Abstract 19434: Early Ischemic Preconditioning Requires Akt- and PKA-Mediated Activation of eNOS via Serine1176 Phosphorylation
Aims: The role of endothelial nitric oxide synthase (eNOS)/NO signaling is well-documented in late preconditioning or postconditioning; however, the role of eNOS and its activation in early IPC remains controversial and poorly defined. This study investigates the role of eNOS in early IPC and what signaling pathways and molecular interactions regulate eNOS activation during early IPC.
Methods and Results: Rat hearts were subjected to 30-min global ischemia and reperfusion (I/R) with or without IPC (3 cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor, L-NAME, PI3K inhibitor, LY294002/LY, or PKA inhibitor, H89, during IPC induction. IPC improved postischemic contractile function and reduced infarction compared to I/R with this abrogated by NOS inhibition. eNOSSer1176, AktSer473, and PKAThr197 phosphorylation was increased following IPC. I/R decreased eNOSSer1176 phosphorylation, while IPC increased it. Mass spectroscopy confirmed eNOSSer1176 phosphorylation and quantative western blots showed that ∼24 % modification of eNOSSer1176 was present following induction of early IPC. Immunoprecipitation demonstrated eNOS, Akt and PKA complexation. Immunohistology showed that IPC induced Akt and PKA phosphorylation in cardiomyocytes and endothelial cells. With eNOS activation, IPC increased NO production as measured by EPR spin trapping and fluorescence microscopy. LY or H89 not only greatly decreased AktSer473 or PKAThr197 phosphorylation, respectively; but also abolished IPC-induced preservation of eNOS and eNOSSer1176 phosphorylation as well as subsequent cardioprotection.
Conclusion: Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection with eNOS-derived NO serving a critical role.
- © 2012 by American Heart Association, Inc.