Muscle Injury In Mice Research Articles

Vitamin A Promotes the Repair of Mice Skeletal Muscle Injury through RARα.

Vitamin A (VitA) is an important fat-soluble vitamin which plays an important role in cell growth and individual development. However, the effect of VitA on the repair process of muscle injury and its molecular mechanism are still unclear. In this study, VitA and RA were first added to the culture medium of differentiated cells. We then detected cell differentiation marker proteins and myotube fusion. Moreover, the effects of VitA on RARα expression and nuclear translocation were further examined. The results showed that VitA significantly promoted the differentiation of C2C12, and the expression of RARα was significantly increased. Furthermore, VitA was injected into skeletal muscle injury in mice. HE staining and Western Blot results showed that VitA could significantly accelerate the repair of skeletal muscle injury and VitA increase the expression of RARα in mice. This study provides a theoretical basis for elucidating the regulation mechanism of VitA-mediated muscle development and the development of therapeutic drugs for muscle diseases in animals.

Low-intensity pulsed ultrasound promotes skeletal muscle regeneration via modulating the inflammatory immune microenvironment.

Background: Low-intensity pulsed ultrasound (LIPUS, a form of mechanical stimulation) can promote skeletal muscle functional repair, but a lack of mechanistic understanding of its relationship and tissue regeneration limits progress in this field. We investigated the hypothesis that specific energy levels of LIPUS mediates skeletal muscle regeneration by modulating the inflammatory microenvironment. Methods: To address these gaps, LIPUS irritation was applied in vivo for 5 min at two different intensities (30mW/cm2 and 60mW/cm2) in next 7 consecutive days, and the treatment begun at 24h after air drop-induced contusion injury. In vitro experiments, LIPUS irritation was applied at three different intensities (30mW/cm2, 45mW/cm2, and 60mW/cm2) for 2 times 24h after introduction of LPS in RAW264.7. Then, we comprehensively assessed the functional and histological parameters of skeletal muscle injury in mice and the phenotype shifting in macrophages through molecular biological methods and immunofluorescence analysis both in vivo and in vitro. Results: We reported that LIPUS therapy at intensity of 60mW/cm2 exhibited the most significant differences in functional recovery of contusion-injured muscle in mice. The comprehensive functional tests and histological analysis in vivo indirectly and directly proved the effectiveness of LIPUS for muscle recovery. Through biological methods and immunofluorescence analysis both in vivo and in vitro, we found that this improvement was attributable in part to the clearance of M1 macrophages populations and the increase in M2 subtypes with the change of macrophage-mediated factors. Depletion of macrophages in vivo eliminated the therapeutic effects of LIPUS, indicating that improvement in muscle function was the result of M2-shifted macrophage polarization. Moreover, the M2-inducing effects of LIPUS were proved partially through the WNT pathway by upregulating FZD5 expression and enhancing β-catenin nuclear translocation in macrophages both in vitro and in vivo. The inhibition and augment of WNT pathway in vitro further verified our results. Conclusion: LIPUS at intensity of 60mW/cm2 could significantly promoted skeletal muscle regeneration through shifting macrophage phenotype from M1 to M2. The ability of LIPUS to direct macrophage polarization may be a beneficial target in the clinical treatment of many injuries and inflammatory diseases.

Melatonin improves muscle injury and differentiation by increasing Pax7 expression.

A balance between muscle injury and regeneration is critical for sustaining muscle function during myogenesis. Melatonin is well recognized for its involvement in neuroprotective activities, immune system regulation and suppression of inflammatory responses. This study set out to provide evidence that melatonin improves muscle regeneration during skeletal muscle differentiation. We began with cloning a stable cell line expressing Pax7 knockdown C2C12 cells. We then investigated markers of muscle degradation and regeneration after treating growth medium and differentiated medium with melatonin. Bioinformatics analysis of RNA sequencing results revealed that melatonin regulates muscle differentiation and that Wnt cascades are involved in the mechanism of muscle differentiation. Screening of miRNA online databases revealed that miR-3475-3p is a specific binding site on Pax7 and acts as a negative regulator of Pax7, which is involved in melatonin-induced muscle differentiation. We then investigated the effects of melatonin treatment in the early stage of glycerol-induced skeletal muscle injury in mice. Rotarod performance, micro-computed tomography and immunohistochemistry findings showed that melatonin-induced increases in Pax7 expression rapidly rescue skeletal muscle differentiation and improve muscle fiber morphology in glycerol-induced muscle injury. Our data support the hypothesis that melatonin rapidly rescues skeletal muscle differentiation and the melatonin/Pax7 axis could therefore serve as an important therapeutic target to optimize muscle healing after injury.

