Anesthetics and Cardiac Signal Transduction

描述（由申请人提供）：这是5R01 h034708 -22的竞争性更新，支持开发一种临床相关的心脏预适应模型，该模型使用来自人类胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）的人类心肌细胞，来源于正常iPSCs （N-iPSCs）和2型糖尿病iPSCs （DM-iPSCs）。在过去的资助周期中，重点是使用在心脏旁路手术中获得的人类心脏组织；在这些研究中，我们研究了挥发性麻醉药对缺血/再灌注（I/R）损伤心脏保护的潜在机制。然而，我们的方法有一些局限性，包括使用来自患有各种疾病的患者的心房标本，并且使用不同的药物治疗。这种人类疾病的体外模型将使正常和糖尿病心肌细胞的发育和比较研究能够解决糖尿病患者预处理效果衰减的遗传和环境机制。重要的是，拟议中的实验还将对糖尿病如何改变干细胞在再生医学中未来使用的潜在功效产生新的见解。目前的假设是，I/R期间线粒体通透性转换（PT）孔的延迟开放是APC的核心，糖尿病通过对线粒体的急性（高血糖）和遗传起源的作用来损害心脏保护。根据我们在开发临床相关的心脏预处理模型方面的进展，我们提出以下具体目标：确定N-iPSCs和DM-iPSCs衍生的人心室心肌细胞的线粒体生物能量学、离子稳态和信号通路。具体目标2。确定sarcKATP通道对麻醉诱导的N-iPSCs和DM-iPSCs衍生的人心肌细胞线粒体保护的贡献。具体目标3。确定麻醉药如何在I/R应激下调节N-iPSCs和DM-iPSCs衍生的完整人心室心肌细胞的PT孔开放。总之，本研究的目的是利用人类胚胎和诱导多能干细胞在体外分化成心脏谱系，来描述APC对糖尿病缺乏疗效的遗传和环境机制。我们的初步数据表明，从正常和糖尿病患者的iPSCs中提取的心肌细胞具有早期人类心肌细胞的功能、结构和分子特性。这些研究将为糖尿病和高血糖通过改变线粒体功能、蛋白磷酸化、ROS形成改变和KATP通道活性来调节麻醉诱导的心脏保护作用提供新的机制信息。具体目标的完成将阐明线粒体在糖尿病期间调节APC的新作用，从长远来看，可能为围手术期干预提供新的治疗靶点。

DESCRIPTION (provided by applicant): This is a competing renewal of 5R01 HL034708-22, which has supported the development of a clinically relevant model of cardiac preconditioning using human cardiomyocytes derived from the human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), derived from both normal iPSCs (N-iPSCs) and type 2 diabetes mellitus iPSCs (DM-iPSCs). In the past funding cycle, focus has been on using human cardiac tissue obtained during cardiac bypass; in those studies we investigated the underlying mechanisms of volatile anesthetic-induced cardiac protection from ischemia/reperfusion (I/R) injury. However, our approach had several limitations, including the use of atrial specimens from patients exhibiting various diseases and in whom different drug therapies were utilized. This in vitro model of human disease will enable developmental and comparative studies of normal and diabetic cardiomyocytes to address genetic and environmental mechanisms responsible for attenuation of preconditioning efficacy in diabetics. Importantly, the proposed experiments will also yield new insights into how diabetes might alter the potential efficacy of stem cells for future use in regenerative medicine. The working hypothesis is that delayed opening of the mitochondrial permeability transition (PT) pore during I/R is central for APC, and that diabetes impairs cardioprotection through actions on mitochondria that are both acute (hyperglycemia) and genetic in origin. On the basis of our progress in developing a clinically relevant model of cardiac preconditioning, we propose the following Specific Aims: Specific Aim 1. Determine mitochondrial bioenergetics, ion homeostasis, and signaling pathways in human ventricular cardiomyocytes derived from N-iPSCs and DM-iPSCs. Specific Aim 2. Determine contributions of sarcKATP channel to anesthetic-induced mitochondrial protection of human cardiomyocytes derived from N-iPSCs and DM-iPSCs. Specific Aim 3. Determine how anesthetics modulate human PT pore opening under I/R stress in intact human ventricular cardiomyocytes derived from N-iPSCs and DM-iPSCs. In summary, the goal of this proposal is to utilize the in vitro differentiation of human embryonic and induced pluripotent stem cells into cardiac lineage to delineate the genetic vs. environmental mechanisms responsible for the lack of efficacy of APC in diabetes. Our preliminary data indicate that the cardiomyocytes derived from iPSCs from normal and diabetic patients exhibit functional, structural, and molecular properties of early-stage human myocytes. These studies will provide novel mechanistic information on the roles of diabetes and hyperglycemia in modulating anesthetic-induced cardioprotection through changes in mitochondrial function, protein phosphorylation, altered ROS formation, and KATP channel activity. Completion of the specific aims will elucidate the novel role of mitochondria to modulate APC during diabetes and in the long run, may suggest new therapeutic targets for perioperative intervention. PUBLIC HEALTH RELEVANCE: The cause of greater cardiac susceptibility to stress in diabetic patients remains unknown. For the first time we are able to make human disease-specific cardiac cells derived from their pluripotent cells. Hence, we can now assess separately the role of genes and environmental factors that are responsible for greater cardiac sensitivity in patients with diabetes. Defects observed during our study may then be targeted via various therapies.