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The critical role of G protein–coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5′-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2−/− platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2−/− platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2−/− platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2−/− mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gβγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 “interactome” that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (βARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, βARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, βARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify βARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and βARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in βARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male βARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the βARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.

Aberrant changes in gene expression underlie the pathogenesis and progression of pressure-overload heart failure, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. Signaling through the G protein Gq triggers maladaptation and heart failure, in part through the activation of G protein-coupled receptor kinase-5 (GRK5). Hypertrophic stimuli induce the accumulation of GRK5 in the nuclei of cardiomyocytes, where it regulates pathological gene expression through multiple transcription factors including NFAT. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that binds to calmodulin (CaM). Here, we sought to prevent GRK5-mediated pathology in pressure-overload maladaptation and heart failure by expressing in cardiomyocytes a peptide encoding the GRK5 NT (GRK5nt) that encompasses the CaM binding domain. In cultured cardiomyocytes, GRK5nt expression abrogated Gq-coupled receptor-mediated hypertrophy, including attenuation of pathological gene expression and the transcriptional activity of NFAT and NF-κB. We confirmed that GRK5nt bound to and blocked Ca(2+)-CaM from associating with endogenous GRK5, thereby preventing GRK5 nuclear accumulation after pressure overload. We generated mice that expressed GRKnt in a cardiac-specific fashion (TgGRK5nt mice), which exhibited reduced cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis after chronic transverse aortic constriction. Together, our data support a role for GRK5nt as an inhibitor of pathological GRK5 signaling that prevents heart failure.

Recent data supporting any benefit of stem cell therapy for ischemic heart disease have suggested paracrine-based mechanisms via extracellular vesicles (EVs) including exosomes. We have previously engineered cardiac-derived progenitor cells (CDCs) to express a peptide inhibitor, βARKct, of G protein-coupled receptor kinase 2, leading to improvements in cell proliferation, survival, and metabolism. In this study, we tested whether βARKct-CDC EVs would be efficacious when applied to stressed myocytes in vitro and in vivo. When isolated EVs from βARKct-CDCs and control GFP-CDCs were added to cardiomyocytes in culture, they both protected against hypoxia-induced apoptosis. We tested whether these EVs could protect the mouse heart in vivo, following exposure either to myocardial infarction (MI) or acute catecholamine toxicity. Both types of EVs significantly protected against ischemic injury and improved cardiac function after MI compared with mice treated with EVs from mouse embryonic fibroblasts; however, βARKct EVs treated mice did display some unique beneficial properties including significantly altered pro- and anti-inflammatory cytokines. Importantly, in a catecholamine toxicity model of heart failure (HF), myocardial injections of βARKct-containing EVs were superior at preventing HF compared with control EVs, and this catecholamine toxicity protection was recapitulated in vitro. Therefore, introduction of the βARKct into cellular EVs can have improved reparative properties in the heart especially against catecholamine damage, which is significant as sympathetic nervous system activity is increased in HF. NEW & NOTEWORTHY βARKct, the peptide inhibitor of GRK2, improves survival and metabolic functions of cardiac-derived progenitor cells. As any benefit of stem cells in the ischemic and injured heart suggests paracrine mechanisms via secreted EVs, we investigated whether CDC-βARKct engineered EVs would show any benefit over control CDC-EVs. Compared with control EVs, βARKct-containing EVs displayed some unique beneficial properties that may be due to altered pro- and anti-inflammatory cytokines within the vesicles.

Significance Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. G protein-coupled receptor (GPCR) kinase 5 (GRK5) has been shown to cause deleterious effects on the cardiomyocyte during HF; however, its effects in cardiac fibroblasts, the crucial cell type responsible for maintaining the structural integrity of the heart, is not understood. Here, we use in vitro and in vivo methods to demonstrate that inhibition of GRK5 inhibits fibroblast activation and attenuates the fibrotic response in the heart., Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

Background and Purpose: The influence of β-adrenoceptor (βAR) signaling on the regulation of exosomes secreted from cardiomyocytes is unknown and since catecholamines are increased in heart failure (HF), there is interest in uncovering whether βARs can induce specific changes in the content of circulating blood exosomes in HF. In this study, we have evaluated whether βAR stimulation by isoproterenol (ISO) on neonatal rat ventricle myocytes (NRVMs) and in vivo in mice can alter the number, size and microRNA (miR) content of secreted exosomes. Methods and Results: ISO treatment of NRVMs did not change exosome number and size of compared than vehicle (PBS). After purifying total RNA from treated myocytes and secreted exosomes, we evaluated the expression level of 37 candidate miR’s, which were selected from previous microarray data. We found that ISO treatment decreased 14 miR’s (miR-222, -106a, -292a, -181b, -210, -489, -214, -1947, -195, -17, -7b, -93, -532 and -19a) in exosomes and in the myocytes themselves, mir-292a and -1947 were up regulated and mir-7b, down regulated. Further, ISO was then used to treat C57 mice via osmotic pump chronically. The number and size of exosomes purified from circulating blood was not changed after 2 and 8 weeks. We evaluated expression of the 14 miRs down-regulated in myocytes as well miR-1 and -21 (important cardiac miRs) both in the blood and hearts of ISO-treated mice. MiR-1 did not change in both blood exosomes and heart tissue and miR-21 was up-regulated in heart tissue but not in blood both at 2 and 8 weeks after ISO treatment. In addition, miRNA-489 and -7b was down-regulated in hearts with miR-214 and -292a up regulated. In blood exosomes, we found only down-regulation of miR-489 and -7b but not miR-214 and 292a. Conclusions: We found that the βAR agonist ISO altered exosomal miR contents but not exosome size and number from myocytes. Importantly, this was found in myocytes in culture and in vivo blood and myocardium that were treated with ISO but several differences were found and changes in blood and myocytes were not homogeneous. However, two miRs, mir-7b and -miR-489, both changes similarly from their origin (NRVMs or mouse hearts) and also in circulating blood exosomes. Therefore, these miRs may represent biomarkers for sympathetic nervous system abnormalities in HF and their therapeutic potential should be evaluated.

