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(Circulation. 2000;102:1814.)
© 2000 American Heart Association, Inc.

Clinical Investigation and Reports

Both ß₂- and ß₁-Adrenergic Receptors Mediate Hastened Relaxation and Phosphorylation of Phospholamban and Troponin I in Ventricular Myocardium of Fallot Infants, Consistent With Selective Coupling of ß₂-Adrenergic Receptors to G_s-Protein

Peter Molenaar, PhD; Sabine Bartel, PhD; Andrew Cochrane, MBBS, FRACS; Donathe Vetter, TA; Homayoun Jalali, MD; Peter Pohlner, MBBS, FRACS; Kylie Burrell, BScHons; Peter Karczewski, PhD; Ernst-Georg Krause, PhD; Alberto Kaumann, MD, PhD

From the Departments of Medicine, The Prince Charles Hospital; Physiology and Pharmacology, University of Queensland, Queensland (P.M.); the Department of Pharmacology, University of Melbourne, Victoria, Australia (P.M., K.B.); Max-Delbrück Center of Molecular Medicine, Cardiology, Berlin, Germany (S.B., D.V., P.K., E.G.K.); Victorian Paediatric Cardiac Surgical Unit, Royal Children’s Hospital, Parkville, Australia (A.C.); the Department of Cardiac Surgery, The Prince Charles Hospital, Queensland (H.J., P.P.); Babraham Institute, Cambridge, and the Department of Physiology, University of Cambridge, UK (A.K.).

Correspondence to Dr Peter Molenaar, Department of Medicine, University of Queensland, The Prince Charles Hospital, Chermside, Queensland, 4032, Australia. E-mail molenaar{at}medicine.uq.edu.au

	Abstract

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Background—In adult human heart, both ß₁- and ß₂-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of ß₁- and ß₂-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot.

Methods and Results—Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 21/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of ß₁-adrenergic receptors with (-)-norepinephrine (NE) and ß₂-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC₅₀ [mol/L] NE 5.5±0.1, n=12; EPI 5.6±0.1, n=13 patients), hastening of the time to reach peak force (-log EC₅₀ [mol/L] NE 5.8±0.2; EPI 5.8±0.2) and 50% relaxation (-log EC₅₀ [mol/L] NE 5.7±0.2; EPI 5.8±0.1). Ventricular membranes from Fallot infants, labeled with (-)-[¹²⁵I]-cyanopindolol, revealed a greater percentage of ß₁- (71%) than ß₂-adrenergic receptors (29%). Binding of (-)-epinephrine to ß₂-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to ß₁-receptors.

Conclusions—Despite their low density, ß₂-adrenergic receptors are nearly as effective as ß₁-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G_s-protein.

Key Words: tetralogy of Fallot • catecholamines • myocardial contraction • receptors, adrenergic, beta

	Introduction

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In adult human atrium and ventricle, stimulation of either ß₁- or ß₂-adrenergic receptors causes an increase in the force of contraction and shortening of the time course of the contractile cycle,^{1 2 3 4} together with increases in cAMP and activation of cAMP-dependent protein kinase (PKA).^{2 5} PKA catalyzes phosphorylation of proteins implicated in cardiac relaxation, phospholamban, and troponin I through both ß₁- and ß₂-adrenergic receptors in atrium² and ventricle.⁴ Unphosphorylated phospholamban is an inhibitor of the Ca²⁺ pump of the sarcoplasmic reticulum, but the pump is disinhibited when phospholamban is phosphorylated, thereby increasing the rate of Ca²⁺ transport into the sarcoplasmic reticulum with subsequent augmentation of contraction and relaxation.⁶ Phosphorylation of troponin I decreases the affinity of troponin C for Ca²⁺, thereby also hastening relaxation.⁷

It is unknown whether the effects of ß₁- and ß₂-adrenergic receptor–mediated hastening of relaxation observed in ventricular myocytes from failing and nonfailing adult human hearts³ and trabeculae from failing heart⁴ can be extrapolated to infant heart. Only in neonatal but not adult rat cardiac myocytes, stimulation of the ß₂-adrenergic receptor caused hastening of calcium transients and cell shortening by a cAMP-dependent mechanism.⁸ Furthermore, ß₂-adrenergic receptors failed to hasten relaxation in ventricular myocytes from adult rats and mice unless coupling to G_i-protein was inhibited with pertussis toxin so that coupling to G_s-protein could be unconcealed.⁹ This is in contrast to adult human ventricular myocytes³ and trabeculae,⁴ in which a smaller population of ß₂-adrenergic receptors were as effective as the larger population of ß₁-adrenergic receptors in mediating hastened relaxation, presumably because of the tighter coupling of ß₂- than ß₁-adrenergic receptors to G_s-protein.^{10 11 12 13 14}

We studied the role of ß₁- and ß₂-adrenergic receptors in ventricular myocardium from nonfailing hearts of infants with Fallot tetralogy, in which the density of ß₂-adrenergic receptors is approximately one third¹⁵ of the density of ß₁-adrenergic receptors.

