Oxidation of myosin heavy chain and reduction in force production in hyperthyroid rat soleus.

We tested the hypothesis that a force reduction in hyperthyroid rat soleus muscle would be associated with oxidative modification in myosin heavy chain (MHC). Daily injection of thyroid hormone [3,5,3'-triiodo-L-thyronine (T3)] for 21 days depressed isometric forces of whole soleus muscle across a range of stimulus frequencies (P < 0.01). In fiber bundles, hyperthyroidism also led to pronounced reductions (P < 0.01) in both K+ - and 4-chloro-m-cresol-induced contracture forces. The degrees of the reductions were similar between these two contractures that were induced by distinct reagents. Treatment with T3 elicited a significant decrease ( approximately 14%; P < 0.05) in the relative content of MHC contained in myofibrillar proteins. The content of carbonyl groups in myofibrillar protein extracts was elevated (P < 0.05) by approximately 50% in T3-treated muscles. Immunoblot analyses on T3-treated muscles showed a greater increase (106%; P < 0.05) of the carbonyl content in MHC than in myofibrillar protein extracts. These data suggest that in hyperthyroidism the decrease in force production of skeletal muscles may stem primarily from failure in myofibrillar protein function resulting from oxidative modification of MHC.     

Contractile and endurance properties of geniohyoid and diaphragm muscles

Despite the wealth of information about the neural control of pharyngeal dilator muscles, little is known about their intrinsic physiological properties. In the present study the in situ isometric contractility and endurance of a pharyngeal dilator, the geniohyoid muscle, were compared with properties of the diaphragm in 12 anesthetized artificially ventilated cats. The contraction time (means +/- SE) of the geniohyoid (27 +/- 2 ms) was shorter than that of the diaphragm (36 +/- 3 ms; P less than 0.0005), as was the half-relaxation time (29 +/- 2 vs. 45 +/- 4 ms; P less than 0.002). The faster contraction and relaxation of the geniohyoid compared with the diaphragm were appropriately reflected in the shape of the force-frequency curves for the two muscles, with that of the geniohyoid located to the right of the diaphragm force-frequency curve. The endurance properties of the two muscles were assessed using repetitive stimulation at 40 Hz in trains lasting 0.33 s, with one train repeated every second. The ratio of force at the end of 2 min of repetitive stimulation to initial force was 0.67 +/- 0.06 for the geniohyoid and 0.15 +/- 0.03 for the diaphragm (P less than 0.00001). After the repetitive stimulation, the muscle force generated in response to a range of stimulus frequencies was reduced to a greater extent for the diaphragm than for the geniohyoid muscle. These results indicate that the geniohyoid muscle has a faster physiological profile than does the diaphragm yet is relatively resistant to fatigue when driven at high rates.     

Diaphragm single-fiber weakness and loss of myosin in congestive heart failure rats

Diaphragm weakness commonly occurs in patients with congestive heart failure (CHF) and is an independent predictor of mortality. However, the pathophysiology of diaphragm weakness is poorly understood. We hypothesized that CHF induces diaphragm weakness at the single-fiber level by decreasing myosin content. In addition, we hypothesized that myofibrillar Ca(2+) sensitivity is decreased and cross-bridge kinetics are slower in CHF diaphragm fibers. Finally, we hypothesized that loss of myosin in CHF diaphragm weakness is associated with increased proteolytic activities of caspase-3 and the proteasome. In skinned diaphragm single fibers of rats with CHF, induced by left coronary artery ligation, maximum force generation was reduced by approximately 35% (P < 0.01) compared with sham-operated animals for slow, 2a, and 2x fibers. In these CHF diaphragm fibers, myosin heavy chain content per half-sarcomere was concomitantly decreased (P < 0.01). Ca(2+) sensitivity of force generation and the rate constant of tension redevelopment were significantly reduced in CHF diaphragm fibers compared with sham-operated animals for all fiber types. The cleavage activity of the proteolytic enzyme caspase-3 and the proteasome were approximately 30% (P < 0.05) and approximately 60% (P < 0.05) higher, respectively, in diaphragm homogenates from CHF rats than from sham-operated rats. The present study demonstrates diaphragm weakness at the single-fiber level in a myocardial infarct model of CHF. The reduced maximal force generation can be explained by a loss of myosin content in all fiber types and is associated with activation of caspase-3 and the proteasome. Furthermore, CHF decreases myofibrillar Ca(2+) sensitivity and slows cross-bridge cycling kinetics in diaphragm fibers.     

