Calcium binding kinetics of troponin C strongly modulate cooperative activation and tension kinetics in cardiac muscle

Tension development and relaxation in cardiac muscle are regulated at the thin filament via Ca2+ binding to cardiac troponin C (cTnC) and strong cross-bridge binding. However, the influence of cTnC Ca2+-binding properties on these processes in the organized structure of cardiac sarcomeres is not well-understood and likely differs from skeletal muscle. To study this we generated single amino acid variants of cTnC with altered Ca2+ dissociation rates (koff), as measured in whole troponin (cTn) complex by stopped-flow spectroscopy (I61Q cTnNWT cTnNL48Q cTn), and exchanged them into cardiac myofibrils and demembranated trabeculae. In myofibrils at saturating Ca2+, L48Q cTnC did not affect maximum tension (Tmax), thin filament activation (kACT) and tension development (kTR) rates, or the rates of relaxation, but increased duration of slow phase relaxation. In contrast, I61Q cTnC reduced Tmax, kACT and kTR by 40–65% with little change in relaxation. Interestingly, kACT was less than kTR with I61Q cTnC, and this difference increased with addition of inorganic phosphate, suggesting that reduced cTnC Ca2+-affinity can limit thin filament activation kinetics. Trabeculae exchanged with I61Q cTn had reduced Tmax, Ca2+ sensitivity of tension (pCa50), and slope (nH) of tension–pCa, while L48Q cTn increased pCa50 and reduced nH. Increased cross-bridge cycling with 2-deoxy-ATP increased pCa50 with WT or L48Q cTn, but not I61Q cTn. We discuss the implications of these results for understanding the role of cTn Ca2+-binding properties on the magnitude and rate of tension development and relaxation in cardiac muscle.