Extracorporeal shockwave therapy (ESWT) is a non-invasive treatment that works by the emission of high-amplitude biphasic pulsed acoustic waves (shockwaves, SW) that rapidly cycle between positive and negative pressures onto the area being treated (Huang et al., 2013; Cheng and Wang, 2015; Simplicio et al., 2020). Thus, acting as a mechanical stimulus, SW can exert its beneficial effects and promote healing processes via mechanotransduction, a process by which cells convert mechanical stimuli into biochemical signals, thereby promoting tissue regeneration and healing (Frairia and Berta, 2012; Notarnicola and Moretti, 2012; Agostino et al., 2015), mainly by modulating inflammation (Sukubo et al., 2015) as well as stimulating angiogenesis (Goertz et al., 2014; Tepeköylü et al., 2018; Wang et al., 2021) and stem cell activities (Tepeköylü, et al., 2013).

Over the last decade, ESWT has become a popular and safe treatment option for various musculoskeletal conditions, ranging from tendinopathies to bone healing disturbances (d’Agostino et al., 2015; Burton, 2021; Kwok et al., 2022; Wuerfel et al., 2022; Lv et al., 2023; Pellegrino et al., 2023; Fulceri et al., 2024a; Majidi et al., 2024). From a clinical perspective, ESWT has proven to b effective also in relieving pain, promoting functional recovery, and reducing disease recurrence in bone and soft-tissue disorders (Ramon et al., 2020; Xu et al., 2019; Liu et al., 2023; Ryskalin et al., 2023; Fulceri et al., 2024b; Xue et al., 2024; Santilli et al., 2025).

On the other hand, its use for acute muscle injuries and chronic muscle conditions is of more recent development (Astur et al., 2015; Ramon et al., 2015; Fleckenstein et al., 2016; Liu et al., 2023; Mazin et al., 2023).

In sports medicine, ESWT has demonstrated benefits in treating injured muscle tissue and painful muscle hematomas and has been associated with reduced layoff times and lower re-injury rates among athletes (Astur et al., 2015; Mazin et al., 2023).

At the same time, preclinical research on the effects of ESWT on muscle tissue has also rapidly expanded, yielding promising results (Kisch et al., 2016; Zissler et al., 2017; Langendorf et al., 2020; Wuerfel et al., 2022; Wang et al., 2024). In vivo studies on animal models suggest that ESWT can improve muscle microcirculation, stimulate tissue regeneration, and accelerate repair processes by affecting humoral and cellular factors (Lee and Cho, 2013; Kisch et al., 2016; Zissler et al., 2017; Huang et al., 2021; Lee et al., 2023; Wang et al., 2024). Moreover, Radial Pressure Waves (RPW) reduced inflammatory response and enhanced muscle regeneration in rabbits with acute biceps femoris injury (Wang et al., 2024). In rabbits, RPW could also alleviate immobilization-induced contracture and reduce the molecular manifestations of muscle fibrosis (Huang et al., 2021). Another study found that capillary blood flow is immediately enhanced after a single session of high-energy SW in the muscles of rats, with even greater effects after repeated sessions (Kisch et al., 2016).

On the contrary, little is known about the biological responses elicited by SW when applied to an in vitro model of skeletal muscle tissue. So far, only a few studies have investigated the rationale for applying SW to muscle tissue using in vitro experimental setups (Hansen et al., 2017; Mattyasovszky et al., 2018; Wang et al., 2017). Therefore, the exact mechanisms underlying ESWT-induced beneficial effects, such as myogenesis, still remain largely unexplored.

Skeletal muscle differentiation, myogenesis, is a finely tuned multistep process in which single mononuclear and elongated cells (i.e., myoblasts) arrest cell division and then start to elongate and fuse, forming multinucleated syncytia delimited by a single plasma membrane called myotubes (Weintraub, 1993; Buckingham et al., 2003). This, in turn, is essential for muscle growth and maintenance and for repairing injured muscles (Kook et al., 2008). This complex sequence of events, which occurs both in muscle development and regeneration, is governed by the coordinated expression of muscle-specific transcription factors belonging to the Myogenic Regulatory Factor (MRF) family, such as the Myoblast Determination Protein 1 (MyoD) and Myogenin (MyoG) (Sabourin and Rudnicki, 2000; Hernández-Hernández et al., 2017). These latter are exclusively expressed in cells committed to the myogenic lineage and become active in a time-correlated manner. MyoD is essential for myoblast identity and acts early in myogenesis to determine myogenic fate, whereas MyoG acts at a later stage, and it is essential to drive the decisive events of myogenic differentiation, such as myoblast fusion (Hasty et al., 1993; Perry and Rudnick, 2000; Zhang et al., 2020).