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.     

Diaphragmatic free radical generation increases in an animal model of heart failure.

Heart failure evokes diaphragm weakness, but the mechanism(s) by which this occurs are not known. We postulated that heart failure increases diaphragm free radical generation and that free radicals trigger diaphragm dysfunction in this condition. The purpose of the present study was to test this hypothesis. Experiments were performed using halothane-anesthetized sham-operated control rats and rats in which myocardial infarction was induced by ligation of the left anterior descending coronary artery. Animals were killed 6 wk after surgery, the diaphragms were removed, and the following were assessed: 1) mitochondrial hydrogen peroxide (H2O2) generation, 2) free radical generation in resting and contracting intact diaphragm using a fluorescent-indicator technique, 3) 8-isoprostane and protein carbonyls (indexes of free radical-induced lipid and protein oxidation), and 4) the diaphragm force-frequency relationship. In additional experiments, a group of coronary ligation animals were treated with polyethylene glycol-superoxide dismutase (PEG-SOD, 2,000 units x kg(-1) x day(-1)) for 4 wk. We found that coronary ligation evoked an increase in free radical formation by the intact diaphragm, increased diaphragm mitochondrial H2O2 generation, increased diaphragm protein carbonyl levels, and increased diaphragm 8-isoprostane levels compared with controls (P < 0.001 for the first 3 comparisons, P < 0.05 for 8-isoprostane levels). Force generated in response to 20-Hz stimulation was reduced by coronary ligation (P < 0.05); PEG-SOD administration restored force to control levels (P < 0.03). These findings indicate that cardiac dysfunction due to coronary ligation increases diaphragm free radical generation and that free radicals evoke reductions in diaphragm force generation.     

Mechanical ventilation-induced oxidative stress in the diaphragm.

Prolonged mechanical ventilation (MV) results in oxidative damage in the diaphragm; however, it is unclear whether this MV-induced oxidative injury occurs rapidly or develops slowly over time. Furthermore, it is unknown whether both soluble (cytosolic) and insoluble (myofibrillar) proteins are equally susceptible to oxidation during MV. These experiments tested two hypotheses: 1). MV-induced oxidative injury in the diaphragm occurs within the first 6 h after the initiation of MV; and 2). MV is associated with oxidative modification of both soluble and insoluble proteins. Adult Sprague-Dawley rats were randomly divided into one of seven experimental groups: 1) control (n = 8); 2) 3-h MV (n = 8); 3). 6-h MV (n = 6); 4). 18-h MV (n = 8); 5). 3-h anesthesia-spontaneous breathing (n = 8); 6). 6-h anesthesia-spontaneous breathing (n = 6); and 7). 18-h anesthesia-spontaneous breathing (n = 8). Markers of oxidative injury in the diaphragm included the measurement of reactive (protein) carbonyl derivatives (RCD) and total lipid hydroperoxides. Three hours of MV did not result in oxidative injury in the diaphragm. In contrast, both 6 and 18 h of MV promoted oxidative injury in the diaphragm, as indicated by increases in both protein RCD and lipid hydroperoxides. Electrophoretic separation of soluble and insoluble proteins indicated that the MV-induced accumulation of RCD was limited to insoluble proteins with molecular masses of approximately 200, 120, 80, and 40 kDa. We conclude that MV results in a rapid onset of oxidative injury in the diaphragm and that insoluble proteins are primary targets of MV-induced protein oxidation.     

Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties.

Initial experiments were conducted using an in situ rat tibialis anterior (TA) muscle preparation to assess the influence of dietary antioxidants on muscle contractile properties. Adult Sprague-Dawley rats were divided into two dietary groups: 1) control diet (Con) and 2) supplemented with vitamin E (VE) and alpha-lipoic acid (alpha-LA) (Antiox). Antiox rats were fed the Con rats' diet (AIN-93M) with an additional 10,000 IU VE/kg diet and 1.65 g/kg alpha-LA. After an 8-wk feeding period, no differences existed (P > 0.05) between the two dietary groups in maximum specific tension before or after a fatigue protocol or in force production during the fatigue protocol. However, in unfatigued muscle, maximal twitch tension and tetanic force production at stimulation frequencies < or = 40 Hz were less (P < 0.05) in Antiox animals compared with Con. To investigate which antioxidant was responsible for the depressed force production, a second experiment was conducted using an in vitro rat diaphragm preparation. Varying concentrations of VE and dihydrolipoic acid, the reduced form of alpha-LA, were added either individually or in combination to baths containing diaphragm muscle strips. The results from these experiments indicate that high levels of VE depress skeletal muscle force production at low stimulation frequencies.     

