4-μM section from the LAA at 10× magnification. Fibrous tissue is stained red, and (atrial) myocardium is stained yellow-orange. Background, perivascular, endocardial fibrosis, and epicardial fibrosis are excluded from quantification (grey areas).


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were candidate independent variables for the multivariate regression. We used generalized linear modeling, and the best multivariate model emerged after ranking of all possible models by the Akaike’s information criterion, which is a measure of the goodness of fit of an estimated statistical model.

For the repeated-measurements analysis involving right- and left-sided atrial fibrosis as separate values in 1 patient, we used a generalized linear mixed-effects modeling with fibrosis as the dependent variable and individual as random intercept, disease with 3 categories (AF + MVD, lone AF, and control), left or right side, and the natural logarithm of fibrosis as independent variables. All statistical calculations were performed with R software (http://www.r-project.org).

ResultsPatient characteristicsTable 1 shows patient characteristics, type of surgery, and medication use for the lone AF and AF + MVD groups. The echocardiographic and hemodynamic data are presented in Table 2, showing as expected, that the AF + MVD group had generally larger atrial and ventricular dimensions and higher pressures than the lone AF group.

Atrial Interstitial FibrosisFigure 2 depicts the amount of fibrous tissue in the LAA and RAA for the different groups. The mean amount of fibrous tissue in the atria (LAA and RAA combined) differs significantly among the AF + MVD, lone AF, and control groups (13.6% ± 5.8%, 9.7% ± 3.2%, and 8.8% ± 2.4%, respectively, P < .01). By using a generalized linear mixed-effects model, the following pattern emerges: The right atrium contains 1.5 times more fibrosis (P < .001) than the left atrium. The atria of the AF + MVD group have 1.4 times more fibrosis than the control group (P = .01) and 1.3 times more fibrosis than the lone AF group (P = .01). No significant difference exists between the amount of atrial fibrosis in the lone AF and control groups. The amount of fibrous tissue in the appendages of the control group did not significantly differ from that in the free wall of the atria (RAA = 10.2% ± 4.0%, right atrial free wall = 12.6% ± 5.4%, P = .3; LAA = 7.5% ± 2.2%, left atrial free wall = 7.3% ± 1.9%, P = .8).

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The amount of fibrous tissue in the RAA and LAA correlated significantly to each other (r = 0.49, P < .0001).

Predictors for Atrial Interstitial FibrosisTable 3 shows the univariate analysis of different variables for the amount of fibrous tissue in the LAA and RAA. The amount of fibrous tissue correlates positively with left and right atrial diameter, left ventricular end-diastolic diameter, right ventricular systolic pressure, pulmonary artery pressures, surgery for MVD, and surgery for tricuspid valve disease, but negatively with statin use.

Figure 3 depicts scatter plots of continuous variables that significantly correlated to the amount of fibrous tissue in the LAA or RAA. Subsequent multivariate

Lone AF (n = 85)

AF + MVD

(n = 26)

Controls (n = 10)

P value

Age (y) 50.7 ± 8.7 59.0 ± 7.6 62.6 ± 15.7 <0.001* <0.001†

0.470‡ Male gender 88% (75/85) 65% (17/26) 50% (5/10) 0.016*

0.007† 0.641‡

BSA (m2) 2.11 ± 0.20 1.96 ± 0.23 0.003 BMI (kg/m2) 27.5 ± 4.28 24.8 ± 3.93 0.005 Diabetes mellitus 2% (2/85) 0% (0/0) 0.958 GFR (ml/min/1.72m2) § 81.4 ± 14.6 72.1 ± 15.6 - 0.011 AF duration (mo) 91.3 ± 61.3 47.6 ± 47.1 - <0.001 Type of AF Paroxysmal Persistent Permanent

72% (61/85) 20% (17/85)

8% (7/85)

31% (8/26) 23% (6/26)

46% (12/26)

-

<0.001

Previous catheter ablation 15% (13/85) 0% (0/26) - 0.076 Type of conc. cardiac surgery Mitral valve Tricuspid valve CABG ASD (all type II)

0% (0/85) 0% (0/85) 0% (0/85) 7% (6/85)

100% (26/26)

15% (4/26) 4% (1/26) 4% (1/26)

-

N/A

0.002 0.529 0.898

Medication use Amiodarone Antihypertensive drugs ACEI Aldosterone-antagonist Statin

62% (53/85) 22% (19/85) 20% (17/85)

0% (0/0) 12% (10/85)

12% (3/26)

54% (14/26) 54% (14/26)

8% (2/26) 0% (0/0)

-

<0.001

0.013 0.002 0.082 0.149

BSA, Body surface area; BMI, body mass index; GFR, glomerular filtration rate; CABG, coronary artery bypass grafting; ACEI, angiotensin-converting enzyme inhibitor; conc, concomitant. ∗ Lone AF vs AF + MVD † Lone AF vs controls ‡ AF + MVD vs controls § Calculated GFR13

Table 1. Patient charecteristics.


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analysis showed that the following factors were independently associated with an increased amount of fibrous tissue in the LAA or RAA: surgery for tricuspid valve disease (factor 1.8, P = .04), right atrial diameter (factor 1.02/mm, P < .01), and surgery for MVD (factor 1.3, P < .01). A protective trend was found for the use of statins with an estimated increase of fibrous tissue of 0.76 (P = .06).

For the average amount of fibrosis (RAA and LAA combined), surgery for MVD was the only remaining significant predictor. Patients with surgery for MVD had a 1.7 estimated increase of fibrous tissue (P < .01).

DiscussionIt is well recognized that fibrous tissue can play an important role in the pathogenesis of AF.2-5 On the other hand, AF itself may result in fibrosis.2 The main finding of this study is that patients with AF and MVD have more atrial fibrosis than patients with lone AF. Furthermore, no difference in atrial fibrosis was found between patients with lone AF and controls. This suggests that AF itself does not result in fibrosis in patients with lone AF and that the mechanism of the arrhythmia in these patients

Lone AF (n = 85)

AF + MVD

(n = 26)

Controls (n = 10)

P value

Left atrial ø (mm) 42.3±7.1 53.7±9.0 - <0.001 Right atrial ø (mm) 53.5±5.6 59.9±11.5 - 0.021 LVEDD (mm) 50.2±5.6 58.8±9.8 - <0.001 LVESD (mm) 35.4±5.2 43.0±8.4 - <0.001 LVEF >50% 40-50% 30-40%

94% (80/85)

4% (3/85) 2% (2/85)

81% (21/26) 15% (4/26) 4% (1/26)

-

0.097

Blood pressure (mmHg) Systolic Diastolic Mean

135 ± 17

83 ± 9 100 ± 11

133 ± 19

79 ± 8 97 ± 11

-

0.695 0.028 0.182

Catheterisation pressures (mmHg) Right atrium mean Wedge RV systolic LV end systolic LV end diastolic Systolic PAP Diastolic PAP Mean PAP

4.6 ± 2.9 9.4 ± 3.9

25.7 ± 6.2 130 ± 22

10.4 ± 4.9 25.1 ± 6.6 9.3 ± 4.1

15.6 ± 4.9

5.3 ± 4.0

19.9 ± 8.0 48.7 ± 25.6

131 ± 29 13.3 ± 6.4

45.0 ± 15.8 19.0 ± 9.0

27.3 ± 10.7

-

0.454

<0.001 0.001 0.940 0.063

<0.001 <0.001 <0.001

ø, Diameter; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; LVEF, left ventricular ejection fraction; RV, right ventricle; LV, left ventricle; PAP, pulmonary artery pressure.

Table 2. Echocardiographic data, blood pressure, and right-sided catheterization pressures.

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is more likely to be related to electrical or autonomic remodeling. This is further endorsed by the multivariate analysis, which found no relation between fibrosis and duration of AF; however, we cannot exclude that structural changes different from fibrosis play a role in lone AF.

In our study, the amount of fibrous tissue in the LAA and RAA did not significantly differ from that of the corresponding left and right atrial free wall. Our findings in the atrial appendages may therefore be extrapolated to the atrial free wall.

This study also showed that the right atrium contains more fibrous tissue than the left atrium. This finding is concordant with the results of Oken and Boucek, who showed that both the right ventricle and the atrium contain more fibrous tissue than the left ventricle and atrium.14 We speculate that the difference in the amount

Figure 2. Amount of fibrous tissue in LAA and RAA per group. Perivascular, endocardial fibrosis, and epicardial fibrosis were excluded. Values are given as percentage with the standard error of the mean (error bars). ∗P < .05. LAA, Left atrial appendage; RAA, right atrial appendage; AF, atrial fibrillation; MVD, mitral valve disease.


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of fibrous tissue between the right and the left atrium has a developmental origin. First, the right atrium has a different embryonic identity than the left atrium.15 Second, the pressures of the right side of the heart are higher than the left side during fetal circulation.16

Estimate

(log)

CI

lower

CI

upper

Missing

P values

Age 0.002 -0.006 0.010 0 0.6798 Male gender -0.174 -0.368 0.020 0 0.0779 BSA -0.175 -0.520 0.170 1 0.3165 BMI -0.010 -0.027 0.007 1 0.2435 Diabetes mellitus 0.077 -0.480 0.633 0 0.7855 GFR 0.003 -0.002 0.007 0 0.3084 AF duration* 0.039 -0.112 0.189 2 0.6107 Type AF† 0.130 -0.020 0.281 0 0.0896 Previous catheter ablation‡ 0.044 -0.186 0.274 0 0.7053 Left atrial ø 0.009 0.001 0.017 1 0.0338 Right atrial ø 0.016 0.006 0.026 12 0.0014 LVEDD 0.012 0.002 0.022 9 0.0224 LVF§ 0.046 -0.141 0.232 0 0.6280 Blood pressure; systolic -0.001 -0.005 0.003 0 0.6723 Blood pressure; diastolic -0.007 -0.016 0.001 0 0.1032 Blood pressure; mean -0.004 -0.011 0.003 0 0.2746 Right atrium pressure 0.004 -0.025 0.033 26 0.7734 Wedge pressure 0.008 -0.004 0.021 22 0.1960 RV systolic pressure 0.005 0.000 0.010 31 0.0427 LV end systolic pressure 0.001 -0.002 0.005 11 0.5202 LV end diastolic pressure 0.007 -0.009 0.023 19 0.3862 PAP systolic 0.008 0.002 0.015 20 0.0127 PAP diastolic 0.007 -0.005 0.020 20 0.2285 PAP mean 0.013 0.002 0.024 28 0.0184 Amiodarone -0.118 -0.265 0.028 0 0.1116 Anti-RR 0.129 -0.033 0.290 0 0.1170 ACE-inhibitor 0.087 -0.077 0.251 0 0.2949 Aldasterone-antagonist -0.136 -0.692 0.420 0 0.6298 Statine -0.264 -0.517 -0.010 0 0.0419 Mitral valve surgery 0.299 0.134 0.464 0 0.0005 Tricuspid valve surgery 0.419 0.030 0.808 0 0.0351 ASD type II closure -0.044 -0.349 0.260 0 0.7727 Estimate denotes beta regression parameter as a measure of association. Values in bold text were applicable for multivariate analysis (P < .1). ø, Diameter; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; LVEF, left ventricular ejection fraction; RV, right ventricle; LV, left ventricle; CI, confidence interval; PAP, pulmonary artery pressure. ∗ History of AF < 5 vs > 5 years. † Paroxysmal versus continuous (persistent and permanent) AF. ‡ Either pulmonary vein isolation or right-sided isthmus ablation in history. § Divided into groups of an ejection fraction of >50%, 40%–50%, or 30%–40%.

Table 3. Univariate analysis for the fibrous content in both atria (pooled data).

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This study showed a trend toward a negative correlation between statins and atrial fibrosis. The protective role of statins in the prevention of AF has been recognized, but the exact pathophysiology of its protective role is unknown.17,18 Fibrosis has been suggested to play a role in this matter.19 To our knowledge, this is the first study to describe a negative relation between statin use and amount of atrial interstitial fibrosis.

Figure 3. Scatter plots of different continuous variables in relation with the amount of interstitial fibrosis in the LAA or RAA. Other continuous variables that correlated significantly with the amount of fibrosis, but not displayed, are as follows: LVEDD: RAA; r = 0.21, P = .03, LAA; r = 0.28, P < .01. Right ventricular systolic pressure: RAA; r = 0.27, P = .02, LAA; r = 0.34, P < .01. Systolic pulmonary artery pressure: RAA; r = 0.25, P = .02, LAA; r = 0.30, P < .01. LAA, Left atrial appendage; RAA, right atrial appendage.


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Substrate and TriggerThis study showed that patients with AF and MVD have more interstitial fibrosis than patients with lone AF. This is probably the explanation for the relatively low success rate of antiarrhythmic surgery in patients with AF and MVD, especially when only a pulmonary vein isolation is performed.7 The greater amount of fibrosis in patients with AF and MVD also potentially explains why those patients have relatively more persistent and permanent AF than patients with lone AF, despite their shorter history of AF (Table 1). The increased amount of fibrous tissue in the atria of patients with AF secondary to MVD might be due to increased wall stress.6,20 The wall stress is dependent on both the pressure and diameter of the atrium (Laplace’s law), and these were both significantly larger in the AF + MVD group compared with the lone AF group. However, increased fibrosis in patients with AF secondary to MVD also may be due to heart failure.21 The AF + MVD group had larger left ventricular dimensions and an impaired glomerular filtration rate, which suggests at least some systolic heart failure.

Age and FibrosisBecause the mean age of patients with AF + MVD exceeded that of patients with lone AF, one could argue that differences in atrial fibrosis between the groups rely on age differences. However, the multivariate analysis showed that MVD is associated with a 72% increase in atrial fibrosis (P < .01) and that age was not related to the amount of fibrosis (P = .7). Even when we limit the analysis to the 5 youngest patients of the control group (aged 50.4 ± 11.2 years), there is no significant difference in the amount of fibrous tissue between the control and lone AF groups (aged 50.7 ± 8.7 years).

Evidence for age-related human atrial fibrosis is scarce and conflicting. Although 2 studies have found a relation between age and atrial fibrosis22,23, others have not.15,21 The relation between age and fibrosis in these studies could be attributed to the increased prevalence of other cardiovascular diseases in elderly people, such as hypertension, coronary artery disease, heart failure, and cardiac valve pathology. Furthermore, it could be that the distribution of age (52 ± 9 years) in this study was too small to find an increase of fibrosis with age, which is often the

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case in animal studies.

Fibrosis and Atrial FibrillationRecent magnetic resonance imaging studies showed that the success after a percutaneous vascular intervention depends on the late enhancement of the left atrial wall, which correlates to the amount of fibrous tissue.24 Our study showed that the right atrium contains more fibrous tissue than the left atrium. This underscores the importance of inclusion of the right atrium in the surgical treatment of AF, especially in patients with MVD. The Maze III operation modifies the substrate in both atria and is still considered as the golden standard for nonpharmacologic treatment of AF with a reported long-term success of more than 90%.7,11

LimitationsBecause of the small wall thickness of the atria, we were unable to make parallel sections and could not discriminate among patchy, diffuse, and compact fibrosis. These different types of fibrosis have different electrophysiologic effects.10 However, no compact fibrosis was encountered in the transverse sections.

