Skeletal muscle force and actomyosin ATPase activity reduced by nitric oxide donor

Perkins, William J., Young-Soo Han, and Gary C. Sieck.Skeletal muscle force and actomyosin ATPase activity reduced bynitric oxide donor. J. Appl. Physiol.83(4): 1326-1332, 1997.Nitric oxide (NO) may exert directeffects on actin-myosin cross-bridge cycling by modulating criticalthiols on the myosin head. In the present study, the effects of the NOdonor sodium nitroprusside (SNP; 100 µM to 10 mM) on mechanicalproperties and actomyosin adenosinetriphosphatase (ATPase) activity ofsingle permeabilized muscle fibers from the rabbit psoas muscle weredetermined. The effects ofN-ethylmaleimide (NEM; 5-250µM), a thiol-specific alkylating reagent, on mechanical properties ofsingle fibers were also evaluated. Both NEM (25 µM) and SNP (1mM) significantly inhibited isometric force and actomyosin ATPaseactivity. The unloaded shortening velocity of SNP-treated single fiberswas decreased, but to a lesser extent, suggesting that SNP effects onisometric force and actomyosin ATPase were largely due to decreased cross-bridge recruitment. The calcium sensitivity of SNP-treated singlefibers was also decreased. The effects of SNP, but not NEM, on forceand actomyosin ATPase activity were reversed by treatment with 10 mMDL-dithiothreitol, athiol-reducing agent. We conclude that the NO donor SNP inhibitscontractile function caused by reversible oxidation of contractileprotein thiols.

     

Force deficits in skeletal muscle after delayed reinnervation

Using a rat hindlimb model, the authors tested the hypothesis that, in muscles reinnervated after long-term denervation, atrophy-dependent and atrophy-independent mechanisms operate independently to produce force deficits. In adult rats, gastrocnemius muscles were subjected to denervation via tibial nerve transection. Reconstruction of the nerve lesion was delayed for periods ranging from 2 weeks to 1 year. After a minimum recovery period of 6 months after nerve repair, muscle mass and maximum isometric tetanic force were measured and specific force was calculated for each muscle (n = 40 muscles from 23 animals). After recovery, observed deficits in muscle mass and maximum tetanic force were directly proportional to the denervation interval. On the other hand, the deficit in specific force was not proportional to the denervation interval; all groups in which the nerve reconstruction was delayed for a month or longer demonstrated a deficit of 30 percent to 50 percent. These data support our hypothesis that, after prolonged denervation followed by reinnervation, the magnitude of the deficit in whole muscle force does not parallel the deficit in specific force. These data support the idea that mechanisms governing muscle atrophy are independent of those resulting in specific force deficits.     

Skeletal muscle protein loss due to D-penicillamine results from reduced protein synthesis

Reports in the literature indicate that the trifunctional amino acid D-penicillamine (D-P) induces a variety of muscle abnormalities, although the mechanisms are unknown. We hypothesised that defects may also arise due to the effects of D-P on rates of protein synthesis, possibly via changes in muscle metal composition. Male Wistar rats were injected with D-P at doses of 50 and 500 mg/kg body weight, i.p. Rats designated as controls were injected with 0.15 mol/l NaCl. After 24 h, there were reductions in muscle protein contents, protein synthetic capacities (RNA:protein ratio), fractional rates of protein synthesis, synthesis rates per unit RNA and synthesis rates per unit DNA in skeletal muscles of D-P treated rats. There were no statistically significant differences between the responses of the muscles containing a predominance of either Type I (represented by the soleus) or Type II (represented by the plantaris) fibres. In general, intracellular amino acids were not significantly affected by D-P treatment. Changes in muscle metals included significant reductions in copper, iron and manganese, without alterations in zinc or magnesium. In liver D-P reduced copper and iron though zinc, manganese and magnesium were unaffected. These effects of D-P on muscle may have been direct, as plasma indices of liver (activities of alkaline phosphatase and alanine aminotransferase) and kidney (urea, creatinine and electrolytes) damage were not significantly altered by D-P treatment. Plasma levels of corticosterone, insulin and free T3 were also not significantly affected by D-P treatment. Muscle protein carbonyl concentrations, an index of free radical activity, were similarly unaffected. This is the first report of reduced rates of muscle protein synthesis in D-P treatment. Our data suggests that the reduced rates of muscle protein synthesis may contribute to, or reflect, the muscle abnormalities observed in patients undergoing D-P treatment.     

