Mouse Tibialis Anterior Muscle Research Articles

Skeletal muscle healing by M1-like macrophages produced by transient expression of exogenous GM-CSF

BackgroundAfter traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing.MethodsTo test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass.ResultsMuscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-β1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p < 0.01) on day 30. uP-mGM electroporation also increased Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill.ConclusionCollectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.

TMEPAI/PMEPA1 Is a Positive Regulator of Skeletal Muscle Mass.

Inhibition of myostatin- and activin-mediated SMAD2/3 signaling using ligand traps, such as soluble receptors, ligand-targeting propeptides and antibodies, or follistatin can increase skeletal muscle mass in healthy mice and ameliorate wasting in models of cancer cachexia and muscular dystrophy. However, clinical translation of these extracellular approaches targeting myostatin and activin has been hindered by the challenges of achieving efficacy without potential effects in other tissues. Toward the goal of developing tissue-specific myostatin/activin interventions, we explored the ability of transmembrane prostate androgen-induced (TMEPAI), an inhibitor of transforming growth factor-β (TGF-β1)-mediated SMAD2/3 signaling, to promote growth, and counter atrophy, in skeletal muscle. In this study, we show that TMEPAI can block activin A, activin B, myostatin and GDF-11 activity in vitro. To determine the physiological significance of TMEPAI, we employed Adeno-associated viral vector (AAV) delivery of a TMEPAI expression cassette to the muscles of healthy mice, which increased mass by as much as 30%, due to hypertrophy of muscle fibers. To demonstrate that TMEPAI mediates its effects via inhibition of the SMAD2/3 pathway, tibialis anterior (TA) muscles of mice were co-injected with AAV vectors expressing activin A and TMEPAI. In this setting, TMEPAI blocked skeletal muscle wasting driven by activin-induced phosphorylation of SMAD3. In a model of cancer cachexia associated with elevated circulating activin A, delivery of AAV:TMEPAI into TA muscles of mice bearing C26 colon tumors ameliorated the muscle atrophy normally associated with cancer progression. Collectively, the findings indicate that muscle-directed TMEPAI gene delivery can inactivate the activin/myostatin-SMAD3 pathway to positively regulate muscle mass in healthy settings and models of disease.

Spermidine is not an independent factor regulating limb muscle mass in mice following androgen deprivation.

Maintaining a critical amount of skeletal muscle mass is linked to reduced morbidity and mortality. In males, testicular androgens regulate muscle mass with a loss of androgens being critical as it is associated with muscle atrophy. Atrophy of the limb muscles is particularly important, but the pathways by which androgens regulate limb muscle mass remain equivocal. We used microarray analysis to identify changes to genes involved with polyamine metabolism in the tibialis anterior (TA) muscle of castrated mice. Of the polyamines, the concentration of spermidine (SPD) was significantly reduced in the TA of castrated mice. To assess whether SPD was an independent factor by which androgens regulate limb muscle mass, we treated castrated mice with SPD for 8 weeks and compared them with sham operated mice. Though this treatment paradigm effectively restored SPD concentrations in the TA muscles of castrated mice, mass of the limb muscles (i.e., TA, gastrocnemius, plantaris, and soleus) were not increased to the levels observed in sham animals. Consistent with those findings, muscle force production was also not increased by SPD treatment. Overall, these data demonstrate for the first time that SPD is not an independent factor by which androgens regulate limb skeletal muscle mass. Novelty: Polyamines regulate growth in various cells/tissues. Spermidine concentrations are reduced in the limb skeletal muscle following androgen depletion. Restoring spermidine concentrations in the limb skeletal muscle does not increase limb muscle mass or force production.

The microRNA miR-133b functions to slow Duchenne muscular dystrophy pathogenesis.

