The mechanisms by which muscular dystrophy-related stress is transduced to the autophagic machinery remain poorly characterized. The formulation of strategies should be based on how disruption of these processes results in the deregulation of signaling pathways that contribute to many pathological effects of the disease. In this study, we investigated the molecular mechanism by which the obestatin/GPR39 system, an autocrine signaling with anabolic impact on normal skeletal muscle, restores autophagy in Duchenne muscular dystrophy (DMD). We report that obestatin integrates 5' AMP-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1) signaling to control ubiquitin proteasome system (UPS), autophagy-lysosome system, and protein synthesis under dystrophic context. The posttranslational modifications of the E3 ligase NEDD4-L emerges as the main switch to activate the autophagy in response to obestatin. This includes NEDD4-L tyrosine phosphorylation and autoubiquitination, which is critical for recruiting the ubiquitin-specific protease 10 to assemble a deubiquitination complex, that orchestrates the unc-51 like autophagy activating kinase 1 (ULK1) and class III PI3K (VPS34) complexes. Reactivation of autophagy through obestatin signaling promotes the recovery of physiological skeletal muscle function. Thus, DMD conditions determine permissiveness to the activation of AMPK that sustain autophagy under anabolic conditions stablished by obestatin signaling through mTORC1.

Abstract Muscle fibrosis is a major driver of progression in diverse myopathies, yet the conserved molecular mediators of this process in humans remain poorly defined. Here, we identify collagen VI as a common regeneration-impairing extracellular matrix (ECM) component across three distinct human myopathies: Duchenne Muscular Dystrophy (DMD), Oculopharyngeal Muscular Dystrophy (OPMD), and Inclusion Body Myositis (IBM). Proteomic profiling of fibrotic biopsies reveals consistent upregulation of collagen VI and laminin γ1, alongside disease-specific alterations. Fibroadipogenic progenitors (FAPs) are the predominant source of these ECM components, including collagen VI and laminin γ1. Functionally, xenotransplantation of patient-derived FAPs into regenerating mouse muscle induces localized collagen deposition, myofiber atrophy, and depletion of Pax7⁺ muscle stem cells. Mechanistic assays demonstrate that FAP-derived collagen VI is sufficient to impair myogenic fusion, while silencing COL6 in patient FAPs restores fusion capacity, directly linking pathological collagen VI deposition to regeneration failure. Our findings uncover collagen VI as a conserved effector of fibrosis and stem cell niche disruption in human myopathies, positioning it as a potential therapeutic target across genetically and clinically distinct muscle diseases.

Duchenne Muscular Dystrophy (DMD) is a severe X-linked disorder characterized by progressive degeneration of skeletal and cardiac muscles caused by mutations in the DMD gene encoding dystrophin, a protein essential for cytoskeletal integrity and muscle function. A truncated dystrophin leads to increased muscle susceptibility to contraction-induced damage, driving chronic inflammation and fibrosis. Although corticosteroids remain the standard of care, novel therapeutic strategies are urgently needed. Niclosamide, a long-established anthelmintic drug, has recently been repurposed in inflammatory and fibrotic conditions, including neuromuscular diseases. We investigated the effects of niclosamide in vitro using primary macrophages from mdx mice, human DMD myoblasts, and murine C2C12 myoblast cultures, and in vivo in a proof-of-concept study in mdx mice. In primary mdx macrophages, niclosamide reduced inflammation and reactive oxygen species production, while promoting an anti-inflammatory/pro-regenerative phenotype. In parallel, niclosamide enhanced the differentiation of human DMD myoblasts, and conditioned medium from niclosamide-treated macrophages significantly improved C2C12 myoblast differentiation. In treated mdx mice, niclosamide improved muscle resistance and reduced muscle damage, as indicated by decreased plasma creatine kinase levels and lower immunoglobulin infiltration. These effects were accompanied by modulation of key markers involved in muscle proliferation and differentiation, supporting a beneficial role of niclosamide in promoting muscle repair in dystrophic muscle. Overall, these findings indicate that niclosamide promotes an anti-inflammatory and pro-regenerative environment, enhancing myoblast differentiation and limiting muscle degeneration, supporting its potential role as a promising therapeutic candidate for Duchenne muscular dystrophy.

