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Abstract Gene therapy using in vivo recombinant adenovirus-mediated gene transfer is an effective technique that offers great potential to improve existing drug treatments for the complex cardiovascular diseases of heart failure and vascular smooth muscle intimal hyperplasia. Cardiac-specific adenovirus-mediated transfer of the carboxyl-terminus of the β-adrenergic receptor kinase (βARKct), acting as a Gβγ-β-adrenergic receptor kinase (βARK)1 inhibitor, improves basal and agonist-induced cardiac performance in both normal and failing rabbit hearts. In addition, βARKct adenovirus infection of vascular smooth muscle is capable of significantly diminishing neointimal proliferation after angioplasty. Therefore, further investigation is warranted to determine whether inhibition of βARK1 activity and sequestration of Gβγ via an adenovirus that encodes the βARKct transgene might be a useful clinical tool for the treatment of cardiovascular pathologies.

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 [beta]AR-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 [beta]AR-mediated dilation in vivo and in situ was enhanced, [[beta].sub.a]AR-mediated vasoconstriction was also increased. Further pharmacological experiments using [[alpha].sub.1]AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced [[alpha].sub.1]DAR vasoconstriction. This is the first study fsda to suggest that VSM [[alpha].sub.1]DARs are a GRK2 substrate in vivo. phenylephrine; hypertension; transgenic mice; gene ablation

More than 30% of the US population has high blood pressure (BP), and less than a third of people treated for hypertension have it controlled. In addition, the etiology of most high BP is not known. Having a better understanding of the mechanisms underlying hypertension could potentially increase the effectiveness of treatment. Because [G.sub.q] signaling mediates vasoconstriction and vascular function can cause BP abnormalities, we were interested in determining the role of vascular smooth muscle (VSM) [G.sub.q] signaling in two divergent models of hypertension: a renovascular model of hypertension through renal artery stenosis and a genetic model of hypertension using mice with VSM-derived high BP. Inhibition of VSM [G.sub.q] signaling attenuated BP increases induced by renal artery stenosis to a similar extent as losartan, an ANG II receptor blocker and current antihypertensive therapy. Inhibition of [G.sub.q] signaling also attenuated high BP in our genetic VSM-derived hypertensive model. In contrast, BP remained elevated 25% following treatment with losartan, and prazosin, an ~-adrenergic receptor antagonist, only decreased BP by 35%. Inhibition of [G.sub.q] signaling attenuated VSM reactivity to ANG II and resulted in a 2.4-fold rightward shift in [EC.sub.50]. We also determined that inhibition of [G.sub.q] signaling was able to reverse VSM hypertrophy in the genetic VSM-derived hypertensive model. These results suggest that [G.sub.q] signaling is an important signaling pathway in two divergent models of hypertension and, perhaps, optimization of antihypertensive therapy could occur with the identification of particular [G.sub.q]-coupled receptors involved. two-kidney, one-clip renovascular hypertension; G protein-coupled receptor kinase 2: seven transmembrane-spanning receptor

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 [micro]g/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 [micro]g/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 [+ o r-] 3.5% (vehicle) to 11.0 [+ or -] 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 [+ or -] 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. myocardial ischemia; ischemia-reperfusion injury; apoptosis doi:10.1152/ajpheart.00227.2007

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of [beta]-adrenergic receptor ([beta]AR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting [alpha][sub.2]AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial [alpha][sub.2]AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein-coupled receptor kinase 2 (GRK2). Adrenal gland-specific GRK2 inhibition reversed [alpha][sub.2]AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac [beta]AR signaling and function, and increased sympatholytic efficacy of a [alpha][sub.2]AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target., Author(s): Anastasios Lymperopoulos [1, 2]; Giuseppe Rengo [1, 2]; Hajime Funakoshi [1]; Andrea D Eckhart [1, 3]; Walter J Koch (corresponding author) [1, 2] Despite recent advances in the prevention [...]

We previously demonstrated that [[Alpha].sub.1B]-adrenergic receptor (AR) gene transcription, mRNA, and functionally coupled receptors increase during 3% [O.sub.2] exposure in aorta, but not in vena cava smooth muscle cells (SMC). We report here that [[Alpha].sub.1B]AR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb [[Alpha].sub.1B]AR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The [[Alpha].sub.1B]AR 5[feet] flanking region was sequenced to -2,460 (relative to ATG + 1). Transient transfection experiments identify the minimal promoter region between -270 and -143 and sequence between -270 and -248 that are required for transcription of the [[Alpha].sub.1B] AR gene in aorta and vena cava SMC during normoxia and hypoxia. An ATTAAA motif within this sequence specifically binds aorta, vena cava, and [DDT.sub.1]MF-2 nuclear proteins, and transcription primarily initiates downstream of this motif at approximately -160 in aorta SMC. Sequence between -837 and -273 conferred strong hypoxic induction of transcription in aorta, but not in vena cava SMC, whereas the cis-element for the transcription factor, hypoxia-inducible factor 1, conferred hypoxia-induced transcription in both aorta and vena cava SMC. These data identify sequence required for transcription of the [[Alpha].sub.1B]AR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the [[Alpha].sub.1B]AR gene also were identified that may confer the differential hypoxic increase in [[Alpha].sub.1B]AR gene transcription in aorta, but not in vena cava SMC.

Research into the regulation of transcription of the alpha-2D/A-AR (adrenergic receptor) gene by vascular smooth muscle cells (SMCs) has involved cloning and sequencing the 2.8-kb 5' flanking region of the rat alpha-2D-AR gene. It was found that the alpha-2D-AR gene is positively regulated by an adenosine 3',5'-cyclic monophosphate (cAMP) response element (CRE)-like motif. This motif gives sensitivity to cAMP in arterial and venous SMCs.

The effects of low-O2 exposure were analyzed on the expression of alpha1B- and alpha1D-adrenoceptors by rat aortic and vena caval smooth muscle cells (SMC). The expression of alpha1B messenger ribonucleic acid and alpha1B gene transcription was upregulated by low O2-environments in vivo by arterial SMC. The upregulation of alpha1B counterbalanced the downregulation of alpha1-adrenoceptor density during hypoxia. Enhanced catecholamine contractile sensitivity was also detected during the expression of alpaha1B-adrenoceptors on arterial SMC.

Persistent elevation of Ca2+ influx due to prolongation of the action potential (AP), chronic activation of the β-adrenergic system and molecular remodeling occurs in stressed and diseased hearts. Increases in Ca2+ 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 β2a subunit of Cav (Cavβ2a). DTG myocytes had increased L-type Ca2+ current (ICa-L), myocyte shortening, and Ca2+ transients. DTG mice had enhanced cardiac performance, but they died suddenly and prematurely. Telemetric electrocardiograms revealed shortened QT intervals. The action potential duration (APD) were 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 Ca2+ influx and increased the peak and tail ICa-L. Enhanced ICa promoted sarcoplasmic reticulum (SR) Ca2+ overload, diastolic Ca2+ 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 Ca2+ overload and fibrosis of AV node and myocardium to induce cellular arrhythmogenicity, arrhythmias and conduction abnormalities. [ABSTRACT FROM AUTHOR]