Impairment of muscle biogenesis contributes to the progression of Duchenne muscular dystrophy (DMD). As a muscle enriched microRNA that has been implicated in muscle biogenesis, the role of miR-133b in DMD remains unknown. To assess miR-133b function in DMD-affected skeletal muscles, we genetically ablated miR-133b in the mdx mouse model of DMD. We show that deletion of miR-133b exacerbates the dystrophic phenotype of DMD-afflicted skeletal muscle by dysregulating muscle stem cells involved in muscle biogenesis, in addition to affecting signalling pathways related to inflammation and fibrosis. Our results provide evidence that miR-133b may underlie DMD pathology by affecting the proliferation and differentiation of muscle stem cells. Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle degeneration. No treatments are currently available to prevent the disease. While the muscle enriched microRNA miR-133b has been implicated in muscle biogenesis, its role in DMD remains unknown. To assess miR-133b function in DMD-affected skeletal muscles, we genetically ablated miR-133b in the mdx mouse model of DMD. In the absence of miR-133b, the tibialis anterior muscle of P30 mdx mice is smaller in size and exhibits a thickened interstitial space containing more mononucleated cells. Additional analysis revealed that miR-133b deletion influences muscle fibre regeneration, satellite cell proliferation and differentiation, and induces widespread transcriptomic changes in mdx muscle. These include known miR-133b targets as well as genes involved in cell proliferation and fibrosis. Altogether, our data demonstrate that skeletal muscles utilize miR-133b to mitigate the deleterious effects of DMD.

Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism.

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3β/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3β activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

Overexpression of Dnmt3a ameliorates diabetic muscle atrophy by modulating the Pten/Akt pathway.

What is the central question of this study? Does Dnmt3a play a crucial role in regulating diabetic muscle atrophy? What is the main finding and its importance? Muscle atrophy is one of the major long-term complications of diabetes mellitus. However, little is known about the molecular mechanism involved. In this paper, we demonstrated that Dnmt3a overexpression effectively improves the diabetic muscle health in mice and documented the underlying mechanisms. DNMT3A might become a promising target to prevent muscle atrophy in patients with diabetes. Muscle atrophy is one of the major long-term complications of diabetes mellitus, which greatly affects the mobility of patients. Epigenetic processes mediated by DNA methyltransferases (DNMTs) play crucial roles in the locomotor system, but little is known about the functions of DNMTs in diabetic muscle atrophy. Here, we investigated the function of Dnmt3a in diabetic muscle atrophy and explored the mechanisms involved. Adeno-associated virus AAV2 overexpressing Dnmt3a or its vector control was injected into the tibialis anterior muscle of streptozotocin-induced diabetic mice. Muscle mass and muscle cross-sectional area were used to evaluate muscle atrophy. In vitro, adeno-associated virus AAV2 overexpressing Dnmt3a or its vector control was transfected into C2C12 myoblasts. Horse serum was used to induce differentiation and palmitate to stimulate the C2C12 myoblasts. The expressions of myogenic regulatory factors were examined by real-time PCR and western blot analysis. Overexpression of Dnmt3a attenuated muscle atrophy in diabetic mice and promoted myotube formation of C2C12 myoblasts. Overexpression of Dnmt3a restored the expressions of myogenic regulatory factors atrogin-1, MuRF1, Pax7, Myod1 and myogenin, both in vivo and in vitro. Moreover, overexpression of Dnmt3a activated the phosphorylation of Akt by inhibiting the activation of Pten. This study demonstrates that overexpression of Dnmt3a prevents diabetic muscle atrophy by modulating the Pten/Akt pathway.

Vascular endothelial growth factor B promotes transendothelial fatty acid transport into skeletal muscle via histone modifications during catch-up growth.

Caloric restriction (CR) followed by refeeding, a phenomenon known as catch-up growth (CUG), results in excessive lipid deposition and insulin resistance in skeletal muscle, but the underlying mechanisms remain elusive. Recent reports have suggested that vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation by regulating endothelial fatty acid transport. Here, we found continuous activation of VEGF-B signaling and increased lipid uptake in skeletal muscle from CR to refeeding, as well as increased lipid deposition and impaired insulin sensitivity after refeeding in the skeletal muscle of CUG rodents. Inhibiting VEGF-B signaling reduced fatty acid uptake in and transport across endothelial cells. Knockdown of Vegfb in the tibialis anterior (TA) muscle of CUG mice significantly attenuated muscle lipid accumulation and ameliorated muscle insulin sensitivity by decreasing lipid uptake. Furthermore, we showed that aberrant histone methylation (H3K9me1) and acetylation (H3K14ac and H3K18ac) at the Vegfb promoter might be the main cause of persistent VEGF-B upregulation in skeletal muscle during CUG. Modifying these aberrant loci using their related enzymes [PHD finger protein 2 (PHF2) or E1A binding protein p300 (p300)] could regulate VEGF-B expression in vitro. Collectively, our findings indicate that VEGF-B can promote transendothelial lipid transport and lead to lipid overaccumulation and insulin resistance in skeletal muscle during CUG, which might be mediated by histone methylation and acetylation.

