MISOPROSTOL ATTENUATES CARDIOMYOCYTE PROLIFERATION IN THE NEONATAL HEART THROUGH A BNIP3-DELTA-EXON3/MEF2-DEPENDENT SIGNALLING PATHWAY

Abstract

Systemic hypoxia resulting from preterm birth, 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 has suggested that the prostaglandin E1 analogue, Misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart through promoting the expression of the BCL-2/adenovirus E1B 19 kd-interacting protein 3 (Bnip3) isoform lacking the third exon (Bnip3-Delta-Exon3). 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 players may be in this pathway. At post-natal day (PND) 5, hypoxia-exposed rat pups demonstrated elevated heart weights, while histological analysis confirmed, significant increases in left-ventricular wall thickness in the absence of fibrosis (P < 0.05) and increased nuclei number (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 hearts and cells, which was also prevented in the presence of Misoprostol (P < 0.05). We further describe a critical role for Bnip3-Delta-Exon3 in the regulation of cardiomyocyte proliferation at the transcriptional level, where this isoform reduced the expression of myocardin, while favoring expression of the cardiac maturation factors, BMP-2 and MEF2A over their proliferative counterparts, BMP-10 and MEF2C. 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 Bnip3-Delta-Exon3. Taken together this data demonstrates a clear mechanism for hypoxia-induced neonatal cardiomyocyte proliferation which can ultimately be reversed pharmacologically with the addition of exogenous PGE1 signaling, through Misoprostol.