THE SKELETAL MUSCLE MOLECULAR CLOCK REGULATES THE TIMING OF SUBSTRATE METABOLISM AND THE CIRCADIAN EXPRESSION OF TITIN-CAP

OF DISSERTATION THE SKELETAL MUSCLE MOLECULAR CLOCK REGULATES THE TIMING OF SUBSTRATE METABOLISM AND THE CIRCADIAN EXPRESSION OF TITIN-CAP Skeletal muscle is a major contributor to whole-body metabolism as it serves as a depot for both glucose and amino acids, and is a highly metabolically active tissue. An intrinsic molecular clock mechanism exists within skeletal muscle that regulates the timing of physiological processes. A key function of the clock is to regulate the timing of metabolic processes to anticipate time of day changes in environmental conditions. The purpose of this study was to identify metabolic genes that are expressed in a circadian manner and determine if these genes are regulated downstream of the intrinsic molecular clock by assaying gene expression in an inducible skeletal muscle-specific Bmal1 knockout mouse model (iMS-Bmal1−/−). The skeletal muscle circadian transcriptome we analyzed was highly enriched for metabolic processes. Acrophase (time of peak expression) analysis of circadian metabolic genes revealed a temporal separation of genes involved in substrate utilization and storage over a 24-h period with many differentially expressed in the skeletal muscle of the iMS-Bmal1−/− mice compared to wildtype. However, the iMSBmal1−/− mice displayed circadian behavioral rhythms indistinguishable from iMSBmal1+/+ mice. We also observed a gene signature indicative of a fast to slow fibertype shift and a more oxidative skeletal muscle in the iMS-Bmal1−/− model. These data provide evidence that the intrinsic molecular clock in skeletal muscle temporally regulates genes involved in the utilization and storage of substrates independent of circadian activity. Disruption of this mechanism caused by phase shifts (that is, social jetlag) or night eating may ultimately diminish skeletal muscle’s ability to efficiently maintain metabolic homeostasis over a 24-h period. The molecular-clock targets genes for circadian expression in a tissuespecific manner, possibly through interactions with tissue-specific factors. In order to identify novel mechanisms responsible for driving circadian gene expression of muscle-specific genes we focused our study on the molecular regulation of the Titincap gene. We choose this gene as it was highly circadian in the skeletal muscle circadian transcriptome, and has previously been shown to be modulated by the clock factor BMAL1 in heart-tissue, and the myogenic regulatory factor MYOD1 in skeletal-muscle. Promoter-reporter experiments demonstrated that BMAL1:CLOCK and MYOD1 work in a synergistic fashion to transactivate the Titin-cap gene in skeletal-muscle. Circadian expression of Titin-cap relied on the normal function of MYOD1 as mutant vectors altered the rhythmic oscillation and expression. We provided evidence that BMAL1 and MYOD1 bind to a tandem E-Box element in the proximal promoter element, and that this element is required for the circadian expression of Titin-cap in skeletal-muscle. These data provide a novel mechanism in which the molecular-clock works with a tissue specific transcription factor to drive circadian gene expression.