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Research ArticleCell biologyMuscle biology
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10.1172/jci.insight.200054
1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
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1Université Paris-Saclay, Université d’Evry, Inserm, IStem, UMR861, Corbeil-Essonnes, France.
2IStem, CECS, Corbeil-Essonnes, France.
3INTEGRARE, Genethon, Inserm, Université d’Evry, Université Paris-Saclay, Evry, France.
4Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, UMRS974, Paris, France.
5Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne université, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France.
6Centre de Référence des maladies Neuromusculaires Nord/Est/Ile-de-France, Institut de Myologie, Hôpital Pitié-Salpêtrière, APHP, Paris, France.
Address correspondence to: Xavier Nissan, 28 rue Henri Desbruères 91100 Corbeil Essonnes, France. Email: xnissan@istem.fr.
Find articles by Nissan, X. in: PubMed | Google Scholar
Published March 23, 2026 - More info
Published in Volume 11, Issue 6 on March 23, 2026
AbstractLimb-girdle muscular dystrophy R2 (LGMD R2) is an autosomal recessive disorder caused by dysferlin deficiency, leading to progressive muscle weakness and wasting. The lack of reliable clinical biomarkers has limited disease monitoring and therapeutic evaluation. Here, we identified Disabled-2 (DAB2) as a molecular and clinical indicator of disease state in LGMD R2. Transcriptomic profiling revealed a significant upregulation of DAB2 in induced pluripotent stem cell–derived (iPSC-derived) myotubes from patients, a finding validated in muscle biopsies from 14 dysferlin-deficient individuals and in dysferlin-deficient Bla/J mice, where DAB2 levels increased with disease progression. Importantly, AAV-mediated expression of full-length dysferlin restored DAB2 levels, supporting its value as a dynamic readout of disease activity for both disease monitoring and therapeutic response. Given the established role of DAB2 in clathrin-mediated endocytosis, particularly in LDL receptor internalization and cholesterol homeostasis, and the pathological lipid accumulation reported in LGMD R2, we investigated its contribution to lipid dysregulation. High DAB2 expression paralleled lipid deposition in patient muscles, iPSC-derived myotubes, and mouse tissue, whereas siRNA-mediated DAB2 knockdown reduced lipid accumulation in LGMD R2 myotubes. Collectively, these findings suggest that DAB2 functions as a mechanistic link between dysferlin deficiency, altered lipid handling, and disease severity, and they highlight its potential as a prognostic marker and therapeutic response measure for LGMD R2.
Graphical Abstract
Introduction
The triggers of muscle degeneration in dysferlinopathies and the molecular pathways underlying disease progression remain poorly understood. Dysferlinopathies comprise a genetically heterogeneous group of muscular dystrophies. Among them, limb-girdle muscular dystrophy R2 (LGMD R2) is characterized by progressive weakness and wasting of the pelvic and scapular girdle muscles. With an estimated prevalence of 5.9–7.4 individuals per million worldwide (1), LGMD R2 is caused by autosomal recessive mutations in the DYSF gene (2, 3), which encodes dysferlin, a 230 kDa transmembrane protein highly expressed in striated muscle and enriched in the transverse tubules. Although several approaches have been described to partially restore muscle strength or motor function in preclinical models (4–16), no curative therapy is currently available, underscoring the need to better understand the molecular mechanisms underlying dysferlin deficiency.
Historically, studies aiming to elucidate the pathological mechanisms underlying dysferlinopathies have focused primarily on the role of dysferlin in plasma membrane repair (PMR) of the injured sarcolemma; a calcium-dependent process critical for maintaining myofiber integrity and function (17). While defects in PMR are observed in muscle fibers from dysferlin-deficient mice (18) and patient myocytes (19), studies also reported that its restoration alone (6) is insufficient to fully prevent or reverse dystrophic phenotypes (7), indicating that additional mechanisms contribute to disease progression. In agreement with these observations, several studies have highlighted a broader role of dysferlin through its involvement in t-tubule formation and maintenance (20–24), intracellular vesicular trafficking (25), and calcium homeostasis (20–22, 24, 26–28).
