Published online before print September 13, 2001, doi: 10.1161/hh1901.098042
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Upregulation of Functional ß3-Adrenergic Receptor in the Failing Canine Myocardium
From the Cardiology Section, Wake Forest University School of Medicine, Winston-Salem, NC.
Correspondence to Che-Ping Cheng, MD, PhD, Cardiology Section, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1045. E-mail ccheng{at}wfubmc.edu
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Abstract |
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Abstract — Altered expression and functional responses to cardiac ß3-adrenergic receptors (ARs) may contribute to progressive cardiac dysfunction in heart failure (CHF). We compared myocyte ß3-AR mRNA and protein levels and myocyte contractile, [Ca2+]i transient, and Ca2+ current (ICa,L) responses to BRL-37344 (BRL, 10-8 mol/L), a selective ß3-AR agonist, in 9 instrumented dogs before and after pacing-induced CHF. Myocytes were isolated from left ventricular myocardium biopsy tissues. Using reverse transcription–polymerase chain reaction, we detected ß3-AR mRNA from myocyte total RNA in each animal. Using a cloned canine ß3-AR cDNA probe and myocyte poly A+ RNA, we detected a single band about 3.4 kb in normal and CHF myocytes. ß3-AR protein was detected by Western blot. ß3-AR mRNA and protein levels were significantly greater in CHF myocytes than in normal myocytes. Importantly, these changes were associated with enhanced ß3-AR–mediated negative modulation on myocyte contractile response and [Ca2+]i regulation. Compared with normal myocytes, CHF myocytes had much greater decreases in the velocity of shortening and relengthening with BRL accompanied by larger reductions in the peak systolic [Ca2+]i transient and ICa,L. These responses were not modified by pretreating myocytes with metoprolol (a ß1-AR antagonist) or nadolol (a ß1- and ß2-AR antagonist), but were nearly prevented by bupranolol or L-748,337 (ß3-AR antagonists). We conclude that in dogs with pacing-induced CHF, ß3-AR gene expression and protein levels are upregulated, and the functional response to ß3-AR stimulation is increased. This may contribute to progression of cardiac dysfunction in CHF.
Key Words: ß3-adrenergic receptor • gene expression • contractility • [Ca2+]i regulation • heart failure
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Introduction |
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Beta-adrenergic receptors (ARs) are integral membrane proteins belonging to the class of G protein–coupled receptors.1–3 It has been well-established that cardiac ß1- and ß2-ARs mediate the catecholamine-induced increase in force and frequency of cardiac contractions, thereby providing a mechanism for myocardial contractile reserve. Congestive heart failure (CHF) is associated with a progressive activation of the sympathetic nervous system (SNS), but a diminished positive inotropic response to ß-AR stimulation, in part because of a selective downregulation of ß1-ARs and an upregulated inhibitory G protein (Gi).1 However, recent observations in human hearts indicate a more complex ß-AR–mediated regulation of myocardial inotropism by catecholamines.
Evidence has been provided that ß3-ARs may also modulate cardiac function.4–7 However, unlike ß1- or ß2-ARs, the function of ß3-ARs is to inhibit cardiac contraction. Gauthier and colleagues5 first demonstrated that ß3-ARs, initially found to be widely expressed in adipose,8 are also expressed in the human heart, and that stimulation of ß3-ARs with a preferential agonist, BRL-37344 (BRL), caused dose-dependent negative inotropic effects. This ß3-AR–mediated negative inotropic effect is not linked to stimulatory G proteins but is coupled to Gi.5,6
Although the precise physiological and pathophysiologic roles of ß3-AR remain uncertain, recent observations suggest that in the normal heart, ß3-AR participates in nitric oxide (NO)–mediated negative feedback control over contractility within the SNS because stimulation of ß3-ARs with BRL resulted in a negative inotropic effect in human donor hearts through NO signaling,9 and ß3-AR deficiency blocked NO-dependent inhibition of myocardial contractility in transgenic mice.10 In failing human hearts, ß3-AR abundance was increased, and the balance between opposing inotropic influences of ß1-ARs and ß3-ARs was significantly altered, providing a potential mechanism for progressive deterioration in cardiac function.4
The role of ß3-ARs could be more clearly elucidated using defined animal models of CHF. Past studies of cardiac ß3-AR function and gene expression performed in normal animals have yielded conflicting findings5–7,11–13 because of the confounding effects of anesthesia, open-chest surgery, tissue preparation, loading conditions, species differences, and use of multicellular preparations. The molecular expression, the functional effect of ß3-ARs on single-cell mechanics, and the dynamics of the cytosolic [Ca2+]i have not been previously assessed in an integrated fashion in normal cardiomyocytes and those from animals with varying degrees of CHF. Virtually no previous studies have specifically examined ß3-AR-induced changes in calcium current (ICa,L).14 Furthermore, the CHF-induced alterations of functional effects and gene expression of cardiac ß3-AR have not been determined in nonhuman species. This has important pharmacological and clinical implications because the vast majority of therapeutic drugs are evaluated in animal models.