A vast body of literature has established G protein–coupled receptor kinase 2 (GRK2; family: β-adrenergic receptor kinases [βARKs]) as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function after injury in numerous animal models. In recent years, discovery of several noncanonical GRK2 targets has expanded our view of this kinase. This article describes the exciting finding that cardiac GRK2 activity can regulate whole-body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgβARKct) display an enhanced obesogenic phenotype when fed a high-fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD-induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD-fed TgβARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics, we found that cardiac GRK2 signaling altered myocardial branched-chain amino acid (BCAA) and endocannabinoid metabolism. In addition, it modulated circulating BCAA and endocannabinoid metabolite profiles on mice fed an HFD. We also found that one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. These results suggest that metabolic changes in the heart due to GRK2 signaling on mice fed an HFD control whole-body metabolism.

Rationale: G protein–coupled receptor kinases (GRKs) acting in the cardiomyocyte regulate important signaling events that control cardiac function. Both GRK2 and GRK5, the predominant GRKs expressed in the heart, have been shown to be upregulated in failing human myocardium. Although the canonical role of GRKs is to desensitize G protein–coupled receptors via phosphorylation, it has been demonstrated that GRK5, unlike GRK2, can reside in the nucleus of myocytes and exert G protein–coupled receptor–independent effects that promote maladaptive cardiac hypertrophy and heart failure. Objective: To explore novel mechanisms by which GRK5 acting in the nucleus of cardiomyocytes participates in pathological cardiac hypertrophy. Methods and Results: In this study, we have found that GRK5-mediated pathological cardiac hypertrophy involves the activation of the nuclear factor of activated T cells (NFAT) because GRK5 causes enhancement of NFAT-mediated hypertrophic gene transcription. Transgenic mice with cardiomyocyte-specific GRK5 overexpression activate an NFAT-reporter in mice basally and after hypertrophic stimulation, including transverse aortic constriction and phenylephrine treatment. Complimentary to this, GRK5 null mice exhibit less NFAT transcriptional activity after transverse aortic constriction. Furthermore, the loss of NFATc3 expression in the heart protected GRK5 overexpressing transgenic mice from the exaggerated hypertrophy and early progression to heart failure seen after transverse aortic constriction. Molecular studies suggest that GRK5 acts in concert with NFAT to increase hypertrophic gene transcription in the nucleus via GRK5’s ability to bind DNA directly without a phosphorylation event. Conclusions: GRK5, acting in a kinase independent manner, is a facilitator of NFAT activity and part of a DNAbinding complex responsible for pathological hypertrophic gene transcription.

Increased abundance of GRK2 [G protein–coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.

Increased abundance of GRK2 [G protein–coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser(670) is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.

Rationale : G protein–coupled receptor kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is upregulated in heart failure. Although GRK5 is a critical regulator of cardiac G protein–coupled receptor signaling, recent data has uncovered noncanonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early heart failure after transverse aortic constriction (TAC) because of GRK5 nuclear accumulation. Objective : In this study, we investigated the role of GRK5 in physiological, swimming-induced hypertrophy (SIH). Methods and Results : Cardiac-specific GRK5 transgenic mice and nontransgenic littermate control mice were subjected to a 21-day high-intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed, including nuclear localization of GRK5, and compared with TAC. Unlike after TAC, swim-trained transgenic GRK5 and nontransgenic littermate control mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC transgenic GRK5 mice was able to preserve cardiac function. Conclusions: These data suggest that although nuclear-localized GRK5 is a pathological mediator after stress, this noncanonical nuclear activity of GRK5 is not induced during physiological hypertrophy.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca(2+)- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca(2+) retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca(2+) and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca(2+)- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.

G protein-coupled receptors (GPCRs) are important regulators of various cellular functions via activation of intracellular signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and subsequent β-arrestin-mediated mechanisms including phosphorylation, internalization, and either receptor degradation or resensitization. The seven-member GRK family varies in their structural composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been highly implicated in various etiologies of cardiovascular disease including hypertension and heart failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in cardiac homeostasis and disease progression, and herein we review the current data on GRKs related to cardiac disease and discuss their potential in the development of novel therapeutic strategies in cardiac diseases including heart failure.

