Functional Autoantibodies in Myocardial Diseases

Central Illustration

Highlights

• •Clinical need: Autoantibodies in myocardial diseases lack validated clinical use.
• •Main message: Cardiac autoantibodies show diverse, context-dependent effects.
• •Future directions: Validate assays, clarify causality, use high-throughput profiling.

Summary

Myocardial autoimmune responses result in distinctive cardiac diseases and are associated with poor clinical outcomes. Autoantibodies (AAbs) play a crucial role in myocardial humoral autoimmunity by targeting endogenous tissue, or “self-antigens.” Myocardial tissue injury or myocyte disarray caused by AAbs triggers a cascade of defensive immunological responses that may lead to worsening biventricular remodeling. However, over the past decade, the concept of AAbs in myocardial diseases has shifted from viewing AAbs as uniformly deleterious to a more nuanced explanation of AAbs influencing downstream intracellular pathways with multiple possible outcomes. This paradigm shift arose from technological advances such as large language models for accelerated epitope screening and high-throughput protein assays that uncover complex autoimmune cross-reactivity and downstream intracellular signaling pathways. In this contemporary review article, we critically appraise the latest translational data on the role of AAbs in myocardial disease and provide a framework to encourage scientific endeavors in the development of new therapeutics for cardiomyopathies.

Autoimmunity emerges from environmental, behavioral, and genetic traits that force the immune system to generate autoantibody (AAb)–stimulating B cells. Upon stimulation by a subset of CD4+ T cells, and in certain contexts type 2 helper (Th2) cells, B cells differentiate into antibody-secreting plasma cells. After antigen binding, B cells bind and internalize these harmful antigens and present them on major histocompatibility complex II molecules that are subsequently recognized by Th2 cells. Clonal expansion of activated B cells and antibody-secreting plasma cells then leads to secretion of high quantities of AAbs. In general, AAbs exert 6 functions¹: activation of the complement system by binding pathogens,² inactivating highly specific toxins,³ opsonizing pathogens that are prepared for phagocytosis,⁴ inducing cell-mediated immune responses,⁵ recruiting cytotoxic cells,⁶ and triggering granulocytes.¹ A strong AAb imbalance may lead to disruption of normal cardiovascular physiological processes, myocardial fibrosis, and adverse outcomes.

AAbs produced by activated B cells have traditionally been divided into cardiac (organ-specific), noncardiac (non–organ specific), or both as they cross-react with comparable molecular structures.^2,3 However, this classification carries important conceptual limitations. In fact, many well-described targets of AAbs, including the β1-adrenergic receptors, M2 muscarinic receptors, and ion channels, are widely expressed across multiple tissues and therefore cannot be considered strictly organ specific. Moreover, cross-reactivity represents a functional entity rather than a well-defined category. Also, isoform-specific expression patterns further complicate rigid classification. In this review, we will use the traditional framework primarily for descriptive purposes, mainly to align with the historical literature. Where possible, we will emphasize classification based on molecular targets and functional aspects, which may provide a more biochemical perspective on their influence on myocardial diseases.

Historically, non–cardiac specific AAbs, also known as antireceptor AAbs, were considered harmful spectators somehow related to pathogenic cardiovascular events.4, 5, 6, 7, 8 Detection of these non–cardiac specific AAbs in serum relied on conventional assays, such as indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA), which inherently carry limitations of cross-reactivity, low specificity, and subjective interpretation. Consequently, these methodological limitations have hindered a comprehensive understanding of the biological mechanisms through which non–cardiac specific AAbs contribute to myocardial inflammation and fibrosis. In addition, earlier studies were largely cross-sectional, lacking the longitudinal data necessary to identify potential causal relationships in autoantibody patterns (Central Illustration). Currently, AAb detection to support clinical decision-making is far more commonly adopted in subdisciplines such as rheumatology (eg, anti-dsDNA in systemic lupus erythematosus) and nephrology (eg, PLA2R in idiopathic membranous nephropathy) than in cardiovascular medicine.^9,10 Table 1 outlines the most clinically relevant AAbs associated with cardiovascular disease processes. However, to date, there is a lack of reproducible pathological AAb to be used clinically for companion diagnostics in cardiology. Bridging this gap requires prospective studies that implement high-throughput assays to define AAb trajectories and their phenotypical correlations in dilated cardiomyopathy (DCM), arrhythmogenic cardiomyopathy (ACM), and myocarditis, and mechanistic demonstration of their pathogenicity and potential benefits in their removal.

Central Illustration.

Historical Perspective on the Assessment and Functionality of Autoantibodies in Myocardial Diseases

Shifting the autoantibody paradigm from a traditional “all-is-harmful” approach toward a more nuanced avenue as they may affect cell-signaling pathways, simultaneously with the development of assays for antibody detection leveraging an increased diagnostic power. Ab = antibody; Ag = antigen; CV = cardiovascular; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; LLMs = large language models; PhIP-Seq = Phage ImmunoPrecipitation Sequencing; SPMs = specialized pro-resolving mediators; Th2 = T helper 2 cells; TGF-β = tissue growth factor beta; TNF-α = tissue necrosis factor alpha; Treg = regulatory T cells.

Table 1.

Contemporary Overview of the Most Important Mechanistic Studies on Autoantibodies in Different Myocardial Phenotypes

	Target protein	Study (Year)	Predominant Phenotype	Methodology Strength; Assay Type; AAb Detection	Key Outcomes
Cardiac-specific AAb
Anti-desmosomal	Desmoglein-2, desmocollin-2, N-cadherin proteins	Chatterjee et al75 (2018)	ACM (ARVC), myocarditis	Moderate; ELISA; presence/absence	In humans: correlation of level of anti-DSG2 antibodies and the burden of premature ventricular contractions (r = 0.70); in vitro: AAbs caused gap junction dysfunction, a common feature of ARVC
Anti-desmoglein-2	Desmoglein-2 protein	Walker et al77 (2023) Giordani et al79 (2024) Borowiec et al80 (2024)	ACM (ARVC), myocarditis ACM (ARVC), DCM, myocarditis ACM (ARVC)	Weak; immunoblot analysis; presence/absence Moderate; ELISA; presence/absence Moderate; ELISA; presence/absence	Strong correlation with ventricular arrhythmias (r = 0.841, P = 0.007) but not total number of ectopic beats (r = 0.383, P = 0.313) of anti-desmoglein-2 AAbs. Yet, AAbs not specific to the disease 56% of ARVC patients, and 48% of DCM/myocarditis patients tested positive for anti-desmoglein-2 AAbs Anti-desmoglein-2 antibody titer was significantly higher in ARVC compared to other right ventricular diseases, and was prognostic for death or HTx
AIDA and anti-α- myosin heavy chain	Intercalated disk and α- and β-myosin (muscle cross-reactivity) heavy chain	Caforio et al78 (2020)	ACM (ARVC), DCM	Weak; indirect immunofluorescence; presence/absence	Anti-intercalated disk and anti-α- and β-myosin heavy chain AAbs where present in the majority (85%) of familial pedigrees and in 45% of sporadic ARVC pedigrees
AIDA and “AHA”	Intercalated disk and “anti-heart”	Caforio et al41 (2021) Caforio et al43 (2021) Baritussio et al100 (2022) Caforio et al101 (2025)	Systemic sclerosis with cardiac involvement Cardiac sarcoidosis Myocarditis Inflammatory CMP	Weak; indirect immunofluorescence; presence/absence Weak; indirect immunofluorescence; presence/absence Weak; indirect immunofluorescence; presence/absence NA	Organ-specific AHA and AIDA at a higher frequency in SSc than in a large control cohort of other non-immune-mediated cardiac diseases, including coronary artery disease, and in normal subjects and is associated with worse prognosis AIDA and “AHA” frequencies were higher in sarcoidosis with cardiac involvement (86%; 62%) than in sarcoidosis without cardiac involvement (0%; 0%) High-titer organ-specific AHA and ANA were independent predictors of death and HTx Serum “high-titer AHA” and ANA were independent predictors of response to immunosuppressive agents in inflammatory CMP patients
Anti-N-cadherin	Desmoglein-2 and N-cadherin	Yeruva et al82 (2023)	ACM	Moderate; ELISA; presence/absence	AAb with catalytic properties and cleavage of ICD adhesion molecules DSG2 and N-cadherin; Activation of p38MAPK contributes to a loss of desmosomal adhesion
Non–cardiac-specific AAb
IgG3 subclass	β1AR	Mohan et al35 (2021) Nagatomo et al34 (2017) Nagatomo et al33 (2016)	DCM DCM DCM	Moderate; ELISA; AAb receptor modulation Moderate; ELISA; AAb presence/absence Moderate; ELISA; AAb presence/absence	IgG3 (+) autoantibody biases beta-blocker toward G-protein coupling while impairing agonist-mediated G-protein activation but promoting G-protein-independent ERK activation Advanced HF group (NYHA functional class III or IV, 81% beta-blocker Rx) lower incidence of the composite endpoint of all-cause death, cardiac transplantation, and hospitalization due to heart failure. IgG3-β1AR-AAb, not total IgG, was associated with more favorable outcomes in advanced HF patients of 96% treated with beta-blockers.
Anti-M2	M2-muscarinic Ach receptor	Baba et al7 (2004) Ma et al37 (2021) Ma et al40 (2019)	DCM DCM DCM	Moderate; ELISA; AAb presence/absence Moderate; ELISA; AAb presence/absence Moderate; ELISA; AAb presence/absence	Increased atrial ectopia and AF burden in DCM patients with abundancy of anti-M2-AAb No differences in all-cause mortality between anti-M2-R (+) and anti-M2-R (−) PPCM patients. Less rehospitalization for HF in anti-M2-R (−) group. PPCM patients M2-R (+) have worse prognosis (OR = 4.7, 95% CI: 1.8-12.2, P = 0.002).
Anti- Kir3.4	Kir3.4 ion channel	Maguy et al67 (2023)	AF	Strong; PEPperCHIP custom peptide microarrays; only detection of Kir3.4 subunit	Kir3.4 autoantibodies significantly reduced atrial refractory period and had 2.8-fold increased susceptibility to AF in mice.
Anti-NaV1.5	NaV1.5 ion channel	Korkmaz et al74 (2013)	SA block/AV block	Moderate; ELISA; presence/absence	More episodes of third-degree atrioventricular and sinoatrial block in every NaV1.5-immunized animal, but not in controls
Cardiac and skeletal muscle cross-reactive AAb
cMyBP-C	cMyBP-C	Lynch et al45 (2017)	Ischemic heart disease	Moderate; ELISA; AAb presence/absence	Increased cMyBP-C-AAb production secondary to myocardial injury
S2-MYH6 and FL-MYH6	α-myosin	Sousa et al47 (2019)	Cardiovascular disease	Moderate; fluid-phase radioimmunoprecipitation; presence/absence	Positivity for ≥2 AAb was associated with increased risk of future CVD events in patients with HbA1c >9% (4/6; HR: 16.1 [95% CI: 3.0-88.2])

AAb = autoantibody; AF = atrial fibrillation; AHA = antiheart antibody; AIDA = anti-intercalated disk antibody; ANA = antinuclear antibody; ARVC = arrhythmogenic rights ventricular cardiomyopathy; AV = atrioventricular; CMP = cardiomyopathy; CVD = cardiovascular disease; DCM = dilated cardiomyopathy; DMT1 = diabetes mellitus type 1; DSC2 = desmoglein-2; DSG2 = desmoglein-2; ELISA = enzyme-linked immunosorbent assay; HbA1c = hemoglobin A1c; HF = heart failure; ICD = intercalated disc; Rx = therapy; SA = sinoatrial.

