Markers Of Muscle Regeneration Research Articles

Asperosaponin VI facilitates the regeneration of skeletal muscle injury by suppressing GSK‐3β‐mediated cell apoptosis

AbstractAsperosaponin VI (ASA VI) is a bioactive triterpenoid saponin extracted from Diptychus roots, of Diptyl, and has previously shown protective functions in rheumatoid arthritis and sepsis. This study investigates the effects and molecular mechanisms of ASA VI on skeletal muscle regeneration in a cardiotoxin (CTX)‐induced skeletal muscle injury mouse model. Mice were subjected to CTX‐induced injury in the tibialis anterior and C2C12 myotubes were treated with CTX. Muscle fiber histology was analyzed at 7 and 14 days postinjury. Apoptosis and autophagy‐related protein expression were evaluated t s by Western blot, and muscle regeneration markers were quantified by quantitative polymerase chain reaction. Docking studies, cell viability assessments, and glycogen synthase kinase‐3β (GSK‐3β) activation analyses were performed to elucidate the mechanism. ASA VI was observed to improve muscle interstitial fibrosis, remodeling, and performance in CTX‐treated mice, thereby increased skeletal muscle size, weight, and locomotion. Furthermore, ASA VI modulated the expression of apoptosis and autophagy‐related proteins through GSK‐3β inhibition and activated the transcription of regeneration genes. Our results suggest that ASA VI mitigates skeletal muscle injury by modulating apoptosis and autophagy via GSK‐3β signaling and promotes regeneration, thus presenting a probable therapeutic agent for skeletal muscle injury.

Stem cell biotherapy: A new remedy for Trichinella spiralis-induced inflammatory myopathy.

Trichinella spiralis (T. spiralis)-induced myopathy is an inflammatory myopathy that is difficult to treat unless the parasite is combated in its early intestinal phase before it reaches the muscles. This study aimed to evaluate the effect of local mesenchymal stem cell (MSC) therapy on T. spiralis-induced inflammatory myopathy in rats. Rats were divided into four groups: Group 1 (non-infected non-treated group); Group 2 (infected non-treated group); Group 3 (infected albendazole (ABZ)-treated group); and Group 4 (infected MSC-treated group). Their muscle status was assessed physiologically with the righting reflex and electromyography (EMG), parasitologically with the total muscle larval count, histopathologically with hematoxylin and eosin and Mallory's trichrome stains, as well as immunohistochemically for myogenin as a marker of muscle regeneration. Additionally, serum muscle enzymes creatine kinase (CK) and lactate dehydrogenase (LDH), as well as muscle matrix metalloproteinases MMP1 and MMP9, were assayed. Finally, the immunological response was assessed by measuring the levels of the muscle inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), and interleukin-4 (IL-4). Our findings revealed that MSC therapy markedly improved muscle EMG and righting reflex, as well as the histopathological appearance of the muscles, reduced inflammatory cellular infiltrates, and increased myogenin immunostaining. It also reduced serum CK and LDH levels, as well as muscle INF-γ, TNF-α, IL-4, MMP1, and MMP9 levels. However, it had no effect on the total muscle larval count. Accordingly, due to its anti-inflammatory properties and muscle-regenerative effect, MSC therapy could be a promising new remedy for T. spiralis-induced myopathy.

Effects of heart failure and diabetes on invasive biomarkers of skeletal muscle wasting

