Endurance training adaptations modulate the redox–force relationship of rat isolated slow-twitch skeletal muscles

1. Studies have shown that, in isolated skeletal muscles, maximum isometric force production (Po) is dependent on muscle redox state. Endurance training increases the antioxidant capacity of skeletal muscles, a factor that could impact on the force-producing capacity following exogenous exposure to an oxidant. We tested the hypothesis that 12 weeks treadmill training would increase anti-oxidant capacity in rat skeletal muscles and alter their response to exogenous oxidant exposure.

2. At the conclusion of the 12 week endurance-training programme, soleus (slow-twitch) muscles from trained rats had greater citrate synthase (CS) and catalase (CAT) activity compared with soleus muscles from untrained rats (P < 0.05).
In contrast, CAT activity of extensor digitorum longus (EDL; fast-twitch) muscles from trained rats was not different to EDL muscles of untrained rats. The CS activity was lower in EDL muscles from trained compared with untrained rats (P < 0.05).

3. Equilibration with exogenous hydrogen peroxide (H2O2, 5 mmol/L) increased the Po of soleus muscles from untrained rats for the duration of treatment (30 min), whereas the Po of EDL muscles was affected biphasically, with a small increase initially (after 5 min), followed by a more marked decrease in Po (after 30 min). The H2O2-induced increase in Po of soleus muscles from trained rats was less than that in untrained rats (P < 0.05), but no differences were observed in the Po of EDL muscles following training.

4. The results indicate that 12 weeks endurance running training conferred adaptations in soleus but not EDL muscles. These adaptations were associated with an attenuation of the oxidant-induced increase in Po of soleus muscles from trained compared with untrained rats. We conclude that endurance training-adapted soleus muscles have a slightly altered redox - force relationship.

Creatine transporter protein content, localization, and gene expression in rat skeletal muscle

The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher (P <= 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater (P <= 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater (P <= 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount (P <= 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle types. CreaT mRNA expression per microgram of total RNA was similar across the three muscle types. These data indicate that rat SOL and RG have an enhanced potential to transport Cr compared with WG, despite a higher TCr in the latter.

Interaction of exercise and diet on GLUT-4 protein and gene expression in type I and type II rat skeletal muscle

We determined the interaction of exercise and diet on glucose transporter (GLUT-4) protein and mRNA expression in type I (soleus) and type II [extensor digitorum longus (EDL)] skeletal muscle. Forty-eight Sprague Dawley rats were randomly assigned to one of two dietary conditions: high-fat (FAT, n =24) or high-carbohydrate (CHO, n =24). Animals in each dietary condition were allocated to one of two groups: control (NT, n =8) or a group that performed 8 weeks of treadmill running (4 sessions week^–1 of 1000 m @ 28 m min^–1 , RUN, n =16). Eight trained rats were killed after their final exercise bout for determination of GLUT-4 protein and mRNA expression: the remainder were killed 48 h after their last session for measurement of muscle glycogen and triacylglycerol concentration. GLUT-4 protein expression in NT rats was similar in both muscles after 8 weeks of either diet. However, there was a main effect of training such that GLUT-4 protein was increased in the soleus of rats fed with either diet (P < 0.05) and in the EDL in animals fed with CHO (P < 0.05). There was a significant diet–training interaction on GLUT-4 mRNA, such that expression was increased in both the soleus (100% ↑P < 0.05) and EDL (142% ↑P < 0.01) in CHO-fed animals. Trained rats fed with FAT decreased mRNA expression in the EDL (↓ 45%, P < 0.05) but not the soleus (↓ 14%, NS). We conclude that exercise training in CHO-fed rats increased both GLUT-4 protein and mRNA expression in type I and type II skeletal muscle. Despite lower GLUT-4 mRNA in muscles from fat-fed animals, exercise-induced increases in GLUT-4 protein were largely preserved, suggesting that control of GLUT-4 protein and gene expression are modified independently by exercise and diet.

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.

Greater effect of diet than exercise training on the fatty acid profile of rat skeletal muscle

We determined the interaction of diet and exercise-training intensity on membrane phospholipid fatty acid (FA) composition in skeletal muscle from 36 female Sprague-Dawley rats. Animals were randomly divided into one of two dietary conditions: high-carbohydrate (64.0% carbohydrate by energy, n = 18) or high fat (78.1% fat by energy, n = 18). Rats in each diet condition were then allocated to one of three subgroups: control, which performed no exercise training; low-intensity (8 m/min) treadmill run training; or high-intensity (28 m/min) run training. All exercise-trained rats ran 1,000 m/session, 4 days/wk for 8 wk and were killed 48 h after the last training bout. Membrane phospholipids were extracted, and FA composition was determined in the red and white vastus lateralis muscles, Diet exerted a major influence on phospholipid FA composition, with the high-fat diet being associated with a significantly (P < 0.01) elevated ratio of n-6/n-3 FA for both red (2.7-3.2 vs. 1.0-1.1) and white vastus lateralis muscle (2.5-2.9 vs. 1.2). In contrast, alterations in FA composition as a result of either exercise-training protocol were only minor in comparison. We conclude that, under the present experimental conditions, a change in the macronutrient content of the diet was a more potent modulator of skeletal muscle membrane phospholipid FA composition compared with either low- or high-intensity treadmill exercise training.

