Abstract 5142: Direct Monitoring of Mitochondrial Calcium Levels in Cultured Cardiomyocytes 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 the cell to death. Mitochondrial matrix calcium ([Ca 2+ ] m ) is considered a critical regulator of MPTP, but direct monitoring of [Ca 2+ ] m has been difficult with previously reported sensors due to limited signal intensity or low targeting efficiency. We developed a novel fluorescent indicator for [Ca 2+ ] m , GCaMP2-mt, by adding the mitochondrial targeting sequence of cytochrome oxidase to a high signal-to-noise Ca 2+ 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 ΔΨ m . We monitored oxidant-induced response of [Ca 2+ ] m and ΔΨ m using time-lapse confocal microscopy. After H 2 O 2 exposure, [Ca 2+ ] 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 ΔΨ m loss. Importantly, there was a cellular heterogeneity in the second increase, and the level of [Ca 2+ ] m overload was variable. However, ΔΨ m loss occurred in an all-or-none manner depending on cellular [Ca 2+ ] m level, with a clear cut-off value. A Ca 2+ chelator BAPTA suppressed both the first and the second increase of [Ca 2+ ] m , and protected the cells against ΔΨ m loss. In contrast, ruthenium red, an inhibitor of mitochondrial Ca 2+ uniporter, suppressed only the second increase of [Ca 2+ ] m , but not the first increase. Despite the suppressed [Ca 2+ ] m overload, ruthenium red was unable to protect against ΔΨ m loss; it caused earlier increase in cytosolic Ca 2+ ([Ca 2+ ] c ) level which might have accelerated the MPTP opening, as measured by an indicator of [Ca 2+ ] c , Fluo-4. These findings support the central role of [Ca 2+ ] m as well as [Ca 2+ ] c overload in the MPTP opening. Conclusion: Direct monitoring of [Ca 2+ ] m using GCaMP2-mt provides deeper insight into the mechanism of cardiomyocyte death.