Biology subjects

Bigot, A.

Publications and source records attributed to Bigot, A..

17 recordsLinked to original sources

bioRxiv2026

Aging remodels the human muscle secretome, impairing stem cell via sphingolipid metabolism

Aging reduces skeletal muscle regenerative capacity, but how the aged environment impairs muscle stem cells (MuSCs) remains unclear. Skeletal muscle acts as a secretory organ, yet age-related changes in its secretome and impact on stem cells are poorly understood. We performed proteomic and lipidomic analyses of conditioned media from primary muscle cells of young (15-25 years) and aged (60-86 years) donors. We identified 127 age-dependent secreted proteins, revealing major remodeling. The lysosomal enzyme acid sphingomyelinase (ASM), which converts sphingomyelin into ceramide, was the most upregulated factor. Recombinant ASM reduced PAX7+ MuSCs in young cultures, showing extracellular ASM impairs stem cell maintenance. Lipidomics revealed disruption of the sphingomyelin-ceramide-S1P axis, with decreased sphingomyelin and S1P, a lipid promoting MuSC proliferation. Exogenous S1P restored the stem cell pool in aged cultures. These findings link secretome remodeling and sphingolipid metabolism to MuSC decline and identify ASM and S1P as targets

cell biology↗

bioRxiv2026

Plasma proteomics identifies an IL-6–associated SAA axis linked to muscle wasting in patients with cancer cachexia

Nearly half of patients with advanced lung cancer develop cachexia, a debilitating syndrome that worsens prognosis. We conducted longitudinal clinical and plasma proteomic profiling of 67 patients with non-small cell lung cancer, with and without cachexia, during first-line treatment. Patients with cachexia at diagnosis exhibited elevated risk of hospitalization and treatment-delaying toxicity. At diagnosis, 128 plasma proteins were upregulated and 67 downregulated in cachectic relative to non-cachectic patients. Longitudinal assessments of body composition, physical performance, metabolism, clinical outcomes, and nutritional risk revealed distinct fat and muscle wasting phenotype trajectories. 71 proteins were associated with fat loss, 92 with muscle loss, and 177 with concurrent muscle and weight loss. We identified and functionally validated 8 plasma proteins linked to muscle loss and adverse clinical outcomes. In a separate cohort of 147 patients with advanced pancreatic cancer receiving the interleukin-6 (IL-6) inhibitor tocilizumab, pharmacological suppression of serum amyloid A (SAA) levels following IL-6 inhibition suggests a systemic IL-6-SAA axis. These results collectively highlight SAA1 and SAA2 as IL-6-driven, cachexia-associated factors that reduce human myotube width. These findings uncover new potential therapeutic targets for cachexia.

Systems Biology↗

bioRxiv2026

Patient-derived 3D engineered human muscle model recapitulates CLCN1 mis-splicing and myotonia in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) lacks human in vitro models that directly link RNA toxicity to mature skeletal muscle function, particularly myotonia. Here, we engineer contractile 3D human skeletal muscle tissues from immortalized myoblasts derived from three DM1 patients representing juvenile, adult, and late-onset subtypes. These tissues reproduce key molecular features of DM1, including nuclear RNA foci, MBNL1 sequestration, and widespread mis-splicing. Functionally, DM1 tissues exhibit impaired calcium handling, subtype-dependent weakness, rapid fatigue, and a fiber-type distribution characterized by increased slow type I fibers and pathological MyHC-I/IIx hybrids. Notably, the 3D environment enables expression and complete pathogenic mis-splicing of CLCN1--undetectable in matched 2D cultures--accompanied by myotonia-like delayed relaxation. Using this model, we assessed therapeutic responses of candidate small-molecule modulators. Phenylbutazone reduced RNA foci and MBNL1 sequestration but failed to rescue spliceopathy or function. In contrast, calcitriol induced coordinated transcriptomic remodeling and robustly rescued myotonia-like relaxation despite persistent CLCN1 mis-splicing. These findings establish a functionally mature human DM1 muscle model and highlight compensatory network activation as a strategy to improve muscle function in DM1.

bioengineering↗

bioRxiv2025

Genomic stress drives activation of interferon signaling and innate immune pathways during SMA Type II myoblasts differentiation