More recently, evidence from patient muscle biopsies and dysferlin-deficient mouse models suggested that progressive skeletal muscle remodeling is preceded by lipid accumulation within myofibers, followed by lipid deposition between myofibers (29–32), consistent with the possibility that altered lipid metabolism may contribute to early stages of LGMD R2 pathogenesis (33). This hypothesis is further supported by several studies (34–37) indicating that cholesterol homeostasis and lipid accumulation are not merely a secondary consequence of muscle degeneration but may represent an early, intrinsic, and therapeutically relevant aspect of the disease.
To dissect how early molecular defects drive progressive muscle damage, robust models that faithfully recapitulate human dysferlinopathy are essential. Over recent years, numerous studies, including studies from our group, have demonstrated the relevance of human induced pluripotent stem cells (hiPSC) identifying molecular mechanisms and pathological phenotypes of muscular diseases (38–45). Building on this approach, we generated skeletal muscle cells (skMC) from 3 LGMD R2 patient-derived hiPSC to investigate cellular and molecular processes associated with this pathology. In our study, we observed that Disabled-2 (DAB2) was consistently upregulated in LGMD R2 hiPSC-derived myotubes using comparative transcriptomic analysis. Measures of DAB2 expression in patient biopsies and in an animal model confirmed its overexpression and suggested correlations with lipid accumulation, disease severity, and disease progression. We also found that dysferlin restoration normalized DAB2 levels, indicating that its expression may reflect both disease burden and therapeutic response. To explore the potential role of DAB2 in lipid accumulation, we performed loss-of-function experiments using siDAB2, which resulted in a reduction of lipid accumulation in LGMD R2 hiPSC-derived myotubes.
Collectively, these observations suggest that DAB2 may act as a clinically relevant tissue-associated marker and a potential regulator of lipid metabolism in LGMD R2, providing insights into disease mechanisms and identifying a candidate target for future gene-based or pharmacological interventions.
ResultsCharacterization of LGMD R2 hiPSC-derived skMC. We previously reported dysferlin expression in healthy hiPSC-derived skMC (44). To investigate the molecular mechanisms underlying LGMD R2, we differentiated 3 dysferlin-mutant hiPSC lines using the same standardized protocol (45). Two lines carry nonsense mutations (LGMD R2 KO_1 and KO_2), resulting in complete loss of dysferlin isoforms, whereas a third line harbors a missense mutation (LGMD R2 MIS) producing an aggregated, misfolded dysferlin protein, as previously reported (46). Pluripotency of all LGMD R2 hiPSC lines was confirmed by measuring SSEA4 and TRA-1-81 expression using flow cytometry (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.200054DS1). Both LGMD R2 and control hiPSC were then differentiated using the established protocol (Figure 1A). qPCR showed no significant differences in OCT4 (Supplemental Figure 2A) or NANOG (Supplemental Figure 2B) pluripotency marker expression between control and LGMD R2 hiPSC lines (day 0). Throughout differentiation, the downregulation of pluripotency markers (Supplemental Figure 2, A and B) and induction of myogenic markers (MYOD, DESMIN, MYOG, TTN; Supplemental Figure 2, C–G) occurred with similar kinetics in both LGMD R2 and control cells, indicating that dysferlin deficiency does not impair early or late stages of myogenic differentiation. Additionally, immunostaining confirmed the ability of LGMD R2 hiPSC to form a well-organized network of striated myotubes positive for titin (Supplemental Figure 2H) and myosin heavy chain (Supplemental Figure 2I), similarly to unaffected control cells. Immunoblotting demonstrated complete absence of dysferlin in myoblasts and myotubes derived from the LGMD R2 nonsense lines and a marked reduction in the missense line compared with controls (Figure 1, B and C). These findings were confirmed by immunostaining, which showed absence of dysferlin in KO myoblasts (Figure 1D) and myotubes (Figure 1E) as well as perinuclear accumulation in MIS myotubes (Figure 1E).
