GRK2 in Cardiovascular Disease and its Potential as a Therapeutic Target

2.1. Cardiac GPCR Distribution and Function in Physiology and Pathology

In the heart, physiological function is primarily mediated by several subfamilies of GPCRs: angiotensin II receptors (AT1R and AT2R), endothelin-1 receptors, and adrenergic (α and β) receptors; conversely, dysregulation of these receptors contributes to the majority of known acquired cardiac pathologies (Figure 1). As this Review focuses on past and future research in the field of cardiac β-adrenergic receptor kinases (bARKs) or GRKs, we will focus on these receptors and their downstream signals. Endothelin-1 (ET-1) is generated by endothelial cells of the vasculature and exerts important actions such as inotropic stimulation on the heart via endothelin receptors; importantly, in HF patients an over-activation of this system is observed concomitantly to elevated circulating levels of ET-1(10). Angiotensin-family receptors, of which the AT1R is of greatest cardiac significance, respond to Angiotensin-II (AngII) produced by the liver and circulated as part of the renin-angiotensin system which regulates blood pressure(11). Despite low sequence homology (~34%), both AT1 and AT2R possess similar affinity for Ang II; however, some FDA-approved drugs to treat heart failure such as losartan display much greater affinity for AT1R than AT2R(12).

Adrenergic receptors (ARs) represent arguably the most important class of cardiac GPCRs; they are the key receptors activated by the sympathetic nervous system, and transduce these signals into physiological cardiovascular responses. Their ligands, norepinephrine and epinephrine, activate differential downstream pathways depending on the receptor type being stimulated(13). There are two subfamilies of ARs: the α-ARs, which are comprised of α1-AR and α2-AR, and the β-ARs made up of β1-AR, β2-AR, and β3-AR (Figure 1). These receptors demonstrate striking differences in G protein partners and downstream signaling pathways, and possess differential stimulation in the heart. The α-ARs are highly responsive to epinephrine and norepinephrine, but β-ARs being less sensitive to epinephrine and norepinephrine but responding with high sensitivity to the synthetic adrenoreceptor agonist isoproterenol(13,14).

The α1-AR is coupled to Gq, whereas the α2-AR is a Gi-coupled receptor; all three β-ARs are coupled to Gs, but β2 and β3 have also been shown to couple to Gi(14,15). Both Gi and Gs target downstream adenylyl cyclase (Figure 1) and trigger degradation and formation of cyclic adenosine monophosphate (cAMP), respectively, controlling such responses as activation of cAMP-dependent protein kinase (PKA), increased cardiac contraction, chronotropy, upregulated cortisol secretion, and breakdown of glycogen and fats(16). The downstream signaling for Gq is phospholipase C (PLC), which catalyzes the production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) from the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂)(17), increasing cytosolic calcium and activity of protein kinase C (PKC). Due to these differences in downstream effectors, it is generally accepted that the Gs-coupled β-ARs increase cardiac contractility and promote pathological hypertrophic growth of myocytes, while the Gi-coupled β-ARs reduce contractility and protect against hypertrophy (HT) (18) (Figure 1). In all cases, dysregulation of cardiac adrenoreceptors or their downstream modulators has been demonstrated to contribute to numerous cardiovascular pathologies.

β-ARs subtypes are primarily expressed on cardiomyocytes (β1-AR), vascular smooth muscle cells (β2-AR) and adipocytes (β3-AR); however all are expressed on cardiomyocytes to varying extent(19,20). In healthy hearts, the β1:β2 receptor ratio is roughly 4:1, with β1-ARs comprising up to 80% β-receptors, β2-ARs comprising 17–20%, and nominal myocardial expression of β3-ARs(20,21). This ratio has significance in the development of cardiac pathologies due to the differing roles these β-ARs fulfill. While both β1-ARs and β2-ARs are activated by both adrenaline and noradrenaline, the downstream β1 signaling (through Gs) is overwhelmingly stimulatory, promoting contractility via activation of protein kinase A (PKA), L-type calcium channels (LTCCs) and troponin I (TnI), regulators of sarcomeric shortening and chronotropy of cardiac contractility(19,21,22). β2-ARs, on the other hand, demonstrate a higher affinity for adrenaline than for noradrenaline and are not only coupled to Gs but also to Gi subunits, which allows them to function in either an activating or an inhibiting manner on the myocardium(19).

