Autoantibodies in Heart Failure and Cardiac Dysfunction

Ziya Kaya, Christoph Leib and Hugo A. KatusAutoantibodies in Heart Failure and Cardiac Dysfunction

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Review

This Review is in a thematic series on Myocardial Inflammation, which includes the following articles:

The Fire Within: Cardiac Inflammatory Signaling in Health and DiseaseInflammation in Myocardial Disease

Autoantibodies in Heart Failure and Cardiac Dysfunction

Regulation of the Inflammatory Response in Cardiac Repair

Inflammation, Endoplasmic Reticulum Stress, Autophagy, and the Monocyte Chemoattractant Protein-1/CCR2 Pathway

Signaling Pathways in Response to Inflammation, Senescence, and Aging in the Heart

Anthony Rosenzweig, Guest Editor

Autoantibodies in Heart Failure and Cardiac DysfunctionZiya Kaya, Christoph Leib, Hugo A. Katus

Abstract: Human heart failure is a disease with multifactorial causes, considerable morbidity, and high mortality.Several circulating autoantibodies, some of them being heart-specific, play a crucial role in the progression andinduction of heart failure. However the precise mechanisms on how these autoantibodies perpetuate or eveninduce an organ specific autoimmune response are not yet fully understood. Also it is being a matter of currentresearch to elucidate a potential pathophysiological role of the innate immune system in generating auto-reactiveantibodies. In this review we will summarize the current available literature on circulating autoantibodies whichare related to human heart failure. We will present clinical and animal studies that demonstrate the occurrenceand pathophysiological relevance of several autoantibodies in heart failure, as well as point out biologicalmechanisms on molecular and cellular level. Finally the beneficial therapeutic effects of numerous clinical studiesthat target the humoral arm of the immune system by using either intravenous immunoglobulins and/orimmunoadsorption will be critically discussed. (Circ Res. 2012;110:145-158.)

Keywords: autoimmunity � autoantibody � myocardial dysfunction � heart failure � immunoadsorption

BackgroundHeart failure is the final clinical entity of many diversedisease causes and mechanisms. Among the modulators ofdisease progression a dysregulation in the immune system ofyet unknown reasons is believed to play a central role indisease progression. The immune system consists of a mul-ticellular highly regulated and complex defense system that ischaracterized by a high interindividual variability in itsresponse to injury and antigens. In its physiological conditionit is programmed to discriminate between self- and foreignconstituents, hereby interacting and eliminating any struc-tures that are recognized as foreign. This process can drift

into a pathological situation in which self-tissue is attacked,resulting in auto-immune disease.

Circulating autoantibodies have been critically linked toheart failure. These autoantibodies are targeted against di-verse self-antigens, which often are not restricted in theirexposition to cardiac muscle. Their prevalence, mode ofaction, and potential therapeutic modulation are intensivelyinvestigated. Although a triggering injury to myocardium isbelieved to be the crucial initiating event, the genetic predis-position, environmental and epigenetic modulators, and otherstill unknown mechanisms are critical for development of thepathological antibody titers observed in peripheral blood and

Original received June 15, 2011; revision received August 31, 2011; accepted September 28, 2011. In October 2011, the average time from submissionto first decision for all original research papers submitted to Circulation Research was 15 days.

From the Department of Internal Medicine III (Z.K., C.L., H.A.K.), University of Heidelberg, Germany.Correspondence to Ziya Kaya or Hugo A. Katus, Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, 69120

Heidelberg, Germany. E-mail [email protected] or [email protected]© 2012 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.111.243360

145 at Universitaet Heidelberg on February 27, 2012http://circres.ahajournals.org/Downloaded from


the intensity of inflammation in myocardial structures. In aprospective study, Caforio et al showed that circulatingantiheart autoantibodies may precede disease manifestationand are independent predictors of disease development.1

Clinical observations on a prognostic relevance of autoanti-bodies have prompted therapeutic trials focused on nonspe-cific removal of autoantibodies from the circulation viaimmunoadsorption. There are first reports on a beneficialoutcome in patients treated by immunoadsorption.2 However,the presence of antiheart specific autoantibodies may notalways be harmful because some antibodies seem to beprotective in chronic heart failure.3 In the following we willdiscuss the pathophysiological role of autoimmunity in heartfailure with focus on the humoral immune response.

Induction of an Immune Responseto Auto-Antigens

The normal consequence of an adaptive immune response toforeign antigens in healthy individuals is the clearance ofthese foreign antigens from the body. Usually, clonal deletionby ubiquitous self-antigens and clonal inactivation by tissue-specific antigens presented in the absence of costimulatorysignals induce self-tolerance, and there is no induction of animmune response to self-antigens. However, a defect in thisselection process results in an adaptive immune response toself-antigens and damage of self-tissue. This injury leads toconstant supply of new autoantigens, which induces persis-tent immune response. Hereby, autoreactive CD4� T cellsupport is required, which are usually selected during theirdevelopment in the thymus (Figure 1).4 Autoimmune diseasescan be mediated by autoreactive antibodies by forming

Non-standard Abbreviations and Acronyms

�-myosin—HC �-myosin heavy chain

ANT adenine-nucleotide transporter

�1-AR �1-adrenoceptors

CHF congestive heart failure

CVB3 coxsackievirus

DCM dilated cardiomyopathy

DC-SIGN dendritic-cell-specific ICAM-3 grabbing non-integrin

hsp-60 heat-shock protein 60

IA immunoadsorption

ICM ischemic cardiomyopathy

IHD ischemic heart disease

IRAK interleukin-1 receptor-associated kinase

IVIG intravenous immunoglobulins

MHC myosin heavy chain

MyD88 myeloid differentiation factor 88

PKA protein kinase A

TLR toll-like receptor

Figure 1. T-cell selection in the thymus.Committed lymphoid progenitors arise inthe bone marrow and migrate to the thy-mus. Early committed T cells lack expres-sion of T-cell receptor (TCR), CD4, andCD8 and are termed double-negative(DN; no CD4 or CD8) thymocytes. DNthymocytes can be further subdividedinto 4 stages of differentiation (DN1,CD44�CD25�; DN2, CD44�CD25�; DN3,CD44�CD25�; and DN4, CD44�CD25�).As cells progress through the DN2 to DN4stages, they express the pre-TCR, whichis composed of the nonrearranging pre-T�chain and a rearranged TCR �-chain.Successful pre-TCR expression leads tosubstantial cell proliferation during theDN4 to double positive (DP) transition andreplacement of the pre-TCR �-chain witha newly rearranged TCR �-chain, whichyields a complete ��-TCR. The��-TCR�CD4�CD8� DP thymocytes theninteract with cortical epithelial cells thatexpress a high density of myosin heavychain (MHC) class I and class II moleculesassociated with self-peptides. The fate ofthe DP thymocytes depends on signalingthat is mediated by interaction of the TCRwith these self-peptide–MHC ligands. Toolittle signaling results in delayed apoptosis(death by neglect). Too much signalingcan promote acute apoptosis (negativeselection); this is most common in the

