Muscle Fiber Repair Research Articles

A Micromanipulation-Actuated Large-Scale Screening to Identify Optimized Microphysiological Model Parameters in Skeletal Muscle Regeneration.

Hydrogel-based 3D cell cultures are extensively utilized to create biomimetic cellular microstructures. However, there is still lack of effective method for both evaluation of the complex interaction of cells with hydrogel and the functionality of the resulting micro-structures. This limitation impedes the further application of these microstructures as microphysiological models (microPMs) for the screening of potential culture condition combinations to enhance the skeletal muscle regeneration. This paper introduces a two-probe micromanipulation method for the large-scale assessment of viscoelasticity and contractile force (CF) of skeletal muscle microPMs, which are produced in high-throughput via microfluidic spinning and 96-well culture. The collected data demonstrate that viscoelasticity parameters (E* and tanδ) and CF both measured in a solution environment are indicative of the formation of cellular structures without hydrogel residue and the subsequent generation of myotubes, respectively. This study have developed screening criterias that integrate E*, tanδ, and CF to examine the effects of multifactorial interactions on muscle fiber repair under hypoxic conditions and within bioprinted bipennate muscle structures. This approach has improved the quality of hypoxic threshold evaluation and aligned cell growth in 3D. The proposed method is useful in exploring the role of different factors in muscle tissue regeneration with limited resources.

Reconstructing vascular networks promotes the repair of skeletal muscle following volumetric muscle loss by pre-vascularized tissue constructs

Current treatment for complex and large-scale volumetric muscle loss (VML) injuries remains a limited success and have substantial disadvantages, due to the irreversible loss of muscle mass, slow muscle regeneration, and rapid formation of non-functional fibrosis scars. These VML injuries are accompanied by denervation and the destruction of native vasculature which increases difficulties in the functional restoration of muscle. Here, reconstruction of the vascular network at the injury site was offered as a possible solution for improving the repair of muscle defects through the timely supply of nutrients and oxygen to surrounding cells. A hydrogel-based tissue construct containing various densities of the vascular network was successfully created in the subcutaneous space of mice by manipulating hydrogel properties, and then implanted into the VML injury site. One month after implantation, the mouse treated with the highly vascularized tissue had extensive muscle repair at the injury site and only spent a shorter time completing the inclined plane tests. These findings suggest that the reconstruction of the functional vascular network at the VML injury site accelerated muscle fiber repair through a timely supply of sufficient blood and avoided invasion by host fibroblasts.

A novel 3D printed type II silk fibroin/polycaprolactone mesh for the treatment of pelvic organ prolapse.

Pelvic organ prolapse (POP) is one of the common diseases in middle-aged and elderly women, caused by weakened pelvic floor muscle ligament tissue support. Pelvic floor reconstruction with mesh implantation has been proven to be an effective treatment for POP. However, traditional non-degradable and inflexible pelvic floor implantation meshes have been associated with pain, vaginal infections, and the need for additional surgeries. In this study, novel meshes with pre-designed structures were fabricated with solution-based electrohydrodynamic printing (EHDP) technology, using a series of polycaprolactone/silk fibroin composites as bioinks. The PCL/SF mesh mechanical performances were particularly enhanced with the addition of silk II, leading it to obtain higher adaptability with soft tissue repair. The mesh containing SF showed more robust degradation performance in the in vitro degradation assay. Furthermore, biocompatibility tests conducted on mouse embryonic fibroblasts (NIH/3T3) revealed enhanced cell affinity. Finally, the biocompatibility and tissue repair properties of PCL/SF mesh were verified through the implantation of meshes in the muscle defect site of mice. The results demonstrated that the 3D printed PCL/SF mesh prepared by EHDP exhibits superior mechanical properties, biocompatibility, biodegradability, as well as ligament and muscle fiber repair ability. The novel implantable meshes are promising for curing POP.

Inhibition of High-Temperature Requirement Protein A2 Protease Activity Represses Myogenic Differentiation via UPRmt.

