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. 2025 Nov 11;8:100173. doi: 10.1016/j.crphys.2025.100173

Differential responses to acute endurance exercise-induced signaling protein phosphorylation and abundance alterations in whole murine gastrocnemius muscle and isolated myofibers

Kazuki Uemichi a,b,c,⁎, Takanaga Shirai c,d,⁎⁎, Shunsuke Sugiyama b, Tohru Takemasa e
PMCID: PMC12663849  PMID: 41321660

Abstract

Skeletal muscle is composed of diverse cell types, including myofibers. In this study, we investigated how acute endurance exercise could induce signaling protein phosphorylation and abundance alterations using protein lysate extracted from whole-muscle tissue or isolated myofibers. We subjected 8-week-old male Institute Cancer Research mice to a single bout of treadmill running-based endurance exercise, and evaluated the signaling protein expression responses in the whole gastrocnemius muscle and isolated myofibers, obtained by incubation in 0.2 % collagenase/medium, immediately after exercise. Our results revealed that exercise activated mitochondrial biogenesis- and protein anabolism-related signaling, including 5′-adenosine monophosphate-activated kinase and Akt/p70 S6 kinases, respectively, as well as increased glucose metabolism-related proteins in whole-muscle samples immediately after exercise. However, these signaling responses were not present in the muscle fiber lysate and mitochondrial oxidative phosphorylation-related protein abundance significantly decreased postexercise. Our data suggest that signaling protein activation and quantitative alterations in myofibers digested in collagenase-dissolved medium post acute exercise do not necessarily reflect the expression responses in whole skeletal muscle tissue.

Keywords: Skeletal muscle, Myofiber, Endurance exercise, Signaling protein

Highlights

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    Exercise stimulates various signaling pathways involved in muscle metabolism.

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    Exercise-induced myofiber-specific signaling responses remain unclear.

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    Simultaneous assessment of signaling alterations using whole-muscle and myofiber lysates.

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    Differential signaling protein status between whole-muscle and isolated myofibers.

1. Introduction

Skeletal muscle is the largest organ in the human body, accounting for approximately 40 % of total body weight (Frontera and Ochala, 2015), possessing a high degree of plasticity, diversity, and heterogeneity (Cai et al., 2023; Martínez Mir et al., 2024; Brook et al., 2016). Recently, studies in skeletal muscle biology have attempted to understand the complex heterogeneity of skeletal muscle with the rise of single-cell ribonucleic acid (RNA) sequencing, which evaluates the transcriptomes of various cell types and investigates cell–cell communications in the skeletal muscle (e.g., endothelial cells, myogenic progenitors, fibroblasts, immune cells, adipocytes) at the single-cell level (Cai et al., 2023; Petrany et al., 2020; Zhang et al., 2024). Moreover, even in the same skeletal muscle, the metabolic phenotype, as defined by myofiber type proportion, reportedly differs between the central portion near the muscle belly and that near the proximal or distal tendon (Martínez Mir et al., 2024), suggesting that fully analyzing the diversity and heterogeneity in skeletal muscle physiological responses and adaptations at the whole-muscle tissue level might be challenging.

Exercise is a well-known and effective tool for maintaining and improving skeletal muscle mass and function, being essential for health and an active lifestyle (Abou Sawan et al., 2023; Zhou et al., 2025). Numerous studies described that various transient exercise modalities induce activation and quantitative changes in diverse physiological responses-associated signaling proteins, including glucose uptake, glycolysis, oxidative metabolism supported by mitochondrial biogenesis and content, and muscle protein anabolism/catabolism (Little et al., 2011; Kido et al., 2016; West et al., 2016). Meanwhile, most of the above-mentioned results were obtained from whole skeletal muscle tissue analysis, and our understanding of exercise-induced signaling responses at the myofiber level, where the effects of other cell types are excluded, remains insufficient.

Myofibers isolation is a widely applied experimental technique for evaluating muscle satellite cell activation (Canibano-Fraile et al., 2021), mitochondrial respiratory function (Pietka and Brookheart, 2024), and contractile apparatus (Tokuda et al., 2023). In particular, isolation by incubation in collagenase-dissolved medium is commonly used for the primary satellite cell cultures (Ono et al., 2012). We recently published that mitochondrial respiratory function increased in satellite cells isolated from murine skeletal muscles that underwent moderate-intensity endurance training, using collagenase treatment (Shirai et al., 2025). Meanwhile, several recent studies have investigated alterations in signaling protein phosphorylation and expression in isolated myofibers obtained by freeze-dried muscle (Campos et al., 2025; Horwath et al., 2025; Deshmukh et al., 2021). However, the differences in the exercise-induced signaling response between isolated myofibers and whole skeletal muscle tissue remain unclear. Therefore, in this study, we aimed to simultaneously examine signaling molecule phosphorylation and expression alterations in protein extracts from whole muscle tissue and isolated myofiber samples after transient exercise to assess a potential myofiber-specific exercise-induced signaling response.

2. Materials and methods

2.1. Experimental animals

Twelve 8-week-old male Institute of Cancer Research mice were obtained from Oriental Yeast Co. Ltd. (Tokyo, Japan), and housed at 22 °C ± 2 °C temperature and 55 % ± 5 % humidity-controlled holding facilities under 12 h light/dark cycle, with ad libitum access to food and water. After 1 week of acclimation, the mice were randomly divided into sedentary control and exercise groups (n = 6 in each group). All experimental procedures conducted in this study were approved by the Institutional Animal Experiment Committee of the University of Tsukuba (animal ethical approval number: 23–380) and based on the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals (National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory, 2011). All experiments conformed to the principles and standards of reporting in animal experiments of Experimental Physiology (Grundy, 2015).

2.2. Acute treadmill running exercise

Treadmill running-based endurance exercise was transiently performed as described previously (Shirai et al., 2020; Ogasawara et al., 2014). The mice were familiarized with running on a rodent treadmill at speeds of 10–20 m/min for 3 days before the experiment. Subsequently, the animals were placed on a flat treadmill and made to run for 60 min at a speed of 25 m/min. Immediately after the exercise intervention, the mice were anesthetized and euthanized by cervical dislocation, and their gastrocnemius muscle was excised and processed for sample preparation.

2.3. Whole-muscle protein lysate preparation

The left leg gastrocnemius muscle protein was extracted in a homogenizing solution containing 50 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH: 7.6), 150 mM sodium chloride, 10 mM ethylenediaminetetraacetic acid, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM sodium orthovanadate, 1 % (v/v) nonidet P-40, 1 % (v/v) Na-deoxycholate, 0.2 % (w/v) sodium dodecyl sulfate (SDS), and 1 % (v/v) complete protease inhibitor cocktail (Nacalai Tesque Inc, Kyoto, Japan). Finally, the homogenized samples were centrifuged at 15,000×g for 20 min at 4 °C, and the supernatants were collected.

2.4. Isolated myofiber protein lysate preparation

The right leg gastrocnemius muscle samples were incubated in 0.2 % collagenase (Worthington) dissolved in high-glucose Dulbecco's Modified Eagle Medium (DMEM) at 37 °C for 90 min, gentle agitated every 15 min, transferred into a DMEM-containing dish, and separated using a wide-mouth Pasteur pipette five times. Isolated myofibers were collected, excess DMEM was removed, and homogenizing solution (the same composition as the solution used for the gastrocnemius muscle protein lysate extraction from the left leg) was added to extract proteins. The homogenized samples were centrifuged at 15,000×g for 20 min at 4 °C, and supernatants were collected.

2.5. Western blots

The supernatant protein concentration was measured using a Protein Assay BCA Kit (FUJIFILM Wako Pure Chemical Co., Osaka, Japan). Prior to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), an aliquot of the extracted protein solution was mixed with an equal volume of sample loading buffer that contained 1 % (v/v) 2-mercaptoethanol, 4 % (w/v) SDS, 125 mM Tris-HCl (pH: 6.8), 10 % (w/v) sucrose, and 0.01 % (w/v) bromophenol blue. After boiling the mixtures at 95 °C for 5 min, 5 μg of protein were loaded onto the gel, separated by SDS-PAGE, and transferred onto an Immuno-Blot PVDF membrane (Bio-Rad Laboratories). The blots were blocked for 1 h at room temperature using a blocking solution (5 % skim milk in TBS containing 0.1 % Tween-20), and the membranes were incubated overnight at 4 °C with primary antibodies in ImmunoShot Platinum (Cosmo Bio Co., LTD.). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (#7074 or #7076, Cell Signaling Technology) for 60 min at room temperature. The signals were visualized using the Immobilon Forte Western HRP Substrate (WBLUF0500, Millipore) and detected using the FUSION Chemiluminescence Imaging System (M&S Instruments, Osaka, Japan). Signal intensities were quantified using the Image J software (National Institutes of Health, Bethesda, MD, USA) and expressed as arbitrary units. Ponceau S staining was used to verify consistent loading.

