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

We studied the effect of alterations in the level of myocardial β-adrenergic receptor kinase (βARK1) in two types of genetically altered mice. The first group is heterozygous for βARK1 gene ablation, βARK1(+/−), and the second is not only heterozygous for βARK1 gene ablation but is also transgenic for cardiac-specific overexpression of a βARK1 COOH-terminal inhibitor peptide, βARK1(+/−)/βARKct. In contrast to the embryonic lethal phenotype of the homozygous βARK1 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), βARK1(+/−) mice develop normally. Cardiac catheterization was performed in mice and showed a stepwise increase in contractile function in the βARK1(+/−) and βARK1(+/−)/βARKct mice with the greatest level observed in the βARK1(+/−)/βARKct 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 βARK1(+/−) and βARK1(+/−)/βARKct 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 βARK1 activity. This has important implications in disease states such as heart failure (in which βARK1 activity is increased) and suggests that βARK1 should be considered as a therapeutic target in this situation. Even partial inhibition of βARK1 activity enhances β-adrenergic receptor signaling leading to improved functional catecholamine responsiveness.

One of the most important mechanisms for rapidly regulating ␤-adrenergic receptor (␤AR) 1 function is agonist-stimulated receptor phosphorylation by G protein-coupled receptor ki-nases (GRKs) resulting in decreased sensitivity to further catecholamine stimulation (1). GRKs phosphorylate only agonistoccupied receptors leading to homologous desensitization (1,2). The ␤-adrenergic receptor kinase (␤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 ␤ARs or other G protein-coupled receptors are activated by agonist, heterotrimeric G proteins dissociate into G ␣ and G ␤␥ subunits, and the G ␤␥ subunit complex, which is membrane anchored by a lipid group (geranylgeranyl), can target ␤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 ␤ARK1 or a peptide inhibitor of ␤ARK1 (␤ARKct), we have recently shown that in vivo, myocardial ␤ 1 -adrenergic and angiotensin II receptors are targets for ␤ARK1 mediated desensitization (7,8). The ␤ARK1 inhibitor utilized is a peptide containing the carboxyl-terminal 194 amino acids of ␤ARK1, which competes with endogenous ␤ARK1 for G ␤␥ binding (7). Evidence suggesting a fundamental role for ␤ARK1 in cardiac development was provided by genetargeted mice in which the ␤ARK1 gene was ablated by homologous recombination (9). Knockout mice, homozygous for the ␤ARK1 deletion, died during mid-gestation with no viable ␤ARK1(Ϫ/Ϫ) 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 ␤ARK1 is required for normal cardiac development (9). In contrast to the complete knock out, ␤ARK1(ϩ/Ϫ) heterozygous animals have no obvious developmental abnormalities despite an approximate 50% reduction in the level of ␤ARK1 protein and GRK activity (9).
In a variety of human and experimental conditions, prominent ␤AR desensitization in response to catecholamine stimulation has recently been shown to be associated with heightened levels of ␤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 ␤AR function were shown to be associated with elevated mRNA levels and activity for ␤ARK1 (10,15). Results from transgenic mice that overexpress ␤ARK1 and GRK5 (7,8) demonstrate how the up-regulation of these molecules in heart failure could markedly alter ␤AR function by enhancing receptor desensitization. Furthermore, chronic treatment with either the ␤AR antagonist bisoprolol in the pig (16) or carvedilol in the mouse 2 (a potent therapeutic agent in human heart failure, see Ref. 18), substantially decreased the level of ␤ARK1 activity. The most compelling evidence demonstrating the importance of ␤ARK1 in heart failure comes from a recent study whereby transgenic mice with cardiac-restricted overexpression of the ␤ARKct were mated into a genetic model of murine heart failure achieved through ablation of the MLP gene (19). Overexpression of the ␤ARK1 inhibitor reversed the heightened ␤AR desensitization in the MLP knockout mice and completely normalized cardiac function. These data strongly implicate abnormal ␤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 ␤ARK1 in disease states. Decreasing the level of myocardial ␤ARK1 in established heart failure is a novel approach to improving impaired ␤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 ␤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 ␤ARK1 activity in the heart, coupled with a physiological assessment of contractile function in the absence and presence of catecholamine stimulation.