Abstract Funding Acknowledgements Type of funding sources: None. Background Muscle wasting is common in patients with diabetes mellitus (DM) and chronic heart failure (HF), which exacerbates cachexia and poor quality of life. However, a gap in clinical translation remains as the mechanisms underlying muscle wasting in DM and HF remain poorly validated, with most evidence assumed from experimental small-animal models. Purpose Validate if markers of muscle wasting (e.g., autophagy, proteolysis, myogenesis) were correlated to muscle phenotype and clinical indices in humans with DM and HF. Method Male patients with DM (n=4), HF (n=4; NYHA=2), DMHF (n=7; NYHA=2) were included. DM was classified as a HbA1c level ≥48mmol/mol and HF classified as a left ventricular ejection fraction (LVEF) ≤40%, with patients on optimised medication. Muscle biopsies of pectoralis major were excised during routine pacemaker surgery and frozen at -80°C for both histological and gene expression analysis. Muscle samples were sectioned, stained, and imaged for fibre cross-sectional area and fibre type. RT-PCR gene expression was conducted for markers of muscle regeneration (myod, myogenin), protein degradation (myostatin and autophagy-related LC3B, ATG7, CTSL), and anabolic signalling (IGF-1). Statistical analysis included ANOVAs and correlation analysis to identify relationships between variables. Values of p<0.05 were accepted as significant. Results Groups (DM, HF, DMHF) were not different (P>0.05) in relation to age (79±9, 72±8, 72±10 years) or BMI (24.5±3.6, 28.5±4.2, 29.5±6.0) respectively. Fibre size or fibre type were not different (P>0.05) between groups. In line with this, gene expression for markers of myogenesis and anabolism as well as myostatin were not different (P>0.05) between groups, but a trend in downregulation of the proteolytic autophagy-related genes (LC3B, ATG7, CTSL) in DMHF vs DM or HF was found on average by 21, 36, and 30% respectively. Autophagy-related genes LC3B, ATG7, CTSL were positively correlated (P<0.05) to type IIa fibre numerical density (R=0.82, 0.86, 0.89) and LVEF (R=0.85, 0.92, 0.83), respectively. Conclusion Our findings indicate that gene expression of autophagy could represent a robust marker of muscle wasting and cardiac dysfunction in humans with DM and HF. Muscle biopsies from patients with DM and HF identified the proteolytic system of autophagy, important for cellular homeostasis, may be inhibited and this was correlated to fibre phenotype and LVEF. However, the process of muscle wasting in DM and HF within the clinical setting may not necessarily reflect those reported in animal models given various other markers were not changed.

Combined application of chondroitinase ABC and photobiomodulation with low-intensity laser on the anal sphincter repair in rabbit

BackgroundPhotobiomodulation with low-intensity laser (LIL) and chondroitinase ABC (ChABC) can repair damaged muscle tissue, so the aim of this study was to investigate the effect of co-administration of these two factors on anal sphincter repair in rabbits.MethodsMale rabbits were studied in 5 groups (n = 7): Control (intact), sphincterotomy, laser, ChABC and laser + ChABC. 90 days after intervention were evaluated resting and maximum squeeze pressures, number of motor units, collagen amount, markers of muscle regeneration and angiogenesis.ResultsResting pressure in the Laser + ChABC group was higher than the sphincterotomy, laser and ChABC groups (p < 0.0001). Maximum squeeze pressure in the all study groups was higher than sphincterotomy group (p < 0.0001). In the laser + ChABC and ChABC groups, motor unit numbers were more than the sphincterotomy group (p < 0.0001). Collagen content was significantly decreased in the laser (p < 0.0001) and laser + ChABC groups. ACTA1 (p = 0.001) and MHC (p < 0.0001) gene expression in the Laser + ChABC group were more than the laser or ChABC alone. VEGFA (p = 0.009) and Ki67 mRNA expression (p = 0.01) in the Laser + ChABC group were more than the laser group, But vimentin mRNA expression (p < 0.0001) was less than the laser group.ConclusionCo-administration of ChABCs and photobiomodulation with LIL appears to improve the tissue structure and function of the anal sphincter in rabbits more than when used alone.

Expression profiles of SLC39A/ZIP7, ZIP8 and ZIP14 in response to exercise-induced skeletal muscle damage

BackgroundZinc transporters are thought to facilitate the mobilization of zinc (Zn) and the role of Zn as a signaling mediator during cellular events. Little is known about the response of Zn movement and zinc transporters during muscle proliferation and differentiation processes after damage. MethodsAfter rats were subjected to one 90-min session of downhill running to cause muscle damage, the gastrocnemius muscles were harvested to assess the expression of zinc transporters SLC39A/ZIP7, ZIP8, ZIP14 and myogenic regulatory factors at the 0 h, 6 h, 12 h, 1 d, 2 d, 3 d, 1 w and 2 w time points after exercise. ResultsSLC39A/ZIP7, ZIP8 and ZIP14 had translocated to different compartments of the cell following damage, and they exhibited differential expression profiles after eccentric exercise. The results regarding the myogenetic regulators showed that nf-κb was upregulated 2 d after exercise, and STAT3 and Akt1 mRNA levels were mostly expressed 2 w after exercise. The upregulation of phosphatidylinositol 3-kinase, catalytic subunit gamma (pik3cg), erk1 and erk2 mostly occurred at the early stage (6 h or 12 h) after exercise. In addition, we found that zip7, zip8 and zip14 expression was moderately correlated with certain markers of muscle regeneration. ConclusionThe zinc transporters SLC39A/ZIP7, ZIP8 and ZIP14 have differential expression profiles upon eccentric exercise, and they might regulate muscle proliferation or differentiation processes through different cellular pathways after exercise-induced muscle damage.

