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Cardiovascular Research 2003 59(2):262-265; doi:10.1016/S0008-6363(03)00477-2
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Negative inotropy starts with the β3-adrenoceptor

Enn K. Seppet*

Department of Pathophysiology, Faculty of Medicine, University of Tartu, 19 Ravila Street, 50411 Tartu, Estonia

* Tel.: +372-7-374-371; fax: +372-7-374-372. enn{at}ut.ee

Received 3 June 2003; revised 6 June 2003; accepted 6 June 2003

See article by Tavernier et al. [1] (pages 288–296) in this issue.

In this issue of Cardiovascular Research, Tavernier et al. describe the effects of β3-adrenergic stimulation on cardiac contractility in mice overexpressing the human β3-adrenoceptor (β3-AR) [1]. This study is a logical sequel to preceding investigations by the same research group that also provided the first evidence of the existence and functional role of this AR subtype in the human heart [2]. That time it was found that in contrast to the classical effects of β1-/β2-adrenergic system—positive inotropy and lusitropy—activation of β3-AR results in negative inotropy via a Gi protein-dependent pathway. Further studies revealed that β3-AR stimulation attenuates cardiac contractility through increased nitric oxide (NO) production and intracellular cGMP [3,4].

To further explore the properties and role of the β3-adrenergic system, Tavernier et al. [1] generated transgenic mice overexpressing the human β3-AR in cardiac cells. The human receptor was selected for two reasons: (i) compared to other species, human cardiac muscle exhibits the strongest negative inotropic effect induced by β3-AR agonists [5] and, (ii) cardiac failure in humans is associated with increased expression of β3-AR (see below). The effectiveness of gene expression was confirmed by polymerase chain reaction and Western blotting, showing human β3-AR mRNA and corresponding protein in transgenic mice, but not in wild-type mice. Then the contractility experiments were performed under the same conditions as those used earlier for human myocardium [2]. The results show that expression of β3-adrenoceptor was accompanied by decreased peak tension in response to low concentrations of β3-AR agonists (CL 316243 and SR 58611A), consistent with their β3-AR-specific action. In parallel, an increase in intracellular cGMP level was observed. Thus, the β3-ARs were functional in that they were coupled to downstream components of the NO-dependent signalling pathway, and thereby capable of reproducing the negative inotropic response qualitatively similar to that observed in human [2], dog [5], and guinea pig [6] cardiac preparations by β3-AR stimulation. These data clearly show that the role of β3-ARs in cardiac muscle is to initiate negative inotropic influence on cardiac contraction. The mechanisms of β3-AR-mediated attenuation of cardiac contractility are largely undiscovered, but the current evidence suggests the involvement of alterations in excitation–contraction coupling and transmembrane ion channels. A β3-AR agonist BRL 37344 is shown to induce a dose-dependent negative inotropic effect, accompanied by inhibition of L-Ca2+ channels and decreased amplitude of the intracellular Ca2+ transient [6,7]. These changes may be caused by increased cGMP levels that, either via activation of cGMP-dependent protein kinases or through attenuation of the cAMP levels by activation of cGMP-stimulated phosphodiesterases, suppress the current through L-Ca2+ channels [8]. NO, another mediator of the pathway, may exert a similar effect by covalent modification of the L-Ca2+ channel protein [8]. On the other hand, the finding that BRL 37344 decreases the amplitude and action potential duration in human endomyocardial biopsies [2] points to potential alterations in repolarizing currents. In line with this, the β3-AR was found to be functionally coupled to cystic fibrosis transmembrane conductance regulator (CFTR) channels. This enables activation of a repolarizing Cl– current under stimulation of the β3-adrenergic pathway [9]. The CFTR protein is found to be present and functional in murine heart [10]. Therefore, it could mediate decreased inotropy under stimulation of β3-ARs in the present study [1]. Controversial data exist regarding the role of K+ currents, however. It has been suggested that β3-ARs are coupled to the K+ channel KvLQT1/MinK [11]. This explains the shortening of the human cardiac action potential [2]. However, others have demonstrated that although the β3-AR is functionally coupled to a K+ channel, its stimulation with BRL 37344 brings about inhibition of IKs, thereby increasing the action potential duration in guinea-pig cardiomyocytes [12]. In experiments of Tavernier et al. [1], the role of IKs may be disregarded due to the lack of IKs in cardiac cells of adult mice [13]. This current functions in guinea pig and human hearts [12], however, and thus the decreased inotropy by β3-agonist could be attributed to changes in altered balance between inward and outward currents. The mechanisms of regulation of cardiac contractility by β3-AR-mediated pathways need to be further addressed in order to test the potential effects of increased cGMP and NO on troponin I, sarcoplasmic reticulum, and enzymes of energy metabolism, e.g., cytochrome oxidase and creatine kinase [8,14].

