Fluorescence Measurements Using Rhodamine-Labeled cTnC Mutants Indicate Little Cooperative Interaction Between Cardiac Thin Filament Regulatory Units

Abstract

Exchange of mixtures of WT cTnC and mutant cTnC(D65A), which cannot bind Ca2+ at N-terminal site II (“dead” cTnC), reduced maximal Ca2+ activated force (Fmax) with little effect on force-Ca2+ relations and force kinetics in skinned cardiac trabeculae (Gillis et al., J Physiol. 580:561-76, 2007), suggesting interaction between structural regulatory units (RUs; 7 actins, 1 troponin, 1 tropomyosin) along cardiac thin filaments is less than in skeletal muscle (Regnier et al., J Physiol. 540:485-97, 2002). To more directly test that this finding, we exchanged skinned cardiac trabeculae with mixtures of mutant cTnC(C35S) and cTnC(C35S,D65A), with one or the other labeled at Cys 84 with 5’tetramethyl rhodamine (IATR) for dichroism measurements. In trabeculae exchanged with 100% cTnC(C35S)-IATR, dichroism increased in response to both Ca2+ and rigor crossbridges, while trabeculae with 100% Tn containing (cTnC(C35S,D65A)-IATR) had no response to Ca2+, but retained a strong response to rigor crossbridge binding. This response to strong crossbridges allows use of cTnC(C35S,D65A)-IATR to determine if isolated regulatory units containing cTnC(C35S,D65A)-IATR are perturbed by Ca2+-induced active contraction in adjacent “live” RUs. To test this, trabeculae were exchanged with a mixture of 20% functional cTnC(C35S)-IATR and 80% unlabeled cTnC(C35S,D65A), to isolate functional RUs. Fmax decreased but there was little change in the Ca2+-dependence of dichroism compared to trabeculae exchanged with 100% functional cTnC(C35S)-IATR. These data indicate minimal or no apparent spread of activation between adjacent RUs in cardiac muscle, indicating that the apparent cooperativity of force production in cardiac muscle results from interactions between myosin and thin filaments within a thin filament structural regulatory unit. Supported by NIH RO1-HL65497 (Regnier).