Protective effects of dietary curcumin and astaxanthin against heat-induced ROS production and skeletal muscle injury in male and female C57BL/6J mice

AimsThis study aimed to: 1) investigate sex differences in heat-induced mitochondrial dysfunction, ROS production, and skeletal muscle injury in mice; 2) evaluate whether curcumin and astaxanthin, alone or together, would prevent those heat-induced changes. Main methodsMale and female C57BL/6J mice were treated with curcumin and astaxanthin for 10 days, then exposed to 39.5 °C heat for up to 3 h. Heat-induced hyperthermia, changes in mitochondrial morphology and function, and oxidative damage to skeletal muscle were evaluated. Key findingsAlthough female mice had a slightly higher basal core body temperature (Tc) than male mice, peak Tc during heat exposure was significantly lower in females than in males. Heat increased ROS levels in skeletal muscle in both sexes; interestingly, the increases in ROS were greater in females than in males. Despite the above-mentioned differences, heat induced similar levels of mitochondrial fragmentation and membrane potential depolarization, caspase 3/7 activation, and injury in male and female skeletal muscle. Individual treatment of curcumin or astaxanthin did not affect basal and peak Tc but prevented heat-induced mitochondrial dysfunction, ROS increases, and apoptosis in a dose-dependent manner. Moreover, a low-dose combination of curcumin and astaxanthin, which individually showed no effect, reduced the heat-induced oxidative damage to skeletal muscle. SignificanceBoth male and female mice can develop mitochondrial dysfunction and oxidative stress in skeletal muscle when exposed to heat stress. High doses of either curcumin or astaxanthin limit heat-induced skeletal muscle injury, but a low-dose combination of these ingredients may increase their efficacy.

Abstract IA18: Clock Control of HIF Activity and Muscle Tissue Regeneration

Abstract Circadian clocks generate rhythmic pulses of glucose metabolism and respiration to divide energy-producing and energy-consuming processes into separate periods in the light-dark cycle. At the cellular level, clocks direct the expression of thousands of genes involved in metabolism, tissue growth, and repair, yet our understanding of how clocks detect signals in the environment to coordinate transcription and metabolic flux at precise stages during development and in post-mitotic cells remains poorly understood. A breakthrough in understanding circadian mechanisms in metabolic plasticity came from our discovery that molecular clock transcription factors respond to changes in environmental oxygen. We uncovered a link between circadian clocks and the control of the oxygen- and mitochondrial stress-responsive hypoxia-inducible factor (HIF) pathway, which controls adaptation to low oxygen environments, such as during tumorigenesis and ischemic injury. Here, we demonstrate a role for the circadian clock in post-muscle injury myogenesis and tissue regeneration. Indeed, loss of Bmal1 in adult muscle stem cell populations leads to impaired regeneration after ischemic muscle injury in mice. Furthermore, we observe time-of-day differences in muscle regeneration rates, with more rapid tissue repair following injury during the dark (active) phase. In addition, myogenesis is reduced in primary Bmal1-/- myoblasts compared to controls, a phenotype that is exaggerated in hypoxic conditions. Finally, we observe that loss of Bmal1 in myoblasts leads to chromatin remodeling that alters transcription of genes involved in metabolic flux and myogenic gene expression. Together, our studies demonstrate that circadian clocks in muscle stem cells control metabolic and genomic reprogramming under hypoxia to regulate in vitro myogenesis and in vivo skeletal muscle regeneration. Citation Format: Clara Bien Peek. Clock Control of HIF Activity and Muscle Tissue Regeneration [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr IA18.

