Skeletal Muscle Injury Research Articles

The role of the cyclooxygenase-2 pathway in tissue ischemia and revascularization following skeletal muscle injury induced by bothropic snake venom

Bothrops asper venom (Bav) contains metalloproteinases that disrupt the microvascular system, impairing muscle tissue regeneration after injury. This study investigated the impact of the cyclooxygenase-2 (COX-2) pathway on vascular injury and revascularization in muscle injuries induced by Bav. Mice were injected with Bav into the gastrocnemius muscle and treated with lumiracoxib, a selective COX-2 inhibitor, 30 min, 2 days, and 6 days post-Bav injection. Muscle tissue was analyzed at 24 h, 7 days, and 21 days post-injection. A decrease in COX-2 expression at 24 h post-Bav injection indicated significant necrosis and tissue loss. Both Bav injection and lumiracoxib treatment influenced the decrease of prostaglandin (PG)D2 and PGE2 production. Seven and 21 days post-Bav injections, COX-2 expression increased, along with PGDs levels unaffected by lumiracoxib, indicating that the other isoform COX-1 pathway could contribute to the release of PGs. Bav/lumiracoxib treated animals presented exacerbated limb ischemia, implying that COX-2-derived prostaglandins preserve vessel integrity. CD31, an angiogenesis marker, initially (24 h) decreased post-Bav injection but increased at 7 and 21 days in Bav/lumiracoxib mice, suggesting a down-modulatory role for COX-2-derived prostaglandins in early angiogenesis and tissue regeneration. Vascular endothelial growth factor (VEGF) production rose 7 days post-Bav injection, supporting its role in angiogenesis. Previous treatment with lumiracoxib promoted release of VEGF levels 21 days post-Bav injury showing that the inhibition of COX-2 pathway in the early stage of revascularization stimulates the neovascularization regulated by elevated release of VEGF. Similarly, metalloproteinases (MMPs), such as MMP-9, MMP-10, and MMP-13, crucial for vascular remodeling, were elevated 21 days after Bav/lumiracoxib treatment. In conclusion, the COX-2 pathway is essential to decrease the high grade of ischemia caused by acute injury induced by Bav. However, the decrease of activity in the COX-2 pathway in the first stages of revascularization contributes to the elevated production of key pro-angiogenic mediators that up-regulate the restoration of microvasculature and blood flow in muscle tissue injured by botropic venoms.

Effect of Porphyromonas gingivalis Infection on Healing of Skeletal Muscle Injury: An In Vivo Study

Background/Objectives: Porphyromonas gingivalis infection has been associated with various systemic diseases and may cause delayed healing of muscle injury. However, the relationship between muscle injury healing and P. gingivalis infection remains unclear. Our hypothesis was that P. gingivalis infection delays the healing of muscle injuries. Methods: Fifty-six 8-week-old male Wistar rats were randomly divided into two groups: sonicated P. gingivalis was intraperitoneally administered in one group (PG group), whereas saline was administered in the other group (CO group). Skeletal muscle injury was induced via cardiotoxin injections in all animals. The cross-sectional area of regenerating muscle cells was evaluated by haematoxylin–eosin staining, and the degree of muscle fibrosis was evaluated by Masson’s trichrome staining. The expression of paired box protein (Pax7) and myoblast determination protein (MyoD) and the identified stages of myocyte regeneration were analysed by immunohistochemical staining. Motion analysis was performed during walking. Results: The cross-sectional area of muscle cells was significantly smaller in the PG group on days 7 and 14 post-injury than in the CO group. The Pax7+/MyoD− ratio was significantly lower in the PG group on day 1 post-injury than in the CO group. Motion analysis of treadmill walking showed that the PG group had a lower minimum calcaneal height on days 3 and 7 post-injury than the CO group. Conclusions: This study suggests that administration of sonicated P. gingivalis in rats can delay the healing process of muscle injury. Further research is needed to understand this mechanism of delay of P. gingivalis.