T-type calcium channel blockade induces apoptosis in C2C12 myotubes and skeletal muscle via endoplasmic reticulum stress activation.

Loss of T-type calcium channel (TCC) function has been reported to result in decreased cell viability and impaired muscle regeneration, but the underlying mechanisms remain largely unknown. We previously found that expression of TCC is reduced in aged pelvic floor muscle of multiple vaginal delivery mice, and this is related to endoplasmic reticulum stress (ERS) activation and autophagy flux blockade. In the present work, we further investigated the effects of TCC function loss on C2C12 myotubes and skeletal muscle, which is mediated by promotion of ERS and ultimately contributes to mitochondrial-related apoptotic cell death. We found that application of a TCC inhibitor induced mitochondria-related apoptosis in a dose-dependent manner and also reduced mitochondrial transmembrane potential (MMP), induced mito-ROS generation, and enhanced expression of mitochondrial apoptosis proteins. Functional inhibition of TCC induced ERS, resulting in disorder of Ca2+ homeostasis in endoplasmic reticulum, and ultimately leading to cell apoptosis in C2C12 myotubes. Tibialis anterior muscles of T-type α1H channel knockout mice displayed a smaller skeletal muscle fiber size and elevated ERS-mediated apoptosis signaling. Our data point to a novel mechanism whereby TCC blockade leads to ERS activation and terminal mitochondrial-related apoptotic events in C2C12 myotubes and skeletal muscles.

Heparan sulfate controls skeletal muscle differentiation and motor functions

BackgroundHeparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear. MethodsWe examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system. ResultsCRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells. SignificanceThese results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.

Induction of a local muscular dystrophy using electroporation in vivo: an easy tool for screening therapeutics

Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose–response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.

Myogenic Cell Expression of Intercellular Adhesion Molecule-1 Contributes to Muscle Regeneration after Injury

This study investigated intercellular adhesion molecule-1 (ICAM-1), a membrane protein that mediates cell-to-cell adhesion and communication, as a mechanism through which the inflammatory response facilitates muscle regeneration after injury. Toxin-induced muscle injury to tibialis anterior muscles of wild-type mice caused ICAM-1 to be expressed by a population of satellite cells/myoblasts and myofibers. Myogenic cell expression of ICAM-1 contributed to the restoration of muscle structure after injury, as regenerating myofibers were more abundant and myofiber size was larger for wild-type compared with Icam1-/- mice during 28 days of recovery. Contrastingly, restoration of muscle function after injury was similar between the genotypes. ICAM-1 facilitated the restoration of muscle structure after injury through mechanisms involving the regulation of myofiber branching, protein synthesis, and the organization of nuclei within myofibers after myogenic cell fusion. These findings provide support for a paradigm in which ICAM-1 expressed by myogenic cells after muscle injury augments their adhesive and fusogenic properties, which, in turn, facilitates regenerative and hypertrophic processes that restore structure to injured muscle.

A subset of microRNAs in the Dlk1-Dio3 cluster regulates age-associated muscle atrophy by targeting Atrogin-1.

