Ischemic preconditioning decreases mitochondrial proton leak and reactive oxygen species production in the postischemic heart

Abstract

Background: Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production.

Materials and methods: Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red.

Results: IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates.

Conclusions: Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.

Figure 1

1A Representative left ventricular developed pressure (LVDP) tracings during Control-IR and IPC experiments. 1B) Recovery of left ventricular function at end-reperfusion expressed as percent of end-equilibration rate pressure product (RPP) in IPC and Control-IR. IPC hearts recovered 63±4% of their RPP vs. 21±2% in Control-IR (p<0.05, n=6/group).

Figure 1

1A Representative left ventricular developed pressure (LVDP) tracings during Control-IR and IPC experiments. 1B) Recovery of left ventricular function at end-reperfusion expressed as percent of end-equilibration rate pressure product (RPP) in IPC and Control-IR. IPC hearts recovered 63±4% of their RPP vs. 21±2% in Control-IR (p<0.05, n=6/group).

Figure 2. Mitochondrial H⁺ leak

2A: H⁺ leak curves in Control-IR and IPC at end-reperfusion and in Non-Ischemic Control mitochondria. H⁺ leak is defined as the respiratory rate required to maintain mΔΨ of -150mV. Respiration and mΔΨ was titrated with malonate (see methods section). Increasing malonate concentrations led to decreasing mΔΨ and state 2 respiration. N=6 for each data points; 6 separate experiments. 2B: H⁺ leak in Control-IR and IPC at end-perfusion and in Non-Ischemic Control extrapolated from 2A curves. IPC had a higher H⁺ leak compared to Non-Ischemic Control, while Control-IR had a higher H⁺ leak compared to both Non-Ischemic and IPC. *p< 0.05 vs. Non-Ischemic, § p<0.05 vs. IPC and Non-Ischemic. 2C: H⁺ leak in Control-IR and IPC at End EQ and in Non-Ischemic Control. There was an increase in H⁺ leak in both Control-IR and IPC mitochondria when compared to Non-Ischemic; however this difference was only marginally significant. H⁺ leak was 49±9 in Control-IR, 47±8 for IPC and 31±1 nanomoles O/mg protein/min for Non-Ischemic (p=0.05 for both IPC and Control-IR vs. Non-Ischemic).

Figure 2. Mitochondrial H⁺ leak

2A: H⁺ leak curves in Control-IR and IPC at end-reperfusion and in Non-Ischemic Control mitochondria. H⁺ leak is defined as the respiratory rate required to maintain mΔΨ of -150mV. Respiration and mΔΨ was titrated with malonate (see methods section). Increasing malonate concentrations led to decreasing mΔΨ and state 2 respiration. N=6 for each data points; 6 separate experiments. 2B: H⁺ leak in Control-IR and IPC at end-perfusion and in Non-Ischemic Control extrapolated from 2A curves. IPC had a higher H⁺ leak compared to Non-Ischemic Control, while Control-IR had a higher H⁺ leak compared to both Non-Ischemic and IPC. *p< 0.05 vs. Non-Ischemic, § p<0.05 vs. IPC and Non-Ischemic. 2C: H⁺ leak in Control-IR and IPC at End EQ and in Non-Ischemic Control. There was an increase in H⁺ leak in both Control-IR and IPC mitochondria when compared to Non-Ischemic; however this difference was only marginally significant. H⁺ leak was 49±9 in Control-IR, 47±8 for IPC and 31±1 nanomoles O/mg protein/min for Non-Ischemic (p=0.05 for both IPC and Control-IR vs. Non-Ischemic).

Figure 2. Mitochondrial H⁺ leak

2A: H⁺ leak curves in Control-IR and IPC at end-reperfusion and in Non-Ischemic Control mitochondria. H⁺ leak is defined as the respiratory rate required to maintain mΔΨ of -150mV. Respiration and mΔΨ was titrated with malonate (see methods section). Increasing malonate concentrations led to decreasing mΔΨ and state 2 respiration. N=6 for each data points; 6 separate experiments. 2B: H⁺ leak in Control-IR and IPC at end-perfusion and in Non-Ischemic Control extrapolated from 2A curves. IPC had a higher H⁺ leak compared to Non-Ischemic Control, while Control-IR had a higher H⁺ leak compared to both Non-Ischemic and IPC. *p< 0.05 vs. Non-Ischemic, § p<0.05 vs. IPC and Non-Ischemic. 2C: H⁺ leak in Control-IR and IPC at End EQ and in Non-Ischemic Control. There was an increase in H⁺ leak in both Control-IR and IPC mitochondria when compared to Non-Ischemic; however this difference was only marginally significant. H⁺ leak was 49±9 in Control-IR, 47±8 for IPC and 31±1 nanomoles O/mg protein/min for Non-Ischemic (p=0.05 for both IPC and Control-IR vs. Non-Ischemic).

Figure 3

Effect of increasing malonate concentrations on mitochondrial respiration in both IPC and Control-IR. Baseline respiration is approximately the same in both groups (n=6/group) and declines at an equal rate with increasing concentrations of malonate.

Figure 4

4A. Effect of increasing concentration of malonate on mitochondrial hydrogen peroxide production. At different respiratory rates, IPC mitochondria produced less H₂O₂. IPC had a lower basal rate of H₂O₂ production (omM malonate point). Inhibition of respiration decreased ROS production in both groups, with IPC producing less at all points. *p<0.05 vs. Control IR, n=6/group. 4B. H₂O₂ production as measured by fluorometry. In the presence of horseradish peroxidase (catalyst), Amplex-red reacts with H₂O₂ to produce the highly fluorescent compound resorufin. Catalase reduces H₂O₂ to H₂O and was used as a negative control. The addition of catalase to the reaction chamber abolishes any increase in fluorometric signal.

Figure 4

4A. Effect of increasing concentration of malonate on mitochondrial hydrogen peroxide production. At different respiratory rates, IPC mitochondria produced less H₂O₂. IPC had a lower basal rate of H₂O₂ production (omM malonate point). Inhibition of respiration decreased ROS production in both groups, with IPC producing less at all points. *p<0.05 vs. Control IR, n=6/group. 4B. H₂O₂ production as measured by fluorometry. In the presence of horseradish peroxidase (catalyst), Amplex-red reacts with H₂O₂ to produce the highly fluorescent compound resorufin. Catalase reduces H₂O₂ to H₂O and was used as a negative control. The addition of catalase to the reaction chamber abolishes any increase in fluorometric signal.

Figure 5

Physiology of the respiratory electron transport chain. Heavy curved arrows indicate the normal flow of electrons through the ETC. Substrates, such as NADH and succinate, normally enter at complex I and II where they are oxidized. The electrons are then transported to complex III by ubiquinol. Cytochrome c transfers electrons from complex III to complex IV which reduces O₂ to form H₂O. In the process, electrons are pumped from the matrix into the intermembrane space at complex I, III and IV generating an electrochemical gradient, the mΔΨ. This gradient is then used by complex V, (ATP synthase), to power the production of ATP from ADP. H⁺ leak is the reentry of protons into the mitochondrial matrix independent of complex V. Complex V is normally the point of reentry for H⁺ back into the matrix, with only minimal H⁺ leak. In the setting of ischemia-reperfusion injury, the respiratory complexes are damaged, and electrons leak from complex I and III (indicated by broken arrows). These electrons bind to O₂ to produce O₂·⁻. O₂·⁻ is dismutated to form H₂O₂ by the enzyme superoxide dismutase (SOD). H₂O₂ inside the matrix crosses the inner mitochondrial membrane to the intermembrane space. In the setting of ischemia-reperfusion injury H⁺ leak increases. Oligomycin-A is a complex V inhibitor; when complex V is inhibited, all reentry of H⁺ into matrix is due to H⁺ leak.