Circadian rhythms modulate the effect of eccentric exercise on rat soleus muscles.

We investigated whether time-of-day dependent changes in the rat soleus (SOL) muscle size, after eccentric exercises, operate via the mechanistic target of rapamycin (mTOR) signaling pathway. For our first experiment, we assigned 9-week-old male Wistar rats randomly into four groups: light phase (zeitgeber time; ZT6) non-trained control, dark phase (ZT18) non-trained control, light phase-trained, and dark phase-trained. Trained animals performed 90 min of downhill running once every 3 d for 8 weeks. The second experiment involved dividing 9-week-old male Wistar rats to control and exercise groups. The latter were subjected to 15 min of downhill running at ZT6 and ZT18. The absolute (+12.8%) and relative (+9.4%) SOL muscle weights were higher in the light phase-trained group. p70S6K phosphorylation ratio was 42.6% higher in the SOL muscle of rats that had exercised only in light (non-trained ZT6). Collectively, the degree of muscle hypertrophy in SOL is time-of-day dependent, perhaps via the mTOR/p70S6K signaling.

Introduction

Light-responsive central clock genes in the suprachiasmatic nucleus (SCN) coordinate circadian rhythms through signaling of peripheral clock genes in other tissues [1]. Glucocorticoids are the primary humoral mediators that send the reset signal from the SCN to the peripheral clock [2]. Hence, organ function varies depending on the time of day. As the main contractile tissue of the body involved in movement, skeletal muscle is not only important for posture but also plays a major role in energy metabolism and glucose uptake [3]. In our previous study, we demonstrated circadian variation in intracellular protein synthesis signaling in rat skeletal muscles [4], similar to what has been observed in body temperature, locomotor activity, and hormone secretion [5, 6]. Specifically, we observed diurnal variation in mechanistic (mammalian) target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) signaling and extracellular signal-regulated kinase (ERK) signaling in skeletal and cardiac muscles. These pathways are key mechanisms for skeletal muscle hypertrophy under mechanical overload (e.g., strength training). Thus, mTOR/p70S6K and ERK signaling pathways are potential targets for maintenance or increase of skeletal muscle mass (muscle hypertrophy). This therapeutic strategy has numerous applications, from improving the health of the elderly to boosting athlete performance.

Given the influence of circadian rhythms, treatment efficacy and the probability of adverse drug reactions will most likely depend on the time of day [7, 8]. Thus, there may be an optimal time for activating mTOR signaling to maximize the effects of exercise on muscle tissue. Recent studies on human skeletal muscle have investigated how exercise at different times of day influences muscle mass (muscle fiber size), with inconsistent results. For instance, following 24 weeks of combined strength and endurance training, evening groups (16:30–18:30) experienced a greater gain in muscle mass than morning groups (07:30–09:30) [9]. In contrast, muscle mass gain did not differ between morning (07:00–09:00) and evening (17:00–19:00) groups after 10 weeks of strength training [10]. Multiple factors could generate such inconsistency, including variation in individual physical fitness, training programs, daily timing of exercises, and diet. Notably, these studies did not describe their criteria for determining when training should occur nor did they consider subject diet during training periods. Therefore, in this study, we designed a rat experiment that controlled the effects of important confounding factors (e.g., individual variation, protein intake, exercise intensity, and exercise timing). Our aims were to examine 1) the effect of exercise on muscle hypertrophy at different times of day and 2) the underlying mechanisms in rat soleus (SOL) muscle. We hypothesized that eccentric exercise at different periods in the day (light vs. dark) will influence mTOR signaling activation and subsequent training-induced skeletal muscle hypertrophy.

Materials and methods

Experimental animals

Nine-week-old male Wistar rats were obtained from a licensed laboratory animal vendor (SLC Inc., Hamamatsu, Shizuoka, Japan). Water and food were provided ad libitum. All rats were housed in an environmentally controlled room (temperature: 23 ± 1°C; relative humidity: 55% ± 5%; 12/12 h light/dark cycle, with lights on at 18:00 and off at 6:00) after acclimation for 1 week, prior to the experiment. All procedures were approved by the Juntendo University Animal Care and Use Committee (H29-07).

Experiment 1: Effect of eccentric exercise on muscle hypertrophy at different times of the day for 8 weeks

Twenty-four male Wistar rats were randomly assigned to two groups: Zeitgeber Time (ZT) 6 (light phase; L, n = 12) and ZT18 (dark phase; D, n = 12) (ZT0: lights on at 18:00, ZT12: lights off at 6:00). Each group was further divided into untrained control (C) or trained (TR) groups (n = 6 per subgroup). The training program was based on previously published methods [11]. Once every 3 d (20 sessions in total), TR animals participated in 90 min of downhill running on an animal treadmill (incline: -16°, speed: 16 m/min). Forty-eight hours (including 12 h of fasting) after the last training session, rats were anesthetized with isoflurane (3%–5%) and pentobarbital sodium (50 mg/kg). Right and left SOL muscles and epididymal white adipose tissue was immediately removed and weighed. Thereafter, muscle samples were gently stretched to near optimal length using a compass before being frozen in liquid nitrogen and stored at -80°C.

Experiment 2: Effect of eccentric exercise on mTOR signaling in SOL at different times of the day

Forty-two male Wistar rats were randomly assigned to two groups: ZT6 (L, n = 21) and ZT18 (D, n = 21). These periods were selected because our previous study showed that mTOR/p70S6K signaling in skeletal muscles is high at ZT6 and low at ZT18 [4]. Rats from each group were further divided into before (Pre), immediately after (Pt0), and 1 h after eccentric exercise (Pt1) (n = 7 per category).

Following 12 h of fasting, Pt0 and Pt1 rats participated in a bout of downhill running for 15 min on an animal treadmill (incline: -16°, speed: 16 m/min). At appropriate time points (Pre, Pt0, Pt1), rats were anesthetized with isoflurane (3%–5%) and pentobarbital sodium (50 mg/kg). Muscle samples were frozen immediately in liquid nitrogen and stored at -80°C until analysis.

Blood samples were collected from the inferior vena cava, centrifuged at 1300 × g for 10 min to obtain serum and stored at -80°C. Corticosterone concentrations were estimated by commercial laboratories (Shibayagi Co., Ltd., Gunma, Japan and Oriental Yeast Co., Ltd., Tokyo, Japan).

Measurement of SOL myofiber cross-sectional area (CSA) and centrally nucleated myofibers

Muscle section preparation and staining were performed following previously described techniques [12]. Briefly, frozen SOL samples were sliced into 10 μm sections using a cryostat (CM3050S, Leica, Wetzlar, Germany) at -20°C, and subsequently stained with hematoxylin and eosin (H&E). Section images were captured using a microscope (10×; BZ-8000; Keyence, Osaka, Japan) and transferred to a computer. CSA of 200–250 muscle fibers were randomly measured using the ImageJ software (NIH, Bethesda, MD, USA). The central nucleus was used as an indicator of muscle regeneration. We evaluated the ratio of the centronucleated fiber, defined by the centrally nucleated muscle fibers divided by the total number of muscle fibers.

Western blotting analysis and immunodetection

SOL samples were frozen in liquid nitrogen, powdered, and then homogenized in ice-cold buffer (50 mM HEPES [pH 7.4], 1 mM EDTA, 1 mM EGTA, 20 mM β-glycerophosphate, 1 mM Na3VO4, 10 mM NaF, and 1% Triton X-100) containing a protease inhibitor cocktail (cOmplete EDTA-free; Roche, Penzberg, Germany) and phosphatase inhibitor cocktail (PhosSTOP; Roche). Homogenates were centrifuged at 12000 × g at 4°C for 15 min. Supernatant protein concentrations were determined using a BCA Protein Assay Kit (Thermo, Rockford, IL, USA). Protein extracts were solubilized in sample buffer (30% glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 62.5 mM Tris–HCl [pH 6.8], and bromophenol blue) and incubated at 95°C for 5 min. Samples containing total protein were subsequently loaded onto 10–12% TGX Fast Cast acrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) and electrophoresed at 150 V for 45 min. Separated proteins were transferred to PVDF membranes (Bio-Rad Laboratories) using a Bio-Rad Mini Trans-Blot cell at 100 V and 4°C for 60 min in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol).

