Anastasios Lymperopoulos

Adrenergic Nervous System in Heart Failure Pathophysiology and Therapy

Heart failure (HF), the leading cause of death in the western world, develops when a cardiac injury or insult impairs the ability of the heart to pump blood and maintain tissue perfusion. It is characterized by a complex interplay of several neurohormonal mechanisms that become activated in the syndrome to try and sustain cardiac output in the face of decompensating function. Perhaps the most prominent among these neurohormonal mechanisms is the adrenergic (or sympathetic) nervous system (ANS), whose activity and outflow are enormously elevated in HF. Acutely, and if the heart works properly, this activation of the ANS will promptly restore cardiac function. However, if the cardiac insult persists over time, chances are the ANS will not be able to maintain cardiac function, the heart will progress into a state of chronic decompensated HF, and the hyperactive ANS will continue to push the heart to work at a level much higher than the cardiac muscle can handle. From that point on, ANS hyperactivity becomes a major problem in HF, conferring significant toxicity to the failing heart and markedly increasing its morbidity and mortality. The present review discusses the role of the ANS in cardiac physiology and in HF pathophysiology, the mechanisms of regulation of ANS activity and how they go awry in chronic HF, methods of measuring ANS activity in HF, the molecular alterations in heart physiology that occur in HF, along with their pharmacological and therapeutic implications, and, finally, drugs and other therapeutic modalities used in HF treatment that target or affect the ANS and its effects on the failing heart.

Myocardial Adeno-Associated Virus Serotype 6–βARKct Gene Therapy Improves Cardiac Function and Normalizes the Neurohormonal Axis in Chronic Heart Failure

Background— The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional &bgr;-adrenergic receptor (&bgr;AR) signaling and cardiac function. The peptide &bgr;ARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve &bgr;AR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term &bgr;ARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results— In HF rats, we delivered &bgr;ARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the &bgr;-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. &bgr;ARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac &bgr;AR signaling. Addition of metoprolol neither enhanced nor decreased &bgr;ARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions— Long-term cardiac AAV6-&bgr;ARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, &bgr;ARKct alone improves outcomes more than a &bgr;-blocker alone, whereas both treatments are compatible. These findings show that &bgr;ARKct gene therapy can be of long-term therapeutic value in HF.

Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of β-adrenergic receptor (βAR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting α2AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial α2AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein–coupled receptor kinase 2 (GRK2). Adrenal gland–specific GRK2 inhibition reversed α2AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac βAR signaling and function, and increased sympatholytic efficacy of a α2AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target.

Reduction of sympathetic activity via adrenal-targeted GRK2 gene deletion attenuates heart failure progression and improves cardiac function after myocardial infarction

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell α2-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function. Herein, we hypothesized that adrenal-targeted GRK2 gene deletion before the onset of HF might be beneficial by reducing sympathetic activation. To specifically delete GRK2 in the chromaffin cells of the adrenal gland, we crossed PNMTCre mice, expressing Cre recombinase under the chromaffin cell-specific phenylethanolamine N-methyltransferase (PNMT) gene promoter, with floxedGRK2 mice. After confirming a significant (∼50%) reduction of adrenal GRK2 mRNA and protein levels, the PNMT-driven GRK2 knock-out (KO) offspring underwent myocardial infarction (MI) to induce HF. At 4 weeks post-MI, plasma levels of both norepinephrine and epinephrine were reduced in PNMT-driven GRK2 KO, compared with control mice, suggesting markedly reduced post-MI sympathetic activation. This translated in PNMT-driven GRK2 KO mice into improved cardiac function and dimensions as well as amelioration of abnormal cardiac β-adrenergic receptor signaling at 4 weeks post-MI. Thus, adrenal-targeted GRK2 gene KO decreases circulating CAs, leading to improved cardiac function and β-adrenergic reserve in post-MI HF. GRK2 inhibition in the adrenal gland might represent a novel sympatholytic strategy that can aid in blocking HF progression.

