Molecular- and Cellular-Level Effects of a Troponin Mutation Linked to Dilated Cardiomyopathy

Abstract

Familial cardiomyopathies are the leading cause of sudden cardiac death among young people, and pediatric-onset disease is particularly devastating. Dilated cardiomyopathy (DCM) is characterized by dilation of the left ventricular chamber and impaired cardiac contractility. DCM can be caused by mutations in proteins that regulate cardiac muscle contraction, including troponin T; however, it is not well-understood how mutation-induced changes in protein biophysics affect cardiac contraction and contribute to the disease phenotype. By understanding the biophysical underpinnings of specific mutations, it might be possible to develop targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. Here, we examined the molecular- and cellular-level impacts of a troponin T mutation implicated in pediatric-onset DCM, R134G. In vitro motility assays revealed decreased calcium sensitivity with the R134G mutant, which would be expected to reduce force production in cardiomyocytes. To probe the molecular mechanism underlying this change in sensitivity, we performed stopped-flow and steady-state fluorescence measurements to determine the equilibrium constants that define thin filament activation. Unexpectedly, these biochemical experiments revealed increased activation of thin filaments containing R134G troponin T at diastolic calcium levels, in contrast to the frequently cited hypothesis that DCM is caused by molecular hypocontractility. We also demonstrate that R134G decreases the cooperativity of myosin binding to regulated thin filaments, which could be responsible for the shift in calcium sensitivity. Consistent with the molecular studies, cardiomyocytes carrying the R134G mutation were hypocontractile; however, they also showed hallmarks of DCM that lie downstream of the initial molecular insult, including disorganized sarcomeres, cellular hypertrophy, and expression of DCM-associated genes. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and suggest the value of precision medicine approaches for the treatment of DCM.