Thereafter, membranes were blocked for 1 h using blocking buffer (5% nonfat dry milk in Tween-Tris-buffered saline [T-TBS: 40 mM Tris-HCl, 300 mM NaCl, and 0.1% Tween 20, pH 7.5]) at room temperature (25–26°C). Membranes were subsequently incubated for 2 h at room temperature (25–26°C) with the following primary antibodies (all from Cell Signaling Technology, Danvers, MA, USA): anti-phospho-mTOR Ser2448 (1:2000; #2971), anti-mTOR (1:2000; #2972), anti-phospho-p70S6K Thr389 (1:2000; #9234), anti-p70S6K (1:2000; #9202), anti-phospho-4EBP1 Thr37/46 (1:2000; #9459), anti-4EBP1 (1:2000; #9644), anti-phospho-p44/42 ERK1/2 Thr202/Thr204 (1:5000; #4370), and anti-p44/42 ERK1/2 (1:5000; #4695) in Can Get Signal, a dilution buffer (Toyobo). Thereafter, membranes were incubated with secondary antibodies (1:5000; #7074) in Can Get Signal for 1 h at room temperature (25–26°C). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation) and recorded with a ChemiDoc™ Touch imaging system (Bio-Rad). Signal intensity was analyzed using Image Lab v.5.2.1 (Bio-Rad). The ratio of phosphorylated to total protein expression was determined using arbitrary units.

Statistical analysis

All values are presented as the mean ± standard error (SE). Absolute muscle weight strongly correlated with body weight; therefore, relative skeletal muscle weight was calculated by dividing absolute muscle weight by body weight. Group differences were analyzed using two-way analysis of variance (ANOVA), one-way analysis of variance (ANOVA), or unpaired t-test. When an interaction (time of day × exercise) was observed, Bonferroni’s post-hoc test was performed. Significance was set at P < 0.05. All analyses were performed using GraphPad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA, USA).

Results

Time of day × exercise interactions were absent for body weight (P = 0.6434; ), fat weight (P = 0.7170; ), and average food intake (P = 0.5487; ). The fat weight in rats in the L(D)TR group was significantly decreased compared with that of rats in the L(D) control (C) group (). No difference was observed in body weight () and average food intake between the groups (). The weekly food intake in the 1st and 2nd weeks was significantly lower in L(D)TR rats than in L(D)C rats ().

Body weight, fat weight, and food intake.

Body weight (a), fat weight (epididymal white adipose tissue) (b), average food intake (c), and weekly food intake (each value represents the mean of 6 days) (d). L, light phase; D, dark phase; C, control; TR, trained group. Values are the mean ± standard error (SE); n = 6 per group. The results of the two-way analysis of variance (ANOVA) are displayed in a, b, c; *P < 0.05, **P < 0.01. The results of the one-way analysis of variance (ANOVA) are displayed in d; ✝LC versus LTR, P < 0.05; ♯DC versus DTR, P < 0.05.

We assessed time of day × exercise interaction effects on the plantaris and gastrocnemius muscle weights (). No such effects could be observed. Time of day × exercise interaction was significant for absolute (P < 0.05; ) and relative (P < 0.05; ) SOL weights, as well as for SOL fiber CSA (P < 0.05; ). Control groups did not differ in absolute and relative SOL weights at different times of the day. However, between the trained animals, LTR rats had significantly heavier muscles than DTR rats (absolute muscle weight, +12.8%, P < 0.001; relative muscle weight, +9.4%, P < 0.05; ). We observed no significant difference in CSA between LTR and DTR rats, although the former was slightly larger (+5.3%, P = 0.4748; ). Muscle fiber CSA was significantly greater in LTR rats than in LC rats (+17.9%, P < 0.0001), but that in DTR and DC rats did not differ (P = 0.5221), as shown in . The representative histological findings of the SOL muscles from each group are shown in . No time of day × exercise interaction effects on the prevalence of centronucleated fibers in soleus muscle could be observed (P = 0.9080; ).

Effect of eccentric exercise on soleus muscle at different times of the day for 8 weeks.

Soleus muscle weight (a), soleus muscle weight relative to body weight (b), soleus cross-sectional area (CSA) (c), hematoxylin and eosin (H&E) staining of rat soleus muscle sections (10× magnification, scale bar = 50 μm) (d), centronucleated fibers of the soleus muscle (e). L, light phase; D, dark phase; C, control; TR, trained group. Values are the mean ± standard error (SE); n = 6 per group. The results of the two-way analysis of variance (ANOVA) are displayed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Time of day and exercise significantly affected (P < 0.05 and P < 0.01, respectively) mTOR phosphorylation, which was higher in LPt0 rats than in LPre rats after acute eccentric exercise (). Although we did not observe time of day × exercise interaction effects on the phosphorylation ratios of mTOR (P = 0.4291; ), 4EBP1 (P = 0.7991; ), and ERK1/2 (P = 0.9119; ), p70S6K phosphorylation significantly changed after exercise (P < 0.001; ) and was higher in LPt0 rats than in DPt0 rats (+42.6%, P < 0.01; ). Exercise significantly affected (P < 0.001) ERK1/2 phosphorylation, which was higher in L(D)Pt0 rats than in L(D)Pre and L(D)Pt1 rats after acute eccentric exercise ().

Effect of eccentric exercise on mTOR signaling in soleus muscle at different times of the day.

Phosphorylation rates of mTOR (a), p70S6K (b), 4EBP1(c), and ERK1/2 (d) in the soleus muscle. Samples were collected before (Pre), immediately after (Pt0), and 1 h after (Pt1) eccentric exercise. L, light phase; D, dark phase. Values are the mean ± standard error (SE); n = 7 per group. The results of the two-way analysis of variance (ANOVA) are displayed. **P < 0.01, ****P < 0.0001.

Time of day and exercise significantly affected (P < 0.001 and P < 0.0001, respectively) serum corticosterone concentration, but we did not observe time of day × exercise interaction effects on the serum corticosterone concentration (P = 0.4379; ). Exercise significantly affected serum corticosterone concentration, which was higher in L(D)Pt0 rats than in L(D)Pre rats and higher in DPt0 rats than in DPt1 (). We observed no significant difference in serum corticosterone concentration between LPt0 and DPt0 rats, but the increased rate of serum corticosterone immediately after eccentric exercise in the light phase group (P < 0.05; ) was significantly lower than that in the dark phase group.

Increased rate of serum corticosterone immediately after eccentric exercise.

Serum corticosterone concentration (a), rate of increase in serum corticosterone (b). L, light phase; D, dark phase. Values are the mean ± standard error (SE); n = 7 per group. The results of the two-way analysis of variance (ANOVA) are displayed in a. The results of the unpaired t-test are displayed in b. *P < 0.05, **P < 0.01, ****P < 0.0001.

Discussion

The main finding of our study was that 8 weeks of eccentric training in the light phase induced a greater increase in SOL mass and size than in the dark phase. This difference may be associated with greater phosphorylation of proteins involved in mTOR/p70S6K signaling. These data support previous research in humans, demonstrating that after 24 weeks of combined strength and endurance training, exercising at different times of day may affect the degree of muscle hypertrophy [9].

In this study, using the same low-intensity downhill running protocol as in the previous study, the same results were seen, with no difference in body weight after training in each group [11]. A significant increase in body fat was observed in the control group but not in the exercise group, confirming the effect of exercise training on suppressing the increase in fat mass. This result is the same as that in the previous study (low-intensity downhill running; incline less than -16°, speed lower than 16 m/min) [13]. During the first two weeks of this study, the average weekly intake in the training group was lower than that in the control group. As was also observed under this situation in previous studies, the intake in the low-intensity exercise group, compared to that in the no-exercise group, first dropped and then returned to the intake in the no-exercise group [14]. It is thought that exercise-induced appetite suppression is due to increased catecholamines associated with exercise stress [15] or increased release of corticotropin-releasing factors (appetite-suppressing peptide) by the hypothalamus [16].