Cyclic GMP is a second messenger by which nitric oxide inhibits diaphragm contraction

We have shown that endogenous nitrogen oxides (NOx) modulate excitation-contraction coupling in diaphragm. Because cyclic GMP (cGMP) is a second messenger for nitric oxide (NO) inhibition of smooth muscle contraction, we rested the hypothesis that NO acts via cGMP in diaphragm. Fiber bundles from rat diaphragm were studied in vitro. Immunohistochemical analysis using a cGMP-specific monoclonal antibody confirmed the presence of cGMP in the subsarcolemmal region, near nitric oxide synthase (NOS). cGMP measured by ELISA in control muscle (0.27 pmol/mg +/- 0.01 SE) was significantly increased by the NO donor S-nitroso-N-acetylcysteine 1 mM (0.55+/-0.05; N = 6; P < 0.001). Contractile studies showed that the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) 10 mM increased submaximal (40 Hz) tetanic force (P < 0.0001). L-NNA effects were exaggerated by the guanylate cyclase inhibitor LY83583 5-10 microM; force at 40 Hz was increased (P < 0.001). L-NNA effects were partially reversed by 8-bromo-cGMP 1 mM (8-Br-GMP; a cell-permeable cGMP analogue; P < 0.0001) or dipyridamole 10 microM (DPM; a phosphodiesterase inhibitor; P < 0.0001). 8-Br-GMP and DPM produced more-complete L-NNA reversal in combination (P < 0.0001). We conclude that cGMP functions as a second messenger by which NO inhibits diaphragm contraction.     

Diaphragm Atrophy and Contractile Dysfunction in a Murine Model of Pulmonary Hypertension

Pulmonary hypertension (PH) causes loss of body weight and inspiratory (diaphragm) muscle dysfunction. A model of PH induced by drug (monocrotaline, MCT) has been extensively used in mice to examine the etiology of PH. However, it is unclear if PH induced by MCT in mice reproduces the loss of body weight and diaphragm muscle dysfunction seen in patients. This is a pre-requisite for widespread use of mice to examine mechanisms of cachexia and diaphragm abnormalities in PH. Thus, we measured body and soleus muscle weight, food intake, and diaphragm contractile properties in mice after 6–8 weeks of saline (control) or MCT (600 mg/kg) injections. Body weight progressively decreased in PH mice, while food intake was similar in both groups. PH decreased (P<0.05) diaphragm maximal isometric specific force, maximal shortening velocity, and peak power. Protein carbonyls in whole-diaphragm lysates and the abundance of select myofibrillar proteins were unchanged by PH. Our findings show diaphragm isometric and isotonic contractile abnormalities in a murine model of PH induced by MCT. Overall, the murine model of PH elicited by MCT mimics loss of body weight and diaphragm muscle weakness reported in PH patients.     

Developmental changes in neuromuscular transmission in the rat diaphragm.

Neuromuscular transmission was studied in diaphragms from rats of three ages, 4-7 days old, 11-12 days old, and adults with the use of an in vitro phrenic nerve-hemidiaphragm preparation. Each hemidiaphragm was stimulated via either muscle or nerve with 1-s stimulus trains at frequencies from 10 to 100 Hz. The patterns of force development obtained in response to the two routes of stimulation were compared for each group. Diaphragms from adults developed maximum force in response to stimulation of approximately 40 Hz with no significant decrease in force at higher frequencies. Within each stimulus train, once peak force was achieved, it was maintained for the remainder of the stimulus and responses to nerve and muscle stimulation were almost identical. In contrast, diaphragms from 4- to 7-day-old rats developed maximum force at approximately 20 Hz; stimulation at greater than or equal to 60 Hz induced significantly less peak force. This decrease in peak force at higher frequencies was significantly larger for nerve than for muscle stimulation. In addition, during each nerve stimulus train diaphragms from 4- to 7-day-old rats were unable to maintain peak force, which decreased at frequencies greater than 20 Hz. The decrease in force reached approximately 50% of peak at stimulation frequencies greater than or equal to 60 Hz. Diaphragms from 11- to 12-day-old rats showed intermediate responses. Based on the responses to phrenic nerve stimulation, we conclude that the neonatal rat diaphragm shows marked neuromuscular transmission failure that is not seen in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of contractile force and mass in the senescent diaphragm with beta(2)-agonist treatment.

