Introduction

Skeletal muscle comprises approximately 40 % of total body mass. It plays a key role in maintaining whole body health due to its role in fat and glucose oxidation, its sensitivity to insulin, and its capacity to store fuel in the form of lipids and glycogen. Skeletal muscle is a highly adaptable tissue that increases its metabolic capacity and its resistance to contraction-induced fatigue in response to endurance exercise. These adaptations involve coordinated increases in mitochondrial biogenesis, angiogenesis, insulin sensitivity, substrate oxidation, fuel storage, and oxidative myosin heavy-chain isoforms [8, 9, 16, 41, 44]. By contrast, physical inactivity combined with an energy-rich diet contributes to perturbed skeletal muscle metabolism and increased muscle fatigue that may lead to metabolic diseases, including type 2 diabetes and obesity [18]. Understanding the molecular factors contributing to the endurance exercise-induced improvement in skeletal muscle metabolic function may identify novel therapeutic targets to treat metabolic disorders.

MicroRNAs (miRNAs) are a class of small (~23 nucleotides) nonconding RNA that negatively regulates gene expression by inducing translational suppression or degradation of target mRNAs [14, 22, 31]. The ability of miRNAs to tightly regulate cellular networks and physiological functions makes them exciting candidates for potential therapeutic targets. MiRNAs play important roles in skeletal muscle health. For example, several muscle-enriched miRNAs including miR-1 and miR-133 are essential for skeletal muscle development [7, 19, 23, 50, 51]. MiR-208a is implicated in muscle metabolic homeostasis [13], miR-378 suppresses mitochondrial fatty acid metabolism [6], whereas miR-499, miR-208b, and miR-23a regulate myosin heavy-chain expression [24, 45, 47]. Skeletal muscle miRNA expression levels are sensitive to endurance exercise [27, 38]. However, the miRNAs influencing exercise-induced skeletal muscle adaptations, such as increases in mitochondrial biogenesis, angiogenesis, and oxidative myosin heavy-chain isoforms, have not been identified.

The regulation of miR-23a may play a role in maintaining skeletal muscle health. We have demonstrated that miR-23a binds to and downregulates peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) in a 3′UTR-dependent manner in vitro [37]. Additionally, transgenic (Tg) mice overexpressing miR-23a have a reduction in skeletal muscle PGC-1α mRNA and protein and several PGC-1α downstream targets required for efficient mitochondrial function [37]. PGC-1α is a transcriptional coactivator that is highly expressed in tissues with high-energy demands such as skeletal muscle, cardiac muscle, brain, and brown adipose tissue [10]. It transcribes gene-encoding proteins that regulate numerous muscle-adaptive responses including mitochondrial biogenesis, angiogenesis, substrate oxidation, and the development of oxidative myosin heavy-chain isoforms [9, 11, 30, 48, 49]. Muscle specific overexpression of PGC-1α increases mitochondrial biogenesis and the amount of oxidative muscle fibers [23], whereas PGC-1α deficiency in skeletal muscle reduces mitochondrial metabolism and oxidative muscle fiber content [15]. Endurance exercise in mice and humans induces skeletal muscle PGC-1α expression [3, 12, 36, 42, 43] and also reduces miR-23a expression [38, 39]. By contrast, skeletal muscle of patients with amyotrophic lateral sclerosis (ALS) presents an elevation in miR-23a and an associated decrease in PGC-1α mRNA and protein and impaired oxidative enzyme activity [37].

Several studies [2, 11, 29], but not all [34], suggest that PGC-1α plays an important role in exercise-induced adaptive mitochondrial biogenesis. Whether the regulation of miR-23a is necessary for exercise-induced skeletal muscle adaptations, particularly those regulated by PGC-1α, is unknown. We therefore hypothesized that forced expression of miR-23a in skeletal muscle would attenuate the endurance exercise-induced increase in PGC-1α and several well-established skeletal muscle adaptations, including increased mitochondrial biogenesis and capillary density as well as transition of myosin heavy-chain IIb to IIa. To test this hypothesis, we investigated changes in skeletal muscle mitochondrial content, angiogenesis, and myosin heavy-chain isoforms at rest and following 4 weeks of voluntary wheel running in wild-type (WT) mice and Tg mice overexpressing miR-23a.

