β₃-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca²⁺ current

β₃-adrenergic receptor (β₃-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of β₃-AR in the human atrium. Unexpectedly, β₃-AR activation increased human atrial tissue contractility and stimulated the L-type Ca²⁺ channel current (I_Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I_Ca,L and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective β₃-AR agonists, SR58611 and BRL37344, and a β₃-AR partial agonist, CGP12177, stimulated I_Ca,L in HAMs with nanomolar potency and a 60%–90% efficacy compared with isoprenaline. The β₃-AR agonists also increased contractility but with a much lower efficacy (~10%) than isoprenaline. The β₃-AR antagonist L-748,337, β₁-/β₂-AR antagonist nadolol, and β₁-/β₂-/β₃-AR antagonist bupranolol were used to confirm the involvement of β₃-ARs (and not β₁-/β₂-ARs) in these effects. The β₃-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I_Ca,L stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, β₃-ARs are positively coupled to L-type Ca²⁺ channels and contractility in human atrial tissues through a cAMP-dependent pathway.

In cardiac myocytes, Ca²⁺ current through the L-type Ca²⁺ channels (I_Ca,L) provides Ca²⁺ for the activation of the contractile apparatus and is a crucial determinant of cardiac contractile activity (1). Among several regulatory pathways that control cardiac Ca²⁺ channel activity, the best described is the β-adrenergic stimulation of I_Ca,L, which contributes to the positive inotropic and part of the positive chronotropic effects of catecholamines (1, 2). Although to date, 3 types of β-adrenergic receptors (β-ARs) have been cloned, β₁-, β₂-, and β₃-ARs, respectively, the effect of catecholamines in human heart is generally attributed to β₁- and β₂-ARs, with opposite effects on hypertrophy (3, 4) and apoptosis (5, 6) and with a respective contribution of each receptor subtype that varies significantly depending on the cardiac tissue, the pathophysiological state, the age, or the developmental stage (7). However, expression of a β₃-AR in human myocardium was also demonstrated both at the mRNA (8, 9) and protein level (9–12).

Like β₁- and β₂-ARs, β₃-ARs are positively coupled to adenylyl cyclase in fat and colon as well as in CHO cells transfected with the cloned receptor (13–15). Therefore, activation of β₃-ARs in human heart would be expected to stimulate I_Ca,L and increase contractility. However, β₃-AR agonists were shown to inhibit contractility in human endomyocardial biopsies from transplanted hearts (8, 16) and in left ventricular samples from failing and nonfailing explanted hearts (11), an effect mediated by G_i/o proteins and NO production via activation of endothelial type-3 NO synthase (8, 16, 17). This effect was accompanied by a shortening of the action potential (8, 18). A similar finding was obtained with BRL37344, a β₃-AR agonist, in Langendorff-perfused isolated guinea pig hearts (19) and was accompanied by a decrease in Ca²⁺ transients. The NO dependency of β₃-AR action was also found in mice, using animals with a homozygous deletion mutation of the β₃-AR (ADRB3^–/–) (20). While NOS3 inhibitors (arginine analogues) potentiated the positive inotropic effect of isoprenaline (ISO) in wild-type mice, this effect was absent in either ADRB3^–/– or NOS3^–/– mice (20). In another study, ISO applied to a culture of neonatal cardiomyocytes isolated from mice with a double homozygous deletion mutation of the β₁-AR and β₂-AR (ADRB1^–/–/ADRB2^–/–) was found to slightly decrease spontaneous beating frequency, a phenomenon mimicked by CL316243, a β₃-AR agonist (21). Moreover, this negative chronotropic effect of ISO turned into a positive one after treatment of the myocytes with Bordetella pertussis toxin (21), confirming the original finding that β₃-AR signaling involves activation of G_i/o proteins (8).

Although the case seems rather strong for a negative inotropic and chronotropic effect of β₃-ARs, a number of studies are inconsistent with such hypothesis. For instance, several reports demonstrate the absence of a functional β₃-AR in mouse heart (22–24). Cardiac overexpression of human β₃-ARs in mice results in G_s activation of adenylyl cyclase and positive inotropy on stimulation with a β₃-AR agonist (25). In human atrium and failing ventricle, several β₃-AR agonists were found to be devoid of any cardiodepressant effect (26–28). These latter findings are of importance, since the amount of β₃-AR proteins was shown to increase up to 3-fold in different models of heart failure (11, 29, 30).

In this study, our aim was to get further insights into the role of β₃-ARs on human cardiac function at the single-cell level. For this, we examined the effects of several β₃-AR ligands on I_Ca,L recorded in isolated human atrial myocytes (HAMs): SR58611 (31) and BRL37344 (32), which are 2 β₃-AR agonists; CGP12177, which is a partial β₃-AR agonist (33) as well as a β₂-AR antagonist and a partial agonist at β₁-AR through a low-affinity site (34, 35); and L-748,337, which is a β₃-AR antagonist (36). These effects were compared with those obtained on the contractility of human atrial strips.

β₃-AR agonists stimulate I_Ca,L in HAMs. To examine the regulation of I_Ca,L by β₃-ARs in HAMs, we used 2 β₃-AR agonists, SR58611 (31) and BRL37344 (32), as well as CGP12177, which is a β₃-AR partial agonist (33), a β₂-AR antagonist, and a partial agonist at β₁-AR through a low-affinity site (34, 35). Typical experiments are shown in Figure 1. In each example, 2 cells were simultaneously exposed to increasing concentrations of SR58611 (Figure 1A), BRL37344 (Figure 1B), and CGP12177 (Figure 1C). Such double-cell experiments provided a simple way to evaluate cell-to-cell variability, since all external parameters (temperature, perfusion, time after isolation, etc.) were strictly identical during the recordings obtained in the 2 cells. As can be seen in Figure 1, all compounds produced a dose-dependent and reversible increase in I_Ca,L. The cumulative dose-response curves shown in Figure 1D were fitted to the Michaelis equation to derive the maximal stimulation (E_max) of I_Ca,L and the concentration required for half-maximal effect (EC₅₀) for each agonist. Thus, E_max and EC₅₀ values, respectively, were as follows: 221% ± 20% and 2.5 ± 0.8 nM (n = 6) for SR58611; 172% ± 26% and 15.2 ± 0.6 nM (n = 10) for BRL37344; and 136% ± 21% and 17.1 ± 3.0 nM (n = 8) for CGP12177 (Figure 1D). The efficacy of SR58611 and BRL37344 on I_Ca,L was comparable to that of ISO (241% ± 47% at 1 μM ISO; n = 5), while that of CGP12177 was somewhat smaller. None of the 3 β₃-AR agonists tested had any stimulatory effect in the nanomolar range of concentrations on I_Ca,L measured in ventricular myocytes isolated from frog and rat hearts (Supplemental Results; supplemental material available online with this article; doi: 10.1172/JCI32519DS1).

