Anesthesia and Cardiomyocyte Signal Transduction

麻醉与心肌细胞信号转导

• 批准号: 6840859
• 负责人: DEREK Scott DAMRON
• 金额: $ 26.78万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6840859
• 关键词: CHO cells; acid base balance; adrenergic receptor; anesthesia; anesthetics; beta adrenergic receptor kinase; biological signal transduction; calcium channel; cardiac myocytes; catecholamines; confocal scanning microscopy; cyclic AMP; electrophysiology; enzyme activity; fibroblasts; heart function; inositol phosphates; isozymes; laboratory rat; myosins; phosphorylation; potassium channel; protein kinase C; sarcoplasmic reticulum; sodium hydrogen exchanger; troponin; western blottings

The overall goal of this application is to identify fundamental cellular mechanisms by which the intravenous anesthetic, propofol, alters cardiac function. Our objectives are: 1) to investigate the actions of propofol on cellular mechanisms involved in steady state regulation of myocardial contractility and 2) to explore the actions of propofol on catecholamine activation of the heart. The rationale for the study is that induction of anesthesia with propofol is frequently associated with cardiovascular depression in patients with and without cardiac disease. The overarching hypothesis is that propofol alters cellular mechanisms involved in the regulation of intracellular free Ca2+ concentration ([Ca2+]i) and/or myofilament Ca2+ sensitivity at the level of the cardiomyocyte via an activation of distinct PKC isoforms. Using freshly dispersed adult rat ventricular myocytes, we will investigate the extent to which PKC activation is involved in propofol-induced changes in the cellular and subcellular mechanisms that regulate ion channel conductances and [Ca2+]i, sarcoplasmic reticulum (SR) Ca2+ handling and myofilament Ca2+ sensitivity. We present compelling preliminary evidence that propofol alters multiple cellular mechanisms involved in the regulation of [Ca2+]i and/or myofilament Ca2+ sensitivity via activation of PKC. Specific Aim 1 will investigate the effects of propofol on cellular mechanisms that regulate myofilament Ca2+ sensitivity (myofibrillar protein phosphorylation and intracellular pH). Specific Aim 2 will identify actions of propofol on cellular mechanisms that regulate [Ca2+]i (K+ and Ca2+ Currents, SR Ca2+ handling). Specific Aim 3 will identify site(s) in the signal transduction pathway and the cellular mechanism by which propofol alters the inotropic response to beta1 adrenoreceptor activation of cardiomyocytes. Specific Aim 4 will identify site(s) in the signal transduction pathway and the cellular mechanism by which propofol alters the inotropic response to alpha1a adrenoreceptor activation of cardiomyocytes. A variety of experimental preparations and techniques are utilized, including: 1) isolated SR vesicles to directly measure "real time" Ca2+ uptake; 2) purified myofibrils to measure contractile protein phosphorylation and myofibrillar actomyosin ATPase; 3) Western blot analysis and confocal microscopy to monitor translocation of PKC isoforms to distinct intracellular sites; 4) field-stimulated cardiomyocytes to simultaneously measure [Ca2+]i or pHi and shortening; 5) measurement of cAMP and IP3 accumulation; 6) electrophysiological measurements of Ca2+ and K+ channel activity; 7) Rat-1 fibroblasts selectively transfected with the alpha1a or beta1 adrenergic receptor for assessment of ligand binding; 8) inhibitor peptides selective for distinct PKC isoforms to identify isoform-specific changes in cellular mechanisms and function. These studies will yield novel information about cellular mechanisms of propofol-induced changes in myocardial regulation.

该应用的总体目标是确定静脉麻醉剂异丙酚改变心脏功能的基本细胞机制。 我们的目标是：1) 研究异丙酚对参与心肌收缩力稳态调节的细胞机制的作用；2) 探索异丙酚对心脏儿茶酚胺激活的作用。 该研究的基本原理是，异丙酚诱导麻醉通常与患有或不患有心脏病的患者的心血管抑郁有关。 总体假设是，异丙酚通过激活不同的 PKC 同工型，改变心肌细胞水平上参与调节细胞内游离 Ca2+ 浓度 ([Ca2+]i) 和/或肌丝 Ca2+ 敏感性的细胞机制。 使用新鲜分散的成年大鼠心室肌细胞，我们将研究 PKC 激活在多大程度上参与异丙酚诱导的细胞和亚细胞机制变化，这些机制调节离子通道电导和 [Ca2+]i、肌浆网 (SR) Ca2+ 处理和肌丝Ca2+ 敏感性。 我们提出了令人信服的初步证据，表明丙泊酚通过激活 PKC 改变了参与 [Ca2+]i 和/或肌丝 Ca2+ 敏感性调节的多种细胞机制。 具体目标 1 将研究异丙酚对调节肌丝 Ca2+ 敏感性（肌原纤维蛋白磷酸化和细胞内 pH）的细胞机制的影响。 具体目标 2 将确定异丙酚对调节 [Ca2+]i 的细胞机制（K+ 和 Ca2+ 电流、SR Ca2+ 处理）的作用。 具体目标 3 将确定信号转导途径中的位点以及异丙酚改变心肌细胞对 β1 肾上腺素受体激活的正性肌力反应的细胞机制。具体目标 4 将确定信号转导途径中的位点以及异丙酚改变心肌细胞 α1a 肾上腺素受体激活的正性肌力反应的细胞机制。 利用了多种实验准备和技术，包括：1）分离的 SR 囊泡直接测量“实时”Ca2+ 吸收； 2)纯化肌原纤维以测量收缩蛋白磷酸化和肌原纤维肌动球蛋白ATP酶； 3) 蛋白质印迹分析和共聚焦显微镜监测 PKC 同种型易位到不同的细胞内位点； 4) 场刺激心肌细胞，同时测量 [Ca2+]i 或 pHi 和缩短； 5)cAMP和IP3积累的测量； 6) Ca2+和K+通道活性的电生理测量； 7) 用α1a或β1肾上腺素受体选择性转染的Rat-1成纤维细胞用于评估配体结合； 8) 对不同 PKC 同工型具有选择性的抑制剂肽，以识别细胞机制和功能中同工型特异性的变化。 这些研究将产生关于异丙酚引起的心肌调节变化的细胞机制的新信息。