Nitric oxide (NO), a potent vasodilating agent, plays an essential role in regulation of mitochondrial respiration, and is crucial for proper skeletal muscle function. In skeletal muscle, neuronal NO synthase (nNOS) produces NO and regulates the nitrate/nitrite reductive pathway. nNOS is anchored to the sarcolemma in skeletal muscle by the dystrophin complex. When nNOS dissociates from cell membrane such as in Duchenne muscular dystrophy, nNOS ceases to fulfil its protective role on muscle function and blood flow regulation. We tested the hypothesis that high nitrate diet could enhance NO in skeletal muscle of nNOS knockout (KO) mice. We used gene expression analysis to identify other potential pathways that may be involved to enhance NO production after 7-day high nitrate diet in mice lacking nNOS compared to the wild type (WT) mice. We provided WT mice and nNOS KO mice with normal chow and either standard or high nitrate water (1 g/L nitrate) for seven days. Following the treatment, we collected quadriceps tissues, and isolated the RNA, before performing bulk RNA-sequencing (Illumina). We performed a differential gene expression analysis using DEseq2, then identified pathways related to significantly differentially expressed genes (DEGs) using ShinyGO enrichment analysis. Finally, we validated candidate genes from potential enriched gene pathways using qPCR. Enrichment analysis of DEGs identified 3 potential enriched gene pathways, along with candidate genes associated with NO production and skeletal muscle development and function: (I) muscle system process, (II) cellular response to endogenous stimulus, and (III) tissue development. qPCR validation confirmed that nitrate supplementation alters gene expression of key markers of skeletal muscle development and function, potentially reversing deficits in nNOS KO mice. While nNOS KO control mice showed a downregulation of Ryr1 and Dmd genes compared to WT mice, nitrate supplementation in nNOS KO mice indicated increased expression of these and other candidate genes, including Pax7, Myc, Notch1, and Vangl2. Our findings suggest that dietary nitrate modulates key pathways involved in skeletal muscle maintenance of development in nNOS deficient mice, compared to control mice. Nitrate supplementation may support muscle homeostasis by upregulating gene programs associated with muscle function and provide a potential molecular basis for therapeutic interventions in diseases characterized by nNOS dysfunction.
Exercise intolerance is a hallmark of heart failure with preserved ejection fraction (HFpEF), particularly in its cardiometabolic form, where obesity, metabolic dysfunction, and impaired peripheral oxygen utilization contribute to functional limitation. Skeletal muscle mitochondrial dysfunction is a recognized contributor to this phenotype, yet whether it is shared across muscles with contrasting oxidative profiles, or accompanied by muscle-specific stress responses, has been little explored. We therefore examined gastrocnemius and soleus from male cardiometabolic HFpEF mice using a descriptive and integrative approach. Male C57BL/6J mice received a 60
Sepsis-induced myopathy (SIM) is characterized by skeletal muscle dysfunction associated with mitochondrial calcium overload. The mitochondria-associated endoplasmic reticulum membrane (MAM) plays a crucial role in mediating mitochondrial calcium uptake. Although MFN2 and the IP3R–GRP75–VDAC1 complex have each been implicated in mitochondrial calcium regulation, their respective contributions and potential interplay in the pathogenesis of SIM remain poorly understood. A murine sepsis model was established by cecal ligation and puncture (CLP). Skeletal muscle function was assessed by compound muscle action potential (CMAP) recording and forelimb grip strength testing. Gastrocnemius muscle histology was evaluated by H E and WGA staining. Tibialis anterior muscles were analyzed by immunofluorescence and transmission electron microscopy (TEM) to characterize MAM ultrastructure. Protein expression was quantified by western blotting, and MFN2 mRNA levels were measured by qPCR. Co-immunoprecipitation was performed to assess GRP75–VDAC1 protein interaction. Mitochondrial and cytoplasmic calcium levels were monitored using Rhod-2 AM and Fluo-4 AM, respectively. Sepsis induced skeletal muscle atrophy and dysfunction both in vivo and in vitro. IP3R-dependent calcium transfer at MAM was associated with pathological mitochondrial calcium overload, and pharmacological IP3R inhibition with 2-APB attenuated mitochondrial calcium uptake and ameliorated muscle atrophy. Concurrently, sepsis-induced MFN2 downregulation was associated with reduced ER-mitochondria distance, further promoting mitochondrial calcium overload. AAV-mediated MFN2 overexpression restored ER-mitochondria distance and was associated with reduced VDAC1 protein expression, attenuated GRP75–VDAC1 interaction, collectively contributing to mitigation of mitochondrial calcium overload and amelioration of skeletal muscle atrophy. MFN2 attenuates sepsis-induced mitochondrial calcium overload through two complementary mechanisms: increasing ER-mitochondria distance and modulating GRP75–VDAC1 interaction and IP3R–GRP75–VDAC1 complex assembly. These findings identify MFN2 as a potential regulator of MAM-mediated mitochondrial calcium homeostasis in SIM and suggest it may represent a therapeutic target for sepsis-induced skeletal muscle dysfunction.
