Calcium regulation in hypertrophy and heart failure

肥厚和心力衰竭中的钙调节

基本信息

批准号：
6564967
负责人：
WILLIAM BARRY
金额：
$ 13.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2003-02-28
项目状态：
已结题

项目摘要

We propose to study alterations in Ca2+ homeostasis and excitation- contraction coupling that occur in isolated ventricular myocytes as a consequences of hypertrophy and/or failure. Our studies will utilize transgenic mice with heart failure; two novel rabbit models of heart failure and hypertrophy (pacing-induced heart failure, and rabbit myocardial infarction); and myocytes isolated from biopsy specimens obtained from human patients with normal ventricular function, and with severe heart failure. The cytosolic Ca2+ concentration will be quantitated by the fluorescent Ca2+ indicator fluo-3, intracellular [Na+] by SBFI, and contraction and relaxation by video motion analysis. Voltage clamp studies in single myocytes will be employed to quantitative Na/Ca exchanger density, L-type Ca2+ channel function, and SR Ca2+ content. Function of the SR CA ATPase will be assessed by the rate of sequestration of Ca2+ in single myocytes when the Na/Ca exchanger is disabled by techniques involving the use of a rapid solution switcher device. Function of the Ca2+ in single myocytes when the Na/Ca exchanger is disabled by techniques involving the use of a rapid solution switcher device. Function of the Ca2+ release channel, the ryanodine receptor, will be assessed by measuring whole cell Ca2+ gain (the rate of change in Ca2+ concentration divided by the magnitude of the L-type Ca2+ current), and Ca2+ spark morphology and probability determined with line scan confocal microscopy. Tissue activity of the calcium- calmodulin dependent kinase, CaM kinase II, will be measured in intact tissue experiments. These techniques will be employed to examine: the mechanisms and time course by which cytoskeletal abnormalities cause hypertrophy and failure; the mechanisms of heart failure produced by packing-induced failure, and the alteration in [Ca2+] homeostasis in peri- infarct myocytes; the extent to which abnormal Ca2+ homeostasis is restored by enhancement of SR Ca ATPase function (phospholamban knockout, transfection with adenoviral vectors driving the expression of adenyl cyclase); the extent to which failing human myocytes have alterations in SR Ca ATPase and Na/Ca exchanger activity similar to those observed in animal models of failure; and the extent to which decreased activation of CaM kinase II, perhaps induced by reduction of the magnitude of the [Ca2+] transient, is an important factor in contributing to the progression of heart failure. These studies build on and extend our previous work with these different systems over the past four years, during the initial cycle of our heart Failure SCOR grant.

我们建议研究Ca2+稳态的改变和激发 - 收缩偶联，这些偶联是肥大和/或衰竭的结果。我们的研究将利用心力衰竭的转基因小鼠。两种新型的心力衰竭和肥大的兔子模型（起搏引起的心力衰竭和兔心肌梗塞）；从从患有正常心室功能且心力衰竭的人类患者中获得的活检标本中分离出的心肌细胞。胞质Ca2+浓度将通过荧光Ca2+指示剂Fluo-3，细胞内[Na+]进行定量，并通过视频运动分析来进行收缩和放松。单个肌细胞中的电压夹具研究将用于定量Na/Ca交换器密度，L型Ca2+通道功能和SR Ca2+含量。当Na/ca交换器通过涉及使用快速解决方案切换器设备的技术禁用Na/Ca交换器时，将通过单个肌细胞中Ca2+的隔离速率评估SR CA ATPase的功能。当Na/Ca交换器通过涉及使用快速解决方案切换器设备的技术禁用Na/Ca交换器时，CA2+的功能。 Ca2+释放通道（Ryanodine受体）的功能将通过测量全细胞Ca2+增益（Ca2+浓度的变化速率除以L型Ca2+电流的大小）以及Ca2+火花形态和概率用线扫描式共焦显微镜确定的概率来评估。钙 - 钙调蛋白依赖性激酶CAM激酶II的组织活性将在完整的组织实验中测量。这些技术将用于检查：细胞骨架异常引起肥大和衰竭的机制和时间过程；填料引起的失败产生的心力衰竭的机制以及梗塞周围肌细胞[Ca2+]稳态的改变；通过增强SR CA ATPase功能（磷脂敲除，用腺病毒载体转染，驱动腺基环酶表达的腺病毒载体转染）来恢复异常Ca2+稳态的程度；失败的人肌细胞在SR CA ATPase和Na/Ca交换器活性中的变化程度与在失败动物模型中观察到的那样。以及降低[Ca2+]瞬态的幅度引起的CAM激酶II激活降低的程度，是有助于心力衰竭进展的重要因素。在过去的四年中，在我们的心力衰竭得分赠款的最初周期中，这些研究在过去的四年中以这些不同的系统为基础并扩展了我们以前的工作。