Skeletal Muscle Fibrosis Research Articles

Role and Interplay of Different Signaling Pathways Involved in Sciatic Nerve Regeneration.

Regeneration of the sciatic nerve is a sophisticated process that involves the interplay of several signaling pathways that orchestrate the cellular responses critical to regeneration. Among the key pathways are the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, cyclic adenosine monophosphate (cAMP), and Janus kinase/signal transducers and transcription activators (JAK/STAT) pathways. In particular, the cAMP pathway modulates neuronal survival and axonal regrowth. It influences various cellular behaviors and gene expression that are essential for nerve regeneration. MAPK is indispensable for Schwann cell differentiation and myelination, whereas PI3K/AKT is integral to the transcription, translation, and cell survival processes that are vital for nerve regeneration. Furthermore, GTP-binding proteins, including those of the Ras homolog gene family (Rho), regulate neural cell adhesion, migration, and survival. Notch signaling also appears to be effective in the early stages of nerve regeneration and in preventing skeletal muscle fibrosis after injury. Understanding the intricate mechanisms and interactions of these pathways is vital for the development of effective therapeutic strategies for sciatic nerve injuries. This review underscores the need for further research to fill existing knowledge gaps and improve therapeutic outcomes.

12582 Electronic-cigarette Exposure Induces Hyperglycemia With A Fibrotic Phenotype In Skeletal Muscle In Mice Subjected To A High-fat Diet

Abstract Disclosure: J. Rivera: None. J. Chavez: None. J. Espinoza-Derout: None. K.M. Hasan: None. X.M. Shao: None. A.P. Sinha-Hikim: None. T.C. Friedman: None. Obesity and electronic cigarette (e-cigarette) use are highly prevalent among youth in the USA. Indeed, young e-cigarette users report less physical activity, leading to a decline in quality of life. Recent studies show e-cigarettes reduce skeletal muscle (SkM) performance in mice; however, the underlying metabolic and SkM cellular effects are unclear. E-cigarettes plus a high-fat diet (HFD) increase mice lipolysis and plasma-free fatty acids. These harmful changes in the liver and heart are prevented by the lipolysis inhibitor Acipimox (Acip). As SkM plays a crucial role in metabolic and chronic disease prevention, understanding the impact of e-cigarettes and HFD on SkM is essential.We exposed C57BL6 mice to e-cigarette inhalation in a free-moving system to deliver 2.4% nicotine in a similar way to humans. Mice were fed with an HFD and divided into 3 groups: Saline (control), e-cigarette (bluCig), and e-cigarette plus Acip (rescue group) for 12 weeks. SkM uptakes 70-80% of the postprandial glucose, so we performed a glucose tolerance test. We found higher glycemia in the e-cigarette compared to the Saline group, while the e-cigarette plus Acip group showed improved glycemia. Histological analysis of SkM with Hematoxylin and eosin (HE) staining revealed large extracellular fibrotic structures in the group with e-cigarettes, suggesting fibrosis. Sirius Red (SR) stain identified collagen deposition. The SR quantification showed an increase in the collagen in the e-cigarette to the saline-exposed group without recovery in the e-cigarette plus Acip group. Fibrosis restricts the SkM hypertrophy. So we analyzed the insulin-like growth factor-1 (IGF-1) (a key anabolic factor) by RT-qPCR. The mRNA levels of IGF-1 were reduced in the SkM of e-cigarettes compared to the Saline group, without recovery of the Acip group. In SkM hypertrophy, Satellite cells (SCs) are stimulated by IGF-1 and SkM fibrosis can affect SCs by restricting them. So we analyzed by western blot the SCs through the transcriptional factor PAX7 (SC marker). We found PAX7 levels were lower in the e-cigarette compared with the Saline group without recovery in the Acip group. These findings suggest the detrimental effects of e-cigarettes on glycemia and SkM health in mice, with implications for long-term health outcomes, particularly among obese individuals and e-cigarette users. Further research is necessary for understanding these adverse effects and promoting youth health. GRANT SUPPORT: U54MD007598, SC2GM135127, U54MD007598-14S2, PR190942, DA050723, and R25DA050723 grants. Presentation: 6/3/2024

12565 Expression Of Sex Hormone Receptors In Levator Ani Muscle Tissue From Patients With Pelvic Organ Prolapse

Abstract Disclosure: T. You: None. A. Halle: None. W. Xu: None. T. Potluri: None. H. Nguyen: None. S.E. Bulun: None. J. Julia Geynisman-Tan: None. H. Zhao: None. Introduction: Women with pelvic floor disorders usually have weakened pelvic floor muscles, which may cause urinary incontinence, fecal incontinence, and pelvic organ prolapse (POP). Histologically, the pelvic floor musculature in POP patients displays increased fibrosis and muscle atrophy. However, the underlying molecular mechanism for POP has not been fully investigated. We and others found that estrogen causes pelvic skeletal muscle fibrosis and inguinal hernias in male animal models. Additionally, clinical trials from the Women's Health Initiative showed women receiving estrogen-only or estrogen plus progestin treatment had statistically significant increases in the risk for urinary incontinence. However, the role of estrogen, progesterone, androgen, and their receptor expression in pelvic floor muscle is not well studied. Methods: TruCut biopsy needles were used to obtain levator ani (LA, the major pelvic floor muscle) biopsies from four groups of patients undergoing surgery: (i) 5 premenopausal women with stage II/III POP (age 38-52 years), (ii) 5 postmenopausal women with stage III POP (59-75 years), (iii) 5 age and parity matched premenopausal controls, and (iv) 5 age and parity matched postmenopausal controls. The control patients did not have any POP (stage 0) or only had sole stress urinary incontinence. H&E staining, Masson’s trichrome staining, and immunohistochemistry (IHC) staining for estrogen receptor α (ERα), progesterone receptor (PGR), and androgen receptor (AR) were performed in LA muscle tissue. Standard patient demographics (age, parity, and diagnosis) were collected. Results: We used H&E staining to morphologically assess stromal cells and myofibers obtained from LA muscle biopsies. Masson’s trichrome staining showed extracellular matrix deposition in the LA muscle from women with or without POP. In addition, we found ERα was present in the stromal cells from nearly every LA biopsy using IHC staining. ERα expression (H-Score) was similar in four groups of patients. ERα IHC staining was absent in LA myofibers. AR was highly expressed in stromal cells in all LA muscles and weakly expressed in myofibers in ∼70% of LA tissues. AR expression (H-Score) was significantly decreased in postmenopausal women with stage III pelvic organ prolapse. PGR was only expressed in a small portion of the LA sample (30%), with PGR exclusively expressed in the stroma of the LA muscle. Conclusion: Our findings indicate that sex hormone receptors (ERα, AR, and PGR) are present in LA muscle stroma, providing the foundation to further define the roles of sex steroid hormones in pelvic organ prolapse. Presentation: 6/3/2024

8658 Role of Progesterone Receptor in Skeletal Muscle Fibrosis and Hernia Formation in Mouse Models and Men with Inguinal Hernias

8658 Role of Progesterone Receptor in Skeletal Muscle Fibrosis and Hernia Formation in Mouse Models and Men with Inguinal Hernias

401P Moderate-term dimethyl fumarate treatment reduces fibrosis of skeletal and cardiac muscle in in the mdx mouse model of Duchenne muscular dystrophy

401P Moderate-term dimethyl fumarate treatment reduces fibrosis of skeletal and cardiac muscle in in the mdx mouse model of Duchenne muscular dystrophy

Single-cell RNA Sequencing Revealed Fibro-adipogenic Progenitors As The Major Contributor To Radiation-induced Skeletal Muscle Fibrosis

Single-cell RNA Sequencing Revealed Fibro-adipogenic Progenitors As The Major Contributor To Radiation-induced Skeletal Muscle Fibrosis

Involvement of lysophosphatidic acid-LPA1-YAP signaling in healthy and pathological FAPs migration

