ATP regulation in adult rat cardiomyocytes: time-resolved decoding of rapid mitochondrial calcium spiking imaged with targeted photoproteins.

The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca(2+) in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP](m)) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP](c)) were much smaller (<5%), indicating that [ATP](c) was effectively buffered in this compartment. Although mitochondrial [Ca(2+)] ([Ca(2+)](m)) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca(2+) transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP](m) occurred over the same time scale as average [Ca(2+)](m) was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca(2+)](m) were observed at 0.9 or 1.3 mum, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca(2+) transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand.