Introduction
Cardiovascular diseases (CVD) involve heart and blood vessels and include coronary artery disease (CAD), stroke, hypertension, congenital heart disease, cardiomyopathy, etc. [1]. CVD is the leading cause of death worldwide that accounts for more than 17.3 million deaths per year [2]. In 2013, CVD represented about one of every three deaths in America. Over 85 million Americans are living with some type of CVD or the after-effects of stroke. Projected costs of CVD including the cost of health care services, medications and loss of productivity totals more than $600 billion in 2015, and it is expected to grow to more than $1200 billion by 2030 [3].
Heart failure (HF) is a chronic, progressive condition in which the heart muscle is unable to pump enough blood to meet the body’s metabolic demands. HF arises as the final manifestation of many CVDs such as coronary artery disease, congenital malformations and hypertension. About 5.7 million adults in the United States are affected by this debilitating disease [3]; HF treatment costs the nation an estimated $30.7 billion each year [4]. Notwithstanding significant advances in HF treatment and management realized with β-adrenergic receptor (β-AR) blockers, angiotensin receptor blockers, angiotensin converting enzyme (ACE) inhibitors, aldosterone inhibitors, and diuretics, conventional pharmacological therapies only impede the progression and death due to HF, but do not cure it causatively [5]. Taking into consideration the steady growth of aging and diabetic populations, deeper understanding of the molecular and cellular processes that contribute to the disease pathogenesis, along with development of innovative therapeutic strategies allowing the causative cure of HF, are indispensable.
Multiple pathophysiological mechanisms contribute to HF development and progression, including neurohumoral activation [6], G-protein-coupled receptor desensitization and down-regulation [7], [8] and [9], and extracellular matrix-mediated pathologic remodeling [10]. Moreover, cardiac pathologies in HF are frequently accompanied by worsening renal function, which is known to be a strong predictor of increased mortality in HF patients [11] and [12]; this is defined as Cardiorenal syndrome (CRS) type II. In the present review, we explore recent advances in exploring GPCR signaling as a possible therapeutic target in cardiac disease and as a potential link between failing heart and kidney, with the particular emphasis on small molecule targeting of G-protein βγ subunit – GPCR kinase 2 (Gβγ-GRK2) components of GPCR signaling.
2. G-protein-coupled receptor signaling.
G-protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, represent a conserved family of receptors that sense molecules outside the cell that activate intracellular signal transduction pathways and consecutive cellular responses. GPCRs are integral proteins comprised of an extracellular N-terminus, seven transmembrane (7-TM) α-helixes (TM-1 to TM-7) connected by three intracellular (IL-1 to IL-3) and three extracellular loops (EL-1 to EL-3), and an intracellular C-terminus [13]. Ligand binding to an extracellular active site of the receptor induces a conformational change in the GPCR which allows for coupling with heterotrimeric guanine-nucleotide regulatory proteins (G-proteins) [14]. G-proteins are heterotrimers of α, β and γ subunits known as Gα, Gβ and Gγ, respectively. The heterotrimeric G-proteins are rendered inactive when reversibly bound to Guanosine diphosphate (GDP) but active when bound to Guanosine triphosphate (GTP) [15]. Receptor activation facilitates the exchange of GDP for GTP on Gα subunits that result in dissociation of the Gα from Gβγ subunits to mediate downstream signaling pathways [16]. Dissociated Gα subunits signal via activation of an effector molecule, such as adenyl cyclase (AC) or phospholipase C β (PLCβ) to produce second messengers such as cyclic adenosine 3′, 5′ monophosphate (cAMP), diacylglycerol (DAG), or inositol 1, 4, 5-triphosphate (IP3), respectively. These second messengers modulate a variety of downstream processes, particularly regulation of contractility, hypertrophy, and apoptosis in the heart [15]. Gα proteins are classified into the families Gαs, Gαi, Gαq, and Gα11/12 [15] with respect to downstream signaling molecules and modulated physiological processes. Dissociated Gβγ subunits target a wide range of signaling pathways involved in receptor desensitization and down-regulation, ion channel activation, enzyme activity modulation, cell division, transcription and cellular organelle function [17], [18], [19] and [20].
