Enhanced charge-independent mitochondrial free Ca2 + and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Abstract

Modulation of mitochondrial free Ca2+ ([Ca2+]m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca2+]m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2mM) on the magnitudes and time-courses of [Ca2+]m and mitochondrial redox state (NADH), membrane potential (ΔΨm), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10mM Na+- or K+-pyruvate/malate (NaPM or KPM) or Na+-succinate (NaSuc) followed by additions of isoflurane, 0.5mM CaCl2 (≈200nM free Ca2+ with 1mM EGTA buffer), and 250μM ADP. Isoflurane stepwise: (a) increased [Ca2+]m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨm and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨm, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨm, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca2+ uptake by inhibiting the Na+/Ca2+ exchanger (NCE), independent of changes in ΔΨm and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca2+]m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.