AAbs are frequently detected in patients with myocardial disease, although their prevalence varies depending the underlying condition, disease stage, and detection method applied. A recent meta-analysis from 38 studies shows a significantly higher prevalence of AAbs in DCM, particularly those targeting β1-adrenergic receptors (OR = 4.96), M2 muscarinic receptors (OR = 4.07) and myosin (OR = 12.26).¹¹ However, reported prevalence rates remain heterogeneous across studies because of differences in assay techniques, population characteristics, and definitions of seropositivity.

The way we approach inflammatory myocardial diseases has also changed tremendously in the past decade. There is a growing appreciation that not all B cells and their AAbs feature pathogenic effects. A subset of regulatory B cells produces the anti-inflammatory cytokine IL-10, which may help reduce inflammation and attenuate reverse cardiac remodeling.¹² Additionally, protective effects of AAbs toward chemokines have been described in patients after SARS-CoV-2 infection.¹³ Also, a historical yet relevant myocarditis treatment trial showed improved cardiac function in patients with higher IgG serum levels.¹⁴

This contemporary review discusses recent advances in our understanding of the complexities of AAbs in myocardial diseases and their downstream responses, which may offer unique diagnostic and therapeutic opportunities in managing specific cardiomyopathies.

Autoantibodies Targeting β1-Adrenergic Receptors

DCM is characterized by left ventricular (LV) dilation and systolic dysfunction in the absence of coronary artery disease.^15,16 AAbs linked to DCM are categorized as either G-protein coupled receptor (GPCR) or non-GPCR–targeting. GPCR-targeting AAbs can adhere to subtypes β1-adrenergic receptors (β1AR), α1-adrenergic receptors, M2 muscarinic receptors, endothelin-1 receptors, or angiotensin II type 1 receptors and disrupt normal physiological processes.17, 18, 19, 20, 21, 22 In healthy individuals, catecholamines bind to β1ARs, activating G-protein signaling and downstream adenylate cyclase, thereby initiating the cyclic adenosine monophosphate–protein kinase A pathway.²⁰ Yet, in patients with DCM, AAbs can target the extracellular domains of β1ARs and uncouple downstream intracellular processes.^22,23 Indeed, in a large cohort of heart failure (HF) patients, a higher burden of β1ARs-AAbs was independently associated with more HF hospitalizations.²⁴ In fact, the loss of cardiomyocytes' structural integrity causes β1AR downregulation and receptor desensitization, thereby promoting negative myocardial remodeling.²⁵ Mechanistically, myocardial stressors prompt B-lymphocytes to differentiate into antibody-secreting plasma cells, producing β1AR-AAbs that alter intracellular downstream signaling pathways.26, 27, 28, 29 Neutralization of anti-β1AR autoantibodies using the cyclic peptide COR-1 ameliorated cardiac dysfunction and prevented myocardial damage in a rat model of autoimmune cardiomyopathy and, in a phase I clinical trial, COR-1 was well tolerated and exhibited favorable pharmacokinetics.³⁰

β1AR AAbs are part of the IgG class of immunoglobulins, which can be further subclassified into IgG1, 2, 3, and 4.³¹ The most prevalent immunoglobulin in DCM is the anti-cardiac IgG3 (IgG3AAb).³² Recent observations in DCM patients harboring the IgG3 subclass β1AR-AAbs showed greater improvement of LV ejection fraction and better clinical outcomes after onset of guideline-directed medical therapy.^33,34 Mechanistically, anti-β1AR IgG3AAbs alter downstream intracellular pathways by weakening the β1AR response through GPCRs in the presence of an agonist (eg, dobutamine) through increased recruitment of β-arrestin (referred to as receptor uncoupling). Conversely, adding β1AR antagonists to patient samples harboring anti-β1AR IgG3AAbs may enhance cAMP-dependent cell signaling (referred to as receptor coupling).³⁵ These results support the intriguing idea of Ig-subclass members causing differential downstream effects on β1AR signaling.

Autoantibodies Targeting Muscarinic Receptors

M2 muscarinic receptor AAbs (M2-AAbs) can attenuate the parasympathetic nervous system.⁷ This may generate positive chronotropic effects through either blockage of the L-type Ca2+ current, enhancement of the cGMP pathway, or accelerating the acetylcholine-regulated potassium outflow current.^7,36 However, compared to the effects of anti-β1AR IgG3Abs in DCM patients, the role of M2-AAbs in myocardial diseases is less recognized.^24,37 The coexistence of anti-β1AR and M2 AAbs was first described in Graves hyperthyroidism, where it was associated with a higher incidence of atrial fibrillation (AF).³⁸ Moreover, in patients with peripartum cardiomyopathy (PPCM), an immune autoreactivity can contribute to the development of DCM. In fact, PPCM patients demonstrate more circulating AAbs compared to healthy pregnant women.³⁹ As such, affected patients harboring both β1AR AAbs and M2-AAbs show higher serum N-terminal pro–B-type natriuretic peptide levels, worse LV remodeling, and greater HF symptom burden than those with just one type of AAb.³⁹ Additionally, when geometric mean serum M2-AAb titers in newly diagnosed PPCM patients were followed over time in a single-arm, prospective, observational study, findings suggested that PPCM patients without serum M2-AAbs can better tolerate guideline-directed medical therapy uptitration and end up having higher dosages, have more pronounced positive LV remodeling, and demonstrate lower HF rehospitalization rates.⁴⁰ Whether this humoral autoimmune response in PPCM patients is triggered by an inherent disarray of the myocardial contractile apparatus (eg, PPCM patients carrying a truncating TTN gene variant) is yet to be determined.