Materials and methods

Ethics statement

All animal experiment protocols were approved by the Animal Care and Use Committees of the University of Tokyo.

Animal experiments

Eight-week-old female miR-23a Tg mice and their WT littermates were used for all experiments. Generation of miR-23a Tg mice were described elsewhere [46]. Briefly, the pCXbG-miR-23a plasmid vector was linearized using SalI–EcoRI sites and injected into C57BL6/J oocytes. Genotyping was carried out by PCR, and fluorescence microscopy was used to confirm transgene expression in adult skeletal muscles. All mice were housed in temperature-controlled quarters (21 °C) with a 12-h light/12-h dark cycle and provided with water and food ad libitum. For voluntary running, animals were subjected to 4 weeks in cage voluntary running. Wheel revolutions per minute were counted to calculate total running distance over the 4-week period [2]. All mice were housed individually and killed at the same time of day after the final running session.

Exercise capacity test was conducted as previously described [20] with minor modifications. Briefly, WT and Tg mouse were placed on a treadmill (Melquest, Japan) at a constant 10° angle. The treadmill was then started at 10 m/min, and the speed was incrementally increased by 1 m/min every 2 min until 24 m/min. Exhaustion was defined as the point where mice were unable to continue running on the treadmill after 10 s of encouragement with a drawing brush.

RNA analysis

Total RNA was extracted from cells and animal tissues using ISOGEN II (WAKO, Osaka, Japan) according to the manufacturer’s protocols, and 1 μg of RNA was reverse transcribed using SuperScript III reverse transcriptase (RT; Invitrogen). Oligo-dT was used to generate cDNA, and an aliquot of the RT reaction was used directly for PCR with Ex Taq HS (TaKaRa, Osaka, Japan) and gene-specific primers. Primer sequences for Dicer, Drosha, Exportin-5, and Ago2 were: Primer sequences were: Dicer, 5′-CAC  ACG  CCT CCT  ACC ACT  ACA ACA  C-3′ and 5′-GGC  TGC  ATC  ATC  GGA   TAG  TAC  ACC-3′; Drosha, 5′-CAA  ATA  CGG  ATC  GGC  AAC  TT-3′, and 5′-CAC  GGG TCT CTT GGT TTT GT-3′; Exportin-5, 5′-TGG AAG  CTC TGG  TTC  TCG  TT-3′ and 5′-GGG  TTA  CGG  AAG  ATG GGA  TT-3′; and Ago2, 5′-ACA  GGG  AAA  TTG  TGG  AGC AC-3′ and 5′-GAT  GGA  AGC CAA  ACC ACA CT-3′.

To measure pri- and pre-miR-23a, 500 ng of total RNA was reverse transcribed with ThermoScriptRT (Invitrogen) as previously reported [46]. Briefly, 500 ng of total RNA, 1 μl of 10 mM of specific RT primer (5′-TGG TAA TCC CTG GCA ATG TG-3′) and 2 μl of 10 mM dNTP were mixed, and distilled water was added to a total volume of 12 μl. The mixture was heated at 85 °C for 5 min, then at 57 °C for 5 min. After chilling on ice for 2 min, 4 μl of 5× cDNA synthesis buffer, 1 μl each of 0.1 M DTT, RNase inhibitor (ToYoBo, Osaka, Japan), and ThermoScript RT (15 U/μl) were added to the mixture. The contents were gently mixed and incubated at 57 °C for 60 min and heated at 85 °C for 5 min to terminate the reaction. For quantification of pri- and pre-miR-23a, 1 μl of RT product was amplified by PCR using Ex Taq HS (TaKaRa) in a total reaction volume of 50 μl. Primer sequences for pri- and pre-miR-23a were: pre-miR-23a-F; 5′-CTG GGG TTC CTG GGG AT-3′, and a common reverse primer with the same sequence as the specific RT primer. PCR conditions for pri- and pre-miR-23a were 35 cycles of denaturation at 98 °C for 10 s, annealing at 57 °C for 15 s, and extension at 72 °C for 15 s for extension. PCR products were electrophoresed on 2 % agarose gels containing 5 × 10–5 % ethidium bromide for 30 min, and fluorescent images were acquired under UV light by LAS3000 (FujiFilm corporation, Tokyo, Japan). Primer sequences for PGC-1α isoforms [35] and GAPDH [46] have been described elsewhere. GAPDH was used as an internal standard.

