Anesthesia and Cardiomyocyte Signal Transduction

这项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)研究异丙酚对稳态调节心肌收缩功能的细胞机制的作用；2)探讨异丙酚对儿茶酚胺激活心脏的作用。这项研究的基本原理是，在有或没有心脏病的患者中，异丙酚麻醉诱导经常与心血管抑郁有关。最重要的假设是，异丙酚通过激活不同的PKC亚型，在心肌细胞水平上改变了参与调节细胞内游离钙浓度([Ca+]i)和/或肌丝钙敏感性的细胞机制。利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引起的细胞和亚细胞机制的变化，这些机制调节离子通道电导和[Ca+]i、肌浆网(SR)钙处理和肌丝钙敏感性。我们提出了令人信服的初步证据，表明异丙酚通过激活PKC改变了参与调节[Ca~(2+)]i和/或肌丝Ca~(2+)敏感性的多种细胞机制。具体目标1将研究异丙酚对调节肌丝钙敏感性(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将确定异丙酚在调节[Ca~(2+)]i(K+和Ca~(2+)电流，SR Ca~(2+)处理)的细胞机制上的作用。特异靶3将确定信号转导通路中的位置(S)以及异丙酚改变心肌细胞对β1肾上腺素受体激活的变力反应的细胞机制。特异靶4将确定信号转导通路中的位置(S)，以及异丙酚改变心肌细胞α1a肾上腺素受体激活的变力反应的细胞机制。应用了多种实验准备和技术，包括：1)分离的SR小泡直接测量“实时”钙摄取；2)纯化的肌原纤维测量收缩蛋白磷酸化和肌原纤维肌球蛋白ATPase；3)蛋白质印迹分析和共聚焦显微镜监测PKC异构体到不同细胞内位置的移位；4)场刺激心肌细胞同时测量[Ca+]i或Phi和缩短；5)测量cAMP和IP3的积累；6)电生理测量Ca+和K+通道的活性；7)选择性地转染α1a或β1肾上腺素能受体的大鼠成纤维细胞用于评估连接和结合；8)选择不同PKC亚型的抑制肽，以确定细胞机制和功能的异构体特异性变化。这些研究将为异丙酚诱导的心肌调节改变的细胞机制提供新的信息。

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.