Level of ;-Adrenergic Receptor Kinase 1 Inhibition Determines Degree of Cardiac Dysfunction After Chronic Pressure Overload–Induced Heart Fa
http://www.100md.com
循环学杂志 2005年第2期
the Departments of Medicine (H.T., S.V.N.P., R.J.L., H.A.R.), Cell Biology (H.A.R.), Molecular Genetics (H.A.R.)
Surgery (W.J.K.)
the Howard Hughes Medical Institute (R.J.L.)
Duke University Medical Center, Durham, NC. Dr Koch is now at the Center for Translational Medicine, Jefferson Medical College, Philadelphia, Pa.
Abstract
Background— Heart failure is characterized by abnormalities in ;-adrenergic receptor (;AR) signaling, including increased level of myocardial ;AR kinase 1 (;ARK1). Our previous studies have shown that inhibition of ;ARK1 with the use of the G; sequestering peptide of ;ARK1 (;ARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of ;ARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of ;ARK1 inhibition correlates with the degree of heart failure.
Methods and Results— Transgenic (TG) mice with varying degrees of cardiac-specific expression of ;ARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial ;ARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of ;ARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low ;ARKct expression developed severe heart failure, whereas mice with high ;ARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of ;ARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal ;AR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in ;AR function, similar to the WT mice.
Conclusions— These data show that the level of ;ARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when ;ARK1 inhibition is considered as a molecular target.
Key Words: receptors, adrenergic, beta ; heart failure ; signal transduction ; mice, transgenic ; gene therapy
Introduction
Catecholamine-stimulated ;-adrenergic receptor (;AR) signaling is one of the most powerful regulators of cardiac function. Abnormalities in ;AR signaling are a prominent characteristic of failing hearts and may contribute to the progressive deterioration in cardiac function.1,2 Chronic stimulation of ;ARs in conditions of heart failure due to high levels of circulating catecholamines leads to desensitization and impaired ;AR responsiveness, in part as a result of increased levels of ;AR kinase 1 (;ARK1) (also known as GRK2).3 Stimulation of ;AR by catecholamines leads to dissociation of heterotrimeric G protein into G and G; subunits. ;ARK1 is recruited to the plasma membrane through its interaction with dissociated membrane-bound G; subunits and phosphorylates the agonist-occupied receptor. ;-Arrestin binds to the phosphorylated ;AR and sterically interdicts further coupling of the receptor with Gs subunit, leading to decreased ;AR signaling.4,5 Thus, phosphorylation of receptor by ;ARK1 is a critical step in regulating ;AR function and is consistent with in vivo studies showing that ;ARK1 is a critical modulator of cardiac function.6
A peptide inhibitor of ;ARK1, ;ARKct is composed of the last 194 amino acids of ;ARK1 and contains the binding site for G; subunits. Overexpression of ;ARKct peptide sequesters the dissociated G; subunits of heterotrimeric G proteins, leading to the inhibition of ;ARK1 recruitment to the membrane.6 Our previous studies have shown that inhibition of ;ARK1 has an important role in the pathophysiology of heart failure with the use of genetically engineered mouse models of heart failure, such as the muscle lim protein–knockout7 model and cardiac-specific overexpression of calsequestrin.8 Overexpression of ;ARKct led to less deterioration in cardiac function and prolonged survival in these genetic models of heart failure.8 Similarly, ;ARKct expression through adenoviral gene delivery in experimental models of myocardial infarction significantly delayed the development of heart failure.9 Importantly, recent experimental data support the concept that normalizing cardiac ;AR function leads to improved in vivo cardiac function in conditions of chronic pressure overload.2 Because the levels of myocardial ;ARK1 have been shown to be elevated in several cardiovascular disorders, including myocardial hypertrophy,10 ischemia,11 hypertension,12 and heart failure,13 ;ARK1 inhibition is a potential novel therapeutic strategy for conditions accompanied by marked ventricular dysfunction.
Although beneficial effects of ;ARKct have been demonstrated, it is not known whether the level of ;ARK1 inhibition correlates directly with the degree of heart failure or its amelioration. In the present study, we tested whether there is a gene-dosage effect of ;ARKct on preservation of cardiac function in heart failure by monitoring cardiac function in ;ARKct transgenic (TG) mice with varying levels of transgene expression that underwent pressure overload–induced heart failure.
Methods
Experimental Animals
TG mice overexpressing ;ARKct peptide were generated as previously described.6 Briefly, the coding sequence for the last 194 aa of the bovine ;ARK1 (;ARK1ct) was fused to the -myosin heavy-chain promoter. ;ARKct TG and wild-type (WT) littermate mice of either sex and 3 months of age were used for this study. Animals were handled according to the approved protocols and animal welfare regulations of the institutional review board at Duke University Medical Center.
Echocardiography
Echocardiography was performed on conscious mice with an HDI 5000 echocardiograph as previously described.7
In Vivo Pressure Overload
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg), and TAC was performed as previously described.2,14 Twelve weeks after surgery, the transstenotic pressure gradient (TSPG) was assessed by recording simultaneous measurements of right carotid and left axillary arterial pressures.
;AR Density and Adenylyl Cyclase Activity
Membrane fractions were prepared as previously described.2,14 Twenty-five micrograms of the membrane fraction was used to perform receptor binding with the use of 250 pmol/L of [125I]cyanopindolol.2,14 Receptor density (fmol) was normalized to milligrams of membrane protein. Adenylyl cyclase assays were performed with the use of 20 μg of the membrane fraction. Generated cAMP was quantified with a liquid scintillation counter (MINAXI;-4000).2,14
Immunoblotting
Immunodetection of myocardial levels of ;ARK1 and ;ARKct was performed on cytosolic extracts with a GRK2 antibody (Santa Cruz Biotechnology) after 12 weeks of TAC. Detection was performed with the use of enhanced chemiluminescence (ECL, Amersham), and the bands were quantified with the use of Bio-Rad Flouro-S Multimage software. ;ARK1 and ;ARKct values were normalized to actin (Santa Cruz Biotechnology) as a loading control. Reproducibility was confirmed by loading the same concentration of protein from each heart on multiple gels.