Skeletal muscles require muscle satellite cell (MuSC) differentiation to facilitate the replenishment and repair of muscle fibers. A key step in this process is called myogenic differentiation. The differentiation ability of MuSCs decreases with age and can result in sarcopenia. Although mitochondria have been reported to be involved in myogenic differentiation by promoting a bioenergetic remodeling, little is known about the interplay of mitochondrial proteostasis and myogenic differentiation. High-temperature-requirement protein A2 (HtrA2/Omi) is a protease that regulates proteostasis in the mitochondrial intermembrane space (IMS). Mice deficient in HtrA2 protease activity show a distinct phenotype of sarcopenia. To investigate the role of IMS proteostasis during myogenic differentiation, we treated C2C12 myoblasts with UCF101, a specific inhibitor of HtrA2 during differentiation process. A key step in this process is called myogenic differentiation. The differentiation ability of MuSCs decreases with age and can result in sarcopenia. Further, CHOP, p-eIF2α, and other mitochondrial unfolded protein response (UPRmt)-related proteins are upregulated. Therefore, we suggest that imbalance of mitochondrial IMS proteostasis acts via a retrograde signaling pathway to inhibit myogenic differentiation via the UPRmt pathway. These novel mechanistic insights may have implications for the development of new strategies for the treatment of sarcopenia.

Structural and ultrastructural changes in the skeletal muscles of dysferlin-deficient mice during postnatal ontogenesis

ABSTRACT A number of sarcolemma proteins are responsible for muscle fiber repair. Dysferlin encoded by the DYSF gene is one of these proteins. Dysferlin promotes membrane repair in striated muscle fibers (MFs). Mutations in DYSF lead to loss of or decreased dysferlin expression, impaired membrane repair in MF, and its destruction, clinically manifesting as dysferlinopathy. Preclinical studies of cell and gene therapies aimed at restoring impaired muscle regeneration require well-characterized small animal models. Our investigation aimed to distinguish the histopathological features of a mouse strain lacking dysferlin expression (Bla/J strain). Ultrastructural changes in the sarcolemma, mitochondria and contractile apparatus were observed. It was shown that postnatal histogenesis of skeletal muscles in genetically determined dysferlin deficiency is characterized by a higher proportion of necrotic muscle fibers, compensatory hypertrophy of muscle fibers with their subsequent atrophy, and decreases in proliferative activity and the level of myogenic differentiation of myogenic progenitor cells compared to wild-type mice (C57Bl/6).

Hypoxia further exacerbates woody breast myopathy in broilers via alteration of satellite cell fate

Woody breast (WB) condition has created a variety of challenges for the global poultry industry. To date, there are no effective treatments or preventative measures due to its unknown (undefined) etiology. Several potential mechanisms including oxidative stress, fiber-type switching, cellular damage, and altered intracellular calcium levels have been proposed to play a key role in the progression of the WB myopathy. In a previous study, we have shown that WB is associated with hypoxia-like status and dysregulated oxygen homeostasis. As satellite cells (SC) play a pivotal role in muscle fiber repair and remodeling under stress conditions, we undertook the present study to determine satellite cell fate in WB-affected birds when reared in either normoxic or hypoxic conditions. Modern random bred broilers from 2015 (n = 200) were wing banded and reared under standard brooding practices for the first 2 wk post-hatch. At 15 d, chicks were divided in 2 body weight-matched groups and reared to 6 wk in either control local altitude or hypobaric chambers with simulated altitude of 6,000 ft. Birds were provided ad libitum access to water and feed, according to the Cobb recommendations. At 6 wk of age, birds were processed and scored for WB, and breast samples were collected from WB-affected and unaffected birds for molecular analyses (n = 10/group). SCs were isolated from normal breast muscle, cultured in vitro, and exposed to normoxia or hypoxia for 2 h. The expression of target genes was determined by qPCR using 2−∆∆Ct method. Protein distribution and expression were determined by immunofluorescence staining and immunoblot, respectively. Data were analyzed by the Student's t test with significance set at P < 0.05. Multiple satellite cell markers, myogenic factor (Myf)-5 and paired box (PAX)-7 were significantly decreased at the mRNA and protein levels in the breast muscle from WB-affected birds compared to their unaffected counterparts. Lipogenic-and adipogenic-associated factors (acetyl-CoA carboxylase, ACCα; fatty acid synthase, FASN, malic enzyme, ME; and ATP citrate lyase, ACLY) were activated in WB-affected birds. These data were supported by an in vitro study where hypoxia decreased the expression of Myf5 and Pax7, and increased that of ACCα, FASN, ME, and ACLY. Together, these data indicate that under hypoxic condition, SC change fate by switching from a myogenic to an adipogenic program, which explains at least partly, the etiology of the WB myopathy.

