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, 2 mM) 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 10 mM Na+- or K+-pyruvate/malate (NaPM or KPM) or Na+-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl2 (≈200 nM free Ca2+ with 1 mM EGTA buffer), and 250 mM 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.

Isoflurane modulates cardiac mitochondrial bioenergetics by selectively attenuating respiratory complexes

“…Complex I was first suggested as a site for VAs effects [21, 22]; this was supported by later findings [23, 24]. Our initial observed effects of ISO [8] to increase the duration of state 3 NADH oxidation and ΔΨ m depolarization also supported complex I as a site of action, but this could alternatively have been due to inhibition of other ETC complexes downstream of complex I, e.g., complexes III (ubiquinol cytochrome c oxidoreductase), IV (cytochrome c oxidase) and V (F 1 F 0 ATP synthase), or complex II (sucinate dehydrogenase) and transport proteins (e.g. adenine nucleotide transporter, ANT); alternatively, as has been reported, ISO might act as an uncoupling agent by promoting a proton leak [25] that could lead to mild ΔΨ m depolarization.…”

“…In our recent study in isolated mitochondria [8] in which we demonstrated that ISO attenuates Ca 2+ extrusion by the mitochondrial Na + /Ca 2+ exchanger, we also found that ISO caused a concentration-dependent decrease in the rate of state 3 NADH oxidation and ADP phosphorylation with the complex I substrate pyruvate/malate (PM). Complex I was first suggested as a site for VAs effects [21, 22]; this was supported by later findings [23, 24].…”

“…Mitochondria were freshly isolated from hearts of Wistar rats (300–350 g) using protocols approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC) as described previously [8]. Briefly, rats were anesthetized with an intraperitoneal injection of inactin (150 mg/kg), and the ventricles were excised and placed in an ice-cold isolation buffer that contained (in mM): 200 manitol, 50 sucrose, 5 KH 2 PO 4 , 5 MOPS, 1 EGTA and 0.1% BSA, with pH adjusted to 7.15 with KOH.…”

“…NADH autofluorescence was measured in the mitochondrial suspension during states 2, 3 and 4 respiration using fluorescence spectrophotometry at excitation wavelength 350 nm and emission wavelengths 395 nm and 456 nm (Photon Technology International, Birmingham, NJ), as we have described before [8, 27–30]. At the end of each experiment (390 s), maximal (fully reduced) NADH (high NADH/NAD + ) was determined with 1 µM ROT, which completely blocks electron transport via FeS centers in complex I at the ubiquinone (Q) binding site so that proton pumping from NADH cannot occur; minimal (fully oxidized) NADH (low NADH/NAD + ) was determined with 20 µM trifluorocarbonylcyanidephenylhydrazone (FCCP), which induce maximal proton leak, and causes an increase in proton pumping and electron transfer between ETC complexes.…”

Molecular pathways of mitochondrial dysfunctions: Possible cause of cell death in anesthesia-induced developmental neurotoxicity

“…For instance, one study reported that sevoflurane preconditioning protects the mitochondria from cerebral ischemia/reperfusion injury by inhibiting Ca 2+ induced mitochondrial permeability transition pore opening (mPTP); thus, long-term neurological deficits are ameliorated (Ye et al, 2012). Subsequent experiments on myocardial protection obtained similar findings: the effects of isoflurane are partly mediated at the mitochondrial level to enhance the net rate of state 2 Ca 2+ uptake by inhibiting the Na + /Ca 2+ exchanger, which is independent of the changes in « m (Agarwal et al, 2012). Similarly, another study confirmed that desflurane-induced post-conditioning against myocardial infarction is mediated by calcium-activated potassium and mPTP in mice in vivo (Stumpner et al, 2012).…”