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(Circulation. 1999;99:65-72.)
© 1999 American Heart Association, Inc.

Clinical Investigation and Reports

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

Alberto Kaumann, MD, PhD; Sabine Bartel, PhD; Peter Molenaar, PhD; Louise Sanders, MA; Kylie Burrell, BScHons; Donathe Vetter; Petra Hempel, MSci; Peter Karczewski, PhD; Ernst-Georg Krause, PhD

From the Babraham Institute, Cambridge, UK (A.K., L.S.); Department of Pharmacology, University of Melbourne, Victoria, Australia (P.M., K.B.); and Max-Delbrück Centre of Molecular Medicine, Cardiology, Berlin, Germany (S.B., D.V., P.H., P.K., E.-G.K.).

Correspondence to Alberto Kaumann, MD, PhD, Babraham Institute, Cambridge CB2 4AT, United Kingdom. E-mail alberto.kaumann{at}bbsrc.ac.uk

	Abstract

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Background—Catecholamines hasten cardiac relaxation through ß-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of ß₁- and ß₂-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation.

Methods and Results—Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 µmol/L) or zinterol (10 µmol/L), mediated through ß₂-adrenergic receptors, and of norepinephrine (10 µmol/L), mediated through ß₁-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17. Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15±3%, 5±2%, and 20±3%, respectively, and reduced the time to half-relaxation by 26±3%, 21±3%, and 37±3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14±3, 12±4, and 12±3; troponin I 40±7, 33±7, and 31±6; and C-protein 7.2±1.9, 9.3±1.4, and 7.5±2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both ß₁- and ß₂-adrenergic receptors.

Conclusions—Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both ß₁- and ß₂-adrenergic receptors, thereby potentially improving diastolic function.

Key Words: heart failure • receptors, adrenergic, beta 2 • catecholamines • phosphoproteins • contractility, diastole

	Introduction

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Activation of cardiac ß-adrenergic receptors by catecholamines augments contractility and hastens relaxation. Catecholamines are thought to promote relaxation (ie, cause lusitropic effects)¹ by inducing the phosphorylation, catalyzed by cAMP-dependent protein kinase A (PKA), of at least 2 proteins, phospholamban^{1 2 3} and troponin I,⁴ and perhaps of C-protein.^{5 6} Phospholamban can also be phosphorylated by a Ca²⁺/calmodulin–dependent protein kinase⁷ (CaM kinase). Nonphosphorylated phospholamban exerts a braking effect on the Ca²⁺ ATPase that pumps Ca²⁺ into the sarcoplasmic reticulum.³ Phospholamban phosphorylation results in liberation of Ca²⁺ ATPase activity, which accelerates its pumping activity, thereby decreasing Ca²⁺ concentrations at the contractile proteins and hastening myocyte relaxation.^{1 2 3} Phosphorylation of troponin I desensitizes contractile proteins to Ca²⁺, thus providing another mechanism for relaxation.^{4 8 9}

Diastolic function deteriorates in congestive heart failure,¹⁰ even when systolic function is unimpaired.¹¹ Relaxation of ventricular myocardium is retarded in heart failure and correlates with prolongation of calcium transients, suggesting a decreased capacity to restore low basal Ca²⁺ levels.¹² One factor that correlates with the prolongation of calcium transients is deficient generation of cAMP, which is observed in heart failure.¹³ Although it has been reported that phosphorylation of phospholamban, troponin I, and C-protein is decreased in failing human ventricle compared with normal myocardium,¹⁴ positive lusitropic responses to the catecholamine dobutamine are still observed in patients with heart failure.¹⁵ The affinity¹⁶ and agonist potency¹⁷ of dobutamine are similar at both ß₁- and ß₂-adrenergic receptors, and it is unknown which subtype is involved. We therefore sought to determine whether both ß₁- and ß₂-adrenergic receptors can mediate positive lusitropic effects and whether implicated proteins are phosphorylated. This is not the case in a variety of animal models in which ß₁- but not ß₂-adrenergic receptors mediate hastening of ventricular relaxation.^{18 19 20 21 22 23} However, there is evidence in human atrium^{24 25} and human ventricular myocytes from normal and failing hearts²⁶ that both ß₁- and ß₂-adrenergic receptors mediate not only inotropic but also lusitropic effects. We now confirm this in ventricular trabeculae from failing ischemic and cardiomyopathic hearts and show it to be associated with phosphorylation of phospholamban, troponin I, and C-protein for both ß₁- and ß₂-adrenergic receptors.