This study shows no increase in fibrous tissue for patients with lone AF compared with controls. We cannot exclude that a different geometry of the fibrous tissue or other types of structural remodeling (eg, Cx43 expression) play a role in these patients.

Differences in age and other baseline characteristics existed between the groups because of selection of the patient population. Meticulous analysis showed no relation between age and fibrosis, but we cannot exclude that unrecorded differences in baseline characteristics interfered with our results.

The amount of fibrous tissue in the lone AF and AF + MVD groups was determined at the base of the atrial appendages and not in the atria itself. Although no difference existed in the control group, a discrepancy in the amount of fibrous tissue between the appendages and atria could exist in these 2 groups.

ConclusionsThe atria of patients with AF secondary to or in combination with MVD contain more fibrous tissue than atria of patients with lone AF, whereas atria of patients


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with lone AF have an equal amount of fibrous tissue as controls. In general, the right atrium contains more fibrous tissue than the left atrium. These findings suggest that AF secondary to or in combination with MVD is dependent on atrial interstitial fibrosis, whereas lone AF is more dependent on electrical or autonomic remodeling. This suggests that for treatment of AF secondary to MVD, a different percutaneous or surgical approach should be applied than in lone AF. Furthermore, statins may reduce the amount of fibrous tissue in the atria and are therefore of interest in pharmacologic therapy for the prevention of AF.

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References

1. Lloyd-Jones DM, Wang TJ, Leip EP, Larson MG, Levy D, Vasan RS, D’Agostino RB, Massaro JM, Beiser A, Wolf PA, Benjamin EJ. Lifetime risk for development of atrial fibrillation: the Framingham Heart Study. Circulation 2004;110:1042-1046

2. Allessie M, Ausma J, Schotten U. Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovasc Res 2002;54:230-246

3. Frustaci A, Chimenti C, Bellocci F, Morgante E, Russo MA, Maseri A. Histological substrate of atrial biopsies in patients with lone atrial fibrillation. Circulation 1997;96:1180-1184

4. Boldt A, Wetzel U, Lauschke J, Weigl J, Gummert J, Hindricks G, Kottkamp H, Dhein S. Fibrosis in left atrial tissue of patients with atrial fibrillation with and without underlying mitral valve disease. Heart. 2004;90:400-405

5. Verheule S, Sato T, Everett T 4th, Engle SK, Otten D, Rubart-von der Lohe M, Nakajima HO, Nakajima H, Field LJ, Olgin JE. Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-beta1. Circ Res 2004;94:1458-1465

6. Verheule S, Wilson E, Everett T 4th, Shanbhag S, Golden C, Olgin J. Alterations in atrial electrophysiology and tissue structure in a canine model of chronic atrial dilatation due to mitral regurgitation. Circulation 2003;107:2615-2622

7. Geuzebroek GS, Ballaux PK, van Hemel NM, Kelder JC, Defauw JJ. Medium-term outcome of different surgical methods to cure atrial fibrillation: is less worse? Interact Cardiovasc Thorac Surg 2008;7:201-206

8. de Bakker JM, van Capelle FJ, Janse MJ, Tasseron S, Vermeulen JT, de Jonge N, Lahpor JR. Slow conduction in the infarcted human heart. ‘Zigzag’ course of activation. Circulation 1993;88:915-926

9. Spach MS, Josephson ME. Initiating reentry: the role on nonuniform anisotropy in small circuits. J Cardiovasc Electrophysiol 1994;5:182-209

10. Kawara T, Derksen R, de Groot JR, Coronel R, Tasseron S, Linnenbank AC, Hauer RN, Kirkels H, Janse MJ, de Bakker JM. Activation delay after premature stimulation in chronically diseased human myocardium relates to the architecture of interstitial fibrosis. Circulation 2001;104:3069-3075

11. Ballaux PK, Geuzebroek GS, van Hemel NM, Kelder JC, Dossche KM, Ernst JM, Boersma LV, Wever EF, Brutel de la Rivière A, Defauw JJ. Freedom from atrial arrhythmias after classic Maze III surgery: a 10-year experience. J Thorac Cardiovasc Surg 2006;132:1433-1440

12. European Heart Rhythm Association (EHRA); European Cardiac Arrhythmia Scoiety (ECAS); American College of Cardiology (ACC); American Heart Association (AHA); Society of Thoracic Surgeons (STS), Calkins H, Brugada J, Packer DL, Cappato R, Chen SA, Crijns HJ, Damiano RJ Jr, Davies DW, Haines DE, Haissaguerre M, Iesaka Y, Jackman W, Jais P, Kottkamp H, Kuck KH, Lindsay BD, Marchlinski FE, McCarthy PM, Mont JL, Morady F, Nademanee K, Natale A, Pappone C, Prystowsky E, Raviele A, Ruskin JN, Shemin RJ. HRS/EHRA/ECAS expert Consensus Statement on catheter and surgical ablation of atrial fibrillation: recommendations for personnel, policy, procedures and follow-up. A report of the Heart Rhythm Society (HRS) Task Force on catheter and surgical ablation of atrial fibrillation. Heart Rhythm 2007;4:816-861


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13. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999;130:461-470

14. Oken DE, Boucek RJ. Quantitation of collagen in human myocardium. Circ Res 1957;5:357-361

15. Franco D, Campione M. The role of Pitx2 during cardiac development. Linking left-right signaling and congenital heart diseases. Trends Cardiovasc Med 2003;13:157-163

16. Abdul-Karim R. Fetal physiology--a review. Obstet Gynecol Surv 1968;23(8):713-745

17. Fauchier L, Pierre B, de Labriolle A, Grimard C, Zannad N, Babuty D. Antiarrhythmic effect of statin therapy and atrial fibrillation a meta-analysis of randomized controlled trials. J Am Coll Cardiol 2008;51:828-835

18. Winchester DE, Wen X, Xie L, Bavry AA. Evidence of pre-procedural statin therapy a meta-analysis of randomized trials. J Am Coll Cardiol 2010;56:1099-1109

19. Kostapanos MS, Liberopoulos EN, Goudevenos JA, Mikhailidis DP, Elisaf MS. Do statins have an antiarrhythmic activity? Cardiovasc Res 2007;75:10-20

20. Schotten U, Neuberger HR, Allessie MA. The role of atrial dilatation in the domestication of atrial fibrillation. Prog Biophys Mol Biol 2003;82:151-162

21. Barasch E, Gottdiener JS, Aurigemma G, Kitzman DW, Han J, Kop WJ, Tracy RP. Association between elevated fibrosis markers and heart failure in the elderly: the cardiovascular health study. Circ Heart Fail 2009;2:303-310

22. Nakai T, Chandy J, Nakai K, Bellows WH, Flachsbart K, Lee RJ, Leung JM. Histologic assessment of right atrial appendage myocardium in patients with atrial fibrillation after coronary artery bypass graft surgery. Cardiology 2007;108:90-96

23. Gramley F, Lorenzen J, Knackstedt C, Rana OR, Saygili E, Frechen D, Stanzel S, Pezzella F, Koellensperger E, Weiss C, Münzel T, Schauerte P. Age-related atrial fibrosis. Age 2009;31:27-38

24. Mahnkopf C, Badger TJ, Burgon NS, Daccarett M, Haslam TS, Badger CT, McGann CJ, Akoum N, Kholmovski E, Macleod RS, Marrouche NF. Evaluation of the Left Atrial Substrate in Patients with Lone Atrial Fibrillation Using Delayed-Enhanced MRI: Implications for Disease Progression and Response to Catheter Ablation. Heart Rhythm 2010;7:1475-1481

Effects of acetylcholine and noradrenalin on action potentials of isolated rabbit sinoatrial and atrial myocytes

6

A.O. Verkerk*

G.S.C. Geuzebroek*

M.W. VeldkampR. Wilders

*These authors have contributed equally to this study

Front Physiol 2012;3:174

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AbstractThe autonomic nervous system controls heart rate and contractility through sympathetic and parasympathetic inputs to the cardiac tissue, with acetylcholine (ACh) and noradrenalin (NA) as the chemical transmitters. In recent years, it has become clear that specific Regulators of G protein Signalling proteins (RGS proteins) suppress muscarinic sensitivity and parasympathetic tone, identifying RGS proteins as intriguing potential therapeutic targets. In the present study, we have identified the effects of 1 µM ACh and 1 µM NA on the intrinsic action potentials of sinotrial (SA) nodal and atrial myocytes. Single cells were enzymatically isolated from the SA node or from the left atrium of rabbit hearts. Action potentials were recorded using the amphotericin-perforated patch-clamp technique in the absence and presence of ACh, NA or a combination of both. In SA nodal myocytes, ACh increased cycle length and decreased diastolic depolarization rate, whereas NA decreased cycle length and increased diastolic depolarization rate. Both ACh and NA increased maximum upstroke velocity. Furthermore, ACh hyperpolarized the maximum diastolic potential. In atrial myocytes stimulated at 2 Hz, both ACh and NA hyperpolarized the maximum diastolic potential, increased the action potential amplitude, and increased the maximum upstroke velocity. Action potential duration at 50 and 90% repolarization was decreased by ACh, but increased by NA. The effects of both ACh and NA on action potential duration showed a dose dependence in the range of 1–1,000 nM, while a clear-cut frequency dependence in the range of 1–4 Hz was absent. Intermediate results were obtained in the combined presence of ACh and NA in both SA nodal and atrial myocytes. Our data uncover the extent to which SA nodal and atrial action potentials are intrinsically dependent on ACh, NA or a combination of both and may thus guide further experiments with RGS proteins.

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IntroductionElectrical activity of cardiac cells is modulated through adrenergic and muscarinic stimulation. The intrinsic pacemaker activity of sinoatrial (SA) nodal cells is accelerated upon noradrenalin-mediated stimulation of the adrenergic β-receptors and decelerated by acetylcholine-mediated stimulation of the muscarinic M2-receptors. Both are G protein-coupled receptors (GPCRs). In recent years, it has become widely appreciated that GPCR signalling in SA nodal pacemaker cells is much more complex than activation of a single membrane current, e.g. activation of the acetylcholine-activated outward potassium current IK,ACh in response to a release of acetylcholine.1,2 If noradrenalin binds to its β-receptor, it activates adenylyl cyclases through its stimulatory G protein α-subunit (Gαs) and thus increases cAMP and protein kinase A levels. Similarly, if acetylcholine binds to its receptor, it decreases these levels through its inhibitory G protein α-subunit (Gαi). In addition, acetylcholine exerts its effects through a Gβγ-mediated activation of the aforementioned IK,ACh. Noradrenalin accelerates the interdependent ‘membrane clock’ and ‘calcium clock’ of the pacemaker cell, whereas these are decelerated by acetylcholine.1,2

It should be kept in mind that noradrenalin not only stimulates the Gs-coupled β1–β3 adrenoceptors but also the Gq-coupled α1 and Gi-coupled α2 adrenoceptors, and that acetylcholine not only stimulates the Gi-coupled M2-receptor but also the other muscarinic receptors, including the Gq-coupled M3-receptor, which is functionally present in the murine sinoatrial node.3 Adding to this complexity, it has also become clear that GPCR signalling is not a simple linear pathway of receptor, G protein and effector. Signalling may be modulated by G protein-binding partners, specifically by members of the family of Regulators of G protein Signalling proteins (RGS proteins). RGS proteins may suppress muscarinic sensitivity and parasympathetic tone and thus affect heart rate and susceptibility to atrial fibrillation. If neuronally expressed, RGS proteins may also affect both sympathetic and parasympathetic output in addition to their direct effects in cardiac myocytes. The function of RGS proteins and their potential as therapeutic targets have recently received attention in several reviews.4-8

Studies with transgenic mice have underlined the important role of specific RGS proteins in the heart. In RGS4 knockout (RGS4−/−) mice, Cifelli et al. observed

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enhanced heart rate responses to the cholinergic agonist carbamylcholine chloride (carbachol), both in intact animals and in isolated hearts.9 In line with these observations, the beating frequency of isolated SA nodal myocytes, as assessed with the perforated patch-clamp technique at room temperature, did not differ between RGS4−/− and control under basal conditions, but showed an enhanced susceptibility to carbachol. This enhanced susceptibility was associated with a reduced desensitization and a slower deactivation of IK,ACh in RGS4−/− myocytes. Similar observations were made by Yang et al. with RGS6 knockout (RGS6−/−) mice.10 An enhanced bradycardia in response to carbachol was found in intact animals, in perfused hearts and in isolated SA nodal myocytes. Whole-cell patch-clamp recordings from cultured RGS6−/− atrial myocytes revealed a significant reduction in the time course of activation and deactivation of IK,ACh as well as the extent of its desensitization. Of note, perfused hearts from RGS6−/− mice showed a normal chronotropic response to the β-adrenergic agonist isoproterenol. Posokhova et al. also studied heart rate and IK,ACh in RGS6−/− mice.11 In intact animals, they found a mild resting bradycardia and an enhanced bradycardic effect of carbachol. In whole-cell patch-clamp recordings from adult SA nodal and cultured neonatal atrial myocytes, IK,ACh showed a slowed deactivation rate, which was also observed in case of deletion of the type 5 G protein β subunit (Gβ5), indicating that the effect is mediated by the RGS6/Gβ5 complex rather than by RGS6 itself.

The modulatory role of endogenous RGS proteins in the muscarinic and adrenergic control of heart rate had already been demonstrated in studies by Fu et al.12,13 Furthermore, Bender et al. had shown that the β-adrenergic effects on deactivation of IK,ACh, i.e. a marked increase in the time constant of its deactivation, are mediated via RGS10 in rat atrial myocytes.14 Also, Tuomi et al. had found that atrial effective refractory periods were smaller in RGS2 knockout mice, likely due to enhanced muscarinic M3-receptor activity, and that these mice had an enhanced susceptibility to pacing-induced atrial tachycardia and fibrillation.15

Given the potential role of RGS proteins as therapeutic means for modulating heart rate and atrial effective refractory period, it is important to know the effects of noradrenalin and acetylcholine on the intrinsic electrical activity of cardiac myocytes, specifically SA nodal and atrial myocytes. Over a period of decades, studies at the whole-heart and tissue levels have provided us with important data


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regarding the effects of sympathetic and parasympathetic stimulation and their interaction.16-21 However, data on action potential configuration from isolated myocytes are not abundant, particularly with the second messenger systems intact. In the present study, we used the amphotericin-perforated patch-clamp technique to record action potentials from isolated rabbit SA nodal and left atrial myocytes and assess the response to noradrenalin, acetylcholine or a combination thereof, thus providing data that may prove useful in the development of RGS protein based therapeutical means.

