This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Manning, B. S. Articles by Koch, W. J. Search for Related Content PubMed PubMed Citation Articles by Manning, B. S. Articles by Koch, W. J. Related Collections Congestive Hypertrophy Gene therapy Animal models of human disease Cell signalling/signal transduction Gene expression Heart failure - basic studies

(Circulation. 2000;102:2751.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Physiological Induction of a ß-Adrenergic Receptor Kinase Inhibitor Transgene Preserves ß-Adrenergic Responsiveness in Pressure-Overload Cardiac Hypertrophy

Brian S. Manning, PhD; Kyle Shotwell, BA; Lan Mao, MD; Howard A. Rockman, MD; Walter J. Koch, PhD

From the Departments of Surgery (B.S.M., K.S., W.J.K.) and Medicine (L.M., H.A.R.), Duke University Medical Center, Durham, NC. Dr Manning is currently with Obesity Molecular Sciences, CVMD Discovery, Pfizer Global Research and Development, Groton Laboratories, Groton, Conn.

Correspondence to Walter J. Koch, PhD, Duke University Medical Center, Box 2606, Room 472 MSRB, Durham, NC 27710. E-mail koch0002{at}mc.duke.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Transgenic mice with constitutive myocardium-targeted expression of a peptide inhibitor of the ß-adrenergic receptor kinase (ßARKct) have increased in vivo cardiac function and enhanced ß-adrenergic receptor (ßAR) responsiveness.

Methods and Results—In the present study, we created transgenic mice with myocardium-targeted ßARKct transgene expression under control of the CARP (cardiac ankyrin repeat protein) promoter, which is active during cardiac development and inactive in the normal adult mouse heart. Consistent with this, adult CARP-ßARKct transgenic mice have normal in vivo cardiac contractility and ßAR responsiveness indistinguishable from their nontransgenic littermates (NLCs). However, because CARP is in a group of fetal genes activated in the adult ventricle during hypertrophy, we subjected animals to transverse aortic constriction (TAC) to induce pressure overload. Seven days after TAC, CARP-ßARKct hearts had elevations in left ventricular mass similar to those in NLCs; however, TAC did induce demonstrable ßARKct expression in the transgenic hearts. TAC in NLC mice resulted in an upregulation of myocardial ßARK1 and a loss of ßAR-mediated inotropic reserve. Importantly, although ßARK1 was increased in the hypertrophic CARP-ßARKct mice, the in vivo loss of ßAR responsiveness was not seen after induced ßARKct expression.

Conclusions—These results demonstrate that acute ßARK1 inhibition can restore lost myocardial ßAR responsiveness and inotropic reserve in vivo. Furthermore, these mice demonstrate the novel utility of the CARP promoter as an inducible element responsive to pathophysiological conditions in the adult heart.

Key Words: receptors, adrenergic, beta • hypertrophy • genetics

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Under normal physiological conditions, the ß-adrenergic receptor (ßAR) signaling system plays a pivotal role in the control of cardiac function, mediating the inotropic, chronotropic, and lusitropic responses to the sympathetic neurotransmitters epinephrine and norepinephrine.¹ These receptors are in turn regulated by ßAR kinase (ßARK1), a member of the G protein–coupled receptor kinase (GRK) family that phosphorylates agonist-occupied receptors that trigger the binding of ß-arrestins and the process of homologous desensitization.^{2 3} The action of the primarily cytosolic ßARK1 (also known as GRK2) toward agonist-occupied receptors, such as ßARs in the heart, is preceded by a membrane-targeting event that is accomplished by the direct interaction between dissociated ß-subunits of activated G proteins (G_ß) and residues within the carboxyl terminus of ßARK1.^{4 5}

ßARK1 expression and activity in the heart appear to constitute a critical regulator of in vivo contractile function.^{6 7} Transgenic mice with myocardium-targeted overexpression of ßARK1 have a loss of ßAR-mediated inotropic reserve.⁶ Enhanced ßARK1 expression and activity are present in the failing human heart⁸ and probably contribute to the well-known ßAR pathology present in this disease.^{8 9} In addition to heart failure, enhanced ßARK1 expression is responsible for the loss of myocardial ßAR responsiveness in pressure-overload cardiac hypertrophy,¹⁰ and it has been shown to be increased in myocardial ischemia^{11 12} and hypertension.¹³ Importantly, reciprocal results (ie, enhanced in vivo cardiac function) have been found after the lowering of ßARK1 activity in the heart.^{6 7 14 15} The activity of ßARK1 can be inhibited in vitro and in vivo by a peptide composed of the G_ß binding domain of ßARK1 contained within the last 194 amino acid residues (ßARKct).^{5 6 15} Interestingly, although complete knockout of the ßARK1 gene leads to embryonic lethality,¹⁶ gene-targeted mice with a loss of 1 ßARK1 allele have enhanced in vivo contractility.⁷ Thus, inhibiting the activity of ßARK1 or lowering its expression appears to offer a novel means to enhance cardiac function.

