Tensin2 deficiency transiently accelerates early-phase regenerative myofiber growth following cardiotoxin-induced injury in mice

Abstract

Tensin2 (TNS2) is a focal adhesion-associated protein that can negatively regulate insulin/insulin-like growth factor 1 (IGF-1) signaling, but its role in skeletal muscle regeneration remains unclear. To address this, we analyzed a TNS2-related immunoreactive signal detected with an anti-phospho-TNS2 (Tyr483) antibody during C2C12 myogenic differentiation and in a cardiotoxin (CTX)-induced tibialis anterior (TA) injury model, and assessed regeneration in Tns2^nph mutant mice. Under basal conditions, Tns2^nph mice showed no obvious abnormalities in muscle histology, myofiber size, or phosphorylation of Akt and p70 ribosomal protein S6 kinase (p70S6K), a canonical downstream effector of mTORC1 signaling, compared with wild-type (WT) mice, although the soleus muscle/body weight ratio was slightly increased. In C2C12 cells, the anti-phospho-TNS2 (Tyr483)-reactive signal was stronger during proliferation than during differentiation. In vivo, this signal increased transiently during the early phase of CTX-mediated regeneration. After injury, Tns2^nph mice showed a greater number of centrally nucleated fibers at day 4, larger myofibers at day 7, and a higher TA muscle/body weight ratio at day 15. In addition, phosphorylation of p70S6K was increased in regenerating muscle in Tns2^nph mice. These findings suggest that TNS2-related signaling may act as a context-dependent modulator of regenerative myofiber growth, possibly through augmented anabolic signaling during early regeneration.

Introduction

Tensin2 (TNS2), also known as C1-Ten, is a focal adhesion-associated member of the tensin family that participates in integrin-linked signaling and cytoskeletal organization [1, 2]. TNS2 is known to be involved in cell adhesion and migration. Previous studies on TNS2 have primarily focused on the kidney. However, its role in skeletal muscle regeneration remains unknown. The Tns2^nph allele, originally identified in the ICGN mouse, carries an 8-bp deletion in exon 18 of Tns2, resulting in a frameshift and premature truncation of the C-terminal region, including the SH2-PTB domain [3]. In the original report, Tns2/TNS2 was not detected by Northern blotting or Western blotting in Tns2^nph mice, although a small amount of transcript was detectable by RT-PCR [3]. Therefore, Tns2^nph mice have been widely regarded as a Tns2-null or TNS2 functional-deficiency model. Subsequent studies demonstrated that the severity of Tns2-deficient nephropathy is strongly influenced by genetic background and by the structural integrity of the SH2-PTB region [2,3,4,5,6,7,8,9,10]. Together, these studies have established TNS2 as a key regulator of podocyte homeostasis, although its function outside the kidney remains poorly understood.

Biochemical studies suggest that TNS2 can negatively regulate insulin/insulin-like growth factor 1 (IGF-1) signaling. TNS2 dephosphorylates insulin receptor substrate 1 (IRS-1), promotes its degradation, and suppresses downstream Akt signaling under catabolic conditions associated with muscle atrophy [11]. In addition, TNS2 undergoes increased p62/SQSTM1-mediated sequestration and degradation during myogenic differentiation [12], and phosphatidylinositol 3,4,5-trisphosphate binding to the SH2 domain modulates the cellular phosphatase activity of TNS2 [13]. Together, these findings suggest that TNS2 may act as a brake on anabolic signaling in skeletal muscle cells.

Skeletal muscle regeneration after acute injury requires tightly coordinated phases of necrosis, inflammation, satellite cell activation, myoblast proliferation, differentiation, fusion, and regenerative growth [14]. The cardiotoxin (CTX)-induced tibialis anterior (TA) injury model is widely used to study these processes in mice [15]. p70 ribosomal protein S6 kinase (p70S6K) is a canonical downstream effector of mechanistic target of rapamycin complex 1 (mTORC1) signaling and promotes protein synthesis, myofiber growth, and regenerative remodeling. Therefore, p70S6K phosphorylation provides a useful readout of anabolic signaling during muscle regeneration. Given its involvement in both focal adhesion signaling and the IRS-1/Akt axis, TNS2 may influence muscle regeneration after injury—a possibility that has not been directly tested.

