Abstract 426: A Novel Method to Study Viral Cardiomyopathy

Abstract

Latent coxsackievirus B3 (CVB3) cardiac infections are implicated in the development of DCM with persistent enteroviral RNA found in up to 66% (30/45) of DCM patient biopsies who are at a 6-fold higher risk of fatality. Current treatments aim at delaying heart failure but none address the viral etiology of the disease. Viral protein presence is thought to disrupt normal cellular signaling leading to tissue dysfunction. The downstream effects of viral perturbations are complex and wide-ranging; especially in proinflammatory contexts seen clinically. Thus, comprehensively understanding the molecular mechanisms of CVB3-mediated disease is key in developing treatments for viral DCM patients. In this study we built a novel in vitro model of chronic CVB3 infection to facilitate high-throughput, systems-level studies of viral cardiomyopathy. Current methods of studying CVB3 rely on animals or animal derived cardiac cells, making large-scale intracellular signaling studies difficult, time intensive, and expensive. To facilitate high-throughput studies we used immortalized human cardiomyocytes to engineer single-cell derived cell lines that express a maturation deficient CVB3 genome. CVB3 RNA expression was validated in each line by two independent methods: gene-specific nested qRT-PCR and single molecule RNA fluorescence in situ hydridization (smFISH). Plaque-assay verified that viral RNA expression did not result in live virus release. Microarray analysis shows that CVB3 expressing cell lines have altered immune cytokine, extracellular matrix protein, and stress-signaling protein expression. Further, differences between CVB3 expressing lines suggest differential responses to viral RNA expression which may identify a set of beneficial adaptations. Future studies will include high-throughput signaling protein activity assays we have developed specifically for phosphatase and kinase activity quantification with the aim of linking immune cytokine signaling dysregulation to pathogenic gene expression. These studies will deepen our knowledge of CVB3-mediated DCM and identify proteins whose targeted modulation could offer new treatment strategies to patients whose current options are either palliative care or heart transplantation.