MicroRNA analysis

The TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA assays (Applied Biosystems, Foster City, CA) were used according to the manufacturer’s protocols for real-time PCR quantification of mature miRNA expression [46]. Product number of TaqMan MicroRNA assay was miR-23a (no. 000399). For miRNA quantification, each reverse RT reaction contained 10 ng of purified total RNA. The reactions were incubated for 30 min at 16 °C, 30 min at 42 °C, and 5 min at 85 °C. Real-time PCR reactions for each miRNA were performed in triplicate in a 10-μl reaction mixture that included 1 μl of the RT product. Reactions were carried out on an Applied Biosystems StepOne Plus Real-Time PCR system in 96-well plates at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A small RNA, U6 was used as the endogenous control.

Western blot

Western blotting was performed as described previously [1] with the following antibodies: anti-PGC-1α (AB3242, Millipore), cytochrome c oxidase complex IV (COX IV; no. 4844, Cell Signaling Technology), cytochrome c (no. 4272, Cell Signaling Technology), γ-tubulin (AK-15, Sigma-Aldrich), and anti-myosin heavy-chain (MyHC) I (BA-F8), IIa (SC-71), and IIb (BF-F3) [40]. The secondary antibodies consisted of a HRP-conjugated anti-rabbit IgG Fab antibody (GE healthcare), an anti-mouse IgG-HRP antibody, an anti-goat IgG-HRP, and an anti-mouse IgM-HRP (Bio-rad).

Immunohistochemistry

Cross-sections from O.C.T.-embedded muscle were fixed with 4 % paraformardehyde in PBS, permeabilized with 0.3 % Triton X, and incubated with the following primary antibodies: anti-myosin heavy-chain (MyHC) I (BA-F8), IIa (SC-71), and IIb (BF-F3); dystrophin (D8043, Sigma-Aldrich); and CD31 antibodies (MCA2388, AbD serotec). Secondary antibodies were DyLight 405-conjugated anti-mouse IgG2b (for MyHC I), DyLight 488-conjugated anti-mouse IgG1 (for MyHC IIa), R-PE-conjugated anti-mouse IgG1 (for dystrophin), DyLight 549-conjugated anti-mouse IgM (for MyHC IIb), and DyLight 488-conjugated anti-rat IgG (for CD31; Jackson Immuno Research). A single anatomical cross-sectional area of the middle portion of each muscle was measured with ImageJ Software.

Enzymatic staining

Frozen tissue section was also used for succinate dehydrogenase (SDH) staining as described previously [25].

Transmission electron microscopy

Transmission electron microscopy analysis was performed on soleus muscle longitudinal sections from the miR-23a Tg and WT mice (n = 2 for each genotype, 10 weeks of age in C57BL6 background) at the Medical Proteomics Laboratory, University of Tokyo.

Statistics

Data are presented as mean ± standard error of the mean (SEM). Statistical significance (P < 0.05) was determined by a Student’s t test for comparisons between two groups. Two-way ANOVA was used to analyze data from the exercise experiments. Post hoc analyses following a significant interaction between genotype × exercise or a main effect was performed using t tests. If multiple t test were performed, a Bonferroni adjustment was used.

Results

Expression of miR-23a in skeletal muscle

We quantified miR-23a expression by real-time RT-qPCR and confirmed that miR-23a was highly expressed in skeletal muscle when compared with other tissues such as liver, kidney, brown fat, and spleen (Fig. 1a). We also observed that endogenous miR-23a expression is higher in slow (soleus) muscle than fast (plantaris) muscle. When comparing Tg mice with WT mice, we confirmed that forced expression of miR-23a resulted in an approximate 3- and 6-fold increase respectively, in mature miR-23a levels in slow oxidative soleus and fast glycolytic plantaris skeletal muscle (Fig. 1a). Primary and precursor miR-23a levels were also increased in Tg mice (Fig. 1b). Subsequently, we confirmed mRNA expression levels of several key members of the miRNA processing complex and observed no difference in the levels of Dicer or Drosha but increases in Exportin-5 and Ago2 in both the soleus and plantaris muscles of the Tg when compared with the WT mice (Fig. 1c).

