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Designer Approaches for G Protein–Coupled Receptor Modulation for Cardiovascular Disease

Summary The new horizon for cardiac therapy may lie beneath the surface, with the downstream mediators of G protein–coupled receptor (GPCR) activity. Targeted approaches have shown that receptor activation may be biased toward signaling through G proteins or through GPCR kinases (GRKs) and β-arrestins, with divergent functional outcomes. In addition to these canonical roles, numerous noncanonical activities of GRKs and β-arrestins have been demonstrated to modulate GPCR signaling at all levels of receptor activation and regulation. Further, research continues to identify novel GRK/effector and β-arrestin/effector complexes with distinct impacts on cardiac function in the normal heart and the diseased heart. Coupled with the identification of once orphan receptors and endogenous ligands with beneficial cardiovascular effects, this expands the repertoire of GPCR targets. Together, this research highlights the potential for focused therapeutic activation of beneficial pathways, with simultaneous exclusion or inhibition of detrimental signaling, and represents a new wave of therapeutic development.

G protein-coupled receptors (GPCRs) have been mainstays of therapeutic drug targeting in the heart for decades, in particular angiotensin II type 1A receptor (AT 1 R) blockers and b-adrenergic receptor (bAR) blockers (b-blockers), which improve symptoms, hemodynamics, and clinical outcomes of heart failure (HF) patients. However, improvements on these classic GPCR blockers have been slow to develop over the last couple of decades and many promising new drugs have failed to reduce morbidity and mortality highlighting the need for novel therapeutics. GPCR signaling systems are comprised of numerous molecular components and multiple pathways are emerging as potential therapeutic targets, which have the potential to shift the paradigm of standard HF treatment. Indeed, a rapidly expanding and exciting area of GPCR research is focused on the differential engagement of proximal signaling pathways in a biased manner to promote beneficial functional or survival effects in the heart while preventing activation of potentially cardiotoxic pathways.
It has been appreciated for some time now that there are at least 2 primary mechanistic signaling pathways engaged following ligand binding to its GPCR, represented by classical G protein-dependent signaling and noncanonical GRK/b-arrestin-mediated signaling (1)(2)(3). For both AT 1 R and bARs, prolonged G protein-dependent signaling is associated with detrimental cardiac outcomes over time, while GRK/ b-arrestin-dependent signaling has been demonstrated in preclinical models to promote beneficial effects in various HF models. Additionally, global gene expression analyses have identified more than 200 GPCRs present in the heart, several at equal or higher expression than AT 1 R and bARs (4), which may represent novel and untapped therapeutic targets by which to improve HF outcomes in either a G proteinor GRK/-b-arrestin-dependent manner. Thus, discovery of compounds, or biased ligands (5,6), that can selectively engage GRK/b-arrestin-dependent signaling is an important and novel area of current GPCR research. Herein, we compare the impact of GRKs and b-arrestins on cardiac function, survival, and remodeling in HF; highlight the latest findings related to biased ligand-mediated engagement of both AT 1 R and bARs; and discuss newly discovered GPCR systems that provide promise for the development of novel HF therapeutics.

