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
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