Myosin cross-bridges do not form precise rigor bonds in hypertrophic heart muscle carrying troponin T mutations |
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Authors: | Midde K Dumka V Pinto J R Muthu P Marandos P Gryczynski I Gryczynski Z Potter J D Borejdo J |
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Affiliation: | a Dept of Molecular Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107, United Statesb Dept of Cell Biology and Genetics and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107, United Statesc Dept of Physics and Astronomy, Texas Christian University, 2800 S. University Dr., Fort Worth, TX 76129, United Statesd Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, Miami, FL 33136, United States |
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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. |
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Keywords: | EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide Tg, Transgenic NTg, Non Transgenic WT, Wild Type MUT, Mutated S, Skewness of distribution K, Kurtosis of distribution SD, Standard Deviation FHC, Familial Hypertrophic Cardiomyopathy TnT, Troponin T SMD, Single Molecule Detection LC1, Myosin Essential Light Chain 1 MF, Myofibril TMRIA, tetramethylrhodamine-5-iodoacetamide dihydroiodide (5-TMRIA) (single isomer) R-LC1, TMRIA labeled LC1 DV, Detection Volume HS, Half Sarcomere RLC, Regulatory Light Chain ACF, AutoCorrelation Function APD, Avalanche PhotoDiode PF, Polarization of Fluorescence FCS, Fluorescence Correlation Spectroscopy S, Skewness K, Kurtosis |
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