Anesthesia and Cardiomyocyte Signal Transduction

本申请的总体目标是确定静脉麻醉剂丙泊酚改变心脏功能的基本细胞机制。 我们的目标是：1）研究丙泊酚对涉及心肌收缩力稳态调节的细胞机制的作用和2）探索丙泊酚对心脏的儿茶酚胺激活的作用。 这项研究的基本原理是，在有或没有心脏病的患者中，丙泊酚诱导麻醉通常与心血管抑郁有关。 总体假设是，丙泊酚通过激活不同的PKC亚型，在心肌细胞水平上改变参与细胞内游离Ca 2+浓度（[Ca 2 +]i）和/或肌丝Ca 2+敏感性调节的细胞机制。 使用新鲜分散的成年大鼠心室肌细胞，我们将调查PKC激活参与丙泊酚诱导的细胞和亚细胞机制，调节离子通道电导和[Ca 2 +]i，肌浆网（SR）Ca 2+处理和肌丝Ca 2+敏感性的变化的程度。 我们提出了令人信服的初步证据，丙泊酚改变了多种细胞机制，涉及调节[Ca 2 +]i和/或肌丝Ca 2+的敏感性，通过激活PKC。 具体目标1将研究丙泊酚对调节肌丝Ca 2+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将确定丙泊酚对调节[Ca 2 +]i（K+和Ca 2+电流，SR Ca 2+处理）的细胞机制的作用。 具体目标3将确定丙泊酚改变心肌细胞对β 1肾上腺素受体激活的变力反应的信号转导途径和细胞机制中的位点。具体目标4将确定丙泊酚改变心肌细胞α 1a肾上腺素受体激活的变力性反应的信号转导途径和细胞机制中的位点。 利用多种实验制备和技术，包括：1）分离的SR囊泡以直接测量“真实的时间”Ca 2+摄取; 2）纯化的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）Western印迹分析和共聚焦显微镜以监测PKC同种型向不同细胞内位点的易位; 4）场刺激心肌细胞以同时测量[Ca 2 +]i或pHi和缩短：5）测量cAMP和IP 3积累：6）电生理学测量Ca 2+和K+通道活性; 7）用α 1a或β 1肾上腺素能受体选择性转染的大鼠-1成纤维细胞，用于评估配体结合; 8）对不同PKC亚型具有选择性的抑制剂肽，以鉴定细胞机制和功能中的亚型特异性变化。 这些研究将产生新的信息丙泊酚诱导的心肌调节的变化的细胞机制。

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.