Link Between Alcohol Consumption And Muscle Damage In Females

New study in animals finds increases in muscle mRNA among females -- but not males  -- in chronic ethanol exposure

NOVEMBER 29, 2003 (Bethesda, MD) – A common manifestation of alcoholism is the degeneration, or wasting away, of skeletal muscle. The condition, known as alcoholic myopathy, affects up to two-thirds of those who excessively consume alcohol; moreover, women appear to be particularly susceptible. The dominant features of this disorder are cramps,impaired muscle strength, and reduced whole body lean tissue mass, all of which are accompanied by reductions in the relative amounts of specific contractile proteins within the muscle itself.

Although there is also some evidence to suggest that malnutrition exacerbates the effects of alcohol on muscle, the mechanisms responsible for myopathy remain elusive. Some studies suggest that acetaldehyde (a toxic intermediate compound formed by the action of alcohol dehydrogenase enzyme during the metabolism of alcohol),rather than alcohol, is the principal pathogenic perturbant.

A New Study

In an attempt to better understand the pathology induced by alcohol on a common muscle, a team of researchers has tested their hypotheses to determine if: (1) increases in c-myc mRNA levels also occur in the muscle of rats chronically exposed to alcohol, (2) muscle of female rats is more sensitive than muscle in their male counterparts, (3) raising acetaldehyde also increases c-myc, (4) prior starvation causes further increases in c-myc mRNA expression in response to ethanol, and (5) other genes involved in apoptosis (i.e., p53 and Bcl-2) are also affected by alcohol.

The researchers are Tatsuo Nakahara, of the Department of Chemistry, Kyushu University, Ropponmatsu, Fukuoka; Kijiro Hashimoto and Makoto Hirano, both from the Center for Emotional and Behavioural Disorders, Hizen National Mental Hospital, Kanzaki, Saga, Japan; Michael Koll and Victor R. Preedy, from the Department of Nutrition and Dietetics, and the Genomics Centre, respectively, King's College London, London, England; and Colin R. Martin, from the Department of Health Sciences, University of York, Heslington, York, United Kingdom. The results of their work, entitled “Acute and Chronic Effects of Alcohol Exposure on Skeletal Muscle c-myc, p53, and Bcl-2 mRNA Expression,” are published in the December 2003 edition of the American Journal of Physiology – Endocrinology and Metabolism. The Journal is one of 14 scientific journals published each month by the American Physiological Society (APS).

Methodology

The experiment was conducted according to the methodology outlined below:

Animals: Wistar rats were maintained for ~1 wk until they weighed ~0.1–0.15 kg then ranked on the basis of weight and divided into appropriate groups of equal mean body weight for one of three experiments.

Study 1: Comparison of the effect of chronic alcohol exposure for 6 wk in male and female rats.  Rats in this experiment were divided as follows: control male (n=10); control female (n=8); ethanol male (n= 10); ethanol female (n=8). The animals were then subjected to an alcohol-feeding regimen in which treated rats were fed a nutritionally complete liquid diet containing 35% of total calories as ethanol ad libitum. Controls were pair-fed the same diet but ethanol was replaced by isocaloric glucose. Mean daily intakes of diets were 49 and 52 ml/day for female and male rats, respectively. Rats were killed, representative skeletal muscle (hindlimb musculature) was dissected into various regions and frozen immediately and stored until analysis.

Study 2: Effects of acute ethanol exposure and of raising endogenous acetaldehyde with cyanamide.  Rats were divided into the following groups: saline+saline (n=7); cyanamide+saline (n=7); saline+ethanol (n=7); cyanamide+ethanol (n=7). The dosage used was 75 mmol/kg body wt for alcohol and 0.5 mmol/kg body wt for cyanamide. Controls were injected with identical volumes of 0.15 mM NaCl. The experimental procedure involved intraperitoneal injection "pretreatment" (30 min) with either saline or cyanamide, followed by intraperitoneal injection "treatment" (150 min) with either saline or ethanol. Rats were killed after 2.5 h of exposure to ethanol.

Study 3: Effects of starvation and acute superimposition of alcohol. Rats were either treated as in the fed state or starved 1-2 days before use and divided as follows: fed+saline (n=8); fed+ethanol (n=8); starved 1 day+saline (n=8); starved 1 day+ethanol (n=9); starved 2 days+saline (n=7); starved 2 days+ethanol (n=8).  The dosage used was 75 mmol ethanol/kg body wt for alcohol; controls were injected with an identical volume of 0.15 mM NaCl. Rats were killed after 2.5 h of exposure to alcohol.

Method for c-myc, p53, Bcl-2, and GAPDH: Total RNA was prepared from the muscle (hindlimb musculature). The levels of c-myc, p53, and Bcl-2 mRNAs were quantified by RT-PCR with an endogenous internal standard, GAPDH. RT was performed on 1 µg of total RNA for 90 min. Multiplexed PCR was carried out in a 20-µl reaction mixture. PCR amplification was performed for 28 (c-myc), 30 (p53), or 34 (Bcl-2) cycles. After eight cycles (c-myc or p53) or twelve cycles (Bcl-2), 0.1 µM of each GAPDH primer pair was added to the reaction mixture, and PCR cycles were further continued for 20 (c-myc) or 22 (p53 or Bcl-2) cycles. PCR products were analyzed by electrophoresis. Gels were stained, visualized with UV transillumination, photographed, and submitted to image analysis. Quantitative image analysis of the PCR fragments was performed and mRNA levels were calculated.

Statistical Analysis: All data were expressed as means ± SE (n = 6–10). In all studies, significance was indicated when P 0.05. 

Results

The results showed that:

• in male rats fed ethanol chronically, there were no increases in c-myc mRNA;

• increases did, however, occur in c-myc mRNA in muscle from female rats who were fed ethanol chronically;  

• raising endogenous acetaldehyde with cyanamide increased c-myc mRNA in acute studies;  

• starvation per se increased c-myc mRNA levels and at 1-day potentiated the acute effects of ethanol, indicative of a sensitization response; and 

• the only effect seen with p53 mRNA levels was a decrease in muscle of rats starved for 1 day compared with fed rats, and there was no statistically

• significant effect on Bcl-2 mRNA in any of the experimental conditions.

Conclusions

Alcohol acutely increases c-myc mRNA in skeletal muscle, possibly reflecting a preapoptotic effect, a compensatory stimulus to induce hypertrophy to counteract the catabolic effects of alcohol, or even a nonspecific cellular stress response to alcohol and/or acetaldehyde. Contrary to expectations, neither p53 nor Bcl-2 mRNA levels were affected by alcohol, even in the presence of cyanamide predosing or starvation. These data are important in increasing our knowledge of the damaging effect alcohol has, for both males and females, on a common muscle.

-end-

Source: December 2003 edition of the American Journal of Physiology – Endocrinology and Metabolism. The Journal is one of 14 scientific journals published each month by the American Physiological Society (APS).

The American Physiological Society (APS) was founded in 1887 to foster basic and applied science, much of it relating to human health. The Bethesda, MD-based Society has more than 10,000 members and publishes 3,800 articles in its 14 peer-reviewed journals every year.

 ***

Editor’s Note: A copy of the research article is available in pdf format to the press. Members of the press are invited to obtain a pdf copy of the study and to interview members of the research team. To do so, please contact Donna Krupa at 703.527.7357 (direct dial), 703.967.2751 (cell) or djkrupa1@aol.com.