2.6. Primary antibodies for western blots

The following primary antibodies were used for western blots: 1:1000 anti-p-AMPK (Thr172, #2535), 1:1000 anti-AMPK (#2793), 1:1000 anti-p-ACC (Ser79, #11818), 1:1000 anti-ACC (#3676), 1:1000 anti-p38MAPK (Thr180/Tyr182, #4511), 1:1000 anti-p38MAPK (#8690), 1:1000 anti-p-DRP1 (Ser616, #3455), 1:1000 anti-p-AS160 (TBC1D4, Ser588, #8730), 1:1000 anti-AS160 (#2670), 1:1000 anti-Hexokinase 1 (#2024), 1:1000 anti-Hexokinase 2 (#2867), 1:1000 anti-PKM2 (#4053), 1:1000 anti-Pyruvate dehydrogenase (#3205), 1:1000 anti-LDHA (#3582), 1:1000 anti-p-Akt (Thr308, #9275), 1:1000 anti-p-Akt (Ser473, #9271), 1:1000 anti-pan-Akt (#4691), 1:1000 anti-p-GSK3β (Ser9, #5558), 1:1000 anti-GSK3β (#9832), 1:1000 anti-p-p70S6K (Thr389, #9234), 1:1000 anti-p-p70S6K (Thr421/Ser424, #9204), 1:1000 anti-p70S6K (#2708), 1:1000 anti-p-ERK1/2 (Thr202/Tyr204, #4376), 1:1000 anti-ERK1/2 (#9102), 1:1000 anti-LC3B (#2775), 1:1000 anti-Ubiquitin (#3936). These antibodies were purchased from Cell Signaling Technology. 1:1000 PGC-1α (516557), purchased from Merck Millipore. 1:1000 anti-OPA1 (612606), purchased from BD Biosciences. 1:1000 anti-OXPHOS (ab110413), 1:1000 anti-Citrate synthetase (ab96600), 1:1000 anti-VDAC1/Porin (ab15895), 1:1000 anti-MFN1 (ab126575), 1:1000 anti-MFN2 (ab124773), 1:1000 anti-DRP1 (ab56788), 1:1000 anti-GLUT4 (ab33786), 1:1000 anti-MuRF1 (ab172479), and 1:1000 anti-Fbx32 (ab168372), purchased from Abcam. 1:1000 anti-PFK1 (sc-377346), purchased from SantaCruz Biotechnology. 1:1000 anti-p62/SQSTM1 (PM045) and 1:1000 anti-Parkin (M230-3), purchased from Medical & Biological Laboratories.

2.7. Statistical analyses

The data are represented as the means ± standard deviation (SD) with individual plots. Unpaired, two-tailed Student's t-tests were performed, and GraphPad Prism 10 (GraphPad, Inc.) was used for all statistical analyses. The statistical significance of the differences was set at p < 0.05 in all cases.

3. Results

Fig. 1a–d presents the mitochondrial biogenesis-related signaling activation and abundance quantifications in whole-muscle or isolated myofibers. 5′-adenosine monophosphate-activated protein kinase (AMPK) is a well-known driver of mitochondrial biogenesis. At the whole-muscle level, the phosphorylated and total AMPK content were significantly higher in the exercise group (Fig. 1a and b). In addition, exercise significantly increased not only acetyl-CoA carboxylase (ACC) phosphorylation, an AMPK activation marker, but also the total ACC content and phosphorylation level (Fig. 1a and b). Moreover, the phosphorylated and phosphorylation levels of p38, an upstream regulator of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), were significantly higher in the exercise group. However, PGC-1α itself remained unchanged (Fig. 1a and b). Conversely, at the isolated myofiber level, AMPK/ACC phosphorylation and total content were downregulated in the exercise group, except for p38 and PGC-1α (Fig. 1c and d).

Fig. 1.