GPCRs respond to extracellular signaling mediated by an extensive amount of agonists such as hormones, proteins and lipids, and participate in a comprehensive variety of physiological processes [21]. In particular, GPCRs play an important role in local and systemic regulation of cardiac function. Specifically, cardiac β-adrenergic receptors (β-ARs) are prominent regulators of cardiovascular chronotropy and inotropy [22] and [23]. Furthermore, GPCRs mediate a variety of functions in the kidney, and inappropriate activation and regulation of GPCRs may lead to kidney disease [24]. In this review, we focus on Gβγ-mediated signaling as a crucial component of HF pathogenesis and as a potential therapeutic target in cardiorenal pathologies.
3. β-Adrenergic receptor signaling in healthy and diseased heart.
As mentioned above, cardiac β-ARs represent crucial regulators of cardiac contractile function. In response to sympathetic nervous system (SNS) activity released via mediators, catecholamines (CA) epinephrine (Epi, also named adrenaline) and norepinephrine (NEpi, also named noradrenaline), β-ARs modulate the rate and force of myocardial contractions [8]. There are three β-AR subtypes identified in mammalian hearts: β1, β2, and β3-ARs [25]. Both β1- and β2-ARs are coupled to the downstream excitatory Gαs protein, which generally results in the activation of adenylyl cyclase (AC) and the generation of cyclic AMP (cAMP), eliciting positive chronotropic and inotropic responses. Upon chronic stimulation, β2-ARs also couple to the inhibitory Gαi protein, which has been reported to exert a cardioprotective effect during cardiac injury [26].
In healthy human myocardium, the predominant β-ARs subtypes are the β1- and β2-ARs, which are present in an approximate 80:20 ratio, respectively with only a relatively minor contribution of β3-ARs [27]. Under physiological conditions, β-ARs account for regulation of both heart rate and contractility [14, 28]. In HF pathogenesis, excess SNS activation and subsequent catecholamine overdrive is initiated as an adaptation to compensate for decreased heart rate and cardiac contractility and to maintain mean arterial pressure (MAP) [29]. Initially, the elevated SNS activity increases heart rate and contractility through β-AR stimulation. However, maladaptive effects of the elevated SNS activity including myocardial ischemia, pathologic hypertrophy, arrhythmogenicity, myocardial necrosis and apoptosis contribute substantially to disease progression [22], [30], [31] and [32]. This maladaptive response results partially from down-regulation and desensitization of cardiac β-ARs due to chronic CA stimulation [15]. In failing hearts, heightened CA β-AR stimulation induces selective down-regulation of β1-ARs and consequent alteration of the β1-AR to β2-AR ratio from an 80:20 distribution to a ratio of 60:40 [27, 33]; the remaining β1-ARs and β2-ARs in failing hearts prevail in a desensitized condition [30].
Cardiac β-AR signaling regulation involves activation-dependent and -independent mechanisms of desensitization [8]. Homologous, agonist-mediated, activation-dependent desensitization is accomplished by an active form of a G-protein-coupled receptor kinase (GRK) that is translocated to the adrenergic receptor after binding with the activated membrane-associated Gβγ subunit to phosphorylate the agonist-occupied receptor [34]. An alternative, activation-independent pathway, known as heterologous desensitization, is accomplished through the activity of a downstream signaling product of β-AR activation or other GPCR signaling events. In both cases, phosphorylated β-AR is bound by β-arrestin molecules which block the access of heterotrimeric G proteins to the receptor thereby uncoupling it and attenuating β-AR signaling in the heart [35, 36].
4. Gβγ-GRK2 signaling manipulation as a strategy to treat cardiac disease
4.1. GRK2: structure, subcellular localization and function in the heart
GRK2 (aka β-adrenergic receptor kinase, βARK) belongs to a family of serine/threonine kinases that share common structural and functional features. Seven mammalian GRKs that have been characterized so far are classified into three subfamilies according to their sequence and structural similarity: (1) the rhodopsin kinase subfamily (GRK1 and GRK7); (2) the βARK subfamily (GRK2 and GRK3); and (3) the GRK4-like subfamily (GRK4, GRK5, GRK6) [37]. Within the cardiovascular system, GRKs 2, 3 and 5 are known to be expressed and play a role in GPCR phosphorylation [38], with GRK2 as a predominant GRK isoform in the heart [39].