In the present study, we analyzed a TNS2-related immunoreactive signal detected with an anti-phospho-TNS2 (Tyr483) antibody during C2C12 differentiation and during CTX-induced skeletal muscle regeneration in vivo. We also evaluated the regenerative phenotype of Tns2^nph mice. Our findings suggest that TNS2 deficiency transiently accelerates early-phase regenerative myofiber growth together with increased p70S6K phosphorylation.

Materials and Methods

Animals

All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Kitasato University (approval no. 20-008). Male mice (Mus musculus) carrying the Tns2^nph mutation congenic on a C57BL/6J background were maintained in-house as described previously [4,5,6,7,8]. The Tns2^nph allele carries the ICGN-derived 8-bp deletion in exon 18 of Tns2. Age-matched 8-week-old male C57BL/6JJcl mice were purchased from CLEA Japan Inc. (Tokyo, Japan) and used as wild-type (WT) controls. Mice were housed in plastic cages in a temperature-controlled room (22 ± 4°C) under a 12-h light/12-h dark cycle (lights on from 08:00 to 20:00). Water and a standard diet (CE-2; CLEA Japan) were provided ad libitum.

Cell culture

C2C12 myoblasts were seeded in collagen-coated 12-well plates (5.0 × 10³ cells/well) and maintained in growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic, and 0.5% gentamicin. After reaching confluence, the medium was replaced with differentiation medium consisting of DMEM supplemented with 2% horse serum, 1% antibiotic-antimycotic, and 0.5% gentamicin. Cells were cultured at 37°C in a humidified atmosphere of 5% carbon dioxide (CO₂).

Muscle injury

Mice were anesthetized by intraperitoneal injection of a mixture of medetomidine (Dorbene; Kyoritsu Pharmaceutical Co., Ltd., Tokyo, Japan), midazolam (Fuji Pharma Co., Ltd., Tokyo, Japan), and butorphanol tartrate (Vetorphale; Meiji Seika Pharma Co., Ltd., Tokyo, Japan). The tibialis anterior (TA) muscle was then injected with 50 µl of 10 µM cardiotoxin (CTX; Latoxan SAS, Valence, France). CTX-injected TA muscles were collected before injection (day 0) and at 2, 4, 7, and 15 days after injection.

Histology and immunofluorescence

Frozen transverse sections (10 µm) of TA muscle were used for both hematoxylin and eosin (HE) staining and immunofluorescence analysis. For HE staining, sections were processed by standard procedures. For immunofluorescence, adjacent sections were fixed in ice-cold acetone for 5 min, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 15 min at room temperature, and blocked with 3% bovine serum albumin, 0.1% Triton X-100, and 0.1% Tween 20 in PBS for 1 h at 4°C. Sections were incubated overnight at 4°C with mouse anti-pan myosin heavy chain (MyHC; clone MF20, 1:50, R&D Systems, Minneapolis, MN, USA) and rabbit anti-dystrophin (1:200, Abcam, Cambridge, UK), followed by rhodamine-conjugated anti-mouse immunoglobulin G (IgG; 1:500, KPL) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, Invitrogen, Carlsbad, CA, USA). Slides were mounted in ProLong Diamond Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI) and observed using a BZ-X810 fluorescence microscope (KEYENCE, Osaka, Japan). For histological quantification, transverse sections were analyzed at the mid-belly level of the TA muscle. For each mouse, three non-overlapping fields within the injured/regenerating region were captured under identical microscope settings. Fields containing tissue folds, tears, sectioning artifacts, large vessels, tendon, tissue edges, or non-muscle tissue were excluded. The same field-selection criteria were applied to all genotypes and time points. Centrally nucleated fibers were counted on HE-stained sections. MyHC staining was used to identify regenerating myofibers and to quantify the MyHC-positive area, which was expressed as the percentage of the total analyzed field area. Dystrophin staining was used to delineate the sarcolemmal borders of individual myofibers, and minimal Feret diameter was measured based on dystrophin-defined fiber outlines. Image analyses were performed using ImageJ.

