GDF11 and its receptor ALK4 independently modulate human myoblast migration and fusion

GDF11 and its receptor ALK4 independently modulate human myoblast migration and fusion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GDF11 and its receptor ALK4 independently modulate human myoblast migration and fusion Rafaella Ferreira Reis, Igor Ferreira da Silva, Beatriz Guimarães Costa-Santos, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7777108/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Skeletal myogenesis GDF11 ALK4 laminin cell adhesion cell fusion Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Tissue and organ regeneration involve structural and functional restoration following an injury or perturbation. Dysregulation of regeneration can lead to abnormal extracellular matrix (ECM) deposition, ultimately resulting in fibrosis (Uezumi et al. 2011 ). In adult skeletal striated muscles, mature cells are cylindrical and contain striated myofibrils and multiple peripheral nuclei. In addition, rare and small progenitor quiescent mononucleated cells are located between the plasma membrane (sarcolemma) and the basement membrane of the muscle fiber. These satellite cells are identified by the expression of PAX7 (Seale et al. 2000 ). Upon activation, they re-enter the cell cycle, proliferate, and give rise to myoblasts. Initially, these cells remain expressing PAX7 and begin to express the myogenic regulatory factors (MRFs) such as MYOD1 and MYF5/6 . Subsequently, those markers are lost, and they express MYOGENIN and MRF4 , which promote the terminal differentiation of skeletal myocytes (Buckingham and Rigby 2014 ). These muscle precursors then fuse to eventually form multinucleated myotubes. Ultimately, they develop into new skeletal muscle fibers or contribute to the recovery of the damaged tissue. A portion of the proliferating myoblasts returns to a quiescent state, exhibiting stemness characteristics and maintaining the pool of satellite cells. Accordingly, the activation of satellite cells is a critical step in skeletal muscle regeneration. The regulatory factors governing stem cell fate and thereby determining the balance between regeneration and repair during tissue healing remain largely elusive. The identification and further characterization of these factors is pivotal for deepening our understanding of functional tissue recovery and can profoundly influence the development of targeted therapeutic strategies for skeletal muscle regeneration. In particular, the use of cell therapy in clinical trials for the treatment of lesions or degenerative diseases has yielded inconsistent results, including the reduced migration capacity of transplanted cells and accelerated cell death (Briggs and Morgan 2013 ). Among the numerous strategies and molecules currently under investigation, the GDF11 signaling pathway has been implicated in the rejuvenation of skeletal muscle, cardiac muscle, and cerebral vasculature, leading to increased neurogenesis and cognitive improvement (Loffredo et al. 2013 ; Katsimpardi et al. 2014 ; Sinha et al. 2014 ). Typically, TGF-β superfamily ligands are synthesized as latent precursors and require cleavage by proprotein convertases for activation (Moustakas and Heldin 2009 ). Once activated, they form homodimers and bind to constitutively active type II serine/threonine kinase receptors. Ligand binding enables these receptors to recruit and phosphorylate type I receptors (Hinck 2012 ). Together, type II and type I receptors assemble into a heterotetrameric complex, wherein type I receptors phosphorylate receptor-activated SMADs (R-SMADs), which then translocate to the nucleus and regulate gene transcription (Horbelt et al. 2012 ; Jurberg et al. 2015 ). In the case of GDF11, SMAD2 and SMAD3 associate with SMAD4 to form a transcriptional activator complex (Andersson et al. 2006 ). Findings with GDF11 are, however, seemingly contradictory (Egerman et al. 2015 ; Rodgers and Eldridge 2015 ; Smith et al. 2015 ; Poggioli et al. 2015 ; Harper et al. 2016 ; Zhou et al. 2017 ). In mouse embryos, for instance, we have previously observed that Gdf11 inactivation leads to an expansion of a group of undifferentiated axial progenitors during tail formation, underscoring its importance in progenitor cell differentiation and tissue patterning (Jurberg et al. 2013 ; Aires et al. 2016 ). In skeletal muscle, Egerman et al. ( 2015 ) reported that Gdf11 impairs muscle regeneration by inhibiting myoblast differentiation and satellite cell expansion, while its administration prior to muscle injury was associated with an increase in nascent myofiber formation. In contrast, studies in aged animals revealed that elevated GDF11 levels may be detrimental. More specifically, Harper et al. ( 2016 ) found that increasing GDF11 impairs skeletal muscle repair, potentially inducing cachexia-like effects. Likewise, GDF11-treatment in older rats promoted tissue fibrosis and compromised the functional recovery of skeletal muscle after injury (Zhou et al. 2017 ). Together, these observations emphasize that timing, dosage, and cellular context critically determine the biological outcomes of GDF11 signaling. Other factors may contribute to the controversies surrounding GDF11 function. Notably, GDF11 shares approximately 90% amino acid sequence identity with its homolog, Myostatin (GDF8) (Nakashima et al. 1999 ). This similarity raises the possibility of antibody cross-reactivity, as suggested by Egerman et al. ( 2015 ). Furthermore, while Sinha et al. ( 2014 ) initially proposed a pro-myogenic role for GDF11, genetic studies have established that Myostatin inhibits muscle growth (Lee and McPherron 2001). Additionally, some in vitro and in vivo studies have reported that the GDF11 propeptide can antagonize the activity of the mature GDF11 peptide (Ge et al. 2005 ; Li et al. 2010 ). In this study, we evaluated the impact of recombinant GDF11 (rGDF11) on chick and human myoblast cultures and used CRISPR-mediated knockout of the ALK4 receptor in human myoblasts to elucidate its role during in vitro skeletal myogenesis. We specifically assessed how GDF11 and ALK4 affect key cellular processes, including adhesion, proliferation, migration, and differentiation. Material and methods Primary chick myogenic cell culture Primary cultures of myogenic cells were derived from pectoral muscle tissue of 11-day-old chick embryos (Granja Tolomei, Brazil) as previously described (Mermelstein et al. 1996 ). The use of chick embryos was approved by the Ethics Committee for Animal Care and Use in Scientific Research at the Federal University of Rio de Janeiro under approval number DAHEICB 081 − 22. In brief, muscle tissue was minced with surgery knives and digested with 0.005% trypsin at 37° C in a 5% CO 2 incubator for 25 min. To stop digestion, a small volume of 8-1-0.5 medium (Minimum Essential Medium with 10% horse serum, 0.5% chick embryo extract, 0.1% L-glutamine, and 1% penicillin-streptomycin) was added to the sample and then centrifuged at approximately 300 × g for 5 min. After discarding the supernatant, the pellet was suspended in the 8-1-0.5 medium and filtered to produce a mononucleated cell suspension. Isolated mononucleated cells were plated at an initial density of 7.5 x 10 5 cells per 35 mm culture dish onto 22-mm Aclar plastic coverslip (Pro-Plastics Inc.) pre-coated with 0.1% gelatin or rat tail-derived collagen. They were cultured in 2 mL of 8-1-0.5 medium under humidified 5% CO 2 atmosphere at 37° C. The percentage of myoblasts was determined by double staining 24-hour cultures with the muscle-specific marker desmin (#8281, Sigma-Aldrich) (Supplementary Table 1) and the nuclear dye DAPI. The number of desmin-positive cells was then counted relative to the total cell count per field of view. On average, myoblasts constituted 80% of each culture, while non-myogenic cells comprised the remaining 20%. All cell culture reagents were purchased from Invitrogen, unless otherwise stated. Human muscle cell culture Immortalized C25-CL48 human myoblasts were previously described elsewhere (Thorley et al. 2016 ). We employed two distinct cell culture conditions. For cell maintenance and proliferation assays, cells were cultured in DMEM high glucose supplemented with 199/EBSS medium, 20% fetal bovine serum (FBS), 5 ng/mL epidermal growth factor (EGF; #PHG0311, ThermoFisher), 25 µg/mL fetuin (#F3385, Sigma-Aldrich), 0.2 µg/mL dexamethasone (#D4902, Sigma-Aldrich), and 50 µg/mL gentamycin (#15750060, ThermoFisher). For differentiation assays, cells were cultured in DMEM/Glutamax supplemented with 10 µg/mL of human recombinant insulin (#91077, Merk), as previously described (Riederer et al. 2012 ). Cell culture treatments Primary chicken or immortalized human muscle cell cultures were maintained for either 12 or 24 hours prior to treatment with 100 ng/mL of recombinant GDF11 (rGDF11; #120 − 11, PeproTech) for the indicated durations. In addition, other myoblast cultures were treated with 1 µM of SB431542 (SB; #S4317, Sigma-Aldrich), a type I receptor inhibitor for ALK4/5/7, diluted in DMSO (#2650, Sigma-Aldrich) as vehicle at a final concentration of 1 µM. Cultures were observed daily through phase-contrast microscopy. Generation of CRISPR-mediated mutant cell lines We used human C25-CL48 myoblasts to inactivate the ACVR1B gene (ALK4) using the CRISPR/Cas9 strategy. In brief, we obtained the sequence of each gene from the "Genome Browser" database ( https://genome.ucsc.edu/ ) and utilized the first exon to design specific guide RNAs (gRNAs) with the CRISPR Design Tool ( http://crispr.mit.edu/ ) (Supplementary Table 2). Commercially synthesized oligonucleotides were subcloned into the expression vector pSpCas9(BB)-2A-GFP (referred herein as pX458) (Ran et al. 2013 ). Positive clones were identified by colony PCR, and successful cloning was confirmed by Sanger sequencing. Next, human cells were electroporated with 20 µg of each plasmid using the Neon® Transfection System (ThermoFisher Scientific), following the manufacturer's protocol. Following electroporation, cells were maintained in culture for 24 hours to allow for cell recovery. Subsequently, GFP + myoblasts were sorted using a FACSAria II flow cytometer and individually plated in a 96-well plate for clonal expansion. Upon cell growth, cellular aliquots were cryopreserved, while other matching samples were used for genomic DNA extraction and genotyping (Supplementary Table 2). Confirmation and characterization of indels were performed through Sanger sequencing. Immunofluorescence Chick muscle cultures were stained following a previously established protocol (Mermelstein et al. 1996 ). In short, cells were fixed with 4% formaldehyde in phosphate buffered saline (PBS) for 3 minutes, washed in PBS, and subsequently incubated with appropriately diluted, unconjugated primary antibodies (Supplementary Table 1) for 1 hour at 37° C. After other PBS washes, cells were incubated with the corresponding diluted secondary antibodies (Supplementary Table 1) for 1 hour at 37° C. Human muscle cells were fixed with analytical-grade ethanol for 10 minutes, followed by three 5-minute PBS washes. Non-specific binding was blocked using PBS containing 2% goat serum, 1% bovine serum albumin (BSA), and 0.3% Triton X-100 for 20 minutes. Unconjugated or conjugated primary antibodies (Supplementary Table 1) diluted in PBS containing 1% BSA and 0.3% Triton X-100 were incubated in a humid chamber either for 1 hour at room temperature or overnight at 4º C. Samples were washed three times with PBS for 5 minutes each, then incubated with the corresponding diluted secondary antibodies (Supplementary Table 1) for 30 minutes. Following antibody incubation, both chick and human samples were washed with PBS again and counterstained with the DNA dye DAPI (#D1306, ThermoFisher Scientific) for 5 minutes. A final round of PBS washes was performed, and slides were mounted with ProLong™ Gold Antifade antifading mounting medium (#P36934, ThermoFisher Scientific). Slides were kept in the dark at room temperature for 72 hours and then transferred to 4ºC for long-term storage. Image acquisition was conducted using a Zeiss Axiovert 100 microscope, a Zeiss AXIO IMAGER A2 fluorescence microscope, or a Leica TCS SP8 confocal microscope. We captured between five to ten fields per condition. Transmigration assay To determine whether rGDF11 treatment or ALK4 deletion alters the migratory capacity of human myoblasts, we performed Transwell migration assays using 24-well polystyrene plates with polycarbonate membranes featuring 8-µm pores (Corning Costar, USA). Initially, both the lower chamber and the upper insert were coated with either 0.5% BSA or 10 µg/mL of recombinant human laminin-111 (LM111; #LN111-02, BioLamina) and incubated for 1 hour at 37º C in 5% CO 2 . Following incubation, the coating solutions were aspirated, and the wells were washed twice with 1× PBS before the plates were allowed to air-dry at room temperature. A blocking step was then carried out using 0.5% BSA for 1 hour at 37º C in 5% CO 2 to minimize nonspecific binding. Thereafter, 10 5 myoblasts were seeded into each insert in migration medium, which comprised DMEM and medium 199 (in a 4:1 ratio) supplemented with 0.5% BSA. The lower chamber was filled with migration medium alone (control) or supplemented with 100 ng/mL rGDF11. In separate experiments, cells in the insert were exposed to migration medium with or without rGDF11 in the upper chamber to evaluate any potential chemorepulsive effects. The plates were incubated for 4 hours at 37° C in a humified atmosphere containing 5% CO 2 . At the end of the incubation period, the supernatant in the insert was carefully removed, and the cells were gently washed twice with 1× PBS. Cells were then fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature, followed by additional gentle washes with 1× PBS. Any cells adhering to the upper surface of the membrane were removed using cotton swabs. Next, cells were permeabilized with 0.1% Triton X-100 for 40 minutes, and the lower surface of the membranes was stained with DAPI at a 1:200 dilution for 30 minutes to label nuclei. Following three washes with 1× PBS, the membranes were carefully excised from the inserts and mounted onto glass slides with coverslips for imaging. Cell adhesion assay To investigate the influence of rGDF11 on muscle cell adhesion, we coated Nunc® Lab-Tek® Chamber Slide™ systems (#S6815, Sigma-Aldrich) with poly-L-lysine (PLL; #P4707, Sigma-Aldrich) as a control for non-specific binding, or with LM111 (#LN111-02, BioLamina). In these experiments, 4x10 3 human wildtype or mutant C25-CL48 cells were resuspended in proliferation medium, with or without 100 ng/mL of rGDF11 (untreated) and allowed to adhere for 120 minutes. Then, the wells were gently washed with PBS, and cells were fixed with 2% PFA for 15 minutes. Subsequently, cells were permeabilized with 0.1% saponin in PBS for 15 minutes and non-specific labeling was blocked using PBS containing 2.5% BSA and 8% FBS for 1 hour. Cells were then labeled with Alexa Fluor® 488-conjugated phalloidin (A12379, ThermoFisher Scientific) for 30 minutes, counterstained with DAPI, and mounted as described above. Image analysis Image processing and quantification were conducted using the FIJI (ImageJ) software (Schindelin et al. 2012 ). This involved quantifying nuclei counts, cell counts, and cell area after performing background subtraction, contrast enhancement, Gaussian blur, and segmentation via a custom-developed macro pipeline, followed by manual curation. The differentiation index was computed as the ratio of nuclei within myosin heavy chain (MyHC)-positive (mono- or multi-nucleated) cells to the total number of nuclei per microscopic field. The fusion index was determined as the percentage of nuclei within MyHC-positive myotubes (specifically, multinucleated cells) per microscopic field. In addition, myotube width was measured along the longest branches of each myotube per microscopic field. The proportion of desmin-positive area was also determined. Flow cytometry Human wild-type and mutant myoblasts were harvested through trypsinization, counted using a hemocytometer, and distributed into U-bottom wells of a 96-well plate. To prevent non-specific labeling, we treated the cells with 50 µL of PBS containing 2% normal mouse serum for 15 minutes. Then, cells were centrifuged at 400 × g for 5 minutes at 4º C and washed with 100 µL of PBS containing 1% BSA (FACS buffer). Subsequently, they were incubated with appropriately diluted conjugated primary antibodies (Supplementary Table 1) for 1 hour at 4º C. After a final round of washing, the cells were resuspended in FACS buffer for analysis via flow cytometry using a FacsCanto II (Becton Dickinson). Statistical analysis We performed at least three independent experiments for each assay, unless otherwise stated. All values were presented as the median with 95% confidence intervals (CI). Statistical analyses were performed using two-tailed unpaired t tests for independent two-group comparisons. Multiple comparisons were analyzed using a linear mixed‑effects model to account for missing values or repeated measures, unless stated otherwise. Statistical significance was defined as p < 0.05. Results Treatment with rGDF11 produces distinct effects depending on the cell type and developmental stage, significantly reducing the number of fibroblasts (desmin-negative cells) in chicken muscle cell cultures We applied embryonic chick primary skeletal myoblasts due to their well-documented autonomous muscle differentiation program in culture (Costa et al. 2021 ). In this model, isolated cells progress through all stages of in vitro skeletal myogenesis without requiring any exogenous stimuli to produce large, multinucleated myotubes with contractile capacity (Mermelstein et al., 1996 ). Moreover, these primary cultures contain muscle fibroblasts, which provide paracrine signals and cell-cell interactions that critically