AMP-activated protein kinase (AMPK) is a master regulator of metabolism. While muscle-specific AMPK 12 double-knockout (12M-KO) mice display alterations in metabolic and mitochondrial capacity, their severe exercise intolerance suggested a secondary contributor to the observed phenotype. We find that tibialis anterior (TA), but not soleus, muscles of sedentary 12M-KO mice display a significant myopathy (decreased myofiber areas, increased split and necrotic myofibers, and increased centrally nucleated myofibers. A mitochondrial- and fiber-type-specific etiology to the myopathy was ruled out. However, 12M-KO TA muscles displayed significant (P<0.05) increases in platelet aggregation and apoptosis within myofibers and surrounding interstitium (P<0.05). These changes correlated with a 45% decrease in capillary density (P<0.05). We hypothesized that the 12M-KO myopathy in resting muscle resulted from impaired AMPK-nNOS signaling, causing increased platelet aggregation, impaired vasodilation, and, ultimately, ischemic injury. Consistent with this hypothesis, AMPK-specific phosphorylation (Ser1446) of nNOS was decreased in 12M-KO compared to wild-type (WT) mice. The AMPK-nNOS relationship was further demonstrated by administration of 5-aminoimidazole-4-carboxamide 1--d-ribofuranoside (AICAR) to 12-MKO muscles and C2C12 myotubes. AICAR significantly increased nNOS phosphorylation and nitric oxide production (P<0.05) within minutes of administration in WT muscles and C2C12 myotubes but not in 12M-KO muscles. These findings highlight the importance of the AMPK-nNOS pathway in resting skeletal muscle.Thomas, M. M., Wang, D. C., D'Souza, D. M., Krause, M. P., Layne, A. S., Criswell, D. S., O'Neill, H. M., Connor, M. K., Anderson, J. E., Kemp, B. E., Steinberg, G. R., and Hawke, T. J. Muscle-specific AMPK 12-null mice display a myopathy due to loss of capillary density in nonpostural muscles.

Cyclic stretch of differentiated myotubes mimics the loading pattern of mature skeletal muscle. We tested a cell culture model of disuse atrophy by the cessation of repetitive bouts of cyclic stretch in differentiated C2C12 myotubes. Myotubes were subjected to cyclic strain (12%, 0.7 Hz, 1 h/d) on collagen-I-coated Bioflex plates using a computer-controlled vacuum stretch apparatus (Flexcell Int.) for 2 (2dSTR) or 5 (5dSTR) consecutive days. Control cultures were maintained in the Bioflex plates without cyclic stretch for 2d or 5d. Additionally, some cultures were stretched for 2 d followed by cessation of stretch for 3d (2dSTR3dCES). Cyclic stretching (5dSTR) increased myotube diameter and overall myotube area by ~2-fold (P<0.05) compared to non-stretched controls, while cessation of stretch (2dSTR3dCES) resulted in ~80% smaller myotubes than 5dSTR cells, and 40-50% smaller than non-stretched controls (P<0.05). Further, the calpain-dependent cleavage products of αII-spectrin (150 kDa) and talin increased (3.5-fold and 2.2-fold, respectively; P<0.05) in 2dSTR3dCES myotubes, compared to non-stretched controls. The 1h cyclic stretching protocol acutely increased the phosphorylation of Akt (+4.5-fold; P<0.05) and its downstream targets, FOXO3a (+4.2-fold; P<0.05) and GSK-3β (+1.8-fold; P<0.05), which returned to baseline by 48 h after cessation of stretch. Additionally, nitric oxide production increased during stretch and co-treatment with the NOS inhibitor, l-NAME, inhibited the effects of stretch and cessation of stretch. We conclude that cessation of cyclic stretching causes myotube atrophy by activating calpains and decreasing activation of Akt. Stretch-induced myotube growth, as well as activation of atrophy signaling with cessation of stretch, are dependent on NOS activity.

