Hyperactive mTORC1 signaling is unaffected by metformin treatment in aged skeletal muscle

Introduction: Appropriate activation of growth signaling pathways, specifically mammalian target of rapamycin complex 1 (mTORC1), is central to muscle mass and metabolism. The goal of these studies was to examine the effects of metformin on mTORC1 signaling in aged skeletal muscle in an attempt to normalize growth signaling. Methods: Aged (23m) and young (3m) male mice were fed a low fat diet without or with 0.5% metformin for up to 8 weeks, then mTORC1‐related signaling was examined in the plantar flexor complex. Results: Metformin had no significant effect on lowering body weight or muscle mass in aged animals, nor altered p70 S6 Kinase 1 (S6K1) and 4E‐binding protein 1 (4E‐BP1) phosphorylation. However, it significantly (P < 0.05) reduced body weight and lowered S6K1 and rpS6 phosphorylation in the young. Conclusions: Collectively, these data suggest metformin is ineffective at normalizing growth signaling in aged skeletal muscle. Muscle Nerve 53 : 107–117, 2016     

Amelioration of ischemia‐reperfusion–induced muscle injury by the recombinant human MG53 protein

Introduction: Ischemia‐reperfusion injury (I‐R) in skeletal muscle requires timely treatment. Methods: Rodent models of I‐R injury were used to test the efficacy of recombinant human MG53 (rhMG53) protein for protecting skeletal muscle. Results: In a mouse I‐R injury model, we found that mg53,?/? mice are more susceptible to I‐R injury. rhMG53 applied intravenously to the wild‐type mice protected I‐R injured muscle, as demonstrated by reduced CK release and Evans blue staining. Histochemical studies confirmed beneficial effects of rhMG53. Of interest, rhMG53 did not protect against I‐R injury in rat skeletal muscle. This was likely due to the fact that the plasma level of endogenous MG53 protein is high in rats. Conclusions: Our data suggest that rhMG53 may be a potential therapy for protection against muscle trauma. A mouse model appears to be a better choice than a rat model for evaluating potential treatments for protecting skeletal muscle. Muscle Nerve 52 : 852–858, 2015     

Effect of recombinant human MG53 protein on tourniquet‐induced ischemia‐reperfusion injury in rat muscle

Introduction: Skeletal muscle ischemia–reperfusion injury (I‐R) is a complex injury process that includes damage to the sarcolemmal membrane, contributing to necrosis and apoptosis. MG53, a muscle‐specific TRIM family protein, has been shown to be essential for regulating membrane repair and has been shown to be protective against cardiac I‐R and various forms of skeletal muscle injury. The purpose of this study was to determine if recombinant human MG53 (rhMG53) administration offered protection against I‐R. Methods: rhMG53 was administered to rats immediately before tourniquet‐induced ischemia and again immediately before reperfusion. Two days later muscle damage was assessed histologically. Results: rhMG53 offered no protective effect, as evidenced primarily by similar Evans blue dye inclusion in the muscles of rats administered rhMG53 or saline. Conclusions: Administration of rhMG53 does not offer protection against I‐R in rat skeletal muscle. Additional studies are required to determine if the lack of a response is species‐specific. Muscle Nerve 49 : 919–921, 2014     

Effects of moderate‐ and high‐intensity chronic exercise on brain‐derived neurotrophic factor expression in fast and slow muscles

Introduction: Brain‐derived neurotrophic factor (BDNF) protein expression is sensitive to cellular activity. In the sedentary state, BDNF expression is affected by the muscle phenotype. Methods: Eighteen Wistar rats were divided into the following 3 groups: sedentary (S); moderate‐intensity training (MIT); and high‐intensity training (HIT). The training protocol lasted 8 weeks. Forty‐eight hours after training, total RNA and protein levels in the soleus and plantaris muscles were obtained. Results: In the plantaris, the BDNF protein level was lower in the HIT than in the S group (P < 0.05). A similar effect was found in the soleus (without significant difference). In the soleus, higher Bdnf mRNA levels were found in the HIT group (P < 0.001 vs. S and MIT groups). In the plantaris muscle, similar Bdnf mRNA levels were found in all groups. Conclusions: These results indicate that high‐intensity chronic exercise reduces BDNF protein level in fast muscles and increases Bdnf mRNA levels in slow muscles. Muscle Nerve 53: 446–451, 2016     