influence skeletal myogenesis, including the composition of the extracellular matrix (Chapman et al. 2016 ). Cultures treated with 100 ng/mL of rGDF11 for two days exhibited a reduction in the median total number of nuclei per 100,000 µm 2 compared with controls (271.7 nuclei, 95% CI: 233.2–310.5 vs. 363.9 nuclei, 95% CI: 316.1–435.7; Figs. 1 A,B). However, no significant differences were observed in the number of nuclei within desmin + cells per 100,000 µm 2 (Figs. 1 A,C) or in the relative area occupied by desmin + myoblasts and myotubes (Fig. 1 A-D). The median proportion of myotube nuclei was slightly higher in the rGDF11-treated cultures than in controls (69.40%, 95% CI: 40.13%–90.37% vs. 55.00%, 95% CI: 41.13%–70.71%; Fig. 1 A-E), although this difference was not statistically significant. As satellite cells are quiescent stem cells with the ability to either generate new differentiating cells or return to their stem cell state, we explored whether treatment with rGDF11 influences cell fate decisions during in vitro skeletal myogenesis. The median total number of Pax7 + cells per 100,000 µm 2 remained consistent under rGDF11 treatment (50.12 cells, 95% CI: 28.05–68.80) compared to control cultures (56.78 cells, 95% CI:41.64–66.44; Fig. 1 A,F). No significant differences in the proportion of Pax7 + cells following rGDF11 incubation (19.60%, 95% CI: 13.07–23.78) in relation to controls were observed (15.59%, 95% CI: 11.59–18.41; Fig. 1 A,G). Because GDF11 signaling has been shown to activate lung and cardiac fibroblasts (Swan et al. 2024 ; Li et al. 2024 ), we hypothesized that it might similarly influence fibroblasts interspersed withing chicken muscle cell cultures. More specifically, we found a reduction in the number of Pax7 − desmin − nuclei per 100,000 µm 2 upon rGDF11 treatment in relation to control cultures (29.74 nuclei, 95% CI: 29.03–30.44 vs. 75.07 nuclei, 95% CI: 66.02–75.07, respectively; Fig. 1 A,H). These results raise the possibility that GDF11 may regulate the proliferation or cell death of skeletal muscle fibroblasts. Given the challenges of prolonged culture maintenance and the intrinsic tendency of primary chick embryonic skeletal muscle cells to undergo autonomous differentiation, we shifted to immortalized human C25-CL48 cells (Thorley et al. 2016 ) for subsequent experiments. This model enables us to further dissect the genetic basis of GDF11 signaling across distinct stages of skeletal myogenesis in vitro . Unlike primary chick embryonic muscle cells, human C25-CL48 cultures permit the study of proliferation and myotube differentiation as two distinct processes, thereby providing greater resolution into the stage-specific roles of GDF11. In addition, these human cultures are composed exclusively of myogenic cells, without fibroblast or other contaminating lineages. rGDF11 inhibits myotube formation in human myoblasts without affecting proliferation Unlike primary chick embryonic muscle cultures, human myoblasts can be maintained in either proliferation or differentiation media, allowing these two fundamental processes to be evaluated independently. As expected, the number of human C25-CL48 myoblasts increased significantly from day 3 to day 6 of culture (Fig. 2 A,B). Incubation with rGDF11 under proliferative conditions did not significantly alter the median total numbers of nuclei per 100,000 µm 2 compared with controls at 3 days (10.60 nuclei, 95% CI: 9.93–14.94 vs. 12.69 nuclei, 95% CI: 8.04–16.57) or 6 days of culture (31.99 nuclei, 95% CI: 30.78–37.19 vs. 30.86 nuclei, 95% CI: 19.83–42.18; Fig. 2 A,B). The proportion of Ki67 + myoblasts increased from day 3 to day 6 of culture, but no significant differences were observed between rGDF11-treated and control cells at either time point (Fig. 2 A,D). Assessment of proliferative capacity by EdU staining similarly revealed no differences between groups (data not shown). The mean population doubling time (PDT) was 2.67 days for rGDF11-treated cultures and 3.13 days for controls (Fig. 2 C). Together, these results indicate that GDF11 does not affect myoblast multiplication. Under differentiation conditions, GDF11 markedly impaired the differentiation of MyHC + cells and myotube formation (Figs. 2 E-J). This effect was not due to differences in the number of cells entering the differentiation program, as no significant differences were observed between GDF11-treated and control groups at either day 3 or day 6 of culture (Fig. 2 F). In controls, differentiation and fusion indices increased from day 3 to day 6, whereas rGDF11-treated myoblasts showed reduced median values (Fig. 2 H,I). Interestingly, rGDF11 treatment particularly blocked the formation of larger myotubes (Fig. 2 J). Together, these findings suggest that GDF11 impairs myotube formation primarily by inhibiting fusion, without affecting myoblast proliferation. By contrast, one-hour pulse of the SB inhibitor in DMSO followed by a medium change to differentiation medium without [SB(1h) in DMSO] or with rGDF11 [SB(1h) + rGDF11] strongly reduced differentiation and fusion indices after six days (Supplementary Fig. 1C,D). Notably, the SB(1h) + rGDF11 condition further decreased both indices relative to controls and DMSO(1h) cultures (Supplementary Fig. 1C,D). These results suggest that GDF11 signal through additional pathways beyond the canonical ALK4, ALK5, and ALK7 receptors (Derynck and Budi 2019 ). Loss of ALK4 impairs muscle differentiation Given that the SB inhibitor targets ALK4, ALK5, and ALK7 receptors, and that DMSO has shown unintended effects on muscle cell cultures, we employed a genetic approach using the CRISPR/Cas9 system for further loss-of-function assays. We have successfully identified homozygous mutant C25-CL48 clones for ACVR1B , herein referred as ALK4-KO. We found that ALK4-KO cells showed a 4-nucleotide deletion within its first exon (Supplementary Fig. 2A). This NHEJ-driven mutation putatively caused a frameshift as inferred by bioinformatic tools to produce a truncated receptor at codon 83. We confirmed the loss of type I receptor ALK4 by confocal microscopy. Wild-type C25-CL48 cells exhibited speckled cytoplasmic distribution of ALK4 receptors, while single mutant cells showed no positive labeling for their truncated receptor (Supplementary Fig. 2B). Incubation of mutant cells under proliferative conditions for 6 days, either in the absence (control) or presence of rGDF11, produced no significant differences in the median total number of nuclei per 100,000 µm 2 among groups (data not shown). However, the inactivation of ALK4 greatly impaired myoblast differentiation and fusion (Figs. 3 A,C-E). After six days in differentiation medium, no significant differences in the median total number of nuclei per 100,000 µm 2 were observed between wild-type and ALK4-KO cells, either untreated (50.47 nuclei, 95% CI: 49.67–54.79 vs. 37.41 nuclei, 95% CI: 37.35–50.21) or treated with rGDF11 (37.54 nuclei, 95% CI: 37.54–52.47 vs. 37.14 nuclei, 95% CI: 36.77–43.72. These results indicates that ALK4 does not affect cell survival under differentiation conditions (Fig. 3 B). By contrast, differentiation and fusion were nearly completed abolished in ALK4-KO cells, and this effect was not altered by rGDF11 treatment. The median differentiation indices of untreated wild-type and ALK4-KO cells were 51.91% (95% CI: 31.39–56.38) and 0.2% (95% CI: 0.14–0.83), respectively. Differently, rGDF11-treated cultures showed markedly reduced differentiation, with median differentiation indices of 0.07% (95% CI: 0.014–1.62) and 0.00% (95% CI: 0.00–0.00) for the respective genotypes (Fig. 3 C). Likewise, the median fusion indices were 42.16% (95% CI: 26.67–46.84) and 0.00% (95% CI: 0.00–0.00) in untreated cells, versus 0.00% (95% CI: 0.00–0.06) and 0.00% (95% CI: 0.00–0.00) following rGDF11 treatment (Fig. 3 D). Analysis of nuclei per MyHC + myocyte/myotube revealed that either rGDF11 or ALK4 inactivation strongly impaired multinucleated myotube formation (Fig. 3 E). Interestingly, MyHC + cells were detected only in untreated ALK4-KO cultures at day 10, and this residual differentiation was further suppressed by rGDF11 (Fig. 3 A,E). These findings highlight two complementary roles: GDF11 suppresses skeletal muscle differentiation independently of ALK4, whereas inactivation of ALK4 alone significantly delays MyHC production and myoblast fusion. GDF11 impairs myoblast migration, while ALK4 is necessary During normal skeletal myogenesis, satellite cell activation and myoblast proliferation are accompanied by an oriented migration, a process critical for proper cell alignment, fusion, and subsequent myotube formation (Kim et al. 2015 ). In cell therapies for muscular dystrophies, the limited migratory capacity of myoblasts poses a significant challenge for muscle regeneration, as cells often remain localized near the injection site (Skuk et al. 1999 , 2006 ; Riederer et al. 2012 ). Given the limited studies addressing the role of GDF11 in myoblast migration, we investigated its impact using LM111 as a substrate in transmigration assays. Consistent with our previous findings (Silva-Barbosa et al. 2008 ; González et al. 2017 ), we showed that wild-type myoblasts migrated efficiently through LM111-coated membranes, whereas migration was limited on BSA-coated control surfaces (data not shown). To investigate whether GDF11 promotes myoblast migration, rGDF11 was added to the lower chamber of the Transwell plates to test its potential chemoattractant effect. However, this treatment did not enhance migration. Conversely, the addition of rGDF11 to the upper chamber significantly reduced myoblast migration toward the lower chamber compared with controls (Fig. 4 A). These findings suggest that rGDF11 impairs myoblast motility, which may account for its inhibitory effect on myotube formation, since migration and cell alignment are prerequisites for myoblast fusion. Moreover, deletion of ACVR1B resulted in a pronounced reduction in the migratory capacity of ALK4-KO cells through LM111 under all tested conditions (Fig. 4 A). ALK4 regulates LM111-dependent myoblast adhesion and integrin expression independently of GDF11 Cell-cell and cell-ECM interactions play fundamental roles in various aspects of skeletal muscle cell biology, particularly in processes such as migration and fusion. To determine whether the effects observed following rGDF11 treatment could be attributed to defects in cell adhesion, we cultured cells on coverslips coated with either poly-L-lysine (PLL) or LM111, in the presence or absence of rGDF11. PLL- or LM111-coated coverslips, with or without rGDF11 treatment, did not significantly affect the average number of nuclei per 100,000 µm 2 in either wild-type or CRISPR-mediated ALK4 mutant cells, after 2 hours in culture (Figs. 4 B,C). In contrast, LM111 coating markedly increased the median cell area of wild-type myoblasts as compared with PLL-coated surfaces (1,781.0 µm 2 , 95% CI [1,542.0, 4,031.0] vs. 405.6 µm 2 , 95% CI [252.9, 1,012.0], respectively). Moreover, rGDF11 treatment did not affect the cell area on either substrate (Fig. 4 B,D; Supplementary Tables 1,2). Notably, LM111 coating promoted robust myoblast-myoblast interactions during the 2-hour adhesion period regardless of rGDF11 treatment, an effect that was significantly more pronounced than that observed with PLL coating (Fig. 4 B-E; Table 1 ). Conversely, loss of ALK4 resulted in reduced cell area and diminished cell-cell contacts on LAM111 (Fig. 4 B-E; Table 1 ). Collectively, these findings suggest that ALK4 may contribute to LM111-mediated cell adhesion and spreading through a GDF11-independent mechanism. Table 1 Cell-cell contact index in adhesion assays. contact index (%) genotype coating treatment # exp median 95% CI (l.l., u.l.) WT PLL untreated 3 4.51 [3.84, 27.74] WT LM111 untreated 3 48.99 [36.14, 60.83] WT PLL rGDF11 3 13.33 [5.51, 20.43] WT LM111 rGDF11 3 54.90 [42.35, 64.54] ALK4-KO PLL untreated 3 23.57 [9.61, 50.11] ALK4-KO LM111 untreated 3 29.31 [13.34, 38.48] ALK4-KO PLL rGDF11 3 39.03 [12.82, 41.92] ALK4-KO LM111 rGDF11 3 27.12 [22.24, 42.42] WT, wild-type; ALK4-KO, ALK4 knockout; PLL, poly-L-lysine; LM111, laminin 111; # exp, number of experiments; 95% CI, 95% confidence interval (CI). Given the observed role of LM111 seen in our experiments, we sought to contextualize its function within the broader framework of muscle regeneration. Together with the dystroglycan complex, the α7β1 integrin serve as a primary receptor for laminins in skeletal muscle. Laminins are key components of the basement membrane that individually surround each muscle fiber (Schüler et al. 2022 ). While mature fibers predominantly express the LM211 and LM221 isoforms, LM111 is enriched in the satellite cell niche during regeneration (Gawlik and Durbeej 2011 ). In particular, multiple LM isoforms, including LM111, can be bound by the versatile integrin α6β1 receptor (Nishiuchi et al. 2006 ). This raises the possibility that distinct LM-integrin interactions may modulate myoblast adhesion and differentiation through a GDF11-dependent mechanism. We further investigated the presence of distinct integrin subunits in wild-type and ALK4-KO myoblasts using flow cytometry to elucidate the alterations in cell adhesion. The relative levels of the common CD29 subunit (β1 integrin) and the LM332-binding CD49c (α3 integrin) exhibited no significant differences between wild-type and ALK4-KO myoblasts (Figs. 4 F,G). In contrast, the fibronectin/VLA4-binding CD49d (α4 integrin) and the LM111/LM332/LM511/LM521-binding CD49f (α6 integrin) exhibited approximately 2- and 4.5-fold downregulation in ALK4-KO myoblasts compared to wild-type cells (Fig. 4 F,G). These findings suggest that ALK4-mediated signaling may play a role in regulating ITGA4 and ITGA6 gene expression, or the stability of CD49d and CD49f integrin subunits through a mechanism that appears to be independent of the GDF11 ligand. The intricate signaling network, where multiple members of the TGF superfamily converge on shared receptors, adds a layer of complexity to the regulatory mechanisms governing skeletal muscle differentiation. Together, our observations highlight promising prospects of both GDF11 and ALK4 as potential candidates in regenerative medicine. Discussion Our study provides new insights into the complex signaling network that governs skeletal myogenesis, particularly by disentangling the role of GDF11 from an ALK4-dependent pathway that regulates myoblast adhesion, migration, and differentiation. Although GDF11 has been widely debated in the literature with reports attributing both stimulatory and inhibitory roles in muscle biology, our data indicate that its direct impact on myoblast proliferation and adhesion is minimal. Instead, we observed a robust inhibitory effect on myoblast differentiation and fusion, which could be partially explained by the reduced cell motility in the presence of GDF11. Surprisingly, pharmacological inhibition of the three known GDF11 receptors (ALK4, ALK5, and ALK7) or genetic knockout of ALK4 in human myoblasts resulted in a robust decrease in cell differentiation and fusion. In both approaches designed to block GDF11 activity, we found reduced myoblast fusion, suggesting the involvement of an additional receptor for GDF11. Furthermore, ALK4-KO myoblasts displayed impaired adhesion and spreading, as well as a marked loss of migratory capacity. These phenotypes could be attributed to the downregulation of the α4 and α6 integrin chains, with probable consequences upon the binding of these cells on fibronectin and laminin, respectively. A critical aspect of our work was the combined use of biochemical stimulation and CRISPR-based genetic approaches, which allowed us to parse the contributions of GDF11 and ALK4 in skeletal muscle development. Despite the well-documented involvement of TGF-β superfamily members in regulating tissue regeneration (Gumucio et al. 2015 ; Derynck and Budi 2019 ; Ren et al. 2023 ), our findings revealed that GDF11 influence on myogenesis is not mediated through alterations in cell proliferation or adhesion per se . Unlike proliferation, adhesion was evaluated at a single early time point with a short assay duration. Although ALK4-KO myoblasts exhibited impaired adhesion within the first two hours, they appeared to recover over time, as indicated by normal proliferation during culture. Nevertheless, transient adhesion defects in an inflammatory or regenerative environment could have significant consequences during critical early phases. Importantly, insufficient substrate adhesion can induce anoikis, a form of programmed cell death (Zhong and Rescorla 2012 ), which may further exacerbate impairments in muscle regeneration. In addition, GDF11 capacity to inhibit differentiation and migration appears to be uncoupled from its potential receptor-mediated signaling events. This divergence from earlier studies that emphasized a proliferative or adhesive role for GDF11 in various cell types (Finkenzeller et al. 2015 ; Zhang et al. 2016 ; Yu et al. 2018 ) underscores the necessity of examining receptor-specific signaling events and their downstream effectors. GDF11 robustly impaired human myoblast differentiation, particularly cell fusion, corroborating recent results on human cells (Egerman et al. 2025 ). Myostatin (GDF8), a closely related ligand with high similarity to GDF11, is also a potent inhibitor of myoblast differentiation (Walker et al. 2016 , 2017 ; Suh and Lee 2020 ). Both factors promote muscle atrophy through Smad2/3-dependent inhibition of Akt signaling (Trendelenburg et al. 2009 ), and via the non-Smad Ras-ERK pathway (Masuzawa et al. 2022 ). Notably, the same concentration of GDF11 that inhibited differentiation also reduced myoblast motility on LM111. This reduction in cell migration may contribute to impaired fusion, as myotube formation requires coordinated migration, alignment, and membrane fusion (Lehka and Rędowicz 2020 ). Alternatively, GDF11 may act on signaling pathways