Anaerobic energy release in skeletal muscle during electrical stimulation in men

The quadriceps femoris muscles of seven men were electrically stimulated under extended anaerobic conditions to quantitate anaerobic energy release and the contribution of the glycolytic system to total ATP production. Muscles were intermittently stimulated 64 times at 20 Hz while leg blood flow was occluded. Each contraction lasted 1.6 s and was followed by 1.6 s of rest. The total contraction time was 102.4 s. Muscle biopsies were taken at rest and following 16, 32, 48, and 64 contractions. The ATP turnover rates during the four 16-contraction periods were 6.12, 2.56, 2.17, and 0.64 mmol X kg dry muscle-1 X s-1 contraction time. Glycolysis provided 58%, phosphocreatine 40% and a decreased ATP store 2% of the consumed energy during the initial 16 contractions. Glycolysis was responsible for 90% of the total ATP production beyond contraction 16. Absolute glycolytic ATP production decreased to 60, 55, and 17% of the amount in the initial 16 contractions during the final three periods, respectively. In conclusion glycolysis produced approximately 195 mmol ATP/kg dry muscle during the initial 48 contractions (76.8 s) and only approximately 15 mmol ATP/kg dry muscle during the final 16 contractions. Equivalent values for total ATP turnover were 278 and 16.5 mmol/kg dry muscle.     

Mechanical and electrical effects of high-frequency and high-intensity stimulation of muscle

A unique tension response can be obtained by stimulating an isometrically held skeletal muscle or a single muscle fiber by a train of high-frequency pulses (2,000 pps) at higher-than-normal intensity, or by a long DC pulse. It is called the tetanoid response, and it is composed of three well-defined stages. Initially, tension develops rapidly, and mechanical output (Po) reaches about 0.35. Subsequently, this tension is maintained at a nearly steady level for the remainder of stimulation. After stimulation, a final increase of tension takes place. Intracellular electrical recordings show that the initial development of tension is elicited by two or three action potentials generated at the beginning of the stimulation, and that no additional action potentials are generated for the remainder of stimulation. During stimulation, part of the fiber membrane (regarded in cross-section) is depolarized, which generates tension, and part of the membrane is hyperpolarized. With termination of stimulation, a single action potential is elicited via anode-break excitation (ABE) on the hyperpolarized portion of the membrane, which gives rise to the final increase of tension.     

Neuromuscular electrical stimulation for skeletal muscle function

Lack of neural innervation due to neurological damage renders muscle unable to produce force. Use of electrical stimulation is a medium in which investigators have tried to find a way to restore movement and the ability to perform activities of daily living. Different methods of applying electrical current to modify neuromuscular activity are electrical stimulation (ES), neuromuscular electrical stimulation (NMES), transcutaneous electrical nerve stimulation (TENS), and functional electrical stimulation (FES). This review covers the aspects of electrical stimulation used for rehabilitation and functional purposes. Discussed are the various parameters of electrical stimulation, including frequency, pulse width/duration, duty cycle, intensity/amplitude, ramp time, pulse pattern, program duration, program frequency, and muscle group activated, and how they affect fatigue in the stimulated muscle.     

Altered muscle force and stiffness of skeletal muscles in alpha-sarcoglycan-deficient mice

Alpha-sarcoglycan (ASG) is a transmembrane protein of the dystrophin-associated complex, and absence of ASG causes limb-girdle muscular dystrophy. We hypothesize that disruption of the sarcoglycan complex may alter muscle extensibility and disrupt the coupling between passive transverse and axial contractile elements in the diaphragm. We determined the length-tension relationships of the diaphragm of young ASG-deficient mice and their controls during uniaxial and biaxial loading. We also determined the isometric contractile properties of the diaphragm muscles from mutant and normal mice in the absence and presence of passive transverse stress. We found that the diaphragm muscles of the null mutants for the protein ASG show 1) significant decrease in muscle extensibility in the directions of the muscle fibers and transverse to fibers, 2) significant reductions in force-generating capacity, and 3) significant reductions in coupling between longitudinal and transverse properties. Thus these findings suggest that the sarcoglycan complex serves a mechanical function in the diaphragm by contributing to muscle passive stiffness and to the modulation of the contractile properties of the muscle.     

Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice

Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor beta superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1(-/-)) and their wild type (WT; i.e., IGF-1(+/+)) littermates. The body weight of IGF-1(-/-) animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1(-/-) animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1(-/-) undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1(-/-) and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1(-/-) and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1(-/-)M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated.     

Effect of electrical stimulation on intracellular triacylglycerol in isolated skeletal muscle

The contribution of intracellular triacylglycerol (TG) as a substrate for skeletal muscle during electrical stimulation is equivocal. Therefore, the purpose of this study was to investigate the effect of electrical stimulation on the TG content in the isolated intact rat flexor digitorum brevis skeletal muscle preparation by use of two different stimulation protocols. Muscles were electrically stimulated for 1 h either continuously at 1 Hz or intermittently (30 s on, 60 s off) at 5 Hz while incubated in 21 degrees C Krebs bicarbonate buffer (pH 7.4) that contained 11 mM glucose. Control muscles were either frozen immediately after excision or incubated for 1 h. TG content was significantly decreased (P less than 0.05) compared with control concentrations in both stimulated muscle groups, with the greatest reduction (60%) occurring after 5-Hz intermittent stimulation. These data indicate that intramuscular TG is hydrolyzed in response to electrical stimulation in the isolated flexor digitorum brevis muscle preparation. In addition, the type of stimulation (higher frequency intermittent vs. lower frequency continuous) employed influences the amount of intracellular TG hydrolyzed.     