Accordingly, we tested the hypothesis that in CHF, ß3-AR expression is increased. This augmentation is proposed to exacerbate the dysfunctional [Ca2+]i regulation, enhance inhibition of cardiac contraction and relaxation, and lead to worsening cardiac failure.
To obviate the limitations in previous studies, we used a more clinically relevant canine CHF model1,3,13 and conducted studies in cardiomyocytes freshly isolated from left ventricular (LV) myocardium obtained by biopsy from the same animals before and after CHF.13 We compared alterations in normal and CHF myocyte steady-state ß3-AR mRNA and protein levels, changes in normal and CHF myocyte contractile performance, [Ca2+]i transient ([Ca2+]iT), and ICa,L responses to ß3-AR stimulation.
The present findings extend our knowledge regarding the cellular and molecular determinants of an enhanced ß3-AR–mediated negative inotropic effect in CHF, provide valuable new insight into the mechanism of the progression of functional impairment in CHF, and may assist in specifically targeting therapy for CHF.
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Materials and Methods |
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Using techniques well-established in our laboratory,13 myocytes were isolated from LV myocardium obtained from 9 instrumented dogs before and after pacing-induced CHF.
Canine ß3-AR Gene Sequencing, Myocyte ß3-AR mRNA, and Protein Levels
Sequencing of Canine ß3-AR Gene
A 525-bp DNA fragment corresponding to canine ß3-AR coding region (bases 115 to 640) was produced by polymerase chain reaction (PCR) with primers: sense, 5'-GCTCCGTGGCCT-CACGGGAA-3', and antisense, 5'-CTTGCTCATGATG-GGCGC-3' and cloned into PCR 2.1 (Invitrogen). DNA sequencing of the recombinant plasmid was performed with T7 and M13 specific primers.
Myocyte Expression of ß3-AR mRNA
Cardiomyocyte total RNA was extracted by RNAqueous phenol-free total RNA isolation kit (Ambion) and treated with RNase-free DNase I (GIBCO BRL). The reverse transcription (RT) reaction was performed with RETROscript first-strand synthesis kit (Ambion) using the antisense primer. The cDNA produced was amplified by PCR.
Northern Blot
Poly A+ RNA was isolated from total RNA using poly (A) Pure mRNA isolation kit (Ambion). ß3-AR mRNA was analyzed by using Northern Max (Ambion). The 525-bp cDNA probe of canine ß3-AR was labeled by random priming with [
-32P]dCTP (3000 Ci/mmol, DuPont) with the use of DECAprime II (Ambion). Hybridization was performed at 42°C overnight in the presence of 500 µg/mL salmon sperm DNA. For standardization, the same blot was reprobed with a GAPDH probe.
Western Blot
LV myocytes were briefly washed with prechilled PBS before the addition of a protein-extraction reagent (Pierce) with proteinase inhibitor cocktail. Cell lysate (50 µg) was blotted to a PVDF membrane. The membrane was incubated with a polyclonal IgG to ß3-AR (1:1500 dilution, Alpha Diagnostic) at 4°C overnight. Following washes, the membrane was incubated with horseradish peroxidase–conjugated anti-rabbit IgG (1:3000 dilution, Sigma). For normalization, the same blot was reprobed with IgG polyclonal antibody to actin at 1:2500 dilution (Santa Cruz Biotechnology Inc).