Statistical Analysis
Data are expressed as mean±SEM. Two-way repeated-measures ANOVA was used to evaluate the echo measurements for analysis of cardiac function after TAC. Post hoc analysis was performed with Newman-Keuls test. Multigroup comparisons were made with 1-way ANOVA and Tukey test. For all analyses, a value of P<0.05 was considered significant.
Results
Quantification of Myocardial ;ARK1 and ;ARKct Protein
We generated standard curves for the level of ;ARK1 protein by loading different amounts of purified ;ARK1 (25 to 150 ng) onto a 10% SDS-PAGE gel and then quantified the amount of ;ARK protein on the Western immunoblot by densitometry using Bio-Rad Flouro-S Multimage software for analysis (Figure 1A). There was a highly linear and reproducible relationship (R2=0.987) between the quantified band and the amount of purified ;ARK1 (Figure 1A). We then quantified the level of myocardial ;ARK1 and ;ARKct protein expression from the standard curves. Interestingly, we found that the level of ;ARKct protein in the cytosol of pressure-overloaded ;ARKct TG mice was quite variable (Figure 1B). Importantly, the highest levels of ;ARKct expression were associated with the greatest percent fractional shortening despite having similar TSPG and ;ARK1 levels (Figure 1B).
We next determined whether the level of ;ARK1 inhibition correlated directly with the degree of cardiac dysfunction by quantifying the level of myocardial ;ARKct protein expression and the associated percent fractional shortening in 24 TG mice after 12 weeks of TAC. TG ;ARKct mice showed a positive linear relationship between the level of ;ARKct protein expression and percent fractional shortening (Figure 2A), as well as when ;ARKct expression was normalized to the level of ;ARK1 (Figure 2B) or actin (Figure 2C). On the basis of ;ARKct transgene expression, mice were divided into high- and low-expression groups by a median of ;ARKct/actin ratio of 61.7 ng/arbitrary units (Figure 2C).
To exclude the possibility that cardiac remodeling by pressure overload might alter the level of ;ARKct protein expression, we measured ;ARKct levels in sham-operated mice and compared them with levels in the 12-week TAC mice. Importantly, we found a variability in the level of ;ARKct transgene expression in sham mice similar to that found in the TAC-operated animals (Figure 2D). In addition, Southern blotting confirmed that high and low ;ARKct expression was due to high and low copy numbers of the transgene (data not shown). These data show that the variability in ;ARKct expression occurred in both sham and TAC-operated mice and that the induction of heart failure by pressure overload did not alter the expression of ;ARKct transgene.
Level of ;ARKct Transgene Expression Directly Regulates In Vivo Cardiac Function in Response to Chronic Pressure Overload
We plotted fractional shortening against the range of TSPG in the low- and high-expressing ;ARKct mice after 12 weeks of banding. Fractional shortening between high- and low-expressing ;ARKct TG mice was clearly different even across a wide range of TSPG (Figure 3A), showing that mice with higher expression of ;ARKct had better cardiac function even after 12 weeks of chronic TAC. As expected, mean TSPG between the high- and low-expressing ;ARKct groups was not significantly different and spread over a broad range of pressures (Table and Figure 3A).
Physiological Parameters in WT and TG ;ARKct Mice
Because equivalence of pressure gradients does not necessarily mean that the average area of stenosis was the same across the groups, we calculated stenotic area across the transverse aorta by approximating stroke volume on the basis of the echocardiographic parameters of chamber size15 and by applying the Gorlin equation.16 TAC stenotic area for the low ;ARKct expressors was 0.4±0.2 mm2, and that for the high ;ARKct expressors was 0.2±0.3 mm2. In addition, pressure proximal to the transverse aortic stenosis was measured and found to be similar for the 3 groups (proximal pressure=170±10 mm Hg for WT, 190±9 mm Hg for high ;ARKct expressors, 173±12 mm Hg for low ;ARKct expressors; P=NS for any of the groups). These data support our assessment that the stenotic area and the load on the left ventricles (LV) were similar across the 3 groups.
To determine the time course for the development of heart failure, we monitored cardiac function in TG ;ARKct and WT littermate control mice by serial echocardiography at 4, 8, and 12 weeks after TAC. Echocardiography showed progressive LV enlargement and deterioration in cardiac function in the WT and TG mice with low level of ;ARKct expression (Figure 3B). These mice showed a 70% reduction in percent fractional shortening, a 270% increase in LV end-systolic dimension, and a 70% increase in LV end-diastolic dimension at 12 weeks after TAC compared with their basal measurements (Figures 3B and 4, Table). In contrast, TG mice with a high level of ;ARKct expression showed a significant preservation of cardiac function over the same period after chronic pressure overload, with only mild increases in LV end-systolic dimension or LV end-diastolic dimension (Figures 3B and 4, Table). Interestingly, chronic pressure overload led to a thinning of the septal and posterior myocardial walls in the WT and low-expressing ;ARKct mice, whereas wall thickness was increased in the high-expressing ;ARKct mice (Figure 3B and Table). At 12 weeks after TAC, the increase in the ratio of LV weight to body weight was significantly blunted in high-expression ;ARKct mice compared with WT and low-expression ;ARKct mice (Table). These morphometric and echocardiographic data show that a high level of ;ARKct protein expression in hearts of TG mice results in a significant blunting in heart size and mass and is associated with preservation of cardiac function in response to chronic pressure overload in vivo.
;AR Signaling in WT and ;ARKct Mice Under Conditions of Chronic Banding
Because previous studies have shown that ;ARK1 inhibition leads to preservation of ;AR function,6 we determined whether inhibition of ;ARK1 through cardiac-specific overexpression of ;ARKct could normalize ;AR function in the mice with a high level of ;ARKct expression under conditions of chronic pressure overload–induced heart failure. ;AR levels and receptor-effector coupling were evaluated in membrane fractions from the WT and TG ;ARKct mice after 12 weeks of banding. ;AR density was significantly reduced by 35% in the hearts of the WT and low-expressing ;ARKct mice on 12 weeks of banding (Figure 5A). In contrast, we found no significant decrease in receptor density after 12 weeks of TAC in the TG mice with high levels of ;ARKct expression (Figure 5A).
