Induced Muscle Injury Research Articles

Jie-Du-Tong-Luo formula protects C2C12 myotubes against high glucose and palmitic acid injury by activating the PI3K/Akt/PPARγ pathway in vitro

IntroductionIn prior reports, Jie-Du-Tong-Luo (JDTL) was reported to help control insulin secretion and blood glucose in patients with diabetes, while also protecting liver and pancreatic islet cells against injury caused by exposure to high glucose (HG) levels. This study was thus developed to assess the effects of JDTL on HG and palmitic acid (PA)-induced muscle injury and to explore the mechanistic basis for these effects. MethodsA model of muscle injury was established using mouse C2C12 myotubes treated with HG + PA. A proteomics approach was used to assess changes in protein levels following JDTL treatment, after which Western immunoblotting was employed to validate significantly affected pathways. ResultsJDTL was able to protect against HG + PA-induced muscle cell injury in this experimental system, altering lipid metabolism and inflammatory activity in these injured C2C12 myotubes. Western blotting suggested that JDTL is capable of activating PI3K/Akt/PPARγ signaling to control lipid metabolism without any corresponding impact on the inflammatory NF-κB pathway. ConclusionsThese data highlight the ability of JDTL to protect against HG + PA-induced injury to muscle cells, and suggest that the underlying basis for such efficacy is related to the PI3K/Akt/PPARγ pathway-mediated modulation of lipid metabolism.

Increase in cathepsin K gene expression in Duchenne muscular dystrophy skeletal muscle.

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

Development of a local controlled release system for therapeutic proteins in the treatment of skeletal muscle injuries and diseases

The present study aims to develop and characterize a controlled-release delivery system for protein therapeutics in skeletal muscle regeneration following an acute injury. The therapeutic protein, a membrane-GPI anchored protein called Cripto, was immobilized in an injectable hydrogel delivery vehicle for local administration and sustained release. The hydrogel was made of poly(ethylene glycol)-fibrinogen (PEG-Fibrinogen, PF), in the form of injectable microspheres. The PF microspheres exhibited a spherical morphology with an average diameter of approximately 100 micrometers, and the Cripto protein was uniformly entrapped within them. The release rate of Cripto from the PF microspheres was controlled by tuning the crosslinking density of the hydrogel, which was varied by changing the concentration of poly(ethylene glycol) diacrylate (PEG-DA) crosslinker. In vitro experiments confirmed a sustained-release profile of Cripto from the PF microspheres for up to 27 days. The released Cripto was biologically active and promoted the in vitro proliferation of mouse myoblasts. The therapeutic effect of PF-mediated delivery of Cripto in vivo was tested in a cardiotoxin (CTX)-induced muscle injury model in mice. The Cripto caused an increase in the in vivo expression of the myogenic markers Pax7, the differentiation makers eMHC and Desmin, higher numbers of centro-nucleated myofibers and greater areas of regenerated muscle tissue. Collectively, these results establish the PF microspheres as a potential delivery system for the localized, sustained release of therapeutic proteins toward the accelerated repair of damaged muscle tissue following acute injuries.

P7C3 ameliorates barium chloride-induced skeletal muscle injury activating transcriptomic and epigenetic modulation of myogenic regulatory factors.

