Dexamethasone potentiates high-affinity beta-agonist binding and g(s)alpha protein expression in airway smooth muscle

Corticosteroids enhance beta-adrenergic responses by actions at both beta-adrenoceptor (beta-AR) and post-beta-AR sites. The present study investigated the effects of dexamethasone on beta-AR density, high-affinity beta-agonist binding, G(s)alpha and G(i)alpha protein expression, and cAMP responses in bovine tracheal smooth muscle (bTSM). Dexamethasone treatment of cultured bTSM cells increased total beta-AR density 1.6- to 1.9-fold as assessed by the saturation binding of [(3)H]CGP-12177 and by displacement of radioligand binding with isoproterenol. Isoproterenol bound to the beta-AR at two sites, a high-affinity site with a density of 5.9 +/- 1.2 fmol/mg protein and a low-affinity site with a density of 16.9 +/- 1. 0 fmol/mg protein. Dexamethasone increased both high- and low-affinity isoproterenol binding sites to 11.1 +/- 2.2 and 25.9 +/- 2.1 fmol/mg protein, respectively, without influencing agonist binding affinities. Dexamethasone also selectively increased G(s)alpha protein levels from 0.99 +/- 0.14 to 1.46 +/- 0.17 microg/mg protein without affecting G(i)alpha levels. The net effect of these changes was a 1.8-fold increase in maximal isoproterenol-induced cAMP generation in dexamethasone-treated bTSM cells. These findings provide new insights into the corticosteroid regulation of beta-adrenergic signaling pathways in airway smooth muscle.     

Effects of forskolin on inotropic performance and phospholamban phosphorylation in exercise-trained hypertensive myocardium.

Beta-adrenergic receptor (beta-AR) responsiveness is downregulated in left ventricular (LV) hypertrophy induced by chronic hypertension. While exercise training in hypertension enhances beta-AR responsiveness, the role of adenylyl cyclase remains unclear. The purpose of the present study was to test whether treadmill running in the spontaneously hypertensive rat (SHR) model improves LV responsiveness to forskolin (FOR) or the combination of FOR + isoproterenol (FOR+ISO). Female SHR (16-wk) were randomly placed into sedentary (SHR-SED; n = 7) or treadmill-trained (SHR-TRD; n = 8) groups. Wistar-Kyoto (WKY; n = 7) animals acted as normotensive controls. Langendorff, isovolumic LV performance was established at baseline and during incremental FOR infusion (1 and 5 micromol/l) and FOR+ISO (5 micromol/l + 1x10(-8) mol/l). Heart rate, systolic blood pressure, and heart-to-body weight ratio were lower in WKY relative to both SHR groups (P < 0.05). LV performance and heart rate significantly increased in all groups to a similar extent with incremental FOR infusion. However, in the presence of 5 micromol/l FOR, ISO increased LV developed pressure, positive change in LV pressure, and negative change in LV pressure to a greater extent in SHR-TRD relative to SHR-SED (P < 0.05). Phospholamban phosphorylation at the Thr17 was greater in SHR-TRD relative to SHR-SED and WKY (P < 0.05). Absolute LV developed pressure was moderately correlated with phospholamban phosphorylation at both the Ser16 (r = 0.64; P < 0.05) and Thr17 (r = 0.52; P < 0.05). Our data suggest that the adenylyl cyclase step in the beta-AR cascade is not downregulated in the early course of hypertension and that the enhanced beta-AR responsiveness with training is likely mediated at levels other than adenylyl cyclase. Our data also suggest that beta-AR inotropic responsiveness in the presence of direct adenylyl cyclase agonism is improved in trained compared with sedentary SHR hearts.     

Enhanced isoproterenol-induced cardiac hypertrophy in transgenic rats with low brain angiotensinogen

We have previously shown that a permanent deficiency in the brain renin-angiotensin system (RAS) may increase the sensitivity of the baroreflex control of heart rate. In this study we aimed at studying the involvement of the brain RAS in the cardiac reactivity to the beta-adrenoceptor (beta-AR) agonist isoproterenol (Iso). Transgenic rats with low brain angiotensinogen (TGR) were used. In isolated hearts, Iso induced a significantly greater increase in left ventricular (LV) pressure and maximal contraction (+dP/dt(max)) in the TGR than in the Sprague-Dawley (SD) rats. LV hypertrophy induced by Iso treatment was significantly higher in TGR than in SD rats (in g LV wt/100 g body wt, 0.28 +/- 0.004 vs. 0.24 +/- 0.004, respectively). The greater LV hypertrophy in TGR rats was associated with more pronounced downregulation of beta-AR and upregulation of LV beta-AR kinase-1 mRNA levels compared with those in SD rats. The decrease in the heart rate (HR) induced by the beta-AR antagonist metoprolol in conscious rats was significantly attenuated in TGR compared with SD rats (-9.9 +/- 1.7% vs. -18.1 +/- 1.5%), whereas the effect of parasympathetic blockade by atropine on HR was similar in both strains. These results indicate that TGR are more sensitive to beta-AR agonist-induced cardiac inotropic response and hypertrophy, possibly due to chronically low sympathetic outflow directed to the heart.     

Diabetes reduces right atrial beta-adrenergic signaling but not agonist stimulation of heart rate in swine

This study assessed the effects of streptozotocin diabetes in swine on the heart rate response to beta-adrenergic stimulation the adenylyl cyclase signal transduction pathway. Diabetic animals (n = 9) were hyperglycemic compared to the control group (n = 10) (12.6 +/- 1.0 vs. 3.53 +/- 0.29 mM). There were no significant differences between the diabetic and nondiabetic groups in the heart rate response to isoproterenol, however, there was a significant reduction (14%) in beta-adrenergic receptor density in the right atrium in the diabetic (61 +/- 3 fmol/mg protein) versus the nondiabetic group (71 +/- 3) (P < 0.05). The content of guanosine triphosphate binding regulatory proteins (Gs and Gi) in the right atrium was not affected by diabetes, nor was adenylyl cyclase activity under unstimulated conditions or with receptor-dependent stimulation with isoproterenol. On the other hand, adenylyl cyclase activity was 34% lower when directly stimulated with forskolin, and it was reduced by 23% when stimulated through Gs with Gpp(NH)p. In conclusion, beta-adrenergic stimulation of heart rate with isoproteronol and the receptor-dependent signal transduction pathway remained intact in the right atrium of diabetic swine despite reduced beta-adrenergic receptor density, G-protein content, and direct stimulation of adenylyl cyclase activity.     

Naturally occurring variation in cardiovascular traits among inbred mouse strains

The objective of this study was to characterize genetic variation in complex cardiovascular traits in two commonly used inbred mouse strains. We performed echocardiography, graded treadmill exercise, tail cuff plethysmography, and telemetry (heart rate, activity, temperature) in age- ( approximately 9 weeks) and sex-matched A/J and C57BL/6J (B6) inbred mice. B6 mice had significantly larger end-diastolic dimension (3.31+/-0.42 mm versus 2.83+/-0.31 mm) and left ventricle mass (46.2+/-14.1 versus 32.7+/-11.5 g) than A/J mice. This relative hypertrophy was eccentric (relative wall thickness ratios: 0.30+/-0.01 versus 0.32+/-0.01) and was not associated with a difference in systolic blood pressure (122.0+/-13.2 versus 123.1+/-20.8 mmHg). Left ventricle fractional shortening (39.1+/-6.2 versus 47.1+/-6.9%) and heart rate (433+/-55 versus 524+/-45 beats per minute) were significantly lower in B6 versus A/J, respectively, resulting in similar resting echocardiographic cardiac indices (0.58+/-0.19 versus 0.50+/-0.17 ml/min/g). Maximum exercise time on a treadmill was significantly greater in B6 than in A/J mice (9.6+/-3.4 versus 4.4+/-1.9 minutes). Telemetry showed that body temperature was generally greater and heart rate lower in B6 than A/J; the relation with activity was more complex. These data suggest that relative to A/J, B6 mice have a phenotype characteristic of the "athlete's heart," that is, eccentric, physiologic hypertrophy, slower heart rates, and increased exercise endurance. This systematic characterization of functionally related cardiovascular traits in A/J and C57BL/6J mice revealed numerous differences whose genetic bases can be dissected with recombinant inbred, recombinant congenic, and chromosome substitution strains.     

