Complex and fractionated electrograms; origin and location

The heart cell

The heart consists of cells (cardiomyocytes) with a length of approximately 100 μm

and a width and thickness of 20 μm. Adult cardiomyocytes are unable to proliferate, if they

die due to heart disease, they will not be replaced. Instead, collagen deposition is usually

initiated to maintain rigidity of the heart. To compensate for reduced cardiac function during

heart failure, cell size may increase (hypertrophy) (figure 1).

The cell membrane of cardiomyocytes consists of a lipid bi-layer which contains ion

channels that are responsible for the depolarizing and repolarizing currents flowing into and

out of the cells. Ion channels consist of complex proteins that form pores (channels) across the

lipid bi-layer. Differences in ion concentrations inside and outside the cell result in a voltage

gradient across the membrane of about - 90 mV (inside negative compared to outside). If the

membrane potential is increased to - 60 mV an action potential is generated. Ion channels are

not static, but gradually open and close. They differ with respect to ion selectivity, that is the

type of ions they allow to pass, and gating, the conditions that influence their opening and

closing. For voltage-gated channels, the probability of being open is controlled by the

membrane potential. Ligand-gated channels are controlled by the binding of a molecule

(ligand) to the channel protein, whereas for stress-activated channels opening is controlled by

a mechanical force applied to the channel.

Voltage-gated ion channels play a major role for the generation of an action potential

under normal conditions. They have specialized charged protein domains, called voltage

sensors, which are sensitive to changes in the membrane potential. Changes above a certain

threshold value exert sufficient electrical force on these domains to encourage the channel to

close or open. Most important voltage-gated ion channels for the cardiac cell are the sodium

(Na+), calcium (Ca

++) channels for depolarization and various potassium (K

+) channels for

repolarization. Sodium and calcium channels give rise to an inward current, while potassium

channels are responsible for outward current that bring the membrane potential back to its

resting value (figure 2). The sodium channels generate, after opening at a membrane potential

of -60 mV, the upstroke of the action potential. They close very quickly after the upstroke, but

then calcium channels have already opened and maintain the membrane potential around 0 V,

the plateau phase of the action potential. Potassium channels are then opened to initiate an

outward current that brings the membrane potential back to its resting value of -90 mV.

Interestingly there is one potassium channel that generates (for a short time) a repolarizing

current (ITo) immediately after the upstroke. Activation of this ITo channel results in a notch of

the action potential,

Coupling between cardiomyocytes

Cardiomyocytes are embedded in a collagen matrix that gives the heart its rigidity and

allows distribution of the cardiac contractility. Contraction of the cells is heralded by

electrical activity that starts in the sinus node and propagates via the atria, AV node and

bundle branches to ventricular myocardium. To allow propagation of the action potential,

cardiomyocytes are electrically coupled by connexins. Connexins are channel proteins that

allow ions and small molecules to pass from a cell to its neighbors. Various connexins are

present in the heart, each with different biophysical characteristics. In the ventricle

connexin43 (Cx43) is the main connexin, whereas atrial cells are connected by Cx43 and

Cx40. Connexins are located at sites where cells are close together (intercalated disk) (figure

3). In adult cardiomyocytes most of the connexins are present at the intercalated disks at the

long ends of the cells; At side-by-side locations of the cells much less connexins are present.

This difference in connexin distribution results in a difference in current flow, which is

greater in the fiber direction than perpendicular to it. Because of this, conduction velocity is

larger in the fiber direction.

Genesis of the extracellular electrogram

Extracellular electrograms arise because current between cardiomyocytes flows partly

through the membrane into extracellular space. Because extracellular space has resistance, the

current will generate an electrical potential, the extracellular electrogram.

In an activated (depolarized) cell the transmembrane potential of the ventricular

cardiomyocyte is about +20 mV. Adjacent cells, which are at rest, have a membrane potential

of -90 mV. Because the extracellular potential is low, there is a voltage gradient between

activated and non-activated cells of about 110 mV. The shape of the potential change from an

activated to a resting cell is as indicated in figure 4 (similar to the upstroke of the action

potential). This potential change (difference) will generate a current. The current strength is

dependent on the voltage difference between two intracellular points and the resistance

between those points. Mathematically the current is the first derivative of the potential divided

by the resistance. The voltage gradient in figure 4 results in an axial current. The amplitude of

this current is small at the front (site C) and the back (site A) of the activation front (1 yellow

arrow). In contrast, current amplitude is high at the site where the activation front is present

