Drinking ‘Off’ the Clock: Effects of Binge Alcohol on the Skeletal Muscle Core Clock

Abstract

IntroductionCircadian rhythms are ~24‐hour oscillations generated by a transcription‐translation feedback loop known as the core molecular clock. The core clock is comprised of a positive arm, which includes the genes circadian locomotor output cycles kaput (Clock) and brain and muscle arnt‐like (Bmal1), and a negative arm, which includes period (Per)1/2/3 and cryptochrome (Cry)1/2. Several physiological processes are ‘Clock’ controlled, and disruptions to these rhythms is linked to many chronic diseases, such as cardiovascular disease, diabetes, depression, and cancer. Previous studies have shown that alcohol (EtOH) intoxication disrupts circadian rhythms within liver, brain and intestines, but the effects of alcohol intoxication on skeletal muscle circadian rhythms has not been assessed. Our objective is to assess the effects of EtOH on regulation of components of the core molecular clock in skeletal muscle.MethodsFemale C57BL6 mice (n=39) were randomized into one of three groups: Baseline (n=3), Control (Saline; n=18), or Alcohol (EtOH; n=18). Mice in Control and EtOH received an IP injection of either saline, or EtOH (5g/kg BW) at the start of the dark cycle (Zeitgeber time (ZT12)), with ab libitum food and water. Baseline mice were euthanized at ZT12, while 3 saline and 3 EtOH mice were euthanized every 4h thereafter. The gastrocnemius was used for RNA isolation, and RT‐PCR was performed to measure circadian gene expression. Data were analyzed by two‐way ANOVA (Time, EtOH) with Fisher’s LSD Post hoc testing (p<0.05).ResultsExpression of the core clock components Bmal1, Cry1/2, and Per1/2/3 was disrupted by EtOH when compared to saline. EtOH increased expression of Per1 and eliminated the normal circadian oscillation for 20 hr post injection. Per2 expression was also increased by alcohol for the first 8h post injection, ZT12–ZT24. EtOH increased Per3 expression at the end of the dark cycle (ZT24), before decreasing it at ZT8 in the light cycle. Alcohol prevented the normal increase in Cry1 throughout the dark phase and the usual suppression during the light cycle (ZT8 and ZT12), while Cry2 expression was increased (>2 fold) over both the dark and light cycles in EtOH when compared to control. In the positive arm of the core clock, EtOH did not significantly disrupt the normal circadian expression of Clock but did cause a rightward shift of the normal oscillation of Bmal1. EtOH also significantly altered gene expression of several downstream targets of the core clock, including D‐box binding protein (DBP), retinoid‐related orphan receptor‐α (ROR‐α), and myogenic differentiation (MyoD), suggesting functional implications of core clock disruption by alcohol. Finally, circadian expression of Redd1 (i.e. regulated in development and DNA damage response‐1), a metabolic regulator, was disrupted by EtOH, as a ~35‐fold induction occurred during the dark cycle compared with controls.ConclusionA single dose of alcohol significantly disrupted several components of the core molecular clock, as well as downstream targets within skeletal muscle. Therefore, dysregulation of the skeletal muscle core clock may contribute to alcoholic myopathy and other co‐morbidities associated with high levels of alcohol consumption.