Carvedilol: the new role of beta blockers in congestive heart failure

The prognosis remains poor for patients with congestive heart failure (CHF), despite reduced mortality rates resulting from the addition of angiotensin converting enzyme inhibitors to traditional treatment regimens. Because much of the myocardial damage that occurs in patients with CHF may be related to sympathetic activation, interest in the use of beta blockers has grown. Recent studies have shown the benefits of beta blocker therapy in many patients with heart failure. Carvedilol, the first beta blocker labeled in the United States specifically for the treatment of heart failure, has been shown to improve left ventricular ejection fraction and may reduce mortality.     

The effects of endothelin-A receptor blockade during the progression of pacing-induced congestive heart failure

OBJECTIVES: We sought to identify the effects of endothelin (ET) subtype-A (ET(A))) receptor blockade during the development of congestive heart failure (CHF) on left ventricle (LV) function and contractility. BACKGROUND: Congested heart failure causes increased plasma levels of ET and ET(A) receptor activation. METHODS: Yorkshire pigs were assigned to four groups: 1) CHF: 240 beats/min for 3 weeks; n=7; 2) CHF/ET(A)-High Dose: paced for 2 weeks then ET(A) receptor blockade (BMS 193884, 50 mg/kg, b.i.d.) for the last week of pacing; n=6; 3) CHF/ET(A)-Low Dose: pacing for 2 weeks then ET(A) receptor blockade (BMS 193884, 12.5 mg/kg, b.i.d.) for the last week, n=6; and 4) Control: n=8. RESULTS: Left ventricle fractional shortening decreased with CHF compared with control (12+/-1 vs. 39+/-1%, p < 0.05) and increased in the CHF/ET(A) High and Low Dose groups (23+/-3 and 25+/-1%, p < 0.05). The LV peak wall stress and wall force increased approximately twofold with CHF and remained increased with ET(A) receptor blockade. With CHF, systemic vascular resistance increased by 120%, was normalized in the CHF/ET(A) High Dose group, and fell by 43% from CHF values in the Low Dose group (p < 0.05). Plasma catecholamines increased fourfold in the CHF group and were reduced by 48% in both CHF/ET(A) blockade groups. The LV myocyte velocity of shortening was reduced with CHF (32+/-3 vs. 54+/-3 microm/s, p < 0.05), was higher in the CHF/ET(A) High Dose group (39+/-1 microm/s, p < 0.05), and was similar to CHF values in the Low Dose group. CONCLUSIONS: ET(A) receptor activation may contribute to the progression of LV dysfunction with CHF.     

Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.     

Role of clathrin-mediated endocytosis in agonist-induced down-regulation of the beta2-adrenergic receptor

Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.     

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.     

Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.     

Denopamine, a beta1-adrenergic agonist, prolongs survival in a murine model of congestive heart failure induced by viral myocarditis: suppression of tumor necrosis factor-alpha production in the heart

OBJECTIVES: This study was designed to examine the effects of denopamine, a selective beta1-adrenergic agonist, in a murine model of congestive heart failure (CHF) due to viral myocarditis. BACKGROUND: Positive inotropic agents are used to treat severe heart failure due to myocarditis. However, sympathomimetic agents have not been found beneficial in animal models of myocarditis. METHODS: In vitro: The effects of denopamine on lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) production was studied in murine spleen cells. In vivo: Four-week-old DBA/2 mice were inoculated with the encephalomyocarditis virus (day 0). Denopamine (14 micromol/kg), denopamine (14 micromol/kg) with a selective beta1-blocker metoprolol (42 micromol/kg), or denopamine (14 micromol/kg) with metoprolol (84 micromol/kg) was given daily, and control mice received the vehicle only. Survival and myocardial histology on day 14 and TNF-alpha levels in the heart on day 6 were examined. RESULTS: In the in vitro study, TNF-alpha levels in treated cells were significantly lower than in controls (p < 0.05). In the in vivo study treatment with denopamine significantly improved the survival of the animals (14 of 25 (56%) treated, vs 5 of 25 (20%) control mice), attenuated myocardial lesions, and suppressed TNF-alpha production (66.5+/-7.5 pg/mg of heart in treated mice vs 113.5+/-15.1 pg/mg of heart in control mice, mean+/-SE). There was a strong linear relationship between mortality and TNF-alpha levels (r=0.98, n=4, p < 0.05). These in vitro and in vivo effects of denopamine were significantly inhibited by metoprolol. CONCLUSIONS: These results suggest that denopamine may exert its beneficial effects, in part, by suppressing the production of TNF-alpha via beta1-adrenoceptors.     

Meta-analysis of the association of Trp64Arg polymorphism of beta 3-adrenergic receptor gene with body mass index

A possible pathogenic polymorphism in the beta 3-adrenergic receptor gene (Trp64Arg) has been reported to be associated with increased body weight, clinical features of insulin resistance, and early development of type 2 diabetes mellitus in several populations. However, such findings have not been consistent among studies, making the hypothesis that this genetic marker is associated with clinical features controversial. To assess the effect of the genotypes on body mass index (BMI), we performed a meta-analysis of the data from the literature using an extension of ANOVA for continuous measures. In a total of 48 subgroups containing subjects with (n = 2447) and without (n = 6789) the Trp64Arg variant, the summary weighted mean difference in BMI was 0.30 (95% confidence interval, 0.13-0.47) kg/m2, indicating that variant carriers exhibited higher BMI (on the average, 0.30 kg/m2 higher) than normal homozygous subjects. In this case, there was no significant evidence against homogeneity of the effect (P = 0.36). This is the first meta-analysis assessing quantitative phenotypes in relation to a genetic polymorphism, and the results support the hypothesis that the Trp64Arg polymorphism is associated with BMI across diverse population groups, suggesting that the beta 3-adrenergic receptor gene locus plays a role in genetic predisposition to increased body weight in a universal manner.     

