NRSA Fellowship for Austin Wellette-Hunsucker: The Myosin Super-Relaxed State and Human Dilated Cardiomyopathy

Project Summary/Abstract: Dilated cardiomyopathies are a frequent cause of heart failure worldwide, distinguished by the reduced systolic function and left ventricular dilation. Dilated cardiomyopathy has been linked to various causes, including viral infections, drug abuse, endocrine dysfunction, and genetic mutations. Since systolic dysfunction presents as a symptom of dilated cardiomyopathy early in disease progression, studying the molecular basis of myosin motors that powers contractions can provide important insight into the mechanism of systolic dysfunction and thus test the effects of therapeutics. Therefore, improving the existing therapeutic strategies that primarily mitigate symptoms (e.g., ACE inhibitors and β-blockers) instead of targeting the sarcomere. At the molecular level, force and power generation in cardiac muscle is determined by the number of myosin motors that bind to the actin thin-filament during the systolic rise of calcium. Once myosin motors bind, myosins hydrolyze ATP and undergo conformational steps to generate force. Recently, attention has focused on the myosin biochemical super-relaxed state, an energy-conserving auto-inhibited state of cardiac myosin that determines the proportion of myosin motors available for recruitment upon activation, modulating the magnitude of myosin motors available to generate sarcomere power. The proportion of myosin motors in the super-relaxed state is controlled by regulatory protein phosphorylation, calcium, and sarcomere length. However, the role of the myosin super-relaxed state as a molecular cause of systolic dysfunction in dilated cardiomyopathy has yet to be investigated. This proposal aims to test the overarching hypothesis that depressed contractile force in dilated cardiomyopathy results from increased myosins in the super-relaxed state, augmented by depressed regulatory protein phosphorylation. Additionally, we hypothesize that therapeutics, such as myosin activators, will reduce the proportion of myosin motors in the super-relaxed state at both short and long muscle lengths to restore calcium sensitivity, sarcomere contractility, and muscle length activation. To test these hypotheses, we will utilize fluorescent ATP pulse-chase and Phos-TagTM biochemical techniques to quantify the proportion of myosins in the super-relaxed state, phosphorylation status of regulatory proteins, and the effects of myosin activators at short and long sarcomere lengths. This proposal will provide novel insight into the effects of myosin activators on the super-relax state, demonstrate fundamental aspects of myosin biochemical states, and contribute to understanding the molecular basis of dilated cardiomyopathy.