Fig. 1
figure 1

Expressions of miR-23a and components of miRNA biogenesis machinery in miR-23a Tg mice. a Expression of miR-23 in various tissues in miR-23a transgenic (Tg) mice (n = 5). Values are relative to the soleus muscle of the wild-type (WT) mice. b Expression of pri- and pre miR-23a was quantified by qRT-PCR (n = 5). Values are relative to the soleus muscle of the WT mice. c Components of the miRNA biogenesis machinery were measured by RT-PCR (n = 6). Data are presented as mean ± SEM. **P < 0.01 vs WT control; ## P < 0.01 vs WT soleus

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Forced overexpression of miR-23a downregulates PGC-1α and its downstream targets in a slow muscle-specific manner

Western blot analysis demonstrated that PGC-1α and mitochondria respiratory chain proteins, such as COX IV and cytochrome c (Cyt-c) were downregulated in the slow soleus but not in the fast plantaris muscle (Fig. 2a, b). We then determined mRNA expression levels of PGC-1α isoforms. No effect of miR-23a overexpression on PGC-1α isoform mRNAs in the soleus or plantaris muscles of the Tg, when compared with WT mice was observed (Fig. 2c). These data suggest that miR-23a suppresses PGC-1α protein expression through translational repression rather than mRNA degradation.

Fig. 2
figure 2

Forced overexpression of miR-23a downregulates PGC-1α and mitochondrial proteins only in slow muscle. a Representative images of PGC-1α and mitochondria respiratory chain proteins from WT and miR-23a Tg mice. b Quantitative data for PGC-1α, COX IV and cytochrome c proteins from WT and miR-23a Tg mice (n = 9). Values are relative to the wild-type (WT) mice. c PGC-1α isoform mRNA expressions were measured by RT-PCR (n = 6). Values are relative to the soleus muscle of the WT mice. Data are presented as mean ± SEM. **P < 0.01; *P < 0.05 vs WT control

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Forced expression of miR-23a did not affect muscle fiber type

Western blot analysis demonstrated no effect of forced overexpression of miR-23a on myosin heavy-chain isoforms (MyHC I, IIa, and IIb) in the soleus or plantaris muscles of the Tg, when compared with WT mice (Fig. 3a). Immunofluorescent imaging of muscle fiber typing was also performed, and again no changes in muscle fiber-type composition in the soleus or plantaris muscles of the miR-23a Tg was observed (Fig. 3b, c), whereas the numbers of MyHC IId/x fibers were slightly decreased in the Tg mice (Fig. 3c). Histological examination revealed reduced SDH staining in type IId/x fibers of soleus muscle (Fig. 3d). Transmission electron microscopy analysis of soleus muscle in the miR-23a Tg mice revealed that mitochondrial structure was generally preserved (Fig. 3d).

Fig. 3
figure 3

Forced expression of miR-23a does not influence skeletal muscle fiber type. a Representative images of myosin heavy chain (MyHC) I, IIa, and IIb in soleus (slow muscle) and plantaris (fast muscle) of WT and transgenic (Tg) mice. b Representative images of soleus and plantaris muscle frozen sections from WT and miR-23a Tg mice immunostained for MyHC I (blue), IIa (green), and IIb (red). Type IId/x fibers remain unstained. Scale bar = 200 μm. c Quantitation of each fiber type is shown (n = 5–6 mice/group). Open bars, WT; black bars, Tg. d Representative images of serial sections with fiber-type staining and succinate dehydrogenase (SDH) staining as well as transmission electron microscopy (EM) for soleus muscle from WT and miR-23a Tg mice. X type IId/x fibers. Scale bar = 1 μm. Data are presented as mean ± SEM. **P < 0.01 vs WT

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Exercise-induced skeletal muscle adaptation in miR-23a Tg mice