Congenital muscular dystrophies (CMDs) are rare, devastating pediatric conditions with no specific treatment or cure. The most common form, LAMA2-related CMD, or LAMA2-CMD (previously known as merosin-deficiency and MDC type 1 A), is caused by myofiber instability from loss of the laminin α2 subunit, a critical extracellular matrix protein in skeletal muscle. Progressive weakness from diffuse skeletal muscle degeneration and fibrosis leads to death in childhood. Animal models significantly enhanced the understanding of LAMA2-CMD and spearheaded innovative rescue strategies. However, limitations of animal models including difficulty recapitulating human muscular dystrophy phenotypes and a lack of validated studies with relevant human tissues have stalled clinical translation. In vitro models of human skeletal muscle exhibiting measurable characteristics of patient disease can significantly improve our ability to study disease mechanisms and further advance therapeutics. Skeletal muscle progenitor cells (SMPCs), mature myoblasts and 3-dimensional (3D) skeletal muscle tissue were derived from induced pluripotent stem cells of a LAMA2-CMD patient and her unaffected parents as controls. The 3D skeletal muscle constructs were embedded in hydrogel with and without co-seeding with human fibroblasts as supporting cells. Cell fusion, tissue remodeling, contractility and attachment were then analyzed. The 3D skeletal muscle model forms into bundles of multinucleated myotubes, with spontaneous and induced contractions mirroring in vivo muscle tissue. During the skeletal muscle progenitor cell differentiation phase, the LAMA2-CMD neuroectoderm clusters were smaller and less complex than control cases. Despite this, SMPCs were robust and purified at equivalent rates from the patient and control lines. With further differentiation to mature myotubes, LAMA2-CMD cells had reduced cellular fusion compared to control cells. In engineered 3D-skeletal muscle tissues anchored to micropillars, the LAMA2-CMD skeletal muscle demonstrated dystrophic features such as poor attachment, reduced remodeling into compact skeletal muscle bundles, and reduced ability to withstand acetylcholine-induced contractions. The LAMA2-CMD skeletal muscle fibers consistently rupture upon contraction. A patient-derived in vitro model of LAMA2-CMD was established that exhibits a dystrophic phenotype closely resembling the human disease. This platform represents a valuable human tissue model to enhance translational research and therapeutic development efforts for CMDs.
