TRPV2 in muscle stem cells is essential for rebuilding skeletal muscle

Skeletal muscles maintain homoeostasis by adapting their form and function in response to external stress [1, 2]. This plasticity is supported by mature myofibres and their associated resident muscle stem cells (MuSCs) [3 –6]. In adult skeletal muscle, MuSCs remain mitotically quiescent under resting conditions but become activated in response to muscle injury or exercise-induced mechanical loading. Upon activation, MuSCs provide essential myoblasts for muscle remodelling, including hypertrophy and regeneration [7, 8]. However, the mechanisms by which MuSCs detect and respond to muscle damage or stress remain unclear.

Ca²⁺ is a ubiquitous intracellular signal that regulates diverse cellular processes [9]. In skeletal muscle, Ca²⁺ signalling governs myogenesis, growth, regeneration, and the maintenance of contraction and plasticity [4]. Excitation–contraction coupling in muscle depends on intracellular Ca²⁺ dynamics mediated by interactions between ryanodine receptors and L-type voltage-gated Ca²⁺ channels, also known as dihydropyridine receptors [10]. The internal Ca²⁺ storage sensor stromal interaction molecule 1 (STIM1) and the store-operated calcium channel Orai1 regulate Ca²⁺ flux during muscle differentiation [11, 12]. Inositol trisphosphate (IP₃) receptors mediate Ca²⁺ release during the early stages of muscle differentiation, whereas ryanodine receptors play a greater role as the sarcoplasmic reticulum matures [4]. Elucidation of Ca²⁺ signalling in MuSCs is needed to understand the processes that underlie muscle formation, growth, and regeneration.

Physical and chemical stimuli activate MuSCs and induce their proliferation. IGF-1 facilitates MuSC proliferation and promotes muscle growth via IP₃ receptors and ryanodine receptors [4]. Additionally, physical stress triggers an extracellular Ca²⁺ response that enhances MuSC proliferation, suggesting the involvement of mechanosensor-mediated mechanotransduction pathways [13]. A recent study demonstrated delayed muscle regeneration in mice lacking the mechanosensitive channel Piezo1, indicating that Piezo1 regulates MuSC function [14].

In addition to Piezo1, members of the transient receptor potential (TRP) family of cation channels, which are candidate mechanosensors, are expressed in MuSCs [15]. However, whether TRP canonical family type 1 (TRPC1), which promotes MyoD expression through elevated intracellular Ca²⁺, exhibits mechanosensitivity remains controversial [16]. Previously, we reported that recombinant TRP vanilloid family type 2 (TRPV2) channels can be activated by hypotonicity- or stretch-induced mechanical loading in ectopic expression systems [17]. In skeletal muscle, TRPV2 is strongly localised at the sarcolemma of dystrophin-deficient mdx mice and δ-sarcoglycan-deficient BIO14.6 hamsters [18]. Although animal models expressing dominant-negative TRPV2 demonstrated amelioration of pathological features [19], the roles of TRPV2 in MuSC activation, proliferation, and fusion are unknown.

In this study, we generated MuSC-specific TRPV2 conditional knockout (cKO) mice to investigate the physiological function of TRPV2 in MuSCs. TRPV2-deficient MuSCs have reduced expression of Pax7 and impaired proliferation, suggesting that TRPV2 regulates an early stage of MuSCs function. Myotube formation in MuSCs was dependent on the expression level of TRPV2, suggesting that TRPV2 contributes to the promotion of myogenesis. MuSC-specific TRPV2 cKO mice also showed significantly attenuated muscle regeneration after cardiotoxin-induced injury and a reduced hypertrophic response to mechanical loading. Additionally, TRPV2 in MuSCs was essential for Ca²⁺ responses induced by mechanical loading. These findings indicate that TRPV2 in MuSCs is crucial for skeletal muscle remodelling.