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of the SMN1 gene. The SMN protein is ubiquitously expressed and has several roles in the cell, including the regulation of RNA metabolism and genome stability. Although SMA is primarily considered a motor neuron disease, because of its ubiquitous expression, loss of SMN leads to systemic pathological consequences, including in skeletal muscle. To better understand the cell-intrinsic effects of SMN loss on myoblast differentiation, we characterized three human SMA Type II myoblast cell lines and controls in vitro. We observed impaired myogenic differentiation, including reduced fusion index and nuclei alignment. RNA sequencing analysis on the SMA myoblasts revealed activation of DNA damage pathways and innate immune activation, confirmed by the presence of 53BP1 foci and increased phosphorylation of the H2AX histones, as well as accumulation of R-loop structures and an increase of single-stranded DNA in the cytoplasm. Resolution of R-loops and DNA damage is known to cause the presence of immunogenic DNA: RNA species in the cytoplasm, activating sterile inflammation pathways. Treating control myoblasts with double-stranded DNA or a cGAS agonist phenocopied the differentiation defects in SMA myotubes. Conversely, treating SMA myoblasts with a STING inhibitor improved fusion. In summary, our results demonstrate that SMN loss exerts a cell-intrinsic impairment in muscle precursor cells, suggesting that the observed muscle dysfunction in Type II patients is not only a consequence of impaired innervation but also of the sterile inflammation caused by the genomic instability after SMN loss.

cell biology↗

bioRxiv2025

Mini-agrin prevents calcium leakage and restores the dystrophin complex.

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 -dystroglycan, -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.

cell biology↗

bioRxiv2025

Collagen VI is a conserved pro-fibrotic signal disrupting muscle regeneration across distinct human myopathies

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 {gamma}1, 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 {gamma}1. 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.

cell biology↗

bioRxiv2025

EMILIN1 emerges as a TGFbeta/SETDB1-regulated secreted biomarker in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is an incurable muscle-wasting disorder characterized by chronic membrane damage, inflammation, and progressive fibrosis. Fibrosis in DMD is driven by sustained TGF{beta} signaling, which promotes extracellular matrix (ECM) accumulation. We previously showed that SETDB1 sustains the TGF{beta}-induced fibrotic response in DMD myotubes. Here, we further show that SETDB1 modulates the TGF{beta}-induced secretome, particularly by regulating ECM-related proteins. Comparison of the basal secretome from DMD patient-derived myotubes and healthy controls revealed a distinct disease-specific profile. Integrating both secretome analyses, we identified EMILIN1, an ECM glycoprotein not previously studied in skeletal muscle, as a robust shared candidate; EMILIN1 is enriched in the DMD secretome, further upregulated by TGF{beta}, and downregulated upon SETDB1 depletion. We confirmed EMILIN1 overexpression in DMD patient muscle biopsies, validating its pathological relevance. Functionally, EMILIN1 depletion modulated myogenic differentiation and reduced expression of the fibrotic marker SERPINE1. These findings establish EMILIN1 as a novel secreted regulator of myogenesis and fibrosis, and implicate SETDB1 in shaping the TGF{beta}-dependent secretome in DMD. Our integrative proteomic approach provides new insights into the molecular drivers of impaired regeneration in DMD and highlights potential therapeutic targets.

genetics↗

bioRxiv2025

Characterisation of DMPK and MBNL1 expression in cell models of Myotonic Dystrophy: A platform for drug screening

Myotonic dystrophy type I (DM1) is caused by CTG repeat expansions in the DMPK gene leading to mRNA toxicity and sequestration of the splicing regulator MBNL1, affecting many tissues. We have developed an in vitro screening platform based on ddPCR and in-cell western to quantify these mRNAs and proteins and characterised more than 20 cell models to define DM1 biomarkers that could be useful for drug screening. DMPK protein levels were reduced in DM1-immortalised myoblasts and myotubes, but not in fibroblasts, while MBNL1 protein was consistently lower in all DM1 myogenic cultures, whether primary or immortalised. Myogenic differentiation of cultures led to an increase in DMPK mRNA expression, which was translated into increased MBNL1 sequestration in foci. We further corroborated the platforms ability to assess therapeutic outcomes, evaluating the effect of a DMPK gapmer ASO and one siRNA: while the gapmer increased MBNL1 protein levels, the siRNA had no significant effect on MBNL1 release. Our platform and the in-depth characterisation of some of the most used models would be of use to the DM1 research community. Significance statementMyotonic dystrophy type I (DM1) is a multisystemic disease with a complex pathogenesis and multiple outcome measures for drug assessment in vitro. In the last years, the increasing number of new potential therapies targeting DM1 in clinical trials has increased the need for robust and rapid evaluation of preclinical candidates, as well as in-depth knowledge of the cell models used. Here, we present a new cell-based platform that enables robust quantification of DMPK and MBNL1 in cell culture for cell model characterisation and drug screening. Indeed, we highlight the differences observed in DMPK and MBNL1 protein quantification in primary fibroblasts and myotubes, immortalised fibroblasts, myoblasts and myotubes. We then, we performed a proof-of-concept drug evaluation of potential therapeutic strategies targeting DMPK, showing the most suitable for targeting the DMPK expanded transcript.