(-)-Norepinephrine and (-)-epinephrine hasten relaxation through ß₁- and ß₂-adrenergic receptors, respectively, with similar potency and efficacy, associated with phosphorylation of phospholamban and troponin I. The relaxation mediated through the smaller ß₂-adrenergic receptor population is probably facilitated by coupling more tightly to G_s-protein than ß₁-adrenergic receptors. In support of this hypothesis, we also demonstrate in Fallot ventricle a greater reduction with guanosine 5-triphosphate (GTP) of (-)-epinephrine binding to ß₂-adrenergic receptors than of (-)-norepinephrine binding to ß₁-adrenergic receptors.

	Methods

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Patients
Myocardium from the right ventricular outflow tract (RVOT) was obtained from patients with tetralogy of Fallot undergoing corrective surgery at the Royal Children’s Hospital (ethics approval numbers 97029A, 951686) and The Prince Charles Hospital (ethics approval numbers EC9876, H/29/Med/PCH/NHMRC/99). Written Informed consent was obtained from the parents of 30 patients with tetralogy of Fallot (19 boys, 11 girls, mean age 15.0 months, Table 1). Six patients had hypoxic spells before surgery. Most patients did have a degree of chronic cyanosis, with mean preoperative hemoglobin saturation of 84±2% (Table 1). None of the patients were in severe heart failure on the basis of a scoring system for grading congestive heart failure in infants and children¹⁶ (score <6).

View this table: [in this window] [in a new window]   Table 1. Patient Details

Preparation of Ventricular Strips
Myocardium from the RVOT was surgically removed from the arrested heart after perfusion with cardioplegic solution. Ventricular muscle was placed immediately into ice-cold preoxygenated solution (mmol/L: Na⁺ 125, K⁺ 5, Ca²⁺ 2.25, Mg²⁺ 0.5, Cl^- 98.5, SO₄^2- 0.5, HCO₃^- 32, HPO₄^2- 1 and EDTA 0.04) and transferred to the laboratory. Ventricular strips (<1.5 mm width) were prepared and mounted into an apparatus with 50-mL organ baths¹⁷ at 37°C, driven with square-wave pulses (5-ms duration, just over threshold voltage) at 1 Hz, and set to an optimal length (L_max),¹¹ with contraction and relaxation measured as described.⁴ The incubation medium was exchanged with solution (above) supplemented with (mmol/L: Na⁺ 15, fumarate 5, pyruvate 5, L-glutamate 5 and glucose 10) together with phenoxybenzamine (5 µmol/L) to irreversibly block -adrenergic receptors and inhibit neuronal and extraneuronal uptake of catecholamines.¹⁰ Some tissues were incubated with the ß₁-adrenergic receptor antagonist CGP 20712A (300 nmol/L),^{4 11} others with the ß₂-adrenergic receptor antagonist ICI 118,551 (50 nmol/L)^{4 11} and others with both CGP 20712A and ICI 118,551. After 90 minutes, the incubation solution was exchanged to remove unbound phenoxybenzamine and supplemented with ascorbic acid (0.2 mmol/L) to prevent catecholamine oxidation and as before with ß-adrenergic receptor antagonists.

Catecholamine Responses
Cumulative concentration-effect curves were established to (-)-norepinephrine in the presence of ICI 118,551 (selective ß₁-adrenergic receptor stimulation⁴ ) or (-)-epinephrine in the presence of CGP 20712A (selective ß₂-adrenergic receptor stimulation⁴ ), followed by a single concentration (200 µmol/L) of (-)-isoproterenol to obtain a maximal effect caused by stimulation of both ß₁- and ß₂-adrenergic receptors.⁴ Experiments were concluded by raising the Ca²⁺ concentration to 9.25 mmol/L. In other experiments, tissues were exposed for 5 minutes to either (-)-norepinephrine (10 µmol/L), (-)-epinephrine (10 µmol/L), or (-)-isoproterenol (200 µmol/L), during which time equilibrium effects were observed. Ventricular strips were then freeze-clamped with liquid nitrogen–precooled tissue clamps and subsequently used for phosphorylation studies. Other tissues were exposed to antagonist but not agonist and freeze-clamped as controls.

