Misoprostol Attenuates Hypoxia-Induced Cardiomyocyte Proliferation Through Bnip3 and Perinuclear Calcium Signaling
Friday, February 14, 2020
5:00 PM – 5:15 PM
Location: Max Bell - MB253
Introduction: Post-natal cardiac development depends on changes in gene expression, promoting maturation and differentiation, while repressing genes linked with immaturity and proliferation. Although the mechanisms remain unclear, neonatal hypoxia impedes these developmental pathways, driving aberrant cardiomyocyte proliferation, and early-life pathological remodeling. Previously we showed that Misoprostol, a prostaglandin E1 analogue, is cardioprotective by increasing the expression of the small Bnip3 isoform (sNip), which alters cardiac transcription factor localization and gene expression in the hypoxia-exposed neonatal heart. Building on this we hypothesized that Misoprostol may also prevent neonatal cardiomyocyte proliferation, and that this would be sNip-dependent.
Methods: Using rat primary ventricular neonatal cardiomyocytes and a cardiac myoblast cell line (H9c2), exposed to environmental hypoxia (10% oxygen) and Misoprostol drug treatments and/or lentiviral sNip transduction, we assessed myocyte proliferation and maturation by fluorescence microscopy, western blot analysis, and RT-PCR (n=3). In addition, we used a rodent model of neonatal hypoxia combining 10% oxygen, with and without Misoprostol from post-natal day 3-10 (n=6). In vivo cardiomyocyte proliferation was assessed by PHH3 immunofluorescence, RT-PCR for cell cycle genes, and gross histology.
Results: In cultured myocytes, molecular markers of proliferation were significantly elevated with hypoxia exposure and were attenuated with Misoprostol drug treatment (p<0.01). Furthermore, sNip promoted cardiomyocyte maturation driving nuclear localization of both calcium and NFATc3, reducing expression of the proliferative MEF2C-myocardin-BMP10 pathway, while favoring cardiac maturation factors, like BMP2 and MEF2A (p<0.01). These observations were supported by knockdown studies, where hypoxia-induced proliferation in Misoprostol-treated cells was restored with siRNA targeting sNip. Finally, hypoxia-exposed rat pups demonstrated significant increases in left ventricular nuclei number concurrent with elevated PHH3 fluorescence, which were both attenuated with the addition of Misoprostol (p<0.05).
Conclusion: Together this data demonstrates a novel mechanism by which Misoprostol drives Bnip3 alternative splicing and myocyte maturation, blocking hypoxia-induced neonatal cardiomyocyte proliferation and pathological remodeling in early life.