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In the above article [Cannavo A, Liccardo D, Lymperopoulos A, Gambino G, D’Amico ML, Rengo F, Koch WJ, Leosco D, Ferrara N, and Rengo G (2016) J Pharmacol Exp Ther 356(2): 503–513; DOI: 10.1124/jpet.115.228411], the following funding information was omitted: This work was funded by the National Institutes of Health [Grant R37 HL061690]. The authors regret this error and any inconvenience it may have caused.

Possible role of VEGF/Akt transduction pathway after beta-2 adrenergic receptors overexpression-promoted angiogenesis.

Exercise upregulates beta-adrenergic receptor expression and this, in turn, stimulates angiogenesis.

Angiogenesis in the failing heart is stimulated beta-1 AR blockade and cardiac beta-2 AR overexpression.

Heart failure is accompanied by severely impaired β-adrenergic receptor (βAR) function, which includes loss of βAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of βAR function is agonist-stimulated receptor phosphorylation by the βAR kinase (βARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in βAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of βARK1 or the β2AR were mated into a genetic model of murine heart failure (MLP−/−). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP−/− and MLP−/−/β2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP−/−/βARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP−/−/βARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP−/− mice but less than controls. Importantly, heightened βAR desensitization in the MLP−/− mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the βARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal βAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit βARK1 as a novel mode of therapy.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and they are responsible for the transduction of extracellular signals, regulating almost all aspects of mammalian physiology. These receptors are specifically regulated by a family of serine/threonine kinases, called GPCR kinases (GRKs). Given the biological role of GPCRs, it is not surprising that GRKs are also involved in several pathophysiological processes. Particular importance is emerging for GRK5, which is a multifunctional protein, expressed in different cell types, and it has been found located in single or multiple subcellular compartments. For instance, when anchored to the plasma membrane, GRK5 exerts its canonical function, regulating GPCRs. However, under certain conditions (e.g., pro-hypertrophic stimuli), GRK5 translocates to the nucleus of cells where it can interact with non-GPCR-related proteins as well as DNA itself to promote “non-canonical” signaling, including gene transcription. Importantly, due to these actions, several studies have demonstrated that GRK5 has a pivotal role in the pathogenesis of chronic-degenerative disorders. This is true in the cardiac cells, tumor cells, and neurons. For this reason, in this review article, we will inform the readers of the most recent evidence that supports the importance of targeting GRK5 to prevent the development or progression of cancer, cardiovascular, and neurological diseases.

BACKGROUND: Increased cardiac G protein-coupled receptor kinase-2 (GRK2) expression has a pivotal role at inducing heart failure (HF)-related β-adrenergic receptor (βAR) dysfunction. Importantly, abnormalities of βAR signalling in the failing heart, including GRK2 overexpression, are mirrored in circulating lymphocytes and correlate with HF severity. Exercise training has been shown to exert several beneficial effects on the failing heart, including normalization of cardiac βAR function and GRK2 protein levels. In the present study, we evaluated whether lymphocyte GRK2 levels and short-term changes of this kinase after an exercise training programme can predict long-term survival in HF patients. METHODS: For this purpose, we prospectively studied 193 HF patients who underwent a 3-month exercise training programme. Lymphocyte GRK2 protein levels, plasma N-terminal pro-brain natriuretic peptide, and norepinephrine were measured at baseline and after training along with clinical and functional parameters (left ventricular ejection fraction, NYHA class, and peak-VO2). Cardiac-related mortality was evaluated during a mean follow-up period of 37 ± 20 months. RESULTS: Exercise was associated with a significant reduction of lymphocyte GRK2 protein levels (from 1.29 ± 0.52 to 1.16 ± 0.65 densitometric units, p

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.

