Abstract
Systemic hypoxia resulting from preterm birth, altered lung development, placental abnormalities, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, stressors that result from systemic hypoxia drive aberrant cardiomyocyte proliferation, which may be initially adaptive, but ultimately can program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the BCL-2/adenovirus E1B 19 kd-interacting protein 3 (Bnip3) resulting in the generation of an isoform lacking the third exon (Bnip3ΔExon3) or small Nip (sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary ventricular neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. At postnatal day (PND) 10, hypoxia-exposed rat pups demonstrated elevated heart weights, while histological analysis confirmed increased nuclei number and the absence of fibrosis (P<0.05), which was completely attenuated with the addition of 10 μg/kg/day misoprostol. Concurrently, molecular markers of proliferation, including Cyclin-D1 were significantly elevated in hypoxia-exposed myocytes, which was also prevented in the presence of misoprostol (P<0.05). We further describe a critical role for sNip in the regulation of cardiomyocyte proliferation at the transcriptional level, where this isoform reduced the expression of a proliferative MEF2C-myocardin-BMP10 pathway, while favoring expression of the cardiac maturation factors, such as BMP2 and MEF2A. These observations were further supported with knockdown studies in H9c2 cells, where we are able to restore hypoxia-induced cardiomyocyte proliferation in misoprostol-treated cells with the addition of an siRNA targeting sNip. Taken together this data demonstrates a mechanism for hypoxia-induced neonatal cardiomyocyte proliferation which can pharmacologically mitigated by misoprostol treatment.