Muscle defects are common in human developmental disorders and often cause severe functional impairment. These defects arise from intricate tissue crosstalk and rare genetic mutations, underscoring the need to systematically identify cell-autonomous mechanisms regulating human myogenesis. Here we show a rationally designed, high-throughput genetic screening platform that integrates human myoblast models, customized CRISPR libraries, and a split-toxin strategy that enables quantitative selection of fusion-defective myocytes. Leveraging this platform, our initial screen uncovers a large group of hits essential for human myoblast fusion. The majority of these hits converge into 23 protein complexes. Notably, mutations in 41 screen hits are associated with human diseases marked by abnormal skeletal-muscle morphology. Applying a new single-cell CRISPR RNA-seq approach, we show that majority of these hits control human myoblast fusion as well as influence early-stage myogenic differentiation. This work establishes a scalable approach to identify cell-autonomous regulators of human muscle differentiation and fusion. Here, the authors present a high-throughput genetic screen to identify genes that regulate human myoblast fusion.

Patient-derived cell models of dystrophic myogenesis and differentiation are valuable preclinical tools for early and mutation-based assessment of candidate therapeutic approaches. Quantitative measurement of gene expression within such models plays a key role in these studies, but normalisation of RT-qPCR data requires a panel of validated stably expressed reference genes. This study aims to identify stable reference genes for RT-qPCR assays in three human derived muscle immortalized cell lines: one healthy WT (from a 16-year-old donor), and two dystrophic lines, DMD1 (from a 11-year-old patient carrying a stop codon mutation on exon 59) and DMD2, from a 14-year-old patient carrying an exon 48-50 deletion. We screened a pool of 14 candidate genes (ACTB, HPRT1, RPL13A, RPS18, GAPDH, ALAS1, UBC, YWHAZ, IPO8, PSMC4, HSP90AB1, NONO, CSNK2A2, AP3D1), investigating stability of expression from proliferation through to 11 days of myogenic differentiation. Data were analysed using four complementary approaches (Bestkeeper, geNorm, Normfinder and DeltaCt) to determine the most appropriate references both within and between cell lines. Our study shows that RPS18, UBC, YWHAZ scored highly across all comparisons, and we therefore suggest that these three genes represent an appropriate reference panel for these human myogenic cell lines, regardless of genotype or differentiation stage.

Zika virus (ZIKV) infection has emerged as a global public health emergency due to its expansion capacity and ability to cause neurological and congenital diseases. Muscle cells are targets for ZIKV, and myalgia and muscle disorders are frequently related symptoms during infection. We have previously demonstrated that myoblasts, the proliferating muscle stem cells essential for muscle repair, are permissive to ZIKV infection, generating infectious viral particles. In contrast, differentiated myotubes, derived from myoblast differentiation and fusion, control ZIKV replication. Nevertheless, little is known about the impact of ZIKV infection on muscle myogenesis. Using an in vitro model of skeletal muscle regeneration, human myoblasts were infected with the ZIKV-Rio-U1 strain, and their proliferation, adhesion, migration, and differentiation/fusion properties were analyzed 72 hours post-infection. We found that ZIKV replicates within myoblasts, promoting biological alterations such as the inhibition of cell cycle progression, preventing cell proliferation. Infected myoblasts exhibit poor adhesion, lack of membrane elongation, a reduced cell area, and decreased migratory capacity. Moreover, infection impaired the fusion of human myoblasts. Although differentiated and fused myotubes control ZIKV infection, proliferating infected myoblasts present an altered myogenic program. These results strongly suggest that ZIKV infection can affect myogenesis, modulating key biological processes crucial for skeletal muscle differentiation and regeneration. Accordingly, it is conceivable that ZIKV infection may impact myogenesis during embryogenesis, growth, and subsequent regenerative episodes during the adult period.

