Abstract 9244: A Novel Phosphorylating Mechanism of STAT3 in Protection against Ischemia/reperfusion by Pharmacological Preconditioning With Tadalafil, a PDE Inhibitor

Abstract

Background: Phosphodiesterase (PDE) inhibition enhances Protein Kinase G-I (PKG-I) activity leading to stem cell survival and cardioprotection against ischemia/reperfusion injury. We previously reported phosphorylation of STAT3 ser727 during late phase of protection. We aimed to investigate the specificity of STAT3 in regulation of PKG-I and cardioprotection using heart specific conditional knockout mice. Methods and Results: Heart specific conditional knockout mice were made by breeding STAT3fl/fl with tamoxifen inducible cre (MCM) transgenic mice. MCM transgene was cloned downstream to heart specific myosin heavy chain promoter (MHC). Cre positive mice (MCM:STAT3fl/fl) were selected from genotyping and tamoxifen (20mg/kg) was administrated for 10 days. KO mice were pretreated with tadalafil (1mg/kg) 1h before LAD coronary artery ligation for 30 min followed by reperfusion. Echocardiographic analysis showed abrogation of cardioprotection in STAT3 knockout mice with decreased ejection fraction and fraction shortening as compared to non-transgenic mice. Mitogen activated protein kinases ERK and P38 were known to phosphorylate STAT3 at ser727. On contrary, western blot analysis with heart tissue lysate showed reduced phosphorylation/activation of ERK both in control and tadalafil treated cells with no change in total ERK (P<0.05). Expression of pP38 and total P38 (p<0.01) were unaltered. Two fold increase in expression of PKG-I (P<0.05) during the late phase of protection was observed with tadalafil. Inhibition of PKG-I with KT5823 or antisense abolished STAT3 phosphorylation at ser727. Results were also replicated in vitro with mesenchymal stem cells isolated from adult male Fischer-344 rats by flushing the cavity of femur and cultured, purity was checked by CD44+ and CD73+ surface markers (flow cytometry). Cells were pretreated 1 h with tadalafil (100μM) then subjected to 2 h hypoxia (1% O2) and reoxygenation for 2 h/24 h. Conclusion: The study showed novel mechanism of phosphorylating STAT3 at ser727 by PKG-I leading to its entry inside nucleus and transcriptional activation of PKG-I by binding onto promoter making it a positive feedback loop. Heart specific STAT3 knockout mice showed abrogation of cardioprotection with tadalafil.