	Methods

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Patients
Informed consent was obtained from all patients (Table 1). All patients had terminal heart failure and underwent cardiac transplant surgery. This study was approved by the ethics committees of the Papworth Hospital, Cambridgeshire (United Kingdom), and Alfred Hospital, Melbourne (Australia).

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

Isolated Right Ventricular Trabecula Carnae
Explanted hearts were obtained immediately (<1 minute) after removal from the patient. The endocardial layer of the right ventricular free wall was rapidly dissected in ice-cold, preoxygenated (95% O₂-5% CO₂) modified Krebs' solution containing (mmol/L) Na⁺ 125, K⁺ 5, Ca²⁺ 2.25, Mg²⁺ 0.5, Cl^- 98.5, SO₄^2- 0.5, HCO₃^- 29, HPO₄^2- 1, and EDTA 0.04 at the surgical theater. Trabeculae (width usually <1 and not >1.3 mm) were dissected, set up at optimum length, and paced to contract isometrically at 60 bpm at 37°C in a bath containing the above solution supplemented with (mmol/L) Na⁺ 15, fumarate 5, pyruvate 5, L-glutamate 5, and glucose 10 as described.²⁷

In all experiments, contractile force and its first derivative were recorded simultaneously. Cross-sectional area of trabeculae that were not snap-frozen was determined from the length and weight of the muscle at the end of the experiment, assuming a density of 1.063. Rapid freezing of trabeculae prevented measurements of length and weight under conditions of contraction.

Specific Activation of ß₁- and ß₂-Adrenergic Receptors
To irreversibly block tissue uptake of catecholamines and -adrenergic receptors, trabeculae were incubated for 90 minutes with phenoxybenzamine followed by washout.²⁸

To establish conditions for selective activation of ß₁- and ß₂-adrenergic receptors, experiments were carried out as described previously on ventricular preparations from hearts without advanced failure²⁷ and ventricular myocytes from a donor heart and hearts in terminal failure.²⁶ To determine ß₁-selective activation, inotropic concentration-effect curves to (-)-norepinephrine in the presence of ICI118551 (50 nmol/L) (to selectively block ß₂-adrenergic receptors) were determined in the absence and presence of the ß₁-selective blocker CGP20712A.^{24 25 26 27} For ß₂-selective activation, inotropic concentration-effect curves to (-)-epinephrine in the presence of CGP20712A (300 nmol/L) were determined in the absence and presence of ICI118551 (50 nmol/L).^{24 25 26 27} In these studies, only a single concentration-effect curve was determined on each trabeculum. After equilibrium effects were reached with the highest catecholamine concentration, (-)-isoproterenol was added at a concentration (200 µmol/L) that surmounts²⁷ the blockade caused by CGP20712A and ICI118551. The experiments were terminated by increasing the CaCl₂ concentration to 9.25 mmol/L in the presence of catecholamines. In 4 additional patients, the effects of 9.25 mmol/L CaCl₂ were also investigated in the absence of catecholamines but in the presence of CGP20712A.