Chronic activation of cardiac β-ARs, such as in hypertension when circulating catecholamine levels are typically elevated, alters the ratio of β1:β2 adrenoreceptors through several mechanisms, changing both downstream G-protein signaling as well as cellular behaviors in the myocardium. This is mostly due to a reduction in β1-AR density on the surface of cardiomyocytes as they are desensitized and internalized for degradation(18), and the subsequent shift in β1:β2 ratio towards 1:1 correlates with progression of HF and severity of disease(23). Contributing further to the loss of adrenergic drive experienced with the loss of β1-AR density on CMs during HF, the corresponding β2-AR density is not only unchanged but Gi levels are often upregulated(24), further biasing β2-AR coupling towards inhibitory Gi signaling and negatively impacting cardiac inotropy and chronotropy. The desensitization and loss of critical β1-ARs in the heart is driven by GRKs.

2.2. GRKs: Regulators of GPCR Signaling in the Heart

The carboxy-terminus tail of GPCRs as well as the 3^rd intracellular loop are regulatory regions for these receptors, being highly targeted for post-translational modifications such as phosphorylation. The desensitization and downregulation of activated GPCRs are primarily mediated by the activity of GRKs on these regions of the receptor which promotes association of arrestin proteins with the GPCR’s carboxy-terminus and prevents re-association of the G-protein(25). The GRK family is comprised of seven serine/threonine kinases (GRK1–GRK7; Figure 2), divided in vertebrate systems into three subfamilies based on receptor type and sequence homology(26): the visual GRKs comprised of GRK1 and GRK7, the βARK GRKs consisting of GRK2 and GRK3, and the GRK4-types including GRK4, GRK5 and GRK6. Incredibly, these 7 kinases are responsible for the regulation of the vast majority of the 800+ known GPCRs(27), highlighting the importance of this family in regulating cellular behavior. There are a very few known GPCRs, such as the b3-AR that are not regulated by GRK-mediated phosphorylation(28).

The first GRK to be discovered nearly 40 years ago was GRK1 or “opsin kinase” which was able to phosphorylate light-activated rhodopsin, and was soon followed by the discovery of GRK2 as the first β-AR-regulating kinase (and was initially known as βARK1 for this reason)(29,30). Of note, despite their concomitant discoveries, the sequence homology between GRK1’s and GRK2’s cognate receptors (β2AR and rhodopsin), which led to the discovery of a greater family of GPCRs, was not revealed until later(30). Phosphorylation of activated rhodopsin by GRK1 was found to be indispensable for its subsequent deactivation, a premise which later held true for other GPCRs regulated by GRKs, such as GRK2 and the β2-AR(31,32). All GRKs are AGC-type protein kinases with high level of homology in their catalytic domain to other AGC kinases such as such as PKA, PKB, and PKC, from which this subfamily derives its name(33).

GRKs are modular proteins with multiple domains(34) consisting of a highly conserved ~30 amino acid amino-terminal region, followed by a regulator of G protein signaling (RGS) homology (RH) domain, followed by the aforementioned AGC-type kinase domain(33) (Figure 2). The first ~500 amino acids are very highly conserved between all known GRKs, with greater structural variability having evolved primarily in the carboxy-terminal tails which dictate their subcellular targeting(33,34) (Figure 2). GRK1 and GRK7 possess short prenylation sequences; GRK2 and GRK3 have evolved a pleckstrin homology (PH) domain which strongly interacts with the βγ-subunits of G proteins (Gbg) as well as lipids, GRK4 and GRK6 contain longer palmitoylation sites, and GRK5 is unique in possessing a positively charged lipid-binding element(35). GRK1 and GRK7, being rhodopsin and cone opsin kinases respectively, are primarily expressed in the photoreceptor cells of the retina; GRK2, GRK3 and GRK5 are more ubiquitously expressed throughout all tissues to varying degrees, and GRK4 is most highly enriched in reproductive organs and lung tissue(36,37).

