Control of Myocardial Contractile Function by the Level of beta -Adrenergic Receptor Kinase 1 in Gene-targeted Mice*

Howard A. RockmanDagger , Dong-Ju ChoiDagger , Shahab A. Akhter§, Mohamed Jaberparallel , Bruno Giros**, Robert J. LefkowitzDagger Dagger §§, Marc G. CaronDagger Dagger §§, and Walter J. Koch§¶¶

From the Dagger  Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599 and Departments of § Surgery,  Cell Biology, and Dagger Dagger  Medicine, §§ Howard Hughes Medical Institute, Duke University, Durham, North Carolina 27710

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We studied the effect of alterations in the level of myocardial beta -adrenergic receptor kinase (beta ARK1) in two types of genetically altered mice. The first group is heterozygous for beta ARK1 gene ablation, beta ARK1(+/-), and the second is not only heterozygous for beta ARK1 gene ablation but is also transgenic for cardiac-specific overexpression of a beta ARK1 COOH-terminal inhibitor peptide, beta ARK1(+/-)/beta ARKct. In contrast to the embryonic lethal phenotype of the homozygous beta ARK1 knockout (Jaber, M., Koch, W. J., Rockman, H. A., Smith, B., Bond, R. A., Sulik, K., Ross, J., Jr., Lefkowitz, R. J., Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), beta ARK1(+/-) mice develop normally. Cardiac catheterization was performed in mice and showed a stepwise increase in contractile function in the beta ARK1(+/-) and beta ARK1(+/-)/beta ARKct mice with the greatest level observed in the beta ARK1(+/-)/beta ARKct animals. Contractile parameters were measured in adult myocytes isolated from both groups of gene-targeted animals. A significantly greater increase in percent cell shortening and rate of cell shortening following isoproterenol stimulation was observed in the beta ARK1(+/-) and beta ARK1(+/-)/beta ARKct myocytes compared with wild-type cells, indicating a progressive increase in intrinsic contractility. These data demonstrate that contractile function can be modulated by the level of beta ARK1 activity. This has important implications in disease states such as heart failure (in which beta ARK1 activity is increased) and suggests that beta ARK1 should be considered as a therapeutic target in this situation. Even partial inhibition of beta ARK1 activity enhances beta -adrenergic receptor signaling leading to improved functional catecholamine responsiveness.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

One of the most important mechanisms for rapidly regulating beta -adrenergic receptor (beta AR)1 function is agonist-stimulated receptor phosphorylation by G protein-coupled receptor kinases (GRKs) resulting in decreased sensitivity to further catecholamine stimulation (1). GRKs phosphorylate only agonist-occupied receptors leading to homologous desensitization (1, 2). The beta -adrenergic receptor kinase (beta ARK1) is a member of a family of at least 6 GRKs, which phosphorylate and regulate a wide variety of receptors that couple to heterotrimeric G proteins (3, 4). When beta ARs or other G protein-coupled receptors are activated by agonist, heterotrimeric G proteins dissociate into Galpha and Gbeta gamma subunits, and the Gbeta gamma subunit complex, which is membrane anchored by a lipid group (geranylgeranyl), can target beta ARK1 to the membrane through a direct physical interaction that facilitates phosphorylation of activated receptors (5, 6).

Using a transgenic based strategy for cardiac-specific overexpression of either beta ARK1 or a peptide inhibitor of beta ARK1 (beta ARKct), we have recently shown that in vivo, myocardial beta 1-adrenergic and angiotensin II receptors are targets for beta ARK1 mediated desensitization (7, 8). The beta ARK1 inhibitor utilized is a peptide containing the carboxyl-terminal 194 amino acids of beta ARK1, which competes with endogenous beta ARK1 for Gbeta gamma binding (7). Evidence suggesting a fundamental role for beta ARK1 in cardiac development was provided by gene-targeted mice in which the beta ARK1 gene was ablated by homologous recombination (9). Knockout mice, homozygous for the beta ARK1 deletion, died during mid-gestation with no viable beta ARK1(-/-) embryos observed past E15.5 (9). Histologic analysis revealed hypoplasia of ventricular myocardium with disorganized trabeculation. Furthermore, in vivo embryonic cardiac function demonstrated significantly impaired left ventricular (LV) ejection fraction compared with wild-type hearts, showing that beta ARK1 is required for normal cardiac development (9). In contrast to the complete knock out, beta ARK1(+/-) heterozygous animals have no obvious developmental abnormalities despite an approximate 50% reduction in the level of beta ARK1 protein and GRK activity (9).