Materials and MethodsCell preparationAll experiments were carried out in accordance with guidelines of the local institutional animal care and use committee. In addition, the investigation complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Both SA nodal and atrial cells were isolated from hearts of male New Zealand White rabbits of 4 months old.

Single SA nodal cells were enzymatically isolated from the entire SA nodal region as described previously.22 Single atrial cells were isolated by enzymatic dissociation from the left atrium using the same protocol as described previously for the isolation of ventricular myocytes23, with the adaptation that the enzymatic solution was complemented with 6.6 µg/mL protease. Small aliquots of either SA nodal or atrial cell suspension were put in a recording chamber on the stage of an inverted microscope. Cells were allowed to adhere for 5 min after which superfusion with Tyrode’s solution was started. Tyrode’s solution had a temperature of 36 ± 0.2°C and contained (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, and HEPES 5.0; pH was set to 7.4 with NaOH. Spindle and elongated spindle-like cells displaying regular contractions were selected for recordings from SA nodal myocytes. Intrinsically quiescent rod-shaped cross-striated myocytes with a smooth surface were selected for recording from atrial myocytes.

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Patch-clamp experimentsAction potentials were recorded by the amphotericin-perforated patch-clamp technique using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). SA nodal action potentials were low-pass filtered (cut-off frequency 1 kHz) and digitized at 2 kHz; atrial action potentials at 5 and 40 kHz, respectively. Potentials were corrected for the estimated liquid junction potential.24 Data acquisition and analysis were accomplished using custom software. For recording from SA nodal myocytes, pipettes (borosilicate glass; resistance 2-3 MΩ) were heat polished and filled with solution containing (in mM): K-gluc 125, KCl 20, NaCl 10, amphotericin-B 0.22, and HEPES 10; pH was set to 7.2 with KOH. For recording from atrial myocytes, the pipette solution contained (in mM): K-gluc 110, KCl 30, NaCl 5, MgCl2 1, amphotericin-B 0.22, and HEPES 10; pH was set to 7.28 with KOH. Action potentials in atrial cells were elicited at 1 to 4 Hz by 2-ms, ≈1.5× threshold current pulses through the patch pipette.

Action potentials were characterized by duration at 20, 50, and 90% repolarization (APD20, APD50, and APD90, respectively), maximum diastolic potential (MDP), action potential amplitude (APA), maximum upstroke velocity (dV/dtmax), and, in case of SA nodal myocytes, cycle length and diastolic depolarization rate measured over the 50-ms time interval starting at MDP +1 mV. Parameter values obtained from 10 consecutive action potentials were averaged. Action potentials were measured in the absence and presence of noradrenalin, acetylcholine or a combination of both in the same myocyte. In case of atrial myocytes, the myocyte was stimulated at a frequency of 2 Hz, unless otherwise stated. In order to obtain steady-state conditions, action potential recordings were started 5 minutes after the addition of noradrenalin or acetylcholine to the Tyrode’s solution.

StatisticsData are presented as mean ± SEM. Paired comparisons were made using a paired t-test. Group comparisons were made using either an unpaired t-test or one-way ANOVA with the Holm-Sidak post hoc test. Frequency dependence and dose dependence were assessed with the Friedman repeated measures ANOVA on ranks, followed by pairwise comparisons with the Student-Newman-Keuls post hoc test. P < 0.05 defined statistical significance.


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ResultsAction potential characteristics of SA nodal and atrial myocytesFirst, we characterized the action potentials of the SA nodal and left atrial myocytes that we used for our study. SA nodal cells were spontaneously active, while atrial cells were quiescent. In atrial cells, action potentials could be elicited by current pulses through the patch pipette. Figure 1A shows typical spontaneous action potentials of an SA nodal myocyte as well as typical action potentials recorded from an atrial myocte that was stimulated at 3 Hz, i.e. with a cycle length similar to that of the SA nodal myocyte. Average action potential parameters are summarized in Figure 1B. All parameters, except APD90, differed significantly between both cell types. Atrial myocytes had a stable resting membrane potential of −79.2 ± 1.1 mV (n = 12), while SA nodal cells showed a spontaneous diastolic depolarization with a maximum diastolic potential of −63.5 ± 1.7 mV (n = 12). In SA nodal cells, this diastolic depolarization resulted in pacemaker activity with an intrinsic cycle length of 302 ± 10 ms. In SA nodal cells, the maximum upstroke velocity was typically low (6.6 ± 1.1 V/s) as opposed to atrial myocytes (297 ± 39 V/s). In both cell types, action potentials overshot the zero potential value, but the APA was higher in atrial myocytes. Action potentials of atrial myocytes repolarized earlier and faster, resulting in shorter APD20 and APD50; however, APD90 did not differ significantly. Atrial action potentials showed a frequency dependence in maximum upstroke velocity, APD20 and APD50. Both APD20 and APD50 increased with an increase in stimulus frequency, whereas maximum upstroke velocity tended to decrease with an increase in frequency (Figure 1C).

Effects of noradrenalin and acetylcholine on SA nodal action potentialsNext, we assessed the effects of noradrenalin, acetylcholine or a combination thereof on the action potentials of our SA nodal myocytes. As illustrated in Figure 2A, application of noradrenalin resulted in an increase in the spontaneous beating frequency, associated with an increase in the diastolic depolarization rate without any striking differences in other action potential parameters. Application of acetylcholine, on the other hand, resulted in a clear hyperpolarization of the maximum diastolic potential, a decrease in the diastolic depolarization rate and a dramatic increase in cycle length (Figure 2B). If acetylcholine was applied in

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the presence of noradrenalin, there was no substantial hyperpolarization of the maximum diastolic potential and the increase in cycle length was less prominent (Figure 2C). Figure 3 summarizes the effects on action potential parameters for a total of 13 cells. These data confirm the aforementioned effects on cycle length (Figure 3A), diastolic depolarization rate (Figure 3B) and maximum diastolic potential (Figure 3C). In addition, Figure 3 shows that both noradrenalin and acetylcholine caused an increase in maximum upstroke velocity, which was larger in case of acetylcholine (Figure 3E). Figure 3 may suggest that acetylcholine increases action potential amplitude (Figure 3D) and action potential duration (Figures 3F–H), but it should be noted that these data are not statistically significant. According to Figure 3, and also Figure 2C, intermediate results were obtained in the combined presence of noradrenalin and acetylcholine, but it should be noted that these observations are based on data from only one cell, as emphasized by the use of ‘bleached’ bars for ‘NA + ACh’ in Figure 3.

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Figure 1. Action potential characteristics of isolated rabbit SA nodal (SAN) and atrial myocytes. (A) Typical action potentials of an isolated SA nodal myocyte and an atrial myocyte. The atrial myocyte was stimulated at 3 Hz. (B) Action potential parameters of SA nodal and atrial myocytes, including maximum diastolic potential (MDP), action potential amplitude (APA), maximum upstroke velocity (dV/dtmax), and action potential duration at 20, 50, and 90% repolarization (APD20, APD50, and APD90, respectively). The atrial myocytes were stimulated at 3 Hz. Asterisks indicate significant differences between both cell types. (C) Frequency dependence of the action potential parameters of the atrial myocytes. Asterisks indicate significant differences between stimulus frequencies.


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A BFigure 3. Change in action potential parameters of isolated rabbit SA nodal myocytes in response to 1 µM noradrenalin (‘NA’, red bars), 1 µM acetylcholine (‘ACh’, blue bars), or a combination of both noradrenalin and acetylcholine (‘NA+ACh’, green bars). Asterisks indicate significant differences between effects of noradrenalin and acetylcholine. Hashes indicate significant differences from control. (A) Change in cycle length. (B) Change in diastolic depolarization rate measured over the 50-ms time interval starting at maximum diastolic potential + 1 mV (DDR50). (C) Change in maximum diastolic potential (MDP). (D) Change in action potential amplitude (APA). (E) Change in maximum upstroke velocity (dV/dtmax). (F) Change in action potential duration at 20% repolarization (APD20). (G) Change in action potential duration at 50% repolarization (APD50). (H) Change in action potential duration at 90% repolarization (APD90).

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Effects of noradrenalin and acetylcholine on atrial action potentialsWe also assessed the effects of noradrenalin, acetylcholine or a combination thereof on the action potentials of our atrial myocytes. The results are shown in Figures 4-7.

As illustrated in Figure 4A, application of noradrenalin resulted in a slight hyperpolarization of the maximum diastolic potential. The same was found for acetylcholine (Figure 4B), but not so much for the combination of noradrenalin and acetylcholine (Figure 4C). In all cases, i.e. application of noradrenalin, acetylcholine or a combination thereof, the action potential amplitude was increased. Effects on action potential duration differed between noradrenalin and acetylcholine, with an increase in case of noradrenalin and a decrease for acetylcholine (Figures 4A,B) and a less pronounced effect for the combination of noradrenalin and acetylcholine (Figure 4C).

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Figure 5 summarizes the effects on action potential parameters for a total of 23 cells. As already observed in the typical examples of Figure 4, there is a slight hyperpolarization of the maximum diastolic potential (Figure 5A) and an increase in action potential amplitude (Figure 5B). Also, under all conditions there is an increase in maximum upstroke velocity (Figure 5C). The effects on APD50 and APD90 are different between the three conditions, with a decrease in case of acetylcholine, an increase in case of noradrenalin and a smaller, but still significant increase in case of acetylcholine combined with noradrenalin (Figures 5E,F).

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In addition to the experiments of Figures 4 and 5, in which the cells were stimulated at 2 Hz, we carried out experiments in which we varied the stimulation frequency over the range of 1-4 Hz. The data, obtained from a total of 12 cells, did not reveal a clear-cut frequency dependence in the effects of acetylcholine or noradrenalin on action potential duration (Figure 6). Data obtained in the combined presence of noradrenalin and acetylcholine are from only two cells and therefore appear ‘grayed out’ in Figure 6.

The above data were obtained with noradrenalin and acetylcholine concentrations of 1 µM (1,000 nM). In a total of 5 cells, we varied these concentrations over the range of 1–1,000 nM. The effects on action potential duration showed a dose dependence, in particular for APD50, with considerably smaller effects at 1 nM than at 1 µM (Figure 7). However, it should be noted that the data with acetylcholine are from one cell, as are the data in the combined presence of noradrenalin and acetylcholine. These data therefore appear ‘grayed out’ in Figure 7.

Figure 6. Frequency dependence of the changes in action potential duration of isolated rabbit left atrial myocytes in response to 1 µM noradrenalin (‘NA’, red symbols), 1 µM acetylcholine (‘ACh’, blue symbols), or a combination of both noradrenalin and acetylcholine (‘NA+ACh’, green symbols). Cells were stimulated at 1-4 Hz. Asterisk indicates significant difference between stimulus frequencies. (A) Action potential duration at 50% repolarization (APD50). (B) Action potential duration at 90% repolarization (APD90).

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DiscussionIn the present study, we have assessed the effects of noradrenalin, acetylcholine or a combination thereof on the action potential configuration of rabbit SA nodal and left atrial myocytes. Action potentials were recorded under close-to-physiological conditions. The temperature of the bath solution was maintained at 36°C and the amphotericin-perforated patch-clamp technique was used to minimize intracellular dialysis, thus maintaining the integrity of second-messenger systems. To the best of our knowledge, the present study is the first in which the effects of noradrenalin, acetylcholine or a combination thereof on the action potential configuration of isolated sinoatrial and atrial myocytes has been tested in detail under such close-to-physiological conditions.

As detailed in Figure 1, our SA nodal and atrial myocytes showed clearly distinct action potential characteristics. We obtained our myocytes from rabbit, which is a widely used animal model in the field of cardiac cellular electrophysiology. In many respects, human action potential morphology is better resembled by rabbit than by mouse.25 Yet, one should be aware that there may be species differences in the underlying mechanisms, as there are, e.g., in calcium handling and adenylate cyclase activity between mouse and man.26

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Figure 7. Dose dependence of the changes in action potential duration of isolated rabbit left atrial myocytes in response to noradrenalin (‘NA’, red symbols), acetylcholine (‘ACh’, blue symbols), or a combination of both (‘NA+ACh’, green symbols).Concentration was varied between 1 and 1,000 nM. Asterisks indicate significant differences between concentrations. (A) Action potential duration at 50% repolarization (APD50). (B) Action potential duration at 90% repolarization (APD90). Note logarithmic abscissa.

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Effects of noradrenalin and acetylcholine on SA nodal action potentialsThe beating rate of our SA nodal cells increases upon application of noradrenalin and decreases upon application of acetylcholine (Figures 2A,B and 3A), which is associated with an increase and a decrease in the rate of diastolic depolarization, respectively (Figure 3B). It is tempting to explain this change in depolarization rate by a cAMP-mediated upregulation or downregulation of the hyperpolarization-activated ‘pacemaker current’ or ‘funny current’ If.

27 However, one should be cautious when drawing conclusions regarding underlying mechanisms from changes in action potentials. This may be illustrated by the increase in maximum upstroke velocity that is observed in response to noradrenalin as well as acetylcholine (Figure 3E). In case of noradrenalin, this increase may well result from an upregulation of the L-type calcium current ICa,L

28, but in case of acetylcholine the even larger increase may be the effect of less voltage-dependent inactivation of ICa,L due to the larger recovery time between consecutive upstrokes and less calcium-dependent inactivation of ICa,L due to the smaller calcium transients.2 Additionally, the activation of a small number of fast sodium channels may have contributed to the increase in maximum upstroke velocity as a result of the acetylcholine-induced hyperpolarization of the maximum diastolic potential which results in incomplete voltage-dependent inactivation of these channels.29 A full exploration of the autonomic modulation of the complex dynamic interactions of voltage- and calcium-dependent processes within the SA nodal pacemaker cell may require in silico experiments.1 Such experiments have already proven useful in a computer simulation study of the autonomic modulation of the electrical activity of bullfrog atrial myocytes.30 Figures 3F-H suggest an increase in action potential duration in response to acetylcholine, but this may reflect, at least in part, the hyperpolarization of the maximum diastolic potential (Figure 3C) rather than a ‘true’ increase in action potential duration. This hyperpolarization can be readily explained by the activation of IK,ACh.

2

A reduction in beating frequency as well as a hyperpolarization of the maximum diastolic potential was also reported for SA nodal myocytes isolated from wild-type mice in response to carbachol.9 These effects were both larger for myocytes from RGS4−/− mice. In the top panel of their Online Figure V, Yang et al. show “representative recordings of spontaneous action potential firing in SA nodal myocytes isolated from wild-type mice”.10 With an increase in cycle length,


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a decrease in diastolic depolarization rate, a hyperpolarization of the maximum diastolic potential and an increase in action potential amplitude, the changes in action potential configuration in response to carbachol are consistent with our data for rabbit SA nodal myocytes in response to acetylcholine (Figure 3).

