Myosin cross-bridges do not form precise rigor bonds in hypertrophic heart muscle carrying troponin T mutations

Abstract

Distribution of orientations of myosin was examined in ex-vivo myofibrils from hearts of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C. Humans are heterozygous for sarcomeric FHC mutations and so hypertrophic myocardium contains a mixture of the wild-type (WT) and mutated (MUT) TnT. If mutations are expressed at a low level there may not be a significant change in the global properties of heart muscle. In contrast, measurements from a few molecules avoid averaging inherent in the global measurements. It is thus important to examine the properties of only a few molecules of muscle. To this end, the lever arm of one out of every 60,000 myosin molecules was labeled with a fluorescent dye and a small volume within the A-band (~1 fL) was observed by confocal microscopy. This volume contained on average 5 fluorescent myosin molecules. The lever arm assumes different orientations reflecting different stages of acto-myosin enzymatic cycle. We measured the distribution of these orientations by recording polarization of fluorescent light emitted by myosin-bound fluorophore during rigor and contraction. The distribution of orientations of rigor WT and MUT myofibrils was significantly different. There was a large difference in the width and of skewness and kurtosis of rigor distributions. These findings suggest that the hypertrophic phenotype associated with the TnT mutations can be characterized by a significant increase in disorder of rigor cross-bridges.