We investigated whether β(2) -adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart.We explored the angiogenic effects of β(2) -adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated β(2) -adrenoceptor overexpression was obtained via direct intramyocardial injection 4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observation to β(2) -adrenoceptor -/- mice undergoing MI.Transgenes were robustly expressed in the LV at 2 weeks post-gene therapy, whereas their expression was minimal at 4-weeks post-gene delivery. In HF rats, cardiac β(2) -adrenoceptor overexpression resulted in enhanced basal and isoprenaline-stimulated cardiac contractility at 2-weeks post-gene delivery. At 4 weeks post-gene transfer, Ad-β(2) -adrenoceptor HF rats showed improved LV remodeling and cardiac function. Importantly, β(2) -adrenoceptor overexpression was associated with a markedly increased capillary and arteriolar length density and enhanced in vivo myocardial blood flow and coronary reserve. At the molecular level, cardiac β(2) -adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway. In β(2) -adrenoceptor-/- mice, we found a ~25% reduction in cardiac capillary density compared with β(2) -adrenoceptor+/+ mice. The lack of β(2) -adrenoceptors was associated with a higher mortality rate at 30 days and LV dilatation, and a worse global cardiac contractility compared with controls.β(2) -Adrenoceptors play an important role in the regulation of the angiogenic response in HF. The activation of VEGF/PKB/eNOS pathway seems to be strongly involved in this mechanism.

Objectives We investigated whether adrenal beta-arrestin 1 (βarr1)-mediated aldosterone production plays any role in post-myocardial infarction (MI) heart failure (HF) progression. Background Heart failure represents 1 of the most significant health problems worldwide, and new and innovative treatments are needed. Aldosterone contributes significantly to HF progression after MI by accelerating adverse cardiac remodeling and ventricular dysfunction. It is produced by the adrenal cortex after angiotensin II activation of angiotensin II type 1 receptors (AT 1 Rs), G protein-coupled receptors that also signal independently of G proteins. The G protein-independent signaling is mediated by βarr1 and βarr2. We recently reported that adrenal βarr1 promotes AT 1 R-dependent aldosterone production leading to elevated circulating aldosterone levels in vivo. Methods Adrenal-targeted, adenoviral-mediated gene delivery in vivo in 2-week post-MI rats, a time point around which circulating aldosterone significantly increases to accelerate HF progression, was performed to either increase the expression of adrenal βarr1 or inhibit its function via expression of a βarr1 C-terminal-derived peptide fragment. Results We found that adrenal βarr1 overexpression promotes aldosterone elevation after MI, resulting in accelerated cardiac adverse remodeling and deterioration of ventricular function. Importantly, these detrimental effects of aldosterone are prevented when adrenal βarr1 is inhibited in vivo, which markedly decreases circulating aldosterone after MI. Finally, the prototypic AT 1 R antagonist losartan seems unable to lower this adrenal βarr1-driven aldosterone elevation. Conclusions Adrenal βarr1 inhibition, either directly or with AT 1 R “biased” antagonists that prevent receptor-βarr1 coupling, might be of therapeutic value for curbing HF-exacerbating hyperaldosteronism.

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 alpha(2)-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 ( approximately 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 beta-adrenergic receptor signaling at 4 weeks post-MI. Thus, adrenal-targeted GRK2 gene KO decreases circulating CAs, leading to improved cardiac function and beta-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.

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 (AT 1 Rs). Until now, the general consensus for AngII signaling to aldosterone production has been that it proceeds via activation of G q/11 -proteins, to which the AT 1 R normally couples. Here, we describe a novel signaling pathway underlying this AT 1 R-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 AT 1 R, 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 AT 1 Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperaldosteronism.

Background— The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function. The peptide βARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve βAR 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 βARKct 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 βARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the β-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. βARKct 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 βAR signaling. Addition of metoprolol neither enhanced nor decreased βARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions— Long-term cardiac AAV6-βARKct 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, βARKct alone improves outcomes more than a β-blocker alone, whereas both treatments are compatible. These findings show that βARKct gene therapy can be of long-term therapeutic value in HF.

Heart failure (HF) still poses an enormous clinical challenge, as its incidence, morbidity, and mortality rates are continuously rising. G protein-coupled receptors (GPCRs) constitute the most ubiquitous superfamily of plasma membrane receptors and represent the single most important type of therapeutic drug target. Because there is overstimulation of the failing heart by various endogenous ligands, such as catecholamines and angiotensin II--which by activating their cognate GPCRs in cardiac muscle induce detrimental effects--therapeutic targeting of these receptors has been pursued. This research has led to the development of successful and useful drug classes, such as angiotensin-converting enzyme inhibitors and beta-adrenergic receptor blockers. However, there still is a need to develop innovative treatments that might be more effective at reversing compromised myocyte function. Over the past several years, much evidence has accumulated indicating that a single GPCR, activated by the same endogenous ligand, can elicit several different signaling pathways with quite different, and often opposite, cellular effects. Because the aforementioned ligands, currently used for HF, target these receptors on their extracellular interface, thus merely preventing the endogenous agonists from binding the receptor, they inhibit all the signaling pathways elicited by the receptor indiscriminately. Importantly, several of these pathways emanating from the same GPCR can actually be beneficial for therapy, so their enhancement rather than their blockade is desirable for HF therapy. This highlights the need for selective targeting of GPCR-induced signaling pathways on the intracellular interface of the receptor, which might produce new and innovative therapies for cardiovascular disease.