Skeletal muscle fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) components and is a hallmark of muscular dystrophies. Fibro-adipogenic progenitors (FAPs) are the main source of ECM, and thus have been strongly implicated in fibrogenesis. In skeletal muscle fibrotic models, including muscular dystrophies, FAPs undergo dysregulations in terms of proliferation, differentiation, and apoptosis, however few studies have explored the impact of FAPs migration.Here, we studied fibroblast and FAPs migration and identified lysophosphatidic acid (LPA), a signaling lipid central to skeletal muscle fibrogenesis, as a significant migration inductor. We identified LPA receptor 1 (LPA1) mediated signaling as crucial for this effect through a mechanism dependent on the Hippo pathway, another pathway implicated in fibrosis across diverse tissues. This cross-talk favors the activation of the Yes-associated protein 1 (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), leading to increased expression of fibrosis-associated genes. This study reveals the role of YAP in LPA-mediated fibrotic responses as inhibition of YAP transcriptional coactivator activity hinders LPA-induced migration in fibroblasts and FAPs.Moreover, we found that FAPs derived from the mdx4cv mice, a murine model of Duchenne muscular dystrophy, display a heightened migratory phenotype due to enhanced LPA signaling compared to wild-type FAPs. Remarkably, we found that the inhibition of LPA1 or YAP transcriptional coactivator activity in mdx4cv FAPs reverts this phenotype. In summary, the identified LPA-LPA1-YAP pathway emerges as a critical driver of skeletal muscle FAPs migration and provides insights into potential novel targets to mitigate fibrosis in muscular dystrophies.

L-Carnitine relieves cachexia-related skeletal muscle fibrosis by inducing deltex E3 ubiquitin ligase 3L to negatively regulate the Runx2/COL1A1 axis.

Cancer cachexia-induced skeletal muscle fibrosis (SMF) impairs muscle regeneration, alters the muscle structure and function, reduces the efficacy of anticancer drugs, diminishes the patient's quality of life and shortens overall survival. RUNX family transcription factor 2 (Runx2), a transcription factor, and collagen type I alpha 1 chain (COL1A1), the principal constituent of SMF, have been linked previously, with Runx2 shown to directly modulate COL1A1 mRNA levels. l-Carnitine, a marker of cancer cachexia, can alleviate fibrosis in liver and kidney models; however, its role in cancer cachexia-associated fibrosis and the involvement of Runx2 in the process remain unexplored. Female C57 mice (48weeks old) were inoculated subcutaneously with MC38 cells to establish a cancer cachexia model. A 5mg/kg dose of l-carnitine or an equivalent volume of water was administered for 14days via oral gavage, followed by assessments of muscle function (grip strength) and fibrosis. To elucidate the interplay between the deltex E3 ubiquitin ligase 3L(DTX3L)/Runx2/COL1A1 axis and fibrosis in transforming growth factor beta 1-stimulated NIH/3T3 cells, a suite of molecular techniques, including quantitative real-time PCR, western blot analysis, co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays, were used. The relevance of the DTX3L/Runx2/COL1A1 axis in the gastrocnemius was also explored in the in vivo model. l-Carnitine supplementation reduced cancer cachexia-induced declines in grip strength (>88.2%, P<0.05) and the collagen fibre area within the gastrocnemius (>57.9%, P<0.05). At the 5mg/kg dose, l-carnitine also suppressed COL1A1 and alpha-smooth muscle actin (α-SMA) protein expression, which are markers of SMF and myofibroblasts. Analyses of the TRRUST database indicated that Runx2 regulates both COL1A1 and COL1A2. In vitro, l-carnitine diminished Runx2 protein levels and promoted its ubiquitination. Overexpression of Runx2 abolished the effects of l-carnitine on COL1A1 and α-SMA. Co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays confirmed an interaction between DTX3L and Runx2, with l-carnitine enhancing this interaction to promote Runx2 ubiquitination. l-Carnitine supplementation restored DTX3L levels to those observed under non-cachectic conditions, both in vitro and in vivo. Knockdown of DTX3L abolished the effects of l-carnitine on Runx2, COL1A1 and α-SMA in vitro. The expression of DTX3L was negatively correlated with the levels of Runx2 and COL1A1 in untreated NIH/3T3 cells. This study revealed a previously unrecognized link between Runx2 and DTX3L in SMF and demonstrated that l-carnitine exerted a significant therapeutic impact on cancer cachexia-associated SMF, potentially through the upregulation of DTX3L.