Exercise training and beta-blocker treatment ameliorate age-dependent impairment of beta-adrenergic receptor signaling and enhance cardiac responsiveness to adrenergic stimulation

Cardiac beta-adrenergic receptor (beta-AR) signaling and left ventricular (LV) responses to beta-AR stimulation are impaired with aging. It is shown that exercise and beta-AR blockade have a favorable effect on cardiac and vascular beta-AR signaling in several cardiovascular diseases. In the present study, we examined the effects of these two different strategies on beta-AR dysregulation and LV inotropic reserve in the aging heart. Forty male Wistar-Kyoto aged rats were randomized to sedentary, exercise (12 wk treadmill training), metoprolol (250 mg.kg(-1).day(-1) for 4 wk), and exercise plus metoprolol treatment protocols. Ten male Wistar-Kyoto sedentary young rats were also used as a control group. Old trained, old metoprolol-treated, and old trained plus metoprolol-treated rats showed significantly improved LV maximal and minimal first derivative of the pressure rise responses to beta-AR stimulation (isoproterenol) compared with old untrained animals. We found a significant reduction in cardiac sarcolemmal membrane beta-AR density and adenylyl cyclase activity in old untrained animals compared with young controls. Exercise training and metoprolol, alone or combined, restored cardiac beta-AR density and G-protein-dependent adenylyl cyclase activation in old rats. Although cardiac membrane G-protein-receptor kinase 2 levels were not upregulated in untrained old compared with young control rats, both exercise and metoprolol treatment resulted in a dramatic reduction of G-protein-receptor kinase 2 protein levels, which is a further indication of beta-AR signaling amelioration in the aged heart induced by these treatment modalities. In conclusion, we demonstrate for the first time that exercise and beta-AR blockade can similarly ameliorate beta-AR signaling in the aged heart, leading to improved beta-AR responsiveness and corresponding LV inotropic reserve.     

Altering the receptor-effector ratio by transgenic overexpression of type V adenylyl cyclase: enhanced basal catalytic activity and function without increased cardiomyocyte beta-adrenergic signalling

The limiting element in beta-adrenergic receptor (betaAR)-G(s)-adenylyl cyclase (AC) signal transduction in the cardiomyocyte is not known, but it has been proposed that the level of adenylyl cyclase expression constrains betaAR signaling. To alter the above equilibrium, type V AC was overexpressed in a myocyte-specific manner in the hearts of transgenic mice using the alpha-myosin heavy chain promoter. Expression of type V AC was approximately 75% over endogenous levels as quantitated by [(3)H]forskolin binding. Functional activity of the transgene product was evident in cardiac membrane AC studies, where basal (45 +/- 11 vs 19 +/- 5 pmol min(-)(1) mg(-)(1)) and forskolin+Mn(2+) (695 +/- 104 vs 386 +/- 34 pmol min(-)(1) mg(-)(1)) stimulated activities were increased compared to activities in nontransgenic (NTG) littermates. However, while isoproterenol stimulated activities were higher (74 +/- 12 vs 46 +/- 9.8 pmol min(-)(1) mg(-)(1)), the fold stimulation over basal was not increased in ACV overexpressors compared to NTG (line 14.3 = 2.29 +/- 0.44-fold, line 15.1 = 1.70 +/- 0.1-fold, NTG = 2.62 +/- 0.18-fold). Similarly, in whole cell patch-clamp studies, betaAR-mediated opening of L-type Ca(2+) channels was not found to be enhanced in transgenic ACV myocytes (225 +/- 15 vs 216 +/- 10% of basal currents). Basal and isoproterenol stimulated PKA activities were elevated in the ACV mice compared to NTG, but again the extent of stimulation over basal was not enhanced. Phosphorylated phospholamban was approximately 2-fold greater in myocytes from ACV hearts compared to NTG, indicating that distal elements of the contractile cascade are activated by AC overexpression. ACV mice displayed increased heart rates and fractional shortening as assessed by echocardiography. However, in vivo hemodynamic studies revealed that heart rate and contractility responses to agonist infusion were not enhanced in ACV mice compared to NTG. We conclude that at native stoichiometries, the levels of adenylyl cyclase influence basal activities and cardiac function, but do not constrain betaAR signaling in the cardiomyocyte.     

