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(Circulation. 2000;101:1707.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Early and Delayed Consequences of ß₂-Adrenergic Receptor Overexpression in Mouse Hearts

Critical Role for Expression Level

Stephen B. Liggett, MD; Nicole M. Tepe, PhD; John N. Lorenz, PhD; Amy M. Canning, BS; Tamara D. Jantz, BS; Sayaka Mitarai, MD; Atsuko Yatani, PhD; Gerald W. Dorn, II, MD

From the Departments of Medicine (S.B.L., A.M.C., T.D.J., G.W.D.) and Pharmacology (S.B.L., N.M.T., J.N.L., S.M., A.Y., G.W.D.), University of Cincinnati Medical Center, Cincinnati, Ohio.

Correspondence to G.W. Dorn II, University of Cincinnati Medical Center, 231 Bethesda Ave, ML 0590, Cincinnati, OH 45267-0590. E-mail dorngw{at}ucmail.uc.edu

	Abstract

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Background—Transgenic cardiac ß₂-adrenergic receptor (AR) overexpression has resulted in enhanced signaling and cardiac function in mice, whereas relatively low levels of transgenically expressed G_s or ß₁AR have resulted in phenotypes of ventricular failure. Potential relationships between the levels of ßAR overexpression and biochemical, molecular, and physiological consequences have not been reported.

Methods and Results—We generated transgenic mice expressing ß₂AR at 3690, 7120, 9670, and 23 300 fmol/mg in the heart, representing 60, 100, 150, and 350 times background ßAR expression. All lines showed enhanced basal adenylyl cyclase activation but a decrease in forskolin- and NaF-stimulated adenylyl cyclase activities. Mice of the highest-expressing line developed a rapidly progressive fibrotic dilated cardiomyopathy and died of heart failure at 25±1 weeks of age. The 60-fold line exhibited enhanced basal cardiac function without increased mortality when followed for 1 year, whereas 100-fold overexpressors developed a fibrotic cardiomyopathy and heart failure, with death occurring at 41±1 weeks of age. Adenylyl cyclase activation did not correlate with early or delayed decompensation. Propranolol administration reduced baseline +dP/dt_max to nontransgenic levels in all ß₂AR transgenics except the 350-fold overexpressors, indicating that spontaneous activation of ß₂AR was present at this level of expression.

Conclusions—These data demonstrate that the heart tolerates enhanced contractile function via 60-fold ß₂AR overexpression without detriment for a period of 1 year and that higher levels of expression result in either aggressive or delayed cardiomyopathy. The consequences for enhanced ßAR function in the heart appear to be highly dependent on which signaling elements are increased and to what extent.

Key Words: receptors, adrenergic, beta • cardiomyopathy • heart failure

	Introduction

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Catecholaminergic activation of cardiac ß-adrenergic receptors (ßARs) regulates the acute hemodynamic response to stress or injury by increasing myocardial contractility and heart rate. A deleterious effect of chronic ßAR stimulation is suggested by clinical heart failure studies in which sympathomimetic agents are associated with a poor outcome,^{1 2 3} by a benefit of ßAR blockade in heart failure,^{4 5 6} and by reports of catecholamine-induced cardiomyopathies.^{7 8 9 10} Increasing cardiac adrenergic signaling by overexpressing ßARs or inhibiting the ßAR kinase, however, appears to have therapeutic potential in heart failure, as demonstrated by favorable effects in genetically modified mice. Transgenic overexpression of ß₂ARs in mouse cardiomyocytes increased basal and isoproterenol-stimulated adenylyl cyclase activity and enhanced resting cardiac systolic function.^{11 12} ß₂AR overexpression normalized the characteristic resting systolic dysfunction in the G_q transgenic mouse model of hypertrophy¹³ but failed to improve a murine genetic dilated cardiomyopathy.¹⁴ Interestingly, augmenting ßAR signaling through overexpressing a peptide inhibitor of the ßAR kinase enhanced ßAR-stimulated cardiac function¹⁵ and significantly improved function in murine dilated cardiomyopathy¹⁴ but not contractile depression in the G_q hypertrophy model.¹³ From these results, it appears that at least in mice, chronic enhancement of ßAR signaling can augment contractile function in the normal heart and provide functional benefit. However, the favorable effects reported in ß₂AR-overexpressing mice contrast with dilated cardiomyopathy and myocardial fibrosis after overexpression of ß₁AR or G_s, the signaling protein that couples ßAR to adenylyl cyclase.^{16 17}

What is lacking in our understanding of pathological effects of enhanced ßAR signaling is a long-term assessment of signaling, structure, and in vivo function of multiple mouse lines expressing a range of a given signaling protein. To address this, transgenic mice were generated with ß₂AR overexpression ranging from 60 to 350 times background. The results reported here confirm a long-term positive inotropic effect of ß₂AR overexpression at 60-fold endogenous ßAR levels but also demonstrate that a long-term consequence of ß₂AR overexpression at higher levels is progressive myocardial fibrosis, ultimately leading to heart failure.