Needling on trigger point promotes muscle regeneration after bupivacaine injection induced injury

Dry needling treatment has a promising relieving effect on Myofascial Pain Syndrome (MPS). In China, acupuncture practitioners use acupuncture needle instead to insert the “A-Shi” acupoint to treat MPS which is defined as the same as the trigger point of dry needling. This method has been applied for thousands of years in China. In this study, bupivacaine injection induced gastrocnemius muscle injury in mice. We applied the clinical improved needling method on animal model by making the angle between the skin and needle less than 30 degree. Animals got needling treatment 24 h later at the point where the bupivacaine was injected. Results of muscle H.E. staining showed that, compared to bupivacaine injection group without needling, acupuncture treatment group showed more intact muscle fibers, less inflammatory cell infiltration and fractured muscle fibers. By RNA sequencing analysis, our work firstly demonstrated that the physical stimulation of needling changed the gene expression of muscle tissue to accelerate the muscular regeneration process. Therefore, our study proved that simple needling at “A-Shi” acupoint promoted muscle regeneration and revealed underlying mechanisms of the beneficial effects of acupuncture and dry needle treatments.

Irgm1 knockout indirectly inhibits regeneration after skeletal muscle injury in mice

Immunity-related GTPase family M1 protein (lRGM1) plays an important role in host resistance to infection, immune inflammation, and tumors, and it is expressed in various tissues and cells, including the central nervous system, cardiovascular system, bone marrow-derived cells, glioma, and melanoma. However, the effect of IRGM1 in the muscles has not been reported to date. In this study, Irgm1−/− mice were used to evaluate the effect of lrgm1 on regeneration after skeletal muscle injury. The tibialis anterior muscle in Irgm1−/− mice was poorly repaired after BaCl2-induced injury, whereas lrgm1 knockout itself had no significant effect on the differentiation of myoblasts. However, the microenvironment of Irgm1−/− mice with a high interferon-gamma level inhibited the differentiation of myoblasts in vivo. These results suggest that lrgm1 knockout indirectly inhibits skeletal muscle regeneration after injury, providing new insights into the biological function of IRGM1.

Local low‐intensity vibration improves healing of muscle injury in mice

Recovery from traumatic muscle injuries is typically prolonged and incomplete. Our previous study demonstrated that whole‐body low‐intensity vibration (LIV) enhances healing in a mouse laceration model. We sought to determine whether locally applied LIV (a) improves muscle repair following injury in mice and (b) is directly transduced by cultured muscle cells, via increased IGF‐1 activity. C57BL/6J mice were subjected to laceration of the gastrocnemius muscle and were treated with LIV applied directly to the lower leg for 30 min/day or non‐LIV sham treatment (controls) for 7 or 14 days. LIV was also applied to differentiating myotubes in culture for 30 min/day for 3 or 6 days. Compared with control mice, LIV increased myofiber cross‐sectional area, diameter, and percent area of peripherally nucleated fibers, and decreased percent damaged area after 14 days of treatment. In cultured myotubes, LIV increased fusion and diameter compared with controls after 6 days of treatment. These LIV‐induced effects were associated with increased total Akt on day 7 in injured muscle and on day 3 in myotubes, whereas phosphorylated‐to‐total Akt ratio increased on day 14 in injured muscle and on day 6 in myotubes but were not associated with increased IGF‐1 levels at any time point. These changes were also associated with LIV‐induced suppression of FOXO1 and Atrogin‐1 gene expression at day 7 in injured muscle. These findings demonstrate that muscle cells can directly transduce LIV signals into increased growth and differentiation, and this effect is associated with increased Akt signaling.

Sustained elevation of MG53 in the bloodstream increases tissue regenerative capacity without compromising metabolic function

MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body.

Systemic and Local Phenotypes of Barium Chloride Induced Skeletal Muscle Injury in Mice.

