PIEZO1 Channels Modulate the Small Extracellular Vesicle Release in C2C12 Cells
Bernareggi Annalisa, Zhang Wen Ru, Sanchez‐Sanchez Laura, Norbedo Alessia, Fracassi Anna, Lucafò Marianna, Sciancalepore Marina, Alberto Griffoni, Russell K. William, Taglialatela Giulio, Lorenzon Paola, Limon Agenor
TL;DR
PIEZO1 channels influence the release of small extracellular vesicles in muscle cells, with different effects in myoblasts and myotubes.
Contribution
This study reveals a novel role of PIEZO1 in modulating extracellular vesicle release in myogenic cells.
Findings
PIEZO1 activation increased extracellular vesicle release in myotubes but not in myoblasts.
Vesicles from both cell types promoted myotube formation but with different capacities.
PIEZO1 activation increased PIEZO1 protein expression in vesicles from myotubes.
Abstract
PIEZO1 are mechanically‐activated ion channels expressed in many cell types. Their pharmacological activation by the selective agonist Yoda1 has been reported to favor skeletal muscle regeneration by controlling the fate of myogenic precursors cells, but the underlying mechanisms remain largely unknown. Hereby, we investigated the possibility that PIEZO1 could control the release of small extracellular vesicles in myogenic C2C12 cells. Myoblasts and differentiated myotubes were treated with the PIEZO1 agonist Yoda1 (5 μM) for 24 hours. Released small extracellular vesicles were isolated by ultracentrifugation methods, and characterized by Western blotting, Nano Tracking and proteomic analysis. Pharmacological activation of PIEZO1 showed cell‐type‐specific effects: In myoblasts, Yoda1 treatment did not significantly affect the size or release of the small extracellular vesicles and…
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Figure 8- —Mass Spectrometry Facility at UTMB
- —NIH10.13039/100000002
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Taxonomy
TopicsErythrocyte Function and Pathophysiology · Extracellular vesicles in disease · Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Introduction
1
Mechanically‐activated (MA) ion channels are the sensors for cellular mechanotransduction, which is the cell capability to convert mechanical force into biological signals. As nicely reviewed by Kefauver et al. (2020), these channels are a large group of proteins expressed in cells across the phylogenetic tree, from prokaryotic to eukaryotic cells. Even though the first evidence of MA channel expression in skeletal muscle cells dates back to the last century (Guharay and Sachs 1984), their role still remains largely unknown. One of the reasons is the lack of specific blockers for MA channels (Kinsella et al. 2024).
In 2010, a new family of MA channels named PIEZO1 (from the Greek “πίεση,” Piezi—pressure) was identified (Coste et al. 2010). The PIEZO family consists of two isoforms: PIEZO1 and PIEZO2. The latter is an essential mechanotransducer for touch, proprioception, and interoception. PIEZO1 is expressed in many cell types such as endothelial cells, cardiac fibroblasts, myogenic precursors, osteoblasts,and stem cells. PIEZO1 is a complex homotrimer composed by subunits named blades, connected to an extracellular cap located above the pore. The specific hallmark of this channel is a propeller‐like structure (about 900 kDa) and a spherical nanodome due to the local curvature of the plasma membrane caused by the blades. The pore conducts a nonselective cation current that slightly favors a Ca^2+^ influx (Coste et al. 2010). PIEZO1 opening is induced both nanodome flattening due to increased membrane tension and reorganization of the channel lipid environment, and by force from filaments through the binding of ECM/cytoskeletal elements (Lacroix and Wijerathne 2025). The open channel probability increases in the presence of Yoda1, a selective agonist that binds to the protein at the extracellular side lowering the threshold for channel opening (Syeda et al. 2015). Despite the identification and the characterization of novel agonist and Yoda1 analogs, Yoda1 still remains the most potent PIEZO1‐selective modulator and, thus, a powerful tool to investigate the effects of PIEZO1 channel activity at cellular level (Kinsella et al. 2024).