Loss of sparc in mouse skeletal muscle causes myofiber atrophy

Introduction: The expression of secreted protein acidic and rich in cysteine (SPARC) in skeletal muscle decreases with age. Here, we examined the role of SPARC in skeletal muscle by reducing its expression. Methods: SPARC expression was suppressed by introducing short interfering RNA (siRNA) into mouse tibialis anterior muscle. Myofiber diameter, atrogin1, and muscle RING‐finger protein 1 (MuRF1) expression, and tumor necrosis factor‐α (TNFα) and transforming growth factor‐β (TGFβ) signaling were then analyzed. Results: Reduced SPARC expression caused decreases in the diameter of myofibers, especially fast‐type ones, accompanied by upregulation of atrogin1, but not MuRF1, at 10 days after siRNA transfection. The expression of TNFα and TGFβ and the phosphorylation status of p38 were not affected by SPARC knockdown, whereas Smad3 phosphorylation was increased at 2 days after siRNA transfection. Conclusions: The loss of SPARC not only upregulates atrogin1 expression but also enhances TGFβ signaling, which may in turn cause muscle atrophy. Muscle Nerve 48:791–799, 2013     

Effects of tail suspension on serum testosterone and molecular targets regulating muscle mass

Introduction: The contribution of reduced testosterone levels to tail suspension (TS)‐induced muscle atrophy remains equivocal. The molecular mechanism by which testosterone regulates muscle mass during TS has not been investigated. Methods: Effects of TS on serum testosterone levels, muscle mass, and expression of muscle atrophy‐ and hypertrophy‐inducing targets were measured in soleus (SOL) and extensor digitorum longus (EDL) muscles after testosterone administration during 1, 5, and 14 days of TS in male mice. Results: TS produced an increase followed by a transient drop in testosterone levels. Muscle atrophy was associated with downregulation of Igf1 and upregulation of Mstn, Redd1, Atrogin‐1, and MuRF1 mRNA with clear differences in Igf1, Mstn, and MAFbx/Atrogin‐1 gene expression between SOL and EDL. Testosterone supplementation did not affect muscle mass or protein expression levels during TS. Conclusions The known anabolic effects of testosterone are not sufficient to ameliorate loss of muscle mass during TS. Muscle Nerve 52 : 278–288, 2015     

Metformin increases peroxisome proliferator–activated receptor γ Co‐activator‐1α and utrophin a expression in dystrophic skeletal muscle

Introduction: Metformin (MET) stimulates skeletal muscle AMP‐activated protein kinase (AMPK), a key phenotype remodeling protein with emerging therapeutic relevance for Duchenne muscular dystrophy (DMD). Our aim was to identify the mechanism of impact of MET on dystrophic muscle. Methods: We investigated the effects of MET in cultured C₂C₁₂ muscle cells and mdx mouse skeletal muscle. Expression of potent phenotypic modifiers was assessed, including peroxisome proliferator–activated receptor (PPAR)γ coactivator‐1α (PGC‐1α), PPARδ, and receptor‐interacting protein 140 (RIP140), as well as that of the dystrophin‐homolog, utrophin A. Results: In C₂C₁₂ cells, MET augmented expression of PGC‐1α, PPARδ, and utrophin A, whereas RIP140 content was reciprocally downregulated. MET treatment of mdx mice increased PGC‐1α and utrophin A and normalized RIP140 levels. Conclusions: In this study we identify the impact of MET on skeletal muscle and underscore the timeliness and importance of investigating MET and other AMPK activators as relevant therapeutics for DMD. Muscle Nerve 52 : 139–142, 2015     

Branched‐chain amino acids protect against dexamethasone‐induced soleus muscle atrophy in rats

