Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.     

Functional properties of human muscle fibers after short-term resistance exercise training

The aim of this study was to assess the relationships between human muscle fiber hypertrophy, protein isoform content, and maximal Ca(2+)-activated contractile function following a short-term period of resistance exercise training. Six male subjects (age 27 +/- 2 yr) participated in a 12-wk progressive resistance exercise training program that increased voluntary lower limb extension strength by >60%. Single chemically skinned fibers were prepared from pre- and posttraining vastus lateralis muscle biopsies. Training increased the cross-sectional area (CSA) and peak Ca(2+)-activated force (P(o)) of fibers containing type I, IIa, or IIa/IIx myosin heavy chain by 30-40% without affecting fiber-specific force (P(o)/CSA) or unloaded shortening velocity (V(o)). Absolute fiber peak power rose as a result of the increase in P(o), whereas power normalized to fiber volume was unchanged. At the level of the cross bridge, the effects of short-term resistance training were quantitative (fiber hypertrophy and proportional increases in fiber P(o) and absolute power) rather than qualitative (no change in P(o)/CSA, V(o), or power/fiber volume).     

Reduced cardiac muscle power with low ATP simulating heart failure

For patients with heart failure, myocardial ATP level can be reduced to one-half of that observed in healthy controls. This marked reduction (from ≈8 mM in healthy controls to as low as 3–4 mM in heart failure) has been suggested to contribute to impaired myocardial contraction and to the decreased pump function characteristic of heart failure. However, in vitro measures of maximum myofilament force generation, maximum shortening velocity, and the actomyosin ATPase activity show effective K_M values for MgATP ranging from ≈10 μM to 150 μM, well below the intracellular ATP level in heart failure. Thus, it is not clear that the fall of myocardial ATP observed in heart failure is sufficient to impair the function of the contractile proteins. Therefore, we tested the effect of low MgATP levels on myocardial contraction using demembranated cardiac muscle preparations that were exposed to MgATP levels typical of the range found in non-failing and failing hearts. Consistent with previous studies, we found that a 50% reduction in MgATP level (from 8 mM to 4 mM) did not reduce maximum force generation or maximum velocity of shortening. However, we found that a 50% reduction in MgATP level caused a 20%–25% reduction in maximal power generation (measured during muscle shortening against a load) and a 20% slowing of cross-bridge cycling kinetics. These results suggest that the decreased cellular ATP level occurring in heart failure contributes to the impaired pump function of the failing heart. Since the ATP-myosin ATPase dissociation constant is estimated to be submillimolar, these findings also suggest that MgATP concentration affects cross-bridge dynamics through a mechanism that is more complex than through the direct dependence of MgATP concentration on myosin ATPase activity. Finally, these studies suggest that therapies targeted to increase adenine nucleotide pool levels in cardiomyocytes might be beneficial for treating heart failure.     

Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers

This study examines the myosin isozyme heterogeneity (in terms of both alkali light chains and myosin heavy chains) among skeletal muscle fibers of the rabbit and correlates these isozyme differences with the differences in a contractile property, the velocity of unloaded shortening, of the fibers. The mean velocities of unloaded shortening (pCa 4.3; 12 degrees C) were as follows: psoas IIb fibers, 2.07 fiber lengths/s (n = 25); tibialis anterior (IIb) fibers, 1.63 fiber lengths/s (n = 18); vastus intermedius IIa fibers, 0.98 fiber lengths/s (n = 15); fibers (IIa) from chronically stimulated tibialis anterior, 0.86 fiber lengths/s (n = 16). Peptide maps of the myosins showed that the myosin heavy chains of the two groups of IIb fibers were indistinguishable from each other, but different from the heavy chains of the IIa fibers. However, the difference in maximal shortening velocity of the two groups of IIb fibers was correlated with a difference in the alkali light chain ratio deduced from the intensity ratio of myosin isoforms separated by gel electrophoresis under nondenaturing conditions. The vastus intermedius (IIa) and chronically stimulated tibialis anterior (IIa) fibers were indistinguishable in terms of either velocities of unloaded shortening or myosin isozyme contents. Soleus fibers contained only slow-twitch myosin. Thus, among fibers that contained a variety of myosin isozymes, differences in shortening velocities were correlated with the alkali light chain ratio, myosin heavy chain type, or a combination of both.     