medulla on encounter with strongly activating self-ligands on hematopoietic cells, particularly dendritic cells. The appropriate, interme-diate level of TCR signaling initiates effective maturation (positive selection). Thymocytes that express TCRs that bind self-peptide–MHC-class–I complexes become CD8� T cells, whereas those that express TCRs that bind self-peptide–MHC-class–II ligands becomeCD4� T cells; these cells are then ready for export from the medulla to peripheral lymphoid sites. SP indicates single positive.Reprinted with permission from Germain R.N. et al.4

146 Circulation Research January 6, 2012



immune complexes, activating the complement system, bind-ing to surface receptors, and influencing downstreamsignaling.5–9

The exact triggers for induction of an autoimmune re-sponses are not well known, but various human autoimmunediseases display a myosin heavy chain (MHC)-linked asso-ciation (multiple sclerosis, graves’ disease, myasthenia gra-vis, rheumatoid arthritis).10–14 Although, during the last years,it has been discovered that autoimmunity plays an importantrole in the pathogenesis of myocarditis and dilated cardiomy-opathy (DCM) in genetically predisposed individuals, to ourknowledge barely any of the available genetic studies infamilial DCM has taken into account the autoimmune phe-notype markers in the characterization of patients and rela-tives. In one study Caforio et al demonstrate that relatives ofDCM patients show higher frequency of circulating autoan-tibodies, possibly predisposing them to developing DCMthemselves.1 However, large genetic association studies withprevalence of cardiac autoantibodies are missing yet. Weknow from animal models of autoimmune myocarditis in-duced by viral infection or immunization with heart-specificautoantigens that there is a genetic predisposition forsusceptibility.15

Cardiac autoimmunity can be triggered by autoantigenspresented to the immune system following cardiac injuryinduced by endogenous or exogenous factors (such as viralinfections). Molecular mimicry and cross-reactivity may playan important role in inducing an autoimmune response,especially in individuals with cardiotropic virus infections(Figure 2).16 The autoantibodies can hereby influence cardiacfunction by negative chronotropic and/or negative inotropiceffects. Furtheremore, they can induce apoptosis of cardio-myocytes and activate complement.16–19

Link Between B-Cells and the InnateImmune System

B-cells represent a crucial link between the innate andadaptive immune system. Besides the antigen-specific B-cellreceptors, B-cells also express toll-like receptors (TLRs) on

their cell surface. These receptors, being highly evolutionaryconserved, are activated by both exogenous (bacterial orviral) or endogenous (cell-derived or ECM-derived) antigens,and thereby constitute an essential part of the innate immunesystem. TLR-signaling in B-cells is critically associated toB-cell activation and tolerance and to diverse pathologicalconditions, such as atherosclerosis, viral myocarditis, andseptic cardiomyopathy (Figure 3).20,21 Stimulation of TLRson B-cells leads to the activation of an intracellular signalingcascade in which the myeloid differentiation factor MyD88and interleukin-1 receptor-associated kinase (IRAK) play adominant role in generating autoreactive plasma cells.21

Signaling through the MyD88/IL-1R axis is linked to cardiacfibrosis during progression to heart failure.22 In coxsackievi-rus B3 (CVB3)-induced myocarditis in mice, the TLR-9mediated activation of MyD88 with subsequent activation ofTNF-� contributes to the development of acute myocarditis.23

Further, the severity of myosin-induced experimental auto-immune myocarditis has been shown to be dependent onTLR-7 triggered MyD88 activation.24 The role of TLR-signaling in human heart failure however is poorly under-stood. Two studies demonstrate that TLR-4 expression isupregulated in human heart failure.25,26 It has to be consideredthat short-term activation of TLRs located in myocardialtissue confers cytoprotection, whereas long-term activationresults in upregulation of proinflammatory cytokines andrecruitment of immunoactive cells (such as neutrophils,monocytes, and dendritic cells) into the myocardium.20 Further-more B cells can activate complement. Complement and com-plement receptors play an important role in both the CVB3-induced and in myosin-induced myocarditis.27–29

�1-Adrenoceptor/M2-Receptor�1-adrenoceptors (�1-AR) play an important role in adren-ergic regulation of myocardial contractility. They belong tothe 7-transmembrane G-protein coupled receptors and stim-ulation by catecholamines activates the adrenoceptor-adeny-lylcyclase–protein kinase A cascade (PKA).30 Activation of�1-AR induces activation of adenylylcyclase, which in turn

Figure 2. Overview on potential interactions in inducing an autoimmune response leading to cardiac damage.

Kaya et al Autoantibodies and Myocardial Dysfunction 147



leads to an increased synthesis of the second messengercyclic adenosinmonophosphate (cAMP). PKA, which is ac-tivated by cAMP, phosphorylates L-Type Ca2�-channelsleading to an increased Ca2�-influx from the extracellularmilieu and Ca2�-release from the sarcoplasmic reticulum.Overall, this �1-receptor mediated signaling cascade in-creases the contractility of the heart. There exists strongevidence that autoantibodies targeted against the �1-receptorplay a pivotal role in human heart failure.

