pH REGULATION IN HEART CELLS

描述（由申请人提供）：这项工作的长期目标是在细胞水平上全面了解pH在心脏功能中的作用。细胞内pH（pHi）和细胞外pH的变化对心脏的电活动、兴奋-收缩偶联和收缩具有深刻的影响，这部分地解释了由心肌缺血引起的心律失常和收缩功能障碍。与心室肌的可用信息相比，对缺乏横小管（浦肯野和心房）的心肌细胞中pHi调节、pH对Ca 2+处理的影响以及细胞内和细胞间H+扩散的了解要少得多。本项目中描述的方案主要关注这两种细胞类型，旨在使用荧光成像和贴片移液器技术帮助填补这一空白。具体目标包括： 1.描述细胞内酸中毒对心房/和浦肯野肌细胞兴奋-收缩偶联的影响。本研究的目的是确定酸中毒对L型和T型钙电流、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的影响。还将研究[Ca2 +] i对pHi空间限制变化的响应。 2.表征心室、心房和浦肯野肌细胞中Na-HCO3共转运的产电特性。这里的目的是测试的假设，Na-HCO3共转运是产电在这些细胞类型。 3.描述心脏浦肯野肌细胞pH调节系统的特性。本文的目的是确定分离的浦肯野肌细胞中Na-H交换（NHE）、Na-HCO3共转运（NBC）和HCO3-Cl交换（AE）的动力学特性和pH依赖性，并检验CI-OH交换（CHE）在这种细胞类型中可操作的假设。 4.表征心房和浦肯野细胞中的细胞内和细胞间H+扩散。这里的目标是确定单细胞内的细胞内H+扩散系数和电耦合的肌细胞对之间的交界处渗透性H+系数，并测试这两个参数的假设是由碳酸酐酶调制。

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.