The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

The influence of myosin regulatory light chain phosphorylation on the contractile performance of fatigued mammalian skeletal muscle

ABSTRACT The myosm regulatory light chain (RLC) of type II fibres is phosphorylated by Ca2+ -calmodulin dependent myosin light chain kinase (skMLCK) during muscular activation. The purpose of this study was to explore the effect of skMLCK gene ablation on the fatigability of mouse skeletal muscles during repetitive stimulation. The absence of myosin RLC phosphorylation in skMLCK knockout muscles attenuated contractile performance without a significant metabolic cost. Twitch force was potentiated to a greater extent in wildtype muscles until peak force had diminished to ~60% of baseline (37.2 ± 0.05% vs. 14.3 ± 0.02%). Despite no difference in peak force (Po) and shortening velocity (Vo), rate of force development (+dP/dt) and shortening-induced deactivation (SID) were almost two-fold greater in WT muscles. The present results demonstrate that myosin RLC phosphorylation may improve contractile performance during fatigue; providing a contractile advantage to working muscles and protecting against progressive fatigue.

Reliability of muscle fiber conduction velocity in the tibialis anterior

This document could not have been completed without the hard work of a number of individuals. First and foremost, my supervisor, Dr. David Gabriel deserves the utmost recognition for the immense effort and time spent guiding the production of this document through the various stages of completion. Also, aiding in the data collection, technical support, and general thought processing were Lab Technician Greig Inglis and fellow members of the Electromyographic Kinesiology Laboratory Jon Howard, Sean Lenhardt, Lara Robbins, and Corrine Davies-Schinkel. The input of Drs. Ted Clancy, Phil Sullivan and external examiner Dr. Anita Christie, all members ofthe assessment committee, was incredibly important and vital to the completion of this work. Their expertise provided a strong source of knowledge and went to ensure that this project was completed at exemplary level. There were a number of other individuals who were an immense help in getting this project off the ground and completed. The donation of their time and efforts was very generous and much needed in order to fulfill the requirements needed for completion of this study. Finally, I cannot exclude the contributions of my family throughout this project especially that of my parents whose support never wavers.

Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction.

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells. (C) 2010 Elsevier Inc. All rights reserved.

Mg(2+) Ions Bind at the C-Terminal Region of Skeletal Muscle alpha-Tropomyosin

Tropomyosin (Tm) is a dimeric coiled-coil protein that polymerizes through head-to-tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may from sites for divalent cation binding. In a previous study, we showed that the Mg(2+)-induced increase in stability of the C-terminal half of Tin is sensitive to imitations near the C-terminus. In the present report, we study the interaction between Mg(2+) and full-length Tin and smaller fragments corresponding to the last 65 and 26 Tin residues. Although the smaller Tin peptide (Tm(259-284(W269))) is flexible and to large extent unstructured, the larger Tm(220-284(W269)) fragments forms a coiled coil in solution whose stability increases significantly in the presence of Mg(2+). NMR analysis shows thin Mg(2+) induces chemical shift perturbations in both Tm(220-284(W269)) and Tm(259-284(W269)) in the vicinity of His276, in which are located several negatively charged residues. (C) 2009 Wiley Periodicals, Inc. Biopolymers 91: 583-590, 2009.

5`-aminoimidazole-4-carboxyamide-ribonucleoside- activated glucose transport is not prevented by nitric oxide synthase inhibition in rat isolated skeletal muscle

