Persistent increases in Ca(2+) influx through Cav1.2 shortens action potential and causes Ca(2+) overload-induced afterdepolarizations and arrhythmias.

Persistent elevation of Ca(2+) influx due to prolongation of the action potential (AP), chronic activation of the ß-adrenergic system and molecular remodeling occurs in stressed and diseased hearts. Increases in Ca(2+) influx are usually linked to prolonged myocyte action potentials and arrhythmias. However, the contribution of chronic enhancement of Cav1.2 activity on cardiac electrical remodeling and arrhythmogenicity has not been completely defined and is the subject of this study. Chronically increased Cav1.2 activity was produced with a cardiac specific, inducible double transgenic (DTG) mouse system overexpressing the ß2a subunit of Cav (Cavß2a). DTG myocytes had increased L-type Ca(2+) current (ICa-L), myocyte shortening, and Ca(2+) transients. DTG mice had enhanced cardiac performance, but died suddenly and prematurely. Telemetric electrocardiograms revealed shortened QT intervals in DTG mice. The action potential duration (APD) was shortened in DTG myocytes due to significant increases of potassium currents and channel abundance. However, shortened AP in DTG myocytes did not fully limit excess Ca(2+) influx and increased the peak and tail ICa-L. Enhanced ICa promoted sarcoplasmic reticulum (SR) Ca(2+) overload, diastolic Ca(2+) sparks and waves, and increased NCX activity, causing increased occurrence of early and delayed afterdepolarizations (EADs and DADs) that may contribute to premature ventricular beats and ventricular tachycardia. AV blocks that could be related to fibrosis of the AV node were also observed. Our study suggests that increasing ICa-L does not necessarily result in AP prolongation but causes SR Ca(2+) overload and fibrosis of AV node and myocardium to induce cellular arrhythmogenicity, arrhythmias, and conduction abnormalities.

Untargeted metabolomic analysis for the clinical screening of inborn errors of metabolism.

Global metabolic profiling currently achievable by untargeted mass spectrometry-based metabolomic platforms has great potential to advance our understanding of human disease states, including potential utility in the detection of novel and known inborn errors of metabolism (IEMs). There are few studies of the technical reproducibility, data analysis methods, and overall diagnostic capabilities when this technology is applied to clinical specimens for the diagnosis of IEMs. We explored the clinical utility of a metabolomic workflow capable of routinely generating semi-quantitative z-score values for ~900 unique compounds, including ~500 named human analytes, in a single analysis of human plasma. We tested the technical reproducibility of this platform and applied it to the retrospective diagnosis of 190 individual plasma samples, 120 of which were collected from patients with a confirmed IEM. Our results demonstrate high intra-assay precision and linear detection for the majority compounds tested. Individual metabolomic profiles provided excellent sensitivity and specificity for the detection of a wide range of metabolic disorders and identified novel biomarkers for some diseases. With this platform, it is possible to use one test to screen for dozens of IEMs that might otherwise require ordering multiple unique biochemical tests. However, this test may yield false negative results for certain disorders that would be detected by a more well-established quantitative test and in its current state should be considered a supplementary test. Our findings describe a novel approach to metabolomic analysis of clinical specimens and demonstrate the clinical utility of this technology for prospective screening of IEMs.

Understanding the apothecaries within: the necessity of a systematic approach for defining the chemical output of the human microbiome.

The human microbiome harbors a massive diversity of microbes that effectively form an "organ" that strongly influences metabolism and immune function and hence, human health. Although the growing interest in the microbiome has chiefly arisen due to its impact on human physiology, the probable rules of operation are embedded in the roots of microbiology where chemical communication (i.e., with metabolites) is a dominant feature of coexistence. Indeed, recent examples in microbiome research offer the impression that the collective microbiome operates as an "apothecary," creating chemical concoctions that influence health and alter drug response. Although these principles are not unappreciated, the majority of emphasis is on metagenomics and research efforts often omit systematic efforts to interrogate the chemical component of the complex equation between microbial community and host phenotype. One of the reasons for this omission may be due to the inaccessibility to high-breadth, high-throughput, and scalable technologies. Since these technologies are now available, we propose that a more systematic effort to survey the host-microbiota chemical output be embedded into microbiome research as there is strong likelihood, and growing precedence, that this component may often be integral to developing our understanding of these ultimate apothecaries and how they impact human health.

Exploiting functional domains of GRK2/3 to alter the competitive balance of pro- and anticontractile signaling in airway smooth muscle.

