l-arginine modulates inflammation and muscle regulatory genes after a single session of resistance exercise in rats.

We investigated the skeletal muscle adaptation to l-arginine supplementation prior to a single session of resistance exercise (RE) during the early phase of muscle repair. Wistar rats were randomly assigned into non-exercised (Control), RE plus vehicle (RE); RE plus l-arginine (RE+L-arg) and RE plus aminoguanidine (RE+AG) groups. Animals received four doses of either vehicle (0.9% NaCl), l-arg (1 g/b.w.), or AG (iNOS inhibitor) (50 mg/b.w.). The animals performed a single RE session until the concentric failure (ladder climbing; 80% overload) and the skeletal muscles were harvested at 0, 8, 24, and 48 hours post-RE. The RE resulted in increased neutrophil infiltrate (24 hours post-RE) (3621 vs 11852; P<.0001) associated with enhanced TNF-α (819.49 vs 357.02; P<.005) and IL-6 (3.84 vs 1.08; P<.0001). Prior, l-arginine supplementation attenuates neutrophil infiltration (5622; P<.0001), and also TNF-α (506.01; P<.05) and IL-6 (2.51, P<.05) levels. AG pretreatment mediated an inhibition of iNOS levels similar to levels found in RE group. RE animals displayed increased of atrogin-1 (1.9 fold) and MuRF-1 (3.2 fold) mRNA levels, reversed by l-arg supplementation [atrogin-1 (0.6 fold; P<.001); MuRF-1 (0.8-fold; P<.001)] at 24 hours post-RE. MyoD up-regulated levels were restricted to l-arg treated animals at 24 hours (2.8 vs 1.5 fold; P<.005) and 48 hours post-RE (2.4 vs 1.1 fold; P<.001). AG pretreatment reversed these processes at 24 hours [atrogin-1 (2.1 fold; P<.0001); MuRF-1 (2.5 fold; P<.0001); MyoD (1.4 fold)]. l-arginine supplementation seems to attenuate the resolution of RE-induced muscle inflammation and up-regulates MyoD expression during the early phase of muscle repair.

Biomimetic Conductive Hydrogel Scaffolds with Anisotropy and Electrical Stimulation for In Vivo Skeletal Muscle Reconstruction.

The nature of the hydrogel scaffold mimicking extracellular matrix plays a crucial role in tissue engineering like skeletal muscle repair. Herein, an anisotropic and conductive hydrogel scaffold is fabricated using gelatin methacryloyl (GelMA) as the matrix hydrogel and silver nanowire (AgNW) as the conductive dopant, through a directional freezing technique for muscle defect repair. The scaffold has an anisotropic structure composed of a directional longitudinal section and a honeycomb cross-section, with high mechanical strength of 10.5 kPa and excellent conductivity of 0.26 S m-1 . These properties are similar to native muscle extracellular matrix (ECM) and allow for cell orientation under the guidance of contact cues and electrical stimulation synergistically. In vitro experiments show that the scaffold's oriented structure combined with electrical stimulation results in enhanced myotube formation, with a length of up to 863 µm and an orientation rate of 81%. Furthermore, the electrically stimulated scaffold displays a promoted muscle reconstruction ability when transplanted into rats with muscle defects, achieving a muscle mass and strength restoration ratio of 95% and 99%, respectively, compared to normal levels. These findings suggest that the scaffold has great potential in muscle repair applications.

Effect of exercise-induced Neutrophil maturation on skeletal muscle repair in vitro.

Neutrophils as first line defender initiate a cascade of healing process immediately after muscle injury. At muscle injury site, neutrophils remove damaged muscle fibers and recruit other immune cells and these functions show in mature neutrophils. In the previous study, physical exercise can mediate neutrophils' functional changes such as phagocytosis and chemotaxis, though there is no research on how exercise-induced neutrophils contribute the muscle regeneration. In this present study, we investigated the maturation of neutrophils after 4 weeks of mouse treadmill exercise and assessed wound healing assay to evaluate whether treatment with exercise-activated neutrophils is effective for skeletal muscle repair in vitro. In the exercise group, significantly higher mRNA levels of maturation markers compared to the sedentary group and exercise-activated neutrophils improved wound healing of mouse muscle cells. To confirm at the human cell level, based on the well-known fact that exercise increases circulating cortisol levels, neutrophil-like cells were treated with dexamethasone (dHL60 + dex) as exercise mimetics. dHL60 + dex had significantly higher mRNA levels of neutrophil maturation marker and improved wound healing of human skeletal muscle cells compared to the control. These findings suggest that exercise affects neutrophil maturation and that exercise-induced neutrophils contribute to skeletal muscle repair in vitro.

