Nanothermometry Measure of Muscle Efficiency.

Despite recent advances in thermometry, determination of temperature at the nanometer scale in single molecules to live cells remains a challenge that holds great promise in disease detection among others. In the present study, we use a new approach to nanometer scale thermometry with a spatial and thermal resolution of 80 nm and 1 mK respectively, by directly associating 2 nm cadmium telluride quantum dots (CdTe QDs) to the subject under study. The 2 nm CdTe QDs physically adhered to bovine cardiac and rabbit skeletal muscle myosin, enabling the determination of heat released when ATP is hydrolyzed by both myosin motors. Greater heat loss reflects less work performed by the motor, hence decreased efficiency. Surprisingly, we found rabbit skeletal myosin to be more efficient than bovine cardiac. We have further extended this approach to demonstrate the gain in efficiency of Drosophila melanogaster skeletal muscle overexpressing the PGC-1α homologue spargel, a known mediator of improved exercise performance in humans. Our results establish a novel approach to determine muscle efficiency with promise for early diagnosis and treatment of various metabolic disorders including cancer.

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II.

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.

1,3-Diethylurea-enhanced Mg-ATPase activity of skeletal muscle myosin with a converse effect on the sliding motility.

We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ≤1M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, Vmax, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover.

Electrophoretic separation of myosin heavy chain isoforms using a modified mini gel system.

The electrophoretic separation of myosin heavy chain isoforms from muscle biopsy homogenates has been widely practiced in the field of exercise physiology to examine how intrinsic (i.e., aging) and extrinsic (i.e., training) factors affect muscle phenotype. In the past, various research groups have used large and mini polyacrylamide gel systems to perform this delicate methodology. As technology has progressed, additional gel formats have been introduced, but available methodologies appear to be lacking. In this investigation, we successfully separated 3 distinct myosin heavy chain isoforms from various muscle samples using a modified mini gel system that can load up to 26 samples per gel. This article will outline our allocated protocol and discuss potential troubleshooting considerations for other researchers performing this intricate methodology. The outlined methodology has resulted in an ability to clearly resolute 3 distinct bands at molecular weights attributed to the myosin heavy chain isoforms in human skeletal muscle at a wide range of human ages (20-78 years). As additional technologies become available, the need to modify and adapt existing electrophoretic protocols for myosin heavy chain isoform separation and other protocols will continue to be evident.

The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation.

Phosphorylation of the myosin regulatory light chain (RLC) in skeletal muscle has been proposed to act as a molecular memory of recent activation by increasing the rate of force development, ATPase activity, and isometric force at submaximal activation in fibers. It has been proposed that these effects stem from phosphorylation-induced movement of myosin heads away from the thick filament backbone. In this study, we examined the molecular effects of skeletal muscle myosin RLC phosphorylation using in vitro motility assays. We showed that, independently of the thick filament backbone, the velocity of skeletal muscle myosin is decreased upon phosphorylation due to an increase in the myosin duty cycle. Furthermore, we did not observe a phosphorylation-dependent shift in calcium sensitivity in the absence of the myosin thick filament. These data suggest that phosphorylation-induced movement of myosin heads away from the thick filament backbone explains only part of the observed phosphorylation-induced changes in myosin mechanics. Last, we showed that the duty cycle of skeletal muscle myosin is strain dependent, consistent with the notion that strain slows the rate of ADP release in striated muscle.

Effect of low pH on single skeletal muscle myosin mechanics and kinetics.

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V(actin)) in the IVM by 36%. Single molecule experiments, at 1 microM ATP, decreased the average unitary step size of myosin (d) from 10 +/- 2 nm (pH 7.4) to 2 +/- 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 microM to 1 mM at pH 6.4 restored d (9 +/- 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V(actin), however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t(on)). Measurement of t(on) as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t(on) by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V(actin) approximately d/t(on)), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V(actin), suggesting a molecular explanation for this component of muscular fatigue.

Counteracting effects of urea and methylamines in function and structure of skeletal muscle myosin.

Myosin is an asymmetric protein that comprises two globular heads (S1) and a double-stranded alpha-helical rod. We have investigated the effects of urea and the methylamines trimethylamine oxide (TMA-O) and glycine betaine (betaine) on activity and structure of skeletal muscle myosin. K(+) EDTA ATPase activity of myosin was almost completely inhibited by urea (2M); TMA-O stimulated myosin activity, whereas betaine had no effect. When combined with urea (0-2M), TMA-O or betaine (1 M) effectively protected the ATPase activity of myosin against inhibition. Intrinsic fluorescence measurements showed that in urea or TMA-O (0-2M), there were no shifts in the center of mass of the fluorescence spectrum of myosin, despite a decrease in fluorescence intensity. However, these osmolytes at concentrations above 2M produced a red shift in the emission spectrum. Betaine alone did not alter the center of mass at any concentration tested up to 5.2M. Thus, modifications in ATPase activity induced by low concentrations of solutes (<2M) are not directly correlated with the modifications in myosin structure detected by fluorescence. Both methylamines (>or=1M) were also able to protect myosin structure against urea-induced effects (2-8M). Protection was not observed for S1, supporting the hypothesis that these osmolytes have a biphasic effect on myosin: at lower concentrations there is an effect on the globular portion (S1), and at higher concentrations there is an effect on the coiled-coil (rod) portion of myosin.