ANKRD1 expression is aberrantly upregulated in the mdm mouse model of muscular dystrophy and induced by stretch through NFκB.

The muscular dystrophy with myositis (mdm) mouse model results in a severe muscular dystrophy due to an 83-amino-acid deletion in the N2A region of titin, an expanded sarcomeric protein that functions as a molecular spring which senses and modulates the response to mechanical forces in cardiac and skeletal muscles. ANKRD1 is one of the muscle ankyrin repeat domain proteins (MARPs) a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. The aberrant over-activation of Nuclear factor Kappa B (NF-κB) and the Ankyrin-repeat domain containing protein 1 (ANKRD1) occurs in several models of progressive muscle disease including Duchenne muscular dystrophy. We hypothesized that mechanical regulation of ANKRD1 is mediated by NF-κB activation in skeletal muscles and that this mechanism is perturbed by small deletion of the stretch-sensing titin N2A region in the mdm mouse. We applied static mechanical stretch of the mdm mouse diaphragm and cyclic mechanical stretch of C2C12 myotubes to examine the interaction between NF-κΒ and ANKRD1 expression utilizing Western blot and qRTPCR. As seen in skeletal muscles of other severe muscular dystrophies, an aberrant increased basal expression of NF-κB and ANKRD1 were observed in the diaphragm muscles of the mdm mice. Our data show that in the mdm diaphragm, basal levels of NF-κB are increased, and pharmacological inhibition of NF-κB does not alter basal levels of ANKRD1. Alternatively, NF-κB inhibition did alter stretch-induced ANKRD1 upregulation. These data show that NF-κB activity is at least partially responsible for the stretch-induced expression of ANKRD1.

Extracellular vesicles from obese and diabetic mouse plasma alter C2C12 myotube glucose uptake and gene expression.

Recent studies have indicated a role for circulating extracellular vesicles (EVs) in the pathogenesis of multiple diseases. However, most in vitro studies have used variable and arbitrary doses of EVs rather than interpreting EVs as an existing component of standard skeletal muscle cell culture media. The current study provides an initial investigation into the effects of circulating EVs on the metabolic phenotype of C2C12 myotubes by replacing EVs from fetal bovine serum with circulating EVs from control mice or mice with obesity and type 2 diabetes (OT2D). We report that EVs associated with OT2D decrease 2-NBDG uptake (a proxy measure of glucose uptake) in the insulin-stimulated state compared to controls. OT2D associated EV treatment also significantly decreased myosin heavy chain type 1 (MHCI) mRNA abundance in myotubes but had no effect on mRNA expression of any other myosin heavy chain isoforms. OT2D-associated circulating EVs also significantly increased lipid accumulation within myotubes without altering the expression of a selection of genes important for lipid entry, synthesis, or catabolism. The data indicate that, in a severely diabetic state, circulating EVs may contribute to insulin resistance and alter gene expression in myotubes in a manner consistent with the skeletal muscle phenotype observed in OT2D.

Loss of NOR-1 represses muscle metabolism through mTORC1-mediated signaling and mitochondrial gene expression in C2C12 myotubes.

Gene expression of the NR4A nuclear orphan receptor NOR-1 is reduced in obesity and in human skeletal muscle during disuse. It has been well established that NOR-1 is highly responsive to both aerobic and resistance exercise and NOR-1 overexpression is coincident with a plethora of metabolic benefits. However, it is unclear whether loss of NOR-1 contributes to inappropriate metabolic signaling in skeletal muscle that could lead to insulin resistance. The purpose of this study was to elucidate the impact of NOR-1 deficiency on C2C12 metabolic signaling. Changes in gene expression after siRNA-mediated NOR-1 knockdown in C2C12 myotubes were determined by qPCR and bioinformatic analysis of RNA-Seq data. Our RNA-Seq data identified several metabolic targets regulated by NOR-1 and implicates NOR-1 as a modulator of mTORC1 signaling via Akt-independent mechanisms. Furthermore, pathway analysis revealed NOR-1 knockdown perturbs the insulin resistance and insulin sensitivity pathways. Taken together, these data suggest skeletal muscle NOR-1 deficiency may contribute to altered metabolic signaling that is consistent with metabolic disease. We postulate that strategies that improve NOR-1 may be important to offset the negative impact that inactivity, obesity, and type 2 diabetes have on mitochondria and muscle metabolism.

