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.

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.

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.

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.

Mitochondria Initiate and Regulate Sarcopenia.

We present the hypothesis that an accumulation of dysfunctional mitochondria initiates a signaling cascade leading to motor neuron and muscle fiber death and culminating in sarcopenia. Interactions between neural and muscle cells that contain dysfunctional mitochondria exacerbate sarcopenia. Preventing sarcopenia will require identifying mitochondrial sources of dysfunction that are reversible.

Genome-wide Mechanosensitive MicroRNA (MechanomiR) Screen Uncovers Dysregulation of Their Regulatory Networks in the mdm Mouse Model of Muscular Dystrophy.

Muscular dystrophies (MDs) are a heterogeneous group of genetic and neuromuscular disorders, which result in severe loss of motor ability and skeletal muscle mass and function. Aberrant mechanotransduction and dysregulated-microRNA pathways are often associated with the progression of MD. Here, we hypothesized that dysregulation of mechanosensitive microRNAs (mechanomiRs) in dystrophic skeletal muscle plays a major role in the progression of MD. To test our hypothesis, we performed a genome-wide expression profile of anisotropically regulated mechanomiRs and bioinformatically analyzed their target gene networks. We assessed their functional roles in the advancement of MD using diaphragm muscles from mdm (MD with myositis) mice, an animal model of human tibial MD (titinopathy), and their wild-type littermates. We were able to show that ex vivo anisotropic mechanical stretch significantly alters the miRNA expression profile in diaphragm muscles from WT and mdm mice; as a result, some of the genes associated with MDs are dysregulated in mdm mice due to differential regulation of a distinct set of mechanomiRs. Interestingly, we found a contrasting expression pattern of the highly expressed let-7 family mechanomiRs, let-7e-5p and miR-98-5p, and their target genes associated with the extracellular matrix and TGF-ß pathways, respectively, between WT and mdm mice. Gain- and loss-of-function analysis of let-7e-5p in myocytes isolated from the diaphragms of WT and mdm mice confirmed Col1a1, Col1a2, Col3a1, Col24a1, Col27a1, Itga1, Itga4, Scd1, and Thbs1 as target genes of let-7e-5p. Furthermore, we found that miR-98 negatively regulates myoblast differentiation. Our study therefore introduces additional biological players in the regulation of skeletal muscle structure and myogenesis that may contribute to unexplained disorders of MD.

Ankyrin repeat domain protein 2 and inhibitor of DNA binding 3 cooperatively inhibit myoblast differentiation by physical interaction.

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB(mdm)) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.

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.

M-cadherin-inhibited phosphorylation of ß-catenin augments differentiation of mouse myoblasts.

ß-Catenin is essential for muscle development because it regulates both cadherin-mediated cell-cell adhesion and canonical Wingless and Int1 (Wnt) signaling. The phosphorylation of ß-catenin by glycogen synthase kinase-3ß (GSK-3ß) at serine31/37/threonine41 regulates its stability and its role in canonical Wnt signaling. In this study, we have investigated whether the N-terminal phosphorylation of ß-catenin is regulated by M-cadherin, and whether this regulation mediates the role of M-cadherin in myogenic differentiation. Our data show that the knockdown of M-cadherin expression by RNA interference (RNAi) in C2C12 myoblasts significantly increases the phosphorylation of ß-catenin at Ser33/37/Thr41 and decreases the protein abundance of ser37/thr41-unphosphorylated active ß-catenin. Furthermore, M-cadherin RNAi promotes TCF/LEF transcription activity but also blunts the initiation of the myogenic progress by Wnt pathway activator lithium chloride or Wnt-3a treatment. Knockdown of ß-catenin expression by RNAi decreases myogenic induction in myoblasts. Forced expression of a phosphorylation-resistant ß-catenin plasmid (S33Y-ß-catenin) fails to enhance myogenic differentiation, but it partially rescues C2C12 cells from M-cadherin RNAi-induced apoptosis. These data show, for the first time, that M-cadherin-mediated signaling attenuates ß-catenin phosphorylation at Ser31/37/Thr41 by GSK-3ß, and that this regulation has a positive effect on myogenic differentiation induced by canonical Wnt signaling.

Loss of desmin triggers mechanosensitivity and up-regulation of Ankrd1 expression through Akt-NF-κB signaling pathway in smooth muscle cells.

Muscle cells, including human airway smooth muscle cells (HASMCs) express ankyrin repeat protein 1 (Ankrd1), a member of ankyrin repeat protein family. Ankrd1 efficiently interacts with the type III intermediate filament desmin. Our earlier study showed that desmin is an intracellular load-bearing protein that influences airway compliance, lung recoil, and airway contractile responsiveness. These results suggest that Ankrd1 and desmin may play important roles on ASMC homeostasis. Here we show that small interfering (si)RNA-mediated knockdown of the desmin gene in HASMCs, recombinant HASMCs (reHASMCs), up-regulates Ankrd1 expression. Moreover, loss of desmin in HASMCs increases the phosphorylation of Akt, inhibitor of κB kinase (IKK)-α, and inhibitor of κB (IκB)-α proteins, leading to NF-κB activation. Treatment of reHASMCs with Akt, IKKα, IκBα, or NF-κB inhibitor inhibits the loss of desmin-induced Ankrd1 up-regulation, suggesting Akt/NF-κB-mediated Ankrd1 regulation. Transfection of reHASMCs with siRNA specific for p50 or p65 corroborates the NF-κB-mediated Ankrd1 regulation. Luciferase reporter assays show that NF-κB directly binds on Ankrd1 promoter and up-regulates Ankrd1 levels. Overall, our data provide a new link between desmin and Ankrd1 regulation, which may be important for ASMC homeostasis.

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.

Desmin regulates airway smooth muscle hypertrophy through early growth-responsive protein-1 and microRNA-26a.

Bronchial biopsies of asthmatic patients show a negative correlation desmin expression in airway smooth muscle cell (ASMC) and airway hyperresponsiveness. We previously showed that desmin is an intracellular load-bearing protein, which influences airway compliance, lung recoil, and airway contractile responsiveness (Shardonofsky, F. R., Capetanaki, Y., and Boriek, A. M. (2006) Am. J. Physiol. Lung Cell. Mol. Physiol. 290, L890-L896). These results suggest that desmin may play an important role in ASMC homeostasis. Here, we report that ASMCs of desmin null mice (ASMCs(Des-/-)) show hypertrophy and up-regulation microRNA-26a (miR-26a). Knockdown of miR-26a in ASMCs(Des-/-) inhibits hypertrophy, whereas enforced expression of miR-26a in ASMCs(Des+/+) induces hypertrophy. We identify that Egr1 (early growth responsive protein-1) activates miR-26a promoter via enhanced phosphorylation of Erk1/2 in ASMCs(Des-/-). We show glycogen synthase kinase-3ß (GSK-3ß) as a target gene of miR-26a. Moreover, induction of ASMCs(Des-/-) hypertrophy by the Erk-1/2/Egr-1/miR-26a/GSK-3ß pathway is consistent in human recombinant ASMCs, which stably suppresses 90% endogenous desmin expression. Overall, our data demonstrate a novel role for desmin as an anti-hypertrophic protein necessary for ASMC homeostasis and identifies desmin as a novel regulator of microRNA.

Induction of Sirt1 by mechanical stretch of skeletal muscle through the early response factor EGR1 triggers an antioxidative response.

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species. The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription. In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging.

Mechanical stretch up-regulates microRNA-26a and induces human airway smooth muscle hypertrophy by suppressing glycogen synthase kinase-3ß.

Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3ß (GSK-3ß), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3'-untranslated repeat of the GSK-3ß mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3ß protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3ß. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy.

Anisotropic regulation of Ankrd2 gene expression in skeletal muscle by mechanical stretch.

The diaphragm muscles in vivo are subjected to mechanical forces both in the direction of the muscle fibers and in the direction transverse to the fibers. However, the effect of directional mechanical forces in skeletal muscle gene regulation is completely unknown. Here, we identified that stretch in the longitudinal and transverse directions to the diaphragm muscle fibers up-regulated Ankrd2 gene expression by two distinct signaling pathways in wild-type (WT) and mdm, a mouse model of muscular dystrophy with early-onset of progressive muscle-wasting. Stretch in the longitudinal direction activated both NF-kappaB and AP-1 transcription factors, whereas stretch in the transverse direction activated only AP-1 transcription factor. Interestingly, longitudinal stretch activated Ankrd2 promoter only by NF-kappaB, whereas transverse stretch activated Ankrd2 promoter by AP-1. Moreover, we found that longitudinal stretch activated Akt, which up-regulated Ankrd2 expression through NF-kappaB. However, transverse stretch activated Ras-GTP, Raf-1, and Erk1/2 proteins, which up-regulated Ankrd2 expression through AP-1. Surprisingly, the stretch-activated NF-kappaB and AP-1 signaling pathways was not involved in Ankrd2 regulation at the basal level, which was high in the mdm mouse diaphragm. Taken together, our data show the anisotropic regulation of Ankrd2 gene expression in the diaphragm muscles of WT and mdm mice via two distinct mechanosensitive signaling pathways.

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.

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.