Although acute signaling response does not always yield chronic effects, previous studies on rat skeletal muscle have demonstrated that the activation of p70S6K signaling is associated with hypertrophic effects of chronic exercise training [17, 18]. Therefore, the fact that mTOR/p70S6K signaling was higher in the light phase than in the dark phase suggests that circadian rhythms are part of the mechanisms underlying exercise-related increase in muscle mass and size. Notably, however, in a previous human study, it was found that time of the day did not influence resistance-exercise-induced p70S6K phosphorylation [19]. Therefore, further research is required to clarify precise mechanisms of exercise-related skeletal muscle hypertrophy, which might explain the discrepancies across studies.

ERK signaling has been suggested to play a role in the signaling network required for regeneration and hypertrophy [20] in the skeletal muscle. Furthermore, ERK signaling pathway is activated by exercise, depending on its intensity [21]. Previous research demonstrated that resistance exercise caused an increase in phosphorylation of the ERK signaling in rat and human skeletal muscle [22, 23]. In this study, ERK signaling was also increased after acute exercise, but no difference was observed at different periods in the day (light vs. dark). This result indicates that ERK signaling is an unlikely pathway for exercise-induced muscle hypertrophy at different periods. Nevertheless, the results of ERK signaling in this study can prove that the intensity of exercise performed at different periods is the same.

In previous studies, it was observed that 4EBP1 signaling after resistance exercise was unchanged compared to that in the case of no exercise [24, 25]. However, recent studies have shown that after resistance exercise, 4EBP1 signaling was significantly higher than the signaling without exercise [26, 27]. Therefore, the effect of exercise on 4EBP1 is still unknown. In the present study, there was no change in 4EPB1 with exercise at different times. This result also indicates that 4EBP1 signaling is unlikely to be the pathway for exercise-induced muscle hypertrophy at different times. In contrast, the result of p70S6K signaling is different in this study; differential activation of p70S6K signaling after eccentric exercise was key in the SOL muscle hypertrophy.

Little is known about the differential p70S6K activation at different periods in the day; however, this may be due to the effect of serum corticosterone. Serum corticosterone may be associated with greater activation of mTOR/p70S6K in the light phase than in the dark phase. Interestingly, in a previous study, it was demonstrated that glucocorticoid (corticosterone in rats, cortisol in humans) levels regulate p70S6K phosphorylation, with higher concentrations suppressing phosphorylation in L6 skeletal myoblasts [28]. There are two possible mechanisms to explain the effect of corticosterone on the mTOR signaling pathway. The first is that glucocorticoids activate glucocorticoid receptors (GR), leading to a decrease in the concentration of intracellular branched-chain amino acids (BCAAs) via increased expression of branched-chain amino transferase 2 (BCAT2) by KLF15, and that via Ras homolog enriched in brain (Rheb) leads to a decrease in the mTOR activity [29, 30]. In the second pathway, glucocorticoids promote the assembly of the TSC1-2 (hamartin-tuberin) complex by regulating the development and DNA damage responses 1 (REDD1) through activation of the GR, and finally reduce the mTOR activity via Rheb [30-32].

The effect of corticosterone in rodents is likely linked to circadian rhythm, as muscle protein breakdown occurs 4 h after the plasma corticosterone concentration peaks [33] at approximately midday (between ZT11 and ZT13, or at the end of the inactive phase and beginning of the active phase) [4, 34]. In this study, the serum corticosterone concentration in the light period (ZT6) group was generally higher than that in the dark period (ZT18) group, possibly due to the interconnection between circadian rhythm and corticosterone concentration that peaked around ZT11-13. In contrast, the corticosterone peak in the dark period (ZT18) group was past and the concentration had lowered. The serum corticosterone levels in the light phase and dark phase groups immediately after exercise were not significantly different, but the rate of increase in corticosterone concentration immediately after exercise was greater in the dark phase than in the light phase group. These observations tentatively suggest that corticosterone has a slightly antagonistic impact on mTOR/p70S6K signaling during the light phase, explaining the increase in exercise-induced p70S6K phosphorylation and subsequent muscle hypertrophy during that time of the day.

Increased circadian dependence of serum corticosterone may be associated with a 1 h exposure to light in the dark phase [35]. This may reduce the effectiveness of exercise in the dark phase under light exposure. However, to block the light in this study, we covered the treadmill using a black shielding bag for 8 weeks during the exercise experiment for the DTR group, and the room temperature was monitored. Therefore, it is believed that the increase in corticosterone due to light exposure at night can be suppressed.

Previous studies have shown a relationship between mTOR/p70S6K and muscle protein synthesis. A mouse study by Ogasawara et al. [36] reported that the promotion of muscle protein synthesis by resistance exercise (transcutaneous electrical stimulation to induce muscle contraction) is regulated by mTOR (both rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC1 or mTORC2). However, a recent study reported that exercise-induced protein synthesis is independent of mTOR/p70S6K. You et al. [37] reported that protein synthesis by mechanical loading is mTORC1-independent in the case of myotenectomy-induced muscle hypertrophy in mice. Different experimental methods of inducing muscle hypertrophy-like electrical stimulation or myotenectomy-induced muscle hypertrophy may also result in different pathways of muscle protein synthesis. Therefore, the relationship between mTOR/p70S6K and muscle protein synthesis remains unclear. In this study also, we have not elucidated the relationship between mTOR/p70S6K as we have not measured muscle protein synthesis.

Our current findings contradict the results of a previous study demonstrating that 6 weeks of aerobic training (swimming for 60 min/bout, 5 bouts/week) increased relative gastrocnemius muscle weight, more so in the dark phase than in the light phase [38]. Differences in exercise type and muscle fiber type between studies could explain the discrepancy. Previous research indicated that different exercises irrefutably activate specific intracellular signaling pathways [39, 40]. Moreover, hormones as indicators of systemic factors also affect muscle mass; for example, differences in hormone levels in the body (testosterone:corticosterone ratio) or differences in hormone levels caused by exercise. Therefore, the effect of systemic factors on muscle mass cannot be ignored [41, 42].

Although previous studies in human subjects have demonstrated that the timing of feeding post-exercise (1–3 hours after training) plays an important role in promoting protein synthesis [43], it has recently been shown that training increases the sensitivity of muscle protein synthesis for at least 24 hours [44, 45]. The intensity and volume of exercise also influence the increase in post-exercise feeding-related protein synthesis [44, 46]. However, previous studies have shown that the effect of post-exercise feeding timing on muscle protein synthesis is attenuated when exercise does not take place until exhaustion [44]. In this study, we used low-intensity exercise [11, 47]; therefore, we believe that increase in muscle mass due to post-exercise feeding might be difficult.

Although previous research found differences in food intake between trained groups at different periods in the day (light vs. dark) [38], the daily food consumption of the trained groups used in this study at different periods in the day did not differ despite the rats having ad libitum access to water and food. Therefore, unlike in the previous study, we can better associate the muscle weight with exercise, rather than with changes in body weight due to differential food intake. As exercise type (endurance or resistance) affects muscles differently, future studies should consider this parameter before investigating whether circadian rhythms definitively affect the exercise-induced elevation of mTOR/p70S6K signaling and associated muscle hypertrophy.

The previous study on rats has shown that most of the fiber components of the soleus muscle are mobilized during downhill running (incline: -16°) [48]. In addition, when low-intensity exercise (10–20 m/min) is performed with downhill running (treadmill grade 15% decline), fast-twitch muscles might be involved to a much lesser extent in the motor unit recruitment [49]. Therefore, the cause of the induced muscle hypertrophy only in the soleus muscle could be determined as only the low-intensity exercise was involved in this study. It is thought that low-intensity exercise may not reflect the effects of circadian rhythm on fast-twitch muscles as the fast-twitch muscles are scarcely stimulated low-intensity exercise. In the future, employment of high-intensity exercise will be required to prove this.