Fig. 1

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Changes in phosphorylation and protein content of 5′-adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), p38, and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) in whole gastrocnemius muscle and isolated myofiber lysates. Representative band images of whole muscle lysates (a), signaling intensities of AMPK Thr172 phosphorylation, total-AMPK, AMPK Thr172/total ratio, ACC Ser79 phosphorylation, total-ACC, ACC Ser79/total ratio, p38 Thr180/Tyr182 phosphorylation, total-p38, p38 Thr180/Tyr182/total ratio, and PGC-1α in whole muscle lysates (b). Representative band images (c) and signaling intensities (d) of AMPK, ACC, p38, and PGC-1α in isolated myofiber lysates. The signaling intensities are expressed relative to the mean value in the Sedentary control group. Values are expressed as means + Standard deviation (SD), and each plot shows the individual values (n = 6 in each group). Significant differences were assessed using a Student's unpaired t-test. Significant differences: P < 0.05 vs. Sed group in each lysate.

To determine how acute exercise affects mitochondrial activation in each lysate, we assessed the content of mitochondrial oxidative phosphorylation (OXPHOS) subunit contents, as well as fusion and fission-related signaling proteins (Fig. 2a–d). Among OXPHOS components in the whole-muscle lysate, only SDHB, a subunit of complex 2, exhibited a slight increase in the exercise group (P = 0.077; Fig. 2b). We observed no significant changes in other OXPHOS parameters or indicators of mitochondrial content indicators, such as citrate synthase (CS) and voltage-dependent anion-selective channel 1 (VDAC1) protein levels, between the groups. Moreover, among the mitochondrial fusion/fission response (i.e., mitochondrial dynamics) regulators in whole-muscle lysates, only phosphorylated dynamin-related protein 1 (DRP1) increased significantly in the exercise group (Fig. 2b). In contrast, mitofusion (MFN) 1/2, optic atrophy 1 (OPA1), and total DRP1 remained unchanged (Fig. 2a and b). On the contrary, exercise markedly reduced certain OXPHOS parameters (NDUFB8-complex 1, UQCRC2-complex 3, ATP5A-complex 5, and total-OXPHOS) as well as phosphorylated and total DRP1 in the isolated myofiber lysates (Fig. 2c and d). Similarly, at the isolated myofiber level, MFN2 and OPA1 were slightly decreased in the exercise group (P = 0.073 and P = 0.075, respectively; Fig. 2d).

Fig. 2.

Fig. 2

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Changes in protein content of mitochondrial content, fusion, and fission-related signals in whole gastrocnemius muscle and isolated myofiber lysates. Representative band images of whole muscle lysates (a), signaling intensities of NADH: ubiquinone oxidoreductase subunit B8 (NDUFB8), succinate dehydrogenase complex iron sulfur subunit B (SDHB), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2), mitochondrially encoded cytochrome c oxidase 1 (MTCO1), ATP synthase F1 subunit alpha (ATP5A), total-oxidative phosphorylation (OXPHOS), citrate synthase (CS), voltage-dependent anion-selective channel 1 (VDAC1), mitofusion (MFN) 1, MFN2, optic atrophy 1 (OPA1), dynamin-related protein 1 (DRP1) Ser616 phosphorylation, and total-DRP1 in whole muscle lysates (b). Representative band images (c) and signaling intensities (d) of mitochondrial content, fusion, and fission-related signals in isolated myofiber lysates. The protein phosphorylation and contents are expressed relative to the mean value in the Sedentary control group. Values are expressed as means + Standard deviation (SD), and each plot shows the individual values (n = 6 in each group). Significant differences were assessed using a Student's unpaired t-test. Significant differences: P < 0.05 vs. Sed group in each lysate.

Fig. 3a–d presents the glucose uptake- and glycolysis-involved signaling protein-related data. Immediately after exercise, glucose transporter 4 (GLUT4)-induced glucose uptake is promoted through the TBC1 domain family member 4 (TBC1D4) phosphorylation following AMPK activation in the skeletal muscle. In the whole-muscle lysates, the phosphorylated TBC1D4, total TBC1D4, and GLUT4 protein content were significantly higher in the exercise group compared with those in the sedentary group (Fig. 3a and b). Similarly, hexokinase (HK) 1/2 and pyruvate kinase M2 (PKM2), key glycolysis-related enzymes, were markedly upregulated compared with those in the sedentary group (Fig. 3a and b). Meanwhile, among the glycolysis-related signals, phosphofructokinase (PFK) 1, pyruvate dehydrogenase (PDH), and lactate dehydrogenase A (LDHA) were unchanged between the groups (Fig. 3a and b). In contrast, phosphorylated and total TBC1D4 were significantly lower in the isolated myofiber lysates of the exercise group compared with those in the sedentary group (Fig. 3c and d). Although statistically nonsignificant, PFK1 and PDH decreased in the exercise compared with those in the sedentary group in the isolated myofiber lysates (P = 0.088 and P = 0.065, respectively; Fig. 3d).

