Gene-mediated inhibition of the b-adrenergic receptor kinase: a new therapeutic strategy for heart failure.

Molecular changes that take place during the evolution of heart failure (HF), especially the well characterized beta-adrenergic receptor (betaAR) signaling abnormalities, represent attractive targets for myocardial gene therapy. The beta-adrenergic receptor kinase (betaARK1 or GRK2) is a cytosolic enzyme that phosphorylates only agonist-occupied betaARs as well as other G protein-coupled receptors (GPCRs), leading to desensitization and functional uncoupling. betaARK1 levels and activity are elevated in the failing heart and therefore, it has recently been evaluated as a potential target for novel HF treatment. This review summarizes recent results obtained in transgenic mouse models as well as in animals where a betaARK1 inhibitor peptide (betaARKct) was delivered via the coronary arteries by exogenous gene transfer. These results strongly suggest that betaARK1 inhibition may represent a significant improvement in HF therapy.

Ventricular dysfunction after cardioplegic arrest is improved after myocardial gene transfer of a beta-adrenergic receptor kinase inhibitor.

BACKGROUND: Acute cardiac contractile dysfunction is common after cardiopulmonary bypass (CPB). A potential molecular mechanism is enhanced beta-adrenergic receptor kinase (betaARK1) activity, because beta-adrenergic receptor (betaAR) signaling is altered in cardiomyocytes after cardioplegia. Therefore, we examined whether adenovirus-mediated intracoronary delivery of a betaARK1 inhibitor (Adv-betaARKct) could prevent post-CPB dysfunction. METHODS AND RESULTS: Rabbits were randomized to receive 5x10(11) total viral particles of Adv-betaARKct or PBS. After 5 days, hearts were arrested with University of Wisconsin solution, excised, and stored at 4 degrees C for 15 minutes or 4 hours before reperfusion on a Langendorff apparatus. Left ventricular (LV) function measured by end-diastolic pressure response to preload augmentation, contractility (LV dP/dt(max)), and relaxation (LV dP/dt(min)) was assessed by use of increasing doses of isoproterenol and compared with a control group of nonarrested hearts acutely perfused on the Langendorff apparatus. In the PBS-treated hearts, LV function decreased in a temporal manner and was significantly impaired compared with control hearts after 4 hours of cardioplegic arrest. LV function in Adv-betaARKct-treated hearts, however, was significantly enhanced compared with PBS treatment and was similar to control nonarrested hearts even after 4 hours of cardioplegia. Biochemically, several aspects of betaAR signaling were dysfunctional in PBS-treated hearts, whereas they were normalized in betaARKct-overexpressing hearts. CONCLUSIONS: Myocardial gene transfer of Adv-betaARKct stabilizes betaAR signaling and prevents LV dysfunction induced by prolonged cardioplegic arrest. Thus, betaARK1 inhibition may represent a novel target in limiting depressed ventricular function after CPB.

Transgenic studies of cardiac adrenergic receptor regulation.

An accumulation of recent data on genetically engineered mouse models suggests that results from studies done in vitro are not necessarily duplicated in vivo. The genetic manipulation of the adrenergic receptor (AR) signaling system in the heart has afforded us the opportunity to not only study the physiological impact of AR signaling manipulation but also to examine how the various components interact with one another in vivo. In particular, although members of the G protein-coupled receptor kinase family do not exhibit substrate selectivity when overexpressed in cell culture, in vivo selectivity is apparent when examined in the cardiovascular system of genetically engineered mice. Additionally, transgenic expression of peptide inhibitors of signaling represents a powerful tool to examine specific targets in order to determine their contribution to a physiologic phenotype following stimulation. Finally, in vivo manipulation of the AR system has provided a broader understanding of the role that various G protein-coupled receptors play in situations where multiple members contribute to a phenotype. Thus, although in vitro studies allow for a more defined environment in which to study the signaling mediated by various receptors, it is essential to verify these findings in vivo to confirm or refute in vitro results.

Vascular beta-adrenergic receptor adenylyl cyclase system in maturation and aging.