Autoantibodies Targeting Cardiac Myosin-Heavy Chain

Cardiac myosin-heavy chain (CMyH) is part of the myocyte's contractile apparatus and converts chemical energy, through ATP hydrolysis, into mechanical energy. Not all antimyosin AAbs are organ specific. In fact, the majority of them found in ischemic, infiltrative, and diabetic patients are often cross-reactive and represent features of an ongoing autoimmune process in the myocardium.41, 42, 43 AAbs specifically targeting CMyH were first described in hearts recovering from acute myocardial infarction.^44,45 Interestingly, patients with coronary artery disease harboring anti-CMyH AAbs have worse LV ejection fraction and higher postinfarct serum levels of natriuretic peptides and cardiac troponin I.⁴⁵ Yet, whether anti-CMyH AAb release after acute myocardial infarction correlates with the magnitude of the infarcted zone or rather reflects myocardial stunning (ie, acute transient contractile dysfunction despite restored perfusion) and/or hibernation (ie, chronic hypoperfusion–associated, causing reversible contractile dysfunction) is unknown.

Recently, a proinflammatory response was detected in a case series of myocarditis patients with underlying myocardial storage disease. The majority of patients with Fabry disease showed a higher burden of anti-CMyH AAbs, which positively correlated with the degree of LV wall thickness.⁴⁶ Also, anti-cardiac myosin IgG AAbs featured strong cross-reactivity to the extracellular loops of βAR in experimental autoimmune myocarditis. In human myocarditis, these cross-reactive AAbs promoted a profibrotic transcriptome, which was associated with poor ventricular recovery and outcomes.³

Cardioimmunologic consequences of longstanding hyperglycemia have recently been described. For example, serum AAbs in patients with type 1 diabetes mellitus featuring optimal (≤7.0%) or suboptimal (≥9.0%) glycosylated hemoglobin were compared. Interestingly, type 1 diabetes patients with poor glycemic control showed higher levels of AAbs targeting subunits of CMyH (eg, sub-fragment 1-MYH6, sub-fragment 2-MYH6) compared with better controlled type 1 diabetes patients, which persisted after both 1 year and 8 years.⁴⁷ Also, Cox proportional hazard analysis showed that cardiovascular disease risk increased 16.1 times when patient serum was positive for 2 or more AAbs targeting subunits of CMyH. Concordantly, lower anti-CMyH AAbs levels significantly decreased the cumulative incidence of cardiovascular disease.⁴⁷ The research group postulated that chronic hyperglycemia (defined as hemoglobin A1c ≥9%) may cause subclinical myocardial injury by CMyH-AAbs targeting specific cardiac antigens.⁴⁷ Moreover, a post hoc cardiac magnetic resonance imaging study demonstrated subclinical myocardial dysfunction (defined as LV dilatation, increased LV mass, and lower ejection fraction) in patients with higher CMyH-AAb burden.⁴⁸

Autoantibodies Targeting Cardiac Troponin I

Troponin is a heterotrimeric protein involved in the excitation-contraction coupling of striated muscle cells. The largest subunit, troponin T, binds tropomyosin of the myofibrillar thin filament, whereas troponin I prevents actin-myosin coupling during muscle cell relaxation. Although both troponin T and troponin I are found in skeletal muscle cells as well as cardiac muscle cells, modern high-sensitivity assays can reliably detect the subtypes without cross-reactivity.^49,50 Recent evidence shows that AAbs targeting cardiac troponin I (cTnI) may lead to cardiomyopathy. Preclinical data showed that programmed cell death-1 (PD-1) knockout mice who developed autoimmune DCM carried significantly higher anti-cTnI AAb titers.⁵¹ Similarly, when wild-type mice were administered monoclonal antibodies against cTnI, they developed DCM through chronic stimulation of Ca2+ influx in cardiomyocytes.^51,52 Also, induction of an immune response against cTnI caused severe myocardial inflammation in mice.⁵³ More recently, a murine post–myocardial infarction HF model showed that cTnI AAbs led to worse cardiac remodeling and worse prognosis, while prompt administration of rapamycin, an immunosuppressant, decreased cTnI AAb titers and ameliorated myocardial inflammation and prognosis.⁵⁴

Furthermore, in a retrospective analysis of 272 DCM and 185 ischemic cardiomyopathy patients, nearly one-third demonstrated positive (≥1:40) IgG AAbs against cTnI. A subgroup of post–myocardial infarction patients (n = 10) featuring high (≥1:160) IgG AAbs targeting cTnI showed less reverse (favorable) remodeling on cardiac magnetic resonance imaging after 6 to 9 months compared to patients without cTnI AAbs.⁵⁵ Also, patients with myocarditis fulfilling the Dallas criteria showed significantly higher cTnI AAb titers compared to healthy volunteers.⁵⁶

However, these circulating autoantibodies may influence serum cardiac troponin detection by high-sensitivity assays and falsely misguide clinical decision-making. In addition to AAb interference, recent data suggest that the molecular form of cTnT may also influence diagnostic interpretation, as measurement of intact or mildly truncated (ie, “long”) cTnT improves discrimination between myocardial infarction and nonischemic “total” troponin elevations. Therefore, this novel assay may reliably rule out myocardial infarction at low concentrations and enhance diagnostic accuracy, particularly for type 1 (ischemic) myocardial infarction.⁵⁷ As such, endogenous autoantibodies (ie, heterophile antibodies) or macrotroponin complexes (ie, cardiac troponin bound to one or more autoantibodies, forming large macrocomplexes) may lead to false-positive results.⁵⁸ Notably, cTnI and cTnT autoantibodies have been detected in up to 10% of healthy individuals without evidence of myocardial injury or myocarditis.⁵⁹

Autoantibodies in Heart Rhythm Disorders and Arrhythmogenic Cardiomyopathy

ACM falls in the realm of inherited cardiac conditions. Currently, 12 genes are linked to the development of ACM. These genes typically affect protein structures integrated into the cardiac myocyte's intercalated disc, such as desmosomal (eg, plakophilin 2 [PKP2], desmoplakin [DSP], plakoglobin [JUP]) and cadherin-type adhesion molecules (eg, desmoglein-2 [DSG2], desmocollin-2 [DSC2]).⁶⁰ Nondesmosomal gene variants encode for gap junction proteins as they orchestrate intercellular electrical transmission. Disruption of gap junction proteins may trigger cardiac conduction delays and/or ventricular arrhythmias.⁶¹ Mechanistically, remodeling of essential intercalated disc protein structures in response to mechanical stress or coexisting infectious or immunologic agents leads to fibrofatty myocardial infiltration and the potential development of life-threatening electrical instability (Figure 1).⁶² The acquired or inherited cardiomyocyte disarray in ACM patients may enable focal autoimmune responses either directly, or secondary to ongoing systemic autoimmune processes. As such, arrhythmogenic AAbs may target ion channels and solid protein structures presumed vital for maintaining intercellular mechanical integrity and/or impede action potentials, which increases the cardiomyocyte's susceptibility for electrical disturbances.

Figure 1.

Pathophysiologic Mechanisms of Different Autoantibodies with Linkage to Phenotypical Features and Outcome

The mechanisms by which various autoantibodies contribute to disease pathology via cell surface receptors such as G-protein coupled receptors or M2 muscarinic receptors, highlighting associations with clinical phenotypes and prognostic outcomes. Key pathways and interactions are depicted, emphasizing the role of autoantibodies in modulating cellular and molecular responses. Biomarkers = N-terminal pro–B-type natriuretic peptide. See text for details. Ab = antibody; AF = atrial fibrillation; B1-R = beta-adrenergic 1-receptor; BCDT = B-cell depletion therapy; CMyH = cardiac myosin heavy chain; DSG-2 = desmoglein-2; EF = ejection fraction; GDMT = guideline-directed medical therapy; HF = heart failure; HSP = heat shock protein; IVIG = intravenous immunoglobulin; LV = left ventricle; LVWT = left ventricular wall thickness; M2R = muscarinic acetylcholine receptor; N-CAD = N-cadherin; PCT = progenitor cell therapy; SCD = sudden cardiac death; VES = ventricular extrasystole.

Autoantibodies and atrial arrhythmias

The role of AAbs in AF is mostly extracted from observational studies. For example, 60% of AF patients in one study seemed to carry circulating AAbs against CMyH.⁶³ Also, DCM patients with AF show a greater abundance of M2-AAbs compared to patients without AF. Along with age and increased levels of atrial natriuretic peptides, the presence of M2-AAbs was found to be the strongest independent predictor of future AF development.^7,64 Also, AAbs that target heat shock proteins (HSPs) may induce persistent or recurrent AF (Figure 1).⁶⁵ In addition, patients with persistent AF who received pulmonary vein isolation demonstrated more circulating anti-HSP70 AAbs compared to patients with paroxysmal AF.⁶⁶ However, whether the abundance of circulating anti-HSP AAbs in AF patients is the cause or a consequence of atrial myocyte damage warrants further investigation.

Recently, patients with unexplained AF showed a higher burden of AAbs targeting the extracellular site of Kir3.4, a GPCR-gated inwardly rectifying potassium channel, compared with age- and sex-matched controls.⁶⁷ The Kir3.4 potassium channel is embedded in atrial tissue and initiates inhibitory postsynaptic potentials, resulting in a negative chronotropic effect (Figure 1).⁶⁸ Accordingly, preclinical models immunized to generate AAbs targeting the Kir3.4 potassium channel feature impaired atrial refractoriness, thus more active re-entry circuits, in the absence of a structural (eg, fibrosis) or electrical substrate.⁶⁷

Autoantibodies and atrioventricular-nodal arrhythmias

Congenital heart block can be provoked by AAbs targeting Ro/SSA and La/SSB self-antigens, which are traditionally recognized in the context of autoimmune connective tissue disorders.⁶⁹ Transplacental passage of anti-Ro/SSA and anti-La/SSB AAbs from mother to fetus confers a 2% to 5% increased risk of delivering a newborn child with high-degree atrioventricular block. In previously affected mothers, the prevalence may increase to 15% to 20%.^69,70 Mechanistically, a consistent body of evidence demonstrates that anti-Ro/SSA AAbs interact with cardiac myocyte L-type Ca2+ channels, causing inhibition of the inward L-type calcium current. These channels are selectively expressed in the atrioventricular and sinoatrial nodes.⁷¹ Prolonged exposure to anti-Ro/SSA AAbs, however, forces downregulation of L-type Ca2+ channels as a result of AAb-crosslinking and a subsequent exposure to cytotoxic and inflammatory substances.⁷² Once infiltrative cardiac conditions (eg, cardiac sarcoidosis or amyloidosis) and infectious causes (eg, Lyme disease and Chagas disease) have been excluded, rapid clinical recognition of immune-mediated third-degree atrioventricular block is essential to allow timely initiation of corticosteroid therapy and optimization of patient outcomes. In this context, measurement of serum anti-Ro/SSA autoantibody levels may support clinical decision-making, and given the potential reversibility of the condition, implantation of permanent cardiac devices may be unnecessary. Immunosuppressive therapy is typically administered for 6 months with gradual tapering, although the optimal duration remains uncertain and should be individualized.⁷³