Fig. 3.

Fig. 3

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Changes in phosphorylation and protein content of glucose uptake- and glycolysis-related signaling in whole gastrocnemius muscle and isolated myofiber lysates. Representative band images of whole muscle lysates (a), signaling intensities of TBC1 domain family member 4 (TBC1D4) Ser588 phosphorylation, total-TBC1D4, TBC1D4 Ser588/total ratio, glucose transporter 4 (GLUT4), hexokinase (HK) 1, HK2, pyruvate dehydrogenase M2 (PKM2), phosphofructokinase (PFK) 1, pyruvate dehydrogenase (PDH), and lactate dehydrogenase A (LDHA) in whole muscle lysates (b), Representative band images (c) and signaling intensities (d) of glucose uptake- and glycolysis-related signaling in isolated myofiber lysates. The protein phosphorylation and contents are expressed relative to the mean value in the Sedentary control group. Values are expressed as means + Standard deviation (SD), and each plot shows the individual values (n = 6 in each group). Significant differences were assessed using a Student's unpaired t-test. Significant differences: P < 0.05 vs. Sed group in each lysate.

We examined the muscle protein anabolism-associated signaling proteins in each lysate (Fig. 4a–d). In the whole-muscle lysates, the phosphorylation of Akt/mechanistic target of rapamycin (mTOR) signaling proteins (Akt, glycogen synthase kinase 3β; GSK3β, p70 ribosomal S6 kinase; p70S6K), well-known muscle protein anabolism indicators, were markedly increased in the exercise compared with those in the sedentary group (Fig. 4a and b). In addition, extracellular signal-regulated kinase (ERK) 1/2, a mitogen-activated protein kinase (MAPK) involved in mTOR activation, was significantly higher in the exercise group than in the sedentary group (Fig. 4a and b). However, no significant change could be observed in any protein anabolism-related signals in the isolated myofiber lysates of the exercise group (Fig. 4c and d). Although statistically nonsignificant, several signals (Ser473 phosphorylation, P = 0.098; phosphorylation level of Akt, P = 0.062; phosphorylated GSK3β, P = 0.077; and total p70S6K, P = 0.057) were downregulated in the exercise compared with those in the sedentary group (Fig. 4c and d).

Fig. 4.

Fig. 4

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Changes in phosphorylation and protein content of protein anabolism and catabolism-related signaling in whole gastrocnemius muscle and isolated myofiber lysates. Representative band images of protein anabolism-related signals in whole muscle lysates (a), signaling intensities of Akt Thr308 phosphorylation, Akt Ser473 phosphorylation, pan-Akt, Akt Thr308/pan ratio, Akt Ser473/pan ratio, glycogen synthase kinase 3β (GSK3β) Ser9 phosphorylation, total-GSK3β, GSK3β Ser9/total ratio, p70 ribosomal S6 kinase (p70S6K) Thr389 phosphorylation, p70S6K Thr421/Ser424 phosphorylation, total-p70S6K, p70S6K Thr389/total ratio, p70S6K Thr421/Ser424/total ratio, extracellular signal-regulated kinase (ERK) 1/2 Thr202/Tyr204 phosphorylation, total-ERK1/2, and ERK1/2 Thr202/Tyr204/total ratio in whole muscle lysates (b), Representative band images (c) and signaling intensities (d) of protein anabolism-related signals in isolated myofiber lysates. The protein phosphorylation and contents are expressed relative to the mean value in the Sedentary control group. Values are expressed as means + Standard deviation (SD), and each plot shows the individual values (n = 6 in each group). Significant differences were assessed using a Student's unpaired t-test. Significant differences: P < 0.05 vs. Sed group in each lysate.