Long periods of skeletal muscle inactivity promote a loss of muscle protein resulting in fiber atrophy. This disuse-induced muscle atrophy results from decreased protein synthesis and increased protein degradation. Recent studies have increased our insight into this complicated process, and evidence indicates that disturbed redox signaling is an important regulator of cell signaling pathways that control both protein synthesis and proteolysis in skeletal muscle. The objective of this review is to outline the role that reactive oxygen species play in the regulation of inactivity-induced skeletal muscle atrophy. Specifically, this report will provide an overview of experimental models used to investigate disuse muscle atrophy and will also highlight the intracellular sources of reactive oxygen species and reactive nitrogen species in inactive skeletal muscle. We then will provide a detailed discussion of the evidence that links oxidants to the cell signaling pathways that control both protein synthesis and degradation. Finally, by presenting unresolved issues related to oxidative stress and muscle atrophy, we hope that this review will serve as a stimulus for new research in this exciting field. Antioxid. Redox Signal. 15, 2519-2528.

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-κB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, l-NAME (200 μM), the COX-2 specific inhibitor NS-398 (100 μM), the NF-κB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10–100 μM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by l-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 μM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-κB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-κB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.

Nitric oxide (NO) induces mitochondrial biogenesis in skeletal muscle cells via upregulation of the peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha). Further, we have shown that nitric oxide interacts with the metabolic sensor enzyme, AMPK. Therefore, we tested the hypothesis that nitric oxide and AMPK act synergistically to upregulate PGC-1 alpha mRNA expression and stimulate mitochondrial biogenesis in culture. L6 myotubes treated with nitric oxide donors, S-nitroso-N-penicillamine (SNAP, 25 mu m) or diethylenetriamine-NONO (DETA-NO, 50 mu m), exhibited elevated AMPK phosphorylation, PGC-1 alpha mRNA and protein, and basal and uncoupled mitochondrial respiration (P < 0.05). Pre-treatment of cultures with the AMPK inhibitor, Compound C, prevented these effects. Knockdown of AMPK alpha 1 in L6 myotubes using siRNA reduced AMPK alpha protein content and prevented upregulation of PGC-1 alpha mRNA by DETA-NO. Meanwhile, siRNA knockdown of AMPK alpha 2 had no effect on total AMPK alpha protein content or PGC-1 alpha mRNA. These results suggest that NO effects on PGC-1 alpha expression are mediated by AMPK alpha 1. Paradoxically, we found that the AMPK-activating compound, AICAR, induced NO release from L6 myotubes, and that AICAR-induced upregulation of PGC-1 alpha mRNA was prevented by inhibition of NOS with NG-nitro-l-arginine methyl ester (l-NAME, 1 mm). Additionally, incubation of isolated mouse extensor digitorum longus (EDL) muscles with 2 mm AICAR for 20 min or electrical stimulation (10 Hz, 13 V) for 10 min induced phosphorylation of AMPK alpha (P < 0.05), which was completely prevented by pre-treatment with the NOS inhibitor, l-NG-monomethyl arginine (l-NMMA, 1 mm). These data identify the AMPK alpha 1 isoform as the mediator of NO-induced effects in skeletal muscle cells. Further, this study supports a proposed model of synergistic interaction between AMPK and NOS that is critical for maintenance of metabolic function in skeletal muscle cells.

Skeletal muscle atrophy can occur as a result of disuse, disease, or aging. Mechanistic pathways among these conditions vary, but in most cases, levels of intracellular calcium (IC Ca2+) increase. This rise in IC Ca2+ stimulates calpain activity and subsequent protein degradation. Calpains cleave the structural proteins that hold the sarcomere together, thereby releasing the contractile proteins for proteasome degradation; this is thought to be the rate‐limiting step in skeletal muscle proteolysis. Little is known about the regulation of calpain activity, but nitric oxide (NO) has been proposed as a possible mediator. This study used the Ca2+ ionophore calcimycin to increase IC Ca2+ and to induce calpain activity in L6 myotubes. We hypothesized that treatment with an NO donor would attenuate calpain proteolysis and myotube atrophy in a dose‐dependent manner following treatment with calcimycin. Results show an attenuation of calpain‐specific talin proteolysis with 1 μM and 10 μM PAPA‐NO after 60 minutes of calcimycin treatment, and preliminary data show prevention of myotube atrophy with DETA‐NO after 24–48 hours of calcimycin treatment. Therefore, moderate doses of NO can prevent calpain proteolysis and possibly skeletal muscle atrophy following a Ca2+ challenge in L6 myotubes. This study was funded by the American Heart Association.