(site B, 4 yellow arrows). This spatial difference in axial current size raises a problem; the

axial current increases from site A to the site B. Because the number of charge carriers per

volume content is constant, current increase must come from current flow through the

membrane. From the point of maximal depolarization (site B) toward the front of the

activation front (site C) the axial current decreases. This means that current must disappear,

which again is only possible through the membrane. Thus we have a transmembrane current

that is inward at the back of the activation front and outward at the front (blue and red arrows

respectively). This is called a current dipole. Current from the dipole is flowing through the

membrane in extracellular space that has resistance and therefore a potential field is generated

in the extracellular space. This potential field can be calculated by simple mathematics. Spach

and coworkers have measured the potential field of a Purkinje strand (figure 5). Calculations

and measurements show that the potential field is positive in front of the activation front and

negative at the back of the activation front. If activation proceeds, this potential field moves.

At a recording site where activation passes, we first observe the positive field of the

approaching wave front. If the activation front is exactly at the recording site, the potential is

zero and if the activation recedes, the potential field becomes negative. At the site where

activation arises, the potential is only negative, whereas the potential is only positive at the

site where activation comes to an end (figure 6).

The amplitude of the extracellular electrogram not only depends on the dipole strength

but also from the distance of the recording point to the bundle. The further away, the smaller

the amplitude. For larger distances, the amplitude of a unipolar electrogram decreases with

the power of two of the distance. Next to a reduction in amplitude the frequency content of

the signal decreases with distance. The downstroke of the electrograms become less sharp

with increasing distance.

The important characteristics of the unipolar electrogram thus are: 1) at a site where

activation is passing, the extracellular electrogram is biphasic; 2) at a site where activation is

arising, the extracellular electrogram is only negative; 3) at a site where activation comes to

an end, the electrogram is positive only. This is the basis of unipolar extracellular signal

morphology in case we are dealing with a simple (1D) myocardial bundle. The heart has,

however, three dimensions as well as fiber orientation, that both may affect electrogram

morphology. Spach et al have recorded electrograms of a 2D sheet of myocardial tissue that

was stimulated in the center. The activation pattern and 3 electrograms are shown in figure 7.

The pattern is elliptically shaped, because activation is faster in the fiber direction than

perpendicular to it. The reason for this is that the electrical coupling is tighter in the fiber

direction because of the abundance of connexins at the long ends of the cells. The authors

observed different configurations for the extracellular electrograms. At site “a” where

activation starts, the electrogram is negative only, compatible with an origin of activation. At

site “b” the electrogram is biphasic consisting of a positive deflection, followed by a negative

one. This is the signal we expect at a site where activation is passing. However, at recording

site “c” activation is passing as well and although a biphasic deflection is recorded, it is

preceded by a small negative one. This initially negative deflection is caused by remote

activation in the fiber direction that in fact runs away from recording site “c”. This activation

front is strong and its negative contribution overrules the positive contribution of the weak

wave front running toward site “c”, at least if this front is at a distance. If this front is close to

site “c” and passes the recording site, the following biphasic deflection is generated.

Activation times

To determine spread of activation, activation times have to be assessed. This

parameter indicates the time myocardial tissue underneath the electrode is activated. There is

a good correlation between the upstroke in the action potential, which marks depolarization of

a cardiomyocyte, and the point of steepest negative deflection in the unipolar extracellular

electrogram. This is even true in ischemic myocardium (figure 8). In a unipolar recording the

different electrode, which is connected to the positive input of an amplifier, is at the recording

site, whereas the negative input is connected with the indifferent electrode, which is far away

from the heart (theoretically at infinity). For a bipolar recording the indifferent electrode is

located close to the different one. If the distance between the electrode terminals is small

(order of magnitude 1 mm) the bipolar electrogram is equal to the first derivative of the

unipolar one (figure 9).

The advantage of a bipolar electrogram is that deflections caused by remote activation

are reduced as to deflections caused by local activation. Disadvantages are the direction

dependence (figure 10) and the interpretation of signal morphology, which is more difficult.

Because of the direction dependence activation time is difficult to assess in a bipolar

electrogram. It should be the positive or negative peak in the electrogram, but never the

beginning of a bipolar electrogram. As with the unipolar electrogram the part of the signal

that marks local activity is preceded and followed by deflections that are remote.

Complex and fractionated electrograms

A major problem of electrograms with multiple deflections, as shown in figure 11, is

selecting the local component (and activation time), the more so because multiple local

components can be present. Several algorithms have been developed in the past to determine

the local component, but all have drawbacks hampering a solid selection. Only if multiple

recordings are present from neighbouring sites, a reliable selection will be possible.

