PROJECT SUMMARY Alcohol exposure is a key risk factor for abnormal heart development and contributes to congenital heart disease, the leading non-infectious cause of infant mortality. Alcohol-induced impaired heart growth and development in early life can also increase the risk of heart disease later in adulthood. Furthermore, adult cardiac system is also sensitive to alcohol exposure, which is an important but underappreciated risk factor contributing to heart disease. Alcohol exposure is associated with ischemic events, arrhythmias and alterations in cardiac function and structure. These pathological consequences could result from complex actions of alcohol including increased cytotoxicity, oxidative stress and abnormal Ca2+ handling. Inflammatory cytokines produced by alcohol exposure could also contribute to alcohol-induced heart injury. However, mechanisms underlying alcohol- induced heart disease are not fully defined and effective therapies are lacking. Traditionally, studies on alcohol exposure have relied on animal models and cells because of limited availability and growth capacity of human primary cardiomyocytes. However, studies in animal models are time consuming, expensive, and not amenable for high-throughput drug screening, and have limitations due to physiological differences from humans. To complement the studies on animal models and cells, we have recently explored the use of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) as a novel and physiologically relevant model to study alcohol-induced cardiotoxicity. hiPSC-CMs have many features similar to human primary CMs, can be engineered into tissue-like structures and maintained in long-term cultures, and can be adapted for high-throughput platforms. We have demonstrated that exposure of hiPSC-CMs with clinically relevant doses of alcohol can recapitulate pathological events caused by alcohol exposure, including oxidative stress, altered gene expression and cardiac dysfunction as indicated by abnormal Ca2+ handling and contractility. We have adapted hiPSC-CMs into high- throughput formats and established high-throughput assays to detect alcohol-induced cardiotoxicity and screen for cardiac proliferative/protective agents. Using this in vitro human cell model and state-of-the-art high- throughput technologies, we propose to investigate underlying molecular mechanisms of alcohol-induced toxicity in human cardiomyocytes and evaluate potential therapies to mitigate alcohol-induced cytotoxicity. In addition, we plan to investigate the involvement of inflammatory cytokines in alcohol-induced cardiac injury and repair. We expect that our established hiPSC-CM model will help understand the molecular and cellular mechanisms underlying alcohol toxicity in human cardiomyocytes and accelerate the development of targeted and effective therapies.