Abstract 15782: Hypertrophic Cardiomyopathy-causing Mutations in Human Beta-Cardiac Myosin Converter Domain Alter Power Output of Sarcomere Function in Vitro
Hypertrophic cardiomyopathy (HCM) affects 1 in 500 in the general population and is an important cause of arrhythmias, heart failure, and sudden cardiac death. beta-cardiac myosin comprises 1/3 of genetic mutations associated with HCM, while the underlying primary effects of these mutations on the biomechanical function of myosin remain elusive. In particular, the converter domain of myosin has been known to be a hot spot for HCM-causing mutations. However, how alterations in this region affect its ability to transduce small changes in the catalytic domain to the lever arm to accomplish the power stroke is unclear. We hypothesize that these mutations affect the biomechanics of human beta-cardiac myosin in different ways (e.g. changing the spring constant of the elastic element of the motor resulting in altered intrinsic force, changing the duty ratio of the myosin and therefore the ensemble force produced, or altering the stroke size of myosin upon interaction with actin), all of which result in changes in the power output of the myosin power stroke. To date, much important biochemical work has involved use of mouse and other non-human cardiac myosin isoforms, as well as skeletal muscle biopsy studies, but these studies have produced mixed results due to isoform differences and lack of homology in amino acid sequence between species. Here we used a recently reported in vitro system to express human beta-cardiac myosin using an adenoviral based expression in a mouse myoblast cell line, and characterized the molecular effects of severe HCM-causing converter domain mutations, R719W and R723G. We also studied HCM-causing mutations at the same amino acid positions which result in more benign clinical phenotypes (normal life span), R719Q and R723C. Using ATPase and in vitro motility assays, we found that severe HCM mutations caused 10~15% increases in actin gliding velocity, no change in ATPase, and loaded in vitro motility assays showed altered power output, while benign mutations showed no significant differences compared to wild type. These observations support the view of altered power output as the primary effect of HCM mutations. Further analysis at the single molecule level is underway to elucidate the precise mechanisms underlying these biomechanical changes.
Author Disclosures: M. Kawana: None. T. Aksel: None. S. Nag: None. S. Sutton: None. K. Ruppel: None. J. Spudich: None.
- © 2014 by American Heart Association, Inc.