Aging is associated with a decrease in diaphragmatic maximal tetanic force production (P(o)) in senescent rats. Treatment with the beta(2)-agonist clenbuterol (CB) has been shown to increase skeletal muscle mass and P(o) in weak locomotor skeletal muscles from dystrophic rodents. It is unknown whether CB can increase diaphragmatic mass and P(o) in senescent rats. Therefore, we tested the hypothesis that CB treatment will increase specific P(o) (i.e., force per cross-sectional area) and mass in the diaphragm of old rats. Young (5 mo) and old (23 mo) male Fischer 344 rats were randomly assigned to one of the following groups (n = 10/group): 1) young CB treated; 2) young control; 3) old CB treated; and 4) old control. Animals were injected daily with either CB (2 mg/kg) or saline for 28 days. CB increased (P < 0.05) the mass of the costal diaphragm in both young and old animals. CB treatment increased diaphragmatic-specific P(o) in old animals (approximately 15%; P < 0.05) but did not alter (P > 0.05) diaphragmatic-specific P(o) in young animals. Biochemical analysis indicated that the improved maximal specific P(o) in the diaphragm of CB-treated old animals was not due to increased myofibrillar protein concentration. Analysis of the myosin heavy chain (MHC) content of the costal diaphragm revealed a CB-induced increase (P < 0.05) in type IIb MHC and a decrease in type I, IIa, and IIx MHC in both young and old animals. These data support the hypothesis that CB treatment can restore the age-associated decline in both diaphragmatic-specific P(o) and muscle mass.     

Effects of uncompensated and compensated metabolic acidosis on canine diaphragm

We investigated the effects of metabolic acidosis and compensated metabolic acidosis on force of contraction of the diaphragm in anesthetized dogs. Mechanically ventilated animals were prepared with an open thorax. A balloon was positioned beneath the diaphragm to measure transdiaphragmatic pressure (Pdi), and a plaster cast was placed around the abdomen to maintain length and geometry of the diaphragm. The force of contraction was evaluated by measuring Pdi during supramaximal phrenic stimulation at different frequencies and also during spontaneous inspiratory efforts. In 13 dogs with an arterial pH (pHa) of 7.38 and arterial PCO2 (PaCO2) of 36.5 Torr, metabolic acidosis was produced by infusion of HCl until pHa equaled 6.98 and PaCO2 equaled 36.4 Torr. Pdi at all frequencies greater than 10 Hz was significantly reduced (P less than 0.05). The dogs were then hyperventilated until pHa was 7.34 and PaCO2 was 12.8 Torr. Pdi was significantly reduced again at all frequencies (P less than 0.05) except 5 Hz. The percent reduction in Pdi by compensated acidosis was significantly greater at low-frequency stimulation than at high (P less than 0.05). Similar qualitative results were observed during spontaneous inspiratory efforts where Pdi was compared at constant magnitudes of diaphragmatic electromyograms. Twitch characteristics revealed that metabolic acidosis led to a significant shortening of twitch relaxation time (P less than 0.05), and compensated metabolic acidosis added to this effect a significant decrease in twitch amplitude (P less than 0.05).     

Effects of buthionine sulfoximine treatment on diaphragm contractility and SR Ca2+ pump function in rats.

The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment (n = 10) for 10 days (20 mM in drinking water) reduced (P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) (n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased (P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm(2)) was increased (P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased (P < 0.05) in BSO compared with CTL. Both Ca(2+) uptake and maximal SERCA activity (mumol.g protein(-1).min(-1)) measured in diaphragm homogenates that were prepared at rest were increased (P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase (P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca(2+) uptake and maximal SERCA activity were increased (P < 0.05) in CTL but not (P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.     