shared by both processes, including IGF-1/Akt, Rho family GTPases, and integrin–FAK signaling (Derynck and Budi 2019 ). We found that both myoblasts and myotubes express GDF11 and its receptors ALK4, ALK5, and ALK7 (data not shown). However, information on GDF11 expression during muscle regeneration remains unavailable. Interestingly, we also observed that macrophage-derived monocytes express GDF11 (data not shown). One possibility is that, during the early pro-inflammatory phase of muscle regeneration, GDF11 acts to inhibit cell differentiation and migration, thereby permitting the expansion of the myoblast pool. In addition, GDF11 has been reported to promote the transition of macrophages from a pro-inflammatory M1 phenotype to a reparative M2 phenotype (Gong et al. 2023 ), which could further support tissue repair. Interestingly, our findings show that GDF11 impairs myoblast fusion, without affecting myoblast proliferation in human muscle cell cultures. Conversely, treatment of chick myogenic cells with GDF11 exhibited a reduction in the total number of nuclei compared to untreated cells. We hypothesize that these differences in the effects of GDF11 on cell proliferation might be related to the presence of fibroblasts in chick myogenic cultures and that GDF11 may influence the proliferation or cell death of skeletal muscle fibroblasts. These results require further investigation. Experiments carried out in ACVR1B -deficient myoblasts further elucidated the molecular underpinnings of this regulatory network. The observed delay in myoblast differentiation, coupled with impaired adhesion and migration onto LM111, points to a critical role for ALK4 in these processes. Importantly, the reduction of surface integrin α6 levels in ACVR1B -deficient cells highlights a novel link between ALK4 signaling and integrin-mediated cell adhesion. CD49f (the α6 integrin chain), a key component of the VLA-6 receptor complex, is known to interact with laminin substrates (Nishiuchi et al. 2006 ; Taniguchi et al. 2009 ; Arimori et al. 2021 ) and facilitate cell–matrix interactions essential for myoblast fusion. Thus, our data suggest that an ALK4/VLA6/LM111 axis may serve as a pivotal modulator of myoblast behavior, independent of GDF11 ligand activity. The identification of this GDF11-independent ALK4 pathway has broad implications for understanding skeletal muscle regeneration. In the context of aging and skeletal muscle degeneration, aberrant signaling through TGF-β family receptors has been implicated in the decline of regenerative capacity (Brack et al.; Gough 2008 ; Carlson et al. 2009 ; Smith et al. 2015 ; Harper et al. 2016 ). Our results indicate that modulation of ALK4 activity, or its downstream effect on integrin α6 expression, could represent a novel therapeutic avenue. By decoupling the effects of GDF11 from the receptor-mediated mechanisms that govern adhesion and differentiation, we open the possibility of selectively targeting ALK4-dependent pathways to enhance muscle regeneration without eliciting the conflicting outcomes associated with GDF11. Nevertheless, our study is not without limitations. While in vitro analyses using chicken and human myoblasts provide valuable mechanistic insights, the translation of these findings to in vivo systems remains to be explored. Future studies will need to assess whether the ALK4/VLA6/LM111 axis similarly influences muscle regeneration in animal models of injury and aging. Moreover, it will be important to determine the extent to which other TGF-β superfamily members and their receptor complexes intersect with or modulate this pathway. Detailed temporal and spatial analyses of receptor expression and downstream signaling events during muscle regeneration will further clarify these interactions. In conclusion, our work delineates a previously underappreciated ALK4-dependent regulatory mechanism that governs myoblast differentiation, adhesion, and migration, independent of direct GDF11 activity. These findings not only reconcile conflicting reports regarding GDF11's role in skeletal muscle biology but also suggest that targeting specific receptor-mediated pathways may offer a more refined strategy for therapeutic intervention in muscle degenerative conditions. Knowledge of the ALK4/VLA6/LM111 axis is crucial for future studies aiming to harness the regenerative potential of skeletal muscle, with the ultimate goal of mitigating age-related muscle decline and promoting effective tissue repair. Declarations Author Contribution R.F.R., M.L.C., V.C.A., C.M., A.D.J., and I.R. designed experiments; R.F.R., I.F.S., B.G.C.S., K.M.B., and A.D.J. performed experiments; R.F.R., I.F.S., B.G.C.S., K.M.B., M.L.C., V.M., W.S., V.C.A., C.M., A.D.J., and I.R. analyzed data; A.D.J., and I.R. wrote the main manuscript text; C.M. contributed to drafting portions of the manuscript; A.D.J., and I.R. prepared figures; M.L.C., W.S., V.C.A., C.M., A.D.J., and I.R. secured funding; All authors reviewed the results, discussed the findings, and approved the final version of the manuscript. Acknowledgement This work is dedicated to the memory of Vania Maria Mello Dias. The research at the Laboratory on Thymus Research is funded by the Brazilian National Institute of Science and Technology on Neuroimmunomodulation (INCT.NIM), MercoSur Fund for Structural Convergence/FOCEM (Project #03/11), Rio de Janeiro Network on Neuroinflammation (FAPERJ) and Inova-IOC network on Neuroimmunomodulation. The work was also supported by fellowships 88882.332573/2019-01 (PROEX/CAPES) to RFR, 160832/2021-7 (CNPq) to IFS, 381427/2025-0 (TDI-B/INCT-CNPq) to BGCS, E-26/201.295/2023 (FAPERJ) to KMB, 302961/2021-6 (CNPq) and E-26/203.930/2024 (CNE/FAPERJ) to CM, 308192/2021-4 (CNPq) and E-26/204.077/2024 (CNE/FAPERJ) to MLC, and E26/202.683/2016 (PD10/FAPERJ) and 101665/2024-5 (PDS/CNPq) to ADJ. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. References Aires R, Jurberg AD, Leal F, et al. Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity. Dev Cell. 2016;38:262–74. https://doi.org/10.1016/j.devcel.2016.06.021 . Andersson O, Reissmann E, Ibáñez CF. Growth differentiation factor 11 signals through the transforming growth factor-beta receptor ALK5 to regionalize the anterior-posterior axis. EMBO Rep. 2006;7:831–7. https://doi.org/10.1038/sj.embor.7400752 . Arimori T, Miyazaki N, Mihara E, et al. Structural mechanism of laminin recognition by integrin. Nat Commun. 2021;12. https://doi.org/10.1038/s41467-021-24184-8 . Brack AS, Conboy MJ, Roy S et al. Increased Wnt Signaling During Aging Alters Muscle Stem Cell Fate and Increases Fibrosis. Briggs D, Morgan JE. Recent progress in satellite cell/myoblast engraftment - Relevance for therapy. FEBS J. 2013;280:4281–93. https://doi.org/10.1111/febs.12273 . Buckingham M, Rigby PWJ. Gene Regulatory Networks and Transcriptional Mechanisms that Control Myogenesis. Dev Cell. 2014;28:225–38. https://doi.org/10.1016/j.devcel.2013.12.020 . Carlson ME, Conboy MJ, Hsu M, et al. Relative roles of TGF-β1 and Wnt in the systemic regulation and aging of satellite cell responses. Aging Cell. 2009;8:676–89. https://doi.org/10.1111/j.1474-9726.2009.00517.x . Chapman MA, Meza R, Lieber RL. Skeletal muscle fibroblasts in health and disease. Differentiation. 2016;92:108–15. https://doi.org/10.1016/j.diff.2016.05.007 . Costa ML, Jurberg AD, Mermelstein C. The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis. Front Physiol. 2021;12:1–15. https://doi.org/10.3389/fphys.2021.668600 . Derynck R, Budi EH. Specificity, versatility, and control of TGF-β family signaling. Sci Signal. 2019;12. https://doi.org/10.1126/scisignal.aav5183 . Egerman MA, Cadena SM, Gilbert JA, et al. GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration. Cell Metab. 2015;22:164–74. https://doi.org/10.1016/j.cmet.2015.05.010 . Egerman MA, Zhang Y, Donne R, et al. ActRII or BMPR ligands inhibit skeletal myoblast differentiation, and BMPs promote heterotopic ossification in skeletal muscles in mice. Skelet Muscle. 2025;15. https://doi.org/10.1186/s13395-025-00373-7 . Finkenzeller G, Stark GB, Strassburg S. Growth differentiation factor 11 supports migration and sprouting of endothelial progenitor cells. J Surg Res. 2015;1–7. https://doi.org/10.1016/j.jss.2015.05.001 . Gawlik KI, Durbeej M. Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies. Skelet Muscle 1; 2011. Ge G, Hopkins DR, Ho W-B, Greenspan DS. GDF11 Forms a Bone Morphogenetic Protein 1-Activated Latent Complex That Can Modulate Nerve Growth Factor-Induced Differentiation of PC12 Cells. Mol Cell Biol. 2005;25:5846–58. https://doi.org/10.1128/mcb.25.14.5846-5858.2005 . Gong M, Yang X, Wang Y, et al. Growth differentiation factor 11 promotes macrophage polarization towards M2 to attenuate myocardial infarction via inhibiting Notch1 signaling pathway. Frigid Zone Med. 2023;3:53–64. https://doi.org/10.2478/fzm-2023-0008 . González MN, de Mello W, Butler-Browne GS, et al. HGF potentiates extracellular matrix-driven migration of human myoblasts: Involvement of matrix metalloproteinases and MAPK/ERK pathway. Skelet Muscle. 2017;7:1–13. https://doi.org/10.1186/s13395-017-0138-6 . Gough NR. Why Aging Muscles Don’t Repair. Sci Signal. 2008. https://doi.org/10.1126/scisignal.130ec272 . 1:. Gumucio JP, Sugg KB, Mendias CL. TGF-β Superfamily Signaling in Muscle and Tendon Adaptation to Resistance Exercise. Exerc Sport Sci Rev. 2015;43:93–9. https://doi.org/10.1249/JES.0000000000000041 . Harper SC, Brack A, MacDonnell S, et al. Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?Response to Harper et al. Circ Res. 2016;118:1143–50. https://doi.org/10.1161/CIRCRESAHA.116.307962 . Hassan SN, Ahmad F. Considering dimethyl sulfoxide solvent toxicity to mammalian cells and its biological effects. Exp Oncol. 2024;46:174–8. https://doi.org/10.15407/exp-oncology.2024.02.174 . Hinck AP. Structural studies of the TGF-βs and their receptors - insights into evolution of the TGF-β superfamily. FEBS Lett. 2012;586:1860–70. https://doi.org/10.1016/j.febslet.2012.05.028 . Horbelt D, Denkis A, Knaus P. A portrait of Transforming Growth Factor β superfamily signalling: Background matters. Int J Biochem Cell Biol. 2012;44:469–74. https://doi.org/10.1016/j.biocel.2011.12.013 . Jurberg AD, Aires R, Varela-Lasheras I, et al. Switching Axial Progenitors from Producing Trunk to Tail Tissues in Vertebrate Embryos. Dev Cell. 2013;25:451–62. https://doi.org/10.1016/j.devcel.2013.05.009 . Jurberg AD, Vasconcelos-Fontes L, Cotta-de-Almeida VV. A Tale from TGF-β Superfamily for Thymus Ontogeny and Function. Front Immunol. 2015;6:1–15. https://doi.org/10.3389/fimmu.2015.00442 . Katsimpardi L, Litterman NK, Schein PA et al. (2014) Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science (1979) 344:630–634. https://doi.org/10.1126/science.1251141 Kim JH, Jin P, Duan R, Chen EH. Mechanisms of myoblast fusion during muscle development. Curr Opin Genet Dev. 2015;32:162–70. Lee SJ, McPherron a C. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci U S A. 2001;98:9306–11. https://doi.org/10.1073/pnas.151270098 . Lehka L, Rędowicz MJ. Mechanisms regulating myoblast fusion: A multilevel interplay. Semin Cell Dev Biol. 2020;104:81–92. Li Q, Li H, Zhu L, et al. Growth Differentiation Factor 11 Evokes Lung Injury, Inflammation, and Fibrosis in Mice through the Activin A Receptor Type II-Like Kinase, 53kDa–Smad2/3 Signaling Pathway. Am J Pathol. 2024;194:2036–58. https://doi.org/10.1016/j.ajpath.2024.07.016 . Li Z, Kawasumi M, Zhao B, et al. Transgenic over-expression of growth differentiation factor 11 propeptide in skeleton results in transformation of the seventh cervical vertebra into a thoracic vertebra. Mol Reprod Dev. 2010. https://doi.org/10.1002/mrd.21252 . Loffredo FS, Steinhauser ML, Jay SM, et al. Growth Differentiation Factor 11 Is a Circulating Factor that Reverses Age-Related Cardiac Hypertrophy. Cell. 2013;153:828–39. https://doi.org/10.1016/j.cell.2013.04.015 . Masuzawa R, Takahashi K, Takano K, et al. DA-Raf and the MEK inhibitor trametinib reverse skeletal myocyte differentiation inhibition or muscle atrophy caused by myostatin and GDF11 through the non-Smad Ras–ERK pathway. J Biochem. 2022;171:109–22. https://doi.org/10.1093/jb/mvab116 . Mermelstein CDS, Costa ML, Filho CC, Neto VM. Intermediate filament proteins in TPA-treated skeletal muscle cells in culture. J Muscle Res Cell Motil. 1996;17:199–206. https://doi.org/10.1007/BF00124242 . Moustakas A, Heldin C-H. The regulation of TGFbeta signal transduction. Development. 2009;136:3699–714. https://doi.org/10.1242/dev.030338 . Nakashima M, Toyono T, Akamine A, Joyner A. Expression of growth/differentiation factor 11, a new member of the BMP/TGFbeta superfamily during mouse embryogenesis. Mech Dev. 1999;80:185–9. Nishiuchi R, Takagi J, Hayashi M, et al. Ligand-binding specificities of laminin-binding integrins: A comprehensive survey of laminin–integrin interactions using recombinant α3β1, α6β1, α7β1 and α6β4 integrins. Matrix Biol. 2006;25:189–97. https://doi.org/10.1016/j.matbio.2005.12.001 . Poggioli T, Vujic A, Yang P, et al. Circulating Growth Differentiation Factor 11/8 Levels Decline with Age. Circ Res. 2015. https://doi.org/10.1161/CIRCRESAHA.115.307521 . Ran FA, Hsu PD, Wright J et al. (2013) Genome engineering using the CRISPR-Cas9 system. 8:2281–308. https://doi.org/10.1038/nprot.2013.143 Ren L-L, Li X-J, Duan T-T, et al. Transforming growth factor-β signaling: From tissue fibrosis to therapeutic opportunities. Chem Biol Interact. 2023;369:110289. https://doi.org/10.1016/j.cbi.2022.110289 . Riederer I, Negroni E, Bencze M, et al. Slowing Down Differentiation of Engrafted Human Myoblasts Into Immunodeficient Mice Correlates With Increased Proliferation and Migration. Mol Ther. 2012;20:146–54. https://doi.org/10.1038/mt.2011.193 . Rodgers BD, Eldridge Ja. (2015) Reduced Circulating GDF11 Is Unlikely Responsible for Age-dependent Changes in Mouse Heart, Muscle, and Brain. Endocrinology en.2015 – 1628. https://doi.org/10.1210/en.2015-1628 Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. https://doi.org/10.1038/nmeth.2019 . Schüler SC, Liu Y, Dumontier S et al. (2022) Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche. Front Cell Dev Biol 10. Seale P, Sabourin LA, Girgis-Gabardo A et al. (2000) Pax7 Is Required for the Specification of Myogenic Satellite Cells skeletal muscle are mitotically quiescent and are acti-vated in response to diverse stimuli, including stretch-ing, exercise, injury, and electrical stimulation (Schultz oblast-specific genes potentially involved in satellite cell. Silva-Barbosa SD, Butler-Browne GS, De Mello W, et al. Human myoblast engraftment is improved in laminin-enriched microenvironment. Transplantation. 2008;85:566–75. https://doi.org/10.1097/TP.0b013e31815fee50 . Sinha M, Jang YC, Oh J et al. (2014) Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science (1979) 344:649–652. https://doi.org/10.1126/science.1251152 Skuk D, Goulet M, Tremblay JP. Use of Repeating Dispensers to Increase the Efficiency of the Intramuscular Myogenic Cell Injection Procedure. Cell Transpl. 2006;15:659–63. Skuk D, Roy B, Goulet M, Tremblay JP. Successful Myoblast Transplantation in Primates Depends on Appropriate Cell Delivery and Induction of Regeneration in the Host Muscle. Exp Neurol. 1999;155:22–30. https://doi.org/10.1006/exnr.1998.6973 . Smith SC, Zhang X, Zhang X, et al. GDF11 Does Not Rescue Aging-Related Pathological Hypertrophy. Circ Res CIRCRESAHA. 2015. https://doi.org/10.1161/CIRCRESAHA.115.307527 . .115.307527. Suh J, Lee Y-S. Similar sequences but dissimilar biological functions of GDF11 and myostatin. Exp Mol Med. 2020;52:1673–93. https://doi.org/10.1038/s12276-020-00516-4 . Swan J, Szabó Z, Peters J, et al. Inhibition of activin receptor 2 signalling ameliorates metabolic dysfunction–associated steatotic liver disease in western diet/L-NAME induced cardiometabolic disease. Biomed Pharmacotherapy. 2024;175. https://doi.org/10.1016/j.biopha.2024.116683 . Taniguchi Y, Ido H, Sanzen N, et al. The C-terminal region of laminin β chains modulates the integrin binding affinities of laminins. J Biol Chem. 2009;284:7820–31. https://doi.org/10.1074/jbc.M809332200 . Thorley M, Duguez S, Mazza EMC, et al. Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines. Skelet Muscle. 2016;6:43. https://doi.org/10.1186/s13395-016-0115-5 . Trendelenburg AU, Meyer A, Rohner D, et al. Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size. Am J Physiol Cell Physiol. 2009;296:C1258–70. https://doi.org/10.1152/ajpcell.00105.2009 . Uezumi A, Ito T, Morikawa D, et al. Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle. J Cell Sci. 2011;124:3654–64. https://doi.org/10.1242/jcs.086629 . Walker RG, Czepnik M, Goebel EJ, et al. Structural basis for potency differences between GDF8 and GDF11. BMC Biol. 2017;15:19. https://doi.org/10.1186/s12915-017-0350-1 . Walker RG, Poggioli T, Katsimpardi L, et al. Biochemistry and Biology of GDF11 and Myostatin. Circ Res. 2016;118:1125–42. https://doi.org/10.1161/CIRCRESAHA.116.308391 . Yu X, Chen X, Zheng XD, et al. Growth Differentiation Factor 11 Promotes Abnormal Proliferation and Angiogenesis of Pulmonary Artery Endothelial Cells. Hypertension. 2018;71:729–41. https://doi.org/10.1161/HYPERTENSIONAHA.117.10350 . Zhang Y, Cheng F, Du X, et al. GDF11/BMP11 activates both smad1/5/8 and smad2/3 signals but shows no significant effect on proliferation and migration of human umbilical vein endothelial cells. Oncotarget. 2016. https://doi.org/10.18632/oncotarget.7642 . Zhong X, Rescorla FJ. Cell surface adhesion molecules and adhesion-initiated signaling: Understanding of anoikis resistance mechanisms and therapeutic opportunities. Cell Signal. 2012;24:393–401. Zhou Y, Sharma N, Dukes D, et al. GDF11 Treatment Attenuates the Recovery of Skeletal Muscle Function After Injury in Older Rats. AAPS J. 2017;19:431–7. https://doi.org/10.1208/s12248-016-0024-x . Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.docx S1.jpg S2.