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Mitochondrial fusion by genetic or pharmacological manipulation protects against ischaemia–reperfusion injury
  1. SB Ong,
  2. S Davidson,
  3. DM Yellon,
  4. DJ Hausenloy
  1. Hatter Cardiovascular Institute, University College London, London, UK

Abstract

Objective Mitochondria are able to change their morphology by undergoing either “fusion” to form elongated interconnected networks or “fission” to generate fragmented disconnected mitochondria. Mitochondrial fission, which can be triggered by apoptosis, oxidative stress and simulated ischaemia, predisposes to the opening of the mitochondrial permeability transition pore (mPTP), a major determinant of cell death. We hypothesised that mitochondrial fusion protects against simulated ischaemia–reperfusion injury by inhibiting mPTP opening.

Methods Mitochondrial fusion was induced in HL-1 cells (a murine cardiac cell-line) using two methods: (1) plasmid transfection with the mitochondrial fusion proteins, Mfn1 and Mfn2, and the dominant negative fission protein Drp1K38A and (2) pharmacological treatment for 40 minutes using mdivi-1 (a small molecule inhibitor of Drp1) or erythropoietin (a cardioprotective growth factor). The cells were then subjected to three experimental protocols: (1) cells, co-transfected with mitochondrial red fluorescent protein, were assigned to two groups using confocal microscopy depending on whether they displayed predominantly (>50%) elongated or fragmented mitochondria; (2) cells, co-transfected with green fluorescent protein, were subjected to 12 h simulated ischaemia and 1 h reperfusion and the percentage cell death was determined by propidium iodide staining; and (3) cells, co-transfected with green fluorescent protein, were loaded with TMRM and subjected to confocal laser-induced oxidative stress to induce mPTP opening.

Results Overexpression of Mfn1, Mfn2 or Drp1K38A resulted in: (1) an increased proportion of cells displaying elongated mitochondria (45.7 ± 5.6% with control vs 65.3 ± 4.1%, 69.1 ± 4.9% and 62.8 ± 6.3%, respectively: N  =  4/group; p<0.05); (2) reduced cell death (41.8 ± 4.1% with control vs 11.6 ± 3.9%, 16.2 ± 3.9% and 12.1 ± 2.9%, respectively; N  =  4/group; p<0.05); and (3) delayed the time taken to induce mPTP opening compared with control by 2–3-fold, respectively (N  =  5/group; p<0.05). Furthermore, treatment with mdivi-1 or erythropoitin (EPO) resulted in: (1) an increased proportion of cells displaying elongated mitochondria (29.0 ± 8.1% with dimethyl sulphoxide (DMSO) control vs 67.3 ± 3.8% for mdivi-1) and (32.3 ± 5.8% with control vs 75.8 ± 3.7% for EPO; N  =  5/treatment; p<0.05); (2) reduced cell death (36.8 ± 5.8% with DMSO control vs 20.5 ± 3.4% for mdivi-1) and (43.1 ± 2.7% with control vs 17.0 ± 2.7% for EPO; N  =  5/treatment; p<0.05).

Conclusions Mitochondrial fusion by genetic or pharmacological manipulation protects cells against simulated ischaemia–reperfusion injury by inhibiting mPTP opening.

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