Nuclear translocation of cardiac G protein-Coupled Receptor kinase 5 downstream of select Gq-activating hypertrophic ligands is a calmodulin-dependent process.

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\u27s 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

In Vivo Stimulation of α- and β-Adrenoceptors in Mice Differentially Alters Small RNA Content of Circulating Extracellular Vesicles

When myocardial function is compromised as in heart failure (HF), there is activation of the sympathetic nervous system with elevated circulating catecholamine levels. These catecholamines activate cardiac and extra-cardiac adrenergic receptors (ARs). Interest in secreted extracellular vesicles (EVs) from the heart is growing and in HF, it is not known whether excessive activation of α- or β-adrenergic receptors (ARs) could induce specific changes in EV content. In this study, we have evaluated, by next generation sequencing, the small RNA content, including micro-RNAs (miRs), of circulating EVs of mice exposed to chronic selective α- or β- AR stimulation. EVs from mouse blood were purified by differential ultracentrifugation resulting in EVs with an average size of 116.6 ± 4.8 nm that by immunoblotting included protein markers of EVs. We identified the presence of miRs in blood EVs using miR-21-5p and -16-5p real-time PCR as known constituents of blood exosomes that make up a portion of EVs. We next performed next generation sequencing (NGS) of small non-coding RNAs found in blood EVs from mice following 7 days of chronic treatment with isoproterenol (ISO) or phenylephrine (PE) to stimulate α- or β-ARs, respectively. PE increased the percent of genomic repeat region reads and decreased the percent of miR reads. In miR expression analysis, PE and ISO displayed specific patterns of miR expression that suggests differential pathway regulation. The top 20 KEGG pathways predicted by differential expressed miRs show that PE and ISO share 11 of 20 pathways analyzed and reveal also key differences including three synapse relative pathways induced by ISO relative to PE treatment. Both α-and β-AR agonists can alter small RNA content of circulating blood EVs/exosomes including differential expression and loading of miRs that indicate regulation of distinct pathways. This study provides novel insight into chronic sympathetic nervous system activation in HF where excessive catecholamines may not only participate in pathological remodeling of the heart but alter other organs due to secretion of EVs with altered miR content

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels

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

Dynamic changes in lymphocyte GRK2 levels in cardiac transplant patients: a biomarker for left ventricular function

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–

Inhibition of Fas-associated death domain-containing protein (FADD) protects against myocardial ischemia/reperfusion injury in a heart failure mouse model

AIM As technological interventions treating acute myocardial infarction (MI) improve, post-ischemic heart failure increasingly threatens patient health. The aim of the current study was to test whether FADD could be a potential target of gene therapy in the treatment of heart failure. METHODS Cardiomyocyte-specific FADD knockout mice along with non-transgenic littermates (NLC) were subjected to 30 minutes myocardial ischemia followed by 7 days of reperfusion or 6 weeks of permanent myocardial ischemia via the ligation of left main descending coronary artery. Cardiac function were evaluated by echocardiography and left ventricular (LV) catheterization and cardiomyocyte death was measured by Evans blue-TTC staining, TUNEL staining, and caspase-3, -8, and -9 activities. In vitro, H9C2 cells transfected with ether scramble siRNA or FADD siRNA were stressed with chelerythrin for 30 min and cleaved caspase-3 was assessed. RESULTS FADD expression was significantly decreased in FADD knockout mice compared to NLC. Ischemia/reperfusion (I/R) upregulated FADD expression in NLC mice, but not in FADD knockout mice at the early time. FADD deletion significantly attenuated I/R-induced cardiac dysfunction, decreased myocardial necrosis, and inhibited cardiomyocyte apoptosis. Furthermore, in 6 weeks long term permanent ischemia model, FADD deletion significantly reduced the infarct size (from 41.20 ± 3.90% in NLC to 26.83 ± 4.17% in FADD deletion), attenuated myocardial remodeling, improved cardiac function and improved survival. In vitro, FADD knockdown significantly reduced chelerythrin-induced the level of cleaved caspase-3. CONCLUSION Taken together, our results suggest FADD plays a critical role in post-ischemic heart failure. Inhibition of FADD retards heart failure progression. Our data supports the further investigation of FADD as a potential target for genetic manipulation in the treatment of heart failure

Differential role of g protein-coupled receptor Kinase 5 in physiological versus pathological cardiac hypertrophy

RATIONALE: G protein-coupled receptor (GPCR) kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is up-regulated in heart failure (HF). Although GRK5 is a critical regulator of cardiac GPCR signaling, recent data has uncovered non-canonical 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 HF after transverse aortic constriction (TAC) due to 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 (TgGRK5) and non-transgenic littermate control (NLC) 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 to TAC. Unlike after TAC, swim-trained TgGRK5 and NLC 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 TgGRK5 mice was able to preserve cardiac function. CONCLUSIONS: These data suggest that while nuclear-localized GRK5 is a pathological mediator after stress, this non-canonical nuclear activity of GRK5 is not induced during physiological hypertrophy

GRK2 Regulates ADP Signaling in Platelets Via P2Y1 and P2Y12.

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for \u3e3 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\u27-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