Binge alcohol disrupts skeletal muscle core molecular clock independent of glucocorticoids

Abstract

Circadian rhythms are central to optimal physiological function as disruption contributes to the development of several chronic diseases. Alcohol (EtOH) intoxication disrupts circadian rhythms within liver, brain, and intestines, but it is unknown whether alcohol also disrupts components of the core clock in skeletal muscle. Female C57BL/6Hsd mice were randomized to receive either saline (control) or alcohol (EtOH) (5g/kg) via intraperitoneal injection at the start of the dark cycle (ZT12), and gastrocnemius was collected every 4hr from Control and EtOH treated mice for the next 48hr following isoflurane anesthetization. In addition, metyrapone was administered prior to alcohol intoxication in separate mice to determine whether the alcohol-induced increase in serum corticosterone contributed to circadian gene regulation. Finally, synchronized C2C12 myotubes were treated with alcohol (100mM) to assess the influence of centrally or peripherally mediated effects of alcohol on the muscle clock. Alcohol significantly disrupted mRNA expression of Bmal1, Per1/2, and Cry1/2 in addition to perturbing the circadian pattern of clock-controlled genes, Myod1, Dbp, Tef, and Bhlhe40 (p<0.05) in muscle. Alcohol increased serum corticosterone levels and glucocorticoid target gene, Redd1, in muscle. Metyrapone prevented the EtOH-mediated increase in serum corticosterone but did not normalize the EtOH-induced change in Per1, Cry1 and Cry2 and Myod1 mRNA expression. Core clock gene expression (Bmal, Per1/2, Cry1/2) was not changed following 4, 8, or 12hrs of alcohol treatment on synchronized C2C12 myotubes. Therefore, binge alcohol disrupted genes of the core molecular clock independently of elevated serum corticosterone or direct effects of EtOH on the muscle.

Keep Your Mask On: The Benefits of Masking for Behavior and the Contributions of Aging and Disease on Dysfunctional Masking Pathways

“…Alcohol also affects entrainment of the SCN to photic and non-photic zeitgebers ( Ruby et al, 2009 ; Prosser and Glass, 2015 ) and impacts the expression of endogenous rhythms in constant conditions ( Ruby et al, 2017 ). Injections of ethanol in mice can also have direct effects on peripheral clocks; in the skeletal muscle and liver, injections changed the expression of core clock genes ( Tice et al, 2021 ), However, the relationship between sleep and alcohol consumption is problematic as alcoholism is also linked to the development of sleep disturbances ( Stein and Friedmann, 2006 ). Chronic alcohol use can directly impact the homeostatic sleep response, wildly altering the expression of sleep architecture ( Brower, 2001 ; Thakkar et al, 2015 ) and disrupting responses to sleep challenge ( Armitage et al, 2012 ).…”

Chronic Alcohol Consumption Disrupts the Skeletal Muscle Circadian Clock in Female Mice

“…Mice were housed on a 12:12 light-dark cycle in a temperature-controlled (25°C) environment with ad libitum access to food and water. Female mice were chosen for experimentation as females have an increased propensity to develop chronic alcohol related injuries at lower cumulative doses of alcohol (Urbano-Marquez et al, 1995; Shenkman et al, 2016; DeGroat et al, 2018) and because the acute circadian studies had been performed in females allowing for comparisons to be made (Tice et al, 2021). This substrain of C57BL/6 mice was used to match our acute alcohol circadian experiments and we are not aware of any differences across substrain in skeletal muscle circadian response to alcohol to validate use of one substrain over another.…”

“…Diets were replaced at the start of the dark cycle each day. At the end of the alcohol treatment period and starting at the beginning of the dark cycle, a subset of mice from the control and alcohol groups ( n = 3/4 per treatment) were euthanized every 4 h for 24 h to complete a 24 h diurnal cycle as described previously (Tice et al, 2021). During this 24-h period, mice continued to have ab libitum access to the liquid diet which was freshly added at the start of the dark cycle.…”

“…Protocol for RNA extraction, cDNA synthesis, and RT-PCR of gastrocnemius muscle tissue were performed as previously described (Tice et al, 2021). All primer sequences used with Powerup SYBR-green master mix (Cat.…”

“…We previously showed that an acute alcohol binge leads to disruption of core clock components and CCGs for at least 48 hours post binge in the skeletal muscle (Tice et al, 2021). The binge style of drinking may not be like that which occurs in alcoholics as it does not account for the cumulative effects of prolonged drinking nor the more moderate intake over the course of the day.…”