Misoprostol Treatment Prevents Hypoxia-Induced Cardiac Dysfunction, Aberrant Cardiomyocyte Mitochondrial Dynamics and Permeability Transition Through Bnip3 Phosphorylation

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 14-3-3{beta}, 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, preventing hypoxia-induced reductions in cardiac ejection fraction and fractional shortening. 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/333666v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@388557org.highwire.dtl.DTLVardef@ba5171org.highwire.dtl.DTLVardef@38dc58org.highwire.dtl.DTLVardef@10a409a_HPS_FORMAT_FIGEXP M_FIG C_FIG