The objective of this study was to determine how maturation and aging affects beta (beta)-adrenergic receptor (AR) control of arterial vasorelaxation. Left ventricular (LV) hemodynamics and arterial vasorelaxation in thoracic artery segments were studied in Brown Norway, Fisher 344 cross rats at 6 weeks, 6 months, and 23 months of age. We defined changes in maturation as occurring between 6 weeks and 6 months of age and changes in aging as occurring between 6 months and 23 months of age. With maturation, isoproterenol resulted in a downward shift in heart rate and an upward shift in both LV dP/dt and peripheral vascular resistance responses. Similar changes were noted with aging except for the downward shift in LV dP/dt isoproterenol response. There was a dose-dependent increase in arterial vasorelaxation in response to isoproterenol in all age groups, but the 6-week-old animals had a 5-fold (P<0.01) increase in vasorelaxation compared to other age groups. The isoproterenol-induced arterial vasorelaxation response was not altered by removal of the endothelium. The vasodilatory responses to nitroglycerin, acetylcholine, and adenosine were diminished (P<0.05) with aging. The vasorelaxation responses to forskolin and IBMX were unchanged with maturation and diminished with aging. Incubation of arterial rings in cholera toxin resulted in a reduction in relaxation only in arteries from 6-week-old rats. Maturation resulted in no change in beta -AR density [20.2+/-0.7 v. 18.5+/-0.5 fmol/mg protein, P=n.s., 6 weeks (n=2, 18 aortas were combined v 6-month-old rats)]. With maturation, there was no change in G alpha(i)level. However, beta ARK1 levels were increased (55. 4+/-2.1 v. 40.8+/-0.4, arbitrary densitometry units) and G alpha(s)levels were decreased (29.5+/-0.8 v. 49.9+/-1.9, arbitrary densitometry units). Aging resulted in no change in beta -AR density (15.3+/-1.7 v. 18.5+/-0.5 fmol/mg membrane protein), but decreases in basal, isoproterenol-, naF-, and forskolin-stimulated AC activities. Compared to 6 week data, 23-month-old rats exhibited no change in either G alpha(i)or beta ARK1, however, G alpha(s) was decreased. In summary, beta -AR-stimulated arterial vasorelaxation is depressed during maturation and aging. Since there is no change in beta -AR density but a decrease in G alpha(s)and in basal/stimulated AC activities, the defect in beta -AR signaling during maturation and aging is probably a post receptor defect, i.e. possibly in the receptor-G protein coupling.

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.

The myocardial beta-adrenergic system in spontaneously hypertensive heart failure (SHHF) rats.

-Responsiveness to beta-adrenergic stimulation is reduced in the failing human myocardium. This results principally from reduced beta-adrenergic receptor (betaAR) density, elevated beta-adrenergic receptor kinase 1 (betaARK1) levels, and functional uncoupling of remaining receptors. The temporal nature of changes in the human myocardial beta-adrenergic system relative to onset of symptomatic heart failure (HF) has been difficult to discern. A relatively new model of HF, the spontaneously hypertensive heart failure (SHHF) rat spontaneously and reproducibly develops left ventricular hypertrophy (LVH) and progresses to HF, thus enabling longitudinal studies to examine the cellular and molecular bases for hypertension-induced cardiac hypertrophy and subsequent HF. The purpose of this study was to examine age-dependent changes in the betaAR system in this model. Lean male SHHF rats at 3, 7, 14, and 20 months were compared with age-matched Sprague-Dawley (SD) control rats ([C]; 4 animals/group). At all ages the SHHF rats had elevated blood pressures and left ventricular end-diastolic pressure relative to the SD control rats (P<0.05). Compared with age-matched SD control rats, LVH was evident by 3 months in SHHF rats; 20-month-old SHHF rats had significantly greater LVH compared with the other SHHF rat groups. beta-adrenergic responsiveness (maximal heart rate to isoproterenol) was reduced only in 20-month-old SHHF rats. betaARK1 protein levels and activity were elevated at 14 months (162+/-10% and 195+/-20% C, respectively), and betaARK1 protein remained elevated at 20 months (140+/-14% C). In contrast, G protein-coupled receptor kinase 5, a second receptor kinase in the heart, remained unchanged at all ages. betaAR density did not change with age in the SD control rats and was similar in the SHHF rats until 20 months of age when the receptor number was reduced (30+/-1%). These data indicate that cardiac dysfunction is coincident with reduced betaAR density. Importantly, cardiac dysfunction was preceded by elevated betaARK1 levels and activity, thus suggesting that betaARK1 may be a precipitating factor in the transition from hypertension-induced compensatory cardiac hypertrophy to HF. Furthermore, these results indicate that the SHHF rat is a powerful model for use in examination of the mechanisms involved in alterations of beta-adrenergic signaling that occur in human HF.