As Tg expression of miR-23a suppressed mitochondrial content in slow muscles, we sought to assess endurance exercise capacity of the mice. Forced overexpression of miR-23a did not influence the spontaneous daily running activity of the mice during 4 weeks of cage voluntary wheel running (Table 1). Following 4 weeks of cage voluntary wheel running, there were no differences in the exercise-induced relative increase in muscle weights of both the soleus and plantaris muscle of the WT and miR-23a Tg mice. Immunofluorescence imaging-based fiber typing showed an increase in MyHC IIa fibers in plantaris muscle both in WT and miR-23a Tg after the voluntary running (Fig. 4a). Myosin heavy-chain IIa protein content and markers of mitochondrial content, including PGC-1α, IV, and cytochrome c (Fig. 4b) were increased in plantaris muscle of both WT and miR-23a Tg mice following 4 weeks of cage voluntary wheel running. There were no changes in these parameters in the soleus muscle of either mouse following exercise (data not shown). However, capillary density increased in both the soleus and plantaris muscles of the WT and miR-23a Tg mice (Fig. 5a, b). Finally, there was no difference in the increase in endurance exercise capacity between the WT or the miR-23a Tg mice (Table 1) following 4 weeks of cage voluntary wheel running.

Table 1 Observations following 4 weeks of voluntary running in WT and Tg mice
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Fig. 4
figure 4

Forced expression of miR-23a has no impact on endurance exercise-induced muscle adaptation. a Quantitation of MyHC IIa fiber in plantaris muscle is shown (n = 5–6 mice/group). b Relative protein expressions of myosin heavy-chain IIa, PGC-1α, and mitochondria respiratory chain proteins in plantaris muscle from WT and miR-23a Tg mice (n = 9). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01

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Fig. 5
figure 5

Forced expression of miR-23a has no impact on endurance exercise-induced angiogenesis. Frozen sections of soleus and plantaris muscles were stained with anti-CD31 antibody. a Representative images of plantaris muscle frozen sections from WT and miR-23a transgenic (Tg) mice immunostained for CD31 (green) and dystrophin (red). Scale bar = 100 μm. b Quantitative data for capillary density in soleus and plantaris muscles from WT and miR-23a Tg mice (n = 5–6). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01

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Discussion

This study showed that forced expression of miR-23a in mice downregulates basal PGC-1α protein expression only in the slow soleus muscle, without a major change in their fiber-type composition. We also confirmed that endurance exercise-induced muscle adaptations occurred in fast muscle plantaris muscle of the Tg mice and that this was comparable to the adaptations in the WT littermates.

Interestingly, we found that Tg expression of miR-23a downregulated PGC-1α only in slow muscle (Fig. 2a, b). A previous study showed that double knockout of muscle-enriched miRNAs (miR-499 and miR-208b) led to greater repression of slow myosin expression in soleus than in tibialis anterior [45]. These results may suggest slow muscle has greater capacity in oxidative metabolic gene expression change. Another possibility could be that PGC-1α 3′UTR length may vary with muscle fiber type. In fact, the 3′-sequence of PGC-1α in the EST database shows two putative poly-adenylation sites in the 3′UTR. Further studies should examine whether PGC-1α 3′UTR variants exist without miR-23a target sites and if this influences the sensitivity of PGC-1α to regulation by miRNAs.

MiR-23a can inhibit myogenesis through translational suppression of fast myosin heavy-chain isoforms (MyHC IIa, IId/x, and IIb) in vitro [47]. We have initially expected that forced expression of miR-23a in mice would attenuate myogenesis for fast MyHC muscle fiber formation. Although we found a slight decrease in MyHC IId/x fibers in soleus muscle, it remains unclear why our in vivo data does not support the in vitro result. It is a possibility that the mild phenotype of the miR-23a Tg mice arises from an insufficient transgene expression. The induction of mature miR-23a in the Tg mice was about 3-fold in slow soleus muscle and 6-fold in fast plantaris muscle, compared with the WT littermates. According to the paper by Wang et al., miR-23a levels were overexpressed by about 5-fold when compared with the negative control in vitro. While this is comparable to that observed in the plantaris muscle in the present study, it is generally recognized that in vitro-cultured myotubes differ considerably from adult myofibers, including the expression profile of MyHC isoforms. In cultured myotubes, the dominant MyHC isoform is embryonic isoform and expressions of MyHC IIa, IIb, or IId/x are very low compared with adult myofiber [26] so that the downregulation of fast myosin heavy-chain isoforms by miR-23a could be prominent in cultured myotubes. Furthermore, miR-23a was transiently overexpressed in vitro while our Tg mouse is a model of chronic miR-23a overexpression. These represent two very different models and therefore comparisons between them are difficult.

