pH REGULATION IN HEART CELLS

描述(由申请人提供)：这项工作的广泛的、长期的目标是在细胞水平上建立对pH在心脏功能中的作用的全面理解。细胞内pH和细胞外pH的变化对心脏的电活动、兴奋收缩偶联和收缩产生深刻的影响，这是心肌缺血引起的心律失常和收缩功能障碍的部分原因。与现有的关于心室肌的信息相比，关于Phi调节、pH对钙处理的影响以及缺乏横管(浦肯野和心房)的心肌细胞内和细胞间的H扩散的了解要少得多。本项目中描述的方案主要集中在这两种细胞类型上，旨在利用荧光成像和贴片移液管技术帮助填补这一空白。具体目标包括： 1.细胞内酸中毒对心房肌细胞和浦肯野肌细胞兴奋收缩偶联的影响目的是确定酸中毒对钙电流(L和T型)的影响，钙瞬变的时空分布，肌浆网对钙的处理以及这些细胞类型的细胞内钙扩散。还将研究[Ca~(2+)]i对PHI空间受限变化的响应。2.研究Na-HCO3共转运在心室肌、心房肌和浦肯野肌细胞中的电生理特性。这里的目的是检验假设，即Na-HCO3共转运在这些类型的细胞中是产生电的。3.研究心脏浦肯野肌细胞pH调节系统的特性。本研究的目的是确定分离的浦肯野心肌细胞Na-H交换(NHE)、Na-HCO3共转运(NBC)和HCO3-CI交换(AE)的动力学特性和pH依赖性，并验证CI-OH交换(CHE)在这类细胞中起作用的假设。4.研究心房和浦肯野细胞内和细胞间的氢扩散。本研究的目的是确定单个细胞内的H扩散系数和成对电耦合的心肌细胞之间的连接通透性H系数，并验证这两个参数都受碳酸酐酶调节的假设。

DESCRIPTION (provided by applicant): The broad, long-term goal of this work is to establish a comprehensive understanding of the role of pH in cardiac function at the cellular level. Changes in intracellular pH (pHi) and extracellular pH have profound effects on electrical activity, excitation-contraction coupling and contraction in the heart, which accounts in part for the arrhythmias and contractile dysfunction elicited by myocardial ischemia. Compared to the information available for ventricular muscle, much less is known concerning pHi regulation, the effects of pH on Ca 2+ handling, and intra- and intercellular H+ diffusion in cardiac myocytes that lack transverse tubules (Purkinje and atrial). The protocols described in this project focus primarily on these two cell types and are designed to help fill this gap using fluorescence imaging and patch pipette techniques. The specific aims include: 1. Characterize the effect of intracellular acidosis on excitation-contraction coupling in atria/and Purkinje myocytes. The goal here is to determine the effects of acidosis on Ca 2+ current (L and T type), the spatiotemporal profile of the Ca2+ transient, Ca 2+ handling by the sarcoplasmic reticulum and intracellular Ca 2+ diffusion in these cell types. The response of [Ca2+]i to spatially confined changes in pHi will also be studied. 2. Characterize the electrogenic properties of Na-HCO3 cotransport in ventricular, atrial and Purkinje myocytes. The objective here is to test the hypothesis that Na-HCO3 cotransport is electrogenic in these cell types. 3.Characterize the properties of pH_ regulatory systems in cardiac Purkinje myocytes. The objective here is to determine the kinetic properties and pH-dependence of Na-H exchange (NHE), Na-HCO3 cotransport (NBC) and HCO3-CI exchange (AE) in isolated Purkinje myocytes and to test the hypothesis that CI-OH exchange (CHE) is operational in this cell type. 4. Characterize intra- and intercellular H+ diffusion in atrial and Purkinje cells. The goal here is to determine the intracellular H+ diffusion coefficient within single cells and the junctional permeability H+ coefficient between pairs of electrically coupled myocytes and to test the hypothesis that both parameters are modulated by carbonic anhydrase.