Comparison of rapid test for high-sensitivity troponin I with laboratory high-sensitivity troponin T in patients with acute chest pain - a prospective monocentric study

Abstract Background and purpose High-sensitivity troponin (hs-Tn) assays are recommended for routine clinical use for diagnosis of acute coronary syndrome (ACS). However, laboratory hs-TnT can be elevated due to non-cardiac conditions, such as pulmonary embolism, skeletal muscle injury or chronic kidney disease. The aim of our study was to compare the diagnostic accuracy of the bed-side rapid hs-troponin I (hs-TnI) assay with hs-TnT measured by routine laboratory. Methods This prospective monocentric study was conducted at a tertiary hospital outpatient cardiac unit with an emergency clinic for patients with acute symptoms. A total of 129 patients were included between May 2021 and August 2022. Hs-TnI was measured via a bed-side test from whole blood directly in the emergency room, while hs-TnT was measured from plasma through the routine laboratory. Definite ACS was confirmed by coronary angiography. Patients were compared by grouping them into definitive diagnosis of ACS or no ACS. Hs-TnI as well as hs-TnT values were compared for accuracy and predictive values. Routine clinical parameters and comorbidities were documented and compared as well. Agreement between hs-TnI and hs-TnT was assessed by likelihood ratios and Bland Altman plot. Results Overall, 129 patients (65.1% male, 61.8 ± 15.6 years) with acute chest pain were analysed. Median hs-TnI was 7.0 ng/L (IQR: 2.7-15.8), while median hs-TnT was 14 ng/L (IQR: 7.0-31.0). Results for TnI were available faster (average: 1:14 h ± 0:54) compared to TnT. Coronary angiography confirmed ACS in 17 patients (13.2%) displaying significant coronary stenoses requiring intervention. The relative risk of ACS patients to have an elevated hs-TnI result was 6.59 compared to that of 2.29 for the hs-TnT test, meaning that a positive hs-TnI test was a better classifier for validated ACS than that of hs-TnT. Hs-TnI exhibited equivalent negative predictive value to hs-TnT (99%) for definite ACS diagnosis, but had comparatively higher positive predictive value (50.0 vs. 25.8%). Bland Altman plot of hs-TnI versus hs-TnT showed an average difference of -4.6 ng/L (95% CI: -12.4 to 3.2) between the tests. In 39 patients with chronic kidney disease (minimum stage 3a CKD) median hs-TnT (27.0 ng/L, IQR: 16.0-55.0) values were higher than hs-TnI (11.2 ng/L, IQR: 7.5-30.7). Conclusion Bed-side hs-TnI is comparable to hs-TnT with a high degree of agreement. Measurement of troponin I using the rapid bed-side hs-TnI assay possesses a shorter blood-draw-to-result-time than routine troponin T measurement.

Evaluating Muscle Damage Biomarkers in Adolescent Athletes: Implications for Public Health in Tacna, Peru—2023

The success of an athlete is influenced by various factors, including nutritional status, physical condition, training level, recovery, psychological factors, and genetics. Studies on athletes often take a preventive approach to evaluate muscle fatigue caused by intense physical exercise, which can impair performance, particularly in soccer players. This study aims to determine the serum levels of biochemical markers of muscle damage in adolescents from sports centers in the city of Tacna. A sample of 326 adolescents who met the inclusion and exclusion criteria was analyzed for muscle damage markers, including CRP, LDH, CK-total, and CK-MB, to assess potential skeletal muscle injury. The results were as follows: for the LDH test, 2.5% had low-level values, 91.4% had values within the normal range, and 6.1% had high values; for the CK-total test, 74.8% were within normal limits, and 25.2% had elevated values; for the CK-MB test, 99.1% had normal values, and 0.9% had elevated values; and for the CRP test, 95.7% were negative, and 4.3% were positive. According to the CK-MB/CK relative index, 7.1% of adolescents were suspected of having muscle damage. This study concludes that there are alterations in the biochemical markers of muscle damage in adolescents from the sports centers in Tacna.

