ALTERED MYOCYTE CALCIUM HOMEOSTATIC MECHANISMS IN HEART FAILURE

心力衰竭中心肌细胞钙稳态机制的改变

• 批准号: 5214258
• 负责人: WILLIAM BARRY
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5214258
• 关键词: calcium channel; heart cell; heart contraction; heart failure; homeostasis; human tissue; laboratory mouse; laboratory rabbit; sarcoplasmic reticulum; single cell analysis; sodium channel

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.

肌质网钙ATPase的表达改变， 据报道，钙交换钠在心室中发生 肥大，可能有功能作用，可能有助于 从肥大到失败的发展。 该项目中描述的工作 将检验假设1）核质网是 减少，2）钙钙交换活性在 完整的单室心肌细胞因腹心室失败而分离 心肌。 心室心肌细胞将从正常和失败中获得 小鼠，兔子和人室心肌。 心室心肌细胞 人体心肌将使用采用一种新技术隔离 在BDM存在下，振动型以切割400 MUM薄切片以抑制 切割伤害。 这有助于Ca2+耐剂的酶促解离 来自小型活检标本的细胞。 钙交换器活性将 通过测量最大钠钙交换电流来定量 电压突然暴露夹紧产生的单元细胞表面积 分离的肌细胞至含有2.7 mm钙的零钠溶液。 一个 新颖的快速解决方案切换器用于在介质中产生变化 在这些实验中，在7毫秒内沐浴一个肌细胞。 孤立的单个肌细胞中的肌质网络功能 通过突然暴露零诱导肌细胞收缩来评估 钠，零钙KCI溶液，然后测量 放松和胞质自由钙的下降速率。 后者 将用Indo-1测量。 这种方法允许评估费率 肌质网可以在一个单一的钙中隔离钙 在初始钙之后的条件下，心室心肌细胞 释放是由细胞外钙的瞬时涌入触发的 空间，通过消除来禁用钠钙交换器 细胞外钠。 另外，在SR功能的心肌细胞中 研究了，孤立的肌细胞的收缩力将由 以不同的起搏速率测量分数缩短，并以a 视频运动检测。 这种方法应允许确定 孤立的肌细胞收缩性能在多大程度上受到损害 从心肌失败获得的心肌细胞以及可能 钙交换器和SR功能改变的贡献。