Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.