In a variety of human and experimental conditions, prominent beta AR desensitization in response to catecholamine stimulation has recently been shown to be associated with heightened levels of beta ARK1 (10-13). In chronic human heart failure, reduced agonist-stimulated adenylyl cyclase activity due to both diminished receptor number and impaired receptor function is a predominant feature (14). In end-stage human heart failure, these changes in beta AR function were shown to be associated with elevated mRNA levels and activity for beta ARK1 (10, 15). Results from transgenic mice that overexpress beta ARK1 and GRK5 (7, 8) demonstrate how the up-regulation of these molecules in heart failure could markedly alter beta AR function by enhancing receptor desensitization. Furthermore, chronic treatment with either the beta AR antagonist bisoprolol in the pig (16) or carvedilol in the mouse2 (a potent therapeutic agent in human heart failure, see Ref. 18), substantially decreased the level of beta ARK1 activity. The most compelling evidence demonstrating the importance of beta ARK1 in heart failure comes from a recent study whereby transgenic mice with cardiac-restricted overexpression of the beta ARKct were mated into a genetic model of murine heart failure achieved through ablation of the MLP gene (19). Overexpression of the beta ARK1 inhibitor reversed the heightened beta AR desensitization in the MLP knockout mice and completely normalized cardiac function. These data strongly implicate abnormal beta AR-G protein coupling in the pathogenesis of the failing heart (19). Taken together, these studies indicate the potential for a therapeutic strategy that aims to modulate the activity level of myocardial beta ARK1 in disease states. Decreasing the level of myocardial beta ARK1 in established heart failure is a novel approach to improving impaired beta AR receptor function and potentially alter the pathogenesis in this disease.

In the present study, we sought to test the hypothesis that the level of cardiac beta ARK1 activity regulates myocardial contractile function in vivo. To test these hypotheses, we used a strategy that utilized mouse genetics to create varying levels of beta ARK1 activity in the heart, coupled with a physiological assessment of contractile function in the absence and presence of catecholamine stimulation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experimental Animals-- The gene-targeted mice used for this study were 1) heterozygous for targeted disruption of the beta ARK1 gene (9), and 2) offspring generated by cross breeding transgenic mice with cardiac-specific overexpression of a beta ARK1 inhibitor (beta ARKct, shown previously to have enhanced basal contractility) (7) with the beta ARK1(+/-) to yield the double gene-targeted line beta ARK1(+/-)/beta ARKct. Offspring were genotyped by Southern blot analysis on DNA extracted from tail biopsies. Mice of either sex, 4-6 months of age were used and compared with wild-type litter mates. The animals in this study were handled according to approved protocols and the animal welfare regulations of the University of North Carolina at Chapel Hill and Duke University.

Hemodynamic Evaluation in Intact Anesthetized Mice-- Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg) and analyzed as described previously (8). Briefly, after endotracheal intubation, mice were connected to a rodent ventilator. Following bilateral vagotomy, the chest was opened and a 1.8-French high fidelity micromanometer catheter (Millar Instruments) was inserted into the left atrium, advanced through the mitral valve, and secured in the LV. Hemodynamic measurements were recorded at baseline and 45-60 seconds after injection of incremental doses of isoproterenol. Doses of isoproterenol were specifically chosen to maximize the contractile response but limit the increase in heart rate. Experiments were then terminated, hearts were rapidly excised, with individual chambers separated, weighed, and frozen in liquid N2 for later biochemical analysis. Ten sequential beats were averaged for each measurement.