Upon the onset of muscle damage, satellite cells are activated; they proliferate and differentiate, eventually fusing with other satellite cells to form myofibers with central nuclei [50, 51]. Therefore, the central nuclei (shown as the ratio of the centronucleated fiber) are used as an indicator of regenerating muscle. It was previously reported that the central nucleus of the soleus muscle of rats was clearly observed after 5 days of a bout of downhill running [51]. However, in this study, there was no difference in the central nucleus in any of the groups because we used the same training intensity and training time for 8 weeks. Therefore, the muscles would be accustomed to the training stimulus, and damage would be less likely to occur.

The clock gene has been reported to be involved in the structure and function of skeletal muscle [52]. It has also been demonstrated that exercise affects the circadian clock (Bmal1/CLOCK) of skeletal muscle [53]. Based on these studies, it may be possible to influence muscles from exercise via the clock genes. As per recent studies, some pathways are likely to influence Per1 by glucocorticoids via REDD1 [54]. Another pathway possibly affects BMAL1 through the activation of mTORC1 [55]. However, further research is needed to confirm whether these pathways affect muscle mass.

We acknowledge that we did not assess changes in mTOR/p70S6K signaling during exercise periods, as we focused only on time points immediately after one exercise session, and at the end of an 8-week session. Future work, including time points during the exercise period, should provide a deeper understanding of the mTOR/p70S6K involvement. We also did not directly measure muscle protein synthesis rate after exercise. Thus, we cannot be certain that enhancement in mTOR/p70S6K signaling directly led to post-exercise muscle hypertrophy. Despite these limitations, our data provide novel insights into the potential influence of circadian rhythms when determining the effectiveness of eccentric exercise in the gaining of muscle mass and size. Future studies are required to clarify the effects of corticosterone administration on protein synthesis (mTOR/p70S6K signaling) following eccentric exercise.

Conclusions

In conclusion, we suggest that eccentric exercise training at different times of the day affects the extent of muscle hypertrophy in rat SOL, a change potentially mediated by differential phosphorylation of the mTOR/p70S6K signaling pathway. The circadian rhythm of corticosterone is a prime candidate for the regulation of differential p70S6K activation in response to eccentric exercise throughout the day.

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11 Jun 2021

PONE-D-21-07409

Circadian rhythms modulate the effect of eccentric exercise on rat skeletal muscles via the mTOR signaling pathway

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Reviewer #1: This study aimed to investigate the effect of circadian rhythms on exercise-mediated muscle anabolism. The authors concluded that the degree of eccentric exercise-mediated muscle hypertrophy in soleus muscle is time-of-day dependent and circadian rhythms of corticosterone and mTOR/p70S6K signaling affect this adaptation. Although this is an interesting project that will bring important insight to our field, there are many concerns.

L46-48: The authors do not follow the current dogma that mTORC1/p70S6K is not a major signaling pathway in the regulation of contraction-induced protein synthesis. Also, ref. 10 does not report the correlation between signaling and muscle hypertrophy. The authors should revise this point and further add the reference that ERK signaling contributes to the contraction-induced protein synthesis.

L68: Why did the authors only investigate soleus muscle? This point significantly reduces the value of this paper. Generally, downhill running affects not only soleus muscle but also gastrocnemius (and plantalis) muscle and therefore the authors should add the data of gastrocnemius at least the results of chronic training.

L84-87: Was exercise intensity constant?

L95-96: Ref. 4 (your previous study) reported that mTOR signaling is high at ZT6 as compared with at ZT18 in plantaris but not soleus.

L100: Why did the authors use different exercise protocols between acute and chronic exercise?

L123-: Is 99% glycerol correct? What does “samples containing total protein” mean?

L156-162: Why is there no difference in body weight between the control and exercise training groups when the control rats eat a lot and do not exercise? Need discussion and add fat mass data if possible.

L187-191: mTOR S2448 phosphorylation does not necessarily associate mTORC1 activity. The authors can evaluate mTORC1 activity more appropriately to measure both p70S6K and 4E-BP1 phosphorylation.

L202-204: The authors report only a %change in serum corticosterone concentration. Please add absolute values at pre- and post-exercise.

L217-226: Resistance exercise increases mTORC1 signaling for more than 24h after exercise while endurance exercise activates mTORC1 signaling only immediately after exercise. The results of this study are in accordance with the signaling responses of endurance exercise rather than resistance exercise, suggesting that factors other than mTORC1 signaling associate downhill running-induced muscle hypertrophy.

L228: Ref. 4 does not report the role of ERK signaling in the mechanisms of muscle hypertrophy.

L236-253: Regarding corticosterone, the authors state that serum corticosterone may be associated mTOR/p70S6K signaling, as it has a role in muscle protein breakdown. Please discuss more specifically with the references. How muscle protein breakdown affects mTOR signaling?

Serum corticosterone response may affect the mTOR signaling response but how about the resting condition? Not resting (absolute value) but only responses affect mTOR signaling?

It is interesting and important to measure REDD1 to know the mechanisms of reduced mTORC1 responses.

L260-265: Obviously, exercise type and fiber type affect the signaling responses. However, the authors suggested that different adaptation to the training is led by serum corticosterone (systemic factor). Therefore, the authors should discuss not only signaling but also systemic factors.

L268: Which paper shows that the timing of feeding is important to induce muscle hypertrophy?

L269: Ref. 30 is a review paper. So please add the original article(s).

Reviewer #2: Please if it is possible to show the circadian clock proteins or the gene expression, since exercise could have modified your circadian clock (i.e Bmal1 / CLOCK). If you can't do this experiment please discuss.

Please detail the food intake in a better way (how much they ate in the night phase, how much they ate in the light phase, adjust the food intake to the weight of the animal ...) and discuss, this is a very important point of the study. I suggest making a table with date of food intake.

Please take better advantage of the H&E, measure the number of membrane nuclei and central nuclei, remembering that eccentric exercise generates high muscle damage.

Since you have individual corticosterone values (changes) and you believe that it is responsible for your cellular results, please make correlations between changes in corticosterone and changes in protein signaling, CSA and strength level.

**********

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4 Oct 2021

PONE-D-21-07409

Circadian rhythms modulate the effect of eccentric exercise on rat skeletal muscles via the mTOR signaling pathway

PLOS ONE

We wish to express our appreciation to the reviewers for their insightful comments on our manuscript. Their comments have helped us significantly improve our paper. We have attempted to address all their concerns. Our point-by-point responses to the comments are given below.

Responses to Reviewer #1

Comment: This study aimed to investigate the effect of circadian rhythms on exercise-mediated muscle anabolism. The authors concluded that the degree of eccentric exercise-mediated muscle hypertrophy in soleus muscle is time-of-day dependent and circadian rhythms of corticosterone and mTOR/p70S6K signaling affect this adaptation. Although this is an interesting project that will bring important insight to our field, there are many concerns.

Comment 1: L46-48: The authors do not follow the current dogma that mTORC1/p70S6K is not a major signaling pathway in the regulation of contraction-induced protein synthesis. Also, ref. 10 does not report the correlation between signaling and muscle hypertrophy. The authors should revise this point and further add the reference that ERK signaling contributes to the contraction-induced protein synthesis.

Response 1: Thank you for your suggestion. There are many signaling pathways that promote muscle growth, such as Insulin/IGF1-AKT-mTOR, TGFβ/myostatin/activin/BMP, β-adrenergic signaling, FGF/zinc ions, and desmosomes/Zinc ions (Sartori et al., 2021). However, until recently, the mTORC1/p70S6K (Kotani et al., 2021; Ashida et al., 2018; Ribeiro et al., 2017) and ERK (Williamson, et al., 2003; Takegaki et al., 2019) signaling have still been used as an indicator for the synthesis of muscle proteins. Therefore, we believe that both mTORC1/p70S6K and ERK signaling are important. We will investigate the other contraction-induced protein synthesis pathways in the future.

We have included the abovementioned information in the revised text and cited the relevant references (p. 3, lines 47–48; p. 16–17, lines 273–280).