Characterization of the alpha1B-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle.

We previously demonstrated that alpha1B-adrenergic receptor (AR) gene transcription, mRNA, and functionally coupled receptors increase during 3% O2 exposure in aorta, but not in vena cava smooth muscle cells (SMC). We report here that alpha1BAR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb alpha1BAR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The alpha1BAR 5' 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 alpha1BAR gene in aorta and vena cava SMC during normoxia and hypoxia. An ATTAAA motif within this sequence specifically binds aorta, vena cava, and DDT1MF-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 alpha1BAR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the alpha1BAR gene also were identified that may confer the differential hypoxic increase in alpha1BAR gene transcription in aorta, but not in vena cava SMC.

CRE-like response element regulates expression of rat alpha 2D-adrenergic receptor gene in vascular smooth muscle.

Contractile and binding studies indicate that alpha 2-adrenergic receptors (ARs) are differentially expressed by vascular smooth muscle cells (SMCs) according to vascular segment (artery, arteriole, vein). In the present study, alpha 2D-AR mRNA was two- to threefold higher in vena cava than in aorta. To understand vascular regulation of alpha 2D-AR expression in these cells, we sequenced 2.8 kb of the 5' flanking region of the alpha 2D-AR gene. Notable features include two potential TATA boxes, an adenosine 3',5'-cyclic monophosphate response element (CRE)-like binding element, and an Sp1 element. Comparison of the rat and human genes revealed an overall homology of 74% over the 1.87-kb sequence 5' to the translation initiator methionine, including complete homology at the distal TATA, CRE-like, and Sp1 sites, alpha 2D-AR transcription starts from the guanine nucleotide 18 base pair downstream from the distal TATA box. Reporter gene constructs demonstrated strong alpha 2D-AR promoter activity, but with several differences in construct activity, in both rat aorta and vena cava SMCs. Analysis of an essential promoter fragment revealed two regions protected by aorta and vena cava SMC nuclear proteins. The core sequences of these protected regions are TGACGCTA and TATAA. The former CRE-like element conferred specific binding of both aorta and vena cava nuclear proteins. In addition, promoter activity was increased 300% by forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate, indicating that the CRE-like element may regulate alpha 2D-AR expression in vascular tissue.

Alpha1D-adrenergic receptors and mitogen-activated protein kinase mediate increased protein synthesis by arterial smooth muscle.

Catecholamines may influence vascular smooth muscle cell (SMC) growth and vascular hypertrophic diseases. We previously demonstrated that stimulation of alpha1-adrenoceptors (AR) causes hypertrophy of vascular SMCs in vitro and in situ. Here, we used adult rat aorta SMCs that express alpha1D- and alpha1B-ARs (but not alpha1A-ARs) in vitro to examine the mechanisms and alpha1-AR subtypes involved. Norepinephrine (NE) increased protein synthesis and content in a time- and dose-dependent manner. To identify the responsible alpha1-AR subtype, we first documented the selectivity of two alpha1-AR subtype antagonists, BMY 7378 (alpha1D-AR antagonist) and chloroethylclonidine (CEC; alpha1B-AR antagonist), using Rat-1 fibroblasts stably transfected with the three different rodent alpha1-AR cDNAs. NE dose-dependently increased protein synthesis in each cell line. In alpha1D fibroblasts, BMY 7378 inhibited growth and protected alpha1D-ARs from CEC alkylation while having little blocking or protecting effect on the growth induced by stimulation of fibroblasts that express alpha1A- or alpha1B-ARs. In rat aorta SMCs, pretreatment with CEC in the presence of BMY 7378 to protect alpha1D-ARs had no effect on NE-induced protein synthesis. BMY 7378 inhibited the SMC growth response with a pKb of 8.4. NE caused rapid and transient p42-p44 mitogen-activated protein kinase (MAPK) activation that was alpha1D-AR dependent. Furthermore, NE caused tyrosine phosphorylation of multiple cellular proteins, phosphorylation of Raf-1, and stimulation of c-fos mRNA expression in aorta SMCs. The selective MAPK kinase inhibitor PD 98059 inhibited NE-induced protein synthesis and MAPK activation with IC50 values of 2.3 and 1.6 microM, respectively. These data demonstrate that SMC growth induced by NE is mediated by alpha1D-ARs that couple to activation of the MAPK cascade.