Myocyte Isolation-- Adult myocytes were isolated as described previously (20, 21). Following anesthesia, the heart was excised and the aorta was cannulated with a 20-gauge needle then mounted on the perfusion apparatus. The perfusion solution was composed of Joklik's minimum essential medium containing (in mM) 113 NaCl, 4.7 KCL, 0.6 KH2PO2, 0.6 Na2PO4, 1.2 MgSO4, 0.5 MgCl2, 10 HEPES, 20 D-glucose, 30 taurine, 2.0 carnitine, 2.0 creatine, and 20 µM Ca2+ at pH 7.4. The aorta was perfused for 2-3 min, then 150 units/ml of type-II collagenase (Worthington) was added and perfused for 15 min. The temperature of perfusate was maintained at 34 °C and all solutions were continuously bubbled with 95% 02, 5% CO2. LV tissue was separated from the great vessels, atria and right ventricle, minced, and allowed to digest in perfusate for 15 min. The digested heart was filtered through 200 µm nylon mesh, placed in a conical tube, and spun at 100 rpm to allow viable myocytes to settle. Serial washes were used to remove nonviable myocytes and digestive enzymes until the concentration of Ca2+ was gradually increased to 1.8 mM in Joklik's minimal essential medium. The operator was blinded to the genotype of the animals.

Evaluation of Myocyte Function-- Myocytes were placed in a 0.5-ml chamber with 1.8 mM Ca2+ Tyrode's solution at room temperature. Myocytes were visualized with a Nikon inverted microscope with a solid state CCD camera attached and displayed on a video monitor. Two platinum electrodes placed in the bathing fluid were connected to a stimulator to field stimulate the myocytes with a pulse duration of 5 ms and a frequency of 0.5 Hz. Myocyte cell edges were enhanced and processed with a video edge motion detection system (Crescent Electronics) at a sampling rate of 240 Hz. Recordings were performed under basal conditions and then 1-2 min after isoproterenol (10-7 M) administration. Calibrated myocyte lengths were converted from analog to digital on-line (MacLab) and stored on computer. All myocytes were studied within 1-2 h after myocyte isolation. Data from 5-8 consecutive contractions were averaged. Contractile parameters measured were: percent cell shortening (%CS) (calculated as percent change in myocyte length from rest (Lmax) to minimum length (Lmin)), rate of shortening (-dL/dt) and rate of relengthening (+dL/dt). 7-15 myocytes from each heart were studied.

GRK Activity by Rhodopsin Phosphorylation-- Myocardial extracts were prepared by homogenization of excised hearts in ice-cold lysis buffer (2 ml) (25 mM Tris-Cl (pH 7.5), 5 mM EDTA, 5 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 48,000 × g for 30 min. The supernatants that contain soluble kinases were concentrated using a Centricon-10 (Amicon) microconcentrator. Protein concentration was determined by the Bradford method. Concentrated cytosolic extracts (200 µg of protein) were incubated with rhodopsin-enriched rod outer segment membranes in reaction buffer (75 µl) containing 10 mM MgCl2, 20 mM Tris-Cl, 2 mM EDTA, 5 mM EGTA, and 0.1 mM ATP (containing [gamma -32P]ATP) as described (9). Reactions were carried out in the absence and presence of purified Gbeta gamma (approx 20 pmol) to maximally activate beta ARK (9). After incubating in white light for 15 min at room temperature, reactions were quenched with ice-cold lysis buffer (300 µl) and centrifuged for 15 min at 13,000 × g. Sedimented proteins were resuspended in 20 µl of protein-gel loading dye and electrophoresed through 12% SDS-polyacrylamide gels. Phosphorylated rhodopsin was visualized by autoradiography of dried polyacrylamide gels and quantified using a Molecular Dynamics PhosphorImager.

