Rapid diaphragm atrophy following cervical spinal cord hemisection.

A cervical (C2) hemilesion (C2Hx), which disrupts ipsilateral bulbospinal inputs to the phrenic nucleus, was used to study diaphragm plasticity after acute spinal cord injury. We hypothesized that C2Hx would result in rapid atrophy of the ipsilateral hemidiaphragm and increases in mRNA expression of proteolytic biomarkers. Diaphragm tissue was harvested from male Sprague-Dawley rats at 1 or 7 days following C2Hx. Histological analysis demonstrated reduction in cross-sectional area (CSA) of type I and IIa fibers in the ipsilateral hemidiaphragm at 1 but not 7 days. Type IIb/x fibers, however, had reduced CSA at 1 and 7 days. A targeted gene array was used to screen mRNA changes for genes associated with skeletal muscle myopathy and myogenesis; this was followed by qRT-PCR validation. Changes in diaphragm gene expression suggested that profound myoplasticity is initiated immediately following C2Hx including activation of both proteolytic and myogenic pathways. We conclude that an immediate myoplastic response occurs in the diaphragm after C2Hx with atrophy occurring in ipsilateral myofibers within 1 day.

Cancer cachexia decreases specific force and accelerates fatigue in limb muscle.

Cancer cachexia is a complex metabolic syndrome that is characterized by the loss of skeletal muscle mass and weakness, which compromises physical function, reduces quality of life, and ultimately can lead to mortality. Experimental models of cancer cachexia have recapitulated this skeletal muscle atrophy and consequent decline in muscle force generating capacity. However, more recently, we provided evidence that during severe cancer cachexia muscle weakness in the diaphragm muscle cannot be entirely accounted for by the muscle atrophy. This indicates that muscle weakness is not just a consequence of muscle atrophy but that there is also significant contractile dysfunction. The current study aimed to determine whether contractile dysfunction is also present in limb muscles during severe Colon-26 (C26) carcinoma cachexia by studying the glycolytic extensor digitorum longus (EDL) muscle and the oxidative soleus muscle, which has an activity pattern that more closely resembles the diaphragm. Severe C-26 cancer cachexia caused significant muscle fiber atrophy and a reduction in maximum absolute force in both the EDL and soleus muscles. However, normalization to muscle cross sectional area further demonstrated a 13% decrease in maximum isometric specific force in the EDL and an even greater decrease (17%) in maximum isometric specific force in the soleus. Time to peak tension and half relaxation time were also significantly slowed in both the EDL and the solei from C-26 mice compared to controls. Since, in addition to postural control, the oxidative soleus is also important for normal locomotion, we further performed a fatigue trial in the soleus and found that the decrease in relative force was greater and more rapid in solei from C-26 mice compared to controls. These data demonstrate that severe cancer cachexia causes profound muscle weakness that is not entirely explained by the muscle atrophy. In addition, cancer cachexia decreases the fatigue resistance of the soleus muscle, a postural muscle typically resistant to fatigue. Thus, specifically targeting contractile dysfunction represents an additional means to counter muscle weakness in cancer cachexia, in addition to targeting the prevention of muscle atrophy.

Inhibition of IkappaB kinase alpha (IKKα) or IKKbeta (IKKß) plus forkhead box O (Foxo) abolishes skeletal muscle atrophy.

Two transcription factor families that are activated during multiple conditions of skeletal muscle wasting are nuclear factor κB (NF-κB) and forkhead box O (Foxo). There is clear evidence that both NF-κB and Foxo activation are sufficient to cause muscle fiber atrophy and they are individually required for at least half of the fiber atrophy during muscle disuse, but there is no work determining the combined effect of inhibiting these factors during a physiological condition of muscle atrophy. Here, we determined whether inhibition of Foxo activation plus inhibition of NF-κB activation, the latter by blocking the upstream inhibitor of kappaB kinases (IKKα and IKKß), would prevent muscle atrophy induced by 7 days of cast immobilization. Results were based on measurements of mean fiber cross-sectional area (CSA) from 72 muscles transfected with 5 different mutant expression plasmids or plasmid combinations. Immobilization caused a 47% decrease in fiber CSA in muscles injected with control plasmids. Fibers from immobilized muscles transfected with dominant negative (d.n.) IKKα-EGFP, d.n. IKKß-EGFP or d.n. Foxo-DsRed showed a 22%, 57%, and 76% inhibition of atrophy, respectively. Co-expression of d.n. IKKα-EGFP and d.n. Foxo-DsRed significantly inhibited 89% of the immobilization-induced fiber atrophy. Similarly, co-expression of d.n. IKKß-EGFP and d.n. Foxo-DsRed inhibited the immobilization-induced fiber atrophy by 95%. These findings demonstrate that the combined effects of inhibiting immobilization-induced NF-κB and Foxo transcriptional activity has an additive effect on preventing immobilization-induced atrophy, indicating that NF-κB and Foxo have a cumulative effect on atrophy signaling and/or atrophy gene expression.

p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting.