GPCR KINASES
GPCR signaling is tightly controlled by cytosolic GRKs.
Canonically, GRKs translocate to or target agonistbound GPCRs where they phosphorylate the receptor, facilitating b-arrestin recruitment for desensitization and internalization of receptors. A thorough review of GRK structure, localization, GPCR activity, cardiac function, and regulation has recently been published (7), highlighting how GRKs control GCPR signal duration and impact. Continued research into the regulation, distribution, and noncanonical signaling of cardiac GRKs has demonstrated expanding roles in both normal cardiac function and cardiovascular disease (Central Illustration) (8)(9)(10)(11). A more thorough understanding of the functional consequences of GRK activities in the heart will allow for targeted approaches for GRK modulation in human therapy.
Despite a high level of shared sequence identity and the same tissue distribution as GRK2, GRK3 has been observed to participate in the regulation of thrombin, endothelin, and a 1 -adrenergic receptor (a 1 AR) activity in the heart. Although cardiac GRK3 levels are not altered during human HF, these data suggest that it may play a role in cardiac growth and hypertrophy (12,13). Transgenic mice overexpressing the C-terminal pleckstrin homology domain of GRK3 exhibit a phenotype of increased systolic function (14)  GPCRs may be protective. This is an important consideration regarding the therapeutic potential of GRK5, and suggests that targeting the cellular localization of GRK5 may be more effective than a kinase inhibitor. In fact, a small molecular inhibitor of GRK5 has already been developed and was used to gain a high-resolution crystal structure for bovine GRK5 (36), which will allow for the investigation of allosteric modulation of GRK5 and may provide a means to differentially target nuclear translocation versus kinase activity. The role of GRK5 in the regulation cardiac signaling in the healthy and diseased myocardium and the pursuit for therapeutic inhibition of GRK5 has recently been reviewed in detail (37).
Despite the wealth of knowledge regarding the role of GRK2 in cardiovascular function, the full scope and impact of GRK2 regulation in cardiac physiology and disease is still being defined. Ongoing research continues to identify new interacting partners and AR ¼ adrenergic receptor; ARKct ¼ adrenergic receptor kinase carboxy-terminal peptide; AT1R ¼ angiotensin II type 1A receptor; beta ¼ beta subunit; G alpha q ¼ G alpha q subunit; gamma ¼ gamma subunit; GRK ¼ G protein-coupled receptor kinase; GRK3ct ¼ G protein-coupled receptor kinase 3 carboxy-terminal peptide; OE ¼ over-expression.
Grisanti et al.  Although these data may suggest cardiac gene transfer of b-arrestin2 as a viable therapy for HF, opposing data suggest that during acute injury, b-arrestin2 overexpression may be detrimental. In this study, b-arrestin2 expression was selectively upregulated in cultured myocytes and rat models of ischemiareperfusion injury, promoting cardiomyocyte death and enhanced ischemia-reperfusion-induced injury, whereas b-arrestin2 knockdown or functional deficiency conferred resistance to ischemia-reperfusion injury (89). The worsening phenotype occurred via an interaction between b-arrestin2 and the p85 subunit of phosphoinositide 3-kinase (PI3K) that negatively regulated p85-PI3K/Cav3 complex formation and subsequently blocked PI3K-mediated activation of Akt and glycogen synthase kinase 3b survival signaling (89). These conflicting reports will need to be resolved to confirm the relevance of b-arrestin2 as a therapeutic target for the treatment of human HF. Some of these differential roles for b-arrestin1 and b-arrestin2 and their therapeutic implications have been recently reviewed (90). This is a cartoon representation outlining the effect on cardiovascular signaling and outcomes when the AT 1 R is differentially affected by direct agonism or antagonism versus the potential beneficial effect of b-arrestin-targeted biased agonism. Ca 2þ ¼ calcium ion; other abbreviations as in Figure 1.

APELIN-APJ SYSTEM
A newly identified GPCR system of therapeutic interest in the cardiovascular system is the apelin-APJ system. Apelin was discovered in 1998 as an endogenous ligand for the previously orphan receptor APJ, which shows homology and similar tissue distribution as the AT 1 R (136,137). Apelin is synthesized as preproapelin and cleaved by angiotensin-converting enzyme into several shorter, active fragments that appear to differ in their ability to activate, internalize, and recycle the receptor (136,138). APJ is a Gi-coupled GPCR, with potential coupling to Gq (136,139,140). In In the heart, apelin is expressed at moderate levels but is a potent inotropic agent through activation of Gq, phospholipase C or protein kinase C, Na þ -H þ sarcolemmal exchange, and Na þ -Ca þþ exchange pathways (140,(145)(146)(147). Endogenous ligands for APJ including apelin fragments appear to exert a G protein bias (139); however, these events still lead to receptor internalization through classical b-arrestin-dependent mechanisms (139). In contrast, activation of APJ through stretch leads to b-arrestin-dependent hypertrophy (147).
Apelin-APJ are also thought to play a role in cardiac dysfunction. Patients show elevations in plasma apelin in patients with early (148,149), which is decreased in later stages (148,150). In murine models of ischemic HF, acute up-regulation of apelin and APJ occurs following ischemic injury and these elevations in expression persist long-term (151)(152)(153). APJ and apelin knockout mice have impairments in contractility (154,155) and impaired healing following ischemia-reperfusion (156). Furthermore, administration of apelin or stable apelin analogs protects against ischemia-reperfusion in rodent models of ischemic heart disease, supporting the therapeutic potential of targeting apelin-APJ for treatment of HF (156)(157)(158)(159)(160). However, the effects of biased ligands in this receptor system remains to be determined.

UROCORTIN-CRF SYSTEM
Another GPCR system that is gaining ground as a handling and cAMP levels to a similar degree as isoproterenol, the CRF peptide effect on myocyte contraction was much less robust (163). Although the mechanism of action of these CRF peptides has not been fully elucidated, it has been hypothesized that