Protein Phosphorylation
Freeze-clamped tissue derived from contracting trabeculae was homogenized in a histidine-buffer containing NaF 25 mmol/L and phenylmethanesulfonyl fluoride 0.1 mmol/L.¹⁸ The homogenates were divided into a particulate fraction (source of phospholamban) and a supernatant fraction (source of troponin I) by centrifugation at 100 000g. Specific antibodies against phosphoserine 16-phospholamban, phosphothreonine 17-phospholamban, and an epitope common to all phospholambar forms were used.^{4 19} The method of protein back-phosphorylation was used as detailed.^{2 4 18}

Radioligand Binding
Snap-frozen ventricular myocardium, stored at -70°C until use, was used for radioligand binding experiments with (-)-[¹²⁵I]-iodocyanopindolol ((-)-[¹²⁵I]-CYP),²⁰ which were analyzed by Prism (Graphpad Software, Inc).

Statistics
Data are expressed as mean±SEM. Parametric (Student’s t test), or in the case of populations with nonuniform distribution of errors, nonparametric (Mann-Whitney test) tests, were performed with the use of In Stat (Graph Pad Software Version 2.0). A value of P0.05 was considered statistically significant.

	Results

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Positive Inotropic and Lusitropic Effects Mediated Through ß₁- and ß₂-Adrenergic Receptors
The functional integrity of ventricular strips was established by observing a positive contractile force-frequency relation between 6 to 120 contractions per minute (n=27 myocardial strips, not shown). Stimulation of ß₁-adrenergic receptors with (-)-norepinephrine, ß₂-adrenergic receptors with (-)-epinephrine, and both ß₁- and ß₂-adrenergic receptors with (-)-isoproterenol increased contractile force and reduced both the time to reach peak force and time to reach 50% relaxation (Figure 1, Tables 1 and 2). The concentration dependence of the effects of (-)-norepinephrine and (-)-epinephrine and their corresponding intrinsic activities were remarkably similar (Figure 2, Table 3).

View larger version (24K): [in this window] [in a new window]   Figure 1. Positive inotropic and lusitropic effects of 10 µmol/L (-)-norepinephrine in the presence of 50 nmol/L ICI 118,551, 10 µmol/L (-)-epinephrine in presence of 300 nmol/L CGP 20712A, or 200 µmol/L (-)-isoproterenol in presence of 300 nmol/L CGP 20712A in right ventricular strips from 29-month-girl (patient 3, Table 1) with complete atresia of pulmonary valve with prior systemic-to-pulmonary shunt. Catecholamine was added to organ bath (arrow) and incubated for 5 minutes. Shown are fast-speed recordings (bar=100 ms) and slow-speed recordings (bar=1 minute). Right panels show overlapping fast-speed recordings of basal contractions and contractions in presence of catecholamine.

View this table: [in this window] [in a new window]   Table 2. Contractile and Relaxant Effects of Catecholamines in Ventricular Strips From Infants With Tetralogy of Fallot

View larger version (27K): [in this window] [in a new window]   Figure 2. Concentration-dependent effects (•) of selective ß1-adrenergic receptor stimulation [(-)-norepinephrine+50 nmol/L ICI 118,551, 16 tissues from 12 patients] and ß2-adrenergic receptor stimulation [(-)-epinephrine+ 300 nmol/L CGP 20712A, 21 tissues from 13 patients] on contractile force, time to reach peak force, and time to reach 50% relaxation (t50) in ventricular strips obtained from infants with tetralogy of Fallot. Contractile force is expressed in terms of absolute force (mN). Basal values are shown by ; effect of 200 µmol/L (-)-isoproterenol is shown by ; effect of high Ca2+ (9.25 mmol/L) in presence of (-)-norepinephrine or (-)-epinephrine and (-)-isoproterenol is shown by . Values given are mean±SEM. Errors not shown when smaller than symbol.

