Anesthetics and Cardiac Signal Transduction

描述（由申请人提供）：这是5 R 01 HL 034708 -22的竞争性更新，其支持使用源自人胚胎干细胞（ESC）的人心肌细胞和源自正常iPSC（N-iPSC）和2型糖尿病iPSC（DM-iPSC）的诱导多能干细胞（iPSC）开发临床相关的心脏预处理模型。在过去的资助周期中，重点一直是使用心脏搭桥术期间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剂诱导的心脏保护免受缺血/再灌注（I/R）损伤的潜在机制。然而，我们的方法有一些局限性，包括使用来自表现出各种疾病的患者的心房样本，以及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，以解决糖尿病患者预处理功效衰减的遗传和环境机制。重要的是，拟议的实验还将产生新的见解，糖尿病可能如何改变干细胞的潜在功效，以供将来用于再生医学。工作假设是I/R期间线粒体渗透性转换（PT）孔的延迟开放是APC的核心，并且糖尿病通过对线粒体的急性（高血糖症）和遗传起源的作用损害心脏保护。基于我们在开发临床相关心脏预适应模型方面的进展，我们提出了以下具体目标：具体目标1。确定来源于N-iPSC和DM-iPSC的人心室心肌细胞中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通道对源自N-iPSC和DM-iPSC的人心肌细胞的麻醉诱导的线粒体保护的贡献。具体目标3。确定麻醉剂如何在I/R应激下调节源自N-iPSC和DM-iPSC的完整人心室心肌细胞中的人PT孔开放。总之，本提案的目标是利用人胚胎干细胞和诱导多能干细胞体外分化为心脏谱系，以描述导致APC在糖尿病中缺乏疗效的遗传与环境机制。我们的初步数据表明，来自正常和糖尿病患者的iPSC的心肌细胞表现出早期人类心肌细胞的功能，结构和分子特性。这些研究将通过线粒体功能、蛋白磷酸化、改变的ROS形成和KATP通道活性的变化，提供关于糖尿病和高血糖在调节麻醉诱导的心脏保护中的作用的新机制信息。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。 公共卫生相关性：糖尿病患者心脏对应激更敏感的原因尚不清楚。我们第一次能够从人类多能细胞中制造出针对人类疾病的心脏细胞。因此，我们现在可以分别评估基因和环境因素对糖尿病患者心脏敏感性的影响。在我们的研究中观察到的缺陷可以通过各种治疗来靶向。

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.