Background: Moderate overexpression of β3AR in cardiomyocytes attenuates hypertrophic remodeling in hearts after neurohormonal stress, without impairement of cardiac function.
Methods: Functional analyses were carried out in wild type (WT) and heterozygote transgenic mice with cardiac myocyte-specific over-expression of human β3AR (hβ3TG) treated for 10 days with isoproterenol (Iso: 30mg/kg/day), angiotensin II (AngII: 2mg/kg/day), with/without L-NAME (2mg/mL). In vitro hypertrophic responses to phenylephrine (PE) were analyzed in neonatal rat ventricular myocytes (NRVM) infected with a recombinant adenovirus expressing the human β3AR (AdVhβ3).
Results: We further dissected the underlying signaling in isolated cardiomyocytes and tissues from hβ3TG mice. Cell fractionation and Proximity Ligation Assay experiments revealed co- localization of β3AR with constitutive NOS isoforms, eNOS and nNOS and with caveolin-3 in lipid rafts/caveolae, which was unperturbed in hypertrophic myocytes. Adult cardiomyocytes from hβ3TG mice co-expressing β3AR with the cGMP-sensitive FRET sensor, redcGES-DE5 exhibited higher constitutive, ODQ-sensitive cGMP production compared with WT, whereas the opposite was observed in cells from sensor-expressing β3AR KO mice. Non-specific NOS inhibition (L-NAME) abrogated the β3AR-mediated protection from hypertrophic remodeling under low dose Iso in vivo and in NRVM. Interestingly, we observed a L-NAME independent increase in protein synthesis in control cells (152±16 (veh)vs230±27 (PE); p<0.01), but not in the AdVhβ3 infected NRVM (130±11 (veh) vs 103±12 (PE); p=ns). As AMP-activated protein kinase (AMPK) is a known inhibitor of cardiac hypertrophy and protein synthesis, we examined its activation by β3AR. First, we observed a colocalization of AMPK with β3AR, eNOS and cav-3. P-Thr162-AMPK was decreased by the PE (0.5 ± 0.1), but restored in β3AR expressing cells (0.9 ± 0.3 of ctl; P<0.05). siRNA targeting of AMPK also partly abrogated the anti-hypertrophic effect of β3AR in response to PE. Moreover, AMPK is also an inducer of autophagy. In NRVM, β3AR overexpression restored autophagy that was decreased by PE in ctl cells in vitro, as measured by LC3-II/LC3-I and p62 abundance; likewise autophagy was restored in hβ3TG mice after TAC compared with WT (LC3II/I ratio: 0.5±0.1 (WT), 1.3±0.3 (hβ3TG), p<0.05).
Conclusions: We conclude that β3AR is part of a caveolar signalosome involving nNOS, eNOS and AMPK in cardiomyocytes, from which it activates both cGMP/PKG and AMPK-dependent pathways to prevent hypertrophic remodeling and maintain autophagy in the face of neurohormonal stress.