Functional Studies
Effects of ß3-AR on Myocyte Contractile Response and [Ca2+]i Regulation
BRL, a frequently studied, selective ß3-AR agonist, causes potent depressions of myocardial contraction in humans, dogs, and rats15 and altered cardiac contractile response in failing human hearts.4 The dosing protocol of BRL (10-8 mol/L) used in this study was identified from our initial dose-response study.
Measurement of Contractile Response
A dose-response study was performed in 5 animals. Briefly, myocyte contractions were elicited by a field stimulation.13 After stabilization, steady-state data were recorded. Myocytes were randomly exposed to BRL (10-10 to 10-7 mol/L), and data were acquired after 3 to 5 minutes of drug exposure and 5 to 15 minutes after drug washout. We found that BRL of 10-8 mol/L was well-tolerated by both normal and CHF myocytes and caused nearly maximum reduction in the percent shortening (ie, systolic amplitude [SA]) (Figure 3A and Table). Higher concentrations of BRL were frequently associated with arrhythmias in CHF myocytes. Thus, the effects of BRL 10-8 mol/L were assessed in the following series of experiments.
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29%), dL/dtmax, and dR/dtmax. After CHF, BRL caused greater reductions in SA (
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Myocyte contractile response to BRL 10-8 mol/L was examined in 9 animals. In 3 subgroups, BRL-mediated actions were determined after pretreatment of myocytes with (1) metoprolol (10-6 mol/L, a ß1-AR antagonist), nadolol (10-5 mol/L, a ß1- and ß2-AR antagonist), and bupranolol (10-6 mol/L, a nonselective ß3-AR antagonist) in 6 animals, or L-748,337 (10-7 mol/L, a selective ß3-AR antagonist) in 2 animals for 5 minutes in random order, (2) with NO synthase inhibitor NGnitro-L-arginine methyl ester (L-NAME) (10-4 mol/L, 30 minutes), and (3) with Gi inhibitor pertussis toxin (PTX) (2 µg/mL, 36°C, 5 hours) inactivated cardiac Gi in 5 animals.2,16
To determine the cellular basis for the ß3-AR–mediated contractile action, the following experiments were performed in two additional series-studies in 9 animals.
Simultaneous Measurement of Calcium Transient and Contractile Response
In the second series of experiments, [Ca2+]i transient ([Ca2+]iT) and contraction responses in a single myocyte were simultaneously measured as described previously.17 After stabilization, the BRL protocol was repeated. When myocytes were loaded with indo-1-AM, compartmentalization of the indicator may have occurred in the mitochondria, thus the absolute value of [Ca2+]i was not used. As described in our past report,17 the ratio of the emitted fluorescence (410/490) was used to represent the relative changes in peak intracellular [Ca2+]i before and after BRL.17 The actual fluorescence ratios were also calibrated as described by O’Neill et al.18
Measurement of Calcium Current Response
In the third series of experiments, the ICa,L response to BRL (10-8 mol/L) was measured by whole-cell patch-clamp technique.2 After baseline ICa,L, data were obtained during the 0 to 10 minutes of BRL superfusion and during the period of washout of BRL.
Statistical Analysis
Data were summarized as mean±SD. Multiple comparisons were performed by analysis of variance. When a significant overall effect was present, intergroup comparisons were performed by using a Bonferroni correction for multiple comparisons. Two-tailed, paired Student’s t tests were used to evaluate mean differences in hemodynamic parameters and cell-functional response indexes before and after CHF. Myocyte functional data were averaged from the number of myocytes studied from each animal. Then myocyte functional data were analyzed by using the mean measurements obtained for each dog before and after CHF. Significance was established as P<0.05.
Drugs
Bupranolol HCL was a gift from Schwarz Pharma (Monheim, Germany). BRL-37344 was obtained from Tocris. L-748,337 was a gift from Merck Research Laboratories (Rahway, NJ). Nadolol, L-NAME, and PTX were obtained from Sigma Chemical Co.