Figure 1

β₃-AR stimulation of I_Ca,L in HAMs. Each experiment shows the time course of I_Ca,L amplitude recorded in 2 HAMs that were simultaneously exposed to increasing concentrations of SR58611 (A), BRL37344 (B), and CGP12177 (C). Each symbol indicates a net amplitude of I_Ca,L measured every 8 seconds at 0 mV membrane potential. The individual current traces were obtained in each experimental condition at the times indicated by the corresponding letters in the main graph. (D) Concentration-response curve for the effects of the 3 agonists on I_Ca,L. The points show the mean ± SEM of 6 (SR58611, filled square), 10 (BRL37344, filled circle), or 8 (CGP12177, open square) cells.

β₃-AR agonists increase I_Ca,L in HAMs via β₃-AR activation. The sub­type selectivity of β-AR ligands is always questionable. For instance, SR58611 and BRL37344 were shown to also activate β₁- and β₂-ARs in the micromolar range of concentrations (13), and CGP12177 was shown to be an agonist of β₁-AR at a low-affinity site (34, 35). Since human cardiomyocytes also possess β₁- and β₂-ARs, we examined the possibility that the stimulatory effects of the 3 agonists on I_Ca,L in HAMs were caused by activation of β₁- and/or β₂-ARs. To do this, we examined whether the β₁-AR/β₂-AR antagonist nadolol was able to antagonize the stimulatory effect of SR58611 and BRL37344. Because the K_d for nadolol on the human β₃-AR is more than 600 nM as compared with 14–40 nM on the β₁- and β₂-ARs (13), most experiments were performed using 200 nM nadolol. At this concentration, nadolol antagonized some of the stimulatory effect of SR58611 (1 μM; data not shown) on I_Ca,L, indicating that the effect of this agonist was at least partly mediated by activation of β₁- and/or β₂-ARs. However, even a 5-fold higher concentration of nadolol (1 μM) had no effect on BRL37344-induced (1 μM) stimulation of I_Ca,L (5.4% ± 3.9%; n = 4; P > 0.05; Figure 2A). In contrast, the effect of BRL37344 was almost completely blocked (by 88.5% ± 5.5%; n = 5; P < 0.001) by 1 μM of the β₃-AR antagonist L-748,337 (36, 37) (Figure 2B). Interestingly, L-748,337 also almost completely antagonized the stimulatory effect of CGP12177 on I_Ca,L (by 87.9% ± 4.4%; n = 4; P < 0.01; Figure 2C), which excludes the possibility that CGP12177 was acting via the low-affinity site of the β₁-AR (34, 35). As seen in Figure 2, B and C, the effect of L-748,337 was fully reversible. In another series of experiments, bupranolol (1 μM), which combines β₁-/β₂-/β₃-AR antagonistic properties (38), was found to strongly antagonize the stimulatory effect of 1 μM CGP12177 (by 79.3% ± 1.6%; n = 5; P < 0.05). Altogether, these results indicate that at the concentrations used the stimulatory effects of BRL37344, CGP12177, and, to a large extent, SR58611 on I_Ca,L in HAMs result from activation of β₃-ARs.

Figure 2

Effects of β₃-AR and β₁-/β₂-AR antagonists on β₃-AR stimulation of I_Ca,L in HAMs. In each experiment, I_Ca,L was recorded in 1 (B and C) or 2 (A) HAMs, which were simultaneously exposed to BRL37344 (1 μM, A; 0.1 μM, B) or CGP12177 (1 μM, C) and then to the β₁-/β₂-AR antagonist nadolol (1 μM, A) or to the β₃-AR antagonist L-748,337 (1 μM, B and C). The dotted lines in A indicates spontaneous rundown.

NO pathway is not involved in the β₃-AR regulation of I_Ca,L. In human ventricle, β₃-AR agonists were shown to produce a negative inotropic effect as a consequence of NOS3 activation (17). Through an activation of the soluble guanylyl cyclase and accumulation of intracellular cGMP, NO can lead to opposite effects on human atrial I_Ca,L: a stimulation at low concentrations due to inhibition of the cGMP-inhibited cAMP-phosphodiesterase (PDE3) (39, 40) and an inhibition at higher concentrations due to activation of the cGMP-stimulated phosphodiesterase (PDE2) (39, 40). To evaluate the contribution of the NO/cGMP pathway in the regulation of I_Ca,L by β₃-AR agonists, we examined the effect of CGP12177 in the presence of the NOS inhibitor N^G-monomethyl-l-arginine (L-NMMA). As shown in Figure 3A, the presence of L-NMMA (1 mM) in both the extracellular and pipette solutions did not modify the stimulatory effect of CGP12177 (1 μM) on I_Ca,L (with L-NMMA, 65% ± 11% over control, n = 4; without L-NMMA, 58% ± 4% over control, n = 4). This indicates that the NO/cGMP pathway is not required for the β₃-AR stimulation of I_Ca,L. To examine whether β₃-AR stimulation can lead to inhibitory effects via this pathway, HAMs were first exposed to serotonin (100 nM), which strongly increased I_Ca,L (by 154% ± 64%; n = 3; Figure 3B) via activation of the 5-HT₄ receptor (41), and then to CGP12177 in the presence of serotonin. As shown in Figure 3B, CGP12177, at concentrations ranging from 0.01 to 10 μM, had no inhibitory effect on prestimulated I_Ca,L (101% ± 1% of serotonin level; n = 3). These experiments indicate that the NO/cGMP pathway is not involved in the activation of I_Ca,L by β₃-AR agonists, and that β₃-AR stimulation does not activate a parallel inhibitory pathway regulating I_Ca,L.