We previously reported that resistance trained (T) and untrained (UT) individuals exhibit similar targeted molecular responses in skeletal muscle following two weeks of leg immobilization and eight weeks of subsequent recovery resistance training (RT). The present analysis examined the skeletal muscle transcriptome, microRNA (miR)-ome, and proteome responses in a subset of these participants (N = 8 T and N = 8 UT). Vastus lateralis biopsies were obtained at baseline (PRE), after the 2-wk immobilization protocol (DIS), and after 8-wk of recovery RT (REC-RT). Tissue RNA and protein were subjected to RNA- and miR-sequencing and proteomics, respectively. Analyses included: (i) group and time differential expression (DE) and pathway enrichment for each independent -ome; (ii) multi-omics integration to reveal modules across the -omes; and (iii) leave one out cross-validated linear regression modeling (LOOCV) to reveal key features predictive of atrophy and hypertrophy. Most differentially expressed (DE) RNAs or differentially abundant (DA) proteins were time-driven rather than training status-driven. In all participants, the transcriptome was most responsive to disuse (PRE-to-DIS: 2047 DE mRNAs; 3 DE miRs; 10 DA proteins) while the transcriptome and proteome were both responsive to recovery RT (DIS-to-REC-RT: 446 DE mRNAs; 0 DE miR; 415 DA proteins). Pathways analyses revealed proteostasis and DNA repair with disuse atrophy, whereas striated muscle contraction, transcriptional regulation, and rRNA expression were enriched with recovery RT. Integrative analyses identified SLIT–ROBO signaling as a potentially novel axis in disuse atrophy, driven predominantly by transcriptomic changes, whereas enrichment of striated muscle contraction pathways in recovery RT was supported by coordinated mRNA and protein regulation. Via LOOCV, the 10 top predictors of disuse atrophy were RNAs (8 protein coding) primarily related to mitochondrial function and protein trafficking, while 9 of the top 10 predictors of recovery RT hypertrophy were RNAs (8 protein coding) related to membrane dynamics, ECM/cytoskeletal organization, and translation initiation. This integrative multi-omic approach reiterates that disuse atrophy and recovery RT-induced hypertrophy elicit similar molecular responses in T and UT individuals, highlighting distinct molecular signatures governing skeletal muscle atrophy and recovery hypertrophy independent of training status.
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used by athletes and those who exercise, yet their influence on the molecular responses to exercise remains unclear. Prior studies have often focused on a limited set of molecular pathways, potentially overlooking broader regulator effects of NSAIDs on skeletal muscle signaling. Therefore, we conducted a systems biology study of skeletal muscle biopsies taken before and after exercise, in combination with NSAID consumption, using transcriptomics and metabolomics, to identify differentially enriched pathways and biofunctions. We conducted a randomized, counterbalanced, double-masked, crossover trial (NCT05512013) in which 12 healthy adults ingested ibuprofen (IBU, 800 mg), celecoxib (CEL, 200 mg), flurbiprofen (FLU, 100 mg), or placebo (PLA) before a 10 × 10 bout of plyometric exercise. Skeletal muscle biopsies were collected before NSAID consumption and three hours post-exercise. Whole transcriptome profiling was performed using RNA-seq, and the metabolomics profile was assessed via untargeted mass spectrometry. Differential expression analysis and pathway enrichment were used to evaluate NSAID-specific effects across biological domains. FLU regulated the largest number of differentially expressed transcripts, followed by IBU and CEL. All NSAIDs activated immune-related gene networks and reversed exercise-induced lipid catabolism, with IBU enhancing adaptive immune signaling and CEL modulating both innate and adaptive pathways. Muscle remodeling pathways, including angiogenesis and cell migration, were activated across all NSAIDs, though cachexia-related genes were also upregulated. Interestingly, FLU uniquely upregulated transcripts involved in neuritogenesis. NSAIDs trigger drug-specific molecular responses in skeletal muscle post-exercise, affecting early recovery through changes in immune, metabolic, and neuronal signaling.
Duchenne muscular dystrophy (DMD) is a devastating disease manifested in skeletal muscle by repetitious myonecrosis and regeneration. Because the regenerative process is closely linked to the cumulative severity of muscle damage, which is variably distributed within and between muscle groups, accurately quantifying muscle regeneration has remained a significant challenge. Myofibers are delineated by immunostaining for laminin, and subsequent image analysis employed to generate a masked outline precisely within each myofiber boundary. Morphometric parameters including minimal Feret’s diameter, cross-sectional area, and circularity were measured for each myofiber. In addition, the number of Pax7-expressing satellite cells were quantified. To evaluate regenerative activity, newly formed myofibers were identified by immunostaining for expression of embryonic myosin heavy chain (eMHC). Necrotic myofibers were enumerated by immunofluorescent detection of immunoglobulin G (IgG) infiltration. The Regenerative Index (RI) was calculated as the number of regenerating (eMHC+) myofibers divided by the number of necrotic (IgG+) myofibers. Determination of RI was performed on muscle biopsies obtained from 10 boys with DMD and 3 age-matched non-DMD controls. A trend toward an increasing minimal Feret’s diameter, cross-sectional area and circularity was observed with increasing age in DMD boys, with circularity showing the strongest trend. Furthermore, compared to DMD boys 7- to 8-years old, the boys 9- to 11-years old had increased myofiber circularity. Pax7-expressing cells per myofiber were elevated in DMD boys compared to control boys of similar ages, without any observation of age-related changes. The Regenerative Index in DMD boys exhibited a decline between 7 and 11 years of age, with an inverse correlation between RI and age. The use of eMHC and IgG immunostaining to calculate RI appears to provide a way to assess regeneration across biopsies that differ in histopathologic severity. Using this approach, RI showed a negative correlation with age in DMD boys aged 7 to 11 years which requires further investigation.
