Ca & InsP3 Receptor Signaling in Cardiac Myocytes

心脏肥大是复杂的遗传预定的电和机械重塑的结果 程序响应机械和神经体液刺激。Ca在激活和激活细胞中起着关键作用， 调节几个肥大转录途径，而钙信号本身经历了深刻的 肥大（Hyp）和心力衰竭（HF）的变化，包括InsP 3依赖性 释放。 拟议研究的具体目标是： 1)表征InsP 3依赖性Ca信号传导在兴奋-收缩偶联（ECC）中的作用， 生理条件下和Hyp/HF中的心房和心室肌细胞。 2)检验InsP 3依赖性Ca释放促进促炎性Ca信号（SR Ca）的假设 钙超载、钙波、钙交替、钙火花频率增加）和膜电位变化 （早期（EAD）和延迟（DAD）后去极化，自发动作电位（AP）和 电交替）。 3)确定细胞和亚细胞Ca信号通路和时空[Ca]，模式 与核Ca/钙调蛋白/钙调神经磷酸酶依赖性NFAT易位相关， 心脏Hyp和HF中的转录和基因表达。 为了实现这些目标，将使用多种实验技术： 在单个心肌细胞中的激光扫描共聚焦显微镜，新型FRET（CFP/YFP）荧光InsP 3 传感器来研究InsP 3的细胞和亚细胞动力学，全细胞电压钳技术来研究InsP 3， 膜电流和膜电位变化，以及分子生物学和药理学工具， 操纵InsP 3/Ca/钙调蛋白/克拉神经磷酸酶/NFAT信号级联。此外，一些动物/细胞 将使用的模型包括野生型小鼠和兔、肥大小鼠、InsPa受体敲除小鼠、 小鼠和心力衰竭兔。 这项研究将产生关于InsP 3R依赖性Ca ~（2+） 在ECC期间心肌细胞中的信号传导，在胚胎发生、电生理变化和核 在正常、Hyp和HF心肌细胞中的转录信号传导。

Cardiac hypertrophy is the result of complex genetically predetermined electrical and mechanical remodeling programs in response to mechanical and neurohumoral stimuli. Ca plays a pivotal role in the activation and regulation of several hypertrophy transcription pathways, while Ca signaling itself undergoes profound changes in hypertrophy (Hyp) and heart failure (HF), including significant contributions from InsP3-dependent Ca release. The specific aims of the proposed study are: 1) characterize the role of InsP3-dependent Ca signaling for excitation-contraction coupling (ECC) in atrial and ventricular myocytes under physiological conditions and in Hyp/HF. 2) test the hypothesis that InsP3-dependent Ca release facilitates arrhythmogenic Ca signals (SR Ca overload, Ca waves, Ca alternans, enhanced Ca spark frequency) and changes of membrane potential (early (EAD) and delayed (DAD) afterdepolarizations, spontaneous action potentials (APs) and electrical alternans) in Hyp/HF ventricular myocytes. 3) identify the cellular and subcellular Ca signaling pathways and spatio-temporal [Ca], patterns relevant for nuclear Ca/calmodulin/calcineurin-dependent NFAT translocation which initiate gene transcription and gene expression in cardiac Hyp and HF. To achieve these aims a multitude of experimental techniques will be used: high resolution Ca imaging by laser scanning confocal microscopy in single cardiac myocytes, novel FRET(CFP/YFP) fluorescent InsP3 sensors to study the cellular and subcellular dynamics of InsP3, whole-cell voltage clamp techniques to study membrane currents and membrane potential changes, and molecular biological and pharmacological tools to manipulate the InsP3/Ca/calmodulin/clacineurin/NFAT signaling cascade. Furthermore, several animal/cell models will be used including wild-type mouse and rabbit, hypertrophy mouse, InsPa receptor knockout mouse and heart failure rabbit. The proposed research will generate fundamental new information on the roles of InsP3R-dependent Ca signaling in cardiac myocytes during ECC, in arrhythmogenesis, electrophysiological changes and nuclear transcription signaling in normal, Hyp and HF cardiac myocytes.