MATERIALS AND METHODS
Experimental Animals-The gene-targeted mice used for this study were 1) heterozygous for targeted disruption of the ␤ARK1 gene (9), and 2) offspring generated by cross breeding transgenic mice with cardiacspecific overexpression of a ␤ARK1 inhibitor (␤ARKct, shown previously to have enhanced basal contractility) (7) with the ␤ARK1(ϩ/Ϫ) to yield the double gene-targeted line ␤ARK1(ϩ/Ϫ)/␤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 N 2 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 KH 2 PO 2 , 0.6 Na 2 PO 4 , 1.2 MgSO 4 , 0.5 MgCl 2 , 10 HEPES, 20 D-glucose, 30 taurine, 2.0 carnitine, 2.0 creatine, and 20 M Ca 2ϩ 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% 0 2 , 5% CO 2 . 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 m 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 Ca 2ϩ 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 Ca 2ϩ 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 Ϫ7 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 (L max ) to minimum length (L min )), rate of shortening (ϪdL/dt) and rate of relengthening (ϩdL/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 g/ml leupeptin, 20 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 48,000 ϫ 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 g of protein) were incubated with rhodopsin-enriched rod outer segment membranes in reaction buffer (75 l) containing 10 mM MgCl 2 , 20 mM Tris-Cl, 2 mM EDTA, 5 mM EGTA, and 0.1 mM ATP (containing [␥-32 P]ATP) as described (9). Reactions were carried out in the absence and presence of purified G ␤␥ (Ϸ20 pmol) to maximally activate ␤ARK (9). After incubating in white light for 15 min at room temperature, reactions were quenched with ice-cold lysis buffer (300 l) and centrifuged for 15 min at 13,000 ϫ g. Sedimented proteins were resuspended in 20 l 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 Ϯ S.E. To examine the effect of isoproterenol on changes in hemodynamic parameters between wild-type controls and the two gene-targeted groups (␤ARK1(ϩ/Ϫ) and ␤ARK1(ϩ/Ϫ)/␤ARKct), a 3 ϫ 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 Ͻ 0.05 was considered significant.
cant difference was observed for any of the measured variables between the three groups (LV/BW; wild type 3.5 Ϯ 0.1, ␤ARK1(ϩ/Ϫ) 3.7 Ϯ 0.1, ␤ARK1(ϩ/Ϫ)/␤ARKct 3.6 Ϯ 0.2, mg/g, p ϭ not significant). In contrast to the embryonic lethal phenotype of the homozygous ␤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 ␤ARKct alone compared with wild type controls (7).
To assess the levels of myocardial ␤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 ␤ARKct is functional, we added purified G ␤␥ to the reactions. As shown in Fig. 1, G ␤␥ -stimulated ␤ARK activity is decreased in a stepwise fashion with the ␤ARK1(ϩ/ Ϫ)/␤ARKct animals having only Ϸ25% of the wild-type myocardial ␤ARK activity. Myocardial extracts from the ␤ARK1(ϩ/Ϫ) animals had 50% of the wild-type activity, which correlates to the 50% decrease in ␤ARK1 protein we have previously described (9). This significant decrease in myocardial ␤ARK1 activity in the double gene-targeted mice could also be demonstrated when expressing the data as fold-stimulation of G ␤␥ over basal rhodopsin phosphorylation activity (ϪG ␤␥ ). In ␤ARK1(Ϯ)/␤ARKct animals, G ␤␥ only stimulated activity by 2.24 Ϯ 0.30-fold (n ϭ 5) compared with 4.48 Ϯ 0.93-fold (n ϭ 6) for the wild-type animals (p Ͻ 0.05). Extracts from ␤ARK1(ϩ/Ϫ) hearts had similar values to wild-type (3.75 Ϯ 0.88-fold, n ϭ 6). Because of the dependence of the ␤ARKct activity on G ␤␥ , as expected, in vitro ␤ARK activity in the absence of G ␤␥ was equivalent between ␤ARK1(ϩ/Ϫ) extracts and ␤ARK1(ϩ/Ϫ)/␤ARKct extracts, which was Ϸ50% of wild-type activity (data not shown).
