Distinct Mutation-Specific Effects on Thin Filament Activation Lead to Dilated Cardiomyopathy Phenotype in Cells

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 of the heart and impaired cardiac contractility. The disease presentation and prognosis depend on the disease-causing mutation. DCM can be caused by mutations in proteins involved in regulating cardiac muscle contraction, including troponin T; however, how these mutations affect cardiac contraction and contribute to the disease phenotype is not well-understood. To address this critical gap in our knowledge, we examined the molecular- and cellular-level impacts of a troponin T mutation implicated in pediatric-onset DCM, R134G. We used stopped-flow and steady-state fluorescence measurements to determine the equilibrium constants that define binding of myosin to regulated thin filaments. Unexpectedly, these biochemical experiments revealed increased activation of thin filaments containing R134G troponin T at the molecular level. This is in contrast to our previous studies of another DCM-causing mutation in troponin T (delK210) and the frequently cited hypothesis that DCM is caused by molecular hypocontractility. Genome edited human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the R134G mutation showed many hallmarks of DCM in hiPSC-CMs and many similarities with delK210 mutant cells. Specifically, R134G cells showed disorganized sarcomeres, decreased cellular contractility, cellular hypertrophy, and expression of DCM-associated genes. These data clearly demonstrate that two mutations within the same molecule can have different underlying biophysical mechanisms at the molecular level but similar effects on cellular contractility. These results reinforce the importance of multiscale studies and suggest the value of precision medicine approaches for the treatment of DCM.