Effects of noradrenalin and acetylcholine on atrial action potentialsIn our atrial myocytes, we found a hyperpolarization of the maximum diastolic potential (or ‘resting’ membrane potential) in response to acetylcholine (Figure 5A), as we did in our SA nodal myocytes (Figure 3C). However, in contrast to our SA nodal myocytes, such hyperpolarization was also observed in response to noradrenalin. This may be explained by the β-adrenergic activation of IK,ACh in atrial myocytes31 and by the β-adrenergic stimulation of the atrial-specific potassium current IKH.32,33 Furthermore, we found an increase in action potential amplitude and, as in our SA nodal myocytes, an increase in maximum upstroke velocity both in response to noradrenalin and in response to acetylcholine (Figures 5B,C). This may be explained by an increase in the fast sodium current, which is responsible for the fast upstroke in atrial cells, due to the more negative maximum diastolic potential and thus larger availability of fast sodium channels (less voltage-dependent inactivation). On the other hand, increased intracellular calcium levels in response to noradrenalin may have inhibitory effects on these sodium channels, but such effects are not apparent in our current data.34

Action potential duration is increased in response to noradrenalin and decreased in response to acetylcholine, with more or less additive effects if both noradrenalin and acetylcholine are present (Figures 5D-F). The shortening of the action potential in response to acetylcholine is in line with data from literature and may be attributed to the activation of IK,ACh.

35,36 Of note, acetylcholine does not inhibit the L-type calcium current of atrial myocytes under basal conditions, in contrast to that of SA nodal myocytes.37

The increase in action potential duration in response to noradrenalin

(Figures 5D-F) may be attributed to the increase in ICa,L in response to β-adrenergic stimulation38,39, which would then be sufficiently large to counteract the action potential shortening effects of the aforementioned β-adrenergic activation of IK,ACh and IKH. However, Yeh et al. observed a prominent decrease in action potential

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duration in a canine left atrial tissue preparation in response to specific β-adrenergic stimulation by isoproterenol (isoprenaline, 5 µM).33 This suggests that the α-adrenergic action of noradrenalin is essential for the increase in action potential duration that we observed in our experiments. A clear increase in action potential duration in response to the selective α1-adrenergic receptor agonist phenylephrine (100 µM) has been observed in left, but not right, atrial preparations from rabbit heart.40 In isolated rat atria, Northover also observed a significant increase in action potential duration in response to phenylephrine (50 µM), whereas the effects of isoproterenol (0.5 µM) were small and rather variable.41 In contrast, phenylephrine (100 µM) only had a minor effect on action potential duration in the experiments by Yeh et al.33 The apparent discrepancies in experimental results may be explained by species- and tissue-dependent differences and/or by differences in cAMP levels due to differences in basal adenylyl cyclase activity.42,43 Furthermore, a dose dependence in the effects of isoproterenol and phenylephrine on the action potential and the underlying transmembrane currents may play a role.30

The data on action potential duration of Figure 5 were obtained at a stimulus frequency of 2 Hz. Figure 6 demonstrates that similar effects were obtained at other frequencies in the range of 1-4 Hz, without a clear-cut frequency dependence, although the increase in APD50 at 1 Hz in the presence of noradrenalin is significantly different from that at 4 Hz. The data at 2 Hz of Figure 6A are in line with the data of Figure 5E. Also, there is consistency between the data at 2 Hz of Figure 6B and the data of Figure 5F. However, the difference between ‘NA’ and ‘NA+ACh’ in Figure 6B is smaller than expected from Figure 5F, where this difference is almost significant (P = 0.06).

CaveatsIn the present study, we have used the amphotericin-perforated patch-clamp technique to test the effects of noradrenalin, acetylcholine or a combination thereof on the action potential configuration of isolated sinoatrial and atrial myocytes under close-to-physiological conditions. Whereas this approach allows a detailed study of the effects of a well-controlled dose of these neurotransmitters on the intrinsic action potential configuration of these isolated myocytes, it does not take into account the complexity and heterogeneity of the intact myocardium.


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Therefore, caution should be applied when translating our in vitro results to the in vivo situation. Furthermore, although human action potential morphology is better resembled by rabbit than by mouse, there may be species differences in the effects of noradrenalin and acetylcholine.

ConclusionOur data show that acetylcholine and noradrenalin can modulate the beating rate of SA nodal myocytes and the action potential duration of atrial myocytes over a wide range. If specific SA nodal RGS protein isoforms exist that suppress muscarinic or adrenergic sensitivity of the SA node, they may prove therapeutical targets for the treatment of SA nodal bradycardia or tachycardia. Atrial-specific RGS protein isoforms may prove useful as therapeutical targets to increase the adrenergic sensitivity of atrial cells, thereby increasing their action potential duration and refractory period and thus decreasing the risk of atrial tachycardia and fibrillation. This will, however, require a large body of research before one can think of clinical translation.

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References

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2. van Borren MM, Verkerk AO, Wilders R, Hajji N, Zegers JG, Bourier J, Tan HL, Verheijck EE, Peters SL, Alewijnse AE, Ravesloot JH. Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells. Basic Res Cardiol 2010;105:73-87

3. Abramochkin DV, Tapilina SV, Sukhova GS, Nikolsky EE, Nurullin LF. Functional M3 cholinoreceptors are present in pacemaker and working myocardium of murine heart. Pflugers Arch 2012;463:523-529

4. Sjögren B, Neubig RR. Thinking outside of the “RGS box”: new approaches to therapeutic targeting of regulators of G protein signaling. Mol Pharmacol 2010;78:550-557

5. Sjögren B, Blazer LL, Neubig RR. Regulators of G protein signaling proteins as targets for drug discovery. Prog Mol Biol Transl Sci 2010;91:81-119

6. Kimple AJ, Bosch DE, Giguère PM, Siderovski DP. Regulators of G-protein signaling and their Gα substrates: promises and challenges in their use as drug discovery targets. Pharmacol Rev 2011;63:728-749

7. Tilley DG. G protein-dependent and G protein-independent signaling pathways and their impact on cardiac function. Circ Res 2011;109:217-230

8. Zhang P, Mende U. Regulators of G-protein signaling in the heart and their potential as therapeutic targets. Circ Res 2011;109:320-333

9. Cifelli C, Rose RA, Zhang H, Voigtlaender-Bolz J, Bolz SS, Backx PH, Heximer SP. RGS4 regulates parasympathetic signaling and heart rate control in the sinoatrial node. Circ Res 2008;103:527-535

10. Yang J, Huang J, Maity B, Gao Z, Lorca RA, Gudmundsson H, Li J, Stewart A, Swaminathan PD, Ibeawuchi SR, Shepherd A, Chen CK, Kutschke W, Mohler PJ, Mohapatra DP, Anderson ME, Fisher RA. RGS6, a modulator of parasympathetic activation in heart. Circ Res 2010;107:1345-1349

11. Posokhova E, Wydeven N, Allen KL, Wickman K, Martemyanov KA. RGS6/Gβ5 complex accelerates IKACh gating kinetics in atrial myocytes and modulates parasympathetic regulation of heart rate. Circ Res 2010;107:1350-1354

12. Fu Y, Huang X, Zhong H, Mortensen RM, D’Alecy LG, Neubig RR. Endogenous RGS proteins and Galpha subtypes differentially control muscarinic and adenosine-mediated chronotropic effects. Circ Res 2006;98:659-666

13. Fu Y, Huang X, Piao L, Lopatin AN, Neubig RR. Endogenous RGS proteins modulate SA and AV nodal functions in isolated heart: implications for sick sinus syndrome and AV block. Am J Physiol Heart Circ Physiol 2007;292:H2532-H2539


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14. Bender K, Nasrollahzadeh P, Timpert M, Liu B, Pott L, Kienitz MC. A role for RGS10 in beta-adrenergic modulation of G-protein-activated K+ (GIRK) channel current in rat atrial myocytes. J Physiol 2008;586:2049-2060

15. Tuomi JM, Chidiac P, Jones DL. Evidence for enhanced M3 muscarinic receptor function and sensitivity to atrial arrhythmia in the RGS2-deficient mouse. Am J Physiol Heart Circ Physiol 2010;298:H554-H561

16. Toda N, Shimamoto K. The influence of sympathetic stimulation on transmembrane potentials in the S-A node. J Pharmacol Exp Ther 1968;159:298-305

17. Grodner AS, Lahrtz HS, Pool PE, Braunwald E. Neurotransmitter control of sinoatrial pacemaker frequency in isolated rat atria and in intact rabbits. Circ Res 1970;27:867-873

18. Levy MN. Sympathetic-parasympathetic interactions in the heart. Circ Res 1971;29:437-445

19. Mackaay AJ, Op’t Hof T, Bleeker WK, Jongsma HJ, Bouman LN. Interaction of adrenaline and acetylcholine on cardiac pacemaker function. Functional inhomogeneity of the rabbit sinus node. J Pharmacol Exp Ther 1980;214:417-422

20. Boyett MR, Kodama I, Honjo H, Arai A, Suzuki R, Toyama J. Ionic basis of the chronotropic effect of acetylcholine on the rabbit sinoatrial node. Cardiovasc Res 1995;29:867-878

21. Brack KE, Coote JH, Ng GA. Interaction between direct sympathetic and vagus nerve stimulation on heart rate in the isolated rabbit heart. Exp Physiol 2004;89:128-139

22. Verkerk AO, den Ruijter HM, Bourier J, Boukens BJ, Brouwer IA, Wilders R, Coronel R. Dietary fish oil reduces pacemaker current and heart rate in rabbit. Heart Rhythm 2009;6:1485-1492

23. Den Ruijter HM, Verkerk AO, Coronel R. Incorporated fish oil fatty acids prevent action potential shortening induced by circulating fish oil fatty acids. Front Physiol 2010;1:149

24. Barry PH, Lynch JW. Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol. 1991;121:101-117

25. Brunner M, Peng X, Liu GX, Ren XQ, Ziv O, Choi BR, Mathur R, Hajjiri M, Odening KE, Steinberg E, Folco EJ, Pringa E, Centracchio J, Macharzina RR, Donahay T, Schofield L, Rana N, Kirk M, Mitchell GF, Poppas A, Zehender M, Koren G. Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome. J Clin Invest 2008;118:2246-2259

26. Jweied E, deTombe P, Buttrick PM. The use of human cardiac tissue in biophysical research: the risks of translation. J Mol Cell Cardiol 2007;42:722-726

27. DiFrancesco D. The onset and autonomic regulation of cardiac pacemaker activity: relevance of the f current. Cardiovasc Res 1995;29:449-456

28. Hagiwara N, Irisawa H, Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol 1988;395:233-253

29. Veldkamp MW, Wilders R, Baartscheer A, Zegers JG, Bezzina CR, Wilde AA. Contribution of sodium channel mutations to bradycardia and sinus node dysfunction in LQT3 families. Circ Res 2003;92:976-983

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30. Shumaker JM, Clark JW, Giles WR. A model of beta-adrenergic effects on calcium and potassium current in bullfrog atrial myocytes. Am J Physiol 1991;261:H1937-H1944

31. Sorota S, Rybina I, Yamamoto A, Du XY. Isoprenaline can activate the acetylcholine-induced K+ current in canine atrial myocytes via Gs-derived betagamma subunits. J Physiol 1999;514:413-423

32. Ehrlich JR, Cha TJ, Zhang L, Chartier D, Villeneuve L, Hébert TE, Nattel S. Characterization of a hyperpolarization-activated time-dependent potassium current in canine cardiomyocytes from pulmonary vein myocardial sleeves and left atrium. J Physiol 2004;557:583-597

33. Yeh YH, Ehrlich JR, Qi X, Hébert TE, Chartier D, Nattel S. Adrenergic control of a constitutively active acetylcholine-regulated potassium current in canine atrial cardiomyocytes. Cardiovasc Res 2007;74:406-415

34. Casini S, Verkerk AO, van Borren MM, van Ginneken AC, Veldkamp MW, de Bakker JM, Tan HL. Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes. Cardiovasc Res 2009;81:72-81

35. Koumi S, Sato R, Hayakawa H. Characterization of the acetylcholine-sensitive muscarinic K+ channel in isolated feline atrial and ventricular myocytes. J Membr Biol. 1995;145:143-150

36. Patterson E, Scherlag BJ, Zhou J, Jackman WM, Lazzara R, Coscia D, Po S. Antifibrillatory actions of cisatracurium: an atrial specific M2 receptor antagonist. J Cardiovasc Electrophysiol 2008;19:861-868

37. Petit-Jacques J, Bois P, Bescond J, Lenfant J. Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes. Pflugers Arch 1993;423:21-27

38. Bean BP. Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology. J Gen Physiol 1985;86:1-30

39. Dun W, Yagi T, Rosen MR, Boyden PA. Calcium and potassium currents in cells from adult and aged canine right atria. Cardiovasc Res 2003;58:526-534

40. Jahnel U, Kaufmann B, Rombusch M, Nawrath H. Contribution of both alpha- and beta-adrenoceptors to the inotropic effects of catecholamines in the rabbit heart. Naunyn Schmiedebergs Arch Pharmacol 1992;346:665-672

41. Northover BJ. Effect of pre-treating rat atria with potassium channel blocking drugs on the electrical and mechanical responses to phenylephrine. Biochem Pharmacol 1994;47:2163-2169

42. Méry PF, Abi-Gerges N, Vandecasteele G, Jurevicius J, Eschenhagen T, Fischmeister R. Muscarinic regulation of the L-type calcium current in isolated cardiac myocytes. Life Sci 1997;60:1113-1120

43. Harvey RD, Belevych AE. Muscarinic regulation of cardiac ion channels. Br J Pharmacol 2003;139:1074-1084


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Neuropeptide substance-P causes action potential prolongation and resting membrane depolarization in rabbit atrial myocytes

7

G.S.C. GeuzebroekA.O. Verkerk

C.A. SchumacherA.C.G. van Ginneken

J.M.T. de BakkerR. Coronel

M.W. Veldkamp

submitted

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AbstractBackground: The cardiac nervous system plays an important role in the genesis of atrial fibrillation (AF). Apart from acetylcholine and norepinephrine, various neuropeptides are released in atrial myocardium of which the direct effects on atrial electrophysiology are largely unknown. We therefore investigated the effects of various neuropeptides including substance-P (SP) on atrial electrophysiology.

Methods and Results: Enzymatically isolated left atrial rabbit myocytes were superfused with SP, neuropeptide-Y, somatostatin-14 or vasoactive intestinal peptide, and studied with patch-clamp and calcium-fluorescence methodologies. With exception of SP, the neurotransmitters did not directly affect atrial action potential (AP) characteristics (at 1 µM). SP reduced both resting membrane potential (RMP) and action potential amplitude, and increased action potential duration at 90% of repolarization (APD90) by 40%. The effects on APD90 were dose-dependent and occurred at concentrations from 10 nM, but did not increase vulnerability to triggered activity. Voltage-clamp analysis revealed that SP significantly diminished the L-type Ca2+ current (ICa,L), the inward rectifier K+ current (IK1) and a steady-state outward current. The transient K+ outward current, the Ca2+-activated Cl- current and the Na+-Ca2+ exchanger current were unaffected, as was the intracellular Ca2+ handling. The reduction in RMP and increase in APD90 are due to the decrease in IK1 and steady-state outward current, respectively. The latter is probably carried by background-like K+ channels.