Statistical Analysis-- Results are expressed as mean value ± S.E. To examine the effect of isoproterenol on changes in hemodynamic parameters between wild-type controls and the two gene-targeted groups (beta ARK1(+/-) and beta ARK1(+/-)/beta ARKct), a 3 × 4 repeated measures analysis of variance (ANOVA) was used. To test for statistical difference in isolated cell contractile parameters and adenylyl cyclase activity, a one factor ANOVA was used. Post hoc analysis with regard to differences in mean values between groups was conducted with either a Newman-Keuls or Duncan test. A Student's t test with Bonferroni correction for 3 comparisons was used to test for statistical difference in the chamber weight parameters. p < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To determine whether altered levels of beta ARK1 influence myocardial growth in the adult mouse, left ventricular weight normalized for body weight (LV/BW) and tibia length was compared in wild type (n = 26), beta ARK1(+/-) (n = 19), and beta ARK1(+/-)/beta ARKct (n = 9) gene-targeted mice. No significant difference was observed for any of the measured variables between the three groups (LV/BW; wild type 3.5 ± 0.1, beta ARK1(+/-) 3.7 ± 0.1, beta ARK1(+/-)/beta ARKct 3.6 ± 0.2, mg/g, p = not significant). In contrast to the embryonic lethal phenotype of the homozygous beta ARK1 knockout, the heterozygote mice developed normally and attained a similar body weight as wild-type adults. Similarly, we previously had not observed any differences in heart weight in the animals overexpressing the beta ARKct alone compared with wild type controls (7).

To assess the levels of myocardial beta ARK activity in the different gene-targeted mice, we prepared soluble myocardial extracts and carried out in vitro GRK phosphorylation assays using rhodopsin as a G protein-coupled receptor substrate. To address whether the beta ARKct is functional, we added purified Gbeta gamma to the reactions. As shown in Fig. 1, Gbeta gamma -stimulated beta ARK activity is decreased in a stepwise fashion with the beta ARK1(+/-)/beta ARKct animals having only approx 25% of the wild-type myocardial beta ARK activity. Myocardial extracts from the beta ARK1(+/-) animals had 50% of the wild-type activity, which correlates to the 50% decrease in beta ARK1 protein we have previously described (9). This significant decrease in myocardial beta ARK1 activity in the double gene-targeted mice could also be demonstrated when expressing the data as fold-stimulation of Gbeta gamma over basal rhodopsin phosphorylation activity (-Gbeta gamma ). In beta ARK1(±)/beta ARKct animals, Gbeta gamma only stimulated activity by 2.24 ± 0.30-fold (n = 5) compared with 4.48 ± 0.93-fold (n = 6) for the wild-type animals (p < 0.05). Extracts from beta ARK1(+/-) hearts had similar values to wild-type (3.75 ± 0.88-fold, n = 6). Because of the dependence of the beta ARKct activity on Gbeta gamma , as expected, in vitro beta ARK activity in the absence of Gbeta gamma was equivalent between beta ARK1(+/-) extracts and beta ARK1(+/-)/beta ARKct extracts, which was approx 50% of wild-type activity (data not shown).


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Fig. 1.   Assessment of in vitro myocardial beta ARK activity. Soluble cytosolic myocardial extracts were prepared and beta ARK activity was assessed using a rhodopsin phosphorylation assay and purified Gbeta gamma to maximally activate beta ARK1 (see "Materials and Methods"). Shown is maximal Gbeta gamma -stimulated beta ARK activity of myocardial extracts from wild-type (WT, n = 6), beta ARK1(+/-) (n = 6), and beta ARK1(+/-)/beta ARKct (n = 5) gene-targeted mice. *, p < 0.005 versus WT; #, p < 0.05 versus beta ARK1(+/-).

We have previously reported that overexpression of the beta ARKct in transgenic mice leads to a significant enhancement of myocardial beta AR signaling and myocardial contractility in vivo under basal conditions and in response to catecholamine infusion (7). To determine whether the level of beta ARK1 modulates catecholamine responsiveness in vivo, cardiac catheterization was performed in intact anesthetized mice before and after infusion of isoproterenol (Fig. 2). Although the isovolumic phase measure of myocardial contractility, LV dP/dtmax, was not increased in the beta ARK1(+/-) at baseline compared to wild type, it was significantly enhanced in the beta ARK1(+/-)/beta ARKct mice. Contractile function was further and significantly augmented in the two gene-targeted groups in response to isoproterenol with the greatest level observed in the beta ARK1(+/-)/beta ARKct (Fig. 2A). Enhanced myocardial relaxation, as assessed by LV dP/dtmin was particularly evident in the beta ARK1(+/-)/beta ARKct animals but not in the beta ARK1(+/-) at baseline (Fig. 2B). As shown in Fig. 2, C and D, LV systolic pressure and heart rate were increased in the gene-targeted animals at baseline, which was further potentiated with isoproterenol stimulation. This is particularly apparent for changes in heart rate (Fig. 2D).


