ELECTROPHYSIOLOGY OF NEONATAL AND ADULT HEART

这项研究的目的是研究Ca 2+作为 在SR的门控Ca 2+释放通道中的传递信号（Ryanodine 受体），并评估受磷蛋白在调节 在哺乳动物心肌细胞中的松弛。 Ca 2+问题的核心 门控是各种肌膜Ca 2+的作用和有效性 在门控钙从SR释放的转运蛋白。我们应该， 因此，首先评估由细胞递送的Ca 2+的门控效率。 Ca ~（2+）通道与Na ~+-Ca ~（2+）交换器和 Ca 2 +-选择性Na+通道（由心钠素转化） 通过定量哺乳动物心脏的心房和心室肌细胞， 穿过肌膜的Ca 2+电荷的积分足以触发 钙释放。 这项研究的核心假设是， Ca 2+通道（DHP受体）和 Ryanodine受体 “特许进入概念”将进一步 通过测试Ryanodine受体阻滞剂的可及性来探索 （钌红和笼状Mg 2+）和笼状Ca 2+缓冲液（DM-硝基酚和Nitrophen） 重氮-2）作用于完整SR释放通道的Ca 2+敏感位点 肌细胞 限制进入假说对 钙离子对Ryanodine受体的失活作用将通过以下方法进行研究： 新的“epi-axial”方法在肌膜下空间光释放Ca 2+。 各种肌膜钙转运蛋白的表达水平 在心肌细胞中的作用对于该评价至关重要，需要 不同Ca 2+递送系统的门控效率的定量 在其他哺乳动物物种中。 因此，Ca 2+信号传导的有效性 通过Na+-Ca 2+交换器，与Ca 2+通道相比，将被探测 在仓鼠肌细胞中，其显示出高10倍的交换器电流， 密度高于大鼠心肌细胞。 两个病理一个发展 E-C耦合模型将被检查，这代表上调 交换器（肌病仓鼠）或Ca 2+通道的表达 （自发性高血压大鼠），SR释放密度低 通道（新生人类和猫心肌细胞）。 详细检查 AT-1心房肿瘤细胞系Ca ~（2+）释放和摄取的研究 这不仅是因为AT-1细胞不表达受磷蛋白（Ca 2+泵 调节蛋白），但也因为这些细胞是很好的候选人， E-C偶联的各种分子组分的遗传操作。 探讨受磷蛋白在调节钙摄取中的作用， 包括评价受磷蛋白磷酸化的动力学 通过注射2D 12抗体和光释放笼状cAMP vs. 改变肌丝的Ca 2+敏感性，调节 异丙肾上腺素对AT-1细胞和正常心室肌的舒张作用 肌细胞 将进行钙吸收调节的研究 在用受磷蛋白的各种片段转染的AT-1细胞中， 明确其结构与功能的关系。 一种新研制的单池荧光分光光度计将用于一些 同时监测一种以上染料活性的实验 并量化笼状Ca 2+的光释放之前和之后 光释放 通过对钙离子释放和吸收特性的研究， 系统在其原生环境中，我们将在某种程度上遵循 信号传导途径的起源点，并表征a） 我们的实验干预的充分性，B）Ca 2+的作用 通道和Na ~+-Ca ~（2+）交换蛋白的表达及其在细胞内的差异表达 不同的动物和病理状态，和c）调节 Ca 2 +-ATP酶（受磷蛋白法）。

The aim of the proposed research is to examine the role of Ca2+ as the transmitter signal in gating Ca2+ release channels of the SR (Ryanodine receptors) and to evaluate the role of phospholamban in regulation of relaxation in mammalian cardiomyocytes. Central to the question of Ca2+ gating is the role and effectiveness of various sarcolemmal Ca2+ transporters in gating the Ca2+ release from the SR. WE shall, therefore, first evaluate the gating efficiency of Ca2+ delivered by the Ca2+ channel versus that transported by the Na+-Ca2+ exchanger and the Ca2+-selective Na+ channel (transformed by Atrionatriuretic Peptide in atrial and ventricular myocytes of mammalian heart by quantifying the integral of Ca2+ charge traversing the sarcolemma sufficient to trigger Ca2+ release. The hypothesis at the core of this study is that there is privileged communication between the Ca2+ channels (DHP receptors) and the Ryanodine receptors. The "privileged access concept" will be further probed by testing the accessibility of Ryanodine receptor blockers (ruthenium red and caged Mg2+) and caged Ca2+-buffers (DM-Nitrophen and Diazo-2) to the Ca2+-sensing site of the SR release channels in intact myocytes. The implications of the limited access hypothesis on the inactivation of the Ryanodine receptor by Ca2+ will be explored by a novel "epi-axial" method to photorelease Ca2+ in sub-sarcolemmal space. The level of expression of various sarcolemmal Ca2+-transporting proteins in cardiac myocytes is critical to this evaluation, requiring quantification of gating efficiency of different Ca2+ delivery systems in other mammalian species. Thus, the effectiveness of Ca2+ signalling via the Na+-Ca2+ exchanger, compared to the Ca2+ channel, will be probed in hamster myocytes, which show a ten-fold higher exchanger current density than the rat myocytes. Two pathological and one development model of E-C coupling will be examined which represent up-regulated expression of either the exchanger (myopathic hamster) or Ca2+ channels (spontaneously hypertensive rats), and the low density of SR release channels (neonatal human and cat cardiomyocytes). Detailed examination of Ca2+ release and uptake in AT-1 atrial tumor cell line will be carried out, not only because AT-1 cells do not express phospholamban (Ca2+ pump regulatory protein), but also because these cells are good candidates for genetic manipulation of various molecular components of E-C coupling. Probing the role of phospholamban in regulation of Ca2+ uptake will include evaluation of the kinetics of phosphorylation of phospholamban by injection of 2D12 antibody and photorelease of caged cAMP vs. alterations in the Ca2+ sensitivity of the myofilaments in regulating the relaxant effects of isoproterenol in AT-1 cells and normal ventricular myocytes. Studies on the regulation of Ca2+ uptake will be carried out in AT-1 cells transfected with various fragments of phospholamban to pinpoint its structure-function relationship. A newly-developed single-cell spectrofluorometer will be used in some experiments to monitor the activity of more than one dye simultaneously and quantify the photorelease of caged Ca2+ prior to and following photorelease. By exploring the properties of Ca2+ release and uptake systems in their native environment, we shall, to some extent, follow the signalling pathways to their points of origin and characterize a) the adequacy of our experimental interventions, b) the role of the Ca2+ channel and Na+-Ca2+ exchanger and their differential expression in different animals and pathological states, and c) the regulation of the Ca2+-ATPase by phospholamban.