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.

Myocardial G protein–coupled receptor kinase (GRK)2 is a critical regulator of cardiac β-adrenergic receptor (βAR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated βAR with a GRK2-derived peptide that binds G β γ (βARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and βARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (αMHC-Cre×GRK2 fl/fl) or only after administration of tamoxifen (αMHC-MerCreMer×GRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved βAR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring βAR signaling by other means to improve outcomes in heart failure.

Aims The G protein-coupled receptor kinase-2 (GRK2 or b-ARK1) regulates b-adrenergic receptors (b-ARs) in the heart, and its cardiac expression is elevated in human heart failure (HF). We sought to determine whether myocardial levels and activity of GRK2 could be monitored using white blood cells, which have been used to study cardiac b-ARs. Moreover, we were interested in determining whether GRK2 levels in myocardium and lymphocytes may be associated with b-AR dysfunction and HF severity. Methods and results In myocardial biopsies from explanted failing human hearts, GRK activity was inversely correlated with b-AR-mediated cAMP production (R 2 ¼ 20.215, P , 0.05, n ¼ 24). Multiple regression analysis confirmed that GRK activity participates with b-AR density to regulate catecholaminesensitive cAMP responses. Importantly, there was a direct correlation between myocardial and lymphocytes GRK2 activity (R 2 ¼ 0.5686, P , 0.05, n ¼ 10). Lymphocyte GRK activity was assessed in HF patients with various ejection fractions (EFs) (n ¼ 33), and kinase activity was significantly higher in patients with lower EFs and was higher with increasing NYHA class (P , 0.001). Conclusion Myocardial GRK2 expression and activity are mirrored by lymphocyte levels of this kinase, and its elevation in HF is associated with the loss of b-AR responsiveness and appears to increase with disease severity. Therefore, lymphocytes may provide a surrogate for monitoring cardiac GRK2 in human HF.

Exercise training and beta-blocker treatment ameliorate age-dependent impairment of beta-adrenergic receptor signaling and enhance cardiac responsiveness to adrenergic stimulation. Am J Physiol Heart Circ Physiol 293: H1596–H1603, 2007. First published June 8, 2007; doi:10.1152/ajpheart.00308.2007.— Cardiac beta-adrenergic receptor (*-AR) signaling and left ventricular (LV) responses to beta-AR stimulation are impaired with aging. It is shown that exercise and beta-AR blockade have a favorable effect on cardiac and vascular *-AR signaling in several cardiovascular diseases. In the present study, we examined the effects of these two different strategies on *-AR dysregulation and LV inotropic reserve in the aging heart. Forty male Wistar-Kyoto aged rats were randomized to sedentary, exercise (12 wk treadmill training), metoprolol (250 mg kg-1 day-1 for 4 wk), and exercise plus metoprolol treatment protocols. Ten male Wistar- Kyoto sedentary young rats were also used as a control group. Old trained, old metoprolol-treated, and old trained plus metoprololtreated rats showed significantly improved LV maximal and minimal first derivative of the pressure rise responses to beta-AR stimulation (isoproterenol) compared with old untrained animals. We found a significant reduction in cardiac sarcolemmal membrane beta-AR density and adenylyl cyclase activity in old untrained animals compared with young controls. Exercise training and metoprolol, alone or combined, restored cardiac beta-AR density and G-protein-dependent adenylyl cyclase activation in old rats. Although cardiac membrane G-proteinreceptor kinase 2 levels were not upregulated in untrained old compared with young control rats, both exercise and metoprolol treatment resulted in a dramatic reduction of G-protein-receptor kinase 2 protein levels, which is a further indication of beta-AR signaling amelioration in the aged heart induced by these treatment modalities. In conclusion, we demonstrate for the first time that exercise and beta-AR blockade can similarly ameliorate beta-AR signaling in the aged heart, leading to improved beta-AR responsiveness and corresponding LV inotropic reserve.