Western blotting

Crushed TA muscles (approximately 50 mg) or C2C12 cells were homogenized in sodium dodecyl sulfate (SDS) buffer containing 10% SDS, 40 mM dithiothreitol, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.1 M Tris-HCl (pH 8.0) supplemented with protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Samples were heated for 3 min in boiling water. Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and samples were adjusted to 8 µg/µl. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% skim milk or 3% bovine serum albumin (for phosphoproteins) in Tris-buffered saline containing Tween 20 and incubated overnight at 4°C with primary antibodies diluted in Can Get Signal Solution 1 (Toyobo, Osaka, Japan). The following antibodies were used: mouse anti-actin (1:10,000, Millipore), rabbit anti-Akt (1:1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-Akt (Ser473; 1:2,000, Cell Signaling Technology), rabbit anti-p70S6K (1:1,000, Cell Signaling Technology), rabbit anti-phospho-p70S6K (Thr389; 1:1,000, Cell Signaling Technology), and rabbit anti-phospho-TNS2 (Tyr483) (1:1,000, OASG07109, Aviva Systems Biology, San Diego, CA, USA). Because a validated pan-TNS2 antibody suitable for mouse skeletal muscle was not available commercially, the anti-phospho-TNS2 (Tyr483) antibody was used to detect a TNS2-related immunoreactive band at the expected molecular weight. In our previous study, FLAG-tagged full-length TNS2 expressed in 293FT cells was detected by both anti-FLAG and anti-phospho-TNS2 (Tyr483) antibodies at a similar molecular size, supporting the ability of this antibody to recognize recombinant TNS2. For the analysis of p70S6K and Akt phosphorylation, phospho-p70S6K and phospho-Akt were detected first. After chemiluminescent detection, the same membranes were stripped with SDS-βME solution and reprobed with antibodies against total p70S6K and total Akt, respectively. Phospho/total ratios were calculated lane by lane using signals obtained from the same biological samples. After incubation with horseradish peroxidase-conjugated secondary antibodies, signals were detected using enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA). Band intensities were quantified with ImageJ. Actin was used as a loading control.

Statistical analysis

Data are presented as mean ± SEM. Sample sizes (n) are indicated in the relevant figure legends and in the text describing Tables 1 and 2. Comparisons between WT and Tns2^nph mice were performed using an unpaired two-tailed Student’s t-test. For time-course experiments, the two genotypes were compared separately at each time point. P<0.05 was considered statistically significant.

Table 1. Basal body and skeletal muscle weights in wild type (WT) and Tns2^nph mice.

	WT	Tns2nph
BW (g)	20.9 ± 0.24	20.8 ± 0.40
Soleus muscle (mg)	14.2 ± 0.38	15.2 ± 0.37
Soleus muscle/BW (mg/g)	0.68 ± 0.01	0.73 ± 0.02*
EDL muscle (mg)	17.0 ± 0.50	16.0 ± 0.60
EDL muscle/BW (mg/g)	0.81 ± 0.02	0.77 ± 0.02
Gastrocnemius muscle (mg)	232.0 ± 11.1	239.2 ± 2.74
Gastrocnemius muscle/BW (mg/g)	11.1 ± 0.48	11.5 ± 0.17

BW, body weight; EDL, extensor digitorum longus. Values are means ± SEM for 5 mice per group. *P<0.05 vs WT.

Table 2. Body weight, tibialis anterior muscle weight, and tibialis anterior muscle-to-body weight ratio after cardiotoxin-induced injury.