The promoter used for these previous myocardium-targeted transgenic mice was the -myosin heavy chain (MyHC) gene promoter, which is not active in the mouse ventricle until around birth and then is constitutively active throughout adulthood.¹⁷ Due to the embryonic lethality found in ßARK1 knockout mice, we sought to express the ßARKct in the fetal heart to investigate potential cardiac requirements for ßARK1 during development. To do this, transgenic mice were generated with ßARKct expression targeted to the developing heart using the gene promoter for the cardiac ankyrin repeat protein (CARP). The CARP gene product is expressed early in cardiac development, detectable at embryonic day 7.5 to 8.0, and functions as a downstream negative regulator in the Nkx2-5 pathway.^{18 19 20} CARP levels remain high throughout cardiac development but decline 2 weeks postnatally, persisting at low or virtually undetectable levels in the adult heart.^{18 20} Transgenic mice harboring the CARP-lacZ transgene have shown that the promoter is developmentally regulated and induced as part of the embryonic gene program in response to hypertrophic stimuli.²⁰ Therefore, in addition to driving ßARKct expression in the developing heart, the present study used CARP-ßARKct mice as a novel model demonstrating the utility of the CARP promoter to activate transgene expression acutely in the adult heart in response to a pathophysiological state.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Transgenic Mice
The CARP-ßARKct transgene was constructed by a 3-way ligation between the 2.2-Kb CARP promoter fragment (a generous gift from Drs Ken Chien and Ju Chen, University of California at San Diego), the 800-bp ßARKct fragment, and the pGEM-SV40 plasmid containing the SV40 intron poly(A) signal.⁶ The resulting transgene construct was linearized and purified before pronuclear injections were performed by the Duke Comprehensive Cancer Center Transgenic Facility. Two lines of mice were established, CARP-ßARKct-3 and CARP-ßARKct-9. Litter sizes and postnatal development were indistinguishable from nontransgenic littermate controls (NLCs). Offspring were screened by standard polymerase chain reaction techniques with forward and reverse primers corresponding to specific regions of the CARP promoter and ßARKct, respectively (Figure 1A). Second-generation adult animals of both sexes (2 to 5 months of age) were used for all physiological studies. Institutional Review Board approval for all mouse experiments was obtained from Duke University.

View larger version (38K): [in this window] [in a new window]   Figure 1. Generation of CARP-ßARKct transgenic mice. A, Organization of CARP-ßARKct transgene construct with positions of oligonucleotides used in polymerase chain reaction screening of transgenic progeny. B, Northern blot of indicated tissues from neonatal CARP-ßARKct mouse showing predominant expression of transgene in heart (GI indicates gastrointestinal tract; BAT, brown adipose tissue; and Skel., skeletal). Levels of 28s ribosomal RNA are also shown as control for RNA loading.

Microsurgical Techniques
In vivo pressure overload was created in the mouse by surgical banding of the transverse aorta as described previously.^{10 21} Transverse aortic constriction (TAC) causes pressure overload on the heart and reproducible cardiac hypertrophy.^{10 21} Sham-operated animals underwent the same surgical procedure without TAC. Operative mortality was <10% in both groups.

Hemodynamic Evaluation in Intact Anesthetized Mice
Initial in vivo hemodynamic evaluation to identify any functional phenotype was performed in anesthetized open-chest adult NLC and CARP-ßARKct animals as described previously.⁶ Subsequently, hemodynamic parameters were evaluated 1 week after TAC in closed-chest anesthetized mice as described previously.¹⁴ Hemodynamic measurements were recorded at baseline and after a graded dose of isoproterenol (50, 500, and 1000 pg). After evaluation, hearts were rapidly excised and individual chambers separated, weighed for hypertrophy measurements, and frozen in liquid N₂ for biochemical analysis.