Changes in the human muscle force-velocity relationship in response to resistance training and subsequent detraining.

Previous studies show that cessation of resistance training, commonly known as "detraining," is associated with strength loss, decreased neural drive, and muscular atrophy. Detraining may also increase the expression of fast muscle myosin heavy chain (MHC) isoforms. The present study examined the effect of detraining subsequent to resistance training on contractile performance during slow-to-medium velocity isokinetic muscle contraction vs. performance of maximal velocity "unloaded" limb movement (i.e., no external loading of the limb). Maximal knee extensor strength was measured in an isokinetic dynamometer at 30 and 240 degrees/s, and performance of maximal velocity limb movement was measured with a goniometer during maximal unloaded knee extension. Muscle cross-sectional area was determined with MRI. Electromyographic signals were measured in the quadriceps and hamstring muscles. Twitch contractions were evoked in the passive vastus lateralis muscle. MHC isoform composition was determined with SDS-PAGE. Isokinetic muscle strength increased 18% (P < 0.01) and 10% (P < 0.05) at slow and medium velocities, respectively, along with gains in muscle cross-sectional area and increased electromyogram in response to 3 mo of resistance training. After 3 mo of detraining these gains were lost, whereas in contrast maximal unloaded knee extension velocity and power increased 14% (P < 0.05) and 44% (P < 0.05), respectively. Additionally, faster muscle twitch contractile properties along with an increased and decreased amount of MHC type II and MHC type I isoforms, respectively, were observed. In conclusion, detraining subsequent to resistance training increases maximal unloaded movement speed and power in previously untrained subjects. A phenotypic shift toward faster muscle MHC isoforms (I --> IIA --> IIX) and faster electrically evoked muscle contractile properties in response to detraining may explain the present results.     

Effects of hindlimb unweighting and aging on rat semimembranosus muscle and myosin.

We tested the hypothesis that lower specific force (force/cross-sectional area) generated by type II fibers from hindlimb-unweighted rats resulted from structural changes in myosin (i.e., a change in the ratio of myosin cross bridges in the weak- and strong-binding state during contraction). In addition, we determined whether those changes were age dependent. Permeabilized semimembranosus muscle fibers from young adult and aged rats, some of which were hindlimb unweighted for 3 wk, were studied for Ca(2+)-activated force generation and maximal unloaded shortening velocity. Fibers were also spin labeled specifically at myosin Cys707 to assess the structural distribution of myosin during maximal isometric contraction using electron paramagnetic resonance spectroscopy. Myosin heavy chain isoform (MHC) expression and the ratio of MHC to actin were evaluated in each fiber. Fibers from the unweighted rats generated 34% less specific force than fibers from weight-bearing rats (P < 0.001), independent of age. Electron paramagnetic resonance analyses showed that the fraction of myosin heads in the strong-binding structural state during contraction was 11% lower in fibers from the unweighted rats (P = 0.019), independent of age. More fibers from unweighted rats coexpressed MHC IIB-IIX compared with fibers from weight-bearing rats (P = 0.049). Unweighting induced a slowing of maximal unloaded shortening velocity and an increase in the ratio of MHC to actin in fibers from young rats only. These data indicate that altered myosin structural distribution during contraction and a preferential loss of actin contribute to unweighting-induced muscle weakness. Furthermore, the age of the rat has an influence on some parameters of changes in muscle contractility that are induced by unweighting.     