To clarify the potential utility of targeting GRK2/3-mediated desensitization as a means of manipulating airway smooth muscle (ASM) contractile state, we assessed the specificity of GRK2/3 regulation of procontractile and relaxant G-protein-coupled receptors in ASM. Functional domains of GRK2/3 were stably expressed, or siRNA-mediated GRK2/3 knockdown was performed, in human ASM cultures, and agonist-induced signaling was assessed. Regulation of contraction of murine tracheal rings expressing GRK2 C terminus was also assessed. GRK2/3 knockdown or expression of the GRK2 C terminus caused a significant (∼ 30-90%) increase in maximal ß-agonist and histamine [phosphoinositide (PI) hydrolysis] signaling, without affecting the calculated EC50. GRK2 C-terminal expression did not affect signaling by methacholine, thrombin, or LTD4. Expression of the GRK2 N terminus or kinase-dead holo-GRK2 diminished (∼ 30-70%) both PI hydrolysis and Ca(2+) mobilization by every Gq-coupled receptor examined. Under conditions of GRK2 C-terminal expression, ß-agonist inhibition of methacholine-stimulated PI hydrolysis was greater. Finally, transgenic expression of the GRK2 C terminus in murine ASM enabled ∼ 30-50% greater ß-agonist-mediated relaxation of methacholine-induced contraction. Collectively these data demonstrate the relative selectivity of GRKs for the ß2AR in ASM and the ability to exploit GRK2/3 functional domains to render ASM hyporesponsive to contractile agents while increasing responsiveness to bronchodilating ß-agonist.

Metabolomics as a key integrator for "omic" advancement of personalized medicine and future therapies.

Investigation into biological complexity, whether for a better understanding of disease or drug process, is a monumental task plaguing investigators. The lure of "omic" technologies for circumventing much of these challenges has led to widespread efforts and adoption. It is becoming clearer that a single "omic" approach (e.g., genomics) is often insufficient for completely defining the complexity in these biological systems. Hence, there is an increasing awareness that a "systems" approach will serve to increase resolution and confidence and provide a strong foundation for further hypothesis-driven investigation. Although certain metabolites are already considered clinically important, the profiling of metabolites via metabolomics (the profiling of metabolites to fully characterize metabolic pathways) is the most recent to mature of these "omic" technologies and has been only recently adopted as compared to genomic or proteomic approaches in systems inquiries. Recent reports suggest that this "omic" may well be a key data stream in systems investigations for endeavors in personalized medicine and biomarker identification, as it seems most closely relevant to the phenotype.

S100A1 genetically targeted therapy reverses dysfunction of human failing cardiomyocytes.

OBJECTIVES: This study investigated the hypothesis whether S100A1 gene therapy can improve pathological key features in human failing ventricular cardiomyocytes (HFCMs). BACKGROUND: Depletion of the Ca²âº-sensor protein S100A1 drives deterioration of cardiac performance toward heart failure (HF) in experimental animal models. Targeted repair of this molecular defect by cardiac-specific S100A1 gene therapy rescued cardiac performance, raising the immanent question of its effects in human failing myocardium. METHODS: Enzymatically isolated HFCMs from hearts with severe systolic HF were subjected to S100A1 and control adenoviral gene transfer and contractile performance, calcium handling, signaling, and energy homeostasis were analyzed by video-edge-detection, FURA2-based epifluorescent microscopy, phosphorylation site-specific antibodies, and mitochondrial assays, respectively. RESULTS: Genetically targeted therapy employing the human S100A1 cDNA normalized decreased S100A1 protein levels in HFCMs, reversed both contractile dysfunction and negative force-frequency relationship, and improved contractile reserve under beta-adrenergic receptor (ß-AR) stimulation independent of cAMP-dependent (PKA) and calmodulin-dependent (CaMKII) kinase activity. S100A1 reversed underlying Ca²âº handling abnormalities basally and under ß-AR stimulation shown by improved SR Ca²âº handling, intracellular Ca²âº transients, diastolic Ca²âº overload, and diminished susceptibility to arrhythmogenic SR Ca²âº leak, respectively. Moreover, S100A1 ameliorated compromised mitochondrial function and restored the phosphocreatine/adenosine-triphosphate ratio. CONCLUSIONS: Our results demonstrate for the first time the therapeutic efficacy of genetically reconstituted S100A1 protein levels in HFCMs by reversing pathophysiological features that characterize human failing myocardium. Our findings close a gap in our understanding of S100A1's effects in human cardiomyocytes and strengthen the rationale for future molecular-guided therapy of human HF.