Just as β1-AR and β2-AR represent the primary GPCRs modulating physiological cardiac responses to catecholamines, GRK2 and GRK5 are the most highly expressed cardiac GRKs at the protein and mRNA level(38). These two kinases are unique in their downstream signaling pathways, yet both contribute to cardiac pathologies and share some underlying traits. Using in vivo models of transgenic mice with cardiac overexpression of either GRK2 and GRK5, for example, it was discovered that both GRKs are able to desensitize β-ARs; however, only GRK2 but not GRK5 was also capable of desensitization of angiotensin II receptors; GRK5 had no effect on these receptors(39). Conversely, GRK5 was found to be capable of reducing cardiac adenosine responses, whereas GRK2 was not(39). During prolonged adrenergic stimulation, which occurs in pathologies such as HF, as well as following acute injury such as myocardial infarction, both the expression and activity of GRK2 and GRK5 are increased(40,41), augmenting AR desensitization and internalization via receptor phosphorylation as described above. In long-term cardiac pathologies where the levels of GRK2 and GRK5 remain elevated, the shift in β-AR ratio and loss of β1-AR stimulation leads to a reduced cardiac reserve as measured in terms of not only inotropy but also measure of chronotropy and lusitropy(42,43). However, the mechanisms involved in the upregulation of cardiac GRKs have more recently been uncovered and are outlined below (and for GRK2 and GRK5, discussed in greater detail by our group in(42,44)). Additionally, little is still known about the mechanisms dictating GRK receptor preferences in the myocardium.

3.1. GRK2 – A Multifunctional Signaling Hub

Along with the aforementioned PH domain which facilitates binding to the dissociated Gβγ subunit of an activated G protein, the carboxy-terminal tail of GRK2 possesses multiple scaffolding and binding regions which indicate its importance as a multi-functional signaling hub in the heart. These binding sites are present in GRK2 and GRK3 and are not shared by GRK5, and include phosphoinositide 3-kinase (PI3K), Akt and clathrin(43). Of note, human HF patients demonstrate an increase in cardiac PI3K activity due to its regulation by GRK2; importantly, this GRK2-mediated PI3K activation was associated with a concomitant downregulation of β-ARs from the plasma membrane as they are internalized into endosomes(45,46). These relationships dictate how GRK2, which is primarily cytosolic in distribution, localizes to the sarcolemma, where it can interact with activated receptors, as GRK2’s carboxy-terminal maintains its association with phospholipids of the cell membrane while the amino-terminal interacts with the β1-AR. This allows for Ser/Thr residues along the receptor’s the cytoplasmic tail to enter the p-loop in GRK2’s catalytic domain once the receptor has been activated, ensuring rapid deactivation of the receptor and accuracy of binding only to receptors which are activated rather than inactive(25).

One of the most clinically significant aspects of GRK2 is its modular structure and numerous binding and scaffolding regions (Figure 2). For example, kinases such as PKA and PKC phosphorylate GRK2 near its carboxyl-terminus at Ser685 which promotes its binding to Gβγ as well as the active GPCR(47) and can preserve downstream contractile signals. Conversely, the nearby Ser670 residue is phosphorylated by ERK, which prevents translocation to the plasma membrane and thus reduces Gβγ interactions(25) and may regulate GRK2 degradation and protein turnover. Thus, it may be possible to target one region of GRK2 while preserving the function of its other domains, allowing for highly selective, context-specific targeting of distinct pathways. In the context of cardiovascular pathologies, preserving downstream pathways which enhance cardiac function while turning “off” deleterious pathways is a feature of GRK2 which should be exploited in the development of therapies for these diseases.

It should also be recognized that not all GRK2 signaling pathways are deleterious. Despite extensive evidence in the literature supporting the premise that elevated GRK2 levels are detrimental across multiple cardiac pathologies, GRK2 expression is critical for embryonic development not only in the heart but elsewhere, as global GRK2 knockout in mice is embryonic lethal; furthermore GRK2-KO embryos present with gross cardiac malformations(48,49). Conditional knockout early in myocyte development appears to be detrimental if not lethal in mouse models as well; postnatally these mice demonstrate an exaggerated response to the negative effects of chronic catecholamine administration(48). This is further supported by studies in Zebrafish which indicate that GRK2 is indispensable for fetal development, possibly through cell cycle regulation via interactions with Hedgehog and associated proteins.(50,51) However, just as GRK2 levels are upregulated across multiple cardiac pathologies, post-developmental reduction of GRK2 to at least 50% globally appears to be generally protective under identical circumstances. These models are detailed below and in (Figure 3).

