Mechanochemistry of myosin mutations that cause cardiomyopathy

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Project Summary Hypertrophic cardiomyopathy (HCM) is a prevalent genetic cardiac disease affecting 1 in every 300-500 people. The disease is characterized by left ventricular hypertrophy, cardiomyocyte disarray, and interstitial fibrosis resulting in impaired diastolic function often with preserved or enhances systolic function. Dilated cardiomyopathy (DCM) has a similar occurrence and is characterized by thinning of one or both ventricular walls producing insufficient systolic function and diminished ejection fraction, hallmarks of a failing heart. Genetic mutations in sarcomere proteins have been identified to be associated with HCM and DCM, with mutations in - cardiac myosin (MYH7) strongly implicated as drivers of both conditions. A widely cited model relating myosin function to disease proposes that HCM arises from myosin mutations that enhance activity yielding hypercontractile myocytes, whereas DCM arises from loss-of-function mutations that diminish activity yielding hypo-contractility. Contractile abnormalities are proposed to be due to MYH7 gene mutations that affect ATPase activity, velocity, force production, the number or availability of motor domains, and thin filament activation. These molecular changes ultimately affect power output in a manner that impacts tissue architecture, electrophysiological signaling, and cardiac performance. Emerging research has provided examples that do not fit clearly into the prevailing model. For example, HCM mutations with decreased activity have been described in molecular assays and at the level of isolated myofibrils. To delineate the mechanisms by which myosin mutations lead to HCM and DCM, it is important to determine how changes in protein sequence lead to changes in activity at both the molecular and ensemble levels. We selected specific HCM and DCM mutations that are predicted to affect the mechanochemistry of the myosin motor. Our goal is to determine the effect of these mutations on myosin activity, and to test whether the HCM gain-of-function and DCM loss-of-function paradigm holds. We will utilize biochemical and biophysical approaches to assess the effect of mutations on the activity of single motors and regulated filament assemblies. Aim 1 will determine the biochemical and mechanical effects of key HCM and DCM mutations in human β-cardiac myosin. We will measure the ensemble kinetics and motility using biochemical and gliding assays. Changes in unitary forces, step-size, and force dependent kinetic steps will be measured using single molecule optical-trapping. Aim 2 will determine the effect of HCM/DCM mutations on heterogenous myosin assembles and thin filament activation. We will examine the effect the regulation of single molecules within a regulated system, and we will construct a myosin nanomachine using DNA origami that mimics cardiac muscle. We will use regulated thin filaments and myofilaments containing defined ratios of WT and mutant myosin. We expect that our approach will result in an unprecedented understanding of the underlying etiology of HCM and DCM.