Muscle-specific AMPK ß1ß2-null mice display a myopathy due to loss of capillary density in nonpostural muscles.

AMP-activated protein kinase (AMPK) is a master regulator of metabolism. While muscle-specific AMPK ß1ß2 double-knockout (ß1ß2M-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 ß1ß2M-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, ß1ß2M-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 ß1ß2M-KO myopathy in resting muscle resulted from impaired AMPK-nNOSµ signaling, causing increased platelet aggregation, impaired vasodilation, and, ultimately, ischemic injury. Consistent with this hypothesis, AMPK-specific phosphorylation (Ser1446) of nNOSµ was decreased in ß1ß2M-KO compared to wild-type (WT) mice. The AMPK-nNOSµ relationship was further demonstrated by administration of 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) to ß1ß2-MKO muscles and C2C12 myotubes. AICAR significantly increased nNOSµ phosphorylation and nitric oxide production (P<0.05) within minutes of administration in WT muscles and C2C12 myotubes but not in ß1ß2M-KO muscles. These findings highlight the importance of the AMPK-nNOSµ pathway in resting skeletal muscle.

Cessation of cyclic stretch induces atrophy of C2C12 myotubes.

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-3ß (+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.

Mechanistic links between oxidative stress and disuse muscle atrophy.

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.

Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts.

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-kappaB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, L-NAME (200 microM), the COX-2 specific inhibitor NS-398 (100 microM), the NF-kappaB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10-100 microM) 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 microM) 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-kappaB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-kappaB 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 and AMPK cooperatively regulate PGC-1 in skeletal muscle cells.

Nitric oxide (NO) induces mitochondrial biogenesis in skeletal muscle cells via upregulation of the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). 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-1α mRNA expression and stimulate mitochondrial biogenesis in culture. L6 myotubes treated with nitric oxide donors, S-nitroso-N-penicillamine (SNAP, 25 µM) or diethylenetriamine-NONO (DETA-NO, 50 µM), exhibited elevated AMPK phosphorylation, PGC-1α 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 AMPKα1 in L6 myotubes using siRNA reduced AMPKα protein content and prevented upregulation of PGC-1α mRNA by DETA-NO. Meanwhile, siRNA knockdown of AMPKα2 had no effect on total AMPKα protein content or PGC-1α mRNA. These results suggest that NO effects on PGC-1α expression are mediated by AMPKα1. Paradoxically, we found that the AMPK-activating compound, AICAR, induced NO release from L6 myotubes, and that AICAR-induced upregulation of PGC-1α mRNA was prevented by inhibition of NOS with N(G)-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 AMPKα (P < 0.05), which was completely prevented by pre-treatment with the NOS inhibitor, L-N(G)-monomethyl arginine (L-NMMA, 1 mM). These data identify the AMPKα1 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.

Endothelial nitric oxide synthase is involved in calcium-induced Akt signaling in mouse skeletal muscle.

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.

Supplemental nitric oxide augments satellite cell activity on cultured myofibers from aged mice.

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.

Nitric oxide facilitates NFAT-dependent transcription in mouse myotubes.

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.

Nitric oxide reverses prednisolone-induced inactivation of muscle satellite cells.

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 microM), 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 microM 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.

Nitric oxide increases GLUT4 expression and regulates AMPK signaling in skeletal muscle.

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.

Increased temperature, not cardiac load, activates heat shock transcription factor 1 and heat shock protein 72 expression in the heart.

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.

Arginine supplementation induces myoblast fusion via augmentation of nitric oxide production.

The semi-essential amino acid, L-arginine (L-Arg), is the substrate for endogenous synthesis of nitric oxide, a molecule that is involved in myoblast proliferation and fusion. Since L-Arg supply may limit nitric oxide synthase (NOS) activity in endothelial cells, we examined L-Arg supplementation in differentiating mouse myoblasts and tested the hypothesis that L-Arg exerts direct effects on myoblast fusion via augmentation of endogenous nitric oxide production. C(2)C(12) myoblasts in differentiation media received one of the following treatments for 120 h: 1 mM L-Arg, 0.1 mM N-nitro-L-arginine methyl ester (L-NAME), L-Arg + L-NAME, 10 mM L-Lysine, or no supplement (Control). Cultures were fixed and stained with hematoxylin and eosin for microphotometric image analysis of myotube density, nuclear density, and fusion index (% of total nuclei in myotubes). Endogenous production of nitric oxide during the treatment period peaked between 24 and 48 h. L-Arg amplified nitric oxide production between 0 and 24 h and increased myotube density, total nuclei number, and nuclear fusion index. These L-Arg effects were prevented by the NOS inhibitor, L-NAME. Further, L-Lysine, a competitive inhibitor of L-Arg uptake, repressed nitric oxide production and reduced myotube density and fusion index. In summary, L-Arg augments myotube formation and increases nitric oxide production in a process limited by cellular L-Arg uptake.

Ibuprofen inhibits skeletal muscle hypertrophy in rats.

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.

In vivo inhibition of nitric oxide synthase impairs upregulation of contractile protein mRNA in overloaded plantaris muscle.