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Canine ß3-AR Gene, ß3-AR mRNA, and Protein Levels in Normal and CHF Myocytes
As presented in Figure 1, a fragment of canine ß3-AR DNA with a predicated size of 525 bp was produced by PCR. The sequence of the fragment was 100% homologous to the corresponding region of the published canine ß3-AR cDNA (GenBank No. U92468). By RT-PCR, the same size of ß3-AR mRNA was detected identically in both normal and CHF groups, with the predicated size of 525 bp.
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Figure 2A shows that in normal myocytes, a single band of ß3-AR mRNA, about 3.4 kb, was present only in the poly A+ RNA samples. The same size of ß3-AR mRNA was detected from each animal before and after CHF. Compared with normal myocytes, CHF myocytes had a significantly higher (73%) signal ratio of ß3-AR mRNA to GAPDH (2.20±0.2 versus 3.8±0.3) (P<0.05) (Figure 2B). With Western blot analysis, a single band of ß3-AR protein, about 65 to 70 kDa, was detected. Compared with normal myocytes, in CHF myocytes, the signal ratio of ß3-AR protein to actin was increased to approximately 247% (1.72±0.9 versus 4.25±2.7) (P<0.05), indicating increased ß3-AR protein levels in CHF (Figure 2C).
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Effect of BRL on Contractile, [Ca2+]iT, and ICa,L Responses in Normal and CHF Myocytes
Consistent with our previous reports, significant myocyte dysfunction was evident in CHF myocytes.13 CHF myocytes were enlarged, and the length-width ratio was greater (58.1±6.3%) (P<0.05) than normal cells. The SA, the velocity of shortening (dL/dtmax) and relengthening (dR/dtmax), peak systolic [Ca2+]iT, and ICa,L in CHF myocytes were significantly decreased (Figures 3 and 4).
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Compared with normal myocytes, in CHF myocytes, superfusion with BRL (10-8 mol/L) caused a more marked decrease in dL/dtmax (25.1% versus 13.3%), SA (44.2% versus 29.1%), and dR/dtmax (23.6% versus 16.7%) (P<0.05) (Figure 3A and Table). As displayed in Figure 3B, BRL (10-10 to 10-7 mol/L) induced dose-dependent inhibition of cell contraction. In the normal myocytes, BRL caused about 14.8%, 25.5%, 31.5%, and 34.2% decreases in SA, respectively. In CHF myocytes, BRL produced significantly greater reductions in SA of 16.6%, 31.5%, 44.2%, and 46.1% (P<0.05), respectively. Thus, CHF myocytes exhibited an increased responsiveness to BRL. Of note, BRL of 10-8 mol/L decreased SA by 31.5% below control value in normal myocytes. Previously, much higher concentrations of BRL (10-7 mol/L) only caused a 26% decrease in peak tension in normal canine ventricular strips,15 suggesting isolated myocytes may be more sensitive to BRL.
As presented in Figure 4 and the Table, these changes in contractile performance were associated with a marked decrease in the peak systolic [Ca2+]iT in both normal (17.6%) and CHF (32.8%) myocytes, whereas the BRL-induced decrease in [Ca2+]iT was significantly greater in CHF myocytes (32%) than that in normal myocytes (17%) (Figure 4A). Compared with normal myocytes, we further found that BRL (10-8 mol/L) caused a much greater decrease in ICa,L in CHF myocytes (32.3% versus 21.6%) (Figure 4B).
As displayed in Figure 5, BRL-induced reductions in SA persisted in normal and CHF myocytes after pretreatment with ß1- or ß1-/ß2-AR antagonists. However, pretreatment with bupranolol, which possesses ß3-AR antagonist properties, completely prevented the action of BRL. Similarly, in the subgroup of 2 animals, ß3-AR–mediated contractile response also was abolished in the presence of a selective ß3-AR antagonist (L-748,337).