Skeletal muscle regeneration in mice has traditionally been studied using local freeze burn or snake venom injection models. More recently, a barium chloride (BaCl2)-induced muscle injury model has been established and is gaining popularity due to the relatively simple procedure and accessibility to required reagents. Here we sought to characterize the local and systemic effects of BaCl2-induced muscle injury. For this study, a 1.2% BaCl2 solution was locally administered to the tibialis anterior (TA) muscle and local and systemic phenotypes were analyzed at different timepoints. When 50 μL of the solution was injected unilaterally in the TA muscle, no mortality was observed. However, when 100 μL of the solution was injected, 50% of the mice died within 24 h. Serum analysis of the mice injected with 50 μL of BaCl2 solution at days 1 and 7 revealed changes resembling rhabdomyolysis. At day 1 post-injection of 50 μL of the BaCl2 solution, acute suppurative inflammation was observed in gross examination of the TA muscle, while extensive hemorrhagic necrosis was revealed on histological examination. At day 7, regenerated myofibers with centralized nuclei appeared with the resolution of acute inflammatory infiltration and the muscle tissue displayed molecular signatures consistent with myofiber differentiation. The overall muscle injury and regeneration phenotypes in the BaCl2-induced muscle injury model were similar to those of the well-established freeze burn or snake venom injection models. Taken together, the BaCl2-induced muscle injury model is comparable to conventional muscle injury and regeneration models, with considerations for possible systemic effects.

Renal Dysfunction Exacerbates Ischemic Muscle Injury in Mice Subjected to Hindlimb Ischemia

Patients with critical limb ischemia (CLI) have multiple concomitant comorbidities which are associated with poorer overall health outcomes. CLI patients with impaired kidney function and/or diabetes are at greater risk for limb amputation and death. While the negative impacts of renal dysfunction and diabetes on CLI outcomes are known clinically, the underlying biologic mechanism(s) responsible are poorly understood. Thus, we sought to evaluate the impact of impaired renal function and diabetes on the skeletal muscle pathology in mice following unilateral hindlimb ischemia (HLI). Renal dysfunction (RD) was induced by dietary administration of adenine which causes renal tubular injury, while diabetes was induced by streptozocin injection. HLI was induced by ligation and excision of the femoral artery, while the contralateral hindlimb was utilized as a control (no surgery). Three groups (n = 15; 5 per group) of C57BL/6J mice were analyzed: control, RD, and RD+ diabetes. Skeletal muscle ischemic injury was assessed using histological measurements of myofiber centralized nuclei, myofiber cross‐sectional area, ischemic lesion areas, and vascular density. High‐resolution O2 consumption was used to examine mitochondrial function in permeablized myofibers. Mice with RD, regardless of diabetes, exhibited greater ischemic lesions within the tibialis anterior muscle (Con = 2.53 ± 0.51% vs. RD = 60.554 ± 24.15% vs. RD+STZ = 79.99 ± 19.99%; P < 0.0001) despite similar capillary density (Con = 466.36 ± 44.98 vs. RD = 455.20 ± 79.55 vs. RD+STZ = 578.20 ± 87.16 capillaries/field; P = 0.9011). Mice with RD exhibited a ~45% decrease in mitochondrial respiratory capacity across numerous substrates, whereas control mice had normal respiration in the ischemic limb muscle seven days post‐HLI. These observations demonstrate that impaired kidney function exacerbates ischemic muscle injury in mice. Future studies are needed to uncover the molecular mechanisms by which RD negatively impacts skeletal muscle.Support or Funding InformationSupported by AHA Grant 18CDA34110044 to TER.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Normal inflammation and regeneration of muscle following injury require osteopontin from both muscle and non-muscle cells

