Characterisation of cold-induced mitochondrial fission in porcine aortic endothelial cells

BACKGROUND Previously, we observed that hypothermia, widely used for organ preservation, elicits mitochondrial fission in different cell types. However, temperature dependence, mechanisms and consequences of this cold-induced mitochondrial fission are unknown. Therefore, we here study cold-induced mitochondrial fission in endothelial cells, a cell type generally displaying a high sensitivity to cold-induced injury. METHODS Porcine aortic endothelial cells were incubated at 4-25 °C in modified Krebs-Henseleit buffer (plus glucose to provide substrate and deferoxamine to prevent iron-dependent hypothermic injury). RESULTS Cold-induced mitochondrial fission occurred as early as after 3 h at 4 °C and at temperatures below 21 °C, and was more marked after longer cold incubation periods. It was accompanied by the formation of unusual mitochondrial morphologies such as donuts, blobs, and lassos. Under all conditions, re-fusion was observed after rewarming. Cellular ATP content dropped to 33% after 48 h incubation at 4 °C, recovering after rewarming. Drp1 protein levels showed no significant change during cold incubation, but increased phosphorylation at both phosphorylation sites, activating S616 and inactivating S637. Drp1 receptor protein levels were unchanged. Instead of increased mitochondrial accumulation of Drp1 decreased mitochondrial localization was observed during hypothermia. Moreover, the well-known Drp1 inhibitor Mdivi-1 showed only partial protection against cold-induced mitochondrial fission. The inner membrane fusion-mediating protein Opa1 showed a late shift from the long to the fusion-incompetent short isoform during prolonged cold incubation. Oma1 cleavage was not observed. CONCLUSIONS Cold-induced mitochondrial fission appears to occur over almost the whole temperature range relevant for organ preservation. Unusual morphologies appear to be related to fission/auto-fusion. Fission appears to be associated with lower mitochondrial function/ATP decline, mechanistically unusual, and after cold incubation in physiological solutions reversible at 37 °C

Composition of ex vivo perfusion solutions and kinetics define differential cytokine/chemokine secretion in a porcine cardiac arrest model of lung preservation

BackgroundEx vivo lung perfusion (EVLP) uses continuous normothermic perfusion to reduce ischemic damage and to improve post-transplant outcomes, specifically for marginal donor lungs after the donation after circulatory death. Despite major efforts, the optimal perfusion protocol and the composition of the perfusate in clinical lung transplantation have not been identified. Our study aims to compare the concentration levels of cytokine/chemokine in different perfusion solutions during EVLP, after 1 and 9 h of cold static preservation (CSP) in a porcine cardiac arrest model, and to correlate inflammatory parameters to oxygenation capacities.MethodsFollowing cardiac arrest, the lungs were harvested and were categorized into two groups: immediate (I-EVLP) and delayed EVLP (D-EVLP), after 1 and 9 h of CSP, respectively. The D-EVLP lungs were perfused with either Steen or modified Custodiol-N solution containing only dextran (CD) or dextran and albumin (CDA). The cytokine/chemokine levels were analyzed at baseline (0 h) and after 1 and 4 h of EVLP using Luminex-based multiplex assays.ResultsWithin 4 h of EVLP, the concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-1α, and IL-1β increased significantly (P < 0.05) in all experimental groups. The CD solution contained lower concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-2, IL-12, IL-10, IL-4, IL-1RA, and IL-18 (P < 0.05) compared with those of the Steen solution. The concentration levels of all experimental groups have correlated negatively with the oxygenation capacity values (P < 0.05). Protein concentration levels did not reach statistical significance for I-EVLP vs. D-EVLP and CD vs. CDA solutions.ConclusionIn a porcine cardiac arrest model, a longer period of CSP prior to EVLP did not result in an enhanced protein secretion into perfusates. The CD solution reduced the cytokine/chemokine secretion most probably by iron chelators and/or by the protecting effects of dextran. Supplementing with albumin did not further reduce the cytokine/chemokine secretion into perfusates. These findings may help in optimizing the preservation procedure of the lungs, thereby increasing the donor pool of organs

Enhancement of iron toxicity in L929 cells by D-glucose: accelerated(re-)reduction.

It has recently been shown that an increase in the cellular chelatable iron pool is sufficient to cause cell damage. To further characterize this kind of injury, we artificially enhanced the chelatable iron pool in L929 mouse fibroblasts using the highly membrane-permeable complex Fe(III)/8-hydroxyquinoline. This iron complex induced a significant oxygen-dependent loss of viability during an incubation period of 5 h. Surprisingly, the addition of L-glucose strongly enhanced this toxicity whereas no such effect was exerted by L-glucose and 2-deoxyglucose. The assumption that this increase in toxicity might be due to an enhanced availability of reducing equivalents formed during the metabolism of L-glucose was supported by NAD(P)H measurements which showed a 1.5-2-fold increase in the cellular NAD(P)H content upon addition of L-glucose. To assess the influence of this enhanced cellular reducing capacity on iron valence we established a new method to measure the reduction rate of iron based on the fluorescent iron(II) indicator PhenGreen SK. We could show that the rate of intracellular iron reduction was more than doubled in the presence of L-glucose. A similar acceleration was achieved by adding the reducing agents ascorbate and glutathione (the latter as membrane-permeable ethyl ester). Glutathione ethyl ester, as well as the thiol reagent N -acetylcysteine, also caused a toxicity increase comparable with L-glucose. These results suggest an enhancement of iron toxicity by L-glucose via an accelerated (re-)reduction of iron with NAD(P)H serving as central electron provider and ascorbate, glutathione or possibly NAD(P)H itself as final reducing agent