Impaired neoangiogenesis in ß2-adrenoceptor gene-deficient mice: restoration by intravascular human ß2-adrenoceptor gene transfer and role of NFκB and CREB transcription factors.

BACKGROUND AND PURPOSE: There is much evidence supporting the role of ß2-adrenoceptors (ß2AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of ß2AR knock-out mice (ß2AR-/-) in vivo and explored possible molecular mechanisms in vitro. EXPERIMENTAL APPROACH: Femoral artery resection (FAR) was performed in wild-type and ß2AR-/- mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis. Intrafemoral delivery of an adenovirus encoding the human ß2AR (ADß2AR) was used to reinstate ß2ARs in ß2AR-/- mice. Molecular mechanisms were investigated in mouse-derived aortic endothelial cells (EC) in vitro, focusing on NFκB activation and transcriptional activity. RESULTS: Angiogenesis was severely impaired in ß2AR-/- mice subjected to FAR, but was restored by gene therapy with ADß2AR. The proangiogenic responses to a variety of stimuli were impaired in ß2AR-/- EC in vitro. Moreover, removal of ß2ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates ßARs) nor TNFα induced NFκB activation in ß2AR(-/-) EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in ß2AR(-/-) ECs. ADß2AR administration restored ß2AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα. CONCLUSIONS AND IMPLICATIONS: Our results suggest that ß2ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.

Calcium influx through Cav1.2 is a proximal signal for pathological cardiomyocyte hypertrophy.

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca(2+)) is implicated in hypertrophic signaling pathways, the specific role of Ca(2+) influx through the L-type Ca(2+) channel (I(Ca-L)) has been controversial and is the topic of this study. To determine if and how sustained increases in I(Ca-L) induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the ß2a subunit of Ca(2+) channels (ß2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a ß2a-GFP were used. In vivo, ß2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume, and cross-sectional area and the expression of PCH markers in a time- and dose-dependent manner. PCH was associated with a hypercontractile phenotype including enhanced I(Ca-L), fractional shortening, peak Ca(2+) transient, at the myocyte level, greater ejection fraction, and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of ß2a in cultured AFVMs increased I(Ca-L), cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of I(Ca-L), intracellular Ca(2+), calcineurin, CaMKII, and SERCA. In conclusion, increasing I(Ca-L) is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca(2+) pools were shown to be involved.

The inotropic peptide ßARKct improves ßAR responsiveness in normal and failing cardiomyocytes through G(ßγ)-mediated L-type calcium current disinhibition.

RATIONALE: The G(ßγ)-sequestering peptide ß-adrenergic receptor kinase (ßARK)ct derived from the G-protein-coupled receptor kinase (GRK)2 carboxyl terminus has emerged as a promising target for gene-based heart failure therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased ß-adrenergic receptor (ßAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by ßARKct and its impact on G(ßγ)-mediated signaling have yet to be fully elucidated. OBJECTIVE: We sought to identify G(ßγ)-regulated targets and signaling mechanisms conveying ßARKct-mediated enhanced ßAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes. METHODS AND RESULTS: Assessing viral-based ßARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca²(+) handling, and site-specific protein phosphorylation, we demonstrate that ßARKct enhances the cardiac L-type Ca²(+) channel (LCC) current (I(Ca)) both in NCs and FCs on ßAR stimulation. Mechanistically, ßARKct augments I(Ca) by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated G(ßγ) subunits downstream of activated ßARs. Despite improved ßAR contractile responsiveness, ßARKct neither increased nor restored cAMP-dependent protein kinase (PKA) and calmodulin-dependent kinase II signaling including unchanged protein kinase (PK)Cε, extracellular signal-regulated kinase (ERK)1/2, Akt, ERK5, and p38 activation both in NCs and FCs. Accordingly, although ßARKct significantly increases I(Ca) and Ca²(+) transients, being susceptible to suppression by recombinant G(ßγ) protein and use-dependent LCC blocker, ßARKct-expressing cardiomyocytes exhibit equal basal and ßAR-stimulated sarcoplasmic reticulum Ca²(+) load, spontaneous diastolic Ca²(+) leakage, and survival rates and were less susceptible to field-stimulated Ca²(+) waves compared with controls. CONCLUSION: Our study identifies a G(ßγ)-dependent signaling pathway attenuating cardiomyocyte I(Ca) on ßAR as molecular target for the G(ßγ)-sequestering peptide ßARKct. Targeted interruption of this inhibitory signaling pathway by ßARKct confers improved ßAR contractile responsiveness through increased I(Ca) without enhancing regular or restoring abnormal cAMP-signaling. ßARKct-mediated improvement of I(Ca) rendered cardiomyocytes neither susceptible to ßAR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca²(+) leakage.

Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms.

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 ß-adrenergic receptor (ßAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the ßARKct, 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 ßARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with ßARKct 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 ßARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via ßARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of ß(2)ARs abolished ßARKct-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 ßARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

Regulation of GPCR signaling in hypertension.

Hypertension represents a complex, multifactorial disease and contributes to the major causes of morbidity and mortality in industrialized countries: ischemic and hypertensive heart disease, stroke, peripheral atherosclerosis and renal failure. Current pharmacological therapy of essential hypertension focuses on the regulation of vascular resistance by inhibition of hormones such as catecholamines and angiotensin II, blocking them from receptor activation. Interaction of G-protein coupled receptor kinases (GRKs) and regulator of G-protein signaling (RGS) proteins with activated G-protein coupled receptors (GPCRs) effect the phosphorylation state of the receptor leading to desensitization and can profoundly impair signaling. Defects in GPCR regulation via these modulators have severe consequences affecting GPCR-stimulated biological responses in pathological situations such as hypertension, since they fine-tune and balance the major transmitters of vessel constriction versus dilatation, thus representing valuable new targets for anti-hypertensive therapeutic strategies. Elevated levels of GRKs are associated with human hypertensive disease and are relevant modulators of blood pressure in animal models of hypertension. This implies therapeutic perspective in a disease that has a prevalence of 65million in the United States while being directly correlated with occurrence of major adverse cardiac and vascular events. Therefore, therapeutic approaches using the inhibition of GRKs to regulate GPCRs are intriguing novel targets for treatment of hypertension and heart failure.

[Activation of sterol regulatory element binding protein and its involvement in endothelial cell migration].

OBJECTIVE: To study the activation of sterol regulatory element binding protein (SREBP) and its critical role in endothelial cell migration. METHODS: Bovine aortic endothelial cells (ECs) were cultured. The expression of SREBP and Cdc42 were determined by Western blot and quantitative real-time PCR. Moreover, outward growth migration model and transwell chamber assay were used to detect ECs migration. RESULTS: (1) SREBP was activated during ECs migration. Western blot analysis demonstrated increased active form SREBP in migrating as compared to non-migrating ECs population. SREBP activation decreased as ECs migration slowed;(2) Coincidental with SREBP activation, mRNA expression of its target genes such as low density lipoprotein receptor, HMG-CoA reductase, and fatty acid synthase also increased in migrating ECs population as detected by real-time PCR; (3) Migration induced SREBP activation in ECs was inhibited by SREBP-acting protein RNAi and pharmacologically by 25-hydroxycholesterol; (4) Inhibition of SREBP led to decreased ECs migration in various models; (5) Cells genetically deficient in SREBP-acting protein, S1P, or S2P, phenotypically exhibited impaired migration; (6) SREBP inhibition in ECs suppressed the activity of small GTPase Cdc42, a key molecule for ECs motility. CONCLUSIONS: SREBP is activated during and plays a critical role in ECs migration. Targeting SREBP could become a novel approach in fighting diseases involving abnormal ECs migration.

G protein-coupled receptor kinase 2 expression and activity are associated with blood pressure in black Americans.

Hypertension occurs with higher prevalence and morbidity in black Americans compared with other groups. Alterations in the signal transduction pathways of 7-transmembrane spanning receptors are found in hypertensive patients. G protein-coupled receptor kinases (GRKs) play an important role in regulating this receptor signaling. The 2 most abundantly expressed GRKs in the cardiovascular system are GRK2 and GRK5, and each has unique substrates. Understanding changes in expression may give us insight into activated receptors in the pathophysiological progression of hypertension. In heart failure and white hypertensives, increased GRK2 expression arises because of neurohormonal stimulation of particular receptors. GRK2 subsequently desensitizes specific receptors, including beta-adrenergic receptors. In blood pressure control, beta-adrenergic receptor desensitization could lead to increased blood pressure. GRK2 and GRK5 mRNA were evaluated in lymphocytes of black Americans via quantitative real-time PCR. GRK2 mRNA expression directly correlated with systolic blood pressure and norepinephrine levels. GRK2 was elevated >30% among those with systolic blood pressure > or =130 mm Hg. No significant correlation between GRK5 mRNA expression and blood pressure or catecholamines was observed. Diabetic status, age, sex, and body mass index were also compared with GRK2 expression using univariate and multivariate analyses. GRK2 protein expression was elevated 2-fold in subjects with higher blood pressure, and GRK activity was increased >40%. Our data suggest that GRK2, but not GRK5, is correlated with increasing blood pressure in black Americans. Understanding the receptors stimulated by increased neurohormonal activation may give insight into the pathophysiology of hypertension in this at-risk population.