BackgroundThe microRNAs (miRNAs) down‐regulated in aged mouse skeletal muscle were mainly clustered within the delta‐like homologue 1 and the type III iodothyronine deiodinase (Dlk1‐Dio3) genomic region. Although clustered miRNAs are coexpressed and regulate multiple targets in a specific signalling pathway, the function of miRNAs in the Dlk1‐Dio3 cluster in muscle aging is largely unknown. We aimed to ascertain whether these miRNAs play a common role to regulate age‐related muscle atrophy.MethodsTo examine anti‐atrophic effect of miRNAs, we individually transfected 42 miRNA mimics in fully differentiated myotubes and analysed their diameters. The luciferase reporter assay using target 3′ untranslated region (UTR) and RNA pull‐down assay were employed to ascertain the target predicted by the TargetScan algorithm. To investigate the therapeutic potential of the miRNAs in vivo, we generated adeno‐associated virus (AAV) serotype 9 expressing green fluorescent protein (GFP) (AAV9‐GFP) bearing miR‐376c‐3p and infected it into the tibialis anterior muscle of old mice. We performed morphometric analysis and measured ex vivo isometric force using a force transducer. Human gluteus maximus muscle tissues (ages ranging from 25 to 80 years) were used to investigate expression levels of the conserved miRNAs in the Dlk1‐Dio3 cluster.ResultsWe found that the majority of miRNAs (33 out of 42 tested) in the cluster induced anti‐atrophic phenotypes in fully differentiated myotubes with increasing their diameters. Eighteen of these miRNAs, eight of which are conserved in humans, harboured predicted binding sites in the 3′ UTR of muscle atrophy gene‐1 (Atrogin‐1) encoding a muscle‐specific E3 ligase. Direct interactions were identified between these miRNAs and the 3′ UTR of Atrogin‐1, leading to repression of Atrogin‐1 and thereby induction of eIF3f protein content, in both human and mouse skeletal muscle cells. Intramuscular delivery of AAV9 expressing miR‐376c‐3p, one of the most effective miRNAs in myotube thickening, dramatically ameliorated skeletal muscle atrophy and improved muscle function, including isometric force, twitch force, and fatigue resistance in old mice. Consistent with our findings in mice, the expression of miRNAs in the cluster was significantly down‐regulated in human muscle from individuals > 50 years old.ConclusionsOur study suggests that genetic intervention using a muscle‐directed miRNA delivery system has therapeutic efficacy in preventing Atrogin‐1‐mediated muscle atrophy in sarcopenia.

SUN-140 TMEPAI Inhibits SMAD 2/3 Mediated Muscle Wasting

Inhibition of myostatin and activin activity using ligand traps, such as soluble receptors, follistatin and propeptides, can markedly increase skeletal muscle mass in healthy mice and ameliorate wasting in models of cancer cachexia and muscular dystrophy. Though effective, clinical translation of these approaches has been hindered by off-target effects. Toward the goal of developing tissue-specific myostatin/activin interventions, we explored the ability of transmembrane prostate androgen-induced (TMEPAI) to promote growth of skeletal muscle. TMEPAI, a transcriptional target of activin in muscle, is a known inhibitor of TGF-β1-mediated SMAD 2/3 signalling. In this study we show that TMEPAI also blocks activin A, activin B, myostatin and GDF-11 in vitro activity. Adeno-associated viral (AAV) gene delivery of TMEPAI into healthy mice increased local muscle mass by as much as 30%. Increased muscle mass was attributed to hypertrophy of fibres in TMEPAI-expressing muscles, and was coincident with an upregulation in markers of protein synthesis (pAkt, pMTOR, p70S6K). The ability of TMEPAI to block activation of the canonical activin/myostatin-SMAD 2/3 axis, was demonstrated by co-injecting AAV6:activin A and AAV6:TMEPAI into healthy mice. In this setting, TMEPAI blocked activin-induced phosphorylation of SMAD3 and associated skeletal muscle wasting. Finally, delivery of AAV6:TMEPAI into tibialis anterior muscles of mice bearing C26 tumours prevented muscle atrophy normally associated with this model. The results support that viral gene delivery of TMEPAI can effectively increase muscle mass via inactivation of the activin/myostatin-SMAD 2/3 pathway.

Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

Understanding the signaling pathways that regulate proliferation and differentiation of muscle progenitors is essential for successful cell transplantation for treatment of Duchenne muscular dystrophy. Here, we report that a γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine tertial butyl ester), which inhibits the release of NICD (Notch intercellular domain), promotes the fusion of human muscle progenitors in vitro and improves their engraftment in the tibialis anterior muscle of immune-deficient mice. Gene expression analysis revealed that DAPT severely down-regulates PTGER2, which encodes prostaglandin (PG) E2 receptor 2 (EP2), in human muscle progenitors in the differentiation condition. Functional analysis suggested that Notch signaling inhibits differentiation and promotes self-renewal of human muscle progenitors via PGE2/EP2 signaling in a cAMP/PKA-independent manner.