Finally, we assessed the signaling proteins associated with muscle protein catabolism in each lysate (Fig. 5a–d). In the whole-muscle lysates, the content of light chain 3B (LC3B)-II protein (a marker of autophagosome formation) and the LC3B-II/I ratio (a marker of autophagy activation) were significantly increased in the exercise group compared with the sedentary group (Fig. 5a and b). Similarly, the level of p62 protein, a selective autophagy marker, and Parkin, a well-known ubiquitin E3 ligase involved in mitophagy, were also higher in the whole-muscle lysates of the exercise compared with those in the sedentary group (Fig. 5a and b). Meanwhile, other ubiquitin E3 ligases involved in the ubiquitin-proteasome system, such as muscle RING-finger protein 1 (MuRF1) and Fbx32 (also known as atrogin-1), as well as ubiquitin-conjugated proteins, remained unchanged (Fig. 5a and b). In addition, we also observed upregulated LC3B-Ⅰ, LC3B-Ⅱ, and ubiquitin-conjugated protein levels in the isolated myofiber lysates of the exercise compared with those in the sedentary group (Fig. 5c and d). Although not statistically significant, p62 protein levels modestly increased in the exercise compared with the sedentary group (P = 0.094; Fig. 5c and d).

Fig. 5.

Fig. 5

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Changes in phosphorylation and protein content of protein catabolism-related signaling in whole gastrocnemius muscle and isolated myofiber lysates. Representative band images of protein catabolism-related signals in whole muscle lysates (a), signaling intensities of light chain 3B (LC3B)-Ⅰ, LC3B-Ⅱ, LC3B-Ⅱ/Ⅰ ratio, p62, Parkin, muscle RING-finger protein 1 (MuRF1), Fbx32, and Ubiquitin-conjugated proteins in whole muscle lysates (b), Representative band images (c) and signaling intensities (d) of protein catabolism-related signals in isolated myofiber lysates. The protein contents are expressed relative to the mean value in the Sedentary control group. Values are expressed as means + Standard deviation (SD), and each plot shows the individual values (n = 6 in each group). Significant differences were assessed using a Student's unpaired t-test. Significant differences: P < 0.05 vs. Sed group in each lysate.

4. Discussion

In this study, we investigated skeletal muscle signaling protein activations and abundances immediately after acute endurance exercise using different lysates extracted from whole gastrocnemius muscle tissue or isolated myofiber samples. Consistent with previous findings, endurance exercise significantly enhanced the phosphorylation and levels of mitochondrial biogenesis- and fission-, as well as glucose uptake-, glycolysis, and protein anabolism and catabolism-associated signaling proteins in whole-muscle lysates. However, interestingly, contrary to the data obtained from the whole-muscle lysates, most of the signaling proteins levels decreased in the isolated myofiber lysates upon exercise. Nevertheless, autophagy- and ubiquitin-conjugated protein-associated signaling proteins, representing major protein catabolism-regulating systems, increased upon exercise regardless of lysate type. Hence, we demonstrated that exercise-responsive signaling proteins exhibit differences in their phosphorylation and abundance in whole-muscle tissue and isolated myofiber lysate samples, suggesting specific signaling protein responses detectable at the myofiber level that are not necessarily reflected in the whole-muscle tissue response immediately after acute endurance exercise.

Our data underscore that careful consideration is warranted when using collagenase-dissociated myofibers to assess exercise-induced physiological responses. In fact, the expected, well-established exercise-induced changes in protein phosphorylation did not occur in the isolated myofiber batches. A recent study has shown that disruption of the extracellular matrix, induced by collagenase treatment, leads to severe acute changes in myofiber structure, function, and cellular signaling (Gineste et al., 2022). During the collagenase-mediated isolation process, myofibers remain intact (presumably viable) for about 2 h, during which time major changes in protein activation and expression may occur. In contrast, whole muscle proteins have been extracted immediately after muscle excision. Thus, collagenase-dissociated myofiber seem not be an ideal model, but at the moment it can be a good substitute to distinguish differences between exercise-induced changes in whole muscle and isolated myofibers.

