The functional significance of the skeletal muscle clock: lessons from Bmal1 knockout models

Abstract

The circadian oscillations of muscle genes are controlled either directly by the intrinsic muscle clock or by extrinsic factors, such as feeding, hormonal signals, or neural influences, which are in turn regulated by the central pacemaker, the suprachiasmatic nucleus of the hypothalamus. A unique feature of circadian rhythms in skeletal muscle is motor neuron-dependent contractile activity, which can affect the oscillation of a number of muscle genes independently of the muscle clock. The role of the intrinsic muscle clock has been investigated using different Bmal1 knockout (KO) models. A comparative analysis of these models reveals that the dramatic muscle wasting and premature aging caused by global conventional KO are not present in muscle-specific Bmal1 KO or in global Bmal1 KO induced in the adult, therefore must reflect the loss of Bmal1 function during development in non-muscle tissues. On the other hand, muscle-specific Bmal1 knockout causes impaired muscle glucose uptake and metabolism, supporting a major role of the muscle clock in anticipating the sleep-to-wake transition, when glucose becomes the predominant fuel for the skeletal muscle.

Keywords: Bmal1 knockout; Circadian rhythms; Glucose metabolism; Glucose uptake; Muscle clock; Muscle denervation; Skeletal muscle.

Fig. 1

Core clock mechanism and control of circadian muscle genes by intrinsic and extrinsic pathways. a Simplified scheme of the core clock mechanism. The clock consists of a stimulatory loop, with the CLOCK-BMAL1 heterodimer stimulating the transcription of Per and Cry genes, and an inhibitory feedback loop with the PER-CRY heterodimer translocating to the nucleus and repressing the transcription of the Clock and Bmal1 genes. An additional loop involves the ROR and RevErb factors. The different isoforms of the clock genes, including the genes coding for PER1 and PER2, CRY1 and CRY2, RORα and RORβ, and RevErbα and RevErbβ, are not indicated in the scheme. b The scheme illustrates how the master clock in the suprachiasmatic nuclei (SCN) of the hypothalamus controls motor activity and other systemic circadian rhythms (including feeding, hormone release, and body temperature), which in turn modulate the circadian rhythm of the muscle clock or directly dictate the oscillation of other muscle circadian genes. Modified from [7]

Fig. 2

Changes in gene expression induced by muscle-specific Bmal1 knockout (mKO), leading to disruption of the muscle clock, or denervation (D), leading to loss of motor activity. Transcript levels were monitored by qPCR every 4 h (0, lights on; 12, lights off). Three representative genes are illustrated. Dbp, a direct target of Bmal1, is strongly repressed by Bmal1 KO but shows only a phase advance of around 4 h in denervated muscles without any change in oscillation amplitude. In contrast, Rcan1.4, a gene controlled by motor neuron activity via calcineurin-NFAT signaling, is essentially unchanged in Bmal1 mKO muscles but is drastically downregulated by denervation. Myod1, coding for the myogenic regulatory factor MyoD, shows an atypical response, with circadian oscillation maintained with increased amplitude in both Bmal1 mKO and denervated muscles. In denervation experiments, the muscles were removed 7 days after sciatic nerve section. The muscles examined were tibialis anterior for Dbp and Myod1 and soleus for Rcan1.4 (data from [27]; changes in Myod1 are unpublished observations)

Fig. 3

Bmal1 and Per2 transcripts do not oscillate in embryonic tissues. Twenty-four-hour expression profiles of Per2 and Bmal1 mRNA in embryonic (E18-E19) and adult mouse heart, as determined by qPCR. Identical results were seen in the liver and kidney, while the skeletal muscle was not analyzed in this study. Note that the embryonic heart shows little circadian variation in Per2 and Bmal1 expression, in contrast with the robust changes seen in the adult tissue (modified from [39])

Fig. 4

The scheme illustrates the role of peripheral clocks in the control of muscle glucose metabolism, as determined using tissue-specific Bmal1 knockout models. The liver clock controls glucose output during the fasting/inactive phase, as shown by the finding that liver-specific Bmal1 KO causes hypoglycemia during this phase [42]. The pancreas β cell clock controls insulin secretion, as β cell-specific Bmal1 KO causes hyperglycemia [–45]. The muscle clock promotes glucose uptake and metabolism at awakening, as skeletal muscle-specific Bmal1 KO causes impaired insulin-dependent glucose uptake and glucose oxidation in skeletal muscle fibers [27]

Fig. 5

Simplified scheme of glucose uptake and metabolism in muscle cells, highlighting two crucial steps controlled by the intrinsic muscle clock: insulin-dependent glucose uptake and pyruvate conversion to acetyl-CoA. Insulin promotes glucose uptake by activating the kinase AKT that phosphorylates the Rab-GTPase-activating protein TBC1D1, thus promoting the translocation of GLUT4 to the plasma membrane. Pyruvate, upon entry into mitochondria (mito), is metabolized to acetyl-CoA by pyruvate dehydrogenase (PDH), whose activity is inhibited by the PDH kinase PDK4 and stimulated by the PDH phosphatase PDP1. The protein expression of GLUT4, and both mRNA and protein levels of TBC1D1, PDK4, and PDP1 vary across the day-night cycle (0, lights on; 12, lights off) and are drastically affected by Bmal1 mKO. Under normal conditions, PDK4 has a peak of expression in the fasting phase (around ZT4), whereas PDP1 peaks around the transition from the fasting to the feeding/active phase (around ZT12). Note that PDK4 starts to decrease and PDP1 to increase during the fasting phase, before awakening, supporting the notion of the anticipatory role of the muscle clock, which prepares the muscles to the upcoming activity period. These circadian adaptations are completely disrupted by Bmal1 mKO, with downregulation of PDP1 and a rightward shift in the peak of PDK4, leading to decrease in PDH activity at awakening (data from [27])