Inhibition of angiotensin II Gq signaling augments beta-adrenergic receptor mediated effects in a renal artery stenosis model of high blood pressure.

Chronic ventricular pressure overload states, such as hypertension, and elevated levels of neurohormones (norepinephrine, angiotensin II, endothelin-1) initiate cardiac hypertrophy and dysfunction and share the property of being able to bind to Gq-coupled 7-transmembrane receptors. The goal of the current study was to determine the role of endogenous cardiac myocyte Gq signaling and its role in cardiac hypertrophy and dysfunction during high blood pressure (BP). We induced renal artery stenosis for 8 weeks in control mice and mice expressing a peptide inhibitor of Gq signaling (GqI) using a 2 kidney, 1 clip renal artery stenosis model. 8 weeks following chronic high BP, control mice had cardiac hypertrophy and depressed function. Inhibition of cardiomyocyte Gq signaling did not reverse cardiac hypertrophy but attenuated increases in a profile of cardiac profibrotic genes and genes associated with remodeling. Inhibition of Gq signaling also attenuated the loss of cardiac function. We determined that Gq signaling downstream of angiotensin II receptor stimulation negatively impacted beta-adrenergic receptor (AR) responses and inhibition of Gq signaling was sufficient to restore betaAR-mediated responses. Therefore, in this study we found that Gq signaling negatively impacts cardiac function during high BP. Specifically, we found that inhibition of AT1-Gq signaling augmented betaAR mediated effects in a renal artery stenosis model of hypertension. These observations may underlie additional, beneficial effects of angiotensinogen converting enzyme (ACE) inhibitors and angiotensin receptor antagonists observed during times of hemodynamic stress.

Negative regulation of VEGF signaling in human coronary artery endothelial cells by G protein-coupled receptor kinase 5.

G protein-coupled receptor kinase 5 (GRK5) is present in endothelial cells (ECs) and has the potential to regulate EC function through seven transmembrane-spanning receptor (7TMR) signaling. Recently, it has been appreciated that GRKs can affect receptor tyrosine kinases (RTKs). VEGF, an RTK, is one of the most potent mediators for EC function and angiogenesis; therefore, we determined the role GRK5 plays in VEGF signaling in human coronary artery ECs (HCAECs). GRK5 levels were increased by VEGF treatment in HCAECs. Adenoviral overexpression of GRK5 inhibited migration and proliferation of HCAECs in response to VEGF. GRK5 overexpression in HCAECs significantly suppressed both acute and late activation of Akt and extracellular signal-related kinase (ERKs) as well as the phosphorylation of GSK-3beta, an endogenous substrate of Akt. Coimmunoprecipitations revealed that GRK5 is physically associated with Akt. This study shows for the first time that GRK5 negatively regulates VEGF signaling in HCAECs and suggests that targeted intervention of GRK5 in ECs might be a novel therapeutic strategy to prevent and treat disorders involving altered EC function.

Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes.

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 G alpha q 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.

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances alpha1D-adrenergic receptor constriction.

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.

GPCR signalling in hypertension: role of GRKs.

Hypertension is a prevalent condition in the developed world and disease severity is directly correlated with additional cardiovascular complications. It is estimated that 30% of the adult population in the United States has hypertension, which is classified as a systolic blood pressure > or =140 mmHg and/or a diastolic blood pressure > or =90 mmHg. A prolonged increase in afterload ultimately leads to congestive heart failure in the majority of cases. Currently, medication designed to treat hypertension is inadequate, thus new therapies need to be explored. Blood pressure is tightly regulated by blood vessel radius, which is established by hormones and/or peptides binding to GPCRs (G-protein-coupled receptors). Catecholamines and peptide hormones, such as AngII (angiotensin II), are elevated in hypertension and, therefore, signalling by these GPCRs is increased. Their signalling is tightly controlled by a class of proteins, the GRKs (GPCR kinases). Elevated levels of either GRK2 or GRK5 in both the lymphocytes and VSM (vascular smooth muscle) are associated with human hypertension and animal models of the disease. The focus of the present review is on the role GRKs, and their regulation of GPCRs, play in high blood pressure.