Desmin degradation in the skeletal muscle of patients with chronic critical illness

Critical illness myopathy (CIM) is a primary myopathy that develops in critically ill patients. Histologic features of CIM include a general decrease in muscle fiber cross-sectional area and a predominant loss of the motor protein myosin. These features are observed in the absence of inflammatory infiltrates but with detectable cytokine activation. The purpose of this study was to examine the state of the extracellular matrix of the human soleus muscle under conditions of CIM caused by chronic impairment of consciousness. Incisional muscle biopsies were taken from the soleus muscle of 6 patients who were in a chronic critical condition and were treated in the Department of Anesthesiology and Reanimation at the A.L. Polenov Russian Research Institute - branch of the Almazov National Medical Research Center. The study included patients with a chronic impairment of consciousness lasting at least 2 months. Muscle biopsies taken from healthy men were used as controls. The biopsies were obtained using needle biopsy under local anesthesia. Using histological staining of tissue sections, it was determined that patients with CIM exhibited a significant increase in collagen area, surpassing the control value by 82%. An increased mRNA content of collagens I, III, and VIa was also observed, along with an increase in the protein content of collagen I and III. At the same time, we did not observe any changes in the content of fibronectin and extracellular tissue growth factor mRNA. However, we did observe an increase in the mRNA of the integrin A7 subunit. The results obtained indicate significant skeletal muscle fibrosis under CIM conditions. Further studies on the signaling pathways that regulate this process are needed.

Increase in cathepsin K gene expression in Duchenne muscular dystrophy skeletal muscle.

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

Acupotomy ameliorates knee osteoarthritis-related collagen deposition and fibrosis in rabbit skeletal muscle through the TGF-β/Smad pathway

ObjectiveTo investigate the effects of acupotomy on skeletal muscle fibrosis and collagen deposition in a rabbit knee osteoarthritis (KOA) model. MethodsRabbits (n = 18) were randomly divided into control, KOA, and KOA + acupotomy (Apo) groups (n = 6). The rabbits in the KOA and Apo groups were modeled using the modified Videman's method for 6 weeks. After modeling, the Apo group was subjected to acupotomy once a week for 3 weeks on the vastus medialis, vastus lateralis, rectus femoris, biceps femoris, and anserine bursa tendons around the knee. The behavior of all animals was recorded, rectus femoris tissue was obtained, and histomorphological changes were observed using Masson staining and transmission electron microscopy. The expression of transforming growth factor-β1 (TGF-β1), Smad 3, Smad 7, fibrillar collagen types I (Col-I) and III (Col-III) was detected using Western blot and real-time polymerase chain reaction (RT-PCR). ResultsHistological analysis revealed that acupotomy improved the microstructure and reduced the collagen volume fraction of rectus femoris, compared with the KOA group (P = .034). Acupotomy inhibited abnormal collagen deposition by modulating the expression of fibrosis-related proteins and mRNA, thus preventing skeletal muscle fibrosis. Western blot and RT-PCR analysis revealed that in the Apo group, Col-I, and Col-III protein levels were significantly lower than those in the KOA group (both P < .01), same as Col-I and Col-III mRNA levels (P = .0031; P = .0046). Compared with the KOA group, the protein levels of TGF-β1 and Smad 3 were significantly reduced (both P < .01), as were the mRNA levels of TGF-β1 and Smad 3 (P = .0007; P = .0011). Conversely, the levels of protein and mRNA of Smad 7 were significantly higher than that in the KOA group (P < .01; P = .0271). ConclusionAcupotomy could alleviate skeletal muscle fibrosis and delay KOA progress by inhibiting collagen deposition through the TGF-β/Smad pathway in the skeletal muscle of KOA rabbits.