In addition, translational research endeavors show another pathway for AAbs to cause AF by targeting the extracellular alpha subunit of cardiac voltage-gated sodium channel NaV1.5. In the absence of ischemic or structural myocardial disease, rats with anti-NaV1.5 AAbs consistently developed third-degree atrioventricular block. However, the proposed mechanism of AAbs downregulating the NaV1.5 channel followed by a reduction of an inward sodium current warrants further investigation.⁷⁴

Autoantibodies and ventricular arrhythmias

Over the past decade, translational research has centered on discovering novel immunologic triggers targeting vulnerable cardiomyocyte adhesion proteins.75, 76, 77, 78 Although ACM patients regularly present with ventricular arrhythmias, a considerable number of patients remain asymptomatic. Myocardial AAbs specifically targeting the cardiomyocytes' intercalated disc (while neglecting skeletal muscle cells) was first discovered in probands and relatives with arrhythmogenic right ventricular cardiomyopathy (ARVC).⁷⁸ Interestingly, patients with a high abundance of AAbs had worse biventricular remodeling with regard to ejection fraction and LV dimensions. Concordantly, AAbs against DSG2 were specifically found in the sera of both humans with ARVC and a boxer dog model featuring an ARVC phenotype and showed a positive correlation with the burden of premature ventricular complexes. Moreover, these anti-DSG2 AAbs were absent in 2 independent batches of control sera.⁷⁵ Mechanistically, these anti-DSG2 AAbs may disrupt normal gap junction function and lower the threshold for the occurrence of life-threatening arrhythmias. Recently, the abundance of anti-DSG2 AAbs was assessed in ARVC and myocarditis/DCM cohorts. Interestingly, more than half of ARVC patients, and almost half of the DCM/myocarditis cohort had significantly elevated anti-DSG2 AAb titers found in their serum.⁷⁹ Also, compared to non-ACM right ventricular involvement, serum anti-DSG2 AAb titers in traditional ARVC patients were significantly higher and appeared to be prognostic for future death and/or heart transplantation.⁸⁰ Similarly, anti-N-cadherin antibodies may attenuate the formation of adherents and gap junctions.⁸¹ Indeed, in a small ACM patient population, IgG immunostaining of human ventricular tissue specimens demonstrated specific AAbs targeting N-cadherin protein structures. Furthermore, destabilization of the cadherin-mediated cohesion with subsequent loss of cell-cell adhesion was caused by catalytic AAbs (eg, proteases) that are able to cleave DSG2 and N-cadherin.⁸²

In addition, Anti-Ro/SSA AAbs have also been linked to acquired long-QT syndrome and/or torsades de pointes. A collection of case series from newborns and children with anti-Ro/SAA–positive mothers and AAb-positive adults consistently show that individuals with higher anti-Ro/SSA AAbs feature longer QTc times.⁷² Mechanistically, circulating anti-Ro/SAA AAbs can bind hERG potassium channels and inhibit an outward potassium current. Physiologically, this results in a delayed restoration of the steady-state membrane potentials along with a prolonged repolarization in myocytes.⁸³

New Insights on Autoantibodies in Myocarditis

Myocardial autoantibodies are increasingly recognized as relevant contributors to the pathophysiology of myocarditis, especially in linking acute inflammatory processes to chronic myocardial damage. Virus-induced AAb responses can trigger autoimmune mechanisms, a concept supported by experimental studies demonstrating that infection-driven immune activation may cause sustained myocardial injury and progression to DCM.^84,85

Preclinical data implementing high-throughput phage libraries has created novel insights on the production of AAbs in viral myocarditis. As such, mice incubated with coxsackievirus B3 demonstrated AAbs against several unknown antigens like COA4 or PIK3AP1. Interestingly, the AAb reactivity to COA4 was consistent in both mouse and human libraries. These findings may accelerate the search for novel diagnostic biomarkers for the detection or exclusion of coxsackievirus B3–mediated viral myocarditis.⁸⁶

Different IgG AAb profiles have been identified in patients with Chagas dilated cardiomyopathy. A Trypanosoma cruzi proteome library was built and incubated against infected serum samples using the phage display immunoprecipitation sequencing (PhIP-Seq) technology. PhIP-Seq is a platform designed to perform an unbiased assessment of antibodies potentially linked to specific conditions by leveraging a high-throughput and highly sensitive assessment of specific antigen-antibody interactions. Intriguingly, out of 1,870 peptides in the T cruzi proteome, only the most relevant peptides showed an adjacent interaction to IgG (eg, a flagellar attachment zone protein, basal body protein, cytoskeleton surface protein, nucleoporin, and the surface protein ToIT). By combining these 5 dominant peptides, the research team created a classifier model for T cruzi infection with 97% accuracy.⁸⁷

Recent attention on myocarditis associated with mRNA vaccines against SARS-CoV-2 have accelerated mechanistic AAb detection studies. Interestingly, young male patients with biopsy-proven myocarditis showed endogenous AAbs against IL-1RA and a hyperphosphorylated IL-1RA isoform after receiving a SARS-CoV-2 mRNA vaccine.⁸⁸ However, in children with either vaccine-associated or SARS-CoV-2–associated myocarditis, there was no significant difference in the degree of myocardial tissue binding by AAbs compared to adults with inflammatory cardiomyopathy, healthy vaccinated adults, or healthy children.⁸⁹ Also, in children and adolescents with mRNA vaccine-associated myocarditis, no differences in AAb and T-cell autoimmune responses were found when compared with asymptomatic, age-matched, vaccinated control subjects.⁹⁰ However, translational data elucidating how AAbs might be implemented in clinical practice for the diagnosis or prognostication of (peri-)myocarditis are currently lacking, mainly because of the absence of supportive longitudinal data from large, well-phenotyped patient cohorts systematically evaluated using high-throughput assays.

