Large variability exists in muscle adaptive response to resistance exercise (RE) training between individuals. Recent studies have revealed a significant role for microRNAs (miRNAs) in skeletal muscle plasticity. In this study, we investigated how RE affects miRNA expression and whether the variability of muscle hypertrophy to RE training may be attributed to differential miRNA regulation in the skeletal muscle. To screen high and low responders to RE, we had 18 young men perform arm curl exercise training. After screening, all the men performed 12 wk of lower body RE training, but only the high or low responders participated in the acute RE test before training. Muscle biopsies were obtained from the vastus lateralis muscle at baseline, 3 h after acute RE, and after the training period. Total RNA was extracted from the skeletal muscle, and miRNA expression (800 miRNAs) was analyzed. RE training increased the cross-sectional area of the biceps brachii (−1.7–26.1%), quadriceps (2.2–16.8%), and hamstrings (1.6–18.4%). Eighty-five and 102 miRNAs were differentially expressed after acute and chronic RE, respectively (P < 0.05). Seventeen miRNAs, especially 23b-3p, 26a-5p, 32-5p, 148b-3p, and 376a-3p, were differentially expressed at baseline, and 23 miRNAs, especially let-7a-5p, 95, 148a-3p, and 376a-3p, and 26 miRNAs, especially 30d-5p and 376a-3p, were differentially regulated after acute and chronic RE, respectively, in the skeletal muscle between high and low responders, indicating that the expression patterns of several miRNAs are altered by acute or chronic RE, and that miRNAs are involved in skeletal muscle adaptation to RE training.
- comprehensive expression analysis
- muscle hypertrophy
resistance exercise (RE) training increases skeletal muscle mass and function and contributes to positive health outcomes, including improvement of fat and glucose metabolism, body composition, and activities of daily living, and prevention of skeletal muscle loss with aging (sarcopenia). Thus, the American College of Sports Medicine and other health-related associations recommend regular RE as well as endurance exercise to improve physical well-being (2). It is generally recognized that there is a large variability in muscle adaptive response to RE (7, 17), although regular RE appears to induce skeletal muscle hypertrophy in the majority of people. A previous large-scale study that examined ∼600 adult men and women reported that ∼10% of the individuals did not exhibit muscle hypertrophy after chronic RE training (7).
A lot of factors, including age, sex, nutritional status, training status, hormonal status, and genotype, influence skeletal muscle responsiveness to RE training (1, 3, 8, 10, 16). Although the mechanisms underlying the adaptive variability of skeletal muscle to RE training are not fully understood, they possibly involve differential protein accumulation, considering the fact that muscle hypertrophy is a result of muscle protein accumulation induced by repeated bouts of RE.
MicroRNAs (miRNAs) are small noncoding RNAs that potently regulate mRNA expression at the posttranscriptional level. They bind to target mRNAs and destabilize and/or degrade them, leading to translational downregulation. Currently, >1,000 miRNAs have been identified in mammals. miRNAs are considered important regulators of cell function. Moreover, the expression levels of miRNA vary significantly in response to metabolic variability and/or diseases (15, 19). Recent studies have revealed a significant role for miRNAs in skeletal muscle plasticity (9, 11). Several studies have determined the impact of an acute bout of RE on miRNA expression in the skeletal muscle (5, 18, 21). Although these studies provided evidence that acute RE affects miRNA expression in the skeletal muscle, there were some limitations. For instance, most of these studies analyzed limited numbers of miRNAs. In fact, very few studies have comprehensively investigated changes in the miRNA expression profile in response to RE. Furthermore, the effect of acute or chronic RE training on miRNA expression is unknown, and whether miRNAs contribute to adaptive variability of the skeletal muscle in response to RE training remains to be investigated.
The purpose of this study was to investigate how acute or chronic RE affects miRNA expression and whether the variability of muscle hypertrophy to RE training may be attributed to differential miRNA regulation in the skeletal muscle. To this end, we used the recently developed digital microRNA high-throughput technology, hybridization-based NanoString nCounter to investigate the expression profiles of >800 miRNAs in response to an acute bout of RE and chronic RE in the skeletal muscle of high and low responders to RE training. This new technology allows direct digital readout of hundreds of miRNAs from small sample without amplification and is highly sensitive compared with older miRNAs detection methods such as microarrays, being comparable to real-time RT-PCR on sensitivity. Consequently, this procedure enabled us to identify candidate miRNAs involved in muscle mass regulation upon resistance training.