3.2. GRK2 in Cardiac Pathology

Myocardial infarction (MI), which comprises a significant proportion of CVD-related deaths(52), does not at first glance appear to represent a model of GRK2 upregulation, which is commonly discussed in terms of chronic sympathetic overdrive; however, sustained adrenergic stimulation is observed in human patients beginning shortly after acute cardiac injury such as ST-elevation myocardial infarction (STEMI), which progresses into long-term remodeling after the acute injury phase(53). A rapid (hours to days) increase in GRK2 levels has been observed in other human cardiac injury studies(54,55), supporting the idea that rapid upregulation in GRK2 levels and activity is an early molecular hallmark of cardiac damage. In a clinical study following patients up to 2 years after diagnosis of acute coronary syndrome, increased GRK2 levels occurred following the event of STEMI, and this observation correlated with decreased cardiac function as measured by LVEF and end systolic volume; which are directly correlated with severity of LV injury and post-injury function(49). In animal models of acute cardiac injury, a similar rapid, sustained GRK2 upregulation occurs(57,58). In a mouse model of transverse aortic constriction (TAC) to induce HT, upregulation of GRK2 levels and activity were observed(39) within the first week after increasing cardiac pressure, preceding morphological and functional changes associated with early or later stages of HF and indeed seem to be powerfully prognostic(2,59). Therefore, a large body of evidence supports the idea that cardiac GRK2 upregulation occurs acutely after myocardial ischemic injury in both clinical and pre-clinical models, with significant correlation to disease severity and progression to HF following acute injury.

Of importance, two cardiomyocyte-specific GRK2 KO mouse models with GRK2 expression maintained during embryonic development and ablated postnatally (αMHC-Cre×GRK2^fl/fl) or with administration of tamoxifen in adult mice following surgical coronary artery ligation (αMHC-MerCreMer×GRK2^fl/fl), resulted in increased β-AR responsiveness, and improved cardiac function, and protected against maladaptive post-infarction remodeling(60). Of clinical significance, GRK2 ablation initiated 10 days following myocardial infarction has similar effect, increasing survival, enhancing cardiac function, and preventing LV remodeling(60). These same two models of GRK2KO mice, when subjected to I/R injury (30 min of myocardial ischemia induced via coronary artery ligation, followed by 24 hrs of reperfusion) demonstrated significantly improved cardiac function afterward as measured by echocardiographic and hemodynamic measurements(61). Additionally, GRK2 ablation in the context of I/R reduced infarct sizes as well as lowered caspase-3, and −9 activities with concomitantly increased levels of the pro-survival proteins Bcl-2 and Bcl-xl(61), demonstrating that reducing GRK2 levels in the context of acute ischemic injury prevented cardiomyocyte apoptosis and implicating GRK2 as a pro-death kinase in the heart.

Regarding cardiac HT, as GRK2 was originally discovered in the setting of cardiac β-AR signaling and that the activity of GRK2 drives receptor deactivation in a rapid manner, it follows that chronic adrenergic signaling from the sympathetic nervous system such as occurs during HF leads to receptor desensitization. However, GRK2 has become increasingly implicated in other cardiac pathologies in the past 10–15 years, supporting its many non-canonical roles and interacting partners. One of these pathologies, maladaptive cardiac HT which precedes cardiac decompensation and failure, appears to be at least partly driven by GRK2. It has been demonstrated in some in vitro models that GRK2 overexpression is sufficient to induce a hypertrophic response(62) as measured by RNA levels of beta-myosin heavy chain (β-MHC), atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP); these small peptides are common markers of cardiac HT and fetal gene re-expression in hypertrophic hearts. Of note, some data indicate that GRK2 promotes cardiac HT via its effects on NFAT activity, possibly via its phosphorylation of Akt and subsequent activation of downstream targets such as NFAT(63). It must be noted however that although GRK2 appears to promote hypertrophic signaling at the molecular level in in vitro models, overexpression of GRK2 in mouse models is not sufficient to induce HT at baseline(64), as cardiac-specific transgenic overexpressing GRK2 mice, which express GRK2 at roughly four times endogenous levels and in line with what is observed in human HF(65) do not demonstrate appreciable differences in whole-heart weight, LV mass or LV wall thickness(64). Additionally, when subjected to a transient aortic constriction (TAC) model of inducing HT, these mice demonstrate maladaptive hypertrophic remodeling to a similar extent as their littermate controls, though they are prone to increased post-surgical mortality and markedly reduced cardiac function as measured by echocardiographic parameters(62,64), suggesting other mechanisms and pathways are regulated by this GRK. These mice with elevated GRK2 also have significantly larger infarcts and myocyte cell death after I/R injury(66).