Receptor-effector coupling was assessed by adenylyl cyclase activity from the membrane fractions of sham and chronic banded WT and TG ;ARKct mice. Hearts from WT and low-expression ;ARKct banded mice showed significant desensitization, as measured by markedly diminished isoproterenol-stimulated membrane adenylyl cyclase activity (Figure 5B). Importantly, however, we found that isoproterenol-stimulated membrane adenylyl cyclase activity was preserved in the TG mice with high ;ARKct expression (Figure 5B). Taken together, these studies show that high levels of ;ARKct protein expression in the heart lead to normalization of ;AR density and isoproterenol-stimulated adenylyl cyclase activity, indicating preservation of ;AR–G protein coupling after chronic banding.
Discussion
In this study we demonstrate that inhibition of ;ARK1 through cardiac-specific overexpression of the ;ARKct peptide ameliorates the development of cardiac dysfunction under conditions of chronic in vivo pressure overload. This amelioration of cardiac dysfunction seems to depend primarily on the level of ;ARKct protein expression in the TG mice. TG mice with high ;ARKct expression showed preserved isoproterenol-stimulated adenylyl cyclase activity and normal ;AR density after 12 weeks of chronic banding. In contrast, WT and TG mice with low ;ARKct expression showed marked abnormalities in ;AR function, similar to that described in human heart failure.1,17 Strikingly, the extent of ;ARK1 inhibition, assessed by the level of ;ARKct protein expression, correlated positively with the attenuation of cardiac dysfunction after 12 weeks of chronic pressure overload. Thus, ;ARK1 as a therapeutic target would have immense clinical implications because ;ARK1 levels are increased in several cardiovascular disorders, including myocardial hypertrophy,10 myocardial ischemia,11 hypertension,12 and heart failure.13
The most likely mechanism for ;ARKct in preventing ;AR abnormalities and cardiac dysfunction is that the transgene sequesters G; subunits and inhibits receptor phosphorylation by blocking the recruitment of ;ARK1 to the agonist-stimulated receptor. Our present study shows that overexpression of the ;ARKct transgene effectively ameliorates heart failure in conditions of a molar ratio of 3:1 for ;ARKct to ;ARK. Previous studies have shown that ;ARK1 plays a critical role in the pathophysiology of heart failure, and inhibition of ;ARK1 with the use of ;ARKct in mouse models of heart failure has been shown to be beneficial.7,8,18 The mouse models of heart failure that have been rescued through ;ARKct include muscle lim protein knockout,7 cardiac overexpression of calsequestrin,8 and mice that developed hypertrophic cardiomyopathy.18 In all 3 models of heart failure, mice containing cardiac-specific overexpression of the ;ARKct showed less deterioration of cardiac function, and the cardiac overexpression of calsequestrin/;ARKct mice even exhibited an increase in mean survival age.8 Interestingly, overexpression of ;ARKct did not prevent the development of heart failure in all genetically modified mice, particularly in those in which ;ARK1 was not elevated, such as the TG Gq mice.19,20 Although these studies suggest that an increase in ;ARK1 level is important for ;ARKct to have an effect on cardiac function, we show in this study that the level of ;ARKct expression is far more important than the upregulation of ;ARK1 in determining response to pressure overload. This point is particularly salient because in our study the level of ;ARK1 did not change significantly among the banded TG hearts.
We show that ;ARKct transgene expression among littermates is variable and is probably due to the number of copies of the transgene in the genome. At the time of generation of TG mice, the integration of the transgene occurs at different loci across the genome as well as in multiple copies at any given loci.21 Therefore, segregation of the number of integrated copies of the transgene among the littermates likely accounts for the variable amounts of the expressed protein in the progeny. One of the standard procedures used to overcome variable transgene expression would be to backcross selected progeny for numerous generations to get uniform expression. Although the littermates have varying levels of transgene expression, this procedure has allowed us to directly correlate the effect of transgene expression with amelioration of heart failure.
Our data in the present study support our hypothesis that a critical determinant for the preservation of cardiac function by ;ARKct is normalization of ;AR function in conditions of chronic overload. This is consistent with our recent data showing that preventing downregulation and desensitization of ;ARs through a mechanism of receptor-localized phosphatidylinositol 3–kinase inhibition also ameliorates the development of heart failure.2 Because multiple pathways are involved in development of heart failure, other possible complementary mechanisms for the beneficial effects of ;ARKct transgene must be considered as well. It is possible that inhibition of other G;-mediated signaling events such as activation of phosphatidylinositol 3–kinase2,22,23 and IK,Ach channels24 may contribute to the mechanism of action of ;ARKct in heart failure. In addition, it is possible that overexpression of ;ARKct inhibits the phosphorylation of other G protein–coupled receptors, such as endothelin and angiotensin receptors, although this would lead to enhanced signaling of the receptor systems.
As discussed earlier, ;ARK-mediated phosphorylation of ;ARs leads to their internalization through a variety of mechanisms, including classic clathrin-coated, pit-mediated processes, caveolae, and noncoated pit mechanisms as well.25–28 It has been observed recently that the agonist-promoted internalization of ;ARs can lead to the assembly of complex signaling cascades that activate cellular growth pathways, such as the various mitogen-activated protein kinase pathways.29–31 We postulate that one of the mechanisms for the beneficial effects of ;ARKct overexpression is by inhibiting the activation of these maladaptive growth pathways of the cardiac myocyte.32
Our previous studies have shown that TG mice develop cardiac hypertrophy to an extent similar to that of the WT littermate controls after 7 days of banding, indicating that the ;ARKct transgene does not alter the development of cardiac hypertrophy.10 These data support our hypothesis that the development of cardiac hypertrophy is not sufficient to preserve cardiac function under conditions of pressure overload; rather, normalization of detrimental signaling pathways such as the ;AR pathway may be the critical determinant.14,33
In summary, we demonstrate that inhibition of ;ARK1 leads to preservation of ;AR function and attenuates deterioration of cardiac function under conditions of chronic pressure overload in vivo. Importantly, the level of ;ARK1 inhibition determines the degree of cardiac preservation, consistent with a gene-dosage effect by ;ARKct on the pathological phenotype. Although multiple mechanisms are likely to be involved in the development of heart failure, we show here that inhibition of ;ARK1 is a novel molecular therapeutic target sufficient to prevent cardiac dysfunction. These findings have important clinical implications in developing future therapeutic strategies for heart failure.