Electrograms may be complex/fractionated because of: 1) remote activation, 2)

changes in conduction velocity, 3) asynchronous conduction in neighbouring myocardial

strands. Remote activation may generate an additional deflection in a “local” electrogram,

because the extensive field of view of the electrode will also pick up the signal of remote

activation. Both the unipolar and bipolar electrogram are subject to remote deflections, but the

effect is greater for unipolar electograms. Remote ventricular activation may cause

deflections in an atrial recording as illustrated in figure 12. The apparently highly fractionated

electrograms in the atrial tracings (MAP) look very wide, but consist of a local (fractionated)

atrial part and a remote ventricular deflection (arrow).

Figure 13 shows that the right superior pulmonary vein (RSPV) is located close to the

superior caval vein (SCV). Therefore, recordings made in the RSPV may pick up signals

generated by activation in the SCV, as illustrated in Figure 14, making a number of

electrograms of the RSPV electrode (LAS 4-5 and 5-6) more complex than they really are.

Deflections indicated by the open arrows are generated by activation in SCV.

Atrial tissue overlays the coronary sinus (CS) and gives rise to double potentials if

electrograms are recorded in the CS (Figure 15). The first deflection is caused by activation

in atrial tissue, whereas the second component is caused by activation in the pericoronary

muscle of the CS. At site 4 coronary sinus and atrium are electrically coupled; the two

deflections are very close together at this site (upper tracing). Separation between the two

components increases towards the distal end of the electrode (direction of LAA).

In infarcted myocardium electrograms usually consist of a remote and a local

component. Figure 16 illustrates epicardial recordings during sinus rhythm over an infarcted

area in an isolated (Langendorff perfused) heart (Durrer 1964). Complexity of electrograms is

caused by the initial Q wave that precedes the local deflection (negative deflection indicated

by red arrow in c3). The local deflection in c3 is indicated by the blue arrow. The simple idea

is that the Q-wave occurs because you are looking at the cavity potential through the infarct

(non-excitable) zone. In fact it is the activation at the subepicardium that runs away from the

recording sites that causes the negative deflection.

Changes in conduction velocity may also cause complex electrograms. Wave front

curvature, tissue discontinuities and fibrillatory conduction may affect the shape of the

extracellular electrogram. Tissue discontinuities may occur in diseased myocardium where

inexcitable tissue as collagen and fat separates myocardial bundles. In such areas myocardial

bundles with a small diameter may be coupled to bundles with a large width. A cell culture

model that illustrates this aspect with coupled myocardial cells, is illustrated in figure 17.

Myocardial cells are grown on a glass substrate in a star shaped pattern with 8 arms.

Stimulation is applied at the upper arm and recordings are made at the entrance of this arm to

the star centre. During basic stimulation (S1), the extracellular signal (upper tracing) is

biphasic, as expected for a passing wave front. The intracellular recording of the lower tracing

shows, however, a tiny nod in the upstroke. After the premature stimulus (S2), this nod

becomes more prominent. In addition the biphasic (almost positive) deflection in the

extracellular electrogram is followed by a negative deflection now. This is caused by

activation delay at the entrance that is due to the fact that the wave front in the small bundle

must activate the large area of the star center. The premature stimulus generates less current

then the basic one because less sodium channels are available for activation, although more

current is needed at the tissue discontinuity to activate the star centre.

Complexity of extracellular electrograms will also increase in case of fibrillatory

conduction. Activation of the pectinate muscle in the right atrium after stimulation of

Bachmann’s bundle (BB) is illustrated in figure 18 (Berenfeld 2002). At stimulation

frequencies less than 6 Hz the activation pattern is rather smooth and activation delay small

(max 30 ms). Electrograms in both Bachman’s bundle and pectinate muscle (PM) are normal

at these frequencies. If stimulation rate is increased to 8 Hz, activation in BB remains similar,

but in the area of PM activation delay increases to 120 ms. Here, the activation pattern

becomes more complex due to areas of functional conduction block and impaired conduction

and the electrograms become very complex due to the ensuing fibrillatory (heterogeneous)

conduction.

Asynchronous activation is another cause of increased complexity of extracellular

electrograms. Asynchronous activation may be caused by electrical barriers as illustrated in

figure 19 for a superfused epicardial sheet of myocardium. A structural barrier was made by

incising the tissue from the right to the center (black line). Recordings were made at the right

side of the barrier at sites above and below the barrier. The tissue was stimulated at the upper

right. Activation moves toward the barrier and gives rise to initial positive deflections in all 3

electrograms. The signal is positive because this wave front is blocked at the barrier.