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Fig. 2.   In vivo assessment of LV contractile function in response to beta -agonist stimulation. Cardiac catheterization was performed in intact anesthetized mice 4-6 months of age, using a 1.8-French high fidelity micromanometer. Parameters measured were LV systolic and end-diastolic pressure, the maximal and minimal first derivative of LV pressure (LV dP/dtmax, dP/dtmin), and heart rate. Four measured parameters are shown at baseline and after progressive doses of isoproterenol in wild-type (open circle ) n = 26, and beta ARK1(+/-) (bullet ) (n = 19), and beta ARK1(+/-)/beta ARKct (black-triangle) (n = 9) mice. A, LV dP/dtmax; B, LV dP/dtmin; C, LV systolic pressure; D, heart rate. Data was analyzed with a repeated measures ANOVA and post hoc analysis by Newman-Keuls, *, p < 0.005 either beta ARK1(+/-) or beta ARK1(+/-)/beta ARKct versus wild type; dagger , p <0.05 beta ARK1(+/-) versus beta ARK1(+/-)/beta ARKct. A significant between-group main effect in response to isoproterenol was found for LV dP/dtmax (p < 0.0001) (A), LV dP/dtmin (p < 0.0001) (B), LV systolic pressure (p < 0.005) (C), and heart rate (p < 0.005) (D). The pattern of change between groups was statistically different for LV dP/dtmax (p < 0.0001) (A) and heart rate (p < 0.0001) (D). Body weight was not significantly different between groups: wild type 27.6 ± 1.2 g; beta ARK1(+/-) 30.7 ± 1.1 g; beta ARK1(+/-)/beta ARKct 29.0 ± 1.5 g.

Heart rate is a powerful determinant of myocardial contractility and can have important influences on LV dP/dtmax (22). To illustrate this point, the data has been plotted for the three groups showing the relationship between LV dP/dtmax and heart rate at baseline (Fig. 3A) and with maximal isoproterenol (Fig. 3B). In general, the animals with the highest heart rate also had the greatest LV dP/dtmax. Taken together these data demonstrate that the level of beta ARK1 activity exerts tight control over the inotropic and chronotropic response to catecholamine stimulation in the heart in vivo.


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Fig. 3.   Influence of Heart Rate on LV dP/dtmax. Plot of the relationship of heart rate on LV dP/dtmax in the 3 groups of mice at baseline (A) and after maximal isoproterenol (Iso, 1000 pg bolus injection) stimulation (B). (open circle ), wild type; (bullet ), beta ARK1(+/-); and (black-triangle), beta ARK1(+/-)/beta ARKct.

Since both loading conditions (LV end-diastolic pressure) and heart rate influence the in vivo measurement of myocardial contractility as measured by LV dP/dtmax (22, 23), studies were performed on adult myocardial cells isolated from both of the gene-targeted mouse strains. To determine whether a decrease in the level of beta ARK1 would affect myocyte contractility in the absence of potential confounding influences such as heart rate and mechanical loading, freshly isolated single adult myocytes were obtained from normal, beta ARK1(+/-), and beta ARK1(+/-)/beta ARKct) gene-targeted mice followed by an assessment of the contractile properties.