Figure 3

NO pathway is not involved in the stimulation of I_Ca,L by β₃-AR agonists. In each experiment, I_Ca,L was recorded in a single HAM exposed to CGP12177 in the presence of either 1 mM L-NMMA in intracellular and extracellular solutions (A) or serotonin (100 nM, B). The dotted line in B indicates spontaneous rundown.

β₃-AR activation of I_Ca,L involves cAMP-dependent protein kinase. Because cAMP-dependent PKA is a classic activator of L-type Ca²⁺ channels and accounts for the stimulatory effects of β₁- and β₂-AR agonists on I_Ca,L, we examined whether PKA was also involved in the β₃-AR activation of I_Ca,L. Figure 4 shows 2 individual experiments in which the cell permeant PKA inhibitor H89 (42) was applied to HAMs on top of BRL37344 (1 μM; Figure 4A) or CGP12177 (1 μM; Figure 4B). In both cases, H89 (1 μM) completely inhibited the stimulatory effect of the β₃-AR agonist. The inhibitory effect of H89 was mostly reversible upon washout. On average, BRL37344 and CGP12177 increased I_Ca,L by 297% ± 84% (n = 4) and 45% ± 6% (n = 4), respectively, in the absence of H89, and the current amplitude differed from basal level by only 3% ± 6% (n = 4) and –7% ± 3% (n = 4) in the presence of the PKA inhibitor. These experiments indicate that β₃-AR agonists increase I_Ca,L in HAMs through activation of the cAMP/PKA pathway.

Figure 4

PKA mediates the stimulation of I_Ca,L by β₃-AR agonists. I_Ca,L was recorded in a single HAM exposed to either 1 μM BRL3744 (A) or 1 μM CGP12177 (B). When the current was at its maximal stimulation, the PKA inhibitor H89 (1 μM) was added to the extracellular solutions in the presence of the β₃-AR agonists, and the current returned to basal levels.

β₃-AR agonists increase force of contraction in human atrial trabeculae. In cardiac myocytes, I_Ca,L provides Ca²⁺ for the activation of the contractile proteins and is a crucial determinant of the cardiac contractile activity. Therefore, any modulation of I_Ca,L amplitude might be expected to induce parallel changes in contractile amplitude. Since our results so far indicate that β₃-ARs are positively coupled to I_Ca,L in HAMs, additional experiments were performed to evaluate the effects of β₃-AR agonists on the force of contraction in trabeculae isolated from human atrial tissues. To eliminate any possible interaction of the agonists with β₁- and/or β₂-ARs (43), the experiments were performed in the presence of nadolol (200 nM), which alone reduced atrial basal contractile amplitude to 82% ± 9% (n = 8) of control level. The individual experiments shown in Figure 5 indicate that CGP12177 (1 μM; Figure 5A), SR58611 (100 nM; Figure 5B), and BRL37344 (1 μM; Figure 5C) induced a modest and transient positive inotropic effect in human atrial muscle. A 10%–20% peak stimulation was observed with all 3 compounds at approximately 2 minutes after drug application, and a steady state was reached at approximately 15 minutes. The summary data in Figure 6C indicate that all 3 β₃-AR agonists induced a significant increase in contractile amplitude at 2 minutes in human atrium, but only BRL37344 maintained a significant positive inotropic effect at 15 minutes (11.7% ± 1.7%; n = 6; P < 0.05). In contrast, the effect of ISO (1 μM) was more prominent (Figure 6C). Because the β₃-AR stimulation of I_Ca,L was mediated by PKA, and because different G_s-coupled receptors generate heterogeneous cAMP signals due to specific functional coupling of phosphodiesterase (PDE) isoforms (44, 45), we questioned whether the modest effect on contractility was due to a limited diffusion of cAMP that might result from a high PDE activity. If this is the case, then PDE inhibition should potentiate the contractile effects of β₃-AR agonists. We tested this hypothesis by reevaluating the contractile effects of β₃-AR agonists in the presence of 10 μM 3-isobutyl-1-methylxanthine (IBMX), a wide-spectrum PDE inhibitor. As shown in Figure 6, the effects of BRL37344 (Figure 6A) and CGP12177 (Figure 6B) were greatly increased by IBMX. While IBMX alone had no significant effect on contraction force (n = 9), it strongly increased the effects of BRL37344 (1 and 10 μM) and CGP12177 (1 μM). Figure 6C shows that in the presence of IBMX, the effects of the 2 β₃-AR agonists were comparable with those produced by ISO (1 μM).

Figure 5

Effect of β₃-AR ligands on contractility in human atrial trabeculae. CGP12177 (A), SR58611 (B), or BRL37344 (C) were applied to human atrial preparations at the concentrations indicated, after 15 minutes of perfusion with 200 nM nadolol followed by the continuous presence of nadolol. The individual contractile traces were obtained in each experimental condition at the times indicated by the corresponding arrows. All 3 compounds significantly (P < 0.05) increased force of contraction.

Figure 6

PDE inhibition potentiates the effects of β₃-AR agonists on contractility in human atrial trabeculae. BRL37344 (A) or CGP12177 (B) were applied to human atrial preparations at the concentrations indicated, in the presence of 200 nM nadolol and in the absence (A) or presence (A and B) of the PDE inhibitor IBMX (10 μM). (C) Summary of the effects of the β₃-AR agonists on contractility. The effects of SR58611 (100 nM), BRL37344 (10 μM), and CGP12177 (1 μM), with or without IBMX (10 μM), are compared with that of ISO (1 μM). The bars show the mean ± SEM of the number of experiments indicated above the bars. *P < 0.05; ***P < 0.005 versus control. ANOVA followed by post-hoc Bonferroni’s test was used to compare the effects of the β₃-AR agonists in the absence or presence of IBMX. ^#P < 0.05.

