Mouse Tibialis Anterior Muscle Research Articles

GABA and glycine synaptic release on axotomized motoneuron cell bodies promotes motor axon regeneration.

Motor axon regeneration after traumatic nerve injuries is a slow process that adversely influences patient outcomes because muscle reinnervation delays result in irreversible muscle atrophy and suboptimal axon regeneration. This advocates for investigating methods to accelerate motor axon growth. Electrical nerve stimulation and exercise both enhance motor axon regeneration in rodents and patients, but these interventions cannot always be easily implemented. A roadblock to uncover novel therapeutic approaches based on the effects of activity is the lack of understanding of the synaptic drives responsible for activity-mediated facilitation of axon regeneration. We hypothesized that the relevant excitatory inputs facilitating axon regrowth originate in GABA/glycine synapses which become depolarizing after downregulation of the potassium chloride cotransporter 2 in motoneurons following axotomy. To test this, we injected tetanus toxin (TeTx) in the tibialis anterior (TA) muscle of mice to block the release of GABA/glycine specifically on TA motoneurons. Thereafter, we axotomized all sciatic motoneurons by nerve crush and analyzed the time-courses of muscle reinnervation in TeTx- treated (TA) and untreated (lateral gastrocnemius, LG) motoneurons. Muscle reinnervation was slower in TA motoneurons with blocked GABA/glycine synapses, as measured by recovery of M- responses and anatomical reinnervation of neuromuscular junctions. Post-hoc immunohistochemistry confirmed the removal of the vesicular associated membrane proteins 1 and 2 by TeTx activity, specifically from inhibitory synapses. These proteins are necessary for exocytotic release of neurotransmitters. Therefore, we conclude that GABA/glycine neurotransmission on regenerating motoneurons facilitates axon growth and muscle reinnervation and discuss possible interventions to modulate these inputs on regenerating motoneurons.

Contractile regulation of the glucocorticoid-sensitive transcriptome in young and aged skeletal muscle.

Elevated glucocorticoids alter the skeletal muscle transcriptome to induce a myopathy characterized by muscle atrophy, muscle weakness, and decreased metabolic function. These effects are more likely to occur and be more severe in aged muscles. Resistance exercise can blunt the development of glucocorticoid myopathy in young muscle, but the potential to oppose the signals initiating myopathy in aged muscle is unknown. To answer this, young (4-mo-old) and aged (24-to 25-mo-old) male C57BL/6 mice were randomized to receive either an intraperitoneal (IP) injection of dexamethasone (DEX; 2 mg/kg) or saline as a control. Two hours postinjections, the tibialis anterior (TA) muscles of mice were subjected to unilateral high-force contractions. Muscles were harvested 4 h later. The glucocorticoid- and contraction-sensitive genes were determined by RNA sequencing. The number of glucocorticoid-sensitive genes was similar between young and aged muscle. Contractions opposed changes to more glucocorticoid-sensitive genes in aged muscle, with this outcome primarily occurring when hormone levels were elevated. Glucocorticoid-sensitive gene programs opposed by contractions were primarily related to metabolism in young mice and muscle size regulation and inflammation in aged mice. In silico analysis implied peroxisome proliferator-activated receptor gamma-1 (PPARG) contributed to the contraction-induced opposition of glucocorticoid-sensitive genes in aged muscle. Increasing PPARG expression in the TA of aged mice using adeno-associated virus serotype 9 partially counteracted the glucocorticoid-induced reduction in runt-related transcription factor 1 (Runx1) mRNA content, recapitulating the effects observed by contractions. Overall, these data contribute to our understanding of the contractile regulation of the glucocorticoid transcriptome in aged skeletal muscle.NEW & NOTEWORTHY We establish the extent to which muscle contractions oppose changes to the glucocorticoid-sensitive transcriptome in both young and aged muscle. We also identify peroxisome proliferator-activated receptor gamma (PPARG) as a transcription factor likely contributing to contraction-induced opposition to the glucocorticoid transcriptome in aged muscle. Overall, these data contribute to our understanding of the contractile regulation of the glucocorticoid transcriptome.