Northern Blot Analysis
Hearts from the various experimental animals were excised as described above, and total RNA was isolated and Northern blot analysis performed by standard methods previously described.⁶

Protein Immunoblotting
Immunodetection of myocardial ßARKct expression was performed on cytosolic extracts prepared from homogenized NLC or CARP-ßARKct hearts (with or without TAC) as described previously.^{6 10} Myocardial ßARK1 levels were detected by immunoblotting for the 80-kDa ßARK1 protein after immunoprecipitation from myocardial extracts with a ßARK1 monoclonal antibody, as described previously.^{10 22}

In Vitro GRK Activity Assays
Assessment of myocardial GRK activity was performed with cytosolic extracts and rhodopsin-enriched rod outer-segment membranes as described previously.^{10 22} The GRK activity found in cardiac cytosolic extracts is almost exclusively due to ßARK1.¹⁰ [³²P] incorporation into the rhodopsin substrate was quantified from dried gels with a Molecular Dynamics PhosphorImager.^{10 22}

Statistical Analysis
Data are expressed as mean±SEM values. Student’s t test was used to test for significance for parameters of hypertrophy, ßARK1 expression, and GRK activity. To test differences in hemodynamic responses between groups with and without TAC, a repeated-measure ANOVA was used. For all analyses, P<0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Generation and Characterization of CARP-ßARKct Transgenic Mice
The 2.2-Kb proximal promoter region of the CARP gene has been previously reported to direct high-level expression of marker transgenes to the developing myocardium.^{18 20} In 2 individual lines of transgenic mice (designated lines 3 and 9), neonatal expression of the ßARKct transcript was confined primarily to the heart, with minor mRNA expression seen in skeletal muscle and very little, if any, expression in other tissues (Figure 1B). Cardiac expression of the ßARKct transgene determined by Northern analysis and protein immunoblotting in neonates demonstrated equivalent expression in both lines (data not shown). Thus, all further experiments were performed on heterozygote CARP-ßARKct-9 mice. Importantly, the expression of ßARKct throughout the developing myocardium did not lead to any obvious pathology in neonates or adults.

A time course to monitor ßARKct expression in the heart from birth to adulthood was performed. CARP-ßARKct mice older than 1 month had significantly lower levels of myocardial ßARKct expression by Northern analysis (Figure 2A). Western blot analysis of the ßARKct peptide produced similar results (Figure 2B). In fact, by day 14, average ßARKct mRNA and peptide expression in CARP-ßARKct hearts was 50% of fetal (e18.0) cardiac levels, and by 30 days, very little, if any, transgene (mRNA or protein) expression was detected (Figure 2). The loss of ßARKct expression within the first 2 months of life is consistent with previously demonstrated properties of the CARP promoter.^{18 20}

View larger version (52K): [in this window] [in a new window]   Figure 2. Time course of CARP-ßARKct mRNA and peptide expression in hearts of transgenic mice. A, Northern blot time course of CARP-ßARKct transgene expression in mouse hearts at indicated times beginning with embryonic day 18 (E18.0). B, Western blot showing expression of CARP-ßARKct peptide in hearts of transgenic mice. Pos. represents cytosolic extract of COS-7 cells infected with ßARKct adenovirus.

In vivo physiological assessment was made in adult mice. Consistent with low and undetectable levels of myocardial ßARKct expression, in vivo cardiac function in adult CARP-ßARKct mice was not different from corresponding NLCs. As shown in Figure 3, basal and isoproterenol-stimulated left ventricular (LV) dP/dt_max responses measured in open-chest anesthetized mice were not altered in CARP-ßARKct animals. Additional hemodynamic parameters were also found to be similar in NLC and CARP-ßARKct mice (data not shown). Consistent with no change in ßAR-mediated in vivo cardiac function, there was no difference in myocardial ßAR density between adult NLC mice (21.0±3.4 fmol per mg membrane protein, n=4) and CARP-ßARKct mice (25.7±2.1 fmol per mg, n=6) (P=NS). Thus, developmental expression of ßARKct does not affect postnatal ßAR density.

View larger version (16K): [in this window] [in a new window]   Figure 3. In vivo hemodynamic assessment in open-chest anesthetized control (NLC) and CARP-ßARKct mice. LV dP/dtmax measured at baseline and after administration of isoproterenol was used as an index of contractile function as described in Methods. No statistical differences were found between NLC and transgenic mice (ANOVA).