Heart failure (HF) is a leading cause of death worldwide and a growing burden on public health, and the underlying mechanisms of cardiac remodeling and decompensation to HF remain a focus of research efforts towards therapeutic development. Signaling via G protein-coupled receptors (GPCRs) is critical for normal heart function and is tightly controlled by GPCR kinases (GRKs) with GRK2 (originally βARK1), being intimately involved in HF progression. In addition to its well-characterized role in regulating GPCRs, ongoing research has demonstrated great diversity in the functional roles of GRK2. I have recently investigated GRK2 amino terminal binding interactions through the generation of transgenic (Tg) mice with cardiac-targeted expression of the amino-terminal peptide βARKnt (residues 50-145). In a murine model of trans-aortic constriction (TAC)-induced pressure overload, echocardiography revealed increased left ventricular (LV) posterior wall thickness (1.57 versus 1.37 ± 0.02; n = 16) and LV mass in TgβARKnt compared to non-transgenic littermate controls (NLC) 4 weeks after TAC or Sham surgery. Interestingly, despite enhanced hypertrophy at baseline and after acute pressure overload, the progression to HF was paradoxically inhibited in TgβARKnt mice during chronic pressure overload with preserved cardiac function (% Ejection Fraction 57.3 versus 37.3 ± 2.0; n = 11, 10). Further, βARKnt expression limited adverse left ventricular remodeling, with reduced interstitial fibrosis (% area fibrosis 4.1 versus 9.2 ± 0.8; n = 11, 9 hearts) and preserved β-adrenergic receptor density 4 weeks after surgery. The effect of cardiac βARKnt expression was not consistent with alterations in GRK2 activity at GPCRs as neither GRK2 peptide inhibition (TgβARKct) nor GRK2 overexpression alter hypertrophy, and overexpression hastens HF development. Further, TgβARKnt mice exhibit reduced epididymal white adipose content and altered mitochondrial respiration, suggesting altered cardiac metabolism. These data support the idea that the βARKnt peptide embodies a distinct functional interaction and that βARKnt-mediated regulation of β-adrenergic receptor density may provide a novel means of cardioprotection during pressure-overload induced HF.

International audience; Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardio-myocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.

Significance: Heart failure (HF) is a common end point for many underlying cardiovascular diseases. Down-regulation and desensitization of β-adrenergic receptors (β-AR) caused by G-protein-coupled receptor (GPCR) kinase 2 (GRK2) are prominent features of HF. Recent Advances and Critical Issues: Significant progress has been made to understand the pathological role of GRK2 in the heart both as a GPCR kinase and as a molecule that can exert GPCR-independent effects. Inhibition of cardiac GRK2 has proved to be therapeutic in the failing heart and may offer synergistic and additional benefits to β-blocker therapy. However, the mechanisms of how GRK2 directly contributes to the pathogenesis of HF need further investigation, and additional verification of the mechanistic details are needed before GRK2 inhibition can be used for the treatment of HF. Future Directions: The newly identified characteristics of GRK2, including the S-nitrosylation of GRK2 and the localization of GRK2 on mitochondria, merit further investigation. They may contribute to it being a pro-death kinase and result in HF under stressed conditions through regulation of intracellular signaling, including cardiac reduction-oxidation (redox) balance. A thorough understanding of the functions of GRK2 in the heart is necessary in order to finalize it as a candidate for drug development. Antioxid. Redox Signal. 21, 2032–2043.

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated and contribute to adverse remodeling and contractile dysfunction, while inhibition via a carboxyl-terminal peptide, βARKct, enhances heart function and can prevent HF. Mounting evidence supports the idea of a dynamic “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for HF therapy. For instance, GRK2 contains a putative amino-terminal R egulator of G protein S ignaling (RGS) domain (βARKrgs) that directly interacts with Gαq and inhibits signaling. Previously, our lab investigated cardiac-specific transgenic expression of a fragment of this RGS domain (βARKnt), that did not reduce acute hypertrophy after pressure overload or demonstrate RGS activity in vivo against Gαq-mediated signaling. In contrast, βARKnt induced hypertrophy and elevated β-adrenergic receptor (βAR) density without altering agonist-induced contractility or adenylyl cyclase activity, due to a compensatory increase in GRK2 activity. Importantly, βAR downregulation in response to chronic agonist administration was attenuated by βARKnt expression, indicating a novel regulation of βAR receptor density. Herein, we investigated the effect of βARKnt expression during chronic pressure overload post trans-aortic constriction (TAC). Echocardiographic analysis revealed increased posterior wall thickness and left-ventricular mass 4 weeks post-TAC compared to non-transgenic littermate controls. Importantly, despite enhanced hypertrophy, the progression to HF was inhibited in βARKnt mice 14 weeks post-TAC. Histological analysis of interstitial fibrosis and cross-sectional area is underway to determine alterations in maladaptive remodeling. Further, cardiomyocyte signaling and βARKnt protein-binding partners are a focus, since our data indicate that βARKnt-mediated regulation of βAR density may provide a novel means of cardioprotection during pressure-overload induced HF.

G protein-coupled receptor (GPCR) kinases (GRKs) play a critical role in cardiac function by regulating GPCR activity. GRK2 suppresses GPCR signaling by phosphorylating and desensitizing active GPCRs, and through protein-protein interactions that uncouple GPCRs from their downstream effectors. Several GRK2 interacting partners, including Gα(q), promote maladaptive cardiac hypertrophy, which leads to heart failure, a leading cause of mortality worldwide. The regulator of G protein signaling (RGS) domain of GRK2 interacts with and inhibits Gα(q) in vitro. We generated TgβARKrgs mice with cardiac-specific expression of the RGS domain of GRK2 and subjected these mice to pressure overload to trigger adaptive changes that lead to heart failure. Unlike their nontransgenic littermate controls, the TgβARKrgs mice exhibited less hypertrophy as indicated by reduced left ventricular wall thickness, decreased expression of genes linked to cardiac hypertrophy, and less adverse structural remodeling. The βARKrgs peptide, but not endogenous GRK2, interacted with Gα(q) and interfered with signaling through this G protein. These data support the development of GRK2-based therapeutic approaches to prevent hypertrophy and heart failure.

