Calcium regulation in hypertrophy and heart failure

我们建议研究Ca2+稳态和兴奋-收缩耦合的改变，发生在分离心室肌细胞肥大和/或衰竭的后果。我们的研究将利用转基因心力衰竭小鼠；两种新的兔心力衰竭和肥厚模型（起搏性心力衰竭和兔心肌梗死）；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。细胞质内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，收缩和松弛通过视频运动分析。单个肌细胞的电压钳研究将用于定量Na/Ca交换器密度，l型Ca2+通道功能和SR Ca2+含量。当Na/ CA交换器被使用快速溶液切换装置的技术禁用时，SR CA atp酶的功能将通过单个肌细胞中Ca2+的隔离率来评估。当Na/Ca交换器通过涉及使用快速溶液切换装置的技术被禁用时，单个肌细胞中Ca2+的功能。Ca2+释放通道的功能，即ryanodine受体，将通过测量全细胞Ca2+增益（Ca2+浓度变化率除以l型Ca2+电流的大小）和Ca2+火花形态和概率来评估，用线扫描共聚焦显微镜确定。钙钙调素依赖性激酶（CaM激酶II）的组织活性将在完整组织实验中测量。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进程；包装诱导心力衰竭的机制和梗死周围肌细胞[Ca2+]稳态的改变；通过增强SR Ca atp酶功能（敲除磷蛋白，转染腺病毒载体驱动腺苷环化酶的表达）恢复异常Ca2+稳态的程度；衰竭的人肌细胞在SR - Ca - atp酶和Na/Ca交换活性方面的改变程度与在衰竭动物模型中观察到的相似；而CaM激酶II的激活降低的程度，可能是由[Ca2+]瞬态的大小减少引起的，是促成心力衰竭进展的重要因素。这些研究建立并扩展了我们在过去四年中使用这些不同系统的工作，在我们心力衰竭SCOR资助的初始周期。

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.