Abstract 13470: Mitochondrial Translocation of Dynamin-Reated Protein 1 Promotes Lethal Ischemia-Reperfusion Injury
Background: Mitochondrial fission is the dynamic process by which mitochondria are cleaved and fragmented. Recent reports suggest that pharmacologic inhibition of fission attenuates lethal ischemia-reperfusion (I-R) injury, thereby implying that fission contributes to cell death. However, the molecular mechanisms by which fission regulates cell fate are unresolved.
Hypothesis: We propose that: (i) I-R elicits the translocation of DRP1 (dynamin-related protein 1: a critical mediator of fission) from the cytosol to the mitochondria. Once in position, DRP1 will constrict the mitochondria, thereby (ii) facilitating release of Cytochrome C (Cyto C) into the cytosol and (iii) triggering apoptosis. If so, (iv) inhibition of DRP1 translocation will attenuate Cyto C release and offer cytoprotection.
Methods: In Protocol 1, cultured HL-1 cells underwent 2 h simulated I + 5, 30 or 120 min reoxygenation or a matched normoxic period. For each group, subcellular localization of DRP1 and Cyto C was resolved by immunoblotting, and cleaved Caspase 3 (harbinger of apoptotic death) was quantified in the cytosol. Protocol 2 was identical, except cells were pretreated with mdivi-1 (specific DRP1 inhibitor; 50 µM) or vehicle. In Protocol 3, cells were pretreated with mdivi-1 or vehicle and viability quantified by Trypan blue staining at 20 h post-R.
Results: Our results revealed a rapid and significant redistribution of DRP1 from the cytosol to the mitochondria post-R, a concomitant release of Cyto C into cytosol, and cleavage of Caspase 3 (Figure). Treatment with mdivi-1 impaired translocation of DRP1 - and, most notably, attenuated Cyto C release, blunted Caspase 3 cleavage (P<0.05 vs vehicle for all variables), and enhanced cell viability (71±2% vs 55+3% in vehicle controls; P < 0.05).
Conclusion: DRP1 translocation (i.e., mitochondrial fission) promotes lethal ischemia-reperfusion injury by facilitating release of Cytochrome C into the cytosol and triggering apoptosis.
- © 2012 by American Heart Association, Inc.