Altered beta-adrenergic signal transduction in nonfailing hypertrophied myocytes from Dahl salt-sensitive rats

Desensitization of the beta-adrenergic receptor (beta-AR) response is well documented in hypertrophied hearts. We investigated whether beta-AR desensitization is also present at the cellular level in hypertrophied myocardium, as well as the physiological role of inhibitory G (G(i)) proteins and the L-type Ca(2+) channel in mediating beta-AR desensitization. Left ventricular (LV) myocytes were isolated from hypertrophied hearts of hypertensive Dahl salt-sensitive (DS) rats and nonhypertrophied hearts of normotensive salt-resistant (DR) rats. Cells were paced at a rate of 300 beats/min at 37 degrees C, and myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) were simultaneously measured. In response to increasing concentrations of isoproterenol, DR myocytes displayed a dose-dependent augmentation of cell shortening and the [Ca(2+)](i) transient amplitude, whereas hypertrophied DS myocytes had a blunted response of both cell shortening and the [Ca(2+)](i) transient amplitude. Interestingly, inhibition of G(i) proteins did not restore beta-AR desensitization in DS myocytes. The responses to increases in extracellular Ca(2+) and an L-type Ca(2+) channel agonist were also similar in both DS and DR myocytes. Isoproterenol-stimulated adenylyl cyclase activity, however, was blunted in hypertrophied myocytes. We concluded that compensated ventricular hypertrophy results in a blunted contractile response to beta-AR stimulation, which is present at the cellular level and independent of alterations in inhibitory G proteins and the L-type Ca(2+) channel.     

Coupling of beta-adrenergic receptors to cardiac L-type Ca2+ channels: preferential coupling of the beta1 versus beta2 receptor subtype and evidence for PKA-independent activation of the channel.

Beta1- and beta2-adrenergic receptors (beta-ARs) co-exist in mammalian heart, and it is generally accepted that both activate adenylyl cyclase (AC), resulting in increased levels of cAMP and subsequent activation of L-type Ca2+ channels (CaCh). To investigate the contribution of each beta-AR subtype in AC and CaCh coupling, we stably expressed cardiac CaCh alpha1 and beta2 subunits along with either beta1-AR or beta2-AR in CHW fibroblasts. Co-expression of either beta-AR with CaCh subunits conferred responsiveness of AC and CaCh to isoproterenol (ISO), which was not observed in non-transfected cells. ISO-promoted cAMP formation occurred at a lower EC50 through the beta2-AR than through the beta1-AR (0.13 +/- 0.01 vs. 0.6 +/- 0.14 nM). In contrast, activation of CaCh was more efficacious via the beta1-AR than the beta2-AR (EC50 for CaCh activation = 238 +/- 33 vs. 1057 +/- 113 nM). Pre-treatment with pertussis toxin (PTX) had no effect upon the responsiveness of either cAMP formation or CaCh activation through either receptor. We conclude (1) that beta1-ARs exhibit preferential coupling to CaCh activation, versus that observed for the beta2-AR; (2) that this preferential coupling cannot be explained solely by cAMP-dependent processes; and (3) that the relative attenuation of beta2-AR-promoted CaCh activation is not due to receptor coupling to PTX-sensitive G proteins. Thus, it is likely that other subtype-specific, cAMP-independent coupling of the beta-AR to CaCh is present.     

