Published online before print January 24, 2005, doi: 10.1161/01.CIR.0000142291.70954.DF
(Circulation. 2005;111:591-597.)
© 2005 American Heart Association, Inc.
Heart Failure |
Level of ß-Adrenergic Receptor Kinase 1 Inhibition Determines Degree of Cardiac Dysfunction After Chronic Pressure Overload–Induced Heart Failure
From 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.), and Surgery (W.J.K.), and 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.
Correspondence to Howard A. Rockman, MD, Duke University Medical Center, Research Dr, DUMC 3104, Durham, NC 27710. E-mail h.rockman{at}duke.edu
Received February 12, 2004; revision received April 22, 2004; accepted May 19, 2004.
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Abstract |
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Background— Heart failure is characterized by abnormalities in ß-adrenergic receptor (ßAR) signaling, including increased level of myocardial ßAR kinase 1 (ßARK1). Our previous studies have shown that inhibition of ßARK1 with the use of the Gß
sequestering peptide of ßARK1 (ßARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of ßARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of ßARK1 inhibition correlates with the degree of heart failure. Methods and Results— Transgenic (TG) mice with varying degrees of cardiac-specific expression of ßARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial ßARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of ßARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low ßARKct expression developed severe heart failure, whereas mice with high ßARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of ßARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal ßAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in ßAR function, similar to the WT mice.
Conclusions— These data show that the level of ßARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when ßARK1 inhibition is considered as a molecular target.
Key Words: receptors, adrenergic, beta • heart failure • signal transduction • mice, transgenic • gene therapy
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Introduction |
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Catecholamine-stimulated ß-adrenergic receptor (ßAR) signaling is one of the most powerful regulators of cardiac function. Abnormalities in ßAR signaling are a prominent characteristic of failing hearts and may contribute to the progressive deterioration in cardiac function.1,2 Chronic stimulation of ßARs in conditions of heart failure due to high levels of circulating catecholamines leads to desensitization and impaired ßAR responsiveness, in part as a result of increased levels of ßAR kinase 1 (ßARK1) (also known as GRK2).3 Stimulation of ßAR by catecholamines leads to dissociation of heterotrimeric G protein into G
and Gß subunits. ßARK1 is recruited to the plasma membrane through its interaction with dissociated membrane-bound Gß subunits and phosphorylates the agonist-occupied receptor. ß-Arrestin binds to the phosphorylated ßAR and sterically interdicts further coupling of the receptor with Gs subunit, leading to decreased ßAR signaling.4,5 Thus, phosphorylation of receptor by ßARK1 is a critical step in regulating ßAR function and is consistent with in vivo studies showing that ßARK1 is a critical modulator of cardiac function.6
A peptide inhibitor of ßARK1, ßARKct is composed of the last 194 amino acids of ßARK1 and contains the binding site for Gß subunits. Overexpression of ßARKct peptide sequesters the dissociated Gß subunits of heterotrimeric G proteins, leading to the inhibition of ßARK1 recruitment to the membrane.6 Our previous studies have shown that inhibition of ßARK1 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 ßARKct led to less deterioration in cardiac function and prolonged survival in these genetic models of heart failure.8 Similarly, ßARKct 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 ßAR function leads to improved in vivo cardiac function in conditions of chronic pressure overload.2 Because the levels of myocardial ßARK1 have been shown to be elevated in several cardiovascular disorders, including myocardial hypertrophy,10 ischemia,11 hypertension,12 and heart failure,13 ßARK1 inhibition is a potential novel therapeutic strategy for conditions accompanied by marked ventricular dysfunction.
Although beneficial effects of ßARKct have been demonstrated, it is not known whether the level of ßARK1 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 ßARKct on preservation of cardiac function in heart failure by monitoring cardiac function in ßARKct transgenic (TG) mice with varying levels of transgene expression that underwent pressure overload–induced heart failure.
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Methods |
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Experimental Animals
TG mice overexpressing ßARKct peptide were generated as previously described.6 Briefly, the coding sequence for the last 194 aa of the bovine ßARK1 (ßARK1ct) was fused to the
-myosin heavy-chain promoter. ßARKct 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.
ßAR 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 (MINAXIß-4000).2,14
Immunoblotting
Immunodetection of myocardial levels of ßARK1 and ßARKct 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. ßARK1 and ßARKct 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.
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Results |
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Quantification of Myocardial ßARK1 and ßARKct Protein
We generated standard curves for the level of ßARK1 protein by loading different amounts of purified ßARK1 (25 to 150 ng) onto a 10% SDS-PAGE gel and then quantified the amount of ßARK 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 ßARK1 (Figure 1A). We then quantified the level of myocardial ßARK1 and ßARKct protein expression from the standard curves. Interestingly, we found that the level of ßARKct protein in the cytosol of pressure-overloaded ßARKct TG mice was quite variable (Figure 1B). Importantly, the highest levels of ßARKct expression were associated with the greatest percent fractional shortening despite having similar TSPG and ßARK1 levels (Figure 1B).
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We next determined whether the level of ßARK1 inhibition correlated directly with the degree of cardiac dysfunction by quantifying the level of myocardial ßARKct protein expression and the associated percent fractional shortening in 24 TG mice after 12 weeks of TAC. TG ßARKct mice showed a positive linear relationship between the level of ßARKct protein expression and percent fractional shortening (Figure 2A), as well as when ßARKct expression was normalized to the level of ßARK1 (Figure 2B) or actin (Figure 2C). On the basis of ßARKct transgene expression, mice were divided into high- and low-expression groups by a median of ßARKct/actin ratio of 61.7 ng/arbitrary units (Figure 2C).
