Abstract 652: Regulation of Cardiac MicroRNA miR-208a by Angiotensin II and the Beta-Adrenergic Receptor Blocker Nebivolol

Abstract

The microRNA miR-208a is a pathologic myo-miR required for hemodynamic pressure overload induced cardiac fibrosis. It is unclear how cardio-deleterious hormones and cardioprotective drugs regulate expression of miR-208a. We hypothesized that the pro-inflammatory hormone angiotensin II (Ang II) and anti-inflammatory drugs rapamycin (Rap: inhibitor of the nutrient sensor kinase mTOR) and nebivolol (Neb: a 3 rd generation β1 adrenergic receptor [β1-AR] blocker that exhibits β3-AR agonism) modulate miR-208a expression in cardiomyocytes. Methods and Results: In mouse atrial cardiomyocyte HL-1 cells, 12 hour treatment with Ang II (100nM) increased miR-208a by 6 fold as determined by real-time RT-PCR analysis (P<0.05; n=5). AT1 receptor (AT1R) blocker losartan (10μM), but not AT2 receptor (AT2R) blocker PD123319 (10μM), inhibited this effect. Pre-treatment (1hr) with Rap (10nM) and Neb (1μM), but not apocynin (500μM: NADPH oxidase inhibitor) or U73122 ((10μM: Phospholipase C inhibitor) attenuated Ang II-induced increases in miR-208a levels (P<0.05). Thus, the AT1R-mediated activation of mTOR up-regulates miR-208a in cardiomyocytes. Immunoblotting revealed that Ang II increased phosphorylation (p) of mTOR (pSer 2448 ), S6K1 (pThr 389 ) and RPS6 (pSer 235/236 ) by 1.5 to 2 fold in HL-1 cells and Rap and Neb suppressed this effect (P<0.05). Neb-induced β3-AR agonism was not involved in miR-208a suppression since β3-AR antagonist SR52930 (1μM) did not reverse Neb-induced suppression of miR-208a and β3-AR mRNA was not detected in HL-1 cells by real time RT-PCR. Interestingly, a 12 hour treatment of HL-1 cardiomyocytes with 1μM Neb attenuated 125 I-Ang II binding to the AT1R by (70%: P<0.05); however a 15 minute pre-incubation with 1μM Neb did not prevent 125 I-Ang II binding to HL-1 cells. Thus, chronic Neb treatment down-regulates AT1R function in cardiomyocytes. Conclusions: We show for the first time that Ang II increases miR-208a expression in cardiomyocytes via activation of mTOR-S6K1 signaling pathway. Nebivolol, a β-blocker, inhibits Ang II-induced miR-208a expression and mTOR signaling. Moreover, chronic Neb treatment down-regulates AT1R in cardiomyocytes. Regulation of AT1R-mTOR-miR-208a axis by Neb is a novel cardioprotective mechanism.