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7777108","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":531691410,"identity":"da2e5eaf-68ad-40a9-92f6-07e02b22c678","order_by":0,"name":"Rafaella Ferreira Reis","email":"","orcid":"","institution":"Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Rafaella","middleName":"Ferreira","lastName":"Reis","suffix":""},{"id":531691411,"identity":"e508b26b-f73b-4405-83bf-850e23141937","order_by":1,"name":"Igor Ferreira da Silva","email":"","orcid":"","institution":"Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Igor","middleName":"Ferreira da","lastName":"Silva","suffix":""},{"id":531691412,"identity":"d2b242bc-10b6-41e2-aee0-ba43d86bd92b","order_by":2,"name":"Beatriz Guimarães Costa-Santos","email":"","orcid":"","institution":"Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"Guimarães","lastName":"Costa-Santos","suffix":""},{"id":531691413,"identity":"00fb8db4-eb53-497f-8567-cf040a3e48a9","order_by":3,"name":"Kayo Moreira Bagri","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Kayo","middleName":"Moreira","lastName":"Bagri","suffix":""},{"id":531691414,"identity":"972e889d-e0a0-45d3-aca5-81461e46cbb5","order_by":4,"name":"Manoel Luis Costa","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Manoel","middleName":"Luis","lastName":"Costa","suffix":""},{"id":531691415,"identity":"0684f329-74a6-4c92-a9b7-b8b04ecdee58","order_by":5,"name":"Vincent Mouly","email":"","orcid":"","institution":"Sorbonne University","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Mouly","suffix":""},{"id":531691416,"identity":"9bf71225-127a-444c-9206-05613c8b8ba1","order_by":6,"name":"Wilson Savino","email":"","orcid":"","institution":"Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Wilson","middleName":"","lastName":"Savino","suffix":""},{"id":531691417,"identity":"bec8c6f3-55e8-4d09-9f93-71ea0e28c9b7","order_by":7,"name":"Vinicius Cotta-de-Almeida","email":"","orcid":"","institution":"Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"Vinicius","middleName":"","lastName":"Cotta-de-Almeida","suffix":""},{"id":531691418,"identity":"61ac3b9e-27e5-4d88-8cf4-756bba34bee7","order_by":8,"name":"Claudia Mermelstein","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Mermelstein","suffix":""},{"id":531691419,"identity":"f4b4e2b8-9700-414d-9fe8-00a889ab647d","order_by":9,"name":"Arnon Dias Jurberg","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYFCCAwzMIIofRCQUkKJFsgGkxYBIe8BaDA6ASSKU8zeeMftcUHHPbvP51YkfHhgwyPOLHcCvReLAGePZM84UJ2+78XazBNBhhjNnJxCwBqiFmbctIdnsxtkNIC0JBrcJaJEHa/mXkGw84+zmH0RpMQBraUiwM+Dv3UacLYYHjhUzzziWkCBxg3ebRYKBBGG/yN04vJm5oCbBnr//7OabPyps5PmlCWgBBhmYSmyQAKuUIKAcBPgbwJQ9A/8BIlSPglEwCkbBiAQAPhBHWDBSBm4AAAAASUVORK5CYII=","orcid":"","institution":"Oswaldo Cruz Foundation","correspondingAuthor":true,"prefix":"","firstName":"Arnon","middleName":"Dias","lastName":"Jurberg","suffix":""},{"id":531691420,"identity":"c3001f37-5324-4b18-a3b1-6e10db408020","order_by":10,"name":"Ingo Riederer","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Ingo","middleName":"","lastName":"Riederer","suffix":""}],"badges":[],"createdAt":"2025-10-04 03:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7777108/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7777108/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94142616,"identity":"510f908b-d682-4e1e-a1a8-6eb0527d8b0a","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3157159,"visible":true,"origin":"","legend":"","description":"","filename":"ReisetalGDF11final.docx","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/a7061d6d9e47cb1acb5bfcc9.docx"},{"id":94142599,"identity":"288770c5-c6bb-4d3b-a7d4-1f22b4aa8874","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12188,"visible":true,"origin":"","legend":"","description":"","filename":"b886bcee32a2467784fe6e0ac375b6be.json","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/a5654ebc4f8dc965ba03392f.json"},{"id":94142837,"identity":"2aab64e6-6dcd-4e79-b2f6-7a4444edc5ff","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15623,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/0d7d21807246182bb9f22320.docx"},{"id":94142613,"identity":"1dd3c7b9-5756-41f2-96f1-4ec07fd9d5db","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154856,"visible":true,"origin":"","legend":"","description":"","filename":"b886bcee32a2467784fe6e0ac375b6be1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/9ea3366fd628a23e91abd7eb.xml"},{"id":94142844,"identity":"968dd0be-ea8e-4477-b7f8-8d183d6eb793","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1106770,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/60aaf483ada914f13bef55f4.jpeg"},{"id":94143422,"identity":"84e70bc1-916a-4cf5-a480-57ff53369801","added_by":"auto","created_at":"2025-10-22 20:45:23","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":733015,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/b20a8a369530a0051a906916.png"},{"id":94142606,"identity":"9d5a3796-3d67-4c6b-b417-19fded2570c1","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":479521,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/576e9ffd17660ed5860558b8.png"},{"id":94142842,"identity":"65757e36-6215-4359-8f34-503be8f6d0d4","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":473285,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/f19d9ae841e0189c4b96e996.jpeg"},{"id":94142611,"identity":"72aaaf49-b5b5-4041-b3e1-6003b7fa00a5","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":430322,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/8ebab986d1f16d2c7e4339bc.png"},{"id":94142840,"identity":"a7e91e12-ecd8-422a-93ea-5927e3f80149","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67581,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/33044cab5965a38347191fd7.png"},{"id":94142847,"identity":"a50ed1e2-735f-41c2-8ae4-ee43eb1dd9e2","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":451084,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/0b21808a50b21ee69619b79a.png"},{"id":94142845,"identity":"4e4ad3c4-b71d-4997-96bd-ae65ec67819f","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202342,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/824e5dbfa51ba1bc3690739b.png"},{"id":94142838,"identity":"21141fef-4bd0-4fbf-8420-f4678bece7d8","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105673,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/8f6cd12b0c5a4ffd1e84f757.png"},{"id":94142618,"identity":"474591b5-6f94-42c7-a16a-d791ea5f339c","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67631,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/2cbc7c3eaee9deb734702c36.png"},{"id":94143423,"identity":"32b713ed-4aa0-428b-b338-32b08cc6e7f2","added_by":"auto","created_at":"2025-10-22 20:45:23","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115294,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/3d79fa54ee299f106ea703a4.png"},{"id":94142621,"identity":"41629baf-2679-4163-a608-0a61cf154f9a","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61569,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/e542cc09c8e8defd9af1a103.png"},{"id":94142846,"identity":"34337468-1ad2-4c32-947a-85af3127ebe7","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19205,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/9cac8f2f3010aff1de925c12.png"},{"id":94143424,"identity":"3dfa4f81-b883-4803-89b5-272f16a9d466","added_by":"auto","created_at":"2025-10-22 20:45:23","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55668,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/bbc41a347cd4afce8577ce59.png"},{"id":94142622,"identity":"1b675e07-76fa-40d8-b3cc-411fac0c8eb0","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154774,"visible":true,"origin":"","legend":"","description":"","filename":"b886bcee32a2467784fe6e0ac375b6be1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/d4e9a81f08e726f4083d0675.xml"},{"id":94142623,"identity":"cb7a2335-c13e-401b-abd4-bd77b596e36a","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165406,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/7d50e6e0d0348efe51838c92.html"},{"id":94142835,"identity":"619f8afc-1096-4096-8593-5048338d4365","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003echick skeletal myogenesis under the influence of GDF11 signaling.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Chick muscle cells were cultured for 72 hours. Following the initial 24 hours, 100 ng/mL of recombinant GDF11 (rGDF11) was administered, or cells were maintained untreated as controls. Cultures were triple labeled with antibodies against desmin to label skeletal muscle cells, Pax7 as a readout of undifferentiated satellite cells, and the nuclear dye DAPI. Cells were visualized using conventional fluorescence microscopy. \u003cstrong\u003e(B–H)\u003c/strong\u003e Tukey box plots illustrating differentiation metrics in control and GDF11-treated cultures. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of the number of nuclei per area. \u003cstrong\u003e(C)\u003c/strong\u003eTotal number of nuclei within desmin-positive cells per area. \u003cstrong\u003e(D)\u003c/strong\u003e Area percentage of desmin-positive cells. \u003cstrong\u003e(E)\u003c/strong\u003e Percentage of nuclei residing in myotubes. \u003cstrong\u003e(F)\u003c/strong\u003e Number of Pax7-labeled skeletal muscle cells as a readout of undifferentiated satellite cells, along with positive nuclei per area. \u003cstrong\u003e(G)\u003c/strong\u003e Percentage of Pax7-positive nuclei. \u003cstrong\u003e(H)\u003c/strong\u003e Number of nuclei negative for both desmin and Pax7 per area. Data are presented as medians with the range indicated (minimum to maximum values). Each dot represents the average value from five to 10 microscopic fields for each independent experiment. Pair-wise comparisons were performed using two-tailed unpaired t-test. ns, not significant (not shown). *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/da009bc1cb61f8133efdd83a.jpg"},{"id":94142596,"identity":"89f6f485-57be-42ef-8156-637547fda82e","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGDF11 modulates human myoblast differentiation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A)\u003c/strong\u003e Representative fluorescence micrographs of human skeletal muscle cells cultured for 3 days or 6 days under proliferative conditions in the absence or presence of 100 ng/mL recombinant GDF11, as indicated. Cultures were fixed and stained with anti-Ki67 (magenta) to detect actively proliferating cells, and nuclei were counterstained with DAPI (blue). \u003cstrong\u003e(B–E)\u003c/strong\u003e Tukey box plots illustrating proliferation metrics in control and GDF11-treated cultures. \u003cstrong\u003e(B)\u003c/strong\u003e Total nuclei per area. \u003cstrong\u003e(C)\u003c/strong\u003e Proliferation doubling time (PDT) in days. \u003cstrong\u003e(D)\u003c/strong\u003e Number of Ki67⁺ nuclei per area. \u003cstrong\u003e(E)\u003c/strong\u003e Representative fluorescence micrographs of human muscle cells cultured under differentiation conditions for 3 days or 6 days in the absence or presence of 100 ng/mL recombinant GDF11, as indicated. Cultures were fixed and stained with anti-MyHC (green) to label differentiated myotubes, and nuclei were counterstained with DAPI (blue). \u003cstrong\u003e(F–I)\u003c/strong\u003e Tukey box plots illustrating differentiation metrics in control and GDF11-treated cultures. \u003cstrong\u003e(F)\u003c/strong\u003e Total nuclei per area. \u003cstrong\u003e(G)\u003c/strong\u003eNumber of MyHC⁺ cells per area. \u003cstrong\u003e(H) \u003c/strong\u003eDifferentiation index. \u003cstrong\u003e(I)\u003c/strong\u003eFusion index. \u003cstrong\u003e(J)\u003c/strong\u003e Number of nuclei residing in MyHC⁺ myotubes per area. Data are presented as medians with the range indicated (minimum to maximum values). Each dot represents the average value from five to 10 microscopic fields for each independent experiment. Pair-wise comparisons were performed using two-tailed unpaired t-test. Multiple comparisons were analyzed using a linear mixed‑effects model due to the presence of missing values. ns, not significant (not shown). *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/151a51d9c0c0c41dbb9bb917.jpg"},{"id":94142602,"identity":"7ceac5d0-33ee-4ee8-9378-9f8e649a4f43","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":143633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALK4 modulates human myoblast differentiation in vitro. (A) \u003c/strong\u003eRepresentative fluorescence micrographs of human wild-type and ALK4 knockout (ALK4-KO) muscle cells cultured under differentiation conditions for 3 days, 6 days or 10 days in the absence or presence of 100 ng/mL recombinant GDF11, as indicated. Cultures were fixed and stained with anti-MyHC (green) to label differentiated myotubes, and nuclei were counterstained with DAPI (blue). \u003cstrong\u003e(B–E)\u003c/strong\u003e Tukey or bar box plots illustrating differentiation metrics in control and GDF11-treated cultures. \u003cstrong\u003e(B)\u003c/strong\u003eTotal nuclei per area. \u003cstrong\u003e(C)\u003c/strong\u003e Differentiation index. \u003cstrong\u003e(D)\u003c/strong\u003e Fusion index. \u003cstrong\u003e(E)\u003c/strong\u003e Number of nuclei residing in MyHC⁺ myotubes. Data are presented as medians with the range indicated (minimum to maximum values). Each dot represents the average value from five to 10 microscopic fields for each independent experiment. Multiple comparisons were analyzed using a linear mixed‑effects model due to the presence of missing values or repeated measures two-ANOVA. ns, not significant (not shown). *, p\u0026lt; 0.05; **, p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/ee5e5a0b06806718ecaa1f05.jpg"},{"id":94142836,"identity":"d55fefac-c49b-432e-b70e-417cbe5cdd34","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":310890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of ALK4 is associated with impaired cell migration and adhesion, coinciding with significant downregulation of CD49d (α4 integrin chain) and CD49f (α6 integrin chain). (A)\u003c/strong\u003e Percentage of migrating cells normalized to WT control cells on laminin 111 (LM111) without recombinant GDF11 (rGDF11). Because the results are normalized, differences in migration induced by LM111 relative to the control cannot be directly assessed. Each dot represents the average number of cells in 10 microscopic fields of independent experiments\u003cstrong\u003e (B)\u003c/strong\u003e Representative fluorescence micrographs of human muscle cells plated on surfaces coated with either poly-L-lysine (PLL) or LM111, treated with 100 ng/mL rGDF11 or left untreated, and labeled with phalloidin (green) to visualize F-actin and DAPI to visualize nuclei. Scale bar = 100 µm. \u003cstrong\u003e(C–E)\u003c/strong\u003eTukey box plots illustrating quantitative analysis of cell adhesion parameters derived from the images in \u003cstrong\u003e(B)\u003c/strong\u003e. \u003cstrong\u003e(C)\u003c/strong\u003e Number of adherent cells per area. \u003cstrong\u003e(D)\u003c/strong\u003e Average cell area (spreading). \u003cstrong\u003e(E)\u003c/strong\u003e Cell-cell contact index, expressed as the percentage of cells forming clusters. Data are presented as medians with the range indicated (minimum to maximum values). Each dot represents the average value from five to 10 fields of view for one independent experiment. \u003cstrong\u003e(F)\u003c/strong\u003e Representative histograms from flow cytometry immunophenotyping showing the expression profiles of integrin subunits in WT and ALK4 KO cells. Markers analyzed include CD29 (β1), CD49c (α3), CD49d (α4), CD49e (α5), and CD49f (α6) integrin chains. The percentage in each histogram indicates the proportion of cells above the gating threshold for the indicated marker. Quantitative analysis of integrin expression based on the median fluorescence intensity (MFI) for each subunit in WT and ALK4 KO cells (N = 2). \u003cstrong\u003e(G)\u003c/strong\u003e Quantification of the flow cytometry data (F). Bar graphs display one representative experiment of positive cells for each marker, as indicated.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/ce6d13ec700a03340f22fe12.jpg"},{"id":100356416,"identity":"6b05e2cb-f236-45b3-9c99-4baa48914a59","added_by":"auto","created_at":"2026-01-16 07:08:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1969403,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/75aa33e2-06c3-45f5-9c13-62557ac765d9.pdf"},{"id":94142597,"identity":"60904a6a-4a97-4772-bd41-79f9de0135b1","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15623,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/53e0b2268334b52cadcd81ed.docx"},{"id":94142834,"identity":"ce033286-26bb-4d10-a52a-1b73c18de3f7","added_by":"auto","created_at":"2025-10-22 20:37:23","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":94322,"visible":true,"origin":"","legend":"","description":"","filename":"S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/607328c0c7d9c83b2e1ba3be.jpg"},{"id":94142605,"identity":"10e8f800-a624-40de-a587-32a3de1e24a0","added_by":"auto","created_at":"2025-10-22 20:29:23","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":119104,"visible":true,"origin":"","legend":"","description":"","filename":"S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7777108/v1/6e36b219513caa02c2c6ac16.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"GDF11 and its receptor ALK4 independently modulate human myoblast migration and fusion","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTissue and organ regeneration involve structural and functional restoration following an injury or perturbation. Dysregulation of regeneration can lead to abnormal extracellular matrix (ECM) deposition, ultimately resulting in fibrosis (Uezumi et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In adult skeletal striated muscles, mature cells are cylindrical and contain striated myofibrils and multiple peripheral nuclei. In addition, rare and small progenitor quiescent mononucleated cells are located between the plasma membrane (sarcolemma) and the basement membrane of the muscle fiber. These satellite cells are identified by the expression of \u003cem\u003ePAX7\u003c/em\u003e (Seale et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Upon activation, they re-enter the cell cycle, proliferate, and give rise to myoblasts. Initially, these cells remain expressing \u003cem\u003ePAX7\u003c/em\u003e and begin to express the myogenic regulatory factors (MRFs) such as \u003cem\u003eMYOD1\u003c/em\u003e and \u003cem\u003eMYF5/6\u003c/em\u003e. Subsequently, those markers are lost, and they express \u003cem\u003eMYOGENIN\u003c/em\u003e and \u003cem\u003eMRF4\u003c/em\u003e, which promote the terminal differentiation of skeletal myocytes (Buckingham and Rigby \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These muscle precursors then fuse to eventually form multinucleated myotubes. Ultimately, they develop into new skeletal muscle fibers or contribute to the recovery of the damaged tissue.\u003c/p\u003e\u003cp\u003eA portion of the proliferating myoblasts returns to a quiescent state, exhibiting stemness characteristics and maintaining the pool of satellite cells. Accordingly, the activation of satellite cells is a critical step in skeletal muscle regeneration. The regulatory factors governing stem cell fate and thereby determining the balance between regeneration and repair during tissue healing remain largely elusive. The identification and further characterization of these factors is pivotal for deepening our understanding of functional tissue recovery and can profoundly influence the development of targeted therapeutic strategies for skeletal muscle regeneration.\u003c/p\u003e\u003cp\u003eIn particular, the use of cell therapy in clinical trials for the treatment of lesions or degenerative diseases has yielded inconsistent results, including the reduced migration capacity of transplanted cells and accelerated cell death (Briggs and Morgan \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Among the numerous strategies and molecules currently under investigation, the GDF11 signaling pathway has been implicated in the rejuvenation of skeletal muscle, cardiac muscle, and cerebral vasculature, leading to increased neurogenesis and cognitive improvement (Loffredo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Katsimpardi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sinha et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Typically, TGF-β superfamily ligands are synthesized as latent precursors and require cleavage by proprotein convertases for activation (Moustakas and Heldin \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Once activated, they form homodimers and bind to constitutively active type II serine/threonine kinase receptors. Ligand binding enables these receptors to recruit and phosphorylate type I receptors (Hinck \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Together, type II and type I receptors assemble into a heterotetrameric complex, wherein type I receptors phosphorylate receptor-activated SMADs (R-SMADs), which then translocate to the nucleus and regulate gene transcription (Horbelt et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Jurberg et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the case of GDF11, SMAD2 and SMAD3 associate with SMAD4 to form a transcriptional activator complex (Andersson et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFindings with GDF11 are, however, seemingly contradictory (Egerman et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rodgers and Eldridge \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Smith et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Poggioli et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Harper et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In mouse embryos, for instance, we have previously observed that \u003cem\u003eGdf11\u003c/em\u003e inactivation leads to an expansion of a group of undifferentiated axial progenitors during tail formation, underscoring its importance in progenitor cell differentiation and tissue patterning (Jurberg et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Aires et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In skeletal muscle, Egerman et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported that Gdf11 impairs muscle regeneration by inhibiting myoblast differentiation and satellite cell expansion, while its administration prior to muscle injury was associated with an increase in nascent myofiber formation. In contrast, studies in aged animals revealed that elevated GDF11 levels may be detrimental. More specifically, Harper et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that increasing GDF11 impairs skeletal muscle repair, potentially inducing cachexia-like effects. Likewise, GDF11-treatment in older rats promoted tissue fibrosis and compromised the functional recovery of skeletal muscle after injury (Zhou et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Together, these observations emphasize that timing, dosage, and cellular context critically determine the biological outcomes of GDF11 signaling.\u003c/p\u003e\u003cp\u003eOther factors may contribute to the controversies surrounding GDF11 function. Notably, GDF11 shares approximately 90% amino acid sequence identity with its homolog, Myostatin (GDF8) (Nakashima et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). This similarity raises the possibility of antibody cross-reactivity, as suggested by Egerman et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, while Sinha et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) initially proposed a pro-myogenic role for GDF11, genetic studies have established that Myostatin inhibits muscle growth (Lee and McPherron 2001). Additionally, some \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies have reported that the GDF11 propeptide can antagonize the activity of the mature GDF11 peptide (Ge et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we evaluated the impact of recombinant GDF11 (rGDF11) on chick and human myoblast cultures and used CRISPR-mediated knockout of the ALK4 receptor in human myoblasts to elucidate its role during \u003cem\u003ein vitro\u003c/em\u003e skeletal myogenesis. We specifically assessed how GDF11 and ALK4 affect key cellular processes, including adhesion, proliferation, migration, and differentiation.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePrimary chick myogenic cell culture\u003c/h2\u003e\u003cp\u003ePrimary cultures of myogenic cells were derived from pectoral muscle tissue of 11-day-old chick embryos (Granja Tolomei, Brazil) as previously described (Mermelstein et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The use of chick embryos was approved by the Ethics Committee for Animal Care and Use in Scientific Research at the Federal University of Rio de Janeiro under approval number DAHEICB 081\u0026thinsp;\u0026minus;\u0026thinsp;22. In brief, muscle tissue was minced with surgery knives and digested with 0.005% trypsin at 37\u0026deg; C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 25 min. To stop digestion, a small volume of 8-1-0.5 medium (Minimum Essential Medium with 10% horse serum, 0.5% chick embryo extract, 0.1% L-glutamine, and 1% penicillin-streptomycin) was added to the sample and then centrifuged at approximately 300 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min. After discarding the supernatant, the pellet was suspended in the 8-1-0.5 medium and filtered to produce a mononucleated cell suspension. Isolated mononucleated cells were plated at an initial density of 7.5 x 10\u003csup\u003e5\u003c/sup\u003e cells per 35 mm culture dish onto 22-mm Aclar plastic coverslip (Pro-Plastics Inc.) pre-coated with 0.1% gelatin or rat tail-derived collagen. They were cultured in 2 mL of 8-1-0.5 medium under humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026deg; C. The percentage of myoblasts was determined by double staining 24-hour cultures with the muscle-specific marker desmin (#8281, Sigma-Aldrich) (Supplementary Table\u0026nbsp;1) and the nuclear dye DAPI. The number of desmin-positive cells was then counted relative to the total cell count per field of view. On average, myoblasts constituted 80% of each culture, while non-myogenic cells comprised the remaining 20%. All cell culture reagents were purchased from Invitrogen, unless otherwise stated.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHuman muscle cell culture\u003c/h3\u003e\n\u003cp\u003eImmortalized C25-CL48 human myoblasts were previously described elsewhere (Thorley et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We employed two distinct cell culture conditions. For cell maintenance and proliferation assays, cells were cultured in DMEM high glucose supplemented with 199/EBSS medium, 20% fetal bovine serum (FBS), 5 ng/mL epidermal growth factor (EGF; #PHG0311, ThermoFisher), 25 \u0026micro;g/mL fetuin (#F3385, Sigma-Aldrich), 0.2 \u0026micro;g/mL dexamethasone (#D4902, Sigma-Aldrich), and 50 \u0026micro;g/mL gentamycin (#15750060, ThermoFisher). For differentiation assays, cells were cultured in DMEM/Glutamax supplemented with 10 \u0026micro;g/mL of human recombinant insulin (#91077, Merk), as previously described (Riederer et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCell culture treatments\u003c/h3\u003e\n\u003cp\u003ePrimary chicken or immortalized human muscle cell cultures were maintained for either 12 or 24 hours prior to treatment with 100 ng/mL of recombinant GDF11 (rGDF11; #120\u0026thinsp;\u0026minus;\u0026thinsp;11, PeproTech) for the indicated durations. In addition, other myoblast cultures were treated with 1 \u0026micro;M of SB431542 (SB; #S4317, Sigma-Aldrich), a type I receptor inhibitor for ALK4/5/7, diluted in DMSO (#2650, Sigma-Aldrich) as vehicle at a final concentration of 1 \u0026micro;M. Cultures were observed daily through phase-contrast microscopy.\u003c/p\u003e\n\u003ch3\u003eGeneration of CRISPR-mediated mutant cell lines\u003c/h3\u003e\n\u003cp\u003eWe used human C25-CL48 myoblasts to inactivate the \u003cem\u003eACVR1B\u003c/em\u003e gene (ALK4) using the CRISPR/Cas9 strategy. In brief, we obtained the sequence of each gene from the \"Genome Browser\" database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and utilized the first exon to design specific guide RNAs (gRNAs) with the CRISPR Design Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.mit.edu/\u003c/span\u003e\u003cspan address=\"http://crispr.mit.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Supplementary Table\u0026nbsp;2). Commercially synthesized oligonucleotides were subcloned into the expression vector pSpCas9(BB)-2A-GFP (referred herein as pX458) (Ran et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Positive clones were identified by colony PCR, and successful cloning was confirmed by Sanger sequencing. Next, human cells were electroporated with 20 \u0026micro;g of each plasmid using the Neon\u0026reg; Transfection System (ThermoFisher Scientific), following the manufacturer's protocol. Following electroporation, cells were maintained in culture for 24 hours to allow for cell recovery. Subsequently, GFP\u003csup\u003e+\u003c/sup\u003e myoblasts were sorted using a FACSAria II flow cytometer and individually plated in a 96-well plate for clonal expansion. Upon cell growth, cellular aliquots were cryopreserved, while other matching samples were used for genomic DNA extraction and genotyping (Supplementary Table\u0026nbsp;2). Confirmation and characterization of indels were performed through Sanger sequencing.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eChick muscle cultures were stained following a previously established protocol (Mermelstein et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). In short, cells were fixed with 4% formaldehyde in phosphate buffered saline (PBS) for 3 minutes, washed in PBS, and subsequently incubated with appropriately diluted, unconjugated primary antibodies (Supplementary Table\u0026nbsp;1) for 1 hour at 37\u0026deg; C. After other PBS washes, cells were incubated with the corresponding diluted secondary antibodies (Supplementary Table\u0026nbsp;1) for 1 hour at 37\u0026deg; C. Human muscle cells were fixed with analytical-grade ethanol for 10 minutes, followed by three 5-minute PBS washes. Non-specific binding was blocked using PBS containing 2% goat serum, 1% bovine serum albumin (BSA), and 0.3% Triton X-100 for 20 minutes. Unconjugated or conjugated primary antibodies (Supplementary Table\u0026nbsp;1) diluted in PBS containing 1% BSA and 0.3% Triton X-100 were incubated in a humid chamber either for 1 hour at room temperature or overnight at 4\u0026ordm; C. Samples were washed three times with PBS for 5 minutes each, then incubated with the corresponding diluted secondary antibodies (Supplementary Table\u0026nbsp;1) for 30 minutes. Following antibody incubation, both chick and human samples were washed with PBS again and counterstained with the DNA dye DAPI (#D1306, ThermoFisher Scientific) for 5 minutes. A final round of PBS washes was performed, and slides were mounted with ProLong\u0026trade; Gold Antifade antifading mounting medium (#P36934, ThermoFisher Scientific). Slides were kept in the dark at room temperature for 72 hours and then transferred to 4\u0026ordm;C for long-term storage. Image acquisition was conducted using a Zeiss Axiovert 100 microscope, a Zeiss AXIO IMAGER A2 fluorescence microscope, or a Leica TCS SP8 confocal microscope. We captured between five to ten fields per condition.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTransmigration assay\u003c/h2\u003e\u003cp\u003eTo determine whether rGDF11 treatment or ALK4 deletion alters the migratory capacity of human myoblasts, we performed Transwell migration assays using 24-well polystyrene plates with polycarbonate membranes featuring 8-\u0026micro;m pores (Corning Costar, USA). Initially, both the lower chamber and the upper insert were coated with either 0.5% BSA or 10 \u0026micro;g/mL of recombinant human laminin-111 (LM111; #LN111-02, BioLamina) and incubated for 1 hour at 37\u0026ordm; C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Following incubation, the coating solutions were aspirated, and the wells were washed twice with 1\u0026times; PBS before the plates were allowed to air-dry at room temperature. A blocking step was then carried out using 0.5% BSA for 1 hour at 37\u0026ordm; C in 5% CO\u003csub\u003e2\u003c/sub\u003e to minimize nonspecific binding. Thereafter, 10\u003csup\u003e5\u003c/sup\u003e myoblasts were seeded into each insert in migration medium, which comprised DMEM and medium 199 (in a 4:1 ratio) supplemented with 0.5% BSA. The lower chamber was filled with migration medium alone (control) or supplemented with 100 ng/mL rGDF11. In separate experiments, cells in the insert were exposed to migration medium with or without rGDF11 in the upper chamber to evaluate any potential chemorepulsive effects. The plates were incubated for 4 hours at 37\u0026deg; C in a humified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. At the end of the incubation period, the supernatant in the insert was carefully removed, and the cells were gently washed twice with 1\u0026times; PBS. Cells were then fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature, followed by additional gentle washes with 1\u0026times; PBS. Any cells adhering to the upper surface of the membrane were removed using cotton swabs. Next, cells were permeabilized with 0.1% Triton X-100 for 40 minutes, and the lower surface of the membranes was stained with DAPI at a 1:200 dilution for 30 minutes to label nuclei. Following three washes with 1\u0026times; PBS, the membranes were carefully excised from the inserts and mounted onto glass slides with coverslips for imaging.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell adhesion assay\u003c/h3\u003e\n\u003cp\u003eTo investigate the influence of rGDF11 on muscle cell adhesion, we coated Nunc\u0026reg; Lab-Tek\u0026reg; Chamber Slide\u0026trade; systems (#S6815, Sigma-Aldrich) with poly-L-lysine (PLL; #P4707, Sigma-Aldrich) as a control for non-specific binding, or with LM111 (#LN111-02, BioLamina). In these experiments, 4x10\u003csup\u003e3\u003c/sup\u003e human wildtype or mutant C25-CL48 cells were resuspended in proliferation medium, with or without 100 ng/mL of rGDF11 (untreated) and allowed to adhere for 120 minutes. Then, the wells were gently washed with PBS, and cells were fixed with 2% PFA for 15 minutes. Subsequently, cells were permeabilized with 0.1% saponin in PBS for 15 minutes and non-specific labeling was blocked using PBS containing 2.5% BSA and 8% FBS for 1 hour. Cells were then labeled with Alexa Fluor\u0026reg; 488-conjugated phalloidin (A12379, ThermoFisher Scientific) for 30 minutes, counterstained with DAPI, and mounted as described above.\u003c/p\u003e\n\u003ch3\u003eImage analysis\u003c/h3\u003e\n\u003cp\u003eImage processing and quantification were conducted using the FIJI (ImageJ) software (Schindelin et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This involved quantifying nuclei counts, cell counts, and cell area after performing background subtraction, contrast enhancement, Gaussian blur, and segmentation via a custom-developed macro pipeline, followed by manual curation. The differentiation index was computed as the ratio of nuclei within myosin heavy chain (MyHC)-positive (mono- or multi-nucleated) cells to the total number of nuclei per microscopic field. The fusion index was determined as the percentage of nuclei within MyHC-positive myotubes (specifically, multinucleated cells) per microscopic field. In addition, myotube width was measured along the longest branches of each myotube per microscopic field. The proportion of desmin-positive area was also determined.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry\u003c/h2\u003e\u003cp\u003eHuman wild-type and mutant myoblasts were harvested through trypsinization, counted using a hemocytometer, and distributed into U-bottom wells of a 96-well plate. To prevent non-specific labeling, we treated the cells with 50 \u0026micro;L of PBS containing 2% normal mouse serum for 15 minutes. Then, cells were centrifuged at 400 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 minutes at 4\u0026ordm; C and washed with 100 \u0026micro;L of PBS containing 1% BSA (FACS buffer). Subsequently, they were incubated with appropriately diluted conjugated primary antibodies (Supplementary Table\u0026nbsp;1) for 1 hour at 4\u0026ordm; C. After a final round of washing, the cells were resuspended in FACS buffer for analysis via flow cytometry using a FacsCanto II (Becton Dickinson).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eWe performed at least three independent experiments for each assay, unless otherwise stated. All values were presented as the median with 95% confidence intervals (CI). Statistical analyses were performed using two-tailed unpaired \u003cem\u003et\u003c/em\u003e tests for independent two-group comparisons. Multiple comparisons were analyzed using a linear mixed‑effects model to account for missing values or repeated measures, unless stated otherwise. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eTreatment with rGDF11 produces distinct effects depending on the cell type and developmental stage, significantly reducing the number of fibroblasts (desmin-negative cells) in chicken muscle cell cultures\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe applied embryonic chick primary skeletal myoblasts due to their well-documented autonomous muscle differentiation program in culture (Costa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this model, isolated cells progress through all stages of \u003cem\u003ein vitro\u003c/em\u003e skeletal myogenesis without requiring any exogenous stimuli to produce large, multinucleated myotubes with contractile capacity (Mermelstein et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Moreover, these primary cultures contain muscle fibroblasts, which provide paracrine signals and cell-cell interactions that critically influence skeletal myogenesis, including the composition of the extracellular matrix (Chapman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCultures treated with 100 ng/mL of rGDF11 for two days exhibited a reduction in the median total number of nuclei per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e compared with controls (271.7 nuclei, 95% CI: 233.2\u0026ndash;310.5 vs. 363.9 nuclei, 95% CI: 316.1\u0026ndash;435.7; Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B). However, no significant differences were observed in the number of nuclei within desmin\u003csup\u003e+\u003c/sup\u003e cells per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,C) or in the relative area occupied by desmin\u003csup\u003e+\u003c/sup\u003e myoblasts and myotubes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). The median proportion of myotube nuclei was slightly higher in the rGDF11-treated cultures than in controls (69.40%, 95% CI: 40.13%\u0026ndash;90.37% vs. 55.00%, 95% CI: 41.13%\u0026ndash;70.71%; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E), although this difference was not statistically significant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs satellite cells are quiescent stem cells with the ability to either generate new differentiating cells or return to their stem cell state, we explored whether treatment with rGDF11 influences cell fate decisions during \u003cem\u003ein vitro\u003c/em\u003e skeletal myogenesis. The median total number of Pax7\u003csup\u003e+\u003c/sup\u003e cells per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e remained consistent under rGDF11 treatment (50.12 cells, 95% CI: 28.05\u0026ndash;68.80) compared to control cultures (56.78 cells, 95% CI:41.64\u0026ndash;66.44; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,F). No significant differences in the proportion of Pax7\u003csup\u003e+\u003c/sup\u003e cells following rGDF11 incubation (19.60%, 95% CI: 13.07\u0026ndash;23.78) in relation to controls were observed (15.59%, 95% CI: 11.59\u0026ndash;18.41; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,G).\u003c/p\u003e\u003cp\u003eBecause GDF11 signaling has been shown to activate lung and cardiac fibroblasts (Swan et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we hypothesized that it might similarly influence fibroblasts interspersed withing chicken muscle cell cultures. More specifically, we found a reduction in the number of Pax7\u003csup\u003e\u0026minus;\u003c/sup\u003edesmin\u003csup\u003e\u0026minus;\u003c/sup\u003e nuclei per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e upon rGDF11 treatment in relation to control cultures (29.74 nuclei, 95% CI: 29.03\u0026ndash;30.44 vs. 75.07 nuclei, 95% CI: 66.02\u0026ndash;75.07, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,H). These results raise the possibility that GDF11 may regulate the proliferation or cell death of skeletal muscle fibroblasts.\u003c/p\u003e\u003cp\u003eGiven the challenges of prolonged culture maintenance and the intrinsic tendency of primary chick embryonic skeletal muscle cells to undergo autonomous differentiation, we shifted to immortalized human C25-CL48 cells (Thorley et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for subsequent experiments. This model enables us to further dissect the genetic basis of GDF11 signaling across distinct stages of skeletal myogenesis \u003cem\u003ein vitro\u003c/em\u003e. Unlike primary chick embryonic muscle cells, human C25-CL48 cultures permit the study of proliferation and myotube differentiation as two distinct processes, thereby providing greater resolution into the stage-specific roles of GDF11. In addition, these human cultures are composed exclusively of myogenic cells, without fibroblast or other contaminating lineages.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003erGDF11 inhibits myotube formation in human myoblasts without affecting proliferation\u003c/h2\u003e\u003cp\u003eUnlike primary chick embryonic muscle cultures, human myoblasts can be maintained in either proliferation or differentiation media, allowing these two fundamental processes to be evaluated independently. As expected, the number of human C25-CL48 myoblasts increased significantly from day 3 to day 6 of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). Incubation with rGDF11 under proliferative conditions did not significantly alter the median total numbers of nuclei per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e compared with controls at 3 days (10.60 nuclei, 95% CI: 9.93\u0026ndash;14.94 vs. 12.69 nuclei, 95% CI: 8.04\u0026ndash;16.57) or 6 days of culture (31.99 nuclei, 95% CI: 30.78\u0026ndash;37.19 vs. 30.86 nuclei, 95% CI: 19.83\u0026ndash;42.18; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). The proportion of Ki67\u003csup\u003e+\u003c/sup\u003e myoblasts increased from day 3 to day 6 of culture, but no significant differences were observed between rGDF11-treated and control cells at either time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,D). Assessment of proliferative capacity by EdU staining similarly revealed no differences between groups (data not shown). The mean population doubling time (PDT) was 2.67 days for rGDF11-treated cultures and 3.13 days for controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Together, these results indicate that GDF11 does not affect myoblast multiplication.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUnder differentiation conditions, GDF11 markedly impaired the differentiation of MyHC\u003csup\u003e+\u003c/sup\u003e cells and myotube formation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-J). This effect was not due to differences in the number of cells entering the differentiation program, as no significant differences were observed between GDF11-treated and control groups at either day 3 or day 6 of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In controls, differentiation and fusion indices increased from day 3 to day 6, whereas rGDF11-treated myoblasts showed reduced median values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH,I). Interestingly, rGDF11 treatment particularly blocked the formation of larger myotubes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Together, these findings suggest that GDF11 impairs myotube formation primarily by inhibiting fusion, without affecting myoblast proliferation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBy contrast, one-hour pulse of the SB inhibitor in DMSO followed by a medium change to differentiation medium without [SB(1h) in DMSO] or with rGDF11 [SB(1h)\u0026thinsp;+\u0026thinsp;rGDF11] strongly reduced differentiation and fusion indices after six days (Supplementary Fig.\u0026nbsp;1C,D). Notably, the SB(1h)\u0026thinsp;+\u0026thinsp;rGDF11 condition further decreased both indices relative to controls and DMSO(1h) cultures (Supplementary Fig.\u0026nbsp;1C,D). These results suggest that GDF11 signal through additional pathways beyond the canonical ALK4, ALK5, and ALK7 receptors (Derynck and Budi \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eLoss of ALK4 impairs muscle differentiation\u003c/h2\u003e\u003cp\u003eGiven that the SB inhibitor targets ALK4, ALK5, and ALK7 receptors, and that DMSO has shown unintended effects on muscle cell cultures, we employed a genetic approach using the CRISPR/Cas9 system for further loss-of-function assays. We have successfully identified homozygous mutant C25-CL48 clones for \u003cem\u003eACVR1B\u003c/em\u003e, herein referred as ALK4-KO. We found that ALK4-KO cells showed a 4-nucleotide deletion within its first exon (Supplementary Fig.\u0026nbsp;2A). This NHEJ-driven mutation putatively caused a frameshift as inferred by bioinformatic tools to produce a truncated receptor at codon 83. We confirmed the loss of type I receptor ALK4 by confocal microscopy. Wild-type C25-CL48 cells exhibited speckled cytoplasmic distribution of ALK4 receptors, while single mutant cells showed no positive labeling for their truncated receptor (Supplementary Fig.\u0026nbsp;2B).\u003c/p\u003e\u003cp\u003eIncubation of mutant cells under proliferative conditions for 6 days, either in the absence (control) or presence of rGDF11, produced no significant differences in the median total number of nuclei per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e among groups (data not shown). However, the inactivation of ALK4 greatly impaired myoblast differentiation and fusion (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,C-E). After six days in differentiation medium, no significant differences in the median total number of nuclei per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e were observed between wild-type and ALK4-KO cells, either untreated (50.47 nuclei, 95% CI: 49.67\u0026ndash;54.79 vs. 37.41 nuclei, 95% CI: 37.35\u0026ndash;50.21) or treated with rGDF11 (37.54 nuclei, 95% CI: 37.54\u0026ndash;52.47 vs. 37.14 nuclei, 95% CI: 36.77\u0026ndash;43.72. These results indicates that ALK4 does not affect cell survival under differentiation conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). By contrast, differentiation and fusion were nearly completed abolished in ALK4-KO cells, and this effect was not altered by rGDF11 treatment. The median differentiation indices of untreated wild-type and ALK4-KO cells were 51.91% (95% CI: 31.39\u0026ndash;56.38) and 0.2% (95% CI: 0.14\u0026ndash;0.83), respectively. Differently, rGDF11-treated cultures showed markedly reduced differentiation, with median differentiation indices of 0.07% (95% CI: 0.014\u0026ndash;1.62) and 0.00% (95% CI: 0.00\u0026ndash;0.00) for the respective genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Likewise, the median fusion indices were 42.16% (95% CI: 26.67\u0026ndash;46.84) and 0.00% (95% CI: 0.00\u0026ndash;0.00) in untreated cells, \u003cem\u003eversus\u003c/em\u003e 0.00% (95% CI: 0.00\u0026ndash;0.06) and 0.00% (95% CI: 0.00\u0026ndash;0.00) following rGDF11 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of nuclei per MyHC\u003csup\u003e+\u003c/sup\u003e myocyte/myotube revealed that either rGDF11 or ALK4 inactivation strongly impaired multinucleated myotube formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Interestingly, MyHC\u003csup\u003e+\u003c/sup\u003e cells were detected only in untreated ALK4-KO cultures at day 10, and this residual differentiation was further suppressed by rGDF11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,E). These findings highlight two complementary roles: GDF11 suppresses skeletal muscle differentiation independently of ALK4, whereas inactivation of ALK4 alone significantly delays MyHC production and myoblast fusion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eGDF11 impairs myoblast migration, while ALK4 is necessary\u003c/h2\u003e\u003cp\u003eDuring normal skeletal myogenesis, satellite cell activation and myoblast proliferation are accompanied by an oriented migration, a process critical for proper cell alignment, fusion, and subsequent myotube formation (Kim et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In cell therapies for muscular dystrophies, the limited migratory capacity of myoblasts poses a significant challenge for muscle regeneration, as cells often remain localized near the injection site (Skuk et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Riederer et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Given the limited studies addressing the role of GDF11 in myoblast migration, we investigated its impact using LM111 as a substrate in transmigration assays.\u003c/p\u003e\u003cp\u003eConsistent with our previous findings (Silva-Barbosa et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), we showed that wild-type myoblasts migrated efficiently through LM111-coated membranes, whereas migration was limited on BSA-coated control surfaces (data not shown). To investigate whether GDF11 promotes myoblast migration, rGDF11 was added to the lower chamber of the Transwell plates to test its potential chemoattractant effect. However, this treatment did not enhance migration. Conversely, the addition of rGDF11 to the upper chamber significantly reduced myoblast migration toward the lower chamber compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These findings suggest that rGDF11 impairs myoblast motility, which may account for its inhibitory effect on myotube formation, since migration and cell alignment are prerequisites for myoblast fusion. Moreover, deletion of \u003cem\u003eACVR1B\u003c/em\u003e resulted in a pronounced reduction in the migratory capacity of ALK4-KO cells through LM111 under all tested conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eALK4 regulates LM111-dependent myoblast adhesion and integrin expression independently of GDF11\u003c/h2\u003e\u003cp\u003eCell-cell and cell-ECM interactions play fundamental roles in various aspects of skeletal muscle cell biology, particularly in processes such as migration and fusion. To determine whether the effects observed following rGDF11 treatment could be attributed to defects in cell adhesion, we cultured cells on coverslips coated with either poly-L-lysine (PLL) or LM111, in the presence or absence of rGDF11. PLL- or LM111-coated coverslips, with or without rGDF11 treatment, did not significantly affect the average number of nuclei per 100,000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e in either wild-type or CRISPR-mediated ALK4 mutant cells, after 2 hours in culture (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,C). In contrast, LM111 coating markedly increased the median cell area of wild-type myoblasts as compared with PLL-coated surfaces (1,781.0 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, 95% CI [1,542.0, 4,031.0] vs. 405.6 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, 95% CI [252.9, 1,012.0], respectively). Moreover, rGDF11 treatment did not affect the cell area on either substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,D; Supplementary Tables\u0026nbsp;1,2). Notably, LM111 coating promoted robust myoblast-myoblast interactions during the 2-hour adhesion period regardless of rGDF11 treatment, an effect that was significantly more pronounced than that observed with PLL coating (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Conversely, loss of ALK4 resulted in reduced cell area and diminished cell-cell contacts on LAM111 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Collectively, these findings suggest that ALK4 may contribute to LM111-mediated cell adhesion and spreading through a GDF11-independent mechanism.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCell-cell contact index in adhesion assays.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003econtact index (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003egenotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecoating\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003etreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e# exp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003emedian\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95% CI (l.l., u.l.)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePLL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003euntreated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[3.84, 27.74]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLM111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003euntreated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e48.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[36.14, 60.83]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePLL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003erGDF11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[5.51, 20.43]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLM111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003erGDF11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e54.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[42.35, 64.54]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALK4-KO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePLL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003euntreated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e23.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[9.61, 50.11]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALK4-KO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLM111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003euntreated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e29.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[13.34, 38.48]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALK4-KO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePLL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003erGDF11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e39.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[12.82, 41.92]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALK4-KO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLM111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003erGDF11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e27.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[22.24, 42.42]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWT, wild-type; ALK4-KO, ALK4 knockout; PLL, poly-L-lysine; LM111, laminin 111;\u003c/p\u003e\u003cp\u003e# exp, number of experiments; 95% CI, 95% confidence interval (CI).\u003c/p\u003e\u003cp\u003eGiven the observed role of LM111 seen in our experiments, we sought to contextualize its function within the broader framework of muscle regeneration. Together with the dystroglycan complex, the α7β1 integrin serve as a primary receptor for laminins in skeletal muscle. Laminins are key components of the basement membrane that individually surround each muscle fiber (Sch\u0026uuml;ler et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While mature fibers predominantly express the LM211 and LM221 isoforms, LM111 is enriched in the satellite cell niche during regeneration (Gawlik and Durbeej \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In particular, multiple LM isoforms, including LM111, can be bound by the versatile integrin α6β1 receptor (Nishiuchi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This raises the possibility that distinct LM-integrin interactions may modulate myoblast adhesion and differentiation through a GDF11-dependent mechanism.\u003c/p\u003e\u003cp\u003eWe further investigated the presence of distinct integrin subunits in wild-type and ALK4-KO myoblasts using flow cytometry to elucidate the alterations in cell adhesion. The relative levels of the common CD29 subunit (β1 integrin) and the LM332-binding CD49c (α3 integrin) exhibited no significant differences between wild-type and ALK4-KO myoblasts (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF,G). In contrast, the fibronectin/VLA4-binding CD49d (α4 integrin) and the LM111/LM332/LM511/LM521-binding CD49f (α6 integrin) exhibited approximately 2- and 4.5-fold downregulation in ALK4-KO myoblasts compared to wild-type cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF,G). These findings suggest that ALK4-mediated signaling may play a role in regulating \u003cem\u003eITGA4\u003c/em\u003e and \u003cem\u003eITGA6\u003c/em\u003e gene expression, or the stability of CD49d and CD49f integrin subunits through a mechanism that appears to be independent of the GDF11 ligand. The intricate signaling network, where multiple members of the TGF superfamily converge on shared receptors, adds a layer of complexity to the regulatory mechanisms governing skeletal muscle differentiation. Together, our observations highlight promising prospects of both GDF11 and ALK4 as potential candidates in regenerative medicine.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study provides new insights into the complex signaling network that governs skeletal myogenesis, particularly by disentangling the role of GDF11 from an ALK4-dependent pathway that regulates myoblast adhesion, migration, and differentiation. Although GDF11 has been widely debated in the literature with reports attributing both stimulatory and inhibitory roles in muscle biology, our data indicate that its direct impact on myoblast proliferation and adhesion is minimal. Instead, we observed a robust inhibitory effect on myoblast differentiation and fusion, which could be partially explained by the reduced cell motility in the presence of GDF11. Surprisingly, pharmacological inhibition of the three known GDF11 receptors (ALK4, ALK5, and ALK7) or genetic knockout of ALK4 in human myoblasts resulted in a robust decrease in cell differentiation and fusion. In both approaches designed to block GDF11 activity, we found reduced myoblast fusion, suggesting the involvement of an additional receptor for GDF11. Furthermore, ALK4-KO myoblasts displayed impaired adhesion and spreading, as well as a marked loss of migratory capacity. These phenotypes could be attributed to the downregulation of the α4 and α6 integrin chains, with probable consequences upon the binding of these cells on fibronectin and laminin, respectively.\u003c/p\u003e\u003cp\u003eA critical aspect of our work was the combined use of biochemical stimulation and CRISPR-based genetic approaches, which allowed us to parse the contributions of GDF11 and ALK4 in skeletal muscle development. Despite the well-documented involvement of TGF-β superfamily members in regulating tissue regeneration (Gumucio et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Derynck and Budi \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ren et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), our findings revealed that GDF11 influence on myogenesis is not mediated through alterations in cell proliferation or adhesion \u003cem\u003eper se\u003c/em\u003e. Unlike proliferation, adhesion was evaluated at a single early time point with a short assay duration. Although ALK4-KO myoblasts exhibited impaired adhesion within the first two hours, they appeared to recover over time, as indicated by normal proliferation during culture. Nevertheless, transient adhesion defects in an inflammatory or regenerative environment could have significant consequences during critical early phases. Importantly, insufficient substrate adhesion can induce anoikis, a form of programmed cell death (Zhong and Rescorla \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which may further exacerbate impairments in muscle regeneration.\u003c/p\u003e\u003cp\u003eIn addition, GDF11 capacity to inhibit differentiation and migration appears to be uncoupled from its potential receptor-mediated signaling events. This divergence from earlier studies that emphasized a proliferative or adhesive role for GDF11 in various cell types (Finkenzeller et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) underscores the necessity of examining receptor-specific signaling events and their downstream effectors. GDF11 robustly impaired human myoblast differentiation, particularly cell fusion, corroborating recent results on human cells (Egerman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Myostatin (GDF8), a closely related ligand with high similarity to GDF11, is also a potent inhibitor of myoblast differentiation (Walker et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Suh and Lee \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Both factors promote muscle atrophy through Smad2/3-dependent inhibition of Akt signaling (Trendelenburg et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and via the non-Smad Ras-ERK pathway (Masuzawa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, the same concentration of GDF11 that inhibited differentiation also reduced myoblast motility on LM111. This reduction in cell migration may contribute to impaired fusion, as myotube formation requires coordinated migration, alignment, and membrane fusion (Lehka and Rędowicz \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Alternatively, GDF11 may act on signaling pathways shared by both processes, including IGF-1/Akt, Rho family GTPases, and integrin\u0026ndash;FAK signaling (Derynck and Budi \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe found that both myoblasts and myotubes express GDF11 and its receptors ALK4, ALK5, and ALK7 (data not shown). However, information on GDF11 expression during muscle regeneration remains unavailable. Interestingly, we also observed that macrophage-derived monocytes express GDF11 (data not shown). One possibility is that, during the early pro-inflammatory phase of muscle regeneration, GDF11 acts to inhibit cell differentiation and migration, thereby permitting the expansion of the myoblast pool. In addition, GDF11 has been reported to promote the transition of macrophages from a pro-inflammatory M1 phenotype to a reparative M2 phenotype (Gong et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which could further support tissue repair.\u003c/p\u003e\u003cp\u003eInterestingly, our findings show that GDF11 impairs myoblast fusion, without affecting myoblast proliferation in human muscle cell cultures. Conversely, treatment of chick myogenic cells with GDF11 exhibited a reduction in the total number of nuclei compared to untreated cells. We hypothesize that these differences in the effects of GDF11 on cell proliferation might be related to the presence of fibroblasts in chick myogenic cultures and that GDF11 may influence the proliferation or cell death of skeletal muscle fibroblasts. These results require further investigation.\u003c/p\u003e\u003cp\u003eExperiments carried out in \u003cem\u003eACVR1B\u003c/em\u003e-deficient myoblasts further elucidated the molecular underpinnings of this regulatory network. The observed delay in myoblast differentiation, coupled with impaired adhesion and migration onto LM111, points to a critical role for ALK4 in these processes. Importantly, the reduction of surface integrin α6 levels in \u003cem\u003eACVR1B\u003c/em\u003e-deficient cells highlights a novel link between ALK4 signaling and integrin-mediated cell adhesion. CD49f (the α6 integrin chain), a key component of the VLA-6 receptor complex, is known to interact with laminin substrates (Nishiuchi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Taniguchi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Arimori et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and facilitate cell\u0026ndash;matrix interactions essential for myoblast fusion. Thus, our data suggest that an ALK4/VLA6/LM111 axis may serve as a pivotal modulator of myoblast behavior, independent of GDF11 ligand activity.\u003c/p\u003e\u003cp\u003eThe identification of this GDF11-independent ALK4 pathway has broad implications for understanding skeletal muscle regeneration. In the context of aging and skeletal muscle degeneration, aberrant signaling through TGF-β family receptors has been implicated in the decline of regenerative capacity (Brack et al.; Gough \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Carlson et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Smith et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Harper et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our results indicate that modulation of ALK4 activity, or its downstream effect on integrin α6 expression, could represent a novel therapeutic avenue. By decoupling the effects of GDF11 from the receptor-mediated mechanisms that govern adhesion and differentiation, we open the possibility of selectively targeting ALK4-dependent pathways to enhance muscle regeneration without eliciting the conflicting outcomes associated with GDF11.\u003c/p\u003e\u003cp\u003eNevertheless, our study is not without limitations. While \u003cem\u003ein vitro\u003c/em\u003e analyses using chicken and human myoblasts provide valuable mechanistic insights, the translation of these findings to \u003cem\u003ein vivo\u003c/em\u003e systems remains to be explored. Future studies will need to assess whether the ALK4/VLA6/LM111 axis similarly influences muscle regeneration in animal models of injury and aging. Moreover, it will be important to determine the extent to which other TGF-β superfamily members and their receptor complexes intersect with or modulate this pathway. Detailed temporal and spatial analyses of receptor expression and downstream signaling events during muscle regeneration will further clarify these interactions.\u003c/p\u003e\u003cp\u003eIn conclusion, our work delineates a previously underappreciated ALK4-dependent regulatory mechanism that governs myoblast differentiation, adhesion, and migration, independent of direct GDF11 activity. These findings not only reconcile conflicting reports regarding GDF11's role in skeletal muscle biology but also suggest that targeting specific receptor-mediated pathways may offer a more refined strategy for therapeutic intervention in muscle degenerative conditions. Knowledge of the ALK4/VLA6/LM111 axis is crucial for future studies aiming to harness the regenerative potential of skeletal muscle, with the ultimate goal of mitigating age-related muscle decline and promoting effective tissue repair.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR.F.R., M.L.C., V.C.A., C.M., A.D.J., and I.R. designed experiments; R.F.R., I.F.S., B.G.C.S., K.M.B., and A.D.J. performed experiments; R.F.R., I.F.S., B.G.C.S., K.M.B., M.L.C., V.M., W.S., V.C.A., C.M., A.D.J., and I.R. analyzed data; A.D.J., and I.R. wrote the main manuscript text; C.M. contributed to drafting portions of the manuscript; A.D.J., and I.R. prepared figures; M.L.C., W.S., V.C.A., C.M., A.D.J., and I.R. secured funding; All authors reviewed the results, discussed the findings, and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work is dedicated to the memory of Vania Maria Mello Dias. The research at the Laboratory on Thymus Research is funded by the Brazilian National Institute of Science and Technology on Neuroimmunomodulation (INCT.NIM), MercoSur Fund for Structural Convergence/FOCEM (Project #03/11), Rio de Janeiro Network on Neuroinflammation (FAPERJ) and Inova-IOC network on Neuroimmunomodulation. The work was also supported by fellowships 88882.332573/2019-01 (PROEX/CAPES) to RFR, 160832/2021-7 (CNPq) to IFS, 381427/2025-0 (TDI-B/INCT-CNPq) to BGCS, E-26/201.295/2023 (FAPERJ) to KMB, 302961/2021-6 (CNPq) and E-26/203.930/2024 (CNE/FAPERJ) to CM, 308192/2021-4 (CNPq) and E-26/204.077/2024 (CNE/FAPERJ) to MLC, and E26/202.683/2016 (PD10/FAPERJ) and 101665/2024-5 (PDS/CNPq) to ADJ.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAires R, Jurberg AD, Leal F, et al. Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity. Dev Cell. 2016;38:262\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.devcel.2016.06.021\u003c/span\u003e\u003cspan address=\"10.1016/j.devcel.2016.06.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAndersson O, Reissmann E, Ib\u0026aacute;\u0026ntilde;ez CF. Growth differentiation factor 11 signals through the transforming growth factor-beta receptor ALK5 to regionalize the anterior-posterior axis. EMBO Rep. 2006;7:831\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.embor.7400752\u003c/span\u003e\u003cspan address=\"10.1038/sj.embor.7400752\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArimori T, Miyazaki N, Mihara E, et al. Structural mechanism of laminin recognition by integrin. Nat Commun. 2021;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-021-24184-8\u003c/span\u003e\u003cspan address=\"10.1038/s41467-021-24184-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrack AS, Conboy MJ, Roy S et al. Increased Wnt Signaling During Aging Alters Muscle Stem Cell Fate and Increases Fibrosis.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBriggs D, Morgan JE. Recent progress in satellite cell/myoblast engraftment - Relevance for therapy. FEBS J. 2013;280:4281\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/febs.12273\u003c/span\u003e\u003cspan address=\"10.1111/febs.12273\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuckingham M, Rigby PWJ. Gene Regulatory Networks and Transcriptional Mechanisms that Control Myogenesis. Dev Cell. 2014;28:225\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.devcel.2013.12.020\u003c/span\u003e\u003cspan address=\"10.1016/j.devcel.2013.12.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarlson ME, Conboy MJ, Hsu M, et al. Relative roles of TGF-β1 and Wnt in the systemic regulation and aging of satellite cell responses. Aging Cell. 2009;8:676\u0026ndash;89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1474-9726.2009.00517.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1474-9726.2009.00517.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChapman MA, Meza R, Lieber RL. Skeletal muscle fibroblasts in health and disease. Differentiation. 2016;92:108\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.diff.2016.05.007\u003c/span\u003e\u003cspan address=\"10.1016/j.diff.2016.05.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCosta ML, Jurberg AD, Mermelstein C. The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis. Front Physiol. 2021;12:1\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphys.2021.668600\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2021.668600\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDerynck R, Budi EH. Specificity, versatility, and control of TGF-β family signaling. Sci Signal. 2019;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/scisignal.aav5183\u003c/span\u003e\u003cspan address=\"10.1126/scisignal.aav5183\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEgerman MA, Cadena SM, Gilbert JA, et al. GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration. Cell Metab. 