ALTERED MYOCYTE CALCIUM HOMEOSTATIC MECHANISMS IN HEART FAILURE

肌浆网钙ATPase表达的变化，以及 据报道，钠钙交换发生在脑室 肥大，并可能有功能影响，这可能有助于 从肥大进展到衰竭。此项目中描述的工作 将检验以下假设：1)肌浆网功能 降低，以及2)钠钙交换活性在 从衰竭的心室中分离出完整的单个心肌细胞 心肌。从正常和衰竭的心肌细胞中获得心肌细胞 小鼠、兔和人的心肌。心肌细胞来源的心肌细胞 将使用一种新技术分离人类心肌，该技术采用了 振动刀切割400微米薄片，在BDM存在下抑制 割伤。这有助于酶促耐钙蛋白的解离 小的活组织标本中的细胞。钠钙交换活性将 通过测量最大钠钙交换电流Per来定量 钳位电压突然暴露产生的单位电池表面积 分离的心肌细胞置于含2.7 mM钙的零钠溶液中。一个 新型快速溶液切换器用于在介质中产生变化 在这些实验中，在7毫秒内洗澡单个心肌细胞。 分离的单个心肌细胞的肌浆网功能将是 通过突然暴露于零引起心肌细胞收缩来评估 钠，零钙氯化钾溶液，然后测量 松弛，细胞内游离钙下降的速率。后者 将使用INDO-1进行测量。这种方法允许评估比率 在这一点上，肌浆网可以将钙隔离在单个 在初始钙离子作用下，心肌细胞 钙的释放是由细胞外的短暂钙内流触发的 空间中，钠钙交换器因消除 胞外钠。此外，在肌细胞中SR功能是 在研究中，分离的心肌细胞的收缩能力将通过 测量不同起搏速率下的短轴缩短率 视频运动检测。这种方法应该允许确定 心肌细胞收缩功能受损的程度 从衰竭心肌中获得的肌细胞，以及可能的 钠钙交换器改变的贡献和SR功能。

Alterations in expression of sarcoplasmic reticulum calcium ATPase, and the sodium calcium exchanger have been reported to occur during ventricular hypertrophy, and could have functional effects which may contribute to the progression from hypertrophy to failure. Work described in this project will test the hypotheses that 1) sarcoplasmic reticulum function is decreased, and 2) that sodium calcium exchange activity is increased in intact single ventricular myocytes isolated from failing ventricular myocardium. Ventricular myocytes will be obtained from normal and failing mouse, rabbit, and human ventricular myocardium. Ventricular myocytes from human myocardium will be isolated using a novel technique which employs a vibratome to cut 400 mum thin sections, in the presence of BDM to inhibit cutting injury. This facilitates enzymatic dissociation of Ca2+-tolerant cells from small biopsy specimens. Sodium calcium exchanger activity will be quantitated by measuring the maximum sodium calcium exchange current per unit cell surface area produced by abrupt exposure of voltage clamped isolated myocytes to zero sodium solution containing 2.7 mM calcium. A novel fast solution switcher is used to produce a change in the medium bathing a single myocyte within 7 milliseconds in these experiments. Sarcoplasmic reticulum function in isolated single myocytes will be assessed by inducing contraction of myocytes by abrupt exposure zero sodium, zero calcium KCI solution, and then measuring the rate of relaxation, and the rate of fall in cytosolic free calcium. The latter will be measured with indo-1. This approach allows assessment of the rate at which the sarcoplasmic reticulum can sequester calcium within a single ventricular myocyte under conditions in which, after initial calcium release is triggered by transient influx of calcium from the extracellular space, the sodium calcium exchanger is disabled by the elimination of extracellular sodium. In addition, in myocytes in which SR function is studied, the contractility of isolated myocytes will be assessed by measurement of fractional shortening at different pacing rates, with a video motion detection. This approach should allow determination of the extent to which isolated myocyte contractile performance is impaired in myocytes obtained from failing myocardium, as well as the possible contributions of altered sodium calcium exchanger and SR function.