To determine relaxation and protein phosphorylation mediated by ß₁-adrenergic receptors, trabeculae were exposed for 2 hours to ICI118551 (50 nmol/L) followed by incubation for 5 minutes with 10 µmol/L (-)-norepinephrine. To assess ß₂-adrenergic receptor–mediated effects for tissues from the same patient, trabeculae exposed for 2 hours to CGP20712A (300 nmol/L) were incubated for 5 minutes with 10 µmol/L (-)-epinephrine or 10 µmol/L zinterol. To assess maximal effects mediated through both ß₁- and ß₂-adrenergic receptors, CGP20712A-treated trabeculae were exposed for 5 minutes to 200 µmol/L (-)-isoproterenol. Basal protein phosphorylation was determined in trabeculae from the same patient incubated with either CGP20712A or ICI118551 but not agonist.

Processing of Trabeculae for Protein Phosphorylation and Immunodetection
Freeze-clamped tissue derived from contracting trabeculae was homogenized in a histidine buffer containing NaF 25 mmol/L and phenylmethanesulfonyl fluoride 100 µmol/L as described.²⁹ The homogenates were centrifuged at 100 000g; the pellet contained phospholamban, and the supernatant contained troponin I and C-protein.

Protein Phosphorylation
The method used, back-phosphorylation, has previously been described²⁹ and adapted to human cardiac tissue.^{14 25} The phosphorylation reaction was started by the addition of [-³²P]ATP and the catalytic subunit of cAMP-dependent protein kinase. We used 30 µg of protein per assay. After 5 minutes of incubation, the reaction was terminated with trichloroacetic acid, the resulting pellets were solubilized and boiled, and proteins were separated by PAGE.²⁹ Radioactive bands corresponding to phospholamban, troponin I, and C-protein were identified according to molecular mass and by immunodetection.¹⁴ The calculated difference in ³²P incorporation between control and agonist-treated trabeculae was considered to reflect agonist-induced endogenous phosphate incorporation into the intact trabeculae and is expressed as picomoles of phosphate per 1 mg protein. Protein was determined³⁰ with BSA as the standard.

Site-Specific Western Blot Analysis of Phospholamban
Specific antibodies against phospholamban phosphoserine 16 and phospholamban phosphothreonine 17 were used as reported.³¹ Crude membrane fractions were solubilized at room temperature in a lysis buffer containing SDS. Blots were incubated with antibodies raised against a synthetic oligopeptide sequence of phospholamban with either a phosphorylated serine 16 or threonine 17 residue³¹ and visualized with an enhanced chemiluminescence-based detection system. To demonstrate the specificity of the immunological reaction, the procedures were also performed in the presence of 0.1 µmol/L of the corresponding 11–amino-acid-residue oligopeptides of phospholamban, phosphorylated at either Ser16 or Thr17.

Statistical Analysis
Data are expressed as sample mean±SE. Student's paired t test or 1-way ANOVA followed by the Bonferroni method was used for multiple comparisons by use of InStat (GraphPad software version 2.0). We used P<0.05 as the limit for statistical significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Positive Inotropic and Lusitropic Effects Mediated Through ß₁- and ß₂-Adrenergic Receptors
(-)-Norepinephrine caused positive inotropic effects in the presence of the ß₂-selective blocker ICI118551 that were antagonized with high potency by the ß₁-selective blocker CGP20712A. The threshold concentrations of (-)-norepinephrine to increase contractile force were 300 nmol/L and 60 µmol/L in the absence and presence of CGP20712A (300 nmol/L), respectively, and were therefore mostly mediated through ß₁-adrenergic receptors. (-)-Epinephrine caused positive inotropic effects in the presence of the ß₁-selective blocker CGP20712A that were antagonized by the ß₂-selective ICI118551. The threshold concentrations of (-)-epinephrine were 60 nmol/L and 20 µmol/L in the absence and presence of ICI118551 (50 nmol/L), respectively, consistent with mediation through ß₂-adrenergic receptors. These results from hearts in terminal failure (data from 22 trabeculae of patients 1 through 3 of Table 1; not shown) agree with previous data from ventricular tissue from hearts in mild failure²⁷ and ventricular myocytes.²⁶

