Abstract 354: Golgi Localized β 1 -adrenergic Receptors Stimulate Golgi PI4P Hydrolysis by PLCε to Regulate Cardiac Hypertrophy

Abstract

Phospholipase C (PLC)-mediated PI4P hydrolysis pathway at the Golgi is important for regulating cardiac hypertrophy. cAMP stimulates this pathway via Epac-mediated activation of Rap1 which directly binds to and activates PLCε. Here, we demonstrate that a membrane permeant β-adrenergic agonist, dobutamine (dob), and the endogenous β-adrenergic agonist, norepinephrine (NE), induce Golgi PI4P hydrolysis in neonatal rat ventricular myocytes (NRVMs) and adult ventricular myocytes. However, a membrane impermeant β-adrenergic agonist, isoproterenol (iso), does not. In addition, a membrane permeant βAR antagonist, metoprolol, but not a membrane impermeant antagonist, sotalol, fully reverses dob-mediated PI4P hydrolysis. Taken together, this suggests that internal β-ARs are required for inducing PI4P hydrolysis. Subsequently,we used YFP-tagged mini Gs proteins that recruit to Gs-coupled receptors upon their activation, to monitor the location of activated receptors. Dob and NE induced robust and rapid recruitment of mini Gs to the plasma membrane and the Golgi, however, iso only induces recruitment to the plasma membrane. Additionally, inhibition of PLCεat the Golgi with either siRNA or the RA1 domain of PLCε, or the use of an Epac inhibitor also inhibits dob-mediated PI4P hydrolysis. This suggests that PI4P hydrolysis by dob requires Golgi-localized PLCεand Epac.Inhibition of the βAR with Golgi-targeted Nb80 prevented PI4P hydrolysis by both dob and NE confirming that Golgi-localized βARs are required for PI4P hydrolysis.Also, Oct3 transporter inhibitors prevent NE-induced PI4P hydrolysis but dyngo, an inhibitor of receptor internalization, had no effect. This indicates that agonist transport and not receptor internalization is required for NE-mediated Golgi βAR activation and PI4P hydrolysis. Furthermore, dob stimulates an increase in cell size and ANF expression in NRVMs that is significantly inhibited by metoprolol but less effectively by sotalol. Taken together, these data suggest that Golgi βARs are involved in mediating cardiomyocyte hypertrophy and may serve as a novel target for treating heart failure.