We hypothesized that targeted mutation of the endothelial nitric oxide synthase (eNOS) gene would reduce Akt-related signaling events in skeletal muscle cells, compared to wild type (WT) controls. Results show that slow myosin heavy chain (type I/beta) expression and the abundance of slow-twitch fibers are reduced in plantaris muscle of eNOS(-/-) mice, compared to WT. Further, basal phosphorylation of Akt (p-Akt (Ser-473)/total Akt) and GSK-3beta (GSK-3beta (Ser-9)/total GSK-3beta) are reduced 60-70% in primary myotubes from eNOS(-/-) mice. Treatment with the calcium ionophore, A23187 (0.4 microM, 1 h), increased phosphorylation of Akt and GSK-3beta by approximately 2-fold (P<0.05) in myotubes from WT mice, but had no effect on phosphorylation of these proteins in eNOS(-/-) myotubes. Additionally, A23187 treatment failed to induce nuclear translocation of the transcription factor, NFATc1, in eNOS(-/-) myotubes. Treatment with the nitric oxide donor, propylamine propylamine NONOate (PAPA-NO; 1 microM for 1 h) increased Akt and GSK-3beta phosphorylation, and induced NFATc1 nuclear translocation in WT and eNOS(-/-) myotubes, and eliminated differences from WT in the NOS knockout cultures. Parallel experiments in C2C12 myotubes found that Akt phosphorylation induced by NO or the guanylate cyclase activator, YC-1, is prevented by co-treatment with either a guanylate cyclase or PI3K inhibitor (10 microM ODQ or 25 microM LY2904002, respectively). These data suggest that eNOS activity is necessary for calcium-induced activation of the Akt pathway, and that nitric oxide is sufficient to elevate Akt activity in primary myotubes. NO appears to influence Akt signaling through a cGMP, PI3K-dependent pathway.

Skeletal muscle is the major site of insulin-stimulated glucose uptake and oxidation processes which are controlled, in part, by activation of the cellular energy-sensor, AMP-activated protein kinase (AMPK). In addition, the transcription coactivator, peroxisome- proliferator activated receptor γ coactivator-1α (PGC-1α), has been considered to be a master regulator of cellular metabolism. Recently, nitric oxide (NO) was shown to be involved in mitochondrial biogenesis in skeletal muscle. Here we aim to find whether NO up-regulates PGC-1α mRNA expression in L6 myotubes and test whether the AMPK-dependent up-regulation of PGC-1α and mitochondrial genes are influenced by nitric oxide synthase (NOS) activity. METHODS: Rat L6 myotubes were differentiated by serum withdrawal to form confluent myotube cultures. Differentiated myotubes were exposed to various treatments and harvested for measurement of specific mRNAs via RT-real time PCR. RESULTS: Diethylenetriamine NONOate (DETA-NO; 50 µM) increased PGC-1α mRNA expression after 3 hr. This was a transient effect, returning to baseline at 6 hrs. Treatment with the AMPK activating compound, 5-aminoimidazole-4-carboxamide-1-ß -D-ribofuranoside (AICAR; 3 mM) increased phospho-to-total (α) AMPK ratio, and expression of mRNAs for PGC-1α, F1 ATP synthase, and citrate synthase, while co-treatment of myotubes with L-Nitroarginine methyl ester (L-NAME; 100µM), prevented these effects. CONCLUSIONS: NO is sufficient to induce PGC-1α mRNA, and NOS activity is required for AMPK activation, and induction of PGC-1α and mitochondrial gene expression.

