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.

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.

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.

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.

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.

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.

MicroRNA-149 inhibits PARP-2 and promotes mitochondrial biogenesis via SIRT-1/PGC-1α network in skeletal muscle.

High-fat diet (HFD) plays a central role in the initiation of mitochondrial dysfunction that significantly contributes to skeletal muscle metabolic disorders in obesity. However, the mechanism by which HFD weakens skeletal muscle metabolism by altering mitochondrial function and biogenesis is unknown. Given the emerging roles of microRNAs (miRNAs) in the regulation of skeletal muscle metabolism, we sought to determine whether activation of a specific miRNA pathway would rescue the HFD-induced mitochondrial dysfunction via the sirtuin-1 (SIRT-1)/ peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) pathway, a pathway that governs genes necessary for mitochondrial function. We here report that miR-149 strongly controls SIRT-1 expression and activity. Interestingly, miR-149 inhibits poly(ADP-ribose) polymerase-2 (PARP-2) and so increased cellular NAD(+) levels and SIRT-1 activity that subsequently increases mitochondrial function and biogenesis via PGC-1α activation. In addition, skeletal muscles from HFD-fed obese mice exhibit low levels of miR-149 and high levels of PARP-2, and they show reduced mitochondrial function and biogenesis due to a decreased activation of the SIRT-1/PGC-1α pathway, suggesting that mitochondrial dysfunction in the skeletal muscle of obese mice may be because of, at least in part, miR-149 dysregulation. Overall, miR-149 may be therapeutically useful for treating HFD-induced skeletal muscle metabolic disorders in such pathophysiological conditions as obesity and type 2 diabetes.