Mouse Tibialis Anterior Muscle Research Articles

Electroporation of Small Interfering RNAs into Tibialis Anterior Muscles of Mice.

Aging and wasting of skeletal muscle reduce organismal fitness. Regrettably, only limited interventions are currently available to address this unmet medical need. Many methods have been developed to study this condition, including the intramuscular electroporation of DNA plasmids. However, this technique requires surgery and high electrical fields, which cause tissue damage. Here, we report an optimized protocol for the electroporation of small interfering RNAs (siRNAs) into the tibialis anterior muscle of mice. This protocol does not require surgery and, because of the small siRNA size, mild electroporation conditions are utilized. By inducing target mRNA knockdown, this method can be used to interrogate gene function in muscles of mice from different strains, genotypes, and ages. Moreover, a complementary method for siRNA transfection into differentiated myotubes can be used for testing siRNA efficacy before in vivo use. Altogether, this streamlined protocol is instrumental for basic science and translational studies in muscles of mice and other animal models.

Satellite cell content and muscle regeneration in a mouse model of NAFLD

ObjectivesMuscle wasting is a common complication in patients with nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effect of NAFLD on satellite cell (SC) content and skeletal muscle repair. MethodsMale CD-1 mice fed a choline-deficient diet for 4 wk were used as an NAFLD model. We performed histologic and mRNA expression analyses, immunochemical staining with single muscle fibers to assess the effect of NAFLD on muscle Pax7+ SCs, and muscle regeneration by intramuscular injection of cardiotoxin. ResultsWe found that the total number of Pax7+ SCs in the extensor digitorum longus and tibialis anterior muscles of mice with NAFLD was significantly decreased when compared with that in the control group, in which the depletion of the SC pool possibly impaired muscle regeneration, as evidenced by the smaller size of the regenerating myofibers. Importantly, we found that NAFLD significantly impaired the differentiation ability of SCs, as shown by a decreased number of SCs expressing a myogenic marker, MyoD. Finally, this study indicated that molecular mechanisms underlying a decline in SC numbers may be attributed to the upregulation of proinflammatory cytokines (tumor necrosis factor α [TNFα]) and an oxidative stress marker (NADPH oxidase-2 [NOX2\\) in mice with NAFLD. ConclusionsThe findings demonstrate that a decrease in SC content in the skeletal muscle is an important factor that contributes to muscle wasting in NAFLD. Thus, preservation of the muscle SC pool is a potential therapeutic strategy to reduce NAFLD-associated muscle wasting.

Adeno-associated virus-mediated expression of an inactive CaMKIIβ mutant enhances muscle mass and strength in mice

A concurrent reduction in muscle mass and strength is frequently observed in numerous conditions, including neuromuscular disease, ageing, and muscle inactivity due to limb immobilization or prolonged bed rest. Thus, identifying the molecular mechanisms that control skeletal muscle mass and strength is fundamental for developing interventions aimed at counteracting muscle loss (muscle atrophy). It was recently reported that muscle atrophy induced by denervation of motor nerves was associated with increased expression of Ca2+/calmodulin-dependent protein serine/threonine kinase II β (CaMKIIβ) in muscle. In addition, treatment with KN-93 phosphate, which inhibits CaMKII-family kinases, partly suppressed denervation-induced muscle atrophy. Therefore, to test a possible role for CaMKIIβ in muscle mass regulation, we generated and injected recombinant adeno-associated virus (AAV) vectors encoding wild-type (AAV-WT), inactive (AAV-K43 M), or constitutively active (AAV-T287D) CaMKIIβ into the left hindlimb tibialis anterior muscle of mice at three months of age. Although AAV-WT infection induced expression of exogenous CaMKIIβ in the hindlimb muscle, no significant changes in muscle mass and strength were observed. By contrast, AAV-K43 M or AAV-T287D infection induced exogenous expression of the corresponding mutants and significantly increased or decreased the muscle mass and strength of the infected hind limb, respectively. Together, these findings demonstrate the potential of CaMKIIβ as a novel therapeutic target for enhancing muscle mass and strength.

Early Changes in Skeletal Muscle of Young C22 Mice, A Model of Charcot-Marie-Tooth 1A.

