Although more than 200 mutations located in the motor domain of the human -cardiac myosin heavy chain (MyHC) cause hypertrophic or dilated cardiomyopathy (HCM or DCM), their molecular effects on the myosin molecule remain elusive. Most studies to determine the functional impact of these mutations on myosin have used non-human myosins and most have not used cardiac myosins. Leinwand and colleagues have recently developed a system for producing recombinant human cardiac myosin motors and we will exploit this system for the first systematic study of the effects HCM and DCM mutations in human -cardiac myosin. Since the majority of mutations that cause disease are located in the myosin motor, we will focus our studies there. We selected for study 10 mutations (5 HCM and 5 DCM), both because of their largely severe clinical phenotypes and because of their locations within the myosin motor domain. We propose to analyze the biochemical properties of the mutations in solution and their biomechanical properties using in vitro motility and laser trap single molecule assays. We hypothesize that mutations that lead to increased force production lead to HCM while those that lead to decreased force production lead to DCM, consistent with recent suggestions. Mechanisms that lead to decreased or increased force production by myosin can be varied. The inherent force-producing capability of the motor, for example, could be increased or decreased by mutations that change the spring constant of the elastic element of the motor. On the other hand, the force- producing capability could be changed in either direction by changes in the duty ratio of the myosin (the fraction of the ATPase cycle that the head is strongly bound to actin). An increase in the duty ratio, for example, would result in more heads bound to actin in a force-producing state at any moment, leading to an overall increase in force. Furthermore, the duty ratio can be changed in more than one way. For example, the ADP release rate (which determines the strongly-bound state time), the ATP hydrolysis step (which defines the recovery stroke time) or the Pi release (the rate of entry into the strongly bound state) can be affected and result in a change in the duty ratio. Thus, it is essential to begin to characterize different mutations, hypothesized to affect different mechanistic aspects of the motor. These studies will begin the process of linking the fundamental changes in motor function to the eventual different clinical outcomes. Therefore, we will determine the biochemical and biomechanical parameters of wild type human ?- and -cardiac myosin and mutant -myosin motors with respect to their steady-state and transient kinetic properties, velocities at ~zero load and under loaded conditions, their duty ratios, their stroke sizes, and the maximum force they produce upon interacting with actin.
Hypertrophic cardiomyopathy is the most common genetic heart disease and is the leading cause of sudden death in the United States. Familial dilated cardiomyopathy is also a very serious genetic heart disease. Both of these diseases can be caused by mutations in the motor protein of the heart, myosin. It is not understood how these motors are affected by these mutations and no treatments exist for the primary defect. This work will for the first time lead to an understanding of what is wrong with these motors and may lead the way to novel treatments.
|Vega, Rick B; Konhilas, John P; Kelly, Daniel P et al. (2017) Molecular Mechanisms Underlying Cardiac Adaptation to Exercise. Cell Metab 25:1012-1026|
|Sung, J; Mortensen, K I; Spudich, J A et al. (2017) How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp. Methods Enzymol 582:1-29|
|Nag, Suman; Trivedi, Darshan V; Sarkar, Saswata S et al. (2017) The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations. Nat Struct Mol Biol 24:525-533|
|Spudich, James A; Aksel, Tural; Bartholomew, Sadie R et al. (2016) Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human ?-cardiac myosin. J Exp Biol 219:161-7|
|Peter, Angela K; Bjerke, Maureen A; Leinwand, Leslie A (2016) Biology of the cardiac myocyte in heart disease. Mol Biol Cell 27:2149-60|
|Pugach, Emily K; Blenck, Christa L; Dragavon, Joseph M et al. (2016) Estrogen receptor profiling and activity in cardiac myocytes. Mol Cell Endocrinol 431:62-70|
|Green, Eric M; Wakimoto, Hiroko; Anderson, Robert L et al. (2016) A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice. Science 351:617-21|
|Hariadi, R F; Sommese, R F; Adhikari, A S et al. (2015) Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments. Nat Nanotechnol 10:696-700|
|Sung, Jongmin; Nag, Suman; Mortensen, Kim I et al. (2015) Harmonic force spectroscopy measures load-dependent kinetics of individual human ?-cardiac myosin molecules. Nat Commun 6:7931|
|Gupte, Tejas M; Haque, Farah; Gangadharan, Binnu et al. (2015) Mechanistic heterogeneity in contractile properties of ?-tropomyosin (TPM1) mutants associated with inherited cardiomyopathies. J Biol Chem 290:7003-15|
Showing the most recent 10 out of 14 publications