The Effect of Gas6‐Axl Double Knockout on Satellite Cell Proliferation and Skeletal Muscle Regeneration After Injury

Growth arrest‐specific 6 (Gas6) is an extracellular signaling molecule that is known to play a role in cell proliferation, inflammation and other processes through its interaction with Tyro3, Axl, and Mer receptors, but little is known about its role in skeletal muscle. The purpose of this study was to examine the effects of Gas6‐Axl double knockout (dKO) in skeletal muscle recovery after injury. Muscle injury was produced by injecting BaCl2 into tibialis anterior (TA) muscles of wild type (WT) and dKO mice. Muscle tissue was harvested 3, 7, and 14 days after injury. Gas6 and Axl expression over the injury timecourse was characterized by western blotting and ELISA. Muscle damage and cross‐sectional fiber areas were assessed on hematoxylin and eosin (H&amp;E) stained sections. Muscle satellite cell content and proliferation were assessed by Pax7/Ki‐67 staining. We found that both Gas6 and Axl protein expression increased substantially after injury in WT muscles, peaked at 3 days post injury, then declined toward basal levels thereafter. TA mass was lower for dKO muscles 14 days after injury, but fiber CSA was elevated in dKO muscles, suggesting a deficit in fiber number leading to compensatory hypertrophy of existing fibers. Pax7‐positive satellite cell number was unaffected by genotype in uninjured control muscles, but was ~3X higher in WT vs. dKO muscles at 3 days post injury, suggesting that the rate of satellite cell proliferation was suppressed or delayed by dKO. These results indicate that Gas6‐Axl signaling plays a role in regulating skeletal muscle regeneration after injury. Impaired Gas6 function may be linked to excess hypertrophy and loss of muscle mass after injury.Support or Funding InformationThis work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI) and a BYU Graduate Research Fellowship.

Molecular Mechanisms of Skeletal Muscle Atrophy in Cerebral Ischemic Stroke

Stroke is the leading cause of death and the top cause of adult disability in patients world‐wide. Skeletal muscle is the main effector organ accounting for disability in stroke patients due to severe muscle atrophy and related weakness. However, the molecular signaling mechanisms that cause post‐stroke muscle atrophy remains elusive. To determine the mechanisms, we used a preclinical model of stroke in which we induced 60 minutes middle cerebral artery occlusion (MCAO) followed by 3 days reperfusion. We found that stroke caused a significant brain lesion and motor deficit. Stroke induced severe skeletal muscle atrophy in the paretic tibialis anterior muscle compared to the corresponding tibialis anterior muscle of sham mice, as evidenced by reduced muscle mass and FCA (fiber cross‐sectional area). Our transcriptome analysis showed that stroke altered mRNA expression profile in skeletal muscle. Some of the genes associated with catabolic pathways are highly dysregulated in post‐stroke muscle. We also found that stroke dramatically repressed the protein deacetylase Sirtuin1 (SirT1) gene expression and its function in skeletal muscle. Interestingly, restoring SirT1 function in post‐stroke muscle notably reversed muscle atrophy, in part, through regulating the key players of autophagy and ubiquitin proteasomal pathway. Collectively, our data uncovered a potential mechanism that causes cerebral ischemic stroke‐induced muscle atrophy and identified SirT1 as a critical regulator of post‐stroke muscle mass.

Minimally Invasive Muscle Embedding Generates Donor-Cell-Derived Muscle Fibers that Express Desmin and Dystrophin.

The aim of this study was to quantify the extent of donor-cell-derived myogenesis achieved by a novel surgical technique known as Minimally Invasive Muscle Embedding (MIME). Through MIME, we implanted a single extensor digitorum longus muscle from donor mice (N=2) that expressed a red fluorescent protein (RFP), into the left tibialis anterior (TA) muscle of immunodeficient host mice (N=4) that expressed a green fluorescent protein (GFP). Soon after MIME, we injected a myotoxin (barium chloride), into the host TA muscle, to trigger concerted muscle degeneration and regeneration. In lieu of MIME, we performed a SHAM procedure on the right TA muscle of the same set of animals. In MIME-treated muscles, 22%±7% and 78%±7% muscle fibers were RFP+ and GFP+, respectively (mean±standard deviation); and all RFP+ fibers were positive for desmin and dystrophin. Conclusion. We conclude that MIME helps generate muscle fibers of donor origin, in host muscle.