Effects of cannabidiol on AMPKα2 /HIF-1α/BNIP3/NIX signaling pathway in skeletal muscle injury.

Cannabidiol: (CBD) is a non-psychoactive natural active ingredient from cannabis plant, which has many pharmacological effects, including neuroprotection, antiemetic, anti-inflammatory and anti-skeletal muscle injury. However, the mechanism of its effect on skeletal muscle injury still needs further research. In order to seek a scientifically effective way to combat skeletal muscle injury during exercise, we used healthy SD rats to establish an exercise-induced skeletal muscle injury model by treadmill training, and systematically investigated the effects and mechanisms of CBD, a natural compound in the traditional Chinese medicine Cannabis sativa L., on combating skeletal muscle injury during exercise. CBD effectively improved the fracture of skeletal muscle tissue and reduced the degree of inflammatory cell infiltration. Biochemical indexes such as CK, T, Cor, LDH, SOD, MDA, and GSH-Px in serum of rats returned to normal. Combining transcriptome and network analysis results, CBD may play a protective role in exercise-induced skeletal muscle injury through HIF-1 signaling pathway. The experimental results implied that CBD could down-regulate the expression of IL-6, NF-κB, TNF-α, Keap1, AMPKα2, HIF-1α, BNIP3 and NIX, and raised the protein expression of IL-10, Nrf2 and HO-1. These results indicate that the protective effect of CBD on exercise-induced skeletal muscle injury may be related to the inhibition of oxidative stress and inflammation, thus inhibiting skeletal muscle injury through AMPKα2/HIF-1α/BNIP3/NIX signal pathways.

SAT1/ALOX15 Signaling Pathway Is Involved in Ferroptosis After Skeletal Muscle Contusion

Skeletal muscle contusion (SMC) is common in daily life and clinical practice, but the molecular mechanisms underlying SMC healing are unclear. Ferroptosis, a regulated cell death type, has gained attention recently. We observed iron overload in skeletal muscle following contusion through HE and Perls staining. Abnormal iron levels are highly likely to induce ferroptosis. Therefore, we aimed to explore whether iron overload after contusion leads to ferroptosis in skeletal muscle and the underlying mechanisms, which will help us understand the effects of iron abnormalities on skeletal muscle repair. Initially, we searched SMC gene expression profiles from the GEO database and used bioinformatics analysis to reveal ferroptosis occurrence. Then, we identified the gene sat1 plays an important role in this process. We further established a rat SMC model and treated rats with ferroptosis inhibitors (Ferrostatin-1, Deferoxamine). Our findings confirmed iron overload from SMC can lead to ferroptosis in rats. We also demonstrated that SAT1 can regulate ferroptosis by affecting ALOX15. Moreover, we constructed a ferroptosis L6 cell model and found that SAT1 knockdown significantly inhibited ALOX15 expression and reduced cellular lipid peroxidation. In conclusion, these results indicated ferroptosis can occur following SMC, and SAT1, as a key regulator, affects skeletal muscle injury healing by mediating high ALOX15 expression, which in turn regulates lipid peroxidation.

Low serum Metrnl levels are associated with increased risk of sarcopenia in the older adults.

