Anti-myogenic and profibrotic effect of serum from patients affected by muscular laminopathies

Emery-Dreifuss Muscular Dystrophy type 2 (EDMD2) and LMNA-related congenital muscular dystrophy (L-CMD) are caused by mutations in LMNA gene. Both pathologies are characterized by joint contractures, muscle weakness and wasting and cardiac involvement. In the last few years, circulating factors have been proposed to play a critical role in the pathogenesis of these diseases. Based on this consideration, we studied the effect of laminopathic serum on the myogenic differentiation in healthy human myoblasts in culture. We observed impaired myogenesis and increased fibrosis in myoblast cultures conditioned with laminopathic serum and a dramatic increase in the level of profibrotic and proinflammatory cytokines in the cell culture supernatants. These results strongly support the pathogenic role of circulating factors in muscular laminopathies and pave the way to a possible therapeutic strategy.

Article

EDMD2 (OMIM#181350) and L-CMD (OMIM#613205) are caused by mutations in LMNA gene, encoding the nuclear lamina proteins lamin A and C, and belong to the group of muscular laminopathies. Both EDMD2 and L-CMD are characterized by the clinical triad of joint contractures in Achilles tendons, elbow and neck, proximal limb and axial muscle wasting and weakness, and cardiac involvement that manifests as heart conduction defects and dilated cardiomyopathy^1,2. High inter and intra-familial clinical variability has been demonstrated for EDMD2: early to very late disease onset, diverse severity and progression of muscular and cardiac disorders, metabolic complications. Regarding L-CMD, clinical spectrum ranges from heart conduction defects and cardiomyopathy to dropped head syndrome, respiratory impairment, severe contractures, delay or absence of motor milestones and loss of ambulation in the first decade ^1,2.

In the last few years, circulating factors have been suggested to play a pivotal role in the pathogenesis of LMNA- related disorders ^3,4. IL17, TGFβ2 and G-CSF have been identified as the most significantly dysregulated cytokines in serum derived from patients affected by muscular laminopathies ^3,4. Interestingly all these cytokines directly or indirectly induce macrophage polarization toward M2 phenotype. M2 macrophages are considered anti-inflammatory cells, they participate in extracellular matrix (ECM) remodeling in tissues wounded by acute and chronic inflammatory stimuli but, if persistently activated, they can be pathogenic, contributing to myofiber necrosis and fibrosis by promoting fibroblast proliferation and connective tissue deposition⁴. Despite this possible explanation of the activation of fibrotic processes in cardiac and skeletal muscle often observed in laminopathic patients, there is few evidence proving the presence of M2 macrophages among immune cells infiltrating laminopathic skeletal muscle tissue. Of note, macrophage infiltration has been observed in dystrophic muscle from patients with early onset muscular laminopathies ⁵.

On the other hand, fibrosis has a deleterious effect on muscle function. ECM is an essential component of skeletal muscle since it provides a scaffold that holds myofibers, blood capillaries and nerves supplying the muscle and plays a key role in maintenance and repair of muscle fibers. However, excessive accumulation of ECM components, especially collagens, leads to an impairment of muscle function, negatively affects muscle regeneration after injury and increases muscle susceptibility to re-injury. Therefore, fibrosis is considered a major cause of muscle weakness ⁶. Moreover, it has been reported that profibrotic molecules, such as TGFbeta 2, induce an increased proliferation of muscle precursors which could reduce myogenic differentiation rate, contributing to muscular dystrophy³.

To elucidate the role of circulating factors in muscular laminopathies, we studied the effect of sera derived from young patients affected by EDMD2 or L-CMD on myogenic differentiation and collagen production in healthy human myoblasts in culture. Myoblast cultures grown at confluence were conditioned for 4 days with healthy donor (control) or laminopathic serum. Immunofluorescence staining of myoblasts grown in laminophatic serum revealed a lower number of myogenin-positive myoblasts with respect to myoblasts grown in the presence of control serum (Fig. 1a). This result demonstrated an impaired myogenesis. Moreover, myoblasts conditioned with laminopathic serum showed an increase in collagen I levels, suggesting the onset of a pro-fibrotic process (Fig.1b). We then evaluated the levels of a panel of cytokines in each cell culture supernatant using a human 24-plex cytokine Luminex performance and the human TGF-beta Luminex immunoassay kits (Bio-techne, Minneapolis, MN, USA). Relative to myoblasts conditioned with control medium, myoblasts cultured in laminopathic serum showed significantly increased levels of the following secreted molecules: VEGF, TGF beta, IL6, IL8, CXCL10/IP10, CCL2, CCL4 and G-CSF (Fig. 2). VEGF and TGF beta are known as a profibrotic factors ^{3, 7} while IL6, which was dramatically increased in laminopathic serum-containing media, is a proinflammatory cytokine also involved in cardiac fibrosis ⁸. CXCL10/IP10, CCL2, G-CSF and CCL4 are pro-regenerative chemokines, which are increased during muscular injury and have been reported to be involved in the chemotaxis of satellite cells for muscle regeneration ⁹. CC chemokine ligand 2 (CCL2), also called macrophage chemoattractant protein 1 (MCP-1), is essential to induce an adequate inflammatory response to repair acute skeletal muscle injury. CCL2 expressed by bone marrow cells, circulating monocytes, and injured muscle tissue cells recruits monocytes or macrophages into injured muscles to conduct phagocytosis and produce IGF-1 which is essential for injury repair ¹⁰. The role of these chemokines is important in the proliferative stage of myoblast activation for tissue repair. However, by increasing myoblast proliferation, these chemokines delay the onset of myogenic differentiation as reported for CCL2, which suppresses the expression of the differentiation marker myogenin ⁹. Moreover, we speculate that hyper-secretion of chemoattractant cytokines may contribute to quiescent satellite cell stimulation and stem cell niche depletion in laminopathic patients.