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Figure 6 illustrates the contribution of NO (Figure 6A) and Gi (Figure 6B) in BRL-induced contractile action. Pretreatment of myocytes with L-NAME or PTX caused no significant changes in baseline SA, but significantly altered the myocyte response to BRL. As shown in Figure 6A, compared with untreated myocytes, in the presence of L-NAME, BRL-induced decreases in SA were significantly reduced in normal (12.3±2.9% versus 28.4±3.0%) and CHF myocytes (15.9±3.0% versus 42.6±3.1%) (P<0.05). However, in this condition, BRL still significantly decreased SA in normal and CHF myocytes (P<0.05). The decreased SA in CHF myocytes remained more marked than that in normal myocytes (15.9% versus 12.3%) (P<0.05). In contrast, pretreatment of PTX prevented BRL caused decreases in SA in both normal (-1.3±2.1%) and CHF myocytes (-1.6±3.0%) (Figure 6B).
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Discussion |
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We investigated the alterations in the cardiac ß3-AR expression and functional effect in an animal model of CHF that mimics many of the functional and neurohormonal changes (especially ß-AR signaling) of clinical CHF.1,3,13 We found that the functional ß3-ARs are expressed in normal LV, and their stimulation induces direct inhibition on [Ca2+]iT and ICa,L and produces a negative inotropic action. Importantly, in CHF, ß3-AR expression is increased. This alteration exacerbates the dysfunctional [Ca2+]i homeostasis and [Ca2+]i regulation, enhances inhibition of cardiac contraction and relaxation, and may lead to worsening cardiac failure.
Expression and Functional Effect of Cardiac ß3-AR Before and After CHF
The distribution and functional effect of ß3-ARs in the heart remains uncertain. Previous experimental studies of cardiac ß3-ARs in a variety of animals and humans have yielded conflicting results. ß3-AR stimulation has been reported variably to have a positive,11 negative,5–7 or no inotropic effect12 on normal LV contractile performance. Both the presence4,5 and absence12 of cardiac ß3-ARs have been documented.
In contrast, we assessed the direct effect of ß3-AR stimulation with BRL in cardiomyocytes isolated from the LV before and after CHF. These studies removed the effects of extracardiac factors, which may influence contractility. We found that in normal myocytes, BRL produced clear dose-dependent depressions of myocyte contraction and relaxation, with significant reductions in SA, dL/dtmax, and dR/dtmax. BRL-induced inhibitions of cell contraction and relaxation were not modified by pretreatment of myocytes with ß1- and ß1-/ß2-AR antagonists, indicating that this effect was not mediated by ß1- and ß2-ARs. By contrast, ß3-AR antagonists completely prevented the BRL-induced contractile action, indicating that the negative inotropic action of BRL is coupled to ß3-ARs.
We further found that the BRL-induced depression in myocyte contraction was accompanied by significant decreases in [Ca2+]iT and ICa,L. Thus, the BRL-evoked cardiac inhibition may be at least partially mediated through [Ca2+]i regulation.
We found the presence of cardiac ß3-ARs, and for the first time, quantified the cardiac ß3-AR mRNA and ß3-AR protein levels by using Northern blot and Western blot analyses in normal and CHF canine LV myocytes. These results are consistent with the observations of Gauthier et al,5 who first demonstrated the presence and functional effect of ß3-ARs in the human donor heart, but contrary to the findings of Tavernier et al,12 who reported no functional or biochemical evidence for the presence of ß3-ARs in the normal dog heart. Their failure to observe functional cardiac ß3-ARs may have attributed to the use of insensitive measures of ß3-AR protein with [125I] CYP and inadequate assessment of LV contractility. The present observation is supported by our previously published report that by using pressure-volume analysis in conscious, instrumented dogs, we have clearly demonstrated that BRL directly inhibited LV contraction and relaxation in the normal heart and caused an enhanced inhibition of LV contraction after CHF. These effects persisted at constant heart rate (by atrial pacing) or after autonomic blockade.
A novel finding in the present study is that, in striking contrast with ß1-ARs, in CHF myocytes, there is an enhanced ß3-AR stimulation-induced negative inotropic response with an exacerbated negative modulation on [Ca2+]i regulation, which is paralleled by an increased ß3-AR expression.