Sarcopenia is a geriatric syndrome characterized by progressive loss of muscle mass and function. Meteorin-like (Metrnl) is a secretory protein that has protective effects on skeletal muscle injury. However, the association of Metrnl level with sarcopenia remains unclear. A total of 772 community-dwelling older adults (median age = 76years), comprising 409 males and 363 females, from both urban and rural areas were enrolled. Serum Metrnl was measured by enzyme-linked immunosorbent assay. Appendicular skeletal muscle mass index (ASMI), grip strength, and gait speed were measured for the assessment of sarcopenia. We found that serum Metrnl levels were lower in patients with sarcopenia [median (IQR) = 180.1 (151.3-220.3) pg/mL] than older adults without sarcopenia [211.9 (163.2-270.0) pg/mL, P < 0.001]. Receiver-operating characteristic curve analysis showed that the optimal cut-off value of serum Metrnl level that predicted sarcopenia was 197.2pg/mL with a sensitivity of 59.2% and a specificity of 63.8% (AUC = 0.63, 95% CI = 0.59-0.67, P < 0.001). Multivariate logistic regression analyses showed that lower serum Metrnl level (< 197.2pg/mL) was significantly associated with increased risk of sarcopenia (adjusted OR = 2.358, 2.36, 95% CI = 1.528-3.685, P < 0.001). Moreover, serum Metrnl concentration was positively correlated with the components of sarcopenia including ASMI (r = 0.135, P < 0.001), grip strength (r = 0.102, P = 0.005), and gait speed (r = 0.106, P = 0.003). Taken together, our findings demonstrate that low serum Metrnl level is correlated with increased risk of sarcopenia in the older adults.

Diagnostic and evaluative efficiency of 68Ga-FAPI-04 in skeletal muscle injury

BackgroundSkeletal muscles are vital for daily function, yet assessing their injuries remain challenging. We aimed to elucidate the effectiveness of 68Ga-FAPI-04 in evaluating skeletal muscle remodeling.ResultsC2C12 cells were subjected to graded H2O2 stimulation in vitro, revealing an initial rise and subsequent decline in fibroblast activation protein (FAP) expression as H2O2 concentration increased. In vivo, a murine triceps surae injury model was created using various solutions to simulate normal repair, mild repair failure, and severe repair failure. Assessments were conducted on days 1, 3, 7, and 14 using PET, MRI, and ultrasound. With 68Ga-FAPI-04, the normal and mild repair failure groups showed significantly higher SUVmax and T/B ratios on day 1 compared to the severe repair failure group. These values gradually decreased in the normal repair group, becoming negligible after day 7. MRI results for the normal repair group showed low to moderate signal intensity by day 7. A clinical study retrospectively evaluated post-hip arthroplasty patient images at intervals of 1 month, 2–3 months, 5–6 months, and over 7 months. In these patients, 18F-FDG SUVmax and volume remained relatively stable over time, while 68Ga-FAPI-04 SUVmax initially increased, then decreased, with a consistent reduction in volume.ConclusionIn skeletal muscle injuries, FAP demonstrates a distinctive mechanism of action, and 68Ga-FAPI-04, in comparison to other tests, more precisely captures alterations in lesion site uptake intensity and volume.Trial registrationTrial registration: ChiCTR2000041204. Registered 22 December 2020, https://www.chictr.org.cn/showproj.html?proj=66211

Characterization of Age-Dependent Changes in Skeletal Muscle Repair and Regeneration Using a Mouse Model of Acute Muscle Injury.

Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging andmuscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.

C2C12 myoblasts differentiate into myofibroblasts via the TGF-β1 signaling pathway mediated by Fibulin2

Myoblast cells play a critical role in the regeneration of skeletal muscle following injury. It has been reported that local elevation of transforming growth factor-β1(TGF-β1) after skeletal muscle injury induces differentiation of myoblast cells into myofibroblasts.However, the mechanisms underlying this differentiation process remain incompletely understood. In this study, we found that Fibulin2 expression significantly increases in myoblast cells in response to TGF-β1 stimulation.Elevated Fibulin2 levels enhance the expression of fibrotic markers, mediated through phosphorylation of Smad2.Conversely, downregulation of Fibulin2 in myoblast cells inhibits the upregulation of fibrotic markers induced by TGF-β1 stimulation.Extracellular secretion of Fibulin2 activates the TGF-β1-Smad2 pathway, thereby promoting the upregulation of fibrotic markers.Hence, Fibulin2 and TGF-β1 form a positive feedback loop that facilitates differentiation of myoblast cells into myofibroblasts.