Skeletal myocyte-specific knockout of Ptpn1 and Ptpn2 enhances inflammatory response in muscle and increases mortality in polymicrobial sepsis

Abstract Background Critically ill patients often suffer from heart and skeletal muscle atrophy and failure (intensive care unit acquired weakness; ICUAW). Inflammation and sepsis are major risk factors for ICUAW. Critically ill patients with sepsis frequently develop insulin resistance that decreases protein synthesis and increases protein degradation in muscle eventually leading to ICUAW. The link between inflammation and insulin resistance is not well understood, but protein tyrosine phosphatases (PTP), that inhibit insulin signaling via insulin receptor (INSR) dephosphorylation as well as inflammation-associated pathways, are implicated. Hypothesis: We hypothesized that PTP1B and TC-PTP mediate inflammation-induced insulin resistance and muscle atrophy in heart and skeletal muscle. Methods and Results qRT-PCR analyses showed that the proinflammatory cytokines tumor necrosis factor (TNF), interleukin (IL)-1β, and IL-6 caused a ~2-fold increase in Ptpn1/PTP1B and Ptpn2/TC-PTP mRNA expression in C2C12 myocytes as well as in neonatal rat ventricular cardiomyocytes. Inhibition of the IL-6/GP130 pathway by siRNA and inhibitors in myocytes in vitro and skeletal muscle specific gp130 knockout mice in vivo attenuated IL-6-induced Ptpn1/PTP1B and Ptpn2/TC-PTP mRNA expression. CLP-induced polymicrobial sepsis was associated with a significant increase in gene expression and protein content of pro-inflammatory cytokines (Tnf, Il1b, Ifng) and leukocyte markers (CD68, CD11c, F4/80) in muscle of skeletal myocyte specific double knockout (Ptpn1+Ptpn2loxP/loxP, MCK cre, dKO) compared to wildtype (Ptpn1+Ptpn2loxP/loxP) mice in sepsis. This also resulted in a lower survival rate of dKO mice in sepsis (42% WT vs. 23% dKO). Immunohistological analyses revealed centralized nuclei, macrophage/monocyte infiltration and enhanced regeneration (Myh3, Pax7, Ki67) in tibialis anterior muscle of septic dKO mice. A strong activation of the NLRP3-inflammasome, as indicated by cleavage of caspase 1, GSDMD, GSDME and caspase 8, indicative for a pronounced inflammation and activated cell death pathways was observed in muscle of septic dKO mice. Conclusion The proinflammatory cytokine IL-6 increases the expression of Ptpn1 and Ptpn2 and enhances overall PTP activity. Selective inhibition of the IL-6R/GP130 signaling pathway by JAK2- and STAT3-inhibition as well as Gp130 deletion in myocytes in vivo attenuates those effects. Muscle-specific deletion of Ptpn1 and Ptpn2 in skeletal muscle leads to increased mortality, enhanced pro-inflammatory response and induced inflammatory, programmed cell death pathways in sepsis. Our data show that Ptpn1 and Ptpn2 play a key anti-inflammatory role in skeletal muscle.

8381 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

9268 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

9291 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

8670 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

Analysis of potential TAK1/Map3k7 phosphorylation targets in hypertrophy and cachexia models of skeletal muscle.

TGFβ-activated kinase-1 (TAK1) is phosphorylated during both muscle growth and muscle wasting. To understand how this can lead to such opposite effects, we first performed multiplex kinase array of mouse embryonic stem cells with and without stimulation of TAK1 to determine its potential downstream targets. The phosphorylation of these targets was then compared in three different models: hypertrophic longissimus muscle of Texel sheep, tibialis anterior muscle of mice with cancer-induced cachexia and C2C12-derived myofibers, with and without blockade of TAK1 phosphorylation. In both Texel sheep and in cancer-induced cachexia, phosphorylation of both TAK1 and p38 was increased. Whereas p90RSK was increased in Texel sheep but not cachexia and the phosphorylation of HSP27 and total Jnk were increased in cachexia but not Texel. To understand this further, we examined the expression of these proteins in C2C12 cells as they differentiated into myotubes, with and without blockade of TAK1 phosphorylation. In C2C12 cells, decreased phosphorylation of TAK1 leads to reduced phosphorylation of p38, JNK, and HSP27 after 16 h and muscle fiber hypertrophy after 3 days. However, continuous blockade of this pathway leads to muscle fiber failure, suggesting that the timing of TAK1 activation controls the expression of context-dependent targets.