BackgroundOsteopontin is secreted by skeletal muscle myoblasts and macrophages, and its expression is upregulated in muscle following injury. Osteopontin is present in many different structural forms, which vary in their expression patterns and effects on cells. Using a whole muscle autograft model of muscle injury in mice, we have previously shown that inflammation and regeneration of muscle following injury are delayed by the absence of osteopontin. The current study was undertaken to determine whether muscle or non-muscle cells provide the source of osteopontin required for its role in muscle regeneration.MethodsThe extensor digitorum longus muscle of wild-type and osteopontin-null mice was removed and returned to its bed in the same animal (autograft) or placed in the corresponding location in an animal of the opposite genotype (allograft). Grafts were harvested at various times after surgery and analysed by histology, flow cytometry and quantitative polymerase chain reaction. Data were analysed using one- or two-way ANOVA or Kruskal-Wallis test.ResultsImmunohistochemistry confirmed that osteopontin was expressed by macrophages in osteopontin-null muscle allografts in wild-type hosts and by myoblasts in wild-type allografts in osteopontin-null hosts. The decrease in muscle fibre number observed in wild-type autografts following grafting and the subsequent appearance of regenerating fibres were delayed in both types of allografts to a similar extent as in osteopontin-null autografts. Infiltration of neutrophils, macrophages and M1 and M2 macrophage subtypes were also delayed by lack of osteopontin in the muscle and/or host. While the proportion of macrophages showing the M1 phenotype was not affected, the proportion showing the M2 phenotype was decreased by osteopontin deficiency. Expression of tumour necrosis factor-α and interleukin-4 was lower in osteopontin-null than in wild-type autografts, and there was no difference between the osteopontin-null graft types.ConclusionsOsteopontins from muscle and non-muscle sources are equally important in the acute response of muscle to injury, and cannot substitute for each other, suggesting that they play distinct roles in regulation of cell behaviour. Future studies of mechanisms of osteopontin’s roles in acute muscle inflammation and regeneration will need to investigate responses to osteopontins derived from both myoblasts and macrophages.

Podocan affects C2C12 myogenic differentiation by enhancing Wnt/β-catenin signaling.

Podocan, a small leucine-rich repeat protein, is a negative regulator of cell proliferation. In this study, we demonstrated that podocan is involved in the differentiation of C2C12 murine myoblasts. Podocan expression increased with the progression of C2C12 differentiation. As expect, siRNA-mediated podocan knockdown inhibited C2C12 differentiation, as indicated by inhibition of MYOG, MYH2, and desmin expression, as well as reductions in the differentiation and fusion indices. Overexpression of podocan using dCas9 technology promoted C2C12 cell differentiation. In addition, supplementation of culture medium with podocan influenced C2C12 differentiation. Podocan knockdown reduced Wnt/β-catenin signaling, characterized by a reduction in the nuclear translocation of β-catenin, whereas podocan overexpression had the opposite effect. Furthermore, treatment with XAV939, an inhibitor of Wnt/β-catenin, reduced the podocan-mediated promotion of C2C12 differentiation. Induction of muscle injury in mice by bupivacaine administration suggested that podocan may play a role in muscle regeneration. In summary, our results suggest that podocan is required for normal C2C12 differentiation and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

Adiponectin promotes muscle regeneration through binding to T-cadherin

Skeletal muscle has remarkable regenerative potential and its decline with aging is suggested to be one of the important causes of loss of muscle mass and quality of life in elderly adults. Metabolic abnormalities such as obesity were linked with decline of muscle regeneration. On the other hand, plasma levels of adiponectin are decreased in such metabolic conditions. However, plasma levels of adiponectin have been shown to inversely correlate with muscle mass and strength in elderly people especially with chronic heart failure (CHF). Here we have addressed whether adiponectin has some impact on muscle regeneration after cardiotoxin-induced muscle injury in mice. Muscle regeneration was delayed by angiotensin II infusion, mimicking aging and CHF as reported. Adiponectin overexpression in vivo decreased necrotic region and increased regenerating myofibers. Such enhanced regeneration by excess adiponectin was also observed in adiponectin null mice, but not in T-cadherin null mice. Mechanistically, adiponectin accumulated on plasma membrane of myofibers both in mice and human, and intracellularly colocalized with endosomes positive for a multivesicular bodies/exosomes marker CD63 in regenerating myofibers. Purified high-molecular multimeric adiponectin similarly accumulated intracellularly and colocalized with CD63-positive endosomes and enhanced exosome secretion in differentiating C2C12 myotubes but not in undifferentiated myoblasts. Knockdown of T-cadherin in differentiating C2C12 myotubes attenuated both adiponectin-accumulation and adiponectin-mediated exosome production. Collectively, our studies have firstly demonstrated that adiponectin stimulates muscle regeneration through T-cadherin, where intracellular accumulation and exosome-mediated process of adiponectin may have some roles.

Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA.

RNA-based vector delivery is a promising gene therapy approach. Recent advances in chemical modification of mRNA structure to form modified mRNA (mmRNA or cmRNA or modRNA) have substantially improved their stability and translational efficiency within cells. However, mmRNA conventionally delivered in solution can be taken up nonspecifically or become cleared away prematurely, which markedly limits the potential benefit of mmRNA therapy. To address this limitation, we developed mmRNA-incorporated nanofibrillar scaffolds that could target spatially localized delivery and temporally controlled release of the mmRNA both in vitro and in vivo. To establish the efficacy of mmRNA therapy, mmRNA encoding reporter proteins such as green fluorescence protein or firefly luciferase (Fluc) was loaded into aligned nanofibrillar collagen scaffolds. The mmRNA was released from mmRNA-loaded scaffolds in a transient and temporally controlled manner and induced transfection of human fibroblasts in a dose-dependent manner. In vitro transfection was further verified using mmRNA encoding the angiogenic growth factor, hepatocyte growth factor (HGF). Finally, scaffold-based delivery of HGF mmRNA to the site of surgically induced muscle injury in mice resulted in significantly higher vascular regeneration after 14 days, compared to implantation of Fluc mmRNA-releasing scaffolds. After transfection with Fluc mmRNA-releasing scaffold in vivo, Fluc activity was detectable and localized to the muscle region, based on noninvasive bioluminescence imaging. Scaffold-based local mmRNA delivery as an off-the-shelf form of gene therapy has broad translatability for treating a wide range of diseases or injuries.

The Chemokine Cxcl10 is Not Needed For Normal Skeletal Muscle Regeneration Following a Toxin-induced Injury

PURPOSE: The CCL2/CCR2 chemokine receptor axis is necessary for successful muscle regeneration following injury. Recently, another chemokine known as CXCL10 (or IP-10) was found to be elevated in human skeletal muscle following muscle-damaging eccentric contractions. This finding may indicate that CXCL10, like CCL2, is important for muscle regeneration. METHODS:To test this, we measured functional and histological markers of muscle regeneration in mice out to 14 days post muscle injury (DPI) in wild type (WT) mice and CXCL10 knockout (KO) mice (n = 13 and 12, respectively). The muscle injury was induced by cardiotoxin (CTX) injection into the tibialis anterior (TA) muscle of a hind limb of each mouse. The opposite limb was injected with saline to serve as a within-animal sham control. At 2, 7 and 14 DPI muscle function of both the CTX- and saline-injected TA muscles was assessed using an in situ contraction preparation. After the functional testing, the TA muscles were excised, weighed, frozen, cut by cross section and mounted on glass slides for histological studies. Mounted muscle samples were stained using hematoxylin and eosin to identify central nucleated fibers and quantify cross sectional area. Samples were also stained immunohistochemically for embryonic myosin heavy chain (eMyHC) to identify the number of fibers undergoing regeneration. RESULTS: Relative to the saline-injected TA, strength significantly decreased (p<0.05) at 2 (WT=46.7±15.9%, KO=44±9.4%) and 7 (WT=65±8.3%, KO= 66±15.9%) DPI, and was recovered by 14 DPI. No significant differences in muscle strength were found between genotypes. Compared to the control TA, the CTX-injected TA muscle mass was significantly reduced (p<0.05) at 7 DPI (WT=86±3%, KO=91±9%) compared to 14 DPI (WT=105±8.3%, KO= 105±11%). No significant differences in muscle mass were found between genotypes. Cross-sectional area of regenerating myofibers was greater at 14 DPI compared to 7 (p<0.05), yet again no significant differences were found between genotypes. The number of myofibers expressing eMyHC was greater at 7 DPI than 14 (p<0.05), with no significant difference between genotypes. CONCLUSIONS: We conclude that CXCL10 is not necessary for normal muscle regeneration from a toxin-induced muscle injury in mice.

Involvement of Transient Receptor Potential Cation Channel Vanilloid 1 (TRPV1) in Myoblast Fusion.

The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc.