Mesenchymal stromal cell transplantation ameliorates fibrosis and microRNA dysregulation in skeletal muscle ischemia.

Peripheral arterial disease (PAD) is associated with lower-extremity muscle wasting. Hallmark features of PAD-associated skeletal muscle pathology include loss of skeletal muscle mass, reduced strength and physical performance, increased inflammation, fibrosis, and adipocyte infiltration. At the molecular level, skeletal muscle ischemia has also been associated with gene and microRNA (miRNA) dysregulation. Mesenchymal stromal cells (MSCs) have been shown to enhance muscle regeneration and improve muscle function in various skeletal muscle injuries. This study aimed to evaluate the effects of intramuscularly delivered human umbilical cord-derived MSCs (hUC-MSCs) on skeletal muscle ischemia. Herein, we report an hUC-MSC-mediated amelioration of ischemia-induced skeletal muscle atrophy and function via enhancement of myofiber regeneration, reduction of tissue inflammation, adipocyte accumulation, and tissue fibrosis. These changes were observed in the absence of cell-mediated enhancement of blood flow recovery as measured by laser Doppler imaging. Furthermore, reduced tissue fibrosis in the hUC-MSC-treated group was associated with upregulation of miR-1, miR-133a, and miR-29b and downregulation of targeted pro-fibrotic genes such as Col1a1 and Fn1. Our results support the use of hUC-MSCs as a novel approach to reduce fibrosis and promote skeletal muscle regeneration after ischemic injury in patients with PAD.

Adenosine A2B receptor activation regulates the balance between T helper 17 cells and regulatory T cells, and inhibits regulatory T cells exhaustion in experimental autoimmune myositis.

Idiopathic inflammatory myopathy (IIM) is a systemic autoimmune disease characterized by skeletal muscle involvement. This study aimed to investigate the role of adenosine receptor signalling pathways in the development of experimental autoimmune myositis (EAM). An ecto-5'-nucleotidase (CD73) inhibitor, adenosine receptor agonists, a hypoxia-inducible factor-1α (HIF-1α) inhibitor or a vehicle were administered to control and EAM mice. Murine splenic CD4+ or regulatory T cells (Tregs) were isolated using magnetic beads and subsequently stimulated with an adenosine A2B receptor agonist, a HIF-1α inhibitor, or vehicle in vitro. In cross-sectional studies, we collected 64 serum samples (69% female, 49±9years), 63 peripheral blood samples (70% female, 50±11years), and 34 skeletal muscle samples (71% female, 63±6years) from patients with IIM. Additionally, 35 serum samples and 30 peripheral blood samples were obtained from age- and sex-matched healthy controls, and six quadriceps muscle samples were collected from patients with osteoarthritis to serve as the normal group. Patients with IIM exhibited increased CD73 [dermatomyositis (DM), polymyositis (PM): P<0.01; immune-mediated necrotizing myopathy (IMNM): P<0.0001] and adenosine deaminase (ADA) expression (DM: P<0.001; PM, IMNM: P<0.0001) in the skeletal muscles, and serum ADA levels [56.7 (95% CI: 53.7, 58.7) vs. 198.8 (95% CI: 186.2, 237.3) ng/μL, P<0.0001]. Intervention with a CD73 inhibitor exacerbated (P=0.0461), whereas adenosine receptor agonists (A1: P=0.0009; A2B: P<0.0001; A3: P=0.0001) and the HIF-1α inhibitor (P=0.0044) alleviated skeletal muscle injury in EAM mice. Elevated expression of programmed cell death protein-1 (PD1: P=0.0023) and T-cell immunoglobulin and mucin-domain containing-3 (TIM3: P<0.0001) in skeletal muscles of patients with IIM were correlated with creatine kinase levels (PD1, r=0.7072, P<0.0001; TIM3, r=0.4808, P=0.0046). PD1+CD4+ (r=0.3243, P=0.0115) and PD1+CD8+ (r=0.3959, P=0.0017) T cells were correlated with Myositis Disease Activity Assessment Visual Analogue Scale scores (muscle) in IIM. The exhausted Tregs were identified in the skeletal muscles of patients with IIM. Activation of the A2B adenosine receptor downregulated HIF-1α (protein or mRNA level, P<0.01), resulting in decreased T helper cell 17 (Th17) (13.58% vs. 5.43%, P=0.0201) and phosphorylated-signal transducer and activator of transcription 3 (p-STAT3)+ Th17 (16.32% vs. 6.73%, P=0.0029), decreased exhausted Tregs (PD1+ Tregs: 53.55% vs. 40.28%, P=0.0005; TIM3+ Tregs: 3.93% vs. 3.11%, P=0.0029), and increased Tregs (0.45% vs. 2.89%, P=0.0006) in EAM mice. The exhausted T cells may be pathogenic in IIM,andthe activation of adenosine A2B receptor signalling pathway can regulate Th17/Treg balance and inhibit Tregsexhaustion, thereby slowing EAM disease progression.