Inhibition of Estrogen Signaling Reverses Established Inguinal Hernias

Background: An inguinal hernia occurs when an intestinal loop or fat pushes through a weak spot in the lower abdominal muscle (LAM), causing a painful bulge that has the potential to cause bowel obstruction. Despite a high prevalence in men (~25%), non-surgical approaches are not available to treat this disease. We recently found a critical role of estrogen and estrogen receptor alpha (ERα) in inguinal hernia formation. To examine this further, we use a humanized aromatase mouse model (Aromhum) where all of the male mice develop scrotal hernias as a pre-clinical model to test the first pharmacological intervention for inguinal hernias. These mice are utilized because their skeletal muscle tissue contains aromatase and produces estradiol (E2), which acts via ERα in the LAM stromal fibroblasts and leads to fibrosis and muscle atrophy. Hypothesis: E2-ERα modulation can inhibit and reverse the formation of inguinal hernias in Aromhum mice by reducing LAM fibrosis and atrophy. Results: We tested three types of treatments to inhibit E2-ERα signaling: letrozole, fulvestrant, and raloxifene. Letrozole, an aromatase inhibitor, was shown to inhibit hernia formation and reversed small (150-175 mm2) scrotal hernias (n = 10-15/group, p<0.0001). The LAM tissues also showed a reduction in fibrosis (n = 5-8/group, p = 0.0004) and a concurrent increase in myofiber cross-sectional area (n = 5-8, p=0.0356) compared to placebo-treated mice. Similarly, fulvestrant and raloxifene, E2-ERα antagonists, also inhibited hernia formation (n = 10-15/group). Most interestingly, both drugs reversed large and severe hernias (>200 mm2, n = 10-15/group), accompanied by a decrease in muscle fibrosis and increase in myofiber cross-sectional area (ongoing study, n = 10-11, p<0.0001) compared to placebo mice. The drug-treated mice had lower expression of pro-fibrotic genes such as Mmp3, Emb, Spon2, Timp1, and Tgfb1 in the LAM tissues compared to placebo-treated LAM. Furthermore, we analyzed the differences in extracellular matrix producing genes and muscle regeneration markers between the placebo and drug-treated muscle tissues. Conclusion: We find that inhibition of the E2-ERα signaling pathway can reverse mild or severe inguinal hernias. Successful treatment is accompanied by decreased skeletal muscle fibrosis and reversal of myocyte atrophy. These interventions are promising non-surgical treatment options for patients suffering from severe and recurrent inguinal hernias.

(-)-Epicatechin induces mitochondrial biogenesis and markers of muscle regeneration in adults with Becker muscular dystrophy.

IntroductionWe conducted an open‐label study to examine the effects of the flavonoid (−)‐epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD).MethodsSeven participants received (−)‐epicatechin 50 mg twice per day for 8 weeks. Pre‐ and postprocedures included biceps brachii biopsy to assess muscle structure and growth‐relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests.ResultsWestern blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5′‐adenosine monophosphate–activated protein kinase). Peroxisome proliferator‐activated receptor gamma coactivator‐1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae‐associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor‐5, myoblast determination protein 1, myocyte enhancer factor‐2, and structure‐associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states.Discussion(−)‐Epicatechin, an exercise mimetic, appears to have short‐term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.

Prolonged Immobilization Exacerbates the Loss of Muscle Mass and Function Induced by Cancer-Associated Cachexia through Enhanced Proteolysis in Mice.

We hypothesized that in mice with lung cancer (LC)-induced cachexia, periods of immobilization of the hindlimb (7 and 15 days) may further aggravate the process of muscle mass loss and function. Mice were divided into seven groups (n = 10/group): (1) non-immobilized control mice, (2) 7-day unloaded mice (7-day I), (3) 15-day unloaded mice (15-day I), (4) 21-day LC-cachexia group (LC 21-days), (5) 30-day LC-cachexia group (LC 30-days), (6) 21-day LC-cachexia group besides 7 days of unloading (LC 21-days + 7-day I), (7) 30-day LC-cachexia group besides 15 days of unloading (LC 30-days + 15-day I). Physiological parameters, body weight, muscle and tumor weights, phenotype and morphometry, muscle damage (including troponin I), proteolytic and autophagy markers, and muscle regeneration markers were identified in gastrocnemius muscle. In LC-induced cachexia mice exposed to hindlimb unloading, gastrocnemius weight, limb strength, fast-twitch myofiber cross-sectional area, and muscle regeneration markers significantly decreased, while tumor weight and area, muscle damage (troponin), and proteolytic and autophagy markers increased. In gastrocnemius of cancer-cachectic mice exposed to unloading, severe muscle atrophy and impaired function was observed along with increased muscle proteolysis and autophagy, muscle damage, and impaired muscle regeneration.