Myosin subunits and contractile properties of single fibers from hypokinetic rat muscles

The effects of prolonged hypokinesia on the contractile properties and myosin isozymes of single fibers from the synergistic fast-twitch plantaris (PL) and slow-twitch soleus (SOL) skeletal muscles of adult rats were studied after 28 days of hindlimb suspension. There was a 31% increase in the mean maximal velocity of unloaded shortening (Vmax) among fibers from SOL with no change in the mean Vmax of fibers from PL after suspension. The myosin heavy and light chain (MHC and MLC) composition of bundles and the MHC composition of single fibers from control and suspended muscles were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase in the relative amount of fast-type MHC's in hypokinetic SOL and a smaller increase in the amount of fast-type MHC's in the PL. Relatively minor changes occurred in the MLC's during hypokinesia. As Vmax increased among individual fibers from control and suspended muscles, the relative amount of fast-type MHC's increased. The results demonstrate that the myosin isozyme composition of skeletal muscle, especially the heavy chains, is altered during hypokinesia, and this finding provides an explanation for changes in Vmax of rat single muscle fibers under the same conditions.     

Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system

Alteration of troponin T (TnT) isoform expression has been reported in human and animal models of myocardial failure. The two adult beef cardiac TnT isoforms (TnT(3) and TnT(4)) were isolated for comparative functional analysis. Thin filaments were reconstituted containing pure populations of the isoforms. The in vitro motility assay was used to directly compare the effect of the two TnT isoforms on force and unloaded shortening as a function of free calcium. We found no significant differences between the two isoforms in terms of calcium sensitivity, cooperativity, or maximal activation (velocity and force) as assessed in a fully calcium-regulated system. Activation by myosin strong binding was similar for thin filaments containing either of the two TnT isoforms. Whereas maximally activated velocity and cooperativity was depressed at pH 6.5, no difference between thin filaments containing the two isoforms was detected. From the small magnitude of the TnT isoform shifts detected in myocardial failure and the lack of significant mechanical effect detected in the motility assay, variable TnT isoform expression is unlikely to be any functional significance in heart failure.     

Beta-adrenergic regulation of cardiac myosin

Calcium-independent regulation of the contractile proteins of cardiac muscle has been studied using hyperpermeable cells from rat ventricles and sections of quickly frozen rat hearts. These preparations have been used to study maximum calcium-activated force, myosin ATPase activity, and the maximum velocity of unloaded shortening. Beta-adrenergic activity increases the amount of force and the ATPase activity in accordance with the concentration of the V1 isozyme of myosin. V3 activity is decreased at the same time. In tissues containing only V1, there is no change in maximum velocity in response to beta-adrenergic stimulation. These results indicate that beta-adrenergic stimulation recruits V1 force generators and probably regulates a transition between a calcium unresponsive and a calcium responsive force generator.     

Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3–2.8 µM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca²⁺ channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca²⁺ sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 µM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca²⁺ influx and activation systems. heart; electrophysiology; permeabilized muscle     

Expression of Green Fluorescent Protein Impairs the Force-Generating Ability of Isolated Rat Ventricular Cardiomyocytes

Green fluorescent protein (GFP) is widely used as a biologically inert expression marker for studying the effects of transgene expression in heart tissue, but its influence on the contractile function of cardiomyocytes has not yet been fully evaluated. We measured the contractile function of isolated rat ventricular myocytes before and after infection with a recombinant adenovirus expressing GFP (Adv-GFP). Myocytes infected with a non-transgene-containing adenovirus (Adv-Null) or uninfected myocytes (UI) served as controls. Using a carbon-fiber-based force-length measurement system for single cardiomyocytes, we evaluated the contractile function over a wide range of loading conditions including the shortening fraction (%FS) and maximal shortening velocity (Vmax) under the unloaded condition, and isometric force. At 24 hours after infection, nearly 80% of the Adv-GFP-infected myocytes expressed GFP. We found that the %FS and Vmax did not differ among the three groups, however, the isometric force showed a mild, but significant, decrease only in Adv-GFP myocytes (Adv-GFP: 29.1 ± 4.0 mN/mm²; Adv-Null: 42.8 ± 6.2 mN/mm²; UI: 47.1 ± 4.8 mN/mm²; p = 0.03). An evaluation of the contractile function of isolated cardiomyocytes under high load conditions revealed impaired isometric contractility by GFP expression. Adv-GFP expression may not be an ideal control for specific gene expression experiments in myocardial tissue.     