Rationale: Coronary artery ligation to induce myocardial infarction (MI) in mice is typically performed by an invasive and time-consuming approach that requires ventilation and chest opening (classic method), often resulting in extensive tissue damage and high mortality. We developed a novel and rapid surgical method to induce MI that does not require ventilation. Objective: The purpose of this study was to develop and comprehensively describe this method and directly compare it to the classic method. Methods and Results: Male C57/B6 mice were grouped into 4 groups: new method MI (MI-N) or sham (S-N) and classic method MI (MI-C) or sham (S-C). In the new method, heart was manually exposed without intubation through a small incision and MI was induced. In the classic method, MI was induced through a ventilated thoracotomy. Similar groups were used in an ischemia/reperfusion injury model. This novel MI procedure is rapid, with an average procedure time of 1.22±0.05 minutes, whereas the classic method requires 23.2±0.6 minutes per procedure. Surgical mortality was 3% in MI-N and 15.9% in MI-C. The rate of arrhythmia was significantly lower in MI-N. The postsurgical levels of tumor necrosis factor-α and myeloperoxidase were lower in new method, indicating less inflammation. Overall, 28-day post-MI survival rate was 68% with MI-N and 48% with MI-C. Importantly, there was no difference in infarct size or post-MI cardiac function between the methods. Conclusions: This new rapid method of MI in mice represents a more efficient and less damaging model of myocardial ischemic injury compared with the classic method.

Background— A salient characteristic of dysfunctional myocardium progressing to heart failure is an upregulation of the adenylyl cyclase inhibitory guanine nucleotide (G) protein α subunit, Gα i2 . It has not been determined conclusively whether increased Gi activity in the heart is beneficial or deleterious in vivo. Gi signaling has been implicated in the mechanism of cardioprotective agents; however, no in vivo evidence exists that any of the Gα subunits are cardioprotective. We have created a novel molecular tool to specifically address the role of Gi proteins in normal and dysfunctional myocardium. Methods and Results— We have developed a class-specific Gi inhibitor peptide, GiCT, composed of the region of Gα i2 that interacts specifically with G protein–coupled receptors. GiCT inhibits Gi signals specifically in vitro and in vivo, whereas Gs and Gq signals are not affected. In vivo expression of GiCT in transgenic mice effectively causes a “functional knockout” of cardiac Gα i2 signaling. Inducible, cardiac-specific GiCT transgenic mice display a baseline phenotype consistent with nontransgenic mice. However, when subjected to ischemia/reperfusion injury, GiCT transgenic mice demonstrate a significant increase in infarct size compared with nontransgenic mice (from 36.9±2.5% to 50.9±4.3%). Mechanistically, this post-ischemia/reperfusion phenotype includes increased myocardial apoptosis and resultant decreased contractile performance. Conclusions— Overall, our results demonstrate the in vivo utility of GiCT to dissect specific mechanisms attributed to Gi signaling in stressed myocardium. Our results with GiCT indicate that upregulation of Gα i2 is an adaptive protective response after ischemia to shield myocytes from apoptosis.

Background— The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results— Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca 2+ )-sensing S100A1, in a rat model of heart failure. The chronic heart failure–rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca 2+ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over β-adrenergic receptor blockade, a current pharmacological heart failure treatment. Conclusions— These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated and contribute to adverse remodeling and contractile dysfunction, while inhibition via a carboxyl-terminal peptide, βARKct, enhances heart function and can prevent HF development. Mounting evidence supports the idea of a dynamic “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for HF therapy. For instance, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain (βARK-RGS) that directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. Previously, our lab investigated cardiac-specific transgenic (Tg) expression of a fragment of this RGS domain (βARKnt). This fragment did not alter acute hypertrophy after pressure overload or demonstrate RGS activity in vivo against Gq-mediated signaling. In contrast, βARKnt induced hypertrophy and elevated β-adrenergic receptor (βAR) density without altering agonist-induced contractility or adenylyl cyclase activity, due to a compensatory increase in GRK2 activity. Importantly, though, βAR downregulation in response to chronic agonist administration was attenuated by βARKnt expression, indicating a novel regulation of βAR receptor density. Given these findings we have recently investigated the effect of βARKnt expression during chronic pressure overload post trans-aortic constriction (TAC). Echocardiographic analysis revealed increased posterior wall thickness and left-ventricular mass 4 weeks post-TAC compared to non-transgenic littermate controls (NLC). Importantly, despite enhanced hypertrophy, the progression to HF was inhibited in βARKnt mice 14 weeks post-TAC (%LV Ejection Fraction of 36.1 ± 0.2 in NLC versus 56.6 ± 0.9 in Tg mice). While mechanistic characterization is underway, these data indicate that βARKnt-mediated regulation of βAR density may provide a novel means of cardioprotection during pressure-overload induced HF.

Heart failure (HF) is a disease of epidemic proportion associated with exceedingly high healthcare costs. Data shows that G protein-coupled receptor (GPCR) kinase 2 (GRK2) plays a critical role in HF progression, promoting dysfunctional adrenergic signaling and myocyte death. We found that the selective serotonin reuptake inhibitor, paroxetine, could selectively inhibit GRK2. In this study wild-type mice underwent myocardial infarction (MI) and following 2 weeks of injury, underwent 4 weeks of treatment with paroxetine or control SSRI, fluoxetine. All post-MI mice exhibited similar left ventricular (LV) dysfunction prior to treatment; however, in contrast to continuing degradation of function, paroxetine produced significant improvement in LV function and structure. Paroxetine enhanced hemodynamic function, blocking the ~2.5 fold increase in LV end diastolic pressure (LVEDP). Paroxetine restored βAR membrane density, returned serum catecholamine and GRK2 levels to baseline, and prevented induction of the ...