Age-related histone H3.3 accumulation associates with a repressive chromatin in mouse tibialis anterior muscle

The present study aimed to investigate age-related changes in histone variant H3.3 and its role in the aging process of mouse tibialis anterior muscle. H3.3 level significantly increased with age and correlated with H3K27me3 level. Acute exercise successfully upregulated the target gene expression in 8-wk-old mice, whereas no upregulation was noted in 53-wk-old mice. H3K27me3 level was increased at these loci in response to acute exercise in 8-wk-old mice. However, in 53-wk-old mice, H3.3 and H3K27me3 levels were increased at rest and were not affected by acute exercise. Furthermore, forced H3.3 expression in the skeletal muscle of 8-wk-old mice led to a gradual improvement in motor function. The results suggest that age-related H3.3 accumulation induces the formation of repressive chromatin in the mouse tibialis anterior muscle. However, H3.3 accumulation also appears to play a positive role in enhancing skeletal muscle function.

Generation of musculoskeletal cells from human urine epithelium-derived presomitic mesoderm cells

BackgroundNumerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally, we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation.ResultsSelective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice, as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover, UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro.ConclusionsUiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders.

Metabolome in Tibialis and Soleus Muscles in Wild-Type and Pin1 Knockout Mice through High-Resolution Magic Angle Spinning 1H Nuclear Magnetic Resonance Spectroscopy.

Skeletal muscles are heterogenous tissues composed of different myofiber types that can be classified as slow oxidative, fast oxidative, and fast glycolytic which are distinguished on the basis of their contractile and metabolic properties. Improving oxidative metabolism in skeletal muscles can prevent metabolic diseases and plays a protective role against muscle wasting in a number of neuromuscular diseases. Therefore, achieving a detailed understanding of the factors that regulate myofiber metabolic properties might provide new therapeutic opportunities for these diseases. Here, we investigated whether peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is involved in the control of myofiber metabolic behaviors. Indeed, PIN1 controls glucose and lipid metabolism in a number of tissues, and it is also abundant in adult skeletal muscles; however, its role in the control of energy homeostasis in this tissue is still to be defined. To start clarifying this topic, we compared the metabolome of the tibialis anterior muscle (mainly glycolytic) and soleus muscle (oxidative) in wild-type and Pin1 knockout mice with High-Resolution Magic Angle Spinning (HR-MAS) NMR on intact tissues. Our analysis reveals a clear demarcation between the metabolomes in the two types of muscles and allows us to decode a signature able to discriminate the glycolytic versus oxidative muscle phenotype. We also detected some changes in Pin1-depleted muscles that suggest a role for PIN1 in regulating the metabolic phenotype of skeletal muscles.

Maintenance of muscle fiber-type memory in a human-to-mouse muscle xenograft model