Drp1 knockdown induces severe muscle atrophy and remodelling, mitochondrial dysfunction, autophagy impairment and denervation.

The maintenance of optimal mitochondrial content and function is critical for muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in skeletal muscle health remains unclear. Here we report knocking down Drp1 (a protein regulating mitochondrial fission) for 4months in adult mouse skeletal muscle resulted in severe muscle atrophy (40-50%). Drp1 knockdown also led to a reduction in ADP-stimulated respiration, an increase in markers of impaired autophagy and increased muscle regeneration, denervation, fibrosis and oxidative stress. Our data indicate that Drp1 is crucial for the maintenance of normal mitochondrial function and that Drp1 depletion severely impairs muscle health. Mitochondria play central roles in skeletal muscle physiology, including energy supply, regulation of energy-sensitive signalling pathways, reactive oxygen species production/signalling, calcium homeostasis and the regulation of apoptosis. The maintenance of optimal mitochondrial content and function is therefore critical for muscle cells. Mitochondria are now well known as highly dynamic organelles, able to change their morphology through fusion and fission processes. Solid experimental evidence indicates that mitochondrial dynamics play key roles in mitochondrial quality control, and alteration in the expression of proteins regulating mitochondrial dynamics have been reported in many conditions associated with muscle atrophy and wasting. However, the exact role that mitochondrial fission plays in skeletal muscle health remains unclear. To address this issue, we investigated the impact of Drp1 (a protein regulating mitochondrial fission) knockdown, introduced via intramuscular injection of adeno-associated virus (AAV) on adult mouse skeletal muscle. Knocking down Drp1 for 4months resulted in very severe muscle atrophy (40-50%). Drp1 knockdown also led to a reduction in ADP-stimulated respiration and increases in markers of muscle regeneration, denervation, fibrosis, oxidative stress and impaired autophagy. Our findings indicate that Drp1 is essential for the maintenance of normal mitochondrial function and that Drp1 suppression severely impairs muscle health.

Satellite Cells and Markers of Muscle Regeneration during Unloading and Reloading: Effects of Treatment with Resveratrol and Curcumin.

We hypothesized that treatment with pharmacological agents known to increase sirtuin-1 activity (resveratrol and curcumin) may enhance muscle regeneration. In limb muscles of mice (C57BL/6J, 10 weeks) exposed to reloading for seven days following a seven-day period of hindlimb immobilization with/without curcumin or resveratrol treatment, progenitor muscle cell numbers (FACS), satellite cell subtypes (histology), early and late muscle regeneration markers, phenotype and morphometry, sirtuin-1 activity and content, and muscle function were assessed. Treatment with either resveratrol or curcumin in immobilized muscles elicited a significant improvement in numbers of progenitor, activated, quiescent, and total counts of muscle satellite cells, compared to non-treated animals. Treatment with either resveratrol or curcumin in reloaded muscles compared to non-treated mice induced a significant improvement in the CSA of both hybrid (curcumin) and fast-twitch fibers (resveratrol), sirtuin-1 activity (curcumin), sirtuin-1 content (resveratrol), and counts of progenitor muscle cells (resveratrol). Treatment with the pharmacological agents resveratrol and curcumin enhanced the numbers of satellite cells (muscle progenitor, quiescent, activated, and total satellite cells) in the unloaded limb muscles but not in the reloaded muscles. These findings have potential clinical implications as treatment with these phenolic compounds would predominantly be indicated during disuse muscle atrophy to enhance the muscle regeneration process.