Skeletal muscle regenerative potential is reduced with aging. We hypothesized that in vitro activation of muscle satellite cells would be compromised, and that nitric oxide (NO) supplementation would improve satellite cell activity in old muscle. Single intact myofibers were isolated from the gastrocnemius muscles of young (2 mo), adult (10 mo), and aged (22 mo) mice. Fibers were centrifuged to stimulate satellite cells and incubated with L-arginine (2mM), the NO donor, diethylenetriamine NONOate (DETA-NO; 10 microM), or control media for 48 h. The number of activated satellite cells after centrifugation was reduced in aged fibers compared to young and adult. L-arginine or DETA-NO treatment increased satellite cell activation in all age groups. However, an age-dependent deficit in satellite cell activity persisted within treatment groups. In separate fibers, exogenous HGF was equally effective in activating satellite cells across age groups, indicating that events downstream of HGF release are intact in aged muscle. These data suggest that l-arginine bioavailability and NO production limit muscle satellite cell activity in response to a submaximal mechanical stimulus, regardless of age. Further, the decline in satellite cell activity in early senescence can be partially abrogated by exogenous L-arginine or an NO donor.

Intracellular calcium transients in skeletal muscle cells initiate phenotypic adaptations via activation of calcineurin and its effector nuclear factor of activated t-cells (NFAT). Furthermore, endogenous production of nitric oxide (NO) via calcium-calmodulin-dependent NO synthase (NOS) is involved in skeletal muscle phenotypic plasticity. Here, we provide evidence that NO enhances calcium-dependent nuclear accumulation and transcriptional activity of NFAT and induces phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) in C2C12 myotubes. The calcium ionophore A23187 (1 microM for 9 h) or thapsigargin (2 microM for 4 h) increased NFAT transcriptional activity by seven- and fourfold, respectively, in myotubes transiently transfected with an NFAT-dependent reporter plasmid (pNFAT-luc, Stratagene). Cotreatment with the NOS-inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 5 mM) or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM) prevented the calcium effects on NFAT activity. The NO donor diethylenetriamine-NONO (DETA-NO; 10 microM) augmented the effects of A23187 on NFAT-dependent transcription. Similarly, A23187 (0.4 microM for 4 h) caused nuclear accumulation of NFAT and increased phosphorylation (i.e., inactivation) of GSK-3beta, whereas cotreatment with L-NAME or ODQ inhibited these responses. Finally, the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA-NO; 1 microM for 1 h) increased phosphorylation of GSK-3beta in a manner dependent on guanylate cyclase activity. We conclude that NOS activity mediates calcium-induced phosphorylation of GSK-3beta and activation of NFAT-dependent transcription in myotubes. Furthermore, these effects of NO are guanylate cyclase-dependent.

PURPOSE: Doxorubicin belongs to a family of anticancer drugs that exert cytotoxic effects by release of reactive oxygen species (ROS) and triggering apoptosis. Since antioxidant enzymes scavenge ROS and defend against apoptosis, we sought to determine whether overexpression of catalase, copper‐zinc superoxide dismutase (CuZnSOD), or manganese SOD (MnSOD) is protective against doxorubicin‐mediated cell death.

