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.

我们建议研究由于肥大和/或衰竭而在离体心室肌细胞中发生的 Ca2+ 稳态和兴奋-收缩耦合的变化。我们的研究将利用患有心力衰竭的转基因小鼠；两种新型兔心力衰竭和肥大模型（起搏诱发的心力衰竭和兔心肌梗死）；以及从心室功能正常和严重心力衰竭患者的活检标本中分离出的肌细胞。细胞质 Ca2+ 浓度将通过荧光 Ca2+ 指示剂 Fluo-3 定量，细胞内 [Na+] 通过 SBFI 定量，收缩和舒张通过视频运动分析定量。单肌细胞的电压钳研究将用于定量 Na/Ca 交换密度、L 型 Ca2+ 通道功能和 SR Ca2+ 含量。当通过使用快速溶液切换装置的技术禁用 Na/Ca 交换器时，将通过单个肌细胞中 Ca2+ 的隔离速率来评估 SR CA ATP 酶的功能。当通过使用快速溶液切换装置的技术禁用 Na/Ca 交换器时，单个肌细胞中 Ca2+ 的功能。 Ca2+ 释放通道（兰尼碱受体）的功能将通过测量全细胞 Ca2+ 增益（Ca2+ 浓度的变化率除以 L 型 Ca2+ 电流的大小）以及用线确定的 Ca2+ 火花形态和概率来评估扫描共焦显微镜。钙钙调蛋白依赖性激酶 CaM 激酶 II 的组织活性将在完整组织实验中进行测量。这些技术将用于检查：细胞骨架异常导致肥大和衰竭的机制和时间过程；堆积引起的心力衰竭的机制，以及梗死周围肌细胞[Ca2+]稳态的改变；通过增强 SR Ca ATP 酶功能（受磷蛋白敲除、用驱动腺苷酸环化酶表达的腺病毒载体转染）恢复异常 Ca2+ 稳态的程度；衰竭的人类心肌细胞 SR Ca ATP 酶和 Na/Ca 交换活性的改变程度与在衰竭动物模型中观察到的相似； CaM 激酶 II 活性降低的程度（可能是由 [Ca2+] 瞬变幅度的降低引起的）是导致心力衰竭进展的重要因素。这些研究建立在我们过去四年心力衰竭 SCOR 资助的初始周期期间对这些不同系统的先前工作的基础上并进行了扩展。