View this table: [in this window] [in a new window]   Table 3. Inotropic and Lusitropic Potencies and Intrinsic Activities of (-)-Norepinephrine and (-)-Epinephrine Through ß1- and ß2-Adrenergic Receptors in RVOT Myocardium

There were no relations between age, preoperative hemoglobin saturation, and potencies (-log EC₅₀ values) or intrinsic activity values for changes in contractile force, time to reach 50% relaxation, and time to reach peak force for (-)-norepinephrine or (-)-epinephrine (r<0.54, Tables 1 and 3). On this basis, the patient population was considered homogeneous.

Phosphorylation of Phospholamban and Troponin I
(-)-Norepinephrine (10 µmol/L), (-)-epinephrine (10 µmol/L), and (-)-isoproterenol 200 µmol/L produced positive inotropic and lusitropic effects (Table 2) and site-specific phosphorylation of serine-16 and threonine-17 of phospholamban (Figures 3 and 4) and troponin I (Figures 5 and 6).

View larger version (51K): [in this window] [in a new window]   Figure 3. Site-specific phosphorylation of phospholamban (PLB) at Ser16 (PSer16-PLB) and Thr17 (PThr17-PLB) caused by 10 µmol/L (-)-epinephrine (EPI) in presence of 300 nmol/L CGP 20712A (CGP). Phosphorylation of Ser16 and Thr17 by EPI in presence of CGP was prevented by ICI 118,551 (50 nmol/L, ICI). Data from patient 7 (Table 1).

View larger version (17K): [in this window] [in a new window]   Figure 4. Site-directed phosphorylation of phospholamban serine-16 and threonine-17 by 10 µmol/L (-)-epinephrine (EPI), 200 µmol/L (-)-isoproterenol (ISO), or 10 µmol/L (-)-norepinephrine (NE) in presence of 300 nmol/L CGP 20712A, 50 nmol/L ICI 118,551, or both antagonists as indicated. Arbitrary units of density (optical density [OD]xmm2) are used for ordinate scale. Data obtained from 3 to 5 tissues from 3 to 5 patients. *P<0.05. P=0.008 for EPI+CGP compared with EPI+CGP+ICI.

View larger version (27K): [in this window] [in a new window]   Figure 5. Autoradiograms showing ß1- and ß2-adrenergic receptor–mediated phosphorylation of troponin I (TNI). A, Selective ß2-adrenergic stimulation (10 µmol/L (-)-epinephrine, EPI+300 nmol/L CGP 20712A, CGP) and ß1-adrenergic stimulation (10 µmol/L (-)-norepinephrine, NE+50 nmol/L ICI 118,551, ICI) reduced in vitro back-phosphorylation catalyzed by PKA, indicating endogenous phosphorylation of proteins in vivo. B, Phosphorylation of troponin I by 10 µmol/L (-)-epinephrine (+300 nmol/L CGP 20712A) was prevented by 50 nmol/L ICI 118,551. Data obtained from patient 2 of Table 1.

View larger version (24K): [in this window] [in a new window]   Figure 6. Phosphorylation of troponin I by 10 µmol/L (-)-epinephrine (EPI), 200 µmol/L (-)-isoproterenol (ISO), or 10 µmol/L (-)-norepinephrine (NE) in presence of 300 nmol/L CGP 20712A, 50 nmol/L ICI 118,551, or both antagonists as indicated. Lower level of phosphate incorporated in vitro (Pi) in presence of catecholamine represents endogenous phosphorylation of proteins. Data from 3 to 8 individual tissues from 7 patients. *P<0.05.

The effects of (-)-epinephrine (in the presence of CGP 20712A) on contractility, hastening of relaxation, and phosphorylation of phospholamban and troponin I were reduced or abolished by ß₂-adrenergic receptor blockade with 50 nmol/L ICI 118,551 (Table 2, Figures 3 to 6), as predicted from the affinity of ICI 118,551 for ß₂-adrenergic receptors.^{4 11} The small residual increases observed for force of contraction (Table 2) and phosphorylation of phospholamban serine-16 (Figure 4) are consistent with an expected 2 log unit rightward shift of concentration-effect curves^{4 11} to (-)-epinephrine (plus CGP 20712A) caused by 50 nmol/L ICI 118,551. Taken together, the evidence with ICI 118,551 demonstrates the specificity of (-)-epinephrine for ß₂-adrenergic receptors.