Functional deficits in nNOSmu-deficient skeletal muscle: myopathy in nNOS knockout mice

Skeletal muscle nNOSmu (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSmu; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSmu. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSmu signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSmu expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention.     

Enhancement of whole body glucose uptake during and after human skeletal muscle low-frequency electrical stimulation.

There is considerable evidence to suggest that electrical stimulation (ES) activates glucose uptake in rodent skeletal muscle. It is, however, unknown whether ES can lead to similar metabolic enhancement in humans. We employed low-frequency ES through surface electrodes placed over motor points of quadriceps femoris muscles. In male subjects lying in the supine position, the highest oxygen uptake was obtained by a stimulation pattern with 0.2-ms biphasic square pulses at 20 Hz and a 1-s on-off duty cycle. Oxygen uptake was increased by approximately twofold throughout the 20-min stimulation period and returned to baseline immediately after stimulation. Concurrent elevation of the respiratory exchange ratio and blood lactate concentration indicated anaerobic glycogen breakdown and utilization during ES. Whole body glucose uptake determined by the glucose disposal rate during euglycemic clamp was acutely increased by 2.5 mg. kg(-1). min(-1) in response to ES and, moreover, remained elevated by 3-4 mg. kg(-1). min(-1) for at least 90 min after cessation of stimulation. Thus the stimulatory effect of ES on whole body glucose uptake persisted not only during, but also after, stimulation. Low-frequency ES may become a useful therapeutic approach to activate energy and glucose metabolism in humans.     

Hematopoietic contribution to skeletal muscle regeneration in acid alpha-glucosidase knockout mice.

Recent studies have shown that cells from bone marrow (BM) can give rise to differentiated skeletal muscle fibers. However, the mechanisms and identities of the cell types involved remain unknown. We performed BM transplantation in acid alpha-glucosidase (GAA) knockout mice, a model of glycogen storage disease type II, and our observations suggested that the BM cells contribute to skeletal muscle fiber formation. Furthermore, we showed that most CD45+:Sca1+ cells have a donor character in regenerating muscle of recipient mice. Based on these findings, CD45+:Sca1+ cells were sorted from regenerating muscles. The cell number was increased with granulocyte colony-stimulating factor after cardiotoxin injury, and the cells were transplanted directly into the tibialis anterior (TA) muscles of GAA knockout mice. Sections of the TA muscles stained with anti-laminin-alpha2 antibody showed that the number of CD45+:Sca1+ cells contributing to muscle fiber formation and glycogen levels were decreased in transplanted muscles. Our results indicated that hematopoietic stem cells, such as CD45+:Sca1+ cells, are involved in skeletal muscle regeneration.     

Delayed angiogenesis and VEGF production in CCR2-/- mice during impaired skeletal muscle regeneration

The regulation of vascular endothelial growth factor (VEGF) levels and angiogenic events during skeletal muscle regeneration remains largely unknown. This study examined angiogenesis, VEGF levels, and muscle regeneration after cardiotoxin (CT)-induced injury in mice lacking the CC chemokine receptor 2 (CCR2). Muscle regeneration was significantly decreased in CCR2-/- mice as was the early accumulation of macrophages after injury. In both mouse strains, tissue VEGF was similar at baseline (no injections) and significantly decreased at day 3 post-CT. Tissue VEGF in wild-type (WT) mice was restored within 7 days postinjury but remained significantly reduced in CCR2-/- mice until day 21. Capillary density (capillaries/mm(2)) within regenerating muscle was maximal in WT mice at day 7 and double that of baseline muscle. In comparison, maximal capillary density in CCR2-/- mice occurred at 21 days postinjury. Maximal capillary density developed concurrent with the restoration of tissue VEGF in both strains. A highly significant, inverse relationship existed between the size of regenerated muscle fibers and capillaries per square millimeter. Although this relationship was comparable in WT and CCR2-/- animals, there was a significant decrease in the magnitude of this response in the absence of CCR2, reflecting the observation that regenerated muscle fiber size in CCR2-/- mice was only 50% of baseline at 42 days postinjury, whereas WT mice had attained baseline fiber size by day 21. Thus CCR2-dependent events in injured skeletal muscle, including impaired macrophage recruitment, contribute to restoration of tissue VEGF levels and the dynamic processes of capillary formation and muscle regeneration.     

