Familial cardiomyopathies are the leading cause of sudden cardiac death among young people, and pediatric-onset disease is particularly devastating. Dilated cardiomyopathy (DCM) is a familial cardiomyopathy characterized by dilation of the left ventricular chamber of the heart. DCM can be caused by mutations in proteins involved in cardiac muscle contraction, including troponin T; however, it is not well understood how these mutations affect cardiac contraction and contribute to the disease phenotype. To address this critical gap in our knowledge, we examined the molecular-level impact of a troponin T mutation implicated in pediatric-onset DCM, R134G. In vitro motility assays, in which troponin/tropomyosin-regulated thin filaments are propelled across a myosin-coated surface, were carried out over a range of physiologically relevant calcium concentrations. These measurements revealed decreased calcium sensitivity in the troponin complex containing the R134G mutant. To elucidate the molecular mechanism underlying the altered calcium sensitivity conferred by the R134G mutation, we used stopped-flow and steady-state fluorescence measurements to determine the equilibrium constants that define binding of myosin to regulated thin filaments. By inputting these equilibrium constants into a computational model of the sarcomeric contraction, we were able to explain the observed changes in calcium-based myosin regulation and make testable predictions about the effect of the mutation on cardiac contractility. Taken together, our results provide new insights into the mechanism of pediatric heart disease.