To drive the overexpression of miR-23a, we used an expression vector containing the chicken β-actin promoter and cytomegalovirus enhancer as well as the β-actin intron and bovine globin poly-adenylation signal. This construct is able to produce whole body Tg expression from pre-implantation embryo to adult stages [28]. Therefore, it is possible that miR-23a can only transiently downregulate adult MyHC expression in a cell culture model. It should also be noted that although we used conventional Tg mice for miR-23a to gain global expression of the gene, we are not able to rule out the possibility that other regulatory factors functioning in a compensatory manner.

Several miRNAs are reported to change upon endurance exercise to modulate skeletal muscle plasticity [27, 38]. The regulation of these miRNAs following endurance exercise is underexplored. As recently reported, endurance exercise decreases miR-23a expression in skeletal muscle in both rodents and humans [38, 39]. Conversely, a number of studies indicate that endurance exercise induces PGC-1α expression [2, 3, 42]. These results may imply a regulatory relationship between the transcription of PGC-1α and its posttranscriptional control by miR-23a. This notion may contribute to fine tuning of gene expression and muscle adaptation.

Following 4 weeks of cage voluntary wheel running, there was no difference in the relative increase in soleus and plantaris muscle weight between the WT or the miR-23a Tg mice (Table 1). The finding that voluntary running increases muscle mass in soleus and plantaris muscles are in agreement with these previous findings [21, 32]. It is generally recognized that the soleus is not as sensitive as the plantaris muscle to voluntary running-induced metabolic and contractile adaptation [5, 17, 33]. However, as miR-23a Tg mice had a reduction in basal levels of PGC-1α and mitochondria respiratory chain proteins in soleus, but not plantaris muscle, it was of interest to investigate if both muscles were sensitive to voluntary running-induced exercise adaptations.

As well as no effect under basal conditions, forced expression of miR-23a in the fast plantaris muscle did not affect mitochondrial or and contractile adaptations to endurance exercise. These results may imply that the endurance exercise induction of PGC-1α is not targeted by miR-23a. As mentioned previously, there is a possibility that PGC-1α 3′UTR variants without miR-23 target sites exist. Ruas et al. recently reported that several PGC-1α isoforms exist that are induced by exercise [35]. The present study observed no changes in the mRNA levels of these isoforms, but this does not rule out their potential role in skeletal muscle exercise-induced adaptations.

PGC-1α is reported to be essential for exercise-induced angiogenesis in skeletal muscle [9]. We confirmed endurance exercise-induced increase in capillary density in miR-23a Tg mice. Capillary density in the Tg mice tended to decrease compared with that in WT mice, although the difference does not reach statistical significance (P = 0.145). This phenomenon is consistent with the changes in muscle properties of miR-23a Tg mice in response to endurance exercise.

MiRNA biogenesis is a complex process requiring coordination of pri-miRNA transcription, their cleavage by endonucleases, exportation from nucleus to cytoplasm, additional cleavage, then incorporation into the RISC complex [4]. Forced expression of miR-23a under control of chicken β-actin promoter increases mRNA for two key components, miRNA export protein, Exportin-5, and Ago2. Pri- and pre-miR23a levels were significantly increased in the skeletal muscle of Tg mice, suggesting that the upregulation of these genes most likely reflects the accumulation of pri- and pre-miR-23a in the Tg mice. Mechanisms regulating the miRNA biogenesis machinery in Tg animals are unclear, and this should be a focus for future investigation.

In summary, this study provides evidence that forced expression of miR-23a in mice decreases basal markers of mitochondrial biogenesis such as PGC-1α, COX IV, and cytochrome c in slow muscle. By contrast, miR-23a Tg mice showed no change in their fiber-type composition and endurance exercise-induced muscle adaptation in fast muscle. This suggests that miR-23a plays a limited role in regulating the skeletal muscle adaptation to endurance exercise.