NOVEL MODULATION OF CARDIAC SARCOPLASMIC RETICULUM

在兴奋-收缩（EC）偶联过程中， 由Ca 2+电流（lca）携带的Ca 2+离子激活大量的 肌浆网Ca ~（2+）释放通道 (or兰尼碱受体（Ryanodine receptor，RYR）。 随后的下降阶段， {Ca2+}，短暂的部分原因是由于SR介导的SR Ca 2+再摄取 Ca ~（2+）ATP酶（SERCA 2A）。 主要调查人员只发现 最近，钙调素激活酶钙调磷酸酶（CaN）单独作用于 和/或与FK 506结合蛋白（FKBP）协同作用，调节 {Ca2+}、瞬态和基本SR Ca 2+释放事件的频率 （“钙火花”）。 钙调神经磷酸酶在L-型钙调神经磷酸酶没有变化的情况下这样做。 Ca 2+电流。 计划的工作将研究这一重要的新调制器 用最先进的技术在细胞和分子水平上 该项目将侧重于以下相互关联的具体目标， 提供了生物物理学和基础分子细胞之间的联系 生物学 (1)CaN在大鼠心室肌中的作用机制 细胞？ 新的结果将扩大与膜片钳肌细胞， 确定CaN活性的升高或抑制如何改变整个细胞 电流、SR Ca 2+负荷、[Ca 2 +]、瞬态和收缩性。 影响 将在生物化学测定中检查抑制剂。 (2)Ca 2+是什么 火花，基本SR钙释放事件，改变CaN？ 共焦 将使用显微镜研究CaN对频率和 Ca 2+火花的动力学，以提供对以下结果的精确补充 (1)以上 (3)什么是SR钙负荷的作用， CaN？ SR负荷将通过使用受磷蛋白缺乏的转基因来改变。 小鼠或通过使用PI的SERCA 2a的瞬时过表达 新型腺病毒组分转染系统。 CaN的影响将 按上述（1）和（2）进行审查。 (4)FKBP如何影响行动 CaN和SR Ca的释放？ （1）、（2）和（3）的结果将用于 在平行研究中比较FKBP活化配体的作用， 正常和CaN抑制细胞。 总而言之，该计划是一个综合性的 生物化学、基因转染、单细胞电压钳、 高分辨率共聚焦Ca 2+成像将定义新的SR调节 心的机制。 该项目将提供一个了解 SR功能的变化是心肌收缩功能障碍的基础， 包括心肌病和肥大的疾病。

During excitation-contraction (EC) coupling a cardiac myocytes in influx of Ca2+ ions carried by the Ca2+ current (lca) activates a large release of Ca2+ from the sarcoplasmic reticulum (SR) vi the SR Ca2+ release channel (or ryanodine receptor, RYR). The subsequent declining phase of this {Ca2+}, transient is due in part, to SR Ca2+ reuptake mediated by the SR Ca2+ ATPase (SERCA2A). The Principal Investigators have discovered only recently that calcineurin (CaN), a calmodulin-activated enzyme, acting alon and/or in concert with FK506 binding proteins (FKBPs), regulates the {Ca2+}, transient and the frequency of elementary SR Ca2+ release events ("Ca sparks"). Calcineurin does this in the absence of changes in L-type Ca2+ current. The planned work will study this important new modulator at the cellular and molecular level by state-of-the-art techniques. The project will focus on the following inter-related specific aims that provide links between the biophysics and the underlying molecular cell biology. (1) How does CaN altar Ca2+ signaling in rat ventricular heart cells? New results will be extended with patch clamped myocytes to determine how elevation or inhibition of CaN activity alter whole cell currents, SR Ca2+ load, the [Ca2+], transient, and contractility. Effects inhibitors will be examined in biochemical assays. (2) How are Ca2+ sparks, the elementary SR Ca-release events, altered by CaN? Confocal microscopy will be used to study effects of CaN on the frequency and kinetics of Ca2+ sparks to provide a precise complement to the results of (1) above. (3) What is the impact of SR calcium load on the action of CaN? SR load will be varied by use of phospholamban-difficient transgenic mice or by transient overexpression of the SERCA2a by the use of the Pl's new novel adenovirus component transfection system. Effects of CaN will be examined as in (1) and (2) above. (4) How does FKBP effect the action of CaN and SR Ca release? The results of (1), (2) and (3) will be used to compare the action of FKBP-activating ligands in parallel studies with normal and CaN-inhibited cells. In summary, the plan is an integrated strategy of biochemistry, gene transfection, single cell voltage clamp, and high resolution confocal Ca2+ imaging that will define novel SR regulatory mechanisms in heart. This project will provide an understanding of the changes in SR function that underlie the contractile dysfunction of diseases including cardiomyopathies and hypertrophy.