Numb is required to prevent p53-dependent senescence following skeletal muscle injury

Regeneration relies on coordinated action of multiple cell types to reconstitute the damaged tissue. Here we inactivate the endocytic adaptor protein Numb in skeletal muscle stem cells prior to chronic or severe muscle injury in mice. We observe two types of senescence in regenerating muscle; a transient senescence in non-myogenic cells of control and Numb mutant mice that partly depends on INK4a/ARF activity, and a persistent senescence in myogenic cells lacking Numb. The senescence levels of Numb-deficient muscle is reduced to wild type levels by an anti-oxidant treatment or p53 ablation, resulting in functional rescue of the regenerative potential in Numb mutants. Ex vivo experiments suggest that Numb-deficient senescent cells recruit macrophages to sustain inflammation and drive fibrosis, two hallmarks of the impaired muscle regeneration in Numb mutants. These findings provide insights into previously reported developmental and oncogenic senescence that are also differentially regulated by p53.

Breaking the co-operation between bystander T-cells and natural killer cells prevents the development of immunosuppression after traumatic skeletal muscle injury in mice.

Nosocomial infections represent serious complications after traumatic or surgical injuries in intensive care units. The pathogenesis of the underlying immunosuppression is only incompletely understood. In the present study, we investigated whether injury interferes with the function of the adaptive immune system in particular with the differentiation of antigen-specific T helper (Th)-cell responses in vivo. We used a mouse model for traumatic gastrocnemius muscle injury. Ovalbumin (OVA), which served as a foreign model antigen, was injected into the hind footpads for determination of the differentiation of OVA-specific Th-cells in the draining popliteal lymph node (pLN). The release of interferon (IFN)-γ from OVA-specific Th-cells was impaired within 24 h after injury and this impairment persisted for at least 7 days. In contrast, the proliferation of OVA-specific Th-cells remained unaffected. Injury did not modulate the function of antigen-presenting cells (APCs) in the pLN. Adoptive transfer of total T-cells from pLNs of injured mice inhibited IFN-γ production by OVA-specific Th-cells in naive mice. Suppressed Th1 priming did not occur in lymphocyte-deficient mice after injury but was restored by administration of T-cells before injury. Moreover, the suppression of Th1 differentiation required the presence of natural killer (NK) cells that were recruited to the pLN after injury; this recruitment was dependent on lymphocytes, toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). In summary, upon traumatic skeletal muscle injury T-cells and NK cells together prevent the development of protective Th1 immunity. Breaking this co-operation might be a novel approach to reduce the risk of infectious complications after injury.

Expression of the dermatomyositis autoantigen transcription intermediary factor 1γ in regenerating muscle.

Autoantibodies against transcription intermediary factor 1γ (TIF1γ) are found in many patients with dermatomyositis (DM). Although TIF1γ is known to play a role in the differentiation of other tissues, its functional role in muscle regeneration has not been elucidated. This study was undertaken to explore the regulation and functional role of this protein during muscle differentiation and regeneration. TIF1γ expression was analyzed in human muscle biopsy specimens using immunofluorescence microscopy. Immunofluorescence microscopy and immunoblotting analyses were used to study TIF1γ expression in a mouse model of muscle injury and repair. The effect of premature TIF1γ silencing on muscle differentiation was studied in cultured mouse myoblasts. In muscle biopsy specimens from DM patients, TIF1γ was expressed at low levels in the nuclei of histologically normal muscle cells but at high levels in the centralized nuclei of atrophic, perifascicular myofibers expressing markers of regeneration. TIF1γ levels were also increased in regenerating myonuclei following muscle injury in mice. Premature silencing of TIF1γ in vitro using small interfering RNA did not accelerate the expression of myogenin, a transcription factor that plays a central role in regulating relatively early stages of muscle differentiation. However, premature silencing of TIF1γ did accelerate myotube fusion and the expression of myosin heavy chain (MyHC), a later marker of muscle differentiation. The DM autoantigen TIF1γ is markedly up-regulated during muscle regeneration in human and mouse muscle cells. Premature silencing of this protein in cultured myoblasts accelerates MyHC expression and myoblast fusion. However, TIF1γ may function independently of, or downstream from, myogenin.