Effect of BCAA Supplementation on Muscle Mass, Muscle Quality and Molecular Markers of Muscle Regeneration in CLD Patients

Effect of BCAA Supplementation on Muscle Mass, Muscle Quality and Molecular Markers of Muscle Regeneration in CLD Patients

Adipose tissue protects against sepsis-induced muscle weakness in mice: from lipolysis to ketones

BackgroundICU-acquired weakness is a debilitating consequence of prolonged critical illness that is associated with poor outcome. Recently, premorbid obesity has been shown to protect against such illness-induced muscle wasting and weakness. Here, we hypothesized that this protection was due to increased lipid and ketone availability.MethodsIn a centrally catheterized, fluid-resuscitated, antibiotic-treated mouse model of prolonged sepsis, we compared markers of lipolysis and fatty acid oxidation in lean and obese septic mice (n = 117). Next, we compared markers of muscle wasting and weakness in septic obese wild-type and adipose tissue-specific ATGL knockout (AAKO) mice (n = 73), in lean septic mice receiving either intravenous infusion of lipids or standard parenteral nutrition (PN) (n = 70), and in lean septic mice receiving standard PN supplemented with either the ketone body 3-hydroxybutyrate or isocaloric glucose (n = 49).ResultsObese septic mice had more pronounced lipolysis (p ≤ 0.05), peripheral fatty acid oxidation (p ≤ 0.05), and ketogenesis (p ≤ 0.05) than lean mice. Blocking lipolysis in obese septic mice caused severely reduced muscle mass (32% loss vs. 15% in wild-type, p < 0.001) and specific maximal muscle force (59% loss vs. 0% in wild-type; p < 0.001). In contrast, intravenous infusion of lipids in lean septic mice maintained specific maximal muscle force up to healthy control levels (p = 0.6), whereas this was reduced with 28% in septic mice receiving standard PN (p = 0.006). Muscle mass was evenly reduced with 29% in both lean septic groups (p < 0.001). Lipid administration enhanced fatty acid oxidation (p ≤ 0.05) and ketogenesis (p < 0.001), but caused unfavorable liver steatosis (p = 0.01) and a deranged lipid profile (p ≤ 0.01). Supplementation of standard PN with 3-hydroxybutyrate also attenuated specific maximal muscle force up to healthy control levels (p = 0.1), but loss of muscle mass could not be prevented (25% loss in both septic groups; p < 0.001). Importantly, this intervention improved muscle regeneration markers (p ≤ 0.05) without the unfavorable side effects seen with lipid infusion.ConclusionsObesity-induced muscle protection during sepsis is partly mediated by elevated mobilization and metabolism of endogenous fatty acids. Furthermore, increased availability of ketone bodies, either through ketogenesis or through parenteral infusion, appears to protect against sepsis-induced muscle weakness also in the lean.

Myositis Autoantigen Expression Correlates With Muscle Regeneration but Not Autoantibody Specificity.

Although more than a dozen myositis-specific autoantibodies (MSAs) have been identified, most patients with myositis are positive for a single MSA. The specific overexpression of a given myositis autoantigen in myositis muscle has been proposed as initiating and/or propagating autoimmunity against that particular autoantigen. The present study was undertaken to test this hypothesis. In order to quantify autoantigen RNA expression, RNA sequencing was performed on muscle biopsy samples from control subjects, MSA-positive patients with myositis, regenerating mouse muscles, and cultured human muscle cells. Muscle biopsy samples were available from 20 control subjects and 106 patients with autoantibodies recognizing hydroxymethylglutaryl-coenzyme A reductase (n = 40), signal recognition particles (n = 9), Jo-1 (n = 18), nuclear matrix protein 2 (n = 12), Mi-2 (n = 11), transcription intermediary factor 1γ (n = 11), or melanoma differentiation-associated protein 5 (n = 5). The increased expression of a given autoantigen in myositis muscle was not associated with autoantibodies recognizing that autoantigen (all q > 0.05). In biopsy specimens from both myositis muscle and regenerating mouse muscles, autoantigen expression correlated directly with the expression of muscle regeneration markers and correlated inversely with the expression of genes encoding mature muscle proteins. Myositis autoantigens were also expressed at high levels in cultured human muscle cells. Most myositis autoantigens are highly expressed during muscle regeneration, which may relate to the propagation of autoimmunity. However, factors other than overexpression of specific autoantigens are likely to govern the development of unique autoantibodies in individual patients with myositis.

Fast and slow myosin as markers of muscle regeneration in mangled extremities: a pilot study.