Figure 1Phenotypic features of LGMD R2 hiPSC-derived skeletal muscle cells reveal dysferlin deficiency. (A) Schematic representation of the skeletal myogenic differentiation protocol. Arrows indicate key stages where specific phenotypes are observed. Growth factors and cytokines used at each differentiation stage are specified, along with the progression of selected gene expression. Schematic created with BioRender. (B and C) Immunoblot analysis of dysferlin expression in LGMD R2 and control hiPSC-derived myoblasts (B) and myotubes (C). GAPDH is used as a loading control. (D and E) Immunostaining of dysferlin (green) in LGMD R2 and control myoblasts (D) and myotubes (E). Nuclei were counterstained with Hoechst (blue). Insets show magnified views of dysferlin staining in myotubes. Scale bar: 20 μm. AA, ascorbic acid; C, CHIR99021; Dex, dexamethasone; E, epidermal growth factor; F, basic fibroblast growth factor; Fe, fetuin; H, hepatocyte growth factor; HS, horse serum; I, insulin; IG, insulin-like growth factor; N, necrosulfonamide; O, oncostatin; P, platelet-derived growth factor; SB, SB431542; hiPSC, human induced pluripotent stem cells.
Transcriptomic profiling reveals gene signatures and disrupted pathways in LGMD R2 myotubes. To investigate global transcriptional changes, we performed RNA-seq on LGMD R2 and control hiPSC-derived myotubes. Principal component analysis (PCA) and heatmap analysis revealed distinct clustering of LGMD R2 and control samples (Supplemental Figure 3A and Figure 2A). Differential expression analysis identified 52 differentially expressed genes (DEGs), including 13 downregulated and 39 upregulated genes in LGMD R2 myotubes (Figure 2B). qPCR analysis validated 20 of 23 selected DEGs, including DYSF itself (Supplemental Figure 3, B and C). Enrichment analysis using EnrichR indicated that downregulated genes were associated with fibrosis-related pathways (e.g., TGF-β signaling) and lipid metabolism (Supplemental Figure 3D), while upregulated genes were linked to extracellular matrix (ECM) remodeling (Supplemental Figure 3E). Gene Ontology (GO) analysis further highlighted relevant biological processes, such as “regulation of intracellular transport” and “transmembrane receptor protein,” with a gene network centered on DAB2 (Figure 2, C and D).
Figure 2Transcriptomic profiling reveals altered gene expression signatures in LGMD R2 hiPSC-derived myotubes. (A) Hierarchical clustering of differentially expressed genes detected in LGMD R2 myotubes compared with controls. Gene expression is color-coded from blue (downregulated) to pink (upregulated). (B) Volcano plot representation of differential gene expression analysis between myotubes derived from 3 control and 3 LGMD R2 hiPSC lines. Downregulated (blue) and upregulated (pink) genes in LGMD R2 myotubes are highlighted. Vertical dashed lines indicate |log2FoldChange|threshold ≥ 0.4, and the horizontal dashed line represents a FDR ≤ 0.05. (C and D) Enriched gene ontology (GO) terms obtained by overrepresentation analysis (ORA) of upregulated genes in LGMD R2 myotubes compared with controls. Each pathway has an adjusted P value of 0.0995. (C) Dot plots show the 6 GO terms shared by at least 3 genes. (D) Cnetplot of the 5 biological processes most significantly shared among GO terms. Bubble size represents the number of enriched genes. DAB2 is highlighted in pink.