Pressure Overload in CARP-ßARKct Mice
We subjected adult mice to LV pressure overload in vivo to determine whether the CARP-ßARKct transgene could be induced. Previously, it has been shown that the endogenous CARP gene is included in a group of fetal genes, such as ANF and skeletal actin, that are silent in the adult ventricle but are activated during hypertrophy.²⁰ Pressure-overload LV hypertrophy was induced by TAC, which we have previously used on mice.^{10 21} Seven days after TAC, mice develop a reproducible and stable LV hypertrophy that interestingly is accompanied by a loss of ßAR responsiveness and inotropic reserve.¹⁰ As shown in Figure 4, morphological and biochemical markers of pressure-overload hypertrophy were identical in both NLC and transgenic mice. Significant pressure-overload LV hypertrophy was achieved after 7 days of TAC as measured by an increase in LV/body weight (BW) ratio in both CARP-ßARKct and NLC mice (Figure 4A). The significant increase in LV/BW ratio was on the order of 43% to 54%, and the TAC-induced increases in LV mass were not statistically different between CARP-ßARKct and NLC animals. The increase in LV/BW ratio was mirrored by an increase in ßARK1 expression after TAC, as measured by protein immunoblotting (Figure 4B and 4C). Consistent with increased ßARK1 protein levels, basal myocardial GRK activity was also significantly enhanced after TAC, and this increased kinase activity in the absence of G_ß (3-fold) was similar in CARP-ßARKct and NLC mice (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of TAC in CARP-ßARKct mice compared with NLC mice. A, LV/BW ratios of NLC and CARP-ßARKct mice. Significant increases in LV/BW ratios were observed in both groups undergoing TAC compared with sham-operated animals (P<0.01, t test). Systolic pressure gradients after TAC were not significantly different between NLC (58±5 mm Hg, n=9) and CARP-ßARKct (65±5 mm Hg, n=19) mice (P=0.4). B, Representative cardiac extract protein immunoblot from 2 mice in each group showing upregulation of myocardial ßARK1 levels after TAC. Purified ßARK1 (right) is used as a control. C, Densitometric analysis of ßARK1 levels from B (n=4 each). Data (mean±SEM) were normalized to basal (sham) levels of ßARK1. *P<0.01 vs sham (t test).

Myocardial Physiological Properties After Hypertrophic Induction of ßARKct
To determine the in vivo physiological response to TAC and hypertrophy, cardiac catheterization was used to measure ßAR responsiveness in intact anesthetized TAC and sham-treated NLC and CARP-ßARKct mice (Figure 5). In these studies, anesthetized mice were analyzed in a closed-chest model, which differs from the open-chest measurements obtained for Figure 3. In response to isoproterenol, LV dP/dt_max in NLC mice was significantly blunted after TAC, which is consistent with enhanced ßARK1 activity. Interestingly, although CARP-ßARKct animals also have hypertrophy and enhanced ßARK1 expression after TAC, their ßAR-mediated cardiac contractility is preserved and, in fact, significantly enhanced compared with NLC-TAC mice (Figure 5). Moreover, basal LV dP/dt_max in CARP-ßARKct-TAC mice is significantly higher than in NLC-sham mice (Figure 5). The Table includes all hemodynamic parameters measured in these mice, including LV dP/dt_min as an index of cardiac relaxation, heart rate, LV systolic pressure, and LV end-diastolic pressure. Interestingly, NLC-TAC mice have a limited relaxation (LV dP/dt_min) response after isoproterenol administration; however, diastolic ßAR responsiveness is restored in CARP-ßARKct mice after pressure overload (Table). The differences between groups for LV dP/dt_min were found to be significant (Table).

View larger version (20K): [in this window] [in a new window]   Figure 5. In vivo contractile function in closed-chest CARP-ßARKct and NLC mice after development of ventricular hypertrophy. Cardiac catheterization was performed in intact anesthetized NLC-sham, NLC-TAC, and CARP-ßARKct-TAC animals (see Methods). Shown are LV dP/dtmax values measured at basal conditions and after graded doses of isoproterenol. Data (mean±SEM) were analyzed with repeated-measures ANOVA. *P<0.05, CARP-ßARKct-TAC vs NLC-sham; #P<0.05, CARP-ßARKct-TAC vs NLC-TAC; P<0.001, CARP-ßARKct-TAC vs NLC-TAC; **P<0.005, NLC-TAC vs NLC-sham. Pattern of change between groups was also statistically significant (P<0.0001, ANOVA).