Acknowledgments
This work was supported by the National Institutes of Health grant HL56687 (Dr Rockman) and the Burroughs Wellcome Fund (Dr Rockman). Dr Rockman is a recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.
Disclosure
Dr Lefkowitz is a founding scientist of and Dr Rockman is a consultant for Norak Biosciences, Inc, a company that is developing drugs that inhibit GRK2.
References
Bristow MR, Minobe W, Rasmussen R, Larrabee P, Skerl L, Klein JW, Anderson FL, Murray J, Mestroni L, Karwande SV, Fowler M, Ginsburg R. Beta-adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms. J Clin Invest. 1992; 89: 803–815.
Nienaber JJ, Tachibana H, Naga Prasad SV, Esposito G, Wu D, Mao L, Rockman HA. Inhibition of receptor-localized PI3K preserves cardiac beta-adrenergic receptor function and ameliorates pressure overload heart fa. J Clin Invest. 2003; 112: 1067–1079.
Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature. 2002; 415: 206–212.
Lefkowitz RJ. G protein–coupled receptor kinases. Cell. 1993; 74: 409–412.
Lefkowitz RJ. G protein–coupled receptors, III: new roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J Biol Chem. 1998; 273: 18677–18680.
Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science. 1995; 268: 1350–1353.
Rockman HA, Chien KR, Choi DJ, Iaccarino G, Hunter JJ, Ross J Jr, Lefkowitz RJ, Koch WJ. Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc Natl Acad Sci U S A. 1998; 95: 7000–7005.
Harding VB, Jones LR, Lefkowitz RJ, Koch WJ, Rockman HA. Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure. Proc Natl Acad Sci U S A. 2001; 98: 5809–5814.
Shah AS, White DC, Emani S, Kypson AP, Lilly RE, Wilson K, Glower DD, Lefkowitz RJ, Koch WJ. In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction. Circulation. 2001; 103: 1311–1316.
Choi DJ, Koch WJ, Hunter JJ, Rockman HA. Mechanism of beta-adrenergic receptor desensitization in cardiac hypertrophy is increased beta-adrenergic receptor kinase. J Biol Chem. 1997; 272: 17223–17229.
Ungerer M, Kessebohm K, Kronsbein K, Lohse MJ, Richardt G. Activation of beta-adrenergic receptor kinase during myocardial ischemia. Circ Res. 1996; 79: 455–460.
Gros R, Benovic JL, Tan CM, Feldman RD. G-protein–coupled receptor kinase activity is increased in hypertension. J Clin Invest. 1997; 99: 2087–2093.
Ungerer M, Bohm M, Elce JS, Erdmann E, Lohse MJ. Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation. 1993; 87: 454–463.
Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, Rockman HA. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation. 2002; 105: 85–92.
Nadeau JH, Burrage LC, Restivo J, Pao YH, Churchill G, Hoit BD. Pleiotropy, homeostasis, and functional networks based on assays of cardiovascular traits in genetically randomized populations. Genome Res. 2003; 13: 2082–2091.
Gorlin R, Gorlin SG. Hydraulic formula for calculation of the area of the stenotic mitral valve, other cardiac valves, and central circulatory shunts. Am Heart J. 1951; 41: 1–29.
Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, Stinson EB. Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. N Engl J Med. 1982; 307: 205–211.
Freeman K, Lerman I, Kranias EG, Bohlmeyer T, Bristow MR, Lefkowitz RJ, Iaccarino G, Koch WJ, Leinwand LA. Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J Clin Invest. 2001; 107: 967–974.
Dorn GW 2nd, Tepe NM, Lorenz JN, Koch WJ, Liggett SB. Low- and high-level transgenic expression of beta2-adrenergic receptors differentially affect cardiac hypertrophy and function in Galphaq-overexpressing mice. Proc Natl Acad Sci U S A. 1999; 96: 6400–6405.
Dorn GW 2nd, Tepe NM, Wu G, Yatani A, Liggett SB. Mechanisms of impaired beta-adrenergic receptor signaling in G(alphaq)-mediated cardiac hypertrophy and ventricular dysfunction. Mol Pharmacol. 2000; 57: 278–287.
Palmiter RD, Brinster RL. Germ-line transformation of mice. Annu Rev Genet. 1986; 20: 465–499.
Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA. Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J Biol Chem. 2000; 275: 4693–4698.
Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, Rockman HA. Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1: a role in receptor sequestration. J Biol Chem. 2001; 276: 18953–18959.
Clapham DE, Neer EJ. G protein beta gamma subunits. Annu Rev Pharmacol Toxicol. 1997; 37: 167–203.
Schmid SL. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu Rev Biochem. 1997; 66: 511–548.
Rapacciuolo A, Esposito G, Prasad SV, Rockman HA. G protein–coupled receptor signalling in in vivo cardiac overload. Acta Physiol Scand. 2001; 173: 51–57.
Naga Prasad SV, Laporte SA, Chamberlain D, Caron MG, Barak L, Rockman HA. Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex. J Cell Biol. 2002; 158: 563–575.
Xiang Y, Rybin VO, Steinberg SF, Kobilka B. Caveolar localization dictates physiologic signaling of beta 2-adrenoceptors in neonatal cardiac myocytes. J Biol Chem. 2002; 277: 34280–34286.
Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK, Caron MG, Lefkowitz RJ. Beta-arrestin–dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science. 1999; 283: 655–661.
Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM. The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem. 2000; 275: 9572–9580.
Miller WE, Lefkowitz RJ. Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking. Curr Opin Cell Biol. 2001; 13: 139–145.
Barki-Harrington L, Rockman HA. Sensing heart stress. Nat Med. 2003; 9: 19–20.