Mangled extremities were classically managed by amputation. But over the past few decades, with the advancement in surgical techniques, an increased number of limb salvages have been possible. As muscles usually get damaged in such grievous injuries, a thorough understanding of muscle regeneration may give a better insight into muscle healing in these injuries. Muscles are composed of slow and fast fibers which can be represented by slow and fast myosin, respectively. There are some animal studies which reported differential regeneration of slow and fast muscle fibers during muscle healing. We conducted this pilot study to find out whether the same holds true for muscle healing in mangled extremities also. This pilot study is designed in 15 patients with lower limb mangled extremities presenting to trauma center of PGIMER, Chandigarh, who were operated within 24h of injury to see whether muscle healing in mangled extremities follows the same pattern. Biopsies were taken during initial surgery conducted within 24h of injury and on the 7th day of injury when patient was posted again for secondary wound closure procedure or revision amputation. The biopsy samples were subjected to histopathological and immunohistochemistry examination using antibodies against fast and slow myosin. We found that the regenerating muscle fibers in the biopsy sample taken on the 7th day of injury showed only slow muscle fibers with the absence of fast muscle fibers when compared with the initial biopsy results showing differential regeneration of slow muscle fibers.

OR20-6 Ketones and Sepsis-Induced Muscle Weakness: Signal or Fuel for Protection?

Background: Septic patients often develop muscle weakness, contributing to ICU morbidity and mortality. We recently observed that obese septic mice and patients do not develop such weakness, explained by increased lipolysis and hepatic conversion of fatty acids into the ketone bodies. Furthermore, in lean septic mice, supplementing nutrition with 3-hydroxybutyrate (3HB) reproduced the protection against muscle weakness seen with obesity. The aim of the current study was to investigate underlying mechanisms of the 3HB-protection. Methods: We performed a mouse study (n=49) in a centrally-catheterized fluid-resuscitated model of prolonged (5 days) abdominal sepsis (cecal ligation and puncture). Lean septic mice received standard parenteral nutrition (PN) supplemented with either D,L-3HB (5 mg/kg/day) or isocaloric glucose (gluc). Healthy pair-fed mice served as controls. The use of 3HB as energy substrate in the muscle was assessed by quantifiying protein, triglyceride and glycogen content and markers of muscle ketolysis, proteolysis and fatty acid oxidation (FAO). Signaling function of 3HB was assessed by markers of muscle autophagy activation, inflammation and regeneration. Results: Although 3HB infusion abated sepsis-induced upregulation of ubiquitin-proteasome system marker Fxbo32 (p=0.002), loss of muscle mass was not prevented (p=0.8). Sepsis reduced triglyceride content (p≤0.01) equally in PN+gluc and PN+3HB mice (p=0.9), but did not affect markers of muscle FAO (Cd36, Cpt1b, Acadl, Hadha mRNA) in PN+gluc and PN+3HB mice (p≥0.2). Muscle glycogen content was unaltered by sepsis (p=0.1), and the illness-induced decrease in plasma glucose concentrations at day 5 (p≤0.004) was similar in PN+gluc and PN+3HB mice (p=1). Gene expression of the ketolysis enzyme Oxct1 was lower in PN+3HB compared to PN+gluc septic mice and healthy controls (p≤0.05). Also, ketone body transporter Mct1 mRNA was elevated by sepsis (p≤0.01) but attenuated by 3HB (p=0.05) compared to glucose. Sepsis-induced elevation of inflammation markers (Tnfα, IL-1β, Nlrp3 mRNA levels; p≥0.4) and autophagy (Atg5, Atg7 mRNA; p≥0.4; LC3 II/I, p-ULK1/ULK1 protein; p≥0.4) were not countered by 3HB infusion compared to glucose. However, PN+3HB septic mice did show enhanced markers of muscle regeneration (Myod1, Myf5 and Myog mRNA; p≤0.04) compared to PN+gluc septic mice and healthy controls. Also, sepsis-induced elevation of expression of Hdac4, an inhibitor of myoblast differentiation stimulator Mef2c, was reduced by 3HB (p=0.03) and resulted in increased Mef2c mRNA levels (p=0.01) compared to glucose. Conclusion: In conclusion, the observed protection against sepsis-induced muscle weakness with 3HB infusion coincided with enhanced markers of muscle regeneration, but not with changes in substrate handling. Supplemented 3HB during sepsis thus acted as a signaling molecule rather than as an energy source.