Abstract Background Muscle passive and active function is dependent on the extracellular matrix (ECM). In diseases characterized by muscle fibrosis, namely Duchenne Muscular Dystrophy (DMD), the ECM contributes to deficits in muscle mechanical function and regeneration. Because fibrosis is often viewed as irreversible in DMD and other muscle diseases, there is great incentive to develop anti-fibrotic therapies to prevent or reverse fibrosis. Methods In this study we tested the effectiveness of intramuscular injections of non-specific Clostridium histolyticum collagenase (CCH) on reducing ECM contents and rescuing muscle mechanical function in D2. mdx mice, models of DMD. We performed unilateral injections of collagenase into the tibialis anterior and gastrocnemius in WT and D2. mdx mice. We measured in vivo plantarflexion strength, ex vivo muscle mechanical function, immunohistochemistry, and total and cross-linked collagen content. Results We found that crude CCH was effective at digesting the muscle ECM but did not provide a therapeutic benefit evidenced by induction of muscle weakness and bleeding within 24 h after CCH injections, and a thickened basal lamina after 7 days. Conclusions We conclude that future studies testing collagenase as an anti-fibrotic should use a collagenase specific to fibrillar collagens and a paired physical therapy protocol to better preserve muscle function while reducing fibrosis.
Abstract Background Prolonged mechanical ventilation is closely associated with ventilator-induced lung injury (VILI) and ventilator-induced diaphragm dysfunction (VIDD). These two conditions occur in parallel and contribute to delayed weaning, prolonged intensive care unit (ICU) stay, and poor clinical outcomes. This study evaluated whether human BM-MSC-derived extracellular vesicles (EVs) can simultaneously alleviate lung and diaphragm abnormalities in a unique rat experimental ICU (ExICU) model. Methods Rats were subjected to 5 days of controlled mechanical ventilation with or without a single intravenous EV dose. Outcomes included lung histopathology, diaphragm single-fiber contractile function, transcriptomics and metabolomics of diaphragm muscle, proteomics and metabolomics of lung tissue, and serial proteomics of bronchoalveolar lavage fluid (BALF). Results Five days of mechanical ventilation in the ExICU model were accompanied by severe lung morphological damage and approximately 50% reductions in diaphragm fiber size and specific force. EV treatment was associated with parallel improvements in lung pathology and diaphragm function. Multi-omics revealed coordinated molecular disturbances across lung, BALF, and diaphragm after mechanical ventilation, the majority of which were reversed by EVs. Conclusion Our findings demonstrate an association between lung injury and diaphragm dysfunction during prolonged mechanical ventilation. BM-MSC-derived EVs exert parallel protective effects on both organs and represent a promising intervention to reduce complications of mechanical ventilation in critically ill patients.