Barki-Harrington L, Luttrell LM, Rockman HA. Dual inhibition of beta-adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor–receptor interaction in vivo. Circulation. 2003; 108: 1611–1618.(Hideo Tachibana, MD, PhD;)
Surgery (W.J.K.)
the Howard Hughes Medical Institute (R.J.L.)
Duke University Medical Center, Durham, NC. Dr Koch is now at the Center for Translational Medicine, Jefferson Medical College, Philadelphia, Pa.
Abstract
Background— Heart failure is characterized by abnormalities in ;-adrenergic receptor (;AR) signaling, including increased level of myocardial ;AR kinase 1 (;ARK1). Our previous studies have shown that inhibition of ;ARK1 with the use of the G; sequestering peptide of ;ARK1 (;ARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of ;ARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of ;ARK1 inhibition correlates with the degree of heart failure.
Methods and Results— Transgenic (TG) mice with varying degrees of cardiac-specific expression of ;ARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial ;ARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of ;ARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low ;ARKct expression developed severe heart failure, whereas mice with high ;ARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of ;ARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal ;AR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in ;AR function, similar to the WT mice.
Conclusions— These data show that the level of ;ARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when ;ARK1 inhibition is considered as a molecular target.
Key Words: receptors, adrenergic, beta ; heart failure ; signal transduction ; mice, transgenic ; gene therapy
Introduction
Catecholamine-stimulated ;-adrenergic receptor (;AR) signaling is one of the most powerful regulators of cardiac function. Abnormalities in ;AR signaling are a prominent characteristic of failing hearts and may contribute to the progressive deterioration in cardiac function.1,2 Chronic stimulation of ;ARs in conditions of heart failure due to high levels of circulating catecholamines leads to desensitization and impaired ;AR responsiveness, in part as a result of increased levels of ;AR kinase 1 (;ARK1) (also known as GRK2).3 Stimulation of ;AR by catecholamines leads to dissociation of heterotrimeric G protein into G and G; subunits. ;ARK1 is recruited to the plasma membrane through its interaction with dissociated membrane-bound G; subunits and phosphorylates the agonist-occupied receptor. ;-Arrestin binds to the phosphorylated ;AR and sterically interdicts further coupling of the receptor with Gs subunit, leading to decreased ;AR signaling.4,5 Thus, phosphorylation of receptor by ;ARK1 is a critical step in regulating ;AR function and is consistent with in vivo studies showing that ;ARK1 is a critical modulator of cardiac function.6
A peptide inhibitor of ;ARK1, ;ARKct is composed of the last 194 amino acids of ;ARK1 and contains the binding site for G; subunits. Overexpression of ;ARKct peptide sequesters the dissociated G; subunits of heterotrimeric G proteins, leading to the inhibition of ;ARK1 recruitment to the membrane.6 Our previous studies have shown that inhibition of ;ARK1 has an important role in the pathophysiology of heart failure with the use of genetically engineered mouse models of heart failure, such as the muscle lim protein–knockout7 model and cardiac-specific overexpression of calsequestrin.8 Overexpression of ;ARKct led to less deterioration in cardiac function and prolonged survival in these genetic models of heart failure.8 Similarly, ;ARKct expression through adenoviral gene delivery in experimental models of myocardial infarction significantly delayed the development of heart failure.9 Importantly, recent experimental data support the concept that normalizing cardiac ;AR function leads to improved in vivo cardiac function in conditions of chronic pressure overload.2 Because the levels of myocardial ;ARK1 have been shown to be elevated in several cardiovascular disorders, including myocardial hypertrophy,10 ischemia,11 hypertension,12 and heart failure,13 ;ARK1 inhibition is a potential novel therapeutic strategy for conditions accompanied by marked ventricular dysfunction.
Although beneficial effects of ;ARKct have been demonstrated, it is not known whether the level of ;ARK1 inhibition correlates directly with the degree of heart failure or its amelioration. In the present study, we tested whether there is a gene-dosage effect of ;ARKct on preservation of cardiac function in heart failure by monitoring cardiac function in ;ARKct transgenic (TG) mice with varying levels of transgene expression that underwent pressure overload–induced heart failure.
Methods
Experimental Animals
TG mice overexpressing ;ARKct peptide were generated as previously described.6 Briefly, the coding sequence for the last 194 aa of the bovine ;ARK1 (;ARK1ct) was fused to the -myosin heavy-chain promoter. ;ARKct TG and wild-type (WT) littermate mice of either sex and 3 months of age were used for this study. Animals were handled according to the approved protocols and animal welfare regulations of the institutional review board at Duke University Medical Center.
Echocardiography
Echocardiography was performed on conscious mice with an HDI 5000 echocardiograph as previously described.7
In Vivo Pressure Overload
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg), and TAC was performed as previously described.2,14 Twelve weeks after surgery, the transstenotic pressure gradient (TSPG) was assessed by recording simultaneous measurements of right carotid and left axillary arterial pressures.
;AR Density and Adenylyl Cyclase Activity
Membrane fractions were prepared as previously described.2,14 Twenty-five micrograms of the membrane fraction was used to perform receptor binding with the use of 250 pmol/L of [125I]cyanopindolol.2,14 Receptor density (fmol) was normalized to milligrams of membrane protein. Adenylyl cyclase assays were performed with the use of 20 μg of the membrane fraction. Generated cAMP was quantified with a liquid scintillation counter (MINAXI;-4000).2,14
Immunoblotting
Immunodetection of myocardial levels of ;ARK1 and ;ARKct was performed on cytosolic extracts with a GRK2 antibody (Santa Cruz Biotechnology) after 12 weeks of TAC. Detection was performed with the use of enhanced chemiluminescence (ECL, Amersham), and the bands were quantified with the use of Bio-Rad Flouro-S Multimage software. ;ARK1 and ;ARKct values were normalized to actin (Santa Cruz Biotechnology) as a loading control. Reproducibility was confirmed by loading the same concentration of protein from each heart on multiple gels.
