Oxidative Stess and Heart Failure by Copper Restriction

铜限制导致的氧化应激和心力衰竭

• 批准号: 6638576
• 负责人: Y James KANG
• 金额: $ 32.18万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-15 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638576
• 关键词: apoptosis; atrial natriuretic peptide; biological signal transduction; cardiac myocytes; cellular pathology; copper; cysteine endopeptidases; cytochrome c; dietary restriction; electron microscopy; free radical oxygen; gene expression; genetically modified animals; heart failure; hypertrophic myocardiopathy; immunocytochemistry; laboratory mouse; metallothionein; molecular pathology; necrosis; northern blottings; nutrition related tag; oxidative stress; pathologic process; terminal nick end labeling; tumor necrosis factor alpha; western blottings

Dietary copper restriction causes cardiac hypertrophy, ultimately leading to heart failure. A proposed mechanism for this cardiomyopathy is the accumulation of reactive oxygen species. Using a cardiac-specific metallothionein (MT)- overexpressing transgenic mouse model, we have observed that elevation of this potent antioxidant in the heart suppresses the progression of heart hypertrophy and likely results in the retarded development of heart failure induced by dietary copper restriction. This observation suggests that oxidative stress may play a crucial role in the pathogenesis of heart failure. We therefore propose to test the hypothesis that oxidative stress triggers the transition from heart hypertrophy to failure induced by copper deficiency. We will use the cardiac-specific MT- overexpressing transgenic mouse model to carry out the following specific aims: (1) To define the role of oxidative stress in the transition from heart hypertrophy to failure, a detailed time- course study of the development of heart failure by dietary copper restriction will be performed. In particular, this study will focus on defining the cause-and-effect relationship between oxidative stress and dynamic changes in cardiomyopathy, cardiac dysfunction and heart failure. (2) To determine cellular events involved in the transition from heart hypertrophy to failure, the significance of cell death in the pathogenesis will be defined by immuno-gold TUNEL and electron microscopy in combination with a novel procedure using cardiac alpha-sarcomeric actin antibody to label necrotic cells. The relative contributions of apoptosis and necrosis to the total cell loss will be analyzed. (3) To investigate signaling pathways leading to myocardial cell death during the transition from heart hypertrophy to failure, oxidative stress-induced mitochondrial cytochrome c release and activation of caspase-9 and -3 will be examined by immunohistochemical method, enzymatic assay and Western blot analysis. The consequence of caspase inhibition will be analyzed in order to dissect major pathways leading to cell death. (4) To examine the role of atrial natriuretic peptide (ANP) and tumor necrosis factor-alpha (TNF-alpha) in the late phase transition from heart hypertrophy to failure, dynamic changes in ANP and TNF-alpha production will be studied. Molecular mechanisms of up-regulation of these cytokines will be analyzed through examining the activation of transcription factors NF-kappaB and AP-1. (5) To explore possible mechanisms by which MT inhibits oxidative stress-mediated myocardial cell death induced by dietary copper deficiency, the effect of MT on oxidative stress- mediated mitochondrial membrane changes that lead to cytochrome c release will be examined. The overall goal of this study is to define the role of oxidative stress in copper deficiency-induced pathogenesis of heart failure. This study will give critical insights into the signaling pathways and molecular mechanisms of failure induced by copper deficiency. Importantly, the data obtained will provide valuable information for novel experimental as well as clinical approaches for possible interventions of the transition from heart hypertrophy to heart failure.

饮食中铜的限制会导致心脏肥厚，最终导致心力衰竭。这种心肌病的机制是活性氧的积累。利用心脏特异性金属硫蛋白（MT）过表达的转基因小鼠模型，我们观察到心脏中这种强抗氧化剂的升高抑制了心脏肥厚的进展，并可能导致饮食铜限制引起的心力衰竭的延迟发展。这一观察结果表明，氧化应激可能在心力衰竭的发病机制中起关键作用。因此，我们建议验证氧化应激触发由缺铜引起的心脏肥厚向衰竭过渡的假设。我们将使用心脏特异性MT-过表达转基因小鼠模型来实现以下具体目的：(1)为了确定氧化应激在心脏肥厚到衰竭转变中的作用，我们将对饮食铜限制导致心力衰竭的发展进行详细的时间过程研究。本研究将重点明确氧化应激与心肌病、心功能障碍和心力衰竭动态变化之间的因果关系。(2)为了确定从心脏肥大到衰竭转变过程中涉及的细胞事件，细胞死亡在发病机制中的意义将通过免疫金TUNEL和电子显微镜来定义，并结合使用心脏α -肌动蛋白抗体标记坏死细胞的新方法。我们将分析细胞凋亡和坏死对总细胞损失的相对贡献。(3)为了研究心肌肥大向衰竭过渡过程中导致心肌细胞死亡的信号通路，我们将采用免疫组织化学、酶法和Western blot分析方法检测氧化应激诱导的线粒体细胞色素c释放和caspase-9和-3的激活。为了剖析导致细胞死亡的主要途径，我们将分析半胱天冬酶抑制的后果。(4)为了研究心房钠素肽（ANP）和肿瘤坏死因子- α (tnf - α)在心脏肥厚到衰竭晚期转变中的作用，我们将研究心房钠素肽和tnf - α生成的动态变化。这些细胞因子上调的分子机制将通过检测转录因子NF-kappaB和AP-1的激活来分析。(5)为探讨MT抑制饮食铜缺乏引起的氧化应激介导的心肌细胞死亡的可能机制，我们将研究MT对氧化应激介导的线粒体膜变化导致细胞色素c释放的影响。本研究的总体目标是确定氧化应激在铜缺乏诱导的心力衰竭发病机制中的作用。这项研究将为铜缺乏诱导的信号通路和分子机制提供重要的见解。重要的是，获得的数据将为新的实验和临床方法提供有价值的信息，为从心脏肥厚到心力衰竭的过渡提供可能的干预措施。