Structure function analysis of cardiac troponin T and myosin heavy chain

Abstract

The basic unit of contraction in striated muscle is the sarcomere. The sarcomere contains thick and thin filaments which are organized in a highly complex crystalline array. The major constituent of the thick filament is the molecular motor myosin. The thin filament is made up of actin, and it is the interaction between myosin and actin which is responsible for contraction. Mutations in sarcomeric proteins have been linked to familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by asymmetric hypertrophy, myofibril disarray and sudden death. Included in these FHC genes are myosin heavy chain (MyHC) and troponin T (TnT), a thin filament protein and member of the troponin complex responsible for calcium-sensitive regulation of contraction. Our laboratory has been interested in the pathogenesis of the disease and has focused on MyHC and TnT to create animal models that would lend insight into the disease process. To assess the impact of FHC mutations on cTnT function, a cell culture model was used. The mouse cTnT was cloned from a mouse cardiac cDNA library and epitope tagged and FHC mutants R95Q, {dollar}\Delta{dollar}163 and a truncation were created. Expression of these mutants in COS cells and neonatal cardiac myocytes showed that these mutant cTnT proteins are stable and, in cardiac myocytes, can incorporate into cardiac sarcomeres. Expression of the {dollar}\Delta{dollar}163 cTnT in adult cardiac myocytes led to a decrease in shortening and increase in relaxation rates when compared to controls. We hypothesized that a lesion in the light chain binding (LCBD) domain in MyHC would behave as a dominant mutation, creating heart disease similar to FHC. To that end, 61 amino acids corresponding to the light chain binding ({dollar}\Delta{dollar}LCBD) were deleted from rat cardiac {dollar}\alpha{dollar}-MyHC. A transgenic mouse expressing a {dollar}\Delta{dollar}LCBD {dollar}\alpha{dollar}MyHC was produced. Heart weights and echocardiography showed that the hearts of these mice were asymmetrically hypertrophied. Histological examination showed marked cellular hypertrophy, myocyte disorganization and valvular pathology. Biochemical analysis showed a decrease in myofibril ATPase. These experiments demonstrate that a myosin incapable of binding MyLCs behaves as a dominant negative mutation and elicits a hypertrophic and cardiomyopathic response.