Muscle weakness following sustained and rhythmic isometric contractions in man

The effects of sustained and rhythmically performed isometric contractions on electrically evoked twitch and tetanic force generation of the triceps surae have been investigated in 4 healthy male subjects. The isometric contractions were performed separately and on different occasions at 30%, 60% and 100% of the force of maximal voluntary contraction (MVC). The area under the maximal voluntary contraction (MVC) force/time curve during the rhythmic and sustained contractions was the same for each experiment. The results showed that following rhythmic isometric exercise there was a small decrease in low (10 and 20 Hz) and high (40 Hz) frequency tetanic tension which was associated with % MVC. However, there was no change in the 20/40 ratio of tetanic forces, MVC or the contraction times and force of the maximal twitch. In contrast, following sustained isometric exercise tetanic forces were markedly reduced, particularly at low frequencies of stimulation. The 20/40 ratio decreased and the induced muscle weakness was greater at 30% than 60% or 100% MVC. The performance of sustained isometric contractions also effected a decrease in contraction time of the twitch and MVC. The results are in accord with previous findings for dynamic work (Davies and White 1982), and show that if isometric exercise is performed rhythmically the effect on tetanic tensions is small and there is no evidence of a preferential loss of electrically evoked force at either high or low frequencies of stimulation following the contractions. For sustained contractions, however, the opposite is true, the ratio of 20/40 Hz forces is markedly reduced and following 30% sustained MVC there is a significant (p less than 0.05) change in the time to peak tension (TPT) of the maximal twitch.     

Sphingomyelinase depresses force and calcium sensitivity of the contractile apparatus in mouse diaphragm muscle fibers

Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2',6'-dimethyl-Tyr-Lys-Phe-NH(2), decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m(2): control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus.     

Increased fatigue of isovelocity vs. isometric contractions of canine diaphragm

A comparison of fatigue as a loss of force with repeated contractions over time was performed in canine respiratory muscle by isometric (nonshortening) and isovelocity (shortening) contractions. In situ diaphragm muscle strips were attached to a linear ergometer and electrically stimulated (30 or 40 Hz) via the left phrenic nerve to produce either isometric (n = 12) or isovelocity (n = 12) contractions (1.5 s) from optimal muscle length (Lo = 8.8 cm). Similar velocities of shortening between isovelocity experiments [0.19 +/- 0.02 (SD) Lo/S] were produced by maximizing the mean power output (Wmax = 210 +/- 27 mW/cm2) that could be developed over 1.5 s when displacement was approximately 0.30 Lo. Initial peak isometric tension was 1.98 kg/cm2, whereas initial peak isovelocity tension was 1.84 kg/mc2 (P less than 0.01) or 93% of initial isometric tension. Fatigue trials of 5 min were conducted on muscles contracting at a constant duty cycle (0.43). At the end of the trials, peak isovelocity tension had fallen to 50% of initial isometric tension (P less than 0.01), whereas peak isometric tension had only fallen by 27%. These results indicate that muscle shortening during force production has a significant influence on diaphragm muscle fatigue. We conclude that the effects of shortening on fatigue must be considered in models of respiratory muscle function, because these muscles typically shorten during breathing.     

ACTN3 and MLCK genotype associations with exertional muscle damage.

Strenuous exercise results in damage to skeletal muscle that is manifested in delayed muscle pain, prolonged strength loss, and increases in muscle proteins in the blood, especially creatine kinase (CK) and myoglobin (Mb). Some individuals experience profound changes in these variables in response to standard laboratory exercise or recreational activities. We proposed that variations in genes coding for two myofibrillar proteins [alpha-actinin 3 (ACTN3) and myosin light chain kinase (MLCK)] may explain the large variability in the response to muscle-damaging exercise. We hypothesized that subjects with specific single nucleotide polymorphisms (SNPs) in ACTN3 and MLCK would show a greater loss in muscle strength and/or a greater increase in blood CK and Mb in response to eccentric exercise. Blood from 157 subjects who performed a standard elbow flexion eccentric exercise protocol was tested for association between genotypes of ACTN3 (1 SNP tested: R577X) and MLCK (2 SNPs tested: C49T and C37885A) and changes in blood CK and Mb and isometric strength. Subjects possessing the ACTN3-deficient genotype (XX) had lower baseline CK compared with the heterozygotes (P = 0.035). After the eccentric exercise, those subjects homozygous for the MLCK 49T rare allele had a significantly greater increase in CK and Mb (P < 0.01) compared with the heterozygotes, and those heterozygous for MLCK C37885A had a significantly greater increase in CK compared with the homozygous wild type (P < 0.05). There was only one subject homozygous for the rare MLCK 37885A allele. MLCK C37885A was also associated with postexercise strength loss (P < 0.05); the heterozygotes demonstrated greater strength loss compared with the homozygous wild type (CC). These results show that variations in genes coding for specific myofibrillar proteins influence phenotypic responses to muscle damaging exercise.     

Reversal of muscle fatigue during 16 h of heavy intermittent cycle exercise.

