The maintenance of muscle mass depends on the balance between protein synthesis and degradation. Aging and various pathological conditions, including denervation, unloading, diabetes, cancer and sepsis, cause progressive muscle atrophy via excessive protein degradation mainly through induction of the ubiquitin-proteasome system . The muscle-specific ubiquitin ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), are upregulated during muscle atrophy and in turn produce atrophy [2, 3]. Several transcription factors, including forkhead box O (FOXOs) [4, 5], nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105 (NFkB) , and myogenin , transcriptionally regulate the gene expression of these ligases during muscle atrophy.
Molecular circadian clock components are evident in most peripheral tissues such as the heart, liver and skeletal muscles, whereas the master clock is located in the hypothalamic suprachiasmatic nucleus (SCN) in mammals . The global knockout (KO) of aryl hydrocarbon receptor nuclear translocator-like (Arntl; also known as Bmal1), a core component of the circadian clock that encodes a basic-helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) transcription factor, results in a shortened lifespan, loss of muscle mass, reduced muscle force, mitochondrial dysfunction and disrupted myofilament architecture in mice [9, 10]. Schroder et al. found that specific tension was reduced and muscle fibrosis was increased in inducible muscle-specific Bmal1 KO (M-KO) mice at the age of 70 weeks . These results imply that clock components are involved in retaining skeletal muscle mass and function. We then postulated that the extent of muscle atrophy is enhanced in M-KO mouse models of atrophy. Therefore, the present study aimed to quantify the effects of a muscle-specific deletion of BMAL1 on denervation-induced and aging-induced muscle atrophy in mice.
Materials and Methods
Animal care and surgical procedures
Muscle-specific Bmal1 knockout (M-KO) mice and conditional Bmal1 flox/flox (f/f) mice (control littermates) were generated as described . In brief, a mouse line with a floxed Bmal1 gene was crossed with mice carrying a Cre recombinase transgene under control of the muscle creatine kinase (Mck) promoter. The mice were housed with access to a standard diet (CE2: CLEA Japan Inc., Tokyo, Japan) and water ad libitum under a 12 h light-12 h dark cycle (LD12:12); lights on at Zeitgeber Time (ZT) 0 and lights off at ZT12. Male mice aged 7 – 8 months (adult) were sacrificed at ZT1 and ZT13 (f/f, n = 6; M-KO, n = 4 at each ZT) to evaluate the effects of a Bmal1 deletion on circadian rhythms in the gastrocnemius (Ga) and soleus (Sol) muscles. The sciatic nerve was dissected from the left hind legs of adult male mice under anesthesia (f/f, n = 11; M-KO, n = 9) to evaluate the effects of deleting muscle Bmal1 on denervation-induced atrophy of the Ga and Sol muscles. The right hind legs remained innervated and served as a control. The mice were sacrificed at seven days after denervation. Adult f/f (n = 10) and M-KO (n = 8) and 23 – 24-month-old (aged) f/f (n = 6) and M-KO (n = 6) mice were sacrificed to evaluate the effects of aging on atrophy of the Ga and Sol muscles. The Animal Care and Use Committees at the National Institute of Advanced Industrial Science and Technology (AIST) approved all experimental protocols described herein (Permission #2015–166).
Real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using RNAiso Plus (Takara Bio Inc., Otsu, Japan). Single-stranded cDNA was synthesized using PrimeScript™ RT reagent kits with gDNA Eraser (Takara Bio Inc.). Real-time RT-PCR proceeded using SYBR® Premix Ex Taq™ II (Takara Bio Inc.) and a LightCycler™ (Roche Diagnostics, Mannheim, Germany) with the primer sequences shown in Supplementary Table 1. The amplification conditions were 95°C for 10 s followed by 45 cycles of 95°C for 5 s, 57°C for 10 s and 72°C for 10 s. The amount of target mRNA was normalized relative to that of Actb. Values for f/f mice at ZT1, the contralateral muscles of f/f, and adult f/f mice are expressed as 1.0 in Figures 123 and 4, respectively.
Data were analyzed using Excel-Toukei 2012 software (Social Survey Research Information Co. Ltd., Osaka, Japan) and all values are expressed as means ± standard error of the mean (SEM). Values were assessed using a two-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison tests. Differences were considered significant at P < 0.05. We showed the ANOVA results in Supplemental Tables 2–6.
Results and Discussion
We initially compared the day/night expression of Bmal1 and other clock genes in the Ga and Sol muscles between f/f and M-KO mice. Rhythmic Bmal1mRNA expression was obviously damped in the Ga muscle of M-KO mice (Figure 1A). The rhythmic expression of BMAL1 target genes such as Per1, Per2, Nr1d1, and Dbp was damped in the Ga muscles (Figure 1A). Levels of Bmal1 mRNA expression were flattened at the intermediate levels in the Sol muscles of M-KO mice, although the expression of BMAL1 target genes such as Nr1d1 and Dbp was significantly decreased (Figure 1B). The activity of Cre recombinase might be weaker in the Sol (mainly type I fibers), than in the Ga (mainly type II fibers) because we used mice expressing a Cre transgene driven by the Mck promoter, which is preferentially activated in type II fibers .