Oxygen modulates alpha 1B-adrenergic receptor gene expression by arterial but not venous vascular smooth muscle.

Blood and tissue O2 levels are major determinants of short-term autoregulatory adjustments in vascular smooth muscle cell (SMC) tension and may effect long-term alterations in SMC catecholamine responsiveness. We examined the hypothesis that prolonged hypoxia altered gene expression of alpha 1-adrenoceptors. After exposure of cultured aortic (in vitro) SMC to 3% O2 for 8 h, alpha 1B mRNA increased to 523% (P = 0.02) of control cells (21% O2) and to 205% (P = 0.04) in in situ organ-cultured aortic SMC. In vivo hypoxic hypoxia (10% inspired O2) similarly increased aortic SMC alpha 1B mRNA 180% (P = 0.02). In contrast, alpha 1D, alpha-actin and beta-actin mRNA levels were not changed in aortic SMC by low O2 in the in vitro, in situ, or in vivo models. Unlike aortic SMC, vena caval SMC alpha 1B mRNA expression did not change with low-O2 exposure in vitro or in vivo, nor did alpha 1D, alpha-actin or beta-actin mRNA. Aortic SMC alpha 1B transcription rate increased 360% (P = 0.02), whereas alpha 1D, alpha-actin, and beta-actin transcription was unchanged. Neither alpha 1B nor alpha 1D mRNA stability was altered by low-O2 exposure. Total alpha 1-adrenoceptor density ([3H]prazosin binding) increased 12% (P = 0.04) after 24 h of 3% O2. This was associated with a 200% increase (P < 0.01) in the chloroethylclonidine (CEC)-sensitive alpha 1-adrenoceptor population and no change in CEC-insensitive alpha 1-adrenoceptor density. Exposure of aortic SMC to 24 h of 3% O2 increased the maximum response of norepinephrine-evoked elevations in intracellular Ca2+ as measured using fura 2. Low O2 did not change responses to another G protein-coupled receptor, angiotensin II. These data suggest that reduced O2, during prolonged hypoxemia or tissue ischemia, may selectively increase expression of functionally coupled alpha 1B-adrenoceptors in arterial blood vessels.

Regulation of vascular smooth muscle growth by alpha 1-adrenoreceptor subtypes in vitro and in situ.

Rat aorta smooth muscle cells which express all three alpha 1-adrenoreceptors (alpha 1A, alpha 1B and alpha 1D) were used to determine the effect of stimulation of alpha 1-adrenergic receptor subtypes on cell growth. "Combined" alpha 1-adrenoreceptor subtype stimulation with norepinephrine alone caused a concentration-dependent, prazosin-sensitive increase in protein content and synthesis: 48 h of stimulation at 1 microM increased cell protein to 216 +/- 40% of time-matched controls (p = 0.008) and RNA to 140 +/- 13% (p = 0.03); protein synthesis increased to 167 +/- 13% (p < 0.01) after 24 h. Stimulation with norepinephrine plus the selective alpha 1A/alpha 1D antagonist 5-methylurapidil produced greater increases in alpha-actin mRNA (270 +/- 40% at 8 h; p = 0.007), total cell protein (220 +/- 45% at 24 h; p = 0.004), and RNA (135 +/- 8% at 24 h; p = 0.01). These effects were prevented by pretreatment with the selective alpha 1B antagonist chloroethylclonidine. Comparable results were obtained for intact aortae. Stimulation with norepinephrine plus 5-methylurapidil increased (p < 0.05) tissue protein, RNA, dry weight, and alpha-actin mRNA; and as in culture cells, combined stimulation with norepinephrine alone attenuated these responses. By comparison, adventitia (fibroblasts) was unaffected. Removal of endothelial cells had no effect. alpha 1B mRNA decreased by 42 +/- 12% (p = 0.01) in cultured cells during combined alpha 1-adrenoreceptor stimulation and by 23 +/- 8% (p = 0.03) for intact aorta. alpha 1D and beta-actin mRNA were unchanged in cultured cells, aorta media, and adventitia. These findings suggest that prolonged stimulation of chloroethylclonidine-sensitive, possibly alpha 1B-adrenoceptors induces hypertrophy of arterial smooth muscle cells and that stimulation of 5-methylurapidil-sensitive, non-alpha 1B-adrenoreceptors attenuates this growth response.