MATERIALS AND METHODS
Eighteen healthy young men volunteered to participate in this study (age 21.4 ± 1.1 yr; standing height 173.1 ± 3.9 m; body mass 66.9 ± 5.6 kg). These individuals had not participated in any regular resistance-training program for at least 1 yr prior to the start of this study and were therefore considered untrained. Before the initiation of this study, all participants were informed of the methods, procedures, and risks, following which they signed an informed-consent document, which was approved by the Ethics Committee of Ritsumeikan University.
Figure 1 shows the design of the experiment. To screen high and low responders, all participants (n = 18) performed supervised arm curl exercise training [10 repetitions at 70% of 1 repetition maximum (RM) for 3 sets with 2 min rest intervals] 3 days per week on alternative days for 6 wk. Training load was renewed after 3 wk of training. We measured the rate of muscle hypertrophy and selected the top and bottom five individuals as high responders and nonresponders, respectively.
After screening, only the high or low responders (n = 10) participated in the acute RE test. The participants were fed the same standard dinner (1,800 kcal) at 18:00 and were allowed only water ad libitum after 22:00. Each individual performed three sets of bilateral knee extension and flexion exercises (10 repetitions at 70% of 1 RM with 3 min rest intervals between sets) in an overnight fasting condition. Muscle biopsies were obtained from the lateral portion of the vastus lateralis using a disposable core biopsy instrument (Bard Max-Core; Bard Peripheral Vascular, Tempe, AZ) before and 3 h after the resistance exercise. The muscle sample was quickly rinsed with ice-cold saline, blotted, frozen immediately in liquid nitrogen, and stored at −80°C until use.
One week after the acute RE test, all individuals (n = 18) started bilateral knee extension and flexion exercise training (10 repetitions at 70% of 1 RM for 3 sets with 2 min rest intervals) 3 days per week on alternative days for 12 wk. To ensure an adequate training load, all training sessions were overseen by a trainer. Training load was reassessed every 3 wk with the 1 RM test, and if the participants could perform 12 repetitions or more at the third set during the training sessions, the load was increased by ∼5% for the next training session. Three or four days after the completion of training, muscle biopsies were obtained from only the high or low responders (n = 10) under conditions similar to those described for the acute RE test.
1 RM strength test.
The 1 RM strength test was performed as described previously (13). In brief, 2–3 wk before training initiation, all participants completed two familiarization sessions with submaximum loads (<50% of the predicted 1 RM), where they received instructions on proper exercise technique. Thereafter, 1 wk before training initiation, the individuals participated in the 1 RM strength test. The participants performed 10 repetitions with a low load as a warm-up. After the warm-up, the load was set at <90% of the predicted 1 RM. Following each successful lift, the load was increased by <5% until the subject failed to lift the load through his entire range of motion. On an average, six trials were required to complete a 1 RM strength test (3–5-min rest between each attempt).
Muscle size measurement.
Multislice magnetic resonance imaging (MRI) of the upper arm and thigh was performed before and after each resistance-training period using a 1.5 T magnetic resonance system (Signa HDxt, GE medical Systems). A T1-weighted, spin-echo, axial plane sequence was performed with a 600 ms repetition time and a 7.6 ms echo time. Continuous transverse images of 10 mm slice thickness were obtained. To prevent the influence of fluid shift within the muscle, the MRI procedure was performed at approximately the same time before and at least 2–3 days after the final exercise session.
miRNA expression profiling.