Effects of radial extracorporeal shock wave with different frequencies on acute skeletal muscle injury in rabbits

To study the efficacy and possible mechanisms of radial extracorporeal shock wave (rESW) with different frequencies for the treatment of acute skeletal muscle injury in rabbits, 48 rabbits of acute injured biceps femoris were randomly divided into 4 groups. Except for the control group, the other groups were treated by rESW with 5 Hz, 10 Hz and 15 Hz, respectively. The injury symptom index scores (ISISs) in the rESW group were significantly lower than those in the control group, with the lowest in the 10 Hz rESW group. Histomorphological features demonstrated a decrease in mononuclear cells and an increase in new myocytes across all groups, with the rESW group showing the most significant changes. The concentrations of PGE2 and IL-1β were significantly lower in all rESW groups by ELISA compared to the control group. Additionally, the 10 Hz group had lower concentrations than the 5 Hz and 15 Hz group. Compared with the control group, MyoD of the rESW groups was significantly increased, and the expression level of the 10 Hz group was higher than that of the other groups. In conclusion, rESW with 5 Hz, 10 Hz and 15 Hz take certain curative effects on acute biceps femoris injury in rabbits, and the 10 Hz rESW takes advantage over 5 Hz and 15 Hz rESW.

Metformin and intermittent fasting mitigate high fat-fructose diet-induced liver and skeletal muscle injury through upregulation of mitophagy genes in rats

BackgroundHigh fat-fructose diet is a proinflammatory diet that increases risk of hepatocytes and myocytes steatosis and fibrosis. Finding anti-inflammatory strategies to fight these harmful effects is paid attention to nowadays. This study compared the effects of two widely anti-inflammatory interventions—metformin and intermittent fasting on myocytes and hepatocyte injury induced by proinflammatory diet and tracking possible underlying mechanisms. In this work, rats fed high fat-fructose diet were subdivided into untreated group, treated by metformin, and/or intermittent fasting.ResultsMetformin (300 mg/kg/day) and intermittent fasting (3 days/week) specially their combination for 4 weeks showed significant improvement in insulin resistance, lipid profile, antioxidants (p < 0.05), as well as enhanced hepatocytes and myocytes repair and reduced collagen deposition through upregulation of mitophagy-related genes: PINK1, PARKIN, LAMP2, and PPAR-α (p < 0.05).ConclusionsIntermittent fasting has beneficial metabolic and molecular therapeutic effects against proinflammatory diet-induced injury. Their results are like those of metformin sparing its adverse effects. Their combination showed additional effects against diet-induced myocytes and hepatocyte injury by upregulation of mitophagy-related genes without the need of increasing the dose of metformin.Graphic abstract

Nutritional approaches applied to recovery of skeletal muscle injury immobilization: a review of nutrition aid for sport trauma.

