Ligand-induced overexpression of a constitutively active beta2-adrenergic receptor: pharmacological creation of a phenotype in transgenic mice.

Transgenic 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.

Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function.

The beta-adrenergic receptor kinase 1 (beta ARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the beta ARK1 gene in mice by homologous recombination. No homozygote beta ARK1-/- embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, beta ARK1-/- embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the "thin myocardium syndrome" observed upon gene inactivation of several transcription factors (RXR alpha, N-myc, TEF-1, WT-1). Lethality in beta ARK1-/- embryos is likely due to heart failure as they exhibit a > 70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in beta ARK1-/- embryos demonstrate that beta ARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Myocardial expression of a constitutively active alpha 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy.

Transgenic 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.

A functional polymorphism in the 5HTR2C gene associated with stress responses also predicts incident cardiovascular events.

Previously we have shown that a functional nonsynonymous single nucleotide polymorphism (rs6318) of the 5HTR2C gene located on the X-chromosome is associated with hypothalamic-pituitary-adrenal axis response to a stress recall task, and with endophenotypes associated with cardiovascular disease (CVD). These findings suggest that individuals carrying the rs6318 Ser23 C allele will be at higher risk for CVD compared to Cys23 G allele carriers. The present study examined allelic variation in rs6318 as a predictor of coronary artery disease (CAD) severity and a composite endpoint of all-cause mortality or myocardial infarction (MI) among Caucasian participants consecutively recruited through the cardiac catheterization laboratory at Duke University Hospital (Durham, NC) as part of the CATHGEN biorepository. Study population consisted of 6,126 Caucasian participants (4,036 [65.9%] males and 2,090 [34.1%] females). A total of 1,769 events occurred (1,544 deaths and 225 MIs; median follow-up time = 5.3 years, interquartile range = 3.3-8.2). Unadjusted Cox time-to-event regression models showed, compared to Cys23 G carriers, males hemizygous for Ser23 C and females homozygous for Ser23C were at increased risk for the composite endpoint of all-cause death or MI: Hazard Ratio (HR) = 1.47, 95% confidence interval (CI) = 1.17, 1.84, p = .0008. Adjusting for age, rs6318 genotype was not related to body mass index, diabetes, hypertension, dyslipidemia, smoking history, number of diseased coronary arteries, or left ventricular ejection fraction in either males or females. After adjustment for these covariates the estimate for the two Ser23 C groups was modestly attenuated, but remained statistically significant: HR = 1.38, 95% CI = 1.10, 1.73, p = .005. These findings suggest that this functional polymorphism of the 5HTR2C gene is associated with increased risk for CVD mortality and morbidity, but this association is apparently not explained by the association of rs6318 with traditional risk factors or conventional markers of atherosclerotic disease.

Intracardiac acoustic radiation force impulse (ARFI) and shear wave imaging in pigs with focal infarctions.

Four pigs, three with focal infarctions in the apical intraventricular septum (IVS) and/or left ventricular free wall (LVFW), were imaged with an intracardiac echocardiography (ICE) transducer. Custom beam sequences were used to excite the myocardium with focused acoustic radiation force (ARF) impulses and image the subsequent tissue response. Tissue displacement in response to the ARF excitation was calculated with a phase-based estimator, and transverse wave magnitude and velocity were each estimated at every depth. The excitation sequence was repeated rapidly, either in the same location to generate 40 Hz M-modes at a single steering angle, or with a modulated steering angle to synthesize 2-D displacement magnitude and shear wave velocity images at 17 points in the cardiac cycle. Both types of images were acquired from various views in the right and left ventricles, in and out of infarcted regions. In all animals, acoustic radiation force impulse (ARFI) and shear wave elasticity imaging (SWEI) estimates indicated diastolic relaxation and systolic contraction in noninfarcted tissues. The M-mode sequences showed high beat-to-beat spatio-temporal repeatability of the measurements for each imaging plane. In views of noninfarcted tissue in the diseased animals, no significant elastic remodeling was indicated when compared with the control. Where available, views of infarcted tissue were compared with similar views from the control animal. In views of the LVFW, the infarcted tissue presented as stiff and non-contractile compared with the control. In a view of the IVS, no significant difference was seen between infarcted and healthy tissue, whereas in another view, a heterogeneous infarction was seen to be presenting itself as non-contractile in systole.

Diminishment of respiratory sinus arrhythmia foreshadows doxorubicin-induced cardiomyopathy.

BACKGROUND: The development of a microcomputer-based device permits quick, simple, and noninvasive quantification of the respiratory sinus arrhythmia (RSA) during quiet breathing. METHODS AND RESULTS: We prospectively and serially measured the radionuclide left ventricular ejection fraction and the RSA amplitude in 34 cancer patients receiving up to nine monthly bolus treatments with doxorubicin hydrochloride (60 mg/m2). Of the eight patients who ultimately developed symptomatic doxorubicin-induced congestive heart failure, seven (87.5%) demonstrated a significant decline in RSA amplitude; five of 26 subjects without clinical symptoms of cardiotoxicity (19.2%) showed a similar RSA amplitude decline. On average, significant RSA amplitude decline occurred 3 months before the last planned doxorubicin dose in patients destined to develop clinical congestive heart failure. CONCLUSION: Overall, RSA amplitude abnormality proved to be a more specific predictor of clinically significant congestive heart failure than did serial resting radionuclide ejection fractions.