GRKs are characterized by a tri-domain structure, with the conserved central catalytic domain and two flanking domains variable in structure in different GRK subfamilies [40]. GRK2’s amino (N)-terminal domain that is responsible for receptor recognition and activity regulation contains a regulator of G protein signaling (RGS) homology (RH) domain that has been demonstrated to interact with Gαq proteins [41]. The carboxyl (C)-terminal domain of GRK2 determines membrane targeting and subcellular localization of the enzyme. This domain contains a pleckstrin homology (PH) domain that binds Gβγ subunits [42]. Under basal conditions, GRK2 is distributed primarily in the cytoplasm. Upon GPCR activation, GRK2 is translocated to the plasma membrane via binding with the activated Gβγ subunits. GRK2-mediated phosphorylation of the GPCR causes β-arrestin recruitment to the receptor and consequent inhibition of dissociated G-proteins from coupling to the receptor/β-arrestin complex and further attenuation of downstream signaling [43]. Moreover, β-arrestin-bound receptors are targeted for clathrin-coated pits in the cell membrane that are internalized and either degraded in intracellular lysosomes or recycled back to the cell surface [44].
Apart from the classical mechanism of modulating GPCR signaling in the heart and extracardiac tissues, GRK2 may have other functions independent of GPCR phosphorylation. Recently emerging data suggest the concept of an extensive “GRK2 interactome” that refers to GRK2 interactions with other intracellular proteins such as α-actinin, clathrin, calmodulin, caveolin, tubulin, Akt, HDAC6 and ERK1/2 [39] and [45]. Investigation of GRK2 functions beyond GPCR desensitization and down-regulation may provide new insights in understanding its role in disease pathogenesis. In the current review, we highlight recent updates relevant for GPCR- Gβγ signaling in HF modulation.
Understanding of the in vivo function of GRK2, particularly its role in cardiovascular system function and development, emerged from gene knockout studies. In 1996, Jaber et al demonstrated lethality of GRK2 homozygous knockout (KO) in mouse models [46]. These animals exhibited hypoplasia of the ventricular myocardium and a 70% decrease in ejection fraction and died by gestational day 15.5, presumably owing to HF. Further studies demonstrated that specific deletion of GRK2 in murine embryonic cardiomyocytes utilizing Cre recombinase expressed under the control of the Nkx2.5 promoter did not cause any apparent developmental abnormalities, suggesting that embryonic lethality of GRK2−/− mice might result from extracardiac or non-cardiomyocyte effects [47].
Cardiomyocytes from adult global heterozygous GRK2 KO mice exhibited significantly enhanced cardiac contractile function compared to wild-type cells [48]. Cardiac-specific overexpression of GRK2 following myocardial ischemia/reperfusion injury (I/R) caused reduced β-adrenergic signaling mediated cardiac contractility and function along with increased apoptosis [49]. These observations demonstrated that cardiac contractile function can be modulated by the level of GRK2 activity. To further evaluate the role of GRK2 in adult cardiac function, two conditional models of GRK2 ablation were generated: αMHC-Cre/GRK2flox/flox for targeted KO of GRK2 specifically in cardiomyocytes in the constitutive way (at birth) and αMHCMerCreMer/GRK2flox/flox for tamoxifen-induced cardic deletion [50]. Both models resulted in positive outcomes following cardiomyocyte-restricted GRK2 ablation post-MI; the αMHC-Cre/GRK2flox/flox mice exhibited prevention of HF development after MI and the αMHCMerCreMer/GRK2flox/flox mice demonstrated improved cardiac function and induced positive reverse remodeling following MI. Moreover, cardiomyocyte GRK2 KO in post-MI mice showed reduced mortality levels. Cardioprotective effects demonstrated by αMHCMerCreMer/GRK2flox/flox mice were significantly better compared to the results when the β-blocker metoprolol was used for treatment of post-MI wild-type (WT) mice over the same time period [50]. Thus, GRK2 gene knockout studies elicited a proposal that GRK2 should be considered as a therapeutic target for HF treatment.
