AimsAutoantibodies against second extracellular loops of β1-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function.Methods and resultsIgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to β1-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed.Conclusions(i) Autoantibodies trigger conformation changes in the β1-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

AimsImmunoadsorption with subsequent immunoglobulin G substitution (IA/IgG) represents a novel therapeutic approach in the treatment of dilated cardiomyopathy (DCM) which leads to the improvement of left ventricular ejection fraction (LVEF). However, response to this therapeutic intervention shows wide inter-individual variability. In this pilot study, we tested the value of clinical, biochemical, and molecular parameters for the prediction of the response of patients with DCM to IA/IgG.Methods and resultsForty DCM patients underwent endomyocardial biopsies (EMBs) before IA/IgG. In eight patients with normal LVEF (controls), EMBs were obtained for clinical reasons. Clinical parameters, negative inotropic activity (NIA) of antibodies on isolated rat cardiomyocytes, and gene expression profiles of EMBs were analysed. Dilated cardiomyopathy patients displaying improvement of LVEF (≥20 relative and ≥5% absolute) 6 months after IA/IgG were considered responders. Compared with non-responders (n = 16), responders (n = 24) displayed shorter disease duration (P = 0.006), smaller LV internal diameter in diastole (P = 0.019), and stronger NIA of antibodies. Antibodies obtained from controls were devoid of NIA. Myocardial gene expression patterns were different in responders and non-responders for genes of oxidative phosphorylation, mitochondrial dysfunction, hypertrophy, and ubiquitin–proteasome pathway. The integration of scores of NIA and expression levels of four genes allowed robust discrimination of responders from non-responders at baseline (BL) [sensitivity of 100% (95% CI 85.8–100%); specificity up to 100% (95% CI 79.4–100%); cut-off value: −0.28] and was superior to scores derived from antibodies, gene expression, or clinical parameters only.ConclusionCombined assessment of NIA of antibodies and gene expression patterns of DCM patients at BL predicts response to IA/IgG therapy and may enable appropriate selection of patients who benefit from this therapeutic intervention.

Dilated cardiomyopathy (DCM) is characterized by contractile dysfunction leading to heart failure. The molecular changes in the human heart associated with this disease have so far mostly been addressed at the gene expression level and only a few studies have analyzed global changes in the myocardial proteome. Therefore, our objective was to investigate the changes in the proteome in patients suffering from inflammatory DCM (iDCM) and chronic viral infection by a comprehensive quantitative approach. Comparative proteomic profiling of endomyocardial biopsies (EMB) from 10 patients with iDCM (left ventricular ejection fraction <40%, symptoms of heart failure) as well as 7 controls with normal left ventricular function and histology was performed by label-free proteome analysis (LC-MS/MS). Mass spectrometric data were analyzed with the Rosetta Elucidator software package. The analysis covered a total of 485 proteins. Among the 174 proteins displaying at least a 1.3-fold change in intensity (p < 0.05), major changes were observed for mitochondrial and cytoskeletal proteins, but also metabolic pathways were affected in iDCM compared to controls. In iDCM patients, we observed decreased levels of mitochondrial proteins involved in oxidative phosphorylation and tricarboxylic acid cycle. Furthermore, deregulation of proteins of carbohydrate metabolism, the actin cytoskeleton, and extracellular matrix remodeling was observed. Proteomic observations were confirmed by gene expression data and immunohistochemistry (e.g. collagen I and VI). This study demonstrates that label-free, mass spectrometry-centered approaches can identify disease dependent alterations in the proteome from small tissue samples such as endomyocardial biopsies. Thus, this technique might allow better disease characterization and may be a valuable tool in potential clinical proteomic studies.

In patients with dilated cardiomyopathy (DCM), cardiac autoantibodies are able to bind with their Fab fragment to epitopes on cardiomyocytes, but thereafter they crosslink through their Fc fragment to cardiac Fc(gamma)-receptor IIa. Polymorphic variability of the Fc(gamma)-receptor IIa is associated with modified affinity of immunoglobin G (IgG) binding and may influence therapeutic effects. In this study, 103 consecutive DCM patients were treated with immunoadsorption (IA) therapy with subsequent IgG substitution (IA/IgG). Echocardiography was performed at baseline and again at 3 and 6 months after IA/IgG. Fc(gamma)-receptor IIa polymorphism R/H131 was genotyped using a nested sequence-specific primer polymerase chain reaction (PCR). Patients with the Fc(gamma)-receptor IIa genotype R/R131 showed significantly greater improvement in left ventricular (LV) function than patients with the R/H131 or H/H131 genotypes did. Irrespective of the Fc(gamma)-receptor polymorphism, patients with shorter disease duration and a more impaired LV function responded with a greater increase in LV ejection fraction (LVEF). Therefore, the Fc(gamma)-receptor polymorphism influences the efficacy of immunomodulatory therapy involving IA/IgG.

Organic cation transporters (OCT1-3 and OCTN1/2) facilitate cardiac uptake of endogenous compounds and numerous drugs. Genetic variants of OCTN2, for example, reduce uptake of carnitine, leading to heart failure. Whether expression and function of OCTs and OCTNs are altered by disease has not been explored in detail. We therefore studied cardiac expression, heart failure-dependent regulation, and affinity to cardiovascular drugs of these transporters. Cardiac transporter mRNA levels were OCTN2>OCT3>OCTN1>OCT1 (OCT2 was not detected). Proteins were localized in vascular structures (OCT3/OCTN2/OCTN1) and cardiomyocytes (OCT1/OCTN1). Functional studies revealed a specific drug-interaction profile with pronounced inhibition of OCT1 function, for example, carvedilol [half maximal inhibitory concentration (IC₅₀), 1.4 μmol/L], diltiazem (IC₅₀, 1.7 μmol/L), or propafenone (IC₅₀, 1.0 μmol/L). With use of the cardiomyopathy model of coxsackievirus-infected mice, Octn2mRNA expression was significantly reduced (56% of controls, 8 days after infection). Accordingly, in endomyocardial biopsy specimens OCTN2 expression was significantly reduced in patients with dilated cardiomyopathy, whereas the expression of OCT1-3 and OCTN1 was not affected. For OCTN2 we observed a significant correlation between expression and left ventricular ejection fraction (r = 0.53, P < 0.0001) and the presence of cardiac CD3⁺ T cells (r = -0.45, P < 0.05), respectively. OCT1, OCT3, OCTN1, and OCTN2 are expressed in the human heart and interact with cardiovascular drugs. OCTN2 expression is selectively reduced in dilated cardiomyopathy patients and predicts the impairment of cardiac function.

IA/IgG therapy induces long-term reduction of negative inotropic antibodies. After 12 months, however, re-increase of negative inotropic antibodies cannot be excluded.

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