Abstract 16938: Transcriptional Repression of Protein Kinase G during Ischemic-Reperfusion Injury in Stem Cells is Restored by Inhibition of Phosphodiesterase 5A with Long Acting Tadalafil
Background: PDE5A activity increases during ischemic reperfusion (IR) injury with concomitantly breakdown of cGMP and PKG deactivation. Pharmacological preconditioning with tadalafil, a PDE5A inhibitor with relatively longer t 1/2 (17.5 h) increases PKG activity and strongly protective against IR injury. However less is known about PKG expressional status and its direct/indirect targets during IR. We aimed to investigate how PKG regulation initiates protection and its signaling pathway.
Methods and Results: Mesenchymal Stem Cells were isolated from adult male Fischer-344 rats by flushing the cavity of femur and cultured in vitro; purity was checked by CD44+ and CD73+ surface markers (flow cytometry). Cells were pretreated 1 h with tadalafil (100uM in DMSO) followed by 1 h ischemia (1% O2) and reperfusion for various durations. Cell death was increased 2 fold (p<0.05) in untreated cells shown by LDH release concomitant with higher caspase activity and TUNEL positivity. Mitochondrial membrane were depolarized shown by tetramethylrhodamine, ethyl ester (TMRE) staining. Electron micrographs study revealed swelling of mitochondria in tadalafil treated cells with IR injury. Western blot analysis showed almost null expression of PKG1 (p<0.001) during early phase of injury together with phosphorylated P38 (p<0.001) whereas increase of PKG1 expression (2.5 fold) was noted during late phase of reperfusion with concomitant increase in expression of PI3K 110α, pAkt, Bcl2, SIRT1 and decrease in the expression of Bax, pERK1/2 with no significant change in pGSK3β and P38 (24 h; p< 0.05) after treatment. Computational analysis showed binding site of promising transcriptional factors like p53, krueppel like zinc finger, heat shock factors, AP2 on PKG (+ strand) with matrix similarity of more than 0.8. Echocardiographic analysis also showed improvement of ejection fraction, fraction shortening and reduced cavity size of hearts which underwent tadalafil treatment.
Conclusion: This study showed for the first time that transcriptional repression of PKG leads to cardiac damage during early phase of IR and treatment with tadalafil restores the protection through PI3K/Akt/mitochondria survival pathway.
- © 2012 by American Heart Association, Inc.