Adenylyl cyclase stimulation assays performed in CHO cells stably transfected with either the human β₁-, β₂-, or β₃-ARs demonstrated that BRL37344, SR58611, and CGP12177 displayed a high β₃-AR selectivity, with EC₅₀ values ranging from 15 to 140 nM (13, 46). In the present study, all 3 ligands increased I_Ca,L in HAMs with similar low EC₅₀ values (3–17 nM; Figure 1). Even though human heart contains more spare β₁- and β₂-AR receptors in the atrial than in the ventricular chambers (47–49), activation of 10%–20% of the total amount of β-ARs would be needed to produce the observed large increase in I_Ca,L (49), which is very unlikely at the nanomolar concentrations of β₃-AR agonists used here. Besides, the effects of SR58611, BRL37344, and CGP12177 on I_Ca,L resembled their effects in β₃-AR adenylyl cyclase assays (13), with SR58611 and BRL37344 behaving as full agonists (by comparison with ISO) and CGP12177 only as partial agonist (Figure 1D) (13, 33). With respect to CGP12177, some caution may be required because the drug is not only a β₃-AR agonist and a β₂-AR antagonist, but is also a partial agonist at β₁-AR through a low-affinity site (34, 35). However, the stimulatory effect of CGP12177 on I_Ca,L (like that of BRL37344) was blocked by L-748,337, a highly potent and selective β₃-AR antagonist (36), at a concentration that had no effect on the stimulation of the current by ISO (data not shown). Moreover, the effect of CGP12177 on I_Ca,L was inhibited by the β₁-/β₂-/β₃-AR antagonist bupranolol but not by the β₁-/β₂-AR antagonist nadolol. Therefore, we are confident that the effects of the agonists tested involved β₃-AR stimulation rather than nonselective β₁- or β₂-AR stimulation and conclude that β₃-AR activation leads to stimulation of the I_Ca,L and contractile activity in human atrium.

Although a number of studies have demonstrated the expression of β₃-ARs in human ventricular (9–12) and atrial tissue (10), little is known about their function in human heart at the single-cell level. A single abstract reports on the lack of β₃-AR modulation of contractile function in human ventricular myocytes (28). Reports on the effects of β₃-AR agonists in isolated cardiomyocytes from other species are limited to 6 studies (29, 50–54). In 3 of these, the effect of a β₃-AR activation was investigated on I_Ca,L in ventricular myocytes isolated from dog (29), rat (53), and guinea pig hearts (50) using BRL37344 as a β₃-AR agonist. In these studies, the drug induced a small, approximately 20%, maximal reduction of basal I_Ca,L, and the effect was increased to approximately 30% when the myocytes were isolated from failing hearts (29, 53). Surprisingly, the effects of BRL37344 persisted after washout of the drug, which is a concern when considering that I_Ca,L is subjected to substantial spontaneous rundown in mammalian cardiomyocytes (55, 56). However, the inhibitory effects of BRL37344 were attenuated by preincubation of the cells with the NOS inhibitor N^G-nitro-l-arginine methyl ester (L-NAME) (50, 53) or after treatment of the myocytes with Bordetella pertussis toxin (53), which is consistent with earlier findings that a β₃-AR signaling cascade involved G_i/o protein and NOS3 activation (8, 16, 17). In our experiments, after rundown subtraction, neither BRL37344, SR58611, or CGP12177, in the range of concentrations at which these ligands act selectively via β₃-ARs (13), had any significant effect on basal I_Ca,L in rat ventricular myocytes (Supplemental Figure 2), while they all stimulated the current in HAMs.

The β₃-AR stimulatory effect on I_Ca,L was not due to NOS activation, because the effect persisted in the presence of L-NMMA. The effect was not mediated by G_i/o proteins, since none of the β₃-AR agonists tested had any effect on I_Ca,L prestimulated by either serotonin or forskolin, while G_i/o protein activation via muscarinic M2 receptors inhibits the current under similar conditions (57). On the contrary, the β₃-AR stimulatory effect on I_Ca,L involved PKA activation, since it was blocked by H89, a cell-permeant PKA inhibitor (42). Therefore, when combining our results in human, rat, and frog cardiomyocytes (Supplemental Figure 1) with those from earlier studies in rat, guinea pig, and dog heart, β₃-AR regulation of I_Ca,L appears clearly different in human heart from any other species explored so far.

Our results raise an interesting possibility that the β₃-AR can change its signaling pathway from a stimulatory, G_s-mediated cAMP/PKA mechanism to an inhibitory, G_i/o-mediated one, depending on the cellular context. Some support for this hypothesis comes from heterologous β₃-AR expression studies. For instance, in CHO cells (14, 58) and murine 3T3-F442A preadipocytes (59) transfected with rat β₃-ARs, the receptors are coupled to G_s proteins, leading to an increase in intracellular cAMP level. However, in rat and mice adipocytes, in addition to their coupling to G_s, β₃-ARs couple to G_i/o proteins and inhibit adenylyl cyclase (60, 61). In the human A549 lung epithelial cell line and in the human colonic carcinoma cell line T84, β₃-ARs appear to be exclusively linked to G_i/o proteins (18, 62). Functional response to β₃-AR activation may also depend on the amount of expressed receptors. Indeed, cardiac overexpression of human β₃-ARs in mice results in G_s activation of adenylyl cyclase and positive inotropy on stimulation with a β₃-AR agonist (25). Thus, if the human atrium expresses a higher amount of β₃-ARs than the ventricle (which would go in line with the higher amount of spare β₁-/β₂-ARs in the atrium; ref. 49), it might favor a G_s- over G_i/o-mediated signaling pathway. Finally, depending on its cellular environment, the β₃-AR might either activate or inhibit the same ion channel, such as the L-type Ca²⁺ channel. Such a situation has been reported for the β₃-AR regulation of the slow component of the delayed rectifier potassium current (I_Ks), a current which like I_Ca,L is activated by cAMP/PKA cascade (63). Indeed, in isolated guinea pig ventricular myocytes, β₃-AR activation by BRL37344 inhibits I_Ks by 20%–40% (51). However, when KvLQT1/MinK channels, which underline I_Ks current, are heterologously coexpressed with the human β₃-AR in Xenopus oocytes, activation of the receptor increases I_Ks (64).

