Many factors play a role in regulating striated muscle contraction; however, the relative importance of the thin filament in this process is still under poorly understood. In this work, the role of troponin C (TnC) on the rate of skeletal muscle contraction and as a regulator of the Ca2+ sensitivity of cardiac force production were studied. To investigate how TnC might affect the rate of contraction, skeletal TnC constructs with altered Ca2+ binding properties were reconstituted into single skinned psoas fibers from rabbits to assess the Ca2+ dependence of force development and the rate of force redevelopment (ktr) at 15°C. This procedure resulted in a sensitization of both force and ktr to Ca2+ for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and ktr to Ca2+, with I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (Fmax) and maximal ktr. Even though V43QTnC and I60QTnC had drastically different effects on the Ca2+ binding properties of TnC, they both exhibited decreases in the cooperativity of force production and elevated ktr at force levels less than 30% Fmax compared to wild-type TnC. However, at matched force levels greater than 30% Fmax, ktr was similar for all the TnC constructs. These results suggest that the TnC mutants primarily affected ktr through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate these results, NEMS-1, a non force generating cross-bridge analogue that activates the thin filament, fully recovered maximal ktr for the I60QTnC at low [Ca2+].
Additionally, a protocol was developed to passively exchange whole human Tn into rat skinned cardiac trabeculae to study how TnC works in conjunction with other thin filament proteins to regulate the Ca2+ sensitivity of force production. To this end, the disease related abnormal Ca2+ sensitivities resulting from a truncation of TnI (residues 1-192) and a single residue K210 TnT deletion on the force-pCa relationship were first chracterized. TnCs were then specifically engineered and combined with these disease related mutations in hopes of correcting their effects on the Ca2+ sensitivity of force. Our results demonstrate that TnC is able to attenuate and even completely restore dysfunctional Ca2+ sensitivity of force generation due to TnI or TnT modifications. Thus, it has been shown that TnC can control the rate of contraction by modulating the level of thin filament activation and is capable of correcting the aberrant Ca2+ binding properties due to modifications in other thin filament proteins.