Skeletal muscle injury affects the quality of life in many pathologies, including volumetric muscle loss, contusion injury, and aging. We hypothesized that the nicotinamide phosphoribosyltransferase (Nampt) activator P7C3 improves muscle repair following injury. In the present study, we tested the effect of P7C3 (1-anilino-3-(3,6-dibromocarbazol-9-yl) propan-2-ol) on chemically induced muscle injury. Muscle injury was induced by injecting 50 µL 1.2% barium chloride (BaCl2) into the tibialis anterior (TA) muscle in C57Bl/6J wild-type male mice. Mice were then treated with either 10 mg/kg body weight of P7C3 or Vehicle intraperitoneally for 7 days and assessed for histological, biochemical, and molecular changes. In the present study, we show that the acute BaCl2-induced TA muscle injury was robust and the P7C3-treated mice displayed a significant increase in the total number of myonuclei and blood vessels, and decreased serum CK activity compared with vehicle-treated mice. The specificity of P7C3 was evaluated using Nampt+/- mice, which did not display any significant difference in muscle repair capacity among treated groups. RNA-sequencing analysis of the injured TA muscles displayed 368 and 212 genes to be exclusively expressed in P7C3 and Veh-treated mice, respectively. There was an increase in the expression of genes involved in cellular processes, inflammatory response, angiogenesis, and muscle development in P7C3 versus Veh-treated mice. Conversely, there is a decrease in muscle structure and function, myeloid cell differentiation, glutathione, and oxidation-reduction, drug metabolism, and circadian rhythm signaling pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (qPCR) and reverse transcription-qPCR analyses identified increased Pax7, Myf5, MyoD, and Myogenin expression in P7C3-treated mice. Increased histone lysine (H3K) methylation and acetylation were observed in P7C3-treated mice, with significant upregulation in inflammatory markers. Moreover, P7C3 treatment significantly increased the myotube fusion index in the BaCl2-injured human skeletal muscle in vitro. P7C3 also inhibited the lipopolysaccharide-induced inflammatory response and mitochondrial membrane potential of RAW 264.7 macrophage cells. Overall, we demonstrate that P7C3 activates muscle stem cells and enhances muscle injury repair with increased angiogenesis.

WITHDRAWN: P4HB Inhibits Cachectic Muscle Regeneration by Restricting Myoblast Migration

Cachexia is a prevalent syndrome in advanced cancer characterized by skeletal muscle atrophy. Impaired muscle regeneration exacerbates the loss of muscle mass. However, the mechanism underlying cachectic impairments in muscle regeneration remains unclear. Previous research revealed that P4HB accumulates in cachexia muscle. In this study, we explored the role of P4HB in muscle regeneration. We induced muscle injury in cachectic mice to demonstrate the impairment of muscle regeneration. Subsequently, we used inducible overexpression cell lines and muscle-specific knockout mice to elucidate the inhibitory role of P4HB in muscle regeneration. Using mass spectrometry, we identified MYH9 as a downstream molecule of P4HB, demonstrating that P4HB affects myoblast migration and thus impacts muscle regeneration. Our results suggest that the accumulation of P4HB in cachectic muscle contributes to regeneration impairment, indicating that P4HB is a potential therapeutic target for cachexia treatment.

Mustn1 is a smooth muscle cell-secreted microprotein that modulates skeletal muscle extracellular matrix composition

ObjectiveSkeletal muscle plasticity and remodeling are critical for adapting tissue function to use, disuse, and regeneration. The aim of this study was to identify genes and molecular pathways that regulate the transition from atrophy to compensatory hypertrophy or recovery from injury. Here, we have used a mouse model of hindlimb unloading and reloading, which causes skeletal muscle atrophy, and compensatory regeneration and hypertrophy, respectively. MethodsWe analyzed mouse skeletal muscle at the transition from hindlimb unloading to reloading for changes in transcriptome and extracellular fluid proteome. We then used qRT-PCR, immunohistochemistry, and bulk and single-cell RNA sequencing data to determine Mustn1 gene and protein expression, including changes in gene expression in mouse and human skeletal muscle with different challenges such as exercise and muscle injury. We generated Mustn1-deficient genetic mouse models and characterized them in vivo and ex vivo with regard to muscle function and whole-body metabolism. We isolated smooth muscle cells and functionally characterized them, and performed transcriptomics and proteomics analysis of skeletal muscle and aorta of Mustn1-deficient mice. ResultsWe show that Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang) is highly expressed in skeletal muscle during the early stages of hindlimb reloading. Mustn1 expression is transiently elevated in mouse and human skeletal muscle in response to intense exercise, resistance exercise, or injury. We find that Mustn1 expression is highest in smooth muscle-rich tissues, followed by skeletal muscle fibers. Muscle from heterozygous Mustn1-deficient mice exhibit differences in gene expression related to extracellular matrix and cell adhesion, compared to wild-type littermates. Mustn1-deficient mice have normal muscle and aorta function and whole-body glucose metabolism. We show that Mustn1 is secreted from smooth muscle cells, and that it is present in arterioles of the muscle microvasculature and in muscle extracellular fluid, particularly during the hindlimb reloading phase. Proteomics analysis of muscle from Mustn1-deficient mice confirms differences in extracellular matrix composition, and female mice display higher collagen content after chemically induced muscle injury compared to wild-type littermates. ConclusionsWe show that, in addition to its previously reported intracellular localization, Mustn1 is a microprotein secreted from smooth muscle cells into the muscle extracellular space. We explore its role in muscle ECM deposition and remodeling in homeostasis and upon muscle injury. The role of Mustn1 in fibrosis and immune infiltration upon muscle injury and dystrophies remains to be investigated, as does its potential for therapeutic interventions.