4.2. GRK2 expression in cardiac disease
The first link between GRK2 and β-adrenergic receptor signaling desensitization and down-regulation was established in 1993, when Ungerer et al demonstrated significant elevation of GRK2 in explanted failing human hearts at mRNA, protein and activity levels [33]. These observations initiated a series of studies performed on animal models or human tissue that aimed to delineate the role of GRK2 in cardiac disease [51]. Particularly, cardiac overexpression of GRK2 was demonstrated to be capable of direct HF induction in experimental animal models; moreover, mice overexpressing GRK2 exhibited decreased isoproterenol(Iso)-stimulated left ventricular contractility in vivo, diminished myocardial AC activity, and reduced functional coupling of β-ARs [52]. Furthermore, GRK2 expression and activity were found to be elevated in human cardiac tissue and in circulating lymphocytes, demonstrating direct correlation with the severity of HF [53] and [54]. More recent studies showed that the changes of GRK2 levels in peripheral lymphocytes mirror changes in the salutary LVAD-supported failing human heart and that these changes correlate strongly with cardiac function such that lower levels of GRK2 are associated with improved β-adrenergic signaling and myocardial function in mechanically supported failing hearts and transplanted human hearts [55] and [56]. The recent study that involves over 200 patients with HF demonstrated that GRK2 lymphocyte level has a prognostic value for outcomes and mortality in HF patients, thus supporting the hypothesis that GRK2 levels in blood can be used as a biomarker in HF [57].
Overall, the aforementioned studies suggest consideration of GRK2 as a therapeutic target for cardiac disease and as a potential biomarker for heart function [58], [59] and [60].
4.3. Recombinant proteins as a way to inhibit Gβγ-GRK2 signaling
Excess cardiac Gβγ-mediated signaling leading to chronic β-AR desensitization and down-regulation is a crucial component of HF pathophysiology [61]. Thus, several approaches, including genetic manipulations and pharmacological targeting, have been explored to interdict pathologic Gβγ-GRK2 signaling.
The first reported approach utilized a recombinant carboxyl (C)-terminal fragment of GRK2 comprised of 194 amino acids encoding the Gβγ binding domain (βARKct) as a presumed inhibitor of Gβγ-GRK2 interactions. In 1994, βARKct was expressed in cells where it attenuated Gβγ mediated signaling with unaffected Gα mediated signaling, indicating its ability to discriminate between Gα and Gβγ pathways [62]. Subsequently, in 1995 Koch et al demonstrated enhanced baseline cardiac contractility in vivo with or without Iso stimulation in transgenic mice with cardiac-specific GRK2 overexpression [52]. Additionally, βARKct was demonstrated to normalize β-adrenergic signaling and cardiac function in hybrid transgenic mice with cardiac-specific concomitant overexpression of both GRK2 and βARKct [63].
Further studies demonstrated salutary effects of βARKct on the recovery of failing myocytes function [64] and [65] and prevention of cardiac dysfunction [66]. Oligonucleotide microarray left ventriclar (LV) gene expression analysis performed in normal, failing and βARKct overexpressing (“rescued”) cardiac samples revealed the ability of βARKct to normalize gene expression changes associated with HF [67]. More recent studies performed in large animal HF models revealed preservation and amelioration of cardiac function along with normalization of CA signaling owing to stable myocardial βARKct gene delivery [68] and [69], suggesting that inhibition of Gβγ-GRK2 interactions with recombinant viral-delivered βARKct peptide is a promising therapy for HF treatment [60].
4.4. Small molecule interdiction of Gβγ-GRK2 signaling
βARKct inhibition of Gβγ-GRK2 interactions has demonstrated salutary effects on cardiac function in both acute and chronic models of HF, however, viral-based gene delivery remains a daunting therapeutic approach. In that perspective, small molecule inhibitors that could be administered systemically may represent an alternative approach to attenuate pathologic components of Gβγ-GRK2 signaling or interactions [58] and [60].
