Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle

Mechanical stimulation has benefits for muscle mass and function. Passive stretching is widely performed in clinical rehabilitation medicine. However, the hypertrophic effects of passive repetitive stretching on senescent skeletal muscles against muscle atrophy remain unknown. We used senescence-accelerated model SAM-P8 mice. The gastrocnemius muscle was passively repetitive stretched by manual ankle dorsiflexion for 15 min, 5 days a week for 2 weeks under deep anesthesia. We examined the effects of passive stretching on muscle mass, myofiber cross-sectional area, muscle fiber type composition, satellite cell and myonuclei content, signaling pathways involved in muscle protein synthesis, and myogenic regulatory factors. The gastrocnemius muscle weight and fiber cross-sectional area of the stretched side was found greater compared with that of the unstretched side. Passive repetitive stretching increased the mRNA expression level of Akt, p70S6K, 4E-BP1, Myf5, myogenin, MuRF1.The phosphorylation level of p70S6K significantly increased in the stretched muscles, whereas of Akt and 4E-BP1 remained unchanged, compared to the unstretched side. The Pax7+ cells and myonuclei content did not differ between the stretched and unstretched muscles. These findings suggest that the hypertrophic or suppressed atrophic observation in the stretched muscles are mainly attributable to the protein turnover provoked by stretching. These findings are applicable to clinical muscle strengthening and sarcopenia prevention.

www.nature.com/scientificreports/ stretching in regulating the myogenic regulatory factors and suppressing denervation-induced atrophy through the Akt/mTOR signaling cascade 6,7,9 . These findings suggest a potential hypertrophic effect of repetitive stretching, which prevents sarcopenic atrophy in senescent skeletal muscles. Disrupted protein homeostasis is well established as an initial process that regulates age-related muscle mass reduction 14 . Skeletal muscle atrophy occurs when protein degradation rates transcend rates of protein synthesis 15 . Hence, strategies that enhance protein synthesis may prevent sarcopenic progression. IGF1/Akt/ mTOR signaling primarily controls muscle protein synthesis 14,15 . Akt stimulates protein synthesis by activating mTOR and inhibits protein degradation through FoxO-mediated ubiquitin-ligases including Muscle Atrophy F-box (MAFbx)/atrogin-1, and Muscle Ring Finger 1 (MuRF1) downregulation 14,15 . Myostatin, a member of the TGF-β superfamily, has been extensively studied as another potential mediator of sarcopenia owing to its potent negative effects on cell growth and intracellular catabolic and anabolic signaling pathways 16 .
Skeletal muscle hypertrophy can be induced by the activation of muscle satellite cells. Once satellite cells are activated, myogenic regulatory factors (MRFs), including MyoD, Myf5, and myogenin, play a pivotal role in skeletal muscle formation and development 17 . Markers of early myogenesis, MyoD and Myf5, contribute to determination, while the marker of late myogenesis, myogenin, plays a downstream role in muscle cell differentiation to form muscle fibers 17 . However, accumulating evidence suggests that satellite cell numbers and responsiveness are altered during aging, which may be associated with sarcopenia 18 . Although satellite cells are indispensable for muscle regeneration, whether they play a key role in the maintenance of skeletal muscle mass and myofiber size throughout life is still controversial.
Thus, the aim of this study was to investigate the impact of passive repetitive stretching on senescent skeletal muscle mass and fiber morphology by using senescence-accelerated mouse prone-8 (SAM-P8), which exhibits characteristics of accelerated muscle aging, and is reported to be valid for muscular aging research 14 . We also compared changes in terms of muscle fiber type composition, satellite cells and myonuclei content, MRFs, and regulatory factors related to muscle protein synthesis to further elucidate the underlying mechanisms of action. We hypothesized that passive repetitive stretching exerted hypertrophic effects against sarcopenic atrophy in SAM-P8 mice.

Results
Muscle mass and muscle fiber cross-sectional area. There was no significant difference in body weight between the pre-and post-stretching animals (Fig. 1A). The gastrocnemius muscle weight of the stretched side was ~ 10.7% greater as compared with that of the unstretched side (P = 0.028; Fig. 1B). In addition, histological analysis using H&E staining showed that the mean gastrocnemius muscle fiber cross-sectional area of the stretched side was ~ 19.9% larger than that of the unstretched side (P = 0.012; Fig. 1C,D). The frequency distribution of the muscle fiber area further revealed that the stretched gastrocnemius muscle had a shift toward larger myofibers as compared with the unstretched side (Fig. 1E).

Muscle fiber type, satellite cells and myonuclei. No clear differences in the distribution of fiber types
were observed when the fiber-type composition was examined with IHC staining ( Fig. 2A). Type 2A fibers were the most common in the gastrocnemius muscle, with lower proportions of type 2B and type 1 fibers (Fig. 2B). This suggests that fiber-type switching is not regulated by passive repetitive stretching for a short duration within 2 weeks. In addition, type 2A fibers were mostly responsible for the difference in the cross-sectional area, which was ~ 27.4% greater on the stretched side compared to the unstretched side (P = 0.045; Fig. 2C). Pax7 expression has been recognized as a marker of satellite cells. However, Pax7+ cells were rare, and there was no significant difference in the number of Pax7+ cells between the stretched and unstretched sides (Fig. 3A,B). The number of myonuclei per fiber determined by H&E staining showed no difference between the stretched and unstretched muscles (Fig. 3C). mRNA expression of Akt, p70S6K, 4E-BP1, MAFbx and MuRF1, MRFs and myostatin. As a housekeeping gene, GAPDH was not significantly altered by the intervention. The messenger RNA expression levels of Akt/mTOR pathway signal molecules, Akt, p70S6K, and 4E-BP1 are affected by passive repetitive stretching (Fig. 4). The mRNA expression of Akt increased by 2.5-fold (P = 0.04995; Fig. 4A) on the stretched side as compared with the unstretched side. 4E-BP1 and p70S6K are two downstream targets of mTORC1. The mRNA expression of p70S6K increased by 4.9-fold (P = 0.012; Fig. 4B) and 4E-BP1 increased by 5.7-fold (P = 0.012; Fig. 4C) in the stretched side as compared with the unstretched side. We then measured the expression of major muscle-specific E3 ubiquitin ligases, including MAFbx and MuRF1. The MAFbx mRNA expression was not altered (Fig. 4D), whereas the MuRF1 mRNA expression increased by 2.1-fold (P = 0.04995; Fig. 4E) on the stretched side as compared with the unstretched side.
Phosphorylation statuses of Akt, p70S6K, and 4E-BP1. The level of Akt phosphorylation in the stretched muscles did not differ significantly from the unstretched side (Fig. 6A). The phosphorylation status of the key mTORC1 activity marker, p70S6K, increased significantly by 22% on the stretched side compared to the unstretched side (P = 0.013, Fig. 6B). The phosphorylation level of another mTORC1 downstream target, 4E-BP1, remained unchanged compared to the unstretched muscles (Fig. 6C)

Discussion
The findings of this study indicate that passive repetitive stretching for 2 weeks attenuated sarcopenic muscle loss in aged mice and were associated with a greater muscle fiber cross-sectional area, particularly in the type www.nature.com/scientificreports/ 2A fiber. To the best of our knowledge, this is the first study to demonstrate muscular hypertrophic adaptation to passive repetitive stretching in aged mice in vivo. The theoretical basis for stretch-induced skeletal muscle hypertrophy dates back to in vitro studies. Goldberg et al. found that repetitive stretching applied to cultured skeletal muscle cells provided mechanical stimuli and triggered cellular biomarkers essential for muscle growth 19 . Indeed, robust hypertrophy was observed after progressive stretch overload of the wing muscles in birds 20 . Stretch parameters such as frequency and duration have been identified as important factors that potentially affect the skeletal muscle adaptive process, as the gene expressions involved in muscle growth and atrophy are responsive to the number of stretch sessions 21,22 . Sarcopenia is an age-related reduction in both muscle mass and quality 1,2 .