To obtain robust phosphorylation signals, we selected a relatively high catecholamine concentration, 10 µmol/L, which, however, under the conditions of the present and previous studies,^{26 27} is receptor subtype–specific and produces nearly maximal effects through ß₁- or ß₂-adrenergic receptors. Marked positive inotropic and lusitropic effects of (-)-norepinephrine and (-)-epinephrine, mediated through ß₁- and ß₂-adrenergic receptors, respectively, were similar to those of (-)-isoproterenol (Figures 1 and 2). Zinterol, a ß₂-selective partial agonist effective in human ventricle³² and atrium,²⁵ also caused positive inotropic and lusitropic effects, but the lusitropic effects tended to be smaller than those of the catecholamines (Figures 1 and 2) and the onset of response tended to be slower than with catecholamines (Figure 1).

View larger version (20K): [in this window] [in a new window]   Figure 1. Positive inotropic and lusitropic effects of ß-adrenergic receptor agonists on 4 right ventricular trabeculae from patient 7 in Table 1. Tracings show recordings of contractile force and, immediately underneath, first derivative. Right-side diagrams are superimposed fast-speed recordings before and 5 minutes after agonist administration. Trabeculae were incubated for 5 minutes with 300 nmol/L CGP20712A and 10 µmol/L (-)-epinephrine or 10 µmol/L zinterol to selectively stimulate ß2-adrenergic receptors, 300 nmol/L CGP20712A and 200 µmol/L (-)-isoproterenol to stimulate ß1- and ß2-adrenergic receptors, or 50 nmol/L ICI118551 and 10 µmol/L (-)-norepinephrine to selectively stimulate ß1-adrenergic receptors. Agonist administration is indicated by arrow. Each agonist caused a positive inotropic response associated with reduction in time to reach peak force and hastened relaxation. Shown are fast-speed recordings of individual contractions (refer to bar calibrated at 100 ms) and slow-speed recordings (refer to bar calibrated at 1 minute).

View larger version (22K): [in this window] [in a new window]   Figure 2. Effects of ß-adrenergic agonists on contractile force, time to peak force, and time to reach 50% relaxation (t50). Trabeculae were incubated with 10 µmol/L (-)-epinephrine (EPI) or 10 µmol/L zinterol (ZINT) in presence of 300 nmol/L CGP20712A to selectively stimulate ß2-adrenergic receptors, 200 µmol/L (-)-isoproterenol (ISO) in presence of 300 nmol/L CGP20712A to stimulate ß1- and ß2-adrenergic receptors, or 10 µmol/L (-)-norepinephrine (NE) in presence of 50 nmol/L ICI118551 to stimulate ß1-adrenergic receptors. Agonist-evoked changes are depicted by black. There were no differences in basal values for force, time to peak force, or t50 between groups (P>0.1). Numbers of patients are given in parentheses under the number of trabeculae. Values given are sample mean±SE. *P<0.05 for effects of ß-adrenergic agonists.

Comparison of Inotropic and Lusitropic Potencies of (-)-Norepinephrine and (-)-Epinephrine Through ß₁- and ß₂-Adrenergic Receptors
To compare the inotropic and lusitropic potency of (-)-norepinephrine through ß₁-adrenergic receptors with the corresponding potencies of (-)-epinephrine through ß₂-adrenergic receptors, we determined concentration-effect curves for the 2 catecholamines under receptor-selective conditions. The lusitropic potencies of both (-)-norepinephrine and (-)-epinephrine were significantly greater than the corresponding inotropic potencies (Figure 3 and Table 2).