One of the described approaches to small molecule pharmacological inhibition of GRK2 signaling is paroxetine, the selective serotonin reuptake inhibitor, identified by Thal et al in 2012 [70]. This antidepressant drug binds to the active site of GRK2 and stabilizes the kinase domain, thereby inhibiting the downstream signaling. This study demonstrated increased contractility in isolated cardiomyocytes in the presence of paroxetine. Further, paroxetine was tested in vivo in a mouse model of MI. Schumacher et al recently showed that paroxetine treatment initiated two weeks post-MI results in improved cardiac function, limited ventricular remodeling and normalized SNS overdrive along with myocardial β-adrenergic system [71]. Thus, direct GRK2 pharmacological inhibition demonstrated salutary effects on HF progression.
Another potential strategy to interdict Gβγ-GRK2 pathologic signaling is targeting Gβγ subunit and inhibiting its protein-protein interactions [5] and [60]. Hence, Bonacci et al in 2006 performed a virtual screening of 1990 compounds from the National Cancer Institute (NCI) chemical library to identify small molecules capable of binding Gβγ protein interaction domain mentioned above [72]. Eighty-five identified compounds were further tested in an enzyme-linked immunosorbent assay (ELISA) for their ability to compete with a phage-displaying SIRK peptide derivative (SIGK) [73] for binding to Gβγ subunit. Among several tested compounds, one termed M119 (cyclohexanecarboxylic acid [2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9Cl)]) demonstrated high apparent affinity for the Gβγ subunit and inhibited Gβγ-SIGK binding in vitro. Moreover, pretreatment of differentiated HL-60 leukocytes with M119 resulted in interference with Gβγ binding to GRK2 and consequent inhibition of GRK2 translocation to the membrane, along with suppression of PLCβ2/3 and PI3Kγ activation by Gβγ. Thus, the small molecule M119 confirmed its ability to interfere with Gβγ-mediated signaling downstream of GPCRs.
Since Gβγ subunits are known to modulate a majority of signaling pathways, the inhibitor that selectively influences a particular subset of Gβγ interactions is needed for Gβγ-GRK2 targeting [28]. Thus, M119 was examined in vivo for efficacy and specificity. It has been demonstrated that inhibition of PLCβ3 that is activated by Gβγ subunits is associated with enhanced morphine-induced antinociception [74]. Co-administration of M119 with μ-opioid receptor agonist morphine resulted in substantial increase of morphine-dependent antinociception in wild-type mice due to M119-induced inhibition of Gβγ-PLCβ3 interactions, whereas M119 alone had no effects on antinociception [72]. Also, M119 demonstrated no effects on morphine-dependent antinociception in PLCβ3–/– mice. Further studies showed that M119 increases analgetic potencies of morphine or μ-selective peptide, whereas it does not have any significant influence on analgesia induced by κ- or δ-opioid receptor agonists [75], corroborating the suggestion that M119 acts as a specific inhibitor of a particular subset of Gβγ-mediated signaling. Accordingly, we have conducted various studies to evaluate the potential of small molecule inhibition of Gβγ-GRK2 associations in different animal HF models.
4.5. Gβγ inhibitory treatment in acute and advanced heart failure models
Taking into account the aforementioned role of β-AR-dependent Gβγ-GRK2 signaling in cardiac disease pathogenesis and the proven efficiency of small molecule inhibitor M119 in selective interdiction of Gβγ-GRK2 interactions, we explored the effects of Gβγ signaling small molecule disruption in myocardial cells and in murine models of HF [76]. In this study, M119 or its highly homologous, more chemically stable analogue gallein was utilized to inhibit Gβγ-GRK2 interactions. M119 pretreatment followed by administration of the β-AR agonist Iso significantly enhanced AC activity and consequent cAMP generation in isolated cardiomyocytes from adult wild-type mice. Moreover, M119 increased the rate of cardiomyocyte contraction alone and in combination with Iso. Importantly, the β-AR antagonist propranolol abolished the effect of M119 and Iso on cardiomyocyte contractility, confirming the selectivity of the compound for β-AR-Gβγ signaling. In addition, both M119 and gallein demonstrated the ability to reduce GRK2 recruitment to the membrane of cardiomyocytes induced by Iso treatment.