Total RNA was extracted from the skeletal muscles of the high or low responders (n = 3 in each group) using ISOGEN II (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The digital multiplexed NanoString nCounter human miRNA expression assay (NanoString Technologies, Seattle, WA) was performed with 10–30 ng of total RNA. Small RNA samples were prepared by ligating a specific DNA tag (miR-tag) onto the 3′-end of each mature miRNA according to the manufacturer's instruction. These tags serve several purposes: they normalize the wide range of melting temperatures of the miRNAs, provide a template to facilitate the use of the NanoString dual probe system, enable single base pair discrimination and specificity of highly homologous miRNA family members, and identify each miRNA species. Excess tags were removed by restriction digestion at 37°C. Hybridizations were carried out by combining 5 μl of each miRNA-miRTag sample with 20 μl of nCounter Reporter probes in hybridization buffer and 5 μl of nCounter Capture probes (for a total reaction volume of 30 μl) overnight at 65°C for 16–20 h. Excess probes were removed by two-step magnetic bead-based purification on the nCounter Prep Station (NanoString Technologies). We quantified the abundances of specific target molecules with the nCounter Digital Analyzer by counting the individual fluorescent barcodes and assessing the target molecules. For each assay, a high-density scan encompassing 843 fields of view was performed. We used the nCounter Digital Analyzer to collect the data after acquiring images of the immobilized fluorescent reporters in the sample cartridge with a charge-coupled device camera.
Analysis of miRNA expression.
miRNA data analysis was performed using the nSolver software, which is freely provided by NanoString Technologies. miRNA profiling data were normalized using the average signals obtained from the top 100 miRNAs and miRNAs that gave significant hybridization signals were used for downstream analysis. The signal from the negative controls + 2 SD was accepted as the cut-off value.
All values were expressed as means ± SD. Student's t-test was used for statistical comparison. Pearson product moment correlation coefficient was used to determine the correlations between select variables. The level of significance was set at P < 0.05.
RESULTS AND DISCUSSION
In this study, RE training increased the cross-sectional area (CSA) of the biceps brachii (10.3%, −1.7–26.1%), quadriceps (6.7%, 2.2–16.8%), and hamstring muscle (9.7%, 1.6–18.4%) in young untrained participants (Fig. 2A). Previous studies have reported that RE training induces muscle hypertrophy in a variety of muscle groups, including the quadriceps, biceps brachii, triceps brachii, pectoralis major, and triceps surae muscle (13, 14, 20). However, the training-induced hypertrophic capacities of different muscle groups have not been investigated. In the current study, we observed significant correlations between the percent changes in muscle CSAs (Fig. 2, B–D) of the upper and lower musculature, indicating that interindividual variability in muscle hypertrophy in response to RE training is common to all muscle groups. Individual factors such as genetic background, dietary habit, or systemic factors may responsible for this interindividual variability in muscle hypertrophy.
To determine how acute or chronic RE modifies miRNA expression in the skeletal muscle, we performed a comprehensive miRNA expression analysis to investigate the expression patterns of 800 miRNAs before and after acute and chronic RE in the skeletal muscle. The results revealed that the expression levels of 85 and 102 miRNAs were altered (P < 0.05, n = 6) after acute and chronic RE, respectively (Supplemental Table S1).1
To our knowledge, three previous studies have investigated the effect of a bout of RE on miRNA expression in the human skeletal muscle. Drummond et al. (5) investigated the effect of a bout of RE on the expression pattern of miRNA-1, 133a, and 206, which are known as muscle enriched miRNAs (myomiRs), and reported that acute RE decreased the expression level of miRNA-1 at 3 and 6 h postexercise in young participants, but not in old participants. Rivas et al. (18) investigated the expression patterns of 60 miRNAs before and after a bout of RE in young and old participants and found that the expression patterns of 17 miRNAs were altered 6 h after exercise in young participants only. Zacharewicz et al. (21) investigated 754 miRNAs before and 2 h after a bout of RE and found that 13 miRNAs were either up- or downregulated in both young and old participants. In the current study, we identified 85 miRNAs that were differentially expressed 3 h after RE (n = 6) and 23 miRNAs that were differentially regulated in young high and low responders (Supplemental Table S2). Out of 23 miRNAs, let-7a-5p, miRNA-95, miRNA-148a-3p, and miRNA-376a-3p were highly expressed (top 100) in skeletal muscle. The expression level of miRNA-95 decreased after exercise only in the high responders; this finding is consistent with the results reported by Rivas et al. (18), who showed that the expression level of miRNA-95-3p decreased 6 h after exercise in young participants, but not in old participants. Surprisingly, no common changes were observed between the present and previous studies. It may be partly due to the different methods used to detect miRNAs.
