β3-adrenoreceptors (AR) are found in the human heart, & their activation couples to nitric oxide (NO) synthesis; however, their role in cardiac function & remodelling is unclear.Methods
Wild type (WT) & heterozygote male mice with cardiomyocyte-specific overexpression of human β3AR (TG) were treated for 10 days with isoproterenol (Iso 30mg/kg/day), angiotensin II (AngII 2mg/kg/day),or saline by osmotic minipump, with/without L-NAME (2mg/mL) in drinking water. In vitro hypertrophic responses to phenylephrine (PE) & Iso were analysed in neonatal rat ventricular myocytes infected with an adenovirus expressing the human β3AR (hβ3-NRVMs). Subcellular localisation of the endogenous β3AR was studied in adult rat ventricular myocytes (ARVMs) using the proximity ligation assay (PLA).Results
LV function was similar between WT & TG at baseline (DP/dt max (mmHg/sec): WT 5894 ± 614 vs TG 8036 ± 1332), & following acute Iso infusion (WT 9803 ± 119 vs TG 10099 ± 1416; n=12-15). 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). Likewise, in hβ3-NRVMs PE or Iso did not induce an increase in cell size (μm2: 1187 ± 30 PBS vs 1232 ± 29 PE vs 1200 ± 35 Iso; p=ns; n=3 isolations, 40 cells/n) or protein synthesis, in contrast to GFP-NRVMs (1175 ± 31 PBS vs 1321 ± 30 PE vs 1392 ± 31 Iso: p < 0.001). Treatmentof hβ3-NRVMs with L-NAME or KT5823 (an inhibitor of cGMP-dependent protein kinase) abolished the protection from PE-induced hypertrophy (1180 ± 28 L-NAME vs 1425 ± 28 L-NAME + PE; p < 0.001; 1274 ± 42 KT vs 1567 ± 43 KT + PE; p < 0.05). PE inhibited AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis & cardiac hypertrophy (activation measured as intensity of phospho-Thr172 AMPK), yet in hβ3-NRVMs AMPK phosphorylation was preserved after PE (1.27 ± 0.3 PBS vs 1.33 ± 0.4 PE; p=ns). In ARVMs, PLA showed colocalisation of the β3AR & eNOS with caveolin-3, which coimmunoprecipitated with AMPK & eNOS.Conclusion
Cardiac-specific overexpression of β3ARs does not alter LV function but inhibits hypertrophy following neurohormone stimulation in vivo and in vitro, partly through a NO-dependent mechanism. This involves a caveolar signalosome including the β3AR, eNOS & AMPK. Cardiac β3ARs may be a therapeutic target in the treatment of hypertrophic cardiomyopathies associated with elevated sympathetic tone.