Background: Natriuretic peptides ProANP and Pro BNP are translated as biologically-inactive pro-hormones that require proteolytic cleavage into biologically active carboxyl-terminal fragments by the serine protease Corin. Activated ANP and BNP bind to GC-A or natriuretic peptide receptor A (NPRA), which increases intracellular second messenger cGMP, and further stimulates three known cGMP effector molecules: cGMP-dependent protein kinases (PKGs), cGMP-dependent phosphodiesterases (PDEs), and cGMP-dependent ion channels. This produces anti-hypertension, anti-hypertrophy and anti-fibrosis effects. The two linked SNP mutations in Corin gene are associated with risk for hypertension and left-ventricular hypertrophy in Blacks. Novel drugs are needed to target this pathway to improve cardiac functions that impaired by the genetic defects and complications caused by many types of heart failure. Methods and Results: We tested a NPR-A agonist PL3994, synthetic molecule made by Palatin Pharmaceutical Company for its potential therapeutic effects on heart failure and cardiac defects. By using our unique Tamoxifen inducible Corin cardiac specific knockout mouse model and Corin T623I/Q636P knockin mouse model, we implant osmotic pumps with PL3994 or PBS into the mice for four weeks while simultaneously constricted mouse aorta artery by TAC by microsurgery. We recorded cardiac function changes by blood pressure, ECHO, and at end stage measured gene expression profiles, hypertrophy, fibrosis and natriuretic pathway molecule: cGMP, cGMP dependent kinases, ANP28/ProANP128, BNP45/proBNP95 in the explanted hearts and plasma. Results. As compared to WT controls, Corin knockout mice and Corin T623I/Q636P knockin mice showed much worse cardiac hypertrophy by ECHO and heart weight and much worse cardiac fibrosis by Trichrome staining. These phenotypes are also supported by the evidences of less signaling cGMP, less PKG kinase activity in the Corin knockout and knockin mice. However, compared to placebo PBS saline, NPR-A agonist PL3994 significantly improved cardiac conditions with less cardiac hypertrophy and fibrosis, also together with increased signaling cGMP, less PKG kinase activity. Conclusions: NPR-A agonist PL3994 is a potential therapeutic drug to treat patients with Corin genetic defects and patients of heart failure caused by malfunctions of natriuretic pathways.

Myocardial infarction (MI) represents an enormous clinical challenge as loss of myocardium due to ischemic injury is associated with compromised left ventricular (LV) function often leading to acute cardiac decompensation or chronic heart failure. S100A1 was recently identified as a positive inotropic regulator of myocardial contractility in vitro and in vivo. Here, we explore the strategy of myocardial S100A1 gene therapy either at the time of, or 2 h after, MI to preserve global heart function. Rats underwent cryothermia-induced MI and in vivo intracoronary delivery of adenoviral transgenes (4 x 10(10) pfu). Animals received saline (MI), the S100A1 adenovirus (MI/AdS100A1), a control adenovirus (MI/AdGFP), or a sham operation. S100A1 gene delivery preserved global in vivo LV function 1 week after MI. Preservation of LV function was due mainly to S100A1-mediated gain of contractility of the remaining, viable myocardium since contractile parameters and Ca(2+) transients of isolated MI/AdS100A1 myocytes were significantly enhanced compared to myocytes isolated from both MI/AdGFP and sham groups. Moreover, S100A1 gene therapy preserved the cardiac beta-adrenergic inotropic reserve, which was associated with the attenuation of GRK2 up-regulation. Also, S100A1 overexpression reduced cardiac hypertrophy 1 week post-MI. Overall, our data indicate that S100A1 gene therapy provides a potential novel treatment strategy to maintain contractile performance of the post-MI heart.

Rationale : G protein–coupled receptor kinases (GRKs) acting in the cardiomyocyte regulate important signaling events that control cardiac function. Both GRK2 and GRK5, the predominant GRKs expressed in the heart, have been shown to be upregulated in failing human myocardium. Although the canonical role of GRKs is to desensitize G protein–coupled receptors via phosphorylation, it has been demonstrated that GRK5, unlike GRK2, can reside in the nucleus of myocytes and exert G protein–coupled receptor–independent effects that promote maladaptive cardiac hypertrophy and heart failure. Objective : To explore novel mechanisms by which GRK5 acting in the nucleus of cardiomyocytes participates in pathological cardiac hypertrophy. Methods and Results : In this study, we have found that GRK5-mediated pathological cardiac hypertrophy involves the activation of the nuclear factor of activated T cells (NFAT) because GRK5 causes enhancement of NFAT-mediated hypertrophic gene transcription. Transgenic mice with cardiomyocyte-specific GRK5 overexpression activate an NFAT-reporter in mice basally and after hypertrophic stimulation, including transverse aortic constriction and phenylephrine treatment. Complimentary to this, GRK5 null mice exhibit less NFAT transcriptional activity after transverse aortic constriction. Furthermore, the loss of NFATc3 expression in the heart protected GRK5 overexpressing transgenic mice from the exaggerated hypertrophy and early progression to heart failure seen after transverse aortic constriction. Molecular studies suggest that GRK5 acts in concert with NFAT to increase hypertrophic gene transcription in the nucleus via GRK5’s ability to bind DNA directly without a phosphorylation event. Conclusions : GRK5, acting in a kinase independent manner, is a facilitator of NFAT activity and part of a DNA-binding complex responsible for pathological hypertrophic gene transcription.

