Project Summary/Abstract Dilated cardiomyopathy (DCM) is the second most common cause of heart failure world-wide, and inherited forms of DCM make up 30% of non-ischemic cases. MYH7, which encodes beta-cardiac myosin (M2β), is one of the more commonly mutated genes and is the molecular motor that powers contraction in ventricular cardiomyocytes. This proposal is focused on examining the structural and functional impact of DCM mutations in human M2β, with an overall goal of determining molecular mechanisms of contractile defects and developing a foundation for therapeutic strategies. The force, velocity, and power generating capacity of muscle is related to the ability to recruit myosin molecules in the thick filament to interact with actin in the thin filament of the muscle sarcomere. The recruited myosin molecules generate force by utilizing a conserved ATPase cycle in which myosin generates a power stroke while interacting with actin. Cardiac myosin can exist in the auto-inhibited state with slow ATP turnover (super relaxed state, SRX) in which head-head and head-tail interactions prevent it from interacting with actin (interacting heads motif, IHM) or the uninhibited state (disordered relaxed state, DRX) that is readily available to produce force. The recruited myosin also impacts the calcium sensitivity of the myofilaments because myosin binding cooperatively activates the actin thin filament. We will test the central hypothesis that DCM mutations impair systolic contraction in the heart by altering the intrinsic force producing ability of individual cardiac myosin molecules, stabilizing the SRX/IHM state, and/or altering cooperative activation of the actin thin filament. In the first Aim we will examine the impact of the DCM mutations on the myosin ATPase cycle, duty ratio, and formation of the SRX state. The structural impact of the mutations will be examined by using a FRET biosensor that monitors the myosin power stroke and another FRET sensor that examines IHM state formation. Electron microscopy will also be used to evaluate the formation of the IHM state which will be directly compared to the fluorescence spectroscopy and biochemical analysis. Aim 2 will examine the impact of DCM mutations on the single molecule mechanical properties of human M2β, including step size and load-dependent detachment, using a load clamped optical trap. In Aim 3 we will utilize a computational model of muscle contraction to predict how the parameters measured in Aims 1&2 will impact ensemble force, velocity, and power. We will then directly examine the impact of the mutations on the force generating properties by incorporating the human M2β constructs into DNA-based “designer” thick filaments, and examining their ability to interact with regulated thin filaments in a calcium dependent manner. The force, velocity, and power measurements will be performed in the “designer” thick filaments, which contain native thick filament-like geometric spacing of myosin molecules. Overall, the completion of the specific aims of this proposal will enhance our understanding of the molecular mechanisms of disease pathogenesis in DCM and provide a foundation for developing therapies for treating DCM.

Project Narrative The heart pumps blood into the systemic system by contracting cardiac muscle during systole, which is initiated by a rise in intracellular calcium concentration that triggers sliding of the thick and thin filaments in the muscle sarcomere. Myosin is the molecular motor that uses the energy from ATP hydrolysis to drive filament sliding and mutations in myosin can lead to dilated cardiomyopathy, a common form of heart failure that disrupts systolic contraction. This proposal will investigate the impact of dilated cardiomyopathy mutations on several aspects of human beta-cardiac myosin structure and function, including the regulatory mechanism, structural mechanism of force generation, and ability to drive filament sliding in a calcium dependent manner.