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 (ΔΨm), 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), ΔΨm, and NADH (or GSH) simultaneously in isolated guinea pig cardiomyocytes. Resting Ca2+ spark frequency increased with each ΔΨm depolarization and decreased with ΔΨm 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 ΔΨm oscillations or prevent Ca2+ spark modulation by ΔΨm. 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.

Research highlights
► This study examines how mitochondrial energy state dynamically modulates Ca2+ sparks.
► Simultaneous measurements of ΔΨm, ROS, and Ca2+sparks.
► The Ca2+ spark rate is inversely correlated with the energy sate of mitochondria.
► The mechanism involves changes in the local oxidative environment induced by ROS.