One of the first studies demonstrating that �-adrenergicreceptor targeted autoantibodies exist in dilated cardiomyop-athy (DCM) was reported by Limas et al31 By use of abio-assay measuring the binding of labeled [3H] dihydroal-prenolol to rat cardiomyocyte membranes they observed acompetitive inhibition of [3H] dihydroalprenolol on incuba-tion with sera from DCM patients. This could not be observedwith sera from ischemic/valvular heart disease patients andhealthy controls. The inhibitory effect was mediated by�-adrenergic autoantibodies of the IgG class. Magnusson et alcompared the sera of patients with DCM or ischemic heartdisease (IHD) to healthy controls to test the antigenic deter-minants of the �1- and �2-AR with regard to the bindingspecificity to the second extracellular loop of the human �1-and �2-adrenergic receptors.32 They found antibodies di-rected to the antigenic �1-second extracellular loop in 31% ofpatients with DCM and only 12% of healthy controls. Thefunctional consequences of �1-AR autoantibody bindinghave first been characterized on spontaneously beating myo-

cytes of neonatal rat heart.33 Immunization of rabbits with asynthetic peptide corresponding to the second extracellularloop of �1-AR leads to production of IgG autoantibodiesagainst this domain.34 As a consequence, these animalsrevealed a decreased density of �1-AR and increased expres-sion of inhibitory G-proteins and G-protein receptors kinase5, leading to reduced intracellular cAMP levels. Six monthsafter immunization a left ventricular hypertrophy and con-tractile dysfunction was observed. The �1-AR desensitizationand functional consequences could be prevented by using theselective �1-AR antagonist bisoprolol. The circulating levelsof �1-AR autoantibodies were markedly increased by induc-tion of experimental heart failure and cardiomyopathy inanimal models.35 In recent years, blocking �1-AR withspecific antagonists has been refined as a potential therapeu-tic strategy to effectively modulate the stimulating anduncoupling effects of �1-AR autoantibodies. Although sev-eral studies show an improved outcome of �-blockers in aclinical setting, more specific agents directed against theactivating site of the autoantibodies are under develop-ment.36–39 Although �1-AR autoantibodies are associatedwith a high risk for progression and prevalence of heartfailure, consistent information on their prevalence, theirfrequency of appearance, formation, and their kinetics inhuman blood is still lacking. The Etiology, Titre-Course, andSurvival study is the largest clinical multicenter trial whichwill investigate the prevalence and kinetics of autoantibodiesin different forms of heart failure until 2013.40 In the

Figure 3. Innate pathways to B-cellactivation and tolerance. Intrinsic toll-like receptor (TLR) signaling in naive andmemory B cells can lead to IgM- andIgG-secreting plasma cell formationthrough interleukin-1 receptor-associatedkinase (IRAK) and myeloid differentiationprimary response gene 88 (MyD88). Mem-ory B cells express increased levels ofTLRs and have a greater capacity to dif-ferentiate into plasma cells via TLR stimu-lation than naive B cells. TLR-activated Bcells can suppress T-cell responses indi-rectly through an IL-10-dependent mech-anism. Type 1 interferon (IFN) productionfrom macrophages and dendritic cellsstimulated with immune complexes orTLR agonists can promote the differentia-tion of B cells into plasma cells. TLR-stimulated dendritic cells and macro-phages can suppress autoreactive B celldifferentiation into plasma cells throughIL6 and soluble CD40L, respectively.B-cell–activating factor (BAFF), producedby autoreactive plasma cells, can alsoinfluence B-cell homeostasis and differen-tiation into plasma cells. In humans,absence of MyD88, UNC-93B, and IRAK4each increase the level of autoreactive Bcells in the periphery. NF�B indicatesnuclear factor-�B; IRF, interferon regula-tory factor. Reprinted with permissionfrom Crampton S.P. et al.21




prospective arm of this trial including 400 patients withmyocarditis or myocardial infarction will be assessed forinduction of �1-AR autoantibodies. The patients will befollowed for cardiac remodeling, heart failure, arrhythmiasand mortality. It is obvious that for treatment of the patho-genic autoantibodies, precise diagnostic assays are required,specifically to monitor treatment effects and to determinecriteria for initiation of treatment. Nikolaev et al haveestablished a highly sensitive detection method which mea-sures �1-receptor-mediated increases in intracellular cAMPlevels by fluorescence resonance energy transfer using ahighly sensitive cAMP sensor, which will be used in theEtiology, Titre-Course, and Survival study.41

In other studies it has been shown that sera of patients withidiopathic DCM react with M2-muscarinic receptor peptide(36% to 39%) and �-AR peptide (31%), respectively.42–44 Asynthetic peptide corresponding to the sequence 169 to 193 ofthe second extracellular loop of the human M2-receptor hasbeen used as a capture antigen to screen sera of patients withidiopathic DCM and healthy blood donors by use of ELISA.42

Only 8% of healthy subjects revealed a signal correspondingto the M2-peptide, whereas 39% of DCM patients werepositive for this peptide. Also, a highly significant coexis-tence has been found of anti-M2–receptor autoantibodies andanti-�1–AR autoantibodies in patient’s sera with idiopathicDCM.42 Antibodies directed against the sequence 169 to 193of M2-receptor have been shown to exert a negative chrono-and ionotropic effect and inhibit adenylcyclase activity.45,46

Immunization of rabbits with peptides corresponding to thesecond extracellular loop of M2-receptor–induced right, butnot left ventricular dilatation.47 On histological analysis, focalmyofibrillar-lysis, loss of myofilaments, mitochondrial swell-ing and condensation, sarcoplasmic vacuolation, depositionof dense granules in the sarcoplasm, and the myofibrils wereobserved after immunization.47 Using specific adrenergic andmuscarinic-blocking agents combined with an in vitro caninePurkinje fiber contractility assay Stavrakis et al studied theopposing effects and interactions of anti-�1–AR autoantibod-ies and anti-M2–receptor autoantibodies on contractility invitro.48 Stavrakis et al showed that circulating anti-M2–receptor autoantibodies derived from sera of DCM patientscompromised the agonistic ionotropic effects of circulatinganti-�–AR-1 autoantibodies, by binding on M2-receptors.The authors therefore propose that circulating activated au-toantibodies to the muscarinic M2-receptor may reduce ven-tricular contractility and thereby promote heart failure inpatients with cardiomyopathy.48 However, circulatinganti-M2 or anti-�1–AR autoantibodies may not be cardiac-specific, which questions their exclusive role in heart failure.

Antimitochondrial AutoantibodiesThere are numerous reports on specific antimitochondrialantibodies and their clinical relevance in a number of patho-logical disorders. The most relevant mitochondrial antigensM1, M2, and M7 are located in the inner mitochondrialmembrane, whereas M3, M4, M5, M6, M8, and M9 arelocated in the outer mitochondrial membrane.49 Anti-M7antibodies are detectable in patients with DCM and there arereports on their functional relevance.50 Thus, anti-M7 anti-

bodies were observed in blood of 31% of DCM patients, 33%of hypertrophic cardiomyopathy patients, and 13% of acutemyocarditis patients. In contrast, no anti-M7 antibodies couldbe found in control patients with other cardiac or immuno-logic disorders. These antibodies react either specifically withheart mitochondria (anti-M7 type a) or reveal cross-reactivitywith antigenic determinants of pig kidney, beef pancreas, orrat lung (anti-M7 type b). Using immobilized submitochon-drial particles derived from the myocardium, immunoadsorp-tion (IA) of anti-M7 autoantibodies was able to abolishanti-M7 type a and type b activity. The authors thereforeproposed cardio-specificity for both types of anti-M7 anti-bodies, although mitochondrial antigens are generally consid-ered not to reveal tissue-specificity.51

The adenine-nucleotide transporter (ANT) is an ADP/ATPcarrier located in the inner mitochondrial membrane.52 Theexistence of autoantibodies directed against ANT in dilatedcardiomyopathy has been shown by Schultheiss et al in1985.53 In their uncontrolled study, functionally active anti-bodies against ANT were observed in 94% of 18 patients withDCM.53 In contrast, in patients with coronary heart disease,suspected alcoholic heart disease or healthy blood donors noanti-ANT antibodies were observed. Antibody titers wereinversely related to the clinical outcome of DCM. Epitopemapping revealed that the antigenic determinants of ANT arelocated in the C-terminal 146 amino acids, corresponding tothe M2 and M3 hydrophilic region allocated to the mitochon-drial matrix space.54 The amino acid sequence of ANTprotein has, to some extent, homology to CVB3.55 Thus it wastempting to postulate a molecular mimicry of autoantibodiesto ANT and CVB-infection, which is an established infec-tious agent causing myocarditis.56 In support of this hypoth-esis, immunization with CVB3 induces a marked productionof ANT autoantibodies in mice, leading to specific alterationsin cellular energy consumption and calcium homeostasis.57,58

The IL-17 producing CD4� Th-cell subset (Th-17) is criti-cally linked to the progression of inflammatory dilatedcardiomyopathy.59 When circulating IL-17 is neutralized,anti-ANT autoantibody production is diminished in a CVB3model of auto-immune myocarditis. It is hypothesized thatthe protective effect of IL-17 inhibition on cardiac inflamma-tion is due to inhibition of CD19(�) B-lymphocyte prolifer-ation and reduction of secretion of anti-ANT autoantibodiesby these cells.60

MyosinNeu et al described in 1987 that immunization of mice withcardiac, but not skeletal myosin, induces severe myocarditisaccompanied by high titers of myosin autoantibodies insera.61 In this study the authors also very elegantly showedthat the genetic background of immunized mice determinedthe prevalence and severity of myocarditis and circulatinglevels of cardiac myosin autoantibodies. Genetic analysisprovided some evidence that susceptibility to myocarditiswas linked to the major histocompatibility complex genotypeand other not well-defined genes. However a more refinedanalysis would have required genome wide association stud-ies in inbred mice strains with and without susceptibility toantibody induced myocarditis. In humans and predominantly




after birth �-myosin heavy chain is strictly expressed in atrialcardiomyocytes, whereas the �-isoform is expressed in bothslow skeletal muscle and ventricular cardiomyocytes.62 Ca-forio et al provided some evidence that myosins may bedetectable in blood of patients with DCM, as assessed byWestern blot.63 Although this is intriguing to explain theprevalence of anti-�- and anti-�-myosin heavy chain antibod-ies in blood, more precise analyses are needed to definitivelytest for the circulating myosin heavy chains in heart failure.Only by the use of high sensitivity troponin assays it waspossible to identify some marker positive patients with DCM.Antimyosin IgG autoantibodies are also detected in patientswith myocarditis.64 ELISA and Western blotting analysisrevealed that 42% patients with myocarditis exhibited auto-antibodies against myosin. These antimyosin autoantibodiesdo not differentiate between myosin prepared from eithercardiac or skeletal muscle, respectively, indicating the knowntissue distribution of �- and �-myosin heavy chains. Anti-myosin autoantibodies can also be detected in experimentalmodels of auto-immune myocarditis induced by CVB3 infec-tion.65 In patients with DCM the presence of antimyosinautoantibodies was associated with deterioration of myocar-dial function.66 To investigate the functional effects of anti-myosin antibodies, Warraich et al affinity purified anticardiacmyosin autoantibodies of patients with DCM or IHD.67 Whenisolated cardiomyocytes were exposed to anticardiac myosinautoantibodies an altering of Ca2�-sensitivity of myofila-ments and reduction of contractility of cardiomyocytes wasobserved. However, affinity-purified autoantibodies were notinternalized by myocytes and had no effect on L-typeCa2�-currents. Because �- and �- myosin isoforms are bothlocated in the sarcomer compartments, the mechanisms ofantibody interaction and intracellular signal transduction areonly poorly understood. A number of studies show thatanticardiac myosin monoclonal antibodies lead to myocardi-tis in some mouse strains.68–70 However these observations donot resolve the molecular mechanism leading to heart failure.Li et al showed that anticardiac myosin autoantibodiescross-react with the �-AR, thereby specifically inducingcAMP-dependent PKA activity in heart cells.71 Therefore theactivation of the �-AR dependent PKA signaling pathwaymight contribute to myocardial dysfunction and apoptosis ofcardiomyocytes.72,73 In support of this, the addition of cardiacmyosin, nonspecific anti-IgG or specific inhibitors of the�1-AR pathway inhibited the anticardiac myosinautoantibody-mediated PKA signaling. Li et al also showedthat transfer of purified anticardiac myosin IgG from immu-nized rats results in myocardial IgG deposition and cardio-myopathy.71 In summary, molecular mimicry between car-diac myosin and the �1-AR is an attractive hypothesis toexplain the anticardiac myosin autoantibody mediated induc-tion of heart failure. The points raised so far are predomi-nantly related to an autoantibody mediated humoral immuneresponse. There are, however, also very convincing data onthe role of the cellular and innate autoimmunity on myocar-dial function. Very recently Lv et al showed that �-myosinheavy chain (�-myosin–HC), although being located strictlyintracellularly in atrial cardiomyocytes, acts as an auto-antigen for CD4� T-cells in myocarditis.74 The authors also

show that �-myosin–HC transcripts are absent in murine andhuman thymus medullary epithelial cells, which are crucialfor development of self-tolerance and autoimmunity.75 Trans-genic expression of �-myosin–HC transcripts in medullaryepithelial cells prevented myocarditis by inducing self-tolerance to �-cardiac–myosin-HC. Further �-myosin–HCspecific Th1 CD4� T-cell clones induced myocarditis afterbeing transferred to DQ8�Rag�/� NOD mice, therebydemonstrating that spontaneous myocarditis is caused by lossof CD4� T-cell tolerance to �-myosin–HC. The currentliterature on antimyosin autoantibodies thus proposes animperfect selection process of (self-) antigens in the thymusduring T-cell development, leading to generation of CD4�Th-cells targeted against �-myosin. Molecular mimicry be-tween �-myosin and �1-AR may then trigger the signaltransduction of �-myosin autoantibody interaction intracellu-larly leading to �1-AR dependent functional impairment ofcardiomyocytes.71–74

Cardiac Troponin ICardiac troponin is part of the regulatory complex of the thinfilament of muscle fibers.76 It consists of the 3 subunitstroponin C, T, and I, which interact in regulation of musclecontraction. Recently, it has been shown that mice deficientof programmed cell death-1 immune inhibitory coreceptorprotein develop severe DCM followed by progressive heartfailure in mice.77 In these mice, antibodies of IgG subtypecould be detected on the surface of cardiomyocytes. Theseantibodies were specific for cardiac troponin I. Interestingly,administration of these anti-TnI–antibodies augmented thevoltage-dependent L-type Ca2�-current of cardiomyocytesand induced ventricular dilatation and heart failure.78 Thesefindings indicated that cardiac troponin I may not be strictlylocalized in the cytoplasm but may also be exposed on thecytoplasmic membrane, making it accessible for antigen–antibody interactions. Recently, we could show that immuni-zation of mice with cardiac troponin I but not with cardiactroponin T induced severe inflammation in the myocardiumfollowed by fibrosis and heart failure with increased mortal-ity,79 although immunization with both troponin I and tro-ponin T induced a strong cellular and humoral immuneresponse. It is likely that differences in troponin expression inthe cytoplasmic membrane may account for the observeddifferences. Elevated troponin I autoantibodies can also bedetected in a virus-induced experimental auto-immune myo-carditis model on infection with CVB3 virus.80 The antigenicdeterminant of the murine troponin I molecule that causessevere inflammation and fibrosis appears to be an 18-merpeptide of troponin I.81 Only mice immunized with residues105 to 122 (referred to as peptide 9) of murine troponin Ideveloped significant inflammation and fibrosis in the myo-cardium with increased expression of proinflammatory cyto-kines, chemokines, and chemokine receptors. This epitope ofmurine troponin I is located in the hydrophilic region oftroponin I and comprises an �-helical structure, making itparticularly susceptible to antigen–antibody interaction.

The clinical significance of circulating antitroponin auto-antibodies as modulators of progression of DCM is not yetclear because there are conflicting results. Although 1 study




indicates an adverse effect of antitroponin autoantibodies oncardiac function and outcome in ischemic cardiomyopathy(ICM),82 other studies report on anti-TnI antibody positivepatients with very similar or even improved outcome ascompared to TnI-antibody negative patients.3,83 Thus, it isstill under debate whether in patients circulating antitroponinI autoantibodies modulate cardiac function or progression inheart failure.

Leuschner et al screened patients with DCM and ICM forthe presence of troponin I autoantibodies and observedantitroponin I IgG antibody titer �1:160 in 7.0% of patientswith DCM and 9.2% with ischemic cardiomyopathy. In thisstudy the presence of anti-TnI antibodies was associated withadverse remodeling post myocardial infarction. Patients withno elevated cardiac troponin I antibody titers showed anincrease in left ventricular ejection fraction and stroke vol-ume 6 to 9 months after acute myocardial infarction.82 Incontrast, improvement of cardiac function and remodelingwas not observed in patients with antitroponin I IgG antibodytiter �1:160. Doesch et al investigated the prognostic valueof circulating TnI autoantibodies in plasma of patients withchronic heart failure.3 This study indicates that the presenceof circulating TnI autoantibodies in plasma is associated withan improved survival in patients with chronic DCM but notICM. The authors thus propose a possible protective effect ofcirculating antitroponin I autoantibodies in DCM. However,further clinical and experimental studies are needed to finallyelucidate the functional role of circulating TnI autoantibodiesand progression of DCM. In a prospective controlled studyHalley et al examined the release of interferon-gamma andinterleukin-10 (IL-10) from peripheral blood mononuclearcells on TnI stimulation in 35 idiopathic DCM patients and 26healthy controls.84 Although there was no difference ininterferon-gamma secretion on TnI stimulation in bothgroups, IL-10 secretion was significantly higher in the pa-tients with DCM. Among DCM subjects, heightened IL-10response to cardiac troponin I was associated with reducedsystemic inflammation (reduced systemic levels of high-sensitivity C-reactive protein) and lower prevalence of ad-vanced diastolic dysfunction compared with those with nor-mal IL-10 response to cardiac troponin I.

