Katie A. McCrink

GRK2 Up-Regulation Creates a Positive Feedback Loop for Catecholamine Production in Chromaffin Cells

Elevated sympathetic nervous system (SNS) activity aggravates several diseases, including heart failure. The molecular cause(s) underlying this SNS hyperactivity are not known. We have previously uncovered a neurohormonal mechanism, operating in adrenomedullary chromaffin cells, by which circulating catecholamine (CA) levels increase in heart failure: severe dysfunction of the adrenal α2-adrenergic receptors (ARs) due to the up-regulation of G protein-coupled receptor-kinase (GRK)-2, the kinase that desensitizes them. Herein we looked at the potential signaling mechanisms that bring about this GRK2 elevation in chromaffin cells. We found that chronic CA treatment of either PC12 or rat primary chromaffin cells can in itself result in GRK2 transcriptional up-regulation through α2ARs-Gi/o proteins-Src-ERK1/2. The resultant GRK2 increase severely enhances the α2AR desensitization/down-regulation elevating not only CA release but also CA biosynthesis, as evidenced by tyrosine hydroxylase up-regulation. Finally, GRK2 knockdown leads to enhanced apoptosis of PC12 cells, indicating an essential role for GRK2 in chromaffin cell homeostasis/survival. In conclusion, chromaffin cell GRK2 mediates a positive feedback loop that feeds into CA secretion, thereby enabling the adrenomedullary component of the SNS to turn itself on.

β-Arrestin2 Improves Post–Myocardial Infarction Heart Failure via Sarco[endo]plasmic Reticulum Ca2+-ATPase–Dependent Positive Inotropy in Cardiomyocytes

Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein–coupled receptor) adapter proteins &bgr;arr (&bgr;-arrestin)-1 and &bgr;arr-2 are functionally distinct in the heart. &bgr;arr1 is cardiotoxic, decreasing contractility by opposing &bgr;1AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post–myocardial infarction (MI). Conversely, &bgr;arr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether &bgr;arr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that &bgr;arr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca2+-ATPase) in vivo and in vitro in a &bgr;1AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. &bgr;arr1 lacks this effect. Moreover, &bgr;arr2 does not desensitize &bgr;1AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to &bgr;arr1. In vivo, post-MI heart failure mice overexpressing cardiac &bgr;arr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, &bgr;arr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac &bgr;AR number or cAMP levels in vivo. In conclusion, enhancement of cardiac &bgr;arr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac &bgr;arr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure.

Impact of CYP2D6 Genetic Variation on the Response of the Cardiovascular Patient to Carvedilol and Metoprolol.

Carvedilol and metoprolol are two of the most commonly prescribed β-blockers in cardiovascular medicine and primarily used in the treatment of hypertension and heart failure. Cytochrome P450 2D6 (CYP2D6) is the predominant metabolizing enzyme of these two drugs. Since the first description of a CYP2D6 sparteinedebrisoquine polymorphism in the mid-seventies, substantial genetic heterogeneity has been reported in the human CYP2D6 gene, with ~100 different polymorphisms identified to date. Some of these polymorphisms render the enzyme completely inactive while others do not modify its activity. Based on all the identified variants, four metabolizer phenotypes are nowadays used to characterize drug metabolism via CYP2D6 in humans: ultra-rapid metabolizer (UM); extensive metabolizer (EM); intermediate metabolizer (IM); and poor metabolizer (PM) phenotypes. As a consequence of these CYP2D6 metabolizer phenotypes, pharmacokinetics and bioavailability of carvedilol and metoprolol can range from therapeutically ineffective levels (in the UM patients) to excessive (overdose) and potentially toxic concentrations (in PM patients). This, in turn, can result in elevated risks for either treatment failure (in terms of blood pressure reduction of hypertensive patients and of improving survival and cardiovascular function of heart failure patients) or for adverse effects (e.g. hypotension and bradycardia). The present review will discuss the impact of these CYP2D6 genetic polymorphisms on the therapeutic responses of cardiovascular patients treated with either of these two β-blockers. In addition, the potential advantages and disadvantages of implementing CYP2D6 genetic testing in the clinic to guide/personalize therapy with these two drugs will be discussed.

Adrenal G protein-coupled receptor kinase-2 in regulation of sympathetic nervous system activity in heart failure

Heart failure (HF), the number one cause of death in the western world, is caused by the insufficient performance of the heart leading to tissue underperfusion in response to an injury or insult. It comprises complex interactions between important neurohormonal mechanisms that try but ultimately fail to sustain cardiac output. The most prominent such mechanism is the sympathetic (adrenergic) nervous system (SNS), whose activity and outflow are greatly elevated in HF. SNS hyperactivity confers significant toxicity to the failing heart and markedly increases HF morbidity and mortality via excessive activation of adrenergic receptors, which are G protein-coupled receptors. Thus, ligand binding induces their coupling to heterotrimeric G proteins that transduce intracellular signals. G protein signaling is turned-off by the agonist-bound receptor phosphorylation courtesy of G protein-coupled receptor kinases (GRKs), followed by βarrestin binding, which prevents the GRK-phosphorylated receptor from further interaction with the G proteins and simultaneously leads it inside the cell (receptor sequestration). Recent evidence indicates that adrenal GRK2 and βarrestins can regulate adrenal catecholamine secretion, thereby modulating SNS activity in HF. The present review gives an account of all these studies on adrenal GRKs and βarrestins in HF and discusses the exciting new therapeutic possibilities for chronic HF offered by targeting these proteins pharmacologically.

Cardiac βarrestin2 Improves Contractility and Adverse Remodeling in Heart Failure, But Is Underexpressed in Humans

The 2 βarrestin isoforms, βarrestin1 and βarrestin2, normally decrease cardiac function and exacerbate post-myocardial infarction (MI) heart failure (HF) by desensitizing the procontractile G protein – dependent signaling of cardiac β1-adrenergic receptors [(1)][1]. Although βarrestin1

Co-IP assays for measuring GPCR–arrestin interactions

βarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The βarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latters roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR-arrestin interaction in cells.

Biased Agonism/Antagonism of Cardiovascular GPCRs for Heart Failure Therapy

G protein-coupled receptors (GPCRs) are among the most important drug targets currently used in clinic, including drugs for cardiovascular indications. We now know that, in addition to activating heterotrimeric G protein-dependent signaling pathways, GPCRs can also activate G protein-independent signaling, mainly via the βarrestins. The major role of βarrestin1 and -2, also known as arrestin2 or -3, respectively, is to desensitize GPCRs, i.e., uncoupled them from G proteins, and to subsequently internalize the receptor. As the βarrestin-bound GPCR recycles inside the cell, it serves as a signalosome transducing signals in the cytoplasm. Since both G proteins and βarrestins can transduce signals from the same receptor independently of each other, any given GPCR agonist might selectively activate either pathway, which would make it a biased agonist for that receptor. Although this selectivity is always relative (never absolute), in cases where the G protein- and βarrestin-dependent signals emanating from the same GPCR result in different cellular effects, pharmacological exploitation of GPCR-biased agonism might have therapeutic potential. In this chapter, we summarize the GPCR signaling pathways and their biased agonism/antagonism examples discovered so far that can be exploited for heart failure treatment. We also highlight important issues that need to be clarified along the journey of these ligands from bench to the clinic.