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Parameters in Closed-Chest Anesthetized Mice at Baseline and After 1000 pg of Isoproterenol

Northern blot analysis of RNA isolated from CARP-ßARKct hearts that had undergone TAC showed the appearance of ßARKct mRNA, which was absent in sham-operated age-matched CARP-ßARKct animals (Figure 6A). Importantly, this induction of transgene mRNA expression was confirmed by protein immunoblotting (Figure 6B), which demonstrated the appearance of detectable levels of the ßARKct peptide in myocardial extracts after TAC in CARP-ßARKct animals. As shown in Figure 6B, there was some variability in the induction of ßARKct expression after TAC. In addition to TAC, we found induction of ßARKct expression in the hearts of adult CARP-ßARKct mice after chronic infusion (7 days) of isoproterenol (data not shown) at doses sufficient to cause myocardial hypertrophy.^{22 23}

View larger version (35K): [in this window] [in a new window]   Figure 6. Induction of ßARKct expression after TAC in CARP-ßARKct mice. A, Representative Northern blot showing induction of ßARKct RNA expression after TAC in CARP-ßARKct mice. RNA from MyHC-ßARKct heart was used as positive control. B, Representative Western blot showing induction of ßARKct peptide expression in hearts of 2 adult CARP-ßARKct mice after TAC. Also shown are extracts from NLC mice (sham and TAC) and sham CARP-ßARKct mice, as well as ßARKct expression from MyHC-ßARKct mouse heart (on left) and positive control for ßARKct expression (on right).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study reports 2 novel findings, including the hypertrophic induction of cardiac ßARKct expression in the adult heart when driven by the CARP promoter. Second, this is the first report of the activity of ßARK1 being inhibited in the developing mouse ventricle. The results of this study demonstrate that induction of ßARKct in the hypertrophied adult heart is able to block myocardial ßAR desensitization associated with pressure-overload hypertrophy and preserve in vivo inotropic reserve. These results are similar to what has been described previously in MyHC-ßARKct mice in which ßARKct was constitutively expressed in the adult heart.¹⁰ However, the present study demonstrates that induced ßARKct expression in the heart can acutely preserve normal ßAR-mediated cardiac signaling and function even in the face of enhanced ßARK1 expression and GRK activity.

Increased ßARK1 expression and activity characterize many forms of cardiac hypertrophy and disease. However, ßARK1 upregulation is not a generalized biochemical marker of hypertrophy because it is not observed in transgenic models of hypertrophy induced by cardiac overexpression of oncogenic ras,¹⁰ or Gq.²⁴ Enhanced ßARK1 expression in the diseased heart may be triggered by heightened sympathetic nervous system activity, which is present in most cardiovascular disorders as an early compensatory mechanism. This hypothesis has been bolstered by recent studies in the mouse^{22 23} in which chronic infusions of the ßAR agonist isoproterenol resulted in increased mRNA, protein, and activity of ßARK1 in the heart. However, this was not seen after phenylephrine-induced hypertrophy, which indicates that catecholamine induction of ßARK1 expression in the heart occurs exclusively via stimulation of ßARs and not -adrenergic receptors.²³

Pressure-overload hypertrophy in vivo has been demonstrated recently to be triggered by signals that activate receptors coupled to the G protein Gq, because targeted inhibition of the myocardial receptor–Gq interface in transgenic mice attenuated myocardial hypertrophy after TAC.²¹ However, it is believed that TAC will increase sympathetic nervous system activity; thus, ßARK1 may be elevated in the heart by catecholamines acting through ßARs during the induction of pressure-overload cardiac hypertrophy via Gq-coupled receptors.

Interestingly, the level of ßARKct expression after TAC did not appear to consistently reach the levels found in the previously described MyHC-ßARKct transgenic mouse (Figure 6). However, this apparently lower ßARKct expression induced in CARP-ßARKct mice was sufficient to preserve myocardial ßAR responsiveness after hypertrophy. These results represent an important advance in the therapeutic potential of ßARK1 inhibition, because previous data demonstrating the reversal of ßAR desensitization in hypertrophy¹⁰ and the prevention of cardiomyopathy in a murine model of heart failure due to MLP(-/-) gene ablation¹⁴ were accomplished by MyHC-ßARKct transgenesis in which ßARKct was expressed in the adult ventricle from birth. Accordingly, it will be interesting to determine in future studies whether CARP-ßARKct mice with inducible ßARK1 inhibition can also "rescue" mouse models of heart failure, including longer-term follow-up after TAC, when decompensation and cardiac dysfunction can occur.