Conclusion: SP directly causes a substantial action potential prolongation due to inhibition of a background K+ current. Since a lengthening of atrial repolarization is potentially anti-arrhythmic, we speculate that SP can prevent and/or terminate AF.

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IntroductionThe intrinsic cardiac nervous system is located in the fat pads of the heart, especially on the atria, and contains highly complex networks of ganglia, also known as ganglionic plexus.1 These ganglia may contain up to 200 neurons1,2, which in addition to the well-known neurotransmitters acetylcholine and norepinephrine, also release a wide range of neuropeptides in the atria of the human heart.3

The cardiac autonomic nervous system has been implicated in the genesis of atrial fibrillation (AF). Indeed, electrical stimulation of the ganglionic plexus in the fatty pads causes a slowing of heart rate and provokes AF.4 Ablation of ganglionic plexus is used to treat patients with lone AF.5-7

Although the effects of noradrenalin (NA) and acetylcholine (ACh) on atrial cellular electrophysiology have been investigated8, little is known of the electrophysiological effects of various other neuropeptides/neurotransmitters on atrial electrophysiology. We therefore examined the electrophysiological response of single atrial myocytes to several neuropeptides, particularly substance-P (SP). Substance-P, the first mammalian tachykinin that was identified9, has been associated with cardiovascular regulation.2 The cardiac (physiological) response to SP has been mainly characterized by a decrease in heart rate and ventricular contractility, through stimulation of cholinergic neurons.10,11 SP has also been implicated in various cardiovascular diseases such as ischemia, heart failure and atrial fibrillation.12 For example, degeneration of SP immuno-reactive nerves has been reported in a dog model of atrial fibrillation.13 In addition, the occurrence of post-operational atrial fibrillation was associated with a decrease in SP serum levels in patients that had undergone coronary artery bypass surgery.14

Thus, we determined the electrophysiological effects of SP, neuropeptide-Y (NPY), somatostatin-14 (SOM-14), and vasoactive intestinal peptide (VIP) on isolated rabbit atrial myocytes. We established that SP significantly modulates atrial action potential (AP) characteristics through alterations in several types of membrane currents, while NPY, SOM-14 and VIP did not elicit a response (supplement figure and table).

MethodsThe study conformed to the ‘Guide for the Care and Use of Laboratory Animals’

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published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the local ethical committee.

Single cell preparation Male New Zealand White specified pathogen free rabbits (2.5-3.5 kg) were anaesthetized with a combination of 20 mg xylazine and 100 mg ketamine (intramuscularly) and heparinized with a bolus of 1000 IU heparin (intravenously). Subsequently, the animals were killed by 200 mg pentobarbital intravenously, after which the heart was quickly excised and mounted on a Langendorff perfusion apparatus. Single atrial cells were isolated from the left atrium by enzymatic dissociation using the protocol that was described previously (for the isolation of ventricular myocytes)15, with a modification consisting of complementing the enzymatic solution with 6.6 µg/mL protease.

Small aliquots of single cell suspension were introduced into a recording chamber on the stage of an inverted microscope. Cells were allowed to adhere for 5 minutes after which superfusion was started. Single quiescent rod-shaped myocytes with clear cross-striations and smooth surfaces were selected for measurements.

Cellular electrophysiologyData acquisition and analysisAPs and membrane currents were recorded at 36.5 °C with the amphotericin-B perforated or ruptured patch-clamp technique, using an Axopatch 200B Clamp amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA). Voltage control, data acquisition, and analysis were performed using custom-made software.

Series resistance was compensated for by 70%-80% and potentials were corrected for liquid junction potential.16 Signals were low-pass filtered (cut-off frequency: 5 kHz) and digitized at 40 kHz. Cell membrane capacitance (Cm) was estimated by dividing the decay time constant of the capacitive transient in response to 5 mV hyperpolarizing voltage clamp steps from a holding potential of –40 mV, by the series resistance.

Current-clamp experimentsAPs were measured with the amphotericin-B-perforated patch clamp technique


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using normal Tyrode’s solution containing (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0, pH 7.4 (NaOH). The patch-pipettes (borosilicate glass; 1-3 MΩ) were filled with a ‘standard’ solution containing (in mM): K-gluconate 110, KCl 30, NaCl 5, MgCl2 1, amphotericin-B 0.22, HEPES 10, pH 7.3 (KOH).

APs were elicited at 1 to 4 Hz by 2-ms (1.5× diastolic stimulation threshold) current pulses applied through the patch pipette. The following action potential characteristics were determined: resting membrane potential (RMP), maximal upstroke velocity (Vmax), AP amplitude (APA), and AP duration (APD) at 20%, 50% and 90% repolarization (APD20, APD50 and APD90, respectively). Values obtained from 10 consecutive APs were averaged.

Atrial myocytes were allowed to equilibrate for a 5-minute period of continuous stimulation (1 Hz) after which the wash-in of the following neurotransmitters and neuropeptides: 1 µM NA, 1 µM ACh, 1 µM Som-14, 1 µM VIP, 1 µM NPY, and 1 µM SP was started. Effects were assessed after equilibration, but not earlier than 3 minutes after application. In dose-response experiments, SP (1 nM – 10 µM) was applied in a cumulative sequence with 5 minutes intervals between increments in concentration. In addition, to assess the arrhythmogenic (triggered activity) effects of SP, atrial myocytes were rapidly paced at a rate of 5-6 Hz by a train of 20 consecutive stimuli, followed by a pause of 10 s. The occurrence of spontaneous depolarizations during the pause was counted in 4 consecutive tracings and averaged.

Voltage-clamp experimentsSteady state currents, L-type Ca2+ current (ICa,L), transient outward K+ current (Ito), Ca2+-activated Cl- current (ICl(Ca)) and Na+-Ca2+ exchange current (INCX) were measured with solutions specified below and using voltage-clamp protocols depicted in the corresponding figures.

Steady state currents, Ito and ICl(Ca) were measured using the amphotericin-B perforated patch clamp technique with standard pipette solution (see above) and normal Tyrode’s solution as external solution. For accurate Ito measurements, 30 µM tetrodotoxin (TTX), 0.25 mM CdCl2, 5 µM E-4031, and 100 µM chromanol 293B was added to the normal Tyrode’s solution. For ICl(Ca) measurements, we added 10 µM TTX and 2 mM 4-aminopyridine (4-AP) to the normal Tyrode’s solution.

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ICa,L was measured using the ruptured patch clamp technique with pipette solution containing (in mM): CsCl 145, K2-ATP 5, EGTA 10, HEPES 10, pH 7.2 (NMDG-OH). The extracellular solution contained (mM): TEA-Cl 145, CsCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0; pH 7.4 (NMDG-OH). ICa,L was measured in the presence of 0.25 mM 4,4’diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS; Sigma-Aldrich, MO, USA) to block ICl(Ca). INCX was measured using the ruptured patch clamp technique with pipette solution containing (in mM): CsCl2 145, NaCl 5, Mg-ATP 10, TEA-Cl 10, EGTA 20, CaCl2 10, HEPES 10, pH 7.2 (NMDG-OH). The bath solution consisted of a K+-free Tyrode’s solution to which 1 mM BaCl2, 2 mM CsCl2, 5 µM nifedipine, 100 µM ouabain, and 200 µM DIDS was added to suppress membrane currents other than INCX. INCX was measured as 10 mM Ni2+-sensitive current during a descending voltage ramp protocol. Since the effects of Ni2+ on INCX are reversible17, INCX measurements in the absence and presence of SP were carried out in the same cell.

Voltage-dependencies of (in)activation were determined by fitting a Boltzmann function (y=A/[1+exp(V-V1/2)/k]) to the individual curves, yielding a half-maximal voltage V1/2 (mV) and a slope factor k (mV). Time constants of inactivation were obtained by fitting current decay with a bi-exponential function y=y0+Afexp(-t/τf)+Asexp(–t/τs), where Af and As are the amplitudes of the fast and slow inactivating components, and τf and τs their respective inactivation time constants. Currents densities were calculated by dividing the current amplitude by Cm.

In voltage clamp experiments the effect of SP on the various membrane currents was assessed at a concentration of 10 µM.

Cytosolic Ca2+ transientsIntracellular Ca2+ (Ca2+

i) was measured in indo-1 loaded myocytes as described previously.18 Dual wavelength emission of indo-1 was recorded ((405-440)/(505-540) nm, excitation at 340 nm) and free Ca2+

i was calculated. Ca2+i transients were

elicited at 5 Hz using field stimulation. For determination of diastolic and systolic Ca2+

i concentrations, and Ca2+i transient amplitudes were, data from 10 consecutive

Ca2+i transients were averaged.

The effect of SP on Ca2+i was assessed at a concentration of 10 µM 3 minutes

after application.


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DrugsAll drugs used for cellular measurements were obtained from Sigma-Aldrich (MO, USA), except for E-4031 (Tocris, MN, USA), SP (Enzo Life Sciences, NY, USA), TTX (Abcam Biomedicals, Cambridge, UK) and NA (Centrafarm, Etten-Leur, The Netherlands).

DIDS was freshly prepared as a 0.5 M and chromanol 293B as a 0.1 M stock solution in DMSO. Nifedipine was prepared as a 5 mM stock solution in ethanol. E-4031 and TTX were prepared as a 5 and 30 mM stock solution in distilled water. All stock solutions were diluted appropriately before use. SP was freshly diluted at its final concentration. DIDS and nifedipine were stored in the dark.

StatisticsData are presented as mean±SEM. A student t-test or Two-Way Repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls method was used where appropriate. P < 0.05 was defined as statistical significant.

ResultsEffect of noradrenalin and acetylcholine on action potential characteristicsSingle atrial myocytes demonstrated an intact signal transduction system as evidenced by their characteristic response to these classical neurotransmitters as shown in supplement figure and table.8 Application of NA slightly hyperpolarized the resting membrane, and increased Vmax, APA and APD. ACh also hyperpolarized the resting membrane and increased Vmax and APA, but decreased APD.

Effect of neuropeptides on action potential characteristics; SP causes membrane depolarization and APD prolongation.Atrial myocytes did not respond to the presence of the neuropeptides Som-14, VIP and NPY at a concentration of 1 µM (supplemt figure and table). Application of SP on the other hand, resulted in substantial alterations in AP morphology in comparison to control conditions (CTRL), particularly a depolarization of the resting membrane and an increase in APD (Figure 1A). Figure 1B summarizes the effects of SP on AP parameters. At 1 µM SP, the initial fast repolarization (APD20) was not

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significantly affected, but APD50 (CTRL: 21.2±7.5 ms vs. SP: 43.4±14.6 ms, n=6) and APD90 (CTRL: 89.0±2.0 ms vs. SP: 125.2±2.5 ms, n=6) were significantly increased (Figure 1BI). The RMP (CTRL: -68.0±1.5 ms vs. SP: -62.0±2.1 ms, n=6) and the APA (CTRL: 108.6±3.4 ms vs. SP: 97.0±6.1 ms, n=6) were both significantly decreased (Figure 1BII). The slight reduction in Vmax was not statistical significant (P=0.3, Figure 1BIII). Furthermore, AP prolongation by SP was present at all stimulation frequencies (1 to 4 Hz) (Figure 1C), and occurred in a dose-dependent fashion from a concentration of 10 nM (Figure 1D).

Effects of substance-P on membrane currentsThe depolarization of the resting membrane and prolongation of the AP can result from a decrease in outward current, an increase in inward current, or both. To establish the mechanism(s) underlying the effect of SP on the AP characteristics, we

Figure 1. Effect of substance-P on action potential (AP) characteristics of rabbit atrial myocytes(A) Atrial action potentials elicited at 1Hz under control conditions and in the presence of 1µM substance-P (SP). (B) Bar histograms showing average values for AP duration at 20,50 and 90% of repolarization (APD20, APD50, and APD90) (BI), resting membrane potential (RMP) and action potential amplitude (APA) (BII), and maximal upstroke velocity (Vmax) (BIII) before (control) and after application of 1 µM SP. (C) Plot showing the frequency-dependence of APD90 before (control) and after application of 1 µM SP. (D) Plot showing concentration-dependence of relative APD90 before (control) and after application of increasing concentrations of SP. * P < 0.05

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investigated the major membrane currents underlying rabbit atrial APs.

Steady-state current. Steady-state currents were measured in the perforated-patch configuration at the end of hyper- and depolarization steps of 500 ms duration (holding potential -60 mV) in the absence of blockers (Figure 2A). Representative examples of current tracings recorded at -120 and +40 mV show a moderate decrease in steady-state in- and outward current in the presence of 10 µM SP (Figure 2B), which was confirmed by the average current-voltage (I-V) relationships in Figure 2C. The steady-state inward current at -120 mV, considered to be carried by IK1 channels, was significantly decreased by SP (CTRL: -6.8±1.1 pA/pF vs SP: -6.0±1.3 pA/pF, n=6). Besides, a significant decrease in steady-state outward current at all voltages positive to -40 mV was seen. At +50 mV the outward current decreased by 20% from 6.9±0.5 pA/pF (CTRL) to 5.5±0.6 pA/pF (SP). At voltages more positive than -40 mV the outward current is considered to be composed of different types of K+ currents, including the sustained component of the transient outward current (Ito), and of the slow, rapid and ultra-rapid components of the delayed rectifier K+ current (IKs, IKr and IKur, respectively).

Figure 2. Effect of substance-P on steady state currents. (A) Voltage protocol. (B) Current tracings recorded at -120 and +40 mV before (left panel) and after application of 10 µM substance-P (SP, right panel). (C) Average current-voltage relationships of the steady-state current before (control) and after application of 10 µM SP. * P < 0.05

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Transient outward K+ current (Ito). Figure 3B shows representative Ito peak currents upon depolarization to 50 mV (protocol as in Figure 3A). On average, Ito peak densities (Fig ure 3C) were not altered by SP, nor was the time course of current decay, as illustrated in Figure 3D. Figures 3E-G summarize the voltage-dependency of Ito activation and inactivation. SP caused a small, but significant shift in V1/2 of inactivation by -3.5 mV (Figure 3F, n=5, P=0.02) as well as a steepening of the slope of the voltage dependency of activation (CTRL: k=14.2±0.9 vs. SP: k=9.8±0.1 mV, Figure 3G, n=5, P<0.01). Figure 3H depicts the average steady state I-V relationships, obtained by current amplitude measurement at the end of the 500 ms voltage steps, in accordance with Figure 2C, but now in the presence of blockers for Na+ current (INa), ICa,L, IKr, and IKs. At -120 mV the steady-state inward current decreased from -6.9±1.6 pA/pF (CTRL) to -5.75±1.5 pA/pF (SP, n=5, P<0.01), while at +50 mV a 25% reduction in steady-state outward current was observed from 6.1±0.85 pA/pF (CTRL) to 4.6±0.46 pA/pF (SP, n=5, P=0.02).