Biology of Tenascin C and its Role in Physiology and Pathology.

Tenascin-C (TNC) is a multimodular extracellular matrix (ECM) protein hexameric with several molecular forms (180-250 kDa) produced by alternative splicing at the pre-mRNA level and protein modifications. The molecular phylogeny indicates that the amino acid sequence of TNC is a well-conserved protein among vertebrates. TNC has binding partners, including fibronectin, collagen, fibrillin-2, periostin, proteoglycans, and pathogens. Various transcription factors and intracellular regulators tightly regulate TNC expression. TNC plays an essential role in cell proliferation and migration. Unlike embryonic tissues, TNC protein is distributed over a few tissues in adults. However, higher TNC expression is observed in inflammation, wound healing, cancer, and other pathological conditions. It is widely expressed in a variety of human malignancies and is recognized as a pivotal factor in cancer progression and metastasis. Moreover, TNC increases both pro-and anti-inflammatory signaling pathways. It has been identified as an essential factor in tissue injuries such as damaged skeletal muscle, heart disease, and kidney fibrosis. This multimodular hexameric glycoprotein modulates both innate and adaptive immune responses regulating the expression of numerous cytokines. Moreover, TNC is an important regulatory molecule that affects the onset and progression of neuronal disorders through many signaling pathways. We provide a comprehensive overview of the structural and expression properties of TNC and its potential functions in physiological and pathological conditions.

Arctigenin derivative A-1 ameliorates motor dysfunction and pathological manifestations in SOD1G93A transgenic mice via the AMPK/SIRT1/PGC-1α and AMPK/SIRT1/IL-1β/NF-κB pathways.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by progressive death of upper and lower motor neurons, leading to generalized muscle atrophy, paralysis, and even death. Mitochondrial damage and neuroinflammation play key roles in the pathogenesis of ALS. In the present study, the efficacy of A-1, a derivative of arctigenin with AMP-activated protein kinase (AMPK) and silent information regulator 1 (SIRT1) activation for ALS, was investigated. A-1 at 33.3 mg/kg was administrated in SOD1G93A transgenic mice orally from the 13th week for a 6-week treatment period. Motor ability was assessed before terminal anesthesia. Muscle atrophy and fibrosis, motor neurons, astrocytes, and microglia in the spinal cord were evaluated by H&E, Masson, Sirius Red, Nissl, and immunohistochemistry staining. Protein expression was detected with proteomics analysis, Western blotting, and ELISA. Mitochondrial adenosine triphosphate (ATP) and malondialdehyde (MDA) levels were measured using an assay kit. A-1 administration in SOD1G93A mice enhanced mobility, decreased skeletal muscle atrophy and fibrosis, mitigated loss of spinal motor neurons, and reduced glial activation. Additionally, A-1 treatment improved mitochondrial function, evidenced by elevated ATP levels and increased expression of key mitochondrial-related proteins. The A-1 treatment group showed decreased levels of IL-1β, pIκBα/IκBα, and pNF-κB/NF-κB. A-1 treatment reduced motor neuron loss, improved gastrocnemius atrophy, and delayed ALS progression through the AMPK/SIRT1/PGC-1α pathway, which promotes mitochondrial biogenesis. Furthermore, the AMPK/SIRT1/IL-1β/NF-κB pathway exerted neuroprotective effects by reducing neuroinflammation. These findings suggest A-1 as a promising therapeutic approach for ALS.