DDAH1 Protects against Cardiotoxin-Induced Muscle Injury and Regeneration.

Nitric oxide (NO) is an important biological signaling molecule affecting muscle regeneration. The activity of NO synthase (NOS) is regulated by dimethylarginine dimethylaminohydrolase 1 (DDAH1) through degradation of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). To investigate the role of DDAH1 in muscle injury and regeneration, muscle-specific Ddah1-knockout mice (Ddah1MKO) and their littermates (Ddah1f/f) were used to examine the progress of cardiotoxin (CTX)-induced muscle injury and subsequent muscle regeneration. After CTX injection, Ddah1MKO mice developed more severe muscle injury than Ddah1f/f mice. Muscle regeneration was also delayed in Ddah1MKO mice on Day 5 after CTX injection. These phenomena were associated with higher serum ADMA and LDH levels as well as a great induction of inflammatory response, oxidative stress and cell apoptosis in the gastrocnemius (GA) muscle of Ddah1MKO mice. In the GA muscle of CTX-treated mice, Ddah1 deficiency decreased the protein expression of M-cadherin, myogenin, Bcl-2, peroxiredoxin 3 (PRDX3) and PRDX5, and increased the protein expression of MyoD, TNFα, Il-6, iNOS and Bax. In summary, our data suggest that DDAH1 exerts a protective role in muscle injury and regeneration.

The Potential Role of Mesenchymal Stem Cells Derived Exosomes on the Healing of Grade II Experimentally Induced Skeletal Muscle Injury in Adult Male Albino Rats. Histological Study

Abstract Background Musculoskeletal injuries are the most common cause of severe longterm pain and physical disability. However, there is still no efficient treatment that leads to prevention of muscle fibrosis. Cell-Free therapy using mesenchymal stem cells (MSCs) derived exosomes could provide a new promising therapy. Aim To study the potential role of MSCs-derived exosomes on the healing of experimentally induced grade II skeletal muscle injury in adult male albino rats. Materials and Methods Forty male albino Wister rats were used in this study. Five out of them were used for isolation of exosomes. The rest were randomly divided into three groups. Group I (Sham group), Group II (muscle injury group left for spontaneous healing), Group III (muscle injury treated with exosomes). Rats from each group were subdivided into two subgroups; subgroup “a and b” in which rats were sacrificed after ten days and twenty days, respectively. Muscle specimens were collected and prepared for proper histological techniques. Immuno-histochemical staining for detection of collagen type I and developmental myosin (MHCd) were also done. Morphometric study and statistical analysis were also performed. Results Granulation tissue and inflammatory cellular infiltration with significant increase in the mean area percentage of collagen fibers were noticed in subgroups of untreated muscle injury compared to the corresponding exosomes treated subgroups. This was confirmed by collagen type I immune histochemical reaction. Group III (treated with exosomes) showed better muscle regeneration and significant decrease in muscle fibrosis. Significant increase in immunohistochemical reaction of developmental myosin was noticed in exosomes treated subgroups compared to corresponding untreated muscle injury subgroups. Conclusions Exosomes enhance muscle regeneration after induced muscle injury. Exosomes also decreased muscle fibrosis and improve muscle healing.

Cell-free DNA as an potential marker of statin induced muscle injury

Cell-free DNA as an potential marker of statin induced muscle injury

MiR-136-5p/FZD4 axis is critical for Wnt signaling-mediated myogenesis and skeletal muscle regeneration.