Measurements of contractile parameters in unloaded isolated adult cells were made in the absence and presence of isoproterenol (10-7 M) at a constant paced stimulation of 0.5 Hz. Adult myocytes isolated from wild-type hearts responded to isoproterenol stimulation with a 12% increase from baseline in %CS and the rate of shortening (-dL/dt) without a change in diastolic cell length (Fig. 4 and Table I). In contrast, adult myocytes isolated from beta ARK1(+/-) heterozygote animals showed an 18% increase in -dL/dt, whereas myocytes from beta ARK1(+/-)/beta ARKct hearts showed an even greater increase in -dL/dt (29%) following isoproterenol administration (Table I and Fig. 4). Similarly, %CS under baseline and isoproterenol conditions was progressively higher in myocytes isolated from the two gene-targeted mouse lines compared with wild-type cells (Table I). Overall cells isolated from beta ARK1(+/-) and beta ARK1(+/-)/beta ARKct mice had a significantly greater, and stepwise, increase in contractile parameters with isoproterenol compared with wild-type litter mates (Table I). These data complement the in vivo assessment of contractile function and show that intrinsic myocyte contractility can be directly influenced by the level of beta ARK1.


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Fig. 4.   Contractile function in isolated mouse myocytes. Representative tracings of myocytes isolated from a wild-type and a beta ARK1(+/-) mouse showing changes in cell length and its first derivative (dL/dt) at baseline and during isoproterenol administration (10-7 M). Changes in cell length were measured in the same cell before and after beta AR stimulation at a paced frequency of 0.5 Hz.

                              
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Table I
Contractile parameters in adult myocytes isolated from gene-targeted mice

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study demonstrates that 1) beta ARK1(+/-) mice that are heterozygote for ablation of the beta ARK1 gene and have a 50% reduction in the level of beta ARK1 in the heart develop normally, 2) the level of chronotropy and inotropy in vivo can be modulated by the level of beta ARK1 expression, and 3) contractile function can be further enhanced through in vivo beta ARK1 inhibition by competing for Gbeta gamma binding and endogenous beta ARK1 translocation and activation.

Complete disruption of the beta ARK1 gene in mice leads to a lethal phenotype with no beta ARK1(-/-) embryos surviving beyond gestational day 15.5 (9). The finding that mice that are heterozygous for the beta ARK1 deletion have no developmental abnormalities and develop into normal adults, suggests that there is a threshold level for beta ARK1 that allows for normal cardiac development. Although the beta ARK1(+/-) mice grow into normal adults, the functional consequence of reduced beta ARK1 levels is a phenotype of decreased desensitization in the heart as shown by the enhanced contractile response to isoproterenol stimulation (Fig. 2).

beta ARK1 requires a membrane-targeting event prior to receptor phosphorylation that occurs through the interaction of membrane anchored Gbeta gamma subunits and the carboxyl terminus of beta ARK1 (5). Preventing beta ARK1 translocation by competing for Gbeta gamma binding in transgenic mice overexpressing a peptide inhibitor (beta ARKct) results in an in vivo phenotype of enhanced basal and agonist-stimulated contractility due to decreased receptor desensitization (7). The mating of these two gene-targeted mice (beta ARK1(+/-) and beta ARKct overexpression) results in a further enhancement of contractility and relaxation (Fig. 2). These data suggest that both the level of beta ARK1 expression and the active process of translocation and activation of beta ARK1, determine the degree of beta AR desensitization and subsequent receptor-G protein coupling.

The isovolumic phase index (LV dP/dtmax) is a sensitive measure of contractility. However, the level of contractility is significantly influenced by heart rate and loading conditions in particular, preload (23). In this regard, it has recently been shown that there is a linear relationship between heart rate and LV dP/dtmax (22). In the present study we show that the significantly enhanced contractile performance of mice with altered levels of beta ARK1, as measured by LV dP/dtmax, is also associated with a modest increase in heart rate. To address this issue, we specifically assessed contractile parameters in single adult ventricular myocytes isolated from both the beta ARK1(+/-) and beta ARK1(+/-)/beta ARKct gene-targeted mice to determine whether the in vivo phenotype in these animals can be attributed to an intrinsic increase in myocyte contractility. The effect of heart rate on contractile function was eliminated by pacing cells at 0.5 Hz. As shown in Table I, a significant augmentation of contractile parameters occurred following beta AR stimulation in the beta ARK1(+/-) cells, which was further enhanced in the beta ARK1(+/-)/beta ARKct cells. Although as a group the cells isolated from the beta ARK1(+/-)/beta ARKct hearts were smaller than the other cells it did not seem to affect indices of contraction, but may have had some influence on the rate of relengthening (+dL/dt). These data show that beta ARK1 can directly influence contractility at a cellular level and confirm the in vivo data showing that reduced beta ARK1 levels leads to enhanced catecholamine responsiveness. These results are also consistent with a recent study on contractile function in single adult myocytes isolated from transgenic mice that overexpress either beta ARK1 or the beta ARKct, which showed that the presence of the beta ARKct resulted in an enhanced contractile response to beta AR stimulation compared with control cells (24).