Muscle fiber size and function in elderly humans: a longitudinal study.

Cross-sectional studies are likely to underestimate age-related changes in skeletal muscle strength and mass. The purpose of this longitudinal study was to assess whole muscle and single muscle fiber alterations in the same cohort of 12 older (mean age: start of study 71.1+/-5.4 yr and end of study 80+/-5.3 yr) volunteers (5 men) evaluated 8.9 yr apart. No significant changes were noted at follow-up in body weight, body mass index, and physical activity. Muscle strength, evaluated using isokinetic dynamometry, and whole muscle specific force of the knee extensors were significantly lower at follow-up. This was accompanied by a significant reduction (5.7%) in cross-sectional area of the total anterior muscle compartment of the thigh as evaluated by computed tomography. Muscle histochemistry showed no significant changes in fiber type distribution or fiber area. Experiments with chemically skinned single muscle fibers (n=411) demonstrated no change in type I fiber size but an increase in IIA fiber diameter. A trend toward an increase in maximal force in both fiber types was observed. Maximum unloaded shortening velocity did not change. In conclusion, single muscle fiber contractile function may be preserved in older humans in the presence of significant alterations at the whole muscle level. This suggests that surviving fibers compensate to partially correct muscle size deficits in an attempt to maintain optimal force-generating capacity.     

Rheology of myocardium. The relation between force, velocity, sarcomere length and activation in rat cardiac muscle

The relations between force, shortening velocity and sarcomere length (F-V-SL) during cardiac contraction, underlie Starling's Law of the Heart. F-V-SL were investigated in isolated, intact and skinned trabeculae and myocytes from rat heart. SL and V were measured with laser diffraction techniques; F was measured with a silicon strain gauge. The "ascending" F-SL relation appeared to result from both length dependent sensitivity of the contractile system to activator calcium ions and the presence of restoring forces (Fr), residing in the collagen skeleton of the muscle. Fr increased exponentially with decreasing SL below slack length to 25% of maximal twitch force (Ft) at SL = 1.60 microns. V was inversely proportional to the load and attained a maximum at zero load (Vo). Vo increased with factors that increased F: [Ca++], SL, and time during the twitch. Vo reached a maximum and remained constant (13.5 microns/s) when F attained or exceeded 50% of its maximum value. Viscous force in the passive muscle increased with V to a maximum of 4% of Ft at V = 40 microns/s. The relation between Vo and these factors could be predicted by a model of contraction in which the measured visco-elastic properties of myocardium were incorporated, while the truly unloaded maximal velocity of sarcomere shortening was assumed to be independent of the level of activation of the contractile filaments. A model of the cardiac cycle which explains the relation between Frank's and Starling's laws is presented.     

Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure

Intrinsic muscle abnormalities affecting skeletal muscle are often reported during chronic heart failure (CHF). Because myosin is the molecular motor of force generation, we sought to determine whether its dysfunction contributes to skeletal muscle weakness in CHF and, if so, to identify the underlying causative factors. Severe CHF was induced in rats by aortic stenosis. In diaphragm and soleus muscles, we investigated in vitro mechanical performance, myosin-based actin filament motility, myosin heavy (MHC) and light (MLC) chain isoform compositions, MLC integrity, caspase-3 activation, and oxidative damage. Diaphragm and soleus muscles from CHF exhibited depressed mechanical performance. Myosin sliding velocities were 16 and 20% slower in CHF than in sham in diaphragm (1.9 +/- 0.1 vs. 1.6 +/- 0.1 microm/s) and soleus (0.6 +/- 0.1 vs. 0.5 +/- 0.1 microm/s), respectively (each P < 0.05). The ratio of slow-to-fast myosin isoform did not differ between sham and CHF. Immunoblots with anti-MLC antibodies did not detect the presence of protein fragments, and no activation of caspase-3 was evidenced. Immunolabeling revealed oxidative damage in CHF muscles, and MHC was the main oxidized protein. Lipid peroxidation and expression of oxidized MHC were significantly higher in CHF than in shams. In vitro myosin exposure to increasing ONOO(-) concentrations was associated with an increasing amount of oxidized MHC and a reduced myosin velocity. These data provide experimental evidence that intrinsic myosin dysfunction occurs in CHF and may be related to oxidative damage to myosin.     

Physiology of a microgravity environment invited review: microgravity and skeletal muscle.

Spaceflight (SF) has been shown to cause skeletal muscle atrophy; a loss in force and power; and, in the first few weeks, a preferential atrophy of extensors over flexors. The atrophy primarily results from a reduced protein synthesis that is likely triggered by the removal of the antigravity load. Contractile proteins are lost out of proportion to other cellular proteins, and the actin thin filament is lost disproportionately to the myosin thick filament. The decline in contractile protein explains the decrease in force per cross-sectional area, whereas the thin-filament loss may explain the observed postflight increase in the maximal velocity of shortening in the type I and IIa fiber types. Importantly, the microgravity-induced decline in peak power is partially offset by the increased fiber velocity. Muscle velocity is further increased by the microgravity-induced expression of fast-type myosin isozymes in slow fibers (hybrid I/II fibers) and by the increased expression of fast type II fiber types. SF increases the susceptibility of skeletal muscle to damage, with the actual damage elicited during postflight reloading. Evidence in rats indicates that SF increases fatigability and reduces the capacity for fat oxidation in skeletal muscles. Future studies will be required to establish the cellular and molecular mechanisms of the SF-induced muscle atrophy and functional loss and to develop effective exercise countermeasures.     

Unilateral lower limb suspension does not mimic bed rest or spaceflight effects on human muscle fiber function.

We used Ca2+-activated skinned muscle fibers to test the hypothesis that unilateral lower leg suspension (ULLS) alters cross-bridge mechanisms of muscle contraction. Soleus and gastrocnemius biopsies were obtained from eight subjects before ULLS, immediately after 12 days of ULLS (post-0 h), and after 6 h of reambulation (post-6 h). Post-0 h soleus fibers expressing type I myosin heavy chain (MHC) showed significant reductions in diameter, absolute and specific peak Ca2+-activated force, unloaded shortening velocity, and absolute and normalized peak power. Fibers obtained from the gastrocnemius were less affected by ULLS, particularly fibers expressing fast MHC isoforms. Post-6 h soleus fibers produced less absolute and specific peak force than did post-0 h fibers, suggesting that reambulation after ULLS induced cell damage. Like bed rest and spaceflight, ULLS primarily affects soleus over gastrocnemius fibers. However, in contrast to these other models, slow soleus fibers obtained after ULLS showed a decrease in unloaded shortening velocity and a greater reduction in specific force.     