The morphological and functional remodelling of skeletal muscle in response to physical activity, ageing, and injury repair is supported by mature multinucleated myofibres and MuSCs residing around the myofibres [1, 2, 5, 6]. Although extensive research elucidated many molecular mechanisms governing the proliferation and activation of mitotically quiescent MuSCs during muscle remodelling [1, 2, 5, 6], the trigger mechanisms that induce MuSC activation remain unclear. This study revealed that TRPV2 in MuSCs is crucial for muscle remodelling, including muscle regeneration and mechanical loading-induced hypertrophy. In cultured MuSCs proliferating at the periphery of isolated myofibres, TRPV2 was expressed in Pax7-positive cells, and these cells displayed a TRPV2-dependent Ca²⁺ response. TRPV2-deficient MuSCs exhibited a substantial reduction in proliferative capacity, a lower number of Pax7-positive cells, and impaired myogenic fusion. MuSC-specific TRPV2-deficient mice demonstrated significantly impaired regeneration after cardiotoxin-induced muscle injury and reduced hypertrophic responses to mechanical loading. Furthermore, MuSCs showed a TRPV2-dependent Ca²⁺ response to mechanical stress. These findings indicate that TRPV2 in MuSCs is essential for promoting the early stage of MuSC function during muscle remodelling. TRPV2 is expressed in both mature myofibres and mitotic MuSCs (Figs. 1 and 3). In the present study, TRPV2 deletion in adult mouse MuSCs did not affect muscle tissue morphology (Fig. 2i) or mouse behaviour, suggesting that MuSC-expressed TRPV2 has a minimal role in muscle function under physiological conditions. In addition, the number of MuSCs sorted by FACS is at the same level in tamoxifen-treated flox and TRPV2-Pax7-cKO mice (Fig. 2f), indicating that TRPV2 does not contribute to the maintenance of MuSC numbers. These results are consistent with the observation that the number of Pax7-positive MuSCs in adult muscle (10 weeks old) treated with tamoxifen at the juvenile stage (5 weeks old) did not differ between the control and TRPV2-Pax7-cKO groups (Fig. 2g, h). Furthermore, TRPV2 expression was barely detectable in MuSCs in both groups (Supplementary Fig. S1). However, TRPV2 was strongly expressed in a subset of skeletal muscle cells undergoing fusion in postnatal Day 1 mice (Supplementary Fig. S2). Here, our study showed that TRPV2 in MuSCs is crucial for the early stage of myogenic progression. Tranilast-mediated inhibition of TRPV2 activity also suppressed muscle fusion (Supplementary Fig. S3). The elimination of TRPV2 during embryonic and early postnatal myogenesis may substantially impact muscle physiology by disrupting myogenesis and skeletal muscle differentiation. Systemic knockout mice lacking the functional domain of TRPV2 exhibit perinatal lethality, such that ~2.5% of offspring survive on a C57BL/6 background [26]. This outcome may partly reflect defects in myogenic progression. On a BL6129SF2/J background, the survival rate increases to ~15%, and no significant abnormalities in reproduction or external appearance are observed, other than a slight reduction in body weight [26]. These observations suggest the presence of compensatory mechanisms that mitigate the phenotypic effects of TRPV2 deficiency. Additional studies of TRPV2-deficient mice that strictly control targeted cells for depletion and timing are required to elucidate the roles of TRPV2 in skeletal muscle cell development, differentiation, muscle physiology, and pathology.