Although β₃-AR activation increased I_Ca,L in HAMs, the positive inotropic effects in human atrium were modest as compared with β₁-/β₂-AR activation with ISO. Because contractility of a trabeculum is a more integrated response than I_Ca,L amplitude in a single myocyte, many other parameters will participate in the inotropic response to β₃-AR agonists. One of these is the action potential duration, which is affected not only by β₃-AR regulation of I_Ca,L but also by the effect of the receptor on other ion channels, such as I_Ks (51), the rapid component of the delayed rectifier potassium current (I_Kr) (52), the hyperpolarization-activated pacemaker current I_f (54), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– current (18). Other downstream factors include intracellular Ca²⁺ transient and sarcoplasmic reticulum function and the phosphorylation status of key proteins such as phospholamban, ryanodine receptor, and contractile proteins. Finally, human cardiac trabeculae are multicellular preparations composed of myocytes and nonmyocytes such as endothelial cells, which also express β₃-ARs (65, 66). Of note, β₃-ARs in endothelial cells activate NO production that may reduce cardiac contractility (67, 68) and counterbalance a stimulatory effect of β₃-ARs in myocytes. We tested this hypothesis in 4 human atrial trabeculae exposed to CGP12177 (100 nM) in the presence or absence of L-NMMA and found that NOS inhibition increased approximately 2-fold the positive inotropic effect of the agonist in atrium (P < 0.05; data not shown). Therefore, the overall effect of a β₃-AR activation on the contractility of human atrium may vary from a stimulation to an inhibition, depending on the chamber, the amount of nonmyocyte endothelial and endocardial cells, and the activity of NOS enzymes. Another level of complexity may result from the ability of the myocyte to distinguish among different stimuli acting on a common signaling cascade. Indeed, even though β₁-, β₂-, and β₃-ARs may all activate the cAMP/PKA pathway, the signaling cascade generated by each receptor may be confined to distinct intracellular compartments, which may result in different functional responses (45). A number of studies, including our own (44, 69), have underlined the importance of cAMP PDEs in generating and maintaining intracellular cAMP domains (45). Our findings that PDE inhibition with IBMX transformed an unimpressively small contractile response to β₃-AR agonists to a large, positive inotropic effect similar to a β₁-AR response strongly suggest that not all cAMP/PKA effectors are normally activated by β₃-AR activation. A similar observation was recently reported for the serotonin 5-HT₄ receptors in failing human ventricular muscle, where these receptors increased contractility only when PDE activity was blocked (70).

In summary, while β₃-AR activation produces negative inotropic effects in human endomyocardial biopsies from transplanted hearts (8, 16) and in left ventricular samples from failing and nonfailing explanted hearts (11, 16) (see also refs. 26–28), our study demonstrates that β₃-AR activation increases contractility in human atrial tissue. This is reminiscent of the contractile effects of the serotonin 5-HT₄ receptors (71), which are also coupled to increases in force of contraction (72) or I_Ca,L (41) in atria but not in healthy ventricles. In human ventricle, the negative inotropic effect of β₃-AR activation was proposed to spare the heart of excessive oxygen demand and may therefore play a role as a “safety-valve” during intense adrenergic stimulation (16). The identification of an opposite effect of β₃-ARs in human atrium is therefore of importance, particularly in the scope of the development of new therapeutic agents acting on these receptors for the control of the adrenergic system in cardiovascular diseases. Finally, the positive coupling of β₃-ARs to I_Ca,L in human atrium, with relatively modest consequences on force of contraction, might confer to these receptors a role in the control of beat frequency. During heart failure, the expression level of β₃-ARs increases (11, 29, 30), and this might change their beneficial effect into a deleterious one, with a persistent negative inotropic effect enhancing myocardial depression. It remains to be determined whether the level of β₃-ARs and their functional role are also modified in human atrial pathologies such as atrial dilation, arrhythmias, or fibrillation. This is particularly relevant, since congestive heart failure and atrial fibrillation are commonly encountered together.

View Supplemental data

This work was supported by a grant from INSERM Programme National de Recherche sur les Maladies Cardiovasculaires, the Fondation Leducq for the Transatlantic Network of Excellence 06 CVD 02 (to R. Fischmeister), and by European Union contract LSHM-CT-2005-018833/EUGeneHeart (to R. Fischmeister). We thank Patrick Lechêne and Antanas Navalinskas for skillful technical assistance, Florence Lefebvre for preparation of the cells, and Bertrand Crozatier for critical reading of the manuscript. V.A. Skeberdis was supported by a fellowship from INSERM (Poste Vert).

Address correspondence to: Rodolphe Fischmeister, INSERM UMR-S 769, Faculté de Pharmacie, Université Paris-Sud 11, 5 Rue Jean-Baptiste Clément, F-92296 Châtenay-Malabry, France. Phone: 33-1-46-83-57-71; Fax: 33-1-46-83-54-75; E-mail: rodolphe.fischmeister@inserm.fr.

Nonstandard abbreviations used: β-AR, β-adrenergic receptor; HAM, human atrial myocyte; IBMX, 3-isobutyl-1-methylxanthine; I_Ca,L, L-type Ca²⁺ channel current; I_Ks, delayed rectifier potassium current; ISO, isoprenaline; L-NMMA, N^G-monomethyl-l-arginine; PDE, phosphodiesterase.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:3219–3227 (2008). doi:10.1172/JCI32519