In this study, the AMPK/ACC/p38 and AMPK/TBC1D4 signaling pathways, stimulating mitochondrial biogenesis and glucose uptake, respectively, were activated in whole-muscle lysates but suppressed in those of isolated myofibers. In skeletal muscles, AMPK is upregulated after whole-body exercise through β-adrenergic receptor activation, associated with hormone secretion, such as epinephrine and norepinephrine (Wang et al., 2021), as well as paracrine actions between muscles mediated by cytokines like interleukin-6 (Ahsan et al., 2022). Meanwhile, in isolated myofibers, AMPK and ACC activation might have been suppressed due to removal of the extracellular muscle cell environment and exogenous factors that influence postexercise signaling responses. Moreover, in the skeletal muscle tissue, AMPK is expressed in nonmuscle cells, including endothelial (Jin et al., 2009), satellite (Furuichi et al., 2021), and immune (Cui et al., 2023) cells, and could be regulated by inflammation or hypoxia (Lyons and Roche, 2018; Sakushima et al., 2020). Therefore, postexercise AMPK activation in the whole-muscle tissue might partially originate from nonmuscle cell types. Since nonmuscle components were removed from the isolated muscle fiber lysates, AMPK activation might not have been observed. Contrary to the results of this study, Wang et al. reported significant AMPK, ACC, and TBC1D4 activation in isolated myofibers immediately after swimming-type endurance exercise (Wang et al., 2019). The total exercise duration in the above-described evidence (four sessions of 30-min exercise bouts) was longer than the protocol used in this study. We previously reported that prolonged endurance exercise duration could represent a factor that increases oxidative stress in whole skeletal muscle (Shirai et al., 2021). Oxidative stress is among the AMPK activity-stimulating factors (Stern and McNew, 2021). In addition, in human myofibers, AMPK, ACC, TBC1D4, and mTOR phosphorylation were reportedly enhanced immediately after acute endurance exercise using 30- or 60-min pedal cycling (Horwath et al., 2025; Kristensen et al., 2015). While pinpointing the factors responsible for the inconsistencies in our data is challenging, variations in endurance exercise parameters, such as exercise duration and modality (e.g., running, swimming, and cycling), might have impacted the results.

We observed no changes in OXPHOS protein levels in the whole-muscle tissue immediately after exercise, whereas the expression levels of several OXPHOS subunits significantly decreased in isolated myofibers. Martin et al. reported that mitochondrial respiratory function, as assessed by high-resolution respirometry, was markedly reduced in permeabilized fibers from rats immediately after endurance exercise compared with the sedentary group (Martins et al., 2018), consistent with our data. Therefore, the decline in mitochondrial function immediately after endurance exercise could be potentially partially explained by the reduced OXPHOS subunit expression levels. Mitophagy selectively removes dysfunctional mitochondria (Seabright and Lai, 2020). However, in this study, Parkin expression did not increase in isolated myofibers immediately after exercise, and the protein levels of CS and VDAC1, indicators of mitochondrial content, were maintained. The OXOHOS subunit reduction in isolated myofibers immediately after exercise might be related to the simultaneous increase in ubiquitin-conjugated protein levels. Still, mitochondrial content itself might have been maintained after exercise. Among the mitochondrial dynamics-regulating factors, the Ser616 phosphorylation of DRP1, a mitochondrial fission modulator and mitophagy induction facilitator (Drake et al., 2016), increased significantly in the whole-muscle tissue immediately after exercise, although it decreased in isolated myofibers. The physiological role of suppressed mitochondrial fission response in isolated myofibers remains unclear. Still, our data suggest that DRP1 Ser616 phosphorylation immediately after endurance exercise might be enhanced in cell types other than muscles.

We detected significant Akt/mTOR and MAPK pathway-involved signaling protein activation, contributing to muscle protein anabolism, only in the whole-muscle lysates: these responses were absent in the isolated myofiber lysates. Several studies demonstrated that acute endurance exercise increases Akt, mTOR, p70S6K, and ERK1/2 phosphorylation in human and rodent whole-muscle tissues immediately after exercise (Hayasaka et al., 2014; Pasiakos et al., 2010; Mascher et al., 2011), supporting the results of this study. Insulin, insulin-like growth factor-1, and other growth factors are well-known to strongly activate Akt/mTOR signals (Liu and Sabatini, 2020). In addition, exercise-induced factors (interleukin-6, interleukin-15, and leukemia inhibitory factor), released from muscle and other organs, activate the mTOR pathway (Gao et al., 2017; Cornish et al., 2020), emphasizing the importance of interorgan crosstalk in postexercise skeletal muscle responses. Furthermore, recent studies have revealed that intramuscular cell–cell communication between muscle and nonmuscle fibers is essential for muscle growth (Zhang et al., 2024; Sakai et al., 2024). In the isolated myofiber lysates used in this study, the influence of humoral factors that could affect myofibers from blood vessels or nonmuscle cells within the whole muscle, was eliminated, suggesting that the protein anabolic response following systemic exercise might be supported not only by muscle cells alone but also by complex signal communication between different organs or cells.