Effective skeletal muscle regeneration requires muscle stem cells (MuSCs) to continuously interpret and respond to signals from their surrounding microenvironment. These niche-derived cues, including inflammatory, extracellular matrix, paracrine, metabolic, and biomechanical signals, direct MuSC progression through quiescence, activation, proliferation, and differentiation by reshaping gene expression programs. Increasing evidence suggests that transcriptional enhancers serve as a key regulatory interface through which environmental information is translated into transcriptional output. Enhancer activity is governed by the coordinated action of lineage-defining transcription factors, histone modifiers, chromatin remodelers, transcriptional coactivators, and architectural proteins that together regulate chromatin accessibility, enhancer-promoter communication, and gene activation. Recent work has shown that enhancer landscapes and three-dimensional genome organization are highly dynamic during muscle regeneration and become altered in aging and disease. In this review, we examine how enhancer-associated mechanisms enable MuSCs to interpret niche-derived signals, highlighting the roles of transcription factor networks, chromatin remodeling complexes, and enhancer-promoter interactions in coordinating gene expression. We further discuss how disruption of enhancer regulation contributes to impaired regeneration in aging and muscular dystrophy, where altered chromatin states and genome organization lead to aberrant transcriptional responses. Understanding how these regulatory elements integrate complex environmental signals will be essential for defining the mechanisms underlying muscle regeneration and may provide new avenues for therapeutic intervention.
Abstract Background Unlike mammals and birds, where new muscle fiber formation (hyperplasia) ceases around birth, large and fast-growing fish such as trout undergo a spectacular post-hatching surge of hyperplasia, followed by a considerably delayed hyperplasia decline. This study investigated the role of muscle stem cells (MuSCs) and their niche in this process by assessing changes in their abundance, myogenic potential and niche functionality. Methods Hyperplasia kinetics were investigated by measuring the total number of fibers and their cross-sectional area (CSA) in white muscle across juvenile stages (10 g to 2 kg). Quantification of MuSCs during growth was performed by pax7 in situ hybridization. To assess the supportive capacity of the MuSCs niche, muscle-derived cells (MDCs) extracted from the Tg(mlc2:gfp) trout line were transplanted into muscle of wild-type trout at different juvenile stages. Expression of GFP in transplanted muscle was measured as an indicator of myogenic progenitor differentiation. Results Histological analysis revealed a significant decrease in hyperplasia and MuSCs density (defined here as pax7 + cells) between 10 and 500 g trout. Transplantation experiments using MDCs from Tg(mlc2:gfp) trout (10 g donors into 10 g to 2 kg recipients) showed alterations in niche functionality as the trout grew from 10 to 500 g. The transplantation of Tg(mlc2:gfp) MDCs from early to late juvenile donor trout (100 g to 2 kg) into 10 g WT recipients showed a decrease in the GFP signal as the donor weight increased. Detailed analyses of GFP + fibers produced after transplantation showed an enrichment of small-CSA GFP + fibers in 10 g but not 100 g trout recipient muscles, indicating a rapid impairment in niche ability to support hyperplasia. In addition, by comparing trout of the same age but different weights, we demonstrated that weight gain, rather than chronological aging, is a key factor driving this decline. Conclusions Overall, these results indicate that the decline in muscle hyperplasia in trout is associated with an early impairment of the MuSC niche, along with a reduced MuSC density. Also, weight gain was found to play a more critical role than aging. These original findings provide new insights into the mechanisms underlying muscle growth as hyperplasia declines in vertebrates.
Dysferlinopathy is an age-dependent muscular dystrophy caused by loss of the membrane-associated protein dysferlin. Disease severity increases with age and selectively affects specific muscle groups, yet the molecular basis for this vulnerability remains unclear. Since lipid remodeling is a hallmark of dysferlinopathy and aging, we investigated how age, sex, and muscle fiber type interact to shape the muscle lipidome in dysferlin-deficient mice. We performed omics-scale lipid profiling across 738 lipid species and 30 lipid classes in quadriceps and gastrocnemius (fast-twitch muscles exhibiting pronounced pathology), soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch, relatively spared) muscles from male and female dysferlin-deficient (BLA/J) and wildtype C57BL/6J (WT) mice aged 3, 10, and 26 months. Normal aging was associated with broad lipid remodeling, however, in the absence of dysferlin markedly amplified this remodeling, leading to elevations in specific triglycerides, diglycerides, cholesterol esters, gangliosides, ceramides, and sphingomyelin compared to WT muscle. Interestingly, we observed minimal sex differences between dysferlin-deficient muscles. Fast-twitch muscles, particularly quadriceps and gastrocnemius, exhibited the most extensive lipid alterations, whereas the slow-twitch soleus muscle showed relative lipid stability even at advanced age. Thus, fast-glycolytic muscles are more susceptible to age- and dysferlin-dependent lipid dysregulation than slow oxidative muscle. The preferential vulnerability of fast-twitch muscles to muscle wasting in dysferlinopathy suggests that fiber-type-dependent lipid handling contributes to selective muscle degeneration. This work defines a comprehensive lipidomic signature of disease progression and provides a framework for understanding how aging, sex, and muscle phenotype interact in muscular dystrophy.