AMP-activated protein kinase activates transcription of the UCP3 and HKII genes in rat skeletal muscle

AMP-activated protein kinase (AMPK) has recently emerged as a key signaling protein in skeletal muscle, coordinating the activation of both glucose and fatty acid metabolism in response to increased cellular energy demand. To determine whether AMPK signaling may also regulate gene transcription in muscle, rats were given a single subcutaneous injection (1 mg/g) of the AMP analog 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR). AICAR injection activated (P < 0.05) AMPK-α₂ (~2.5-fold) and transcription of the uncoupling protein-3 (UCP3, ~4-fold) and hexokinase II (HKII, ~10-fold) genes in both red and white skeletal muscle. However, AICAR injection also elicited (P < 0.05) an acute drop (60%) in blood glucose and a sustained (2-h) increase in blood lactate, prompting concern regarding the specificity of AICAR on transcription. To maximize AMPK activation in muscle while minimizing potential systemic counterregulatory responses, a single-leg arterial infusion technique was employed in fully conscious rats. Relative to saline-infused controls, single-leg arterial infusion of AICAR (0.125, 0.5, and 2.5 µg · g^-1 · min^-1 for 60 min) induced a dose-dependent increase (2- to 4-fold, P < 0.05) in UCP3 and HKII transcription in both red and white skeletal muscle. Importantly, AICAR infusion activated transcription only in muscle from the infused leg and had no effect on blood glucose or lactate levels. These data provide evidence that AMPK signaling is linked to the transcriptional regulation of select metabolic genes in skeletal muscle.

Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle

The purpose of this study was to determine whether nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no L-NAME ingestion and acute exercise, rest plus L-NAME, and rest without L-NAME. The exercised rats ran on a treadmill for 53 ± 2 min and were then killed 4 h later. NOS inhibition significantly (P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor-{gamma} coactivator 1beta (PGC-1beta) mRNA levels and tended (P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or beta-hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.

Activated calcineurin ameliorates contraction-induced injury to skeletal muscles of mdx dystrophic mice

Utrophin expression is regulated by calcineurin and up-regulating utrophin can decrease the susceptibility of dystrophic skeletal muscle to contraction-induced injury. We overexpressed the constitutively active calcineurin-A α in skeletal muscle of mdx dystrophic mice (mdx CnA*) and examined the tibialis anterior muscle to determine whether the presence of activated calcineurin promotes resistance to muscle damage after lengthening contractions. Two stretches (10 s apart) of 40% strain relative to muscle fibre length were initiated from the plateau of a maximal isometric tetanic contraction. Muscle damage was assessed 1, 5 and 15 min later by the deficit in maximum isometric force and by quantifying the proportion of muscle fibres staining positive for intracytoplasmic albumin. The force deficit at all time points after the lengthening contractions was approximately 80% in mdx muscles and 30% in mdxCnA* muscles. The proportion of albumin-positive fibres was significantly less in control and injured muscles from mdxCnA* mice than from mdx mice. Compared with mdx mice, mean fibre cross-sectional area was 50% less in muscles from mdxCnA* mice. Furthermore, muscles frommdxCnA* mice exhibited a higher proportion of fibres expressing the slow(er) myosin heavy chain (MyHC) I and IIa isoforms, prolonged contraction and relaxation times, lower absolute and normalized maximum forces, and a clear leftward shift of the frequency–force relationship with greater force production at lower stimulation frequencies. These are structural and functional markers of a slower muscle phenotype. Taken together, our findings show that muscles from mdxCnA* mice have a smaller mean fibre cross-sectional area, a greater sarcolemmal to cytoplasmic volume ratio, and an increase in utrophin expression, promoting an attenuated susceptibility to contraction-induced injury. We conclude that increased calcineurin activity may confer functional benefits to dystrophic skeletal muscles.

Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency-force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency-force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

Statin-induced increases in atrophy gene expression occur independently of changes in PGC1α protein and mitochondrial content

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg(-1)·day(-1)) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles.

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.

Alterations in the proportions of skeletal muscle proteins following a unilateral lesion to the substantia nigra pars compacta of rats

It is well established that mammalian skeletal muscles exhibit a considerable degree of plasticity and one of the main determining factors of this plasticity is the activity pattern and duration of motoneurone discharge. Lesions to the right substantia nigra pars compacta (SNpc) of six adult rats were made to determine whether altered output from the SNpc ultimately leads to a change in the expression of proteins in contralateral skeletal muscles. After 4 months, altered motor performance was identified by the administration of amphetamine. After 7 months, 30–70% of dopaminergic cells in the SNpc had been destroyed. The protein content of muscles was then quantified from densitometric scans of gels, and expressed as a % of the amount of actin (the protein used as a reference in this study). The lesion affected the expression of different protein isoforms in the fast- and slow-twitch muscles. In slow-twitch soleus muscles, the lesion decreased the proportion of α-tropomyosin and increased the proportion of β-tropomyosin. In the fast-twitch extensor digitorum longus muscles, the lesion increased the proportion of the fast isoform of troponin-T_1f, and decreased the proportions of the two isoforms of myosin light chain. This study establishes a connection between the chronic effects of a lesion to the SNpc, with a loss of dopaminergic neurones, impaired motor performance, and altered expression of proteins in skeletal muscle. The implication of these results is that the altered motor function observed in Parkinson’s disease may be associated with alterations to the expression of skeletal muscle proteins.