Identification and Validation of Alcohol Biomarker Signatures by Proteomics

描述(申请人提供)：三分之一的慢性酗酒者表现出心脏功能障碍，导致扩张性心肌病，最终导致充血性心力衰竭。酒精性心肌病(ACM)在功能上以可逆性和不可逆性改变为特征。酒精中毒还会导致骨骼肌萎缩，称为酒精性肌病，与ACM密切相关。骨骼肌无力的进展与左心室射血分数的降低平行。酒精对肌肉结构和功能的慢性影响已被证明会导致几种蛋白质和途径的变化。这项拨款申请的目标是确定人类ACM可靠的诊断和预后蛋白生物标记物。本应用的具体目的是：1)确定慢性饮酒大鼠心脏和血清中早发性ACM相关蛋白的生物标志物。因为在人类中，三角肌可能至少部分地作为心脏组织的替代品，ii)研究慢性酒精中毒患者肌肉活检和血清中蛋白质的表达，并将其与无症状酒精患者和非酒精对照组进行比较。 将研究一种独特的心脏组织库，该库来自不可逆的脑损伤的酒精中毒患者，这些患者的心脏无法移植并在生命支持终止后被冻结，iii)通过比较患有和不患有急性心肌梗死的慢性酒精中毒患者和非酒精对照组的左心室蛋白质谱，发现ACM蛋白生物标志物。此外，来自相同受试者的相应肌肉和血清样本将被评估生物标记物。Iv)进行生物统计学和生物信息学数据挖掘，对大鼠和人类样本的蛋白质组表达进行差异分析，将全球蛋白质变化与临床数据相关联，以获得用于验证的疾病特异性蛋白质模式，将数据整合到关系数据库中，用于未来与其他心肌病的比较研究，并绘制受影响的蛋白质相互作用组路径中的蛋白质变化簇，以开发ACM和肌病发病机制的机制模型。V)验证新的血清和肌肉样本中新出现的生物标记物蛋白质组板的特异性、准确性和有用性，这将导致ACM生物标记物的临床背景验证和应用。ACM蛋白质生物标志物的全面搜索和发现将包括血清蛋白质组和组织亚细胞成分。 我们将结合2D电泳法、多维液相色谱和串联质谱学来研究蛋白质的变化，以及蛋白质翻译后的修饰，包括那些与酒精代谢物直接相关的修饰。还将开发用于验证目的的免疫分析方法。美国有1800万酗酒者。这些人中的许多人患有酒精性心肌病，这是可以确定病因的扩张型心肌病的唯一主要原因。通过特定生物标志物的早期诊断将为治疗和预防心脏增大、充血性心力衰竭和死亡提供机会。

DESCRIPTION (provided by applicant): One third of chronic alcohol abusers display cardiac dysfunction, leading to dilated cardiomyopathy and eventually to congestive heart failure. Functionally, alcoholic cardiomyopathy (ACM) is characterized by reversible and irreversible alterations. Alcoholism also causes atrophy of skeletal muscle, termed alcoholic myopathy, which is closely correlated with ACM. Skeletal muscle weakness progresses in parallel with depression of the left ventricle ejection fraction. A chronic effect of alcohol on the structure and function of the muscles has been shown to induce changes in several proteins and pathways. The goal of this grant application is to identify reliable diagnostic and prognostic protein biomarkers of ACM in humans. The specific aims of this application are i) to identify early onset ACM-related protein biomarkers in heart and serum of rats fed alcohol chronically. Since in humans, the deltoid muscle may serve at least in part, as a surrogate for cardiac tissue, ii) to study protein expression in muscle biopsies and serum of chronic alcoholics with clinical ACM and myopathy and compare it to asymptomatic alcoholics and nonalcoholic controls. A unique heart tissue bank, from irreversibly brain damaged alcoholic patients whose hearts could not be transplanted and were frozen after termination of life support, will be investigated, iii) To discover ACM protein biomarkers by comparing protein profiles of the left ventricle of chronic alcoholics with and without ACM, and nonalcoholic controls. Additionally, corresponding muscle and serum samples from the same subjects will be evaluated for biomarkers. iv) To perform biostatistical and bioinformatics data mining for differential analysis of proteomic expression from rat and human samples, correlate global protein changes with clinical data to obtain disease-specific protein patterns for validation, integrate data in a relational database for future comparative studies with other cardiomyopathies, and map clusters of protein changes in affected pathways of the protein interactome in order to develop a mechanistic model of the pathogenesis of ACM and myopathy. v) To validate the specificity, accuracy and usefulness of emergent proteomic panels of biomarkers in new serum and muscle samples that will lead to clinical context validation and application of ACM biomarkers. The comprehensive search and discovery of protein biomarkers of ACM will encompass the proteomes of sera, and of subcellular components of tissues. A combination of 2D electrophoresis with multidimensional liquid chromatography and tandem mass spectroscopy will be employed to characterize protein changes, as well as, post-translational protein modifications, including those directly related to alcohol metabolites. Immunoassays will be also developed for validation purposes. There are 18 million alcohol abusers in the US. Many of these persons suffer from alcoholic cardiomyopathy, which is the single leading cause of dilated cardiomyopathy in which an etiology can be determined. Early diagnosis of this condition by specific biomarkers will offer the opportunity for treatment and prevention of heart enlargement, congestive heart failure and death.