Background:The C22 mouse is a Charcot-Marie-Tooth 1A transgenic model with minimal axonal loss.Objective:To analyse early skeletal muscle changes resulting from this dysmyelinating neuropathy.Methods:Histology of tibialis anterior muscles of C22 mice and wild type litter mate controls for morphometric analysis and (immuno-)histochemistry for known denervation markers and candidate proteins identified by representational difference analysis (RDA) based on mRNA from the same muscles; quantitative PCR and Western blotting for confirmation of RDA findings.Results:At age 10 days, morphometry was not different between groups, while at 21 days, C22 showed significantly more small diameter fibres, indicating the onset of atrophy at an age when weakness becomes detectable. Neither (immuno-)histochemistry nor RDA detected extrajunctional expression of acetylcholine receptors by age 10 and 21 days, respectively. RDA identified some mRNA up-regulated in C22 muscles, among them at 10 days, prior to detectable weakness or atrophy, integral membrane protein 2a (Itm2a), eukaryotic initiation factor 2, subunit 2 (Eif2s2) and cytoplasmic phosphatidylinositol transfer protein 1 (Pitpnc1). However, qPCR failed to measure significant differences. In contrast, Itm2a and Eif2s2 mRNA were significantly down-regulated comparing 21 versus 10 days of age in both groups, C22 and controls. Western blotting confirmed significant down-regulation of ITM2A protein in C22 only.Conclusion:Denervation-like changes in this model develop slowly with onset of atrophy and weakness at about three weeks of age, before detection of extrajunctional acetylcholine receptors. Altered Itm2a expression seems to begin early as an increase, but becomes distinct as a decrease later.

Impaired Skeletal Muscle Development and Regeneration in Transglutaminase 2 Knockout Mice.

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C−CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.

Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR)

BackgroundDuchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by mutations in the dystrophin gene. Transplantation of myogenic stem cells holds great promise for treating muscular dystrophies. However, poor engraftment of myogenic stem cells limits the therapeutic effects of cell therapy. Mesenchymal stem cells (MSCs) have been reported to secrete soluble factors necessary for skeletal muscle growth and regeneration.MethodsWe induced MSC-like cells (iMSCs) from induced pluripotent stem cells (iPSCs) and examined the effects of iMSCs on the proliferation and differentiation of human myogenic cells and on the engraftment of human myogenic cells in the tibialis anterior (TA) muscle of NSG-mdx4Cv mice, an immunodeficient dystrophin-deficient DMD model. We also examined the cytokines secreted by iMSCs and tested their effects on the engraftment of human myogenic cells.ResultsiMSCs promoted the proliferation and differentiation of human myogenic cells to the same extent as bone marrow-derived (BM)-MSCs in coculture experiments. In cell transplantation experiments, iMSCs significantly improved the engraftment of human myogenic cells injected into the TA muscle of NSG-mdx4Cv mice. Cytokine array analysis revealed that iMSCs produced insulin-like growth factor-binding protein 2 (IGFBP2), urokinase-type plasminogen activator receptor (uPAR), and brain-derived neurotrophic factor (BDNF) at higher levels than did BM-MSCs. We further found that uPAR stimulates the migration of human myogenic cells in vitro and promotes their engraftment into the TA muscles of immunodeficient NOD/Scid mice.ConclusionsOur results indicate that iMSCs are a new tool to improve the engraftment of myogenic progenitors in dystrophic muscle.

Rational engineering of a functional CpG-free ITR for AAV gene therapy.

Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors. Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication. To study the biological potency, we made micro-dystrophin (μDys) AAV vectors carrying either the wild-type ITR or the CpG-free ITR. We delivered the CpG-free μDys vector to one side of the tibialis anterior muscle of dystrophin-null mdx mice and the wild-type μDys vector to the contralateral side. Evaluation at four months after injection showed no difference in the vector genome copy-number, μDys expression, and muscle histology and force. Our results suggest that the complete elimination of the CpG motif in the ITR does not affect the biological activity of the AAV vector. CpG-free ITRs could be useful in engineering therapeutic AAV vectors.

AMP deamination is sufficient to replicate an atrophy-like metabolic phenotype in skeletal muscle

BackgroundSkeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. MethodsAMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. ResultsTA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (−25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (−16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (−24%), phosphorylation of AMPK substrates (−14%), and PGC-1α protein (−22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (−55%), basal ATP synthase-dependent (−13%), and maximal uncoupled oxygen consumption (−15%). ConclusionsIncreased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting.

