Diversity of mitochondrial Ca²⁺ signaling in rat neonatal cardiomyocytes: evidence from a genetically directed Ca²⁺ probe, mitycam-E31Q.

I(Ca)-gated Ca(2+) release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca(2+), but the extent to which mitochondria contribute to the overall Ca(2+) signaling remains controversial. To examine the possible role of mitochondria in Ca(2+) signaling, we developed a low affinity mitochondrial Ca(2+) probe, mitycam-E31Q (300-500 MOI, 48-72h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca(2+) in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca(2+) while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca(2+)) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na(+) increased global cytosolic and mitochondrial Ca(2+) signals in one population of mitochondria, but unexpectedly decreased it (release of Ca(2+)) in another mitochondrial population. Such mitochondrial Ca(2+) release signals were seen not only during long lasting Na(+) withdrawal, but also when Ca(2+) loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca(2+) transients appear to activate with a delay following the cytosolic rise of Ca(2+) and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca(2+) signaling.