Although prior studies have demonstrated that senescent muscles preserved the capacity to undergo hypertrophy, the ability to perceive and respond to mechanical inputs and translate them into biochemical signals, which is called "mechano-transduction, " was reportedly blunted during aging 11 . The impact of aging on the cellular mechano-transduction process is rooted in multiple factors such as modifications in cell cytoskeleton structures, alterations in mechanosensitive signaling, and the extracellular matrix environment 11 . Zotz et al. found that 1 week of intermittent stretching in aged rats resulted in an unchanged muscle mass, accompanied by reduced fiber size 12 . Hotta et al. reported an increase in soleus and plantaris muscle weights after 4 weeks of continuous stretching without further histological analysis of muscle characteristics 13 . As the rats were awake during continuous muscle stretching, it became difficult to isolate the effects of stretching from isometric contractile activity. In another study, contractions were eliminated by animal anesthesia during stretching, while muscle weight and fiber area remained unaltered 23 . Our results here extend those of previous observations by demonstrating that clinically feasible protocol of passive repetitive stretching is effective in preserving or improving muscle mass and fiber area in the senescent muscles. Sarcopenia predominantly affects type 2 muscle fibers, whereas type 1 fibers are less affected 2 . In SAM-P8 mice aging from 6 to 10 months, the proportion of fiber type 2A became greater from 53 to 66%, whereas that of muscle fiber type 2B decreased from 42 to 30%, as reported by Guo et al 24 . In addition, the type 2A fiber area www.nature.com/scientificreports/ increased from 6 to 8 months, followed by a sharp reduction up to 10 months 24 . The remarkable amelioration of the type 2A fiber area with passive repetitive stretching in the present study is notable considering that the preferential atrophy of these high-power generation muscle fibers is a hallmark of sarcopenia progression. As a countermeasure, passive repetitive stretching with a short duration may mitigate or delay the characteristic age-related muscle loss. However, it appears that muscle fiber-type composition seemed not to be susceptible to regulation within 2 weeks of stretching. Fiber-type plasticity within skeletal muscle is regulated by a sophisticated signaling network with two major pathways, calcineurin signaling and AMP-activated protein kinase (AMPK) signaling with a major mediator, PGC-1α 25 . Our observations suggest that the hypertrophic effect of passive repetitive stretching occurred without modifying the fiber-type composition within a short duration of 2 weeks. The regulation of muscle mass and fiber size substantially reflects changes in protein homeostasis, i.e. the balance between protein synthesis and degradation 15 . Therefore, to decipher the mechanism of action behind the hypertrophic effect of passive repetitive stretching, we first verified whether the Akt/mTOR pathway, which predominantly controls muscle protein synthesis, was involved in muscular adaptation. Akt is an upstream regulator of mTOR, and it is widely recognized that signaling by mTOR is a core module of the pathway through which mechanical stimuli regulate protein synthesis and muscle growth 26 . The regulation is primarily mediated by two downstream targets of the mTOR complex 1 (mTORC1), translational suppressor 4E-BP1 and ribosomal protein p70S6K 27 . Skeletal muscle stretching activates these signaling molecules with elevated phosphorylation levels, including Akt, p70S6K, and 4E-BP1, in vitro 28,29 . Enhanced Akt, p70S6K, and 4E-BP1 phosphorylation were observed in denervated mice soleus muscles when subjected to repetitive stretching in vivo 9 . Several studies have demonstrated that the responsiveness of Akt/mTOR signaling is diminished in overload-induced muscle growth during aging, suggesting limited plasticity for aged muscle hypertrophy 30 . The present study showed that repetitive stretching strongly increased the mRNA expressions of Akt, p70S6K, and 4E-BP1. However, the phosphorylation level of p70S6K was elevated, whereas that of Akt and 4E-BP1 remained unchanged, compared to the unstretched muscles. Previous studies have shown that the phosphorylation status of the key signaling proteins implicated in the regulation of protein synthesis exerted time-course changes in response to a period of acute stretching 8,9 . Thus, a time course study of the acute effect of a single bout of passive repetitive stretching on phosphorylation would be useful to further elucidate protein synthesis regulation induced by stretching of the senescent skeletal muscles.
