Abstract 5142: Direct Monitoring of Mitochondrial Calcium Levels in Cultured Cardiomyocytes 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 (ΔΨm), is the earliest event that commits the cell to death. Mitochondrial matrix calcium ([Ca2+]m) is considered a critical regulator of MPTP, but direct monitoring of [Ca2+]m has been difficult with previously reported sensors due to limited signal intensity or low targeting efficiency. We developed a novel fluorescent indicator for [Ca2+]m, GCaMP2-mt, by adding the mitochondrial targeting sequence of cytochrome oxidase to a high signal-to-noise Ca2+ sensor protein GCaMP2, and monitored the dynamic changes in oxidant-induced cardiomyocyte death.
Methods and Results: GCaMP2-mt was transduced in neonatal rat cardiomyocytes using a recombinant adenovirus. We confirmed that GCaMP2-mt colocalized well with tetramethylrhodamine ethyl-ester, a fluorescent indicator of ΔΨm. We monitored oxidant-induced response of [Ca2+]m and ΔΨm using time-lapse confocal microscopy. After H2O2 exposure, [Ca2+]m remained unchanged for about 20 min, followed by kinetically-distinct two-step increase; the first increase lasted for about 5 min, and the second increase was slower and persisted until cells underwent irreversible ΔΨm loss. Importantly, there was a cellular heterogeneity in the second increase, and the level of [Ca2+]m overload was variable. However, ΔΨm loss occurred in an all-or-none manner depending on cellular [Ca2+]m level, with a clear cut-off value. A Ca2+ chelator BAPTA suppressed both the first and the second increase of [Ca2+]m, and protected the cells against ΔΨm loss. In contrast, ruthenium red, an inhibitor of mitochondrial Ca2+ uniporter, suppressed only the second increase of [Ca2+]m, but not the first increase. Despite the suppressed [Ca2+]m overload, ruthenium red was unable to protect against ΔΨm loss; it caused earlier increase in cytosolic Ca2+ ([Ca2+]c) level which might have accelerated the MPTP opening, as measured by an indicator of [Ca2+]c, Fluo-4. These findings support the central role of [Ca2+]m as well as [Ca2+]c overload in the MPTP opening.
Conclusion: Direct monitoring of [Ca2+]m using GCaMP2-mt provides deeper insight into the mechanism of cardiomyocyte death.