MiR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle.

Our knowledge of the coordination of fuel usage in skeletal muscle is incomplete. Whether and how microRNAs are involved in the substrate selection for oxidation is largely unknown. Here we show that mice lacking miR-183 and miR-96 have enhanced muscle oxidative phenotype and altered glucose/lipid homeostasis. Moreover, loss of miR-183 and miR-96 results in a shift in substrate utilization toward fat relative to carbohydrates in mice. Mechanistically, loss of miR-183 and miR-96 suppresses glucose utilization in skeletal muscle by increasing PDHA1 phosphorylation via targeting FoxO1 and PDK4. On the other hand, loss of miR-183 and miR-96 promotes fat usage in skeletal muscle by enhancing intramuscular lipolysis via targeting FoxO1 and ATGL. Thus, our study establishes miR-183 and miR-96 as master coordinators of fuel selection and metabolic homeostasis owing to their capability of modulating both glucose utilization and fat catabolism. Lastly, weshow that loss of miR-183 and miR-96 can alleviate obesity andimprove glucose metabolism in high-fat diet-induced mice, suggesting that miR-183 and miR-96 may serve as therapeutic targets for metabolic diseases.

Dystrophin deficiency disrupts muscle clock expression and mitochondrial quality control in mdx mice.

Impaired oxidative capacity and mitochondrial function contribute to the dystrophic pathology in muscles of patients with Duchenne muscular dystrophy (DMD) and in relevant mouse models of the disease. Emerging evidence suggests an association between disrupted core clock expression and mitochondrial quality control, but this has not been established in muscles lacking dystrophin. We examined the diurnal regulation of muscle core clock and mitochondrial quality control expression in dystrophin-deficient C57BL/10ScSn-Dmdmdx (mdx) mice, an established model of DMD. Male C57BL/10 (BL/10; n = 18) and mdx mice (n = 18) were examined every 4 h beginning at the dark cycle. Throughout the entire light-dark cycle, extensor digitorum longus (EDL) muscles from mdx mice had decreased core clock mRNA expression (Arntl, Cry1, Cry2, Nr1d2; P < 0.05) and disrupted mitochondrial quality control mRNA expression related to biogenesis (decreased; Ppargc1a, Esrra; P < 0.05), fission (increased; Dnm1l; P < 0.01), fusion (decreased; Opa1, Mfn1; P < 0.05), and autophagy/mitophagy (decreased: Bnip3; P < 0.05; increased: Becn1; P < 0.05). Cosinor analysis revealed a decrease in the rhythmicity parameters mesor and amplitude for Arntl, Cry1, Cry2, Per2, and Nr1d1 (P < 0.001) in mdx mice. Diurnal oscillations in Esrra, Sirt1, Map1lc3b, and Sqstm1 were absent in mdx mice, along with decreased mesor and amplitude of Ppargc1a mRNA expression (P < 0.01). The expression of proteins involved in mitochondrial biogenesis (decreased: PPARGC1A, P < 0.05) and autophagy/mitophagy (increased: MAP1LC3BII, SQSTM1, BNIP3; P < 0.05) were also dysregulated in tibialis anterior muscles of mdx mice. These findings suggest that dystrophin deficiency in mdx mice impairs the regulation of the core clock and mitochondrial quality control, with relevance to DMD and related disorders.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Vector-mediated expression of muscle specific kinase restores specific force to muscles in the mdx mouse model of Duchenne muscular dystrophy.