Moreover, Akt normally blocks the upregulation of several ubiquitin-proteasome genes related to protein degradation in skeletal muscles by negatively regulating FoxO transcription factors 15 . In skeletal muscles, the major muscle-specific ubiquitin ligases include MAFbx/atrogin-1 and MuRF1, which are associated with  31 . These divergent findings suggest that proteasome activity is potentially influenced by stretching protocols or responds differently under physiological and pathological conditions. The alternation of MAFbx and MuRF1 expression in aged skeletal muscle has been reported to be inconsistent. Several studies found that the expression levels of MAFbx and MuRF1 increased in skeletal muscles with aging, which may contribute to sarcopenia 32,33 . However, unaltered and even decreased expression levels of MAFbx and MuRF1 have been shown in other studies 34,35 . In our study, MuRF1 mRNA expression was elevated, indicating the involvement of the cellular degradation pathway in aged skeletal muscle adaptation to passive stretching. Combined with the greater muscle mass and fiber size, this may suggest that hypertrophic or suppressed atrophic observations in the senescent muscles may result from relatively enhanced overall rates of protein synthesis, which possess a superior position in protein homeostasis in the experimental period. Likewise, differential expression patterns of myostatin in stretching have been observed in previous reports 21,22,36 . Myostatin negatively regulates skeletal muscle growth, primarily by acting via activin type II receptors (ActRII), resulting in the activation of Smad signaling 14,15 . Smad signaling suppresses Akt signaling and its downstream effectors such as mTOR and FoxO to regulate muscle growth 14,15 . Alterations in myostatin expression and signaling activity in the context of aging are not completely understood. We could speculate that passive stretching is a potential intervention to counter, at least in part, sarcopenia via myostatin inhibition. However, myostatin expression was not affected by stretching in our study. A further time-course study may help to define the myostatin expression alternation in response to passive repetitive stretching of senescent skeletal muscles. In addition to protein turnover within individual myofibers, as stated previously, skeletal muscle hypertrophy can also be induced by the activation of muscle satellite cells. In mature muscles, satellite cells are generally quiescent but become activated in response to various stimuli or under muscle regeneration to form new myofibers 37 .  17 . Previous studies have shown that mechanical stretching can induce activation of skeletal muscle satellite cells 5 . Elevated expression levels of MRFs have also been observed after short-term passive repetitive stretching 7 . Pax7 expression has been recognized as a marker of satellite cells. To elucidate whether passive repetitive stretching triggered an active regenerative process that may contribute to senescent skeletal muscle hypertrophy, we first sought to detect Pax7+ cells by immunohistochemical analysis. Pax7+ cells were rare, and there was no significant difference in the number of Pax7+ cells between the stretched and unstretched muscles. Therefore, satellite cell content was not stimulated by passive repetitive stretching. As also observed in a human study, satellite cell response during post-exercise recovery is blunted with aging 38 . The expression of MyoD was unchanged, whereas the Myf5 and myogenin mRNA expressions were upregulated in the stretched side when compared with the unstretched side. It has also been reported that MRFs mRNA increases occur in muscles, even in the absence of proliferating satellite cells 39 . Finally, the number of nuclei per fiber was measured in each muscle section to determine whether or not stretching result in satellite cell activation and incorporation of new nuclei. As a result, the stretched and unstretched muscles did not differ in SAM-P8 mice. We are not convinced of the possibility of stretch-induced myogenesis without an increase in MyoD, Pax7 expression levels and new nuclei addition. Overload-induced muscle hypertrophy requires the involvement of satellite cells in growing mice, whereas it is not necessary for hypertrophic growth in mature adult mice 40 . Our study had some limitations. Considering that SAMP8 mice muscle was not collected before stretching intervention as controls, it remains unclear that how sarcopenic morphological changes progressed during stretching intervention. Therefore, we cannot draw a robust conclusion regarding the extent of the effect of passive repetitive stretching on the muscle mass and fiber area: suppressing the progression of atrophy or inducing hypertrophy. It would also be useful to add time-matched senescence-accelerated mouse resistant-strain 1 (SAMR1) with/without stretching intervention to highlight the difference in muscular adaptation in response to stretching in aged and normal mice. Although pax7+ cells and MRFs related to muscle satellite cells were assessed after the final section of stretching, it is better detected at an early time point in the protocol because of potential time course changes. The time course of the acute effect of a single bout of passive repetitive stretching on the phosphorylation status of the key signaling molecules would be useful to further elucidate protein synthesis regulation induced by stretching of the senescent skeletal muscles.

