Muscle Clock Research Articles

In vitro and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous

The notion that the circadian rhythm is exclusively regulated by a central clock has been challenged by the discovery of peripheral oscillators. These peripheral clocks are known to have a direct influence on the biological processes in a tissue or cell. In fish, several peripheral clocks respond directly to light, thus raising the hypothesis of autonomous regulation. Several clock genes are expressed with daily rhythmicity in Atlantic cod (Gadus morhua) fast skeletal muscle. In the present study, myosatellite cell culture and short-term cultured fast skeletal muscle explant models were developed and characterized, in order to investigate the autonomy of the clock system in skeletal muscle of Atlantic cod. Myosatellite cells proliferated and differentiated in vitro, as shown by the changes in cellular and myogenic gene markers. The high expression of myogenic differentiation 1 during the early days post-isolation implied the commitment to myogenic lineage and the increasing mRNA levels of proliferating cell nuclear antigen (pcna) indicated the proliferation of the cells in vitro. Transcript levels of myogenic marker genes such as pcna and myogenin increased during 5 days in culture of skeletal muscle explants, indicating that the muscle cells were proliferating and differentiating under ex vivo conditions. Transcript levels of the clock gene aryl hydrocarbon receptor nuclear translocator-like 2 (arntl2) in myosatellite cells showed no daily oscillation regardless of photoperiod manipulation. On the other hand, mRNA levels of the clock gene circadian locomotor output cycles kaput (clock) showed circadian rhythmicity in 5-day-old skeletal muscle explant under different photoperiod regimes. The expression of arntl2, cryptochrome2 (cry2), period 2a (per2a) and nuclear receptor subfamily 1, group D, member 1 was not rhythmic in muscle explants but photoperiod manipulation had a significant effect on mRNA levels of cry2 and per2a. Taken together, the lack of rhythmicity of molecular clocks in vitro and ex vivo indicate that the putative peripheral clock in Atlantic cod fast skeletal muscle is not likely to be autonomous.

Exercise influences circadian gene expression in equine skeletal muscle

Circadian rhythms are endogenously generated 24-h oscillations that coordinate numerous aspects of mammalian physiology, metabolism and behaviour. The existence of a molecular circadian clock in equine skeletal muscle has previously been demonstrated. This study investigates how the circadian 24-h expression of exercise-relevant genes in skeletal muscle is influenced by a regular exercise regime. Mid-gluteal, percutaneous muscle biopsies were obtained over a 24-h period from six Thoroughbred mares before and after an 8-week exercise programme. Real-time qPCR assays were used to assess the expression patterns of core clock genes ARNTL, PER2, NR1D1, clock-controlled gene DBP, and muscle genes MYF6, UCP3, VEGFA, FOXO1, MYOD1, PPARGC1A, PPARGC1B, FBXO32 and PDK4.Two-way repeated measures ANOVA revealed a significant interaction between circadian time and exercise for muscle genes MYF6, UCP3, MYOD1 and PDK4. A significant effect of time was observed for all genes with the exception of VEGFA, where a main effect of exercise was observed. By cosinor analysis, the core clock genes, ARNTL (P < 0.01) and NR1D1 (P < 0.05), showed 24-h rhythmicity both pre- and post-exercise, while PER2 expression was rhythmic post-exercise (P < 0.05) but not pre-exercise. The expression profiles of muscle genes MYOD1 and MYF6 showed significant fits to a 24-h cosine waveform indicative of circadian rhythmicity post-exercise only (P < 0.01). This study suggests that the metabolic capacity of muscle is influenced by scheduled exercise and that optimal athletic performance may be achieved when exercise times and competition times coincide.