To examine cardiac-specific effects of Gβγ inhibitory treatment initiated at the onset of HF, an acute pharmacologic murine model of HF [77] was implemented. Chronic β-AR stimulation by Iso delivered via implantable miniosmotic pumps was started simultaneously with systemic administration of M119 or vehicle and continued for 7 days. M119 treatment mitigated HF progression; particularly, M119-treated mice maintained essentially normal cardiac function and showed significantly reduced cardiac hypertrophy along with decreased level of interstitial and perivascular fibrosis, compared to vehicle-treated animals. Considering these data, Gβγ small molecule inhibition was administered after the onset of HF, in a transgenic mouse model of established HF generated by cardiac restricted calsequestrin (CSQ) overexpression [78]. Importantly, the CSQ transgenic mouse model recapitulates essential hallmarks of HF, including pathologic β-AR signaling [76]. One month of daily gallein administration resulted in prevention of HF progression, especially in normalized echocardiographic parameters, reduced pathologic cardiac hypertrophy and diminished expression of HF molecular markers. Moreover, M119 and gallein significantly reduced pathologically increased cardiac GRK2 protein level in Iso-pumped and CSQ animals, respectively. Overall, this study demonstrated salutary effects of Gβγ small molecule inhibitory treatment on both manifestation and progression of HF.
Beneficial effects of small molecular inhibitors observed in acute pharmacological and transgenic mouse models of HF elicited further interest to investigation of cardiac and systemic effects of Gβγ signaling inhibition. Consequently, Kamal and colleagues in 2014 examined outcomes of utilizing the small molecule Gβγ inhibitor gallein in a transverse aortic constriction (TAC) mouse model of pressure-overload induced cardiac hypertrophy and HF [79]. The TAC surgical model, firstly validated by Rockman et al [80], is considered a relatively clinically relevant model of HF [81] and [82]. In this study, vehicle-treated mice developed the decline in cardiac function at 8 weeks post-TAC with the concomitant worsening at 12 weeks post-TAC. Daily gallein administration for eight weeks was initiated after the establishment of HF (four weeks post-TAC). This treatment regimen, initiated after the onset of HF, alleviated cardiac dysfunction and hypertrophy along with significantly enhanced survival in the gallein-treated group compared to the vehicle-treated group. Preservation of cardiac function was accompanied by the recovery of β-AR density and reduction of GRK2 gene expression and membrane translocation. Furthermore, membrane recruitment of phosphoinositide 3-kinase γ (PI3Kγ), which GPCR-induced activation was implicated in maladaptive cardiac hypertrophy and dysregulated β-AR function [28], [83], [84], [85] and [86], was reduced in gallein-treated mice compared with vehicle-treated mice. Gallein treatment also resulted in attenuated progression of cardiac hypertrophy and reduced myocardial fibrosis. Interestingly, ameliorated cardiac remodeling was accompanied by decreased phosphorylation of cardiac Akt (aka protein kinase B) and its downstream signal GSK-3β. As mentioned above, PI3Kγ-mediated GPCR dependent Akt activation and subsequent GSK-3β Ser-9 phosphorylation lead to cardiac hypertrophy [85], [87] and [88]. Of note, authors attributed the significantly reduced expression of the fetal genes atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) detected in gallein-treated TAC mice to elevated levels of nonphosphorylated GSK-3β that was suggested to negatively regulate transcription and protein translation of the hypertrophic genes [89]. Other detected beneficial effects of gallein treatment on TAC HF mice were attenuated cardiac inflammatory cytokine expression and reduced myocardial apoptosis. Collectively, these data corroborate the suggestion that Gβγ inhibitory treatment preserves cardiac function and halts HF progression in different small animal models of HF, in part via downstream inhibitory effects on cardiac fibrosis, hypertrophic gene expression and inflammation along with promotion of cell survival.