	Methods

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Transgenic Mice
The wild-type human ß₂AR cDNA was ligated into the Sal-1 site (exon 3) of the full-length 5.5-kb -myosin heavy chain promoter essentially as previously described.¹² The linearized constructs were injected into male pronuclei of fertilized FVB/N mouse oocytes and implanted into pseudopregnant female oviducts. Thirteen founders were identified by genomic Southern analysis.

Analysis of Cardiac Function
In vivo ventricular function was measured with invasive and noninvasive techniques essentially as previously described^{12 18 19} in lightly anesthetized (ketamine/thiobutabarbital), spontaneously breathing, closed-chest mice. Noninvasive cardiac function was assessed by 2D guided M-mode echocardiography of tribromoethanol-anesthetized mice. In some cases, studies were performed before and after intraperitoneal administration of 100 ng/g isoproterenol.

Assessment of Cardiac Hypertrophy
Hypertrophy was assessed gravimetrically by an analytical balance and by RNA dot-blot Northern analysis of hypertrophy-associated genes as previously described.^{18 19}

ßAR Signaling Studies
Radioligand binding with ¹²⁵I-cyanopindolol to ventricular membranes was performed as previously described.^{12 13} For adenylyl cyclase activities, ventricular membranes (10 µg) were coincubated with (mmol/L) phosphoenolpyruvate 2.8, GTP 0.06, ATP 0.12, cAMP 0.1, and ascorbic acid 0.1; and 4 U/mL myokinase, 10 U/mL pyruvate kinase, and 3x10⁶ dpm [-³²P]ATP for 10 minutes at 37°C with various concentrations of isoproterenol, 10 mmol/L NaF, or 100 µmol/L forskolin. Reactions were stopped by dilution with 1.0 mL of a 4°C solution containing excess ATP and cAMP and 25 000 dpm/mL [³H]cAMP (used for column recovery). [³²P]cAMP was separated by chromatography over alumina columns. GRK2 and G_i2/3 were measured in whole-heart homogenates by immunoblotting using standard techniques and antibodies from Santa Cruz Biotechnology as we have previously described in detail.^{20 21}

Measurement of Calcium Currents
Whole-cell patch clamp studies were performed on ventricular cardiomyocytes as described previously.^{22 23} In the studies of basic Ca²⁺ channel kinetics, cells were dialyzed with 5 mmol/L EGTA. For isoproterenol experiments, EGTA was replaced with 10 mmol/L BAPTA to prevent Ca²⁺-dependent inactivation.²⁴

Statistical Analysis
Unless stated otherwise, data are presented as mean±SEM. Transgenic mice from a single line and nontransgenic littermates were compared by 2-tailed Student’s t test. Multiple comparisons between different lines or at different ages within 1 line were performed with 1-way ANOVA followed by a Bonferroni procedure. In vivo dose-response data were analyzed by a mixed-factor ANOVA, with repeated measures on the second factor. A value of P<0.05 was considered significant.

	Results

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To better understand the effects of cardiac ß₂AR overexpression, we created a series of ß₂AR-overexpressing mouse lines in the FVB/N background with a range in transgene copy number. Of 13 founder mice, 1 failed to breed; 3 failed to transmit the transgene to progeny, suggesting that they were chimeric; independent lines were established for 5 founders; 4 other founders died before breeding; and 1 established line was lost before the present studies. The 4 founders that died had massive cardiac enlargement with dilated atria (Figure 1), pulmonary congestion, and pleural effusions.