Skeletal muscle (SM) injuries are a very common occurrence among athletes during training and/or competitive periods, resulting in time loss and absence from sports. Of several existing interventions to improve SM recovery, the nutritional approach has emerged as integral part to improve the physiological response and maintain the body composition to promote a rapid and safe return to play. Growing evidence supports that the physiological response to SM injuries results in a number of changes, suggesting the relevance of a personalized nutritional approach, according to the degree of immobilization, SM mass loss, strength and function. Therefore, we aimed to review the current evidence of the nutritional approach applied to SM injuries recovery in athletes, highlighting energy intake, use the main nutrients and elements for the SM recovery, such as proteins, carbohydrates, vitamins and omega-3 fatty acids. Currently, evidence from injured athletes is scarce, and so specific guidelines cannot be established, calling from greater research into nutritional interventions in SM injury recovery in athletes.

Aerobic Exercise Protects against Cardiotoxin-Induced Skeletal Muscle Injury in a DDAH1-Dependent Manner.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a critical enzyme that regulates nitric oxide (NO) signaling through the degradation of asymmetric dimethylarginine (ADMA). Previous studies have revealed a link between the beneficial effects of aerobic exercise and the upregulation of DDAH1 in bones and hearts. We previously reported that skeletal muscle DDAH1 plays a protective role in cardiotoxin (CTX)-induced skeletal muscle injury and regeneration. To determine the effects of aerobic exercise on CTX-induced skeletal muscle injury and the role of DDAH1 in this process, wild-type (WT) mice and skeletal muscle-specific Ddah1-knockout (Ddah1MKO) mice were subjected to swimming training for 8 weeks and then injected with CTX. In WT mice, swimming training for 8 weeks significantly promoted skeletal muscle regeneration and attenuated inflammation, oxidative stress, and apoptosis in the gastrocnemius (GA) muscle after CTX injection. These phenomena were associated with increases in the protein expression of PAX7, myogenin, MEF2A, eNOS, SOD2, and peroxiredoxin 5 and decreases in iNOS expression in GA muscles. Swimming training also decreased serum ADMA levels and increased serum nitrate/nitrite (NOx) levels and skeletal muscle DDAH1 expression. Interestingly, swimming training in Ddah1MKO mice had no obvious effect on CTX-induced skeletal muscle injury or regeneration and did not repress the CTX-induced inflammatory response, superoxide generation, or apoptosis. In summary, our data suggest that DDAH1 is important for the protective effect of aerobic exercise on skeletal muscle injury and regeneration.

Promoting and accelerating muscle regeneration through cell therapy in a mouse model

ObjectivesSkeletal muscle injuries and disorders are universal clinical challenges with direct and indirect mechanisms and notable residual effects, such as prolonged, intense pain and physical disability. Stem cells, an innovative tool for cell therapy for musculoskeletal disorders, specifically promote skeletal muscle regeneration. This study was aimed at investigating the use of mesenchymal stem cells (MSCs) and their differentiated myocytes as a cell-based therapy to promote regeneration in damaged or diseased skeletal muscle. MethodsBone marrow mesenchymal stem cells (BM-MSCs) were isolated from the bone marrow of adult mice and grown in tissue culture flasks. The BM-MSCs were positive for CD90 and CD105, and negative for CD45 and CD34. These cells were induced with specific differentiation medium in vitro to differentiate into a skeletal muscle cell lineage over 7 days. Skeletal muscle differentiation was characterized according to morphology through hematoxylin and eosin staining, and scanning electron microscopy. Immunostaining for Myf-6, myosin heavy chain (MHC), and desmin—specific factors for skeletal muscle development—was performed to confirm skeletal muscle differentiation. An in vivo study in a muscle injury model was used to evaluate cell therapy based on naïve stem cells and differentiated myocytes. ResultsCultured mouse BM-MSCS were positive for CD90 and CD105, and negative for CD45 and CD34. These cells developed into skeletal muscle with strong skeletal muscle differentiation potential, as confirmed by immunohistochemistry for the markers Myf6, MHC, and desmin. The differentiated myocytes showed better repair enhancement than undifferentiated stem cells after transplantations into a mouse model of skeletal muscle atrophy. ConclusionsMyocytes derived from BM-MSCs may be incorporated into muscular atrophy treatment as a biological strategy for managing skeletal muscle diseases and injuries, thus advancing cell-based clinical treatments.

Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin-B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3-6months) were bred for EC-specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non-injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5days post-injury (dpi); both recovered to uninjured (0dpi) level by 10dpi. At 0dpi, tibialis anterior isometric force of CKO was less than CON. At 10dpi, isometric force was reduced by half in both groups. During intermittent contractions (75Hz, 330mss-1, 120s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. KEY POINTS: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult. To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle. Capillary regeneration was reduced at 5days post-injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups. Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function.

Acute Kidney Injury in Rhabdomyolysis: A 5-Year Children's Hospital Network Study.

Rhabdomyolysis is a skeletal muscle injury that can cause myoglobinuria and acute kidney injury (AKI). Risk factors for AKI in children are not clearly understood with no standardized treatment guidelines for rhabdomyolysis. Our study explores factors associated with AKI and management of pediatric patients with rhabdomyolysis. Medical records from a children's hospital network over a 5-year period were retrospectively reviewed. The results are described with respect to the presence or absence of AKI. Of the 112 patients who met the inclusion criteria, AKI incidence was 7.1% (n = 8), with all affected patients having exertional etiology. The overall mean age was 13.5 years; patients without AKI were younger than patients with AKI (13.3 versus 17; p < 0.001). Using regression models for hypothesis generation, we found that patients with AKI were more likely to be older (OR = 1.44, 95%CI [1.11-2.19]; p = 0.03), have myoglobinuria (OR = 22.98, 95%CI [2.05-432.48]; p = 0.02), and have received intravenous bicarbonate (OR = 16.02, 95%CI [1.44-228.69]; p = 0.03). In our study, AKI was uncommon and associated with older age, myoglobinuria and bicarbonate treatment. Larger, prospective studies are needed to further understand AKI risk factors and optimal management of pediatric rhabdomyolysis.

Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Mice.

Chronic kidney disease (CKD) is associated with various pathologic changes, including elevations in serum phosphate levels (hyperphosphatemia), vascular calcification, and skeletal muscle atrophy. Elevated phosphate can damage vascular smooth muscle cells and cause vascular calcification. Here, we determined whether high phosphate can also affect skeletal muscle cells and whether hyperphosphatemia, in the context of CKD or by itself, is associated with skeletal muscle atrophy. As models of hyperphosphatemia with CKD, we studied mice receiving an adenine-rich diet for 14 weeks and mice with deletion of Collagen 4a3 (Col4a3-/-). As models of hyperphosphatemia without CKD, we analyzed mice receiving a high-phosphate diet for three and six months as well as a genetic model for klotho deficiency (kl/kl). We found that adenine, Col4a3-/-, and kl/kl mice have reduced skeletal muscle mass and function and develop atrophy. Mice on a high-phosphate diet for six months also had lower skeletal muscle mass and function but no significant signs of atrophy, indicating less severe damage compared with the other three models. To determine the potential direct actions of phosphate on skeletal muscle, we cultured primary mouse myotubes in high phosphate concentrations, and we detected the induction of atrophy. We conclude that in experimental mouse models, hyperphosphatemia is sufficient to induce skeletal muscle atrophy and that, among various other factors, elevated phosphate levels might contribute to skeletal muscle injury in CKD.