Background Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disease characterized by progressive muscle degeneration, exhaustion of the muscle stem cell pool, and extensive fibrotic remodelling, ultimately leading to loss of function and reduced quality of life. Although conventional cultures and mouse models have provided valuable insights into the pathogenesis of DMD, their mild phenotypes and prolonged disease progression require large sample sizes and lengthy experimental timelines. In turn, the field lacks experimental models that recapitulate the complexity of the dystrophic muscle phenotype in vitro for disease modelling or drug screening. Methods We developed a three-dimensional (3D) construct of dystrophic skeletal muscle using a scaffold-free approach, starting from primary cells isolated from the mdx(4cv) mouse strain, a widely used model of Duchenne muscular dystrophy (DMD). To assess the pathological fidelity of the DMD 3D model, we conducted a thorough morphological and functional characterization. Taking advantage of the controlled and isolated nature of the system, we explored the paracrine role of the derived muscle-extracellular vesicles (EVs), investigating their potential contribution to disease progression. Results The heterogeneous 3D skeletal muscle model of DMD faithfully reproduced the hallmark pathological features observed in patient-derived muscle tissue, including progressive muscle degeneration, fibrotic remodelling, and defective regenerative capacity. Furthermore, it enabled mechanistic investigations of muscle-derived EVs, revealing their ability to propagate both regenerative and catabolic signals. Conclusions This 3D model provides a physiologically relevant and reproducible tool for studying the molecular mechanisms underlying DMD and evaluating potential therapeutic interventions, reducing the use of animal models. Its capacity to replicate key aspects of the muscle pathology holds significant potential for identifying novel biomarkers and therapeutic targets, with broad implications for translational research.
Background The PAX7::FOXO1 subtype of alveolar rhabdomyosarcoma (aRMS) is both understudied and an unmet clinical need. The biology of PAX7::FOXO1 aRMS is significantly different than PAX3::FOXO1 aRMS, presenting an opportunity to gain biological, clinical and therapeutic insights by murine genetic modeling of Pax7::Foxo1 for comparison to the existing conditional genetic mouse model of Pax3::Foxo1 aRMS. Methods Using gene targeting, a mouse strain harboring a Cre-LoxP activated conditional knock-in of Pax7::Foxo1 targeted to the murine Pax7locus has been generated. Results Activation of the conditional knock-in of Pax7::Foxo1 allele in the prenatal Pax7lineage recapitulates the birth defects (stunting and maxillofacial deformities) known for the conditional knock-in of Pax3::Foxo1 allele. Activation of the conditional knock-in of Pax7::Foxo1 allele in the prenatal Myf6 lineage is viable, fertile, and results in ongoing expression from Pax7 promoter in myofibers. Conclusions This mouse strain is a resource for the rhabdomyosarcoma research community to explore lineage (cell) of origin, to define sufficiency for tumor initiation or the necessity of cooperative tumor initiating mutations, to uncover the biology of the PAX7::FOXO1 subtype of alveolar rhabdomyosarcoma, and to test therapies including immunotherapies.