Overexpression of beta2-adrenergic receptors in mouse liver alters the expression of gluconeogenic and glycolytic enzymes

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.     

Transgenic replacement of type V adenylyl cyclase identifies a critical mechanism of beta-adrenergic receptor dysfunction in the G alpha q overexpressing mouse.

Chronic activation of Gq coupled receptors, or overexpression of G alpha q, in cardiomyocytes results in hypertrophy, enhanced expression of fetal genes, decreased basal and beta-adrenergic receptor (beta AR) stimulated adenylyl cyclase (AC) activities, and depressed cardiac contractility in vivo. Among several abnormalities of the beta AR-Gs-AC pathway that occur in G alpha q overexpressing transgenic mice, we have investigated whether the observed approximately 45% decrease in type V AC expression and function compared to non-transgenic (NTG) is the basis of the above phenotype. Transgenic mice were generated that overexpressed by approximately 50% the rat type V AC in the heart using the alpha-myosin heavy chain promoter. These mice were mated with the G alpha q transgenics resulting in animals (ACV/G alpha q) that had restored levels of forskolin stimulated AC activities in cardiac membranes. In addition, basal cardiac AC activities were normalized in the ACV/G alpha q mice (NTG=23+/-4.4, G alpha q=14+/-3.6, ACV/G alpha q=29+/-5.3 pmol/min/mg) as were maximal isoproterenol stimulated activities (59+/-8.9, 34+/-4.6, 52+/-6.7 pmol/min/mg respectively). Cardiac contractility was also improved by ACV replacement, with increased fractional shortening (51+/-2%, 36+/-6%, 46+/-3% respectively). In contrast, hypertrophy and expression of hypertrophy associated fetal genes were not affected. Thus the observed decrease in type V AC that accompanies the development of the cardiac phenotype in the G alpha q model is the dominant mechanism of dysfunctional beta AR signalling and contractility. In contrast, the decrease in type V AC or beta AR signalling to cAMP is not the basis of the hypertrophic response.     

Thermic effect of food and beta-adrenergic thermogenic responsiveness in habitually exercising and sedentary healthy adult humans.

The thermic effect of food (TEF) is an important physiological determinant of total daily energy expenditure (EE) and energy balance. TEF is believed to be mediated in part by sympathetic nervous system activation and consequent beta-adrenergic receptor (beta-AR) stimulation of metabolism. TEF is greater in habitually exercising than in sedentary adults, despite similar postprandial sympathetic nervous system activation. We determined whether augmented TEF in habitually exercising adults is associated with enhanced peripheral thermogenic responsiveness to beta-AR stimulation. In separate experiments in 22 sedentary and 29 habitually exercising adults, we measured the increase in EE (indirect calorimetry, ventilated hood) during beta-AR stimulation (intravenous isoproterenol: 6, 12, and 24 ng x kg fat-free mass(-1) x min(-1)) and EE before and after a liquid meal (40% of resting EE; 53% carbohydrate, 32% fat, 15% protein). The increase in EE during incremental isoproterenol administration was greater (P = 0.01) in habitual exercisers (0.34 +/- 0.03, 0.54 +/- 0.04, 0.81 +/- 0.05 kJ/min; means +/- SE) than in sedentary adults (0.26 +/- 0.03, 0.40 +/- 0.03, 0.64 +/- 0.04 kJ/min). The area under the TEF response curve was also greater (P = 0.04) in habitual exercisers (160 +/- 9 kJ) than in sedentary adults (130 +/- 11 kJ) and was positively related to beta-AR thermogenic responsiveness (r = 0.32, P = 0.02). We conclude that TEF is related to beta-AR thermogenic responsiveness and that the greater TEF in habitual exercisers is attributable in part to their augmented beta-AR thermogenic responsiveness. Our results also suggest that peripheral thermogenic responsiveness to beta-AR stimulation is a physiological determinant of TEF and hence energy balance in healthy adult humans.     