View larger version (34K): [in this window] [in a new window]   Figure 3. Concentration-dependent effects of (-)-norepinephrine in presence of 50 nmol/L ICI118551 (selective stimulation of ß1-adrenergic receptors) and (-)-epinephrine in presence of 300 nmol/L CGP20712A (selective stimulation of ß2-adrenergic receptors) on contractile force, time to peak force, and time to reach 50% relaxation (t50). shows basal levels; , effects of (-)-norepinephrine or (-)-epinephrine; , maximal stimulation of ß1- and ß2-adrenergic receptors with 200 µmol/L (-)-isoproterenol; and , effect of raising extracellular Ca2+ to 9.25 mmol/L. Values given are sample mean±SE from 11 patients. Basal values for force and t50 were not different between tissues used for (-)-norepinephrine and (-)-epinephrine (P>0.2). Basal time to peak force values were shorter in tissues used for (-)-norepinephrine concentration-response curves (P=0.01), possibly because of stimulation of ß1-adrenergic receptors by endogenous (-)-norepinephrine. Errors not shown when smaller than symbol.

View this table: [in this window] [in a new window]   Table 2. Inotropic and Lusitropic Potencies and Intrinsic Activities of (-)-Norepinephrine and (-)-Epinephrine Through ß1- and ß2-Adrenergic Receptors

High Ca²⁺ Does Not Hasten Relaxation
High extracellular calcium concentration has been shown to abbreviate the duration of Ca²⁺ transients in canine cardiomyocytes²³ and to hasten relaxation in guinea pig cardiomyocytes.³³ To examine the effects of a high Ca²⁺ concentration, we compared the effects of 9.25 mmol/L CaCl₂ with those of basal conditions (2.25 mmol/L) in trabeculae from patients 8 and 15 through 17 in Table 1. Increasing extracellular Ca²⁺ to 9.25 mmol/L failed to hasten relaxation, whereas (-)-norepinephrine, administered as a positive control, hastened relaxation (Figure 4). In 11 trabeculae from 4 patients, Ca²⁺ 9.25 mmol/L increased contractile force from 1.2±0.4 (SE from 4 patients) to 6.6±2.0 mN/mm² but did not change relaxation parameters. The t₅₀ and time to peak values were 127±7 and 126±5 ms, and 179±15 and 186±7 ms at 2.25 and 9.25 mmol/L CaCl₂, respectively.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effects of raising extracellular Ca2+ to 9.25 mmol/L (in presence of 300 nmol/L CGP20712A and from 2.25 mmol/L Ca2+) of 10 µmol/L (-)-norepinephrine (in presence of 50 nmol/L ICI118551 and 2.25 mmol/L Ca2+) on individual trabeculae from patient 16. Effects on contractile force are shown above the differentiated signal. Right-side diagrams are superimposed fast-speed recordings before and after stimulation. Note hastening of relaxation onset with (-)-norepinephrine but not with high Ca2+ concentration (9.25 mmol/L). Arrow indicates administration of Ca2+ or (-)-norepinephrine. Shown are fast-speed recordings of individual contractions (refer to bar calibrated at 100 ms) and slow-speed recordings (bar calibrated at 1 minute).

Phosphorylation of Phospholamban, Troponin I, and C-Protein
Both ß₁- and ß₂-adrenergic agonists reduced the capacity of tissues to incorporate ³²P-phosphate into phospholamban, troponin I, and C-protein in vitro, as assessed after back-phosphorylation and shown representatively in Figure 5. This result indicates a higher endogenous phosphorylation state of each protein in the intact tissue after 5 minutes of exposure to the agonists compared with that of non–agonist-exposed tissues. The amount of protein phosphorylation in the intact ventricular trabeculae caused by the agonists was calculated from the back-phosphorylation reaction. (-)-Norepinephrine, (-)-epinephrine, and (-)-isoproterenol caused similar phosphorylation of phospholamban, troponin I, and C-protein through ß₁-, ß₂-, or ß₁- plus ß₂-adrenergic receptors, respectively (Figure 6). Zinterol also induced phosphorylation of the 3 proteins through ß₂-adrenergic receptors (Figure 6).