As discussed previously, compensatory sympathetic nervous system activation and subsequent systemic release of catecholamines (CA) from the adrenal gland medullary chromaffin cells increase the rate and the intensity of cardiac contractions in response to diminished cardiac output of the failing heart [90]. Adrenal chromaffin cell α2-ARs that belong to GPCR family are essential regulators of sympathetic outflow in HF, providing the feedback inhibition for CA release [32] and [91]. Lymperopoulos and colleagues showed that, similar to the dysregulation of β-AR signaling in failing hearts, the adrenal α2-ARs undergo desensitization and down-regulation in response to catecholamine overdrive and concomitant adrenal GRK2 upregulation, thus contributing to HF pathogenesis [92]. Further studies revealed that delivery of adenoviral vectors containing GRK2 to adrenal glands resulted in enhancement of plasma CA levels and failure of adrenal α2-ARs to inhibit CA secretion, whereas GRK2 inhibition using βARKct or chromaffin cell specific GRK2 gene deletion recovers adrenal α2-AR function, reduces CA release and attenuates HF progression [31], [92] and [93]. More recently, Jafferjee et al demonstrated that CA treatment of rat pheochromocytoma-derived or primary chromaffin cells results in GRK2 gene transcription upregulation and subsequent enhancement of α2-AR desensitization and down-regulation accompanied by elevated CA biosynthesis and release [94].
Taking into account the aforementioned role of adrenal α2-ARs in regulation of SNS activity in HF, we recently assessed effects of small molecule Gβγ signaling disruption on the adrenal gland in pathogenesis of pressure-overload induced HF [79]. Significantly decreased CA production and release, alleviated adrenal medulla hypertrophy and restored α2-AR feedback inhibition was observed 12 weeks post-TAC in the gallein-treated group compared to the vehicle-treated group. Additionally, cultured in vitro adrenal glands from gallein-treated mice exhibited significantly decreased levels of basal CA secretion. Moreover, the effects of gallein on CA generation and GRK2 expression were examined in cultured human pheochromocytoma tissue, a tumor characterized by increased CA production. Gallein treatment significantly reduced CA production in cultured pheochromocytoma slices as reflected by lowered expression of tyrosine hydroxylase, an enzyme that catalyzes the rate limiting step in CA synthesis [95], and chromogranin A, a neurokine that is synthesized and co-secreted with vesicular CA [96]. Importantly, authors observed downregulation of both GRK2 protein level and membrane translocation in cultured pheochromocytoma slices treated with gallein. Taken together, this study provides deeper insights into understanding of pathological mechanisms contributing to HF progression. In addition, the study suggested small molecule Gβγ inhibition as a potential systemic therapy that attenuates HF progression due to simultaneous inhibition of cardiac and adrenal Gβγ-GRK2 interactions [79] and [58] (see overview of approaches in Figure 1).
5. GPCR-Gβγ-GRK2 signaling in cardiorenal pathologies.
The kidney performs essential regulatory roles in the body, including waste excretion, homeostasis maintenance, fluid volume and blood pressure regulation, as well as hormone secretion. GPCRs are widely expressed in the kidney, exemplified by arginine vasopressin receptor (AVP), dopamine-1 receptor (D1-R), angiotensin II receptors and endothelin (ET)-1 receptors [24]. GPCRs are involved in regulation of numerous kidney functions including water and electrolyte transport in renal tubules, maintenance of acid-base balance and renal blood flow and filtration [24] and [59]. Dysregulation of GPCR signaling is associated with severe kidney and systemic disorders such as renal fibrosis [97], [98] and [99], acute kidney injury (AKI) [100] and [101], hypertension [102], [103] and [104], and chronic kidney disease (CKD) [105].
Combined heart and kidney disorders, characterized by pathological interactions (“crosstalk”) between affected organs, are defined as cardiorenal syndrome (CRS) [106]. Different approaches have been applied to characterization and classification of CRS [107]; according to a classification proposed by Ronco et al in 2008, CRS is discriminated into five types with respect to the acute or chronic pathogenesis and the initiating event [106]. While the specific mechanisms behind this pathologic crosstalk between heart and kidney remain poorly understood, CRS is associated with exacerbated dysfunction of either or both organs and reduced survival [108] and [109]. Essentially, kidney maladaptive remodeling and impaired function serve as a strong predictor of mortality in HF patients [11] and [12]. Thus, investigation of mechanisms of pathologic crosstalk between failing heart and kidney may contribute to development of novel therapeutic strategies for HF, kidney dysfunction and CRS.