Na-K–ATPaseImpairment of sarcolemmal Na-K–ATPase activity in pa-tients with idiopathic DCM has been reported previously.85 Ina controlled study by Baba et al 100 patients with DCM and100 age matched controls have been screened for anti-Na-K-ATPase autoantibodies using ELISA.86 Anti-Na–K-ATPaseautoantibodies were detected in 26% of DCM patients and2% of controls. Patients who were positive for anti-Na–K-ATPase autoantibodies showed more frequent prematureventricular complexes and nonsustained ventriculartachycardia. Na-K–ATPase activity has been measured invitro in the presence of purified IgG from patients with andwithout anti-Na–K-ATPase autoantibodies. IgG from patientswith anti-Na–K-ATPase autoantibodies lowered the activityof Na-K–ATPase in vitro, thereby demonstrating the biolog-ical activity of these autoantibodies. Further, Western blottingand radio-ligand binding analysis revealed that the Na-K–

ATPase � subunit is bound by corresponding autoantibodiesand that these autoantibodies exert an inhibitory effect.86 Theauthors therefore suggested that binding of Na-K–ATPaseautoantibodies results in a conformational change leading toa low-affinity of the ATPase. They further hypothesize thatthe reduction of Na-K–ATPase activity due to correspondingantibody–antigen interactivity leads to abnormal intracellularCa2� handling and delayed afterdepolarizations via reverse-mode operation of the Na�/Ca2� exchanger resulting fromincreased intracellular Na� concentrations.86 Further, it hasbeen shown that immunization of rabbits with sarcolemmalNa-K–ATPase results in myocardial hypertrophy due to leftventricular pressure overload and myocardial fibrosis.87 Im-munoblotting showed that expression of the �3-isoform ofNa-K–ATPase was selectively reduced in myocardium inimmunized animals. Taken together the presence of autoan-tibodies against Na-K–ATPase has been linked to clinicaloutcome of DCM. However, little is known on the specificity,role, and mechanism of Na-K–ATPase autoantibodies inother forms of heart failure.

Other Heart AntibodiesLatif et al screened sera of 45 patients with DCM and 43patients with IHD for antiheart antibodies.88 Western blottingand 2-dimensional SDS-gel electrophoresis followed byN-terminal protein sequencing revealed that DCM patients, incontrast to IHD patients, showed significantly more fre-quently elevated autoantibodies. The most prevalent antigenswere myosin light chain1, MHC, actin, tropomyosin, andheat-shock protein 60 (hsp-60). Anti-hsp–60 autoantibodieswere found in 85% of the DCM patients, whereas only 42%of patients with IHD were positive. Heat-shock proteins arehighly conserved immunogenic molecules that are locatedintracellulary. They are part of the chaperone system, areinvolved in protein folding, and are upregulated in myocar-dial stress.89 In the study by Latif et al antimyosin autoanti-bodies were not the predominant autoantibodies in sera ofDCM patients, as proposed by Caforio et al,63 67% DCMpatients were positive for antimyosin autoantibodies whereas85% were positive for anti-hsp–60 autoantibodies. Also,Latif et al were not able to detect autoantibodies against�1-AR, ANT, or mitochondrial M7 antigen, neither in DMCnor in IHD patients. A difference that is explained by the useof the unfractionated myocardial homogenate in their stud-ies.90 In CBV3-induced murine experimental auto-immunemyocarditis antimyosin autoantibodies but not anti-hsp–60autoantibodies are the predominant antibody fraction.91 Thepathogenic role of anti-hsp–60 autoantibodies in the devel-opment of heart failure is still unknown. Very recently, Lin etal induced heart failure in rats by coronary ligation of leftanterior descending artery.92 They could show that hsp-60after induction of heart failure was translocated to the plasmamembrane and, more importantly, it was also detectable onthe cell-surface, thus being a potential target for antibodies orthe innate immune system. Membrane hsp-60 correlated withincreased apoptosis. The authors concluded that abnormal traf-ficking of hsp-60 to the cell surface may be an early trigger formyocyte loss and the progression of heart failure. Hsp-60might also be pathologically significant by inducing cell lysis




through activating complement.88 However, anti-hsp–60 au-toantibodies are not cardiac-specific, and it remains to beelucidated whether anti-hsp–60 autoantibodies may serve as adiagnostic marker or as a specific target against heart failure. Anumber of other antigenic targets for autoantibodies havebeen described, but their pathogenic role is not understood(Table 1).93–103

Autoantibodies as a Clinical Target forTherapy of Heart Failure

Most therapeutic efforts target the humoral immune systemby eliminating circulating autoantibodies by IA104–107 (Table2, Figure 4). A number of these clinical studies have providedsome encouraging results, but clearly more robust data andrandomized trials are needed. One of the clinical pilot studiesusing IA strategy for treatment of DCM in patients was doneby Wallukat et al: They first showed that IA removedcirculating IgG3 antibodies targeted against �1-AR108 andlater that IA may improve cardiac function in patients.109,110

18 patients with DCM (NYHA: III–IV; LVES ��30%) wererandomly assigned either to IA with subsequent IgG substi-tution (IA/IgG) or control.110 In contrast to the control groupIA/IgG patients showed significant improvement in hemody-namic parameters (cardiac index, stroke volume index, sys-temic vascular resistance) three months after therapy. How-ever, this study did not report specifically on the changes ofautoantibody levels following IA and was not adequatelycontrolled and blinded. In a following retrospective study,patients with congestive heart failure (CHF) or DCM (NYHAclass II–III; LVES ��35%) who received IA therapyshowed significantly decreased hospitalization rate comparedto CHF- or DCM- controls during 3 years follow up.106

In further studies, it has been shown that apart fromimproving cardiac hemodynamic parameters in DCM pa-tients, IA influences the cellular component of the adaptiveimmune system.111 From 25 DCM patients (ejection fraction[EF] �30%), 12 patients were randomized for IA therapy andsubsequent IgG substitution at 1-month intervals until month

Table 1. Overview of Autoantibodies Associated With Heart Failure and Cardiac Dysfunction

Autoantibody CardiomyopathySuggested Antigenic

localization Suggested Pathomechanism Reference

Anti-�1–receptor DCM Cell surface? Negative ionotropic Limas et al. 38

DCM Cell surface? Positive ionotropic Wallukat et al. 108

Anti-M2–receptor IDCM Second extracellular loop M2(amino acids 163–169)

Negative ionotropic Fu et al. 42, 43

Antimitochondrial M7 DCM acute myocarditis Inner mitochondiral membrane Unknown Klein et al. 50

Adenine-nucleotidetransporter

IDCM, DCM C-terminal 146 amino acidsallocated in the mitochondrial matrix

Alterations in energy metabolism Schultheiss et al. 53Manchado et al. 54

Myosin heavy chainalpha and beta

DCM Alpha: atrial cardiomyocytes Beta Negative ionotropic Caforio et al. 63

Myosin heavy chain alpha Myocarditis (mouse) Intracellularly Deterioration of self-toleranceduring thymic selection processes

Lv et al. 74

Antitroponin I DCM (mouse) Cell surface Okazaki et al. 78

Myocarditis/DCM (mouse) Cell surface Negative ionotropic Goeser et al. 79

Myocarditis/DCM (mouse) Amino acid residues 105–122 Negative ionotropic Kaya et al. 81

Anti-Na-K–ATPase IDCM (human) Alpha3-subunit Antiarrhythmogenic effect dueto a conformational change of

Na-K-ATPase from a high-affinityto low-affinity state.