Electrograms recorded at site “a” and “b” are local deflections, whereas electrogram at site

“c”, distal from the barrier, is remote. Activation induced by the stimulus also curves around

the edge of the barrier and arrives at the distal side of the barrier (electrode “c”) after about 14

ms. The corresponding (second) deflection in “c” is biphasic, because activation is passing;

the positive part of this deflection is smaller than the negative one because the arriving wave

front virtually starts at the edge of the barrier. Similar deflections are present at the same

instant in the electrograms recorded at the sites “a” and “b”, but these are remote. The

amplitude of the signal is lower the further the electrode is away from the barrier.

The left panel of figure 20 shows activation maps of an epicardial sheet of myocardial

tissue stimulated at the centre at BCL 600 ms (S1 upper panel) and after a (second) premature

stimulus of 320 ms (S3). Activation pattern after the basic stimulus is smooth, without zones

of conduction block. After the premature stimulus, a zone of (functional) conduction block

(black line) arises at the left, 30 to 40 ms after onset of activation. Extracellular electrograms

around the line of block (dashed red line) are illustrated in the right panel. Signals close to the

line of block show double deflections caused by activation on either side of the line. For

electrograms proximal to the line of block, the first deflection is shaper (local activity) than

the second one (remote activity). For electrograms distal from the line of block the second

deflection is the local one and sharper than the first one (Schalij 1988).

Fractionated electrograms arise in areas where myocardial and collagen bundle

intermingle and may happen in infarcted myocardium where myocardial bundles survive

(figure 21). The signal in the lower panel is a bipolar electrogram of which the first deflection

is caused by remote activation and the second deflection by local activation in the surviving

myocardial bundles. The local activation time is difficult to assess because the local deflection

reveals multiple components.

Intermingling of myocardial and collagen bundles is also illustrated in figure 22. The

left panel shows the histology of an infarcted papillary muscle. Red marks collagen, yellow

myocardial tissue. The papillary muscle was superfused in a tissue bath. Recorded, highly

fractionated, extracellular electrograms are shown at the right. Of the selected white area

histology is indicated at different levels in the right panel. Note that separated bundles (like

“d” and “e”) may merge at a certain level (210 μm), while others merge and diverge again

(“f”).

An infarcted area with surviving myocardial bundles is schematically illustrated in

figure 23 showing a number of separated myocardial bundles of which adjacent ones are

interconnected at some sites. Activation that proceeds from “a” to “b” has to follow the

indicated route (black line). An electrogram recorded at the black dot will be fractionated as

indicated at the right lower part of the slide. Fractionation arises because the signal is

composed of deflections generated by (remote) activation in the different bundles (signals at

the right). Only the deflection caused by activation directly underneath the black electrode is

local. Because activation in the different bundles is asynchronous, due to time delay,

deflections are shifted in time. The preparation was stimulated at the left lower corner and

activation has to follow the indicated zig-zag course perpendicular to the fiber direction.

Interestingly, the electrogram at the center becomes smooth if all the bundles are stimulated

simultaneously at the left and activation runs parallel to the fiber direction (figure 24) Again,

the electrogram is the sum of all the deflections generated by activation in the various

bundles, but now deflections occur simultaneously. The sum of these signals is illustrated by

the electrogram at the lower right. It shows a standard biphasic deflection in the ideal case. In

reality, this smooth deflection will not occur, but activation in the fiber direction will give rise

to less fractionation than activation that runs perpendicular to the fibers.

In case multiple bundles are present, local activation is reflected by the deflection

generated by activation in the bundle directly underneath the recording electrode. In case the

surface of the recording electrode covers more than one bundle, multiple local deflections are

in fact present.

Assessing local activation time in a single fractionated electrogram is often difficult.

Figure 25 shows recordings made from an infarcted papillary muscle. An electrode array with

5 electrode terminals was positioned perpendicular to the fiber direction. If only one recording

had been available, it would be difficult to assess the local activation times in the tracings.

Activation times (presumed to be the point of maximal negative dV/dt) in the electrograms

are indicated by coloured dots. All local deflections have (remote) deflections in neighbouring

tracings that occur at exactly the same time (see deflections along the dotted vertical lines).

Note that the amplitude of the remote deflections decrease the further the electrode is away

from the electrode with the local deflection. These observations indicate that the preparation

consists of multiple isolated bundles (right panel) that are, however, coupled at some sites.