During adult myogenesis, satellite cells, the quiescent adult stem population of myogenic precursors located between the basal membrane and the sarcolemma of skeletal muscle, re‐enter the cell cycle and become activated; a fraction of them proliferate as myoblasts to then differentiate into multinucleated myotubes, while the rest return into a quiescent state (reviewed in Chal and Pourquié 2017). Channel expression has been detected in myogenic myoblasts, myotubes and skeletal muscle fibers. It has been suggesting that PIEZO1 channels have a role during myogenesis since Yoda1 promotes myoblast fusion and myotube formation (Bosutti et al. 2021), and restores the myogenic potential of dystrophic satellite cells (Ortuste Quiroga et al. 2022). Moreover, Yoda1 counters the skeletal muscle atrophy produced by immobilization in adults (Hirata et al. 2022). Although the role of PIEZO1 channels in muscle regeneration is well documented (Bernareggi et al. 2022; Hirano et al. 2022), the mechanism by which their effects are elicited is still largely unknown.
Extracellular vesicles (EV) are a heterogenous population of vesicles released by cells, usually classified by their size (small EV < 200 nm, and medium/large EV > 200 nm), biogenesis mechanism (from endosome or plasmalemma), and biochemical composition (expression of markers and cargo composition). Small EV (sEV) include exosomes (50–150 nm) but also ectosomes smaller than 200 nm (Mathieu et al. 2021). Exosomes originate as intraluminal vesicles within late endosomal compartments called multivesicular bodies; the multivesicular bodies must be docked to the plasma membrane before they fuse and release exosomes into the extracellular space (endocytic pathway). Ectosomes originate by direct budding from the plasma membrane, and they are usually enriched with lipid raft proteins involved in membrane curvature (Sapoń et al. 2023). The specific pool of biological molecules sorted inside of sEV (cargo), includes proteins, lipids, metabolites, RNA and DNA. The cargo content is then taken up by target cells, where it influences gene expression and phenotype. Therefore, both exosomes and ectosomes are powerful mediators for the short‐ and long‐range signaling between cells. Very recently, PIEZO1 channels have been detected in sEV (Cronemberger Andrade et al. 2025), suggesting that they could act as gatekeepers for functional and structural integrity of the extracellular vesicles (Sanghvi et al. 2025). Moreover, even though PIEZO1 channels are mainly located at the plasma membrane, there is evidence of their intracellular localization and role in controlling endosome trafficking (Carrillo‐Garcia et al. 2021).
In skeletal muscle, sEV play a pivotal role in controlling muscle development and regeneration in response to physical exercise (Jia et al. 2025). Although the existence of a mechanotransduction machinery able to control biogenesis and release of sEV is still a matter of investigation, it is well known that sEV released by skeletal muscle cells act mostly within the microenvironment, where they tend to accumulate (Watanabe et al. 2022). In particular, sEV released by myogenic precursors modulate gene expression to control myogenesis and, thus, skeletal muscle regeneration (Forterre et al. 2014; Ji et al. 2022).
Here, we tested the possibly that PIEZO1 channel activity might influence sEV released from myogenic precursor cells. To do this, we studied the effect of Yoda1 treatment on sEV release and cargo in undifferentiated myogenic precursors (myoblasts) and multinucleated myotubes. Moreover, we evaluated the effect of Yoda1 treatment on PIEZO1 expression in both sEV and cellular lysates derived from myogenic precursors. To that aim, we used C2C12 cells, a well‐known myogenic cell line in which the expression of the PIEZO1 protein is well documented (Tsuchiya et al. 2018).
Materials and Methods
2
Cell Culture
2.1
Myoblasts derived from the C2C12 cell line (RRID: CVCL_0188, ATCC‐CRL‐1772, LCG Standards S.r.l, MI, Italy) were kept in growth media (GM) containing Dulbecco's Modified Eagle Medium‐High Glucose (DMEM, Sigma‐Aldrich, St. Louis, Missouri, USA), 20% Fetal Bovin Serum (FBS; EuroClone, Pero, MI, Italy), 4 mM l‐Glutamine (EuroClone, Perom MI, Italy), 1% Penicillin‐Streptomycin (EuroClone, Pero, MI, Italy), at 37°C in humidified air containing 5% CO_2_. The EV from myoblasts were obtained from cells maintained in GM until 50% of confluence. Myotubes derived from myoblasts at 80% of confluence, were kept in differentiation medium (DM) containing DMEM, 2% Horse Serum (EuroClone, Pero, MI, Italy), 1% GlutaMAX 1X (Gibco, Grand Island, NY, USA) and 1% Penicillin‐Streptomycin Solution for about 4–5 days, since when well elongated myotubes were clearly visible.
Extracellular Vesicle Isolation
2.2
Control and Yoda1‐treated cells (Yoda1, 5 μM, Sigma‐Aldrich, St. Louis, Missouri, USA) were incubated with GM or DM containing EV‐depleted FBS or HS, respectively. The medium collected at the end of the treatment (24 h), was filtered (pore size 0.2 μm) and centrifuged at 300g for 10 min, and the supernatant stored a − 80°C until use. To isolate EV, the supernatant was centrifuged (4°C) at 2000g for 10 min, 10,000g for 10 min, and at 100,000g for 3 h. The resulting pellet was resuspended in 2 ml PBS and ultracentrifuged at 182,000g for 1 h at 4°C. The EV‐enriched pellets were resuspended in filtered phosphate‐buffered saline (PBS) and immediately used for TEM and nanoparticle tracking analyzes. Part of them were stored at − 20°C for Western blot and proteomic analysis.
Transmission Electron Microscopy (TEM)
2.3
Ultrastructure analysis of exosomes was performed with 5 μl drops adsorbed on a 200‐mesh coated resin grid (FCF 200–CU Formvar/Carbon, Electron Microscopy Sciences) for 10 min at RT. Grids were blotted with filter paper and stained with 2% aqueous uranyl acetate (catalog #541‐09‐3, Electron Microscopy Sciences) for negative staining for 1 min at RT. The uranyl acetate was then removed using filter paper, and the grids were dried with warm regular light for 2 min. Images were acquired with a Philips CM‐100 transmission electron microscope at 60 kV using an Orius SC2001 digital camera.
Nano Tracking Analysis (NTA)
2.4
NTA was performed using the NanoSight N300 system and NTA 2.1 operating system (Malvern) according to manufacturer's instructions. Briefly, sEV preparation was diluted in PBS and injected into the NanoSight for analysis of both particle size and concentration.
Western Blot Analysis
2.5
Exosome markers: Western blot analyzes were conducted using 15 μL of sEV preparation lysed in RIPA buffer supplemented with protease inhibitors. Protein separation was achieved via SDS‐PAGE, followed by transfer onto a nitrocellulose membrane. The membrane was incubated overnight at 4°C with primary antibodies targeting CD63, CD81, and Hsp70 (cat number EXOAB‐KIT‐1, SBI Biotechnology, Palo Alto, CA, USA). Detection and quantification were performed using IR Dye 680 nm and 800 nm secondary antibodies (cat numbers 926‐68071 and 926‐32211, LI‐COR Biosciences, Lincoln, NE, USA).
PIEZO1: 10 µg of cell lysate and 5 µg of sEV lysate proteins were separated in SDS‐PAGE and transblotted onto nitrocellulose membranes. The incubation with primary antibodies was performed overnight at 4°C: rabbit anti‐human PIEZO1 (Proteintech, Rosemont, IL, USA), rabbit anti‐human actin (Abcam, Cambridge, UK). After 5 washes Tris‐buffered saline buffer for 5 min, the membranes were incubated for 1 h at 4°C with secondary antibody anti‐rabbit (Sigma‐Aldrich, Milan, Italy). Immunocomplexes were visualized by chemiluminescence using the LiteABLOT Turbo Chemiluminescent Substrate (EuroClone, Milan, Italy) and the ChemiDoc MP Imaging System (Bio‐Rad Laboratories). All membranes were analyzed using ImageJ software.
Proteomic Analysis
2.6
Aliquots from each sEV sample (10 μL) were brought to a final concentration of 50 mM TEAB and 4% SDS. Benzonase treatment was applied to degrade nucleic acids, follow by reduction, alkylation, and digestion. The samples underwent a reduction step at 65°C for 30 min, and alkylation was performed at room temperature for 30 min to prevent the formation of disulfide bonds. The samples were then loaded onto a Strap column for a 2‐h enzymatic digestion. After digestion, peptide fluorometric quantification was conducted to assess the concentration of peptides in each sample (as shown in the Table 1 of Supporting Information). Following quantification, samples were dried using a Speedvac concentrator and then resuspended to a concentration of 0.2 μg/mL. For the Mass Spectrometry Analysis, 2 μL of each sample were injected onto the Thermo Scientific Orbitrap Eclipse LCMS, using 8 Da staggered windows DIA (Data‐Independent Acquisition). The resulting data were processed and analyzed using Fragpipe with a library‐free approach. The details of the analysis are in SI.
Quantification of Myotube Formation
2.7
Quantification of myotube formation was performed on 2‐day differentiating C2C12 cell cultures, to which the DM medium was replaced with DM containing EV‐depleted HS, supplemented with sEV isolated from (i) untreated myoblasts, (ii) untreated myotubes, (iii) Yoda1‐treated myoblasts, or (iv) Yoda1‐treated myotubes. A set of parallel 2‐day differentiating C2C12 cell cultures were maintained in DM as controls.
After 2 days of treatment (at day 4 of differentiation), sEV‐treated and control cultures were fixed in freshly prepared 3.7% paraformaldehyde in phosphate‐buffered saline (PBS) for 15 min at room temperature (20°C). Following extensive and gentle washing with PBS (3 × 10 min, 20°C), staining was performed by incubating the cells in PBS containing 10 µM DAPI (60 min, 4°C). Cells were then rinsed in PBS (3 × 5 min, 20°C). Fluorescence images were acquired using a Nikon ECLIPSE Ti epifluorescence microscope, equipped with a 360 nm laser line for excitation and a 445–475 nm bandwidth emission filter. Images were captured using a 40X air objective. Fluorescence images were analyzed using ImageJ software (NIH, Bethesda, Maryland). The number of myotubes (defined as cells with more than 2 nuclei) was calculated in randomly selected optical fields (OF) and normalized to the average of total myotubes in control condition. For each experimental point, at least 40 randomly selected OFs from three different cell culture dishes were analyzed, ensuring a minimum of 4 myotubes per OF.
Statistical Analysis
2.8
GraphPad Prism 5.00 (GraphPadSoftware) and OriginPro2021 were used to analyze the data. Sample data were drawn from a Gaussian‐distributed population. The mean ± standard error of the mean (SEM) was used to compare the results, with a p < 0.05 for the statistical significance (unpaired t‐test).
Results
3
The myogenic precursors used in this study were derived from the C2C12 cell‐line, a well‐established cell model that recapitulates in vitro the myogenic cellular processes occurring in skeletal muscle in vivo (McMahon et al. 1994). We carried out our experiments during two of the key steps of the myogenic process. Specifically, when C2C12 cells are undifferentiated and proliferating myoblasts and at a later step of myogenesis, that is, when they are fused into multinucleated myotubes. The capability of Yoda1 (5 μM) to activate PIEZO1 channels in these two differentiation phases has already been reported (Tsuchiya et al. 2018).
Characterization of sEV Released From Myoblasts and Myotubes Following Yoda1 Treatment
3.1
To investigate the effect of PIEZO1 on sEV release, myoblasts and myotubes were incubated in EV‐depleted medium for 24 h in the absence (control) or in the presence of 5 μM Yoda1 (Supporting Information Figure S1A). At the end, conditioned media were collected and used to isolate sEV. In accordance with the International Society of Extracellular Vesicles (Minimal Information for Studies of Extracellular Vesicles, MISEV, Welsh. 2024), the presence of sEV was confirmed by Western blotting (WB), transmission electron microscopy (TEM) and Nano Tracking analysis (NTA). In addition, a proteomic analysis was performed to verify whether Yoda1 could have altered the proteomic cargo profile of the sEV (Supporting Information Figure S1B).
In the first set of experiments, three specific exosome markers, namely Hsp70, CD63, and CD81 were investigated by WB in particles isolated from control and Yoda1‐treated myoblasts. In vesicles from both control and Yoda1‐treated cells, Hsp70 was only weakly detected, while they were positive for CD63 and negative for CD81 (Figure 1A). Yoda1 did not alter the morphology of the particles, which displayed a rounded shape and a size typical of the sEV released by control myoblasts in our experimental conditions (Figure 1B), and by C2C12 myoblasts as reported by others (Watanabe et al. 2022). As determined by NTA, the particle size distribution exhibited a bell‐shaped curve profile indicative of a homogeneous population in control conditions and after Yoda1 treatment (Figure 1C). The size values (mean diameter), ranged from 100 to 120 nm (mean: 107.40 ± 3.94 nm, n = 5 independent measurements) for sEV released by control myoblasts, and from 95 to 154 nm (mean: 123.20 ± 12.24 nm, n = 5 independent cell measurements) for sEV released by Yoda1‐treated myoblasts, respectively (Figure 1D). The mean number of particles normalized to the cellular protein concentration revealed that the amount of sEV released was only slightly different in the two experimental conditions (Figure 1E, 6.74 × 10^11^ ± 3.00 × 10^11^ vs 6.31 × 10^11^ ± 2.84 × 10^11^, n = 5, p > 0.05).
Yoda1 effects on sEV released by myoblasts. (A) Expression of sEV markers (e.g. Hsp70, CD63 and CD81) in vesicles from control (cMB‐sEV) and treated myoblasts (yMB‐sEV). (B) TEM images of sEV. Yoda1 did not significantly change either concentration (C) or size (D) of the released vesicles in treated myoblasts (yMB‐sEV) as compared to control (cMB‐sEV). (E) Number of released sEV normalized to the concentration of cell protein. Data are from 5 independent cell measurements (unpaired t‐test). Box plot represents median, 25th and 75th percentiles‐interquartile range; whiskers range within 1.5 IQR.
Protein level analysis for specific sEV markers Hsp70, CD63 and CD81 was also performed in vesicle preparations from control and Yoda1‐treated myotubes (Figure 2A). In this case, the TEM analysis revealed the co‐presence of round and cup‐shaped morphologies in both experimental conditions (Figure 2B). Similarly to myoblasts, the particle distribution confirmed the presence of a single homogeneous population of sEV in the two experimental conditions (Figure 2C). As in myoblasts, the size of sEV released by Yoda1‐treated myotubes (121.37 ± 14.73 nm, range 90–173 nm, n = 6 independent measurements) tended to be larger than the size of sEV released by control myotubes (101.35 ± 10.54 nm, range 63.3–130 nm, n = 6 independent measurements). However, the difference was not statistically significant (p = 0.29) (Figure 2D). The quantification of sEV released by Yoda1‐treated myotubes with respect to control myotubes revealed that the release significantly increased by Yoda1 treatment (2.86 × 10^11^ ± 7.14 × 10^10^ vs 8.57 × 10^11^ ± 9.60 × 10^10^, n = 6, **p = 0.002, Figure 2E). Thus, our finding suggested PIEZO1 activity having a role in controlling the sEV release specifically in myotubes.
Yoda1 effects on sEV released by myotubes. (A) Expression of sEV markers (e.g. Hsp70, CD63 and CD81) in vesicles from control (cMT‐sEV) and treated myotubes (yMT‐sEV). (B) TEM images of sEV. (C) Concentration of released vesicles increased following Yoda1‐treatment. (D) Yoda1 treatment did not affect the size. (E) Number of released sEV normalized to the concentration of cell protein. Data are from 6 independent measurements (unpaired t‐test). Box plot represents median, 25th and 75th percentiles‐interquartile range; whiskers range within 1.5 IQR.
Proteomic Profiling of sEVs From Myoblasts and Myotubes Following Yoda1 Treatment
3.2
It is known that the sEV proteomic profile of myogenic precursors changes in accordance with the stage of myogenic differentiation (Forterre et al. 2014a; Watanabe et al. 2022). In line with these studies, the analysis of the proteomic profile of sEV derived from untreated cells, tended to change during myogenic differentiation (Figure 3A,B, see also Supporting Information Figures S2–S3) confirming the reliability of our experimental procedures.
Proteome analysis overview of sEV isolated in control condition. (A) Volcano plot indicating the log2‐fold change in protein expression in cMT‐sEV vs cMB‐sEV. Black dots represent proteins significantly downregulated (left) and upregulated (right), while those similar are in gray. Dotted lines indicate cutoffs (see in Materials and methods). (B) Enriched ontology clusters of proteins differently expressed in the two populations of sEV.
The proteomic cargo profile induced by the PIEZO1 agonist on sEV released by myoblasts revealed a variation in the expression of a few proteins (Figure 4).
Comparison of proteomic sEV profile from control and Yoda1‐treated myoblasts. (A) Volcano plot indicating the log2‐fold change in protein expression. Black dots represent proteins significantly downregulated (left) and upregulated (right) in cMB‐sEV in respect to yMB‐sEV, while those similar are in gray. Dotted lines indicate cutoffs (see in Materials and methods). (B) Gene Ontolgy (GO) terms in up and downregulated genes due to Yoda1 treatment. (C) Overlap between genes lists, identical genes are reflected by purple lines, while blue curves link genes that belong to the same enriched ontology term.
