Sex-dependent molecular alterations during development of alcoholic cardiomyopathy in rats
Open Access
- Author:
- Fogle, Rachel LaDean
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 02, 2010
- Committee Members:
- Thomas C Vary, Dissertation Advisor/Co-Advisor
Willard M Freeman, Committee Member
Patricia Sue Grigson, Committee Member
Christopher Hollenbeak, Committee Member
Christopher J Lynch, Committee Member
Charles H Lang, Committee Chair/Co-Chair - Keywords:
- meta-analysis
random effects
alcoholic heart muscle disease
mass spectrometry
chronic ethanol
iTRAQ
proteomics - Abstract:
- Chronic alcohol abuse contributes to the development of alcoholic heart muscle disease, which may ultimately result in heart failure. This disease is characterized by alterations in the ability of the heart to pump efficiently as well as structural changes resulting in myocardial dysfunction. The functional and structural changes observed with the progression of the disease to a dilated cardiomyopathy are consistent with alterations in protein content secondary to an inhibition of protein synthesis. Although the effects of chronic alcohol intake are frequently studied in males, females are also susceptible to alcohol-induced myocardial defects. There are discrepancies among investigations regarding sex and the toxic effects of chronic alcohol intake on the myocardium. To date, only selected candidate proteins have been investigated and, therefore, the full extent to which myocardial proteins are affected by chronic alcohol consumption across the sexes remains unresolved. The purpose of this research was to utilize proteomic technology in order to examine the global effect of chronic alcohol abuse on the profile of cardiac proteins. Rats were maintained for 16-18 weeks on a 40% alcohol-containing diet in which ethanol was provided both in drinking water and agar blocks. Echocardiography evaluation allowed non-invasive detection of structural and functional changes as a result of the alcohol insult. Two different proteomic labeling techniques were used in these studies. First, ICAT (isotope coded affinity tags) was used to determine alterations in protein content in male alcohol-fed rats compared to control rats. Next, iTRAQ (isobaric tags for relative and absolute quantitation) reagents were utilized to compare protein profiles between male and female rats. The 4-plex iTRAQ technology allowed the four experimental groups (male control, male alcohol, female control, and female alcohol) to be analyzed simultaneously with each iTRAQ application. Enriched subcellular fractions of mitochondrial, myofibrillar, nuclear, and sarcoplasmic proteins were generated to reduce sample complexity and permit detection of low abundance proteins via mass spectrometry-based approaches. A random effects model of meta-analysis, developed and applied to sample iTRAQ datasets, allowed the overall summary effect of protein content across multiple iTRAQ experiments investigating the effect of long-term alcohol abuse on protein levels to be determined. Prolonged feeding of an ethanol-containing diet for 16-18 weeks resulted in decreased left ventricular weight as well as alterations in structural and functional parameters, including stroke volume, cardiac output, and end-diastolic diameter in male rats, but not female rats. Initial proteomic analysis via either labeling technique, i.e. ICAT or iTRAQ, identified and quantified hundreds of myocardial proteins. Specifically, the ICAT study resulted in 77 significant proteins of 448 total identified proteins. In this study, myofibrillar proteins involved in contraction, such as α-myosin and actin, were down-regulated in males as a consequence of long-term alcohol consumption, consistent with previous reports. Application of the random effects model allowed determination of statistically significant proteins within each subcellular fraction across the multiple iTRAQ datasets generated for that fraction. The number of statistically significant proteins was greatly reduced (-89% to -96%) from the original number of identifications within that fraction. A total of 45, 95, 98, and 113 proteins were determined as significant alcohol-induced alterations in protein levels in the mitochondrial, myofibrillar, nuclear, and sarcoplasmic fractions, respectively. Pathway and network analysis revealed several sexual dimorphisms. A network of proteins involved in cardiovascular system development and function highlighted such a sexual dimorphism. Alcohol-induced up-regulation of the troponins, key contractile regulatory proteins, was reported in females consuming alcohol in comparison with non-drinking females, while concomitant down-regulation was observed in alcohol-receiving males when compared to non-drinking male counterparts. These findings were validated by Western blot analysis. Pathway analysis of proteins involved in energy metabolism, e.g., beta-oxidation and oxidative phosphorylation, revealed another sex-dependent difference. Males consuming alcohol showed increased expression of proteins in complex I (NADH dehydrogenase), complex III (cytochrome b-c1), complex IV (cytochrome c oxidase), and complex V (ATPase), whereas females showed no change or even a decrease in expression. As determined by echocardiography evaluation, long-term alcohol intake demonstrated more significant alterations in the structural and functional parameters of the heart in male rats compared to female rats. Based on these observations and the corresponding changes in proteins, we speculate that females may be protected from the toxic effects of alcohol due to their adaptive ability to maintain contractile function, maintain efficiency of force generation, and minimize oxidative stress experienced by the cell. The adaptive response to the alcohol insult observed in males is indicative of a decreased efficiency of contraction and increased energy production, as demonstrated by increased expression of troponin as well as proteins involved in mitochondrial energy production. However, males lack the ability to overcome the alcohol-induced insult to the myocardial tissue, leading to increased production of reactive oxygen species and structural abnormalities. This is consistent with the alterations in myocardial structure and function that are observed in males, but not females, following assessment via echocardiography that suggest male myocardium is more susceptible to the toxic effects of alcohol abuse. Based on the changes in echocardiography parameters and protein content, we speculate there is a difference in the alcoholic phenotype that may explain the dichotomy in the development and progression of alcoholic heart muscle disease observed between male and female rats.