An adrenal β-arrestin 1-mediated signaling pathway underlies angiotensin II-induced aldosterone production in vitro and in vivo

Aldosterone produces a multitude of effects in vivo, including promotion of postmyocardial infarction adverse cardiac remodeling and heart failure progression. It is produced and secreted by the adrenocortical zona glomerulosa (AZG) cells after angiotensin II (AngII) activation of AngII type 1 receptors (AT1Rs). Until now, the general consensus for AngII signaling to aldosterone production has been that it proceeds via activation of Gq/11-proteins, to which the AT1R normally couples. Here, we describe a novel signaling pathway underlying this AT1R-dependent aldosterone production mediated by β-arrestin-1 (βarr1), a universal heptahelical receptor adapter/scaffolding protein. This pathway results in sustained ERK activation and subsequent up-regulation of steroidogenic acute regulatory protein, a steroid transport protein regulating aldosterone biosynthesis in AZG cells. Also, this βarr1-mediated pathway appears capable of promoting aldosterone turnover independently of G protein activation, because treatment of AZG cells with SII, an AngII analog that induces βarr, but not G protein coupling to the AT1R, recapitulates the effects of AngII on aldosterone production and secretion. In vivo, increased adrenal βarr1 activity, by means of adrenal-targeted adenoviral-mediated gene delivery of a βarr1 transgene, resulted in a marked elevation of circulating aldosterone levels in otherwise normal animals, suggesting that this adrenocortical βarr1-mediated signaling pathway is operative, and promotes aldosterone production and secretion in vivo, as well. Thus, inhibition of adrenal βarr1 activity on AT1Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperaldosteronism.

Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure.

Exercise training has been reported to exert beneficial effects on cardiac function and to reduce morbidity and mortality of chronic heart failure (HF). Augmented sympathetic nervous system (SNS) activity, leading to elevated circulating catecholamine (CA) levels, is a hallmark of chronic HF that significantly aggravates this disease. Exercise training has been shown to also reduce SNS overactivity in HF, but the underlying molecular mechanism(s) remain unidentified. We recently reported that adrenal G protein-coupled receptor kinase-2 (GRK2), an enzyme that regulates the sympathoinhibitory alpha(2)-adrenoceptors (alpha(2)-ARs) present in the CA-producing adrenal medulla, is upregulated in HF, contributing to the chronically elevated CA levels and SNS activity of the disease. In the present study, we tested whether exercise training can affect the adrenal GRK2-alpha(2)-AR-CA production system in the context of HF. For this purpose, a 10-wk-long exercise training regimen of adult male Sprague-Dawley rats starting at 4 wk postmyocardial infarction (post-MI) was employed, and examination at the end of this treatment period revealed significant amelioration of beta-AR-stimulated contractility in response to exercise training, accompanied by cardiac GRK2 reduction and restoration of circulating plasma CA levels. Importantly, adrenal GRK2 expression (72 + or - 5% reduction vs. post-MI untrained) and alpha(2)-AR number were also restored after exercise training in post-MI animals. These results suggest that exercise training restores the adrenal GRK2-alpha(2)-AR-CA production axis, and this might be part of the mechanism whereby this therapeutic modality normalizes sympathetic overdrive and impedes worsening of the failing heart.

Negative Impact of β-Arrestin-1 on Post-Myocardial Infarction Heart Failure via Cardiac and Adrenal-Dependent Neurohormonal Mechanisms