References:

Sartori, R., Romanello, V., & Sandri, M. (2021). Mechanisms of muscle atrophy and hypertrophy: Implications in health and disease. Nature Communications, 12(1), 1-12.

Kotani, T., Takegaki, J., Tamura, Y., Kouzaki, K., Nakazato, K., & Ishii, N. (2021). The effect of repeated bouts of electrical stimulation-induced muscle contractions on proteolytic signaling in rat skeletal muscle. Physiological Reports, 9(9), e14842.

Ashida, Y., Himori, K., Tatebayashi, D., Yamada, R., Ogasawara, R., & Yamada, T. (2018). Effects of contraction mode and stimulation frequency on electrical stimulation-induced skeletal muscle hypertrophy. Journal of Applied Physiology, 124(2), 341-348.

Ribeiro, M. B. T., Guzzoni, V., Hord, J. M., Lopes, G. N., de Cássia Marqueti, R., de Andrade, R. V., ... & Durigan, J. L. Q. (2017). Resistance training regulates gene expression of molecules associated with intramyocellular lipids, glucose signaling and fiber size in old rats. Scientific Reports, 7(1), 1-13.

Williamson, D., Gallagher, P., Harber, M., Hollon, C., & Trappe, S. (2003). Mitogen-activated protein kinase (MAPK) pathway activation: effects of age and acute exercise on human skeletal muscle. The Journal of Physiology, 547(3), 977-987.

Takegaki, J., Sase, K., & Fujita, S. (2019). Repeated bouts of resistance exercise attenuate mitogen-activated protein-kinase signal responses in rat skeletal muscle. Biochemical and Biophysical Research Communications, 520(1), 73-78.

Comment 2: L68: Why did the authors only investigate soleus muscle? This point significantly reduces the value of this paper. Generally, downhill running affects not only soleus muscle but also gastrocnemius (and plantalis) muscle and therefore the authors should add the data of gastrocnemius at least the results of chronic training.

Response 2: Thank you for your suggestion. We have presented the observations made in the gastrocnemius and plantalis muscles in Table 1 (p. 11–12, lines 192–196). We have indicated that only soleus, for which interaction was found, was used in the analysis because there was no interaction or other effect of downhill training on gastrocnemius or plantalis.

Comment 3: L84-87: Was exercise intensity constant?

Response 3: Yes, it was constant. The program for each training session was 16 m/min (incline: -16°) and 90 minutes of exercise time.

Comment 4: L95-96: Ref. 4 (your previous study) reported that mTOR signaling is high at ZT6 as compared with at ZT18 in plantaris but not soleus.

Response 4: In the SOL, there was no significant difference, but there was a 49.5% difference observed between the two points near the highest and lowest values; thus, even if there was no difference at rest (or even if the difference was not large), the effects of exercise could still be different.

Comment 5: L100: Why did the authors use different exercise protocols between acute and chronic exercise?

Response 5: Although the increase and duration of the signal may be different when the time is shortened, the time is shortened because it helps obtain an understanding of the signaling pathways involved. We decided that it is not necessary to use the same protocol in order to see the response reflected one time because each response will be different during the adaptation process.

Comment 6: L123-: Is 99% glycerol correct? What does “samples containing total protein” mean?

Response 6: Thank you for pointing this out. The final concentration was 30% glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 62.5 mm Tris–HCl pH 6.8, and bromphenol blue. We have corrected the relevant text in the revised manuscript (p. 8, lines 128–129).

“Samples containing total protein” are samples of mixtures of protein extracts and sample buffer.

Comment 7: L156-162: Why is there no difference in body weight between the control and exercise training groups when the control rats eat a lot and do not exercise? Need discussion and add fat mass data if possible.

Response 7: We agree with the reviewer’s contention, and acknowledge that the suggestion made by the reviewer is valuable. We have added the fat mass data (revised Fig. 1) and have discussed this issue in the Discussion section of the revised manuscript (p. 15, lines 248–260).

We noticed that the information on food intake in the original manuscript was incorrect. We apologize for this oversight and have corrected it in the revised manuscript. The food intake in the control group was almost the same as that in the training group; however, rats in the control group in this study had more body fat (epididymal white adipose tissue) than those in the training group.

Comment 8: L187-191: mTOR S2448 phosphorylation does not necessarily associate mTORC1 activity. The authors can evaluate mTORC1 activity more appropriately to measure both p70S6K and 4E-BP1 phosphorylation.

Response 8: We agree with the valuable comment made by the reviewer. We added 4E-BP1 data and modified the relevant text in the revised manuscript (revised Fig. 3 and p. 17, lines 281–290).

Comment 9: L202-204: The authors report only a %change in serum corticosterone concentration. Please add absolute values at pre- and post-exercise.

Response 9: Thank you for your suggestion. We have added the serum corticosterone concentration data (revised Fig. 4 and p. 14, lines 225–239).

Comment 10: L217-226: Resistance exercise increases mTORC1 signaling for more than 24h after exercise while endurance exercise activates mTORC1 signaling only immediately after exercise. The results of this study are in accordance with the signaling responses of endurance exercise rather than resistance exercise, suggesting that factors other than mTORC1 signaling associate downhill running-induced muscle hypertrophy.

Response 10: Thank you for your keen analysis. The signaling pattern described in the comment is certainly true for humans, but that in animals can also be degraded in a relatively short time in response to hypertrophy-inducing movements, such as electrical stimulation (Bolster et al., 2003). However, we believe that the timing of the exercise is also important. In this study, we assessed the effects of a bout of exercise on Wistar rats to check whether exercising at different times (ZT6 or ZT18) had different effects on mTOR/p70S6K signaling.

References:

Bolster, D. R., Kubica, N., Crozier, S. J., Williamson, D. L., Farrell, P. A., Kimball, S. R., & Jefferson, L. S. (2003). Immediate response of mammalian target of rapamycin (mTOR)‐mediated signalling following acute resistance exercise in rat skeletal muscle. The Journal of physiology, 553(1), 213-220.

Comment 11: L228: Ref. 4 does not report the role of ERK signaling in the mechanisms of muscle hypertrophy.

Response 11: Ref. 4 was not cited on line 228. In Ref. 4, we described that the physiological significance of circadian variation in ERK phosphorylation in the cardiac and plantaris muscles remains unclear. Based on the results of transient exercise at different times in this study, there was no difference in ERK signaling, suggesting that this signaling is less likely as a pathway for exercise-induced muscle hypertrophy in the light period. Nevertheless, the results of ERK signaling in this study can prove that the intensity of exercise performed at different times is the same.

We have modified the relevant text in the revised manuscript because our explanation may be inadequate (p. 16–17, lines 277–280).

Comment 12: L236-253: Regarding corticosterone, the authors state that serum corticosterone may be associated mTOR/p70S6K signaling, as it has a role in muscle protein breakdown. Please discuss more specifically with the references. How muscle protein breakdown affects mTOR signaling?

Serum corticosterone response may affect the mTOR signaling response but how about the resting condition? Not resting (absolute value) but only responses affect mTOR signaling?

It is interesting and important to measure REDD1 to know the mechanisms of reduced mTORC1 responses.

Response 12:

Thank you for your suggestion. Considering muscle protein degradation affects mTOR signaling, we have added a discussion to the revised manuscript (p. 17–18, lines 297–306).

The serum corticosterone concentration in the light period (ZT6) group was generally higher than that in the dark period (ZT18) group, possibly due to the interconnection between circadian rhythm and corticosterone concentration that peaked around ZT11-13. In contrast, the corticosterone peak in the dark period (ZT18) group was past and the concentration had lowered. The serum corticosterone levels in LTR and DLR groups immediately after exercise were not significantly different, but the rate of increase in corticosterone concentration immediately after exercise was greater in the dark-phase group than in the light-phase group (revised Fig. 4). These observations tentatively suggest that corticosterone has a slightly antagonistic impact on mTOR/p70S6K signaling during the light phase, explaining the increase in exercise-induced p70S6K phosphorylation and subsequent muscle hypertrophy during that time of day (p. 18–19, lines 310–317). However, the underlying mechanisms are unknown, and would be investigated in the future.