Oxidative stress is a primary trigger of cachectic muscle wasting, but the signaling pathway(s) that links it to the muscle wasting processes remains to be defined. Here, we report that activation of p38 mitogen-activated protein kinase (MAPK) (phosphorylation) and increased oxidative stress (trans-4-hydroxy-2-nonenal protein modification) in skeletal muscle occur as early as 8 h after lipopolysaccharide (1 mg/kg) and 24 h after dexamethasone (25 mg/kg) injection (intraperitoneal) in mice, concurrent with upregulation of autophagy-related genes, Atg6, Atg7, and Atg12. Treating cultured C2C12 myotubes with oxidant hydrogen peroxide (4 h) resulted in increased p38 phosphorylation and reduced FoxO3 phosphorylation along with induced Atg7 mRNA expression without activation of NF-kappaB or FoxO3a transcriptional activities. Furthermore, inhibition of p38alpha/beta by SB202190 blocked hydrogen peroxide-induced atrophy with diminished upregulation of Atg7 and atrogenes [muscle atrophy F-box protein (MAFbx/Atrogin-1), muscle ring finger protein 1 (MuRF-1), and Nedd4]. These findings provide direct evidence for p38alpha/beta MAPK in mediating oxidative stress-induced autophagy-related genes, suggesting that p38alpha/beta MAPK regulates both the ubiquitin-proteasome and the autophagy-lysosome systems in muscle wasting.

Calpain-1 is required for hydrogen peroxide-induced myotube atrophy.

Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 microM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (-81%), alpha-actinin (-40%), desmin (-79%), talin (-37%), and troponin I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate alphaII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 microM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress.

Antioxidants attenuate oxidative damage in rat skeletal muscle during mild ischaemia.

We have previously shown oxidative stress and oedema, caused by both xanthine oxidase-derived oxidants and infiltrating neutrophils, within skeletal muscle after contractile-induced claudication. The purpose of this study was to determine whether supplementation with antioxidant vitamins attenuates the oxidative stress, neutrophil infiltration and oedema associated with an acute bout of contractile-induced claudication. Rats received vehicle, vitamin C, vitamin E or vitamin C + E for 5 days prior to contractile-induced claudication. Force production was significantly reduced in the claudicant limbs of all groups compared with the control (sham) limb of control animals. Contractile-induced claudication caused a significant increase in protein oxidation, lipid peroxidation, neutrophil infiltration and oedema compared with sham muscles. Supplementation with vitamin C, E or C + E prevented the increases in each of these, and there were no differences between groups. These findings suggest that, in an animal model of exercise-induced claudication, neutrophil chemotaxis is caused by oxidizing species and that antioxidant supplementation can prevent oxidative damage, neutrophil infiltration and oedema following an acute bout of contractile-induced claudication.

Xanthine oxidase and activated neutrophils cause oxidative damage to skeletal muscle after contractile claudication.

We previously showed oxidative damage and edema within skeletal muscle after contractile claudication. To investigate the sources of this oxidative damage in the gastrocnemius muscle, we administered allopurinol (Allo, to inhibit xanthine oxidase) and cyclophosphamide (Cyclo, to deplete neutrophils) before inducing contractile claudication in male Sprague Dawley rats. Contractile claudication (ligated stimulated, LS) caused a significant increase in xanthine oxidase activity [sham ligated stimulated (SS) = 2.57 +/- 0.07; LS = 3.22 +/- 0.07] and neutrophil infiltration (SS = 0.47 +/- 0.03; LS = 0.91 +/- 0.10) compared with controls (SS), and this was associated with increased lipid peroxidation, protein oxidation, muscle damage, and edema. Pretreatment with Allo attenuated the increase in xanthine oxidase activity and attenuated lipid hydroperoxides (control LS = 12.85 +/- 0.50; Allo LS = 9.96 +/- 0.71), muscle damage, and neutrophil infiltration (control LS = 0.91 +/- 0.10; Allo LS = 0.61 +/- 0.07). This latter finding suggests that xanthine oxidase-derived oxidants are chemotactic to neutrophils. Pretreatment with Cyclo reduced neutrophil infiltration (control LS = 0.91 +/- 0.10; Cyclo LS = 0.55 +/- 0.02) and attenuated lipid peroxidation (control LS = 12.85 +/- 0.50; Cyclo LS = 6.462 +/- 0.62), protein oxidation (control LS = 2.59 +/- 0.47; Cyclo LS = 1.77 +/- 0.60), muscle damage, and edema. Together, these data indicate that contractile claudication causes an increase in xanthine oxidase activity and neutrophils in muscle and that inhibition of these oxidant sources protects against oxidative stress, muscle damage, and edema.

Oxidative damage to skeletal muscle following an acute bout of contractile claudication.

The purpose of this study was to determine the extent and sources of oxidative stress within skeletal muscle following an acute bout of contractile claudication. Twenty-four hours after unilateral ligation of the femoral artery, rat hind limbs were stimulated in vivo for 30 min, and force production measured. One-hour post-stimulation, animals were sacrificed and soleus and gastrocnemius muscles removed. There was significant reduction in force in the control limb (sham ligated/stimulated (SS)), while force in the ligated limb (ligated/stimulated (LS)) was reduced by 72%. There was an increase in skeletal muscle lipid hydroperoxides (53 and 47%) and protein carbonyls (57 and 54%) in the soleus and gastrocnemius muscles, respectively, and the muscle wet/dry weight ratio was increased in the gastrocnemius muscles. Total glutathione (GHS) was reduced, while xanthine oxidase (XO) activity and neutrophil levels were increased, in LS compared to SS in both soleus and gastrocnemius muscles. These data suggest that an acute bout of contractile claudication causes significant oxidative damage and edema to skeletal muscle. This is associated with both an increase in the activity of the radical-producing enzyme xanthine oxidase and an increase in activated neutrophils.