Pathophysiological Regulation of Atrial Myocyte Excitation-Contraction Coupling and Calcium Signaling

项目总结/摘要 心房肌细胞兴奋收缩偶联（ECC）和肌浆网（SR）钙释放 具有由于横小管膜的缺乏或不规则组织而产生的独特特征 系统心房肌细胞具有两种类型的SR，连接型（j-SR）和非连接型（nj-SR）。钙从j- SR由通过电压门控L-型Ca通道的Ca进入控制，而从nj-SR的释放则通过以下方式发生： 随后传播Ca诱导的Ca释放（CICR）。在心房ECC中， 未回答：心脏ryanodine受体（RyR）SR Ca释放通道具有固有的低胞质Ca-2 +- 敏感性导致难题如何从nj-SR CICR甚至可以被激活。本次调查旨在 建立了一种新心房ECC综合模型。在心力衰竭（HF）中，心脏经历结构性和 旨在维持足够心输出量的功能变化（心脏重塑）。我们 将研究在HF发展的不同阶段，心房重塑如何导致深刻的 心房Ca信号传导、ECC和收缩力的变化有助于维持心输出量。 具体目标1.确定心房肌细胞中钙释放的独特性质， CICR。我们将测试心房钙释放的“火焰-弥散-摄取-火焰”（FDUF）范式的新假设， ECC。通过这种机制，胞质和腔Ca（串联RyR）调节RyR的协调作用被抑制。 在单个SR Ca释放单位以及整个SR网络的水平上， 肌浆网/内质网Ca ATP酶和SR内Ca扩散确保了强大和有效的CICR。 具体目标2：确定HF进展期间ECC的心房重构和Ca释放 优化心输出量我们将检验以下假设，即在HF的不同阶段，阶段特异性心房肌收缩和舒张功能不全， 重构通过改变调节心房SR Ca释放的分子机制来决定心输出量， ECC和收缩性。拟议的研究将涉及分子、细胞、完整器官和体内整体 动物实验。 我们将使用多种实验技术：高分辨率[Ca]i，[Ca]mito和[Ca]SR共聚焦 显微镜，细胞缩短测量，全细胞电压和电流钳技术，单个RyR 通道记录，笼状化合物的亚细胞光解，腺病毒基因转移， 免疫组织化学和生物化学技术。实验工作是由计算 Ca释放和ECC的建模。一个核心作用是在兔慢性HF模型中， HF在人类中的进展。兔子实验将与转基因动物的研究相辅相成 对CICR和ECC至关重要的Ca处理蛋白的表达水平发生了特异性改变。细胞研究 将在动物中进行完整心脏血流动力学研究和体内超声心动图研究 在HF的发展过程中。

PROJECT SUMMARY/ABSTRACT In atrial myocytes excitation-contraction coupling (ECC) and Ca release from the sarcoplasmic reticulum (SR) have unique features that result from the lack or the irregular organization of the transverse tubule membrane system. Atrial myocytes have two types of SR, junctional (j-SR) and non-junctional (nj-SR). Ca release from j- SR is controlled by Ca entry through voltage-gated L-type Ca channels whereas release from nj-SR occurs by subsequent propagating Ca-induced Ca release (CICR). In atrial ECC a fundamental question has remained unanswered: The cardiac ryanodine receptor (RyR) SR Ca release channel has an inherently low cytosolic Ca- sensitivity resulting in a conundrum how CICR from the nj-SR can even be activated. This investigation aims to establish a novel comprehensive model of atrial ECC. In heart failure (HF) the heart undergoes structural and functional changes (cardiac remodeling) that are aimed towards maintaining an adequate cardiac output. We will investigate how at different stages during the development of HF, remodeling of the atria leads to profound changes in atrial Ca signaling, ECC and inotropy that contribute to maintaining cardiac output. Specific aim 1. Determine the unique properties of Ca release in atrial myocytes that ensure robust CICR. We will test a novel hypothesis of a 'fire-diffuse-uptake-fire' (FDUF) paradigm for atrial Ca release and ECC. By this mechanism the coordinated action of RyR regulation by cytosolic and luminal Ca (tandem RyR activation) at the level of individual SR Ca release units as well as the entire SR network, Ca uptake by sarco/endoplasmic reticulum Ca ATPase and intra-SR Ca diffusion assure robust and efficient CICR. Specific aim 2: Determine how atrial remodeling of ECC and Ca release during the progression of HF optimizes cardiac output. We will test the hypothesis that at different stages of HF, stage-specific atrial remodeling determines cardiac output by altering molecular mechanisms that regulate atrial SR Ca release, ECC and contractility. The proposed studies will involve molecular, cellular, intact organ and in-vivo whole animal experiments. We will use a multitude of experimental techniques: high resolution [Ca]i, [Ca]mito and [Ca]SR confocal microscopy, cell shortening measurements, whole-cell voltage and current clamp techniques, single RyR channel recordings, subcellular photolysis of caged compounds, adenoviral gene-transfer, immunohistochemistry and biochemistry techniques. Experimental work is paralleled by computational modeling of Ca release and ECC. A central role plays a rabbit chronic HF model that recapitulates the progression of HF in humans. Rabbit experiments will be complemented with studies on transgenic animals with specific alterations in expression levels of Ca handling proteins crucial to CICR and ECC. Cellular studies will be paralleled with intact heart hemodynamic studies and in-vivo echocardiographic studies in animals during progression of HF.