Skeletal muscle can undergo a regenerative process in response to injury or disease to maintain muscle quality and function. Myogenesis depends on the proliferation and differentiation of myoblasts, and miRNAs can maintain the balance between them by precisely regulating many key factors in the myogenic network. Here, we found that miR-136-5p was significantly upregulated during the proliferation and differentiation of C2C12 cells. We demonstrate that miR-136-5p acts as a myogenic negative regulator during the development of mouse C2C12 myoblasts. In terms of mechanism, miR-136-5p inhibits the formation of β-catenin/LEF/TCF DNA-binding factor transcriptional regulatory complex by targeting FZD4, a gating protein in the Wnt signaling pathway, thereby enhancing downstream myogenic factors and finally promoting myoblast proliferation and differentiation. In addition, in BaCl2 -induced muscle injury mouse model, miR-136-5p knockdown accelerated the regeneration of skeletal muscle after injury, and further led to the improvement of gastrocnemius muscle mass and muscle fiber diameter, while being suppressed by shFZD4 lentivirus infection. In summary, these results demonstrate the essential role of miR-136-5p/FZD4 axis in skeletal muscle regeneration. Given the conservation of miR-136-5p among species, miR-136-5p may be a new target for treating human skeletal muscle injury and improving the production of animal meat products.

Single cell analysis reveals satellite cell heterogeneity for proinflammatory chemokine expression

Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood. Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence. Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury. Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.

Division of loading time in reloading the disused atrophic soleus muscle induces proximal muscle injury.

[Purpose] This study aimed to compare the effects of loading time division in reloading atrophied muscles in different muscle long-axis regions. [Materials and Methods] We divided 8-week-old male Wistar rats into control (CON), 14-day hindlimb suspension (HS), 7-day hindlimb suspension followed by 60-min reloading for 7 consecutive days (WO), and 7-day hindlimb suspension followed by 60-min reloading on two separate occasions for 7 days (WT) groups. After the experimental period, muscle fibre cross-sectional area and necrotic fibre/central nuclei fibre ratio were measured in the soleus muscle's proximal, middle, and distal regions. [Results] The necrotic fibre/central nuclei fibre ratio was higher in the WT group than in the other groups in the proximal region. Proximal muscle fibre cross-sectional area was higher in the CON group than in the other groups. In the middle region, only HS group had muscle fibre cross-sectional area lower than the CON group. Similarly, muscle fibre cross-sectional area of the HS group was lower than the CON and WT groups in the distal region. [Conclusion] When reloading atrophied muscles, dividing the loading time can inhibit atrophy in the distal region but induce muscle injury in the proximal region.

Myonecrosis Induction by Intramuscular Injection of CTX.

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress. Graphical abstract.

Induction of Skeletal Muscle Injury by Intramuscular Injection of Cardiotoxin in Mouse.

Skeletal muscle is the most abundant tissue in the human body and has a tremendous capability to regenerate in response to muscle injuries and diseases. Induction of acute muscle injury is a common method to study muscle regeneration in vivo. Cardiotoxin (CTX) belongs to the family of snake venom toxins and is one of the most common reagents to induce muscle injury. Intramuscular injection of CTX causes overwhelming muscle contraction and lysis of myofibers. The induced acute muscle injury triggers muscle regeneration, allowing in-depth studies on muscle regeneration. This protocol describes a detailed procedure of intramuscular injection of CTX to induce acute muscle injury that could be also applied in other mammalian models.

Etiology, risk factors and complications of exercise induced muscle injury

Healthy lifestyle includes physical activity as the basic component however, exercises with eccentricity have been associated to a higher risk of muscle injury and slower recovery. The development of exercise-induced muscle injury is a phenomenon that results from atypical or unaccustomed activity; the intensity of the pain and the extent of the damage steadily deteriorate over time, and when the exercise session contains an eccentric component, they may last for several days. Increasing levels of muscle-specific circulating protein are present, along with increased levels of muscular pain and decreased muscle strength. The extent of severity of the symptoms following eccentric exercise varies greatly. Exercise-induced muscle injury is influenced by a number of factors including intensity, duration, mode, muscle group, age, gender, genetics, and dietary state. Numerous scientific studies illustrate the circumstances under which exercise causes muscle injury. It is widely acknowledged that exercise that is unfamiliar, particularly exercise that involves eccentric contractions, causes damage. exercise-induced muscle injury is accompanied by an inflammatory response involving numerous mediators, including the production of muscle-specific creatine kinase and interleukins and interleukins receptor antagonist and acute phase proteins. Complications of exercise-induced muscle injury include temporary muscle inflammation, loss of strength, muscle discomfort, restricted range of motion, elevated passive tension, discomfort, and a momentary decline in insulin sensitivity and can also lead to rhabdomyolysis. The purpose of this research is to review the available information about an etiology, risk factors and complications of exercise-induced muscle injury.