This study also represents the first report of developmental cardiac expression of ßARKct in transgenic mice. Importantly, there were no overt alterations in the size or function of the heart due to fetal ventricular ßARKct expression driven by the CARP promoter. These results actually are consistent with the hearts of heterozygous ßARK1 knockout mice that develop normally.^{7 16} An aspect of fetal cardiac ßARKct expression that might present a problem in development involves other G_ß-dependent processes. We have previously shown that G_ß can activate the p21^ras–mitogen-activated protein kinase pathway²⁵ and have recently used ßARKct to inhibit pathological in vivo vascular smooth muscle cell proliferation.^{26 27 28} Thus, ßARKct has the potential to inhibit any role G_ß may have in the developing heart. Our results with myocardial ßARKct expression throughout gestation suggest that G_ß does not play a major role in developmental cardiac mitogenesis, which in itself is a significant finding.

A potential novel application of the results of this study concerns gene therapy. In development of future cardiac gene transfer vectors (adenoviral or adeno-associated virus), the CARP promoter may offer advantages over other cardiac promoters. The CARP promoter not only targets transgene expression primarily to the heart but also will only be active during pathophysiological states. For example, a therapeutic transgene could be delivered to the failing heart to produce a beneficial effect, and if the disease process stops, the transgene will also be eliminated via the functional "turnoff" of the CARP promoter. However, transgene expression could be reestablished if the need arises, such as a relapse of the cardiac pathology. This could be a powerful application of ßARKct, because it appears that the acute inhibition of ßARK1 is a novel therapeutic target in conditions of compromised heart function. Importantly, this can be accomplished either by a gene therapy approach or the development of small molecules that can inhibit ßARK1 activity.

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants HL-61690 (Dr Koch) and HL-56687 (Dr Rockman).

Received May 25, 2000; revision received June 21, 2000; accepted June 22, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Brodde O-E. ß-adrenergic receptors in failing human myocardium. Basic Res Cardiol. 1996;91:35–40.
   
2. Inglese J, Freedman NJ, Koch WJ, et al. Structure and mechanism of the G protein-coupled receptor kinases. J Biol Chem. 1993;268:23735–23738.[Free Full Text]
   
3. Lefkowitz RJ. G protein-coupled receptors, III: new roles for receptor kinases and ß-arrestins in receptor signaling and desensitization. J Biol Chem. 1998;273:18677–18680.[Free Full Text]
   
4. Pitcher JA, Inglese J, Higgins JB, et al. Role of ß-subunits of G proteins in targeting the ß-adrenergic receptor kinase to membrane-bound receptors. Science. 1992;257:1264–1267.[Abstract/Free Full Text]
   
5. Koch WJ, Inglese J, Stone WC, et al. The binding site for the ß subunits of heterotrimeric G proteins on the ß-adrenergic receptor kinase. J Biol Chem. 1993;268:8256–8260.[Abstract/Free Full Text]
   
6. Koch WJ, Rockman HA, Samama P, et al. Cardiac function in mice overexpressing the ß-adrenergic receptor kinase or a ßARK inhibitor. Science. 1995;268:1350–1353.[Abstract/Free Full Text]
   
7. Rockman HA, Akhter SA, Choi D-J, et al. Regulation of myocardial contractile function by the level of ß-adrenergic receptor kinase-1 in gene targeted mice. J Biol Chem. 1998;273:18180–18184.[Abstract/Free Full Text]
   
8. Ungerer M, Bohm M, Elce JS, et al. Altered expression of ß-adrenergic receptor kinase and ß₁-adrenergic receptors in the failing heart. Circulation. 1993;87:454–463.[Abstract/Free Full Text]
   
9. Bristow MR, Ginsburg R, Minobe W, et al. Decreased catecholamine sensitivity and ß-adrenergic receptor density in failing human hearts. N Engl J Med. 1982;307:205–211.[Abstract]
   
10. Choi D-J, Koch WJ, Hunter JJ, et al. Mechanism for ß-adrenergic receptor desensitization in cardiac hypertrophy is increased ß-adrenergic receptor kinase. J Biol Chem. 1997;272:17223–17229.[Abstract/Free Full Text]
    
11. Ungerer M, Kessebohm K, Kronsbein K, et al. Activation of ß-adrenergic receptor kinase during myocardial ischemia. Circ Res. 1996;79:455–460.[Abstract/Free Full Text]
    
12. Maurice JP, Shah AS, Kypson AP, et al. Molecular ß-adrenergic signaling abnormalities in failing rabbit hearts after infarction. Am J Physiol. 1999;276:H1853–H1860.
    