Tuning in to the rhythm of clock genes in skeletal muscle

Circadian rhythms and metabolic homeostasis are closely entangled biological processes. Clock genes refer to a group of genes which regulate a variety of biological responses through their periodic expression. Genes encoding the core clock mechanism include the positive transcriptional regulators brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput protein (CLOCK) and the transcriptional repressors PERIOD (PER) and CRYPTOCHROME (CRY) [1]. Together, these factors ultimately activate or repress their own expression and therefore constitute a self-sustained transcriptional feedback loop. Impairment of the clock system has been associated with various diseases including obesity, cardiovascular dysfunction and cancer [2]. In mice, inactivation of Bmal1 and Clock was shown to impair both glucose tolerance and insulin secretion [3]. Notably, inactivation of the clock system in pancreatic islets leads to overt diabetes mellitus, emphasizing the importance of clock genes in beta-cell function. While the critical role of clock genes in metabolism is now well appreciated, many questions remain to be solved about the peripheral and tissue-specific action of circadian regulators in insulin-sensitive tissues. For instance, mice deficient in hepatic expression of Bmal1 exhibited hypoglycemia in their resting phase whereas whole body Bmal knockout mice had normal resting blood sugar levels [4]. Presumably such differences stem from failed synchronization of the intact central circadian timing system, which resides in the brain's suprachiasmatic nucleus (SCN), and peripheral, cell-autonomous clocks in the liver. In fact, peripheral pacemakers of the clock system may largely determine basic metabolic functions in response to environmental inputs. In this issue of Molecular Metabolism, Schiaffino and coworkers present convincing evidence for the physiological importance of a functional muscle clock [5]. Using Cre-loxP recombination technology, the authors inactivated Bmal1 specifically in skeletal muscle of mice, either constitutively (mKO) or through an inducible construct (imKO). While whole-body knockout of Bmal1 was associated with reduced activity, degenerative loss of skeletal muscle mass and accelerated aging, Bmal1 mKO mice had normal life span and the weight of certain muscle types was even increased. The circadian rhythm of locomotor activity was unaltered in the knockout mice, but surprisingly activity levels were increased during the dark phase in Bmal1 mKO mice. Importantly, insulin-stimulated glucose uptake into skeletal muscle was substantially reduced in the knockout mice whereas whole-body glycemia was unaltered. In Bmal-deficient muscles, the abundance of the insulin-regulated glucose transporter protein GLUT4 was reduced to approximately half of the normal levels, similar to the levels found in heterozygous muscle-specific GLUT4 knockout mice [6]. Interestingly, expression of the RabGAP protein TBC1D1, a direct target of AKT and AMP-dependent kinase and a potential regulator of insulin- and contraction-stimulated GLUT4 translocation was also substantially reduced in skeletal muscle of mKO mice. Ablation of Tbc1d1 or knockout of the related Tbc1d4 (AS160) also results in reduced abundance of GLUT4 protein in muscle whereas mRNA levels of GLUT4 in skeletal muscle were not altered [7,8]. In skeletal muscle of mKO mice, the mRNA of GLUT4 was also unchanged, indicating that the loss of GLUT4 in Bmal1 mKO mice likely results from post-translational events, perhaps missorting and degradation of the protein. As both GLUT4 and TBC1D1 display oscillation in their expression in muscle, this study makes a case by linking diurnal expression of signaling proteins to the regulated trafficking of insulin effectors. Analysis of muscle metabolites further revealed a reduced activity of pyruvate dehydrogenase (PDH), leading to impaired glucose oxidation and redirection of glycolytic intermediates to alternative metabolic pathways. Altogether, these results point towards a central role of the circadian clock for skeletal muscle metabolism by regulating metabolic switching between fasting and feeding state. Further studies are needed to follow up on the molecular mechanisms that integrate chronobiology and metabolism in peripheral insulin-sensitive tissues. The relation of reduced Tbc1d1 levels and diminished GLUT4 protein in skeletal muscle remains to be investigated, as the lack of an effect on expression of the related Tbc1d4. Because inactivation of Bmal1 in skeletal muscle alters circadian expression of most other core clock and clock-associated genes, it also remains to be determined which factors are ultimately responsible for the altered expression of insulin effectors and signaling proteins. Even though whole-body glycemia of Bmal1 mKO mice apparently was normal, a subtle desynchronization of the central circuitry in the SCN and the periphery could lead to impaired glucose disposal and might impose a risk for developing type 2 diabetes. An open question remains whether overexpression of Bmal1 might improve insulin action in skeletal muscle. Thus, adjusting the clock in insulin-resistant skeletal muscle could constitute a future option for treating metabolic dysfunction. Hopefully time will tell, so we stay tuned for more exciting research on clock genes.

Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock

Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.

Scheduled Exercise Phase Shifts the Circadian Clock in Skeletal Muscle

It has been well established in mammals that circadian behavior as well as the molecular clockwork can be synchronized to the light-dark cycle via the suprachiasmatic nucleus of the hypothalamus (SCN). In addition to light, it has been demonstrated that nonphotic time cues, such as restricting the time of food availability, can alter circadian behavior and clock gene expression in selected peripheral tissues such as the liver. Studies have also suggested that scheduled physical activity (exercise) can alter circadian rhythms in behavior and clock gene expression; however, currently, the effects of exercise alone are largely unknown and have not been explored in skeletal muscle. Period2::Luciferase (Per2::Luc) mice were maintained under 12 h of light followed by 12 h of darkness then exposed to 2 h of voluntary or involuntary exercise during the light phase for 4 wk. Control mice were left in home cages or moved to the exercise environment (sham). A second group of mice had restricted access to food (4 h · d(-1) for 2 wk) to compare the effects of two nonphotic cues on PER2::LUC bioluminescence. Skeletal muscle, lung, and SCN tissue explants were cultured for 5-6 d to study molecular rhythms. In the exercised mice, the phase of peak PER2::LUC bioluminescence was shifted in the skeletal muscle and lung explants but not in the SCN suggesting a specific synchronizing effect of exercise on the molecular clockwork in peripheral tissues. These data provide evidence that the molecular circadian clock in peripheral tissues can respond to the time of exercise suggesting that physical activity contributes important timing information for synchronization of circadian clocks throughout the body.

Circadian expression of clock and putative clock-controlled genes in skeletal muscle of the zebrafish

To identify circadian patterns of gene expression in skeletal muscle, adult male zebrafish were acclimated for 2 wk to a 12:12-h light-dark photoperiod and then exposed to continuous darkness for 86 h with ad libitum feeding. The increase in gut food content associated with the subjective light period was much diminished by the third cycle, enabling feeding and circadian rhythms to be distinguished. Expression of zebrafish paralogs of mammalian transcriptional activators of the circadian mechanism (bmal1, clock1, and rora) followed a rhythmic pattern with a ∼24-h periodicity. Peak expression of rora paralogs occurred at the beginning of the subjective light period [Zeitgeber time (ZT)07 and ZT02 for roraa and rorab], whereas the highest expression of bmal1 and clock paralogs occurred 12 h later (ZT13-15 and ZT16 for bmal and clock paralogs). Expression of the transcriptional repressors cry1a, per1a/1b, per2, per3, nr1d2a/2b, and nr1d1 also followed a circadian pattern with peak expression at ZT0-02. Expression of the two paralogs of cry2 occurred in phase with clock1a/1b. Duplicated genes had a high correlation of expression except for paralogs of clock1, nr1d2, and per1, with cry1b showing no circadian pattern. The highest expression difference was 9.2-fold for the activator bmal1b and 51.7-fold for the repressor per1a. Out of 32 candidate clock-controlled genes, only myf6, igfbp3, igfbp5b, and hsf2 showed circadian expression patterns. Igfbp3, igfbp5b, and myf6 were expressed in phase with clock1a/1b and had an average of twofold change in expression from peak to trough, whereas hsf2 transcripts were expressed in phase with cry1a and had a 7.2-fold-change in expression. The changes in expression of clock and clock-controlled genes observed during continuous darkness were also observed at similar ZTs in fish exposed to a normal photoperiod in a separate control experiment. The role of circadian clocks in regulating muscle maintenance and growth are discussed.

MiR-206-mediated dynamic mechanism of the mammalian circadian clock.