The effect of maternal high fat diet on offspring muscle regeneration

BackgroundSkeletal muscle fibres are post‐mitotic cells. As such, they exclusively rely on regenerative processes to restore muscle structure and function when damage has occurred through direct trauma or everyday tasks. Skeletal muscle quality relies on its development during the pre‐natal period. Foetal muscle development is a highly plastic process that is influenced by the intrauterine environment. Maternal behaviour, such as diet or exercise, can alter this environment. Using a maternal high fat diet model, we fed C57Bl/6 female mice with a low or high fat diet prior to and during gestation and lactation. The offspring were then switched to a low or high fat diet. We assessed whether maternal diet hindered offspring muscle regeneration after injury, and whether post‐natal diet could reverse these effects.ResultsAdiposity levels were higher in C57Bl/6 female mice consuming a high fat diet. There was no difference in offspring weight born to lean or obese dams at weaning; however, in adulthood, offspring born to obese dams were smaller than their control counterparts regardless of post‐natal diet. Markers of muscle regeneration including Pax7, MyoG, Myf5 and MRF4 were downregulated in offspring born to an obese mother or fed a high‐fat diet when compared to offspring experiencing normal growth.ConclusionAn obesogenic maternal diet negatively affects offspring muscle growth and markers of muscle regeneration. These effects could not be offset by offspring diet, highlighting the importance of the pre‐natal environment in muscle development and function.Support or Funding InformationAustralian Research Council (DE150100538)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electrically‐stimulated acupuncture improves muscle function and increases renal blood flow through exosomes‐carried miR‐181

Introductionour previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) can prevent muscle atrophy by increasing muscle protein anabolism in mouse models of chronic kidney disease, diabetes and denervation. Previous other studies have found that Acu/LFES improves renal blood flow and GFR in nephropathy patients and animals. We hypothesized that Acu/LFES benefits both skeletal muscle and kidney possibly through exosome carried microRNA.MethodsNormal C57BL6 mice were awake without any anesthesia and appeared to be comfortable throughout the Acu/LFES. Acupuncture points selected were according to the WHO Standard Acupuncture guidelines. The mice were treated with Acu/LFES (anode: Yang Ling Quan, GB34 and cathode: Zu San Li, ST36) daily for 15 days. Renal plasma flow was measured by p‐Aminohippuric acid infusion. Glomerular filtration rate was determined using inulin infusion and measured with a FIT‐GFR kit. Exosomes were isolated by serial centrifugations. A miR deep sequencing assay and qPCR were used to identify microRNA expression in exosomes.ResultsSkeletal muscle grip function and muscle regeneration markers were increased after Acu/LFES treatment. Acu/LFES increased muscle cross‐sectional area in normal mice vs. sham Acu‐LFES mice. The abundance of Pax7, MyoD, myogenin and embryo myosin heavy chain was significantly increased by Acu‐LFES in soleus, extensor digitorum longus (EDL) and gastrocnemius muscle. The number of central nuclei was increased in Acu‐LFES treated muscle fibers. Interestingly, PAH clearance was increased by 45% in the mice after 15 minutes of muscle Acu/LFES versus mice with sham Acu/LFES (sham 6.1 ± 1.3 vs. Acu/LFES 8.9 ± 2.7 ml/min/g BW, P = 0.009, n=8/group). Inulin clearance was increased 39.8% (sham 65.9 ± 16.3 vs. Acu/LFES108.9 ± 19.7 ul/min/BW, P = 0.014). We found that Acu/LFES increases serum exosome abundance. When exosome secretion was blocked using GW4869, the Acu/LFES‐induced increase in renal blood flow and GFR were limited. To identify how exosomes regulate renal blood flow, we performed microRNA deep sequencing of exosomes isolated from mouse serum and found that 34 microRNAs were altered by Acu/LFES. Notably, miR‐181d‐5p, a microRNA that is involved in regulating translation of renin, was significantly increased in the serum exosomes of Acu/LFES treated mice. Using a luciferase reporter assay, we demonstrated that miR‐181 directly inhibits angiotensinogen, which provides potential evidence of Acu/LFES regulation of renal blood flow.ConclusionsAcu/LFES not only improves muscle function, but also increases miR‐181 in serum exosomes and increases renal blood flow, suggesting that miR181 may regulate renal blood flow possibly by influencing the renin‐angiotensin system. This study provides new insights about the mechanism(s) of muscle‐organ cross talk through exosome‐delivered microRNA.Support or Funding InformationNIH R01 AR060268 and AHA 17IBDG33780000This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regenerative biomarkers for Duchenne muscular dystrophy.

