Recovery from cold-induced mitochondrial fission in endothelial cells requires reconditioning temperatures of ≥ 25C

Mitochondrial integrity and function constitute a prerequisite for cellular function and repair processes. We have previously shown that mitochondria of different cell types exhibit pronounced fragmentation under hypothermic conditions. This fission, accompanied by a decline of cellular ATP content, showed reversibility at 37C. However, it is unclear whether other temperatures as currently discussed for reconditioning of organs allow this reconstitution of mitochondria. Therefore, we here study in a model of cultured porcine aortic endothelial cells how different rewarming temperatures affect mitochondrial re-fusion and function. After 48 h cold incubation of endothelial cells in Krebs-Henseleit buffer with glucose (5 mM) and deferoxamine (1 mM) at 4C pronounced mitochondrial fission was observed. Following 2 h rewarming in cell culture medium, marked fission was still present after rewarming at 10 or 15C. At 21C some re-fusion was visible, which became more marked at 25C. Networks of tubular mitochondria similar to control cells only re-appeared at 37C. ATP content decreased at 4C from 3.6 ± 0.4 to 1.6 ± 0.4 nmol/106 cells and decreased even further when rewarming cells to 10and 15C. Values after rewarming at 21C were similar to the values before rewarming while ATP gradually increased at higher rewarming temperatures. Metabolic activity dropped to 5 ± 11% of control values during 4C incubation and recovered with increasing temperatures to 36 ± 10% at 25C and 78 ± 17% at 37C. Integrity of monolayers, largely disturbed at 4C (large gaps between endothelial cells; cell injury ≤ 1%), showed partial recovery from 15C upwards, complete recovery at 37C. Endothelial repair processes (scratch assay) at 25C were clearly inferior to those at 37C. These data suggest that reconditioning temperatures below 21C are not optimal with regard to reconstitution of mitochondrial integrity and function. For this goal, temperatures of at least 25C appear required, with 30C being superior and 37C yielding the best results.


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