		WT	Tns2nph
Day4	BW (g)	22.4 ± 0.39	20.4 ± 0.58*
	TA muscle (mg)	58.4 ± 2.1	49.1 ± 3.3
	TA muscle/BW (mg/g)	0.26 ± 0.013	0.24 ± 0.017
Day7	BW (g)	22.1 ± 0.29	21.5 ± 0.82
	TA muscle (mg)	59.0 ± 3.1	67.5 ± 5.7
	TA muscle/BW (mg/g)	0.27 ± 0.015	0.33 ± 0.020
Day15	BW (g)	22.1 ± 0.41	21.8 ± 0.10
	TA muscle (mg)	45.4 ± 3.4	54.0 ± 1.9
	TA muscle/BW (mg/g)	0.21 ± 0.012	0.25 ± 0.008*

BW, body weight; TA, tibialis anterior. Values are means ± SEM for 4–5 mice per group at each time point. *P<0.05 vs wild type (WT) at the same time point.

Results

Basal muscle phenotype of Tns2^nph mice

We first examined body and muscle weights in adult mice to assess whether TNS2 dysfunction affects skeletal muscle under basal conditions. Absolute weights of the soleus, extensor digitorum longus, and gastrocnemius muscles did not differ significantly between WT and Tns2^nph mice (Table 1; n=5 mice per group). The soleus muscle/body weight ratio was slightly but significantly higher in Tns2^nph mice, whereas the other relative muscle weights were unchanged (Table 1).

Hematoxylin and eosin (HE) staining of TA muscle sections revealed no obvious histological abnormalities in Tns2^nph mice (Fig. 1A). In line with this finding, the minimal Feret diameter of TA myofibers was comparable between genotypes (Fig. 1B). We next examined signaling molecules related to anabolic growth. The phosphorylation levels of Akt and p70 ribosomal protein S6 kinase (p70S6K) in uninjured TA muscle were similar between genotypes (Figs. 1C and D). Thus, Tns2^nph mice exhibit no overt skeletal muscle phenotype under basal conditions.

Fig. 1.

Basal skeletal muscle phenotype of wild type (WT) and Tns2^nph mice. (A) Representative hematoxylin and eosin (HE)-stained transverse sections of tibialis anterior (TA) muscle from WT and Tns2^nph mice. Scale bar=100 µm. (B) Minimal Feret diameter of TA myofibers (n=5 mice per group). (C) Representative immunoblots of phospho-Akt (Ser473), total Akt, phospho-p70S6K (Thr389), and total p70S6K in TA muscle. Phosphorylated proteins and the corresponding total proteins were detected on the same membranes after stripping and reprobing. (D) Quantification of phospho-Akt/Akt and phospho-p70S6K/p70S6K ratios (n=5 mice per group). Values are means ± SEM.

Anti-phospho-TNS2 (Tyr483)-reactive signal during myogenic differentiation and muscle regeneration

Because previous studies suggested that TNS2 is regulated during myogenic differentiation [11,12,13], we examined a TNS2-related immunoreactive signal detected with an anti-phospho-TNS2 (Tyr483) antibody in C2C12 cells. Consistent with the morphological changes seen in phase-contrast images (Fig. 2A), the anti-phospho-TNS2 (Tyr483)-reactive signal was stronger in proliferating cells cultured in growth medium than in differentiated cells cultured in differentiation medium (Fig. 2B).

Fig. 2.

Anti-phospho-TNS2 (Tyr483)-reactive signal during C2C12 differentiation and cardiotoxin (CTX)-induced muscle regeneration. (A) Representative phase-contrast images of C2C12 cells at 24 and 72 h in growth medium (GM) and at 144 and 216 h after induction of differentiation in differentiation medium (DM). Scale bar=50 µm. (B) Representative immunoblot of the anti-phospho-TNS2 (Tyr483)-reactive signal and actin in C2C12 cells cultured in GM or DM. Representative of three independent experiments. (C) Representative hematoxylin and eosin (HE)-stained sections of tibialis anterior (TA) muscle in wild type mice at days 0, 2, 4, 7, and 15 after CTX injection. Scale bar=100 µm. (D) Representative immunoblot of the anti-phospho-TNS2 (Tyr483)-reactive signal and actin in TA muscle at days 0, 2, 4, 7, and 15 after CTX injection. Representative of three independent experiments.