Conclusion
Passive repetitive stretching attenuate sarcopenic muscle loss in aged model mouse and are associated with a greater muscle fiber cross-sectional area. Repetitive stretching promotes the gene expression of signal molecules and phosphorylate p70S6K involved in muscle protein synthesis in the senescent skeletal muscles of SAM-P8 mice. Our study suggests that passive repetitive stretching is an effective measure for sarcopenia prevention and maintenance of skeletal muscle mass and function in patients who are exposed to prolonged inactivity, unconscious, or paralyzed ("Supplementary data").
They were housed in plastic cages in a temperature-controlled room with a 12-h light-dark cycle and provided free access to water and standard food. The experimental procedures were approved by the Animal Care and Use Committees of Hokkaido University. The study protocol was carried out in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions, under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The study was carried out in compliance with the ARRIVE guidelines.
Stretching protocol. The mice were anesthetized with isoflurane solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) using inhalation anesthesia equipment (NARCOBIT-E-II KN-1071, Natsume Seisakusho, Tokyo, Japan). The right gastrocnemius muscles were stretched by manual ankle dorsiflexion with knee extended position and within the natural range of motion to avoid muscle damage. Stretching was performed repeatedly, 15 times/min for 15 min daily, 5 days a week for 2 weeks (Fig. 7). Contralateral unstretched muscles were examined as controls.
Tissue collection and histology. Twenty-four hours after the final stretch session, the gastrocnemius muscles of both legs of the mice were removed under deep anesthesia. After weighing, the gastrocnemius muscles were divided into two blocks. Samples for RNA extraction and western blot analysis were immediately preserved in liquid nitrogen and stored at − 80 °C. Samples intended for histology were collected in Tissue-Tek ® O.C.T. compound (Sakura Finetek, Tokyo, Japan) for frozen sections. SAM-P8 mice gastrocnemius muscle transversal cross-section (8 μm) frozen sections were cut using Cryostat HM550 (Thermo Fisher Scientific, Waltham, MA, USA) and stained with hematoxylin and eosin (H&E) to determine the muscle fiber size and frequency distribution. Tissue slides were observed using PALM MicroBeam IV (ZEISS, Oberkochen, Germany). The cross-sectional area of the myofibers and the number of myonuclei was calculated from 200 myofibers per muscle sample using the ImageJ software (http:// imagej. nih. gov/ ij/).

RNA isolation and real-time polymerase chain reaction. A hand homogenizer with Trizol Reagent
(Thermo Fisher Scientific) was used to homogenize the muscle tissues, followed by the addition of 0.2-fold chloroform (FUJIFILM Wako Pure Chemical Corporation), a precipitator to extract total RNA from the supernatant, and the removal of protein and deoxyribonucleic acid. One-step real-time reverse transcription polymerase chain reaction was performed using the Power SYBR Green RNA-to-CT™ 1-Step Kit (Applied Biosystems, Waltham, MA, USA) with the following steps: 48 °C for 30 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, with an annealing and extension step at 60 °C for 1 min. The primers used are shown in Table 1. GAPDH was used as a housekeeping gene.
Statistics. The Student's t-test or Wilcoxon signed-rank test were used as appropriate after ascertaining normality of distribution via the Shapiro-Wilk test. A P-value < 0.05 was considered to be statistically significant. Statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA) and SPSS Statistics version 26 (IBM Corp., Armonk, NY, USA). Data are expressed as mean ± standard error of the mean (SEM).