Abstract 3986: Activation of the AKT Pathway Mediates Upregulation of Hif-1α and Sirt1 via Downregulation of miR-199a and is Neutralized by Beta-adrenergic Stimulation
We have recently reported that upregulation of hypoxia-inducible factor-1alpha (Hif-1α) and Sirt1 during early hypoxia preconditioning (HPC) required downregulation of miR-199a and are both essential for preconditioning cardiac myocytes against hypoxic conditions. Conversely, others and we have reported that miR-199a is upregulated during cardiac hypertrophy and failure. The objective of this study was to delineate the signaling pathways that regulate the expression of miR-199a, Hif-1α, and Sirt1, and their role in myocyte protection against hypoxic damage. Since preconditioning is mainly associated with activation of the AKT pathway, we questioned if it is sufficient for inducing downregulation of miR-199a and upregulation of its targets Hif-1α and Sirt1. Indeed, our results show that overexpression of a constitutively active AKT mutant in myocytes induced 70% downregulation of miR-199a that was associated with a robust increase in Hif-1α (10±2 fold) and Sirt1 (4±0.8 fold). Moreover, this effect was completely abrogated by overexpression of miR-199a, proving that its downregulation is required for the increases seen in those molecules. On the other hand, inhibition of AKT by wortmannin or dominant negative AKT confirmed that its activation is required for HPC-induced Hif-1α and Sirt1. Consequently, we examined whether receptor activation of the PI-3kinase-AKT pathway would also enhance Hif-1α expression and whether it was miR-199a dependent. Our results revealed that insulin induced 95% downregulation of miR-199a and upregulation of Hif-1α (20±8 fold) and Sirt1 (4±1 fold), which were both completely abrogated by overexpression of miR-199a. In contrast, β1 and β2 adrenergic receptor overexpressing-mice hearts and isoproterenol-stimulated myocytes, exhibited 4–5 fold increase in miR-199a, which suggested a likely antagonism of the effects induced by the AKT pathway. To test this idea, we pretreated the myocytes with isoproterenol for 16 h before applying HPC or caAKT. This resulted in 90±15 % reduction in HPC- or caAKT-induced Hif-1α and Sirt1 expression and their protectiveness against hypoxic damage. Thus, activation of the beta-adrenergic pathway counteracts the survival effects of the AKT pathway via upregulating miR-199a.