DAB2 as a marker of LGMD R2 pathophysiology. DAB2 overexpression in dysferlin-deficient myotubes was first confirmed at the transcript level by qPCR (Figure 3A) and validated at the protein level by Western blotting (Figure 3B). Confocal imaging further demonstrated increased cytoplasmic accumulation of DAB2, as evidenced by a higher number of DAB2+ puncta in LGMD R2 myotubes compared with control (Figure 3C and Supplemental Videos 1 and 2). To assess the relevance of these findings in human disease, we analyzed DAB2 expression in skeletal muscle biopsies from 14 genetically confirmed dysferlin-deficient patients and 2 unaffected controls (Supplemental Table 1). Dysferlin pathogenic variants were distributed across the protein without clustering at specific hot spots (Figure 3D). In most patient samples, DAB2 mRNA was significantly elevated in LGMD R2 patient biopsies compared with controls (Figure 3E). These observations were further supported by the analysis of a previously published RNA-seq dataset from 10 patients with LGMD R2 and 13 unaffected controls (47, 48), in which we noted an increased DAB2 expression in most patient muscle biopsies (Supplemental Figure 4). Importantly, our analysis confirmed that DAB2 expression did not correlate with patient sex (Supplemental Figure 5A), clinical subtype of dysferlinopathy (Supplemental Figure 5B), or serum creatine kinase (CK) levels (Supplemental Figure 5C). Instead, DAB2 levels appeared to reflect the severity of muscle pathology, as patients with higher Walton scale scores (Figure 3F) and more pronounced muscle morphological alterations (Supplemental Figure 5D) exhibited the highest DAB2 expression. Protein-level analysis confirmed these findings: DAB2 was markedly increased in the muscle biopsy from patient 2, who exhibited clear dystrophic features, while no modulation was observed in patient 6, whose muscle appeared morphologically normal and comparable with a healthy control (Figure 3G). Collectively, these data support the use of DAB2 as a marker of dysferlin deficiency and disease progression.
Figure 3Dysregulation of Disabled-2 (DAB2) in human dysferlin-deficient myotubes and muscle biopsies. (A–C) Analysis of DAB2 in LGMD R2 hiPSC-derived myotubes. (A) qPCR analysis of DYSF and DAB2 expression in myotubes from 3 control (gray) and 3 LGMD R2 (pink) hiPSC lines. Data represent mean ± SD of 3 independent differentiations per line (n = 9), normalized to the mean of control myotubes. **P ≤ 0.001 (unpaired 2-tailed t test with Welch’s correction on log-transformed data); ###P ≤ 0.001 (unpaired 2-tailed t test). (B) Immunoblots of dysferlin (red) and DAB2 (green) in control and LGMD R2 myotubes. GAPDH served as loading control. (C) Immunostaining of DAB2 (red) and α-actinin (green) in control (top) and LGMD R2 (bottom) myotubes. Nuclei were counterstained with Hoechst (blue). Insets show magnified regions. Scale bar: 10 μm. (D–G) DAB2 expression in muscle biopsies from 14 dysferlin-deficient patients. (D) Map of patient mutations in DYSF gene (exons separated by dotted lines). Protein domains are indicated. Each patient is numbered; clinical phenotype and sex are indicated by shape and color (Supplemental Table 1). (E) DAB2 mRNA levels from QuantSeq (Illumina) in control (gray) and patient (pink) muscle biopsies. Each dot represents a biopsy; bars indicate mean. *P ≤ 0.05 (Mann-Whitney U test). (F) Correlation between DAB2 expression and patient involvement (mildly affected 0 ≤ Walton scale ≤ 10 severely affected). Dots represent biopsies, color-coded from gray (low DAB2) to pink (high DAB2). Patients with the lowest (patient 6) and highest (patient 2) DAB2 expression are highlighted. *P ≤ 0.05 (Pearson correlation). (G) HPS-stained deltoid sections from patients 6 and 2 with immunostaining for dysferlin (red) and DAB2 (yellow). Scale bar: 500 μm. HPS, hematoxylin phloxine saffron; CdysfF, dysferlin domain C-terminal region; Fer, Ferlin domain; IdysfF, inner dysferlin domain; NdysfF, dysferlin domain N-terminal region; TM, transmembrane domain.
