Hypertrophic cardiomyopathy-causing mutations in both the head and lever arm regions of human beta-cardiac myosin disrupt myosin's ability to form a critical regulatory autoinhibited state

Hypertrophic cardiomyopathy (HCM), a genetic disease largely associated with autosomal dominant mutations in sarcomeric proteins expressed in the left ventricle of the human heart, has been postulated to arise due to sarcomeric hypercontractility, leading to eventual hypertrophy and downstream defects including myocyte disarray and cardiac fibrosis. Most of these mutations are found in two proteins: -cardiac myosin and the cardiac isoform of myosin binding protein-C (MyBP-C). Historically, -cardiac myosin mutations were thought to contribute to hypercontractility by enhancing the fundamental motor characteristics of myosin, including its kcat, step size d, actin gliding velocity, and force output. However, it has become increasingly clear that for the majority of mutations, this model does not hold. Instead, a more attractive model has recently gained traction: many -cardiac myosin mutations appear to instead disrupt the ability of myosin to form a folded-back autoinhibited state. This autoinhibited state has long been documented in smooth muscle myosin but has only recently been recognized to be a common feature across the entire conventional myosin class (myosin II). In this dissertation, I describe two separate works providing supporting evidence for the hypothesis that HCM-causing -cardiac myosin mutations primarily function to reduce the autoinhibited state. In the first study, we showed that two mutations in the myosin motor domain, R403Q and R663H, increased activity by reducing the autoinhibited state. These two mutations potentially disrupt the autoinhibited state by interfering with a necessary interface between the two heads of the myosin dimer that is involved in forming the autoinhibited state. Additionally, MyBP-C was shown to stabilize the autoinhibited state, while both R403Q and R663H prevent MyBP-C from increasing autoinhibition, suggesting that mutations may have compounding impacts to increase contractility. In the second study, we investigated HCM-causing mutations in the lever arm region of myosin. The five mutations studied, D778V, L781P, S782N, A797T, and F834L, showed varying impacts on myosin function. Their effects naturally segregated them into two categories: the mutations in the light chain binding regions of the lever arm (A797T and F834L), had clear impacts on the autoinhibited state, indicating that they function much like R403Q and R663H to reduce autoinhibition. Mutations in the N-terminal "pliant" region of the lever arm (D778V, L781P, and S782N), however, showed a unique, novel phenotype: they only affected the autoinhibited state in the presence of actin, but had no effect in the absence of actin. Additionally, they showed varying impacts on myosin's fundamental contractile parameters. Collectively, these studies point to the relevance of the autoinhibited state in the pathogenesis of HCM, but they also suggest that reduction of the autoinhibited state isn't the only mechanism resulting in HCM. However, the general mechanism of increased contractility as a result of HCM mutations does appear to hold, supported by evidence from studies in iPSC-derived cardiomyocytes containing HCM-causing mutations. Regardless, a drug (mavacamten) that results in increases in the autoinhibited state usually results in reversal of the exact molecular defect (when that defect is reduced autoinhibition), and otherwise results in reduced contractility, correcting defects caused by all sarcomeric mutations. Thus, elucidation of the molecular mechanism behind HCM has garnered important insights that have buoyed the development of the first specific treatment for HCM. .

Keywords: Biochemistry.
Advisor: Spudich James A.
Krasnow Mark
Yeh Ellen