We next examined the same anti-phospho-TNS2 (Tyr483)-reactive signal during muscle regeneration in vivo using the CTX injury model [14, 15]. Histological changes after injury were confirmed by HE staining (Fig. 2C). The anti-phospho-TNS2 (Tyr483)-reactive signal was low in uninjured TA muscle, increased at days 2 and 4 after CTX injection, and decreased thereafter (Fig. 2D). These findings indicate that the anti-phospho-TNS2 (Tyr483)-reactive signal is dynamically associated with the early phase of muscle regeneration in vivo.

Regenerative phenotype of Tns2^nph mice after muscle injury

Because the anti-phospho-TNS2 (Tyr483)-reactive signal increased after CTX injury, we next evaluated the regenerative response of Tns2^nph mice. The TA muscle/body weight ratio was significantly higher in Tns2^nph mice at day 15 after injury, whereas no significant difference was observed at days 4 or 7 (Table 2; n=4–5 mice per group at each time point). Absolute TA muscle weight also tended to be higher in Tns2^nph mice at days 7 and 15 (Table 2).

To characterize regeneration histologically, we examined HE-stained and immunofluorescent sections of TA muscle at days 4, 7, and 15 after CTX injury (Fig. 3A). Centrally nucleated fibers are a hallmark of newly formed or regenerating myofibers after muscle injury and therefore provide an index of early regenerative myofiber formation. The number of centrally nucleated fibers was significantly higher in Tns2^nph mice at day 4 after CTX injection (Fig. 3B). The MyHC-positive area reflects the extent to which regenerating myofibers occupy the injured region; this area did not differ significantly between genotypes (Fig. 3C). Minimal Feret diameter and fiber size distribution were used as indicators of regenerative myofiber growth and maturation. Minimal Feret diameter analysis showed that regenerating myofibers were significantly larger in Tns2^nph mice at day 7, although this difference was no longer evident by day 15 (Fig. 3D). Fiber size distribution analysis also showed a rightward shift toward larger fibers in Tns2^nph mice during regeneration (Figs. 3E and F). Together, these data suggest that the Tns2^nph mutation transiently accelerates early-phase regenerative myofiber growth rather than causing a major change in steady-state muscle structure.

Fig. 3.

Regenerative phenotype of Tns2^nph mice after cardiotoxin (CTX) injury. (A) Representative hematoxylin and eosin (HE) and immunofluorescence images of regenerating tibialis anterior (TA) muscle at days 4, 7, and 15 after CTX injection. Higher-magnification insets are shown for the day-4 and day-7 images to facilitate identification of centrally nucleated fibers. Arrowheads indicate regenerating myofibers with centrally located nuclei. Scale bar=50 µm. (B) Number of centrally nucleated fibers (n=5 mice per group at each time point). (C) Quantification of the area occupied by myosin heavy chain (MyHC)-positive fibers based on the immunofluorescence images shown in (A) (n=5 mice per group at each time point). (D) Minimal Feret diameter of regenerating myofibers measured using dystrophin-defined fiber outlines (n=5 mice per group at each time point). (E, F) Fiber size distribution of regenerating myofibers at days 7 and 15 after CTX injection (n=5 mice per group at each time point). Values are means ± SEM. *P<0.05.

Anabolic signaling during regeneration

To explore the mechanism underlying this phenotype, we analyzed Akt and p70S6K phosphorylation during regeneration. Phosphorylation of p70S6K was significantly higher in Tns2^nph muscle at day 4 after CTX injury, whereas no clear genotype-dependent change in Akt phosphorylation was detected (Figs. 4A and B). These findings suggest that TNS2 dysfunction is associated with enhanced activation of a p70S6K-linked anabolic pathway during the early phase of muscle regeneration.

Fig. 4.

Akt and p70S6K signaling during muscle regeneration. (A) Representative immunoblots of phospho-Akt (Ser473), total Akt, phospho-p70S6K (Thr389), and total p70S6K in regenerating TA muscle at the indicated days after CTX injection. Phosphorylated proteins and the corresponding total proteins were detected on the same membranes after stripping and reprobing. (B) Quantification of phospho-Akt/Akt and phospho-p70S6K/p70S6K ratios (n=5 mice per group at each time point). Values are means ± SEM. *P<0.05.