L-type Ca2+ current (ICa,L). ICa,L was measured using a 2 step protocol (Figure 4A). During the first depolarizing pulses (P1) ICa,L activates, while the second pulse (P2) is used for measurement of voltage dependency of inactivation. The representative ICa,L recordings upon a depolarizing pulse to 0 mV (Figure 4B), and the average I-V relationships of ICa,L in Figure 4C, show a decrease in peak ICa,L in the presence of SP. At 0 mV, the reduction in peak ICa,L amounted to ~20%, from -21±3.3 pA/pF in control conditions to –16.6±2.3 pA/pF in the presence of SP (n=6, P<0.05). Neither the voltage dependencies of (in)activation (Figure 4D), nor the time course of current decay (Figure 4B, inset) were affected by SP.

Ca2+-activated Cl- current (ICl(Ca)). ICl(Ca) was elicited by depolarizing steps to +30, +40 and +50 mV from a holding potential of -60 mV (Figure 5A), and defined as the early transient peak (Figure 5B). SP did not affect ICl(Ca) as peak amplitudes were similar under control conditions and in the presence of SP (Figure 5C). Comparable to the data shown in Figure 2C and 3H, SP reduced steady-state outward currents at the end of the 250 ms depolarizing steps by ~25% (Figure 5D, at +50 mV: CTRL 7.2±0.8 pA/pF vs. SP 5.7±0.5, n=6, P=0.01) in the presence of 2 mM 4-AP. This


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rules out the possibility that the SP effect is mediated by a reduction in sustained component of Ito or IKur.

Figure 3. Effect of 10 µM substance-P (SP) on transient outward K+ current (Ito). (A) Voltage protocol. (B) Current tracings recorded at -120 and +50 mV before (left panel) and after application of 10 µM substance-P (SP, right panel). (C) Average current-voltage (I-V) relationships of Ito peak currents before (control) and after application of 10 µM SP. (D) Bar histogram showing average values for time constants of fast and slow inactivation before (control) and after application of SP. (E) Plot showing average voltage-dependencies of activation and steady-state inactivation before (control) and after application of 10 µM SP. Solid lines indicate the Boltzmann fits of the average data (F,G) Bar histograms showing average values for half-maximal voltage (V1/2) and slope factor (K) of voltage-dependence of inactivation and activation resp. before (control) and after application of 10 µM SP. (H) Average I-V relationships of steady-state current before (control) and after application of 10 µM SP. Blockers for Na+-, Ca2+-, rapid and slow delayed rectifier K+ currents were continuously present. * P < 0.05

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Na+-Ca2+ exchange current (INCX)). INCX was measured in Ca2+-buffered conditions as the Ni2+-sensitive current during a descending voltage ramp protocol (Figure 6A, inset). Figure 6B shows a representative example of INCX in control conditions and in the presence of SP. On average, SP had no effect on the reverse (outward) mode, nor on the forward (inward) mode of INCX (Figure 6B).


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Figure 4. Effect of 10 µM substance-P on L-type Ca2+ current (ICa,L). (A) Voltage protocol. (B) Current tracings recorded under control conditions and in the presence of 10 µM substance-P (SP). Insets: voltage protocol (left) and bar histogram showing the average time constants of fast and slow inactivation before (control) and after application of SP (right). (C) Average current-voltage relationships of ICa,L before (control) and after application of 10 µM SP. (D) Average voltage-dependencies of activation and steady-state inactivation before (control) and after application of SP. Solid lines indicate the Boltzmann fits of the average data. * indicates P < 0.05


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Figure 5. Effect of 10 µM substance-P on Ca2+ activated Cl- current (ICa(Cl)). (A) Voltage protocol. (B) Current tracings recorded during a depolarizing step to +30mV under control conditions and in the presence of 10 µM substance-P (SP). (C,D) Average current-voltage relationships of peak ICa(Cl) (C) and steady-state current at t=300 ms (D) before (control) and after application of 10 µM SP. Blockers for Na+-, transient outward and ultra-rapid delayed rectifier K+ currents were continuously present. * indicates P < 0.05.


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SP sensitive steady-state outward current. To obtain more insight in the nature of the steady-state outward current inhibited by SP, we deduced the SP sensitive current by subtracting the current amplitude in the presence of SP from the current amplitude measured under control conditions, using the data shown in Figure 2, 3 and 5. Figure 7 shows that the resultant SP sensitive I-V curves are largely similar, despite the presence of the various ion channel blockers. They are virtually linear and reverse sign at ~ -75 mV, close to the calculated Nernst potential for K+ ions (-85mV), indicating that an outward background (or leakage) K+ current is involved. The additional observation that the SP sensitive current in the voltage-range where ICa,L is active, is less pronounced in the presence of a Ca2+ channel blocker (open circles vs. black-filled circles, Figure 7), may point also to a contribution of a Ca2+-regulated K+ current.

Effects of SP on spontaneous action potentials and calcium homeostasisTo investigate the effects of SP on the incidence of spontaneous APs on the one hand, and modifications in Ca2+

i homeostasis on the other hand, atrial myocytes were rapidly paced at a rate of 5 Hz by a train of 20 consecutive stimuli, followed by a pause of 10 s. Figure 8 shows representative examples of the effects of SP on transmembrane potentials (Figure 8A) and Ca2+

i concentration (Figure 8B,C) measured in separate atrial myocytes during and after rapid pacing. The last 2 stimulated APs (Figure 8A) and Ca2+ transients (Figure 8B) of the rapid pacing protocol are shown. Under control conditions (Figure 8A, black line), burst pacing

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Figure 7. Effects of various membrane current blockers on the substance-P sensitive steady-state current. Substance-P (SP) sensitive currents were extracted from Figure 2 (black-filled circles), Figure 3 (open circles) and Figure 5 (grey filled circles), by subtraction of the current in the presence of SP from the current measured under control conditions.


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resulted in a transient depolarization directly after termination of stimulation and incidentally induced a spontaneous AP. In the presence of 10 µM SP (Figure 8A, grey line), there was an increase in the amplitude of the transient depolarization following the last stimulated AP and a depolarization of the resting membrane. Rapid pacing did not result in Ca2+ aftertransients (Figure 8B). Figure 8C,D shows that the diastolic, systolic and peak Ca2+

i were not different under control conditions and in presence of SP.

These findings indicate that 1) the SP induced membrane depolarization does not result in an increased incidence of spontaneous AP’s and 2) the reduction ICa,L does not affect Ca2+

i handling.

DiscussionThe cardiac autonomic nervous system plays a significant role in the induction of atrial fibrillation. Particularly, alterations in the sympatho-vagal balance and excessive intrinsic cardiac nerve activity have been implicated to trigger atrial arrhythmias.19-21 In addition to the classical neurotransmitters, the wide range of neuropeptides harboured by the atrial intracardiac ganglia and nerve fibers

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Figure 8. Effect of substance-P on triggered activity and intracellular calcium concentration. (A) Action potential (AP) recordings after burst pacing under control conditions and in the presence of 10 µM substance-P (SP). Insets: (left) Stimulation protocol (a train of 20 consecutive stimuli at 5 or 6 Hz was followed by a pause of 10 s, (right) Bar histogram showing the total number of triggered APs in 4 consecutive tracings before (control) and after application of 10 µM SP. (B) Intracellular calcium (Ca2+

i) transients measured after burst pacing before (control) and after application of 10 µM SP. (C) Ca2+

i transients under control conditions and in the presence of 10 µM SP. (D) Bar histograms showing average diastolic, systolic and peak Ca2+

i before (control) and after application of 10 µM SP.* P < 0.05

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potentially modulate atrial electrophysiology.3,12,22,23 To date however, the efferent actions on the atrial myocyte directly mediated by the various neuropeptides have scarcely been defined.

In this study we therefore explored the effects of several neuropeptides, most importantly SP, on the electrical properties of single rabbit atrial myocytes. Our study shows that SP exerts a significant effect on atrial action potential morphology, whereas other neuropeptides (i.e. NPY, SOM-14, VIP) do not. Alterations in action potential configuration by SP were characterized by a subtle reduction in resting membrane potential and action potential amplitude, together with a profound increase in action potential duration. The AP prolonging effect was dose-dependent and occurred from a concentration as low as 10 nM, suggesting that the electrophysiological actions of SP on atrial myocytes are receptor-mediated involving the neurokinin cell-surface receptors (NK1-NK3).24 Remarkably, the AP prolongation was also present at high pacing rates and was effectuated without an increase in ICa,L current and facilitation of spontaneous Ca2+ aftertransients. SP therefore may be a powerful anti-fibrillatory agent that is efficacious in the atrium.

Mechanisms of reduced resting membrane potential and action potential prolongation by substance PThe effects of SP on membrane currents are diverse and highly tissue-specific. Data on the effect of SP on membrane currents largely stem from studies on central and peripheral neurons, and to a lesser degree from studies on smooth muscle cells originating from the gastro-intestinal tract. To our knowledge no data are available concerning the actions of SP on membrane currents in cardiac myocytes.

We observed a small but significant decrease of steady-state inward current in the negative potential range (considered to be IK1), as well as a ~25% reduction in steady-state outward current in the positive potential range in the presence of SP. A reduction in IK1 is consistent with the observed depolarization of the resting membrane, and to the reduced APA and Vmax (although the latter was not statistically significant). Similar to our findings, SP mediated reduction of IK1 has also been reported in central and peripheral neurons from rat and guinea pig.25-29 Alternatively, the small decrease in steady-state inward current at negative potentials, could also reflect inhibition of constitutively active (i.e. in the absence


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of an agonist) acethylcholine-activated K+ current (IKACh).30 Indeed, IKACh carried by

GIRK1/GIRK4 channels expressed in oöcytes, is inhibited by SP through a protein kinase C dependent pathway.31 The reduction in steady-state outward current in the positive potential range complies with the observed AP prolongation in the presence of SP. The ionic mechanism underlying this decrease in steady-state outward current, however, remains to be elucidated. The well-known outward currents that are active in this voltage range in atrial myocytes are IKs, IKr and IKur.

32 Yet, our experiments do not indicate a decrease in any of these current types by SP, since the reduction in steady-state outward current persists in the presence of their respective blockers (Figure 7). Changes in outward ICa(Cl) and Ito do not contribute to the observed action potential prolongation, as peak amplitudes of both current types are unchanged in the presence of SP, despite minor kinetic alterations in the latter.

Finally, the reduction of ICa,L is expected to lead to a shortening of the AP. Therefore, the reduction in steady-state outward current by far outweighs the effect of ICa,L reduction on AP duration.

The nature of the SP sensitive steady state outward currentExamination of the SP sensitive current (Figure 7) indicates that an outward K+ current is involved, since the reversal potential is close to the calculated Nernst potential for K+ ions (-85mV). A potential candidate to be at the basis of this K+ current is the so-called background or leakage K+ current, which was found to be inhibited by SP in neurons.33-37 Although it has proven difficult to designate the channel type underlying this background K+ current, recent evidence suggests that members of the two-pore-domain K+ (K2P) channel family generate such a K+ background conductance.38 At least two members of the K2P family have been identified in cardiac myocytes, TASK-1 and TREK-1, and there is clear evidence that these channels may regulate AP duration.39,40 Furthermore, the data from Figure 7 implicate the involvement of another K+ current. That is, the SP sensitive current in the voltage-range where ICa,L is active, is less pronounced in the presence of a Ca2+ channel blocker (open circles vs. filled circles), which may point to a contribution of a Ca2+-activated K+ current (IK,Ca) as well. Small-conductance K,Ca channels (SK1-3) have been demonstrated to underly IK,Ca in atrial myocytes41 and shown to have a

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significant role in atrial repolarization and fibrillation.42,43

ConclusionThe results from this study represent the first evidence that SP can act as a neurotransmitter directly to the atrial myocyte, leading to significant AP prolongation through inhibition of an outward potassium background current. The lengthening of atrial repolarization with consequent increase in the effective refractory period, is considered anti-arrhythmic for re-entrant arrhythmias and may prevent or terminate atrial fibrillation. The AP-prolonging effect of SP was unabated at higher frequencies, contrary to many class III antiarrhythmic drugs known to display reverse rate-dependent effects on cardiac repolarization.44

We speculate that stimulation of SP release may be protective against AF (this study), whereas reduced SP levels may facilitate AF development.14,43


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C

Control1 µM Som-14

Control1 µM VIP

1 µM NPYControl

Control

1 µM ACh

Control

1 µM NA

25 ms

40 pA

0 mV

0 mV

0 mV

0 mV

0 mV

A

B

D

E

Supplement figure. Effects of various neurotransmitters and neuropeptides on atrial action potential configuration.

(A) Representative examples of APs stimulated at 1Hz under control conditions and in the presence of 1 µM noradrenalin (NA), (B) acetylcholine (ACh), (C) somatostatin-14 (Som-14), (D) neuropeptide-Y (NPY), (E) and vasoactive peptide (VIP).

NA

(n=12, 2 Hz)

ACh

(n=6, 2 Hz)

SOM-14

(n=3, 1 Hz)

NPY

(n=2, 1 Hz)

VIP

(n=1, 1 Hz)

CTRL NT CTRL NT CTRL NT CTRL NT CTRL NT

RMP (mV)

-77.8 ±

1.5

-80.9**

± 1.3

-77.1 ±

1.7

81.5* ±

1.4

-79.0 ±

1.6

-78.3 ±

2.1

-82.8 ±

0.4

-83.1 ±

0.4

-80.7 -79.4

Vmax

(V/s)

361.5 ±

47.5

418.3**

± 41.8

268.3 ±

40.4

370.4*

± 31.2

374.2 ±

30.6

372.9 ±

88.1

192.8 ±

30.4

200.7 ±

33.7

542.0 616.1

APA (mV)

113.6 ±

2.4

122.0**

± 2.2

107.1 ±

5.6

116.8*

± 3.9

113.8 ±

1.9

114.4 ±

4.1

102.5 ±

6.6

101.9 ±

5.5

123.2 124.6

APD90

(ms) 78.5

± 7.6

109.7**

± 5.1

88.1 ±

14.4

54.7*

± 8.7

106.2 ±

12.4

109.6 ±

14.5

89.3 ±

8.3

89.7 ±

5.7

82.1 79.3

RMP, resting membrane potential; Vmax, maximal upstroke velocity; APA, action potential amplitude; APD90, action potential duration at 90% of repolarization. * indicates P<0.05 ** indicates P<0.01

Supplement table. Effects of NA, ACh, SOM-14, NPY and VIP on action potential characteristics.