MiR-21 regulates skeletal muscle atrophy and fibrosis by targeting TGF-beta/SMAD7-SMAD2/3 signaling pathway

Long-term denervation-induced atrophy and fibrosis of skeletal muscle due to denervation leads to poor recovery of muscle function. Studies have shown that the transforming growth factor-β1 (TGF-β1)-Smad signaling pathway plays a central role in muscle atrophy and fibrosis. Recent studies demonstrate the role of microRNAs (miRs) in various pathological conditions, including muscle regeneration. miR-21 has been shown to play a dynamic role in inflammatory responses and in accelerating injury responses to fibrosis. We used both RNA sequencing and quantitative RT-PCR strategies to examine the alternations of miRNAs during denervation-induced gastrocnemius muscle atrophy and fibrosis. Our data showed that MiR-21 was upregulated in denervated gastrocnemius muscle tissue, and TGF-β1treatment increased miR-21 expression. Inhibition of miR-21 reduced gastrocnemius muscle fibrosis and significantly downregulated the expression of p-SMAD2/3 and the fibrosis-associated markers TGF-β1, connective tissue growth factor, alpha smooth muscle actin. Masson's trichrome staining revealed that atrophy and fibrosis in gastrocnemius muscle tissue were reduced in the miR-21 inhibition group compared to the control group. We confirmed that SMAD7 is a direct target of miR-21 using a dual luciferase assay. Furthermore, Immunofluorescence and Western blot analyses revealed that miR-21 inhibition reduced SMAD2/3 phosphorylation and nuclear translocation. While SMAD7-siRNA abolished the effect. Consequently, the discovery that miR-21 regulates the atrophy and fibrosis of the gastrocnemius muscle offers a possible therapeutic approach for their management.

Targeted Antisense Oligonucleotide-Mediated Skipping of Murine Postn Exon 17 Partially Addresses Fibrosis in D2.mdx Mice.

Periostin, a multifunctional 90 kDa protein, plays a pivotal role in the pathogenesis of fibrosis across various tissues, including skeletal muscle. It operates within the transforming growth factor beta 1 (Tgf-β1) signalling pathway and is upregulated in fibrotic tissue. Alternative splicing of Periostin's C-terminal region leads to six protein-coding isoforms. This study aimed to elucidate the contribution of the isoforms containing the amino acids encoded by exon 17 (e17+ Periostin) to skeletal muscle fibrosis and investigate the therapeutic potential of manipulating exon 17 splicing. We identified distinct structural differences between e17+ Periostin isoforms, affecting their interaction with key fibrotic proteins, including Tgf-β1 and integrin alpha V. In vitro mouse fibroblast experimentation confirmed the TGF-β1-induced upregulation of e17+ Periostin mRNA, mitigated by an antisense approach that induces the skipping of exon 17 of the Postn gene. Subsequent in vivo studies in the D2.mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that our antisense treatment effectively reduced e17+ Periostin mRNA expression, which coincided with reduced full-length Periostin protein expression and collagen accumulation. The grip strength of the treated mice was rescued to the wild-type level. These results suggest a pivotal role of e17+ Periostin isoforms in the fibrotic pathology of skeletal muscle and highlight the potential of targeted exon skipping strategies as a promising therapeutic approach for mitigating fibrosis-associated complications.

Lipidomics and single-cell RNA sequencing reveal lipid and cell dynamics of porcine glycerol-injured skeletal muscle regeneration model

AimsIntramuscular fat (IMF) infiltration and extracellular matrix (ECM) deposition are characteristic features of muscle dysfunction, such as muscular dystrophy and severe muscle injuries. However, the underlying mechanisms of cellular origin, adipocyte formation and fibrosis in skeletal muscle are still unclear. Main methodsPigs were injected with 50 % glycerol (GLY) to induce skeletal muscle injury and regeneration. The acyl chain composition was analyzed by lipidomics, and the cell atlas and molecular signatures were revealed via single-cell RNA sequencing (scRNA-seq). Adipogenesis analysis was performed on fibroblast/fibro-adipogenic progenitors (FAPs) isolated from pigs. Key findingsThe porcine GLY-injured skeletal muscle regeneration model was characterized by IMF infiltration and ECM deposition. Skeletal muscle stem cells (MuSCs) and FAP clusters were analyzed to explore the potential mechanisms of adipogenesis and fibrosis, and it was found that the TGF-β signaling pathway might be a key switch that regulates differentiation. Consistently, activation of the TGF-β signaling pathway increased SMAD2/3 phosphorylation and inhibited adipogenesis in FAPs, while inhibition of the TGF-β signaling pathway increased the expression of PPARγ and promoted adipogenesis. SignificanceGLY-induced muscle injury and regeneration provides comprehensive insights for the development of therapies for human skeletal muscle dysfunction and fatty infiltration-related diseases in which the TGF-β/SMAD signaling pathway might play a primary regulatory role.

