Effect of Varying Mutant Troponin C Content on Contractile Properties of Striated Muscle

Genetic therapies show much potential as novel, long-term treatments for skeletal and cardiac muscle diseases. Troponin C (TnC), a subunit of troponin, is a particularly promising target for genetic therapies, as it is involved in thin filament activation, Ca2+ signaling and Ca2+ handling. Specifically, expression of engineered TnC mutants with altered Ca2+ binding properties has the potential to restore normal contractile function in diseased/damaged muscle with abnormal Ca2+ properties. We are, therefore, studying skeletal TnC (sTnC) and cardiac TnC (cTnC) mutants that we, and others, have demonstrated affect Ca2+ signaling: F27W, M80Q sTnC and I60Q sTnC, and cTnC L48Q and I61Q. Since various amounts of mutant proteins are incorporated into the myofilaments following viral vector administration of mutant TnC cDNA, the goal of this study was to determine the minimal amount of protein expression necessary to alter contractility in striated muscle. In preliminary studies, different ratios of F27W, M80Q to WT sTnC (100:1, N=4; 50:50, N=4; 25:75, N=4; 1:100, N=6; C0=1mg/mL for all TnCs) were reconstituted into demembranated skeletal muscle fibers. Following reconstitution, the maximal Ca2+ activated force (Fmax) was 90±0.02% (WT), 83±0.03% (100:1), 92±0.04% (50:50) and 90±0.01% (25:75) of pre-extracted Fmax (216±15 mN/mm2). Ca2+ sensitivity was significantly enhanced for all ratios (100:1, pCa50=6.17±0.02, p<0.001; 50:50, pCa50=5.90±0.03, p<0.05; 25:75, pCa50=6.01±0.01, p<0.01) as compared to WT (pCa50=5.76±0.04). These preliminary data indicate that <25% of the mutant protein is required to alter contractility without changing the maximal Ca2+ activated force. Continuing work will identify the minimal level of mutant TnC incorporation in striated muscle that significantly alters pCa50 and the rate force develops.