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References

1. Pauza DH, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart. Anat Rec 2000;259:353-382

2. Hoover DB, Chang Y, Hancock JC, Zhang L. Actions of tachykinins within the heart and their relevance to cardiovascular disease. Jpn J Pharmacol 2000;84:367-373


4. He B, Scherlag BJ, Nakagawa H, Lazzara R, Po SS. The intrinsic autonomic nervous system in atrial fibrillation: a review. ISRN Cardiol 2012:490674

5. Krul SPJ, Driessen AHG, van Boven WJ, Linnenbank AC, Geuzebroek GSC, Jackman WM, Wilde AAM, de Bakker JMT, de Groot JR. Thoracoscopic video-assisted pulmonary vein antrum isolation, ganglionated plexus ablation, and periprocedural confirmation of ablation lesions: first results of a hybrid surgical-electrophysiological approach for atrial fibrillation. Circ Arrhythm Electrophysiol 2011;4:262-270

6. Nakagawa H, Scherlag BJ, Patterson E, Ikeda A, Lockwood D, Jackman WM. Pathophysiologic basis of autonomic ganglionated plexus ablation in patients with atrial fibrillation. Heart Rhythm 2009;6:S26-S34

7. Yilmaz A, Geuzebroek GSC, Van Putte BP, Boersma LVA, Sonker U, De Bakker JMT, Van Boven WJ. Completely thoracoscopic pulmonary vein isolation with ganglionic plexus ablation and left atrial appendage amputation for treatment of atrial fibrillation. Eur J Cardiothorac Surg 2010;38:356-360

8. Verkerk AO, Geuzebroek GSC, Veldkamp MW, Wilders R. Effects of acetylcholine and noradrenalin on action potentials of isolated rabbit sinoatrial and atrialmyocytes. Front Physiol 2012;3:174

9. V Euler US, Gaddum JH. An unidentified depressor substance in certain tissue extracts. J Physiol 1931;72:74-87

10. Chiao H, Caldwell RW. Local cardiac effects of substance P: roles of acetylcholine and noradrenaline. Br J Pharmacol 1995;114:283-288

11. Hoover DB. Effects of substance P on rate and perfusion pressure in the isolated guinea pig heart. J Pharmacol Exp Ther 1990;252:179-184

12. Ejaz A, LoGerfo FW, Pradhan L. Diabetic neuropathy and heart failure: role of neuropeptides. Expert Rev Mol Med 2011;13:e26

13. Yu Y, Liu L, Jiang JY, Qu XF, Yu G. Parasympathetic and substance P-immunoreactive nerve denervation in atrial fibrillation models. Cardiovasc Pathol 2012;21:39-45

14. Guler N, Ozkara C, Dulger H, Kutay V, Sahin M, Erbilen E, Gumrukcuoglu HA. Do cardiac neuropeptides play a role in the occurrence of atrial fibrillation after coronary bypass surgery? Ann Thorac Surg 2007;83:532-537


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15. Den Ruijter HM, Verkerk AO, Coronel R. Incorporated fish oil fatty acids prevent action potential shortening induced by circulating fish oil fatty acids. Front Physiol 2010;1:149

16. Barry PH, Lynch JW. Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol 1991;121:101-117

17. Hinde AK, Perchenet L, Hobai IA, Levi AJ, Hancox JC. Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 ºC, dialysed internally with cAMP-free and cAMP-containing solutions. Cell Calcium 1999;25:321-331

18. Baartscheer A, Schumacher CA, van Borren MMGJ, Belterman CNW, Coronel R, Fiolet JWT. Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model. Cardiovasc Res 2003;15:1015-1024

19. Coumel P. Autonomic influences in atrial tachyarrhythmias. J Cardiovasc Electrophysiol 1996;7:999-1007

20. Fioranelli M, Piccoli M, Mileto GM, Sgreccia F, Azzolini P, Risa MP, Francardelli RL, Venturini E, Puglisi A. Analysis of heart rate variability five minutes before the onset of paroxysmal atrial fibrillation. Pacing Clin Electrophysiol 1999;22:743-749

21. Sharifov OF, Fedorov VV, Beloshapko GG, Glukhov AV, Yushmanova AV, Rosenshtraukh LV. Roles of adrenergic and cholinergic stimulation in spontaneous atrial fibrillation in dogs. J Am Coll Cardiol 2004;43:483-490

22. Beaulieu P, Lambert C. Peptidic regulation of heart rate and interactions with the autonomic nervous system. Cardiovasc Res 1998;37:578-585

23. Steele PA, Gibbins IL, Morris JL. Projections of intrinsic cardiac neurons to different targets in the guinea-pig heart. J Auton Nerv Syst 1996;56:191-200

24. Almeida TA, Rojo J, Nieto PM, Pinto FM, Hernandez M, Martín JD, Candenas ML. Tachykinins and tachykinin receptors: structure and activity relationships. Curr Med Chem 2004;11:2045-2081

25. Koyano K, Velimirovic BM, Grigg JJ, Nakajima S, Nakajima Y. Two signal transduction mechanisms of substance P-induced depolarization in locus coeruleus neurons. Eur J Neurosci 1993;5:1189-1197

26. Li YW, Guyenet PG. Effect of substance P on C1 and other bulbospinal cells of the RVLM in neonatal rats. Am J Physiol 1997;273:R805-R813

27. Shen KZ, North RA. Substance P opens cation channels and closes potassium channels in rat locus coeruleus neurons. Neuroscience 992;50:345-353

28. Stanfield PR, Nakajima Y, Yamaguchi K. Substance P raises neuronal membrane excitability by reducing inward rectification. Nature 1985;315:498-501

29. Wang DS, Xu TL, Li JS. Modulation of glycine-activated chloride currents by substance P in rat sacral dorsal commissural neurons. Sheng Li Xue Bao 1999;51:361-370

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30. Dobrev D, Friedrich A, Voigt N, Jost N, Wettwer E, Christ T, Knaut M, Ravens U. The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation. Circulation 2005;112:3697-3706

31. Mao J, Wang X, Chen F, Wang R, Rojas A, Shi Y, Piao H, Jiang C. Molecular basis for the inhibition of G protein-coupled inward rectifier K+ channels by protein kinase C. Proc Natl Acad Sci U S A 2004;101:1087-1092

32. Ravens U, Cerbai E. Role of potassium currents in cardiac arrhythmias. Europace 2008;10:1133-1137

33. Ishimatsu M. Substance P produces an inward current by suppressing voltage-dependent and -independent K+ currents in bullfrog primary afferent neurons. Neurosci Res 1994;19:9-20

34. Paul K, Cox CL. Excitatory actions of substance P in the rat lateral posterior nucleus. Eur J Neurosci 2010;31:1-13

35. Shen KZ, Surprenant A. Common ionic mechanisms of excitation by substance P and other transmitters in guinea-pig submucosal neurones. J Physiol 1993;462:483-501

36. Vanner S, Evans RJ, Matsumoto SG, Surprenant A. Potassium currents and their modulation by muscarine and substance P in neuronal cultures from adult guinea pig celiac ganglia. J Neurophysiol 1993;69:1632-1644

37. Yasuda K, Robinson DM, Selvaratnam SR, Walsh CW, McMorland AJC, Funk GD. Modulation of hypoglossal motoneuron excitability by NK1 receptor activation in neonatal mice in vitro. J Physiol 2001;534:447-464

38. Enyedi P, Czirják G. Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev 2010;90:559-605

39. Limberg SH, Netter MF, Rolfes C, Rinné S, Schlichthörl G, Zuzarte M, Vassiliou T, Moosdorf R, Wulf H, Daut J, Sachse FB, Decher N. TASK-1 channels may modulate action potential duration of human atrial cardiomyocytes. Cell Physiol Biochem 2011;28:613-624

40. Zhang H, Shepherd N, Creazzo TL. Temperature-sensitive TREK currents contribute to setting the resting membrane potential in embryonic atrial myocytes. J Physiol 2008;586:3645-3656

41. Xu Y, Tuteja D, Zhang Z, Xu D, Zhang Y, Rodriguez J, Nie L, Tuxson HR, Young JN, Glatter KA, Vázquez AE, Yamoah EN, Chiamvimonvat N. Molecular identification and functional roles of a Ca2+-activated K+ channel in human and mouse hearts. J Biol Chem 2003;278:49085-49094

42. Li N, Timofeyev V, Tuteja D, Xu D, Lu L, Zhang Q, Zhang Z, Singapuri S, Albert TR, Rajagopal AV, Bond CT, Periasamy M, Adelman J, Chiamvimonvat N. Ablation of a Ca2+-activated K+ channel (SK2 channel) results in action potential prolongation in atrial myocytes and atrial fibrillation. The Journal of Physiology 2009;587:1087-1100

43. Yu T, Deng C, Wu R, Guo H, Zheng S, Yu X, Shan Z, Kuang S, Lin Q. Decreased expression of small-conductance Ca2+-activated K+ channels SK1 and SK2 in human chronic atrial fibrillation. Life Sci 2012;90:219-227

44. Dorian P, Newman D. Rate dependence of the effect of antiarrhythmic drugs delaying cardiac repolarization: an overview. Europace 2000;2:277-285


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Summary

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Chapter 8

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Chapter 1 reviews the epidemiology, risk factors and symptomatology of atrial fibrillation, the history of the underlying mechanisms of atrial fibrillation and the history of surgery for atrial fibrillation.

Many consider the classical Maze III procedure as the most successful treatment and the golden standard for the treatment of drug-refractory symptomatic atrial fibrillation. It was the first procedure that could overcome the thrombogenic, arrhythmogenic and partially the negative hemodynamic effects of atrial fibrillation. In Chapter 2 we show that the classical Maze III procedure is safe and effective for both atrial fibrillation without and with underlying structural heart disease (lone and concomitant atrial fibrillation). In a cohort of 203 consecutive patients who underwent a Maze III procedure no in-hospital mortality was noted. Freedom from atrial fibrillation after a mean follow-up of 4 years was 90% and 69% for patients who underwent a Maze III procedure for lone and concomitant atrial fibrillation respectively. None of the patients who underwent a Maze III procedure suffered from a stroke during follow-up.

Because the classical Maze III procedure is a complex and time-consuming operation, different modified Maze procedures have been developed over years. These modified Maze procedures differ in the lesion set and/or technique of creating non-conductive lines. Chapter 3 describes a cohort of 66 patients with mainly concomitant atrial fibrillation who underwent either a pulmonary vein isolation only, a left atrial Maze or a bi-atrial Maze procedure. While a left atrial Maze and bi-atrial Maze procedure are quite successful, pulmonary vein isolation only was not.

Surgical therapy for lone atrial fibrillation has been focusing on the pulmonary veins after the recognition that ectopic foci, originating from the pulmonary veins, play an important role in the pathophysiology of atrial fibrillation.1 The Sint Antonius Hospital in Nieuwegein was one of the first centers who performed a completely thoracoscopic isolation of the pulmonary veins in combination with ganglionic plexus ablation for lone atrial

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fibrillation. Chapter 4 describes the results of the first 30 patient treated in this way for lone atrial fibrillation. After a follow-up period of almost a year, the mortality was 0% and 77% of the patients were free from atrial fibrillation. Two patients required a conversion to a sternotomy to control a bleeding. The mean hospital stay was 5 days. This shows that a completely thoracoscopic pulmonary veins isolation with ganglionic plexus ablation is a safe and effective procedure. This minimal invasive approach for lone atrial fibrillation has made the classical Maze III procedure almost completely obsolete in the treatment of drug-refractory symptomatic atrial fibrillation.

The success of the surgical therapy of atrial fibrillation and of the Maze III procedure in particular depends on the left atrial diameter. The bigger the left atrium, the smaller the chance of a successful outcome. Especially in case of mitral valve pathology the diameter of the atrium is large and surgery for atrial fibrillation is less successful.2 This is probably due to the presence of a larger arrhythmogenic substrate. Because fibrosis plays an important role in establishing the arrhythmogenic substrate, we have scored the amount of fibrosis in different groups of patients. Chapter 5 shows that the left and right atrial appendages of patients with atrial fibrillation in combination with mitral valve disease contain more fibrosis than the appendage of patients with atrial fibrillation without mitral valve disease. Appendages of patients with lone fibrillation did not contain significantly more fibrosis than controls. Overall, right atrial appendages contained more fibrosis than left atrial appendages.

The importance of the autonomic nervous system in the pathophysiology of atrial fibrillation has been underscored years ago by Coumel and recently physicians are targeting the ganglionic plexus in the treatment of atrial fibrillation.3-6 The ‘classical’ neurotransmitters of the autonomic nervous system are noradrenalin and acetylcholine. Chapter 6 describes the effect of these 2 ‘classical’ neurotransmitters at a concentration of 1µM on the action potential of isolated left atrial cells and of sinus node

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cells of rabbits. Both acetylcholine and noradrenalin increase the maximal diastolic potential (hyperpolarization) and increase the maximum upstroke velocity of isolated left atrial rabbit myocytes. While noradrenalin increases the action potential duration at 90% of repolarization (APD90), acetylcholine shortens it. The effect of noradrenalin on the action potential of sinus node cells was a decreased cycle length and an increased maximal diastolic potential (hyperpolarization) and the opposite was the case for acetycholine.

Apart from the ‘classical’ neurotransmitters noradrenalin and acetylcholine, the atria contain many more neurotransmitters.7 Chapter 7 describes the effect of various neurotransmitters on the action potential characteristics of isolated left atrial rabbit myocytes. While Neuropeptide-Y, Somatostatin-14 and Vasoactive Intestinal Peptide did not elicit an effect on the action potential, Substance-P (1 µM) increased the action potential duration and decreased the maximum diastolic potential (depolarization). The effect on the action potential duration was dose dependent and present at the tested frequencies of 1-4 Hz. The voltage clamp experiments suggested that a non-specific current, probably a potassium current, is responsible.

In this thesis we have investigated the results of various surgical procedures for atrial fibrillation which have been performed in the last 2 decades in the Sint Antonius Hospital, Nieuwegein, The Netherlands. In the 1990s the classical Maze III procedure was the main surgical technique for drug-refractory symptomatic lone atrial fibrillation at the Sint Antonius Hospital. A better understanding of the pathophysiology of atrial fibrillation has enabled an evolution in the surgical treatment of atrial fibrillation. Nowadays a completely thoracoscopic procedure is the main surgical therapy for lone atrial fibrillation at the Sint Antonius Hospital. Because knowledge and therapy of atrial fibrillation go hand in hand, this thesis deals also with the structural and humoral remodeling associated with atrial fibrillation, in particular with the amount of atrial fibrosis in patients with and without atrial fibrillation

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and with the electrophysiological effects of different neurotransmitters. This may help in developing more effective and probably tailored, patient specific and mechanism (substrate/trigger) specific, therapeutic procedures for atrial fibrillation.

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References:


2. Gillinov AM, Sirak J, Blackstone EH, McCarthy PM, Rajeswaran J, Pettersson G, Sabik FJ 3rd, Svensson LG, Navia JL, Cosgrove DM, Marrouche N, Natale A. The Cox Maze procedure in mitral valve disease: predictors of recurrent atrialfibrillation. J Thorac Cardiovasc Surg 2005;130:1653-1660






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Samenvatting

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Hoofdstuk 1 geeft een overzicht van de epidemiologie, risicofactoren en symptomen van boezemfibrilleren. Verder bevat het de geschiedenis van de verschillende theorieën over de onderliggende mechanismen en de chirurgische behandelingen van boezemfibrilleren.