INTRODUCTION. Muscle tissue can regenerate through muscle stem cells known as muscle satellite cells (MuSCs). MIME is a technique developed in our laboratory, through which, donor muscle tissue with MuSCs can be implanted into a host muscle to promote donor-cell-derived myogenesis. OBJECTIVE. We studied whether or not MuSCs maintain memory of the type of muscle fibers (fast or slow) with which they were associated. We hypothesized that MuSCs do not maintain memory of the fibers with which they are associated and form slow or fast muscle fibers based on the host environment. METHODS. We tested our hypothesis by implanting donor muscle, which contained mostly slow fibers, into host muscle, which contained mostly fast fibers. Implantation of donor tissue was performed by MIME. Following MIME, we injected a myotoxin (1.2% barium chloride, BaCl2) into the host muscle, which induced concerted degeneration followed by MuSC-dependent regeneration. Specifically, we studied mouse tibialis anterior muscles, which were implanted with segments of human donor tibialis anterior muscle. The human TA is comprised mostly of slow fibers (~60%; i.e., slow myosin heavy chain positive [sMyHC+]). The mouse TA is comprised 100% of fast fibers (i.e., fast myosin heavy chain positive [fMyHC+] fibers). This experimental paradigm is appropriate to ascertain whether or not satellite cells retain memory of the muscle fibers with which they were originally associated. We collected host muscles after 12 weeks of regeneration to ascertain if there was an increase in the percentage of slow muscle fibers. We used antibodies specific to sMyHC and fMyHC to stain frozen TA muscle sections. Host mice were immunodeficient and ubiquitously expressed enhanced green fluorescent protein (NSG-EGFP, The Jackson Laboratory Strain #021937). Since host mouse muscle fibers exhibited green fluorescence, but donor human muscle fibers did not, host and donor muscle fibers could be easily differentiated under fluorescence optics. RESULTS. Quantitative data confirmed that the cluster of human muscle fibers, which regenerated in the host mouse TA muscle had more slow (~50%) than fast (~30%) fibers (T-test, p<0.01, N = 4 TA muscles from 4 different host mice). CONCLUSION. Contrary to our hypothesis, our data suggest that MuSCs do maintain memory of the type of muscle fibers with which they were originally associated. MuSCs give rise to fast or slow muscle fibers based on their donor origin when they are implanted along with their original donor muscle tissue. This work was supported by NIH R03HD091648, Pitch Award from the Alliance for Regenerative Rehabilitation Research and Training (AR3T) from NIH P2CHD086843, and a subcontract from NIH R01AR079884-01 (Peter L. Jones PI) to JAR. We thank the Wayne State University DPT Program for supporting student research and providing research training through coursework and research experiences. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Concerted regulation of skeletal muscle metabolism and contractile properties by the orphan nuclear receptor Nr2f6.

The maintenance of skeletal muscle plasticity upon changes in the environment, nutrient supply, and exercise depends on regulatory mechanisms that couple structural and metabolic adaptations. The mechanisms that interconnect both processes at the transcriptional level remain underexplored. Nr2f6, a nuclear receptor, regulates metabolism and cell differentiation in peripheral tissues. However, its role in the skeletal muscle is still elusive. Here, we aimed to investigate the effects of Nr2f6 modulation on muscle biology in vivo and in vitro. Global RNA-seq was performed in Nr2f6 knockdown C2C12 myocytes (N=4-5). Molecular and metabolic assays and proliferation experiments were performed using stable Nr2f6 knockdown and Nr2f6 overexpression C2C12 cell lines (N=3-6). Nr2f6 content was evaluated in lipid overload models in vitro and in vivo (N=3-6). In vivo experiments included Nr2f6 overexpression in mouse tibialis anterior muscle, followed by gene array transcriptomics and molecular assays (N=4), ex vivo contractility experiments (N=5), and histological analysis (N=7). The conservation of Nr2f6 depletion effects was confirmed in primary skeletal muscle cells of humans and mice. Nr2f6 knockdown upregulated genes associated with muscle differentiation, metabolism, and contraction, while cell cycle-related genes were downregulated. In human skeletal muscle cells, Nr2f6 knockdown significantly increased the expression of myosin heavy chain genes (two-fold to three-fold) and siRNA-mediated depletion of Nr2f6 increased maximal C2C12 myocyte's lipid oxidative capacity by 75% and protected against lipid-induced cell death. Nr2f6 content decreased by 40% in lipid-overloaded myotubes and by 50% in the skeletal muscle of mice fed a high-fat diet. Nr2f6 overexpression in mice resulted in an atrophic and hypoplastic state, characterized by a significant reduction in muscle mass (15%) and myofibre content (18%), followed by an impairment (50%) in force production. These functional phenotypes were accompanied by the establishment of an inflammation-like molecular signature and a decrease in the expression of genes involved in muscle contractility and oxidative metabolism, which was associated with the repression of the uncoupling protein 3 (20%) and PGC-1α (30%) promoters activity following Nr2f6 overexpression in vitro. Additionally, Nr2f6 regulated core components of the cell division machinery, effectively decoupling muscle cell proliferation from differentiation. Our findings reveal a novel role for Nr2f6 as a molecular transducer that plays a crucial role in maintaining the balance between skeletal muscle contractile function and oxidative capacity. These results have significant implications for the development of potential therapeutic strategies for metabolic diseases and myopathies.

