AMP-activated protein kinase dependent signaling pathways and modulation of autophagy are involved in the anti-hypertrophic effect of the human beta 3 adrenoreceptor

Abstract

Background: The human heart contains β3-adrenoreceptors (β3AR) which are coupled to nitric oxide (NO) synthesis; however, their role in cardiac remodeling is unclear Methods: Mice with cardiomyocyte-specific overexpression of human β3AR (TG) & wild-type littermates (WT) were treated with isoproterenol (Iso30mg/kg/d), angiotensin II (AngII 2mg/kg/d), or saline by osmotic minipump, with/without L-NAME (2mg/mL). In vitro hypertrophic response, signaling & NO production (EPR) were analyzed in neonatal rat ventricular myocytes infected with an adenovirus expressing the human β3AR (hβ3-NRVM). Subcellular localization of β3AR was studied in adult ventricular myocytes using the proximity ligation assay (PLA). Results: PLA showed colocalization of the β3AR & eNOS with caveolin-3, which co-segregated with AMP-activated protein kinase (AMPK) & eNOS after sucrose gradient fractionation. Following 10 day Iso or AngII, cardiac myocyte hypertrophy & an upregulation of βMHC mRNA expression were observed in WT (μm2: 682±18 Sal vs 914±54 Iso; p<0.001; n=9), but not TG mice (697±16 Sal vs 783±39 Iso; p=ns; n=9). L-NAME treatment abrogated the protection from Iso-induced hypertrophy in TG mice (728±30 L-NAME vs 963±60 L-NAME+Iso; p<0.001; n = 9). In vitro, when compared to GFP-NRVM, hβ3-NRVM showed higher NO production (GFP 2108±245; hβ3 3631±341; n =4; p<0.01) & failed to show a cardiac hypertrophic response (assessed by measurement of cell size, protein synthesis & NFAT activation) following PE stimulation. Treatment of hβ3-NRVM with L-NAME abolished the protection from PE-induced hypertrophy. In GFP-NRVM, PE inhibited AMPK (phospho-Thr172 AMPK), an inhibitor of protein synthesis & cardiac hypertrophy, yet in hβ3-NRVM AMPK phosphorylation was preserved after PE. Transfection of hβ3-NRVM with siRNA against AMPK abolished the protection from PE-induced hypertrophy (μm2: Scr: hβ3-veh 1088±64; hβ3-PE 1139±56, p=ns; siRNA: hβ3-veh 1143±57; hβ3- PE 1594±101, n=3; p<0.01). AMPK is also known to activate autophagy. In GFP-NRVM, PE inhibited autophagy (LC3 II/I ratio and p62 abundance), yet in hβ3-NRVM autophagy was preserved after PE. Conclusion: Cardiac-specific overexpression of β3AR inhibits hypertrophy following neurohormone stimulation in vivo & in vitro, partly through NO-dependent & AMPK-dependent mechanisms, consistent with colocalization of hβ3AR with NOS and AMPK in caveolae. Protection from PE-induced hypertrophy may involve AMPK-mediated maintenance of autophagy. Cardiac β3ARs may thus provide a therapeutic target in the treatment of cardiac remodeling associated with elevated sympathetic tone.