Skeletal muscle and motor deficits in Neurofibromatosis Type 1

Neurofibromatosis Type 1 (NF1) is a genetic neurocutaneous disorder with multisystem manifestations, including a predisposition to tumor formation and bone dysplasias. Studies over the last decade have shown that NF1 can also be associated with significant motor deficits, such as poor coordination, low muscle tone, and easy fatigability. These have traditionally been ascribed to developmental central nervous system and cognitive deficits. However, recent preclinical studies have also illustrated a primary role for the NF1 gene product in muscle growth and metabolism; these findings are consistent with clinical studies demonstrating reduced muscle size and muscle weakness in individuals with NF1. Currently there is no evidence-based intervention for NF1 muscle and motor deficiencies; this review identifies key research areas where improved mechanistic understanding could unlock new therapeutic options.     

Denervated muscle fibers explain the deficit in specific force following reinnervation of the rat extensor digitorum longus muscle

The authors tested the hypothesis that, after denervation and reinnervation of skeletal muscle, observed deficits in specific force can be completely attributed to the presence of denervated muscle fibers. The peroneal nerve innervating the extensor digitorum longus muscle in rats was sectioned and the distal stump was coapted to the proximal stump, allowing either a large number of motor axons (nonreduced, n = 12) or a drastically reduced number of axons access to the distal nerve stump (drastically reduced, n = 18). A control group of rats underwent exposure of the peroneal nerve, without transection, followed by wound closure (control, n = 9). Four months after the operation, the maximum tetanic isometric force (Fo) of the extensor digitorum longus muscle was measured in situ and the specific force (sFo) was calculated. Cross-sections of the muscles were labeled for neural cell adhesion molecule (NCAM) protein to distinguish between innervated and denervated muscle fibers. Compared with extensor digitorum longus muscles from rats in the control (295 +/- 11 kN/m2) and nonreduced (276 +/- 12 kN/m2) groups, sFo of the extensor digitorum longus muscles from animals in the drastically reduced group was decreased (227 +/- 15 kN/m2, p < 0.05). The percentage of denervated muscle fibers in the extensor digitorum longus muscles from animals in the drastically reduced group (18 +/- 3 percent) was significantly higher than in the control (3 +/- 1 percent) group, but not compared with the nonreduced (9 +/- 2 percent) group. After exclusion of the denervated fibers, sFo did not differ between extensor digitorum longus muscles from animals in the drastically reduced (270 +/- 20 kN/m2), nonreduced (301 +/- 13 kN/m2), or control (303 +/- 10 kN/m2) groups. The authors conclude that, under circumstances of denervation and rapid reinnervation, the decrease in sFo of muscle can be attributed to the presence of denervated muscle fibers.     

Skeletal muscle response to spaceflight, whole body suspension, and recovery in rats

Comparisons of soleus and extensor digitorum longus (EDL) muscles from male Sprague-Dawley rats (350-400 g) after 7 days of weightlessness, 7 and 14 days of whole body suspension (WBS), and 7 days of recovery from WBS and from vivarium controls were made. Muscle mass loss of approximately 30% was observed in soleus after 7 and 14 days of WBS. Measurement of slow- and fast-twitch fibers showed significant alterations. Reductions in cross-sectional areas and increases in fiber densities in soleus after spaceflight and WBS were related to previous findings of muscle atrophy during unloading. Capillary density also showed a marked increase with unloading. Seven days of weightlessness were sufficient to effect a 20 and 15% loss in absolute muscle mass in soleus and EDL, respectively. However, the antigravity soleus was more responsive in terms of cross-sectional area reductions. After 7 days of recovery from WBS, with normal ambulatory loading, the parameters studied showed a reversal to control levels. Muscle plasticity, in terms of fiber and capillary responses, indicated differences in responses in the two types of muscles and further amplified that antigravity posture muscles are highly susceptible to unloading. Studies of recovery from spaceflight for both muscle metabolism and microvascular modifications are further justified.     

Skeletal muscle ouabain binding sites are reduced in rats with chronic heart failure.

Intrinsic skeletal muscle abnormalities decrease muscular endurance in chronic heart failure (CHF). In CHF patients, the number of skeletal muscle Na(+)-K(+) pumps that have a high affinity for ouabain (i.e., the concentration of [(3)H]ouabain binding sites) is reduced, and this reduction is correlated with peak oxygen uptake. The present investigation determined whether the concentration of skeletal muscle [(3)H]ouabain binding sites found during CHF is related to 1) severity of the disease state, 2) muscle fiber type composition, and/or 3) endurance capacity. Four muscles were chosen that represented slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and mixed fiber types. Measurements were obtained 8-10 wk postsurgery in 23 myocardial infarcted (MI) and 18 sham-operated control (sham) rats. Eighteen rats had moderate left ventricular (LV) dysfunction [LV end-diastolic pressure (LVEDP) < 20 mmHg], and five had severe LV dysfunction (LVEDP > 20 mmHg). Rats with severe LV dysfunction had significant pulmonary congestion and were likely in a chronic state of compensated congestive failure as indicated by an approximately twofold increase in both lung and right ventricle weight. Run time to fatigue and maximal oxygen uptake (VO(2 max)) were significantly reduced ( downward arrow39 and downward arrow28%, respectively) in the rats with severe LV dysfunction and correlated with the magnitude of LV dysfunction as indicated by LVEDP (run time: r = 0.60, n = 21, P < 0.01 and VO(2 max): r = 0.93, n = 13, P < 0.01). In addition, run time to fatigue was significantly correlated with VO(2 max) (r = 0.87, n = 15, P < 0.01). The concentration of [(3)H]ouabain binding sites (B(max)) was significantly reduced (21-28%) in the three muscles comprised primarily of oxidative fibers [soleus: 259 +/- 14 vs. 188 +/- 17; plantaris: 295 +/- 17 vs. 229 +/- 18; red portion of gastrocnemius: 326 +/- 17 vs. 260 +/- 14 pmol/g wet tissue wt]. In addition, B(max) was significantly correlated with VO(2 max) (soleus: r = 0.54, n = 15, P < 0.05; plantaris: r = 0.59, n = 15, P < 0.05; red portion of gastrocnemius: r = 0.65, n = 15, P < 0.01). These results suggest that downregulation of Na(+)-K(+) pumps that possess a high affinity for ouabain in oxidative skeletal muscle may play an important role in the exercise intolerance that attends severe LV dysfunction in CHF.     

Sprint training attenuates the deficits of force and dynamic stiffness in rat soleus muscle caused by eccentric contractions

We investigated whether sprint training attenuates the deficits in force and dynamic stiffness caused by eccentric contractions to the soleus muscles of Wistar rats. Two groups of male rats were analyzed: sedentary (C, n=8) and trained (T, n=8). T rats were sprint trained for 10 weeks. Subsequently, the right soleus muscles of rats were freed under anesthesia, leaving the bone insertion and blood supply intact. Eccentric contractions were induced by lengthening muscles during tetanic contractions. Force and dynamic stiffness were tested before and after 20 rounds of eccentric contractions. Tension decline was analyzed using a two-state model (first-order kinetics) in the context of Kramer's theory. Training improved the twitch tension (C, 6.44+/-0.6N/cm(2); T, 10.90+/-0.8N/cm(2)), tetanic force (C, 61.74+/-0.6N/cm(2); T, 85.62+/-0.8N/cm(2)), and increased the dynamic stiffness (C, 41.28+/-1.0N/cm(2); T, 49.56+/-3.2N/cm(2)). Twitch tension after eccentric contractions declined to 73% and 75% in C and T groups, respectively, while tetanic tension decreased to 60% and 36% in C and T groups, respectively. After eccentric contractions, dynamic stiffness decreases were smaller in T rats (from 49.56+/-3.2 to 36.09+/-2.1N/cm(2)) than in C rats (from 41.28+/-1.0 to 20.73+/-1.8N/cm(2)). Sprint training increased the dynamic stiffness and tetanic tension of the soleus muscle and protected against the attenuation induced by eccentric contractions. Finally, the two-state model provided evidence that the number of force-generating cross-bridges increases in trained muscle.     

