Browsing by Author "Rockman, HA"
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Item Open Access Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.(Circulation, 2001-11-13) Kohout, TA; Takaoka, H; McDonald, PH; Perry, SJ; Mao, L; Lefkowitz, RJ; Rockman, HABACKGROUND: Stimulation of beta(1)- and beta(2)-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the beta(3)AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. METHODS AND RESULTS: To better understand the role of beta(3)ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human beta(3)AR (TGbeta(3) mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous beta(1)- and beta(2)ARs, isoproterenol resulted in an increase in contractility in the TGbeta(3) mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human beta(3)AR agonist L-755,507 significantly increased contractility in the TGbeta(3) mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGbeta(3) mice was investigated in terms of beta(3)AR-G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGbeta(3) mice with L-755,507 resulted in a pertussis toxin-insensitive 1.33-fold increase in [(35)S]GTPgammaS loading and a 1.6-fold increase in adenylyl cyclase activity. CONCLUSIONS: Cardiac overexpression of human beta(3)ARs results in positive inotropy only on stimulation with a beta(3)AR agonist. Overexpressed beta(3)ARs couple to G(s) and activate adenylyl cyclase on agonist stimulation.Item Open Access 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, 2001-05-08) Harding, VB; Jones, LR; Lefkowitz, RJ; Koch, WJ; Rockman, HAChronic human heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased levels of betaAR kinase 1 (betaARK1), which seems critical to the pathogenesis of the disease. To determine whether inhibition of betaARK1 is sufficient to rescue a model of severe heart failure, we mated transgenic mice overexpressing a peptide inhibitor of betaARK1 (betaARKct) with transgenic mice overexpressing the sarcoplasmic reticulum Ca(2+)-binding protein, calsequestrin (CSQ). CSQ mice have a severe cardiomyopathy and markedly shortened survival (9 +/- 1 weeks). In contrast, CSQ/betaARKct mice exhibited a significant increase in mean survival age (15 +/- 1 weeks; P < 0.0001) and showed less cardiac dilation, and cardiac function was significantly improved (CSQ vs. CSQ/betaARKct, left ventricular end diastolic dimension 5.60 +/- 0.17 mm vs. 4.19 +/- 0.09 mm, P < 0.005; % fractional shortening, 15 +/- 2 vs. 36 +/- 2, P < 0.005). The enhancement of the survival rate in CSQ/betaARKct mice was substantially potentiated by chronic treatment with the betaAR antagonist metoprolol (CSQ/betaARKct nontreated vs. CSQ/betaARKct metoprolol treated, 15 +/- 1 weeks vs. 25 +/- 2 weeks, P < 0.0001). Thus, overexpression of the betaARKct resulted in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with beta-blocker therapy. These data demonstrate a significant synergy between an established heart-failure treatment and the strategy of betaARK1 inhibition.Item Open Access Catecholamines, cardiac beta-adrenergic receptors, and heart failure.(Circulation, 2000-04-11) Lefkowitz, RJ; Rockman, HA; Koch, WJItem Open Access Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.(J Clin Invest, 1996-04-01) Rockman, HA; Hamilton, RA; Jones, LR; Milano, CA; Mao, L; Lefkowitz, RJTo assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.Item Open Access 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, 1998-06-09) Rockman, HA; Chien, KR; Choi, DJ; Iaccarino, G; Hunter, JJ; Ross, J; Lefkowitz, RJ; Koch, WJHeart failure is accompanied by severely impaired beta-adrenergic receptor (betaAR) function, which includes loss of betaAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of betaAR function is agonist-stimulated receptor phosphorylation by the betaAR kinase (betaARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in betaAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of betaARK1 or the beta2AR 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-/-/beta2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP-/-/betaARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP-/-/betaARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP-/- mice but less than controls. Importantly, heightened betaAR 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 betaARK1 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 betaAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit betaARK1 as a novel mode of therapy.Item Open Access In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.(Circulation, 1999-08-10) Akhter, SA; Eckhart, AD; Rockman, HA; Shotwell, K; Lefkowitz, RJ; Koch, WJBACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.Item Open Access Ligand-induced overexpression of a constitutively active beta2-adrenergic receptor: pharmacological creation of a phenotype in transgenic mice.(Proc Natl Acad Sci U S A, 1997-01-07) Samama, P; Bond, RA; Rockman, HA; Milano, CA; Lefkowitz, RJTransgenic overexpression (40- to 100-fold) of the wild-type human beta2-adrenergic receptor in the hearts of mice leads to a marked increase in cardiac contractility, which is apparently due to the low level of spontaneous (i.e., agonist-independent) activity inherent in the receptor. Here we report that transgenic mice expressing a mutated constitutively active form of the receptor (CAM) show no such phenotype, owing to its modest expression (3-fold above endogenous cardiac beta-adrenergic receptor levels). Surprisingly, treatment of the animals with a variety of beta-adrenergic receptor ligands leads to a 50-fold increase in CAM beta2-adrenergic receptor expression, by stabilizing the CAM beta2-adrenergic receptor protein. Receptor up-regulation leads in turn to marked increases in adenylate cyclase activity, atrial tension determined in vitro, and indices of cardiac contractility determined in vivo. These results illustrate a novel mechanism for regulating physiological responses, i.e., ligand-induced stabilization of a constitutively active but inherently unstable protein.Item Open Access Myocardial expression of a constitutively active alpha 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy.(Proc Natl Acad Sci U S A, 1994-10-11) Milano, CA; Dolber, PC; Rockman, HA; Bond, RA; Venable, ME; Allen, LF; Lefkowitz, RJTransgenic mice were generated by using the alpha-myosin heavy chain promoter coupled to the coding sequence of a constitutively active mutant alpha 1B-adrenergic receptor (AR). These transgenic animals demonstrated cardiac-specific expression of this alpha 1-AR with resultant activation of phospholipase C as shown by increased myocardial diacylglycerol content. A phenotype consistent with cardiac hypertrophy developed in adult transgenic mice with increased heart/body weight ratios, myocyte cross-sectional areas, and ventricular atrial natriuretic factor mRNA levels relative to nontransgenic controls. These transgenic animals may provide insight into the biochemical triggers that induce hypertrophy in cardiac disease and serve as a convenient experimental model for studies of this condition.Item Open Access Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.(Proc Natl Acad Sci U S A, 1996-09-03) Rockman, HA; Choi, DJ; Rahman, NU; Akhter, SA; Lefkowitz, RJ; Koch, WJTransgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.