What is the central question of this study? The (dystrophin-deficient) muscles ofmdxmice generate less contractile force per cross-sectional area (specific force) than those of healthy wild-type mice: what is the influence of muscle specific kinase (MuSK) upon the properties of the tibialis anterior (TA) muscle inmdxmice? What is the main finding and its importance? Injection of adeno-associated viral vector encoding MuSK into the TA muscle of youngmdxmice increased the specific force of the muscle, suggesting the MuSK signalling system has the potential to restore healthy growth to dystrophin-deficient muscles. In the mdx mouse model of Duchenne muscular dystrophy, muscle fibres are fragile and prone to injury and degeneration. Compared to wild-type mice, muscles of mdx mice also develop less specific force (contractile force/cross-sectional area). We recently reported that injecting adeno-associated viral vector encoding muscle specific kinase (AAV-MuSK) into muscles of mdx mice increased utrophin expression and made the muscles more resistant to acute stretch-induced injury. Here we injected AAV-MuSK unilaterally into the tibialis anterior muscle of mdx mice at a younger age (4weeks), and recorded contraction force from the muscles in situ at 12weeks of age. Compared to contralateral empty-vector control muscles, muscles injected with AAV-MuSK produced 28% greater specific force (P=0.0005). They did not undergo the compensatory hypertrophy that normally occurs in muscles of mdx mice. Injection of AAV encoding rapsyn (a downstream effector of MuSK signalling) caused no such improvement in muscle strength. Muscles injected with AAV-MuSK displayed a 10% reduction in the number of fibres with centralized nuclei (P=0.0015). Our results in mdx mice suggest that elevating the expression of MuSK can reduce the incidence of muscle fibre regeneration and improve the strength of dystrophin-deficient muscles.

Metformin Increases Sarcolemma Integrity and Ameliorates Neuromuscular Deficits in a Murine Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease characterized by progressive muscle weakness and wasting. Stimulation of AMP-activated protein kinase (AMPK) has been demonstrated to increase muscle function and protect muscle against damage in dystrophic mice. Metformin is a widely used anti-hyperglycemic drug and has been shown to be an indirect activator of AMPK. Based on these findings, we sought to determine the effects of metformin on neuromuscular deficits in mdx murine model of DMD. In this study, we found metformin treatment increased muscle strength accompanied by elevated twitch and tetanic force of tibialis anterior (TA) muscle in mdx mice. Immunofluorescence and electron microscopy analysis of metformin-treated mdx muscles revealed an improvement in muscle fiber membrane integrity. Electrophysiological studies showed the amplitude of miniature endplate potentials (mEPP) was increased in treated mice, indicating metformin also improved neuromuscular transmission of the mdx mice. Analysis of mRNA and protein levels from muscles of treated mice showed an upregulation of AMPK phosphorylation and dystrophin-glycoprotein complex protein expression. In conclusion, metformin can indeed improve muscle function and diminish neuromuscular deficits in mdx mice, suggesting its potential use as a therapeutic drug in DMD patients.

Cancer-derived exosome miRNAs induce skeletal muscle wasting by Bcl-2-mediated apoptosis in colon cancer cachexia

Cancer cachexia is a kind of whole-body metabolic disorder syndrome accompanied by severe wasting of muscle tissue in which cancer exosomes may be involved. Analysis of clinical samples showed that the serum exosome concentrations were correlated with the development of cancer cachexia. Exosomes secreted by C26 cells could decrease the diameter of C2C12 myotubes in vitro and decrease mouse muscle strength and tibialis anterior (TA) muscle weight in vivo. GW4869, an inhibitor of exosome excretion, ameliorated muscle wasting in C26 tumor-bearing mice. MicroRNA (miRNA) sequencing (miRNA-seq) analysis suggested that miR-195a-5p and miR-125b-1-3p were richer in C26 exosomes than in exosomes secreted from MC38 cells (non-cachexic). Both miR-195a-5p and miR-125b-1-3p mimics could induce atrophy of C2C12 myoblasts. Downregulation of Bcl-2 and activation of the apoptotic signaling pathway were observed in C2C12 myoblasts transfected with miR-195a-5p and miR-125b-1-3p mimics, in the gastrocnemius muscle of C26 tumor-bearing mice and in the TA muscle injected with C26 exosomes. Results of dual-luciferase assay confirmed the targeting of miR-195a-5p/miR-125b-1-3p to Bcl-2. Overexpression of Bcl-2 successfully reversed atrophy of C2C12 myoblasts induced by the two miRNA mimics. These results suggested that cancer exosome enriched miRNAs might induce muscle atrophy by targeting Bcl-2-mediated apoptosis.

Iron accumulation in skeletal muscles of old mice is associated with impaired regeneration after ischaemia-reperfusion damage.