Interestingly, unlike other signaling pathways, protein catabolic signals (particularly autophagy-related signals) increased immediately after exercise both in the whole muscle and isolated myofiber lysate samples, consistent with the results of a previous rodent study (Vainshtein et al., 2015). However, since the LC3B ratio, an autophagosome formation indicator (Mizushima and Yoshimori, 2007), increased only in the whole-muscle lysate samples, autophagy activation might occur at the whole skeletal muscle level immediately after exercise. Meanwhile, in contrast with our data, several human studies described that LC3B-Ⅱ suppression in whole skeletal muscles or myofibers immediately after exercise (Horwath et al., 2025; Mazo et al., 2021; Schwalm et al., 2015), suggesting that actual autophagy flux should be interpreted with caution. We previously observed that endurance exercise training using treadmill running significantly increased ubiquitinated protein levels without altering MuRF1 and Fbx32 protein abundance in murine whole-muscle tissue (Shirai et al., 2021). Therefore, postendurance exercise ubiquitinated protein level increase is not necessarily accompanied by ubiquitin ligase expression increase and might be mediated by an unknown mechanism, specifically enhancing ubiquitination at the myofibers.

5. Limitations

We analyzed protein abundance and phosphorylation levels to assess the signaling molecule quantity and activation status. However, at the time points several hours after a single bout of exercise, mRNA expression alterations are more pronounced than those in protein content (Egan and Zierath, 2013). Therefore, integrating protein and mRNA expressions would be necessary to understand the signaling responses. In addition, we cannot rule out that metabolic, mechanical, and environmental stresses associated with the isolation procedures themselves (e.g., temperature changes during collagenase treatment, mechanical stress during isolation, and changes in oxygen supply) might have influenced signaling proteins in myofiber lysates. In future studies, standardization of the protein extraction process will be necessary for whole-muscle tissue and isolated myofiber samples under the same conditions to ensure consistency. In this context, several studies have detected myofiber-specific exercise responses using muscle fibers dissected from freeze-dried whole-muscle tissue (Horwath et al., 2025; Deshmukh et al., 2021; Wang et al., 2019; Kristensen et al., 2015). Although freeze-drying might enable more rigorous myofiber signaling response detection than collagenase treatment, both methods should be directly compared. Moreover, in this study, dead myofibers could not be detected or removed during the isolation process, and floating myofibers were collected as much as possible by visual inspection. Therefore, the number of pooled myofibers was not quantified. Finally, this study was limited to an analysis of the gastrocnemius muscle, muscle fiber type-specific exercise responses might thus have been masked. Comparisons between the red and white portions of the gastrocnemius muscle (Yoshihara et al., 2024) or between the slow-twitch oxidative and fast-twitch glycolytic muscles, respectively (Takahashi et al., 2024), after acute exercise should be noted as a potential objective for future research.

6. Conclusion

In summary, our data proved that despite increased activation and abundance of multiple signaling proteins associated with mitochondrial biogenesis, glucose metabolism, mitochondrial fission, and muscle protein anabolism in the whole skeletal muscle of mice immediately after acute endurance exercise, the above-described signaling responses did not occur in myofibers isolated by enzymatic digestion. These discoveries suggest that acute exercise-induced signaling responses in myofiber lysates might not necessarily be consistent with those in whole-muscle tissue lysates, depending on the isolation procedure used. The results of this study could have important implications for understanding the myofiber-specific signaling events underlying transient physiological responses to exercise.

CRediT authorship contribution statement

Kazuki Uemichi: Data curation, Formal analysis, Funding acquisition, Investigation, Validation, Visualization, Writing – original draft, Writing – review and editing. Takanaga Shirai: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Investigation, Validation, Writing – review and editing. Shunsuke Sugiyama: Formal analysis, Investigation, Validation. Tohru Takemasa: Funding acquisition, Methodology, Project administration, Resources, Supervision.

Funding information

This work was supported by Japan Society for the Promotion of Science (Grant Number: 23H03266 to T.T., 23KJ0237 to K.U., and 23KJ2060 to T.S.) and Daiichi-Sankyo “Habataku” Support Program for the Next Generation of Researchers (Grant Number: 24FAFA10002 to K.U.).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We would like to thank Dr. Satoshi Fujita (Faculty of Sport and Health Science, Ritsumeikan University) for arranging discussions and providing meaningful comments to our research.

Contributor Information

Kazuki Uemichi, Email: kazuem@fc.ritsumei.ac.jp.

Takanaga Shirai, Email: wtk-0598di@kanagawa-u.ac.jp.

Data availability

Data will be made available on request.

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