Baba et al. 86

DCM (rabbit) Sarcolemmal transmembrane Cardiac hypertrophy Baba et al. 87

Anti-hsp60 DCM/ischemic heart disease(human)

. . . Unknown Latif et al. 90

Experimental coronary arteryligation (rat)/DCM/ICM (human)

Cell-surface Abnormal trafficking of hsp-60 tothe cell surface to trigger myocyte

loss after LAD-ligation

Lin et al. 92

Antiserotoninergic 5-HT4receptor

Congenital heart block (human) Cell surface Unknwon Kamel et al. 95

Anti-SR–Ca2�-ATPase Experimental myocarditis (mouse) . . . Metabolic interactions Khaw et al. 96

Antiacetylcholine receptor Bradycardia Cell surface Unknown Goin et al. 94

Antilaminin DCM/myocarditis Extracellularly Unknown Wolff et al. 103

Antitropomyosin DCM Intracellularly Unknown Latif et al. 90

Antiactin DCM . . . Unknown Latif et al. 90

Anti- myosin light chain-1 DCM . . . Unknown Latif et al. 90

DCM indicates dilated cardiomyopathy; ICM, ischemic cardiomyopathy; hsp60, heat-shock protein 60; IDCM, idiopathic dilated cardiomyopathy; LAD, left anteriordescending artery.




3. In this study a beneficial effect of IA/IgG therapy onmyocardial inflammation was observed with decrease in thenumber of CD4� and CD8� T lymphocytes and totalnumber of leukocytes in the myocardial biopsies. Further,IA/IgG treatment reduced the expression of HLA class IIantigens. Differential expression of HLA antigens, a compo-nent of the major compatibility complex (MHC), has beencritically linked to pathogenesis of DCM.112,113 Also, anti-�1–AR autoantibodies were significantly reduced in IA/IgGtreated patients.111 Removal of anti-�1–AR IgG3 autoanti-bodies may not be linked to the beneficial hemodynamicoutcome after IA therapy, because hemodynamic improve-ment after IA therapy was similar among patients positiveand negative for �1-AR autoantibodies, as shown in anunblinded clinical study including 22 DCM patients (NYHAIII–IV, EF �30%) by Mobini et al114 Opara et al showed in

a case-control study with 6 DCM patients (LVEF �40%,NYHA II–III) that IA affects gene expression of the type IIIintermediate filament protein desmin, in that expressionlevels of desmin in endomyocardial biopsies were signifi-cantly decreased in patients after IA therapy.115 The authorssuggest that production of cardiac autoantibodies is linked toDCM-associated changes in myocardial gene expression andthat removal of these antibodies by IA therapy may modulatemyocardial gene expression. Another study by Bulut et alshows the effect of IA on the concentration of endothelialmicroparticles (eMP) in blood of 13 patients with advancedCHF (NYHA III and IV) secondary due to chronic iDCM (EF�35%).116 Microparticles, which include exosomes, mi-crovesicles, apoptotic bodies, and apoptotic microparticles,are small (0.1–1 �m), membranous vesicles that can containDNA, RNA, miRNA, intracellular proteins, and express

Table 2. Overview of Clinical Studies for Treatment and Their Clinical Outcome

Autoantibody Targeted Study TypeType of Clinical

InterventionPatients and

Cardiomyopathy Clinical Outcome Reference

Not specified Prospective uncontrolled(n�4) 6 mo

Immunoadsorption chronic DCM and NYHAClass II–III CHF

Improvement in hemodynamicparameters

Cooper et al.104

Not specified Retrospective controlled(n�17 each group)

Immunoadsorption DCM, left ventricularejection fraction less

than 35%, NYHAclasses II–III

Improvement in hemodynamicparameters and reduction of

morbidity

Knebel et al.106

Not specified Randomized uncontrolled 6mo (Group 1: n�11 with

four IA courses at monthlyintervals. Group 2: n�11

with one IA course withoutrepetition)

Immunoadsorption DCM (left ventricularejection fraction

�35%)

One course of IA treatment iscomparable to multiple IA

courses

Staudt et al.107

Beta-1-AR-autoantibody Prospective uncontrolled(n�22)

SpecificImmunoadsorption

DCM (NYHA III–IV, EF�30%, stable

medication)

The beneficial hemodynamiceffects induced by IA are notdirectly associated with the

removal of beta(1)ARautoantibodies

Mobini et al.114

Not specified Analysis of myocardialgene expression of the

intermediate cytoskeletalfilament desmin (n�6)

3 mo

Immunoadsorption DCM (LVEF �40%,NYHA II–III)

Myocardial desmin geneexpression is significantly

decreased upon IA therapy

Kallwellis-Operaet al. 115

Not specified Prospective uncontrolled(n�3) 6 mo

Immunoadsorption CHF (NYHA III and IV)secondary due to

chronic iDCM with EF�35%

Improvement in endothelialfunction and significantdecrease of circulating

microparticles

Bulut et al. 116

Not specified Prospective randomizeddoubled blinded Not

specified (n�20)

Intravenousimmunoglobulins

chronic symptomaticCHF and LVEF of

�40%

Antiinflammatory effect oncytokine level which

correlates to improved LVEFupon immunoglobulin

treatment

Gullestad et al.120

Not specified Prospective double blindedplacebo controlled (n�20)

