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.

Impact of Musashi-1 and Musashi-2 Double Knockdown on Notch Signaling and the Pathogenesis of Endometriosis.

The stem cell marker and RNA-binding protein Musashi-1 is overexpressed in endometriosis. Musashi-1-siRNA knockdown in Ishikawa cells altered the expression of stem cell related genes, such as OCT-4. To investigate the role of both human Musashi homologues (MSI-1 and MSI-2) in the pathogenesis of endometriosis, immortalized endometriotic 12-Z cells and primary endometriotic stroma cells were treated with Musashi-1- and Musashi-2-siRNA. Subsequently, the impact on cell proliferation, cell apoptosis, cell necrosis, spheroid formation, stem cell phenotype and the Notch signaling pathway was studied in vitro. Using the ENDOMET Turku Endometriosis database, the gene expression of stem cell markers and Notch signaling pathway constituents were analyzed according to localization of the endometriosis lesions. The database analysis demonstrated that expression of Musashi and Notch pathway-related genes are dysregulated in patients with endometriosis. Musashi-1/2-double-knockdown increased apoptosis and necrosis and reduced stem cell gene expression, cell proliferation, and the formation of spheroids. Musashi silencing increased the expression of the anti-proliferation mediator p21. Our findings suggest the therapeutic potential of targeting the Musashi-Notch axis. We conclude that the Musashi genes have an impact on Notch signaling and the pathogenesis of endometriosis through the downregulation of proliferation, stemness characteristics and the upregulation of apoptosis, necrosis and of the cell cycle regulator p21.