Myosin structure and function in health and disease - Myosin molecular motors play important roles in many cellular processes, including migration, transport of intracellular contents, and contraction. A variety of heritable diseases owe their origins to defects in the myosin family of molecular motors. One of the most severe examples is inherited familial hypertrophic cardiomyopathy (HCM), which leads to hyper-contractility and impaired relaxation of the heart. Untreated, this hyper-contractility causes significant thickening (hypertrophy) of the walls of the left ventricle of the heart, which can lead to heart rhythm disorders, heart failure, and even sudden death. For most patients, therapeutic interventions for HCM are currently limited to symptomatic relief, which in severe cases requires invasive procedures such as heart muscle reduction surgery, defibrillator placement, or even heart transplantation. HCM results from mutations in various cardiac muscle proteins, with mutations in -cardiac myosin, the motor that drives ventricular contraction, and myosin binding protein C accounting for about 90% of these cases. HCM is not rare, affecting as many as 1 in 500 people. Studies using human -cardiac myosin have shown that HCM mutations induce variable changes in the basic biochemical and biomechanical parameters of the myosin motor such as force production and velocity of moving actin filaments. However, these variable changes do not adequately account for the cardiac hyper-contractility that is a clinical hallmark of HCM. Rather, it has recently been shown that HCM- causing mutations in the myosin motor domain disrupt intramolecular interactions that stabilize a folded-back, off state of myosin. This results in an increase in the number of heads functionally accessible to interact with actin, which in turn may lead to hyper-contractility. In this proposal, the effects of HCM-causing point mutations in different regions of human -cardiac myosin and myosin binding protein C will be explored to determine if disruption of the off-state is a common mechanism driving HCM. A small molecule direct cardiac myosin inhibitor was recently approved to treat a subset of HCM patients. It is thought to work, at least in part, by stabilizing the folded back, off-state. Whether HCM mutations that affect the stability of the folded back state alter the efficacy of the drug will be tested, as variable clinical responses to the drug have been reported. The effects of HCM mutations on the efficacy of two other drugs in clinical trials/development that inhibit myosin in different ways will be compared as well. Finally, a variety of approaches will be employed to determine the effects of both HCM mutations and small molecule myosin inhibitors on the high-resolution structure of the myosin motor domain and the structure of the folded-back state of myosin, including X-ray crystallography and cryo-electron microscopy. FRET probes will be placed on normal and mutant -cardiac myosin to observe the effects of HCM mutations on the transition between the on-and-off states in the presence and absence of the cardiac myosin inhibitors. These studies will provide important insights into the disease pathogenesis of HCM and the efficacy of small molecule myosin inhibitors in treating the underlying molecular pathology.