&bgr;-Arrestin (&bgr;arr)-1 and &bgr;-arrestin-2 (&bgr;arrs) are universal G-protein–coupled receptor adapter proteins that negatively regulate cardiac &bgr;-adrenergic receptor (&bgr;AR) function via &bgr;AR desensitization and downregulation. In addition, they mediate G-protein–independent &bgr;AR signaling, which might be beneficial, for example, antiapoptotic, for the heart. However, the specific role(s) of each &bgr;arr isoform in cardiac &bgr;AR dysfunction, the molecular hallmark of chronic heart failure (HF), remains unknown. Furthermore, adrenal &bgr;arr1 exacerbates HF by chronically enhancing adrenal production and hence circulating levels of aldosterone and catecholamines. Herein, we sought to delineate specific roles of &bgr;arr1 in post–myocardial infarction (MI) HF by testing the effects of &bgr;arr1 genetic deletion on normal and post-MI cardiac function and morphology. We studied &bgr;arr1 knockout (&bgr;arr1KO) mice alongside wild-type controls under normal conditions and after surgical MI. Normal (sham-operated) &bgr;arr1KO mice display enhanced &bgr;AR-dependent contractility and post-MI &bgr;arr1KO mice enhanced overall cardiac function (and &bgr;AR-dependent contractility) compared with wild type. Post-MI &bgr;arr1KO mice also show increased survival and decreased cardiac infarct size, apoptosis, and adverse remodeling, as well as circulating catecholamines and aldosterone, compared with post-MI wild type. The underlying mechanisms, on one hand, improved cardiac &bgr;AR signaling and function, as evidenced by increased &bgr;AR density and procontractile signaling, via reduced cardiac &bgr;AR desensitization because of cardiac &bgr;arr1 absence, and, on the other hand, decreased production leading to lower circulating levels of catecholamines and aldosterone because of adrenal &bgr;arr1 absence. Thus, &bgr;arr1, via both cardiac and adrenal effects, is detrimental for cardiac structure and function and significantly exacerbates post-MI HF.

Modulation of adrenal catecholamine secretion by in vivo gene transfer and manipulation of G protein-coupled receptor kinase-2 activity

We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.

Cardiomyocyte lipids impair β-adrenergic receptor function via PKC activation.

Normal hearts have increased contractility in response to catecholamines. Because several lipids activate PKCs, we hypothesized that excess cellular lipids would inhibit cardiomyocyte responsiveness to adrenergic stimuli. Cardiomyocytes treated with saturated free fatty acids, ceramide, and diacylglycerol had reduced cellular cAMP response to isoproterenol. This was associated with increased PKC activation and reduction of β-adrenergic receptor (β-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced β-AR insensitivity and the accompanying reduction in cell surface β-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARγ, or acyl-CoA synthetase-1 or high-fat diet showed reduced inotropic responsiveness to dobutamine. This was associated with activation of protein kinase C (PKC)α or PKCδ. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in β-AR responsiveness. This can be attributed to PKC activation and reduced β-AR levels.

Targeting the β-Adrenergic Receptor System Through G-Protein–Coupled Receptor Kinase 2: A New Paradigm for Therapy and Prognostic Evaluation in Heart Failure From Bench to Bedside

G-protein–coupled receptors (GPCRs) are a superfamily of more than 1000 membrane proteins that respond to a wide spectrum of extracellular signals, modulating various physiopathological processes.1,2 Several GPCRs, such as adrenergic, angiotensin, endothelin, and adenosine receptors, are expressed in cardiovascular (CV) tissues to maintain CV homeostasis. Importantly, GPCR-mediated adrenergic deregulation has been shown to both cause and contribute to the onset and progression of major CV diseases ultimately leading to heart failure (HF). Thus, GPCRs have become salient targets of current pharmacotherapy in CV disorders, and in past decades, many efforts have been made to better clarify their role in the pathophysiology of cardiac disease, focusing not only on receptor functions but also on postreceptor components that mediate or regulate their responses. Among the latter, a relevant role has been attributed to G-protein–coupled receptor kinases (GRKs). In this review, we focus on GRK2, the most abundant and versatile GRK expressed on CV system, tracing the way from initial experimental evidence to more recent data suggesting a potential role for this kinase in the clinical management of HF.1,2 ### GPCR Signaling and GRK Functions: Pathophysiological Background On stimulation, GPCRs interact with heterotrimeric G proteins, which in turn dissociate into 2 functional monomers, namely Gα and Gβγ, both of which modulate different effector systems. Agonist binding to GPCR promotes the activation of complex regulatory mechanisms to protect the receptor from both acute and chronic stimulation, a process termed desensitization. As extensively described, GPCR desensitization involves 3 main events in chronological order: receptor phosphorylation and uncoupling from G proteins, internalization of membrane-bound receptors, and downregulation through reduced mRNA and protein synthesis or increased degradation of internalized receptors.1,2 The desensitization process is mediated by 3 families of proteins: second-messenger–dependent protein kinases, GRKs and arrestins. The defining feature of GRKs is that …