Activation of skeletal carbohydrate-response element binding protein (ChREBP)-mediated de novo lipogenesis increases intramuscular fat content in chickens

The intracellular lipids in muscle cells of farm animals play a crucial role in determining the overall intramuscular fat (IMF) content, which has a positive impact on meat quality. However, the mechanisms underlying the deposition of lipids in muscle cells of farm animals are not yet fully understood. The purpose of this study was to determine the roles of carbohydrate-response element binding protein (ChREBP) and fructose in IMF deposition of chickens. For virus-mediated ChREBP overexpression in tibialis anterior (TA) muscle of chickens, seven 5-d-old male yellow-feather chickens were used. At 10 d after virus injection, the chickens were slaughtered to obtain TA muscles for analysis. For fructose administration trial, sixty 9-wk-old male yellow-feather chickens were randomly divided into 2 groups, with 6 replicates per group and 5 chickens per replicate. The chickens were fed either a basal diet or a basal diet supplemented with 10% fructose (purity ≥ 99%). At 4 wk later, the chickens were slaughtered, and breast and thigh muscles were collected for analysis. The results showed that the skeletal ChREBP mRNA levels were positively associated with IMF content in multiple species, including the chickens, pigs, and mice (P < 0.05). ChREBP overexpression increased lipid accumulation in both muscle cells in vitro and the TA muscles of mice and chickens in vivo (P < 0.05), by activation of the de novo lipogenesis (DNL) pathway. Moreover, activation of ChREBP by dietary fructose administration also resulted in increased IMF content in mice and notably chickens (P < 0.05). Furthermore, the lipidomics analysis revealed that ChREBP activation altered the lipid composition of chicken IMF and tented to improve the flavor profile of the meat. In conclusion, this study found that ChREBP plays a pivotal role in mediating the deposition of fat in chicken muscles in response to fructose-rich diets, which provides a novel strategy for improving meat quality in the livestock industry.

Histological and biochemical changes in lymphatic vessels after skeletal muscle injury induced by lengthening contraction in male mice.

Lymphatic vessels are actively involved in the recovery process of inflamed tissues. However, the changes in intramuscular lymphatic vessels during inflammation caused by skeletal muscle injury remain unclear. Therefore, the purpose of this study was to clarify the changes in lymphatic vessels after skeletal muscle injury. The left tibialis anterior muscles of male mice were subjected to lengthening contractions (LC) for inducing skeletal muscle injury, and samples were collected on Days 2, 4, and 7 for examining changes in both the skeletal muscles and intramuscular lymphatic vessels. With hematoxylin-eosin staining, the inflammatory response was observed in myofibers on Days 2 and 4 after LC, whereas regeneration of myofibers was found on Day 7 after LC. The number and area of intramuscular lymphatic vessels analyzed by immunohistochemical staining with an antibody against lymphatic vessel endothelial hyaluronan receptor 1 were significantly increased only on Day 4 after LC. Based on the abovementioned results, intramuscular lymphatic vessels undergo morphological changes such as increase under the state of muscle inflammation. This study demonstrated that the morphology of intramuscular lymphatic vessels undergoes significant changes during the initial recovery phase following skeletal muscle injury.

Human myofiber-enriched aging-induced lncRNA FRAIL1 promotes loss of skeletal muscle function.

