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 ([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 ΔΨ_m. We monitored oxidant-induced responses of [Ca ²⁺]_m and ΔΨ_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 [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 ΔΨ_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 ΔΨ_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.