Greater GTP Sensitivity of Agonist Binding to ß₂- Than to ß₁-Adrenergic Receptors
(-)-[¹²⁵I]-CYP bound to 16.0±2.8 fmol/mg protein ß₁-+ß₂-adrenergic receptors with -log K_D of 11.2±0.1 (n=5 individual experiments, not shown). The ratio of ß₁-:ß₂-adrenergic receptors determined by (-)-[¹²⁵I]-CYP binding remaining in the presence of 50 nmol/L ICI 118,551 (ß₁-adrenergic receptors) or 300 nmol/L CGP 20712A (ß₂-adrenergic receptors) was 71.3:28.7±2.5 (n=5 individual experiments). GTP produced a marked rightward shift of the binding-inhibition curve for (-)-epinephrine but only a small shift of the curve for (-)-norepinephrine. The binding inhibition curve to (-)-epinephrine in the absence of 0.1 mmol/L GTP but not that of (-)-norepinephrine could be resolved into high and low affinity populations (Figure 7, Table 4).

View larger version (16K): [in this window] [in a new window]   Figure 7. Guanine nucleotide sensitivity of catecholamine binding. Figure shows mean radioligand binding competition curves between (-)-[125I]-CYP and (-)-norepinephrine (in presence of 50 nmol/L ICI 118,551) and (-)-epinephrine (in presence of 300 nmol/L CGP 20712A) in absence or presence of 0.1 mmol/L GTP.

View this table: [in this window] [in a new window]   Table 4. Summary of Data From Competition Binding Experiments Between (-)-[125I]-CYP and (-)-Norepinephrine or (-)-Epinephrine in Absence or Presence of GTP

	Discussion

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(-)-Epinephrine Can Hasten Relaxation Through ß₂-Adrenergic Receptors Through PKA
Despite their lower density, agonist-occupied ß₂-adrenergic receptors appear nearly as effective as ß₁-adrenergic receptors in increasing contractile force and relaxation velocity of ventricular myocardium from infant patients with tetralogy of Fallot without advanced heart failure. Consistent with these observations, stimulation of ß₁-adrenergic receptors with (-)-norepinephrine or ß₂-adrenergic receptors with (-)-epinephrine caused PKA-dependent phosphorylation of phospholamban and troponin I, two important proteins involved in the production of positive inotropic and lusitropic effects of the catecholamines.^{6 7} We propose that the effectiveness of human ß₂-adrenergic receptors in mediating positive inotropic and lusitropic effects of (-)-epinephrine is due to selective coupling of these receptors to G_s-protein compared with human ß₁-adrenergic receptors.

Catecholamine-evoked relaxation of ventricular myocardium from nonfailing hearts from infants with tetralogy of Fallot from the age of 21/2 months is quantitatively similar to that found in ventricular myocardium from adult patients in terminal heart failure,⁴ despite lower basal contractile force in Fallot than adult myocardium. These results are fundamentally different from those reported for nonfailing ventricular myocardium of adult rats⁹ in which only ß₁- but not ß₂-adrenergic receptors mediate positive lusitropic effects. ß₂-Adrenergic receptors in the hearts of adult rats couple to G_i-protein, and only inactivation of G_i-protein with pertussis toxin uncovers coupling to G_s-protein with PKA-dependent phosphorylation of phospholamban and hastening of relaxation.⁹ ß₂-Adrenergic receptors of adult mice as well as human ß₂-adrenergic receptors genetically overexpressed in mouse heart also couple to G_i-protein.⁹ Previous evidence in ventricular myocardium from adult failing human heart is, however, consistent with functional coupling of ß₂-adrenergic receptors to G_s-protein, as demonstrated with PKA-catalyzed phosphorylation of phospholamban and troponin I and ensuing hastened relaxation,⁴ despite reported increases in G_i-protein and mRNA levels.^{21 22 23} Taken together, our evidence in infant (present work) and adult⁴ ventricle as well as atrium² is consistent with selective functional coupling of human cardiac ß₂-adrenergic receptors to G_s-protein compared with ß₁-adrenergic receptors, at least from 21/2 months of age onward to adulthood. Our results stress the importance of obtaining evidence directly from human cardiac tissues because the conclusions gained from the hearts of adult mice and rats cannot be extrapolated to humans.

