PT - JOURNAL ARTICLE AU - Matthew D. Martens AU - Nivedita Seshadri AU - Lucas Nguyen AU - Donald Chapman AU - Elizabeth S. Henson AU - Bo Xiang AU - Arielys Mendoza AU - Sunil Rattan AU - Spencer B. Gibson AU - Ayesha Saleem AU - Grant M. Hatch AU - Christine A. Doucette AU - Jason M. Karch AU - Vernon W. Dolinsky AU - Ian M. Dixon AU - Adrian R. West AU - Christof Rampitsch AU - Joseph W. Gordon TI - Misoprostol Treatment Prevents Hypoxia-Induced Cardiac Dysfunction, Aberrant Cardiomyocyte Mitochondrial Dynamics and Permeability Transition Through Bnip3 Phosphorylation AID - 10.1101/2020.10.09.333666 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.10.09.333666 4099 - http://biorxiv.org/content/early/2020/10/21/2020.10.09.333666.short 4100 - http://biorxiv.org/content/early/2020/10/21/2020.10.09.333666.full AB - Systemic hypoxia, a major complication associated with reduced gestational time, affects more 60% of preterm infants and is a known driver of hypoxia-induced Bcl-2-like 19kDa-interacting protein 3 (Bnip3) expression in the neonatal heart. At the level of the cardiomyocyte, Bnip3 activity plays a prominent role in the evolution of necrotic cell death, disrupting subcellular calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests both a cardioprotective role for protein kinase A (PKA) through stimulatory prostaglandin (PG) E1 signalling during prolonged periods of hypoxia, and a cytoprotective role for Bnip3 phosphorylation, indicating that post-translational modifications of Bnip3 may be a point of convergence for these two protective pathways. Using a combination of in vivo and multiple cell models, including human iPSC-derived cardiomyocytes, we tested if the PGE1 analogue misoprostol is cardioprotective during neonatal hypoxic injury by altering the phosphorylation status of Bnip3. Here we report that hypoxia exposure significantly increases Bnip3 expression, mitochondrial-fragmentation, -ROS, -calcium accumulation and -permeability transition, while reducing mitochondrial membrane potential, all of which were restored to control levels with the addition of misoprostol, despite elevated Bnip3 protein expression. Through both gain- and loss-of-function genetic studies we further show that misoprostol-induced protection directly affects Bnip3, preventing mitochondrial perturbations. We demonstrate that this is a result of PG EP4 receptor signalling, PKA activation, and direct Bnip3 phosphorylation at threonine-181. Furthermore, when this PKA phosphorylation site within Bnip3 is neutralized, the protective misoprostol effect is lost. We also provide evidence that misoprostol traffics Bnip3 away from the ER through a physical interaction with 14-3-3β, thereby preventing aberrant ER calcium release and MPT. In vivo studies further demonstrate that misoprostol treatment increases Bnip3 phosphorylation at threonine-181 in the mouse heart, while both misoprostol treatment and genetic ablation of Bnip3 prevented hypoxia-induced reductions in contractile function. Taken together, our results demonstrate a foundational role for Bnip3 phosphorylation in the molecular regulation of cardiomyocyte contractile and metabolic dysfunction and identifies EP4 signaling as a potential pharmacological mechanism to prevent hypoxia-induced neonatal cardiac injury.Competing Interest StatementThe authors have declared no competing interest.