The expression of myocardial heat shock protein 72 (HSP72) postexercise is initiated by the activation of heat shock transcription factor 1 (HSF1). However, it remains unknown which physiological stimuli govern myocardial HSF1 activation during exercise. These experiments tested the hypothesis that thermal stress and mechanical load, concomitant with simulated exercise, provide independent stimuli for HSF1 activation and ensuing cardiac HSP72 gene expression. To elucidate the independent roles of increased temperature and cardiac workload in the exercise-mediated upregulation of left-ventricular HSP72, hearts from adult male Sprague-Dawley rats were randomly assigned to one of five simulated exercise conditions. Upon reaching a surgical plane of anesthesia, each experimental heart was isolated and perfused using an in vitro working heart model, while independently varying temperatures (i.e., 37 degrees C vs. 40 degrees C) and cardiac workloads (i.e., low preload and afterload vs. high preload and afterload) to mimic exercise responses. Results indicate that hyperthermia, independent of cardiac workload, promoted an increase in nuclear translocation and phosphorylation of HSF1 compared with normothermic left ventricles. Similarly, hyperthermia, independent of workload, resulted in significant increases in cardiac levels of HSP72 mRNA. Collectively, these data suggest that HSF1 activation and HSP72 gene transcriptional competence during simulated exercise are linked to elevated heart temperature and are not a direct function of increased cardiac workload.

PURPOSE: Despite the therapeutic benefits of corticosteroids as anti-inflammatory agents, pharmacological doses of these drugs induce a catabolic state in skeletal muscle, and inhibit satellite cell-dependent regeneration and recovery of skeletal muscle following injury. Therefore, we hypothesized that corticosteroid exposure would inhibit satellite cell activity in skeletal muscle fibers. METHODS: Male Swiss-Webster mice (9 mo. old) were injected i.p. daily for 8 wks with prednisolone (2mg/kg/d; GC, n=8) or vehicle (Control, n=8). Single myofibers were isolated from the gastrocnemius by collagenase digestion (n=4 mice/group), and plated on 24-well culture plates, one fiber/well. Plates containing myofibers were centrifuged (1500g, 30min) to mechanically activate satellite cells, and maintained in culture (37°C, 5% CO2) for 48h. Separately, protein lysates and were isolated from gastrocnemius samples (n=4 mice/group) for analysis of nitric oxide synthase (NOS) protein isoforms by western blot. RESULTS: Expression of both constitutive NOS isoforms was significantly reduced in muscle from the GC group compared to Control (nNOS: −30%, eNOS: −34%). Following centrifugation, the Control group had MyoD+ mononuclear cells emanating from 54% of myofibers, compared to 25% of fibers in the GC group. Supplementation of culture media with the nitric oxide donor, DETA-NO (5-50μM), during the 48h following centrifugation caused a dose-dependent increase in the number of fibers with emanating MyoD+ cells. Further, supplementation of fiber cultures with 50μM DETA-NO eliminated the GC-induced deficit in the proportion of fibers with activated satellite cells (Control=76% vs. GC=74%). CONCLUSIONS: Prednisolone treatment in vivo inhibits mechanical activation of skeletal muscle satellite cells. This is likely related to the prednisolone-induced down-regulation of NOS expression, since supplementation of myofibers with DETA-NO restores satellite cell activity to control levels.

Long‐term corticosteroid therapy causes myopathy and can inhibit regeneration of skeletal muscle. Therefore, we hypothesized that corticosteroid exposure reduces satellite cell activity in skeletal myofibers. Male Swiss–Webster mice were injected daily for 8 weeks with prednisolone (GC) or vehicle (control). Single myofibers were isolated from the gastrocnemius, centrifuged to mechanically activate satellite cells, and maintained in culture for 48 h. Both constitutive nitric oxide synthase (NOS) isoforms were reduced in muscle by GC treatment (nNOS: −30%, eNOS: −34%). Fewer myogenic (myoD+) cells emanated from GC myofibers compared to control (−61%, P < 0.05). Supplementation of culture media with the nitric oxide donor, diethylenetriamine NONOate (DETA‐NO; 5–50 μM), caused a dose‐dependent increase in the number of myoD+ cells arising from both control and GC myofibers (P < 0.05), and 10 and 50 μM DETA‐NO eliminated the GC‐induced deficit in myogenic cells (P > 0.05). Therefore, supplementation of GC myofibers with DETA‐NO restores satellite cell activity to control levels. Nitric oxide production could be an important therapeutic target for the prevention of corticosteroid myopathy. Muscle Nerve, 2007

