Confocal Imaging of Both Mitochondrial and Cytosolic Free Ca²⁺ in Cardiac Myocytes Co-Loaded with Rhod 2 and Fluo 3: Inhibition by Ruthenium Red of Mitochondrial but not Cytosolic Ca²⁺ Transients

Extract

Previously, we showed rapid mitochondrial Ca²⁺ transients in adult rabbit cardiac myocytes during the contractile cycle. Ruthenium red, an inhibitor of mitochondrial Ca²⁺ uptake by the Ca²⁺ uniporter, inhibits mitochondrial Ca²⁺ transients in adult rabbit cardiac myocytes during electrical stimulation. Here, we extend this finding to show that ruthenium red inhibition is specific for mitochondrial Ca2+ transients and not cytosolic Ca²⁺ transients.

Ca ²⁺-tolerant adult rabbit cardiac myocytes were isolated by collagenase and hyaluronidase digestion and loaded in the cold with 2.5 μM Rhod 2-AM for 30 minutes. Subsequently, the cells were incubated in nutrient medium at 37°C for 4-6 hours during which time cytosolic but not mitochondrial Rhod 2 was lost. Subsequently, the myocytes were loaded with 10 μM Fluo 3-AM for 15 minutes at 37°C, conditions that lead to predominantly cytosolic localization of Fluo 3. In sequential scans, the red fluorescence of Rhod 2 and the green fluorescence of Fluo 3 were imaged using a Zeiss LSM 410 laser scanning confocal microscope.