Effects of protein kinase C dependent phosphorylation and a familial hypertrophic cardiomyopathy-related mutation of cardiac troponin I on structural transition of troponin C and myofilament activation

In experiments reported here, we compared tension and thin filament Ca2+ signaling in preparations containing either wild-type cardiac troponin I (cTnI) or a mutant cTnI with an R146G mutation [cTnI(146G)] linked to familial hypertrophic cardiomyopathy. Myofilament function is altered in association with cTnI phosphorylation by protein kinase C (PKC), which is activated in hypertrophy. Whether there are differential effects of PKC phosphorylation on cTnI compared to cTnI(146G) remains unknown. We therefore also studied cTDI and cTnl(146G) with PKC sites mutated to Glu, which mimics phosphorylation. Compared to cTnI controls, binary complexes with either cTnl(146G) or cTnl(43E/45E/144E) had a small effect on Ca2+-dependent structural opening of the N-terminal regulatory domain of cTnC as measured using Forster resonance energy transfer. However, this structural change was significantly reduced in the cTnC-cTnI(43E/45E/144E/146G) complex. Exchange of cTnI in skinned fiber bundles with cTnl(146G) induced enhanced Ca2+ sensitivity and an elevated resting tension. Exchange of cTnI with cTnI(43E/45E/144E) induced a depression in Ca2+ sensitivity and maximum tension. However, compared to cTnl(146G), cTnl(43E/45E/144E/146G) had little additional effects on myofilament response to Ca2+. By comparing activation of tension to the open state of the N-domain of cTnC with variations in the state of cTnI, we were able to provide data supporting the hypothesis that activation of cardiac myofilaments is tightly coupled to the open state of the N-domain of cTnC. Our data also support the hypothesis that pathological effects of phosphorylation are influenced by mutations in cTnI.