Abstract 16644: An Obligatory Role for Dynamin-Related Protein 1-Mediated Mitochondrial Fission in Oxygen-Sensing and Anatomical Closure of the Human Ductus Arteriosus

Introduction: Closure of the ductus arteriosus (DA) is essential for the transition from fetal to neonatal circulation. Oxygen initiates DA constriction by increasing mitochondrial-derived H 2 O 2 in ductal smooth muscle cells (DASMC). H 2 O 2 acts as a redox second messenger which increases intracellular calcium triggering vasoconstriction. The mechanism by which oxygen alters mitochondrial redox signaling is unknown, however we observed that oxygen rapidly fragments the mitochondrial network. We hypothesized that mitochondrial fission, mediated by dynamin-related protein 1 (Drp1), is required for O 2 -induced vasoconstriction and DA closure. Methods: We assessed the effects of inhibiting Drp1 on DA constriction and closure using DA rings from term rabbits (n=48) and human infants (n=30). Drp1 was inhibited by using Mdivi-1, a specific, inhibitor of Drp1 assembly and Drp1 siRNA. DASMC metabolism was measured using a Seahorse analyzer. Mitochondrial fission was quantified using 2-photon confocal microscopy in cells transfected with mitochondrial-targeted photoactivated GFP. Results: Increasing PO 2 from 40 to 140 mmHg fragments the DASMC mitochondrial network within 10-15 minutes by activating Drp1. Mdivi-1 selectively eliminates O 2 -constriction in rabbit DA ([[Unable to Display Character: △]]Tension O 2 : 1548 ± 100 to 275 ± 63 mg, n=8) and human DA (700 ± 102 to 337 ± 92 mg, N=6). Fission increases H 2 O 2 production by activating pyruvate dehydrogenase, and increasing oxidative metabolism. Both are inhibitable by Mdivi-1. Inhibition of Drp1 (or elimination of mitochondrial H 2 O 2 , by mitochondrial-targeted catalase) selectively eliminates O 2 -induced increases in cytosolic calcium. O 2 activates Drp1 by a cyclin-dependent kinase (CDK)-mediated phosphorylation of Drp1 at serine 616. Inhibition of Drp1 decreases O 2 -induced proliferation of DASMC. In a tissue culture model, O 2 -induced DA closure occurs after 3 days in rabbits and 6-14 days in humans. Mdivi-1 preserves the DA lumen versus control (luminal area 4.10 ± 1.29 mm 2 vs 0.04 ± 0.03 mm 2 , human) and prevents anatomical closure. Conclusion: Mitochondrial fission is an obligatory, early step in mammalian O 2 -sensing that is required for DA constriction and closure. Drp1 is a promising target for modulating DA patency.