Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt

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• 作者：Moritake Iguchi^a ; Masashi Kato^a ; Junichi Nakai^b ; Toshihiro Takeda^a ; Madoka Matsumoto-Ida^a ; Toru Kita^a ; Takeshi Kimura^a ; Masaharu Akao^a ; ^{akao@kuhp.kyoto-u.ac.jp}
• 关键词：Mitochondrial calcium ; Cytosolic calcium ; Mitochondrial permeability transition pore ; Oxidative stress
• 刊名：International Journal of Cardiology
• 出版年：2012
• 期刊代码：128_01675273
• 类别：med
• 出版时间：12 July, 2012
• 卷：158
• 期：2
• 页码：225-234
• 文件大小：2352 K

摘要

Background

An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (螖唯_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 螖唯_m. We monitored oxidant-induced responses of [Ca²⁺]_m and 螖唯_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 螖唯_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 螖唯_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.