View larger version (55K): [in this window] [in a new window]   Figure 1. Cardiac phenotype of ß2AR-overexpressing mice. A, A 25-week-old ß2-60 mouse has normal size heart and no gross or microscopic abnormalities. B, A 25-week-old ß2-350 mouse exhibits 4-chamber enlargement with severe left ventricular dilatation and atrial mural thrombi. Histological examination of this heart revealed severe fibrotic replacement of left ventricular myocardium (see Figure 4A). C, One of 4 ß2AR-overexpressing founder mice that died of heart failure. Left atrium (LA) is massively enlarged with extensive laminar thrombus. Left ventricle (LV) is enlarged and hypertrophied. RV indicates right ventricle.

Pharmacological Properties
The 4 successfully propagated -myosin heavy chain (MHC)–ß₂AR transgenic lines exhibited a range of ß₂AR expression (¹²⁵I-cyanopindolol binding; Table 1) 60-, 100-, 150-, and 350-fold greater than nontransgenic and were so designated (ß₂-60, etc). Results from adenylyl cyclase studies are shown in Figure 2 and Table 1. When normalized to NaF stimulation (Figure 2B), basal activities were increased 3-fold in the 2 lowest-expressing lines and 2-fold in the 2 higher-expressing lines. The extent of maximal isoproterenol stimulation was also increased with each line compared with nontransgenic. However, the maximal increase was not commensurate with the increase in basal, so the fold stimulation over basal was in fact decreased. Absolute levels of activity (pmol · min^-1 · mg^-1) are shown in Figure 2A and Table 1. As can be seen, only the ß₂-60 line had higher basal activities when the data are expressed in this way. Both NaF- and forskolin-stimulated adenylyl cyclase activities were depressed to similar extents in all transgenic lines (Figure 2, C and D), suggesting that a compensatory event distal to the receptor occurs with chronic overexpression of ß₂AR at these levels. We therefore assessed by immunoblotting possible changes in expression of ßAR kinase (GRK2) or G_i, both of which are known to alter ßAR signaling when increased in the heart. Neither ßAR kinase nor G_i2/3 levels were increased in any of the ß₂AR overexpressors (data not shown). Immunoreactivity of adenylyl cyclase type V/VI was not sufficient for accurate determination, so it is not possible to exclude a change in adenylyl cyclase expression with ß₂AR overexpression.

View this table: [in this window] [in a new window]   Table 1. Characteristics of ß2AR-Overexpressing Mouse Lines

View larger version (30K): [in this window] [in a new window]   Figure 2. Agonist stimulation of cardiac adenylyl cyclase activity in ß2AR-overexpressing mice. A, Agonist-stimulated adenylyl cyclase activity of transgenic and nontransgenic animals. B, Isoproterenol-stimulated adenylyl cyclase activity in cardiac membranes of nontransgenic and ß2AR overexpressing mice normalized to NaF activation. Shown are means of 4 to 7 experiments per line. Error bars are omitted for clarity (see Table 1). C and D, NaF- and forskolin-stimulated activities.

Early Effects
Initial phenotypic characterizations and interline comparisons of mice 10 to 15 weeks old demonstrated pathology in ß₂-350 mice. Gross morphological analysis revealed a 25% increase in heart weight of the ß₂-350 line compared with the other ß₂AR lines or with nontransgenic littermates (Table 1). Whereas echocardiographic left ventricular fractional shortening was enhanced in the 3 lower-expressing ß₂AR lines (Table 1), ß₂-350 exhibited impaired left ventricular function with ventricular enlargement (Table 1). Increased cardiac ßMHC and atrial natriuretic factor gene expression, characteristic features of cardiac hypertrophy, were also detected only in ß₂-350 mice (see Table 1 and below). Furthermore, whereas the 3 lower-expressing ß₂AR lines showed no histological evidence of fibrosis or cardiomyocyte hypertrophy (not shown), ß₂-350 hearts showed replacement fibrosis (see below). Thus, young adult mice expressing ß₂AR at 350 times normal levels developed cardiomegaly, increased expression of hypertrophy-associated genes, and depressed systolic function, whereas multiple transgenic lines with lower ß₂AR expression levels had normal cardiac size and gene expression with enhanced systolic function.