A comparison of the properties of the cardiac beta-adrenergic receptor adenylyl cyclase system in the rat and the marmoset monkey

1. A comparison was made between adrenergic receptor binding properties and catecholamine-stimulated adenylyl cyclase activity in cardiac membrane fractions from the rat and the marmoset monkey. 2. [125I]HEAT and [125I]ICYP were used to determine respectively, the alpha- and beta-adrenergic receptor binding in cardiac membrane fractions. 3. Greatest adrenergic receptor density and degree of specific binding was evident using membranes sedimenting between 6000 and 46,000 g. 4. In rat heart, the ratio of beta- to alpha-adrenergic receptors was 57:43, while for the marmoset this ratio was 92:8. 5. Basal, isoproterenol, sodium fluoride and forskolin-stimulated adenylyl cyclase activities in the rat and marmoset monkey were investigated in several different cardiac membrane fractions. 6. The highest-fold stimulation of adenylyl cyclase activity was present in membranes sedimenting between 0 and 500 g. 7. Adenylyl cyclase activities were higher in the marmoset heart membrane preparations, however the rat heart adenylyl cyclase exhibited greater sensitivity to isoproterenol; ED50 3.8 X 10(-7) M compared with 7.5 X 10(-7) M for the marmoset. 8. Differences between rat and marmoset catecholamine-sensitive adenylyl cyclase activity were apparent when a variety of adrenergic agonists and antagonists were tested. 9. In the marmoset but not the rat, adrenergic antagonists alone stimulated basal adenylyl cyclase activity. 10. Differences in the activation of cardiac adenylyl cyclase by GTP and GMP-PNP were also evident between the rat and the marmoset monkey, particularly with regard to basal and isoproterenol-stimulated activity.     

Functional desensitization to isoproterenol without reducing cAMP production in canine failing cardiocytes

To corroborate alterations in the functional responses to beta-adrenergic receptor (beta-AR) stimulation with changes in beta-AR signaling in failing cardiomyocytes, contractile and L-type Ca(2+) current responses to isoproterenol along with stimulated cAMP generation were compared among cardiomyocytes isolated from canines with tachycardia-induced heart failure or healthy hearts. The magnitude of shortening of failing cardiomyocytes was significantly depressed (by 22 +/- 4.4%) under basal conditions, and the maximal response to isoproterenol was significantly reduced (by 45 +/- 18%). Similar results were obtained when the responses in the rate of contraction and rate of relaxation to isoproterenol were considered. The L-type Ca(2+) current amplitude measured in failing cardiomyocytes under basal conditions was unchanged, but the responses to isoproterenol were significantly reduced compared with healthy cells. Isoproterenol-stimulated cAMP generation was similar in sarcolemmal membranes derived from the homogenates of failing (45 +/- 6.8) and healthy cardiomyocytes (52 +/- 8.5 pmol cAMP. mg protein(-1). min(-1)). However, stimulated cAMP generation was found to be significantly reduced when the membranes were derived from the homogenates of whole tissue (failing: 67 +/- 8.1 vs. healthy: 140 +/- 27.8 pmol cAMP. mg protein(-1). min(-1)). Total beta-AR density was not reduced in membranes derived from either whole tissue or isolated cardiomyocyte homogenates, but the beta(1)/beta(2) ratio was significantly reduced in the former (failing: 45/55 vs. healthy: 72/28) without being altered in the latter (failing: 72/28, healthy: 77/23). We thus conclude that, in tachycardia-induced heart failure, reduction in the functional responses of isolated cardiomyocytes to beta-AR stimulation may be attributed to alterations in the excitation-contraction machinery rather than to limitation of cAMP generation.     