On the contrary, the same analysis on sEV released by myotubes detected proteins that were down (left) and up (right) in Yoda1 compared control differentiated cells (Figure 5A). Indeed, the enriched ontology clusters showed differences in pathways related to extracellular matrix organization (GO: 0005201, down regulated by Yoda1 treatment), or chromosome reorganization (nucleosomal DNA binding, GO: 0031492, up regulated by Yoda1 treatment; Figure 5B,C).
Comparison of proteomic sEV profile from control and Yoda1‐treated myotubes. (A) Volcano plot indicating the log2‐fold change in protein expression. Black dots represent proteins significantly downregulated (left) and upregulated (right) in cMT‐sEV with respect to yMT‐sEV, while those similar are in gray. Dotted lines indicate cutoffs (see in Materials and methods). (B) Gene ontolgy (GO) terms in up and downregulated genes due to Yoda1 treatment. (C) Overlap between genes lists, identical genes are reflected by purple lines, while blue curves link genes that belong to the same enriched ontology term.
PIEZO1 Content in sEV From Myoblasts and Myotubes Following Yoda1 Treatment
3.3
Besides the well‐known markers for sEV, Hsp70, CD63, and CD81, WB analysis performed on sEV revealed the presence of PIEZO1 in the sEV derived from both myoblasts and myotubes in control conditions, with a higher protein content level detected in myotubes (Figure 6A, PIEZO1/Actin in cMB‐sEV: 78.98 ± 10.95, n = 3; Figure 6B in cMT‐sEV: 263.11 ± 20.78, n = 3, **p < 0.001 not shown). Moreover, we also observed that the Yoda1 treatment did not affect the PIEZO1 protein level in sEV derived from myoblasts (Figure 6A, yMB‐sEV: 92.76.48 ± 4.82, n = 3, p > 0.05 vs cMB‐sEV) but that it did in sEV derived from myotubes (Figure 6B, in yMT‐sEV: 380.48 ± 6.61, n = 3, ***p < 0.001 vs cMT‐sEV). In parallel, WB analysis performed on myoblasts and myotubes, from which sEV were derived, showed that the Yoda1 treatment enhanced the PIEZO1 protein level more in myoblasts than in myotubes (Figure 6C, PIEZO1/Actin in cMB: 60.95 ± 0.74; yMB: 89.92 ± 2.25, n = 3, ***p < 0.001; Figure 6D, PIEZO1/Actin in cMT: 60.95 ± 0.74; yMT: 89.92 ± 2.25, n = 3, *p < 0.05). The above mentioned Yoda1 effect on PIEZO1 expression in myogenic cells is in line with previous studies where both mechanical activation and pharmacological activation of PIEZO1 by Yoda1 were found to stimulate the expression of the protein in other cell models (Kang et al. 2024).
PIEZO1 expression in sEV and cellular lysate. Representative Western blot revealing PIEZO1 in sEV derived from myoblasts (A), and myotubes (B). Note that the pharmacological activation of PIEZO1 by Yoda1 increases the protein expression only in sEV derived from myotubes. PIEZO1 was also detected in myoblast (C) and myotube lysate (D), and its expression is significantly increased by treatment Yoda1 both in myoblasts (yMB, 3 replicates each, unpaired t‐test) and myotubes (yMT, 3 replicates each, unpaired t‐test) as compared to the corresponding controls (cMB and cMT).
Yoda1‐induced sEV Promotes Myotube Formation
3.4
The ability of sEV to affect myotube formation was evaluated in myogenic‐committed cells (2 days in DM) cultured for further 2 days in medium containing exclusively exogenous sEV derived from untreated or Yoda1‐treated myoblasts or myotubes. To compare these effects, the number of myotubes measured in the presence of “exogenous” sEV was normalized to the one observed in DM medium without exogenous sEV addition. As shown in Figure 7, the myotube formation was similar after treatment with sEV derived from untreated myoblasts or myotubes (cMB and cMT). Conversely, the sEV from Yoda1‐treated myoblasts (yMB) or myotubes (yMT) enhanced the myotube formation, with a more pronounced effect observed for sEV derived from yMT. These findings indicate that the Yoda1 treatment enhances the pro‐myogenic potential of sEV, especially when secreted by myotubes.