Late Effects
Because cardiomegaly, fetal gene expression, and fibrosis in young adult mice were observed only in the highest-expressing ß₂AR transgenic line, we considered that a longitudinal analysis of ß₂AR overexpressors might detect additional phenotypic features related to duration of transgene expression. Cohorts of 20 to 30 mice from each of 3 lines (ß₂-60, -100, and -350) were therefore prospectively followed for 1 year. No increase in all-cause mortality was found in ß₂-60 mice compared with nontransgenic littermates during this period (Figure 3). Early mortality was, however, observed in ß₂-350 mice, which died at 25±1 weeks (Figure 3), suggesting an association between high levels of ß₂AR expression and early death. The cause of death appeared to be left heart failure, as shown by pulmonary congestion (increased lung weights) and massive cardiac enlargement (Table 2). ß₂-100 mice also developed cardiac enlargement, heart failure, and premature death, but death occurred at 41±1 weeks (Figure 3, Table 2). Thus, these longitudinal mortality data demonstrate delayed deleterious effects of ß₂AR overexpression that are proportional in severity and rapidity of progression to the level of cardiac ß₂AR.

View larger version (20K): [in this window] [in a new window]   Figure 3. Impaired survival of high-level ß2AR overexpressors. Kaplan-Meier survival curve of ß2AR-overexpressing mouse lines followed for 50 weeks.

View this table: [in this window] [in a new window]   Table 2. Characteristics of ß2AR-Overexpressing Mice That Develop Heart Failure

Because these studies indicated that mortality in ß₂-350 mice was a function of the age of the animal, histological, morphometric, and molecular analyses were performed at 7, 11, and 17 weeks of age. These studies showed that the decline in left ventricular function was associated with cardiomyocyte dropout and fibrotic replacement. Fibrosis was not evident at 7 weeks, was apparent at 11 weeks, and was marked by 17 weeks of age (Figure 4A). The pattern of fetal cardiac gene expression remained constant over time (Figure 4B).

View larger version (110K): [in this window] [in a new window]   Figure 4. Cardiac deterioration with high levels of ß2AR. A, Representative Masson’s trichrome stains (magnification x100) of full-thickness apical left ventricular free wall from 7-, 11-, 17-, and 25-week-old ß2-350 overexpressors showing progressive cardiomyocyte loss and fibrotic replacement with increasing age. The 25-week heart depicted is same as that shown in Figure 1B. B, RNA dot-blot analysis of cardiac gene expression shows a pattern of increased fetal gene expression in 7-, 11-, and 17-week-old ß2-350 mice. SERCA indicates sarcoplasmic reticulum calcium ATPase; ANF, atrial natriuretic factor; sk, skeletal; card, cardiac; and PLB, phospholamban.

In Vivo Hemodynamics
To more rigorously assess the effects of ß₂AR density and duration of expression on cardiac functional status, we performed in vivo hemodynamic studies on 12- and 20- to 24-week ß₂-350 mice compared with 12-week ß₂-60 mice. Whereas baseline heart rates were significantly increased in ß₂-60, heart rates were not increased in either 12- or 20-week-old ß₂-350 mice (Table 3). Basal +dP/dt was doubled in ß₂-60 mice and increased in 12-week-old but not 20-week-old ß₂-350 mice (Table 3). Thus, enhanced basal cardiac systolic function was observed in mice expressing lower levels of ß₂AR and in younger mice expressing high levels of ß₂AR. With development of cardiomegaly in the latter mice, resting systolic function decreased.

View this table: [in this window] [in a new window]   Table 3. Invasive Hemodynamic Studies of ß2AR Overexpressors

Because cardiac phenotypes resulting from ß₂AR overexpression may be a consequence of either enhanced agonist-stimulated ßAR function or an increase in spontaneous receptor activation,¹¹ we assessed hemodynamic responsiveness to isoproterenol. As shown in Table 3 and Figure 5A, inotropic and chronotropic responses to isoproterenol were generally blunted in the ß₂AR-overexpressing mice. In ß₂-60 mice, this was because basal heart rate and +dP/dt were already near maximal. Likewise, echocardiographic left ventricular fractional shortening was increased by isoproterenol in nontransgenic mice (44±3% basal, 61±3% isoproterenol, P<0.001) but not in ß₂-60 mice (54±3% basal, 56±4% isoproterenol, P=NS). However, in ß₂-350 mice, there was no dP/dt response to isoproterenol, whether basal values were increased (12 week) or normal (20 week) (Table 3, Figure 5A). The response to nonselective ßAR blockade with intravenous propranolol was used to distinguish between agonist-dependent and -independent effects of overexpressed ß₂AR (Figure 5B). Propranolol prevented isoproterenol-mediated increases in +dP/dt in nontransgenic mice, demonstrating the adequacy of the dose used. Basal +dP/dt, which was elevated in the ß₂-60 mice, was normalized by propranolol. At 12 weeks of age, when ß₂-350 mice had significantly elevated basal dP/dt, propranolol failed to normalize contractility. Thus, a critical difference between the ß₂AR overexpressors may be endogenous agonist participation in enhanced cardiac function at lower levels of ß₂AR but predominantly agonist-independent ß₂AR effects at higher levels of receptor expression.