View larger version (58K): [in this window] [in a new window]   Figure 5. Autoradiograms demonstrating the effects of (-)-epinephrine (E, 10 µmol/L) and (-)-isoproterenol (ISO, 200 µmol/L) in presence of CGP20712A (300 nmol/L) on phosphorylation of phospholamban (PLB; A) and of C-protein and troponin I (B) in intact human ventricular trabeculae. (-)-Epinephrine, acting through ß2-adrenergic receptors, and (-)-isoproterenol, acting through both ß1- and ß2-adrenergic receptors, reduced in vitro back-phosphorylation catalyzed by PKA, indicating that phosphorylation of these proteins occurred in vivo. Molecular weight (Mr) standards are shown on right. CGP indicates ventricular trabeculae incubated in the presence of CGP20712A only.

View larger version (32K): [in this window] [in a new window]   Figure 6. Effects of ß-adrenergic agonists, 10 µmol/L (-)-epinephrine (EPI) and 10 µmol/L zinterol (ZIN), in presence of 300 nmol/L CGP20712A (selective stimulation of ß2-adrenergic receptors), 200 µmol/L (-)-isoproterenol (ISO) in presence of 300 nmol/L CGP20712A (stimulation of ß1- and ß2-adrenergic receptors,) and 10 µmol/L (-)-norepinephrine (NE) in presence of 50 nmol/L ICI118551 (selective stimulation of ß1-adrenergic receptors) on phosphorylation of phospholamban, troponin I, and C-protein in individual trabeculae. Trabeculae were incubated with agonists for 5 minutes. Numbers refer to individual patients. *P<0.05.

Site-Specific Phosphorylation of Phospholamban
Both (-)-norepinephrine and (-)-epinephrine induced site-specific phosphorylation of Ser16 and Thr17 residues of phospholamban through ß₁- (2 patients) and ß₂-adrenergic receptors (5 patients), respectively (Figure 7), as demonstrated in competition assays with synthetic phospho-oligopeptides (Figure 7). The (-)-epinephrine-evoked phosphorylation of phospholamban at both Ser16 and Thr17 was prevented by ICI118551 (50 nmol/L) (Western blots from 1 patient; not shown).

View larger version (48K): [in this window] [in a new window]   Figure 7. Left, Site-specific phosphorylation of phospholamban (PLB) at Ser16 (PSer-PLB, top) and Thr17 (PThr-PLB, bottom) evoked in presence of CGP20712A (300 nmol/L, CGP) by (-)-epinephrine (10 µmol/L, E) through ß2-adrenergic receptors, by (-)-isoproterenol (200 µmol/L, ISO) through both ß1- and ß2-adrenergic receptors, and by (-)-norepinephrine (NE, 10 µmol/L) in presence of ICI118551 (ICI, 50 nmol/L) through ß1-adrenergic receptors. Phosphorylated phospholamban was probed with antibodies specifically directed against either PSer-PLB or PThr-PLB. Right, To confirm immune specificity, immunoreaction was quenched by addition of 0.1 µmol/L PSer16-PLB oligopeptide (top) or PThr17-PLB oligopeptide (bottom).

A trabeculum of 1 patient (patient 8 in Table 1) was exposed to high Ca²⁺ (9.25 mmol/L), which increased contractile force (basal, Ca²⁺ 2.25 mmol/L) from 1.4 to 10.5 mN but did not shorten the t₅₀ of relaxation, shorten the time to the onset of relaxation, or produce phosphorylation at Ser16 or Thr17 of phospholamban (not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Both ß₁- and ß₂-Adrenergic Receptors Hasten Relaxation Through cAMP Pathways
Our results show that both the ß₁- and ß₂-adrenergic receptors mediate hastening of relaxation of human ventricular myocardium, consistent with an involvement of cAMP-dependent pathways for both receptors. (-)-Norepinephrine and (-)-epinephrine had similar lusitropic potencies through ß₁- and ß₂-adrenergic receptors, respectively, and the lusitropic intrinsic activity of (-)-epinephrine was nearly that of (-)-norepinephrine. As expected from cAMP pathways, the PKA substrates implicated in relaxation,^{1 2 3 4 5 6} Ser16 phospholamban, troponin I, and C-protein were phosphorylated by (-)-norepinephrine through ß₁-adrenergic receptors and by (-)-epinephrine and zinterol through ß₂-adrenergic receptors. These findings are at variance with evidence in all other species studied so far (cat, sheep, rat, dog),^{18 19 20 21 22 23} in which ß₂-adrenergic receptors, unlike ß₁-adrenergic receptors, appear reluctant to couple to the cAMP pathway that hastens relaxation, and stress the importance of direct work on human myocardium.

