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: 3737339
• 负责人: WILLIAM BARRY
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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/3737339
• 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.

肌浆网钙ATP酶表达的改变， 据报道，钠钙交换器在心室 肥大，并可能有功能影响，这可能有助于 从肥大进展到衰竭。 本项目中描述的工作 将检验以下假设：1）肌浆网功能是 降低，和2）钠钙交换活性增加， 从衰竭心室肌分离的完整单个心室肌细胞 心肌 将从正常和衰竭的心室肌细胞中获得心室肌细胞。 小鼠、兔和人心室心肌。 心室肌细胞 将使用一种新的技术分离人类心肌， 振动切片机切割400 μ m薄切片，在BDM存在下抑制 切割伤 这促进了耐Ca 2+的 小活检标本中的细胞。 钠钙交换器活性将 通过测量最大钠钙交换电流进行定量， 电压箝位突然暴露产生的晶胞表面积 分离的肌细胞加入到含有2.7mM钙的零钠溶液中。 一 一种新型的快速溶液切换器被用于在介质中产生变化， 在这些实验中，在7毫秒内浸泡单个肌细胞。 分离的单个肌细胞中的肌浆网功能将被 通过突然零暴露诱导肌细胞收缩来评估 钠，零钙KCl溶液，然后测量 松弛和细胞溶质游离钙的下降速率。 后者 将用indo-1测量。 这种方法可以评估比率 肌浆网可以将钙隔离在一个 心室肌细胞的条件下，在初始钙 释放是由细胞外钙离子的瞬时内流触发的 空间，钠钙交换器被禁用的消除 胞外钠 此外，在SR功能受损的心肌细胞中， 在研究中，分离的肌细胞的收缩性将通过 测量不同起搏频率下的短轴缩短分数， 视频运动检测 这种方法应允许确定 分离的肌细胞收缩性能受损的程度， 从衰竭心肌中获得的心肌细胞，以及可能的 改变钠钙交换器和SR功能的贡献。