Consistent with past reports,3,13 we found that after pacing-induced CHF, there is a primary defect in the cardiomyocyte force-generating capacity and relaxation process, with impaired [Ca2+]i homeostasis. BRL exacerbated the dysfunctional cell contraction and [Ca2+]i regulation. BRL caused much greater decreases in [Ca2+]iT and ICa,L in CHF myocytes than in normal myocytes, with resultant decreased SA, dL/dtmax, and dR/dtmax. In these CHF myocytes, ß3-AR mRNA and protein levels were significantly increased by 73% and 147%, respectively.
Our finding of the upregulation of both ß3-AR gene expression and functional response in CHF LV myocytes should be compared with a recent study. Moniotte et al4 reported a similar upregulation of ß3-AR expression but blunted negative inotropic response to BRL in human failing myocardium. This discrepancy might result from several confounding factors such as species difference in ß3-ARs,15 different severity of CHF, different measurements of contractility, and different tissue (or cell) preparations.
It is likely that the very different observations in ß3-AR–mediated contractile response in CHF between the two studies are mainly owing to the fact that Moniotte et al4 studied ventricular strips, whereas we examined single LV myocytes. It has been shown that there are different distributions of ß3-ARs in different cardiac chambers.19 Thus, in their study, the use of ventricular strips of multiple cells preparation, as well as the clinical use of multiple drugs in the cardiomyopathic patients, may have complicated the accurate assessment of the degree and functional significance of alterations of the LV myocyte response to ß3-AR stimulation.
In contrast, in our study, BRL-induced contractility changes were compared in isolated single LV myocytes obtained from the same animal before and after advanced CHF. Myocyte cell SA and dL/dtmax were used as a measure of contractility, which may provide a more sensitive means of detecting CHF caused changes in myocardial contractile response to ß3-AR stimulation. Although the ß3-AR density per myocyte was not measured in the current study, it is possible the significant increase in ß3-AR gene expression in CHF LV myocytes may be associated with increased ß3-AR numbers per cell.2 Thus, the enhanced contractile response to BRL in CHF myocytes may reflect the presence of an increase in the number of ß3-AR per cell.
Nevertheless, the findings of Moniotte et al4 and the present study all demonstrated a similar, potentially detrimental functional consequence with ß3-AR activation in CHF. In CHF, when marked increases in sympathetic tone and cardiac norepinephrine release have rendered the positive inotropic ß1-AR system relatively unresponsive, the upregulated ß3-AR pathways would continue to exhibit a negative inotropic effect. This altered balance between opposing inotropic influences of ß1-ARs and ß3-ARs in CHF may contribute to progressive cardiac dysfunction in CHF. However, a definitive statement regarding the importance of ß3-ARs in the pathogenesis of CHF cannot be drawn from the described altered ß3-ARs in CHF. Whether ß3-ARs are a contributing cause or merely a result of ventricular dysfunction remains an unresolved question. More insight will be gained from future work demonstrating beneficial actions of ß3-AR antagonists or adverse actions of ß3-AR activation in response to endogenous catecholamines, which are currently ongoing.
Potential Mechanism(s) of the Upregulated ß3-ARs in CHF
In the present investigation, both molecular and functional assessments provide evidence that ß3-AR is upregulated in the failing canine heart. Although the mechanism for this effect has not yet been elucidated, several lines of evidence suggest that the overexpression of ß3-ARs in our animal model may be related to the advanced stage of CHF. The prolonged stimulation by catecholamine causes ß1-AR downregulation and uncoupling.1 In contrast, the ß3-ARs are relatively resistant to long-term, agonist-induced desensitization processes20 and may play an important role in the diminished numbers of ß1-ARs. Furthermore, norepinephrine, the primary neurotransmitter released by SNS, has relatively high affinity for the ß3-AR (unlike the ß2-AR).21 In addition, Gi, implicated in the ß3-AR signaling, is elevated in failing myocardium. Decreased ß1-receptor and Gs density with increased (50%) Gi protein has been reported in pacing-induced CHF.3
In this investigation, we demonstrated that the altered myocyte inotropic response to BRL is owing to a ß3-AR stimulation caused by an enhanced negative modulation on [Ca2+]i regulation. After CHF, ß3-AR stimulation exacerbates the dysfunctional [Ca2+]i homeostasis, resulting in further reduction in peak systolic [Ca2+]iT and ICa,L. The stimulation of an increased number of ß3-ARs could have caused the amplified response in CHF myocytes.