We tried to analyze the expression of REDD1 (35 kDa) using antibodies against it (10638-1-AP) purchased from Proteintech Group, Inc., but could not detect clear bands (as shown in the figure below).

Comment 13: L260-265: Obviously, exercise type and fiber type affect the signaling responses. However, the authors suggested that different adaptation to the training is led by serum corticosterone (systemic factor). Therefore, the authors should discuss not only signaling but also systemic factors.

Response 13:

Thank you for your suggestion. We have added the following discussion to the revised manuscript (p. 19, lines 334–338):

Moreover, hormones as indicators of systemic factors also affect muscle mass; for example, differences in hormone levels in the body (testosterone:corticosterone ratio) or differences in hormone levels caused by exercise. Therefore, the effect of systemic factors on muscle mass cannot be ignored (Crowley & Matt, 1996; Guzzoni, 2019).

Comment 14: L268: Which paper shows that the timing of feeding is important to induce muscle hypertrophy?

Response 14: Thank you for this query. We have cited the following reference (p. 20, line 341):

Phillips, S. M., Tipton, K. D., Aarsland, A., Wolf, S. E., & Wolfe, R. R. (1997). Mixed muscle protein synthesis and breakdown after resistance exercise in humans. American Journal of Physiology, 273(1), E99-E107.

Comment 15: L269: Ref. 30 is a review paper. So please add the original article(s).

Response 15: Thank you for your suggestion. We have cited the following original article (p. 20, line 344):

Burd, N. A., West, D. W., Moore, D. R., Atherton, P. J., Staples, A. W., Prior, T., ... & Phillips, S. M. (2011). Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up to 24 h after resistance exercise in young men. The Journal of Nutrition, 141(4), 568-573.

Responses to Reviewer #2

Comment 1: Please if it is possible to show the circadian clock proteins or the gene expression, since exercise could have modified your circadian clock (i.e Bmal1 / CLOCK). If you can't do this experiment please discuss.

Response 1: Thank you for your suggestion. Unfortunately, we could not measure the gene expression in this study because of limitation of the samples. Therefore, we have added relevant discussion about it in the revised manuscript. (p. 21, lines 367–373).

The clock gene has been reported to be involved in the structure and function of skeletal muscle (Andrews et al., 2010). It has also been demonstrated that exercise affects the circadian clock (Bmal1/CLOCK) of skeletal muscle (Wolff et al., 2012). Based on these studies, it may be possible to influence muscles from exercise via the clock genes. As per recent studies, some pathways are likely to influence Per1 by glucocorticoids via REDD1 (Saracino et al., 2019). Another pathway possibly affects BMAL1 through the activation of mTORC1 (Dadon-Freiberg et al., 2021). However, further research is needed to confirm whether these pathways affect the muscle mass.

Comment 2: Please detail the food intake in a better way (how much they ate in the night phase, how much they ate in the light phase, adjust the food intake to the weight of the animal ...) and discuss, this is a very important point of the study. I suggest making a table with date of food intake.

Response 2: Thank you for your comments. We measured food intake, without food and water restrictions, after a single training session of 3 days. The weekly food intake for each group is shown in the revised figure 1. Unfortunately, we are unable to present the detailed feed intake. To avoid disrupting the biological rhythms and to minimize non-training stresses (entering and exiting the animal house, turning lights on and off, etc.) in the experimental animals, we took only a limited number of measurements. It has also been previously reported that exposure to light and food restriction alters the periodicity of endocrine, body temperature, and activity in experimental animals (Mohawk et al., 2007; Wideman et al., 2009; Depres-Brummer et al., 1995; Krieger et al., 1974). In addition, it is reported that factors like exercise and food restriction were stressful for experimental animals and affected the secretion of corticosterone (Fediuc, 2006; Heiderstadt, 2000). However, although we do not know exactly when the animals ate, there was no notable difference observed in food intake.

Comment 3: Please take better advantage of the H&E, measure the number of membrane nuclei and central nuclei, remembering that eccentric exercise generates high muscle damage.

Response 3: We are thankful for the valuable suggestion. We have added the data on the central nucleus as an indicator of regenerating muscle (revised Fig. 2) and have discussed this issue in the Discussion section of the revised manuscript (p. 21, lines 358–366).

Upon the onset of muscle damage, satellite cells are activated; they proliferate and differentiate, eventually fusing with other satellite cells to form myofibers with central nuclei (Sasaki et al., 2007; Yu et al., 2017). Therefore, the central nuclei (shown as the ratio of the centronucleated fiber) are used as an indicator of regenerating muscle. It was previously reported that the central nucleus of the soleus muscle of rats was clearly observed after 5 days of a bout of downhill running (Yu et al., 2017). However, in this study, there was no difference in the central nucleus in any of the groups because we used the same training intensity and training time for 8 weeks. Therefore, the muscles would be accustomed to the training stimulus, and damage would be less likely to occur.

Comment 4: Since you have individual corticosterone values (changes) and you believe that it is responsible for your cellular results, please make correlations between changes in corticosterone and changes in protein signaling, CSA and strength level.

Response 4: Thank you for your suggestion. We acknowledge that the analyses suggested by you would provide great insights. However, we could not determine the correlations between the variables because these data were obtained from different rats (i.e., they are not matching data).

Submitted filename: PONE_Responses_to_Reviewer20210701-5.docx

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6 Dec 2021

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Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

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Reviewer #2: Yes

**********

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Reviewer #1: (No Response)

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comment 1: L46-48: The authors do not follow the current dogma that mTORC1/p70S6K is not a major signaling pathway in the regulation of contraction-induced protein synthesis. Also, ref. 10 does not report the correlation between signaling and muscle hypertrophy. The authors should revise this point and further add the reference that ERK signaling contributes to the contraction-induced protein synthesis.

Response 1: Thank you for your suggestion. There are many signaling pathways that promote muscle growth, such as Insulin/IGF1-AKT-mTOR, TGFβ/myostatin/activin/BMP, β-adrenergic signaling, FGF/zinc ions, and desmosomes/Zinc ions (Sartori et al., 2021). However, until recently, the mTORC1/p70S6K (Kotani et al., 2021; Ashida et al., 2018; Ribeiro et al., 2017) and ERK (Williamson, et al., 2003; Takegaki et al., 2019) signaling have still been used as an indicator for the synthesis of muscle proteins. Therefore, we believe that both mTORC1/p70S6K and ERK signaling are important. We will investigate the other contraction-induced protein synthesis pathways in the future.

We have included the abovementioned information in the revised text and cited the relevant references (p. 3, lines 47–48; p. 16–17, lines 273–280).

It’s not scientific. They might use mTORC1 and ERK as an indicator but mTORC1/p70S6K is not a major signaling pathway in the regulation of contraction-induced protein synthesis (PMID: 26227152, 30509128. Please note that PMID: 30509128 reported that while the contraction-induced protein synthesis is mTORC1-independent, muscle hypertrophy is mTORC1-dependent).

Also, there is no evidence that ERK regulates contraction-induced muscle protein synthesis, and rather that is denied (PMID: 23077579).

Therefore, of course, the authors can use mTORC1 as an indicator but state and discuss carefully based on the fact because the authors did not measure muscle protein synthesis.

Comment 2: L68: Why did the authors only investigate soleus muscle? This point significantly reduces the value of this paper. Generally, downhill running affects not only soleus muscle but also gastrocnemius (and plantalis) muscle and therefore the authors should add the data of gastrocnemius at least the results of chronic training.

Response 2: Thank you for your suggestion. We have presented the observations made in the gastrocnemius and plantalis muscles in Table 1 (p. 11–12, lines 192–196). We have indicated that only soleus, for which interaction was found, was used in the analysis because there was no interaction or other effect of downhill training on gastrocnemius or plantalis.