DAB2 expression inversely correlates with dysferlin content in vivo. We next assessed Dab2 expression in a dysferlin-deficient Bla/J mouse model (4). Homozygous Bla/J mice develop a progressive muscular dystrophy and recapitulate the main histopathological features observed in patients, including inflammation, fiber degeneration, and centrally nucleated fibers. Dystrophic changes are detectable at the age of 8 weeks and worsen by the age of 4 months, with widespread muscle impairment by 8 months. To investigate Dab2 dynamics at early disease stages, we analyzed psoas and gluteus muscles at 3 and 6 months of age. Tibialis anterior, which remains preserved in this model, was used as a control. At 3 months, histological analysis revealed that the psoas was the most affected muscle, with few centrally nucleated fibers and inflammatory areas (Figure 4A). By 6 months, both psoas and gluteus, but not tibialis anterior, showed significant morphological alterations (Figure 4A and Supplemental Figure 6). qPCR analysis of Dab2 confirmed the correlation with disease progression, as shown by the significant increase in Dab2 mRNA in the psoas at 3 months, with no changes in gluteus or tibialis anterior (Figure 4B), and its upregulation at 6 months in both psoas and gluteus but not in tibialis anterior (Figure 4C). These results were confirmed at the protein level by immunoblotting, which showed overexpression of Dab2 isoforms in psoas and gluteus muscles of 6-month-old Bla/J mice compared with controls (Figure 4D). To evaluate the potential use of Dab2 expression as a therapeutic response indicator, we next analyzed its expression in dysferlin-deficient presymptomatic mice treated with a dual AAV vector delivering full-length dysferlin (8) (Supplemental Figure 7A). Histological analysis showed that dysferlin rescue prevented dystrophic features in psoas and gluteus at both 1 and 6 months after injection, compared with saline-treated mice (Figure 4E and Supplemental Figure 7B). Correspondingly, qPCR revealed that dysferlin rescue (Figure 4F and Supplemental Figure 7C) was associated with a significant normalization of Dab2 mRNA levels (Figure 4G and Supplemental Figure 7D) in 1- and 6-month–treated mice. These results suggest that Dab2 may be used to monitor disease progression and therapeutic efficacy in vivo.
Figure 4DAB2 is upregulated in severely affected muscles of dysferlin-deficient mice and restored upon dysferlin gene therapy. (A–D) Analysis of Dab2 expression in dysferlin-deficient mice model (Bla/J). (A) Histological comparison of psoas and gluteus muscles from 3-month-old (left) and 6-month-old (right) control and Bla/J mice using HPS staining. Scale bar: 50 μm. Associated quantification of Dab2 mRNA by qPCR in psoas, gluteus, and tibialis anterior muscles of control (gray) and Bla/J (pink) mice at 3 (B) and 6 months (C). Expression levels are normalized to control mice. Data represent mean ± SD (n = 3–5 per group). *P ≤ 0.05, ***P ≤ 0.001 (unpaired 2-tailed t test with Welch’s correction). (D) Immunoblots of murine Dab2 isoforms (green) in psoas (left) and gluteus (right) muscles from 6-month-old control and Bla/J mice. Actin is a loading control. (E–G) Dab2 expression following AAV-mediated dysferlin gene therapy in Bla/J mice. (E) Representative HPS-stained psoas sections from 1-month-old Bla/J mice after 1 month (left) or 6 months (right) of PBS or AAV dysferlin treatment. Scale bar: 200 μm. Associated measure of Dysf (F) and Dab2 (G) mRNA by qPCR in psoas muscles of PBS-treated (pink) or AAV-dysferlin-treated (gray) Bla/J mice. Expression levels are normalized to PBS-treated mice. Data represent mean ± SD (n = 5 per group). *P ≤ 0.05, ***P ≤ 0.001 (unpaired 2 tailed t test with Welch’s correction). Pso, psoas muscle; Glu, gluteus muscle; Ta, tibialis anterior muscle.