1. Bers, D.M. 2002. Cardiac excitation-contraction coupling. Nature. 415:198-205.
   View this article via: CrossRef PubMed Google Scholar
2. Brette, F., Leroy, J., Le Guennec, J.Y., Sallé, L. 2006. Ca²⁺ currents in cardiac myocytes: old story, new insights. Prog. Biophys. Mol. Biol. 91:1-82.
   View this article via: CrossRef PubMed Google Scholar
3. Morisco, C., Zebrowski, D.C., Vatner, D.E., Vatner, S.F., Sadoshima, J. 2001. β-Adrenergic cardiac hypertrophy is mediated primarily by the β₁-subtype in the rat heart. J. Mol. Cell. Cardiol. 33:561-573.
   View this article via: CrossRef PubMed Google Scholar
4. Ahmet, I., et al. 2004. Beneficial effects of chronic pharmacological manipulation of β-adrenoreceptor subtype signaling in rodent dilated ischemic cardiomyopathy. Circulation. 110:1083-1090.
   View this article via: CrossRef PubMed Google Scholar
5. Communal, C., Singh, K., Sawyer, D.B., Colucci, W.S. 1999. Opposing effects of β₁- and β₂-adrenergic receptors on cardiac myocyte apoptosis - Role of a pertussis toxin-sensitive G proteins. Circulation. 100:2210-2212.
   View this article via: PubMed Google Scholar
6. Zaugg, M., et al. 2000. β-Adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation. 102:344-350.
   View this article via: PubMed Google Scholar
7. Brodde, O.E., Bruck, H., Leineweber, K. 2006. Cardiac adrenoceptors: physiological and pathophysiological relevance. J. Pharmacol. Sci. 100:323-337.
   View this article via: CrossRef PubMed Google Scholar
8. Gauthier, C., Tavernier, G., Charpentier, F., Langin, D., Lemarec, H. 1996. Functional β₃-adrenoceptor in the human heart. J. Clin. Invest. 98:556-562.
   View this article via: JCI CrossRef PubMed Google Scholar
9. Moniotte, S., et al. 2001. Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies. J. Mol. Cell. Cardiol. 33:2121-2133.
   View this article via: CrossRef PubMed Google Scholar
10. Chamberlain, P.D., et al. 1999. The tissue distribution of the human β₃-adrenoceptor studied using a monoclonal antibody: direct evidence of the β₃-adrenoceptor in human adipose tissue, atrium and skeletal muscle. Int. J. Obes. Relat. Metab. Disord. 23:1057-1065.
    View this article via: CrossRef PubMed Google Scholar
11. Moniotte, S., et al. 2001. Upregulation of β₃-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation. 103:1649-1655.
    View this article via: PubMed Google Scholar
12. De Matteis, R., et al. 2002. Immunohistochemical identification of the β₃-adrenoceptor in intact human adipocytes and ventricular myocardium: effect of obesity and treatment with ephedrine and caffeine. Int. J. Obes. Relat. Metab. Disord. 26:1442-1450.
    View this article via: CrossRef PubMed Google Scholar
13. Blin, N., Camoin, L., Maigret, B., Strosberg, D. 1993. Structural and conformational features determining selective signal transduction in the β3-adrenergic receptor. Mol. Pharmacol. 44:1094-1104.
    View this article via: PubMed Google Scholar
14. Emorine, L.J., et al. 1989. Molecular characterization of the human β₃-adrenergic receptor. Science. 245:1118-1121.
    View this article via: CrossRef PubMed Google Scholar
15. Strosberg, A.D. 1997. Structure and function of the β₃-adrenergic receptor. Annu. Rev. Pharmacol. Toxicol. 37:421-450.
    View this article via: CrossRef PubMed Google Scholar
16. Rozec, B., Gauthier, C. 2006. β₃-Adrenoceptors in the cardiovascular system: Putative roles in human pathologies. Pharmacol. Ther. 111:652-673.
    View this article via: CrossRef PubMed Google Scholar
17. Gauthier, C., et al. 1998. The negative inotropic effect of β₃-adrenoreceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle. J. Clin. Invest. 102:1377-1384.
    View this article via: JCI CrossRef PubMed Google Scholar
18. Leblais, V., et al. 1999. β₃-adrenoceptor control the cystic fibrosis transmembrane conductance regulator through a cAMP/Protein kinase A-independent pathway. J. Biol. Chem. 274:6107-6113.
    View this article via: CrossRef PubMed Google Scholar
19. Kitamura, T., et al. 2000. The negative inotropic effect of β₃-adrenoceptor stimulation in the beating guinea pig heart. J. Cardiovasc. Pharmacol. 35:786-790.
    View this article via: CrossRef PubMed Google Scholar
20. Varghese, P., et al. 2000. β₃-adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility. J. Clin. Invest. 106:697-703.
    View this article via: JCI CrossRef PubMed Google Scholar
21. Devic, E., Xiang, Y., Gould, D., Kobilka, B. 2001. β-adrenergic receptor subtype-specific signaling in cardiac myocytes from β₁ and β₂ adrenoceptor knockout mice. Mol. Pharmacol. 60:577-583.
    View this article via: PubMed Google Scholar
22. Kaumann, A.J., et al. 1998. (-)-CGP 12177 causes cardiostimulation and binds to cardiac putative β₄-adrenoceptors in both wild-type and β₃-adrenoceptor knockout mice. Mol. Pharmacol. 53:670-675.
    View this article via: PubMed Google Scholar
23. Oostendorp, J., Kaumann, A.J. 2000. Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through β₁- and putative β₄-adrenoceptors in mouse left atria: no evidence for β₂- and β₃-adrenoceptor function. Naunyn Schmiedebergs Arch. Pharmacol. 361:134-145.
    View this article via: CrossRef PubMed Google Scholar
24. Heubach, U.F., Rau, T., Eschenhagen, T., Ravens, U., Kaumann, A.J. 2002. Physiological antagonism between ventricular β₁-adrenoceptors and α₁-adrenoceptors but no evidence for β₂- and β₃-adrenoceptor function in murine heart. Br. J. Pharmacol. 136:217-229.
    View this article via: CrossRef PubMed Google Scholar
25. Kohout, T.A., et al. 2001. Augmentation of cardiac contractility mediated by the human β₃-adrenergic receptor overexpressed in the hearts of transgenic mice. Circulation. 104:2485-2491.
    View this article via: CrossRef PubMed Google Scholar
