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

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+)) is considered to be a critical regulator of MPTP, but direct monitoring of Ca(2+) is difficult with previously-reported sensors. We developed a novel fluorescent indicator for Ca(2+), 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.

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 ΔΨ(m). We monitored oxidant-induced responses of Ca(2+) and ΔΨ(m) using time-lapse confocal microscopy. The response of Ca(2+) was synchronous with that of cytosolic calcium and was divided into three kinetically-distinct phases; the first phase, during which Ca(2+) maintained its baseline level; the second phase, during which Ca(2+) showed a rapid and sudden increase; and the third phase, during which Ca(2+) 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+) level with a clear cut-off value.

CONCLUSIONS

Direct monitoring of Ca(2+) using GCaMP2-mt provides deeper insight into the mechanism of cardiac myocyte death.