Inherited myopathies are genetic disorders characterised by declining motor function due to progressive muscle weakening and wasting. Recently, pathogenic variants in PAX7, the master transcriptional regulator of muscle stem cells, have been associated with myopathies of variable severity, arguing for impaired satellite cell function as the main pathogenic driver. Here, we report the characterisation of two missense PAX7 variants in a patient with asymmetric, progressive muscle weakness affecting facial, upper and lower body muscles, and myopathic changes on muscle pathology. Despite this disorder closely phenocopying the clinical presentation of Facioscapulohumeral muscular dystrophy (FSHD), genetic, epigenetic and transcriptomic profiling indicated that FSHD was unlikely. However, exome sequencing revealed two heterozygous variants in PAX7: c.335 C > T, (p.Pro112Leu) and c.1328 G > A (p.Cys443Tyr). Modelling these PAX7 variants in human myoblasts resembled the transcriptomic findings found in the muscle biopsy from the patient. Specifically, these PAX7 variants caused upregulation of splicing factors, an increase in mitochondrial reactive oxygen species levels and reduced cell proliferation. The phenotypic cell changes caused by the PAX7 variants support a pathomechanism whereby diminished satellite cell function impairs muscle homoeostasis. Together, multimodal investigation suggests that these variants in PAX7 are likely causative of an FSHD-like autosomal recessive myopathy and expand the spectrum of neuromuscular disorders originating from impaired satellite cell function.

Muscle disorders such as myofibrillar myopathies and Duchenne muscular dystrophy involve mutations in key cytoskeletal proteins and lead to progressive muscle degeneration. Yet, the mechanical characterization of affected muscle cells has relied mainly on immature or non-human models. Here, we introduce a human in vitro platform based on patient-derived immortalized myoblasts differentiated into myotubes on nanogrooved substrates, which promote alignment and organotypic maturation. Using immunostaining and atomic force microscopy (AFM), we show that desmin- and dystrophin-mutated myotubes exhibit distinct morphological and mechanical phenotypes compared to wild-type myotubes. We developed an AFM stiffness pipeline to quantify cell body stiffness across myotubes of variable thickness. Desmin- and dystrophin-mutated myotubes are stiffer than controls, with desmin mutants also displaying cytoskeletal disorganization. A dynamic fatigue assay (cyclic AFM indentations over time) further revealed impaired stiffening and faster mechanical fatigue in desmin mutants, while dystrophin mutants preserved resilience. This set of results establishes a reproducible and human-relevant system to probe muscle mechanics in disease, offering a unique intermediate model between conventional immortalized lines and complex iPSC-derived tissues, and enabling future quantitative screening and translational applications.

Background: Three-dimensional skeletal muscle organoids (3D SkMO) are becoming of increasing interest for preclinical studies in Duchenne muscular dystrophy (DMD), provided that the used platform demonstrates the possibility to form functional and reproducible 3D SkMOs, to investigate on potential patient-related phenotypic differences. Methods: In this study, we employed fibrin-based 3D skeletal muscle organoids derived from immortalized myogenic precursors of DMD patients carrying either a stop codon mutation in exon 59 or a 48–50 deletion. We compared dystrophic lines with a healthy wild-type control (HWT) by assessing microtissue formation ability, contractile function at multiple timepoints along with intracellular calcium dynamics via calcium imaging, as well as expression of myogenic markers. Results: We found patient-specific structural and functional differences in the early stages of 3D SkMO development. Contractile force, measured as both single twitch and tetanic responses, was significantly lower in dystrophic 3D SkMOs compared to HWT, with the most pronounced differences observed at day 7 of differentiation. However, these disparities diminished over time under similar culturing conditions and in the absence of continuous nerve-like stimulation, suggesting that the primary deficit lies in delayed myogenic maturation, as also supported by gene expression analysis. Conclusions: Our results underline that, despite the initial maturation delay, DMD muscle precursors retain the capacity to form functional 3D SkMOs once this intrinsic lag is overcome. This suggests a critical role of dystrophin in early myogenic development, while contraction-induced stress and/or an inflammatory microenvironment are essential to fully recapitulate dystrophic phenotypes in 3D SkMOs.