Statistical Analysis
Data are expressed as mean±SEM. Two-way repeated-measures ANOVA was used to evaluate the echo measurements for analysis of cardiac function after TAC. Post hoc analysis was performed with Newman-Keuls test. Multigroup comparisons were made with 1-way ANOVA and Tukey test. For all analyses, a value of P<0.05 was considered significant.
Results
Quantification of Myocardial ;ARK1 and ;ARKct Protein
We generated standard curves for the level of ;ARK1 protein by loading different amounts of purified ;ARK1 (25 to 150 ng) onto a 10% SDS-PAGE gel and then quantified the amount of ;ARK protein on the Western immunoblot by densitometry using Bio-Rad Flouro-S Multimage software for analysis (Figure 1A). There was a highly linear and reproducible relationship (R2=0.987) between the quantified band and the amount of purified ;ARK1 (Figure 1A). We then quantified the level of myocardial ;ARK1 and ;ARKct protein expression from the standard curves. Interestingly, we found that the level of ;ARKct protein in the cytosol of pressure-overloaded ;ARKct TG mice was quite variable (Figure 1B). Importantly, the highest levels of ;ARKct expression were associated with the greatest percent fractional shortening despite having similar TSPG and ;ARK1 levels (Figure 1B).
We next determined whether the level of ;ARK1 inhibition correlated directly with the degree of cardiac dysfunction by quantifying the level of myocardial ;ARKct protein expression and the associated percent fractional shortening in 24 TG mice after 12 weeks of TAC. TG ;ARKct mice showed a positive linear relationship between the level of ;ARKct protein expression and percent fractional shortening (Figure 2A), as well as when ;ARKct expression was normalized to the level of ;ARK1 (Figure 2B) or actin (Figure 2C). On the basis of ;ARKct transgene expression, mice were divided into high- and low-expression groups by a median of ;ARKct/actin ratio of 61.7 ng/arbitrary units (Figure 2C).
To exclude the possibility that cardiac remodeling by pressure overload might alter the level of ;ARKct protein expression, we measured ;ARKct levels in sham-operated mice and compared them with levels in the 12-week TAC mice. Importantly, we found a variability in the level of ;ARKct transgene expression in sham mice similar to that found in the TAC-operated animals (Figure 2D). In addition, Southern blotting confirmed that high and low ;ARKct expression was due to high and low copy numbers of the transgene (data not shown). These data show that the variability in ;ARKct expression occurred in both sham and TAC-operated mice and that the induction of heart failure by pressure overload did not alter the expression of ;ARKct transgene.
Level of ;ARKct Transgene Expression Directly Regulates In Vivo Cardiac Function in Response to Chronic Pressure Overload
We plotted fractional shortening against the range of TSPG in the low- and high-expressing ;ARKct mice after 12 weeks of banding. Fractional shortening between high- and low-expressing ;ARKct TG mice was clearly different even across a wide range of TSPG (Figure 3A), showing that mice with higher expression of ;ARKct had better cardiac function even after 12 weeks of chronic TAC. As expected, mean TSPG between the high- and low-expressing ;ARKct groups was not significantly different and spread over a broad range of pressures (Table and Figure 3A).
Physiological Parameters in WT and TG ;ARKct Mice
Because equivalence of pressure gradients does not necessarily mean that the average area of stenosis was the same across the groups, we calculated stenotic area across the transverse aorta by approximating stroke volume on the basis of the echocardiographic parameters of chamber size15 and by applying the Gorlin equation.16 TAC stenotic area for the low ;ARKct expressors was 0.4±0.2 mm2, and that for the high ;ARKct expressors was 0.2±0.3 mm2. In addition, pressure proximal to the transverse aortic stenosis was measured and found to be similar for the 3 groups (proximal pressure=170±10 mm Hg for WT, 190±9 mm Hg for high ;ARKct expressors, 173±12 mm Hg for low ;ARKct expressors; P=NS for any of the groups). These data support our assessment that the stenotic area and the load on the left ventricles (LV) were similar across the 3 groups.
To determine the time course for the development of heart failure, we monitored cardiac function in TG ;ARKct and WT littermate control mice by serial echocardiography at 4, 8, and 12 weeks after TAC. Echocardiography showed progressive LV enlargement and deterioration in cardiac function in the WT and TG mice with low level of ;ARKct expression (Figure 3B). These mice showed a 70% reduction in percent fractional shortening, a 270% increase in LV end-systolic dimension, and a 70% increase in LV end-diastolic dimension at 12 weeks after TAC compared with their basal measurements (Figures 3B and 4, Table). In contrast, TG mice with a high level of ;ARKct expression showed a significant preservation of cardiac function over the same period after chronic pressure overload, with only mild increases in LV end-systolic dimension or LV end-diastolic dimension (Figures 3B and 4, Table). Interestingly, chronic pressure overload led to a thinning of the septal and posterior myocardial walls in the WT and low-expressing ;ARKct mice, whereas wall thickness was increased in the high-expressing ;ARKct mice (Figure 3B and Table). At 12 weeks after TAC, the increase in the ratio of LV weight to body weight was significantly blunted in high-expression ;ARKct mice compared with WT and low-expression ;ARKct mice (Table). These morphometric and echocardiographic data show that a high level of ;ARKct protein expression in hearts of TG mice results in a significant blunting in heart size and mass and is associated with preservation of cardiac function in response to chronic pressure overload in vivo.
;AR Signaling in WT and ;ARKct Mice Under Conditions of Chronic Banding
Because previous studies have shown that ;ARK1 inhibition leads to preservation of ;AR function,6 we determined whether inhibition of ;ARK1 through cardiac-specific overexpression of ;ARKct could normalize ;AR function in the mice with a high level of ;ARKct expression under conditions of chronic pressure overload–induced heart failure. ;AR levels and receptor-effector coupling were evaluated in membrane fractions from the WT and TG ;ARKct mice after 12 weeks of banding. ;AR density was significantly reduced by 35% in the hearts of the WT and low-expressing ;ARKct mice on 12 weeks of banding (Figure 5A). In contrast, we found no significant decrease in receptor density after 12 weeks of TAC in the TG mice with high levels of ;ARKct expression (Figure 5A).