Cyclo His-Pro Attenuates Muscle Degeneration in Murine Myopathy Models.

Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in inflammation and fibrosis that ultimately affect the overall muscle biomechanics. At the opposite end of the spectrum of muscle diseases, age-related sarcopenia is a common condition that affects an increasing proportion of the elderly. Although characterized by different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. Here, the therapeutic effects of Cyclo Histidine-Proline (CHP) against DMD and sarcopenia are evaluated. In the mdx mouse model of DMD, it is shown that CHP restored muscle contractility and force production, accompanied by the reduction of fibrosis and inflammation in skeletal muscle. CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age-related sarcopenia. These findings from two different models of muscle dysfunction hence warrant further investigation into the effects of CHP on muscle pathologies in animal models and eventually in patients.

Muscle Steatosis and Fibrosis in Older Adults, From the AJR Special Series on Imaging of Fibrosis.

The purpose of this article is to review steatosis and fibrosis of skeletal muscle, focusing on older adults. Although CT, MRI, and ultrasound are commonly used to image skeletal muscle and provide diagnoses for a variety of medical conditions, quantitative assessment of muscle steatosis and fibrosis is uncommon. This review provides radiologists with a broad perspective on muscle steatosis and fibrosis in older adults by considering the public health impact, biologic mechanisms, and evaluation with CT, MRI, and ultrasound. Promising directions in clinical research that entail artificial intelligence algorithms and the imaging assessment of biologic age are also reviewed. The imaging methods presented hold promise for improving the evaluation of common conditions affecting older adults, including sarcopenia, frailty, and cachexia.

Estrogen Signaling Modulation Prevents and Even Reverses Skeletal Muscle Fibrosis

Background: Skeletal muscle fibrosis represents accumulation of extracellular matrix (ECM), often leading to muscle weakness and atrophy. Notably, lower abdominal muscle (LAM) fibrosis and atrophy cause inguinal herniation—a prevalent condition lacking pharmacological treatment. We developed a herniation mouse model, Aromhum, characterized by spontaneous scrotal hernias due to local estradiol (E2) production within LAM as well as hernia-associated fibroblasts (HAFs) that express platelet-derived growth factor alpha (PDGFRA) and estrogen receptor alpha (ESR1). Objectives &amp; Hypothesis: Our objective was to investigate the mechanism of ESR1 signaling on LAM HAFs and subsequent development of hernias. Methods: We manipulated estrogen signaling in two ways: first, we generated fibroblast-specific estrogen receptor alpha knockout mice (fEsr1−/−-Aromhum) capable of local E2 production but incapable of signaling through ESR1 (n = 5-10 mice/group). Second, we blocked E2 signaling pharmacologically using the potent ESR1 antagonist fulvestrant (0.15mg/kg, 90 days, n=10-15 mice/group). We conducted in vitro experiments on primary LAM HAFs exposed to 10nM E2 ± 100nM Fulvestrant (24-48h). These cells underwent analysis via ESR1 ChIP, ATAC, and RNA-seq in 3 technical replicates (3-5 mice each). Additionally, to demonstrate clinical relevance, we probed human herniated LAM tissues from patients (n=25 samples, 21-76 years). Results: All Aromhum mice develop scrotal hernias by 6 weeks. However, fEsr1−/−-Aromhum mice did not develop hernias, while littermate controls exhibited hernias. Thus, HAF ESR1 depletion mitigated LAM fibrosis and atrophy. Similarly, administering fulvestrant prior to hernia onset prevented hernia development. Remarkably, fulvestrant treatment in mice that had developed large scrotal hernias (&gt;200mm2) led to complete hernia regression with reversed muscle fibrosis and fiber atrophy (collagen levels comparable to wild-type mice). In vitro HAF culturing and subsequent multiomic analyses unveiled a core set of 58 genes directly influenced by E2/ESR1, including crucial ECM genes like fibulins ( Fbln5, Fbln7), metalloproteases ( Adamts3, Adamts6), and signaling molecules ( Ltbp1, Ncam1, Piezo2). Pathways such as TGFβ, WNT, and N-Glycan biosynthesis were significantly upregulated by E2. Human herniated LAM tissues exhibited substantial fibrosis, along with stromal HAF markers PDGFRA and ESR1. We also validated expression of the core E2-modulated genes in human LAM tissues ( NCAM1, LTBP1, ADAMTS6, PIEZO2). Collectively, these findings provide compelling evidence of ESR1 pathway activation and downstream gene involvement in both Aromhum scrotal hernias and human inguinal hernias. Conclusion: Our research underscores the central role of E2 in skeletal muscle fibrosis. Notably, we demonstrated that fibrosis can be entirely reversed by modulation of ESR1 signaling. Our study offers valuable insights into downstream genes and pathways that may serve as therapeutic targets for inguinal hernias and other fibrotic disorders. Funding: NIH: R01DK121529, Department of Veterans Affairs Research Career Scientist Award: IK6 RX003351. 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.