Inhibition of nitric oxide synthase (NOS) activity in vivo impedes hypertrophy in the overloaded rat plantaris. We investigated the mechanism for this effect by examining early events leading to muscle growth following 5 or 12 days of functional overload. Male Sprague-Dawley rats (approximately 350 g) were randomly divided into three treatment groups: control, N(G)-nitro-L-arginine methyl ester (L-NAME; 90 mg.kg(-1).day(-1)), and 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM; 10 mg.kg(-1).day(-1)). Unilateral removal of synergists induced chronic overload (OL) of the right plantaris. Sham surgery performed on the left hindlimb served as a normally loaded control. L-NAME and TRIM treatments prevented OL-induced skeletal alpha-actin and type I (slow) myosin heavy chain mRNA expression at 5 days. Conversely, neither L-NAME nor TRIM affected hepatocyte growth factor or VEGF mRNA responses to OL at 5 days. However, OL induction of IGF-I and mechanogrowth factor mRNA was greater (P < 0.05) in the TRIM group compared with the controls. Furthermore, the phosphorylated-to-total p70 S6 kinase ratio was higher in OL muscle from NOS-inhibited groups, compared with control OL. At 12 days of OL, the cumulative proliferation of plantaris satellite cells was assessed by subcutaneous implantation of time release 5'-bromo-2'-deoxyuridine pellets during the OL-inducing surgeries. Although OL caused a fivefold increase in the number of mitotically active (5'-bromo-2'-deoxyuridine positive) sublaminar nuclei, this was unaffected by concurrent NOS inhibition. Therefore, NOS activity may provide negative feedback control of IGF-I/p70 S6 kinase signaling during muscle growth. Moreover, NOS activity may be involved in transcriptional regulation of skeletal alpha-actin and type I (slow) myosin heavy chain during functional overload.

Effects of increased dietary fat and exercise on skeletal muscle lipid peroxidation and antioxidant capacity in male rats.

BACKGROUND: Elevated dietary fat increases oxidative metabolism and has been linked to increased oxidative stress, while exercise training may augment antioxidant capacity. Most studies examining oxidative stress in skeletal muscle employ extremely high levels of dietary fat and/or intense exercise training that may not adequately model human diet and activity patterns. AIM: The purpose of this study was to examine the interaction between an elevated (40% of calories) monounsaturated fat diet and a moderate-intensity exercise program similar to recommended human exercise prescriptions, on skeletal muscle oxidative stress and antioxidant defenses. METHODS: Twenty-four male Sprague-Dawley rats (approximately 500 g) were randomly divided into 4 groups (n = 6/group): Standard Diet-Sedentary (SD-Sed), Standard Diet-Exercise (SD-Ex), Elevated Fat Diet-Sedentary (EFD-Sed), and Elevated Fat Diet-Exercise (EFD-Ex). The SD groups consumed 76% of calories from CHO, 14% from protein, and 10 % from fat, while the EFD groups received a diet of 46% of calories from CHO, 14% from protein, and 40 % from fat (high oleic sunflower oil). The exercise groups were progressively treadmill trained at 20 m/min, 4 days/week increasing from 15 min/day to 35 min/day by the end of 4 wks. RESULTS AND CONCLUSION: Antioxidant adaptations associated with exercise training or an elevated fat diet individually reduced basal lipid peroxidation levels in the plantaris muscle. However, the combination of exercise plus a monounsaturated fat diet increased lipid peroxidation levels above that with either treatment alone. This suggests an exhaustion of the antioxidant capacity in the plantaris muscle when both exercise and increased dietary fat diet are combined.

Chronic exercise and the pro-inflammatory response to endotoxin in the serum and heart.

PURPOSE: To examine the effects of moderate intensity chronic exercise on tumor necrosis factor-alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) responses to endotoxin in female Sprague-Dawley rats. METHODS: Rats were divided into two groups, exercise (n=17) and sedentary (n=24). Exercise (Ex) rats completed 12 weeks of motorized treadmill running 3 days/week for 15-25 min at 22-25 m/min, while sedentary (Sed) rats remained in their cages. Twenty-four hrs after the last exercise session, animals were subdivided into three groups. One subgroup served as baseline controls. These rats received an injection of saline (s) and were killed immediately (Sed-s and Ex-s), while the other groups received an injection of lipopolysaccharide (LPS). LPS animals were killed 2 h (Sed-L2 and Ex-L2) or 4 h after the LPS injection (Sed-L4 and Ex-L4). RESULTS: Serum TNFalpha was elevated 2 h after LPS injection in both Sed and Ex groups, but was significantly higher in the chronic exercise group (Ex-L2 versus. Sed-L2). Similarly, serum beta glucuronidase activity, an indicator of tissue damage, was elevated 2 and 4 h after LPS injection, and was significantly higher in the exercise groups. Post-treatment left ventricular TNFalpha and iNOS activity, as well as stable nitric oxide derivatives in the serum (NOx), were significantly higher in LPS-injected groups compared to saline groups, but no difference in LPS effect was observed between sedentary and exercise groups. CONCLUSIONS: Moderate intensity chronic exercise stress caused an exaggerated serum TNFalpha response to endotoxin and an elevation in a serum marker of LPS-induced tissue damage.