13. Gros R, Benovic JL, Tan M, et al. G-protein-coupled receptor kinase activity is increased in hypertension. J Clin Invest. 1997;99:2087–2093.[Medline] [Order article via Infotrieve]
    
14. Rockman HA, Chien KR, Choi D-J, et al. Expression of a ß-adrenergic receptor kinase 1 inhibitor prevents the development of heart failure in gene targeted mice. Proc Natl Acad Sci U S A. 1998;95:7000–7005.[Abstract/Free Full Text]
    
15. Akhter SA, Eckhart AD, Rockman HA, et al. In vivo inhibition of elevated myocardial ß-adrenergic receptor kinase activity in hybrid transgenic mice restores normal ß-adrenergic signaling and function. Circulation. 1999;100:648–653.[Abstract/Free Full Text]
    
16. Jaber M, Koch WJ, Rockman HA, et al. Essential role of ß-adrenergic receptor kinase 1 in cardiac development and function. Proc Natl Acad Sci U S A. 1996;93:12974–12979.[Abstract/Free Full Text]
    
17. Subramaniam A, Jones WK, Gulick J, et al. Tissue-specific regulation of the -myosin heavy chain promoter in transgenic mice. J Biol Chem. 1991;266:24613–24620.[Abstract/Free Full Text]
    
18. Jeyaseelan R, Poizat C, Baker RK, et al. A novel cardiac-restricted target for doxorubicin: CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes. J Biol Chem. 1997;272:22800–22808.[Abstract/Free Full Text]
    
19. Zou Y, Evans S, Chen J, et al. CARP, a cardiac ankyrin repeat protein, is downstream in the NKx2.5 homeobox gene pathway. Development. 1997;124:793–804.[Abstract]
    
20. Kuo H-C, Chen J, Ruiz-Lozano P, et al. Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis. Development. 1999;126:4223–4234.[Abstract]
    
21. Akhter SA, Luttrell LM, Rockman HA, et al. Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science. 1998;280:574–577.[Abstract/Free Full Text]
    
22. Iaccarino G, Tomhave ED, Lefkowitz RJ, et al. Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by ß-adrenergic receptor stimulation and blockade. Circulation. 1998;98:1783–1789.[Abstract/Free Full Text]
    
23. Iaccarino G, Dolber PC, Lefkowitz RJ, et al. ßARK1 levels in catecholamine-induced myocardial hypertrophy: regulation by ß but not ₁ adrenergic stimulation. Hypertension. 1999;33:396–401.[Abstract/Free Full Text]
    
24. Dorn GW, Tepe NM, Lorenz JN, et al. Low- and high-transgenic expression of ß₂-adrenergic receptors differentially affects cardiac hypertrophy and function in Gq overexpressing mice. Proc Natl Acad Sci U S A. 1999;96:6400–6405.[Abstract/Free Full Text]
    
25. Koch WJ, Hawes BE, Allen LF, et al. Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G_ß activation of p21^ras. Proc Natl Acad Sci U S A. 1994;91:12706–12710.[Abstract/Free Full Text]
    
26. Davies MG, Huynh TTT, Fulton GJ, et al. G-protein signaling and vein graft intimal hyperplasia: reduction of intimal hyperplasia in vein grafts by a G_ß inhibitor suggests a major role of G-protein signaling in lesion development. Arterioscler Thromb Vasc Biol. 1998;18:1275–1280.[Abstract/Free Full Text]
    
27. Huynh TTT, Iaccarino G, Davies MG, et al. Adenoviral mediated inhibition of G_ß signaling limits the development of vein graft intimal hyperplasia. Surgery. 1998;124:177–186.[Medline] [Order article via Infotrieve]
    
28. Iaccarino G, Smithwick LA, Lefkowitz RJ, et al. Targeting G_ß signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis. Proc Natl Acad Sci U S A. 1999;96:3945–3950.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				S. J. Matkovich, A. Diwan, J. L. Klanke, D. J. Hammer, Y. Marreez, A. M. Odley, E. W. Brunskill, W. J. Koch, R. J. Schwartz, and G. W. Dorn II Cardiac-Specific Ablation of G-Protein Receptor Kinase 2 Redefines Its Roles in Heart Development and {beta}-Adrenergic Signaling Circ. Res., October 27, 2006; 99(9): 996 - 1003. [Abstract] [Full Text] [PDF]

