Heart failure is accompanied by severely impaired ␤-adrenergic receptor (␤AR) function, which includes loss of ␤AR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of ␤AR function is agonist-stimulated receptor phosphorylation by the ␤AR kinase (␤ARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in ␤AR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of ␤ARK1 or the ␤ 2 AR were mated into a genetic model of murine heart failure (MLP ؊͞؊ ). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP ؊͞؊ and MLP ؊͞؊ ͞␤ 2 AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP ؊͞؊ ͞␤ARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP ؊͞؊ ͞␤ARKct mice, as measured by LV dP͞dtmax, was increased significantly compared with the MLP ؊͞؊ mice but less than controls. Importantly, heightened ␤AR desensitization in the MLP ؊͞؊ mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the ␤ARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal ␤AR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit ␤ARK1 as a novel mode of therapy.One of the most important mechanisms for rapidly regulating ␤-adrenergic receptor (␤AR) function is agonist-stimulated receptor phosphorylation by G protein-coupled receptor kinases (GRKs) resulting in decreased sensitivity to further catecholamine stimulation (1, 2). ␤ARK1 is a member of the multigene GRK family that regulates a wide variety of receptors that couple to heterotrimeric G proteins (1, 2). Desensitization of agonistoccupied receptors by the cytosolic ␤AR kinase (␤ARK1) requires a membrane-targeting event before its activation and receptor phosphorylation, which is mediated by a direct physical interaction between residues within the carboxyl terminus of ␤ARK1 and the dissociated membrane-anchored ␤␥ subunits of G proteins (G␤␥) (3, 4).Heart failure is a disease characterized by left ventricular (LV) dysfunction associated with a complex of symptoms that relate to inadequate perfusion of tissues and pulmonary congestion. Although the fundamental molecular abnormality that causes this progressive deterioration in cardiac function is unknown, one of the leading candidates is abnormal ␤AR signaling. Chronic human heart failure is characterized by severely attenuated ␤AR signaling, resulting from diminished receptor number and impaired receptor function (5...

Pressure overload cardiac hypertrophy in the mouse was achieved following 7 days of transverse aortic constriction. This was associated with marked ␤-adrenergic receptor (␤-AR) desensitization in vivo, as determined by a blunted inotropic response to dobutamine. Extracts from hypertrophied hearts had Ϸ3-fold increase in cytosolic and membrane G protein-coupled receptor kinase (GRK) activity. Incubation with specific monoclonal antibodies to inhibit different GRK subtypes showed that the increase in activity could be attributed predominately to the ␤-adrenergic receptor kinase (␤ARK). Although overexpression of a ␤ARK inhibitor in hearts of transgenic mice did not alter the development of cardiac hypertrophy, the ␤-AR desensitization associated with pressure overload hypertrophy was prevented. To determine whether the induction of ␤ARK occurred because of a generalized response to cellular hypertrophy, ␤ARK activity was measured in transgenic mice homozygous for oncogenic ras overexpression in the heart. Despite marked cardiac hypertrophy, no difference in ␤ARK activity was found in these mice overexpressing oncogenic ras compared with controls. Taken together, these data suggest that ␤ARK is a central molecule involved in alterations of ␤-AR signaling in pressure overload hypertrophy. The mechanism for the increase in ␤ARK activity appears not to be related to the induction of cellular hypertrophy but to possibly be related to neurohumoral activation.The regulation of myocardial ␤-adrenergic receptors (␤-ARs) 1 involves a process characterized by a rapid loss of receptor responsiveness despite continued presence of agonist. Two classes of kinases regulate receptors through rapid receptor phosphorylation; the second messenger activated protein kinases, such as cAMP-dependent kinase A and protein kinase C (1), and the G protein-coupled receptor kinases (GRK), which phosphorylate only activated receptors leading to a process termed homologous desensitization (2, 3). Of the six known members of the emerging GRK family, GRK2 (commonly known as ␤ARK1) and GRK5 appear to be dominantly expressed in the heart (4, 5). Desensitization of agonist-occupied receptors by the primarily cytosolic ␤ARK1 requires a membrane-targeting event prior to receptor phosphorylation by a direct physical interaction between residues within the carboxyl terminus of ␤ARK and the dissociated, membrane-anchored ␤␥ subunits of G proteins (6, 7). Unlike ␤ARK1, GRK5 does not undergo agonist-dependent translocation from cytosol to membrane but rather is constitutively membrane-bound (5).Decreased responsiveness to ␤-AR agonists is a characteristic of chronic heart failure. In heart failure, ␤-AR desensitization is due to both diminished receptor number (receptor down-regulation) and impaired receptor function (receptor uncoupling) (8), in part related to enhanced ␤ARK activity (9, 10). Recent data suggest a step wise increase in plasma norepinephrine levels in individuals from normal cardiac function to asymptomatic left ventricular (LV) dysfunction...

We studied the effect of alterations in the level of myocardial ␤-adrenergic receptor kinase (␤ARK1) in two types of genetically altered mice. The first group is heterozygous for ␤ARK1 gene ablation, ␤ARK1(؉/؊), and the second is not only heterozygous for ␤ARK1 gene ablation but is also transgenic for cardiac-specific overexpression of a ␤ARK1 COOH-terminal inhibitor peptide, ␤ARK1(؉/؊)/␤ARKct. In contrast to the embryonic lethal phenotype of the homozygous ␤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), ␤ARK1(؉/؊) mice develop normally. Cardiac catheterization was performed in mice and showed a stepwise increase in contractile function in the ␤ARK1(؉/؊) and ␤ARK1(؉/؊)/␤ARKct mice with the greatest level observed in the ␤ARK1(؉/؊)/␤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 ␤ARK1(؉/؊) and ␤ARK1(؉/؊)/␤ARKct myocytes compared with wildtype cells, indicating a progressive increase in intrinsic contractility. These data demonstrate that contractile function can be modulated by the level of ␤ARK1 activity. This has important implications in disease states such as heart failure (in which ␤ARK1 activity is increased) and suggests that ␤ARK1 should be considered as a therapeutic target in this situation. Even partial inhibition of ␤ARK1 activity enhances ␤-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 kinases (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 end...

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