AMP-activated protein kinase inhibition in fibro-adipogenic progenitors impairs muscle regeneration and increases fibrosis.

Following muscle injury, fibro-adipogenic progenitors (FAPs) are rapidly activated and undergo apoptosis at the resolution stage, which is required for proper muscle regeneration. When excessive FAPs remain, it contributes to fibrotic and fatty infiltration, impairing muscle recovery. Mechanisms controlling FAP apoptosis remain poorly defined. We hypothesized that AMP-activated protein kinase (AMPK) in FAPs mediates their apoptosis during the muscle regeneration. To test, AMPKα1fl/fl PDGFRαCre mice were used to knock out AMPKα1 in FAPs. Following AMPKα1 knockout, the mice were injected with phosphate-buffered saline or glycerol to induce muscle injury. Tibialis anterior muscle and FAPs were collected at 3, 7 and 14days post-injury (dpi) for further analysis. We found that AMPKα1 deletion in FAPs enhanced p65 translocation to the nuclei by 110% (n=3; P<0.01). AMPKα1 knockout group had a higher gene expression of MMP-9 (matrix metalloproteinase-9) by 470% (n=3; P<0.05) and protein level by 39% (n=3; P<0.05). Loss of AMPKα1 up-regulated the active TGF-β1 (transforming growth factor-β1) levels by 21% (n=3; P<0.05). TGF-β promoted apoptotic resistance, because AMPKα1-deficient group had 36% lower cleaved Caspase 3 (cCAS3) content (n=3; P<0.05). Fibrotic differentiation of FAPs was promoted, with increased collagen protein level by 54% (n=3; P<0.05). Moreover, obesity decreased phosphorylation of AMPK by 54% (n=3; P<0.05), which decreased cCAS3 in FAPs by 44% (n=3; P<0.05) and elevated collagen accumulation (52%; n=3; P<0.05) during muscle regeneration. These data suggest that AMPK is a key mediator of FAPs apoptosis, and its inhibition due to obesity results in fibrosis of regenerated muscle.

Effect of Platelet Rich Plasma Versus Spontaneous Healing in Induced Muscle Injury in Senile Male Albino Rats.

Effect of Platelet Rich Plasma Versus Spontaneous Healing in Induced Muscle Injury in Senile Male Albino Rats.

Local application of a transcutaneous carbon dioxide paste prevents excessive scarring and promotes muscle regeneration in a bupivacaine-induced rat model of muscle injury.

In postoperative patients with head and neck cancer, scar tissue formation may interfere with the healing process, resulting in incomplete functional recovery and a reduced quality of life. Percutaneous application of carbon dioxide (CO2 ) has been reported to improve hypoxia, stimulate angiogenesis, and promote fracture repair and muscle damage. However, gaseous CO2 cannot be applied to the head and neck regions. Previously, we developed a paste that holds non-gaseous CO2 in a carrier and can be administered transdermally. Here, we investigated whether this paste could prevent excessive scarring and promote muscle regeneration using a bupivacaine-induced rat model of muscle injury. Forty-eight Sprague Dawley rats were randomly assigned to either a control group or a CO2 group. Both groups underwent surgery to induce muscle injury, but the control group received no treatment, whereas the CO2 group received the CO2 paste daily after surgery. Then, samples of the experimental sites were taken on days 3, 7, 14, and 21 post-surgery to examine the following: (1) inflammatory (interleukin [IL]-1β, IL-6), and transforming growth factor (TGF)-β and myogenic (MyoD and myogenin) gene expression by polymerase chain reaction, (2) muscle regeneration with haematoxylin and eosin staining, and (3) MyoD and myogenin protein expression using immunohistochemical staining. Rats in the CO2 group showed higher MyoD and myogenin expression and lower IL-1β, IL-6, and TGF-β expression than the control rats. In addition, treated rats showed evidence of accelerated muscle regeneration. Our study demonstrated that the CO2 paste prevents excessive scarring and accelerates muscle regeneration. This action may be exerted through the induction of an artificial Bohr effect, which leads to the upregulation of MyoD and myogenin, and the downregulation of IL-1β, IL-6, and TGF-β. The paste is inexpensive and non-invasive. Thus, it may be the treatment of choice for patients with muscle damage.