Please discuss why exercise in this study induced muscle hypertrophy only in soleus muscle despite eccentric exercise predominantly activates fast-twitch fibers. Based on this result, the author should change the title from “rat skeletal muscle” to “rat soleus muscle”

Comment 4: L95-96: Ref. 4 (your previous study) reported that mTOR signaling is high at ZT6 as compared with at ZT18 in plantaris but not soleus.

Response 4: In the SOL, there was no significant difference, but there was a 49.5% difference observed between the two points near the highest and lowest values; thus, even if there was no difference at rest (or even if the difference was not large), the effects of exercise could still be different.

Even if there was a relatively large difference, if there was no significant difference, the authors should not be stated that there is a difference.

Comment 10: L217-226: Resistance exercise increases mTORC1 signaling for more than 24h after exercise while endurance exercise activates mTORC1 signaling only immediately after exercise. The results of this study are in accordance with the signaling responses of endurance exercise rather than resistance exercise, suggesting that factors other than mTORC1 signaling associate downhill running-induced muscle hypertrophy.

Response 10: Thank you for your keen analysis. The signaling pattern described in the comment is certainly true for humans, but that in animals can also be degraded in a relatively short time in response to hypertrophy-inducing movements, such as electrical stimulation (Bolster et al., 2003). However, we believe that the timing of the exercise is also important. In this study, we assessed the effects of a bout of exercise on Wistar rats to check whether exercising at different times (ZT6 or ZT18) had different effects on mTOR/p70S6K signaling.

Bolster et al 2003 used a squat model. They used ES to impose a jump but not to directly stimulate muscle activation. This squat model does not induce muscle hypertrophy and is currently not used as a resistance training model.

Comment 12: L236-253: Regarding corticosterone, the authors state that serum corticosterone may be associated mTOR/p70S6K signaling, as it has a role in muscle protein breakdown. Please discuss more specifically with the references. How muscle protein breakdown affects mTOR signaling?

Serum corticosterone response may affect the mTOR signaling response but how about the resting condition? Not resting (absolute value) but only responses affect mTOR signaling?

It is interesting and important to measure REDD1 to know the mechanisms of reduced mTORC1 responses.

Response 12:

Thank you for your suggestion. Considering muscle protein degradation affects mTOR signaling, we have added a discussion to the revised manuscript (p. 17–18, lines 297–306).

The main point was that how muscle protein breakdown affects mTOR. It is well known that corticosterone regulates protein breakdown and mTOR, respectively, but it is unclear how protein breakdown affects mTOR. Please delete the following sentence: “as it has a well-known role in the breakdown of muscle proteins.

Comment 14: L268: Which paper shows that the timing of feeding is important to induce muscle hypertrophy?

Response 14: Thank you for this query. We have cited the following reference (p. 20, line 341):

Phillips, S. M., Tipton, K. D., Aarsland, A., Wolf, S. E., & Wolfe, R. R. (1997). Mixed muscle protein synthesis and breakdown after resistance exercise in humans. American Journal of Physiology, 273(1), E99-E107.

They did not investigate the timing of feeding. Also, they did not measure muscle size.

Finally, please consider changing the title from “via the mTOR…” to “possibly via the mTOR…” or delete “via the mTOR…” because the authors have not proven causation.

Reviewer #2: (No Response)

**********

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15 Jan 2022

We appreciate the insightful comments from the reviewers. Their comments have helped us improve our paper. We have attempted to address all their concerns. Our point-by-point responses to the comments are given below.

Reviewer #1:

Comment 1: L46-48: The authors do not follow the current dogma that mTORC1/p70S6K is not a major signaling pathway in the regulation of contraction-induced protein synthesis. Also, ref. 10 does not report the correlation between signaling and muscle hypertrophy. The authors should revise this point and further add the reference that ERK signaling contributes to the contraction-induced protein synthesis.

Response 1: Thank you for your suggestion. There are many signaling pathways that promote muscle growth, such as Insulin/IGF1-AKT-mTOR, TGFβ/myostatin/activin/BMP, β-adrenergic signaling, FGF/zinc ions, and desmosomes/Zinc ions (Sartori et al., 2021). However, until recently, the mTORC1/p70S6K (Kotani et al., 2021; Ashida et al., 2018; Ribeiro et al., 2017) and ERK (Williamson, et al., 2003; Takegaki et al., 2019) signaling have still been used as an indicator for the synthesis of muscle proteins. Therefore, we believe that both mTORC1/p70S6K and ERK signaling are important. We will investigate the other contraction-induced protein synthesis pathways in the future.

We have included the abovementioned information in the revised text and cited the relevant references (p. 3, lines 47–48; p. 16–17, lines 273–280).

Comment 1-1: It’s not scientific. They might use mTORC1 and ERK as an indicator but mTORC1/p70S6K is not a major signaling pathway in the regulation of contraction-induced protein synthesis (PMID: 26227152, 30509128. Please note that PMID: 30509128 reported that while the contraction-induced protein synthesis is mTORC1-independent, muscle hypertrophy is mTORC1-dependent). Also, there is no evidence that ERK regulates contraction-induced muscle protein synthesis, and rather that is denied (PMID: 23077579). Therefore, of course, the authors can use mTORC1 as an indicator but state and discuss carefully based on the fact because the authors did not measure muscle protein synthesis.

Response 1-1:

We thank the reviewer for this suggestion. We acknowledge that in the study by You et al. (2019), protein synthesis by mechanical loading is mTORC1-independent, but muscle hypertrophy is mTORC1-dependent. However, some previous studies have shown that exercise-induced protein synthesis involves mTORC1. A study in mice by Ogasawara et al. (2018) reported that resistance exercise (transcutaneous electrical stimulation to induce muscle contraction) increases muscle protein synthesis via rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC1 or mTORC2. The difference in these results might be due to the differences in the pathways of muscle protein synthesis induced by different stimuli leading to muscle hypertrophy like electrical stimulation or myotenectomy. In this study, we did not measure muscle protein synthesis; therefore, the relationship between mTORC1 and muscle protein synthesis by downhill running is currently unknown. Following the reviewer's advice, we have described this in the Discussion section.

We have added the following discussion to the revised manuscript (p. 19–20, lines 327–339):

“Previous studies have shown a relationship between mTOR/p70S6K and muscle protein synthesis. A mouse study by Ogasawara et al. (2018) reported that the promotion of muscle protein synthesis by resistance exercise (transcutaneous electrical stimulation to induce muscle contraction) is regulated by mTOR (both rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC1 or mTORC2). However, a recent study reported that exercise-induced protein synthesis is independent of mTOR/p70S6K. You et al. (2019) reported that protein synthesis by mechanical loading is mTORC1-independent in the case of myotenectomy-induced muscle hypertrophy in mice. Different experimental methods of inducing muscle hypertrophy-like electrical stimulation or myotenectomy-induced muscle hypertrophy may also result in different pathways of muscle protein synthesis. Therefore, the relationship between mTOR/p70S6K and muscle protein synthesis remains unclear. In this study also we have not elucidated the relationship between mTOR/p70S6K as we have not measured muscle protein synthesis.”

References:

Ogasawara, R., & Suginohara, T. (2018). Rapamycin‐insensitive mechanistic target of rapamycin regulates basal and resistance exercise‐induced muscle protein synthesis. The FASEB Journal, 32(11), 5824-5834.

You, J. S., McNally, R. M., Jacobs, B. L., Privett, R. E., Gundermann, D. M., Lin, K. H., ... & Hornberger, T. A. (2019). The role of raptor in the mechanical load‐induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy. The FASEB Journal, 33(3), 4021-4034.

Comment 2: L68: Why did the authors only investigate soleus muscle? This point significantly reduces the value of this paper. Generally, downhill running affects not only soleus muscle but also gastrocnemius (and plantalis) muscle and therefore the authors should add the data of gastrocnemius at least the results of chronic training.

Response 2: Thank you for your suggestion. We have presented the observations made in the gastrocnemius and plantalis muscles in Table 1 (p. 11–12, lines 192–196). We have indicated that only soleus, for which interaction was found, was used in the analysis because there was no interaction or other effect of downhill training on gastrocnemius or plantalis.