Nanotopographical Cues Tune the Therapeutic Potential of Extracellular Vesicles for the Treatment of Aged Skeletal Muscle Injuries.

Skeletal muscle regeneration relies on the tightly temporally regulated lineage progression of muscle stem/progenitor cells (MPCs) from activation to proliferation and, finally, differentiation. However, with aging, MPC lineage progression is disrupted and delayed, ultimately causing impaired muscle regeneration. Extracellular vesicles (EVs) have attracted broad attention as next-generation therapeutics for promoting tissue regeneration. As a next step toward clinical translation, strategies to manipulate EV effects on downstream cellular targets are needed. Here, we developed an engineering strategy to tune the therapeutic potential of EVs using nanotopographical cues. We found that EVs released by young MPCs cultured on flat substrates (fEVs) promoted the proliferation of aged MPCs while EVs released by MPCs cultured on nanogratings (nEVs) promoted myogenic differentiation. We then employed a bioengineered 3D muscle aging model to optimize the administration protocol and test the therapeutic potential of fEVs and nEVs in a high-throughput manner. We found that the sequential administration first of fEVs during the phase of MPC proliferative expansion (i.e., 1 day after injury) followed by nEV administration at the stage of MPC differentiation (i.e., 3 days after injury) enhanced aged muscle regeneration to a significantly greater extent than fEVs and nEVs delivered either in isolation or mixed. The beneficial effects of the sequential EV treatment strategy were further validated in vivo, as evidenced by increased myofiber size and improved functional recovery. Collectively, our study demonstrates the ability of topographical cues to tune EV therapeutic potential and highlights the importance of optimizing the EV administration strategy to accelerate aged skeletal muscle regeneration.

Regenerating Skeletal Muscle Compensates for the Impaired Macrophage Functions Leading to Normal Muscle Repair in Retinol Saturase Null Mice.

Skeletal muscle repair is initiated by local inflammation and involves the engulfment of dead cells (efferocytosis) by infiltrating macrophages at the injury site. Macrophages orchestrate the whole repair program, and efferocytosis is a key event not only for cell clearance but also for triggering the timed polarization of the inflammatory phenotype of macrophages into the healing one. While pro-inflammatory cytokines produced by the inflammatory macrophages induce satellite cell proliferation and differentiation into myoblasts, healing macrophages initiate the resolution of inflammation, angiogenesis, and extracellular matrix formation and drive myoblast fusion and myotube growth. Therefore, improper efferocytosis results in impaired muscle repair. Retinol saturase (RetSat) initiates the formation of various dihydroretinoids, a group of vitamin A derivatives that regulate transcription by activating retinoid receptors. Previous studies from our laboratory have shown that RetSat-null macrophages produce less milk fat globule-epidermal growth factor-factor-8 (MFG-E8), lack neuropeptide Y expression, and are characterized by impaired efferocytosis. Here, we investigated skeletal muscle repair in the tibialis anterior muscle of RetSat-null mice following cardiotoxin injury. Our data presented here demonstrate that, unexpectedly, several cell types participating in skeletal muscle regeneration compensate for the impaired macrophage functions, resulting in normal muscle repair in the RetSat-null mice.

Loss of Ptpn11 (Shp2) drives satellite cells into quiescence.

The equilibrium between proliferation and quiescence of myogenic progenitor and stem cells is tightly regulated to ensure appropriate skeletal muscle growth and repair. The non-receptor tyrosine phosphatase Ptpn11 (Shp2) is an important transducer of growth factor and cytokine signals. Here we combined complex genetic analyses, biochemical studies and pharmacological interference to demonstrate a central role of Ptpn11 in postnatal myogenesis of mice. Loss of Ptpn11 drove muscle stem cells out of the proliferative and into a resting state during muscle growth. This Ptpn11 function was observed in postnatal but not fetal myogenic stem cells. Furthermore, muscle repair was severely perturbed when Ptpn11 was ablated in stem cells due to a deficit in stem cell proliferation and survival. Our data demonstrate a molecular difference in the control of cell cycle withdrawal in fetal and postnatal myogenic stem cells, and assign to Ptpn11 signaling a key function in satellite cell activity.