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

Moritake Iguchi, Masashi Kato, Junichi Nakai, Toshihiro Takeda, Madoka Matsumoto-Ida, Toru Kita, Takeshi Kimura, Masaharu Akao

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

Background: An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨm), 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 ΔΨm. We monitored oxidant-induced responses of [Ca 2+]m and ΔΨm 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 [Ca 2+]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 ΔΨm. 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 ΔΨm 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.

Original languageEnglish
Pages (from-to)225-234
Number of pages10
JournalInternational Journal of Cardiology
Volume158
Issue number2
DOIs
Publication statusPublished - 2012 Jul 12

Keywords

  • Cytosolic calcium
  • Mitochondrial calcium
  • Mitochondrial permeability transition pore
  • Oxidative stress

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

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