cell biology↗

bioRxiv2024

Identification of bazedoxifene for the treatment of LGMD R2 by high throughput screening.

LGMD R2 is a rare genetic disorder characterized by progressive proximal muscle weakness and wasting caused by a recessive loss of function of dysferlin, a transmembrane protein controlling plasma membrane repair in skeletal muscles. We report here the development of an in vitro high-throughput assay using immortalized myoblasts and monitored reallocation of an aggregated mutant form of dysferlin (DYSFL1341P). Using this assay, we screened a library of 2239 drugs and identified two autophagy inducers, namely saracatinib and bazedoxifene, as potential drugs to repurpose for LGMD R2 patients carrying the DYSFL1341P mutation. Functional characterization of these drugs revealed that saracatinib and bazedoxifene had a protective effect on the plasma membrane in osmotic shock assay. While saracatinib restores functionality in membrane resealing through a specific rescue of L1341P dysferlin from degradation, bazedoxifene demonstrates an additional protective effect on dysferlin KO mice muscle fibers. Finally, further investigations into the molecular mechanism of action of bazedoxifene revealed an induction of autophagy flux, which may underlie the molecules effect on the survival of LGMD R2 myofibers.

pharmacology and toxicology↗

bioRxiv2023

Fibroblasts-dependent maturation and phenotype exacerbation of dystrophic hiPSC-derived MYOtissues enables muscle strength evaluation for gene therapy screening

Current gene therapy approaches for Duchenne muscular dystrophy (DMD) using AAV-mediated delivery of microdystrophin ({micro}Dys) have shown limited efficacy in patients, contrasting with the favorable outcomes observed in animal models. This discrepancy is partly due to the lack of models that replicate key pathogenic features associated with the severity of the human disease, such as fibrosis and muscle dysfunction. To tackle the translational gap, we develop a human disease model that recapitulates these critical hallmarks of DMD for a more predictive therapeutic investigation. Using a muscle engineering approach, we generate MYOrganoids from iPSC-derived muscle cells co-cultured with fibroblasts that enable functional maturation for muscle force analysis upon contractions. Incorporation of DMD fibroblasts within DMD iPSC-derived muscle cells allows phenotypic exacerbation by unraveling of fibrotic signature and fatiguability through cell-contact-dependent communication. Although {micro}Dys gene transfer partially restores muscle resistance, it fails to fully restore membrane stability and reduce profibrotic signaling. These findings highlight the persistence of fibrotic activity post-gene therapy in our human DMD system, an unparalleled aspect in existing DMD models, and provide the opportunity to explore the underlying mechanisms of dysregulated cellular communication to identify anti-fibrotic strategies empowering gene therapy efficacy.

bioengineering↗

bioRxiv2022

Dystrophin is a mechanical tension modulator

Duchenne muscular dystrophy (DMD) represents the most common inherited muscular disease, where progressing muscle weakness leads to loss of ambulation and premature death. DMD is caused by mutations in the dystrophin gene, and is known to reduce the contractile capacity of muscle tissue both in vivo, and in reconstituted systems in vitro. However, these observations are based on mechanical studies that focused on stimulated contractions of skeletal muscle tissue. Seemingly paradoxical, upon evaluating bioengineered skeletal muscles produced from DMD patient derived myoblasts we observe an increase in myosin motor mediated homeostatic tissue tension that strongly correlates with decreased stimulated tissue strength, suggesting the involvement of dystrophin in regulating the baseline homeostatic tension level of tissue. This was confirmed by three independent pairs of isogenic cell lines, one of each lacking dystrophin compared to the isogenic dystrophin-expressing control. From this we speculate that the protective function of dystrophin also supports cellular fitness via active participation in the mechanosensation to achieve and sustain an ideal level of tissue tension. Hence, this study reveals fundamental novel insights into skeletal muscle biomechanics and into a new key mechanical aspect of DMD pathogenesis, provided by increased homeostatic tissue tension.

