Identification of the circadian transcriptome in adult mouse skeletal muscle

Abstract

Circadian rhythms are approximate 24-h behavioral and physiological cycles that function to prepare an organism for daily environmental changes. The basic clock mechanism is a network of transcriptional-translational feedback loops that drive rhythmic expression of genes over a 24-h period. The objectives of this study were to identify transcripts with a circadian pattern of expression in adult skeletal muscle and to determine the effect of the Clock mutation on gene expression. Expression profiling on muscle samples collected every 4 h for 48 h was performed. Using COSOPT, we identified a total of 215 transcripts as having a circadian pattern of expression. Real-time PCR results verified the circadian expression of the core clock genes, Bmal1, Per2, and Cry2. Annotation revealed cycling genes were involved in a range of biological processes including transcription, lipid metabolism, protein degradation, ion transport, and vesicular trafficking. The tissue specificity of the skeletal muscle circadian transcriptome was highlighted by the presence of known muscle-specific genes such as Myod1, Ucp3, Atrogin1 (Fbxo32), and Myh1 (myosin heavy chain IIX). Expression profiling was also performed on muscle from the Clock mutant mouse and sarcomeric genes such as actin and titin, and many mitochondrial genes were significantly downregulated in the muscle of Clock mutant mice. Defining the circadian transcriptome in adult skeletal muscle and identifying the significant alterations in gene expression that occur in muscle of the Clock mutant mouse provide the basis for understanding the role of circadian rhythms in the daily maintenance of skeletal muscle.

Fig. 1.

Real-time PCR verification of core clock gene expression in skeletal muscle. Real-time PCR was performed to validate the circadian expression of core clock genes identified by COSOPT from the array results. Graphical presentation of the results shows brain muscle arnt-like 1 (Bma1, A), period 2 (Per2, B), and cryptochrome (Cry2, C) transcript levels oscillate over 48 h with a circadian period indicative of a functioning circadian clock in adult skeletal muscle. Independent duplicate samples were run at each of 12 time points and normalized to GAPDH expression as described in METHODS. The light gray bars across the top of each graph represent the presumptive night with the intervening white spaces representing presumptive day.

Fig. 2.

Temporal distribution of circadian genes by peak expression. Cycling genes were clustered according to their respective circadian time of peak expression. Genes were equally distributed between subjective day [circadian time 2 (CT2), CT6, and CT10: n = 103] and subjective night (CT14, CT18, and CT22: n = 112). Almost one-third (n = 68) of the cycling transcripts had peak expression at CT18, a time period of high physical activity and feeding.

Fig. 3.

Gene ontology of skeletal muscle circadian transcriptome. A pie chart showing the distribution of circadian genes by biological process. Gene annotation was performed using GNF SymAtlas and literature searches. The “others” designation represents biological processes that occurred infrequently in the circadian transcriptome and included (frequency in parentheses) blood coagulation (2), chitin metabolism (1), DNA repair (1), meiosis (2), methyl-transferase (1), and defense response (2). The list of genes within each biological cluster is shown in Supplemental Table S1.

Fig. 4.

Altered expression of circadian genes in Clock mutant. Approximately 30% of the circadian genes in the Clock mutant showed a loss in cycling or a phase shift in peak expression. Graphs comparing wild-type vs. Clock mutant expression shows a loss of cycling (A–C) or a shift in the time of peak expression (D, E). The absence of circadian oscillation in the Clock mutant is shown for Per2 (A), Dbp (B), and Myod1 (C), three known clock-controlled genes. A phase shift in peak expression in the Clock mutant is shown for Ucp3 (D) and Pdk4 (E), both muscle-enriched circadian genes. The light gray bars across the top of each graph represent the presumptive night with the intervening white spaces representing presumptive day.