The modulation of myosin function: How small molecules and sarcomeric mutations impact actomyosin interactions

Abstract

The subcellular mechanisms that regulate striated muscle function have been studied by biophysicists and physiologists for over a century. Numerous scientists have made significant findings in the field of muscle biology with fundamental discoveries regarding sarcomeric specific proteins, lattice-spacing architecture, and mechanisms of sarcomeric regulation. With these essential findings as the foundation of our field, there are still many sarcomere-based mechanisms of regulation that remain unresolved. The body of work presented here is aimed at extending the knowledge of sarcomeric contraction and regulation by leveraging recent advances in structural, mechanical, and biochemical assays utilized in the field of muscle biology. Specifically, the presented studies demonstrate an integrated approach to elucidating the myofilament specific processes that underlie striated muscle function by addressing (1) the underlying regulatory role of myosin through differences in structural recruitment and biochemical cycling, (2) the structural and biochemical pathways that small molecules alter sarcomeric proteins as mechanisms of therapy for individuals with congenital heart failure, and (3) underlying mechanisms of dysfunction in hypercontractile models of striated muscle diseases.The presented work focuses primarily on the thick filament protein myosin and the regulatory role the protein plays in sarcomeric function. In comparison to the vastly studied thin filament, significantly less is known about the modulation of sarcomeric function through myosin. First, we assessed the structural organization and position of myosin within the sarcomere and compared those results to sophisticated biochemical measurements of nucleotide turnover kinetics. We show that the diffraction-based measurements of myosin recruitment as a parameter for myosin regulation is independent from biochemical regulation of myosin head cycling kinetics. Second, we determined the mechanistic pathway of a novel myosin-specific small molecule as a therapeutic agent in hypo-contractile models of cardiac diseases. We show how inotropic interventions can modulate myosin cycling and recruitment through product release inhibition; a mechanistic pathway that can rescue a transgenic murine model harboring a loss-of-function troponin mutation that causes dilated cardiomyopathy. Lastly, we utilize a transgenic porcine model harboring the first reported familial hypertrophic cardiomyopathy mutation to describe how perturbations in specific regions of myosin can lead to sarcomeric dysfunction. We show that isolated protein measurements do not fully recapitulate the phenotypic disease expression observed in myofibril-level and tissue-level preparations, suggesting that certain myosin mutations require the interaction of other sarcomeric proteins to manifest. To summarize, the work presented here expands the understanding of sarcomeric regulation though myosin specific pathways. We address topics ranging from basic molecular regulation of sarcomeric function to fundamental mechanistic pathways of different diseases and novel sarcomere-specific therapeutic agents.