26. Kaumann, A.J., Molenaar, P. 1997. Modulation of human cardiac function through 4 β-adrenoceptor populations. Naunyn Schmiedebergs Arch. Pharmacol. 355:667-681.
    View this article via: CrossRef PubMed Google Scholar
27. Molenaar, P., Sarsero, D., Kaumann, A.J. 1997. Proposal for the interaction of non-conventional partial agonists and catecholamines with the ‘putative beta 4-adrenoceptor’ in mammalian heart. Clin. Exp. Pharmacol. Physiol. 24:647-656.
    View this article via: CrossRef PubMed Google Scholar
28. Harding, S.E. 1997. Lack of evidence for β₃-adrenoceptor modulation of contractile function in human ventricular myocytes [abstract]. Circulation. 96(Suppl. S):284.
29. Cheng, H.J., et al. 2001. Upregulation of functional β₃-adrenergic receptor in the failing canine myocardium. Circ. Res. 89:599-606.
    View this article via: CrossRef PubMed Google Scholar
30. Morimoto, A., Hasegawa, H., Cheng, H.J., Little, W.C., Cheng, C.P. 2004. Endogenous β₃-adrenoreceptor activation contributes to left ventricular and cardiomyocyte dysfunction in heart failure. Am. J. Physiol. Heart Circ. Physiol. 286:H2425-H2433.
    View this article via: CrossRef PubMed Google Scholar
31. Simiand, J., et al. 1992. Antidepressant profile in rodents of SR 58611A, a new selective agonist for atypical β-adrenoceptors. Eur. J. Pharmacol. 219:193-201.
    View this article via: CrossRef PubMed Google Scholar
32. Muzzin, P., Seydoux, J., Giacobino, J.P., Venter, J.C., Fraser, C. 1988. Discrepancies between the affinities of binding and action of the novel β-adrenergic agonist BRL 37344 in rat brown adipose tissue. Biochem. Biophys. Res. Commun. 156:375-382.
    View this article via: CrossRef PubMed Google Scholar
33. Staehelin, M., Simons, P. 1982. Rapid and reversible disappearance of β-adrenergic cell surface receptors. EMBO. J. 1:187-190.
    View this article via: PubMed Google Scholar
34. Granneman, J.G. 2001. The putative beta4-adrenergic receptor is a novel state of the beta1-adrenergic receptor. Am. J. Physiol. Endocrinol. Met. 280:E199-E202.
    View this article via: PubMed Google Scholar
35. Joseph, S.S., Lynham, J.A., Colledge, W.H., Kaumann, A.J. 2004. Binding of (-)-[³H]-CGP12177 at two sites in recombinant human β₁-adrenoceptors and interaction with β-blockers. Naunyn Schmiedebergs Arch. Pharmacol. 369:525-532.
    View this article via: CrossRef PubMed Google Scholar
36. Candelore, M.R., et al. 1999. Potent and selective human β₃-adrenergic receptor antagonists. J. Pharmacol. Exp. Ther. 290:649-655.
    View this article via: PubMed Google Scholar
37. Kozlovski, V.I., Chlopicki, S., Gryglewski, R.J. 2003. Effects of two β₃-agonists, CGP 12177A and BRL 37344, on coronary flow and contractility in isolated guinea pig heart. J. Cardiovasc. Pharmacol. 41:706-713.
    View this article via: CrossRef PubMed Google Scholar
38. Galitzky, J., et al. 1993. Coexistence of β₁-, β₂-, and β₃-adrenoceptors in dog fat cells and their differential activation by catecholamines. Am. J. Physiol. 264:E403-E412.
    View this article via: PubMed Google Scholar
39. Kirstein, M., et al. 1995. Nitric oxide regulates the calcium current in isolated human atrial myocytes. J. Clin. Invest. 95:794-802.
    View this article via: JCI CrossRef PubMed Google Scholar
40. Vandecasteele, G., Verde, I., Rucker-Martin, C., Donzeau-Gouge, P., Fischmeister, R. 2001. Cyclic GMP regulation of the L-type Ca²⁺ channel current in human atrial myocytes. J. Physiol. 533:329-340.
    View this article via: CrossRef PubMed Google Scholar
41. Ouadid, H., Seguin, J., Dumuis, A., Bockaert, J., Nargeot, J. 1992. Serotonin increases calcium current in human atrial myocytes via the newly described 5-hydroxytryptamine₄ receptors. Mol. Pharmacol. 41:346-351.
    View this article via: PubMed Google Scholar
42. Chijiwa, T., et al. 1990. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5- isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265:5267-5272.
    View this article via: PubMed Google Scholar
43. Pott, C., et al. 2003. The preferential β₃-adrenoceptor agonist BRL 37344 increases force via β₁-/β₂-adrenoceptors and induces endothelial nitric oxide synthase via β₃-adrenoceptors in human atrial myocardium. Br. J. Pharmacol. 138:521-529.
    View this article via: CrossRef PubMed Google Scholar
44. Rochais, F., et al. 2006. A specific pattern of phosphodiesterases controls the cAMP signals generated by different G_s-coupled receptors in adult rat ventricular myocytes. Circ. Res. 98:1081-1088.
    View this article via: CrossRef PubMed Google Scholar
45. Fischmeister, R., et al. 2006. Compartmentation of cyclic nucleotide signaling in the heart: The role of cyclic nucleotide phosphodiesterases. Circ. Res. 99:816-828.
    View this article via: CrossRef PubMed Google Scholar
46. Howe, R. 1993. β₃-adrenergic agonists. Drugs Future. 18:529-549.
47. Kaumann, A.J., Lemoine, H., Morris, T.H., Schwederski, U. 1982. An initial characterization of human heart beta-adrenoceptors and their mediation of the positive inotropic effects of catecholamines. Naunyn Schmiedebergs Arch. Pharmacol. 319:216-221.
    View this article via: CrossRef PubMed Google Scholar
48. Schwinger, R.H., Bohm, M., Erdmann, E. 1990. Evidence against spare or uncoupled beta-adrenoceptors in the human heart. Am. Heart J. 119:899-904.
    View this article via: CrossRef PubMed Google Scholar
49. Brown, L., et al. 1992. Spare receptors for beta-adrenoceptor-mediated positive inotropic effects of catecholamines in the human heart. J. Cardiovasc. Pharmacol. 19:222-232.
    View this article via: PubMed Google Scholar
50. Au, A.L., Kwan, Y.W. 2002. Modulation of L-type Ca²⁺ channels by β₃-adrenoceptor activation and the involvement of nitric oxide. J. Card. Surg. 17:465-469.