Lysophosphatidic acid (LPA) utilizes a heterotrimeric guanine nucleotide regulatory (G) protein-coupled receptor to activate the mitogen-activated protein kinase pathway and induce mitogenesis in fibroblasts and other cells. A single cell assay system was used to examine the functional interaction of the LPA receptor with G proteins in intact mouse fibroblasts, by measuring LPA-stimulated induction of the immediate-early gene, c-fos, as read out by a stably expressed fos-lacZ reporter gene. Pretreatment of these cells with pertussis toxin at 100 ng/ml almost completely abolished LPA-stimulated c-fos induction. Western blotting revealed that two pertussis toxin (PTX)-sensitive G proteins, G alpha i2 and G alpha i3, were present in membranes prepared from these cells, and Northern blotting confirmed the absence of message for other PTX-sensitive subunits. Microinjection of an alpha il/alpha i2-specific antibody into living cells decreased LPA-stimulated induction of c-fos by 60%, whereas introduction of antibodies to either alpha i3 or alpha 16, a subtype not present in these cells but used as a control, decreased LPA-stimulated c-fos induction by only 19%. In contrast, the alpha i1/alpha i2-specific antibody had no effect on insulin-induced c-fos expression, which is thought to utilize a G protein-independent mechanism of signaling. In addition, cellular expression of an epitope-tagged PTX-resistant mutant of G alpha i2, but not PTX-resistant G alpha i3, restored LPA-stimulated c-fos induction in cells in which endogenous G protein a subunits were uncoupled from the receptor by pretreatment with PTX. Together, these results provide conclusive in vivo evidence that G alpha i2 is the PTX-sensitive G protein a subunit which mediates LPA-stimulated c-fos induction and perhaps mitogenesis in these cells.

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving G protein-coupled receptors (GPCRs) or nitric oxide (NO). These regimens are thought to target distinct molecular pathways. We showed that these pathways were interdependent and converged on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function. Ischemic injury coupled to GPCR activation, including GPCR desensitization and myocyte loss, required GRK2 activation, and we found that cardioprotection mediated by inhibition of GRK2 depended on endothelial nitric oxide synthase (eNOS) and was associated with S-nitrosylation of GRK2. Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other, the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into the mechanism of action of classic drugs used to treat heart failure and new therapeutic approaches to ischemic heart disease.

cAMP-dependent protein kinase (PKA) regulates the L-type calcium channel, the ryanodine receptor, and phospholamban (PLB) thereby increasing inotropy. Cardiac contractility is also regulated by p38 MAPK, which is a negative regulator of cardiac contractile function. The aim of this study was to identify the mechanism mediating the positive inotropic effect of p38 inhibition. Isolated adult and neonatal cardiomyocytes and perfused rat hearts were utilized to investigate the molecular mechanisms regulated by p38. PLB phosphorylation was enhanced in cardiomyocytes by chemical p38 inhibition, by overexpression of dominant negative p38α and by p38α RNAi, but not with dominant negative p38β. Treatment of cardiomyocytes with dominant negative p38α significantly decreased Ca(2+)-transient decay time indicating enhanced sarco/endoplasmic reticulum Ca(2+)-ATPase function and increased cardiomyocyte contractility. Analysis of signaling mechanisms involved showed that inhibition of p38 decreased the activity of protein phosphatase 2A, which renders protein phosphatase inhibitor-1 phosphorylated and thereby inhibits PP1. In conclusion, inhibition of p38α enhances PLB phosphorylation and diastolic Ca(2+) uptake. Our findings provide evidence for novel mechanism regulating cardiac contractility upon p38 inhibition.

G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5's newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca(2+)/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

Rationale: Phosphorylation of β 2 -adrenergic receptor (β 2 AR) by a family of serine/threonine kinases known as G protein–coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein–coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. Objective: We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of β 2 AR to the receptor coupling to G i signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. Methods and Results: Overexpression of GRK2 led to a G i -dependent decrease of contractile response to βAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant β 2 AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type β 2 AR (WT-TG) or a mutant β 2 AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G i signaling with pertussis toxin restores cardiac function in heart failure associated with increased β 2 AR to G i coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of β 2 AR by GRK and resultant increase in G i -biased β 2 AR signaling play an important role in the development of heart failure. Conclusions: Our data show that enhanced β 2 AR phosphorylation by GRK, in addition to PKA, leads the receptor to G i -biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G i -biased β 2 AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.

Maladaptive ventricular hypertrophy often precedes the pathological endpoint of Heart Failure (HF). G protein Coupled Receptors (GPCRs), such as the β-adrenergic receptor, play a crucial role in the development of cardiac hypertrophy. GPCR Kinase 5 (GRK5) is a primary GRK expressed in cardiomyocytes. Traditionally, it phosphorylates and desensitizes agonist-bound GPCRs. Recent, exciting data from our lab shows that GRK5 translocates into the nucleus and phosphorylates Histone Deacetylase 5 (HDAC5) in response to hypertrophic stimuli. This novel kinase activity of GRK5 causes nuclear export of HDAC5, enhancing transcription of hypertrophic genes regulated by myocyte elongation factor 2 (MEF2). We hypothesize that that nuclear GRK5 activity plays a critical role in cardiomyocytes, facilitating pathological hypertrophy and HF, and that hypertrophic GPCR signaling induces GRK5 nuclear translocation. To test this hypothesis, we utilized neonatal rat ventricular cardiomyocytes (NRVMs) to determine GRK5 localization following the hypertrophic stimulus of phenylephrine (PE) treatment. To determine the mechanism causing nuclear translocation, we analyzed the effects targeted inhibition of key post-GPCR hypertrophic signaling pathways on GRK5 cellular localization. Treatment of NRVMs with PE for 1 hour increases nuclear GRK5 accumulation by 100%. Chronic infusion of PE also lead to a 50% increase in nuclear GRK5 in the hearts of mice overexpressing cardiac-specific GRK5. Calmodulin inhibition (CaM) in NRVMs via calmidizolium chloride decreases nuclear GRK5 accumulation, while inhibition of PKC and PKD shows no significant effect. Using a luciferase assay, we have shown that overexpression of CaM increases MEF2 activity 3-fold and causes increased nuclear accumulation of GRK5. Further, overexpression of a GRK5 mutant that lacks CaM binding activity, results in a lack of PE- and CaM-induced nuclear translocation. We have now found that hypertrophic stimuli increases myocyte GRK5 nuclear translocation via a CaM-dependent process. Defining the role of CaM signaling in pathological GRK5 nuclear localization may offer a new therapeutic target for maladaptive hypertrophy and HF.