biophysics↗

bioRxiv2022

Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, caveolae composed of caveolin-3 and Bin1 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Overexpression of Bin1 lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations cause defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1.

cell biology↗

bioRxiv2021

Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip For Modelization of Muscular Dystrophies

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for muscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which is problematic in the context of limited patient sample. Here we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments. Spontaneous contractions in myotubes derived from primary human myoblasts were observed. Analysis of nuclear morphology confirmed a similar organization between obtained myotubes and in vivo myofibers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modelled with successful development of mutant 3D myotubes displaying contractile dysfunction. This technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should allow to substantially improve drug screening capability.

bioengineering↗

bioRxiv2019

The SMYD3 methyltransferase promotes myogenesis by activating the myogenin regulatory network

The coordinated expression of myogenic regulatory factors, including MyoD and myogenin, orchestrates the steps of skeletal muscle development, from myoblast proliferation and cell-cycle exit, to myoblast fusion and myotubes maturation. Yet, it remains unclear how key transcription factors and epigenetic enzymes cooperate to guide myogenic differentiation. Proteins of the SMYD (SET and MYND domain-containing) methyltransferase family participate in cardiac and skeletal myogenesis during development in zebrafish, Drosophila and mice. Here, we show that the mammalian SMYD3 methyltransferase coordinates skeletal muscle differentiation in vitro. Overexpression of SMYD3 in myoblasts promoted muscle differentiation and myoblasts fusion. Conversely, silencing of endogenous SMYD3 or its pharmacological inhibition impaired muscle differentiation. Genome-wide transcriptomic analysis of murine myoblasts, with silenced or overexpressed SMYD3, revealed that SMYD3 impacts skeletal muscle differentiation by targeting the key muscle regulatory factor myogenin. The role of SMYD3 in the regulation of skeletal muscle differentiation and myotube formation, partially via the myogenin transcriptional network, highlights the importance of methyltransferases in mammalian myogenesis.

cell biology↗

bioRxiv2019

A gold standard method for human cell quantification after xenotransplantation

Xenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell-and gene-based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space.\n\nThese two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.

cell biology↗

bioRxiv2018

Clathrin plaques form mechanotransducing platforms

Large flat clathrin plaques are stable features of the plasma membrane associated with sites of strong adhesion suggesting that they could also play a role in force transduction. Here, we analyzed how clathrin plaques interact with the cytoskeleton and how they respond to mechanical cues in skeletal muscle myotubes. We show that branched actin networks surrounding clathrin plaques are directly regulated by dynamin 2, anchor intermediate filaments and sequester YAP at the plasma membrane. Dynamin 2, clathrin and desmin intermediate filaments are all required for basal YAP nucleocytoplasmic distribution and efficient nuclear translocation in response to mechanical stimuli. Dynamin 2 mutations that are responsible for centronuclear myopathy in humans disorganize the desmin network and deregulate YAP signaling both in vitro and in vivo. Thus, clathrin plaques and associated dynamin 2 are defined here as a new sensor conveying mechanical cues and integrate cell signaling with cytoskeletal regulation.

cell biology↗

bioRxiv2018

Lack of functional caveolae in Cav3 mutated human dystrophic myotubes results in deficient mechanoprotection and IL6/STAT3 mechanosignaling.

Caveolin-3 is the major structural protein of caveolae in muscle cells. Mutations in the CAV3 gene cause different type of muscle disorders mostly characterized by defects in membrane integrity and repair, deregulation in the expression of various muscle proteins and deregulation of several muscle associated signaling pathways. We show here that myotubes derived from patients bearing the CAV3 P28L and R26Q mutations present a lack of functional caveolae at the plasma membrane which results in an abnormal mechanoresponse. Mutant myotubes can no longer buffer the increase of membrane tension induced by mechanical stress and present an hyperactivation of the IL6/STAT3 signaling pathway at rest and under mechanical stress. The impaired mechanical regulation of the IL6/STAT3 signaling pathway by caveolae leads to chronic activation and a higher expression of muscle specific genes. These defects could be reversed by reassembling a pool of functional caveolae through expression of wild type Cav3. Our findings bring more mechanistic insight into human Cav3 associated muscle disorders and show a general defect in the mechanoresponse of CAV3 P28L and R26Q myotubes.

cell biology↗