    View this article via: PubMed Google Scholar
51. Bosch, R.F., et al. 2002. β₃-Adrenergic regulation of an ion channel in the heart-inhibition of the slow delayed rectifier potassium current I_Ks in guinea pig ventricular myocytes. Cardiovasc. Res. 56:393-403.
    View this article via: CrossRef PubMed Google Scholar
52. Karle, C.A., et al. 2002. Rapid component I_Kr of the guinea-pig cardiac delayed rectifier K⁺ current is inhibited by β₁-adrenoreceptor activation, via cAMP/protein kinase A-dependent pathways. Cardiovasc. Res. 53:355-362.
    View this article via: CrossRef PubMed Google Scholar
53. Zhang, Z.S., et al. 2005. Enhanced inhibition of L-type Ca²⁺ current by β₃-adrenergic stimulation in failing rat heart. J. Pharmacol. Exp. Ther. 315:1203-1211.
    View this article via: CrossRef PubMed Google Scholar
54. Sartiani, L., et al. 2006. Functional remodeling in post-myocardial infarcted rats: focus on beta-adrenoceptor subtypes. J. Mol. Cell. Cardiol. 40:258-266.
    View this article via: CrossRef PubMed Google Scholar
55. Belles, B., Malécot, C.O., Hescheler, J., Trautwein, W. 1988. ‘Run-down’ of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium. Pflügers Arch. 411:353-360.
    View this article via: CrossRef PubMed Google Scholar
56. Kepplinger, K.J.F., et al. 2000. Molecular determinant for run-down of L-type Ca²⁺ channels localized in the carboxyl terminus of the a_1C subunit. J. Physiol. 529:119-130.
    View this article via: CrossRef PubMed Google Scholar
57. Vandecasteele, G., Eschenhagen, T., Fischmeister, R. 1998. Role of the NO-cGMP pathway in the muscarinic regulation of the L-type Ca²⁺ current in human atrial myocytes. J. Physiol. 506:653-663.
    View this article via: CrossRef PubMed Google Scholar
58. Granneman, J.G., Lahners, K.N., Chaudhry, A. 1991. Molecular cloning and expression of the rat β₃-adrenergic receptor. Mol. Pharmacol. 40:895-899.
    View this article via: PubMed Google Scholar
59. Feve, B., et al. 1991. Atypical β-adrenergic receptor in 3T3-F442A adipocytes. J. Biol. Chem. 266:20329-20336.
    View this article via: PubMed Google Scholar
60. Chaudhry, A., MacKenzie, R.G., Georgic, L.M., Granneman, J.G. 1994. Differential interaction of β₁- and β₃-adrenergic receptors with G_i in rat adipocytes. Cell. Signal. 6:457-465.
    View this article via: CrossRef PubMed Google Scholar
61. Begin-Heick, N. 1995. β₃ -adrenergic activation of adenylyl cyclase in mouse white adipocytes: modulation by GTP and effect of obesity. J. Cell. Biochem. 58:464-473.
    View this article via: CrossRef PubMed Google Scholar
62. Robay, A., Toumaniantz, G., Leblais, V., Gauthier, C. 2005. Transfected β₃- but not β₂-adrenergic receptors regulate cystic fibrosis transmembrane conductance regulator activity via a new pathway involving the mitogen-activated protein kinases extracellular signal-regulated kinases. Mol. Pharmacol. 67:648-54.
    View this article via: CrossRef PubMed Google Scholar
63. Kurokavva, J., Chen, L., Kass, R.S. 2003. Requirement of subunit expression for cAMP-mediated regulation of a heart potassium channel. Proc. Natl. Acad. Sci. U. S. A. 100:2122-2127.
    View this article via: CrossRef PubMed Google Scholar
64. Kathöfer, S., et al. 2000. Functional coupling of human β₃-adrenoreceptors to the KvLQT1/MinK potassium channel. J. Biol. Chem. 275:26743-26747.
    View this article via: PubMed Google Scholar
65. Dessy, C., et al. 2005. Endothelial β₃-adrenoreceptors mediate nitric oxide-dependent vasorelaxation of coronary microvessels in response to the third-generation β-blocker nebivolol. Circulation. 112:1198-205.
    View this article via: CrossRef PubMed Google Scholar
66. Dessy, C., et al. 2004. Endothelial β₃-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endothelium-dependent hyperpolarization. Circulation. 110:948-954.
    View this article via: CrossRef PubMed Google Scholar
67. Massion, P.B., Feron, O., Dessy, C., Balligand, J.L. 2003. Nitric oxide and cardiac function — Ten years after, and continuing. Circ. Res. 93:388-398.
    View this article via: CrossRef PubMed Google Scholar
68. Fischmeister, R., Castro, L., Abi-Gerges, A., Rochais, F., Vandecasteele, G. 2005. Species- and tissue-dependent effects of NO and cyclic GMP on cardiac ion channels. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 142:136-143.
    View this article via: CrossRef PubMed Google Scholar
69. Jurevicius, J., Skeberdis, V.A., Fischmeister, R. 2003. Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following β₂- adrenergic stimulation of I_Ca,L in frog ventricular myocytes. J. Physiol. 551:239-252.
    View this article via: CrossRef PubMed Google Scholar
70. Brattelid, T., et al. 2004. Functional serotonin 5-HT₄ receptors in porcine and human ventricular myocardium with increased 5-HT₄ mRNA in heart failure. Naunyn Schmiedebergs Arch. Pharmacol. 370:157-166.
    View this article via: PubMed Google Scholar
71. Schoemaker, R.G., Du, X.Y., Bax, W.A., Bos, E., Saxena, P.R. 1993. 5-Hydroxytryptamine stimulates human isolated atrium but not ventricle. Eur. J. Pharmacol. 230:103-105.
    View this article via: CrossRef PubMed Google Scholar
72. Jahnel, U., Rupp, J., Ertl, R., Nawrath, H. 1992. Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle. Naunyn Schmiedebergs Arch. Pharmacol. 346:482-485.
    View this article via: PubMed Google Scholar
73. Rucker-Martin, C., et al. 1993. Behaviour of human atrial myocytes in culture is donor age dependent. Neuromusc. Disord. 3:385-390.
    View this article via: CrossRef PubMed Google Scholar
74. Rivet-Bastide, M., et al. 1997. cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes. J. Clin. Invest. 99:2710-2718.
    View this article via: JCI CrossRef PubMed Google Scholar