Additionally, there are multiple non-GPCR substrates for, and thus non-canonical functions of, GRK2 that have recently come to light and which provide new avenues to develop molecular and pharmacological inhibitors of GRK2 signaling in the heart. Among these novel interacting proteins, which include non-GPCRs such as the receptor tyrosine kinase, platelet-derived growth factor (PDGF) receptor, ion channels such as the ENaC sodium channel, critical signaling hub kinases like p38 MAPK and PI3K/AKT, signal transducers and second messengers such as IRS1, RKIP, Pin1, PDE and eNOS(67,68). IRS1 and PDE represent significant GRK2 signaling discoveries, as treatment of diabetic (db/db) mice with a peptide inhibitor of GRK2 prevented the insulin resistance and diabetic cardiomyopathy typically observed in these mice, due to reductions in fetal gene expression, oxidative stress and cytokine levels which GRK2 negatively regulates in this pathology(69). It is now known that GRK2 also phosphorylates insulin receptor substrate-1 (IRS1), which in ischemic cardiomyocytes with increased GRK2 levels negatively regulates insulin signaling and ultimately inhibiting membrane translocation of glucose transporter 4 (GLUT4)(70). Finally, high-fat diet feeding in mice increases cardiac expression of phosphodiesterase 4D (PDE4D), which drives contractile dysfunction and is a downstream effect of GRK2’s action on IRS1 which was prevented via pharmacological inhibition of GRK2 with paroxetine(71). Thus, GRK2 has emerged as a critical driver of diabetic cardiomyopathy as a result of a novel, non-GPCR interaction.

A large number of recently identified GRK2 interacting partners are vital components of cell cycle regulation, protein turnover and degradation, quality control and import; these include transcription factors and modulators like Smad-2/3, HDACs, Period-1/2, IκBα, and the calcium-binding protein DREAM; E3 ubiquitin ligases and chaperones such as Mdm2 and Hsp90, membrane components such as clathrins and caveolins; and mitochondrial proteins such as Mitofusin(67). The GRK2-Hsp90 relationship has been investigated with great interest recently and uncovered a role for GRK2 in regulation of mitochondrial function, as it has been demonstrated that stress-induced phosphorylation of ser670 in the carboxy-terminus of GRK2 by the mitogen-activated protein kinase ERK promotes recruitment and binding of the chaperonin Hsp90(72). Moreover, both in vitro models of oxidative stress and in vivo mouse models of ischemic injury induced GRK2 localization to mitochondria preceded pro-death signaling such as cleavage of Caspase-3 and increased mitochondrial susceptibility to Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP)(72). Importantly, blocking localization of GRK2 to mitochondria has been demonstrated to be cardioprotective, and will be discussed in more detail below.

3.3. Molecular and Pharmacological Therapies Targeting GRK2

Currently, there are no clinically utilized molecular or small-molecule inhibitors on the market that directly targets the activity of GRK2, though research is ongoing in this field and appears highly promising. Current strategies that target GRK2 levels or activity rely on indirect regulation of the kinase by modulation of upstream effectors. One well-known example of this indirect regulation is β-AR attenuation with β-blockers(73); these drugs are commonly prescribed in combination with Angiotensin-II receptor blockers (ARBs) and are considered the standard first-line treatment for patients with HF(74,75). While blocking β-AR activation and signaling provides improvement of cardiac function in HF patients via multiple mechanisms, one critical off-target effect is that β-blockade appears to not only maintain receptor sensitivity but also down-regulates GRK2 levels and activity in the heart and restores β-AR density(76,77). Despite the importance of these drugs in treating HF by slowing its progression and offering some preservation of cardiac function, their utilization has not significantly reduced mortality in HF patients over the past several decades and thus necessitates further research into novel treatments for these patients and targeting GRK2 appears promising in several pre-clinical animal models. Importantly, literature to date indicate that long-term targeting of GRK2 by pharmacological inhibition or genetic manipulation has not demonstrated any drawbacks or limitations such as compensatory GRK2 upregulation or loss of efficacy over time; the data thus far support the premise that targeting GRK2 in chronic cardiac pathologies such as HF is one of its greatest strengths.