Species-dependent differences between the functions of ß₁- and ß₂-adrenergic receptors may possibly be due to differences in coupling to the G_s-protein/cAMP pathway and differential coupling to G_s and G_i, guanine nucleotide–sensitive transducer proteins that cause activation and inhibition of the cAMP pathway, respectively. In cat heart, stimulation of the G_s/cAMP pathway through ß₁- and ß₂-adrenergic receptors is proportional to the corresponding receptor densities,³⁴ suggesting similar coupling of these 2 receptors, but only ß₁-adrenergic receptors hasten relaxation.¹⁸ In contrast, human cardiac ß₂-adrenergic receptors are more tightly coupled to the G_s/cAMP pathway^{27 28 35 36} than are ß₁-adrenergic receptors, and this was later confirmed with human recombinant receptors.^{37 38} The selective coupling of human ß₂-adrenergic receptors probably contributes to the marked lusitropic effects of (-)-epinephrine.³⁶ In ventricular myocytes from nonfailing rat hearts, the ß₂-adrenergic–G_s/cAMP pathway leading to relaxation can be demonstrated only in the presence of pertussis toxin,²² presumably after inactivation of functional G_i. This suggests that coupling of the rat cardiomyocyte ß₂-adrenergic receptor is tighter to G_i than to G_s. The tighter coupling of human ß₂-adrenergic receptors to the G_s/cAMP pathway compared with ß₁-adrenergic receptors may explain why, even in failing human ventricle (this work) in which G_i function and mRNA levels (G_ia2) are increased,^{39 40 41} ß₂-adrenergic receptor–mediated relaxation can still occur through the G_s/cAMP pathway. It is thus possible that in nonfailing hearts with unaltered G_i function, ß₂-adrenergic receptors may mediate even more marked relaxant effects than in failing hearts.

One limitation of this study is that the magnitude of lusitropic effects did not correlate (not shown) with the degree of protein phosphorylation in the same ventricular preparation. Protein phosphorylation peaks may have preceded the observed lusitropic response, an issue that can be clarified only with future kinetic experiments. Our findings of significant PKA-dependent phosphorylation of phospholamban, troponin I, and C-protein through both ß₁- and ß₂-adrenergic receptors agree, however, with an earlier report of PKA stimulation⁴² suggesting a causal relationship. Unlike the situation in many other species,^{18 19 20 21 22 23} human ventricular (this work) and atrial²⁵ ß₂-adrenergic receptors appear to function mainly through a PKA-dependent pathway. Support for this concept has also recently been provided for the ß₂-adrenergic receptor–mediated increases in L-type Ca²⁺ current with an obligatory involvement of PKA in human atrial myocytes.⁴³ Zinterol increased the L-type Ca²⁺ current and appeared to dissociate slowly from the ß₂-adrenergic receptors.⁴³ Correspondingly, we attribute the relatively slow inotropic and lusitropic onset observed in human ventricular trabeculae (Figure 1) to slow equilibration of zinterol with ß₂-adrenergic receptors.