We have previously reported that overexpression of the FIG. 2. In vivo assessment of LV contractile function in response to ␤-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/dt max , dP/dt min ), and heart rate. Four measured parameters are shown at baseline and after progressive doses of isoproterenol in wild-type (E) n ϭ 26, and ␤ARK1(ϩ/Ϫ) (q) (n ϭ 19), and ␤ARK1(ϩ/Ϫ)/␤ARKct (OE) (n ϭ 9) mice. A, LV dP/dt max ; B, LV dP/dt min ; C, LV systolic pressure; D, heart rate. Data was analyzed with a repeated measures ANOVA and post hoc analysis by Newman-Keuls, *, p Ͻ 0.005 either ␤ARK1-(ϩ/Ϫ) or ␤ARK1(ϩ/Ϫ)/␤ARKct versus wild type; †, p Ͻ0.05 ␤ARK1(ϩ/Ϫ) versus ␤ARK1(ϩ/Ϫ)/␤ARKct. A significant between-group main effect in response to isoproterenol was found for LV dP/dt max (p Ͻ 0.0001) (A), LV dP/dt min (p Ͻ 0.0001) (B), LV systolic pressure (p Ͻ 0.005) (C), and heart rate (p Ͻ 0.005) (D). The pattern of change between groups was statistically different for LV dP/dt max (p Ͻ 0.0001) (A) and heart rate (p Ͻ 0.0001) (D). Body weight was not significantly different between groups: wild type 27.6 Ϯ 1. ␤ARKct in transgenic mice leads to a significant enhancement of myocardial ␤AR signaling and myocardial contractility in vivo under basal conditions and in response to catecholamine infusion (7). To determine whether the level of ␤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/dt max , was not increased in the ␤ARK1(ϩ/Ϫ) at baseline compared to wild type, it was significantly enhanced in the ␤ARK1(ϩ/Ϫ)/␤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 ␤ARK1(ϩ/Ϫ)/ ␤ARKct ( Fig. 2A). Enhanced myocardial relaxation, as assessed by LV dP/dt min was particularly evident in the ␤ARK1(ϩ/Ϫ)/ ␤ARKct animals but not in the ␤ARK1(ϩ/Ϫ) 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).
Heart rate is a powerful determinant of myocardial contractility and can have important influences on LV dP/dt max (22). To illustrate this point, the data has been plotted for the three groups showing the relationship between LV dP/dt max 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/dt max . Taken together these data demonstrate that the level of ␤ARK1 activity exerts tight control over the inotropic and chronotropic response to catecholamine stimulation in the heart in vivo.
Since both loading conditions (LV end-diastolic pressure) and heart rate influence the in vivo measurement of myocardial contractility as measured by LV dP/dt max (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 ␤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, ␤ARK1(ϩ/Ϫ), and ␤ARK1(ϩ/Ϫ)/␤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 Ϫ7 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 (ϪdL/dt) without a change in diastolic cell length ( Fig. 4 and Table I). In contrast, adult myocytes isolated from ␤ARK1(ϩ/Ϫ) heterozygote animals showed an 18% increase in ϪdL/dt, whereas myocytes from ␤ARK1(ϩ/Ϫ)/␤ARKct hearts showed an even greater increase in ϪdL/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 ␤ARK1(ϩ/Ϫ) and ␤ARK1(ϩ/Ϫ)/␤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 ␤ARK1.

