Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes

Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially-targeted FRET-based calcium indicator (4 mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca²⁺]_m) was measured at different stimulation frequencies (0.1-4 Hz) and external calcium concentrations (1.8-3.6 mM) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura-4AM. The increases in [Ca²⁺]_m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca²⁺]_m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca²⁺]_m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium-calcium exchanger (mNCE) resulted in a rise in [Ca²⁺]_m at baseline and -paradoxically- in an acceleration of Ca²⁺-release. In conclusion: rapid increases in [Ca²⁺]_m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat-to-beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.

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