Drinking Off(s) the Clock: Chronic Alcohol Suppresses Skeletal Muscle Molecular Clock

Abstract

Circadian rhythms generated both centrally in the suprachiasmatic nucleus (SCN) and peripherally at the tissue level assist in the maintenance of physiological homeostasis, while circadian disruption is a characteristic of several diseases. The intrinsic skeletal muscle core clock has emerged as a key feature of metabolic control and influences several aspects of muscle physiology including contractile strength and mitochondrial content. Alcoholic muscle disease or myopathy is characterized by skeletal muscle weakness and myofiber atrophy resulting from the prolonged consumption of alcohol. Despite alcoholic myopathy being more prevalent than alcoholic liver disease, the entirety of mechanisms leading to its onset remains unknown. Alcohol disrupts normal cyclic expression of several core clock genes within the brain and liver, and we previously reported that acute alcohol intoxication disrupts the skeletal muscle core clock. Thus, the goal of this work was to determine whether the chronic consumption of alcohol dysregulates the skeletal muscle core molecular clock and clock-controlled genes (CCGs) in concert with alcoholic myopathy. Methods: C57BL/6Hsd female mice (12wks old) were pair fed a control liquid diet (CON) or an isocaloric ethanol (EtOH) containing liquid diet of increasing concentration (up to 32% kcal EtOH) for 7 weeks. Gastrocnemius muscles were collected from CON (n=4) and EtOH (n=3-4) mice every 4-hours for 24-hours. RNA was isolated, cDNA synthesized, and RT-PCR was performed. Results: Body weight and fat mass between groups was similar at sacrifice while liver mass was increased by alcohol. Lean mass assessed by MRI tended to be decreased by alcohol (p=0.06). Chronic alcohol consumption disrupted genes of the core clock including suppressing the rhythmic peak in expression of brain muscle ARNT like-1 (Bmal1), period (Per) 1 and 2, and cryptochrome 2 (Cry2). Genes involved in the regulation of Bmal1 also exhibited lower rhythmic peaks including Rev-erb (NR1D1) and myoblast determination protein 1 (MyoD), with significant reductions apparent at several of the 4-hour time points. The CCG D-box binding PAR BZIP transcription factor (DBP) was also suppressed at the end of the light phase and beginning of the dark active phase (ZT8 and ZT12). Skeletal muscle atrophy related genes forkhead box O1 (Foxo1), regulated in development and DNA damage response 1 (Redd1) and muscle RING-finger protein 1 (Murf1) were also altered by alcohol at the start of the dark (active) cycle. These data contrasted our previously described acute model in which subtle rhythmic shifts and large changes in the expression of several genes were observed specifically when blood alcohol levels (~4-12hr) were elevated. Therefore, there is a differential response of the skeletal muscle to acute and chronic alcohol consumption with prolonged intake leading to a lower amplitude of change over the 24-hour cycle of several core clock related genes. Future work will focus on determining the contribution of these disruptions to metabolic and functional perturbations characteristic of chronic alcoholic myopathy and the impact of these disruptions on the development of alcoholic myopathy.