The ß₂-adrenergic receptor was nearly as effective as the ß₁-adrenergic receptor in mediating positive inotropic and relaxant effects and in the extent of phosphorylation of phospholamban and troponin I. However, ß₂-adrenergic receptors comprise the minor fraction of receptors in human ventricle from children with tetralogy of Fallot (this study and Reference 15¹⁵ ), suggesting that ß₂-adrenergic receptors are coupled more tightly to the G_s-protein/cAMP pathway than ß₁-adrenergic receptors in infant heart. Selective coupling of ß₂-adrenergic receptors to the G_s-protein/adenylyl cyclase system was first reported in human atrium¹⁰ and ventricle^{11 12} and later confirmed for recombinant receptors.^{13 14} Furthermore, evidence for selective stimulation of adenylyl cyclase through ß₂-adrenergic receptors, compared with ß₁-adrenergic receptors, has also been reported to occur in atria from children with Fallot tetralogy.²⁴ The finding of considerably greater GTP-evoked decrease of binding affinity of (-)-epinephrine through ß₂-adrenergic receptors compared with (-)-norepinephrine binding through ß₁-adrenergic receptors is consistent with selective coupling of Fallot ventricular ß₂-adrenergic receptors. The magnitude of the GTP shift appears to be proportional to the tightness of coupling between receptor and G-protein.²⁵ Taken together with the relaxation and phosphorylation data, this evidence is consistent with the hypothesis that the smaller population of ß₂-adrenergic receptors is nearly as effective as the considerably greater population of ß₁-adrenergic receptors in mediating positive inotropic and lusitropic effects caused by selective G_s-protein coupling in infant Fallot heart, as previously suggested for adult human heart.⁴

Role of Serine-16 and Threonine-17 Phosphorylation at Phospholamban
The current view is that phosphorylation of phospholamban serine-16 is the main mechanism for ß-adrenergic receptor-mediated hastening of relaxation,^{26 27 28} and our results from infant Fallot ventricle are consistent with this. Phosphorylation of serine-16 is obligatory for phosphorylation of threonine-17.²⁶ We showed that stimulation of both ß₁- and ß₂-adrenergic receptors caused calcium calmodulin–dependent kinase (CaMkinase II)-dependent phosphorylation at threonine-17 of phospholamban in Fallot ventricle, possibly because of increased Ca²⁺ from L-type Ca²⁺ channels^{29 30} and from ryanodine channels.

Possible Clinical Implications
The relatively high heart rate in infants demands faster ventricular relaxation to allow diastolic filling. Our results suggest that ventricular relaxation could be facilitated by endogenous (-)-norepinephrine and (-)-epinephrine through both ß₁-and ß₂-adrenergic receptors.

Catecholamines also may cause harmful effects. Isoproterenol enhances right-to-left shunt and decreases pulmonary flow,³¹ and similar effects may occur with endogenous catecholamines through ß₁- and ß₂-adrenergic receptors. This mechanism may explain the beneficial effects of ß-blockers for the acute treatment of hypoxic spells that have been reported to enhance pulmonary blood flow and decrease right-to-left shunts.^{31 32} Besides causing increased oxygen consumption, catecholamines also may be involved in arrhythmias observed in Fallot hearts.³³ Our finding that activation of the PKA-dependent pathway through both ß₁- and ß₂-adrenergic receptors is similar in infant Fallot (this report) and adult heart⁴ resembles the similar properties of L-type calcium channels in ventricular myocytes obtained from Fallot infants and adult humans.³⁰ The similarities include increases in calcium current density through ß-adrenergic receptors and production of proarrhythmic afterdepolarizations in ventricular myocytes from Fallot infants. It is possible that arrhythmias in infant Fallot ventricle could be elicited by norepinephrine and epinephrine through ß₁- and ß₂-adrenergic receptors, as observed in vitro in isolated atrium from adult nonfailing hearts³⁴ and experimentally through ß₂-adrenergic receptors in dogs.³⁵ For the treatment of both hypoxic spells and arrhythmias, nonselective ß-blockers may be more effective than ß₁-selective blockers.

Study Limitations
Myocardium from the RVOT of infants with tetralogy of Fallot was used in the present study. It is still unknown whether our findings in Fallot myocardium differ from the function of ß₁- and ß₂-adrenergic receptors in ventricle from healthy infants. The relaxation pathway, however, seems to work in a remarkably similar way in nonfailing infant Fallot ventricle and failing adult ventricle.