ABSTRACT Background Cystinosis is a rare multisystem, autosomal recessive disease caused by dysfunction or loss of cystinosin (CTNS), which results in lysosomal cystine accumulation, primarily affecting the kidneys. Advances in renal transplantation, cysteamine treatment and improved medical care have increased life expectancy, revealing additional systemic phenotypes like myopathy later in life. Muscle weakness is a major concern leading to life‐threatening events in patients, and yet the aetiology of cystinosis myopathy remains to be elucidated. Methods We generated human muscle cell‐based models using CRISPR technology to explore the pathophysiology of cystinosis myopathy with the potential to develop new therapies. We used a 4‐day differentiation protocol of myoblasts into myotubes to study the effect of CTNS loss in key regulators of myogenic differentiation using western blot analysis. Afterwards, we used lentiviral (LV)‐mediated CTNSWT cDNA addition in CTNS−/− cells to corroborate the CTNS‐specific effect. As a next step, we performed multiomic analysis (proteomics, transcriptomics and metabolomics) to gain in‐depth knowledge of affected mechanisms. Results The polyclonal, isogenic human CTNS knock‐out (KO; CTNS−/−) myoblasts exhibited unaltered growth characteristics and accumulated cystine. Early‐stage differentiation of myoblasts into myotubes showed a mild reduction in the fusion index of CTNS−/− myotubes. Upon examination of several key regulators of myogenic differentiation, we observed significantly decreased myosin heavy chain (MyHC) and ryanodine receptor (RyR) protein levels in CTNS−/− myotubes compared to WT cells. Complementation with CTNSWT cDNA addition in CTNS−/− cells rescued the fusion index, cystine and altered protein levels to WT. In addition, proteomic analysis showed no differences at myoblast level upon the loss of CTNS, but following myotube differentiation, CTNS deletion led to an increase of five protein groups mainly involved in oxidative stress pathways, and a decrease of 18 protein groups biologically connected in myofibril assembly and muscle cell differentiation processes. Importantly, LV‐mediated CTNS addback reverted protein levels to WT levels. Moreover, metabolomics revealed a distinct clustering resulting from CTNS loss. Conclusions Muscle‐specific complications are often overlooked in systemic cystinosis treatment. We show that defective CTNS function impairs effective cystine mobilization from lysosomes, thereby affecting the protein levels of myogenic regulators. A deeper understanding of the molecular mechanisms underlying cystinosis myopathy holds promise for the development of targeted, personalized therapies to improve the quality of life for patients living with cystinosis.

Muscle cell death in muscular dystrophies depends upon calcium ion (Ca++) leakage through sarcolemma and sarcoplasmic reticulum, triggered by muscle stretch during eccentric contraction. We show here that Ca++ spikes are detected in dystrophic myogenic cells in culture since early differentiation, before sarcomere assembly and contraction. Healthy and genetically corrected dystrophic myotubes do not display Ca++ spikes which are blocked by co-culturing DMD myogenic cells with embryonic mouse motoneurons or treating them with agrin proteoglycan. Same effect is elicited by a muscle spliced, COOH peptide of agrin (termed here mini-agrin) that interacts with dystroglycan, favouring its binding to the basal lamina. Lack of dystrophin in DMD myotubes results in decreased expression of CaV1.1 (CACNA1S), a Ca++ sensor component of the Dihydropyridine Receptor (DHPR) complex, known to regulate Ryanodine Receptor 1 (RyR1). These events explain the emergence of Ca++ spikes. Mini-agrin addition to medium, or lentivector-mediated mini-agrin expression in transplanted cells in vivo, stabilize the expression of CaV1.1 on the membrane. This leads to disappearance of Ca++ spikes and to reappearance of α-dystroglycan, α-sarcoglycan and n-NOS, indicating the reconstitution of the dystrophin complex in the absence of dystrophin. These findings unveil a novel regulatory mechanism and offer a new therapeutic opportunity for targeting calcium ion influx as a co-treatment strategy. ### Competing Interest Statement The authors have declared no competing interest. Medical Research Council, https://ror.org/03x94j517, “MR/X00466X/1, MICA-Strategy for heart repair in Duchenne Muscular Dystrophy using genetically engineered autologous Mesoangioblasts”