2015;22:164\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cmet.2015.05.010\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2015.05.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEgerman MA, Zhang Y, Donne R, et al. ActRII or BMPR ligands inhibit skeletal myoblast differentiation, and BMPs promote heterotopic ossification in skeletal muscles in mice. Skelet Muscle. 2025;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13395-025-00373-7\u003c/span\u003e\u003cspan address=\"10.1186/s13395-025-00373-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFinkenzeller G, Stark GB, Strassburg S. Growth differentiation factor 11 supports migration and sprouting of endothelial progenitor cells. J Surg Res. 2015;1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jss.2015.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jss.2015.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGawlik KI, Durbeej M. Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies. Skelet Muscle 1; 2011.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGe G, Hopkins DR, Ho W-B, Greenspan DS. GDF11 Forms a Bone Morphogenetic Protein 1-Activated Latent Complex That Can Modulate Nerve Growth Factor-Induced Differentiation of PC12 Cells. Mol Cell Biol. 2005;25:5846\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mcb.25.14.5846-5858.2005\u003c/span\u003e\u003cspan address=\"10.1128/mcb.25.14.5846-5858.2005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong M, Yang X, Wang Y, et al. Growth differentiation factor 11 promotes macrophage polarization towards M2 to attenuate myocardial infarction via inhibiting Notch1 signaling pathway. Frigid Zone Med. 2023;3:53\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2478/fzm-2023-0008\u003c/span\u003e\u003cspan address=\"10.2478/fzm-2023-0008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez MN, de Mello W, Butler-Browne GS, et al. HGF potentiates extracellular matrix-driven migration of human myoblasts: Involvement of matrix metalloproteinases and MAPK/ERK pathway. Skelet Muscle. 2017;7:1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13395-017-0138-6\u003c/span\u003e\u003cspan address=\"10.1186/s13395-017-0138-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGough NR. Why Aging Muscles Don\u0026rsquo;t Repair. Sci Signal. 2008. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/scisignal.130ec272\u003c/span\u003e\u003cspan address=\"10.1126/scisignal.130ec272\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 1:.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGumucio JP, Sugg KB, Mendias CL. TGF-β Superfamily Signaling in Muscle and Tendon Adaptation to Resistance Exercise. Exerc Sport Sci Rev. 2015;43:93\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1249/JES.0000000000000041\u003c/span\u003e\u003cspan address=\"10.1249/JES.0000000000000041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarper SC, Brack A, MacDonnell S, et al. Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?Response to Harper et al. Circ Res. 2016;118:1143\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/CIRCRESAHA.116.307962\u003c/span\u003e\u003cspan address=\"10.1161/CIRCRESAHA.116.307962\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHassan SN, Ahmad F. Considering dimethyl sulfoxide solvent toxicity to mammalian cells and its biological effects. Exp Oncol. 2024;46:174\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15407/exp-oncology.2024.02.174\u003c/span\u003e\u003cspan address=\"10.15407/exp-oncology.2024.02.174\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHinck AP. Structural studies of the TGF-βs and their receptors - insights into evolution of the TGF-β superfamily. FEBS Lett. 2012;586:1860\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.febslet.2012.05.028\u003c/span\u003e\u003cspan address=\"10.1016/j.febslet.2012.05.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHorbelt D, Denkis A, Knaus P. A portrait of Transforming Growth Factor β superfamily signalling: Background matters. Int J Biochem Cell Biol. 2012;44:469\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biocel.2011.12.013\u003c/span\u003e\u003cspan address=\"10.1016/j.biocel.2011.12.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJurberg AD, Aires R, Varela-Lasheras I, et al. Switching Axial Progenitors from Producing Trunk to Tail Tissues in Vertebrate Embryos. Dev Cell. 2013;25:451\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.devcel.2013.05.009\u003c/span\u003e\u003cspan address=\"10.1016/j.devcel.2013.05.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJurberg AD, Vasconcelos-Fontes L, Cotta-de-Almeida VV. A Tale from TGF-β Superfamily for Thymus Ontogeny and Function. Front Immunol. 2015;6:1\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2015.00442\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2015.00442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatsimpardi L, Litterman NK, Schein PA et al. (2014) Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science (1979) 344:630\u0026ndash;634. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1251141\u003c/span\u003e\u003cspan address=\"10.1126/science.1251141\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim JH, Jin P, Duan R, Chen EH. Mechanisms of myoblast fusion during muscle development. Curr Opin Genet Dev. 2015;32:162\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee SJ, McPherron a C. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci U S A. 2001;98:9306\u0026ndash;11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.151270098\u003c/span\u003e\u003cspan address=\"10.1073/pnas.151270098\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLehka L, Rędowicz MJ. Mechanisms regulating myoblast fusion: A multilevel interplay. Semin Cell Dev Biol. 2020;104:81\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Q, Li H, Zhu L, et al. Growth Differentiation Factor 11 Evokes Lung Injury, Inflammation, and Fibrosis in Mice through the Activin A Receptor Type II-Like Kinase, 53kDa\u0026ndash;Smad2/3 Signaling Pathway. Am J Pathol. 2024;194:2036\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ajpath.2024.07.016\u003c/span\u003e\u003cspan address=\"10.1016/j.ajpath.2024.07.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Z, Kawasumi M, Zhao B, et al. Transgenic over-expression of growth differentiation factor 11 propeptide in skeleton results in transformation of the seventh cervical vertebra into a thoracic vertebra. Mol Reprod Dev. 2010. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mrd.21252\u003c/span\u003e\u003cspan address=\"10.1002/mrd.21252\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoffredo FS, Steinhauser ML, Jay SM, et al. Growth Differentiation Factor 11 Is a Circulating Factor that Reverses Age-Related Cardiac Hypertrophy. Cell. 2013;153:828\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2013.04.015\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2013.04.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMasuzawa R, Takahashi K, Takano K, et al. DA-Raf and the MEK inhibitor trametinib reverse skeletal myocyte differentiation inhibition or muscle atrophy caused by myostatin and GDF11 through the non-Smad Ras\u0026ndash;ERK pathway. J Biochem. 2022;171:109\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jb/mvab116\u003c/span\u003e\u003cspan address=\"10.1093/jb/mvab116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMermelstein CDS, Costa ML, Filho CC, Neto VM. Intermediate filament proteins in TPA-treated skeletal muscle cells in culture. J Muscle Res Cell Motil. 1996;17:199\u0026ndash;206. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00124242\u003c/span\u003e\u003cspan address=\"10.1007/BF00124242\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoustakas A, Heldin C-H. The regulation of TGFbeta signal transduction. Development. 2009;136:3699\u0026ndash;714. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/dev.030338\u003c/span\u003e\u003cspan address=\"10.1242/dev.030338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakashima M, Toyono T, Akamine A, Joyner A. Expression of growth/differentiation factor 11, a new member of the BMP/TGFbeta superfamily during mouse embryogenesis. Mech Dev. 1999;80:185\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNishiuchi R, Takagi J, Hayashi M, et al. Ligand-binding specificities of laminin-binding integrins: A comprehensive survey of laminin\u0026ndash;integrin interactions using recombinant α3β1, α6β1, α7β1 and α6β4 integrins. Matrix Biol. 2006;25:189\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.matbio.2005.12.001\u003c/span\u003e\u003cspan address=\"10.1016/j.matbio.2005.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePoggioli T, Vujic A, Yang P, et al. Circulating Growth Differentiation Factor 11/8 Levels Decline with Age. Circ Res. 2015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/CIRCRESAHA.115.307521\u003c/span\u003e\u003cspan address=\"10.1161/CIRCRESAHA.115.307521\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRan FA, Hsu PD, Wright J et al. (2013) Genome engineering using the CRISPR-Cas9 system. 8:2281\u0026ndash;308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nprot.2013.143\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2013.143\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRen L-L, Li X-J, Duan T-T, et al. Transforming growth factor-β signaling: From tissue fibrosis to therapeutic opportunities. Chem Biol Interact. 2023;369:110289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cbi.2022.110289\u003c/span\u003e\u003cspan address=\"10.1016/j.cbi.2022.110289\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiederer I, Negroni E, Bencze M, et al. Slowing Down Differentiation of Engrafted Human Myoblasts Into Immunodeficient Mice Correlates With Increased Proliferation and Migration. Mol Ther. 2012;20:146\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/mt.2011.193\u003c/span\u003e\u003cspan address=\"10.1038/mt.2011.193\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRodgers BD, Eldridge Ja. (2015) Reduced Circulating GDF11 Is Unlikely Responsible for Age-dependent Changes in Mouse Heart, Muscle, and Brain. Endocrinology en.2015\u0026thinsp;\u0026ndash;\u0026thinsp;1628. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/en.2015-1628\u003c/span\u003e\u003cspan address=\"10.1210/en.2015-1628\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.2019\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.2019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSch\u0026uuml;ler SC, Liu Y, Dumontier S et al. (2022) Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche. Front Cell Dev Biol 10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeale P, Sabourin LA, Girgis-Gabardo A et al. (2000) Pax7 Is Required for the Specification of Myogenic Satellite Cells skeletal muscle are mitotically quiescent and are acti-vated in response to diverse stimuli, including stretch-ing, exercise, injury, and electrical stimulation (Schultz oblast-specific genes potentially involved in satellite cell.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva-Barbosa SD, Butler-Browne GS, De Mello W, et al. Human myoblast engraftment is improved in laminin-enriched microenvironment. Transplantation. 2008;85:566\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/TP.0b013e31815fee50\u003c/span\u003e\u003cspan address=\"10.1097/TP.0b013e31815fee50\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSinha M, Jang YC, Oh J et al. (2014) Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science (1979) 344:649\u0026ndash;652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1251152\u003c/span\u003e\u003cspan address=\"10.1126/science.1251152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkuk D, Goulet M, Tremblay JP. Use of Repeating Dispensers to Increase the Efficiency of the Intramuscular Myogenic Cell Injection Procedure. Cell Transpl. 2006;15:659\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkuk D, Roy B, Goulet M, Tremblay JP. Successful Myoblast Transplantation in Primates Depends on Appropriate Cell Delivery and Induction of Regeneration in the Host Muscle. Exp Neurol. 1999;155:22\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/exnr.1998.6973\u003c/span\u003e\u003cspan address=\"10.1006/exnr.1998.6973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSmith SC, Zhang X, Zhang X, et al. GDF11 Does Not Rescue Aging-Related Pathological Hypertrophy. Circ Res CIRCRESAHA. 2015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/CIRCRESAHA.115.307527\u003c/span\u003e\u003cspan address=\"10.1161/CIRCRESAHA.115.307527\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. .115.307527.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuh J, Lee Y-S. Similar sequences but dissimilar biological functions of GDF11 and myostatin. Exp Mol Med. 2020;52:1673\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s12276-020-00516-4\u003c/span\u003e\u003cspan address=\"10.1038/s12276-020-00516-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSwan J, Szab\u0026oacute; Z, Peters J, et al. Inhibition of activin receptor 2 signalling ameliorates metabolic dysfunction\u0026ndash;associated steatotic liver disease in western diet/L-NAME induced cardiometabolic disease. Biomed Pharmacotherapy. 2024;175. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2024.116683\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2024.116683\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaniguchi Y, Ido H, Sanzen N, et al. The C-terminal region of laminin β chains modulates the integrin binding affinities of laminins. J Biol Chem. 2009;284:7820\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M809332200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M809332200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThorley M, Duguez S, Mazza EMC, et al. Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines. Skelet Muscle. 2016;6:43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13395-016-0115-5\u003c/span\u003e\u003cspan address=\"10.1186/s13395-016-0115-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrendelenburg AU, Meyer A, Rohner D, et al. Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size. Am J Physiol Cell Physiol. 2009;296:C1258\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/ajpcell.00105.2009\u003c/span\u003e\u003cspan address=\"10.1152/ajpcell.00105.2009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUezumi A, Ito T, Morikawa D, et al. Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle. J Cell Sci. 2011;124:3654\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jcs.086629\u003c/span\u003e\u003cspan address=\"10.1242/jcs.086629\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalker RG, Czepnik M, Goebel EJ, et al. Structural basis for potency differences between GDF8 and GDF11. BMC Biol. 2017;15:19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12915-017-0350-1\u003c/span\u003e\u003cspan address=\"10.1186/s12915-017-0350-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalker RG, Poggioli T, Katsimpardi L, et al. Biochemistry and Biology of GDF11 and Myostatin. Circ Res. 2016;118:1125\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/CIRCRESAHA.116.308391\u003c/span\u003e\u003cspan address=\"10.1161/CIRCRESAHA.116.308391\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu X, Chen X, Zheng XD, et al. Growth Differentiation Factor 11 Promotes Abnormal Proliferation and Angiogenesis of Pulmonary Artery Endothelial Cells. Hypertension. 2018;71:729\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/HYPERTENSIONAHA.117.10350\u003c/span\u003e\u003cspan address=\"10.1161/HYPERTENSIONAHA.117.10350\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Cheng F, Du X, et al. GDF11/BMP11 activates both smad1/5/8 and smad2/3 signals but shows no significant effect on proliferation and migration of human umbilical vein endothelial cells. Oncotarget. 2016. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18632/oncotarget.7642\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.7642\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhong X, Rescorla FJ. Cell surface adhesion molecules and adhesion-initiated signaling: Understanding of anoikis resistance mechanisms and therapeutic opportunities. Cell Signal. 2012;24:393\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou Y, Sharma N, Dukes D, et al. GDF11 Treatment Attenuates the Recovery of Skeletal Muscle Function After Injury in Older Rats. AAPS J. 2017;19:431\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1208/s12248-016-0024-x\u003c/span\u003e\u003cspan address=\"10.1208/s12248-016-0024-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Skeletal myogenesis, GDF11, ALK4, laminin, cell adhesion, cell fusion","lastPublishedDoi":"10.21203/rs.3.rs-7777108/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7777108/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMuscle 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 \u003cem\u003eACVR1B\u003c/em\u003e (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 \u003cem\u003eACVR1B\u003c/em\u003e-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\u0026rsquo;s paradoxical role in muscle cell biology and may inform novel therapeutic strategies to counteract skeletal muscle degeneration and age-related decline.\u003c/p\u003e","manuscriptTitle":"GDF11 and its receptor ALK4 independently modulate human myoblast migration and fusion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 20:29:18","doi":"10.21203/rs.3.rs-7777108/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4c482565-bc57-491c-9153-7ff44e0075e1","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-06T00:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-22 20:29:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7777108","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7777108","identity":"rs-7777108","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}