TRPV2 deletion in MuSCs significantly impaired both cardiotoxin-induced muscle regeneration and mechanical loading-induced hypertrophy (Figs. 6 and 7). The extent of myofibre damage differs between these models, and the niche environments for MuSCs are distinct [6]. Therefore, the mechanisms triggering activation of mitotically quiescent MuSCs likely differ between these models. In regenerating muscle, the loss of components of the MuSC niche, including myofibres, may partially contribute to quiescent MuSC activation [27, 28]. In contrast, the MuSC niche is preserved in overloaded muscle [29]. The activation and proliferation of MuSCs in this context would require either an increase in factors promoting MuSC function through myofibre-dependent mechanotransduction or the direct sensing of mechanical forces by MuSCs themselves. A novel aspect of this study is the finding that MuSCs directly sense mechanical stress, in a TRPV2-dependent manner (Fig. 8). Our experiments showed that skeletal muscle from MuSC-specific TRPV2 cKO mice exhibited a substantially reduced number of pax7-positive cells and proliferation of MuSCs in both cardiotoxin-induced muscle regeneration and mechanical loading-induced hypertrophy models (Figs. 6 and 7). A similar decrease in proliferation was observed in isolated myofibre cultures lacking direct contact with the basement membrane, where pax7-positive cells were greatly reduced (Figs. 3 and 4). These findings suggest that TRPV2 plays a central role in the early stage of MuSC function for myogenesis and proliferation, independent of myofibre integrity or niche status. In future studies, the role of TRPV2 in MuSC proliferation and fusion during muscle remodelling could be examined in greater detail by isolating and culturing MuSCs from damaged and hypertrophic muscle fibres via FACS. In this study, DAPI-positive cells lacking Pax7 expression were observed on Day 3 in the control EDL culture (Fig. 3a). To characterise these cells, double staining with anti-Pax7 and anti-MyoD antibodies revealed the presence of Pax7⁻/MyoD⁺ cells on Day 3, although they were absent on Day 2 (Supplementary Fig. S4). Thus, the number of MuSCs on fibres is considerably reduced after TRPV2 deletion. A small number of Pax7⁻/MyoD⁻ cells were also detected; we aim to elucidate their identity in future studies.

Hepatocyte growth factor, basic fibroblast growth factor, and insulin-like growth factor-1 (IGF-1) are potential regulators of MuSC proliferation during muscle regeneration [30 –33]. IGF-1 promotes the translocation of TRPV2 to the plasma membrane [18, 34] and enhances its activity [34]. Because the MuSC-specific TRPV2-deficient mice used in this study retain intact myofibres, the levels of IGF-1 derived from damaged myofibres and the surrounding niche environment should be comparable to those in controls. TRPV2 elimination reportedly downregulates the IGF-1 receptor/phosphoinositide 3-kinase pathway in cardiomyocytes [21]. In this context, the impaired muscle regeneration we observed in TRPV2-deficient MuSCs may result from downregulation of IGF-1 receptor signalling in MuSCs. It is also possible that MuSCs regulate IGF-1 production in an autocrine manner, depending on muscle conditions. For example, in cardiomyocytes and ventricular fibroblasts, IGF-1 is secreted extracellularly in response to mechanical stimuli [35, 36]. We previously showed that the IGF-1 secretion of cardiomyocytes depends on TRPV2 [21]. Even during muscle regeneration not directly triggered by mechanical stress, the physical environment surrounding MuSCs is likely to be dynamically altered by myofibre degradation [37]. Although the specific source of IGF-1 during muscle regeneration remains unclear, TRPV2 elimination in MuSCs may lead to the impairment of the regeneration process mediated by IGF-1.

Serum response factor-dependent interleukin-6 paracrine signalling within myofibres activates MuSCs, leading to muscle hypertrophy [38]. Silent mating type information regulation 2 homologue 1 (Sirt1)-dependent humoral factors in myofibres may function as MuSC growth factors [39]. However, the mechanosensor molecules that activate serum response factor or Sirt1 in myofibres remain unknown. In the MuSC-specific TRPV2 cKO model used in this study, the production and secretion of interleukin-6 or Sirt1-dependent factors are expected to be similar to levels in control mice because myofibres remain intact. Thus, the absence of a mechanical load-dependent hypertrophic response in TRPV2-deficient MuSCs may result from a failure of MuSCs to respond to myofibre stimulation. Alternatively, mechanotransduction in MuSCs may play a pivotal role in the mechanical load-dependent hypertrophic response via TRPV2. These findings suggest that TRPV2 has a crucial function in regulating the myogenic function of MuSC in response to mechanical loading.

