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-科学家培训拟用于模拟心脏骤停复苏后致死性心脏再灌注损伤的体外和体内机制以及治疗性低温（TH）心脏保护作用。申请人最近的工作涉及小鼠心脏骤停和缺血/再灌注（IR）损伤的小鼠心肌细胞模型，表明在复苏的几分钟内快速、早期冷却保护线粒体免受氧化剂损伤并保持收缩功能。在这两种互补模型中，TH心脏保护作用与Akt 1激活相关。最后，心肌细胞似乎在几分钟内通过Akt定位到线粒体亚细胞组分来适应氧化应激。 该建议的中心假设是心脏骤停后TH心脏保护作用是Akt活化和定位于线粒体以及随后GSK-32的抑制和己糖激酶II的活化的结果。所提出的目标和候选人职业发展计划将有助于确定：目标#1）心肌细胞I/R期间Akt 1活化对于TH保护线粒体完整性是必要的，并且与Akt亚细胞定位、GSK-32抑制和己糖激酶II活化相关;目标#2）核靶向Akt 1相对于线粒体靶向Akt 1的过表达将增强或复制TH心脏保护;和目标#3）在目标#1和#2中观察到的Akt 1磷酸化和亚细胞信号传导变化也是心脏骤停小鼠模型中TH心脏保护所需的。拟议的工作是夏普博士先前在心脏细胞生物学和急诊医学培训方面的背景的自然发展，并将为他提供几种新的工具，用于线粒体功能和氧化应激措施，共聚焦成像，靶向转染策略以及体内和体外心肌I/R损伤研究方法。该计划将确保从初级教师成功过渡到独立资助的终身教职。 该提案解决了一个重大的公共卫生问题，该问题构成美国死亡的主要原因，每年影响估计335，000人。非裔美国人的心脏骤停率高得不成比例，年轻女性的心脏骤停率也在上升。虽然有希望，但在心肺复苏（CPR）期间有效和快速实施TH，特别是在院外环境中，在技术上具有挑战性。了解Akt介导的改善心血管功能和TH生存的途径可能会导致复制或增强TH保护作用的药理学药物的开发。

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.