ABSTRACT Background Systemic muscle wasting is a prevalent condition that predicts adverse health outcomes in aging and disease. Despite its clinical relevance, the development of predictive biomarkers and effective pharmacological therapies remains limited. Peptides have recently gained attention for their diverse bioactive functions, positioning them as promising biomarkers and therapeutic agents for muscle wasting. Methods This scoping review systematically identifies studies examining the direct association between well‐defined peptides and clinical components of muscle wasting: muscle mass, strength and physical performance. The review follows the Preferred Reporting Items for Systematic Reviews and Meta‐Analysis for Scoping Reviews (PRISMA‐ScR) guidelines. A comprehensive search of Embase, PubMed and Web of Science was conducted up to 31 October 2024, focusing on original human or animal studies. Studies involving congenital or inherited muscle disorders, inflammatory myopathies and neurodegenerative diseases, such as Parkinson's disease, were excluded. A snowball approach was used to synthesize the presumed cellular pathways of identified peptides. Results A total of 126 studies were included: 71 (56.3%) focused on a single muscle wasting component (48 on mass, 16 on strength and 7 on performance), 31 (24.6%) examined two, 16 (12.7%) analysed all three separately, and 8 (6.3%) assessed sarcopenia as a categorical variable. Eighty‐seven distinct peptides linked to muscle wasting were identified, ranging from collagen tripeptide (3 amino acids) to insulin (51 amino acids). The most studied peptides are ghrelin (14.3%), brain natriuretic peptide (BNP, 11.1%), C‐peptide (11.1%), insulin (10.3%) and Szeto‐Schiller 31 (SS‐31, 6.3%). Most (62.1%) influence one or more of four key muscle homeostasis pathways (PI3K/Akt/mTOR, ActR/SMAD, IKK/NF‐κB and AMPK/PGC1α), which regulate atrophy (via FOXO, NF‐κB, SMAD2/3, glucocorticoid receptor and GSK‐3β) and hypertrophy (via androgen receptors, PGC‐1α and S6K). Flaws in study design and reporting were prevalent, hindering clinical translation. Sex bias was evident, with females comprising 23.9% of participants in human interventional studies and only 9.1% and 12.4% of mice and rats in rodent studies, respectively. Clinical, pre‐analytical and analytical reporting gaps were common: 56.6% documented diurnal timing, food intake and activity around peptide collection; none specified storage‐to‐analysis duration; and only 11.5% reported detection limits for peptide measurements. Conclusion This scoping review highlights the potential of peptides as biomarkers and intervention targets for muscle wasting. It connects the cellular receptors and signaling pathways linking peptides with skeletal muscle wasting. Improving clinical translation requires addressing study design limitations, incorporating more representative study populations and adhering to standardized reporting guidelines. The application of machine learning can support the identification of novel bioactive peptides.

Recently, the repertoire of human small nucleolar noncoding RNAs (snoRNAs) and their potential functions has expanded with the discovery of new snoRNAs and messenger RNA (mRNA) targets, for which snoRNA-guided modifications may influence their stability, translatability, and splicing. We previously identified snoRNAs that are abundant in healthy human muscle progenitor cells. In this study, we demonstrated that SNORA40 and SNORA70 loss-of-function impairs myogenic differentiation. Interestingly, gain-of-function can rescue impaired differentiation muscle progenitor cells in myotonic dystrophy type 1 (DM1). We identified cyclin D3 (CCND3) mRNA, which is partially located in the nucleolus, as a target for SNORA40 and SNORA70, which are required for its pseudouridylated status. Expression of the CCND3 protein is required for muscle progenitors to exit the cell-cycle when they are induced to differentiate. We revealed that this switch requires SNORA40/70. Finally, we observed that DM1 cells show reduced levels of SNORA40/70 and undetectable CCND3 protein. However, restoring normal levels of SNORA40/70 partially restored CCND3 protein expression, coinciding with improved cell fusion capacity in DM1 muscle progenitors. Collectively, these data suggest that this effect may stem from SNORA40/70-dependent pseudouridylation of CCND3 mRNA, emphasizing snoRNAs as key players in normal and pathological muscle differentiation.