Lnc-GD2H Promotes Proliferation by Forming a Feedback Loop With c-Myc and Enhances Differentiation Through Interacting With NACA to Upregulate Myog in C2C12 Myoblasts.

In the present study, the roles of a novel long non-coding RNA (lncRNA), lnc-GD2H, in promoting C2C12 myoblast proliferation and differentiation and muscle regeneration were investigated by quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine (EdU), immunofluorescence staining, luciferase reporter, mass spectrometry, pulldown, chromatin immunoprecipitation, RNA immunoprecipitation assay, wound healing assays, and cardiotoxin (CTX)-induced muscle injury assays. It was observed that lnc-GD2H promoted myoblast proliferation as evidenced by the enhancement of the proliferation markers c-Myc, CDK2, CDK4, and CDK6, percentage of EdU-positive cells, and rate of cell survival during C2C12 myoblast proliferation. Additional experiments confirmed that c-Myc bound to the lnc-GD2H promoter and regulated its transcription. lnc-GD2H promoted cell differentiation with enhanced MyHC immunostaining as well as increased expression of the myogenic marker genes myogenin (Myog), Mef2a, and Mef2c during myoblast differentiation. Additional assays indicated that lnc-GD2H interacted with NACA which plays a role of transcriptional regulation in myoblast differentiation, and the enrichment of NACA at the Myog promoter was impaired by lnc-GD2H. Furthermore, inhibition of lnc-GD2H impaired muscle regeneration after CTX-induced injury in mice. lnc-GD2H facilitated the expression of proliferating marker genes and formed a feedback loop with c-Myc during myoblast proliferation. In differentiating myoblasts, lnc-GD2H interacted with NACA to relieve the inhibitory effect of NACA on Myog, facilitating Myog expression to promote differentiation. The results provide evidence for the role of lncRNAs in muscle regeneration and are useful for developing novel therapeutic targets for muscle disorders.

Effects Of Icing With Or Without Heat Stress On Skeletal Muscle Fibrosis Following Injury

PURPOSE: The aims of the present study were to investigate 1) the effects of icing treatment as first aid following skeletal muscle injury on fibrosis in the regenerating muscle, and 2) whether the different start timing of heat treatment impacts fibrosis followed by the icing treatment after skeletal muscle injury. METHODS: Male Wistar rats (10 weeks of age) were randomly assigned to injured, injured with icing (ICE), and injured with icing and heat treatments (I + H) groups. The I + H group was further divided into two groups depending on the difference of timing to start the heat treatment (d1 and d2). Bupivacaine (BPVC) was injected into slow soleus muscles bilaterally in order to induce muscle injury in all rats. Application of icing treatment (ice pack, 0 °C for 20 min) was performed in the ICE and I + H groups only once immediately after the BPVC injection. Rats in the I + H group further received intermittent heat treatment (42 °C for 30 min on alternating days) started from 1- or 2-day after the injury until the day 14. At 1, 3, 7, 14, and 28 days after the injury, soleus muscles were dissected and analyzed. RESULTS: At 28 days after BPVC injection, the fibrotic area in regenerating soleus muscle was significantly higher in ICE than in injured groups (P < 0.05). In addition, mRNA microarray analysis showed that the expression level of genes involved in fibrosis, myogenesis, and muscle differentiation was upregulated when icing treatment was applied immediately following the injury at 3, 7, and 14 days after BPVC injection (P < 0.05). On the other hand, there was no significant difference in the fibrotic area in injured soleus between injured and both I + H groups at 28 days following the injury. The fibrotic area in the I + Hd1 group was also significantly lower than that in the ICE and I + Hd2 groups (P < 0.05). CONCLUSIONS: These results revealed that icing treatment applied immediately following skeletal muscle injury facilitated fibrosis in the regenerating muscle via, in part, inducing a change in gene expression dynamics of some specific gene groups. Our results also suggested that application of intermittent heat treatment, especially when started from 1-day after skeletal muscle injury, followed by icing treatment could be a potential strategy to attenuate the icing-related progression of fibrosis.