Effects Of Adipose Mesenchymal Stem Cells Injected Intramuscularly On Myostatin Signaling Skeletal Muscle In Rats After Eccentric Exercise

PURPOSE: To observe the effect of adipose mesenchymal stem cells (ASCs) on myostatin signal of skeletal muscle in rats at different time points after eccentric exercise, and to explore the mechanism of ASCs injection on skeletal muscle injury and repair after eccentric exercise. METHODS: Phosphate buffer saline (PBS) were injected into the gastrocnemius muscle in the left leg, and ASCs were injected into the gastrocnemius muscle of the right leg of SD rats after eccentric exercise. The rats were randomly divided into four groups: one day D1, three days D3, seven days W1 and fourteen days W2 after exercise. Skeletal muscle ultrastructure was observed by electron microscopic. The content of serum creatine kinase (CK), skeletal muscle troponin I (sTnI), myostatin (MSTN), follistatin (FST) were measured by ELISA. Real-time PCR was used to detect the expressions of MSTN, ACVR2B, FST mRNA in skeletal muscle. The expression of MSTN, ACVR2B, FST, p-Smad2/3 were detected by Western Blot. RESULTS: Compared with group PBS, adipose mesenchymal stem cell injection significantly promoted the repair of muscle fibers. Compared with group D1, the level of CK in group W2 was significantly decreased, the content of sTnI level in group D3 and group W1 were remarkably increased, the serum MSTN contents in group W1 were significantly decreased, which in group W2 was remarkably increased. Compared with group PBS, the relative expression quantities of MSTN mRNA were significantly decreased at time point D3 and which were extremely significantly down-regulated at time point W2. Compared with group PBS, the expression of MSTN protein were significantly decreased at time point D3, the expression of ACVR2B protein were remarkably increased at time point D1,D3 and W1, however, which was significantly decreased at time point W2. Compared with group PBS, the phosphorylation of Smad2/3 shown significantly decreased at the time point W2 of group ASCs. CONCLUSIONS: After eccentric exercise, allogeneic adipose mesenchymal stem cells injected intramuscularly can decreased the transcription of MSTN in skeletal muscle. Adipose mesenchymal stem cells injected intramuscularly may improve the regeneration and repair of skeletal muscle after eccentric exercise through affecting the downstream signaling pathway of MSTN.

Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair.

Dystrophic muscle is characterized by chronic injury and a steady recruitment of inflammatory Ly6Chi monocytes. Recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. Here, we investigated the contribution of splenic Ly6Chi monocytes to dystrophic muscle pathology. Using the mdx mouse model of muscular dystrophy, we show that Ly6Chi monocytes accumulate in great numbers in the spleen over the course of the disease. The chemokine receptor CCR2 was upregulated on Ly6Chi monocytes in mdx spleen before disease onset, thereby enabling their recruitment to dystrophic muscle. Splenectomy performed before disease onset significantly reduced the number of Ly6Chi monocytes infiltrating dystrophic limb muscle. Moreover, in the absence of splenic Ly6Chi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. However, during late disease, a lack of splenic Ly6Chi monocytes adversely affected muscle fiber repair, due to a delay in the phenotypic shift of proinflammatory F4/80+Ly6ChiCD206lo to antiinflammatory F4/80+Ly6CloCD206+ macrophages. Overall, we show that the spleen is an indispensable source of Ly6Chi monocytes in muscular dystrophy and that splenic monocytes are critical players in both muscle fiber injury and repair.

A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.

Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.

Retinoic acid regulates the activity of satellite cells and promotes skeletal muscle regeneration impaired due to obesity in mice