Altered single cell force-velocity and power properties in exercise-trained rat myocardium.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. The ability of the myocardium to perform external work is a critical aspect of ventricular function, but previous studies of myocardial adaptation to exercise training have been limited to measurements of isometric tension or unloaded shortening velocity, conditions in which work output is zero. We measured force-velocity properties in single permeabilized myocyte preparations to determine the effect of exercise training on loaded shortening and power output. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise trained (T) groups. T rats underwent 11 wk of progressive treadmill exercise. Myocytes were isolated from T and C hearts, chemically skinned, and attached to a force transducer. Shortening velocity was determined during loaded contractions at 15 degrees C by using a force-clamp technique. Power output was calculated by multiplying force times velocity values. We found that unloaded shortening velocity was not significantly different in T vs. C myocytes (T = 1.43 muscle lengths/s, n = 46 myocytes; C = 1.12 muscle lengths/s, n = 43 myocytes). Training increased the velocity of loaded shortening and increased peak power output (peak power = 0.16 P/P(o) x muscle length/s for T myocytes; peak power = 0.10 P/P(o) x muscle length/s for C myocytes, where P/P(o) is relative tension). We found no effect of training on myosin heavy chain isoform content. These results suggest that training alters power output properties of single cardiac myocytes and that this adaptation may improve the work capacity of the myocardium.     

TNF-alpha acts via TNFR1 and muscle-derived oxidants to depress myofibrillar force in murine skeletal muscle

Tumor necrosis factor-alpha (TNF) diminishes specific force of skeletal muscle. To address the mechanism of this response, we tested the hypothesis that TNF acts via the type 1 (TNFR1) receptor subtype to increase oxidant activity and thereby depress myofibrillar function. Experiments showed that a single intraperitoneal dose of TNF (100 microg/kg) increased cytosolic oxidant activity (P < 0.05) and depressed maximal force of male ICR mouse diaphragm by approximately 25% within 1 h, a deficit that persisted for 48 h. Pretreating animals with the antioxidant Trolox (10 mg/kg) lessened oxidant activity (P < 0.05) and abolished contractile losses in TNF-treated muscle (P < 0.05). Genetic TNFR1 deficiency prevented the rise in oxidant activity and fall in force stimulated by TNF; type 2 TNF receptor deficiency did not. TNF effects on muscle function were evident at the myofibrillar level. Chemically permeabilized muscle fibers from TNF-treated animals had lower maximal Ca2+-activated force (P < 0.02) with no change in Ca2+ sensitivity or shortening velocity. We conclude that TNF acts via TNFR1 to stimulate oxidant activity and depress specific force. TNF effects on force are caused, at least in part, by decrements in function of calcium-activated myofibrillar proteins.     

What limits the velocity of fast-skeletal muscle contraction in mammals?

In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5 M KCl, 22 degrees C for kinetic assays of myosin, 200 mM ionic strength, 12 degrees C to measure Vo). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, KAD) and the rate of ATP-induced acto-HMM dissociation (controlled by K1k+2) under the same experimental condition used to measure Vo). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit Vo, whereas K1k+2 was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit Vo for both isoforms.     

Diaphragm contractile dysfunction in MyoD gene-inactivated mice

MyoD is one of four myogenic regulatory factors found exclusively in skeletal muscle. In an effort to better understand the role that MyoD plays in determining muscle contractile properties, we examined the effects of MyoD deletion on both diaphragmatic contractile properties and myosin heavy chain (MHC) phenotype. Regions of the costal diaphragm from wild-type and MyoD knockout [MyoD (-/-)] adult male BALB/c mice (n = 8/group) were removed, and in vitro diaphragmatic contractile properties were measured. Diaphragmatic contractile measurements revealed that MyoD (-/-) animals exhibited a significant (P < 0.05) downward shift in the force-frequency relationship, a decrement in maximal specific tension (P(o); -33%), a decline in maximal shortening velocity (V(max); -37%), and concomitant decrease in peak power output (-47%). Determination of MHC isoforms in the diaphragm via gel electrophoresis revealed that MyoD elimination resulted in a fast-to-slow shift (P < 0.05) in the MHC phenotype toward MHC types IIA and IIX in MyoD (-/-) animals. These data indicate that MyoD deletion results in a decrease in diaphragmatic submaximal force generation and P(o), along with decrements in both V(max) and peak power output. Hence, MyoD plays an important role in determining diaphragmatic contractile properties.