We assessed the effects of denervation-induced muscle atrophy in M-KO mice to determine the functional role of muscle BMAL1. The relative weight of the contralateral Ga muscle to body weight was significantly higher in M-KO, than in f/f mice (Figure 2A). This hypermuscular phenotype might be associated with a decrease in the expression of myostatin, a potent negative regulator of skeletal muscle growth, in M-KO mice (Wada et al., manuscript in preparation). The weight of denervated Ga muscles from f/f and M-KO mice decreased by 18.3% and 17.0%, respectively, compared with the contralateral Ga muscles at seven days after denervation (Figure 2A). The ratio of the loss of muscle mass was essentially identical between f/f and M-KO mice, suggesting that M-KO mice were not resistant and/or susceptible to denervation-induced muscle atrophy. In accordance with this finding, the mRNA expression of MAFbx was increased about 2.5-fold in the denervated Ga muscles from both f/f and M-KO mice (Figure 2B). Denervation also significantly induced MuRF1 mRNA expression in both mouse genotypes, although to a slightly, but significantly attenuated degree in M-KO, compared with f/f mice (Figure 2B). Denervation upregulated the mRNA expression of myogenic differentiation 1 (Myod1; a master regulator of myogenesis) 3.5-fold in both f/f and M-KO mice (Figure 2B), although Myod1 is transcriptionally regulated by BMAL1 [10, 14]. Myogenin (Myog) transcription factor transcriptionally regulates MAFbx and MuRF1 gene expression through E-box elements on their promoter regions during denervation-induced muscle atrophy . The expression of Myog mRNA was 25-fold upregulated in denervated muscle from f/f, but not M-KO mice (Figure 2B), suggesting that BMAL1 is involved in denervation-induced Myog expression independently of its function of facilitating muscle atrophy. The reduced Myog expression in denervated M-KO mice might have suppressed the expression of MuRF1, but not of MAFbx. Transcription factors, including FOXOs, might compensate for decreased Myog expression in M-KO mice. The denervation-induced mRNA expression of embryonic myosin heavy chain (eMHC) that is associated with muscle regeneration was similar between the mouse genotypes (Figure 2B). Although sustained denervation causes fibrosis in skeletal muscle [15, 16], the effect of denervation on the expression of genes associated with fibrosis and fatty degeneration such as fibroblast growth factor receptor 1 (Fgfr1), collagen type I alpha 2 (Col1a2), and fatty acid binding protein 4 (Fabp4) was not significant in either genotype (Figure 2B). We also measured the mass of Sol muscles that mainly comprises slow-twitch muscle fibers. Consistent with the Ga, the contralateral Sol muscle weighed significantly more in M-KO than in f/f mice (Figure 3A). The weight of denervated Sol muscles decreased by 6.9% compared with the contralateral muscles in f/f mice, but the difference did not reach significance. Denervation decreased the weight of the Sol by 12.7% in M-KO mice. These results suggest that muscle Bmal1 marginally affects the regulation of muscle mass in both fast- and slow-twitch muscles in M-KO mice, although the expression of muscle atrophy- or fibrosis-related genes was partly attenuated (Figure 3). The mRNA expression of MAFbx and MuRF1 was upregulated by denervation in the Ga, but not in the Sol muscle. The extent of Myog upregulation also differed between the Ga and Sol muscles. It has been reported that the effects of denervation on the level of these transcripts are greater in the Ga, than in the Sol muscle [17, 18]. Susceptibility to denervation-induced atrophy might differ between the Ga and Sol muscles.
Dyar et al. found that muscle histology was normal in M-KO even at the age of 26 months , which is in contrast to global Bmal1 KO mice. We also confirmed that the ablation of muscle Bmal1 scarcely affected the extent of the aging-induced loss of muscle mass (sarcopenia). The relative weight of the Ga and Sol muscles did not significantly differ between aged (23 – 24 months) f/f and M-KO mice (Figure 4A and C). Furthermore, the expression of eMHC, MAFbx and MuRF1 mRNA was also scarcely affected by Bmal1 ablation (Figure 4B and D).
The present study showed that denervation and aging resulted in a similar progression of muscle atrophy between M-KO and f/f mice, whereas the global deletion of Bmal1 caused extreme muscle loss in younger mice . Furthermore, untreated (contralateral) Ga and Sol muscles weighed significantly more in M-KO, than in f/f mice, although global KO of Bmal1 causes severe muscle atrophy [9, 10]. We recently described that many genes involved in lipid metabolism and protein degradation are transcriptionally regulated in a circadian manner by systemic neural signals in skeletal muscle . Therefore, the extreme muscle loss in global Bmal1 KO seemed to be caused by disruption of the central clock in the SCN.