Comment 2-1: Please discuss why exercise in this study induced muscle hypertrophy only in soleus muscle despite eccentric exercise predominantly activates fast-twitch fibers. Based on this result, the author should change the title from “rat skeletal muscle” to “rat soleus muscle”

Response 2-1:

We thank the reviewer for this suggestion. We have added the following description to the revised manuscript (p. 21–22, lines 368–377) and changed the title to ‘rat soleus muscle’.

The previous study on rats has shown that most of the fiber components of the soleus muscle are mobilized during downhill running (incline: -16°) (Smith et al., 1997). In addition, when low-intensity exercise (10-20 m/min) is performed with downhill running (treadmill grade 15% decline), fast-twitch muscles might be involved to a much lesser extent in the motor unit recruitment (Dudley et al., 1982). Therefore, the cause of the induced muscle hypertrophy only in the soleus muscle could be determined as only the low-intensity exercise was involved in this study. It is thought that low-intensity exercise may not reflect the effects of circadian rhythm on fast-twitch muscles as the fast-twitch muscles are scarcely stimulated by low-intensity exercise. In the future, employment of high-intensity exercise will be required to prove this.”

References:

Smith, H. K., Plyley, M. J., Rodgers, C. D., & McKee, N. H. (1997). Skeletal muscle damage in the rat hindlimb following single or repeated daily bouts of downhill exercise. International journal of sports medicine, 18(02), 94-100.

Dudley, G. A., Abraham, W. M., & Terjung, R. L. (1982). Influence of exercise intensity and duration on biochemical adaptations in skeletal muscle. Journal of applied physiology, 53(4), 844-850.

Comment 4: L95-96: Ref. 4 (your previous study) reported that mTOR signaling is high at ZT6 as compared with at ZT18 in plantaris but not soleus.

Response 4: In the SOL, there was no significant difference, but there was a 49.5% difference observed between the two points near the highest and lowest values; thus, even if there was no difference at rest (or even if the difference was not large), the effects of exercise could still be different.

Comment 4-1: Even if there was a relatively large difference, if there was no significant difference, the authors should not be stated that there is a difference.

Response 4-1:

We apologize for the inadequate explanation in our previous reply. The statistically significant difference described in our previous study (Reference no. 4) was calculated using a statistical method of the modified cosinor analysis to determine the existence of a circadian rhythm. This is different from the tests of differences between means that are usually used (e.g., t-test). Therefore, we think that the statement in our previous publication, "Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p < 0.05), but not in the soleus muscle." is reasonable. In addition, in the cited study (Reference no. 4), we do not claim that the mTOR signaling is high at ZT6 as compared with at ZT18 in plantaris but not soleus.

Comment 10: L217-226: Resistance exercise increases mTORC1 signaling for more than 24h after exercise while endurance exercise activates mTORC1 signaling only immediately after exercise. The results of this study are in accordance with the signaling responses of endurance exercise rather than resistance exercise, suggesting that factors other than mTORC1 signaling associate downhill running-induced muscle hypertrophy.

Response 10: Thank you for your keen analysis. The signaling pattern described in the comment is certainly true for humans, but that in animals can also be degraded in a relatively short time in response to hypertrophy-inducing movements, such as electrical stimulation (Bolster et al., 2003). However, we believe that the timing of the exercise is also important. In this study, we assessed the effects of a bout of exercise on Wistar rats to check whether exercising at different times (ZT6 or ZT18) had different effects on mTOR/p70S6K signaling.

Comment 10-1: Bolster et al 2003 used a squat model. They used ES to impose a jump but not to directly stimulate muscle activation. This squat model does not induce muscle hypertrophy and is currently not used as a resistance training model.

Response 10-1:

We apologize for the lack of explanation. As pointed out by the reviewer, the study by Bolster et al. (2003) is an experiment in which rats were made to jump using electrical stimulation. In addition, a previous study by Nader et al. (2001) used low-frequency electrical stimulation to increase mTORC1 signaling (p70S6K), which returned to baseline 6 hours after stimulation. We believe that different exercise intensities cause distinct changes in signaling.

Reference:

Nader, G. A., & Esser, K. A. (2001). Intracellular signaling specificity in skeletal muscle in response to different modes of exercise. Journal of applied physiology, 90(5), 1936-1942.

Comment 12: L236-253: Regarding corticosterone, the authors state that serum corticosterone may be associated mTOR/p70S6K signaling, as it has a role in muscle protein breakdown. Please discuss more specifically with the references. How muscle protein breakdown affects mTOR signaling?

Serum corticosterone response may affect the mTOR signaling response but how about the resting condition? Not resting (absolute value) but only responses affect mTOR signaling?

It is interesting and important to measure REDD1 to know the mechanisms of reduced mTORC1 responses.

Response 12:

Thank you for your suggestion. Considering muscle protein degradation affects mTOR signaling, we have added a discussion to the revised manuscript (p. 17–18, lines 297–306).

Comment 12-1: The main point was that how muscle protein breakdown affects mTOR. It is well known that corticosterone regulates protein breakdown and mTOR, respectively, but it is unclear how protein breakdown affects mTOR. Please delete the following sentence: “as it has a well-known role in the breakdown of muscle proteins.

Response 12-1:

As per the reviewer’s suggestion, we have now deleted the text (p. 17, lines 291–293).

Comment 14: L268: Which paper shows that the timing of feeding is important to induce muscle hypertrophy?

Response 14: Thank you for this query. We have cited the following reference (p. 20, line 341):

Phillips, S. M., Tipton, K. D., Aarsland, A., Wolf, S. E., & Wolfe, R. R. (1997). Mixed muscle protein synthesis and breakdown after resistance exercise in humans. American Journal of Physiology, 273(1), E99-E107.

Comment 14-1: They did not investigate the timing of feeding. Also, they did not measure muscle size.

Response 14-1:

Thank you for pointing this out. We have now replaced the references. We have also revised the text to avoid misunderstanding in the revised manuscript as follows (p. 20, lines 350–352):

“Although previous studies in human subjects have demonstrated that the timing of feeding post-exercise (1-3 hours after training) plays an important role in promoting protein synthesis (Rasmussen et al., 2000),…..”

Reference:

Rasmussen, B. B., Tipton, K. D., Miller, S. L., Wolf, S. E., & Wolfe, R. R. (2000). An oral essential amino acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise. Journal of applied physiology, 88(2), 386-392.

Finally, please consider changing the title from “via the mTOR…” to “possibly via the mTOR…” or delete “via the mTOR…” because the authors have not proven causation.

Response:

As per the reviewer's suggestion, we have now changed the title to “Circadian rhythms modulate the effect of eccentric exercise on rat soleus muscles.”

Reviewer #2: (No Response)

Submitted filename: PONE_Responses_to_Reviewer20220116-1.docx

Click here for additional data file.

7 Feb 2022

Circadian rhythms modulate the effect of eccentric exercise on rat soleus muscles

PONE-D-21-07409R2

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Reviewers' comments:

16 Feb 2022

PONE-D-21-07409R2

Circadian rhythms modulate the effect of eccentric exercise on rat soleus muscles

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Table 1

Effect of eccentric exercise on plantaris and gastrocnemius muscle weight at different times of the day for 8 weeks.

	L		D
	C	TR	C	TR
Plantaris muscle weight, mg	330.7 (17.3)	343.7 (15.5)	324.5 (13.4)	331.4 (24.0)
Plantaris muscle weight, mg g BW-1 §	0.99 (0.03)	1.05 (0.03)	0.99 (0.02)	1.05 (0.03)
Gastrocnemius muscle weight, mg	1612.8 (44.2)	1643.4 (73.8)	1536.6 (75.7)	1595.8 (100.2)
Gastrocnemius muscle weight, mg g BW-1 §	4.8 (0.1)	5.0 (0.1)	4.8 (0.1)	5.1 (0.1)

Values are means (standard error; SE). Two-way ANOVA demonstrated that there was no time of day × exercise interaction. n = 6 per group. § represents a main effect of training. L, light phase; D, dark phase; C, control; TR, trained group.