Pre-ischaemic mitochondrial substrate constraint by inhibition of malate-aspartate shuttle preserves mitochondrial function after ischaemia-reperfusion

Abstract

Key points: Pre-ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against ischaemia-reperfusion injury. The underlying mechanism remains unknown. We examined whether transient inhibition of the MAS during ischaemia and early reperfusion by AOA treatment could prevent mitochondrial damage at later reperfusion. The AOA treatment preserved mitochondrial respiratory capacity with reduced mitochondrial oxidative stress during late reperfusion to the same extent as ischaemic preconditioning (IPC). However, AOA treatment, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion. The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation of mitochondrial function during late reperfusion in an IR-injured heart.

Abstract: Mitochondrial dysfunction plays a central role in ischaemia-reperfusion (IR) injury. Pre-ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against IR injury, although the underlying mechanism remains unknown. We hypothesized that a transient inhibition of the MAS during ischaemia and early reperfusion could preserve mitochondrial function at later phase of reperfusion in the IR-injured heart to the same extent as ischaemic preconditioning (IPC), which is a well-validated cardioprotective strategy against IR injury. In the present study, we show that pre-ischaemic administration of AOA preserved mitochondrial complex I-linked state 3 respiration and fatty acid oxidation during late reperfusion in IR-injured isolated rat hearts. AOA treatment also attenuated the excessive emission of mitochondrial reactive oxygen species during state 3 with complex I-linked substrates during late reperfusion, which was consistent with reduced oxidative damage in the IR-injured heart. As a result, AOA treatment reduced infarct size after reperfusion. These protective effects of MAS inhibition on the mitochondria were similar to those of IPC. Intriguingly, the protection of mitochondrial function by AOA treatment appears to be different from that of IPC because AOA treatment, but not IPC, downregulated myocardial tricarboxilic acid (TCA)-cycle intermediates at the onset of reperfusion. MAS inhibition thus preserved mitochondrial respiratory capacity and decreased mitochondrial oxidative stress during late reperfusion in the IR-injured heart, at least in part, via metabolic regulation of TCA cycle intermediates in the mitochondria at the onset of reperfusion.

Keywords: heart; ischemia-reperfusion; malate-aspartate shuttle; mitochondria; oxidative stress.

© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Figures

Figure 1. A schematic of the MAS

Various substrates are involved in the MAS, although the main role of this shuttle is the transfer of cytosolic NADH to the mitochondrial matrix through the mitochondrial inner membrane, which is impermeable to NADH. Inhibition of the MAS results in a reduced concentration of NADH in the mitochondrial matrix. AST, aspartate amino transferase; MDH, malate dehydrogenase.

Figure 2. Study design

Two different perfusion solutions containing glucose or glucose + FFA as substrates for metabolism during the isolation procedures of rat hearts were used. Isolated perfused rat hearts were divided into seven groups (n = 7–8 in each group): Sham‐operated hearts (Sham group), IR‐injured hearts by 30 min of global no‐flow ischaemia and reperfusion (IR group), IR‐injured hearts with IPC induced by 2 × 5min cycles of IR prior to global no‐flow ischaemia (IPC group), IR‐injured hearts with a pre‐ischaemic administration of AOA to inhibit the MAS during ischaemia and early reperfusion (Pre‐AOA group), IR‐injured hearts co‐perfused with 0.2 mmol L−1 AOA and 5 mmol L−1 dimethyl succinate for 5 min prior to global no‐flow ischaemia (AOA + DiSuc group), IR‐injured hearts co‐perfused with 0.2 mmol L−1 AOA for 5 min during global no‐flow ischaemia (per‐AOA group) and IR‐injured hearts treated with pre‐ischaemic AOA and IPC (AOA + IPC group). Three experimental series were conducted: Experimental series I was conducted using both glucose and glucose + FFA, and experimental series II and III were conducted using only glucose as the substrate for perfusion solutions.

Figure 3. The mitochondrial enzymatic activities and the protein content of mitochondrial ETC complexes in the cardiac muscle during late reperfusion

A, enzymatic activities of CS and HAD. B, representative blots of ETC complexes I–V. C, summarized data of protein content of mitochondrial ETC complexes I–V (relative to the Sham). Data are the mean ± SEM.

Figure 4. Mitochondrial respiratory capacity in the cardiac muscle during late reperfusion

A, representative graph of mitochondrial respiratory capacity in permeabilized muscle fibres from a sham‐operated heart. B, summarized data of mitochondrial respiratory capacity in each respiratory state. C, RCR with complex I‐linked substrates. D, RCR with complex I+II‐linked substrates. GM, state 2 respiration with glutamate + malate; GM3, state 3 respiration with glutamate and malate; GMS3, state 3 respiration with glutamate, malate and succinate; 4o, state 4 respiration with oligomycin; ROX, residual oxygen consumption evaluated after adding rotenone and antimycin A. Data are the mean ± SEM. *P < 0.05 vs. Sham. †P < 0.05 vs. IR.

Figure 5. The capacity of mitochondrial fatty acid oxidation in the cardiac muscle during late reperfusion

A, mitochondrial state 3 respiration with fatty acids. B, RCR with fatty acids. MOc, state 2 respiration with malate and octanoyl‐l‐carnitine; MOc3, state 3 respiration with malate and octanoyl‐l‐carnitine. Data are the mean ± SEM. *P < 0.05 vs. Sham. †P < 0.05 vs. IR.

Figure 6. The mitochondrial ROS emission and oxidative damage in the cardiac muscle during late reperfusion

A, mitochondrial H2O2 emission during state 3 with complex I‐linked substrates. B, mitochondrial H2O2 emission per O2 consumption during state 3 with complex I‐linked substrates. C, oxidative damage. Data are the mean ± SEM. *P < 0.05 vs. Sham. †P < 0.05 vs. IR.

Figure 7. Myocardial interstitial concentrations of metabolites

The myocardial interstitial concentrations of lactate, TCA intermediates (citrate, glutamate, succinate, fumarate and malate) and purine metabolites (adenosine, hypoxanthine, inosine and xanthine) during stabilization, global no‐flow ischaemia and reperfusion. Data are the mean ± SEM. The area under the curve has been calculated for each period and compared using one‐way ANOVA. †P < 0.05 IPC or AOA vs. IR. ‡P < 0.05 IPC vs. AOA.

Figure 8. Infarct size after reperfusion

AAR, area‐at‐risk; DiSuc, dimethyl succinate; IS, infarct size. Data are the mean ± SEM. †P < 0.05 vs. IR.

Figure 9. A possible mechanism of preservation of the mitochondria during late reperfusion in the IR‐injured heart by inhibition of the MAS

Transient inhibition of the MAS aiming at ischaemia and early reperfusion works like a break against the acceleration of mitochondrial activity at the onset of reperfusion via a limited entry of NADH and respiratory substrates into the mitochondria. The gradual wake‐up of the mitochondria induced by the inhibition of the MAS during early reperfusion may reduce the excess emission of mitochondrial ROS, which results in the preservation of the mitochondria during late reperfusion.