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

Leonard Quiring,Ursula Rauen,Niklas Lohaus,Dhanusha Schwan,Anja Rech,Andrea Dlugos,Björn Walter

doi:10.1186/s10020-021-00430-z

Abstract

BackgroundPreviously, 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.MethodsPorcine 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).ResultsCold-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.ConclusionsCold-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.

Highlights

Organs are stored under hypothermic conditions to preserve integrity and function for transplantation
The cold incubation solution was added at room temperature, and cells were kept under a 5% C O2, 21% O2 and 74% N2 atmosphere at 48 h of cold incubation (4 °C) or at other temperatures specified in the results section (Rauen et al 1999)
Cold‐induced mitochondrial fission Under normothermic control conditions, porcine aortic endothelial cells stained with MitoTracker Red showed a network of long filamentous mitochondria (Fig. 1A)

Summary

Introduction

Organs are stored under hypothermic conditions (in special preservation solutions) to preserve integrity and function for transplantation. As an additional change occurring in cells incubated under hypothermic conditions, we observed a pronounced mitochondrial fission after cold incubation of liver endothelial cells for 18 h at 4 °C in University of Wisconsin solution (Kerkweg et al 2003) This fission occurred both in the absence and in the presence of the iron chelator deferoxamine. Marked fission was observed under hypothermic conditions in many cell types relevant in transplantation medicine, such as hepatocytes (Pless et al 2012; Rauen et al 2003), corneal endothelial cells (Rauen et al 2006), and kidney epithelial cells (Hendriks et al 2017; Zhang et al 2010)/renal tubules (Bienholz et al 2017) In these studies, mitochondrial fission was observed after different periods of cold incubation (18 h–5 days) at 4 °C or 10 °C and in many different storage solutions. We here study cold-induced mitochondrial fission in endothelial cells, a cell type generally displaying a high sensitivity to cold-induced injury

Methods

Results

Discussion

Conclusion