#40 Defending the Neonatal Heart: Misoprostol Prevents Bnip3-Induced Cardiometabolic Dysfunction during Hypoxia
Matthew D Martens, University of Manitoba; Jared T Field, University of Manitoba; Yan Hai, CHRIM; Wajihah Mughal, University of Manitoba; Simone da Silva Rosa, University of Manitoba; Tammy Ivanco, University of Manitoba; William Diehl-Jones, Athabasca University; Joseph Gordon, University of Manitoba
Systemic hypoxia affects more than 60% of preterm infants and is associated with both impaired cardiac metabolism and the development of persistent pulmonary hypertension. While the mechanism for injury remains unclear, it appears that the genetically conserved, pro-death Bnip3 pathway may play a central role. We hypothesize that misoprostol, an FDA-approved prostaglandin receptor agonist, inhibits Bnip3 PKA-induced phosphorylation, thereby protecting infants from hypoxia-induced cardiometabolic dysfunction.
Both environmental hypoxia (10% oxygen) and misoprostol were applied to primary neonatal cardiomyocytes to assess their effects on cardiometabolic dysfunction. Mitochondrial membrane potential and superoxide production were measured via fluorescence microscopy (n>80 cells). Concurrently, a metabolic flux analyzer (Seahorse XF-24) was used to assess mitochondrial respiration, which was compared to control treatments (normoxia and/or drug vehicle) (n=5). The secondary outcome of this study used a human cell line (HCT-116) (n>45 cells) to focus on the underlying mechanism, assessed through fluorescent imaging with a plasmid-based PKA biosensor, as well as by expression of wild-type and non-phosphorylatable constructs of Bnip3.
In primary neonatal cardiomyocytes, hypoxia exposure reduced mitochondrial membrane potential (p<0.01), and drove mitochondrial superoxide production (p<0.01). However, both measures were completely attenuated with the application of misoprostol (p<0.01). Under the same conditions, hypoxia exposure significantly reduced basal and maximal mitochondrial respiration, which were rescued with misoprostol (p<0.05). In parallel overexpression studies, wild-type Bnip3 depolarized mitochondria by more than 50%, which was blocked with the application of misoprostol (p<0.01). However, when Bnip3 phosphorylation was inhibited with a neutral alanine mutation, misoprostol-induced cardiometabolic protection was lost (p<0.01). Finally, using a PKA-biosensor we showed that misoprostol triggers a 3-fold increase (p<0.01) in intracellular PKA activation.
Taken together, our data demonstrates that misoprostol activates cAMP/PKA, causing the down-stream inhibition of Bnip3, which may serve to prevent hypoxia-induced cardiometabolic dysfunction in the neonatal heart.