In conclusion, we can infer the secretion of proinflammatory molecules in EDMD2 and L-CMD serum activates pro-regenerative and pro-fibrotic processes, favoring satellite cell recruitment and proliferation, while impairing myogenic differentiation. The onset of fibrosis further exacerbates the aberrant muscle remodeling process. In this context, elevated IL6 levels are a main trigger of inflammation and fibrosis. Our data strongly suggest a pivotal role of circulating factors in the pathogenesis of muscular laminopathies and suggest the balanced use of anti-inflammatory drugs as a possible therapeutic strategy for patients with early onset LMNA-related muscular dystrophies¹⁰. However, as cytokines represent an extremely sensitive system, a personalized medicine approach aimed at monitoring individual response to treatment and muscle inflammatory processes must be considered in view of therapeutic interventions. The use of in vitro test systems as the one here proposed could help patient follow-up in clinical trials avoiding invasive analyses and providing accurate data.

Acknowledgements

This study has been presented at the “Pisa Muscle Award 2024” meeting. The authors thank patients for their kind collaboration. The authors acknowledge the useful discussion of data within the Italian Network for Laminopathies (https://www.igm.cnr.it/laminopatie/en/) and Associazione Italiana di Miologia AIM (https://www.miologia.org/). The authors thank Michele Martinelli for administrative support and Patrizia Sabatelli for technical support.

Funding

This research was funded by ECOSISTER Project. cod. ECS_00000033-CUP B89I22000650001, Progetto AIFA TREAT-LMNA 2019-004426-24, Progetto AIDMED e Associazione Alessandra Proietti MIF-EDMD.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript or in the decision to publish the results.

Authors contributions

ES, GR, GS: study conception and design; AP, GR: patient evaluation and serum collection; PC: secretome analysis. ES; sample collection, immunofluorescence staining and image analysis; ES: interpretation of results and manuscript preparation. All authors reviewed the results and approved the final version of the manuscript.

Ethical consideration

All patients or parents/legal tutors signed informed consent to anonymous data analysis. The study was approved by the local ethics committees according to the declaration of Helsinki and EU ethical rules. The BioLaM Biobank that provided primary myoblast cultures was approved by the Rizzoli Orthopedic Institute Ethics Committee on 5 September 2016, Prot. Gen. 0018250-01-13.

History

Received: March 11, 2025

Accepted: June 12, 2025

Figures and tables

Figure 1. Effect of laminopathic serum on myogenic differentiation and collagen I production in control myoblasts. (a) Immunofluorescence staining of desmin (green), myogenin (red) and DAPI (blue) in control myoblasts conditioned for 4 days with serum derived from healthy donors (control serum), EDMD2 (EDMD2-serum) or L-CMD (L-CMD-serum) patients. The percentage of myogenin-positive myoblasts is reported in the graph. (b) Immunofluorescence staining of collagen I (red) of the same samples showed in (a). The Mean Fluorescence Intensity (MFI) of collagen I is reported in the graph. Scale bars: 10μm. 30 cells per sample were analyzed in triplicate experiments. Data are reported as means ± SD. Statistically significant differences are indicated in the graphs (* p < 0.05, **p < 0.01 or **** p < 0.001).

Figure 2. Secretome analysis in conditioned media. Levels of VEGF, IL6, IL8, CCL10/IP10, G-CSF, TGF beta, CCL2, and CCL8 measured by a human cytokine Luminex performance immunoassay kit, and of TGF-beta 1 measured by the human TGF-beta Luminex immunoassay kit (both from Bio-techne, Minneapolis, MN, USA) in the supernatants of myoblasts conditioned with serum derived from healthy donors (control-serum), EDMD2 (EDMD2-serum) or L-CMD (L-CMD-serum) patients. Data are reported as means ± SEM of triplicate experiments. Statistically significant differences are indicated in the graphs (* p < 0.05, **p < 0.01, *** p< 0.05).