The loss of skeletal muscle mass during aging is a significant health concern linked to adverse outcomes in older individuals. Understanding the molecular basis of age-related muscle loss is crucial for developing strategies to combat this debilitating condition. Long noncoding RNAs (lncRNAs) are a largely uncharacterized class of biomolecules that have been implicated in cellular homeostasis and dysfunction across a many tissues and cell types. To identify lncRNAs that might contribute to skeletal muscle aging, we screened for lncRNAs whose expression was altered in vastus lateralis muscle from older compared to young adults. We identified FRAIL1 as an aging-induced lncRNA with high abundance in human skeletal muscle. In healthy young and older adults, skeletal muscle FRAIL1 was increased with age in conjunction with lower muscle function. Forced expression of FRAIL1 in mouse tibialis anterior muscle elicits a dose-dependent reduction in skeletal muscle fiber size that is independent of changes in muscle fiber type. Furthermore, this reduction in muscle size is dependent on an intact region of FRAIL1 that is highly conserved across non-human primates. Unbiased transcriptional and proteomic profiling of the effects of FRAIL1 expression in mouse skeletal muscle revealed widespread changes in mRNA and protein abundance that recapitulate age-related changes in pathways and processes that are known to be altered in aging skeletal muscle. Taken together, these findings shed light on the intricate molecular mechanisms underlying skeletal muscle aging and implicate FRAIL1 in age-related skeletal muscle phenotypes.

Transcriptional dysregulation of autophagy in the muscle of a mouse model of Duchenne muscular dystrophy

It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.

Remedial effects of tilapia skin peptides against dexamethasone-induced muscle atrophy in mice by modulation of AKT/FOXO3a and Sirt1/PGC-1α signaling pathways

Tilapia skin peptides (TSP) possess a range of physiological activities. This study aimed to explore the effects of TSP on Dexamethasone (DEX)-induced muscle atrophy. In vitro, C2C12 myotube myotube diameter and expression levels of the muscle-specific E3 ubiquitin ligases F-box only protein 32 (Atrogin-1) and muscle ring finger protein 1 (MuRF1) challenged with DEX were reversed by TSP. In vivo, DEX was injected subcutaneously to build muscle atrophy model mice. TSP enhanced grip strength, running distance, body lean muscle content, cross-sectional area of the gastrocnemius and tibialis anterior muscles of DEX-induced mice. Moreover, TSP inhibited the expression levels of Atrogin-1 and MuRF1. Mechanically, TSP improved DEX-induced muscle atrophy by regulating the Protein Kinase B α (AKT)/Forkhead box O3 protein (FOXO3a), Sirtuin 1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling pathway, and downstream factors such as nuclear respiratory factor (NRF)1/2 and mitochondrial transcription factor A (TFAM).

Yap/Taz activity is associated with increased expression of phosphoglycerate dehydrogenase that supports myoblast proliferation

In skeletal muscle, the Hippo effector Yap promotes satellite cell, myoblast, and rhabdomyoblast proliferation but prevents myogenic differentiation into multinucleated muscle fibres. We previously noted that Yap drives expression of the first enzyme of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (Phgdh). Here, we examined the regulation and function of Phgdh in satellite cells and myoblasts and found that Phgdh protein increased during satellite cell activation. Analysis of published data reveal that Phgdh mRNA in mouse tibialis anterior muscle was highly expressed at day 3 of regeneration after cardiotoxin injection, when markers of proliferation are also robustly expressed and in the first week of synergist-ablated muscle. Finally, siRNA-mediated knockdown of PHGDH significantly reduced myoblast numbers and the proliferation rate. Collectively, our data suggest that Phgdh is a proliferation-enhancing metabolic enzyme that is induced when quiescent satellite cells become activated.

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model.

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived limb muscles, the orofacial muscles originate from the branchial arches, with exclusive contributions from the cranial neural crest. A recent study has revealed that regeneration is also different in the orofacial muscle group. However, the underlying regulatory mechanism remains to be uncovered. Current skeletal muscle regeneration models mainly focus on the limb and trunk muscle. In this protocol, dry ice was used to induce freezing injury in the mouse masseter muscle and tibialis anterior muscle to create an orofacial muscle fibrosis model. The temporal dynamics of muscle satellite cells and fibro-adipogenic progenitors were different between the two muscles, leading to impaired myofiber regeneration and excessive extracellular matrix deposition. With the help of this model, a deeper investigation into muscle regeneration in the orofacial area could be carried out to develop therapeutic approaches for patients with orofacial diseases.