Discussion

The present study demonstrates that TNS2 deficiency transiently accelerates early-phase regenerative growth after acute muscle injury. In uninjured muscle, Tns2^nph mice were largely similar to WT controls with respect to histology, myofiber size, and phosphorylation of Akt and p70S6K. Although the soleus muscle/body weight ratio was slightly higher in Tns2^nph mice, overall basal muscle morphology was preserved. The slight increase in the soleus muscle/body weight ratio under basal conditions should be interpreted cautiously. The soleus is a predominantly slow-twitch, postural muscle and may be more sensitive than fast-twitch muscles to subtle changes in loading, metabolic state, or insulin/IGF-1-related signaling. Because TNS2 is linked to focal adhesion and insulin/IGF-1 signaling, TNS2 dysfunction could theoretically exert muscle-type-specific effects. However, the absolute soleus weight was not significantly different, and no obvious abnormalities were observed in TA muscle histology, myofiber size, or Akt/p70S6K phosphorylation under basal conditions. Therefore, the increased soleus muscle/body weight ratio is unlikely to indicate a robust basal hypertrophic phenotype. Rather, it may reflect a subtle muscle-type-specific adaptation or the effect of normalization to body weight. Further analyses of fiber-type composition and muscle function would be required to clarify its physiological significance. After CTX injury, Tns2^nph mice showed more centrally nucleated fibers at day 4, larger regenerating myofibers at day 7, and a higher TA muscle/body weight ratio at day 15. These changes were accompanied by increased phosphorylation of p70S6K, supporting the interpretation that TNS2 deficiency may facilitate regenerative growth signaling.

The present study extends the biology of TNS2 beyond the kidney. Previous studies established TNS2 as the causative gene for nephropathy in the ICGN mouse and showed that the renal phenotype is highly dependent on genetic background, modifier loci, and the integrity of the SH2-PTB region [3,4,5,6,7,8,9,10, 16]. Although previous studies of TNS2 have focused mainly on podocytes, the signaling functions of TNS2 are not necessarily kidney-specific. TNS2 is a focal adhesion-associated protein that links cytoskeletal organization to intracellular signaling pathways. In podocytes, TNS2 contributes to focal adhesion-related homeostasis and has been linked to mTORC1-associated signaling. In skeletal muscle cells, TNS2 has been reported to regulate IRS-1/Akt signaling under catabolic conditions. Because p70S6K is a canonical downstream effector of mTORC1 that promotes protein synthesis and myofiber growth, the enhanced p70S6K phosphorylation observed in regenerating Tns2^nph muscle may reflect altered regulation of a growth-related signaling axis shared across different cell types.

Notably, the severity of glomerulosclerosis and podocyte injury caused by TNS2 deficiency is strongly influenced by genetic background; 129, FVB, and ICGN strains are highly susceptible [3,4,5, 7, 10], whereas C57BL/6 mice are relatively resistant to these renal pathologies [9, 16]. Genetic determinants on chromosome 2 have been implicated in this resistance in the C57BL/6 background [16]. Thus, although subtle systemic effects cannot be completely excluded, the present C57BL/6-based model argues against a major contribution of overt secondary renal disease to the regenerative phenotype observed here. In contrast, little was known about the role of TNS2 in skeletal muscle. Our findings suggest that TNS2-related signaling correlates with proliferative and reparative states in muscle.

The pattern observed in this study should be interpreted in light of the antibody used. Koh et al. showed that TNS2 dephosphorylates IRS-1 and suppresses Akt signaling under catabolic conditions [11], whereas p62/SQSTM1-mediated degradation of TNS2 increases during myogenic differentiation [12]. Kim et al. further demonstrated that phosphatidylinositol 3,4,5-trisphosphate binding to the SH2 domain regulates cellular phosphatase activity of TNS2 [13]. In the present study, the immunoreactive band detected with an anti-phospho-TNS2 (Tyr483) antibody was stronger in proliferating C2C12 cells, decreased during differentiation, and increased transiently during the early phase of CTX-induced regeneration. This transient increase should not be interpreted simply as evidence that TNS2 promotes myofiber growth. Rather, it may represent a context-dependent regulatory response induced during periods of active proliferation and repair. One possible interpretation is that TNS2-related signaling acts as a negative feedback mechanism that limits excessive anabolic signaling or helps coordinate the transition from proliferation to differentiation. In this model, loss or impairment of TNS2-related regulatory mechanisms would weaken this brake, resulting in enhanced p70S6K phosphorylation and a transient increase in regenerative myofiber growth. However, the effect size was modest, indicating that TNS2 is more likely to fine-tune than to dominate skeletal muscle growth responses.