As far as we know, the current understanding of SW-induced effects mainly relies on gene expression analyses and biochemical data, with limited literature on the ultrastructural changes related to its positive clinical results.

Therefore, the present study aims to investigate for the first time whether SW can promote ultrastructural changes indicative of myogenesis. For this purpose, a primary line of murine myoblasts, namely, the C2C12 cell line, was used, which represents a cell line derived from mouse satellite cells that faithfully mimics skeletal muscle differentiation (Tang et al., 2018); thus, it represents an excellent and well-established model to study myogenic regulation and its responses to stimulation (Luo et al., 2019). In particular, the myogenic response to SW stimulation was studied by evaluating the morpho-functional and ultrastructural changes in fusion-competent myoblast cells towards the formation of multinucleate syncytia as well as the time course expression pattern of the myogenic markers, MyoD and MyoG.

Through these experiments, we seek to provide novel morphological/ultrastructural evidence of the potential of SW in promoting myoblast transition into a multinucleated cell, syncytium. This, in turn, holds the significant therapeutic promise of ESWT for promoting muscle healing and regeneration.

In the last decades, extracorporeal shockwave therapy (ESWT) has emerged as a non-invasive and safe therapeutic modality for treating several orthopedic and musculoskeletal pathologies (Moya et al., 2018). While ESWT has proven effective in promoting healing in bones, tendons, and joint pathologies, its impact on muscle tissue repair and regeneration remains less understood. Indeed, there is limited evidence of its effectiveness both in clinical and experimental settings (Astur et al., 2015; Fleckenstein et al., 2016; Zissler et al., 2017; Huang et al., 2021; Lee et al., 2023). Remarkably, some findings on animal models indicate that ESWT may promote an acceleration of muscle healing (Kisch et al., 2016; Zissler et al., 2017; Wang et al., 2024). However, the biological mechanisms underlying shockwaves (SW) have remained largely unexplored, and only a few studies on the in vitro effects of SW on myoblast cells are currently available (Hansen et al., 2017; Mattyasovszky et al., 2018; Wang et al., 2017). At the same time, as far as we know, a detailed ultrastructural analysis of myoblast cells exposed to SW and morphometric analysis of protein subcellular distribution following SW treatment has never been performed. In particular, in the present study, we demonstrate that SW is capable of promoting in vitro C2C12 myoblast fusion and differentiation, as evidenced by morphological and ultrastructural changes. Moreover, we identified that the nuclear translocation of myogenic markers MyoD and MyoG contributes to this promising effect mediated by SW (Figure 8). These findings highlight the potential of SW as a stimulus to influence the biology of myoblast cells and promote their differentiation, which could have important implications for regenerative medicine and muscle repair.

A crucial event in myogenesis is the cell-cell fusion of mononucleated myoblasts to form multinucleated syncytia (Sampath et al., 2018; Lehka and Rędowicz, 2020). This, in turn, can be divided into several key stages where myoblasts undergo remodeling, including, for instance, cell elongation, membrane apposition, fusion pore formation, and ultimately, membrane merging (Abmayr and Pavlath, 2012; Leikina et al., 2013). Remarkably, data from the present research showed that SW treatment can induce a significant increase in cell size and elongation, both of which are key events during in vitro myoblast differentiation and precede myoblast fusion (Ohtake et al., 2006). As shown by previous studies, upon appropriate stimuli including mechanical stimulation, C2C12 cells typically undergo a remodeling process during differentiation, transitioning towards a more fusiform and elongated morphology (Burattini et al., 2004; Casadei et al., 2009; Grossi et al., 2011; Wang et al., 2014; Espinoza-Álvarez et al., 2024). Similarly, in our study SW-treated cells displayed noticeable changes in shape, with a marked increase in cell size, as quantified through morphometric analysis. The morphological changes induced by SW exposure were significantly more pronounced compared to controls by 7d post-treatment, suggesting that SW may accelerate the differentiation process.