The enhanced response to ß3-AR stimulation in CHF may also be related to increased numbers of ß3-ARs or an altered signal transduction. Although the intracellular pathway coupling ß3-AR stimulation is still incompletely characterized, it has been reported that ß3-AR stimulation decreases cardiac contractility through activation of NO synthase pathway.6,10,15 In CHF, the NO-cGMP signaling may be altered,22 thereby altering CHF myocyte response to ß3-AR stimulation. In agreement with previous studies, we found that in the presence of L-NAME, the BRL-induced negative inotropic response was strongly reduced, indicating an involvement of the NO pathway in ß3-AR–mediated action. However, our present observation indicates that the NO pathway may not be fully responsible for the altered LV myocyte response to BRL because we found that the myocyte response to BRL was only partially inhibited by pretreatment myocytes with NO synthase inhibitor. In the presence of L-NAME, myocyte contractility was still significantly reduced, indicating ß3-AR activation is not mediated exclusively through the NO pathway. A similar observation has also been made in beating guinea pig hearts.6
Our study indicates that ß3-AR–mediated contractile action couples to Gi because pretreatment of PTX completely prevented BRL contractile effects in normal and CHF myocytes. This is not consistent with the past report of Gauthier et al.5 In their study, it was shown that in human ventricular strips treated with PTX, the effect of BRL on contractility was attenuated but was not completely suppressed. This discrepancy is owing to an incomplete blockade of Gi by PTX in their study because an inadequate amount of PTX and duration (0.5 µg/mL for 2 hours) was used. It has been reported that PTX treatment for 3 to 5 hours at 5 µg/mL is required to inactivate human cardiac Gi.16 Therefore, our present study indicated that the enhanced contractile response to ß3-AR stimulation in CHF myocytes may be coupled to Gi through both NO-dependent and NO-independent mechanisms. The activation of Gi also has the potential to couple ß3-ARs to other important signaling pathways such as the MAP kinase.23 The increase in other neurohormonal activation, such as tumor necrosis factor-, endothelin 1, and angiotensin II, may also differentially modulate ß3-AR expression and function. Clearly, further studies are needed to fully characterize intracellular pathway coupling ß3-AR stimulation.
Clinical Implications
Our observation and those of others5,6,10 support the view that in the normal heart, the ß3-AR pathway may exert a negative counterregulation against excessive positive inotropy. However, increased ß3-AR expression in CHF myocytes may underlie the progressive nature of functional impairment in CHF. Both the expression and the function of ß3-ARs are increased after pacing-induced CHF. This finding is a striking contrast with the changes in ß1- and ß2-ARs. The upregulated ß3-ARs in CHF exacerbates the dysfunctional [Ca2+]i homeostasis and [Ca2+]i regulation and, thus, exhibits a greater inhibition on cardiac contraction and relaxation and might lead to worsening cardiac failure. The implications of these findings are important to our understanding of the pathophysiology of CHF. They may also suggest several novel therapeutic strategies for the treatment of CHF, such as the use of ß3-AR blockers or Gi inhibitors. Our findings also indicate that using ß3-AR agonists for the treatment of obesity and diabetes24 may have cardiac side effects, especially in CHF patients.
In conclusion, we found that in dogs with pacing-induced CHF, the gene expression and functional responses of cardiac ß3-AR are upregulated, which may potentially contribute to the progression of CHF.
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Acknowledgments |
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This study was supported by grants from NIH (HL45258, HL53541, and T32HL07868) and AHA (9640189N). We acknowledge the technical assistance of Dr P. Tan and M. Cross and the secretarial assistance of A. Burnette.
Received March 28, 2001; accepted August 21, 2001.
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