BackgroundAs a group of highly conserved small non-coding RNAs with a length of 21~23 nucleotides, microRNAs (miRNAs) regulate the gene expression post-transcriptionally by base pairing with the partial or full complementary sequences in target mRNAs, thus resulting in the repression of mRNA translation and the acceleration of mRNA degradation. Recent work has revealed that miRNAs are essential for the development and functioning of the skeletal muscles where they are. In particular, miR-206 has not only been identified as the only miRNA expressed in skeletal muscles, but also exhibited crucial roles in regulation of the muscle development. Although miRNAs are known to regulate various biological processes ranging from development to cancer, much less is known about their role in the dynamic regulation of the mammalian circadian clock.ResultsA detailed dynamic model of miR-206-mediated mammalian circadian clock system was developed presently by using Hill-type terms, Michaelis-Menten type and mass action kinetics. Based on a system-theoretic approach, the model accurately predicts both the periodicity and the entrainment of the circadian clock. It also explores the dynamics properties of the oscillations mediated by miR-206 by means of sensitivity analysis and alterations of parameters. Our results show that miR-206 is an important regulator of the circadian clock in skeletal muscle, and thus by study of miR-206 the main features of its mediation on the clock may be captured. Simulations of these processes display that the amplitude and frequency of the oscillation can be significantly altered through the miR-206-mediated control.ConclusionsMiR-206 has a profound effect on the dynamic mechanism of the mammalian circadian clock, both by control of the amplitude and control or alteration of the frequency to affect the level of the gene expression and to interfere with the temporal sequence of the gene production or delivery. This undoubtedly uncovers a new mechanism for regulation of the circadian clock at a post-transcriptional level and provides important insights into the normal development as well as the pathological conditions of skeletal muscles, such as the aging, chronic disease and cancer.

Working around the clock: circadian rhythms and skeletal muscle

The study of the circadian molecular clock in skeletal muscle is in the very early stages. Initial research has demonstrated the presence of the molecular clock in skeletal muscle and that skeletal muscle of a clock-compromised mouse, Clock mutant, exhibits significant disruption in normal expression of many genes required for adult muscle structure and metabolism. In light of the growing association between the molecular clock, metabolism, and metabolic disease, it will also be important to understand the contribution of circadian factors to normal metabolism, metabolic responses to muscle training, and contribution of the molecular clock in muscle-to-muscle disease (e.g., insulin resistance). Consistent with the potential for the skeletal muscle molecular clock modulating skeletal muscle physiology, there are findings in the literature that there is significant time-of-day effects for strength and metabolism. Additionally, there is some recent evidence that temporal specificity is important for optimizing training for muscular performance. While these studies do not prove that the molecular clock in skeletal muscle is important, they are suggestive of a circadian contribution to skeletal muscle function. The application of well-established models of skeletal muscle research in function and metabolism with available genetic models of molecular clock disruption will allow for more mechanistic understanding of potential relationships.

The effect of denervation on the molecular clock in skeletal muscle

We recently identified the circadian transcriptome in mouse skeletal muscle. The primary objective of this study was to test the hypothesis that innervation is required to maintain oscillation of the circadian molecular clock in muscle. A second objective was to determine if the phase of the molecular clock is the same in different muscles. Using the Period‐2:Luciferase mouse, the tibialas anterior was denervated (d‐TA) by sectioning the common peroneal nerve on one leg with the contralateral leg serving as a sham operated control. Following two weeks of denervation, various muscles and liver were collected from three animals every four hours for 24 hr. RT‐PCR analysis revealed that the expression pattern of components of the molecular clock (Bmal1, Per2 and Clock) as well as Myod1, a clock‐controlled gene, was altered in the d‐TA compared to i‐TA. Luciferase activity, reflecting Per2 protein expression, of the d‐TA was significantly elevated at each time point in comparison to inneravted‐TA (i‐TA), though the time of peak expression remained unchanged. In the heart, diaphragm and liver, peak luciferase activity peaked four hours later than what was observed in hindlimb muscles suggesting the phase of the clock is slightly different in these tissues. Collectively, these results indicate loss of neural input causes alterations in the molecular clock in adult skeletal muscle.KAE (AR045617) and JJM (AR053641)

T.P.2 02 The mitotic clock in skeletal muscle: Immortalization of human myoblasts and consequences for studying muscle disease

T.P.2 02 The mitotic clock in skeletal muscle: Immortalization of human myoblasts and consequences for studying muscle disease