TY - JOUR
T1 - Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt
AU - Iguchi, Moritake
AU - Kato, Masashi
AU - Nakai, Junichi
AU - Takeda, Toshihiro
AU - Matsumoto-Ida, Madoka
AU - Kita, Toru
AU - Kimura, Takeshi
AU - Akao, Masaharu
N1 - Funding Information:
This work was supported by research grants (Grants-in-Aid in Scientific Research, Leading Project for Biosimulation) from the Ministry of Education, Culture, Science, and Technology of Japan , and a grant from the Takeda Science Foundation , from the Kanae Foundation for the Promotion of Medical Science and from the Suzuken Memorial Foundation . The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing of International Journal of Cardiology [37] .
PY - 2012/7/12
Y1 - 2012/7/12
N2 - 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.
AB - 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.
KW - Cytosolic calcium
KW - Mitochondrial calcium
KW - Mitochondrial permeability transition pore
KW - Oxidative stress
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U2 - 10.1016/j.ijcard.2011.01.034
DO - 10.1016/j.ijcard.2011.01.034
M3 - Article
C2 - 21295866
AN - SCOPUS:84862198234
VL - 158
SP - 225
EP - 234
JO - International Journal of Cardiology
JF - International Journal of Cardiology
SN - 0167-5273
IS - 2
ER -