Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt
Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt
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DOI：
10.1016/j.ijcard.2011.01.034
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发表时间：
2012-07-12
影响因子：
3.5
通讯作者：
Akao, Masaharu
中科院分区：
文献类型：
--
作者：
Iguchi, Moritake;Kato, Masashi;Akao, Masaharu
背景资料：线粒体渗透性转换孔（MPTP）的打开导致线粒体膜电位（Delta Psi（m））的损失，这是使细胞死亡的最早事件。线粒体基质钙（[Ca 2 +]（m））被认为是MPTP的关键调节剂，但用先前报道的传感器直接监测[Ca 2 +]（m）是困难的。我们通过在高信噪比的Ca ~（2+）传感蛋白GCaMP 2上添加线粒体靶向序列，开发了一种新的[Ca ~（2+）]（m）荧光指示剂GCaMP 2-mt，并监测了氧化剂诱导的心肌细胞死亡的动态变化。我们证实GCaMP 2-mt与四甲基罗丹明乙酯共定位，所述四甲基罗丹明乙酯是Delta Psi（m）的荧光指示剂。我们使用延时共聚焦显微镜监测氧化剂诱导的[Ca 2 +]（m）和Δ Psi（m）的反应。[Ca ~（2+）]（m）的变化与胞浆Ca ~（2+）的变化同步，并可分为3个不同的动态时相：第一时相[Ca ~（2+）]（m）维持在基线水平，第二时相[Ca ~（2+）]（m）迅速突然升高，第三时相[Ca ~（2+）]（m）迅速升高，第四时相[Ca ~（2+）]（m）迅速升高，第四时相[Ca ~（2+）]（m）迅速升高，第四时相[Ca ~（2+）]（m）迅速升高，第五时相[Ca ~（2+）]（m）迅速升高，第四时相[Ca ~（2+）]（m）迅速升高，第五时相[Ca ~（2+）]（m）迅速升高。第三阶段，[Ca ~（2+）]（m）继续缓慢增加，直至Δ Psi（m）崩溃。第三阶段可能是通过线粒体Ca 2+单向转运体介导的，因为它是由单向转运体作用药物调节的。重要的是，有一个显着的细胞异质性在第三阶段，和Delta Psi（m）的损失发生在一个全或无的方式取决于细胞[Ca 2 +]（m）水平与一个明确的cut-off value.Conclusions：直接监测[Ca 2 +]（m）使用GCaMP 2-mt提供了更深入的了解心肌细胞死亡的机制。(C)2011爱思唯尔爱尔兰有限公司保留所有权利。
Background: An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (Delta Psi(m)), is the earliest event that commits a cell to death. Mitochondrial matrix calcium ([Ca2+](m)) is considered to be a critical regulator of MPTP, but direct monitoring of [Ca2+](m) is difficult with previously-reported sensors. We developed a novel fluorescent indicator for [Ca2+](m), GCaMP2-mt, by adding a mitochondrial targeting sequence to a high signal-to-noise Ca2+ sensor protein GCaMP2, and monitored dynamic changes in oxidant-induced cardiac myocyte death.Methods and results: GCaMP2-mt was transduced into neonatal rat cardiac myocytes using a recombinant adenovirus. We confirmed that GCaMP2-mt colocalized with tetramethylrhodamine ethyl-ester, a fluorescent indicator of Delta Psi(m). We monitored oxidant-induced responses of [Ca2+](m) and Delta Psi(m) using time-lapse confocal microscopy. The response of [Ca2+](m) was synchronous with that of cytosolic calcium and was divided into three kinetically-distinct phases; the first phase, during which [Ca2+](m) maintained its baseline level; the second phase, during which [Ca2+](m) showed a rapid and sudden increase; and the third phase, during which [Ca2+](m) continued to increase at a slower rate until the collapse of Delta Psi(m). The third phase was likely to be mediated through a mitochondrial Ca2+ uniporter, because it was modulated by uniporter-acting drugs. Importantly, there was a remarkable cellular heterogeneity in the third phase, and Delta Psi(m) loss occurred in an all-or-none manner depending on the cellular [Ca2+](m) level with a clear cut-off value.Conclusions: Direct monitoring of [Ca2+](m) using GCaMP2-mt provides deeper insight into the mechanism of cardiac myocyte death. (C) 2011 Elsevier Ireland Ltd. All rights reserved.