The catecholamine (-)-isoproterenol can induce phosphorylation of phospholamban at both Ser16 (through PKA) and Thr17 (through CaM kinase) in rodent ventricle.^{7 44} The CaM kinase–catalyzed phosphorylation of phospholamban can contribute to increased contractility and hastened relaxation, provided dephosphorylation is negligible.⁴⁴ Type 1 phosphatase catalyzes this dephosphorylation, and the activity of type 1 phosphatase can, in turn, be inhibited by isoproterenol through PKA-catalyzed phosphorylation of protein phosphatase inhibitor-1 in guinea pig ventricular myocytes.⁴⁵ Our results show for the first time that Thr17 phosphorylation of phospholamban occurs in failing human ventricle through both ß₁- and ß₂-adrenergic receptors. We suggest that the CaM kinase–catalyzed phosphorylation of Thr17 of phospholamban in human ventricle can be demonstrated because its dephosphorylation is retarded by simultaneous inhibition of type 1 phosphatase by the ß₁- or ß₂-adrenergic receptor–mediated phosphorylation of protein phosphatase inhibitor-1. High extracellular Ca²⁺ concentration does not appear to result in activation of sarcoplasmic reticulum CaM kinase because, in contrast to the catecholamines, it does not hasten relaxation in trabeculae from nonfailing¹² or failing hearts (Figure 4) and can actually prolong contractions and Ca²⁺ transients of trabeculae from failing human ventricle.¹² In agreement with this, we have seen in the trabeculae of patient 8 in Table 1 that high Ca²⁺ concentration did not hasten relaxation and did not induce phosphorylation at Thr17 or Ser16 of phospholamban, whereas (-)-epinephrine caused these effects through ß₂-adrenergic receptors, in line with an indirect role of PKA but not necessarily of high Ca²⁺ concentration per se. In contrast, concentrations of (-)-norepinephrine (through ß₁-adrenergic receptors) and (-)-epinephrine (through ß₂-adrenergic receptors) and Ca²⁺, which cause matching increases in contractile force, are associated with marked hastening of relaxation with the catecholamines only (even in the presence of high Ca²⁺ concentration; Figure 3) but not with Ca²⁺ alone.

Possible Clinical Relevance
We have conclusively shown that (-)-epinephrine, acting through ß₂-adrenergic receptors, and (-)-norepinephrine, acting through ß₁-adrenergic receptors, hasten relaxation with similar potency and efficacy and cause phosphorylation of proteins implicated in the relaxation process. These results require verification in myocardium from normal hearts of donors not treated with drugs, such as ACE inhibitors. It seems reasonable, however, to suggest that diastolic function of failing heart may be improved by the action of endogenous catecholamines, mediated through both ß₁- and ß₂-adrenergic receptors. For example, it is conceivable that during stress endogenous plasma epinephrine surges elicit not only tachycardia but also beneficial hastening of ventricular relaxation, mediated at least partly through ß₂-adrenergic receptors, thus producing a relative lengthening of diastole.

Because ß₂-adrenergic receptors hasten relaxation in failing human ventricular myocardium, it could be clinically desirable to selectively improve diastolic function under conditions in which ß₁-adrenergic receptors are blocked. This may happen in patients with chronic heart failure undergoing treatment with ß₁-selective blockers.⁴⁶ It is plausible that in these patients endogenous epinephrine may actually contribute to an improvement in diastolic function via ß₂-adrenergic receptors. The likelihood of this occurring is enhanced by the observation that at least in human atrium ß₂-adrenergic receptor function is increased by long-term ß₁-adrenergic receptor blockade.²⁴

	Acknowledgments

 
Dr Kaumann is grateful to the British Heart Foundation and Dr Molenaar to the NHMRC (Australia) for support. Petra Hempel was supported by the Sonnenfeld-Stiftung, Berlin, Germany. We thank Dr David Kaye for patient details and the surgeons of Papworth-Everard Hospital (Cambridgeshire, United Kingdom) and Alfred Hospital (Melbourne, Australia) for explanted hearts.

Received May 26, 1998; revision received August 19, 1998; accepted September 22, 1998.

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