Background: Previous studies have shown that both cardiac-specific GRK2 transgenic (TG) (αMHC-GRK2) mice and eNOS knockout (KO) mice have larger infarcts after I/R compared to control mice. In contrast, cardiac-targeted eNOS TG mice and cardiac specific GRK2 KO mice show cardioprotection after I/R, suggesting a dynamic interaction between the two proteins. Mechanistically, GRK2 can be inhibited by cellular NO through S-nitrosylation with Cys340 being the major site; meanwhile GRK2 has been shown to directly bind to and inhibit Akt, which is a strong activator of eNOS. Aim: to investigate the potential novel interaction between GRK2 and eNOS, and the consequent functional impact on the protein activity and cardiac phenotype after I/R injury. Methods: αMHC-GRK2 mice were crossed with either eNOS TG or eNOS KO mice. All mice were subjected to sham or 30min myocardial ischemia via coronary artery ligation followed by 24hrs of reperfusion. Infarct size, cardiac function, and tissue apoptosis were examined. Co-IP was used to test the interaction between GRK2 and eNOS, and phosphorylation of eNOS was studied in neonatal myocytes. Results: 1).αMHC-GRK2/eNOS TG hybrid mice showed a significantly reduced infarct size after I/R compared to αMHC-GRK2 mice, accompanied by improved cardiac function measured by echocardiography and hemodynamics, and significantly less apoptosis tested by TUNEL assay, implying a rescue effect by eNOS. 2). αMHC-GRK2/eNOS KO mice exhibited a bigger infarct size compared to either αMHC-GRK2 mice or eNOS KO mice. 3). CO-IP confirmed the interaction between GRK2 and eNOS in cardiac tissue, which was increased upon β-AR agonist treatment. 4). In neonatal ventricular myocytes, GRK2 overexpression significantly decreased eNOS phosphorylation at Ser1177 after exposure to H2O2, while GRK2 knockdown by siRNA led to slight increase in pSer1177. Conclusions: eNOS interacts with and may be a downstream target of GRK2 in the heart. Decreased activation of eNOS may mediate the deleterious effect of GRK2 overexpression during cardiac I/R injury.

Background— Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein–coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the β-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results— Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. Conclusions— Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through β-adrenergic receptors and metabolism through the insulin receptor.

G protein‐coupled receptor kinase 2 (GRK2), which is upregulated in the failing human myocardium, appears to have a role in heart failure (HF) pathogenesis. In peripheral lymphocytes, GRK2 expression has been shown to reflect myocardial levels. This study represents an attempt to define the role for GRK2 as a potential biomarker of left ventricular function in HF patients. We obtained blood from 24 HF patients before and after heart transplantation and followed them for up to 1 year, also recording hemodynamic data and histological results from endomyocardial biopsies. We determined blood GRK2 protein by Western blotting and enzyme‐linked immunosorbent assay. GRK2 levels were obtained before transplant and at first posttransplant biopsy. GRK2 levels significantly declined after transplant and remained low over the course of the study period. After transplantation, we found that blood GRK2 signifi cantly dropped and remained low consistent with improved cardiac function in the transplanted heart. Blood GRK2 has potential as a biomarker for myocardial function in end‐stage HF. Clin Trans Sci 2010; Volume #: 1–5

Rationale: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein–coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. Objective: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac β-adrenergic receptor (βAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the βARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. Methods and Results: We demonstrate, using cardiac-specific GRK2 and βARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with βARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in βARKct mice (16.8±1.3%, P 2 ARs abolished βARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. Conclusion: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the βARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

G protein-coupled receptor (GPCR) kinases (GRKs) are critical regulators of cellular signaling and function. In cardiomyocytes, GRK2 and GRK5 are two GRKs important for myocardial regulation, and both have been shown to be up-regulated in the dysfunctional heart. We report that increased levels and activity of GRK5 in failing myocardium may have unique significance due to its nuclear localization, a property not shared by GRK2. We find that transgenic mice with elevated cardiac GRK5 levels have exaggerated hypertrophy and early heart failure compared with control mice after pressure overload. This pathology is not present in cardiac GRK2-overexpressing mice or in mice with overexpression of a mutant GRK5 that is excluded from the nucleus. Nuclear accumulation of GRK5 is enhanced in myocytes after aortic banding in vivo and in vitro in myocytes after increased Gαq activity, the trigger for pressure-overload hypertrophy. GRK5 enhances activation of MEF2 in concert with Gq signals, demonstrating that nuclear localized GRK5 regulates gene transcription via a pathway critically linked to myocardial hypertrophy. Mechanistically, we show that this is due to GRK5 acting, in a non-GPCR manner, as a class II histone deacetylase (HDAC) kinase because it can associate with and phosphorylate the myocyte enhancer factor-2 repressor, HDAC5. Moreover, significant HDAC activity can be found with GRK5 in the heart. Our data show that GRK5 is a nuclear HDAC kinase that plays a key role in maladaptive cardiac hypertrophy apparently independent of any action directly on GPCRs.