Trolox attenuates mechanical ventilation-induced diaphragmatic dysfunction and proteolysis.

Prolonged mechanical ventilation results in diaphragmatic oxidative injury, elevated proteolysis, fiber atrophy, and reduced force-generating capacity. We tested the hypothesis that antioxidant infusion during mechanical ventilation would function as an antioxidant to maintain redox balance within diaphragm muscle fibers and therefore prevent oxidative stress and subsequent proteolysis and contractile dysfunction. Sprague-Dawley rats were anesthetized, tracheostomized, and mechanically ventilated with 21% O(2) for 12 hours. The antioxidant Trolox was intravenously infused in a subset of ventilated animals. Compared with acutely anesthetized, nonventilated control animals, mechanical ventilation resulted in a significant reduction (-17%) in diaphragmatic maximal tetanic force. Importantly, Trolox completely attenuated this mechanical ventilation-induced diaphragmatic contractile deficit. Total diaphragmatic proteolysis was increased 105% in mechanical ventilation animals compared with controls. In contrast, diaphragmatic proteolysis did not differ between controls and mechanical ventilation-Trolox animals. Moreover, 20S proteasome activity in the diaphragm was elevated in the mechanical ventilation animals (+76%); Trolox treatment attenuated this mechanical ventilation-induced rise in protease activity. These results are consistent with the hypothesis that mechanical ventilation-induced oxidative stress is an important factor regulating mechanical ventilation-induced diaphragmatic proteolysis and contractile dysfunction. Our findings suggest that antioxidant therapy could be beneficial during prolonged mechanical ventilation.

Cumulative effects of aging and mechanical ventilation on in vitro diaphragm function.

STUDY OBJECTIVE: Unloading the diaphragm, via mechanical ventilation (MV), results in significant diaphragmatic atrophy, contractile dysfunction, and oxidative stress in young adult animals. Since aging increases skeletal muscle susceptibility to atrophy and injury, we tested the hypothesis that MV-induced diaphragmatic contractile dysfunction would be exacerbated in aging rats. METHODS: Fisher 344/Brown Norway hybrid rats (4 months old [young] and 30 months old [old]) were assigned to either control or MV groups. MV rats were anesthetized, tracheostomized, and ventilated with 21% O(2) for 12 h. Arterial BP, pH, and blood gas homeostasis were maintained in the MV animals throughout the experimental period. Animals in the control group were acutely anesthetized, and the diaphragms were immediately removed. Muscle strips from the mid-costal diaphragm were removed from each experimental animal, and contractile properties were studied in vitro. RESULTS: Compared to young control animals, aging (old control animals) was associated with a 13% decrease in maximal isometric tension (24.5 N/cm(2) vs 21.3 N/cm(2)). Although, MV induced similar relative losses (24%) in diaphragmatic isometric tension in both young and old animals receiving MV, the combined effects of aging and MV resulted in a 34% decrement in diaphragmatic isometric tension compared to young control animals (24.5 N/cm(2) vs 16.1 N/cm(2)). CONCLUSIONS: These data do not support the hypothesis that aging exacerbates the relative MV-induced impairment in diaphragmatic isometric tension. Nonetheless, the additive effects of aging and MV have dramatic effects on diaphragmatic force reserve. This could exacerbate weaning difficulties in older individuals receiving MV.

Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload.

The purpose of this study was to determine the necessity of nitric oxide (NO) for hypertrophy and fiber-type transition in overloaded (OL) skeletal muscle. Endogenous NO production was blocked by administering N(G)-nitro-L-arginine methyl ester (L-NAME; 0.75 mg/ml; approximately 100 mg x kg-1 x day-1) in drinking water. Thirty-eight female Sprague-Dawley rats (approximately 250 g) were randomly divided into four groups: control-nonoverloaded (Non-OL), control-OL, L-NAME-Non-OL, and L-NAME-OL. Chronic overload of the plantaris was induced bilaterally by surgical removal of the gastrocnemius and soleus. Rats in the Non-OL groups received sham surgeries. L-NAME treatment began 24 h before surgery and continued until the rats were killed 14 days postsurgery. Although OL induced hypertrophy in both control (+76%) and L-NAME (+39%) conditions (P < 0.05), mean plantaris-to-body mass ratio in the L-NAME-OL group was significantly lower (P < 0.05) than that in the control-OL group. Microphotometric analysis of histochemically determined fiber types revealed increases in cross-sectional area (P < 0.05) for all fiber types (types I, IIA, and IIB/X) in the OL plantaris from control rats, whereas L-NAME-OL rats exhibited increases only in type I and IIB/X fibers. SDS-PAGE analysis of myosin heavy chain (MHC) composition in the plantaris indicated a significant (P < 0.05) OL effect in the control rats. Specifically, the mean proportion of type I MHC increased 6% (P < 0.05), whereas the proportion of type IIb MHC decreased approximately 9% (P < 0.05). No significant OL effects on MHC profile were observed in the L-NAME rats. These data support a role of NO in overload-induced skeletal muscle hypertrophy and fiber-type transition.