The role of succinate and ROS in reperfusion injury - A critical appraisal

Abstract

We critically assess the proposal that succinate-fuelled reverse electron flow (REF) drives mitochondrial matrix superoxide production from Complex I early in reperfusion, thus acting as a key mediator of ischemia/reperfusion (IR) injury. Real-time surface fluorescence measurements of NAD(P)H and flavoprotein redox state suggest that conditions are unfavourable for REF during early reperfusion. Furthermore, rapid loss of succinate accumulated during ischemia can be explained by its efflux rather than oxidation. Moreover, succinate accumulation during ischemia is not attenuated by ischemic preconditioning (IP) despite powerful cardioprotection. In addition, measurement of intracellular reactive oxygen species (ROS) during reperfusion using surface fluorescence and mitochondrial aconitase activity detected major increases in ROS only after mitochondrial permeability transition pore (mPTP) opening was first detected. We conclude that mPTP opening is probably triggered initially by factors other than ROS, including increased mitochondrial [Ca²⁺]. However, IP only attenuates [Ca²⁺] increases later in reperfusion, again after initial mPTP opening, implying that IP regulates mPTP opening through additional mechanisms. One such is mitochondria-bound hexokinase 2 (HK2) which dissociates from mitochondria during ischemia in control hearts but not those subject to IP. Indeed, there is a strong correlation between the extent of HK2 loss from mitochondria during ischemia and infarct size on subsequent reperfusion. Mechanisms linking HK2 dissociation to mPTP sensitisation remain to be fully established but several related processes have been implicated including VDAC1 oligomerisation, the stability of contact sites between the inner and outer membranes, cristae morphology, Bcl-2 family members and mitochondrial fission proteins such as Drp1.

Keywords: Calcium; Heart; Hexokinase; Ischemia/reperfusion injury; Mitochondria; Permeability transition pore; Reactive oxygen species; Succinate.

Graphical abstract

Fig. 1

Time course of ROS production measured using surface fluorescence in the Langendorff-perfused heart subject to ischemia and reperfusion. Both control and IP hearts were pre-loaded with either 5-cH₂DCFDA as its di-(acetoxymethyl ester) or PO1 and then subject to 30 min index ischemia followed by reperfusion for the time shown. Data are expressed as means ± SEM (error bars) for 6 (5-cH₂DCFDA) or 9–11 (PO1) separate heart perfusions and are taken from where further details may be found. Significant differences between control and IP hearts, calculated by unpaired t-test, are shown by horizontal lines (*p < 0.05; **p < 0.01).

Fig. 2

Time course of changes in flavoprotein and NAD(P)H surface fluorescence in the Langendorff-perfused heart subject to ischemia and reperfusion. Surface fluorescence data (normalised as % of values at the end of ischemia) are only shown for the start of ischemia (− 30 min) and reperfusion (0 min). Data are taken from where further details may be found, and are presented as means ± SEM (error bars) of 7 control and IP hearts. Significant differences between control and IP hearts calculated by unpaired t-test are shown by horizontal lines (*p < 0.05; **p < 0.02, ***p < 0.01).

Fig. 3

IP does not attenuate succinate accumulation in ischemia while dimethyl malonate impairs the hemodynamic performance of the Langendorff-perfused heart. Panel A shows the succinate content (nmol per mg dry weight; mean ± SEM, n = 6) of freeze clamped control (CI) and IP (IPI) hearts following 30 min global ischemia; values in normoxic hearts were below our limit of detection (< 0.5 nmol per mg). Data are taken from . Panel B shows that the Rate Pressure Product (RPP) of normoxic Langendorff-perfused rat hearts was decreased by the presence of increasing concentrations of dimethyl malonate (DMM). Data are expressed as a percentage of values in the absence of DMM (29,900 ± 2800 mmHg*beat/min) and are given as means ± SEM (error bars) of 7 hearts. Statistical significance was determined by paired Student's t-test (*p < 0.01).

Fig. 4

Succinate is transported by MCT1 as the monocarboxylate anion. MCT1 was expressed in Xenopus laevis oocytes proton-linked monocarboxylate transport measured in real time using the pH-sensitive fluorescent dye BCECF as described previously . Panel A shows that 30 mmol/L l-lactate (L) is transported into and out of Xenopus laevis oocytes expressing MCT1 at both pH 7.4 and 6.0 but not control oocytes. By contrast, 30 mmol/L succinate (S) is only transported after the external pH has been decreased to 6.0 consistent with its transport as a monocarboxylate (i.e. in the monoprotonated form). Panel B shows that transport of both succinate and l-lactate at pH 6.0 is blocked by prior exposure of the oocyte to 1 μmol/L AR-C155858, a specific MCT1 inhibitor .

Fig. 5

The [Ca²⁺]-sensitivity of mPTP opening in isolated heart mitochondria is reduced by succinate despite greater ROS production both before and after pore opening. Panel A shows the effects of sequential additions of Ca²⁺ (100 μmol/L) on isolated rat heart mitochondria incubated with 5 mmol/L l-glutamate + 2 mmol/L l-malate (GM) or GM plus 5 mmol/L succinate (GMS) on mPTP opening and ROS production. Opening of the mPTP was detected as a loss of Δψ (Rhd-123 fluorescence increase) or accumulated Ca²⁺ (Fura-FF fluorescence increase) and a decrease in LS while ROS production was monitored using Amplex Red in a parallel experiment on the same mitochondria. Further details may be found elsewhere . Panel B presents mean rates of ROS production (± SEM of 6 different mitochondrial preparations) before Ca^2 + addition, after the penultimate Ca²⁺ addition and following pore opening.

Fig. 6

The extent of HK2 dissociation from mitochondria during ischemia is strongly correlated with infarct size following reperfusion. HK activity was measured in mitochondria isolated after 30 min ischemia and the infarct size after 2 h reperfusion. The changes in HK activity was confirmed to be due exclusively to HK2 dissociation by Western blotting. The pre-treatments used to modify HK2 binding at the end of ischemia were: Control (CP), ischemic preconditioned (IP) and perfusion with acetate (Ac), high [Ca²⁺] (CaC) or high glucose (HG) with or without IP. Data are taken from .

Fig. 7

Proposed sequence of events in ischemia and reperfusion that lead to mPTP opening and reperfusion injury and their attenuation by IP. This scheme is a slightly modified version of those presented previously , .