S100A1, a Ca 2+ -binding protein of the EF-hand type, is known to modulate sarcoplasmic reticulum Ca 2+ handling in skeletal muscle and cardiomyocytes. Recently, S100A1 has been shown to be expressed in endothelial cells (ECs). Because intracellular Ca 2+ ([Ca 2+ ] i ) transients can be involved in important EC functions and endothelial NO synthase activity, we sought to investigate the impact of endothelial S100A1 on the regulation of endothelial and vascular function. Thoracic aortas from S100A1 knockout mice (SKO) showed significantly reduced relaxation in response to acetylcholine compared with wild-type vessels, whereas direct vessel relaxation using sodium nitroprusside was unaltered. Endothelial dysfunction attributable to the lack of S100A1 expression could also be demonstrated in vivo and translated into hypertension of SKO. Mechanistically, both basal and acetylcholine-induced endothelial NO release of SKO aortas was significantly reduced compared with wild type. Impaired endothelial NO production in SKO could be attributed, at least in part, to diminished agonist-induced [Ca 2+ ] i transients in ECs. Consistently, silencing endothelial S100A1 expression in wild type also reduced [Ca 2+ ] i and NO generation. Moreover, S100A1 overexpression in ECs further increased NO generation that was blocked by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenylborate. Finally, cardiac endothelial S100A1 expression was shown to be downregulated in heart failure in vivo. Collectively, endothelial S100A1 critically modulates vascular function because lack of S100A1 expression leads to decreased [Ca 2+ ] i and endothelial NO release, which contributes, at least partially, to impaired endothelium-dependent vascular relaxation and hypertension in SKO mice. Targeting endothelial S100A1 expression may, therefore, be a novel therapeutic means to improve endothelial function in vascular disease or heart failure.

Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 μg/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 μg/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 ± 3.5% (vehicle) to 11.0 ± 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 ± 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.

Heart physiology depends on oxidative metabolism that likely requires dynamic and permanent reorganization of mitochondria by fusion and fission. To directly evaluate mitochondrial fusion dynamics in cardiomyocytes (CM), mitochondrial matrix-targeted photoactivatable GFP and DsRed were introduced either in vitro or in vivo by adenovirus and were followed by confocal microscopy. Four conditions were analyzed: 24 to 48 h cultured neonatal and in vitro transduced adult CM, and CM from in vivo infected rat hearts. In the latter case, CM were isolated 7-10 days after infection and were imaged promptly or 24-48 h post harvesting. Neonatal CM mitochondria form a highly connected network, whereas both in vitro and in vivo transformed cultured CM displayed only some connectivity. Impressively, in vivo transduced adult CM that were imaged promptly after harvesting, unveiled a significantly higher connectivity among mitochondria than the 24-48h cultured adult CM. Furthermore, fusion events (f.e./75 square micrometers/min) were almost absent in cultured in vitro transduced CM, meanwhile in vivo transduced and cultured CM showed 0.4±0.2 f.e./min, whereas isolated, freshly-imaged CM displayed 1.4± 0.1 f.e./min. Imaging in perfused whole heart ex vivo, showed considerable mitochondrial continuity and fusion activity in ventricular CM. To study more directly the role of CM's contractile activity in mitochondrial fusion, CM were incubated with Verapamil (10μM), that blocked spontaneous contraction and partially suppressed the fusion activity of mitochondria. Also, mitochondrial fusion activity appeared to be higher after spontaneous contraction or short term field stimulation in isolated freshly-imaged CM. Thus, mitochondria are dynamic in both neonatal and adult CM, but under culture conditions, adult CM lose mitochondrial fusion activity. This might be at least in part, because cardiomyocyte contractile activity is altered in culture and contractions likely provide some factors to support mitochondrial fusion activity.

5-HT1A receptors couple to many signaling pathways in CHO-K1 cells through pertussis toxin-sensitive G proteins. The purpose of this study was to determine which members of the Gi/o/z family mediate 5-HT1A receptor-activated Na+/H+ exchange as measured by microphysiometry of cell monolayers. The method was extensively validated, showing that proton efflux was sodium-dependent, inhibited by amiloride analogs, and activated by growth factors, phorbol ester, calcium ionophore, and hypertonic stress. 5-HT and the specific agonist (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide rapidly stimulated proton efflux that was blocked by a specific receptor antagonist, amiloride analogs or pertussis toxin. The activation by 5-HT depended upon extracellular sodium and could be demonstrated under conditions of imposed intracellular acid load, as well as in the presence and absence of glycolytic substrate. Acceleration of proton efflux was not inhibited by sequestration of G protein betagamma-subunits, a maneuver that blocked 5-HT1A receptor activation of mitogen-activated protein kinase. Transfection of Gzalpha and pertussis toxin-resistant mutants of Goalpha and Gialpha1 did not reverse the blockade induced by pertussis toxin. In contrast, pertussis toxin-resistant mutants of Gialpha2 and Gialpha3 "rescued" the ability of 5-HT to increase proton efflux after pertussis toxin treatment. These experiments demonstrate clearly that Gialpha2 and Gialpha3 can specifically mediate rapid agonist-induced acceleration of Na+/H+ exchange.