#20 Targeting Bnip3 Splicing: Misoprostol Switches Pro-death BNIP3 to Pro-Survival to Safeguard Mitochondria during Hypoxic Injury
Jared Field, University of Manitoba; Matthew Martens, University of Manitoba; Yan Hai, University of Manitoba; Simone da Silva Rosa, University of Mantioba; Wajihah Mughal, University of Mantioba; William Diehl-Jones, Athabasca University; Tammy Ivanco, University of Manitoba; Joseph Gordon, University of Manitoba
Perinatal hypoxia negatively impacts neonatal growth and development, which has been shown to impact cardiometabolic health. Bnip3 is a central regulator of cell death during hypoxic injury. Additionally, Bnip3 is subjected to alternative splicing, producing pro-death and pro-survival isoforms. We previously observed that misoprostol (a prostaglandin E2 agonist) has a cytoprotective effect and can alter Bnip3 expression; thus, I hypothesize that targeting BNIP3 splicing with misoprostol, promoting Bnip3ΔExon3, can reverse the detrimental effects of cellular hypoxia through genetic and mitochondrial pathways.
Misoprostol-induced splicing of full length Bnip3 (Bnip3FL) into the splice variant Bnip3ΔExon3 was identified in neonatal rat pups by RT-PCR. Secondly, primary rat neonatal cardiomyocytes (NCM), and two cell lines (H9C2, HCT-116) expressing Bnip3 isoforms were used to determine the mechanisms of Bnip3ΔExon3 cellular protection. Gain-of-function experiments (n= 10) used fluorescent biosensors to assess the effects of Bnip3ΔExon3 on cell viability, calcium dynamics, and transcription-factor activation. Finally, localization of pro-survival Bcl-2 protein was assessed by subcellular fractionation and immunoblotting.
In rat pup hearts, misoprostol markedly increased Bnip3ΔExon3 expression. Expression of Bnip3ΔExon3 Mitochondria of NCM were protected from hypoxia-induced depolarization (p< 0.01). In cells expressing Bnip3 isoforms, Bnip3FL caused an 8-fold increase in cell death, an effect rescued by Bnip3ΔExon3 (46%; p< 0.01). Additionally, Bcl-2 translocation to the mitochondrial, were it has a protective role, was enhanced by Bnip3ΔExon3. Moreover, Bnip3ΔExon3 protected cells against Bnip3-induced mitochondrial calcium overloading (p< 0.05) and mitochondrial permeability transition. Finally, Bnip3ΔExon3 regulated transcription factor activity, evidenced by a 5-fold increase in active NFAT and a 50% deactivation of HDAC5.
Together these data uncover the potential for drug-targeted splicing of the hypoxia-inducible death gene, BNIP3, into a robust pro-survival factor, Bnip3ΔExon3, that can operate through calcium signaling, transcription factor regulation and Bcl-2 translocation to protect life when challenged with hypoxia.