G protein βγ subunits regulate cardiomyocyte hypertrophy through a perinuclear Golgi phosphatidylinositol 4-phosphate hydrolysis pathway

We recently identified a novel GPCR-dependent pathway for regulation of cardiac hypertrophy that depends on Golgi phosphatidylinositol 4-phosphate (PI4P) hydrolysis by a specific isoform of phospholipase C (PLC), PLCε, at the nuclear envelope. How stimuli are transmitted from cell surface GPCRs to activation of perinuclear PLCε is not clear. Here we tested the role of G protein βγ subunits. Gβγ inhibition blocked ET-1–stimulated Golgi PI4P depletion in neonatal and adult ventricular myocytes. Blocking Gβγ at the Golgi inhibited ET1–dependent PI4P depletion and nuclear PKD activation. Translocation of Gβγ to the Golgi stimulated perinuclear Golgi PI4P depletion and nuclear PKD activation. Finally, blocking Gβγ at the Golgi or PM blocked ET-1–dependent cardiomyocyte hypertrophy. These data indicate that Gβγ regulation of the perinuclear Golgi PI4P pathway and a separate pathway at the PM is required for ET-1–stimulated hypertrophy, and the efficacy of Gβγ inhibition in preventing heart failure maybe due in part to its blocking both these pathways. INTRODUCTION G protein–coupled receptors (GPCRs) regulate many aspects of cardiac function, including cardiac contractility and hypertrophic growth. One of the key GPCR-regulated pathways in cardiomyocytes is the phosphoinositide-specific phospholipase C (PI-PLC) signaling pathway, which hydrolyzes plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3-dependent calcium release in the nucleus and DAG-dependent activation of protein kinase D (PKD) are two of the critical signals involved in regulation of cardiac hypertrophic growth (Vega et al., 2004; Wu et al., 2006). Gq has been shown to drive cardiac hypertrophy (Knowlton et al., 1993; D’Angelo et al., 1997) and is known to activate PLCβ (Smrcka et al., 1991; Taylor et al., 1991). Indeed, PLCβ1 has been directly implicated in α1-adrenergic receptor–dependent hypertrophic growth of neonatal rat ventricular myocytes (NRVMs; Filtz et al., 2009). Our laboratory has identified unique roles for a distinct isoform of PI-PLC, PLCε, in regulation of various aspects of cardiac function (Smrcka et al., 2012). PLCε provides a link between β-adrenergic receptor activation and type 2 ryanodine receptor (Ryr2)–dependent calcium release in the regulation of cardiac contractility (Wang et al., 2005; Oestreich et al., 2007; Oestreich et al., 2009). PLCε is also required for development of the pressure overload model of cardiac hypertrophy in mice and is downstream of multiple receptor-dependent stimuli in isolated ventricular myocytes (Zhang et al., 2011, 2013). Like all PI-PLCs, PLCε hydrolyzes PIP2, but it has distinct regulatory features, in that it is directly activated by small GTPases, including Ras, Rap, and Rho (Smrcka et al., 2012). PLCε can also be activated by G protein βγ subunits, although direct interactions have not been demonstrated (Wing et al., 2001). These regulatory Monitoring Editor Thomas F. J. Martin University of Wisconsin Received: Oct 24, 2014 Revised: Dec 23, 2014 Accepted: Jan 14, 2015 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E14-10-1476) on January 21, 2015. Address correspondence to: Alan V. Smrcka (Alan_Smrcka@urmc.rochester.edu). © 2015 Malik et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: ANF, atrial natriuretic factor; AVM, adult ventricular myocyte; cpTOME, 8-4-(chlorophenylthio)-2′-O-methyladenosine-3′,5′-monophosphate; DAG, diacylglycerol; Epac, exchange factor activated by cAMP; ET-1, endothelin-1; FAPP-PH, four-phosphate adaptor protein–pleckstrin homology domain; GPCR, G protein–coupled receptor; GRK2ct, C terminus of G protein–coupled receptor kinase 2; mAKAP, muscle-specific A kinase anchoring protein; nDKAR, nuclear targeted D kinase reporter; NRVM, neonatal rat ventricular myocyte; PI4P, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5 trisphosphate; PI-PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; PKD, protein kinase D; PM, plasma membrane; PTX, pertussis toxin. http://www.molbiolcell.org/content/suppl/2015/01/18/mbc.E14-10-1476v1.DC1. html Supplemental Material can be found at: