Novel Mitochondrial Mechanisms of Ethanol-Induced Cardiac Injury in Aged Females

摘要：心血管对酒精中毒的反应存在性别差异，女性对酒精的毒性作用明显比男性更敏感，尽管这一现象背后的机制尚不清楚，也没有系统的研究。有证据表明，绝经后妇女心肌梗死后的死亡率高于老年男性，这加剧了女性对酒精性心脏病的易感性，目前尚不清楚衰老和雌激素（E2）缺乏的影响是否与酒精诱导的心脏缺血/再灌注（I/R）损伤有关。由于线粒体在维持细胞存活和心脏保护中发挥着关键作用，因此乙醇和年龄相关的I/R损伤增加可能是由线粒体亚蛋白质组的反应改变引起的。为了解决这个问题，我们将采用体外分离心脏和体内线粒体呼吸研究，结合新出现的iTRAQ 8plex蛋白质组学方法，LC MS/MS和PANTHER本体学分析来表征慢性酒精摄入后雌性大鼠心肌衰老和E2缺乏时线粒体信号的变化。特别感兴趣的是与代谢、氧化应激和细胞死亡相关的蛋白质的适应性变化。具体目的如下：1)慢性乙醇摄入会加剧与年龄相关的I/R损伤增加，这与已知心脏保护信号和雌激素受体（ER）亚型的线粒体靶向性降低有关；2)慢性乙醇摄入会加剧E2缺乏对年龄依赖性线粒体保护信号中断的影响。在成年和老年卵巢完整和去性腺的雌性大鼠中，评估兰根多夫灌注心脏在整体I/R后的梗死面积和凋亡细胞死亡；呼吸研究将在分离的线粒体中进行。Western blotting和qRT- PCR将用于线粒体ER亚型靶向和下游信号；iTRAQ和多反应监测（MRM）用于蛋白变化和er翻译后修饰。我们在这里描述的高通量、靶向iTRAQ蛋白质组学方法将使我们能够在相对较短的时间内，就女性在老年和成年大鼠酒精相关心功能障碍发展中的作用提供关键和新颖的机制见解。此外，从拟议的研究中收集的信息应该为治疗绝经后妇女酒精性心脏病的新治疗靶点提供有力、直接的支持，并为未来实验研究的设计提供动力和理论基础。

DESCRIPTION (provided by applicant): ABSTRACT Sex differences in the cardiovascular response to alcoholism exist such that women are apparently more sensitive to the toxic effects of alcohol vs men, although the mechanisms which underlie this phenomenon are poorly understood and have not been systematically investigated. Greater vulnerability to alcoholic heart disease in women is exacerbated by evidence indicating that post-menopausal women have higher mortality rates after myocardial infarction vs aged men, and it is unclear whether the effects of aging and estrogen (E2) deficiency are additive with regard to ethanol-induced cardiac ischemia/reperfusion (I/R) injury. Because of the pivotal role played by the mitochondria in the maintenance of cell survival and cardioprotection, it is logical that ethanol and age-associated increases in I/R injury might arise from altered responses in the mitochondrial subproteome. To address this problem, we will employ in vitro isolated heart and in vivo mitochondrial respiration studies in conjunction with the newly emerging iTRAQ 8plex proteomics approach, LC MS/MS and PANTHER ontological analysis to characterize alterations in mitochondrial signaling occurring with aging and E2 deficiency in the female rat myocardium following chronic ethanol ingestion. Of particular interest are adaptive changes in proteins associated with metabolism, oxidative stress, and cell death. Specific aims are as follows: 1) chronic ethanol ingestion will exacerbate age-related increases in I/R injury in association with reduced mitochondrial targeting of known cardioprotective signals and estrogen receptor (ER) subtypes, and 2) chronic ethanol ingestion will exacerbate the effects of E2 deficiency on age dependent disruptions in protective mitochondrial signaling. Infarct size and apoptotic cell death will be assessed in Langendorff-perfused hearts following global I/R in adult and aged ovary-intact and gonadectomized female rats; respiration studies will be performed in isolated mitochondria. Western blotting and qRT- PCR will be employed for mitochondrial ER subtype targeting and downstream signals; iTRAQ and multiple reaction monitoring (MRM) for protein changes and post-translational modifications of ERs. The high throughput, targeted iTRAQ proteomics approach we describe here will allow us to provide critical and novel mechanistic insight, in a relatively short time, on the role of female sex in the development of alcohol related cardiac dysfunction in aged and adult rats. Moreover, information gleaned from the proposed studies should provide strong, direct support for new therapeutic targets for treating alcoholic heart disease in postmenopausal women, as well as provide impetus and rationale for the design of future experimental studies. PUBLIC HEALTH RELEVANCE: Current experimental evidence supports a J- or U-shaped association between alcohol consumption and heart attack and/or high blood pressure. As such, while light-to-moderate drinking has been associated with protection from heart disease, heavy drinking is associated with the development of heart failure, which remains a major public health crisis and a leading cause of morbidity and mortality in aged men and women. The CDC has most recently estimated that ~75,000 deaths per year can be attributed to the harmful effects of alcohol. Moreover, nearly 33% of chronic alcohol-dependent individuals exhibit cardiac dysfunction, which often becomes manifest as an alcohol-specific heart failure. Interestingly, women appear to more sensitive to the toxic effects of alcohol on the heart when compared to men, even though total alcohol consumption appears to be less in alcoholic women vs men. It is also important to note that heart disease remains the leading cause of death in post-menopausal women, with mortality rates surpassing those of age-matched men. The specific mechanisms which produce this gender-specific cardiac dysfunction and enhanced susceptibility to ischemic injury in the aged female heart following chronic ethanol ingestion have not been directly examined, and the studies proposed herein will fill this information gap. We propose a comprehensive and novel approach that will identify proteins that comprise the functional sequelae of complex cardiac phenotypes of chronic ethanol ingestion, and will further characterize possible targets for therapeutic intervention in the aged female population.