Myosin regulatory light chain is phosphorylated by myosin light chain kinase at conserved serine and threonine residues in a number of species. Phosphorylation of myosin regulatory light chain regulates smooth muscle contraction, but appears to have a modulatory role in striated muscle contraction. We assessed the in vivo role of myosin regulatory light chain phosphorylation in the striated muscles of Drosophila melanogaster by substituting alanine at each or both conserved myosin light chain kinase-dependent phosphorylation sites, serine 66 and serine 67. We report here that myosin light chain kinase-dependent phosphorylation is not required for myofibrillogenesis or for the development of maximal isometric force in indirect flight muscles. However, mutants with substitutions at the major phosphorylation site (serine 66) or with the double substitutions had reduced power output in isolated flight muscle fibres and reduced flight ability, showing that myosin regulatory light chain phosphorylation is a key determinant of the stretch activation response in Drosophila.
Myocardial failure in dilated cardiomyopathy may result from subcellular alterations in contractile protein function, excitation-contraction coupling processes, or recovery metabolism. We used isometric force and heat measurements to quantitatively investigate these subcellular systems in intact left ventricular muscle strips from nonfailing human hearts (n=14) and from hearts with end-stage failing dilated cardiomyopathy (n = 13). In the failing myocardium, peak isometric twitch tension, maximum rate of tension rise, and maximum rate of relaxation were reduced by 46%o (p =0.013), 51% (p =0.003), and 46% (p=0.018), respectively (37°C, 60 beats per minute). Tension-dependent heat, reflecting the number of crossbridge interactions during the isometric twitch, was reduced by 61% in the failing myocardium (p=0.006). In terms of the individual crossbridge cycle, the average crossbridge force-time integral was increased by 33% (p=0.04) in the failing myocardium. In the nonfailing myocardium, the crossbridge force-time integral was positively correlated with the patient's age (r=0.86, p<0.02), whereas there was no significant correlation with age in the failing group. The amount and rate of excitation-contraction coupling-related heat evolution (tension-independent heat) were reduced by 69%1 (p=0.024) and 71% (p=0.028), respectively, in the failing myocardium, reflecting a considerable decrease in the amount of calcium released and in the rate of calcium removal. The efficiency of the metabolic recovery process, as assessed by the ratio of initial heat to total activity-related heat, was similar in failing and nonfailing myocardium (0.54±0.03 versus 0.50±0.02, p =0.23). Thus, in failing dilated cardiomyopathy, contractile protein function is altered with an increased force-time integral of the individual crossbridge cycle. However, this alteration does not explain failure since the same changes are present in nonfailing myocardium from older patients. The findings suggest that reduced tension generation in failing dilated cardiomyopathy primarily results from disturbed excitation-contraction coupling processes with a reduced amount of calcium released and a reduced rate of calcium removal. (Circulation Research 1992;70:1225-1232 The recent development of a method for dissecting thin viable muscle strips from larger pieces of human myocardium13 makes it possible to investigate these subcellular systems in the intact muscle by means of a myothermal method14 under physiological conditions (37°C, 60 beats per minute). Heat evolution represents the entire metabolic energy turnover during the isometric twitch. Total activity-related heat can be partitioned into the heat evolution of the contractile proteins, the excitation-contraction coupling system, and the recovery system. These measurements in conjunction with the mechanical performance provide quantitative information on the extent and rate of the reactions involved
Familial hypertrophic cardiomyopathy (FHC) is a disease of the sarcomere. In the beta-myosin heavy chain gene, which codes for the mechanical enzyme myosin, greater than 40 point mutations have been found that are causal for this disease. We have studied the effect of two mutations, the R403Q and L908V, on myosin molecular mechanics. In the in vitro motility assay, the mutant myosins produced a 30% greater velocity of actin filament movement (v(actin)). At the single molecule level, v(actin) approximately d/t(on), where d is the myosin unitary step displacement and t(on) is the step duration. Laser trap studies were performed at 10 microM MgATP to estimate d and t(on) for the normal and mutant myosin molecules. The increase in v(actin) can be explained by a significant decrease in the average t(on)'s in both the R403Q and L908V mutants (approximately 30 ms) compared to controls (approximately 40 ms), while d was not different for all myosins tested (approximately 7 nm). Thus the mutations affect the kinetics of the cross-bridge cycle without any effect on myosin's inherent motion and force generating capacity. Based on these studies, the primary signal for the hypertrophic response appears to be an apparent gain in function of the individual mutant myosin molecules.
A method for measuring the molar stoichiometry of myosin light chain phosphorylation in intact smooth muscle has been developed. Antiserum to the 20,000-Da light chains of bovine aortic smooth muscle was harvested from rabbits and used to label light chains by a radioimmunoblotting procedure. In the initial characterization it was found that the 20,000-Da light chains could be transferred by electroblotting from polyacrylamide gels to nitrocellulose paper with an efficiency of approximately 80% over a protein range of 0.1-5.0 micrograms. At a dilution of 1:500, the unpurified light chain antiserum required approximately 10-12 h at 22 degrees C to reach equilibrium binding to the transferred light chains. Moreover, equilibrium labeling of the light chain-antibody complex with 125I-protein A required 4-6 h of incubation at 22 degrees C. By using these conditions, a radioimmunoassay for the 20,000-Da light chains was developed that was linear over a protein range of 0.1-5.0 micrograms (5-250 pmol). As little as 20 ng of light chains could be measured if a second antibody procedure (goat anti-rabbit immunoglobulin G Fab fragments) was used. Phosphorylated and unphosphorylated myosin light chains were separated by glycerol-urea polyacrylamide gel electrophoresis. This procedure, combined with radioimmunoblot, gave similar estimates of phosphorylation levels when compared with direct assay for phosphate or scanning of Coomassie blue-stained gels. Moreover, when applied to intact uterine smooth muscle, the glycerol-urea gel radioimmunoblot gave values of myosin light chain phosphorylation for relaxed and contracted muscles that were not statistically different from those obtained with a two-dimensional electrophoretic method.(ABSTRACT TRUNCATED AT 250 WORDS)
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