We investigated the utility of branched‐chain amino acids (BCAA) in dexamethasone‐induced muscle atrophy. Dexamethasone (600 μg/kg, intraperitoneally) and/or BCAA (600 mg/kg, orally) were administered for 5 days in rats, and the effect of BCAA on dexamethasone‐induced muscle atrophy was evaluated. Dexamethasone decreased total protein concentration of rat soleus muscles. Concomitant administration of BCAA reversed the decrease. Dexamethasone decreased mean cross‐sectional area of soleus muscle fibers, which was reversed by BCAA. Dexamethasone increased atrogin‐1 expression, which has been reported to play a pivotal role in muscle atrophy. The increased expression of atrogin‐1 mRNA was significantly attenuated by BCAA. Furthermore, dexamethasone‐induced conversion from microtubule‐associated protein 1 light chain 3 (LC3)‐I to LC3‐II, which is an indicator of autophagy, was blocked by BCAA. These findings suggest that BCAA decreased protein breakdown to prevent muscle atrophy. BCAA administration appears to be useful for prevention of steroid myopathy. Muscle Nerve, 2010     

A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling,thereby reducing energy requirements during ischemia

Polyphenol resveratrol (RSV) has been associated with Silent Information Regulator T1 (SIRT1) and AMP‐activated protein kinase (AMPK) metabolic stress sensors and probably responds to the intracellular energy status. Our aim here was to investigate the neuroprotective effects of RSV and its association with SIRT1 and AMPK signaling in recurrent ischemia models. In this study, elderly male Wistar rats received a combination of two mild transient middle cerebral artery occlusions (tMCAOs) as an in vivo recurrent ischemic model. Primary cultured cortical neuronal cells subjected to combined oxygen–glucose deprivation (OGD) were used as an in vitro recurrent ischemic model. RSV administration significantly reduced infarct volumes, improved behavioral deficits and protected neuronal cells from cell death in recurrent ischemic stroke models in vivo and in vitro. RSV treatments significantly increased the intracellular NAD⁺/NADH ratio, AMPK and SIRT1 activities, decreased energy assumption and restored cell energy ATP level. SIRT1 and AMPK inhibitors and specific small interfering RNA (siRNA) for SIRT1 and AMPK significantly abrogated the neuroprotection induced by RSV. AMPK‐siRNA and inhibitor decreased SIRT1 activities; however, SIRT1‐siRNA and inhibitor had no impact on phospho‐AMPK (p‐AMPK) levels. These results indicated that the neuroprotective effects of RSV increased the intracellular NAD⁺/NADH ratio as well as AMPK and SIRT1 activities, thereby reducing energy ATP requirements during ischemia. SIRT1 is a downstream target of p‐AMPK signaling induced by RSV in the recurrent ischemic stroke model.     

SIRT1 inhibition by sirtinol aggravates brain edema after experimental subarachnoid hemorrhage

Secondary brain injury following subarachnoid hemorrhage (SAH) is poorly understood. We utilized a rat model of SAH to investigate whether SIRT1 has a protective role against brain edema via the tumor suppressor protein p53 pathway. Experimental SAH was induced in adult male Sprague‐Dawley rats by prechiasmatic cistern injection. Brain SIRT1 protein levels were examined in the sham controls and in rats 6, 12, 24, 48, and 72 hr after SAH induction. The SIRT1 inhibitor sirtinol was administered by intracerebroventricular infusion. Neurological functions, blood–brain barrier (BBB) disruption, and brain water content were assessed. Endothelial cell apoptosis, caspase 3 protein expression, p53 acetylation, and matrix metalloproteinase‐9 (MMP‐9) activity were examined. Compared with the control, SIRT1 protein expression increased remarkably, reaching a maximum at 24 hr after SAH. Sirtinol treatment significantly lowered SIRT1 expression, accompanied by deteriorated neurologic function, BBB disruption, brain edema, increased endothelial cell apoptosis, and increased MMP‐9 gelatinase activity compared with the rats treated with vehicle only. Our results suggest that increased expression of endogenous SIRT1 may play a neuroprotective role against brain edema after SAH. © 2014 Wiley Periodicals, Inc.     