Receptor-effector coupling was assessed by adenylyl cyclase activity from the membrane fractions of sham and chronic banded WT and TG ;ARKct mice. Hearts from WT and low-expression ;ARKct banded mice showed significant desensitization, as measured by markedly diminished isoproterenol-stimulated membrane adenylyl cyclase activity (Figure 5B). Importantly, however, we found that isoproterenol-stimulated membrane adenylyl cyclase activity was preserved in the TG mice with high ;ARKct expression (Figure 5B). Taken together, these studies show that high levels of ;ARKct protein expression in the heart lead to normalization of ;AR density and isoproterenol-stimulated adenylyl cyclase activity, indicating preservation of ;AR–G protein coupling after chronic banding.
Discussion
In this study we demonstrate that inhibition of ;ARK1 through cardiac-specific overexpression of the ;ARKct peptide ameliorates the development of cardiac dysfunction under conditions of chronic in vivo pressure overload. This amelioration of cardiac dysfunction seems to depend primarily on the level of ;ARKct protein expression in the TG mice. TG mice with high ;ARKct expression showed preserved isoproterenol-stimulated adenylyl cyclase activity and normal ;AR density after 12 weeks of chronic banding. In contrast, WT and TG mice with low ;ARKct expression showed marked abnormalities in ;AR function, similar to that described in human heart failure.1,17 Strikingly, the extent of ;ARK1 inhibition, assessed by the level of ;ARKct protein expression, correlated positively with the attenuation of cardiac dysfunction after 12 weeks of chronic pressure overload. Thus, ;ARK1 as a therapeutic target would have immense clinical implications because ;ARK1 levels are increased in several cardiovascular disorders, including myocardial hypertrophy,10 myocardial ischemia,11 hypertension,12 and heart failure.13
The most likely mechanism for ;ARKct in preventing ;AR abnormalities and cardiac dysfunction is that the transgene sequesters G; subunits and inhibits receptor phosphorylation by blocking the recruitment of ;ARK1 to the agonist-stimulated receptor. Our present study shows that overexpression of the ;ARKct transgene effectively ameliorates heart failure in conditions of a molar ratio of 3:1 for ;ARKct to ;ARK. Previous studies have shown that ;ARK1 plays a critical role in the pathophysiology of heart failure, and inhibition of ;ARK1 with the use of ;ARKct in mouse models of heart failure has been shown to be beneficial.7,8,18 The mouse models of heart failure that have been rescued through ;ARKct include muscle lim protein knockout,7 cardiac overexpression of calsequestrin,8 and mice that developed hypertrophic cardiomyopathy.18 In all 3 models of heart failure, mice containing cardiac-specific overexpression of the ;ARKct showed less deterioration of cardiac function, and the cardiac overexpression of calsequestrin/;ARKct mice even exhibited an increase in mean survival age.8 Interestingly, overexpression of ;ARKct did not prevent the development of heart failure in all genetically modified mice, particularly in those in which ;ARK1 was not elevated, such as the TG Gq mice.19,20 Although these studies suggest that an increase in ;ARK1 level is important for ;ARKct to have an effect on cardiac function, we show in this study that the level of ;ARKct expression is far more important than the upregulation of ;ARK1 in determining response to pressure overload. This point is particularly salient because in our study the level of ;ARK1 did not change significantly among the banded TG hearts.
We show that ;ARKct transgene expression among littermates is variable and is probably due to the number of copies of the transgene in the genome. At the time of generation of TG mice, the integration of the transgene occurs at different loci across the genome as well as in multiple copies at any given loci.21 Therefore, segregation of the number of integrated copies of the transgene among the littermates likely accounts for the variable amounts of the expressed protein in the progeny. One of the standard procedures used to overcome variable transgene expression would be to backcross selected progeny for numerous generations to get uniform expression. Although the littermates have varying levels of transgene expression, this procedure has allowed us to directly correlate the effect of transgene expression with amelioration of heart failure.
Our data in the present study support our hypothesis that a critical determinant for the preservation of cardiac function by ;ARKct is normalization of ;AR function in conditions of chronic overload. This is consistent with our recent data showing that preventing downregulation and desensitization of ;ARs through a mechanism of receptor-localized phosphatidylinositol 3–kinase inhibition also ameliorates the development of heart failure.2 Because multiple pathways are involved in development of heart failure, other possible complementary mechanisms for the beneficial effects of ;ARKct transgene must be considered as well. It is possible that inhibition of other G;-mediated signaling events such as activation of phosphatidylinositol 3–kinase2,22,23 and IK,Ach channels24 may contribute to the mechanism of action of ;ARKct in heart failure. In addition, it is possible that overexpression of ;ARKct inhibits the phosphorylation of other G protein–coupled receptors, such as endothelin and angiotensin receptors, although this would lead to enhanced signaling of the receptor systems.
As discussed earlier, ;ARK-mediated phosphorylation of ;ARs leads to their internalization through a variety of mechanisms, including classic clathrin-coated, pit-mediated processes, caveolae, and noncoated pit mechanisms as well.25–28 It has been observed recently that the agonist-promoted internalization of ;ARs can lead to the assembly of complex signaling cascades that activate cellular growth pathways, such as the various mitogen-activated protein kinase pathways.29–31 We postulate that one of the mechanisms for the beneficial effects of ;ARKct overexpression is by inhibiting the activation of these maladaptive growth pathways of the cardiac myocyte.32
Our previous studies have shown that TG mice develop cardiac hypertrophy to an extent similar to that of the WT littermate controls after 7 days of banding, indicating that the ;ARKct transgene does not alter the development of cardiac hypertrophy.10 These data support our hypothesis that the development of cardiac hypertrophy is not sufficient to preserve cardiac function under conditions of pressure overload; rather, normalization of detrimental signaling pathways such as the ;AR pathway may be the critical determinant.14,33
In summary, we demonstrate that inhibition of ;ARK1 leads to preservation of ;AR function and attenuates deterioration of cardiac function under conditions of chronic pressure overload in vivo. Importantly, the level of ;ARK1 inhibition determines the degree of cardiac preservation, consistent with a gene-dosage effect by ;ARKct on the pathological phenotype. Although multiple mechanisms are likely to be involved in the development of heart failure, we show here that inhibition of ;ARK1 is a novel molecular therapeutic target sufficient to prevent cardiac dysfunction. These findings have important clinical implications in developing future therapeutic strategies for heart failure.
