Surgical Revascularization's Impact on Mitochondria in Hibernating Myocardium

描述(由申请人提供)： 背景：冬眠心肌是一种存活的、持续功能不全的心肌，可因持续或反复的心肌缺血而发生，其特点是低灌注量而无坏死迹象。本课题组已成功建立了一种可复制的猪冬眠心肌模型，并对该模型进行了广泛的研究。作为这项工作的延伸，我们现在已经建立了一种新的方法，通过这种方法：(A)我们进行非体外循环下存活的单血管搭桥手术，以重建缺血心肌的血运；(B)我们使用生理学和蛋白质组学方法测试这种手术在恢复心肌功能方面的成功。手术重建存活的慢性缺血心肌的动物模型从未被研究过，因此它对了解心力衰竭和恢复的潜力具有重要的意义。方法：在猪冬眠心肌模型上，采用心脏不停跳非体外循环技术，利用左乳内动脉绕过慢性闭塞的左前降支，行血管重建术(假手术)。在12周和20周时，两组患者分别行搭桥前后多层螺旋CT(MDCT)和经胸超声心动图(TTE)检查，以确定LIMA通畅率、局部室壁增厚的恢复情况和心内膜下血流灌注的恢复情况。同时，对冬眠心肌血运重建、线粒体生物能量学和蛋白质变化的研究也进行了分析，以更准确地了解心肌功能恢复的频谱。血运重建后的蛋白质组线粒体反应将与慢性缺血(冬眠)对照组进行比较，使用蛋白质印迹分析和蛋白质组学分析[相对和绝对量化的等量标记(ITRAQ)]。目的：本研究的目的是了解猪模型冬眠心肌血运重建成功后对冬眠心肌的影响。在这个模型中，我们将通过测量血流量(微球分析)和心功能(2D ECHO，MDCT)来评估手术血运重建是否在静息和增加工作负荷(多巴酚丁胺负荷)时使局部血流量正常化。第二个目的是了解冬眠心肌血运重建后氧耗的变化。我们将通过比较血运重建心肌和冬眠心肌的生物能量状态，量化两组线粒体中的蛋白质组变化，并根据蛋白质组变化评估酶蛋白活性来评估这一目标。结论：冬眠心肌潜在恢复的临床认识受限于缺乏合适的动物模型来识别和测试预测手术益处的所谓标记物。对冬眠的适应是复杂的，涉及线粒体的生物能量学和蛋白质组学的变化，这些变化允许组织在牺牲心肌功能的同时保持其生存能力。冬眠心肌似乎对持续的缺血应激有动态的蛋白质组反应，这与晚期心力衰竭时能量底物代谢和功能的全球变化有相似之处。这些蛋白质组学变化可能限制氧化损伤和细胞凋亡，并影响血管重建后的功能恢复。我们认为冬眠心肌是一种促进生存的适应，但以收缩为代价。临床上，冬眠心肌血运重建可导致从功能几乎完全恢复到致密梗死的广泛预后。通过冠状动脉血运重建重建血流后冬眠心肌恢复完整可靠的心肌功能和线粒体生物能量学的机制仍不清楚，需要研究。

DESCRIPTION (provided by applicant): Background: Hibernating myocardium is viable, persistently dysfunctional myocardium that occurs in response to continuing or repetitive myocardial ischemia and is characterized by hypoperfusion without evidence of necrosis. Our group has successfully established a reproducible swine model of hibernating myocardium and has studied this model extensively. As an extension of this work, we have now established a novel method whereby (a) we perform off-pump single vessel bypass surgery with survival to revascularize ischemic myocardium and (b) we test the success of such surgery in recovering myocardial function using physiologic and proteomic approaches. Surgical revascularization of chronically ischemic myocardium with survival has never been studied in an animal model and so it has significant implications in understanding heart failure and the potential for recovery. Methods: In a swine model of hibernating myocardium, surgical revascularization (vesus sham operation) will be performed using the left internal mammary artery to bypass the chronically obstructed left anterior descending artery using a beating heart, off-pump technique, which is identical to the clinical technique. At 12 and 20 weeks, a prebypass and postbypass multi-detector computer tomography (MDCT) and transthoracic echocardiogram (TTE) in both groups will be obtained to determine LIMA patency, recovery of regional wall thickening, and recovery of subendocardial perfusion. Also, studies of the recovery induced by surgical revascularization of hibernating myocardial blood flow, mitochondrial bioenergetics and protein alterations are simultaneous analyzed at the terminal study to more accurately understand the spectrum of myocardial functional recovery. Proteomic mitochondrial responses following revascularization will be compared to the control group of chronic ischemia (hibernation) using, western blot analysis and proteomic analysis [isobaric Tags for Relative and Absolute Quantification (iTRAQ)]. Aims: The aim of this study is to understand the changes that are induced upon the hibernating myocardium of the swine model when it is successfully surgically revascularized. In this model we will evaluate whether surgical revascularization normalizes regional blood flow at rest and with increased work load (dobutamine stress) by measuring blood flow (microsphere analysis) and cardiac function (2D ECHO, MDCT). A second aim is to understand the alteration in the oxygen consumption of the hibernating myocardium following revascularization. We will evaluate this aim by comparing the bioenergic state of the revascularized myocardium to the hibernating myocardium, quantifying the proteomic changes in the mitochondria of both groups and evaluating enzymatic protein activity as directed by the proteomic changes. Conclusion: Clinical understanding of potential recovery of hibernating myocardium is limited by the lack of an appropriate animal model to identify and test alleged markers predicting benefit with surgery. Adaptations to hibernation are complex and involve alterations in the bioenergetics and proteomics of the mitochondria that allow the tissue to maintain its viability while sacrificing myocardial function. Hibernating myocardium appears to have dynamic proteomic responses to persistent ischemic stress, which has similarities to the global changes in energy substrate metabolism and function seen in advanced heart failure. These proteomic changes may limit oxidative injury and apoptosis and impact functional recovery after revascularization. We believe that hibernating myocardium is an adaptation to promote survival but at the expense of contractility. Clinically, revascularization of hibernating myocardium results in a wide spectrum of outcomes from near total recovery of function to dense infarction. The mechanisms of hibernating myocardium to restore complete and reliable myocardial function and mitochondrial bioenergetics following the reestablishing blood flow through coronary revascularization remain unknown and require study.