Abstract Background Denervation triggers dramatic atrophy of skeletal muscle, accompanied by synaptic, contractile and metabolic changes. Several factors were shown to contribute to genetic reprogramming and proteostasis changes after denervation. However, the mechanisms underlying the coordinated regulation of denervation-induced muscle fiber remodeling remain misunderstood. Methods We investigated the role of the transcriptional co-repressor CtBP1 in the regulation of denervation-induced responses in muscle fibers. To this end, we analyzed its expression and localization in innervated and denervated muscles and assessed the consequences of its knockdown induced in vivo with AAV9, on synaptic, contractile and metabolic properties of muscle fibers. Results CtBP1 was present both in sub- and non-synaptic myonuclei in innervated muscle. Although CtBP1 levels remained unchanged in denervated muscle, CtBP1 accumulated transiently in myonuclei after 2 days of denervation in TA/EDL muscles. Ctbp1 knockdown perturbed the expression of a large set of activity-independent and -dependent genes in innervated and denervated skeletal muscles. Reducing CtBP1 levels had limited effect on the expression of most synaptic genes, but increased transcript levels of Chrne, encoding the adult ε sub-unit of acetylcholine receptors (AChR). However, it did not affect AChR turnover or maintenance of the post-synaptic compartment upon denervation. Importantly, we uncovered that Ctbp1 knockdown exacerbates denervation-induced changes in metabolic gene expression, including most genes encoding proteins of the respiratory chain complexes. Consistently, it induced a contractile shift towards slower fibers in innervated fast muscle, mimicking denervation, and enhanced the denervation-induced metabolic transition towards oxidative slow-twitch fibers. Moreover, Ctbp1 knockdown precipitated the profound ultrastructural remodeling of mitochondria network induced after denervation. Conclusions Our study unveils that CtBP1 sustains the innervated muscle pattern and antagonizes the effect of denervation on synaptic, contractile and metabolic muscle properties, with important implications for CtBP1-related muscle diseases.
Abstract Background Irisin which is encoded by FNDC5 gene, has emerged as a promising therapeutic candidate for alleviating sarcopenia; however, its long-term therapeutic application in muscle regeneration remains insufficiently developed. Methods Our study investigated the potential of FNDC5 gene delivery in both cellular and animal levels via a virus vector. It was indicated that FNDC5 overexpression promoted myoblast proliferation while inhibiting myogenic differentiation, and vice versa. Mechanistically, we confirmed that the product of FNDC5 gene, irisin binds to integrin αVβ5 on the cell membrane of myoblasts, leading to activation of the downstream FAK/SRC complex using coimmunoprecipitation (co-IP) and confocal microscopy. RNA-Seq and qPCR analyses revealed that irisin exposure activated genes related to the extracellular matrix (ECM) and its ligands, thereby promoting the cell cycle and enhancing the proliferation of myoblast. Additionally, treatment with the integrin αVβ5 inhibitor cilengitide enhanced myogenic differentiation but suppressed cell proliferation, highlighting the crucial role of integrin αVβ5 in determining myoblasts’ cell fate. Furthermore, in sarcopenia mouse models, AAV-mediated FNDC5 supplementation activated integrin αVβ5-related signaling as expected, resulting in increased muscle mass and alleviation of sarcopenia symptoms. Results Our findings demonstrate that integrin αVβ5 acts as a key switch in regulating the proliferation and differentiation of myoblasts under FNDC5 gene delivery. When integrin αVβ5 is active, FNDC5 over-expression enhances cell proliferation rather than differentiation. Conversely, when integrin αVβ5 is inhibited, FNDC5 over-expression promotes myogenic differentiation over proliferation. Moreover, the binding of irisin to integrin αVβ5 induces the expression of ECM proteins in myoblasts and activates the Bgn-TLR4 signaling pathway, which further enhances cell proliferation. This FNDC5-ITGAVB5-FAK-mTOR-Bgn-TLR4 signaling axis was also validated in a sarcopenia mouse model. Conclusion Integrin αVβ5 is a critical regulator of myoblast proliferation and differentiation in response to FNDC5 gene supplement. These findings provide new insights into FNDC5 biological functions and suggest potential therapeutic strategies for enhancing muscle regeneration and treating sarcopenia.
