Structural and Kinetic Effects of HCM Related Mutations R146G/Q and R163W of Cardiac Troponin I on cTnI-cTnC Interaction within Reconstituted Thin Filament

Abstract

Mutations in cardiac troponin I (cTnI) that cause hypertrophic cardiomyopathy (HCM) have been reported to change the contractility of cardiac myofilaments, but the underlying molecular mechanism remains elusive. In this study, fluorescence spectroscopy was used to investigate the specific structural and kinetic effects that HCM related cTnI mutations R146G/Q and R163W exert on Ca2+ and myosin S1 dependent conformational transitions in cTn structure while in the reconstituted thin filament. Fluorescently labeled mutants of cTnC and cTnI were prepared for this purpose. Ca2+-induced changes in interactions between cTnC and the inhibitory region (cTnIIR) and regulatory region (cTnIRR) of cTnI were individually monitored using steady-state and time-resolved Förster resonance energy transfer, and kinetics were determined using FRET stopped-flow. It was found that R146G/Q and R163W all changed the molecular distances between cTnC and cTnIIR and cTnIRR in unique and various ways. However, kinetic rates of conformational transitions induced by Ca2+-dissociation were universally slowed when R146G/Q and R163W were present, except that R146Q sped up changes in the cTnIRR−cTnC interaction. This explained the universal trend of increased Ca2+-sensitivity. Interestingly, the kinetic rates of changes in the cTnIIR−cTnC interaction were always slower than that of the cTnIRR−cTnC interaction, suggesting that the flycasting mechanism that normally underlies deactivation is preserved in spite of mutation. Thus our results revealed that R146G/Q and R163W each impact regulatory switching in their own unique way, but with a common kinetic outcome: the slowing of thin filament deactivation. Furthermore, R146Q and R163W disrupted the blocked state, causing the thin filament to behave as if it is in the fourth “pre-relax” state of regulation.