Yoda1‐induced sEV promotes myotube formation. (A) Representative optical field areas of 4‐day differentiating C2C12 cells in control conditions (Ctrl; DM medium), treated with exogenous sEV derived from MB and MT cultures (cMB‐sEV and cMT‐sEV) or with exogenous sEV derived from Yoda1‐treated MB and MT cultures (yMB‐sEV and yMT‐sEV). Fluorescence images (DAPI staining) were overlaid on the corresponding bright‐field images. (B) The number of myotubes in each optical field (OF) was normalized to the average number of myotubes observed in Ctrl condition. For each experimental point, at least 40 randomly selected.
Discussion
4
The main outcomes reported in this study may be summarized as follows: (i) a specific enhancing effect of Yoda1 on sEV release in myotubes; (ii) a different controlling effect of Yoda1 on the proteomic cargo profile of sEV released by myoblasts and myotubes; (iii) a specific modulating effect of Yoda1 in sEV biogenesis in myotubes; (iv) a potentiated effect of the Yoda1‐induced sEV on myotube formation. Altogether, our findings indicate that PIEZO1 activity controls myotube fusion modulating the sEV release in myogenic cell precursors.
The characteristics of the particles isolated in our study, closely resemble those of sEV derived from C2C12 cells reported in previous studies, confirming the reliability of our experimental procedures. Expression of the exosome markers Hsp70 and CD63 was found in both myoblast‐ and myotube‐derived‐sEV, and CD81 only in those derived from myotubes (Watanabe et al. 2022; Forterre et al. 2014). Concerning the particle size, our samples were enriched with small particles having a diameter smaller than 200 nm, in line with a previous study conducted on C2C12 cells (Watanabe et al. 2022).
In a previous study (Bosutti et al. 2021), we already observed a “time window” for the effect of Yoda1‐evoked PIEZO1 activity during myogenesis. Briefly, PIEZO1 channels were detected starting from quiescent satellite cells up to fully differentiated skeletal muscle fibers. However, in the same study, we also reported that Yoda1 enhances the “window” for the transition from a predominantly proliferative state to MyoG‐positive myogenic precursors as well as for cell fusion into myotubes. Here we show that Yoda1 treatment potentiated the sEV release only in myotubes. This phenomenon could have at least three different explanations. Firstly, myotubes expressed more PIEZO1 than myoblasts, as evidenced by the WB analysis on cell lysates. Secondly, myotubes generate spontaneous contractions in our culture conditions that could potentiate channel activity in the presence of Yoda1. Indeed, a similar increase in sEV release has been reported also in electrically‐stimulated contracting C2C12 myotubes (Murata et al. 2023; Vilchinskaya et al. 2025). Thirdly, the potentiated sEV release observed in myotubes could be due to alterations of the cell membrane composition during the myogenic differentiation program (Briolay et al. 2013). Indeed, variations in membrane fluidity could potentiate PIEZO1 channel activity and enhance the Yoda1 effect (Ridone et al. 2020). Regardless of the mechanism involved, the Yoda1‐potentiating effect on sEV release, which is limited to myotubes, confirms the dependence of the functional role of PIEZO1 channels on the phase of myogenesis.
Our results also showed that Yoda1 differently modified the cargo in sEV derived from myoblasts and myotubes. Interestingly, in myoblasts, the effect of PIEZO1 activity involved pathways related to protein synthesis (GO: 0006412–translation) and cytoskeleton remodeling (GO: 0097435–supramolecular fiber organization), both required for myotube differentiation. In myotubes, the proteomic analysis revealed downregulation of proteins controlling the extracellular matrix (ECM) organization and an upregulation of proteins controlling the nucleosome DNA binding. Both effects could contribute to the regulation of the fate of myogenic precursor cells, within their microenvironment. Indeed, ECM remodeling induced by exercise facilitates adaptation, regeneration and repair of skeletal muscles (Kritikaki et al. 2021). Moreover, ECM dysregulation influences the activity of satellite cells: for instance, an increase in ECM stiffness is correlated with an impairment in muscle regeneration observed during aging (Lacraz et al. 2015), while a reduction in ECM stiffness favors myoblast fusion and promotes myotube differentiation (Engler et al. 2004). With regard to the upregulation of proteins controlling the nucleosome DNA binding, this could play a role in controlling gene expression towards muscle specific gene expression, thereby promoting the myogenic differentiation (Bai and Morozov 2010). All the above pathways could contribute to myotube formation by Yoda 1‐induced sEV (from myoblasts and myotubes).
