title: Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt.
abstract: BACKGROUND
  An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨ(m)), is the earliest event that commits a cell to death. Mitochondrial matrix calcium ([Ca(2+)](m)) is considered to be a critical regulator of MPTP, but direct monitoring of [Ca(2+)](m) is difficult with previously-reported sensors. We developed a novel fluorescent indicator for [Ca(2+)](m), GCaMP2-mt, by adding a mitochondrial targeting sequence to a high signal-to-noise Ca(2+) sensor protein GCaMP2, and monitored dynamic changes in oxidant-induced cardiac myocyte death.
  
  
  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 ΔΨ(m). We monitored oxidant-induced responses of [Ca(2+)](m) and ΔΨ(m) using time-lapse confocal microscopy. The response of [Ca(2+)](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 [Ca(2+)](m) showed a rapid and sudden increase; and the third phase, during which [Ca(2+)](m) continued to increase at a slower rate until the collapse of ΔΨ(m). The third phase was likely to be mediated through a mitochondrial Ca(2+) uniporter, because it was modulated by uniporter-acting drugs. Importantly, there was a remarkable cellular heterogeneity in the third phase, and ΔΨ(m) loss occurred in an all-or-none manner depending on the cellular [Ca(2+)](m) level with a clear cut-off value.
  
  
  CONCLUSIONS
  Direct monitoring of [Ca(2+)](m) using GCaMP2-mt provides deeper insight into the mechanism of cardiac myocyte death.
authors.affiliation: Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Moritake
authors.initials: M
authors.lastname: Iguchi
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakai
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Takeda
authors.forename: Madoka
authors.initials: M
authors.lastname: Matsumoto-Ida
authors.forename: Toru
authors.initials: T
authors.lastname: Kita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kimura
authors.forename: Masaharu
authors.initials: M
authors.lastname: Akao
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008929:Mitochondria, Heart / Q000737:chemistry / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D005456:Fluorescent Dyes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D002118:Calcium