PROJECT SUMMARY An estimated 3.1 million cases of myocarditis/cardiomyopathy were diagnosed in 2017. Patients with myocarditis are at risk of sudden death from acute heart failure and progression to dilated cardiomyopathy, often necessitating a heart transplant. The incidence and severity of myocarditis is higher in men than women. Additionally, there are no disease-specific therapies to reduce myocarditis. Enteroviruses including coxsackievirus group B3 (CVB3) are a common cause of myocarditis in the US. Viruses are frequently found in endomyocardial biopsies from patients with myocarditis. There is currently no clear understanding of why an enterovirus like CVB3 would target the heart or the mechanism for how a relatively mild viral infection like CVB3 leads to heart failure. Previously it was hypothesized that an overwhelming lytic CVB3 infection damages the heart. It was recently published that the predominant mode of viral egress for CVB3 from cardiomyocytes is in extracellular vesicle-like mitochondrial autophagosomes, and that CVB3 requires mitochondrial fission for viral replication. Interestingly, other viruses that cause myocarditis have been found to use mitochondria to promote viral replication including influenza A, HIV, poliovirus, hepatitis C virus and SARS-CoV-2. The high energy demands of cardiac muscle and abundant mitochondria in the heart provide for the first time an explanation for why such disparate types of viruses, with no obvious cardiac tropism, replicate in the heart. The overall goal of this proposal is to determine whether the CVB3 replicative cycle through mitochondria in cardiomyocytes induces sex differences in cardiac inflammation during myocarditis and to determine whether microRNA and/or protein from mitochondrial-derived extracellular vesicles influence viral replication and inflammation in a sex-specific manner.