Ultrastructural investigations corroborate the morphological evidence of an early myogenic commitment of C2C12 cells following SW stimulation. Furthermore, the high-resolution TEM analysis provided compelling evidence that SW stimulation can trigger a sequence of membrane-associated events that culminate in the fusion of C2C12 myoblasts. Unlike untreated controls, SW-treated cells display key features of impending fusion, such as membrane protrusions and localized contact sites, which precede the formation of nascent multinucleated syncytial structures. In detail, at early time points (24 h) following SW stimulation, we could detect the occurrence of finger-like protrusions at the fusion site of C2C12 cells, which in contrast are absent in fusion-incompetent control cells. This feature allowed us to document “en face” the SW-induced fusion process as these structures are key intermediates of early fusion events as they facilitate the alignment of opposing membranes, a crucial prerequisite for successful cell-cell fusion. This observation resonates with findings by Le Clainche and Carlier (Le Clainche and Carlier, 2008), who described the critical role of acting remodeling and filipodia-like structures in membrane apposing during fusion, as well as more recent works that emphasize the functional role of cytoskeletal protrusion in mediating cell-cell recognition, contact stabilization, and thus efficient fusion (Randrianarison-Huetz et al., 2018; Cong et al., 2019; Lu et al., 2024a; 2024b).

As fusion progresses, the plasma membrane of opposing aligned myoblasts begins to merge, forming localized cell-cell contacts and membrane-associated electron-dense plaques. Over time, multiple fusion sites appear, which then expand laterally, leading to membrane breakdown and the establishment of full cytoplasmic continuity between adjacent cells. Furthermore, at 72 h post-treatment, we observed an increased number of discrete fusion points characterized by aligned plasma membranes and the occurrence of electron-dense plaques, indicative of nascent fusion machinery assembly. These sites later (7d post-treatment) expanded to form wider zones of membrane continuity, leading to the dissolution of intervening membranes and the formation of nascent syncytial structures. The identification of these features at TEM, especially in a sequential and time-dependent manner, provides unique ultrastructural evidence of the dynamic fusion landscape induced by this mechanical stimulation. Interestingly, while most studies on myoblast fusion focus on the biochemical and genetic modulation of fusogenic proteins, only a few reports have addressed the morphological and ultrastructural underpinnings of this process, and particularly in response to a mechanical stimulus such as shockwave. Within this frame, the results of the present study bridge this gap offering a detailed ultrastructural framework for understanding how SW exposure can prime and accelerate myogenic fusion through the induction of membrane protrusion and the establishment of localized fusion domains. These findings are in line with emerging literature suggesting that mechanical forces and membrane tension are key modulators of fusion competence in muscle cells (Kim et al., 2015; Chakraborty et al., 2022; Millay, 2022). The prolonged presence and maturation of fusion sites observed at later time points (up to 7d) suggest that SW stimulation not only can initiate early membrane contact but also sustains the fusion process, possibly through the modulation of membrane and cytoskeletal dynamics. Further studies on the possible effects of SW in the coordination of known fusogenic proteins may bring novel insights into the biophysical regulation of myoblast fusion and differentiation.

Additionally, the TEM results observed in this study align with the evidence of an increased proportion of multinucleated cells following SW exposure, which are detectable as early as 24 h post-treatment and significantly increased at later stages. This further supports the progression toward myogenic differentiation and the formation of syncytial structures. However, our data contrast with those of Hansen et al. who found no significant effect on the in vitro differentiation of primary isolated human myoblasts treated with low-intensity SW (Li-SW) (Hansen et al., 2017). Interestingly, when performing the in vivo experiment, the same authors reported a significantly increased expression of myogenic genes in the early phase of regeneration in the Li-SW-treated hind limbs after acute injury. On the other hand, Mattyasovszky et al. documented morphological changes similar to those in our study, where Li-SW was shown to enhance in vitro myotube formation in L6 rat myoblasts (Mattyasovszky et al., 2018). The inconsistencies between the above-reported data are not so unexpected since a significant body of literature highlights the challenges of comparing results due to the high variability in treatment protocols and type of SW devices used. This issue does not only apply to in vivo studies but is even more evident when comparing in vitro data. Indeed, the controversial data from the in vitro studies may be explained by the heterogeneous experimental setting including differences in cell types and above all the variable application method of the shockwaves. This variability underscores the need for the establishment of standardized in vitro treatment protocols to improve the reproducibility and comparability of future studies.