We have previously shown that adenoviral-mediated gene transfer of the beta ARKct can restore beta AR signaling in failing myocytes isolated from chronically paced rabbits (25). In that in vitro study we showed that the biochemical defects in beta AR signaling in isolated failing myocytes could be reversed by gene transfer of the beta ARKct (25). In this study, we extend those findings by showing that in vivo contractile function and isoproterenol responsiveness in the intact animal is related to the level of beta ARK1. Furthermore, we not only demonstrate that an inhibitor of beta ARK1 (beta ARKct) can affect cellular contractility but also that the contractile state of the myocyte can be directly influenced by the level of beta ARK1 expression.

There is increasing evidence that elevated levels of beta ARK1 contribute to impaired catecholamine responsiveness observed in disease states of cardiac hypertrophy and heart failure (26). Elevated levels of beta ARK1 have been shown to be present in heart extracts from human end-stage heart failure (15) and in circulating lymphocytes from patients with mild to moderate essential hypertension (11). An essential role for beta ARK1 leading to impaired catecholamine responsiveness has been shown in various animal models of cardiac disease including cardiac hypertrophy (12) and myocardial ischemia (13). In this regard it is worthwhile to note that the use of angiotensin-converting enzyme inhibitors (17) in experimental heart failure was associated with a reduction in myocardial beta ARK1 activity. Furthermore in a mouse model of pressure overload hypertrophy, impaired beta AR signaling that occurs with the development of modest myocardial hypertrophy, could be completely reversed in the presence of the beta ARK inhibitor (beta ARKct) (12). In this study, we show that not only is beta ARK1 a critical modulator of in vivo cardiac function (7), but the level of beta ARK1 activity is important and can directly influence the degree to which the beta AR-signaling pathway is activated. This has important implications in disease states of increased beta ARK activity when considering beta ARK1 as a therapeutic target, since even partial inhibition of beta ARK1 function will effectively enhance beta AR signaling leading to improved catecholamine responsiveness. This study is of particular significance given our recent data showing the dramatic beneficial effect of overexpression of a beta ARK inhibitor in a mouse model of heart failure (19).

    ACKNOWLEDGEMENTS

We gratefully acknowledge the expert help from Dr. Lan Mao in the microsurgical techniques in the mouse, Kyle Shotwell for technical assistance with beta ARK activity assays, Mark Griswold with isolated cell measurements, and Susan Suter and Sandy Duncan for maintenance of the mouse colonies.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL 56687 (to H. A. R.), HL 16037 (to R. J. L.), and NS 19576 (to M. G. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Present address: CNRS U-5541, University Bordeaux II, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France.

** Present address: Unite INSERM 288, CHU Pitie-Salpetriere, Paris, France.

¶¶ To whom correspondence should be addressed: Dept. of Surgery, Duke University Medical Center, Box 2606, Room 472 MSRB, Durham, NC 27710. Tel.: 919-684-3007; Fax: 919-684-5714; E-mail: koch0002{at}mc.duke.edu.

1 The abbreviations used are: beta AR, beta -adrenergic receptor; GRK, G protein-coupled receptor kinase; beta ARK, beta AR kinase; LV, left ventricular; ANOVA, analysis of variance; BW, body weight; %CS, percent cell shortening; -dL/dt, rate of shortening; +dL/dt, rate of relengthening.

2 G. Iaccarino, E. D. Tomhave, R. J. Lefkowitz, and W. J. Koch, submitted.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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