Background Denervation occurs as a consequence of disease or injury and is typically accompanied by skeletal muscle atrophy. Although the relationship between nerves and muscle atrophy has been studied, the direct molecular contributions of nerves remain unclear. Methods We used the axolotl (Ambystoma mexicanum), a vertebrate with robust regenerative capacity and optically accessible musculature, to investigate the role of nerve-derived signals in muscle maintenance. Results Forelimb denervation produced significant muscle atrophy in axolotls. Quantitative imaging showed that reduced muscle fiber size-rather than fiber loss-underlies this atrophy. To investigate the molecular basis of denervation-induced atrophy, we focused on fibroblast growth factor 2 (FGF2), a nerve-derived factor previously implicated in axolotl limb regeneration. Electroporation of FGF2 into denervated muscles significantly preserved muscle fiber size compared with controls. Conversely, pharmacological inhibition of FGF signaling with SU5402 reduced fiber size, supporting a requirement for FGF signaling in maintaining muscle mass. Conclusions These findings demonstrate that FGF2 is sufficient to mitigate denervation-induced muscle atrophy and support a requirement for FGF signaling in the maintenance of muscle mass. Together with the neural expression of Fgf2, our data support a model in which nerve-derived FGF2 contributes to muscle maintenance. This work positions the axolotl as a tractable model for dissecting neuromuscular signaling and identifies FGF2 as a promising therapeutic candidate for neuromuscular atrophy. Understanding this mechanism may inform strategies to preserve muscle mass after nerve injury or in neurodegenerative diseases.
Filaminopathies, caused by pathogenic FLNC variants, are rare neuromuscular disorders characterized by protein aggregation, z-disk pathology and lead to progressive muscle weakness and/or cardiomyopathies. To address the lack of existing filaminopathy models in skeletal muscle, we developed a patient-specific cellular platform using induced pluripotent stem cells (iPSCs) harboring two truncating filamin C (FLNc) variants (p.Q1662X, p.Y2704X). Employing a developmental human skeletal muscle organoid hSMO model, we enrich for myogenic progenitor cells that are further differentiated into functional myotubes through 2D and 3D approaches (myotubes and musculoids). The 2D myotubes exhibited poor sarcomeric organization and hallmarks of filaminopathies, including protein aggregation and proteostatic dysfunction, marked by elevated aggresome formation and an increased basal autophagic flux. The 3D musculoids revealed ultrastructural abnormalities and enabled the identification of novel disease-associated proteins involved in ER stress and protein folding (e.g. DNAJC10) through proteomic analysis. Proteomic findings were additionally validated in 2D cultures and in corresponding patient-derived muscle biopsies enhancing the model’s translational value. Our model is suitable to monitor aspects of filaminopathies’ pathogenesis and to investigate possible therapeutic interventions with quantitative readouts.
BackgroundHuman primary muscle cell (HPMC) lines derived from skeletal muscle biopsies are potentially powerful tools to interrogate the molecular pathways underlying fundamental muscle mechanisms. HPMCs retain their genome in culture, but many endogenous circulating factors are not present in the in vitro environment, or at concentrations that do not mirror physiological levels. To address the assumption that HPMCs are valid models of age and sex-specificity in human muscle research, we examined to what extent differentiated HPMC lines retain their source phenotype in culture.MethodsBiopsies from the vastus lateralis muscle were collected from ten males aged 18-30, ten females aged 18-30 and ten males aged 60-75 recruited from a healthy population. A portion of the muscle was used for the establishment of 30 individual HMPC lines. The remaining sample was immediately snap frozen and stored for further analysis. RNA was extracted from muscle tissue samples and their corresponding, fully differentiated HMPCs and analysed using RNA Sequencing. To compare their transcriptomic signature, principal component analysis (PCA), differential expression analysis, single-cell deconvolution and pathway enrichment analysis were conducted in R.ResultsA comparison of the transcriptomic signature of 30 human muscle biopsies and their corresponding differentiated HPMCs indicated a near-complete lack of retention of the genes and pathways differentially regulated in vivo when compared to their in vitro equivalent, with the exception of several genes encoded on the Y-chromosome.ConclusionsThe diversity of resident cell populations in muscle tissue and the lack of sex- and age-dependent circulating factors in the cellular milieu likely contribute to these observations, which call for caution when using differentiated HPMCs as an experimental model of human muscle sex or age.