	Acknowledgments

 
This study was supported by the National Health and Medical Research Council (Australia, Dr Molenaar) and the British Heart Foundation (Dr Kaumann). Dr Molenaar thanks the theater staff at Royal Children’s Hospital (Parkville, Australia) and The Prince Charles Hospital (Chermside, Australia), who coordinated tissue collection, and Dr Robert Justo for discussions.

Received March 20, 2000; revision received May 8, 2000; accepted May 16, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Hall JA, Kaumann AJ, Brown MU. Selective ß₁-adrenoceptor blockade enhances positive inotropic effects of endogenous catecholamines through ß₂-adrenoceptors in human atrium. Circ Res. 1990;66:1610–1623.[Abstract/Free Full Text]

2. Kaumann AJ, Sanders L, Lynham JA, et al. ß₂-Adrenoceptor activation by zinterol causes protein phosphorylation, contractile effects and relaxant effects through a cAMP pathway in human atrium. Mol Cell Biochem. 1996;163/164:113–123.

3. DelMonte F, Kaumann AJ, Poole-Wilson PA, et al. Coexistence of functional ß₁- and ß₂-adrenoceptors in single myocytes from human ventricle. Circulation. 1993;88:889–893.

4. Kaumann AJ, Bartel S, Molenaar P, et al. Activation of ß₂-adrenergic receptors hastens relaxation and mediates phosphorylation of phospholamban, troponin I and C protein in ventricular myocardium from patients with terminal heart failure. Circulation. 1999;99:65–72.[Abstract/Free Full Text]

5. Kaumann AJ, Hall JA, Murray JA, et al. A comparison of the effects of adrenaline and noradrenaline on human heart: the role of ß₁- and ß₂-adrenoceptors in the stimulation of adenylate cyclase and contractile force. Eur Heart J. 1989;10(suppl B):29–37.

6. Koss KL, Kranias EG. Phospholamban: a prominent regulator of myocardial contractility. Circ Res. 1996;79:1059–1063.[Free Full Text]

7. Zhang R, Zhao J, Mandveno A, et al. Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation. Circ Res. 1995;76:1028–1035.[Abstract/Free Full Text]

8. Steinberg SF. The molecular basis for distinct ß-adrenergic receptor subtype actions in cardiomyocytes. Circ Res. 1999;85:1101–1111.[Free Full Text]

9. Xiao R-P, Cheng H, Zhou Y-Y, et al. Recent advances in cardiac ß₂-adrenergic signal transduction. Circ Res. 1999;85:1092–1100.[Abstract/Free Full Text]

10. Gille E, Lemoine H, Ehle B, et al. The affinity of (-)-propranolol for ß₁- and ß₂-adrenoceptors of human heart: differential antagonism of the positive inotropic effects and adenylate cyclase stimulation by (-)-noradrenaline and (-)-adrenaline. Naunyn Schmiedebergs Arch Pharmacol. 1985;331:60–70.[Medline] [Order article via Infotrieve]

11. Kaumann AJ, Lemoine H. ß₂-Adrenoceptor-mediated positive inotropic effects of adrenaline in human ventricular myocardium: quantitative discrepancies between binding and adenylate cyclase stimulation. Naunyn Schmiedebergs Arch Pharmacol. 1987;335:403–411.[Medline] [Order article via Infotrieve]

12. Bristow MR, Hershberger RE, Port JD, et al. ß₁- and ß₂-Adrenergic receptor-mediated adenylate cyclase stimulation in nonfailing and failing human ventricular myocardium. Mol Pharmacol. 1989;35:295–303.[Abstract]

13. Green SA, Holt BD, Liggett SB. ß₁- And ß₂-adrenergic receptors display subtype-selective coupling to G_s. Mol Pharmacol. 1992;41:889–893.[Abstract]

14. Levy FO, Zhu X, Kaumann AJ, et al. Efficacy of ß₁-adrenergic receptors is lower than that of ß₂-adrenergic receptors. Proc Natl Acad Sci U S A. 1993;90:10798–10802.[Abstract/Free Full Text]

15. Sun LS, Du SF, Quaegebeur JM. Right ventricular infundibular ß-adrenoceptor complex in tetralogy of Fallot patients. Pediatr Res. 1997;42:12–16.[Medline] [Order article via Infotrieve]

16. Ross RD, Bollinger RO, Pinsky WW. Grading the severity of congestive heart failure in infants. Pediatr Cardiol. 1992;13:72–75.[Medline] [Order article via Infotrieve]

17. Blinks JR. Convenient apparatus for recording contractions of isolated muscle. J Appl Physiol. 1965;20:755–757.[Abstract/Free Full Text]