We then investigated whether the interindividual variability of muscle hypertrophy to RE training may be attributed to different miRNA expression patterns in the skeletal muscle and found that some miRNAs were differentially expressed at the baseline condition (17 miRNAs) and out of those miRNAs, miRNA-23b-3p, miRNA-26a-5p, miRNA-32-5p, miRNA-148b-3p, and miRNA-376a-3p were highly expressed (top 100) in skeletal muscle. Because there is no information on the relationship between resting miRNA expression and muscle responsiveness to exercise, the causal relationship between these two parameters could not be determined in this study. However, some factors that affect muscle responsiveness to RE are known to alter baseline miRNA expression. For example, aging is generally associated with a reduced capacity to induce muscle hypertrophy in response to RE training, and a previous study reported that older individuals exhibited reduced miRNA expression compared with young individuals (18). Interestingly, miRNA-26a-5p, which was expressed at higher levels at baseline in high responders compared with low responders, was expressed at lower levels in older individuals compared with young individuals (18). Additional studies need to be performed to investigate the effect of baseline miRNA expression levels on muscle anabolic response to RE training and its association with aging.
In the present study, we identified 102 miRNAs that exhibited altered expression patterns after chronic RE training and 26 miRNAs that were differentially regulated in young high or low responders. Out of 26 miRNAs, miRNA-30d-5p and miRNA-376a-3p were highly expressed (top 100) in skeletal muscle. Although a limited number of previous studies have investigated the effect of chronic RE on miRNA expression, chronic RE has been demonstrated to alter miRNA expression in the skeletal muscle. Davidsen et al. (4) investigated the expression patterns of 21 miRNAs before and after 12 wk of RE training and found that four miRNAs (miRNA-26a, 29a, 378, and 451) were differentially expressed between high and low responders among young men. In elderly men and women, chronic RE training decreased the expression levels of miRNA-1 (12) and miRNA-133b (22). miRNA-29a was commonly identified as being deferentially regulated in both the present and previous studies. However, although miRNA-29a expression decreased after training only in high responders in the present study, it decreased after training only in low responders in the previous study (4). The reason for this discrepancy and the role of miRNA-29a in muscle adaptation to RE training are unclear. However, a recent study demonstrated that miRNA-29 expression is upregulated with age in rodent skeletal muscle and that overexpression of miRNA-29 alters the expression levels of markers of myogenesis, muscle growth, and senescence (IGF-1 and p85α were downregulated, while SA-βgal, p16Ink4A, and p53 were upregulated) (6), indicating that an increase in miRNA-29 expression contributes to loss of muscle mass and senescence. Thus, the decrease in miRNA-29a expression observed in the present study may positively contribute to muscle hypertrophy. It will be interesting to investigate whether changes in miRNA-29 expression contribute to muscle hypertrophy in future studies.
In the present study, we investigated how acute or chronic RE influences miRNA expression and whether the variability of muscle hypertrophy to RE training may be attributed to differential miRNA regulation in the skeletal muscle. We identified 151 miRNAs that were differentially expressed after acute or chronic RE and 57 miRNAs that were differentially expressed at the baseline condition or regulated by acute or chronic RE between high and low responders. These results indicate that several miRNAs are involved in skeletal muscle adaptation to RE training, although the precise roles of these miRNAs are currently unclear. In particular, miRNA-136-5p and miRNA-376a-3p not only were differentially expressed at baseline but were differentially regulated after both acute and chronic RE. It will be of great interest to investigate in the future study the possibility of specific biomarkers where these two miRNAs may contribute to adaptive variability of the skeletal muscle in response to RE training. Additional validation studies investigating the roles of these miRNAs will provide insights into the mechanisms underlying the effects of differential miRNA expression on muscle hypertrophy.
This work was supported by JSPS KAKENHI Grant No. 25560379 to S. Fujita and Grant No. 23240097 to Katsuhiko Suzuki.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: R.O., T.A., and S.F. conception and design of research; R.O., T.A., and S.S. analyzed data; R.O., T.A., and S.F. interpreted results of experiments; R.O. prepared figures; R.O. and S.F. drafted manuscript; R.O., T.A., and S.F. edited and revised manuscript; R.O., T.A., and S.F. approved final version of manuscript; T.A., T.U., S.S., T.H., and S.F. performed experiments.
↵1 The online version of this article contains supplemental material.
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