One potential avenue of GRK2 targeting in several models of CVD is off-target use of paroxetine hydrochloride, an FDA-approved selective serotonin reuptake inhibitor (SSRI). Paroxetine was identified in 2012 as a GRK2 inhibitor from a large (~40,000 compounds) screen using an aptamer displacement assay, followed by additional testing of GRK2 inhibition by the top 1,200 FDA-approved drugs(78). Not only is paroxetine able to inhibit GRK2 kinase activity against known substrates with an IC₅₀ of ~30 μM, but it possessed the ability to cross the cell membrane and inhibits agonist-induced β-AR phosphorylation, β-arrestin 2 recruitment and β-AR receptor internalization(78,79). It was discovered that paroxetine fits inside GRK2’s active site and inhibits kinase activity of the p-loop, and its protective effects on β-AR signaling were directly due to kinase inhibition as opposed to inhibiting Gβγ binding. In a mouse model of MI, paroxetine treatment initiated within 2 weeks of injury at a dose of 5 mg/kg per day protected against decompensation of cardiac function versus mice that received vehicle or fluoxetine, another SSRI, which does not bind to GRK2 or inhibit its activity(41). Paroxetine treatment increased basal and isoprenaline-induced contractility, prevented gross cardiac remodeling as well as HT and fibrosis, improved LVEF hemodynamics and reduced circulating catecholamine levels(41). It also was shown to be more effective at improving post-MI HF than the β-blocker metoprolol and, given concomitant with metoprolol, cardiac function was also greatly rescued in this group(41).

In a recent very small clinical study, (~38 patients) where paroxetine was administered at 20 mg/day within 7 days of percutaneous coronary revascularization following hospital admission for STEMI, paroxetine treatment improved some but not all parameters of cardiovascular function observed in preclinical models(80). There were no significant differences observed between paroxetine and placebo groups with respect to improvement of LVEF, LVEDV or LVESV, but extent of LV fibrosis as measured by late gadolinium enhancement was significantly decreased in the patients who were administered paroxetine(80). Given that a significant proportion of HF patients are concurrently prescribed SSRIs such as paroxetine to treat depression and anxiety disorders(81), these data suggest some FDA-approved compounds may have off-target cardioprotective effects that influence their utilization in these patient groups.

Another promising small-molecule inhibitor of GRK2 is gallein and its closely related analogue M119(82). Gallein is a small xanthene-family GPCR antagonist which disrupts Gβγ binding interactions, including Gβγ-GRK2 binding(83). In a mouse model of TAC to induce pressure-overload HT, gallein treatment at a dose of 10 mg/kg per day initiated 4 weeks after TAC and continuing for 8 weeks improved survival, prevented cardiac remodeling and improved cardiac function(83). The cardioprotection offered by gallein appears to be two-fold; treatment restored β-AR density in cardiomyocytes via attenuation of Gβγ-mediated GRK2 recruitment to the membrane as well as reduced reduced protein expression of GRK2 and prevented activation of downstream GRK2 phosphorylation targets such as Akt and glycogen synthase kinase 3β (GSK3β)(83). Significantly, gallein also reduced circulating plasma catecholamine levels, fetal gene-re-expression and circulating inflammatory markers(83). Similar to gallein, M119 was identified from a small-molecule screening of Gβγ disrupters(82). In cardiomyocytes, M119 was sufficient to block GRK2 localization to the membrane following β-AR stimulation(84) and increased β-AR-stimulated cAMP production and sarcomeric shortening. In a murine model of acute HF utilizing 30 mg/kg per day of isoproterenol over 7 days, M119 also prevented cardiac dysfunction, reduced cardiac HT and fibrosis, and lowered GRK2 expression as well as markers of HT such as ANF and β-MHC(84). Importantly, some newer small-molecule inhibitors of GRK2 are currently in development or in preclinical testing as potential therapeutics for HF with promising preliminary data(27,85,86).