Acknowledgments
This work was supported by the National Institutes of Health grant HL56687 (Dr Rockman) and the Burroughs Wellcome Fund (Dr Rockman). Dr Rockman is a recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.
Disclosure
Dr Lefkowitz is a founding scientist of and Dr Rockman is a consultant for Norak Biosciences, Inc, a company that is developing drugs that inhibit GRK2.
References
Bristow MR, Minobe W, Rasmussen R, Larrabee P, Skerl L, Klein JW, Anderson FL, Murray J, Mestroni L, Karwande SV, Fowler M, Ginsburg R. Beta-adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms. J Clin Invest. 1992; 89: 803–815.
Nienaber JJ, Tachibana H, Naga Prasad SV, Esposito G, Wu D, Mao L, Rockman HA. Inhibition of receptor-localized PI3K preserves cardiac beta-adrenergic receptor function and ameliorates pressure overload heart fa. J Clin Invest. 2003; 112: 1067–1079.
Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature. 2002; 415: 206–212.
Lefkowitz RJ. G protein–coupled receptor kinases. Cell. 1993; 74: 409–412.
Lefkowitz RJ. G protein–coupled receptors, III: new roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J Biol Chem. 1998; 273: 18677–18680.
Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science. 1995; 268: 1350–1353.
Rockman HA, Chien KR, Choi DJ, Iaccarino G, Hunter JJ, Ross J Jr, Lefkowitz RJ, Koch WJ. Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc Natl Acad Sci U S A. 1998; 95: 7000–7005.
Harding VB, Jones LR, Lefkowitz RJ, Koch WJ, Rockman HA. Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure. Proc Natl Acad Sci U S A. 2001; 98: 5809–5814.
Shah AS, White DC, Emani S, Kypson AP, Lilly RE, Wilson K, Glower DD, Lefkowitz RJ, Koch WJ. In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction. Circulation. 2001; 103: 1311–1316.
Choi DJ, Koch WJ, Hunter JJ, Rockman HA. Mechanism of beta-adrenergic receptor desensitization in cardiac hypertrophy is increased beta-adrenergic receptor kinase. J Biol Chem. 1997; 272: 17223–17229.
Ungerer M, Kessebohm K, Kronsbein K, Lohse MJ, Richardt G. Activation of beta-adrenergic receptor kinase during myocardial ischemia. Circ Res. 1996; 79: 455–460.
Gros R, Benovic JL, Tan CM, Feldman RD. G-protein–coupled receptor kinase activity is increased in hypertension. J Clin Invest. 1997; 99: 2087–2093.
Ungerer M, Bohm M, Elce JS, Erdmann E, Lohse MJ. Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation. 1993; 87: 454–463.
Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, Rockman HA. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation. 2002; 105: 85–92.
Nadeau JH, Burrage LC, Restivo J, Pao YH, Churchill G, Hoit BD. Pleiotropy, homeostasis, and functional networks based on assays of cardiovascular traits in genetically randomized populations. Genome Res. 2003; 13: 2082–2091.
Gorlin R, Gorlin SG. Hydraulic formula for calculation of the area of the stenotic mitral valve, other cardiac valves, and central circulatory shunts. Am Heart J. 1951; 41: 1–29.
Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, Stinson EB. Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. N Engl J Med. 1982; 307: 205–211.
Freeman K, Lerman I, Kranias EG, Bohlmeyer T, Bristow MR, Lefkowitz RJ, Iaccarino G, Koch WJ, Leinwand LA. Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J Clin Invest. 2001; 107: 967–974.
Dorn GW 2nd, Tepe NM, Lorenz JN, Koch WJ, Liggett SB. Low- and high-level transgenic expression of beta2-adrenergic receptors differentially affect cardiac hypertrophy and function in Galphaq-overexpressing mice. Proc Natl Acad Sci U S A. 1999; 96: 6400–6405.
Dorn GW 2nd, Tepe NM, Wu G, Yatani A, Liggett SB. Mechanisms of impaired beta-adrenergic receptor signaling in G(alphaq)-mediated cardiac hypertrophy and ventricular dysfunction. Mol Pharmacol. 2000; 57: 278–287.
Palmiter RD, Brinster RL. Germ-line transformation of mice. Annu Rev Genet. 1986; 20: 465–499.
Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA. Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J Biol Chem. 2000; 275: 4693–4698.
Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, Rockman HA. Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1: a role in receptor sequestration. J Biol Chem. 2001; 276: 18953–18959.
Clapham DE, Neer EJ. G protein beta gamma subunits. Annu Rev Pharmacol Toxicol. 1997; 37: 167–203.
Schmid SL. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu Rev Biochem. 1997; 66: 511–548.
Rapacciuolo A, Esposito G, Prasad SV, Rockman HA. G protein–coupled receptor signalling in in vivo cardiac overload. Acta Physiol Scand. 2001; 173: 51–57.
Naga Prasad SV, Laporte SA, Chamberlain D, Caron MG, Barak L, Rockman HA. Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex. J Cell Biol. 2002; 158: 563–575.
Xiang Y, Rybin VO, Steinberg SF, Kobilka B. Caveolar localization dictates physiologic signaling of beta 2-adrenoceptors in neonatal cardiac myocytes. J Biol Chem. 2002; 277: 34280–34286.
Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK, Caron MG, Lefkowitz RJ. Beta-arrestin–dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science. 1999; 283: 655–661.
Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM. The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem. 2000; 275: 9572–9580.
Miller WE, Lefkowitz RJ. Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking. Curr Opin Cell Biol. 2001; 13: 139–145.
Barki-Harrington L, Rockman HA. Sensing heart stress. Nat Med. 2003; 9: 19–20.
Barki-Harrington L, Luttrell LM, Rockman HA. Dual inhibition of beta-adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor–receptor interaction in vivo. Circulation. 2003; 108: 1611–1618.(Hideo Tachibana, MD, PhD;)