Laminin 111 (LM-111) is an extracellular matrix (ECM) glycoprotein found in basement membranes and proposed for muscle disease therapy. LM-111 treatment reduces muscle damage, restores muscle strength, alleviates inflammation, and promotes regeneration in murine and canine dystrophic models. LM-111 also improves myoblast transplantation (MT) efficacy by inducing higher proliferation, survival, dispersion, and differentiation of transplanted myoblasts. LM can undergo partial proteolysis and produce peptides called matrikines that modulate cell activity and trigger distinct biological responses from the full-length glycoproteins. In this study, we investigated the biological activity of the HuAG73 peptide, derived from the human LM, on human myoblasts, both in vitro and in vivo, using immunodeficient mice. The HuAG73 peptide offers a significant advantage over LM-111 due to its smaller size and simpler structure. HuAG73 promoted adhesion, proliferation, migration, and fusion of human myoblasts in culture. It also mimicked LM-111 in an MT assay. In conclusion, HuAG73 is a novel, relevant therapeutic candidate molecule for treating muscle diseases.

Muscle fibrosis is a major driver of progression in diverse myopathies, yet the conserved molecular mediators of this process in humans remains poorly defined. Here, we identify collagen VI as a common pro-fibrotic and regeneration-impairing extracellular matrix (ECM) component across three distinct human myopathies: Duchenne Muscular Dystrophy (DMD), Oculopharyngeal Muscular Dystrophy (OPMD), and Inclusion Body Myositis (IBM). Proteomic profiling of fibrotic biopsies revealed consistent upregulation of collagen VI and laminin γ1, alongside disease-specific alterations involving other ECM proteins. Fibro-adipogenic progenitors (FAPs) are the predominant source of these ECM components, including collagen VI and laminin γ1. Functionally, xenotransplantation of patient-derived FAPs into regenerating mouse muscle induced localized collagen deposition, myofiber atrophy, and depletion of Pax7⁺ muscle stem cells. Mechanistic assays demonstrated that FAP-derived collagen VI is sufficient to impair myogenic fusion, while silencing COL6 in patient FAPs restores fusion capacity, directly linking pathological collagen VI deposition to regeneration failure. Our findings uncover collagen VI as a conserved effector of fibrosis and stem cell niche disruption in human myopathies, positioning it as a potential therapeutic target across genetically and clinically distinct muscle diseases.

Background/Objectives: Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named “on-target, off-tumor toxicity”. As tumors generate an acidic microenvironment, we investigated whether we could generate pH-dependent antibodies to increase their tumor specificity. For this proof-of-concept study, we selected the tyrosine kinase receptor AXL because we already developed several antibodies against this target. Methods: To generate a pH-dependent anti-AXL antibody, we performed classical panning of a single-chain variable fragment (scFv) library using phage display at an acidic pH throughout the process. Results: After the third round of panning, 9 scFvs, among the 96 picked clones, bound to AXL at acidic pH and showed very low binding at a neutral pH. After reformatting them into IgG, two clones were selected for further study due to their strong pH-sensitive binding. Using molecular docking and alanine scanning, we found that their binding strongly depended on two histidine residues present on AXL at positions 61 and 116. Conclusions: To conclude, we set-up an easy process to generate pH-dependent antibodies that may increase their tumor-binding specificity and potentially decrease toxicity towards healthy tissues.