				M. A. Nordlie, L. E. Wold, B. Z. Simkhovich, C. Sesti, and R. A. Kloner Molecular Aspects of Ischemic Heart Disease: Ischemia/Reperfusion-Induced Genetic Changes and Potential Applications of Gene and RNA Interference Therapy Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2006; 11(1): 17 - 30. [Abstract] [PDF]

				C. R. Bridges, K. Gopal, D. E. Holt, C. Yarnall, S. Cole, R. B. Anderson, X. Yin, A. Nelson, B. W. Kozyak, Z. Wang, et al. Efficient myocyte gene delivery with complete cardiac surgical isolation in situ J. Thorac. Cardiovasc. Surg., November 1, 2005; 130(5): 1364 - 1364. [Abstract] [Full Text] [PDF]

				J. W. Holmes Candidate mechanical stimuli for hypertrophy during volume overload J Appl Physiol, October 1, 2004; 97(4): 1453 - 1460. [Abstract] [Full Text] [PDF]

				M. A Movsesian Altered cAMP-mediated signalling and its role in the pathogenesis of dilated cardiomyopathy Cardiovasc Res, June 1, 2004; 62(3): 450 - 459. [Abstract] [Full Text] [PDF]

				M. L. Williams, J. A. Hata, J. Schroder, E. Rampersaud, J. Petrofski, A. Jakoi, C. A. Milano, and W. J. Koch Targeted {beta}-Adrenergic Receptor Kinase ({beta}ARK1) Inhibition by Gene Transfer in Failing Human Hearts Circulation, April 6, 2004; 109(13): 1590 - 1593. [Abstract] [Full Text] [PDF]

				J.R. Keys and W.J. Koch The Adrenergic Pathway and Heart Failure Recent Prog. Horm. Res., January 1, 2004; 59(1): 13 - 30. [Abstract] [Full Text]

				J. G. Dobson, J. Fray, J. L. Leonard, and R. E. Pratt Molecular mechanisms of reduced {beta}-adrenergic signaling in the aged heart as revealed by genomic profiling Physiol Genomics, October 17, 2003; 15(2): 142 - 147. [Abstract] [Full Text] [PDF]

				J. A. Hata and W. J. Koch Phosphorylation of G Protein-Coupled Receptors: GPCR Kinases in Heart Disease Mol. Interv., August 1, 2003; 3(5): 264 - 272. [Abstract] [Full Text] [PDF]

				V. de Waard, T. A.E. van Achterberg, N. J. Beauchamp, H. Pannekoek, and C. J.M. de Vries Cardiac Ankyrin Repeat Protein (CARP) Expression in Human and Murine Atherosclerotic Lesions: Activin Induces Carp in Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 64 - 68. [Abstract] [Full Text] [PDF]

				J. Kim, A. D. Eckhart, S. Eguchi, and W. J. Koch beta -Adrenergic Receptor-mediated DNA Synthesis in Cardiac Fibroblasts Is Dependent on Transactivation of the Epidermal Growth Factor Receptor and Subsequent Activation of Extracellular Signal-regulated Kinases J. Biol. Chem., August 23, 2002; 277(35): 32116 - 32123. [Abstract] [Full Text] [PDF]

				S. F. Steinberg G protein-coupled receptor kinases: gotta real kure for heart failure? J. Am. Coll. Cardiol., August 1, 2001; 38(2): 541 - 545. [Full Text] [PDF]

				X.-J. Du Sympathoadrenergic mechanisms in functional regulation and development of cardiac hypertrophy and failure: findings from genetically engineered mice Cardiovasc Res, June 1, 2001; 50(3): 443 - 453. [Full Text] [PDF]

				C. L. Antos, N. Frey, S. O. Marx, S. Reiken, M. Gaburjakova, J. A. Richardson, A. R. Marks, and E. N. Olson Dilated Cardiomyopathy and Sudden Death Resulting From Constitutive Activation of Protein Kinase A Circ. Res., November 23, 2001; 89(11): 997 - 1004. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Manning, B. S. Articles by Koch, W. J. Search for Related Content PubMed PubMed Citation Articles by Manning, B. S. Articles by Koch, W. J. Related Collections Congestive Hypertrophy Gene therapy Animal models of human disease Cell signalling/signal transduction Gene expression Heart failure - basic studies