Fibrotic Fibro-Adipogenic Progenitors Produce an Altered Extracellular Matrix that Impairs Myogenesis

Introduction: Fibrosis is the pathological accumulation of extracellular matrix (ECM) components, impairing tissue function. In skeletal muscle, the main source of ECM deposition are fibro-adipogenic progenitors (FAPs). FAPs can signal to muscle satellite cells (MuSCs) through soluble factors and ECM deposition. FAPs are more highly activated into myofibroblasts in fibrotic conditions, but how this affects their signaling to MuSCs is unknown. Cell-derived matrices (CDMs) can be synthesized from FAPs to investigate their ECM deposition. The goal of this study was to understand how FAPs are signaling to MuSCs through their ECM deposition. We hypothesized that FAPs isolated from mdx mice, a model for skeletal muscle fibrosis, would have increased collagen deposition compared to wt FAPs and would produce a matrix that reduces myogenesis. Methods: FAPs were isolated from the lower limb muscles of wt and mdx mice. FAPs were plated in media containing 50μM ascorbic acid to promote collagen deposition for 2 weeks in order to synthesize CDMs. CDMs were imaged using second harmonic generation (SHG) imaging to visualize collagen organization. Some CDMs were solubilized and replated to create a flat protein coating of matrix. MuSCs were then plated on the CDMs, solubilized matrix, in a transwell co-culture with FAPs, and on plastic in differentiation media for 5 days. Results: There was a significant negative correlation between the level of FAP activation into myofibroblasts and the MuSC differentiation index, independent of if the FAPs were isolated from wt or mdx mice. SHG images revealed CDMs derived from mdx FAPs had less aligned collagen fibrils compared to wt CDMs. Conclusions: High activation into myofibroblasts is often an indicator of a fibrotic FAP phenotype. The negative correlation between myofibroblasts and MuSC differentiation found here indicates this phenotype impairs myogenesis. FAPs in transwell co-culture did not have a significant effect on MuSC differentiation, highlighting the importance of ECM deposition rather than soluble signaling is important in FAP signaling to MuSCs. Targeting FAPs activation into myofibroblasts provides a therapeutic target to attenuate fibrosis and improve regeneration in skeletal muscle. Research reported in this publication was supported by the National Institute Of Arthritis And Musculoskeletal And Skin Diseases of the National Institutes of Health under Award Number F31AR082700. The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. 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.