Dissecting the Molecular Mechanism for Familial Cardiomyopathies

Abstract

The heart needs to generate sufficient force and power to pump blood to the body and to adjust its power output in response to stresses. Mutations of contractile proteins, such as cardiac troponin T, can lead to familial cardiomyopathies, the leading cause of sudden cardiac death in people under the age of 30. Two common forms of cardiomyopathy, hypertrophic (HCM) and dilated (DCM) are characterized by thickening or thinning of the ventricular wall, respectively. However, how changes in contractile proteins at the molecular level lead to the disease phenotype is not well understood. To address this problem, we examined two point mutations in human cardiac troponin T (TnT), R92Q and ΔK210, which cause hypertrophic and dilated cardiomyopathy, respectively, in human patients. We have reconstituted the calcium-based regulation of actomyosin in vitro using tissue purified and recombinant cardiac proteins. Similar to previous studies, in vitro motility assays which reconstitute the calcium-dependent interactions between myosin and regulated thin filaments revealed that R92Q is shifted towards submaximal calcium activation while ΔK210 is shifted towards supermaximal calcium activation. We dissected the molecular mechanism of these differential changes using a combination of fluorescence equilibrium titrations and stopped-flow experiments, and show that these effects are due to alterations in the free energies of the biochemical transitions that regulate muscle activation. In particular, we see that these mutations differentially affect calcium-dependent positioning of tropomyosin along the thin filament. We propose that these changes affect the regulation of force production by the molecular motor myosin, leading to alterations in mechanosensing by heart tissue.