The skeletal muscle tissue has a remarkable capacity of growth and regeneration. Fusion of myoblasts and myotubes elongation are fundamental processes in muscle development. Previous studies have depicted impaired myogenic processes in animal models and myoblast from human patients with muscle diseases. Here, we evaluated the myogenesis in patients with Limb-girdle Muscle Dystrophy 2B (LGMD2B). Aiming to explain why dysferlin-deficient muscle cells lose its myogenic potential, we used immortalized myoblasts from LGMD2B patients and a cellular DYSF knocking-out model. Myotubes from patients were smaller and containing less myonuclei than control myotubes. Main muscle regulatory factors expression were not altered in these cells. The analysis of the expression of newly described genes associated with muscle fusion and growth, such as MYMK, MYMX, PALMD, SHISA2, COL25A, didn´t show any difference with controls, which is a novel finding. It was also observed that dysferlin deficiency doesn’t alter the expression of FAM65B and HDAC6 genes, components of a proposed protein complex that needs to be formed to allow muscle differentiation. Interestingly, morphometric analysis of DYSF knock-out myotubes induced by CRISPR/Cas9 also revealed reduced myogenic capacity with formation of smaller myotubes. These findings suggest that the absence of DYSF itself is sufficient to impair muscle formation in vitro, and that downstream gene and protein expression related to muscle development might depend on the presence and proper function of dysferlin.

Duchenne muscular dystrophy (DMD) is a genetic, progressive neuromuscular disease caused by mutations in the dystrophin protein which compromise the integrity of the sarcolemma. Current care of DMD involves both supportive and targeted disease modifying medications. Obestatin, a peptide derived from preproghrelin, is a potential candidate to enhance existing treatments for DMD. This study was conducted to analyse the molecular mechanism by which obestatin acts on myofiber metabolism and muscle restructuring in DMD. Through human and animal models of DMD, we identify the calcium-activated protein phosphatase 3 (PPP3) as key node in obestatin signalling for restoration of muscle homeostasis and activation of membrane repair. In particular, we describe how obestatin signalling recovers muscle function by coordinated activation of the transcription factor EB (TFEB) and the nuclear factor of activated T cell (NFATc1) in which PPP3 is a core component. TFEB dephosphorylation triggers its nuclear translocation and the activation of macroautophagic/autophagic and mitochondrial biogenesis. NFATc1 promotes the slow myofiber phenotype fibre marker utrophin. Overall, obestatin treatment ameliorates distinctive dystrophic features of DMD, including muscle contractile damage, elevated serum creatine kinase levels, and reduced muscle force. Hence, obestatin represents a promising therapeutic approach for treating DMD, not only as monotherapy but also as part of combinatorial treatment strategies aimed at overcoming the barriers that limit the efficacy of gene or cell therapy.

Muscle regeneration is governed by a complex interplay between immune cells and satellite cells (muscle progenitors), orchestrated by signaling molecules of the TGF-β superfamily. Among these, the role of GDF11 activity in skeletal muscle remains contentious, with conflicting evidence suggesting both stimulatory and inhibitory effects. This functional divergence may emerge from the combinatorial activities of its shared type I receptors and context-dependent activation of downstream SMADs. To dissect the role of GDF11 in skeletal myogenesis, we employed a combination of biochemical stimulation and CRISPR-based genetic approaches in chicken or human myoblasts. Analysis of cell proliferation, differentiation, adhesion, and migration revealed that GDF11 does not affect myoblast proliferation or adhesion, but strongly inhibits myotube differentiation and myoblast migration. Furthermore, loss of ACVR1B (ALK4) strongly delays myoblast differentiation, and impairs cell adhesion and migration on laminin-111 (LM111), a known ligand of the integrin VLA-6. Notably, flow cytometry phenotyping demonstrated that ACVR1B -deficient myoblasts exhibit reduced surface levels of the integrin α6 subunit (CD49f) compared to wild-type cells. Together, our findings suggest a GDF11-independent ALK4/VLA6/LM111 axis governing skeletal myoblast adhesion and fusion. Knowledge of these receptor interactions is critical for understanding GDF11’s paradoxical role in muscle cell biology and may inform novel therapeutic strategies to counteract skeletal muscle degeneration and age-related decline.