Succinate accumulation drives ischaemia-reperfusion injury during organ transplantation

Abstract

During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient^1,2. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human. We identify common metabolic alterations during WI and those affected by CI, thereby elucidating mechanisms underlying the benefits of CI, and how WI causes damage. Succinate accumulation is a major feature within ischaemic hearts across species, and CI slows succinate generation, thereby reducing tissue damage upon reperfusion caused by the production of mitochondrial reactive oxygen species (ROS)^3,4. Importantly, the inevitable periods of WI during organ procurement lead to the accumulation of damaging levels of succinate during transplantation, despite cooling organs as rapidly as possible. This damage is ameliorated by metabolic inhibitors that prevent succinate accumulation and oxidation. Our findings suggest how WI and CI contribute to transplant outcome and indicate new therapies for improving the quality of transplanted organs.

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Fig. 1. The cardiac ATP/ADP ratio during WI and CI

a, A schematic illustrating the experimental design mimicking ischaemia during transplantation. The whole mouse heart (~150 – 180 mg), or sections from the transected apex of the pig or human heart (~120 mg) were either immediately clamped frozen at liquid nitrogen temperature to generate a baseline sample under fully oxygenated “normoxic” conditions, or incubated to induce warm ischaemia (WI, 37°C) or cold ischaemia (CI) (~2°C) for the indicated times prior to freeze clamping. Tissues were then extracted and metabolites assayedATP and ADP were determined in heart tissue after various times of WI or CI. b-d, The ATP/ADP ratio for b mouse, c pig and d human. e-g, The sum of ATP and ADP (nmol/mg wet weight) for e mouse, f pig and g human. Data are means ± SEM, N = 5 (pig), 4 (human). For b and e data are means ± SEM, WI 6 and CI 240 min, N = 11; WI 0, 12, 30 and CI 0 min, N = 9; CI 6 min, N = 7; CI 12 min, N = 4; CI 30 min, N = 3.

Fig. 2. Volcano plots of metabolites that change significantly between 30 min WI and CI.

Volcano plots showing -log₁₀ of the adjusted p value plotted against the Log₂ of the fold-change in metabolite abundance between 30 min WI and 30 min CI for a mouse (N = 5), b pig (n = 5) and c human (N = 4). Fold-changes and p-values between time points were calculated by using linear modelling with an empirical Bayes approach as implemented in the 'limma' package of R, adjusting for multiple-testing by using the Benjamini-Hochberg procedure. Metabolites that changed in the same way in all three species are in red. Metabolites that changed in the same way in two of the species but were not detected, or did not change markedly, in the third are in green. Proprionylcarnitine, propcarn; acetyl carnitine, acetylcarn; succinyl adenosine, succaden; 3-hydroxybutyrate, 3HB; pyroglutamic acid, pyroglu; cysteine, cys.

Fig. 3. Succinate levels in the heart during WI and CI.

a-d, Fold change in succinate abundance under WI or CI, relative to normoxic heart tissue in a, d mouse (n = 6), b pig (n = 4) c human (n = 4). Data are means ± SEM. For a-c significant difference between groups were measured by two way ANOVA with multiple comparisons at individual points by Sidak test (* P < 0.05, ** P < 0.01 *** P < 0.001, **** P < 0.0001). e, Absolute succinate concentrations as described in a – d. Data are means ± SEM.Mouse (n = 6), pig (n = 4) human (n = 4). f-h, Relative change in succinate/fumarate ratio under WI or CI, compared to normoxic heart tissue. Data are means ± SEM.Mouse (n = 6), pig (n = 4) human (n = 4). These data are presented as relative changes in the ratio of the ion current for these metabolites, which is proportional to, but not the same as, the true ratio of the metabolite levels.

Fig. 4. Preventing pathological consequences of succinate metabolism during heart transplantation.

a, Surface and core temperature of pig heart during flush with cold UW solution. Following retrieval the heart was flushed with cold solution and immersed in slushed ice. The mean temperature (n = 4) every 10 s is shown ± SEM (shaded). The inset shows the temperature probes. b, Surface and core succinate of the pig heart treated as in a. Core and surface tissue biopsies were taken at 0, 6, 12 and 30 minutes, frozen and succinate concentration measured (n = 3, Data are means ± SEM). *P < 0.05, by a two-way ANOVA with Sidak's multiple comparison test. c, Succinate concentration in the mouse heart after standard retrieval and 30 min CIT (‘Isch.’), after standard retrieval and 30 min CIT and anastomosis (‘Isch. + Impl.’), and finally after 5 min reperfusion (‘Reper.’). Data mean ± SEM (Cont and Isch + Impl, n = 6; Isch, n = 4; Reper, n = 3). Compared to control (Cont.) by one-way ANOVA with Bonferroni multiple comparisons tests (***P < 0.001, ****P < 0.0001). d,Schematic of heterotopic heart transplantation in mice, with a further 12 min WI after retrieval, followed by 30 min cold storage. e-g, Hearts were infused with DMM (3.4 mg) or saline prior to cardioplegia and then exposed to 12 min WI, and then stored cold for 30 min, or infused with cardioplegia solution ± AMS (3.2 mg), followed by cold storage for 30 min. All hearts were then transplanted into recipients and 24 h later serum troponin e, mtDNA f, or mtDNA/nDNA ratio g, were measured. h, mtDNA damage in the donor heart was measured 24 h after transplantation by a PCR assay in which the greater the ratio of amplification the less damage to the mtDNA. e -g (-WI, n=20; +AMS, n=5; +WI, n= 24; +DMM, n= 4) h (Cont, n=8; -WI, n=4; +AMS, n=5; +WI, n= 7; +DMM, n= 8). Data are means ± SEM). * P < 0.05, **P < 0.01, ****P < 0.0001 by one-sided unpaired t-test with Welch’s correction.