Recovery from FOLFOX chemotherapy-induced systemic and skeletal muscle metabolic dysfunction in mice.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is used to treat colorectal cancer and can acutely induce metabolic dysfunction. However, the lasting effects on systemic and skeletal muscle metabolism after treatment cessation are poorly understood. Therefore, we investigated the acute and lasting effects of FOLFOX chemotherapy on systemic and skeletal muscle metabolism in mice. Direct effects of FOLFOX in cultured myotubes were also investigated. Male C57BL/6J mice completed four cycles (acute) of FOLFOX or PBS. Subsets were allowed to recover for 4 wk or 10 wk. Comprehensive Laboratory Animal Monitoring System (CLAMS) metabolic measurements were performed for 5 days before study endpoint. C2C12 myotubes were treated with FOLFOX for 24 hr. Acute FOLFOX attenuated body mass and body fat accretion independent of food intake or cage activity. Acute FOLFOX decreased blood glucose, oxygen consumption (V̇o2), carbon dioxide production (V̇co2), energy expenditure, and carbohydrate (CHO) oxidation. Deficits in V̇o2 and energy expenditure remained at 10 wk. CHO oxidation remained disrupted at 4 wk but returned to control levels after 10 wk. Acute FOLFOX reduced muscle COXIV enzyme activity, AMPK(T172), ULK1(S555), and LC3BII protein expression. Muscle LC3BII/I ratio was associated with altered CHO oxidation (r = 0.75, P = 0.03). In vitro, FOLFOX suppressed myotube AMPK(T172), ULK1(S555), and autophagy flux. Recovery for 4 wk normalized skeletal muscle AMPK and ULK1 phosphorylation. Our results provide evidence that FOLFOX disrupts systemic metabolism, which is not readily recoverable after treatment cessation. FOLFOX effects on skeletal muscle metabolic signaling did recover. Further investigations are warranted to prevent and treat FOLFOX-induced metabolic toxicities that negatively impact survival and life quality of patients with cancer.NEW & NOTEWORTHY The present study demonstrates that FOLFOX chemotherapy induces long-lasting deficits in systemic metabolism. Interestingly, FOLFOX modestly suppressed skeletal muscle AMPK and autophagy signaling in vivo and in vitro. The FOLFOX-induced suppression of muscle metabolic signaling recovered after treatment cessation, independent of systemic metabolic dysfunction. Future research should investigate if activating AMPK during treatment can prevent long-term toxicities to improve health and quality of life of patients with cancer and survivors.

Mitochondria transplant therapy improves regeneration and restoration of injured skeletal muscle.

BACKGROUND: Injection of exogenous mitochondria has been shown to improve the ischaemia-damaged myocardium, but the effect of mitochondrial transplant therapy (MTT) to restore skeletal muscle mass and function has not been tested following neuromuscular injury. Therefore, we tested the hypothesis that MTT would improve the restoration of muscle function after injury. METHODS: BaCl2 was injected into the gastrocnemius muscle of one limb of 8-12-week-old C57BL/6 mice to induce damage without injury to the resident stem cells. The contralateral gastrocnemius muscle was injected with phosphate-buffered saline (PBS) and served as the non-injured intra-animal control. Mitochondria were isolated from donor mice. Donor mitochondria were suspended in PBS or PBS without mitochondria (sham treatment) and injected into the tail vein of BaCl2 injured mice 24 h after the initial injury. Muscle repair was examined 7, 14 and 21 days after injury. RESULTS: MTT did not increase systemic inflammation in mice. Muscle mass 7 days following injury was 21.9 ± 2.1% and 17.4 ± 1.9% lower (P < 0.05) in injured as compared with non-injured intra-animal control muscles in phosphate-buffered saline (PBS)- and MTT-treated animals, respectively. Maximal plantar flexor muscle force was significantly lower in injured as compared with uninjured muscles of PBS-treated (-43.4 ± 4.2%, P < 0.05) and MTT-treated mice (-47.7 ± 7.3%, P < 0.05), but the reduction in force was not different between the experimental groups. The percentage of collagen and other non-contractile tissue in histological muscle cross sections, was significantly greater in injured muscles of PBS-treated mice (33.2 ± 0.2%) compared with MTT-treated mice (26.5 ± 0.2%) 7 days after injury. Muscle wet weight and maximal muscle force from injured MTT-treated mice had recovered to control levels by 14 days after the injury. However, muscle mass and force had not improved in PBS-treated animals by 14 days after injury. The non-contractile composition of the gastrocnemius muscle tissue cross sections was not different between control, repaired PBS-treated and repaired MTT-treated mice 14 days after injury. By 21 days following injury, PBS-treated mice had fully restored gastrocnemius muscle mass of the injured muscle to that of the uninjured muscle, although maximal plantar flexion force was still 19.4 ± 3.7% (P < 0.05) lower in injured/repaired gastrocnemius as compared with uninjured intra-animal control muscles. CONCLUSIONS: Our results suggest that systemic mitochondria delivery can enhance the rate of muscle regeneration and restoration of muscle function following injury.