1. The nucleoside intermediate 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) activates skeletal muscle AMP-activated protein kinase (AMPK) and increases glucose uptake. The AMPK phosphorylates neuronal nitric oxide synthase (nNOS)µ in skeletal muscle fibres. There is evidence that both AMPK and nNOSµ may be involved in the regulation of contraction-stimulated glucose uptake.
2. We examined whether both AICAR- and contraction-stimulated glucose uptake were mediated by NOS in rat skeletal muscle.
3. Rat isolated epitrochlearis muscles were subjected in vitro to electrically stimulated contractions for 10 min and/or incubated in the presence or absence of AICAR (2 mmol/L) or the NOS inhibitor NG-monomethyl-l-arginine (l-NMMA; 100 µmol/L).
4. Muscle contraction significantly (P < 0.05) altered the metabolic profile of the muscle. In contrast, AICAR and l-NMMA had no effect on the metabolic profile of the muscle, except that AICAR increased muscle 5'-aminoimidazole-4-carboxyamide-ribonucleotide (ZMP) and AICAR content. Nitric oxide synthase inhibition caused a small but significant (P < 0.05) reduction in basal 3-O-methylglucose transport, which was observed in all treatments. 5'-Aminoimidazole-4-carboxyamide-ribonucleoside significantly increased (P < 0.05) glucose transport above basal, with NOS inhibition decreasing this slightly (increased by 209% above basal compared with 184% above basal with NOS inhibition). Contraction significantly increased glucose transport above basal, with NOS inhibition substantially reducing this (107% increase vs 31% increase). 5'-Aminoimidazole-4-carboxyamide-ribonucleoside plus contraction in combination were not additive on glucose transport.
5. These results suggest that NO plays a role in basal glucose uptake and may regulate contraction-stimulated glucose uptake. However, NOS/nitric oxide do not appear to be signalling intermediates in AICAR-stimulated skeletal muscle glucose uptake.

Carbohydrate ingestion does not alter skeletal muscle AMPK signaling during exercise in humans

There is evidence that increasing carbohydrate (CHO) availability during exercise by raising preexercise muscle glycogen levels attenuates the activation of AMPK{alpha}2 during exercise in humans. Similarly, increasing glucose levels decreases AMPK{alpha}2 activity in rat skeletal muscle in vitro. We examined the effect of CHO ingestion on skeletal muscle AMPK signaling during exercise in nine active male subjects who completed two 120-min bouts of cycling exercise at 65 ± 1% VO2 peak. In a randomized, counterbalanced order, subjects ingested either an 8% CHO solution or a placebo solution during exercise. Compared with the placebo trial, CHO ingestion significantly (P < 0.05) increased plasma glucose levels and tracer-determined glucose disappearance. Exercise-induced increases in muscle-calculated free AMP (17.7- vs. 11.8-fold), muscle lactate (3.3- vs. 1.8-fold), and plasma epinephrine were reduced by CHO ingestion. However, the exercise-induced increases in skeletal muscle AMPK{alpha}2 activity, AMPK{alpha}2 Thr172 phosphorylation and acetyl-CoA Ser222 phosphorylation, were essentially identical in the two trials. These findings indicate that AMPK activation in skeletal muscle during exercise in humans is not sensitive to changes in plasma glucose levels in the normal range. Furthermore, the rise in plasma epinephrine levels in response to exercise was greatly suppressed by CHO ingestion without altering AMPK signaling, raising the possibility that epinephrine does not directly control AMPK activity during muscle contraction under these conditions in vivo.

Stimulation of calcineurin Aα activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin A transgene (CnA) was overexpressed in skeletal muscles of mdx (mdx CnA*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnA* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnA* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnA* than mdx mice. In the diaphragm, despite a slower phenotype and a 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnA* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.

ß2-Agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle

Aging is associated with a slowing of skeletal muscle contractile properties, including a decreased rate of relaxation. In rats, the age-related decrease in the maximal rate of relaxation is reversed after 4-wk administration with the β2-adrenoceptor agonist (β2-agonist) fenoterol. Given the critical role of the sarcoplasmic reticulum (SR) in regulating intracellular Ca2+ transients and ultimately the time course of muscle contraction and relaxation, we tested the hypothesis that the mechanisms of action of fenoterol are mediated by alterations in SR proteins. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) kinetic properties were assessed in muscle homogenates and enriched SR membranes isolated from the red (RG) and white (WG) portions of the gastrocnemius muscle in adult (16 mo) and aged (28 mo) F344 rats that had been administered fenoterol for 4 wk (1.4 mg/kg/day ip, in saline) or vehicle only. Aging was associated with a 29% decrease in the maximal activity (Vmax) of SERCA in the RG but not in the WG muscles. Fenoterol treatment increased the Vmax of SERCA and SERCA1 protein levels in RG and WG. In the RG, fenoterol administration reversed an age-related selective nitration of the SERCA2a isoform. Our findings demonstrate that the mechanisms underlying age-related changes in contractile properties are fiber type dependent, whereas the effects of fenoterol administration are independent of age and fiber type.