Autoantibodies Linking Myocardial Involvement in Systemic Autoimmune Rheumatic Diseases

Systemic autoimmune rheumatic diseases (SARDs) hold heterogeneous clinical features with potential myocardium and pericardium tissue involvement.^41,43 Myocardial injury in this context is driven by a complex interplay of immune-mediated mechanisms, including immune-complex deposition, complement activation, endothelial dysfunction, and microvascular inflammation. In fact, persistent inflammatory signaling induces cytokine-derived fibroblast formation, leading to myocardial collagen deposition and subsequent myocardial fibrosis.⁹¹ Although the chronic inflammation seen in autoimmune diseases often exhibits a latent and subclinical cardiac phenotype, there is a clear link with increased cardiovascular morbidity and mortality.⁹² In contrast to premature AAb detection models in the diagnosis of inflammatory myocardial disease, more advanced AAb assays are currently applied in clinical practice to diagnose SARD. Indeed, AAb detection is currently embedded in the diagnosis of specific SARD phenotypes (eg, anti-Sm and dsDNA AAbs in systemic lupus erythematosus, anti-Jo-1 AAbs in polymyositis/dermatomyositis, and anti-topoisomerase I and RNA polymerase III in systemic sclerosis) and leverages high diagnostic specificity, even in a preclinical setting.⁹³ Beyond diagnostic value, emerging evidence suggests that AAbs against anticitrullinated proteins in rheumatoid factor–positive rheumatoid arthritis are highly correlated with a greater risk of ischemic and HF events.⁹² As such, autopsy studies show an increased histological staining for citrullination at the level of the myocardial interstitium in patients with rheumatoid arthritis compared with other autoimmune diseases.⁹⁴ Furthermore, in systemic lupus erythematosus, endothelial dysfunction and vascular stiffness may arise from oxidative stress, proinflammatory cytokine activation, and AAb-mediated injury. Autoantibodies such as antiphospholipid AAbs further contribute by inducing a proinflammatory endothelial phenotype while facilitating microvascular thrombosis. In systemic sclerosis, AAbs may induce early microvascular injury, leading to progressive vasculopathy and vascular stiffness and eventually causing tissue hypoxia and fibrosis.⁹⁵

Beyond classic autoimmune diseases, the role of AAbs in myocardial injury has also been highlighted in other clinical contexts. Since the implementation of targeted immune checkpoint inhibitors as a cancer-related therapy, secondary immune-related adverse events have been increasingly studied with the coexistence of AAbs. In particular, the phenotypical triad of myositis, myasthenia gravis, and myocarditis tends to show concordant AAb involvement, with notably elevated levels of anti–Mi-2, anti-PM/Scl, and anti-SRP AAbs.⁹⁶

Therapeutic Reduction of Cardiac Autoantibodies: A Phenotype-Based Approach

Although increasing evidence supports the reduction of serum AAbs across the spectrum of cardiovascular diseases, therapeutic strategies to lower the burden of cardiac AAbs in serum have not yet been widely accepted in clinical practice. Table 2 summarizes the different therapeutic approaches to remove specific deleterious AAbs, for which several clinical trials are ongoing. Indeed, AAb reduction therapies largely remain a clinical case-by-case decision and may be used as rescue therapy or as a bridge to a ventricular assist device or heart transplantation.

Table 2.

Overview of Therapeutic Approaches to Remove AAbs

AAb Removal Technique	Mechanism of Action	Research Output	Phenotype	Outcomes
Immunoadsorption	Therapeutic plasma exchange with an immunoadsorption column containing protein A which binds IgG3 and selectively removes it from plasma	•Small-scale, single-arm, longitudinal trial in DCM102 •Multicenter, randomized, parallel-group comparative study103 •Retrospective analysis in β1ARs-AAbs positive patients listed for heart tx5 •Multicenter, double-blind, RCT (NCT00558584) •Multicenter, randomized, double-blind trial104	•DCM •DCM •DCM •DCM •DCM	•Better QoL, NYHA class, and positive LV remodeling up to 6 mo after treatment20,105, 106, 107, 108 •Improvement of symptoms, VO2 max, 6-minute walking distance after 3 mo; Improvement LVEF in patients with higher AAbs levels (subgroup analysis)103 •Significantly prolonged survival rate after 5 y •Ongoing trial •Completed
Intravenous immunoglobulin G	In vivo treatment of IVIG or usage of active binding and direct neutralizing agents (eg, peptides, aptamers)	•Randomized, placebo-controlled study109	•DCM and parvovirus-associated myocarditis	•No significant improvement of ventricular function or functional capacity; potential benefit in patients with high viral load (>500 copies/μg DNA), miRNA patterns of active viral replication or coinfection of human herpesvirus?109,110
B cell depletion therapy	Disrupting autoimmune B cell production	•Preclinical: Anti-CD20 monoclonal antibody (eg, rituximab)111 •Proof-of-concept study anti-CD20 monoclonal antibody (eg, rituximab)112 •Case series of SSc patients receiving rituximab add-on therapy113	•Mice with HCM phenotype •DCM patients with EMB-proven anti-CD20 B-lymphocyte infiltration •SSc patients with primary heart involvement (signs of myocardial injury based on biomarkers and MRI)	•In vivo improvement of cardiac function reduction of myocardial hypertrophy and fibrosis111 •Significant improvement of LV ejection fraction, LV end-diastolic diameters and cardiac biomarkers 4 wk after treatment in steroids/azathioprine refractory patients112 •Normalization of cTnI and decrease of T2 (not T1) mapping time on cardiac MRI; mixed response on ventricular arrhythmia burden113