BackgroundEctopic lipid accumulation in skeletal muscle induced by obesity leads to muscle atrophy and impaired regenerative capacity. Retinoic acid (RA) has been reported to enhance myogenesis in embryonic stem cells and promotes regeneration in adult skeletal muscle. However, the effects of RA on muscle regeneration impaired due to obesity have not been addressed. The objective of this study is to explore the effects of RA on activation and myogenic differentiation of satellite cells (SCs) in obese mice, which promote muscle regeneration.MethodsTransgenic Pax7cre/ROSAmT/mG mice were used for lineage tracing of SCs. Obesity was induced by feeding 60% high fat diet for 10 weeks. Two days following tamoxifen injection, 10 mg/kg body weight (BDW) RA or equivalent vehicle were injected subcutaneously into the tibialis anterior (TA) muscle of mice once per two days before injury, and TA muscle injury was induced by cardiotoxin injection. The effects of RA supplementation on SCs and corresponding skeletal muscle regeneration were evaluated at different times post‐injury.ResultsHistological analysis showed that RA supplementation improved muscle fiber regeneration and recovery in obese mice at day 7 post‐injury. The number of SCs in the muscle of obese mice at 3 days post‐injury were increased and the fusion of SCs into regenerated muscle fibers at 7 days post‐injury were promoted in RA treated group. Correspondingly, RA supplementation increased the expression of retinoic acid receptor α (RARα) in the TA muscle samples. The expression of myogenesis genes such as Pax7, Myf5, MyoD and myogenin were increased in muscles with RA treatment. Compared with the vehicle treated group, inflammatory responses including expression of IL6 and TNFα at day 3 and IL1β and IL18 at day 7 were decreased in the skeletal muscle of mice with RA treatment. RA supplementation also increased the expression of insulin like growth factor 1 (IGF1) in muscle at day 3 post‐injury.ConclusionsRetinoic acid promoted regeneration and repair of muscle fibers after injury in obese mice accompanied by increased retinoic acid signaling, elevated expression of IGF1 and resolution of inflammation in the regenerating muscle. These benefits could be attributed to enhanced proliferation and myogenic differentiation of SCs after RA treatment.Support or Funding InformationSupported by NIH Grant R01HD067449. Effects of retinoic acid (RA) supplementation on skeletal muscle regeneration. H&amp;E staining (A, D) and fluorescence images (B, E) of tibialis anterior (TA) muscle at 3‐ and 7‐days post‐injury (dpi). Green fluorescence tracked Pax7+ satellite cells and derived muscle fibers (regenerated) after tamoxifen induction while red fluorescence showed original cells. C, F. mRNA expression. *p&lt;0.05, ** p&lt;0.01. Data are expressed as mean ±SEM (n=3).imageEffects of retinoic acid (RA) supplementation on skeletal muscle regeneration. H&amp;E staining (A, D) and fluorescence images (B, E) of tibialis anterior (TA) muscle at 3‐ and 7‐days post‐injury (dpi). Green fluorescence tracked Pax7+ satellite cells and derived muscle fibers (regenerated) after tamoxifen induction while red fluorescence showed original cells. C, F. mRNA expression. *p&lt;0.05, ** p&lt;0.01. Data are expressed as mean ±SEM (n=3).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of warm acupuncture on pathological morphology and pain-induced inflammatory mediators in rats with myofascial pain trigger

Based on the establishment of a rat model of trigger point, this study was to intervene with warm acupuncture, and to evaluate the effect on pathological morphology and pain-induced inflammation of the rat model by microscopic pathology and microdialysis. Sixty-four SD rats were randomly divided into group A (blank control), group B (model control) and group C (model and intervention control). Groups A and B were divided into 3 groups (A0, A1, A2 and B0, B1, B2), the group C was divided into 2 groups (C1 and C2). The MTrPs model was established in both groups B and C, warm acupuncture intervention were given to the C1 group for 7 days and the C2 group for 15 days. Rats were sacrificed in batches. MTrPs were locally sampled and stained with hematoxylin-eosin after the preparation. The pathological changes were observed under light microscopy. The iocal interleukin-1β and prostaglandin E2 were detected by microdialysis technique. Microscopically, the muscle fibers of the model were arranged disorderly, broken, twisted, local fibrosis, contracture thickening and so on; macrophage and other inflammatory cell invasion in local area and a large area of adhesion occurred on the contracture nodule, the pathological state of local muscle fibers was significantly improved after warm needle intervention, local microvascular formation and maturation, local muscle fiber repair. After successful modeling, the amount of interleukin-1β and prostaglandin E2 in group B0 was significantly higher than that in group A0 before warm needle intervention (P<0.01). After warming intervention for 7 days, there was no significant difference in the amount of interleukin-1β and prostaglandin E2 between group C1 and group B1 (P>0.05). Group C1 and B1 were significantly higher than group A1 (P<0.01); warm needle intervention for 15 days, the amount of interleukin-1β and prostaglandin E2 in group C2 were lower than those in group B2 (P<0.05), but those in group C2 and B2 were significantly higher than group A2 (P<0.01), and the amount of interleukin-1β and prostaglandin E2 in group C2 was lower than group C1 (P<0.05). The modeling method of exercise combined hitting used in this study was proved to be effective by histopathology; warm acupuncture can improve the pathological and inflammatory state of local muscle fiber in myofascial pain trigger of rat, promote local microvascular formation and maturation, and help the trigger point local muscle fiber repair.

Skeletal muscle fibrosis: an overview.

Extracellular matrix (ECM) is an essential component of skeletal muscle. It provides a framework structure that holds myofibers and blood capillaries and nerves supplying the muscle. In addition, it has a principal role in force transmission, maintenance and repair of muscle fibers. Excessive accumulation of ECM components, especially collagens, either due to excessive ECM production, alteration in ECM-degrading activities, or a combination of both is defined as fibrosis. Skeletal muscle fibrosis impairs muscle function, negatively affects muscle regeneration after injury and increases muscle susceptibility to re-injury, therefore, it is considered a major cause of muscle weakness. Fibrosis of skeletal muscle is a hallmark of muscular dystrophies, aging and severe muscle injuries. Thus, a better understanding of the mechanisms of muscle fibrosis will help to advance our knowledge of the events that occur in dystrophic muscle diseases and develop innovative anti-fibrotic therapies to reverse fibrosis in such pathologic conditions. This paper explores an overview of the process of muscle fibrosis, as well as different murine models for studying fibrosis in skeletal muscles. In addition, factors regulating fibrosis and strategies to inhibit muscle fibrosis are discussed.

Co-delivery of a laminin-111 supplemented hyaluronic acid based hydrogel with minced muscle graft in the treatment of volumetric muscle loss injury.

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.

Safety and toxicological evaluation of a novel, fermented, peptide-enriched, hydrolyzed swine placenta extract powder

Placenta is an important organ that connects the developing fetus to allow nutrient uptake, antibody provisions and gas exchange via the blood supply of the mother. We developed a novel, standardized, stable, water-soluble, peptide-enriched hydrolyzed, Horus fermented placenta powder (HFPEP) from healthy, pathogen-free, swine placenta. Earlier studies demonstrated that HFPEP significantly improves physical fatigue, hepatic functions and repair of muscle fibers. We examined the broad safety of HFPEP in various toxicology models in Good Laboratory Practices-approved laboratories. The acute oral toxicity study was conducted in female Sprague-Dawley rats, and the acute oral LD50 was found to be greater than 5000 mg/kg body weight. Ames’ bacterial reverse mutation assay was conducted to determine the ability of HFPEP to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of a metabolic activation system (S9) at the doses of 50, 15, 4.5, 1.35 and 0.41 mg/ml. No mutagenic potential was observed. Mutagenic potential was also evaluated using in vivo micronucleus test, and no mutagenic potential of HFPEP was observed. Repeated dose 28-d oral toxicity study was performed in male and female rats with 14-d recovery period at the dose levels of 250, 500 or 1000 mg/kg. No abnormal clinical signs or toxicity were detected. No observed adverse effect level of HFPEP was found to be greater than 1000 mg/kg body weight. These studies affirm that HFPEP has broad spectrum safety for human consumption.

Preliminary time‐course study of antiinflammatory macrophage infiltration in crush‐injured skeletal muscle

Muscle damage induces massive macrophage infiltration of the injury site, in which activated pro-inflammatory and anti-inflammatory phenotypes (currently classified as M1 and M2, respectively) have been documented as distinct functional populations predominant at different times after the conventional acute injury by intramuscular injection of snake venoms (cardiotoxin, notexin) or chemicals (bupivacaine hydrochloride, barium chloride). The present study employed a muscle-crush injury model that may better reflect the physiologic damage and repair processes initiated by contusing a gastrocnemius muscle in the lower hind-limb of adult mice with hemostat forceps, and examined the time-course invasion of M1 and M2 macrophages during muscle regeneration by immunocytochemistry of CD197 and CD206 marker proteins. CD197-positive M1 macrophages were observed exclusively at 1-4 days after crush followed by the alternative prevalence of CD206-positive M2 at 7 days of myogenic differentiation, characterized by increasing levels of myogenin messenger RNA expression. Preliminary PCR analysis showed that M2 may produce hepatocyte growth factor (HGF) in culture, providing additional benefit to understanding that M2 populations actively promote regenerative myogenesis (muscle fiber repair) and moto-neuritogenesis (re-attachment of motoneuron terminals onto damaged fibers) through their time-specific infiltration and release of growth factor at the injury site early in muscle regeneration.