DISCUSSION
The present study demonstrates that 1) ␤ARK1(ϩ/Ϫ) mice that are heterozygote for ablation of the ␤ARK1 gene and have a 50% reduction in the level of ␤ARK1 in the heart develop normally, 2) the level of chronotropy and inotropy in vivo can be modulated by the level of ␤ARK1 expression, and 3) contractile function can be further enhanced through in vivo ␤ARK1 inhibition by competing for G ␤␥ binding and endogenous ␤ARK1 translocation and activation.
Complete disruption of the ␤ARK1 gene in mice leads to a lethal phenotype with no ␤ARK1(Ϫ/Ϫ) embryos surviving beyond gestational day 15.5 (9). The finding that mice that are heterozygous for the ␤ARK1 deletion have no developmental abnormalities and develop into normal adults, suggests that there is a threshold level for ␤ARK1 that allows for normal cardiac development. Although the ␤ARK1(ϩ/Ϫ) mice grow into normal adults, the functional consequence of reduced  ␤ARK1 levels is a phenotype of decreased desensitization in the heart as shown by the enhanced contractile response to isoproterenol stimulation (Fig. 2). ␤ARK1 requires a membrane-targeting event prior to receptor phosphorylation that occurs through the interaction of membrane anchored G ␤␥ subunits and the carboxyl terminus of ␤ARK1 (5). Preventing ␤ARK1 translocation by competing for G ␤␥ binding in transgenic mice overexpressing a peptide inhibitor (␤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 (␤ARK1(ϩ/Ϫ) and ␤ARKct overexpression) results in a further enhancement of contractility and relaxation (Fig. 2). These data suggest that both the level of ␤ARK1 expression and the active process of translocation and activation of ␤ARK1, determine the degree of ␤AR desensitization and subsequent receptor-G protein coupling.
The isovolumic phase index (LV dP/dt max ) 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/dt max (22). In the present study we show that the significantly enhanced contractile performance of mice with altered levels of ␤ARK1, as measured by LV dP/dt max , 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 ␤ARK1(ϩ/Ϫ) and ␤ARK1(ϩ/Ϫ)/␤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 ␤AR stimulation in the ␤ARK1(ϩ/Ϫ) cells, which was further enhanced in the ␤ARK1(ϩ/Ϫ)/␤ARKct cells. Although as a group the cells isolated from the ␤ARK1(ϩ/Ϫ)/␤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 (ϩdL/dt). These data show that ␤ARK1 can directly influence contractility at a cellular level and confirm the in vivo data showing that reduced ␤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 ␤ARK1 or the ␤ARKct, which showed that the presence of the ␤ARKct resulted in an enhanced contractile response to ␤AR stimulation compared with control cells (24).
We have previously shown that adenoviral-mediated gene transfer of the ␤ARKct can restore ␤AR signaling in failing myocytes isolated from chronically paced rabbits (25). In that in vitro study we showed that the biochemical defects in ␤AR signaling in isolated failing myocytes could be reversed by gene transfer of the ␤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 ␤ARK1. Furthermore, we not only demonstrate that an inhibitor of ␤ARK1 (␤ARKct) can affect cellular contractility but also that the contractile state of the myocyte can be directly influenced by the level of ␤ARK1 expression.
There is increasing evidence that elevated levels of ␤ARK1 contribute to impaired catecholamine responsiveness observed in disease states of cardiac hypertrophy and heart failure (26). Elevated levels of ␤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 ␤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 ␤ARK1 activity. Furthermore in a mouse model of pressure overload hypertrophy, impaired ␤AR signaling that occurs with the development of modest myocardial hypertrophy, could be completely reversed in the presence of the ␤ARK inhibitor (␤ARKct) (12). In this study, we show that not only is ␤ARK1 a critical modulator of in vivo cardiac function (7), but the level of ␤ARK1 activity is important and can directly influence the degree to which the ␤AR-signaling pathway is activated. This has important implications in disease states of increased ␤ARK activity when considering ␤ARK1 as a therapeutic target, since even partial inhibition of ␤ARK1 function will effectively enhance ␤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 ␤ARK inhibitor in a mouse model of heart failure (19).