Despite the observed morphological and ultrastructural changes, our study found no significant alteration in the total levels of the myogenic markers between SW-treated cells and controls. This finding is noteworthy, as MyoD and MyoG are pivotal in regulating myoblast differentiation and fusion (Bentzinger et al., 2012). MyoD is known to initiate the myogenic program, while MyoG regulates the later stages of differentiation (Hernández-Hernández et al., 2017). Previous studies have reported variable effects of mechanical stimuli on the expression of these markers, with some studies showing increased MyoD and MyoG expression in response to external stimuli (Grossi et al., 2007; Abe et al., 2009; Chan et al., 2023), while others have observed no significant changes (Kook et al., 2008; Kuang et al., 2009; Wang et al., 2020). Our results suggest that while SW treatment does not significantly alter the overall levels of the myogenic markers, it may still facilitate myogenesis by promoting the transition from mononucleated to multinucleated cells. This is likely due to the mechanical cues provided by SW, which may activate signaling pathways involved in myoblast fusion without altering MyoD and MyoG levels.

Indeed, one of the most striking findings in the present study was the enhanced nuclear localization of MyoD and MyoG following SW treatment. Nuclear translocation is critical for both of these transcription factors to exert their function (Bentzinger et al., 2012). Previous studies by Ferri et al. (2009) showed that myogenic regulatory factors (MRFs) nucleo-cytoplasmic trafficking is involved in muscle differentiation, suggesting that, besides their expression levels, MRF subcellular localization, related to their functional activity, also plays a key role as a regulatory step in transcriptional control mechanisms. Consistent with their transcriptional activity, immunogold electron microscopy data revealed that SW may have the potential to influence the biology of myoblast cells by modulating MRF subcellular localization. In detail, we observed a significant increase in the nuclear localization of both markers in SW-treated cells, with MyoD showing a more prominent shift compared to MyoG. Remarkably, the increase in nuclear MyoD localization observed in the present study is similar to that reported by Chandorkar et al. (2022), who demonstrated that mechanical stimulation enhances the nuclear accumulation of this transcription factor in muscle cells. In contrast, MyoG nuclear shuttling was less pronounced and occurred at later time points, which again aligns with the sequential roles of these myogenic markers.

Thus, it can be speculated that SW was not capable of increasing the expression of myogenic markers, but rather acted by favoring the nuclear localization of these transcription factors to drive the myogenic program. Again, the sequential pattern of MRFs’ nuclear subcellular localization suggests that SW treatment can accelerate the differentiation process by facilitating the early stages of myogenesis, leading to the rapid formation of multinucleated cells.

At present, there is still limited evidence specifically addressing the mechanobiology of shockwave therapy concerning upstream signaling pathways and nucleocytoplasmic shuttling. Although mechanistic investigations were beyond the scope of the present study, insights can be drawn from related research on mechanical stimuli and related cellular responses, which allow us to hypothesize potential upstream pathways, including YAP/TAZ, MAPK, and integrin-mediated signaling pathways, and which warrant further exploration in future studies (Figure 9).

