Abstract P232: Cardiac Excitation Contraction Coupling Stimulates Mitochondrial Respiration and Superoxide Flash Activity

Abstract

Normal cardiac excitation-contraction (EC) coupling relies on highly coordinated intracellular calcium (Ca2+) handling and adequate mitochondrial energy metabolism. Whether mitochondria can sense the oscillating Ca2+ signals during EC coupling to adjust its respiration status and other functions is largely unknown. Recently, we discovered stochastic and transient mitochondrial superoxide production events, named superoxide flashes, in resting cardiac myocytes. Superoxide flashes are coupled with mitochondrial respiration and triggered by physiological relevant transient permeability transition pore (mPTP) openings. Using superoxide flash as the optical determination of mitochondrial respiration status in living myocytes, we found that EC coupling promotes mitochondrial respiration and reactive oxygen species (ROS) production as evidenced by the significantly increased superoxide flash activity by electrical stimulation (pacing frequency 0.5 - 2 Hz). Pre-incubation with Ru360 (50-100 uM), a mitochondrial Ca2+ uniporter inhibitor, or cyclosporine A (1 uM), an mPTP blocker, prevented this increase. Simultaneous monitoring of mitochondrial Ca2+ (by rhod-2) and superoxide (by mt-cpYFP, a mitochondrial targeted superoxide indicator) revealed step-wise accumulation of mitochondrial Ca2+ during cytosolic Ca2+ transients in individual mitochondria, which is followed by superoxide flash production and matrix Ca2+ release. Importantly, mitochondrial respiration only responds to transient Ca2+ signals (acute addition of ionophore or pacing) but not chronic manipulations of steady state Ca2+ (depleting or elevating resting cytosolic Ca2+). Through spatiotemporal mapping of superoxide flash and local sarcoplasmic reticulum (SR) Ca2+ releasing events, Ca2+ sparks, we found that significant amount of Ca2+ sparks clustered near the flashing mitochondria. Antioxidant pretreatment attenuated the superoxide flash associated Ca2+ sparks. In summary, mitochondrial Ca2+ uptake during cardiac EC coupling leads to stochastic acceleration of mitochondrial respiration, mPTP activity and free radical production, which in turn modulate local Ca2+ signaling through promoting SR Ca2+ release.