Compartmentalized Phospholipase C Signaling as a Hub That Mediates -adrenergic Receptor Crosstalk in Cardiac Hypertrophy

Abstract

β-adrenergic receptors are G protein coupled receptors that are critical regulators of cardiac output during sympathetic stimulation. Chronic stimulation of the adrenergic system of the heart by sympathetic nerves or circulating catecholamines under conditions of cardiac stress leads to cardiac hypertrophy and ultimately heart failure. One of the first line therapies for the treatment of heart failure is β-blockers to prevent the activation of β-adrenergic receptors. β-blockers ameliorate the symptoms of heart failure but still have side effects. Therefore, novel insights for designing selective therapies will provide substantial therapeutic impact. Previously, our laboratory characterized one isoform of phospholipase C, PLCε, and showed that it is tightly associated with the pathogenesis of cardiac hypertrophy. Further investigations revealed a novel pro-hypertrophic pathway where PLCε at the nuclear envelope/Golgi interface, hydrolyzes Golgi phosphatidylinositol 4-phosphate (PI4P), generates local diacylglycerol (DAG), mediates nuclear protein kinase D (PKD) activation and hypertrophic gene expression. This pathway can be activated by multiple upstream signals including, EPAC-selective cAMP analog, cpTOME or adenylyl cyclase activator forskolin. However, stimulation of β-ARs with membrane impermeant agonist isoproterenol does not activate this pathway despite strongly raising intracellular cAMP. It was reported that β1ARs are present at Golgi in HeLa cells and can generate cAMP there. These studies lead to our overall hypothesis that compartmentalized PLCε activity is regulated distinctly by discrete spatially distributed βARs in cardiomyocytes. In my dissertation research, we tested the role of internal β1ARs in the stimulation of Golgi PLCε in neonatal ventricular myocytes. By utilizing a fluorescent biosensor that recognize an active form of β-ARs, we detected a pre-existing pool of Golgi-localized β1AR in cardiomyocytes that can be activated by the permeant agonist, dobutamine, or the endogenous ligand, norepinephrine (NE) through an organic cation transporter in cardiomyocytes. The activation of Golgi PLCε is uniquely regulated by Golgi-β1-AR stimulation but not cell surface β1AR stimulation. Importantly, blockade of Golgi β1ARs prevents NE induced cardiomyocyte hypertrophy. This defines a functional role of Golgi-β1ARs in a highly biologically relevant system. I further investigated other mechanisms associated with Golgi PLCε in the regulation of cardiac hypertrophy. β1-AR and β2-ARs are two major subtypes of β-ARs present in the heart. However, the two β-AR subtypes elicit different or opposite effects on cardiac function and hypertrophy. We showed that β2-AR protects against hypertrophy through inhibition of hypertrophic Golgi PLCε signaling. Using pharmacological approaches, we found that endosomal Gβγ released by internalized β2ARs inhibited the activition of Golgi PLCε by angiotensin II and Golgi-β1-ARs, ultimately resulting in decreased PKD and histone deacetylase 5 (HDAC5) phosphorylation and protection against cardiac hypertrophy. This supports a novel potential mechanism for β2-AR antagonism of the PLCε pathway that may contribute to the known protective effects of β2-AR signaling on the development of heart failure. We used a genetically-encoded Golgi-targeted β1AR inhibitor to study the functional role of Golgi-β1ARs in animals. By analysis of the heart tissues expressing Golgi-β-blocker or negative control undergoing transaortic constriction induced heart failure, we found promising statistical trends supporting our overall hypothesis that Golgi-β1ARs is involved in the promotion of pathological cardiac hypertrophy. Together this study provides evidence of functionally active subcellular βARs that regulate distinct compartmentalized signaling in cardiac muscle cells. Our work informs a novel therapeutic approach of combining a β2 agonist with hydrophobic β1 selective blocker in the treatment of heart failure.