Beyond pharmacological and small-molecule inhibitors, there exist genetic and peptide-based mechanisms of targeting detrimental effects of GRK2 signaling in cardiac injury and HF, some of which have shown promise. One of the first and most-studied of these is the βARKct peptide(87), comprised of the carboxy-terminus 194 amino acids of GRK2(88). This peptide encompasses both the Gβγ binding domain and the ERK phosphorylation site ser670, which can block GRK2-mediated GPCR desensitization at the sarcolemmal membrane as well as mitochondrial localization. The first demonstration that this peptide showed promise as a therapeutic was in cardiac-specific transgenic mice with expression of βARKct. This peptide was able to prevent endogenous GRK2 interaction with β-Ars, with increased cAMP levels following stimulation, improved myocardial contractility and LV function compared to non-transgenic control mice(89,90). Following this, multiple in vivo models of cardiac dysfunction including viral-mediated gene therapy have corroborated the protective effects of βARKct expression. For example, a large-animal model of MI-induced HF demonstrated that cardiac-targeted, adeno-associated viral expression of βARKct 2 weeks after injury resulted in significantly improved echocardiographic parameters, LV hemodynamics and circulating norepinephrine levels; this improvement in cardiac function continued for up to 6 weeks(91). A rat model of HF utilizing AAV6-targeted delivery of βARKct via intra-cardiac injection as a therapeutic provided long-term (>12 weeks) LV expression of the βARKct transgene and significantly improved cardiac contractility and circulating catecholamine levels. In this model, βARKct was able to reverse LV remodeling, which current therapies including administration of β-blockers cannot do(92). Lastly, similar findings have been observed in murine models of I/R, MI, and TAC(66,87,93,94). One drawback of these studies is that, unlike pharmacological inhibitors of GRK2, utilizing βARKct as a treatment for cardiac injury requires protein expression by cardiomyocytes. This presents a hurdle for clinical applicability, as treatment would require viral-mediated gene delivery of βARKct. However, these data are highly promising and more research is needed to develop βARKct as a clinical therapeutic.

An exciting aspect of βARKct modulation of endogenous GRK2 signaling, which has expanded on research into novel functions for this protein, is the discovery that not only does βARKct rescue cardiac function by blocking GRK2 interaction with β-ARs and arrestins at the cell membrane but appears to mediate effects separate from adrenergic signaling such as bioenergetics, substrate utilization and apoptosis. Following the discovery that ERK phosphorylation at GRK2-ser670 is indispensable for mitochondrial translocation by the chaperonin Hsp90(72,95), it was determined that this stress-induced mitochondrial GRK2 pool (mtGRK2) is not only present in all mitochondrial compartments, but is associated with increased Ca²⁺-induced opening of the mPTP and reduced mitochondrial function. Of note, overexpression of GRK2 in isolated mouse cardiomyocytes increased in vitro production of mitochondrial superoxide (O₂^−•) and conversely impaired fatty acid oxidation (FAO) and ATP production. Inhibition of GRK2 increased oxygen consumption rates (OCR) and ATP production(96). Inhibition of GRK2 in a global heterozygous knockdown mouse model (GRK2^+/−), furthermore, increased OCR and ATP production over WT mice or those with cardiac-specific overexpression of GRK2 (TgGRK2) and was sufficient to protect FAO and mitochondrial OCR in a murine TAC model of HF(97).