BackgroundOxidative stress is implicated in the insidious loss of muscle mass and strength that occurs with age. However, few studies have investigated the role of iron, which is elevated during ageing, in age‐related muscle wasting and blunted repair after injury. We hypothesized that iron accumulation leads to membrane lipid peroxidation, muscle wasting, increased susceptibility to injury, and impaired muscle regeneration.MethodsTo examine the role of iron in age‐related muscle atrophy, we compared the skeletal muscles of 3‐month‐old with 22‐ to 24‐month‐old 129SvEv FVBM mice. We assessed iron distribution and total elemental iron using laser ablation inductively coupled plasma mass spectrometry and Perls' stain on skeletal muscle cross‐sections. In addition, old mice underwent ischaemia–reperfusion (IR) injury (90 min ischaemia), and muscle regeneration was assessed 14 days after injury. Immunoblotting was used to determine lipid peroxidation (4HNE) and iron‐related proteins. To determine whether muscle iron content can be altered, old mice were treated with deferiprone (DFP) in the drinking water, and we assessed its effects on muscle regeneration after injury.ResultsWe observed a significant increase in total elemental iron (+43%, P < 0.05) and lipid peroxidation (4HNE: +76%, P < 0.05) in tibialis anterior muscles of old mice. Iron was further increased after injury (adult: +81%, old: +135%, P < 0.05) and associated with increased lipid peroxidation (+41%, P < 0.05). Administration of DFP did not impact iron or measures of lipid peroxidation in skeletal muscle or modulate muscle mass. Increased muscle iron concentration and lipid peroxidation were associated with less efficient regeneration, evident from the smaller fibres in cross‐sections of tibialis anterior muscles (−24%, P < 0.05) and an increased percentage of fibres with centralized nuclei (+4124%, P < 0.05) in muscles of old compared with adult mice. Administration of DFP lowered iron after IR injury (PRE: −32%, P < 0.05 and POST: −41%, P < 0.05), but did not translate to structural improvements.ConclusionsMuscles from old mice have increased iron levels, which are associated with increased lipid peroxidation, increased susceptibility to IR injury, and impaired muscle regeneration. Our results suggest that iron is involved in effective muscle regeneration, highlighting the importance of iron homeostasis in muscle atrophy and muscle repair.

Muscle follistatin gene delivery increases muscle protein synthesis independent of periodical physical inactivity and fasting.

Blocking of myostatin and activins effectively counteracts muscle atrophy. However, the potential interaction with physical inactivity and fasting in the regulation of muscle protein synthesis is poorly understood. We used blockade of myostatin and activins by recombinant adeno-associated virus (rAAV)-mediated follistatin (FS288) overexpression in mouse tibialis anterior muscle. To investigate the effects on muscle protein synthesis, muscles were collected 7days after rAAV-injection in the nighttime or in the daytime representing high and low levels of activity and feeding, respectively, or after overnight fasting, refeeding, or ad libitum feeding. Muscle protein synthesis was increased by FS288 independent of the time of the day or the feeding status. However, the activation of mTORC1 signaling by FS288 was attenuated in the daytime and by overnight fasting. FS288 also increased the amount of mTOR colocalized with lysosomes, but did not alter their localization toward the sarcolemma. This study shows that FS288 gene delivery increases muscle protein synthesis largely independent of diurnal fluctuations in physical activity and food intake or feeding status, overriding the physiological signals. This is important for eg cachectic and sarcopenic patients with reduced physical activity and appetite. The FS288-induced increase in mTORC1 signaling and protein synthesis may be in part driven by increased amount of mTOR colocalized with lysosomes, but not by their localization toward sarcolemma.

The Role of Septin7 in Skeletal Muscle Regeneration

Septins have recently been acknowledged as the fourth component of the cytoskeleton. In humans 13 isoforms have been identified and classified into 4 homology groups. While their role was extensively studied in neurons and cancer cells, their role in skeletal muscle remains unexplored. Here the Tibialis anterior muscle of mice was injected with BaCl2 to induce a mild damage and regeneration was followed for two weeks. The expression of Septin7 was upregulated on the fourth day following injection (by 60±28%) as compared to the contralateral leg; p<0.04) and its level correlated with the expression of Pax7 (r2=0.5273; p<0.01) the marker of satellite cell activation and the appearance of central nuclei of regenerating fibers. By the end of the second week the expression of both Septin7 and Pax7 decreased, in parallel, close to control levels. The migration of control and Septin7 knock-down (KD) C2C12 myoblasts was followed in a migration assay. Although the total distance covered by the cells during the migration period (3h) was identical (256±9 µm in control (n=61), while 269±7 µm in KD (n=60)), Septin7 KD cells tended not to change the direction of migration which was reflected in the larger distance between the initial and final positions (91±11, and 165±9 µm for control and KD cells, respectively; p<0.05). In parallel, KD cells displayed an altered cell shape with less protrusions. Septin filament reorganisation during migration was followed live by expressing a fluorescent Septin7 fusion protein. These findings indicate that Septin7 plays an essential role in the ability of myoblasts to migrate to the site of muscle damage and thus in the regeneration process of skeletal muscle. Supported by GINOP-2.3.2-15-2016-00044, Thematic Excellence Programme of the Ministry for Innovation and Technology in Hungary (ED_18-1-2019-0028).