Defective beta-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice with calsequestrin overexpression.

Calsequestrin is a high capacity Ca(2+)-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca(2+)-induced Ca(2+) release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998) J. Clin. Invest. 101, 1385-1393). To investigate whether alterations in intracellular Ca(2+) trigger changes in the beta-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired beta-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total beta-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased beta-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in beta-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.     

Vascular beta-adrenergic receptor system is dysfunctional after myocardial infarction

We identified abnormalities in the vascular beta-adrenergic receptor (beta-AR) signaling pathway in heart failure after myocardial infarction (MI). To examine these abnormalities, we measured beta-AR-mediated hemodynamics, vascular reactivity, and the vascular beta-AR molecular signaling components in rats with heart failure after MI. Six weeks after MI, these rats had an increased left ventricular (LV) end-diastolic pressure, decreased LV systolic pressure, and decreased rate of LV pressure change (dP/dt). LV dP/dt responses to isoproterenol were shifted downward, although the responses for systemic vascular resistance were shifted upward in heart failure rats (P < 0.05). Isoproterenol- and IBMX-induced vasorelaxations were blunted in heart failure rats (P < 0.05) with no change in the forskolin-mediated vasorelaxation. These changes were associated with the following alterations in beta-AR signaling (P < 0.05): decreases in beta-AR density (aorta: 58.7 +/- 6.0 vs. 35.7 +/- 1.9 fmol/mg membrane protein; carotid: 29.6 +/- 5.6 vs. 18.0 +/- 3.9 fmol/mg membrane protein, n = 5), increases in G protein-coupled receptor kinase activity levels (relative phosphorimage counts of 191 +/- 39 vs. 259 +/- 26 in the aorta and 115 +/- 30 vs. 202 +/- 7 in the carotid artery, n = 5), and decreases in cGMP and cAMP in the carotid artery (0.85 +/- 0.10 vs. 0.31 +/- 0.06 pmol/mg protein and 2.3 +/- 0.3 vs. 1.2 +/- 0.1 pmol/mg protein, n = 5) with no change in Galpha(s) or Galpha(i )in the aorta. Thus in heart failure there are abnormalities in the vascular beta-AR system that are similar to those seen in the myocardium. This suggests a common neurohormonal mechanism and raises the possibility that treatment in heart failure focused on the myocardium may also affect the vasculature.     

Hypoxia-induced downregulation of beta-adrenergic receptors in rat heart.

To test the desensitization hypothesis of cardiac beta-adrenergic receptors (beta-AR) in chronic hypoxia, the effect of 1, 3, 7, 15, and 21 days of exposure to hypobaric hypoxia (380 Torr) was evaluated in Wistar rats. Exposure to hypoxia for 1-15 days did not induce any change in right and left ventricular beta-AR density (Bmax) determined with [125I]iodocyanopindolol or in antagonist affinity. After 21 days, Bmax decreased by 24% in the left ventricle. In contrast, no change in beta-AR was shown in the right hypertrophied ventricle. Agonist affinity in the left ventricle was not altered, as shown by the analysis of displacement curves of isoproterenol (normoxia 185 +/- 26 nM, hypoxia 170 +/- 11 nM). Moreover, there was no significant decrease in adenylate cyclase activity (pmol.mg-1.min-1) in the left ventricle. In the right ventricle, a 21-day exposure to hypoxia led to a decrease in basal and maximal activity when stimulated by isoproterenol. A decrease in tissue norepinephrine content was observed after 7 days of hypoxia. In conclusion, these data support the beta-AR downregulation hypothesis as one of the mechanisms of myocardial adaptation to high altitude occurring after 2-3 wk of exposure to hypoxia. The regulation pathways of beta-AR may differ between left nonhypertrophied and right hypertrophied ventricles. No evidence of profound abnormality of signal transduction was shown.     