Skeletal Muscle Gene Expression Profile in Response to Caloric Restriction and Aging: A Role for SirT1.

SirT1 plays a crucial role in the regulation of some of the caloric restriction (CR) responsive biological pathways. Aging suppresses SirT1 gene expression in skeletal muscle, suggesting that aging may affect the role of CR in muscle. To determine the role of SirT1 in the regulation of CR regulated pathways in skeletal muscle, we performed high-throughput RNA sequencing using total RNA isolated from the skeletal muscles of young and aged wild-type (WT), SirT1 knockout (SirT1-KO), and SirT1 overexpression (SirT1-OE) mice fed to 20 wk ad libitum (AL) or 40% CR diet. Our data show that aging repressed the global gene expression profile, which was restored by CR via upregulating transcriptional and translational process-related pathways. CR inhibits pathways linked to the extracellular matrix and cytoskeletal proteins regardless of aging. Mitochondrial function and muscle contraction-related pathways are upregulated in aged SirT1 KO mice following CR. SirT1 OE did not affect whole-body energy expenditure or augment skeletal muscle insulin sensitivity associated pathways, regardless of aging or diet. Overall, our RNA-seq data showed that SirT1 and CR have different functions and activation of SirT1 by its activator or exercise may enhance SirT1 activity that, along with CR, likely have a better functional role in aging muscle.

The Effects of Calcium-ß-Hydroxy-ß-Methylbutyrate on Aging-Associated Apoptotic Signaling and Muscle Mass and Function in Unloaded but Nonatrophied Extensor Digitorum Longus Muscles of Aged Rats.

Beta-hydroxy-beta-methylbutyrate (HMB), a naturally occurring leucine metabolite, has been shown to attenuate plantar flexor muscle loss and increase myogenic stem cell activation during reloading after a period of significant muscle wasting by disuse in old rodents. However, it was less clear if HMB would alter dorsiflexor muscle response to unloading or reloading when there was no significant atrophy that was induced by unloading. In this study, we tested if calcium HMB (Ca-HMB) would improve muscle function and alter apoptotic signaling in the extensor digitorum longus (EDL) of aged animals that were unloaded but did not undergo atrophy. The EDL muscle was unloaded for 14 days by hindlimb suspension (HS) in aged (34-36 mo.) male Fisher 344 × Brown Norway rats. The rats were removed from HS and allowed normal cage ambulation for 14 days of reloading (R). Throughout the study, the rats were gavaged daily with 170 mg of Ca-HMB or water 7 days prior to HS, then throughout 14 days of HS and 14 days of recovery after removing HS. The animals' body weights were significantly reduced by ~18% after 14 days of HS and continued to decline by ~22% during R as compared to control conditions; however, despite unloading, EDL did not atrophy by HS, nor did it increase in mass after R. No changes were observed in EDL twitch contraction time, force production, fatigue resistance, fiber cross-sectional area, or markers of nuclear apoptosis (myonuclei + satellite cells) after HS or R. While HS and R increased the proapoptotic Bax protein abundance, BCL-2 abundance was also increased as was the frequency of TUNEL-positive myonuclei and satellite cells, yet muscle mass and fiber cross-sectional area did not change and Ca-HMB treatment had no effect reducing apoptotic signaling. These data indicate that (i) increased apoptotic signaling preceded muscle atrophy or occurred without significant EDL atrophy and (ii) that Ca-HMB treatment did not improve EDL signaling, muscle mass, or muscle function in aged rats, when HS and R did not impact mass or function.

Muscle-specific sirtuin1 gain-of-function ameliorates skeletal muscle atrophy in a pre-clinical mouse model of cerebral ischemic stroke.

Stroke causes severe long-term disability in patients due to the induction of skeletal muscle atrophy and weakness, but the molecular mechanisms remain elusive. Using a preclinical mouse model of cerebral ischemic stroke, we show that stroke robustly induced atrophy and significantly decreased SirT1 gene expression in the PTA (paralytic tibialis anterior) muscle. Muscle-specific SirT1 gain-of-function mice are resistant to stroke-induced muscle atrophy and this protective effect requires its deacetylase activity. Although SirT1 counteracts the stroke-induced up-regulation of atrogin1, MuRF1 and ZNF216 genes, we found a mechanism that regulates the ZNF216 gene transcription in post-stroke muscle. Stroke increased the expression of the ZNF216 gene in PTA muscle by activating PARP-1, which binds on the ZNF216 promoter. The SirT1 gain-of-function or SirT1 activator, resveratrol, reversed the PARP-1-mediated up-regulation of ZNF216 expression at the promoter level, suggesting a contradicted role for SirT1 and PARP-1 in the regulation of ZNF216 gene. Overall, our study for the first-time demonstrated that (a) stroke causes muscle atrophy, in part, through the SirT1/PARP-1/ZNF216 signaling mechanism; (b) SirT1 can block muscle atrophy in response to different types of atrophic signals via different signaling mechanisms; and (c) SirT1 is a critical regulator of post-stroke muscle mass.

Transcriptome Analysis of Skeletal Muscle Reveals Altered Proteolytic and Neuromuscular Junction Associated Gene Expressions in a Mouse Model of Cerebral Ischemic Stroke.

Stroke is a leading cause of mortality and long-term disability in patients worldwide. Skeletal muscle is the primary systemic target organ of stroke that induces muscle wasting and weakness, which predominantly contribute to functional disability in stroke patients. Currently, no pharmacological drug is available to treat post-stroke muscle morbidities as the mechanisms underlying post-stroke muscle wasting remain poorly understood. To understand the stroke-mediated molecular changes occurring at the transcriptional level in skeletal muscle, the gene expression profiles and enrichment pathways were explored in a mouse model of cerebral ischemic stroke via high-throughput RNA sequencing and extensive bioinformatic analyses. RNA-seq revealed that the elevated muscle atrophy observed in response to stroke was associated with the altered expression of genes involved in proteolysis, cell cycle, extracellular matrix remodeling, and the neuromuscular junction (NMJ). These data suggest that stroke primarily targets muscle protein degradation and NMJ pathway proteins to induce muscle atrophy. Collectively, we for the first time have found a novel genome-wide transcriptome signature of post-stroke skeletal muscle in mice. Our study will provide critical information to further elucidate specific gene(s) and pathway(s) that can be targeted to mitigate accountable for post-stroke muscle atrophy and related weakness.

A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.

Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.

The role of SIRT1 in skeletal muscle function and repair of older mice.

