Calcium regulation in hypertrophy and heart failure

我们建议研究在钙离子稳态和兴奋-收缩偶联发生在分离的心室肌细胞肥大和/或失败的后果的改变。我们的研究将利用转基因小鼠心力衰竭;两种新的兔心力衰竭和肥大模型（起搏诱导的心力衰竭和兔心肌梗死）;和心肌细胞分离的活检标本获得的人类患者与正常的心室功能，并与严重的心力衰竭。细胞溶质Ca 2+浓度将通过荧光Ca 2+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。将采用单个心肌细胞的电压钳研究来定量Na/Ca交换密度、L型Ca 2+通道功能和SR Ca 2+含量。当通过涉及使用快速溶液切换装置的技术禁用Na/Ca交换器时，SR CA ATP酶的功能将通过单个肌细胞中Ca 2+的螯合率来评估。当Na/Ca交换器通过涉及使用快速溶液转换装置的技术被禁用时，单个肌细胞中Ca 2+的功能。通过测量全细胞Ca 2+增益（Ca 2+浓度的变化率除以L型Ca 2+电流的幅度）评估Ca 2+释放通道（ryanodine受体）的功能，并使用线扫描共聚焦显微镜确定Ca 2+火花形态和概率。将在完整组织实验中测量钙-钙调蛋白依赖性激酶CaM激酶II的组织活性。这些技术将被用来研究：细胞骨架异常引起肥大和衰竭的机制和时间过程;由填塞诱导的衰竭产生的心力衰竭的机制，以及在心肌梗死心肌细胞中[Ca ~（2+）]稳态的改变;通过增强SR Ca ATP酶功能恢复异常Ca 2+稳态的程度（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）;衰竭的人肌细胞在SR Ca ATP酶和Na/Ca交换剂活性中具有与在衰竭动物模型中观察到的那些相似的改变的程度;以及可能由[Ca 2 +]瞬变幅度降低诱导的CaM激酶II活化降低的程度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，在我们的心力衰竭SCOR补助金的初始周期中。

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.