This study examined the effects of extended sessions of heavy intermittent exercise on quadriceps muscle fatigue and weakness. Twelve untrained volunteers (10 men and 2 women), with a peak oxygen consumption of 44.3 +/- 2.3 ml.kg(-1).min(-1), exercised at approximately 91% peak oxygen consumption for 6 min once per hour for 16 h. Muscle isometric properties assessed before and after selected repetitions (R1, R2, R4, R7, R12, and R15) were used to quantitate fatigue (before vs. after repetitions) and weakness (before vs. before repetitions). Muscle fatigue at R1 was indicated by reductions (P < 0.05) in peak twitch force (135 +/- 13 vs. 106 +/- 11 N) and by a reduction (P < 0.05) in the force-frequency response, which ranged between approximately 53% at 10 Hz (113 +/- 12 vs. 52.6 +/- 7.4 N) and approximately 17% at 50 Hz (324 +/- 27 vs. 270 +/- 30 N). No recovery of force, regardless of stimulation frequency, was observed during the 54 min between R1 and R2. At R2 and for all subsequent repetitions, no reduction in force, regardless of stimulation frequency, was generally found after the exercise. The only exception was for R2, where, at 20 Hz, force was reduced (P < 0.05) by 18%. At R15, force before repetitions for high frequencies (i.e., 100 Hz) returned to R1 (333 +/- 29 vs. 324 +/- 27 N), whereas force at low frequency (i.e., 10 Hz) was only partially (P < 0.05) recovered (113 +/- 12 vs. 70 +/- 6.6 N). It is concluded that multiple sessions of heavy exercise can reverse the fatigue noted early and reduce or eliminate weakness depending on the frequency of stimulation.     

Muscle shortening increases sensitivity of fatigue to severe hypoxia in canine diaphragm.

The effects of inspired O2 on diaphragm tension development during fatigue were assessed using isovelocity (n = 6) and isometric (n = 6) muscle contractions performed during a series of exposures to moderate hypoxia [fraction of inspired O2 (FIO2) = 0.13], hyperoxia (FIO2 = 1), and severe hypoxia (FIO2 = 0.09). Muscle strips were created in situ from the canine diaphragm, attached to a linear ergometer, and electrically stimulated (30 Hz) to contract (contraction = 1.5 s/relaxation = 2 s) from optimal muscle length (Lo = 8.9 cm). Isovelocity contractions shortened to 0.70 Lo, resulting in a mean power output of 210 mW/cm2. Fatigue trials of 35 min duration were performed while inspired O2 was sequentially changed between the experimental mixtures and normoxia (FIO2 = 0.21) for 5-min periods. In this series, severe hypoxia consistently decreased isovelocity tension development by an average of 0.1 kg/cm2 (P less than 0.05), which was followed by a recovery of tension (P less than 0.05) on return to normoxia. These responses were not consistently observed in isometric trials. Neither isovelocity nor isometric tension development was influenced by moderate hypoxia or hyperoxia. These results demonstrate that the in situ diaphragm is relatively insensitive to rapid changes in O2 supply over a broad range and that the tension development of the shortening diaphragm appears to be more susceptible to severe hypoxia during fatigue. This may reflect a difference in either the metabolic or blood flow characteristics of shortening contractions of the diaphragm.     

Changes in rat muscle with compensatory overload occur in a sequential manner

The present study was initiated to determine the time course of changes in the profile of selected skeletal muscle myofibril proteins during compensatory overload. Whole muscle isometric contractile properties were measured to assess the physiological consequences of the overload stimulus. Compensatory overload of plantaris muscle of rats was induced by surgical ablation of the synergistic soleus and gastrocnemius muscles. Myosin light chain (LC) and tropomyosin (TM) compositions of control (CP) and overloaded plantaris (OP) muscles were determined by electrophoresis and myofibrillar ATPase assays were performed to assess changes in contractile protein interactions. Within one week of overload decreases in the alpha:beta TM ratio and myofibrillar ATPase activity were observed. Following 30 days of overload, a transition in type II to type I fibres was associated with an increase in slow myosin LC1. Interestingly, after 77 days of overload, the TM subunit ratio returned to one resembling a fast twitch muscle. It is proposed that the early and transitory changes in the TM subunits of OP, as well as the rapid initial depression in maximum tetanic isometric force and myofibrillar ATPase activity may be explained as a result of muscle fibre degeneration-regeneration. We propose that alterations in protein expression induced by compensatory overload reflect both degenerative-regenerative change and increased neuromuscular activity.