Skeletal muscle has an extraordinary capacity to regenerate after injury and trauma. The muscle repair mechanism is a complex process orchestrated by multiple steps. In neuromuscular disorders such as Duchenne muscular dystrophy (DMD), the pathological consequences of the lack of dystrophin and the loss of the dystrophin-associated protein complex are dramatic, with a progressive cascade of events, such as continual influx of inflammation, repeated cycles of degeneration and impaired regeneration. Thus, muscle regeneration is a hallmark of the disease and careful monitoring of regenerative processes with robust markers should provide useful information to the field. Since decades, several indices of regeneration such as centronucleation and fibre size have been commonly used. In the present review, we discuss the impaired regenerative process in DMD, the common and new indices of regeneration and their associated methodologies. We notably highlight the regenerative marker embryonic myosin as a robust indicator of muscle regeneration. We also describe new quantitative methodologies offering the possibility of using a panel of translational regenerative biomarkers to obtain a more complete view of the regeneration processes. Upregulation of utrophin, an autosomal and functional paralogue of dystrophin, is one of the most promising therapeutic strategies as it targets the primary cause of the disease and is applicable to all DMD patients regardless their genetic defects. As utrophin is a regeneration associated protein increased in dystrophic muscle, we discuss the correlation of utrophin levels after drug treatment with regeneration markers. The recent advances in technologies and complementary markers of muscle regeneration described in this review, provide an unprecedented opportunity to develop more robust utrophin DMD based strategies for all DMD patients.

Embryonic myosin is a regeneration marker to monitor utrophin-based therapies for DMD.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. Constitutive utrophin expression, a structural and functional paralogue of dystrophin, can successfully prevent the dystrophic pathology in the dystrophin-deficient mdx mouse model. In dystrophic muscles, utrophin is increased as part of the repair process and localized at the sarcolemma of regenerating myofibers. The presence of developmental myosin such as embryonic myosin (MyHC-emb) and neonatal represents a useful marker of muscle regeneration and a meaningful indicator of muscle damage, which correlates with the clinical severity of milder Becker muscular dystrophy and DMD patients. In the present study, we demonstrate that MyHC-emb is a robust marker of regeneration at different ages and in different skeletal muscles. We also evaluate the correlation between utrophin, dystrophin and MyHC-emb in wild-type (wt) and regenerating dystrophic muscles. Restoration of dystrophin significantly reduced MyHC-emb levels. Similarly, overexpression of utrophin in the transgenic mdx-Fiona mice reduced the number of MyHC-emb positive fibers to wt level, prevented the regenerative process and rescued the muscle function. In contrast, the absence of utrophin in the dystrophin-deficient double-knockout mice resulted in a higher MyHC-emb content and in a more severe dystrophic pathophysiology than in mdx mice. These data illustrate the importance of monitoring utrophin and MyHC-emb levels in the preclinical evaluation of therapies and provide translational support for the use of developmental myosin as a disease biomarker in DMD clinical trials.

Role of accelerated aging in limb muscle wasting of patients with COPD.

PurposeSkeletal muscle wasting is an independent predictor of health-related quality of life and survival in patients with COPD, but the complexity of molecular mechanisms associated with this process has not been fully elucidated. We aimed to determine whether an impaired ability to repair DNA damage contributes to muscle wasting and the accelerated aging phenotype in patients with COPD.Patients and methodsThe levels of phosphorylated H2AX (γH2AX), a molecule that promotes DNA repair, were assessed in vastus lateralis biopsies from 10 COPD patients with low fat-free mass index (FFMI; COPDL), 10 with preserved FFMI and 10 age- and gender-matched healthy controls. A panel of selected markers for cellular aging processes (CDKN2A/p16ink4a, SIRT1, SIRT6, and telomere length) were also assessed. Markers of oxidative stress and cell damage and a panel of pro-inflammatory and anti-inflammatory cytokines were evaluated. Markers of muscle regeneration and apoptosis were also measured.ResultsWe observed a decrease in γH2AX expression in COPDL, which occurred in association with a tendency to increase in CDKN2A/p16ink4a, and a significant decrease in SIRT1 and SIRT6 protein levels. Cellular damage and muscle inflammatory markers were also increased in COPDL.ConclusionThese data are in keeping with an accelerated aging phenotype as a result of impaired DNA repair and dysregulation of cellular homeostasis in the muscle of COPDL. These data indicate cellular degeneration via stress-induced premature senescence and associated inflammatory responses abetted by the senescence-associated secretory phenotype and reflect an increased expression of markers of oxidative stress and inflammation.