De klassieke Maze III procedure wordt nog steeds gezien als de meest succesvolle behandeling van medicamenteus onbehandelbaar, symptomatisch boezemfibrilleren. Het was de eerste procedure die alle negatieve effecten van boezemfibrilleren (trombo-embolieën, onregelmatige hartslag en verminderde hemodynamiek) kon tenietdoen. In hoofdstuk 2 wordt aangetoond dat de klassieke Maze III procedure veilig en effectief is voor zowel boezemfibrilleren met en zonder onderliggende structurele hartziekten. In een cohort van 203 patiënten die een Maze III procedure ondergingen, was er geen sterfte gedurende de ziekenhuisopname. Gemiddeld 4 jaar na de operatie was 90% van de patiënten met boezemfibrilleren zonder structurele hartafwijking nog vrij van boezemfibrilleren. Dit percentage was 69% voor patiënten met boezemfibrilleren in combinatie met een structurele hartziekte. Geen van de patiënten die een Maze III procedure had ondergaan maakte een beroerte door gedurende de vervolgperiode.

Omdat de klassieke Maze III procedure technisch complex en tijdrovend is, zijn er de afgelopen jaren verschillende gemodificeerde Maze procedures ontwikkeld. Deze gemodificeerde Maze procedures verschillen van de klassieke Maze III procedure in de gebruikte (ablatie) lijnen en/of de techniek voor het maken van deze niet-geleidende lijnen. Hoofdstuk 3 beschrijft een cohort van 66 patiënten met boezemfibrilleren (overwegend met onderliggende structurele hartziekte) die óf een isolatie van de pulmonaalvenen alleen, óf een linkszijdige Maze procedure, óf een bi-atriale Maze procedure ondergingen. De resultaten van zowel de linkszijdige Maze, als de bi-atriale Maze waren bevredigend. Een pulmonaalvenenisolatie alleen was voor deze groep patiënten echter minder succesvol.

Chirurgische behandeling van boezemfibrilleren zonder onderliggende structurele hartafwijking heeft zich de laatste jaren meer en meer gericht op de isolatie van de pulmonaalvenen nadat men tot de ontdekking kwam dat prikkels uit deze longvenen een belangrijke rol spelen in het ontstaan van boezemfibrilleren.1

Het Sint Antonius Ziekenhuis in Nieuwegein was een van de eerste centra ter wereld die een compleet thoracoscopische isolatie van de pulmonaalvenen

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met modificatie van het autonome zenuwstelsel verrichte bij patiënten met boezemfibrilleren zonder structurele hartafwijking. Hoofdstuk 4 beschrijft de resultaten van de eerste 30 patiënten die op deze manier werden behandeld. Na een vervolgperiode van bijna een jaar was de mortaliteit 0%, en was 77% van de patiënten vrij van boezemfibrilleren. Bij twee patiënten was uiteindelijk een sternotomie noodzakelijk in verband met een bloeding. De gemiddelde duur van de ziekenhuisopname was 5 dagen. Deze resultaten illustreren dat een thoracoscopische isolatie van de pulmonaalvenen met modificatie van het autonome zenuwstelsel, een veilige en effectieve behandelmethode is. Deze minimaal invasieve benaderingswijze voor boezemfibrilleren heeft er voor gezorgd dat de klassieke Maze III procedure vrijwel overbodig is geworden voor de behandeling van medicamenteus onbehandelbaar, symptomatische boezemfibrilleren zonder onderliggende structurele hartafwijking.

Het operatiesucces van chirurgie voor boezemfibrilleren is afhankelijk van de diameter van de linker boezem. Hoe groter deze diameter, hoe kleiner de kans op een goede uitkomst. Juist bij mitraliskleplijden is de diameter van de boezem in de regel groot en een chirurgische behandeling van boezemfibrilleren is in die categorie patiënten dan ook minder succesvol.2 Waarschijnlijk is het verminderd succes toe te schrijven aan ongunstige karaktereigenschappen van het boezemweefsel. Omdat fibrose een belangrijke rol speelt in deze karaktereigenschappen, onderzochten wij de hoeveelheid fibrose in de boezems van verschillende patiëntengroepen. In hoofdstuk 5 is te lezen dat patiënten met boezemfibrilleren in combinatie met mitraliskleplijden meer fibrose in hun linker en rechter boezems hadden dan patiënten met boezemfibrilleren zonder bijkomende structurele hartziekte. De boezems van patiënten met boezemfibrilleren zonder structurele hartziekte bevatten niet meer fibrose dan controlepatiënten. Algemeen genomen, bevatten de rechter boezems meer fibrose dan de linker boezems.

Jaren geleden erkende Coumel al dat het autonome zenuwstelsel een belangrijke rol speelt in de pathofysiologie van boezemfibrilleren. Pas sinds enkele jaren richten clinici zich nu ook op het autonome zenuwstelsel in de behandeling van boezemfibrilleren.3-6 De ‘klassieke’ neurotransmitters van het autonome zenuwstelsel zijn noradrenaline en acetylcholine. In hoofdstuk 6 staat een beschrijving van de effecten van deze twee ‘klassieke’ neurotransmitters op de

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actiepotentiaal. Dit onderzoek werd uitgevoerd aan enzymatisch geïsoleerde cellen uit het linker atrium en de sinusknoop van het konijnenhart. In cellen afkomstig uit het linker atrium geven zowel acetylcholine als noradrenaline (bij een concentratie van 1µM) een toename van het maximaal diastolische potentiaal (hyperpolarisatie) en een toename van de maximale stijgsnelheid van de actiepotentiaal. Noradrenaline geeft verder een toename van de actiepotentiaalduur gemeten op 90% van de repolarisatie (APD90), terwijl acetylcholine die juist doet afnemen. In cellen afkomstig uit de sinusknoop geeft noradrenaline een afname van de cycluslengte en een toename van de maximaal diastolische potentiaal (hyperpolarisatie), acetylcholine doet juist het tegenovergestelde.

Naast de ‘klassieke’ neurotransmitters zijn er nog vele andere neurotransmitters in het atrium aanwezig.7 In hoofdstuk 7 worden de effecten beschreven van diverse neurotransmitters op actiepotentiaalkarakteristieken van cellen geïsoleerd uit het linker atrium van het konijnenhart. ‘Neuropeptide-Y’, ‘Somatostatin-14’ en ‘Vasoactive Intestinal Peptide’ hadden geen effect op de actiepotentiaal. Substance-P (1µM) daarentegen gaf een toename van de actiepotentiaalduur en een afname van de maximale diastolische potentiaal (depolarisatie). Het effect op de duur van een actiepotentiaal was dosisafhankelijk en werd gemeten bij frequenties van 1-4 Hz. De ‘voltage-clamp’ experimenten suggereren dat de afname in een kalium-selectieve achtergrondstroom, verantwoordelijk is voor het ‘substance-P’-effect.

Dit proefschrift bevat de resultaten van diverse chirurgische behandelingen zoals die in de afgelopen 20 jaar in het Sint Antonius Ziekenhuis te Nieuwegein zijn uitgevoerd. In de jaren negentig was in het Sint Antonius Ziekenhuis de klassieke Maze III procedure de meest toegepaste chirurgische techniek voor medicamenteus onbehandelbaar, symptomatisch boezemfibrilleren. Verbeterd inzicht in de pathofysiologie van boezemfibrilleren zorgde ervoor dat er innovaties plaats vonden op het gebied van chirurgische behandeling van deze aandoening. Tegenwoordig wordt in het Sint Antonius Ziekenhuis een volledig thoracoscopische procedure het meest toegepast bij boezemfibrilleren zonder onderliggende structurele hartafwijking. Aangezien verbeteringen in de behandeling van boezemfibrilleren samengaan met de kennis hierover, behandeld dit proefschrift

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zowel structurele als neuro-humorale remodelering. Dit proefschrift richt zich in het bijzonder op de hoeveelheid atriale fibrose bij patiënten met of zonder boezemfibrilleren. Tevens geeft het inzicht in de elektrofysiologische effecten van verschillende neurotransmitters. Deze nieuw verworven kennis kan hopelijk een bijdrage leveren aan het ontwikkelen van effectievere behandelmethoden voor boezemfibrilleren, die idealiter afgepast zijn op individuele patiëntkarakteristieken en op het onderliggende mechanisme.

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References:1. Haïssaguerre M, Jaïs P, Shah DC, Takahashi A, Hocini M, Quiniou G, Garrigue S, Le Mouroux

A, Le Métayer P, Clémenty J. Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins. N Engl J Med 1998;339:659-666

2. Gillinov AM, Sirak J, Blackstone EH, McCarthy PM, Rajeswaran J, Pettersson G, Sabik FJ 3rd, Svensson LG, Navia JL, Cosgrove DM, Marrouche N, Natale A. The Cox maze procedure in mitral valve disease: predictors of recurrent atrialfibrillation. J Thorac Cardiovasc Surg 2005;130:1653-1660






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Hierbij wil ik mijn familie, vriendin, vrienden en collega’s bedanken. Zonder jullie was dit niet mogelijk geweest. Terwijl ik dit schrijf weet ik dat ik mensen vergeet te bedanken. Bij dezen wil ik alvast iedereen bedanken die ik vergeten ben om bij naam te noemen, maar die hoe klein dan ook, een bijdrage hebben geleverd aan dit proefschrift.

Allereerst wil ik beginnen mijn vader, moeder en zusje te bedanken. Pap en mam, bedankt voor jullie onvoorwaardelijke steun. Zonder jullie was dit nooit gelukt. Viv, kom je wat vaker langs? Ik mis je.

Ik begon in mijn derde jaar van Geneeskunde met onderzoek samen met Philippe Ballaux en prof dr. Norbert van Hemel. Beste Philippe, mag ik je bedanken voor het fundament van mijn proefschrift, namelijk de resultaten van de klassieke Maze III operatie. Het heeft veel tijd gekost, maar het is een mooi stuk geworden. Ik wil ook je vrouw, Bernadette, bedanken voor de goede zorgen. Beste Norbert, bedankt voor je steun, je kritische blik en je tijd. Altijd stond je klaar om een manuscript te herzien. Niet te vergeten, ben jij diegene die mij liet kennismaken met het laboratorium van de experimentele cardiologie. Jij en Philippe staan aan de basis van dit proefschrift.

Graag wil ik mijn promotor prof. dr. ir. Jacques de Bakker en co-promotors dr. Ruben Coronel en dr. Marieke Veldkamp bedanken. Ik denk dat ik geen beter team aan mijn zijde kon hebben. Beste Jacques, naast een briljante wetenschapper, ben je ook een zeer integer persoon. Jouw persoonlijkheid is uniek. Beste Ruben, na mijn snuffelstage bij de experimentele cardiologie was het al snel duidelijk dat ik jou als co-promotor wenste. Na het voltooien van mijn proefschrift ben ik nog steeds dezelfde mening toegedaan. Je bent een purist in hart en nieren. Beste Marieke, ook jij was een onmisbare schakel in de keten. Menig uurtje hebben wij samen gespendeerd om de manuscripten in orde te krijgen. En we zijn nog niet klaar.

Iedereen van de Experimentele Cardiologie uit het AMC wil ik bedanken voor hun steun en bijdrage. In het bijzonder wil ik noemen; Cees, Shirley, Ton, Antoni, André, Arie, Charly, Amin, Tobias en Jan. Bedankt voor alles. Cees, wat hebben wij gelachen. Ik zal het gaan missen! En natuurlijk bedankt voor de cellen. Shirley,

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bedankt voor je ondersteuning. Ruim 1000 digitale foto’s hebben we genomen en geanalyseerd op fibrose, wat een werk! Ton, het lijkt wel of de tijd geen invloed op jou heeft. Antoni, bedankt voor je ondersteuning met het patch-clampen. Arie, niemand kan patch-clampen zoals jij. Bedankt dat ik gebruik mocht maken van jouw kennis en kunde. Charly en Amin, bedankt voor de ondersteuning bij het offeren van de dieren. Jan en Tobias, bedankt voor de discussies en de gezelliheid.

Bas, ik wil jou ook bedanken voor de vriendschap. Volgende keer zoek ik eerst uit waar het congres precies is, voordat we er naar toe rijden.

Mark, we promoveren allebei op dezelfde dag. Dit heeft voor ons beide als een stok achter de deur gefungeerd, en het heeft gewerkt. Bedankt voor je steun. Je stond altijd voor me klaar.

Ik wil de maatschap Cardio-Thoracale Chirurgie uit het Sint Antonius Ziekenhuis bedanken voor hun steun. Zonder jullie steun was dit onmogelijk geweest. Er is een rijke historie tussen de experimentele cardiologie uit het AMC en de Cardio-Thoracale Chirurgie uit het Sint Antonius Ziekenhuis. Ritmechirurgie werd verricht door o.a. Jo Defauw in samenwerking met de Experimentele Cardiologie van het AMC. Helaas lijken deze dagen vervlogen. Ik hoop dat we in de toekomst nieuwe vlakken van samenwerking weten te vinden.

Helga, bedankt voor je uitmuntende ondersteuning. Mimount, samen hebben we heel wat holters moeten regelen van de mini-Maze patiënten. Bedankt voor je hulp.

De gehele assistenten-groep van de Cardio-Thoracale Chirurgie wil ik bedanken voor de samenwerking. Vikash, Thomas, Geoffrey en Nabil, ik heb mooie jaren met jullie gekend. Er komen er vast nog veel meer.

De Maatschap en assistenten-groep Cardiologie wil ik bedanken voor hun bijdrage. In het bijzonder Hans Kelder. Bedankt voor je geduld en samenwerking op het gebied van de statistiek.

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Niet te vergeten de Maatschap en assistenten-groep Heelkunde. Bedankt voor 2 fantastische jaren. Ik heb niet alleen veel geleerd van jullie, maar ook zijn er nieuwe vriendschappen tot stand gekomen.

Sander Minnoye, bedankt voor de fantastische lay-out van dit proefschrift. Ik denk dat er meer uren in zijn gaan zitten dan we van te voren hadden gedacht, maar het resultaat is iets om trots op te zijn.

Bleijer en Tegel, door mijn baan en promotie heb ik ook veel van jullie moeten eisen. Vaak moest ik ‘nee’ verkopen. En als ik dan wel kon komen, viel ik vaak in slaap van vermoeidheid. Hopelijk is er meer tijd vanaf nu.

Thijs bedankt!

Als laatste wil ik mijn lieve vriendin Caroline bedanken. Een onvoorstelbare steun in de rug. Grotendeels komt dit proefschrift op jouw conto. Vanaf nu hebben we hopelijk wat meer ‘quality time’ samen.

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UvA-DARE (Digital Academic Repository) Modulation of ... · op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde