Abstract 3995: Differential Roles of Akt and Extracellular Signal-Regulated Kinase Signaling Pathways on Autophagy During Alpha1-Adrenergic Agonist-Mediated Cardioprotection
Introduction: We and others previously reported that alpha1-adrenergic signaling pathway protected cardiac myocytes from apoptosis. However, the role of cardiac autophagy, which has been recently known for its activation in the failing heart, on this cardioprotective state is not well known.
Hypothesis: We assessed the hypothesis that autophagy contributed to the alpha1-adrenergic agonist-mediated cardioprotection.
Methods and Results: Using transgenic mice expressing GFP- microtubule-associated protein 1 light chain 3 (LC3), we implanted osmotic pumps for administrating alpha1-adrenergic agonist phenylephrine (PE, 5mg/kg/day), norepinephrine (NE, 5mg/kg/day), or saline, for 4 weeks. Echocardiogram revealed no change in cardiac systolic function of mice treated with PE despite the severe systolic failure induced by NE. Wide field fluorescence microscopy with z-stack method showed that PE markedly increased the GFP-LC3-labeled autophagosomes in myocardium compared with NE or saline treatment (p<0.05, p<0.01, respectively). Cardiac autophagy induced by PE in vivo was also confirmed by monodansylcadaverine staining and western blotting with anti-LC3 antibody. Using cultured neonatal rat cardiac myocytes transfected with GFP-tagged LC3, we observed that PE (10−5M) activated cardiac autophagy in vitro with stimulating autophagic flux (p<0.01 vs control). TUNEL staining and cytofluorimetric analysis showed that blocking autophagy either by knockdown of autophagy-related 5 (ATG5) via RNAi or by 3-methyladenine negated the anti-apoptotic effect of PE. Using knockdown of Akt or extracellular signal-regulated kinase (ERK) via RNAi or the inhibitors against PI3-kinase or MEK1, we observed that both PI3-kinase/Akt and MEK/ERK signaling pathways were required for anti-apoptotic action of PE. PI3-kinase/Akt was also required for PE-induced autophagy. In contrast, however, the inhibition of MEK/ERK did not negate but further enhanced autophagy in cardiac myocytes.
Conclusions: In conclusion, cardiac autophagy is required for alpha1-adrenergic agonist-mediated cardio-protection against apoptosis. The results also indicate that Akt and ERK signaling pathways differentially regulate cardiac autophagy in the cardioprotection.