Therapeutic effect of microcurrent on calf muscle atrophy in immobilized rabbit

Introduction: Immobilization causes significant muscle loss. In this study we assessed the regenerative effect of microcurrent electrical stimulation (MES) on gastrocnemius muscle (GCM) atrophy induced by immobilization by cast (IC) in rabbits. Methods: Fifteen rabbits were divided into 3 groups: IC (group 1); IC and free re‐ambulation for 2 weeks after cast removal (CR) (group 2); and IC and MES for 2 weeks after CR (group 3). We evaluated clinical parameters (calf circumference, compound muscle action potential of tibial nerve, and thickness of GCM by ultrasound), histomorphometric data (muscle composition and cross‐sectional area), and immunohistochemistry. Results: Mean atrophic changes in clinical parameters in group 3 were significantly less than those in groups 1 and 2 (P < 0.05). Histomophometric and immunohistochemical parameters in group 3 were significantly greater than those in groups 1 and 2, respectively (P < 0.05). Discussion: MES prevents muscle atrophy and facilitates regeneration of muscle. Muscle Nerve 58 : 270–276, 2018     

New mouse model of skeletal muscle atrophy using spiral wire immobilization

Introduction: Disuse‐induced skeletal muscle atrophy is a serious concern; however, there is not an effective mouse model to elucidate the molecular mechanisms. We developed a noninvasive atrophy model in mice. Methods: After the ankle joints of mice were bandaged into a bilateral plantar flexed position, either bilateral or unilateral hindlimbs were immobilized by wrapping in bonsai steel wire. Results: After 3, 5, or 10 days of immobilization of the hip, knee, and ankle, the weight of the soleus and plantaris muscles decreased significantly in both bilateral and unilateral immobilization. MAFbx/atrogin‐1 and MuRF1 mRNA was found to have significantly increased in both muscles, consistent with disuse‐induced atrophy. Notably, the procedure did not result in either edema or necrosis in the fixed hindlimbs. Conclusions: This method allows repeated, direct access to the immobilized muscle, making it a useful procedure for concurrent application and assessment of various therapeutic interventions. Muscle Nerve 54 : 788–791, 2016     

Isometric skeletal muscle force measurement in primary myopathies

Introduction: In myopathy patients, it is useful to measure skeletal muscle forces. Conventional methods require voluntary muscle activation, which can be unreliable. We evaluated a device for nonvoluntary force assessment. Methods: We tested 8 patients (unknown myopathy n = 2, inflammatory myopathy, facioscapulohumeral muscular dystrophy, mitochondrial myopathy, dysferlinopathy, multi‐minicore disease, Becker‐Kiener muscular dystrophy, n = 1 each). Isometric twitch torques of ankle dorsiflexors were measured after fibular nerve stimulation. Results: Six patients had decreased torques vs. 8 controls (men: median Newton‐meter 1.6 vs. 5.7, women: 0.2 vs. 3.9, both P < 0.0001). Values correlated with Manual Muscle Test results (r = 0.73; r² = 0.53; P < 0.0001). In weak dorsiflexors, torque could be measured despite lower signal‐to‐noise ratios. In 2 patients with hypertrophy, we measured increased torques. Conclusions: Nonvoluntary muscle force assessment can be used in patients with myopathies, and values correlate with voluntary forces determined by traditional methods. Muscle Nerve 53 : 913–917, 2016     

Adaptive responses of TRPC1 and TRPC3 during skeletal muscle atrophy and regrowth

Introduction: We assessed the time‐dependent changes of transient receptor potential canonical type 1 (TRPC1) and TRPC3 expression and localization associated with muscle atrophy and regrowth in vivo. Methods: Mice were subjected to hindlimb unloading for 7 or 14 days (7U, 14U) followed by 3, 7, or 14 days of reloading (3R, 7R, 14R). Results: Soleus muscle mass and tetanic force were reduced significantly at 7U and 14U and recovered by 14R. Recovery of muscle fiber cross‐sectional area was observed by 28R. TRPC1 mRNA was unaltered during the unloading‐reloading period. However, protein expression remained depressed through 14R. Decreased localization of TRPC1 to the sarcolemma was observed. TRPC3 mRNA and protein expression levels were decreased significantly during the early phase of reloading. Conclusions: Given the known role of these channels in muscle development, changes observed in TRPC1 and TRPC3 may relate closely to muscle atrophy and remodeling processes. Muscle Nerve 49 : 691–699, 2014