BACKGROUND: Sirtuin 1 (SIRT1) is a NAD+ sensitive deacetylase that has been linked to longevity and has been suggested to confer beneficial effects that counter aging-associated deterioration. Muscle repair is dependent upon satellite cell function, which is reported to be reduced with aging; however, it is not known if this is linked to an aging-suppression of SIRT1. This study tested the hypothesis that Sirtuin 1 (SIRT1) overexpression would increase the extent of muscle repair and muscle function in older mice. METHODS: We examined satellite cell dependent repair in tibialis anterior, gastrocnemius, and soleus muscles of 13 young wild-type mice (20-30 weeks) and 49 older (80+ weeks) mice that were controls (n = 13), overexpressed SIRT1 in skeletal muscle (n = 14), and had a skeletal muscle SIRT1 knockout (n = 12) or a satellite cell SIRT1 knockout (n = 10). Acute muscle injury was induced by injection of cardiotoxin (CTX), and phosphate-buffered saline was used as a vector control. Plantarflexor muscle force and fatigue were evaluated before or 21 days after CTX injection. Satellite cell proliferation and mitochondrial function were also evaluated in undamaged muscles. RESULTS: Maximal muscle force was significantly lower in control muscles of older satellite cell knockout SIRT1 mice compared to young adult wild-type (YWT) mice (P < 0.001). Mean contraction force at 40 Hz stimulation was significantly greater after recovery from CTX injury in older mice that overexpressed muscle SIRT1 than age-matched SIRT1 knockout mice (P < 0.05). SIRT1 muscle knockout models (P < 0.05) had greater levels of p53 (P < 0.05 MKO, P < 0.001 OE) in CTX-damaged tissues as compared to YWT CTX mice. SIRT1 overexpression with co-expression of p53 was associated with increased fatigue resistance and increased force potentiation during repeated contractions as compared to wild-type or SIRT1 knockout models (P < 0.001). Muscle structure and mitochondrial function were not different between the groups, but proliferation of satellite cells was significantly greater in older mice with SIRT1 muscle knockout (P < 0.05), but not older SIRT1 satellite cell knockout models, in vitro, although this effect was attenuated in vivo after 21 days of recovery. CONCLUSIONS: The data suggest skeletal muscle structure, function, and recovery after CTX-induced injury are not significantly influenced by gain or loss of SIRT1 abundance alone in skeletal muscle; however, muscle function is impaired by ablation of SIRT1 in satellite cells. SIRT1 appears to interact with p53 to improve muscle fatigue resistance after repair from muscle injury.

Effects of hindlimb suspension and reloading on gastrocnemius and soleus muscle mass and function in geriatric mice.

Reloading of atrophied muscles after hindlimb suspension (HLS) can induce muscle injury and prolong recovery after disuse in old rats, especially in fast contracting muscles. Less is known about the responses in mice and whether fast and slow muscles from geriatric mice will respond in a similar fashion to HLS unloading and recovery (HLS + R). Furthermore, while slow muscles undergo atrophy with disuse, they typically are more resistant to sarcopenia than fast contracting muscles. Geriatric (28 mo. of age) male C57BL/6 mice were randomly placed into 3 groups. These included HLS for 14 days n = 9, and HLS followed by 14 days of reloading recovery (HLS + R; n = 9), or normal ambulatory cage controls (n = 9). Control mice were not exposed to unloading. Electrically evoked maximal muscle function was assessed in vivo in anesthetized mice at baseline, after 14 days of HLS or HLS + R. As expected, HLS significantly reduced body weight, wet weight of gastrocnemius and soleus muscles and in vivo maximal force. There were no differences in vivo fatigability of the plantar flexor muscles and overall fiber size. There were only minor fiber type distribution and frequency distribution of fiber sizes that differ between HLS + R and control gastrocnemius and soleus muscles. Soleus muscle wet weight had recovered to control levels after reloading, but type I/IIA fibers in the soleus muscles were significantly smaller after HLS + R than control muscles. In contrast, gastrocnemius muscle wet weight did not recover to control levels after reloading. Plantar flexion muscle force (primarily influenced by the gastrocnemius muscles) did not recover in HLS + R conditions as compared to HLS conditions and both were lower than control force production signaling for apoptosis, autophagy and anabolic markers were not different between control and HLS + R gastrocnemius and soleus muscles in geriatric mice. These results suggest that molecular signaling does not explain attenuated ability to regain muscle wet weight, fiber size or muscle force production after HLS in geriatric mice. It is possible that fluid shifts, reduced blood flow, or shortened muscle fibers which failed to regain control lengths contributed to the attenuation of muscle wet weight after HLS and reloading and this affected force production. Further work is needed to determine if altered/loss of neural activity contributed to the inability of geriatric mice to regain gastrocnemius muscle weight and function after HLS and reloading.

