Structural and kinetic effects of hypertrophic cardiomyopathy related mutations R146G/Q and R163W on the regulatory switching activity of rat cardiac troponin I

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, Förster resonance energy transfer (FRET) was used to investigate the specific structural and kinetic effects that HCM related rat cTnI mutations R146G/Q and R163W exert on Ca2+ and myosin S1 dependent conformational transitions in rat cTn structure. Ca2+-induced changes in interactions between cTnC and cTnI were individually monitored in reconstituted thin filaments using steady state and time resolved FRET, and kinetics were determined using stopped flow. R146G/Q and R163W all changed the FRET distances between cTnC and cTnI 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. Interestingly, the kinetic rates of changes in the inhibitory region of cTnI were always slower than that of the regulatory region, suggesting that the fly casting mechanism that normally underlies deactivation is preserved in spite of mutation. In situ rat myocardial fiber studies also revealed that FRET distance changes indicating mutation specific disruption of the cTnIIR−actin interaction were consistent with increased passive tension.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (225 K)Download as PowerPoint slideHighlights► FRET study of effects of HCM-related cTnI mutations in reconstituted thin filaments. ► R146G/Q and R163W each uniquely impact interactions between cTnI and cTnC or actin. ► Fly casting mechanism underlying deactivation preserved in spite of mutation. ► R146G/Q and R163W cause TF to enter pre relax like state in the absence of S1-ADP. ► In situ effects on force−Ca2+ relationship of myocardial fibers match in vitro.