Role of plasminogen activator inhibitor-1 in muscle wasting induced by a diabetic state in female mice.

Muscle wasting is a complication in patients with diabetes and leads to a reduced quality of life. However, the detailed mechanisms of diabetes-induced muscle wasting remain unknown. Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that suppresses plasminogen activator activity, is involved in the pathophysiology of various diseases, including diabetes. In the present study, we examined the role of endogenous PAI-1 in the decrease in muscle mass and the impaired grip strength induced by the diabetic state by employing streptozotocin (STZ)-treated PAI-1-deficient female mice. The analyses of skeletal muscles and grip strength were performed in PAI-1-deficient and wild-type mice 4 weeks after the induction of a diabetic state by STZ administration. PAI-1 deficiency did not affect muscle mass in the lower limbs measured by quantitative computed tomography or tissue weights of the tibialis anterior, gastrocnemius and soleus muscles of female mice with or without STZ treatment. On the other hand, PAI-1 deficiency significantly aggravated grip strength decreased by STZ in female mice. PAI-1 deficiency did not affect the mRNA levels of Pax7, MyoD, myogenin or myosin heavy chain in either the tibialis anterior or soleus muscles of female mice with or without STZ treatment. In conclusion, we revealed for the first time that PAI-1 deficiency aggravates grip strength impaired by the diabetic state in female mice, although it did not affect diabetes-decreased muscle mass.

μ-Crystallin in Mouse Skeletal Muscle Promotes a Shift from Glycolytic toward Oxidative Metabolism.

μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6–147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism.

Histological Analysis of Tibialis Anterior Muscle of DMDmdx4Cv Mice from 1 to 24 Months.

The mdx-C57/B6 mouse model does not show the clinical signs of Duchenne muscular dystrophy (DMD), although muscles exhibit hallmarks of permanent regeneration and alterations in muscle function. The DMDmdx4Cv strain exhibits very few revertant dystrophin positive myofibers, making that model suitable for studies on gene and cell therapies. The study appraises the histological evolution of the Tibialis Anterior muscle of WT and DMD mdx4Cv mutant from 1 to 24 months. Histological analysis included a series of immunostainings of muscle sections for assessing tissue features (fibrosis, lipid deposition, necrosis) and cellular characteristics (size of myofibers, number and distribution of myonuclei, number of satellite cells, vessels, macrophages). None of the investigated cell types (satellite cells, endothelial cells, macrophages) showed variations in their density within the tissue in both WT and DMD mdx4Cv muscle. However, analyzing their number per myofiber showed that in DMD mdx4Cv, myofiber capillarization was increased from 1 to 6 months as compared with WT muscle, then dropped from 12 months. Macrophage number did not vary in WT muscle and peaked at 6 months in DMD mdx4Cv muscle. The number of satellite cells per myofiber did not vary in WT muscle while it remained high in DMD mdx4Cv muscle, starting to decrease from 12 months and being significantly lower at 24 months of age. Myofiber size was not different in DMD mdx4Cv from WT except at 24 months, when it strongly decreased in DMD mdx4Cv muscle. Necrosis and lipid deposition were rare in DMD mdx4Cv muscle. Fibrosis did not increase with age in DMD mdx4Cv muscle and was higher than in WT at 6 and 12 months of age. As a whole, the results show a strong decrease of the myofiber size at 24 months, and an increased capillarization until 6 months of age in DMD mdx4Cv as compared with the WT. Thus, DMD mdx4Cv mice poorly recapitulates histological DMD features, and its use should take into account the age of the animals according to the purpose of the investigation.