The figure also shows that electrode “c” covers 2 bundles, which means that there are two

activation times in tracing “c”.

Thus, depending on the size of the recording electrode and the diameter of myocardial

bundles, multiple (local) activation times can be present. The model of the superfused

infarcted papillary muscle is, however, a rather simple example, because the model is

virtually 2 D and myocardial and collagen bundles run parallel.

The ventricular heart cell

100 μm

healthy

heart failure

1)

Action potential and ion currents

–90 mV

0 mV

+20 mV

Ito

IK1

ICa

IKs

IKr

100 ms

INa

depolarizing current

repolarizing current

2)

Cell 1 Cell 2

Cell 1

Cell 2

- - - -

+ + + + +

Cell-cell coupling

activated at rest

Current flow

Intercalated disk connexins

3)

+ + + + - - - - -

transmembrane current

membrane

+20 mV

-90 mV

A B C

current dipole

propagation

extracellular

intracellular

I

I

I

Generation of transmembrane current 4)

Extracellular potential distribution

Single Purkinje strand

Spach et al Circ Res 1973

200

400

600

800

500

300

100

700

m

200 400 600 800 1000 1200 1400 200 400 600 800 1000 1200 1400 m

-60 V

-200 V

-100 V

-500 V

+ 60 V

+ 200 V

+ 100 V

+ 500 V

0 V

membrane

propagation

-1 mV +1 mV

current dipole

extracellular

intracellular

5)

Unipolar extracellular electrogram

myocardial bundle

propagation potential field

6)

2 4 6 8 10 12 14 16 18 20 ms

1 mm

1 mV

1 mm

Activation in 2D

Spach 1979

a b

c

7)

Activation time Activation time

t = 0 ms

t = 0 ms

ischemia control

intracellular

recordings

extracellular

recordings

50 mV

20 mV

Activation times in unipolar electrograms

200 ms

8)

Activation time in bipolar recording

+-+-

unipolar

bipolar Wave front

+ -

activation time

activation time

9)

+

-

Bipolar recording: direction dependence

activation time

activation time

wave front wave front

wav

e fr

on

t

10)

output

Complex electrograms

Local and remote deflections

11)

III

HRA

MAP bipolar

MAP unipolar

V A

Ventricular complex in atrial recording

ventricular deflection

atrium

12)

RSPV

RMPV

RIPV

LSPV

SCV

Left atrium

Right

atrium

LSPV

RIPV LIPV

RSPV

SCV

ICV

CS

adjacent structures

de Bakker Wittkampf 2012

RSPV - SCV

13)

Sinus Rhythm

SCV

RSPV

20 ms

SCV activity recorded in RSPV

de Bakker Wittkampf 2012

14)

LAA

cs

LA

atrium

coronary sinus

Double potentials in CS

a

cs

1

2

3

4

1 2

3 4

15)

Epicardial unipolar electrogram

Infarcted area

Durrer D Am Heart J 1964

16)

100 µ

Tissue discontinuities

ENDO; Patient: ?, diagnosis: ?, date: 30-03-2000

Q:\D30035.d00: {\it18}

1350...1550 ms 3 mV/s, 1197 mm/s, 0.025 mV/cm [-0.35,0.25 mV]

0.25 mV

140 ms S1 S2

Intracellular recording

Extracellular recording

17)

Fibrillatory conduction Tissue discontinuities in pectinate muscle network in RA

Berenfeld O, Circ Res. 2002;90:1173-1180

0 30 60 90 120 ms 6 Hz 8 Hz

6 Hz 8 Hz

18)

a

b

c

a

b

c

5 7 9 11 13

5 ms s

Structural conduction block

barrier

19)

Double potentials

Schalij M, 1988

20)

10 20 30 50 60 40 70 80 90 100 ms

20

80

30

10

S1

S3

Intermingling myocardial bundles

& collagen fibers

fibrosis

myocardium

1 mV

50 ms

bipolar recording

21)

e

c

d

b

c

e

d

b

c

e

d

b

e

d

b

c

0 μm 70 μm 140 μm 210 μm

200 μm

Infarcted papillary muscle

surviving myocardium

fibrosis

f f f

22)

c

d

e

b

a

b

c

d

e

Fractionated electrogram

fibrosis

myocardium

Asynchronous conduction

a

b

+

23)

electrogram fibrosis

myocardium

Synchronous conduction

activation

front +

24)

Activation times

in fractionated electrograms

fibrosis

a

b

c

d

e ?

?

25)