View larger version (28K): [in this window] [in a new window]   Figure 5. Hemodynamic consequences of high- and low-level ß2AR overexpression. A, Left ventricular dP/dtmax for ß2-60 and ß2-350 mice (12 and 20 weeks) as a function of intravenous isoproterenol dose. Each point represents mean of results from 4 animals per group. B, ßAR blockade with intravenous propranolol reverses basal enhancement of dP/dt in 12-week-old ß2-60 mice but not 12-week-old ß2-350 mice.

Ca²⁺ Channel Activity
To confirm that the observed perturbations in cardiac function, myocyte signaling, and adenylyl cyclase activity did not simply reflect increased fibrotic content of ß₂-350 hearts, patch-clamp studies of inward calcium currents (I_Ca) were performed on ventricular cardiomyocytes from 12- and 24-week-old ß₂-350 mice. Cardiomyocyte capacitance, a measure of cell size, was significantly increased compared with nontransgenic siblings at 12 weeks (159.1±4.2 pF, n=129 versus 143.7±4.8 pF, n=67) and 24 weeks (274.7±14 pF, n=41 versus 145.4±5.8 pF, n=59; P<0.05), consistent with the molecular and morphometric indices of cardiac hypertrophy noted above. Whereas the voltage dependence of I_Ca in ß₂-350 cells was not altered, I_Ca density (shown in Figure 6A) was significantly reduced compared with that in nontransgenic cells (5.0±0.3 pA/pF, n=24 versus 8.9±0.4 pA/pF, n=38 at 12 weeks; 5.2±0.5 pA/pF, n=12 versus 10.3±0.6 pA/pF, n=26 at 24 weeks, P<0.05). Myocytes from 24-week ß₂-350 mice showed significantly reduced isoproterenol responsiveness (50% of control) without any change in EC₅₀ (Figure 6B). These results demonstrate progressive cardiomyocyte ßAR-Ca²⁺ dysfunction in aging ß₂-350 mice.

View larger version (19K): [in this window] [in a new window]   Figure 6. ß2-350 cardiomyocyte electrophysiological properties. A, Representative whole-cell ICa recorded in nontransgenic (NTG) and ß2-350–overexpressing myocytes at 24 weeks of age. B, Concentration-dependent effects of isoproterenol (Iso) on ICa for NTG and ß2AR-overexpressing myocytes. EC50s were 26.5±6.9, 8.2±4.7, and 43.0±30 nmol/L for NTG, ß2-350–overexpressing mice at 12 weeks, and the same at 24 weeks, respectively. Data are mean±SEM from 6 to 40 cells.

	Discussion

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The objective of these studies was to establish a "transgene dose-response" for ß₂AR expression in the heart. In pursuit of this objective, we created a series of cardiac-specific ß₂AR transgenic mice and found that the range of ß₂AR expression was associated with a spectrum of phenotypes: (1) enhanced contractile function in young mice with ß₂AR levels 60 times normal; (2) rapidly progressive fibrosis, cardiac hypertrophy, and heart failure with ß₂AR levels 350 times normal; and (3) early enhanced function with delayed development of fibrotic cardiomyopathy at intermediate expression levels. Enhanced baseline function accruing from ß₂AR overexpression at levels 60 times normal occurred in the absence of any detectable pathological consequences over a 1-year period. Although the ß₂-60 overexpressing mice are similar to ß₂AR transgenic mice initially reported by Lefkowitz and coworkers¹¹ and by us,¹² it is of course not possible to exclude the possibility that pathological characteristics may eventually develop in ß₂-60 mice as they continue to age. The particularly important findings of the present studies, however, pertain to the ß₂-100 and -350 lines, which exhibited progressive ventricular dysfunction, directly related in severity and rapidity of progression to the level of ß₂AR expression. With all lines, we observed a decrease in NaF- and forskolin-stimulated activities, but the ßAR signaling appears to be even further dampened in the higher-expressing lines. Physiologically, this is manifested as lower systolic function with a lack of responsiveness to agonist, which is common in both. This further loss of ventricular function may be due to a decrease in L-type Ca²⁺ channel density, as observed in the ß₂-350 mice, or some other post–receptor-effector derangement.

