Hypothermia in Cardiac Arrest: Akt Preservation of Mitochondrial Integrity

描述(由申请人提供)：项目摘要/摘要研究和医生-科学家培训被建议在体外和体内模拟心脏骤停复苏和治疗性低温(TH)心脏保护后致命性心脏再灌注损伤的机制。申请人最近关于小鼠心脏骤停和小鼠心肌细胞缺血/再灌注(IR)损伤模型的研究表明，复苏后几分钟内的快速、早期降温可以保护线粒体免受氧化损伤，并保留收缩功能。在这两种互补模型中，心肌保护与Akt1的激活有关。最后，通过将Akt定位于线粒体亚细胞部分，心肌细胞似乎在几分钟内就能适应氧化应激。这一建议的中心假设是，心脏骤停后TH的心脏保护是Akt激活和定位到线粒体的结果，随后GSK-32被抑制，己糖激酶II被激活。所提出的目标和候选的职业发展计划将有助于确定：目的1)心肌细胞I/R期间Akt1的激活对于保护线粒体的完整性是必要的，并与Akt亚细胞定位、GSK-32抑制和己糖激酶II激活有关；目标2)以核和线粒体为靶点的Akt1的过表达将增强或复制TH的心脏保护；和目的#3)AIMS#1和#2中的Akt1磷酸化和亚细胞信号变化也是心脏骤停小鼠心脏保护所必需的。这项拟议的工作是夏普博士先前心脏细胞生物学背景和急救医学培训的自然发展，将使他拥有几种新的工具，用于线粒体功能和氧化应激测量、共聚焦成像、靶向转导策略，以及用于研究心肌I/R损伤的体内和体外方法。该计划将确保在独立资助下，成功地从初级教员过渡到终身教职。这项提案解决了一个主要的公共卫生问题，该问题是美国主要的死亡原因，估计每年影响33.5万人。在非裔美国人中，心脏骤停的发生率高得不成比例，在年轻女性中，这一比例正在上升。尽管前景看好，但在心肺复苏(CPR)期间，尤其是在院外环境下，有效和快速地实施TH在技术上是具有挑战性的。了解Akt介导的改善心血管功能和TH存活的通路可能会导致复制或增强TH保护作用的药理辅助药物的开发。

DESCRIPTION (provided by applicant): Project Summary/Abstract Studies and physician-scientist training are proposed to model in vitro and in vivo mechanisms of lethal cardiac reperfusion injury following cardiac arrest resuscitation and therapeutic hypothermia (TH) cardiac protection against this injury. Recent work by the applicant involving mouse cardiac arrest and mouse cardiomyocyte models of ischemia/reperfusion (IR) injury suggests that rapid, early cooling within minutes of resuscitation protects against mitochondrial oxidant injury and preserves contractile function. TH cardioprotection in both these complementary models is associated with Akt1 activation. Finally, cardiomyocytes appear to adapt to oxidant stress within minutes by localization of Akt to the mitochondrial subcellular fraction. The central hypothesis of this proposal is that TH cardioprotection following cardiac arrest is the result of Akt activation and localization to the mitochondria with subsequent inhibition of GSK-32 and activation of hexokinase II. The proposed aims and candidate career development plan will help determine whether: Aim #1) Akt1 activation during cardimyocyte I/R is necessary for TH protection of mitochondrial integrity and is associated with Akt subcellular localization, GSK-32 inhibition and hexokinase II activation; that Aim #2) Overexpression of nuclear versus mitochondrial targeted Akt1 will enhance or replicate TH cardioprotection; and Aim #3) Akt1 phosphorylation and subcellular signaling changes seen in Aims #1 and #2 will also be required for TH cardioprotection in a mouse model of cardiac arrest. The proposed work is a natural progression of Dr Sharp's prior background in cardiac cell biology and training in emergency medicine, and will equip him with several new tools for mitochondrial functional and oxidant stress measures, confocal imaging, targeted transfection strategies, and in vivo as well as in vitro approaches to studies of myocardial I/R injury. The plan will ensure a successful transition from junior faculty to tenure track with independent funding. This proposal addresses a major public health problem that constitutes a leading cause of death in the United States, affecting an estimated 335,000 individuals every year. Rates of cardiac arrest are disproportionately high among African Americans and are rising among young women. Although promising, the effective and rapid implementation of TH during cardiopulmonary resuscitation (CPR) particularly in the out of hospital setting is technically challenging. Understanding the Akt-mediated pathways that improve cardiovascular function and survival with TH could lead to the development of pharmacologic adjuncts that replicate or enhance the protective effects of TH.