The role and regulation of stars in skeletal muscle

STARS is a muscle specific protein that is upregulated in response to endurance exercise and may potentially increase skeletal muscle cell sensitivity to muscle contraction. STARS enhances the activation of intracellular signalling pathways involved in skeletal muscle growth, regeneration and oxidative metabolism.

Muscle redox signalling pathways in exercise. Role of antioxidants

Recent research highlights the importance of redox signalling pathway activation by contraction-induced reactive oxygen species (ROS) and nitric oxide (NO) in normal exercise-related cellular and molecular adaptations in skeletal muscle. In this review, we discuss some potentially important redox signalling pathways in skeletal muscle that are involved in acute and chronic responses to contraction and exercise. Specifically, we discuss redox signalling implicated in skeletal muscle contraction force, mitochondrial biogenesis and antioxidant enzyme induction, glucose uptake and muscle hypertrophy. Furthermore, we review evidence investigating the impact of major exogenous antioxidants on these acute and chronic responses to exercise. Redox signalling pathways involved in adaptive responses in skeletal muscle to exercise are not clearly elucidated at present, and further research is required to better define important signalling pathways involved. Evidence of beneficial or detrimental effects of specific antioxidant compounds on exercise adaptations in muscle is similarly limited, particularly in human subjects. Future research is required to not only investigate effects of specific antioxidant compounds on skeletal muscle exercise adaptations, but also to better establish mechanisms of action of specific antioxidants in vivo. Although we feel it remains somewhat premature to make clear recommendations in relation to application of specific antioxidant compounds in different exercise settings, a bulk of evidence suggests that N-acetylcysteine (NAC) is ergogenic through its effects on maintenance of muscle force production during sustained fatiguing events. Nevertheless, a current lack of evidence from studies using performance tests representative of athletic competition and a potential for adverse effects with high doses (>70 mg/kg body mass) warrants caution in its use for performance enhancement. In addition, evidence implicates high dose vitamin C (1 g/day) and E (≥260 IU/day) supplementation in impairments to some skeletal muscle cellular adaptations to chronic exercise training. Thus, determining the utility of antioxidant supplementation in athletes likely requires a consideration of training and competition periodization cycles of athletes in addition to type, dose and duration of antioxidant supplementation.

Pelvic floor muscle evaluation in incontinent patients

The aim of this study was to assess pelvic floor muscle (PFM) strength and perception and its correlation with stress urinary incontinence (SUI). One hundred and one women were divided into two groups according to the presence (G1=51 patients) or absence (G2=50 patients) of SUI. Subjective [urine stream interruption test (UST), visual survey of perineal contraction and transvaginal digital palpation to assess pelvic muscle contraction] and objective evaluations of pelvic floor muscles in all patients were performed (vaginal manometry). During the UST, 25.5% of G1 patients and 80% of G2 patients were able to interrupt the urine stream (p<0.05). Digital evaluation of pelvic muscular contraction showed higher strength in G2 than in G1 patients (p<0.0001). Perineometer evaluation of PFM strength was significantly higher in the continent group (p<0.001). Pelvic floor muscle weakness in incontinent patients demonstrates the importance of functional and objective evaluation of this group of muscles.

Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

THE RATE of FORCE DEVELOPMENT OBTAINED AT EARLY CONTRACTION PHASE IS NOT INFLUENCED BY ACTIVE STATIC STRETCHING

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)