Calcium regulation in hypertrophy and heart failure

我们建议研究作为肥厚和/或衰竭的后果，在分离的心室肌细胞中发生的钙稳态和兴奋-收缩偶联的变化。我们的研究将利用转基因心力衰竭小鼠；两种新的心力衰竭和肥厚兔模型(起搏诱导的心力衰竭和兔心肌梗死)；以及从心功能正常和严重心力衰竭患者的活检标本中分离的心肌细胞。细胞内钙离子浓度用荧光指示剂Fluo-3定量，细胞内[Na+]浓度用SBFI定量，收缩和松弛用视频运动分析。单个心肌细胞的电压钳研究将用于定量Na/Ca交换器密度、L型钙通道功能和肌浆网钙离子含量。当钠/钙交换器被使用快速溶液切换装置的技术禁用时，SR CA ATPase的功能将通过单个心肌细胞中钙离子的隔离速率来评估。当钠/钙交换器被使用快速溶液转换装置的技术禁用时，单个心肌细胞中钙离子的功能。钙释放通道兰尼定受体的功能将通过测量全细胞钙增益(钙浓度变化率除以L型钙电流的大小)以及在行扫描共聚焦显微镜下确定的钙放电形态和概率来评估。在完整的组织实验中，将测量钙调素依赖的激酶，CaM激酶II的组织活性。这些技术将被用来检测：细胞骨架异常导致肥大和衰竭的机制和时间进程；由填充性衰竭引起的心力衰竭的机制，以及梗死灶周围心肌细胞[Ca+]稳态的改变；通过增强SR Ca ATPase功能(磷蛋白基因敲除，转染腺病毒载体驱动腺苷环化酶的表达)，异常的Ca+稳态恢复的程度；衰竭的人类心肌细胞SR Ca ATPase和Na/Ca交换活性的变化程度，类似于在衰竭动物模型中观察到的变化；而CaM激酶II活性降低的程度可能是由[Ca~(2+)]瞬变幅度降低引起的，这是导致心力衰竭进展的一个重要因素。这些研究是我们在过去四年心力衰竭SCOR资助的初始周期中对这些不同系统进行的先前工作的基础和扩展。

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.