The Tns2^nph allele is a truncating mutation caused by an 8-bp deletion in exon 18 [3]. Although this allele is not an engineered complete deletion allele, previous molecular analyses showed no detectable Tns2/TNS2 signal by Northern blotting or Western blotting, with only a small amount of transcript detected by RT-PCR [3]. Thus, the present findings are interpreted in the context of TNS2 functional deficiency rather than a partial missense or hypomorphic mutation. This interpretation is also consistent with previous work showing that deletion of the SH2-PTB domain, but not catalytic inactivation of the protein tyrosine phosphatase domain alone, is sufficient to induce podocyte injury [10]. Therefore, loss of full-length TNS2 and disruption of SH2-PTB-dependent signaling are likely to be central to the phenotype observed in this model.

Several limitations of this study should be noted. We did not directly assess satellite cell behavior, inflammatory cell infiltration, fiber-type composition, transcriptional responses, or muscle function during regeneration. In addition, a validated pan-TNS2 antibody suitable for detecting endogenous TNS2 in mouse skeletal muscle was not available. In our previous study, FLAG-tagged full-length TNS2 expressed in 293FT cells was detected by both anti-FLAG and anti-phospho-TNS2 (Tyr483) antibodies at a similar molecular size, supporting the ability of this antibody to recognize recombinant TNS2. Nevertheless, in the present skeletal muscle samples, the identity of the endogenous band was inferred from its apparent molecular weight and antibody reactivity. The anti-phospho-TNS2 (Tyr483)-reactive band could not be directly validated in Tns2^nph regenerating muscle, because archived day-4 muscle samples or corresponding protein lysates were not available. The Tns2^nph mutation is characterized by an 8-bp deletion in exon 18, leading to a frameshift at Ser516 and a premature stop codon. While the Tyr483 phosphorylation site remains upstream of this truncation point, the resulting truncated protein lacks the essential C-terminal SH2 and PTB domains. Such a severely truncated protein is likely to be biochemically unstable and subject to rapid degradation, or the transcript may undergo nonsense-mediated mRNA decay. This provides a logical basis for the disappearance of the TNS2-related immunoreactive signal in Tns2^nph muscle, supporting the specificity of the observed signal as endogenous TNS2. The temporal pattern of the phenotype also suggests that Tns2^nph-associated TNS2 dysfunction primarily accelerates the early phase of regeneration rather than producing sustained myofiber hypertrophy. Increased p70S6K phosphorylation and a greater number of centrally nucleated fibers were observed at day 4, followed by larger regenerating myofibers at day 7. The increase in the TA muscle/body weight ratio at day 15 may reflect the cumulative tissue-level consequence of this earlier regenerative acceleration. However, because the difference in myofiber diameter was no longer evident by day 15 and muscle function was not assessed, we cannot conclude that the phenotype leads to a lasting functional benefit. Further long-term and functional analyses will be required to determine whether this early acceleration has physiological significance.

In conclusion, TNS2 deficiency transiently accelerates early-phase regenerative myofiber growth and is associated with increased p70S6K phosphorylation after acute muscle injury. The transient increase in the anti-phospho-TNS2 (Tyr483)-reactive signal during early regeneration supports the view that TNS2-related signaling may act as a context-dependent modulator of skeletal muscle repair. These findings support a model in which TNS2 functional deficiency accelerates early regenerative growth, although the long-term functional significance of this effect remains to be determined.

Conflict of Interest

The authors declare no conflict of interest.