Nitric oxide (NO) and 5'-AMP-activated protein kinase (AMPK) are involved in glucose transport and mitochondrial biogenesis in skeletal muscle. Here, we examined whether NO regulates the expression of the major glucose transporter in muscle (GLUT4) and whether it influences AMPK-induced upregulation of GLUT4. At low levels, the NO donor S-nitroso-N-penicillamine (SNAP, 1 and 10 microM) significantly increased GLUT4 mRNA ( approximately 3-fold; P < 0.05) in L6 myotubes, and cotreatment with the AMPK inhibitor compound C ablated this effect. The cGMP analog 8-bromo-cGMP (8-Br-cGMP, 2 mM) increased GLUT4 mRNA by approximately 50% (P < 0.05). GLUT4 protein expression was elevated 40% by 2 days treatment with 8-Br-cGMP, whereas 6 days treatment with 10 microM SNAP increased GLUT4 expression by 65%. Cotreatment of cultures with the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one prevented the SNAP-induced increase in GLUT4 protein. SNAP (10 microM) also induced significant phosphorylation of alpha-AMPK and acetyl-CoA carboxylase and translocation of phosphorylated alpha-AMPK to the nucleus. Furthermore, L6 myotubes exposed to 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) for 16 h presented an approximately ninefold increase in GLUT4 mRNA, whereas cotreatment with the non-isoform-specific NOS inhibitor N(G)-nitro-l-arginine methyl ester, prevented approximately 70% of this effect. In vivo, GLUT4 mRNA was increased 1.8-fold in the rat plantaris muscle 12 h after AICAR injection, and this induction was reduced by approximately 50% in animals cotreated with the neuronal and inducible nitric oxide synthases selective inhibitor 1-(2-trifluoromethyl-phenyl)-imidazole. We conclude that, in skeletal muscle, NO increases GLUT4 expression via a cGMP- and AMPK-dependent mechanism. The data are consistent with a role for NO in the regulation of AMPK, possibly via control of cellular activity of AMPK kinases and/or AMPK phosphatases.

PURPOSE We sought to determine whether cyclooxygenase (COX) activity is necessary for overload-induced growth of adult rat skeletal muscle, and whether nitric oxide synthase (NOS) activity is involved in upregulation of COX messenger RNA (mRNA) expression in skeletal muscle. METHODS Unilateral surgical removal of the gastrocnemius and soleus was performed on the right hindlimb of 16 female Sprague-Dawley rats (approximately 230 g) to induce chronic overload (OL) of the plantaris for 14 d, with sham surgeries performed on the contralateral leg as a normally loaded (NL) control. Half of the rats were treated with the nonspecific COX inhibitor, ibuprofen (0.2 mg.mL(-1) in drinking water; approximately 20 mg.kg(-1).d(-1)). In a second experiment, the plantaris was unilaterally overloaded for 5 or 14 d in male rats (approximately 350 g; N = 16 rats per time point) and half of the animals were treated with the NOS inhibitor, L-NAME (0.75 mg.mL(-1) in drinking water; approximately 90 mg.kg(-1).d(-1)). RESULTS Ibuprofen treatment inhibited plantaris hypertrophy by approximately 50% (P < 0.05) following 14 d of OL, as did L-NAME treatment (P < 0.05). COX-1 and COX-2 mRNA did not differ between any groups at 5 d. At 14 d, however, L-NAME caused a 30-fold increase in plantaris COX-1 mRNA expression independent of loading condition. Additionally, OL induced a 20-fold increase in COX-2 mRNA expression compared with NL (P < 0.05) at 14 d, without affecting COX-1 mRNA level. L-NAME treatment significantly inhibited OL-induced expression of COX-2 mRNA. CONCLUSION COX activity is important for in vivo muscle hypertrophy, and plantaris overload is associated with NOS activity-dependent COX-2 expression.