DAB2 expression is associated with lipid accumulation in dysferlin-deficient muscle and myotubes. Based on the reported defective lipid metabolism in LGMD R2 and the role of DAB2 in low-density lipoprotein receptor (LDLR) endocytosis and cholesterol-rich LDL uptake (49, 50), we assessed the relation between dysferlin deficiency, lipid accumulation, and DAB2 expression. We first temporally and spatially characterized these parameters in Bla/J mice. Histological analysis confirmed that lipid deposition occurred predominantly in affected muscles, with strong Oil Red O staining in psoas and gluteus, but not tibialis anterior, at 6 months (Supplemental Figure 8). Dysferlin gene therapy markedly reduced lipid accumulation in the psoas (Figure 5A) and gluteus (Supplemental Figure 9) after 6 months of treatment compared with PBS-treated controls, consistent with previous findings. These observations were then assessed in patient muscles with low, high, and very high DAB2 expression. Lipid accumulation measured by Oil Red O staining indicated that patients with high or very high DAB2 content showed increased lipid deposition, whereas patients with low DAB2 levels and healthy controls displayed minimal lipid accumulation (Figure 5B). These findings were finally confirmed in vitro by analyzing lipid uptake in hiPSC-derived myotubes treated with fatty acids. Analysis of LGMD R2 and control cell lines revealed that LGMD R2 myotubes exhibited significantly greater lipid accumulation than controls, as shown by immunostaining (Figure 5C, Supplemental Figure 10, A–E, and Supplemental Videos 3 and 4) as well as by the quantification of lipid droplet volume (Figure 5D) and number (Supplemental Figure 10F) in myotubes. Finally, siRNA-mediated knockdown of DAB2 in hiPSC-derived myotubes (Supplemental Figure 10G) revealed reduced lipid deposition (Figure 5E and Supplemental Figure 10H and Supplemental Video 5). Quantitative analyses confirmed significant decreases in lipid droplet volume (Figure 5F and Supplemental Figure 10J) upon DAB2 silencing, consistent with the observed downregulation of DAB2 protein levels (Supplemental Figure 10, H and I). Together, these results support a functional link between DAB2 expression and pathological lipid accumulation in various dysferlin-deficient models.
Figure 5DAB2 expression is associated with lipid accumulation in dysferlin-deficient muscle and myotubes. (A) Oil Red O–stained sections of psoas muscles from 1-month-old Bla/J mice treated for 6 months with PBS or AAV-dysferlin (AAV DYSF). Scale bar: 500 μM. (B) Deltoid biopsies from a healthy control and dysferlin-deficient patients with low (patient 4), high (patient 1), or very high (patient 12) DAB2 expression, stained for DAB2, HPS, and ORO. Scale bar: 50 μm. (C–F) Lipid accumulation in hiPSC-derived myotubes supplemented with fatty acids. (C) Immunostaining of lipid droplets (green) in myotubes (red) from 1 control and 3 LGMD R2 cell lines. Nuclei were counterstained with Hoechst (blue). White boxes indicate magnified regions. Scale bar: 20 μm. (D) Associated quantification of lipid volume in control (gray) and LGMD R2 myotubes (pink). Data represent mean ± SD of 1 representative experiment from n=3 independent experiments. ***P ≤ 0.001 (1-way ANOVA with Dunnett’s multiple comparisons test after transformation). (E) Immunostaining of lipid droplets (green) in LGMD R2 KO_1 myotubes (red) transfected with siScramble (left) or siDAB2 (right). White boxes indicate magnified regions. Scale bar: 20 μm. (F) Associated quantification of lipid volume in LGMD R2 KO_1 myotubes after treatment with siScramble (pink) or siDAB2 (gray). Data represent mean ± SD of 1 representative experiment from n = 3 independent experiments. ***P ≤ 0.001 (unpaired 2-tailed t test with Welch’s correction). HPS, hematoxylin phloxine saffron; ORO, Oil Red O; siSCR, siScramble.
DiscussionIn this study, we investigated the molecular
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