18. Karczewski P, Bartel S, Krause E-G. Differential sensitivity to isoproterenol of troponin I and phospholamban phosphorylation in isolated rat heart. Biochem J. 1990;266:115–122.[Medline] [Order article via Infotrieve]

19. Drago GA, Colyer J. Discrimination between two sites of phosphorylation on adjacent amino acids by phosphorylation site-specific antibodies to phospholamban. J Biol Chem. 1994;269:25073–25077.[Abstract/Free Full Text]

20. Kaumann AJ, Lynham JA, Sanders L, et al. Contribution of differential efficacy to the pharmacology of human ß₁- and ß₂-adrenoceptors. Pharmacol Commun. 1995;6:215–222.

21. Neumann J, Schmitz W, Scholz H, et al. Increase of myocardial G_i-proteins in human heart failure. Lancet. 1988;2:936–937.[Medline] [Order article via Infotrieve]

22. Feldman AM, Cates AE, Veazey WB, et al. Increase in the 40,000-mol wt pertussis toxin substrate (G-protein) in the failing human heart. J Clin Invest. 1988;82:189–197.

23. Eschenhagen T, Mende U, Nose M, et al. Increased messenger RNA level of the inhibitory G-protein -subunit G_ia2 in human end-stage heart failure. Circ Res. 1992;70:688–696.[Abstract/Free Full Text]

24. Kozlik-Feldmann R, Kramer RF, Wicht HH, et al. Distribution of myocardial beta-adrenoceptor subtypes and coupling to the adenylate cyclase in children with congenital heart disease and implication for treatment. J Clin Pharmacol. 1993;33:588–595.[Abstract]

25. Kent RS, DeLean A, Lefkowitz RJ. A quantitative analysis of beta-adrenergic receptor interactions: resolution of high and low affinity binding states of the receptor by computer modelling of ligand binding data. Mol Pharmacol. 1980;17:14–23.[Abstract/Free Full Text]

26. Luo W, Chu G, Sato Y, et al. Transgenic approaches to define the functional role of dual site phospholamban phosphorylation. J Biol Chem. 1998;273:4734–4739.[Abstract/Free Full Text]

27. Calaghan SC, White E, Colyer J. Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca²⁺ and contraction following ß-adrenergic stimulation of rat heart. Pflugers Arch. 1998;436:948–956.[Medline] [Order article via Infotrieve]

28. Kuschel M, Karczewski P, Hempel P, et al. Ser16 prevails over Thr17 phospholamban phosphorylation in the ß-adrenergic regulation of cardiac relaxation. Am J Physiol. 1999;276:H1625–H1633.[Abstract/Free Full Text]

29. Skeberdis VA, Jurevicius J, Fischmeister R. ß₂-Adrenergic activation of L-type Ca²⁺ current in cardiac myocytes. J Pharmacol Exp Ther. 1997;282:452–461.[Abstract/Free Full Text]

30. Pelzmann B, Schaffer P, Bernhart E, et al. L-type calcium current in human ventricular myocytes at a physiological temperature from children with tetralogy of Fallot. Cardiovasc Res. 1998;38:424–432.[Abstract/Free Full Text]

31. Diekmann L, Hilgenberg F. Hämodynamische Untersuchungen mit Isoprenalin und ß-Rezeptorenblockern bei Kindern mit Fallotscher Tetralogie. Z Kreislaufforsch. 1972;61:40–52.[Medline] [Order article via Infotrieve]

32. Cumming GR, Carr W. Relief of dyspnoeic attacks in Fallot’s tetralogy with propranolol. Lancet. 1966;1:519–522.

33. Ross BA. From the bedside to the basic science laboratory: arrhythmias in Fallot’s tetralogy. J Am Coll Cardiol. 1993;21:1738–1740.[Medline] [Order article via Infotrieve]

34. Kaumann AJ, Sanders L. Both ß₁- and ß₂-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium. Naunyn Schmiedebergs Arch Pharmacol. 1993;311:219–236.

35. Billman GE, Castillo LC, Hensley J, et al. ß₂-Adrenergic receptor antagonists protect against ventricular fibrillation: in vivo and in vitro evidence for enhanced sensitivity to ß₂-adrenergic stimulation in animals susceptible to sudden death. Circulation. 1997;96:1914–1922.[Abstract/Free Full Text]

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