Effect of TnC with altered Ca2+ binding kinetics on force generation kinetics in striated muscle

Thin filament activation and force development in striated muscle result from coupling between Ca2+ binding to troponin C (TnC) and crossbridge formation. We study how altered TnC Ca2+ binding kinetics determine activation kinetics in demembranated psoas fibers and myofibrils. Ca2+ dissociation rates from isolated WT sTnC (WT) and M80Q,F27W sTnC (QW) are 725 ± 2 and 18.3 ± 0.6 s–1 at 5C, respectively, and will be characterized in whole Tn complexes. Extracting sTnC from fibers and reconstituting Tn complexes with QW greatly increases Ca2+ sensitivity of force (pCa50) by 0.42 vs. WT at SL 2.5 lm, 15C. While the maximal rate of force redevelopment (ktr) does not change (13.5 ± 1.1 vs. 13.2 ± 0.9 s–1), ktr slightly increases for QW at low force levels. To better examine activation kinetics, we use myofibril preparations with rapid solution changes. Despite a large difference in pCa50 with QW, preliminary results in myofibrils show no change in maximal force generation (pCa 3.5): Fmax = 308 ± 35 vs. 272 ± 26 mN/mm2; activation rate (kact) = 9.9 ± 0.9 vs. 10.3 ± 0.7 s–1; and ktr = 9.5 ± 0.8 vs. 8.5 ± 0.5 s–1 for WT vs. QW, respectively. Relaxation kinetics can also be measured with this method, and we will use whole Tn exchange to enhance replacement of sTnC. Ongoing studies will examine activation and relaxation at low Ca2+ levels and compare results to studies in cardiac muscle. HL65497, PRIN2004.