The other finding which merits discussion is the detection of the PIEZO1 protein in sEV released by myogenic precursors. To our knowledge, this is the first evidence for the PIEZO1 protein in sEV released by skeletal muscle cells. As recently hypothesized for other ion channels located in extracellular vesicles (Sanghvi et al. 2025), the presence of PIEZO1 could ensure a safety transport of molecules under mechanical stimulations such as osmotic stress. The higher PIEZO1 content detected in sEV released by myotubes compared to those released by myoblasts also suggests a different sEV biogenesis in the two cell types. As mentioned in Introduction, the small particles include an heterogenous population made up of a mix of multivesicular‐derived (exosome) and plasmalemma‐derived vesicles (or small ectosome). Exosomes originate as intraluminal vesicles from the late endosomal compartment, called multivesicular bodies; they dock and fuse to the plasma membrane to be released into the extracellular space. Ectosomes originate from direct budding of the plasma membrane (Figure 8A). While the tetraspanin CD63 is considered a marker for exosomes, CD81 is also present in ectosomes (Rome et al. 2019; Mathieu et al. 2021). In our experimental conditions, we detected CD81 and a high content of PIEZO1 in myotube‐derived sEV. Assuming the channels are mainly localized at the plasma membrane level, the co‐presence of CD81 and PIEZO1 proteins suggests a prevalent membrane budding origin of the sEV released by myotubes. This hypothesis is in line with the idea of a specific type of sEV biogenesis during different phases of the myogenic program (Romancino et al. 2013). The fact that PIEZO1 protein was highly detected in myotube‐derived sEV after Yoda1 treatment, and not in those derived from treated myoblasts, further supports the idea that PIEZO1 channel activity promotes the release by membrane budding in myotubes (this hypothetical mechanism is described in Figure 8B,C).
Hypothetical mechanism of sEV release controlled by PIEZO1 activity. (A) sEV are a heterogeneous population of vesicles made of exosomes and ectosomes. Since PIEZO1 is mostly located at the plasma membrane, which is where ectosomes originate through plasmalemma budding, they are more likely to contain the mechanically‐activated protein compared to exosomes. (B) In myoblasts, pharmacological stimulation of PIEZO1 with Yoda1 promotes myotube formation by altering the cargo, without affecting the extent of the release. (C) In myotubes, Yoda1 enhances sEV release and alters their cargo. The increased presence of PIEZO1 in Yoda1‐induced sEV in myotubes suggests a prevalent effect of Yoda1 in promoting ectosome than exosome release. The cartoon also shows the greatest effect on myotube formation of sEV delivered by Yoda1‐treated myotubes (Created in BioRender. Bernareggi (2026) https://BioRender.com/hmpy402).
In conclusion, we show that PIEZO1 activity modulates the secretion levels, cargo content and biogenesis of sEV depending on the differentiation state of myogenic cells. In addition, our study reveals a novel effect of PIEZO1‐induced sEV on myotube formation, which represents a key step in myogenesis. This evidence makes PIEZO1 a key element in controlling skeletal muscle regeneration. Although the interpretation of our results is limited by the use of cell lines and needs to be confirmed in primary precursors, they suggest a potential new role of PIEZO1 channels in cell communication during the myogenic process.
Author Contributions
Annalisa Bernareggi: conceptualization, investigation, methodology, validation, visualization, formal analysis, data curation, supervision, writing‐original draft, writing – review and editing. Zhang Wen Ru: methodology, data curation. Laura Sanchez‐Sanchez: methodology, validation, data curation, formal analysis. Alessia Norbedo: methodology, validation, data curation, formal analysis. Anna Fracassi: writing – review and editing. Marianna Lucafò: formal analysis, data curation, writing – review and editing. Marina Sciancalepore: writing – review and editing. Alberto Griffoni: data curation, formal analysis, methodology. William Russell: funding acquisition, methodology, validation, visualization, and editing. Giulio Taglialatela: conceptualization, supervision, funding acquisition, and editing. Paola Lorenzon: conceptualization, investigation, methodology, visualization, writing – review and editing. Agenor Limon: supervision, data curation, visualization, investigation, funding acquisition, writing – review and editing.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Supplementary information.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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