Misoprostol attenuates hypoxia‐induced neonatal cardiomyocyte proliferation through Bnip3 and perinuclear calcium signaling

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. Although the molecular mechanisms are still unknown, metabolic stressors resulting from neonatal hypoxia drive aberrant glycolysis and cardiomyocyte proliferation, which may be initially adaptive, but ultimately can program the heart for early life pathological remodeling. Recent evidence suggests the prostaglandin E1 analogue misoprostol is cytoprotective in the hypoxia‐exposed neonatal heart by promoting the alternative splicing of Bnip3, resulting in the generation of an isoform lacking the third exon (Bnip3ΔExon3) known as small Nip (sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary ventricular neonatal cardiomyocytes and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In cultured myocytes, molecular markers and fluorescent indicators of proliferation, such as cylcin‐D1 and PHH3, were significantly elevated with hypoxia exposure and were attenuated with misoprostol treatment (p<0.01). Moreover, hypoxia reduced mitochondrial respiration and increased cytosolic production of NADH, which were restored by misoprostol treatment. In addition, both sNip and misoprostol promoted cardiomyocyte maturation by increasing nuclear calcium accumulation, and drove the nuclear localization of NFATc3 (p<0.01). Gene expression studies identified that misoprostol and sNip reduced the expression of the proliferative MEF2C‐myocardin‐BMP10 pathway, while favoring expression of the cardiac maturation factors BMP2 and MEF2A (p<0.01). Furthermore, an siRNA targeting sNip restored hypoxia‐induced proliferation in misoprostol‐treated cells. Finally, we observed a significant increase in left ventricular nuclei number and PHH3 staining in hypoxia‐exposed rat pups, which were also attenuated with misoprostol treatment (p<0.05). These findings suggest a novel mechanism by which misoprostol promotes Bnip3 alternative splicing and myocyte maturation, thereby blocking hypoxia‐induced neonatal cardiomyocyte proliferation. Support or Funding Information This work is supported by the Natural Science and Engineering Research Council of Canada, the Heart and Stroke Foundation of Canada, and the Children’s Hospital Research Institute of Manitoba.