24 wk

Intravenousimmunoglobulins

Reduction of MIP-1alpha,MIP-1beta and IL-8 proteinlevels correlates with LVEF

Damas et al.129

Beta1-AR-auto-antibody Prospective randomizedplabeo controlled (CAD

n�21) (DCM�12) 6 mo

Anti-idiotypicantibodies

CHF (NYHA functionalclass II/III)

Improvement of cardiacfunction by intravenous

immunoglobulins is not due toneutralization of beta1AR

autoantibodies

Larsson et al.130

DCM indicates dilated cardiomyopathy; IA, immunoadsorption; CAD, coronary artery disease; ICM, ischemic cardiomyopathy; IL, interleukin; CHF, congestive heartfailure; EF, ejection fraction; MIP, monocyte inhibitory protein.




extracellular surface markers from the parental cells.117 Ele-vated levels of eMP are found in patients with heart failure.118

Bulut et al reported reduced levels of circulating eMP inDCM patients after IA treatment. However, a causal relation-ship between IA treatment and decreased eMP levels was notproven in this study and the authors therefore cannot rule outconfounding (and yet unknown) factors that might havecaused the observed changes in endothelial function afterIA.116 Taken together IA therapy holds the potential forclinical care of DCM or heart failure, yet larger controlledtrials are urgently needed.

Immunoglobulin mixtures as clinically available in IVIGpreparations are composed of diverse immunoactive immu-noglobulins, mostly of IgG subtype, made from humanplasma of a batch of healthy donors.119 In a double-blindedcontrolled study with 40 patients with chronic symptomaticCHF and LVEF of �40% IVIG, but not placebo treatment,changed the balance between inflammatory and anti-inflammatory cytokines in chronic heart failure in humans,which correlated with improved hemodynamic cardiac pa-rameters.120 The potential of IVIG to prevent CHF and toreduce proinflammatory cytokines has been shown in anexperimental model of virus induced autoimmune myocardi-tis in experimental Chagas disease and in human DCM.121–126

IVIG preparations are believed to bind to and neutralizecirculating autoantibodies of diverse specifity.

IVIG consist of natural polyreactive antibodies that exhibitanti-idiotypicity and autoantibody neutralizing capacity.127,128

It has been reported that IVIG treatment has influence oncytokine balance and downregulates monocyte inhibitoryprotein (MIP)-1�, MIP-1�, and IL-8 protein levels in chronicheart failure.120,129 Whether IVIG treatment modulates circu-lating autoantibodies that are related to heart failure is still amatter of speculation because a direct proof for such aninteraction is still lacking. In a prospective randomizedplacebo controlled study by Larsson et al rather questions thehypothesis that IVIG treatment neutralizes or modulatescirculating autoantibodies because beneficial outcome of

IVIG treatment in DCM was not due to neutralization ofanti-�1–AR autoantibodies but was dependent on not yetclearly specified mechanisms.130 However, in other autoim-mune diseases IVIG were capable of neutralizing autoanti-bodies against factor VIII, DNA, platelet, glycoproteins,cardiolipin, and cytoplasmic antineutrophil autoantibod-ies.131–134 Furthermore, animal studies propose that IVIG iscapable of downregulating specific autoreactive B-cells.135

The molecular and cellular mechanism on how IVIG areable to reduce or inhibit autoimmune responses is a matter ofcurrent research. A minor population of IVIG belonging tothe IgG crystallizable fragments with glycans terminating inalpha2,6 sialic acids specifically bind to the lectin dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) onmyeloid associated cells.136 Binding of sFC on DC-SIGNstimulates the very recently discovered DC-SIGN–Th2-pathway.137 The IVIG stimulated DC-SIGN–Th2-pathwayleads to an upregulation of FCgammaRIIB receptors oneffector macrophages. FCgammaRIIB receptors are trans-membrane proteins that play a major role in autoimmunityand infection.138 Activation of FCgammaRIIB increases theactivation threshold for an immune response by inhibiting thefunctions of activating Fcgamma receptors. Further, FCgam-maIIRB is able to inhibit autoreactive B-cells function bydecreasing antibody production. Application of IVIG thusmay inhibit effector macrophages by activation of the DC-SIGN–Th2-pathway that leads to cross-linking FC-gammaRIIB receptors after stimulation with antigen-antibodycomplexes and subsequent proinflammatory cytokine produc-tion. However further experimental studies are needed tofurther elucidate the molecular action pattern of IVIG. Insummary IVIG treatment reflects, besides immunoadsorp-tion, a promising novel therapeutic option in order to treatheart failure with a pathological autoimmune mechanisticcause. However, it needs to be evaluated whether IVIGinfluences circulating autoagressive autoantibodies in heartfailure in any way. Further experimental or clinical data onsuch potential mechanisms are necessary in order to better

Figure 4. Potential mechanisms of inducing an autoimmune response after cardiomyocyte damage with production of patho-genic autoantibodies leading to myocardial dysfunction and potential therapeutic options. �1-AR indicates �1-adrenoceptors;ANT, adenine-nucleotide transporter; HSP, heat-shock protein.




understand a possible interaction between IVIG and autoan-tibodies in heart failure.

Future DirectionsThere should be more focused research on studying the roleof antibodies and autoimmunity in the pathogenesis of notonly myocarditis but also in the development of dilatedcardiomyopathy even its role in postinfarct remodeling.Genetic studies in patients should be performed to identifygenetic association for prevalence of cardiac autoantibodiesand for susceptibility for autoimmune induced cardiomyopa-thy. Immunoadsorption, immunosuppression, and/or immu-nomodulation may be beneficial in some of these patients andcardiac-specific autoantibodies, which are shown to bedisease-specific, could be used as biomarkers for identifyingthem and their relatives at risk. However, to get thesetherapeutic approaches established, more controlled, blinded,multicenter clinical studies are needed.

AcknowledgmentsThe authors thank Theresa Tretter for critically reading the manu-script and thank Simone Fleck for her help in drafting the figure.

Sources of FundingThis work was supported by the Deutsche Forschungsgemeinschaftresearch grants KA 1797/3-1, KA 1797/4-1 to Dr Kaya.

DisclosuresDr Katus developed the troponin T assay and holds a patent on thisassay jointly with Roche Diagnostics. The remaining authors reportno conflicts.

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Autoantibodies in Heart Failure and Cardiac Dysfunction