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

Abstract

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.