Considering the role of GPCR signaling in normal physiology and pathology of both heart and kidney, we recently scrutinized the role of GPCR-Gβγ-GRK2 in CRS type 2 (CRS2), which is characterized as a chronic heart failure (CHF) accompanied by the development of CKD [106]. The study proposed that elevated activity of SNS and endothelin (ET) system causes desensitization and down-regulation of renal GPCRs owing to pathologic Gβγ-GRK2 interactions, resembling the dysregulation of β-ARs observed in HF. To recapitulate the clinical features of CRS2 progression, a non-ischemic TAC mouse model of pressure-overload induced HF was utilized. To determine the role of Gβγ-GRK2 signaling in kidney dysfunction besides the crosstalk with the heart, a direct bilateral ischemia reperfusion (I/R) acute kidney injury (AKI) model was also implemented [110]. Development of CKD secondary to TAC was reflected by elevated serum creatinine levels, emerged morphological and molecular signs of tubular damage and increased focal tubulo-interstitial and perivascular fibrosis in the kidneys at 12 weeks post-TAC. Development of CKD in the chronic phase of HF is consistent with clinically observed consequence of CRS2 progression. Importantly, observed maladaptive changes were accompanied by elevated levels of ET-1 along with increased protein expression and membrane localization of ET receptors (ETA and ETB), that corroborates the proposed role of ET system in CRS. Furthermore, authors detected the elevation of Gβγ-GRK2 signaling in kidneys at 12 weeks post-TAC; essentially, upregulation of both ET and Gβγ-GRK2 signaling was attenuated by small molecule Gβγ inhibitor gallein treatment. Gallein pretreatment of mice subjected to AKI revealed protective effects of small molecule Gβγ-GRK2 inhibition on kidney function. Importantly, GRK2, ET-1 and ETA gene expression was elevated in kidneys of both CHF and AKI I/R mice.
Overall, these data suggest the role of Gβγ-GRK2 interactions in both acute and chronic kidney injury and the potential mechanism underlying pathologic crosstalk between heart and kidney in CRS2. Moreover, the study provides mechanistic insight into fibrotic tissue remodeling, demonstrating the role of Gβγ signaling in mouse embryonic fibroblasts (MEFs) endothelin-1 induced activation and migration [111] and [112].
A study performed by White et al aimed to explore the role of Gβγ subunits in kidney repair after AKI I/R injury. Their study suggested that Gβγ inhibition with gallein may delay the recovery process [113]. In this study, rats were treated with low (30 mg/kg) or high (100 mg/kg) dose of gallein during three days after I/R. However, the low dose far exceeds those reported in our dose-response study [79], and the high dose may in fact represent toxicity. As described above, our study with a lower dose of Gallein was indeed renoprotective [110].
Taking into consideration the bidirectional nature of the crosstalk between heart and kidney, Polhemus and colleagues recently examined whether catheter-based renal denervation (RDN) that is thought to reduce blood pressure owing to the disruption of sympathetic signaling [114] and [115] possesses cardioprotective effects [116]. To model the heart injury, authors subjected spontaneously hypertensive rats (SHR) representing a model of established hypertension to myocardial I/R at 4 weeks after either bilateral radiofrequency-RDN (RF-RDN) or Sham-RDN. Rats treated with RF-RDN exhibited a significant reduction in myocardial infarct size and substantially improved left ventricular function 24 hours following the I/R. Moreover, RF-RDN treated rats demonstrated the attenuation of myocardial oxidative stress and elevation of cytoprotective nitric oxide (NO) signaling [117] compared to the Sham-RDN treated group. Importantly, RF-RDN treatment caused the reduction in myocardial GRK2 gene expression level, particularly the decrease in GRK2 Ser670 phosphorylated protein level. Phosphorylation at residue Ser670 of GRK2 results in the activation of downstream mitochondrial cell death pathways [118]. Interestingly, neither significant cardioprotective effects nor alterations in myocardial GRK2 signaling were detected in normotensive rats following RF-RDN treatment. These findings highlighted the importance of CA signaling for the communication between heart and kidney, suggesting involvement of GRK2 signaling in these interactions (see overview of cardiorenal crosstalk in Figure 2).