Cromolyn sodium delays disease onset and is neuroprotective in the SOD1G93A Mouse Model of amyotrophic lateral sclerosis

Accumulating evidence suggests that neuroinflammatory processes are implicated in the initiation and progression of amyotrophic lateral sclerosis (ALS). Previous reports have demonstrated an increase in microgliosis and astrogliosis in the lumbar spinal cord of SOD1G93A transgenic mice before the onset of symptoms, a neuroinflammatory response which correlated with disease progression. Importantly, early stage homeostatic microglia enhanced motor neuron survival, while pro-inflammatory microglia were toxic to motor neurons in the SOD1G93A mice. Recent studies from our group have demonstrated that cromolyn sodium, an FDA approved compound, exerts neuroprotective effects in mouse models of Alzheimer’s disease by altering microglial cell activation. Here, we tested the neuroprotective and anti-inflammatory effects of cromolyn sodium in the SOD1G93A mouse model of ALS. Our results indicate that cromolyn sodium treatment significantly delayed the onset of neurological symptoms, and improved deficits in PaGE performance in both male and female mice, however, there was only an effect on survival in female mice. Furthermore, there was a significant increase in motor neuron survival in the lumbar spinal cord as well as a significant decrease in the denervation of the neuromuscular junction of the tibialis anterior muscle in cromolyn treated transgenic SOD1G93A mice. Lastly, cromolyn treatment decreased the expression of pro-inflammatory cytokines/chemokines in the lumbar spinal cord and plasma and decreased mast cell degranulation in the tibialis anterior muscle of transgenic SOD1G93A mice. Together, these findings suggest that cromolyn sodium provides neuroprotection in the SOD1G93A mice by decreasing the inflammatory response.

Conditional Deletion of Dicer in Adult Mice Impairs Skeletal Muscle Regeneration.

Skeletal muscle has a remarkable regenerative capacity, which is orchestrated by multiple processes, including the proliferation, fusion, and differentiation of the resident stem cells in muscle. MicroRNAs (miRNAs) are small noncoding RNAs that mediate the translational repression or degradation of mRNA to regulate diverse biological functions. Previous studies have suggested that several miRNAs play important roles in myoblast proliferation and differentiation in vitro. However, their potential roles in skeletal muscle regeneration in vivo have not been fully established. In this study, we generated a mouse in which the Dicer gene, which encodes an enzyme essential in miRNA processing, was knocked out in a tamoxifen-inducible way (iDicer KO mouse) and determined its regenerative potential after cardiotoxin-induced acute muscle injury. Dicer mRNA expression was significantly reduced in the tibialis anterior muscle of the iDicer KO mice, whereas the expression of muscle-enriched miRNAs was only slightly reduced in the Dicer-deficient muscles. After cardiotoxin injection, the iDicer KO mice showed impaired muscle regeneration. We also demonstrated that the number of PAX7+ cells, cell proliferation, and the myogenic differentiation capacity of the primary myoblasts did not differ between the wild-type and the iDicer KO mice. Taken together, these data demonstrate that Dicer is a critical factor for muscle regeneration in vivo.

Role of Mutant TBP in Regulation of Myogenesis on Muscle Satellite Cells.

In polyglutamine (PolyQ) diseases, mutant proteins cause not only neurological problems but also peripheral tissue abnormalities. Among all systemic damages, skeletal muscle dystrophy is the severest. Previously by studying knock-in (KI) mouse models of spinal cerebellar ataxia 17 (SCA17), it was found that mutant TATA box binding protein (TBP) decreases its interaction with myogenic differentiation antigen, thus reducing the expression of skeletal muscle structural proteins and resulting in muscle degeneration. In this paper, the role of mutant TBP in myogenesis was investigated. Single myofibers were isolated from tibialis anterior muscles of wild type (WT) and SCA17KI mice. The 1TBP18 staining confirmed the expression of mutant TBP in muscle satellite cells in SCA17KI mice. In the BaCl2-induced TA muscle injury, H&E cross-section staining showed no significant change in myofibril size before and after BaCl2 treatment, and there was no significant difference in centralized nuclei between WT and SCA17KI mice, suggesting that mutant TBP had no significant effect on muscle regeneration. In the cultured primary myoblasts from WT and SCA17KI mice in vitro, representative BrdU immunostaining showed no significant difference in proliferation of muscle satellite cells. The primary myoblasts were then induced to differentiate and immunostained for eMyHC, and the staining showed there was no significant difference in differentiation of primary myoblasts between WT and SCA1KI mice. Our findings confirmed that mutant TBP had no significant effect on myogenesis.