Within this frame, our ultrastructural data are in line with previous studies showing that mechanical cues can enhance the nuclear import of specific proteins and transcription factors, thereby influencing multiple essential pathways in the cell (Tothova et al., 2006; Montel et al., 2019; Kong et al., 2021; García-García et al., 2022). For instance, research on vascular smooth cells has shown that nuclear protein import was significantly stimulated in stretched cells when compared with control cells through a mitogen-activated protein kinase (MAPK)-mediated mechanism, facilitating cell growth (Richard et al., 2007). Of note, MAPK/extracellular signal-regulated kinase (ERK) signaling pathway is known to be involved in mechanically induced signaling in various cell types, including skeletal muscle, where MAPK/ERK pathway has been reported to mediate the transmission of extracellular signals to the nucleus in response to contraction and passive stretch (Martineau and Gardiner, 1985). At the same time, in vitro studies on smooth muscle cells have demonstrated that the MAPK pathway is involved in the stretch-induced stimulation of nuclear protein import and increased nuclear pore expression (Richard et al., 2007). In line with this, a few studies have suggested that shockwaves produce mechanical stimulation through pressure change, thus initiating a serial cellular function by triggering various intracellular signaling events that involve MAPK signaling (Weihs et al., 2014; Catalano et al., 2017; Shen et al., 2021). For instance, research on porcine articular chondrocytes revealed that the transient production of ROS induced by SW can activate the MAPK/Nfr2 signaling, thus leading to the phosphorylation of ERK1/2 and p38, Nrf2 nuclear translocation, and subsequent activation of the expression of genes encoding various antioxidant enzymes (Shen et al., 2021). By contrast, pretreatment of chondrocytes with the specific inhibitors of MEK1/2 and p38, respectively, mitigated the SW-induced nuclear translocation of Nrf2 and ECM synthesis. Again, Nrf2 inhibition by both small hairpin RNA knockdown and brusatol reduced the SW-enhanced ECM synthesis (Shen et al., 2021).

Moreover, a number of studies have demonstrated that low-intensity SW interacts with integrin and activates FAK by phosphorylation mediated by integrin α5 and β1, thereby triggering a series of cellular signaling and related biological changes, which are known to be relevant for myoblast differentiation by regulating promyogenic factors (Han et al., 2011). Again, the Wnt/β-catenin was shown to be an additional mediator in the integrin-FAK signaling pathway in response to SW in a cell culture model of osteoblasts (Xu et al., 2012).

Finally, another potential network that may link shockwave-mediated mechanosensing to gene transcription is the Hippo signaling pathway, whose central components are Yes-associated protein (YAP) and the homologous transcriptional coactivator with PDZ-binding motif (TAZ), which exhibits a strong structural similarity and genetic redundancy (Low et al., 2014). In particular, YAP/TAZ plays an important role as a mechanosensitive effector that induces cell-specific transcriptional programs as an adaptation to dynamic mechanical cues (Dupont et al., 2011; Aragona et al., 2013; Li H. X. et al., 2021; Pan et al., 2024). In line with this, previous mechanistic in vitro studies on subchondral bone stem/progenitor cells (SCB-SPCs) have demonstrated that YAP undergoes activation and nuclear translocation after radial SW stimulation, promoting self-renewal of SCB-SPCs (Zhao et al., 2021). Furthermore, a recent study by Li Y. et al. (2021) demonstrated that low-intensity ESWT upregulates the expression level and nuclear translocation of YAP, which acts as an integrated transcriptional complex for related gene expression modification, thereby promoting the activation of rat Schwann cells (SCs). The following SCs activation process promotes nerve axon regeneration, which might lead to functional recovery after peripheral nerve injury. Contrariwise, in vitro knockdown of TAZ by small interfering RNA (siRNA) significantly attenuated this process.

Although the present data increase our understanding of SW-induced myoblast differentiation, the picture is still far from clear, and further mechanistic studies are needed to fully understand the mechanisms induced by SW.

The present study brings new insights into the SW-induced regulation of myoblast fusion and early myogenic differentiation, while raising intriguing questions regarding the contribution of mechanical stimulation to the modulation of myogenesis. However, some limitations should be noted. First, we used an immortalized myoblast cell line, namely, the C2C12 cell line. Although this cell line is a well-established and widely used model for studying skeletal muscle cell biology, it does have some limitations, particularly regarding the translatability of findings to human muscle physiology. Therefore, experiments using also primary murine or human myoblast cells should be considered for future investigations to further extend our findings. Another limitation is the therapy schedule. In the present research, cells were stimulated only once with SW, whereas in clinical practice, multiple weekly sessions of shockwave treatment are generally recommended for the patients. Therefore, in the follow-up studies, additional frequencies, number of impulses, and/or applications should be investigated, in order to better translate the in vivo schedule treatment to our in vitro model. Furthermore, research is required to determine the upstream signaling cascade of SW-induced myoblast commitment toward early myogenic differentiation.