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To exclude the possibility that cardiac remodeling by pressure overload might alter the level of ßARKct protein expression, we measured ßARKct 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 ßARKct 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 ßARKct expression was due to high and low copy numbers of the transgene (data not shown). These data show that the variability in ßARKct 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 ßARKct transgene.
Level of ßARKct 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 ßARKct mice after 12 weeks of banding. Fractional shortening between high- and low-expressing ßARKct TG mice was clearly different even across a wide range of TSPG (Figure 3A), showing that mice with higher expression of ßARKct had better cardiac function even after 12 weeks of chronic TAC. As expected, mean TSPG between the high- and low-expressing ßARKct groups was not significantly different and spread over a broad range of pressures (Table and Figure 3A).
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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 ßARKct expressors was 0.4±0.2 mm2, and that for the high ßARKct 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 ßARKct expressors, 173±12 mm Hg for low ßARKct 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 ßARKct 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 ßARKct 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 ßARKct 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 ßARKct mice, whereas wall thickness was increased in the high-expressing ßARKct 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 ßARKct mice compared with WT and low-expression ßARKct mice (Table). These morphometric and echocardiographic data show that a high level of ßARKct 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.
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P<0.001 for high-expression ßARKct mice vs WT or low-expression mice.
ßAR Signaling in WT and ßARKct Mice Under Conditions of Chronic Banding
Because previous studies have shown that ßARK1 inhibition leads to preservation of ßAR function,6 we determined whether inhibition of ßARK1 through cardiac-specific overexpression of ßARKct could normalize ßAR function in the mice with a high level of ßARKct expression under conditions of chronic pressure overload–induced heart failure. ßAR levels and receptor-effector coupling were evaluated in membrane fractions from the WT and TG ßARKct mice after 12 weeks of banding. ßAR density was significantly reduced by 
35% in the hearts of the WT and low-expressing ßARKct 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 ßARKct expression (Figure 5A).
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Receptor-effector coupling was assessed by adenylyl cyclase activity from the membrane fractions of sham and chronic banded WT and TG ßARKct mice. Hearts from WT and low-expression ßARKct 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 ßARKct expression (Figure 5B). Taken together, these studies show that high levels of ßARKct protein expression in the heart lead to normalization of ßAR density and isoproterenol-stimulated adenylyl cyclase activity, indicating preservation of ßAR–G protein coupling after chronic banding.
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Discussion |
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In this study we demonstrate that inhibition of ßARK1 through cardiac-specific overexpression of the ßARKct 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 ßARKct protein expression in the TG mice. TG mice with high ßARKct expression showed preserved isoproterenol-stimulated adenylyl cyclase activity and normal ßAR density after 12 weeks of chronic banding. In contrast, WT and TG mice with low ßARKct expression showed marked abnormalities in ßAR function, similar to that described in human heart failure.1,17 Strikingly, the extent of ßARK1 inhibition, assessed by the level of ßARKct protein expression, correlated positively with the attenuation of cardiac dysfunction after 12 weeks of chronic pressure overload. Thus, ßARK1 as a therapeutic target would have immense clinical implications because ßARK1 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 ßARKct in preventing ßAR abnormalities and cardiac dysfunction is that the transgene sequesters Gß subunits and inhibits receptor phosphorylation by blocking the recruitment of ßARK1 to the agonist-stimulated receptor. Our present study shows that overexpression of the ßARKct transgene effectively ameliorates heart failure in conditions of a molar ratio of 3:1 for ßARKct to ßARK. Previous studies have shown that ßARK1 plays a critical role in the pathophysiology of heart failure, and inhibition of ßARK1 with the use of ßARKct 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 ßARKct 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 ßARKct showed less deterioration of cardiac function, and the cardiac overexpression of calsequestrin/ßARKct mice even exhibited an increase in mean survival age.8 Interestingly, overexpression of ßARKct did not prevent the development of heart failure in all genetically modified mice, particularly in those in which ßARK1 was not elevated, such as the TG Gq mice.19,20 Although these studies suggest that an increase in ßARK1 level is important for ßARKct to have an effect on cardiac function, we show in this study that the level of ßARKct expression is far more important than the upregulation of ßARK1 in determining response to pressure overload. This point is particularly salient because in our study the level of ßARK1 did not change significantly among the banded TG hearts.
We show that ßARKct 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 ßARKct is normalization of ßAR function in conditions of chronic overload. This is consistent with our recent data showing that preventing downregulation and desensitization of ßARs 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 ßARKct transgene must be considered as well. It is possible that inhibition of other Gß-mediated signaling events such as activation of phosphatidylinositol 3–kinase2,22,23 and IK,Ach channels24 may contribute to the mechanism of action of ßARKct in heart failure. In addition, it is possible that overexpression of ßARKct 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, ßARK-mediated phosphorylation of ßARs 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 ßARs 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 ßARKct 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 ßARKct 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 ßAR pathway may be the critical determinant.14,33
In summary, we demonstrate that inhibition of ßARK1 leads to preservation of ßAR function and attenuates deterioration of cardiac function under conditions of chronic pressure overload in vivo. Importantly, the level of ßARK1 inhibition determines the degree of cardiac preservation, consistent with a gene-dosage effect by ßARKct on the pathological phenotype. Although multiple mechanisms are likely to be involved in the development of heart failure, we show here that inhibition of ßARK1 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.
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Acknowledgments |
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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.
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References |
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 A. Curcio, T. Noma, S. V. Naga Prasad, M. J. Wolf, A. Lemaire, C. Perrino, L. Mao, and H. A. Rockman Competitive displacement of phosphoinositide 3-kinase from beta-adrenergic receptor kinase-1 improves postinfarction adverse myocardial remodeling Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1754 - H1760. [Abstract] [Full Text] [PDF]
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