Sandel, Clayton

Abstract Background Previous studies suggest that autoantibodies against cardiac myosin lead to dilated cardiomyopathy (DCM). Anti-cardiac myosin antibodies cross-react with the beta adrenergic receptor (βAR) and signal cAMP-dependent protein kinase A (PKA) in cardiomyocytes leading to apoptosis, fibrosis, dilated cardiomyopathy and arrhythmias. Purpose To determine if cross-reactive anti-cardiac myosin/anti-βAR autoantibodies which signal cardiomyocytes through PKA might play a role to establish DCM by promoting remodeling, apoptosis, and fibrosis. Methods Forty-one adults with DCM were enrolled <6 months from symptom onset and followed for 12 months. Serum and myocarditis/DCM-derived human mAb were analyzed by ELISA for autoantibodies, and a PKA assay measured anti-HCM/βAR antibody-mediated signaling of cardiomyocytes (ATCC primary heart cell line H9c2). The top 50 genes differentially expressed in the cardiomyocytes treated with sera or human mAb were identified and compared to genes differentially expressed in blood of DCM patients to identify shared disease-specific genes. Results Anti-HCM autoantibodies including autoantibody responses against 32 overlapping synthetic peptides of the S2 fragment of HCM were significantly elevated in patients whose ejection fraction did not improve over 1-year compared to those with improved ejection fraction. The human mAb confirmed our results with HCM, βAR, specific HCM peptides, and PKA signaling. RNA sequencing revealed differentially expressed genes in serum/mAb-treated cardiomyocytes compared to genes identified after RNA sequencing of peripheral blood of patients (n=10) with DCM for >1 year from onset. A primary heart cell line (H9c2-ATCC) treated with myocarditis/DCM patient sera or human mAb revealed differentially expressed genes associated with cardiac hypertrophy and heart failure, and included inflammasome component NLRP3 and complement factor H. Ingenuity Pathway Analyses revealed 27, 7, and 1 differentially expressed genes related to apoptosis, fibrosis, and hypoxia, respectively. Gene expression of CASZ1, a transcription factor important in protection against DCM, was strongly correlated with PKA signaling (r=0.89). The KDM6B gene for lysine demethylase associated with hypoxia and apoptosis pathways and was shared between cardiomyocyte and peripheral blood analysis of DCM patients. Overall, 5 genes were shared in heart failure vs in vitro Ab-treated cardiomyocyte RNA sequencing analysis: CYP4F3, KDM6B, MBOAT7, SMAP2, and DDIT4, which affects phosphorylation of mTOR to promote autophagy and cell death, cardiac hypertrophy and dysfunction. Conclusions Significantly higher responses to cardiac myosin in patients with DCM were related to lack of left ventricular function improvement and to differential expression of genes promoting apoptosis, fibrosis and disease severity. These studies identify autoantibody-directed gene signaling as a potential novel therapeutic target in DCM. Acknowledgement/Funding National Heart, Lung, and Blood Institute, Bethesda, MD, USA

Abstract Background Cardiomyopathy may occur due to viral infections or drug induced heart damage. Cardiac myosin released from damaged heart has been shown to be a damage associated molecular pattern which binds to TLR2 or TLR8 and can act as an adjuvant to induce a strong autoimmune response against the heart. The result is autoimmunity against the heart which can lead to apoptosis, fibrosis and heart failure. Purpose Immune biomarkers of the early stages of heart failure are needed to identify individuals who develop progressive heart failure, do not recover their ejection fraction and may be candidates for immunotherapies. Methods Forty-one patients with myocarditis and heart failure <6 months after onset were followed for 12 months and compared to age matched controls. Peripheral blood mononuclear cells were analyzed by FACS analysis and serum analyzed by ELISA for autoantibodies and cytokines. Statistical analysis was determined by Mann Whitney test. Peripheral blood of 10 patients with dilated cardiomyopathy (DCM) vs 19 healthy controls were analyzed for gene expression by RNA sequencing and pathway analysis using Reactome. Results Autoantibodies against human cardiac myosin and the beta-adrenergic receptor were significantly elevated in our cohort and functionally acted on cardiomyocytes to activate protein kinase A. Concomitantly, a Th17+ immunophenotype was significantly elevated in blood as well as in cardiac biopsies. CD4+IL17+ T cells (p=0.0008) and Th17-promoting cytokines TGF beta (p<0.0001), IL-6 (p<0.0001), IL-23 (p=0.0001), GMCSF (p=0.0336) and GMCSF-secreting CD4+ T cells (p=0.0006) were significantly elevated in blood. A Th17 immunophenotype was significantly associated with heart failure primarily in males (p=0.029). Persistent heart failure (NYHA class III and IV) and non-recovery of left ventricular function were associated with significantly higher percentages of IL17A-producing T cells at baseline, 6 and 12 months after onset, and IL-17A (p=0.019) and elevated Th17-promoting cytokines IL-6 (p=0.0001) and TGF-beta (p=0.0076). Decreased T regulatory immunosuppressive cells were significantly (p=0.0006) decreased and correlated with elevated Th17 cytokines in heart failure. Overrepresentation analysis of differentially expressed genes (adj p<0.05) in blood of patients with DCM >1year were identified using Reactome which revealed significant (FDR = 1.52E-13) enrichment of neutrophil degranulation (48 genes). Conclusion Our study illustrates a strong Th17 signature in more severe heart failure early in disease with elevated anti-cardiac myosin autoantibodies in non-recovery of left ventricular function. We observed a strong correlation with Th17-related neutrophil degranulation pathways in later disease, which may be biomarkers of fibrosis progression and disease severity in patients with heart failure. Cardiomyopathy with a Th17 signature might be treated with preventive immunomodulatory therapies such as anti-IL17A. Acknowledgement/Funding National Heart Lung and Blood Institute, Bethesda, MD, USA