Given the significance of these findings, identifying therapeutic targets to protect failing or injured hearts from the effects of mtGRK2 has emerged as a promising field, and one of the earliest therapeutics tested has been βARKct, to significant success. Expression of exogenous βARKct either via adenoviral infection or in a novel knock-in (KI) mouse line where endogenous GRK2 was replaced by a Ser-to-Ala mutation at the 670 position (GRK2-S670A) resulted in increased basal respiratory rate as well as ATP-linked respiration(96). These effects are likely due to stoichiometric inhibition of endogenous mtGRK2 accumulation by the presence of βARKct acting as a decoy, since the recombinant peptide also possesses the consensus ERK phosphorylation site at ser670. Significantly, mitochondrial parameters such as maximal respiration rate, spare capacity, coupling efficiency and an indirect measurement of glycolytic activity, the extracellular acidification rate (ECAR), were not altered at baseline between WT and βARKct-expressing cardiomyocytes(96); these results strongly suggest that the beneficial phenotype seen with βARKct is due to the limiting of GRK2’s mitochondrial accumulation following oxidative stress and that further mechanisms for mtGRK2 regulation should be investigated.

The generation of the GRK2-S670A KI mouse resistant to ERK phosphorylation was an exciting recent tool for investigating mtGRK2 signaling(72,96). When this GRK2 mutant is overexpressed in cardiomyocytes, utilization of exogenous fatty acids (FAs) such as palmitate was comparable to WT, with a normal basal respiratory phenotype(96). These experiments established that mtGRK2’s deleterious effects require competent kinase activity, as cardiomyocyte expression of a kinase-dead mutant (GRK2-K220R) demonstrated normal FA utilization and basal respiration with a trend towards increased palmitate-induced ATP production compared to WT GRK2 and in stark contrast to GRK2-OE(96). The clinical implications of these findings are significant, as it has been demonstrated that cardiac GRK2 levels are elevated in mouse models of diabetes (T2DM) and high-fat diet(98) and that heterozygous knockdown of GRK2 confers resistance to weight gain in mice fed a high-fat diet(98,99). Additionally, has been well-established that in animal models of MI and post-infarction human patients, myocardial substrate delivery and glucose consumption are negatively altered; these parameters are link to extent of cell death, infarct size and survival of the area injured by ischemia(100). Linking these deficits to the negative role which mtGRK2 plays in GRK2-mediated substrate utilization and mitochondrial bioenergetics, it was recently discovered that GRK2-S670A KI mice demonstrate significantly reduced cardiomyocyte death and preserved cardiac function post-IR injury compared to WT mice; importantly, GRK2-S670A expressing cardiomyocytes subjected to an in vitro model of I/R had enhanced glucose-mediated mitochondrial respiration that was the result of improved glucose oxidation(101). Thus, significant evidence indicates that mtGRK2 is a viable target for protecting the heart against metabolic changes which result from acute ischemic injury or during HF.

Lastly, the N-terminus of GRK2 possesses a regulator of G protein signaling (RGS) homology (RH) domain (see Figure 2). Interestingly, the amino acid sequence of GRK2’s RH domain is considered atypical and less-conserved compared to other RGS proteins, possessing a unique region which can directly interact with Gαq to regulate signaling without alteration of GTPase activity whereas typical RGS proteins bind and accelerate GTPase (GTP hydrolysis) activity in Gα subunits(102). Given the contribution of aberrant Gαq signaling and its effectors such as downstream PLC activity and Ca²⁺ levels, to the development of pressure-overload cardiac HT, GRK2-RGS presents another possible therapeutic target. It has been demonstrated that expression of a GRK2-RGS-like peptide (βARKrgs) containing GRK2 amino acid residues 50–185 may indeed provide protection against HT due to its binding to Gαq in place of endogenous GRK2(64). Mice with α-MHC-driven cardiac overexpression of this peptide (TgβARKrgs) possess similar cardiac phenotype and function to WT mice, with a trend toward concentric HT but normal cardiac function(64,103), at baseline. However, at 4 weeks (and up to 14 weeks) following TAC-induced pressure overload, TgβARKrgs mice demonstrated reduced hypertrophic response and improved cardiac function as measured by LV mass, HW/TL, cardiomyocyte cross-sectional area and hypertrophic gene expression (ANF, BNP, βMHC)(64). Significantly, it was also shown that endogenous GRK2 expression was significantly lower in TgβARKrgs compared to controls following induction of CHT, which may confer more global cardioprotection against the typical increase in GRK2 levels seen in HF patients(41,103). These data indicate that βARKrgs not only protects against maladaptive HT in failing hearts, but may also prevent the transition to decompensation and represents a viable therapeutic in this model of cardiac pathology.