Targeted disruption of the beta2 adrenergic receptor gene.

beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism.     

Modulation of {beta}-adrenoceptor signaling in the hearts of 4-wk simulated weightlessness rats.

The modulation of beta-adrenoceptor signaling in the hearts of hindlimb unweighting (HU) simulated weightlessness rats has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness; then the effects of simulated microgravity on beta-adrenoceptor signaling were studied. Mean arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (-dP/dtmax) were monitored in the course of the in vivo experiment. Single rat ventricular myocyte was obtained by the enzymatic dissociation method. Hemodynamics, myocyte contraction, and cAMP production in response to beta-adrenoceptor stimulation with isoproterenol or adenylyl cyclase stimulation with forskolin were measured, and Gs protein was also determined. Compared with the control group, no significant changes were found in heart weight, body weight and ABP, while LVP and +/-dP/dtmax were significantly reduced. The ABP decrease, LVP increase, and +/-dP/dtmax in response to isoproterenol administration were significantly attenuated in the HU group. The effects of isoproterenol on electrically induced single-cell contraction and cAMP production in myocytes of ventricles in the HU rats were significantly attenuated. The biologically active isoform, Gsalpha (45 kDa) in the heart, was unchanged. Both the increased electrically induced contraction and cAMP production in response to forskolin were also significantly attenuated in the simulated weightlessness rats. Above results indicated that impaired function of adenylyl cyclase causes beta-adrenoceptor desensitization, which may be partly responsible for the depression of cardiac function.     

Effects of exercise training on acclimatization to hypoxia: systemic O2 transport during maximal exercise.

Acclimatization to hypoxia has minimal effect on maximal O2 uptake (Vo2 max). Prolonged hypoxia shows reductions in cardiac output (Q), maximal heart rate (HR-max), myocardial beta-adrenoceptor (beta-AR) density, and chronotropic response to isoproterenol. This study tested the hypothesis that exercise training (ET), which attenuates beta-AR downregulation, would increase HRmax and Q of acclimatization and result in higher Vo2 max. After 3 wk of ET, rats lived at an inspired Po2 of 70 Torr for 10 days (acclimatized trained rats) or remained in normoxia, while both groups continued to train in normoxia. Controls were sedentary acclimatized and nonacclimatized rats. All rats exercised maximally in normoxia and hypoxia (inspired Po2 of 70 Torr). Myocardial beta-AR density and the chronotropic response to isoproterenol were reduced, and myocardial cholinergic receptor density was increased after acclimatization; all of these receptor changes were reversed by ET. Normoxic Vo2 max (in ml.min-1.kg-1) was 95.8 +/- 1.0 in acclimatized trained (n = 6), 87.7 +/- 1.7 in nonacclimatized trained (P < 0.05, n = 6), 74.2 +/- 1.4 in acclimatized sedentary (n = 6, P < 0.05), and 72.5 +/- 1.2 in nonacclimatized sedentary (n = 8; P > 0.05 acclimatized sedentary vs. nonacclimatized sedentary). A similar distribution of Vo2 max values occurred in hypoxic exercise. Q was highest in trained acclimatized and nonacclimatized, intermediate in nonacclimatized sedentary, and lowest in acclimatized sedentary groups. ET preserved Q in acclimatized rats thanks to maintenance of HRmax as well as of maximal stroke volume. Q preservation, coupled with a higher arterial O2 content, resulted in the acclimatized trained rats having the highest convective O2 transport and Vo2 max. These results show that ET attenuates beta-AR downregulation and preserves Q and Vo2 max after acclimatization, and support the idea that beta-AR downregulation partially contributes to the limitation of Vo2 max after acclimatization in rats.