Morphological changes in rat skeletal muscle following reinnervation

Morphological changes appearing in the course of muscle regeneration after reinnervation of denervated M. soleus (slow) and M. tibialis anterior (fast) rat skeletal muscle were investigated. It was found that pathological changes typical for denervation atrophy (seen on the 10th day after crushing the sciatic nerve) and symptoms of regeneration (beginning about the 15th day) were much more pronounced in the soleus than in the tibialis muscle. Some stages of regeneration in the soleus muscle could be distinguished. The contractile material destructions were the first pathological changes that disappeared after the beginning of regeneration. In the second stage other denervation changes disappeared and intensive regeneration of muscle fibres was observed. In the next stage regeneration slowed down, and the reduction of the excess of muscle nuclei was visible. Four months after crushing the nerve, regeneration proceeded to completion with only some traces of the passed processes: in the soleus muscle, chains of sarcolemmal nuclei, satellite cells and newly formed muscle fibres were more often seen than in contralateral muscle; in the tibialis, collagen depots were present around the vessels and between muscle fascicles.     

Unloading of juvenile muscle results in a reduced muscle size 9 wk after reloading.

The role of satellite cells and DNA unit size in determining muscle size was examined by inhibiting postnatal skeletal muscle development by using hindlimb suspension. Satellite cell mitotic activity and DNA unit size were determined in the soleus muscles from hindlimb-suspended and age-matched weight-bearing rats before the initiation of hindlimb suspension, at the conclusion of a 28-day hindlimb-suspension period, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-suspended rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb suspension, but they were the same (P > 0. 05) as those of weight-bearing rats 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, nuclei per millimeter, and DNA unit size for the hindlimb-suspended rats were significantly (P < 0.05) smaller than for the weight-bearing rats at all recovery times. Satellite cell mitotic activity was significantly (P < 0.05) higher in the soleus muscles from hindlimb-suspended rats 2 wk after reloading, but it was the same (P > 0.05) as in weight-bearing rats 9 wk after reloading. Juvenile soleus muscles failed to achieve normal muscle size 9 wk after reloading because there was incomplete compensation for the hindlimb-suspension-induced interruptions in myonuclear accretion and DNA unit size expansion.     

Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and die during the neonatal period

All four of the muscle actins (skeletal, cardiac, vascular, and enteric) in higher vertebrates show distinct expression patterns and display highly conserved amino acid sequences. While it is hypothesized that each of the muscle isoactins is specifically adapted to its respective tissue and that the minor variations among them have developmental and/or physiological relevance, the exact functional and developmental significance of these proteins remains largely unknown. In order to begin to assess these issues, we disrupted the skeletal actin gene by homologous recombination. All mice lacking skeletal actin die in the early neonatal period (day 1 to 9). These null animals appear normal at birth and can breathe, walk, and suckle, but within 4 days, they show a markedly lower body weight than normal littermates and many develop scoliosis. Null mice show a loss of glycogen and reduced brown fat that is consistent with malnutrition leading to death. Newborn skeletal muscles from null mice are similar to those of wild-type mice in size, fiber type, and ultrastructural organization. At birth, both hemizygous and homozygous null animals show an increase in cardiac and vascular actin mRNA in skeletal muscle, with no skeletal actin mRNA present in null mice. Adult hemizygous animals show an increased level of skeletal actin mRNA in hind limb muscle but no overt phenotype. Extensor digitorum longus (EDL) muscle isolated from skeletal-actin-deficient mice at day 2 to 3 showed a marked reduction in force production compared to that of control littermates, and EDL muscle from hemizygous animals displayed an intermediate force generation. Thus, while increases in cardiac and vascular smooth-muscle actin can partially compensate for the lack of skeletal actin in null mice, this is not sufficient to support adequate skeletal muscle growth and/or function.     

Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial marker enzyme expression in mice

LKB1 has been identified as a component of the major upstream kinase of AMP-activated protein kinase (AMPK) in skeletal muscle. To investigate the roles of LKB1 in skeletal muscle, we used muscle-specific LKB1 knockout (MLKB1KO) mice that exhibit low expression of LKB1 in heart and skeletal muscle, but not in other tissues. The importance of LKB1 in muscle physiology was demonstrated by the observation that electrical stimulation of the muscle in situ increased AMPK phosphorylation and activity in the wild-type (WT) but not in the muscle-specific LKB1KO mice. Likewise, phosphorylation of acetyl-CoA carboxylase (ACC) was markedly attenuated in the KO mice. The LKB1KO mice had difficulty running on the treadmill and exhibited marked reduction in distance run in voluntary running wheels over a 3-wk period (5.9 +/- 0.9 km/day for WT vs. 1.7 +/- 0.7 km/day for MLKB1KO mice). The MLKB1KO mice anesthetized at rest exhibited significantly decreased phospho-AMPK and phospho-ACC compared with WT mice. KO mice exhibited lower levels of mitochondrial protein expression in the red and white regions of the quadriceps. These observations, along with previous observations from other laboratories, clearly demonstrate that LKB1 is the major upstream kinase in skeletal muscle and that it is essential for maintaining mitochondrial marker proteins in skeletal muscle. These data provide evidence for a critical role of LKB1 in muscle physiology, one of which is maintaining basal levels of mitochondrial oxidative enzymes. Capacity for voluntary running is compromised with muscle and heart LKB1 deficiency.     

Skeletal muscle performance determined by modulation of number of myosin motors rather than motor force or stroke size

Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.     

失重状态下肌萎缩研究进展

失重条件下人和动物生理状态会发生一系列的变化,其中骨骼肌萎缩和力量下降较为显著,目前其发生的机制仍不明确且缺少特效的干预措施。本文从肌肉湿重及肌纤维横截面积的变化、肌纤维类型的变化、肌纤维超微结构的变化、肌梭的适应性变化四个方面进行简要阐述,探讨肌肉萎缩的可能发生机制。     

Skeletal muscle dressed in SOCs

Store-operated Ca(2+) channels (SOCs) are activated in response to Ca(2+) release from the endoplasmic reticulum (ER). The stromal interaction molecule 1 (STIM1) is the ER sensor that transmits the stored Ca(2+) content to the pore-forming SOCs Orai and TRPC channels. Recent studies reveal high levels of Orai1 and STIM1 in skeletal muscle, and a prominent role of SOCs in muscle development and function.     

Skeletal muscle enlargement with weight-lifting exercise by rats

A rat model of weight lifting that produces skeletal muscle enlargement utilizing regimens of resistance training similar to those employed in human training programs is described. The model consists of electrically stimulating the lower leg muscles to contract against a weighted pulley bar. Animals were subjected to training protocols employing low-frequency repetitions with high training loads within a training session. Initial maximum loads of between 200 and 800 g were progressively increased during the 16 wk of training. Work done at the end of the training period increased to an average value 66% higher than that performed at the start of training. The gastrocnemius wet weight and protein content increased (P less than 0.001) by 18 and 17%, respectively, in the stimulated loaded leg in all but one training protocol, a program in which rats were exercised more frequently. RNA content, but not concentration, was increased in the trained gastrocnemius muscle from each protocol, resulting in muscle enlargement. These data indicate that the basic model presented here provides a suitable vehicle for future studies into the biochemical events that may cause skeletal muscle enlargement during resistance training but, based on limited data, suggests that an increased frequency of training days may hinder muscle enlargement in this model.