AAb = autoantibody; CDC = cardiosphere-derived cells; CSC = cardiac stem cell; cTnI = cardiac troponin I; DCM = dilated cardiomyopathy; EMB = endomyocardial biopsy; HCM = hypertrophic cardiomyopathy; HFpEF = heart failure with preserved ejection fraction; IVIG = intravenous immunoglobulin G; MCS = mesenchymal stem cells; MRI = magnetic resonance imaging; LV = left ventricle; LVEF = left ventricular ejection fraction; SSc = systemic sclerosis; QoL = quality of life; RCT = randomized controlled trial; tx = transplantation.

Challenges of AAbs Predicting Disease Onset and Outcome

The majority of cardiac phenotypes feature a unique AAb blueprint, although there is some overlap for abundant AAb subtypes (Figure 2). However, there are well-known challenges that hamper broad clinical implementation of these blueprints. First, AAb-subclasses feature a wide variety of characteristics. The complexity of different cellular interactions following autoimmune processes challenges the discernment of their true pathogenic features. Second, it remains challenging to divide AAbs into either disease-causing entities or a component of a complex framework of counter-regulatory mechanisms in cardioimmunologic disease. In fact, the current literature makes it difficult to distinguish whether AAbs play a causal role in disease pathogenesis or merely represent an associated epiphenomenon. Accordingly, semiquantification strategies for AAbs through immunoassays (eg, ELISA, hemagglutination inhibition [HAI], and the complement fixation test [CFT]) are often irreproducible, insensitive, and not well standardized, with significant cross-reactivity between immune targets, epitopes, and different parts of the antibody (eg, Fc region) undervalued.³ This may either lead to inconsistent results of AAb presence/absence or create depthless ideas on how AAbs interact with their target receptors, not to mention the downstream cell-signaling actions they may provoke.

Figure 2.

Specific Autoantibodies Related to Distinctive Cardiac Phenotypes

The relationship between specific autoantibodies and various cardiac conditions, including dilated cardiomyopathy, peripartum cardiomyopathy, diabetic cardiomyopathy, cardiovascular disease (CVD), arrhythmogenic cardiomyopathy, and conduction disorders. Highlights known autoantibodies implicated in myocardial diseases and heart failure. Ab = antibody; CMyH = cardiac myosin heavy chain; DSC-2 = desmocollin-2; DSG-2 = desmoglein-2; HSP = heat shock protein.

A promising accelerator in the field is the creation of extensive AAb repertoires using libraries of distinct human peptides, such as Phage ImmunoPrecipitation Sequencing (PhIP-Seq). Applying a T7 bacteriophage displayed peptide system facilitates the search of AAbs to a wide range of autoantigens while screening large libraries that contain peptides covering all open reading frames.⁹⁷ Rapid extracellular antigen profiling (REAP) is another high-throughput technology used to identify AAbs that target the human exoproteome; it utilizes genetically barcoded yeast to display thousands of extracellular proteins to detect functional AAb by converting antibody binding into quantifiable sequencing data.⁹⁸ Recently, large language models (LLMs) have been applied to predict antibody epitope overlap, using both sequence-based thresholds and advanced antibody language models.⁹⁹ These approaches, including AbLang-RBD and AbLang-PDB, accurately identified overlapping epitopes across diverse antibodies, revealed the important contribution of light-chain features, and outperformed existing sequence- and structure-based methods. Additionally, these models were then externally validated on therapeutically relevant antibodies, such as HIV-1 broadly neutralizing antibodies, and showed promise for large-scale antibody screening and candidate prioritization for diagnostic testing.⁹⁹ Despite the diagnostic utility of AAb detection in fields like rheumatology and nephrology, developing similar tests for myocardial phenotypes remains challenging. New modalities of AAb detection should first be validated against gold standards represented by clinically validated autoimmune serology AAb tests. Reconsidering the one-sided harmful characteristics of AAbs in myocardial disease by implementing high-throughput peptide assays may be the first step in closing this gap.

Conclusions

Mechanistic insights into how cardiac AAbs are crucial initiators of myocardial disease hold the promise of unveiling new diagnostic and therapeutic avenues. Appreciating that not all AAbs in myocardial disease cause pathologic features requires following the paradigm shift of prior and ongoing translational research efforts. Considering the subtility of how AAbs target their receptors and meticulously modify downstream cellular effects, expanding the use of high-throughput techniques is crucial to understanding their effects and advocating for their use in daily clinical practice.

Funding Support and Author Disclosures

Dr Vanhentenrijk is supported by a grant from the Belgian American Educational Foundation (BAEF), a Pfizer Fellowship Grant, and the Myocarditis Foundation Fellowship Award. Dr Liu is supported by the G. Harold & Leila Y. Mathers Charitable Foundation (Mathers2023-CFL). Dr Tang has served as consultant for Cardiol Therapeutics, CardiaTec Biosciences, Alleviant Medical, AstraZeneca, Salubris Biotherapeutics, BioCardia, Tenax Therapeutics, BridgeBio Pharma, and Vasa Therapeutics, and has received honoraria from Springer and Belvoir Media Group. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.