Cyclooxygenase-2 expression in skeletal muscle of knockout mice suffering Duchenne muscular dystrophy

The purpose of the present study was to investigate the role of cyclooxygenase-2 (COX-2) expression in fibrotic lesion in mdx mice. A total of six male C57BL/10 mice and six C57BL/10-DMD/mdx were distributed into two groups: control and animals with Duchenne muscular dystrophy (DMD). The medial part of gastrocnemius muscle was evaluated being the specimens stained with hematoxylin and eosin (H&E) and Sirius Red under normal and polarized light to differentiate type I (red and yellow) and III (green) collagen. COX-2 expression was assessed by immunohistochemistry. The results revealed histopathological changes in C57BL/10-DMD/mdx as depicted by regenerating fibers. Sirius Red stain showed a substantial increase in the amount of type I collagen of mdx mice. DMD induced a strong COX-2 immunoexpression in intercellular space. Taken together, our results are consistent with the notion that necrotic and fibrotic lesions are able to increase COX-2 expression in DMD.     

The effects of low frequency electrical stimulation on satellite cell activity in rat skeletal muscle during hindlimb suspension

Background  

The ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. It has been shown that chronic hindlimb unloading downregulates the satellite cell activity. This study investigated the role of low-frequency electrical stimulation on satellite cell activity during a 28 d hindlimb suspension in rats.     

Phase transition in force during ramp stretches of skeletal muscle.

Active glycerinated rabbit psoas fibers were stretched at constant velocity (0.1-3.0 lengths/s) under sarcomere length control. As observed by previous investigators, force rose in two phases: an initial rapid increase over a small stretch (phase I), and a slower, more modest rise over the remainder of the stretch (phase II). The transition between the two phases occurred at a critical stretch (LC) of 7.7 +/- 0.1 nm/half-sarcomere that is independent of velocity. The force at critical stretch (PC) increased with velocity up to 1 length/s, then was constant at 3.26 +/- 0.06 times isometric force. The decay of the force response to a small step stretch was much faster during stretch than in isometric fibers. The addition of 3 mM vanadate reduced isometric tension to 0.08 +/- 0.01 times control isometric tension (P0), but only reduced PC to 0.82 +/- 0.06 times P0, demonstrating that prepowerstroke states contribute to force rise during stretch. The data can be explained by a model in which actin-attached cross-bridges in a prepowerstroke state are stretched into regions of high force and detach very rapidly when stretched beyond this region. The prepowerstroke state acts as a mechanical rectifier, producing large forces during stretch but small forces during shortening.     

Conformation of the myosin motor during force generation in skeletal muscle

Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide.     

The role of p53 in vivo during skeletal muscle post-natal development and regeneration: studies in p53 knockout mice

The tumour suppressor gene p53 is recognised as a central regulator of the cell cycle and apoptosis. Post-natally, p53 mutations are associated with many cancers and mice lacking p53 are prone to spontaneous tumour formation. The present study examines skeletal muscle formation in post-natal mice lacking p53 using two different models of skeletal muscle regeneration. The level of endogenous myogenic cell proliferation in mature skeletal muscle was examined and the time course of muscle regeneration after whole muscle transplantation or crush injury were compared in p53 (-/-) and control C57Bl/6J adult mice, using desmin and proliferating cell nuclear antigen (PCNA) immunohistochemistry and histological analysis. The pattern of inflammation, myoblast proliferation and myotube formation in regenerating p53 (-/-) skeletal muscles appears normal and similar to those in control C57Bl/6J muscle. These data indicate that p53 is not required for the regulation of myoblast proliferation, differentiation and myotube formation in vivo during myogenesis of adult skeletal muscle.     

Anaerobic energy provision in aged skeletal muscle during tetanic stimulation

The effect of age on skeletal muscle anaerobic energy metabolism was investigated in adult (11 mo) and aged (25 mo) Fischer 344 rats. Hindlimb skeletal muscles innervated by the sciatic nerve were stimulated to contract with trains of supramaximal impulses (100 ms, 80 Hz) at a train rate of 1 Hz for 60 s, with an occluded circulation. Soleus, plantaris, and red and white gastrocnemius (WG) were sampled from control and stimulated limbs. All muscle masses were reduced with age (9-13%). Peak isometric tensions, normalized per gram of wet muscle, were lower throughout the stimulation in the aged animals (28%). The potential for anaerobic ATP provision was unaltered with age in all muscles, because resting high-energy phosphates and glycogen contents were similar to adult values. Anaerobic ATP provision during stimulation was unaltered by aging in soleus, plantaris, and red gastrocnemius muscles. In the WG, containing mainly fast glycolytic (FG) fibers, ATP and phosphocreatine contents were depleted less in aged muscle. In situ glycogenolysis and glycolysis were 90.0 +/- 4.8 and 69.3 +/- 2.6 mumol/g dry muscle (dm) in adult WG and reduced to 62.3 +/- 6.9 and 51.5 +/- 5.5 mumol/g dm, respectively, in aged WG. Consequently, total anaerobic ATP provision was lower in aged WG (224.5 +/- 20.9 mumol/g dm) vs. adult (292.6 +/- 7.6 mumol/g dm) WG muscle. In summary, the decreased tetanic tension production in aged animals was associated with a decreased anaerobic energy production in FG fibers. Reduced high-energy phosphate use and a greater energy charge potential after stimulation suggested that the energy demand was reduced in aged FG fibers.(ABSTRACT TRUNCATED AT 250 WORDS)