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: 6110439
• 负责人: WILLIAM BARRY
• 金额: $ 18.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110439
• 关键词: calcium channel; cardiac myocytes; 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.

肌浆网钙 ATP 酶表达的改变，以及 据报道，钠钙交换发生在心室期间 肥大，并可能产生功能影响，可能有助于 从肥大到衰竭的进展。 本项目中描述的工作 将检验以下假设：1) 肌浆网功能是 减少，2）钠钙交换活性增加 从衰竭心室中分离出完整的单心室肌细胞 心肌。 心室肌细胞取自正常和衰竭的心室肌细胞 小鼠、兔和人的心室心肌。 心室肌细胞来自 将使用一种新技术分离人类心肌，该技术采用 振动切片机切割 400 微米薄片，在 BDM 存在的情况下抑制 切割伤。 这有利于 Ca2+ 耐受的酶解 来自小活检标本的细胞。 钠钙交换活性将 通过测量每个最大钠钙交换电流来定量 突然暴露钳位电压产生的晶胞表面积 将心肌细胞分离到含有 2.7 mM 钙的零钠溶液中。 一个 新颖的快速溶液切换器用于产生介质的变化 在这些实验中，在 7 毫秒内沐浴单个肌细胞。 分离的单个肌细胞的肌浆网功能将 通过突然零暴露诱导心肌细胞收缩来评估 钠、零钙KCI溶液，然后测定 松弛，以及胞质游离钙的下降率。 后者 将使用 indo-1 进行测量。 这种方法可以评估速率 肌浆网可以在一个单一的时间内隔离钙 心室肌细胞在初始钙治疗后的条件下 释放是由细胞外钙的瞬时流入触发的 空间，钠钙交换器通过消除而被禁用 细胞外钠。 此外，在 SR 功能处于 研究中，分离的肌细胞的收缩性将通过以下方式评估 测量不同起搏速率下的缩短分数， 视频运动检测。 这种方法应该允许确定 孤立的心肌细胞收缩性能受损的程度 从衰竭的心肌中获得的心肌细胞，以及可能的 钠钙交换器和 SR 功能改变的贡献。