Another important distinction between the rapidly progressive ß₂-350 and ß₂-60 lines is the evidence for intrinsic receptor signaling activity. Catheterization-based hemodynamic studies in ß₂-350 mice showed no effect of ß₂AR blockade with propranolol, whereas in ß₂-60 mice, propranolol normalized the basal enhanced contractile function. As a neutral antagonist, propranolol would be expected to block endogenous catecholamine activation of the receptor but not the spontaneous transition of a proportion of ß₂AR to the active (R*) conformation. It is interesting to speculate that the unrestricted signaling activity of R* in the ß₂-350 line contributes to its aggressive cardiomyopathy. Thus, expression of ß₂AR at a level that enhances the in vivo response to agonists but does not cause in vivo ligand-independent signaling may improve cardiac function without deleterious effects. However, exceeding the putative threshold for ligand-independent ß₂AR receptor signaling may cause rapid development of the cardiomyopathic syndrome reported here, as well as additional counterregulatory effects not observed with the other lines.

The pathological and physiological characteristics exhibited by the older ß₂-350 and ß₂-100 mice reproduce some features of catecholamine cardiomyopathy as described in human subjects with pheochromocytomas.^{7 8 9 10} The pathophysiology of catecholamine-mediated cardiomyopathy has been postulated to result from ischemic necrosis secondary to intense vasospasm or from direct toxic effects of oxidized catecholamines. Our studies demonstrate that none of these putative mechanisms are necessary for development of cardiomyopathy, because ß₂ARs were selectively overexpressed in cardiac myocytes, and there is no reason to believe that circulating or local catecholamine levels are increased in these mice. Rather, these studies and those with the previously reported ß₁AR- and G_s-overexpressing models^{16 17} support a direct effect of chronic unrestricted ßAR signaling on cardiomyocytes, a notion consistent with catecholamine cardiomyocyte toxicity demonstrated in some tissue culture studies.²⁵

The variability in adenylyl cyclase responsiveness in the various ß₂AR overexpressors suggests that regulatory mechanisms may be evoked by certain levels of long-term overexpression that seem to partially desensitize receptor signaling. Changes in the expression of ßAR kinase,²⁶ G_i, and adenylyl cyclase²⁷ in the heart have been reported to be associated with decreased ßAR signaling. An increase in ßAR kinase would be expected to exclusively alter receptor-mediated stimulation, which is not the case in these ß₂AR overexpressors, in which we find forskolin- and NaF-stimulated activities also depressed, and ßAR kinase protein expression was not altered in any of the ß₂AR lines. An increase in G_i could theoretically alter basal, ßAR-mediated, and NaF-mediated signaling; however, we found no evidence of such an increase. Finally, we also considered that a decrease in adenylyl cyclase expression could serve to decrease signaling at baseline and in response to agonist, forskolin, and NaF. Indeed, given the above, a decrease in adenylyl cyclase expression seems to be quite a reasonable candidate. However, we are unable to quantitatively assess type V/VI adenylyl cyclase expression in the mouse heart, so we cannot reach a conclusion in this regard.

We have shown that cardiac ß₂AR overexpression 60 times background results in enhanced in vitro and in vivo signaling without apparent pathological consequences in mice up to 1 year old. Higher levels of expression result in delayed (ß₂-100) or rapidly progressive (ß₂-350) cardiomyopathies. That deleterious effects can occur at some level of ß₂AR expression is not altogether surprising. The obverse finding, that moderate levels of overexpression are apparently not detrimental, is contrary to the notion heralded by some that enhanced ßAR-G_s–adenylyl cyclase signaling by any means is universally deleterious. Indeed, it is notable that cardiomyopathy caused by ß₁AR overexpression is observed at 5 to 15 times endogenous ßAR levels,¹⁷ whereas a 60-fold increase in ß₂AR appears to be well tolerated. Thus, overly broad generalizations regarding potential deleterious effects of ßAR signaling via increased ß₁AR, ß₂AR, or G_s or via inhibition of ßARK may be overly simplistic, because there appear to be fundamental differences in signaling evoked by these mechanisms.

	Acknowledgments

 
This study was supported by grants GM-54169, HL-58010, HL 22619, and P50-HL-52318 from the National Institutes of Health.

Received August 13, 1999; revision received October 18, 1999; accepted November 5, 1999.

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