Signaling Mechanisms in Mammalian Myoblast Fusion

Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion.

Ultrasound assessment of asymmetric hypertrophy of the rectus abdominis muscle and prevalence of associated injury in professional tennis players

To assess rectus abdominis (RA) thickness and injury prevalence using ultrasound in a group of professional tennis players. Observations with regard to muscle fiber repair is described. We likewise studied the potential link between RA volume asymmetry and the risk of muscle strain. The degree of asymmetry between the different RA slices was assessed using ultrasound in 61 professional tennis players. The history of RA injury in these tennis players was likewise studied, taking into account the following factors: dominant vs non-dominant arm, history of RA strains, duration thereof and number of recurrences. Ultrasound examination was performed with an 8- to 12-MHz linear multi-frequency transducer. Ultrasound revealed the presence of fibrous scar tissue in the RA muscle in 18 cases (29.5%). In all instances, the lesion was located in the RA on the side of the nondominant arm. In 16 of the cases, the lesion was infra-umbilical and L2 was affected in two cases. The mean maximum width of the fibrous repair tissue was 9 mm (range 5-16). The mean distance between the umbilicus and the scar was 5.8 cm (range 2.9-11.4). Statistical study of the ultrasound measurements obtained for the different slices revealed statistically significant differences between the different depths and according to arm dominance. In the series studied, the prevalence of RA muscle lesion in professional tennis players was 29.5%. Asymmetric hypertrophy of the RA muscle appears to constitute a risk factor for suffering an injury in this location.

Deubiquitinating enzyme A20 negatively regulates NF‐κB signaling in skeletal muscle inmdxmice

Duchenne muscular dystrophy (DMD) is caused by the lack of a functional dystrophin protein that results in muscle fiber membrane disruption and, ultimately, degeneration. Regeneration of muscle fibers fails progressively, and muscle tissue is replaced with connective tissue. As a result, DMD causes progressive limb muscle weakness and cardiac and respiratory failure. The absence of dystrophin from muscle fibers triggers the chronic activation of the nuclear factor of kappa B (NF-κB). Chronic activation of NF-κB in muscle leads to infiltration of macrophages, up-regulation of the ubiquitin-proteosome system, and down-regulation of the helix-loop-helix muscle regulatory factor, MyoD. These processes, triggered by NF-κB activation, promote muscle degeneration and failure of muscle regeneration. A20 (TNFAIP3) is a critical negative regulator of NF-κB. In this study, we characterize the role of A20 in regulating NF-κB activation in skeletal muscle, identifying a novel role in muscle regeneration. A20 is highly expressed in regenerating muscle fibers, and knockdown of A20 impairs muscle differentiation in vitro, which suggests that A20 expression is critically important for regeneration of dystrophic muscle tissue. Furthermore, down-regulation of the classic pathway of NF-κB activation is associated with up-regulation of the alternate pathway in regenerating muscle fibers, suggesting a mechanism by which A20 promotes muscle regeneration. These results demonstrate the important role of A20 in muscle fiber repair and suggest the potential of A20 as a therapeutic target to ameliorate the pathology and clinical symptoms of DMD.

Expression and Functional Roles of Angiopoietin-2 in Skeletal Muscles

BackgroundAngiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed.Methodology/Principal FindingsPrimary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H2O2, but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation.Conclusion/SignificanceSkeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair.

Impaired muscle regeneration and myoblast differentiation in mice with a muscle‐specific KO of IGF‐IR

IGF-I and its receptor IGF-IR are seen as critical effectors of muscle hypertrophy, a notion recently questioned. Using MKR transgenic mice that express a dominant negative IGF-IR only in skeletal muscle, we have examined the role of the IGF-IR signaling in differentiation and repair of muscle fibers after damage-induced muscle regeneration. This process is impaired in MKR muscle, with incomplete regeneration, persistence of infiltrating cells and sustained expression of differentiation markers. Analysis of MKR and WT muscle-derived progenitor stem cells and myoblasts showed twice as many such cells in MKR muscle and an incomplete in vitro differentiation, that is, despite similar levels of myogenin expression, the level of fusion of MKR myoblasts was significantly reduced in comparison to WT myoblasts. These data show IGF-IR signaling is not only required at early hyperplasia stages of muscle differentiation, but also for late stages of myofiber maturation and hypertrophy.