Learning needs of congestive heart failure patients

Congestive heart failure (CHF) affects approximately one-half million Canadians and five million Americans. Patient education is a vital component of nursing care of this population, with the goals of improving the CHF patient's quality of life, minimizing symptoms and hospital admissions, and reducing length of hospital stay. A review of the literature related to the educational needs of CHF patients reveals minimal research. The purpose of this study was to compare the perceived learning needs of CHF patients by patients and nurses, and to identify existing gaps between their perceptions. Fifty (50) CHF patients and 47 cardiac nurses were surveyed using a modified version of the CHF Patient Learning Needs Inventory developed by Hagenhoff et al. This instrument measured the importance of specific learning topics within the categories of anatomy and physiology, medications, diet, risk factors, activity, psychological factors, and other pertinent information. The results indicated that both groups found most information "moderately" to "very" important to learn. The patients generally rated all information items higher than nurses did. The most significant finding was that the nurses rated the diet category as second in importance, while the patients rated it last. The results from the study will be incorporated into a needs-based educational program for CHF patients.     

Functional analysis of myocardial performance in murine hearts overexpressing the human beta 2-adrenergic receptor

Transgenic mice overexpressing the human beta 2-adrenergic receptor gene were compared with wild mice type in terms of cardiac function, using a modified work-performing isolated murine heart preparation and on-line computer analysis. A preload-dependent experiment was performed, in which venous return was gradually increased in 5 mmHg increments from 5 mmHg to 25 mmHg. At each preload, aortic flow, left atrial pressure and aortic pressure were measured in all hearts, and from these parameters stroke volume, contractility, and cardiac index (cardiac output divided by body weight in g) were calculated and compared between groups. At increasing preload levels, the heart rates ranged from 322 beats/min (+/-29) to 369 beats/min (+/-39) in control mice and from 469 beats/min (+/-36) to 540 beats/min (+/-39) in transgenic mice. Cardiac index increased from 138 microliters/min/g (+/-13) and 48 microliters/min/g (+/-5) for transgenic and control mice, respectively at 5 mmHg preload to 262 microliters/min/g (+/-51) and 167 microliters/min/g (+/-15), respectively at 20 mmHg preload. The contractility in the transgenic mice were significantly increased at lower preload levels compared to control mice (1420 mmHg/s +/- 204 v 1187 mmHg/s +/- 127). An increase in myocardial adrenergic receptor density (100-200 fold) leads to significantly higher indices of cardiac function in transgenic mice compared to control mice. The increased heart rate leading to a positive inotropic effect in the hearts of transgenic mice is, at least in part, due to the overexpression of adrenergic receptors. These findings suggest a possible alternative method of establishing a positive chronotropic and inotropic state without the use of pharmacological agents.     

Palmitoylated cysteine 341 modulates phosphorylation of the beta2-adrenergic receptor by the cAMP-dependent protein kinase

We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human beta2-adrenergic receptor (Gly341beta2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with Gs. In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341beta2AR) restored normal phosphorylation and receptor-Gs coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified beta2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346beta2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341beta2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoylation restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the beta2AR and by doing so modulates the responsiveness of the receptor.     

The current role of amiodarone in patients with congestive heart failure

Amiodarone in low to moderate doses is generally safe in controlling arrhythmias in patients with congestive heart failure (CHF). However, its role is uncertain, because it did not affect the overall mortality rate in three out of four large-scale studies. Whether some subgroups might benefit is a matter of speculation. In patients with sustained ventricular tachycardia, or in those who have survived an episode of sudden death, implantation of a cardioverter-defibrillator may be a better strategy.     

Ace inhibitors: treatment of congestive heart failure

Nasal passage geometry was measured by acoustic rhinometry in 8 healthy medical students (5 males and 3 females, 21-29 years old; mean age 24 years) after 6 min in different postures of head and body. The minimum cross-sectional area (A-min) and volume between the nostril and 7 cm posteriorly were measured on both sides. When changing from sitting to horizontal the total airway dimension (i.e., the sum of A-min for the two sides) decreased by about 16% (Mean +/- SD = 0.19 +/- 0.14 cm2), and when standing up it increased by about 12% (0.14 +/- 0.13 cm2). A-min seemed more sensitive than volume to detecting postural changes. Including the variation between the cavities, the coefficient of variation (CV = SD/Mean) for area was 24.8 +/- 6.7 and for volume 22.4 +/- 6.4 for the 8 subjects. For the total nasal airway passage the corresponding figures were 12.9 +/- 3.9 and 10.9 +/- 5.5. These figures are considerably higher than for subjects measured only in the sitting position under comparable circumstances. In conclusion, our findings indicate a composite response of the nasal cavity mucosa to both systemic (hydrostatic) and local conditions, probably induced by vascular and cutaneous reflexes. These factors must be taken into account in studies of environmental, clinical, and pharmacological conditions.