In this study, we showed that TRPV2 is involved in the Ca²⁺ response of MuSCs to mechanical stimulation. However, we cannot conclude that TRPV2 directly induces Pax7 expression because no data currently support its role as a primary transducer of mechanotransduction in vivo. Nonetheless, TRPV2 was expressed in Pax7-positive MuSCs under continuous mechanical loading (Fig. 7f–h). When mechanical loading induces TRPV2 expression in MuSCs, the PI3K/Akt pathway may be activated downstream of TRPV2-mediated Ca²⁺ signalling (Figs. 4k and 5e), promoting cell proliferation and contributing to muscle hypertrophy through enhanced MuSC fusion. Thus, TRPV2 may act as an amplifier of the muscle hypertrophy pathway triggered by mechanical loading.

Matrix elasticity reportedly directs stem cell lineage specification, as described by Engler et al. [40]. There is evidence that both biochemical and physical properties of the microenvironment play key roles in cell fate determination [41 –43]. However, the molecular identity of mechanosensors in MuSCs remains unclear. Piezo1 channels exert stage-dependent functions in myogenesis and promote MuSC function during muscle regeneration [14]. Additionally, experiments with C2C12 cells showed that PIEZO1-mediated Ca²⁺ influx activates RhoA/ROCK-mediated actomyosin assembly at the lateral cortex of myotubes [44]. Among the TRPV family members, TRPV2 and TRPV4 reportedly respond to hypotonic cell swelling, shear stress, and membrane stretching [15]. TRPV2 is expressed in human cancer stem cells [45 –47] and progenitor cells [48]; its overexpression is reported to suppress stemness [49]. Conversely, TRPV2 elimination substantially upregulates cancer stem cell markers while enhancing spheroid and hepatoma colony formation in human hepatoma HEpG2 cells [47]. These experimental results are consistent with our findings that TRPV2 promotes MuSC function and is essential for muscle remodelling in vivo. Considering that TRPV2 functions as a polymodal mechanosensor responding to both chemical and physical stimuli, it may interact with the dynamic microenvironment at various stages of MuSC function to integrate multiple biological signals. Further studies are warranted to identify specific stages at which TRPV2 interacts with other regulatory molecules.

Multiple mechanosensitive ion channels may play critical roles in the activation, proliferation, and differentiation of MuSCs [4, 14]. It is possible that these channels provide functional compensation in the absence of TRPV2. However, in our study, MuSCs lacking TRPV2 failed to exhibit a Ca²⁺ response to mechanical stimuli (Fig. 8d, e). This observation suggests that other channels are unlikely to compensate for the loss of TRPV2 in mediating mechanosensitive Ca²⁺ responses in TRPV2-Pax7-cKO MuSCs. Recently, the TRPM7 channel, a candidate mechanosensitive ion channel, has been reported to mediate Mg²⁺ influx that promotes MuSC activation [50]. Efforts to clarify the distinct roles of various mechanosensitive ion channels in MuSC activation, proliferation, and differentiation, as well as their potential functional interactions, remain important for future studies.

This study suggests that the number of Pax7-positive cells in muscle remodelling may be regulated by TRPV2. An increased number of Pax7-positive cells has also been observed in the muscle of patients with Duchenne muscular dystrophy and dystrophin-deficient mdx mice [51, 52]. However, functional defects in dystrophic satellite cells impair asymmetric cell division and myogenic commitment, leading to reduced muscle regeneration in Duchenne muscular dystrophy patients [53]. Because TRPV2 expression may be upregulated in MuSCs of mdx mice as a compensatory mechanism for functional deficits, further studies are necessary to elucidate the mechanisms that underlie TRPV2 upregulation in dystrophic muscle.

This study demonstrated that TRPV2 is essential for MuSC function in muscle remodelling and offered a therapeutic strategy targeting TRPV2 in MuSCs. Although the physiological role of TRPV2 in myogenesis and mature myofibres remains unclear, further functional analyses will clarify its potential as a therapeutic target in muscle pathology.

Supplemental data