Dynamic modulation of Ca²⁺ sparks by mitochondrial oscillations in isolated guinea pig cardiomyocytes under oxidative stress

Local control of Ca²⁺-induced Ca²⁺ release (CICR) depends on the spatial organization of L-type Ca²⁺ channels and ryanodine receptors (RyR) in the dyad. Analogously, Ca²⁺ uptake by mitochondria is facilitated by their close proximity to the Ca²⁺ release sites, a process required for stimulating oxidative phosphorylation during changes in work. Mitochondrial feedback on CICR is less well understood. Since mitochondria are a primary source of reactive oxygen species (ROS), they could potentially influence the cytosolic redox state, in turn altering RyR open probability. We have shown that self-sustained oscillations in mitochondrial inner membrane potential (ΔΨ_m), NADH, ROS, and reduced glutathione (GSH) can be triggered by a laser flash in cardiomyocytes. Here, we employ this method to directly examine how acute changes in energy state dynamically influence resting Ca²⁺ spark occurrence and properties. Two-photon laser scanning microscopy was used to monitor cytosolic Ca²⁺ (or ROS), ΔΨ_m, and NADH (or GSH) simultaneously in isolated guinea pig cardiomyocytes. Resting Ca²⁺ spark frequency increased with each ΔΨ_m depolarization and decreased with ΔΨ_m repolarization without affecting Ca²⁺ spark amplitude or time-to-peak. Stabilization of mitochondrial energetics by pretreatment with the superoxide scavenger TMPyP, or by acute addition of 4'-chlorodiazepam, a mitochondrial benzodiazepine receptor antagonist that blocks the inner membrane anion channel, prevented or reversed, respectively, the increased spark frequency. Cyclosporine A did not block the ΔΨ_m oscillations or prevent Ca²⁺ spark modulation by ΔΨ_m. The results support the hypothesis that mitochondria exert an influential role on the redox environment of the Ca²⁺ handling subsystem, with mechanistic implications for the pathophysiology of cardiac disease.