Voluntary wheel running increases satellite cell abundance and improves recovery from disuse in gastrocnemius muscles from mice.

Reloading of atrophied muscles after hindlimb suspension unloading (HSU) can induce injury and prolong recovery. Low-impact exercise, such as voluntary wheel running, has been identified as a nondamaging rehabilitation therapy in rodents, but its effects on muscle function, morphology, and satellite cell activity after HSU are unclear. This study tested the hypothesis that low-impact wheel running would increase satellite cell proliferation and improve recovery of muscle structure and function after HSU in mice. Young adult male and female C57BL/6 mice ( n = 6/group) were randomly placed into five groups. These included HSU without recovery (HSU), normal ambulatory recovery for 14 days after HSU (HSU+NoWR), and voluntary wheel running recovery for 14 days after HSU (HSU+WR). Two control groups were used: nonsuspended mouse cage controls (Control) and voluntary wheel running controls (ControlWR). Satellite cell activation was evaluated by providing mice 5-bromo-2'-deoxyuridine (BrdU) in their drinking water. As expected, HSU significantly reduced in vivo maximal force, decreased in vivo fatigability, and decreased type I and IIa myosin heavy chain (MHC) abundance in plantarflexor muscles. HSU+WR mice significantly improved plantarflexor fatigue resistance, increased type I and IIa MHC abundance, increased fiber cross-sectional area, and increased the percentage of type I and IIA muscle fibers in the gastrocnemius muscle. HSU+WR mice also had a significantly greater percentage of BrdU-positive and Pax 7-positive nuclei inside muscle fibers and a greater MyoD-to-Pax 7 protein ratio compared with HSU+NoWR mice. The mechanotransduction protein Yes-associated protein (YAP) was elevated with reloading after HSU, but HSU+WR mice had lower levels of the inactive phosphorylated YAPserine127, which may have contributed to increased satellite cell activation with reloading after HSU. These results indicate that voluntary wheel running increased YAP signaling and satellite cell activity after HSU and this was associated with improved recovery. NEW & NOTEWORTHY Although satellite cell involvement in muscle remodeling has been challenged, the data in this study suggest that voluntary wheel running increased satellite cell activity and suppressed Yes-associated protein (YAP) protein relative to no wheel running and this was associated with improved muscle recovery of force, fatigue resistance, expression of type I myosin heavy chain, and greater fiber cross-sectional area after disuse.

Anisotropic mechanosensitive pathways in the diaphragm and their implications in muscular dystrophies.

The diaphragm is the "respiratory pump;" the muscle that generates pressure to allow ventilation. Diaphragm muscles play a vital function and thus are subjected to continuous mechanical loading. One of its peculiarities is the ability to generate distinct mechanical and biochemical responses depending on the direction through which the mechanical forces applied to it. Contractile forces originated from its contractile components are transmitted to other structural components of its muscle fibers and the surrounding connective tissue. The anisotropic mechanical properties of the diaphragm are translated into biochemical signals that are directionally mechanosensitive by mechanisms that appear to be unique to this muscle. Here, we reviewed the current state of knowledge on the biochemical pathways regulated by mechanical signals emphasizing their anisotropic behavior in the normal diaphragm and analyzed how they are affected in muscular dystrophies.

Dietary resveratrol confers apoptotic resistance to oxidative stress in myoblasts.

High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 µM) or very high (1 mM) levels of H2O2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H2O2. Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H2O2. Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 µM of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H2O2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts.

Resveratrol Enhances Exercise-Induced Cellular and Functional Adaptations of Skeletal Muscle in Older Men and Women.

Older men (n = 12) and women (n = 18) 65-80 years of age completed 12 weeks of exercise and took either a placebo or resveratrol (RSV) (500 mg/d) to test the hypothesis that RSV treatment combined with exercise would increase mitochondrial density, muscle fatigue resistance, and cardiovascular function more than exercise alone. Contrary to our hypothesis, aerobic and resistance exercise coupled with RSV treatment did not reduce cardiovascular risk further than exercise alone. However, exercise added to RSV treatment improved the indices of mitochondrial density, and muscle fatigue resistance more than placebo and exercise treatments. In addition, subjects that were treated with RSV had an increase in knee extensor muscle peak torque (8%), average peak torque (14%), and power (14%) after training, whereas exercise did not increase these parameters in the placebo-treated older subjects. Furthermore, exercise combined with RSV significantly improved mean fiber area and total myonuclei by 45.3% and 20%, respectively, in muscle fibers from the vastus lateralis of older subjects. Together, these data indicate a novel anabolic role of RSV in exercise-induced adaptations of older persons and this suggests that RSV combined with exercise might provide a better approach for reversing sarcopenia than exercise alone.

MicroRNA-434-3p regulates age-related apoptosis through eIF5A1 in the skeletal muscle.

Increased activation of catabolic pathways, including apoptosis causes sarcopenia. However, the precise molecular mechanism that initiates apoptosis during aging is not well understood. Here, we report that aging alters miRNA expression profile in mouse skeletal muscle as evidenced by miRNA microarray and real-time PCR. We identified miR-434-3p as a highly downregulated miRNA in the skeletal muscle of aging mice. Myocytes transfected with miR-434-3p mimic prevents apoptosis induced by various apoptotic stimuli, and co-transfection of miR-434-3p antagomir abolishes the inhibitory role of miR-434-3p. We found that miR-434-3p inhibits apoptosis by targeting the eukaryotic translation initiation factor 5A1 (eIF5A1). Overexpression of miR-434-3p in myocytes reduces the loss of mitochondrial transmembrane potential, and activation of caspases-3, -8 and -9 by suppressing eIF5A1 in response to various apoptotic stimuli whereas inhibition of miR-434-3p reversed this scenario. Skeletal muscles from aging mice exhibit low levels of miR-434-3p and high levels of eIF5A1, suggesting a possible role for miR-434-3p in the initiation of apoptosis in aging muscle. Overall, our data identified for the first time that miR-434-3p is an anti-apoptotic miRNA that may be therapeutically useful for treating muscle atrophy in various pathophysiological conditions, including sarcopenia.

Epigallocatechin-3-gallate increases autophagy signaling in resting and unloaded plantaris muscles but selectively suppresses autophagy protein abundance in reloaded muscles of aged rats.

We have previously found that Epigallocatechin-3-gallate (EGCg), an abundant catechin in green tea, reduced apoptotic signaling and improved muscle recovery in response to reloading after hindlimb suspension (HS). In this study, we investigated if EGCg altered autophagy signaling in skeletal muscle of old rats in response to HS or reloading after HS. Fischer 344×Brown Norway inbred rats (age 34months) were given 1ml/day of purified EGCg (50mg/kg body weight), or the same sample volume of the vehicle by gavage. One group of animals received HS for 14days and the second group of rats received 14days of HS, then the HS was removed and they were allowed to recover by ambulating normally around the cage for two weeks. EGCg decreased a small number of autophagy genes in control muscles, but it increased the expression of other autophagy genes (e.g., ATG16L2, SNCA, TM9SF1, Pink1, PIM-2) and HS did not attenuate these increases. HS increased Beclin1, ATG7 and LC3-II/I protein abundance in hindlimb muscles. Relative to vehicle treatment, EGCg treatment had greater ATG12 protein abundance (35.8%, P<0.05), but decreased Beclin1 protein levels (-101.1%, P<0.05) after HS. However, in reloaded muscles, EGCg suppressed Beclin1 and LC3-II/I protein abundance as compared to vehicle treated muscles. EGCg appeared to "prime" autophagy signaling before and enhance autophagy gene expression and protein levels during unloading in muscles of aged rats, perhaps to improve the clearance of damaged organelles. However, EGCg suppressed autophagy signaling after reloading, potentially to increase the recovery of hindlimb muscles mass and function after loading is restored.