The results of the present study [1] disagree with another recent report that addressed the influence of overexpression of human β3-ARs on cardiac contractility in mice in situ [15]. In this latter study, the administration of the selective human β3-adrenoceptor agonist, L-755,507, resulted in an increased LV dP/dtmax together with a shift of end-systolic points of the pressure–volume relations upward and to the left, both changes indicating the increased contractility by an agonist. The reasons for these discordant results are not obvious. They may be related to different contraction frequencies (6.6–7.3 Hz [15] vs. 1.66 Hz [1]), owing to the rate-dependency of the developed force. In addition, the potential influence of anesthetic regimen on cardiac function in situ [16,17] should not be overlooked. For example, ketamine/xylazine is known to produce significant reductions in heart rate [16,17]. Notably, this was confirmed, as the heart rate decreased from 687 to 436 beats per min under the same type of anesthesia [15]. When added at this lower baseline heart rate, the β3-AR agonist (L755,507) significantly increased the heart rate in β3-transgenic mice, but not in wild-type mice [15]. In a recent study on murine heart in situ [18], an increase in heart rate from 420 to 600 beats per min caused an upward and leftward shift of pressure–volume loops, i.e., towards increased contractility, due to an increased role of L-Ca2+ channel in providing activator Ca2+ within this frequency region [18]. Therefore, it is plausible that the changes in pressure–volume loops observed after addition of L755,507 in β3-transgenic mice [15] could result from the effects of increased heart rate rather than from the influence of that agonist on the contractile properties per se. This problem was avoided in studies of Tavernier et al. [1], as the contraction frequency was kept constant. Nevertheless, further studies are needed to estimate the cardiac function of β3-transgenic mice at physiological contraction frequencies in situ. It is also important to notice that the β3-transgenic mice used in the present study [1] exhibited higher basal heart rate compared to wild-type animals, whereas both groups of animals used in the experiments in situ [15] had similar heart rates. These differences may be related to diverse genetic backgrounds of the animals used, which should also be taken into consideration while comparing the results of different studies.

It is well known that development of cardiac failure is associated with increased activation of the sympathetic nervous system in order to stimulate contractility and maintain cardiac output and systemic blood pressure [19]. However, chronic increase in circulating epinephrine and norepinephrine results in a decreased inotropic and lusitropic response of the myocardium to these catecholamines. The underlying mechanisms comprise downregulation of β1-AR density, upregulation of Gi protein together with RGS3 and RGS4 proteins [20], reduced activity of adenylate cyclase, and desensitization and internalization of β1- and β2-ARs (reviewed in Refs. [19,21]). Altogether, these alterations result in decreased phosphorylation of phospholamban in sarcoplasmic reticulum and of L-Ca2+ channels, thus contributing to decreased contractile function. Aside from the suppressed function of the β1/β2-AR-mediated signalling system, the expression of β3-AR increases in the course of development of cardiac failure, in both human and dog [7,22]. The upregulated AR is functionally active, for it is tightly coupled to Gi protein and its stimulation with β3-AR agonists results in decreased intracellular Ca2+ transients and contractile function in situ [7,22]. The persistence of β3-AR's function is explained by the lack of phosphorylation sites for βARK1, causing a relative resistance to agonist-induced desensitization [23].

The role of upregulation of the β3-AR-mediated signalling system in the pathophysiology of failing heart is still unclear. Given the complexity of structural and functional alterations, such as hypertrophy of cardiomyocytes, proliferation of fibroblasts and fibrosis, inflammatory processes, neurohumoral activation, and compromised mitochondrial function, it is difficult to understand whether it represents the cause or consequence of these changes. Here, the article by Tavernier et al. [1] offers several clear answers to this question. First, it shows that increased abundance of β3-AR is sufficient to activate a whole cascade of the signalling system that finally, via increased cGMP and NO production, suppresses cardiac contraction. This conclusion concurs with the results of a recent study that addressed the influence of G{alpha}i2 gene transfer on the myocardial contractile response to isoproterenol [24]. It was found that overexpression of G{alpha}i2 blunted the β-adrenergic response in a dose-dependent manner [24]. When viewed in the context of cardiac failure, these results show that the combination of increased density of β3-ARs and enhanced Gi protein levels provides the cardiomyocytes with a potent system to inhibit contractility and/or suppress the effects of β1/β2-adrenergic pathway on contractile function. Moreover, they make it easier to understand the causal links between the changes in expression of the components of the β3-adrenergic pathway and the contractile function of failing heart. For example, the observation that the contractile response to isoproterenol was more attenuated in dilated cardiomyopathic human hearts than in failing ischemic hearts can be attributed to corresponding differences in Gi protein expression [22]. Second, the finding that β3-transgenic mice did not exhibit myocyte hypertrophy and fibrogenesis [1] suggests that an activated β3-adrenergic system is not primarily responsible for triggering these processes in the failing heart. This is contradictory to the effect of overexpression of β1-ARs, causing cardiac hypertrophy, fibrosis, and heart failure in mice [25].

When put together, these results support the hypothesis that upregulation of the β3-AR-mediated pathway may serve to prevent myocardial damage under excess catecholamines, particularly in early stages of disease [21]. The type of protection can be envisaged as an "intracellular β-blockade" (a term used to emphasize the role of overexpressed G{alpha}i2 [24]), which starts with β3-AR activation. Such a blockade may limit the development of intracellular Ca2+ overload by suppressing L-type Ca2+ channels and improve diastolic function by producing NO [14,21]. β3-adrenergic attenuation of the contractility could also preserve intracellular ATP and phosphocreatine, thus contributing to the survival of the cardiomyocytes. It is possible that the β3-adrenergic system modulates cardiac contractility not only in a steady-state but also on a beat-to-beat basis, owing to cyclical changes in intracellular NO and cGMP production correspondingly to the cardiac cycle (reviewed in [14]). However, in later stages of the disease, when decreased eNOS expression [22] starts to limit the NO production, the protective role of the β3-adrenergic pathway may fade away, leaving the myocardium under the influence of a full set of factors promoting cardiac failure.

Aside from these scenarios, the activation of β3-AR-mediated system may switch on in other, hitherto unknown signalling pathways [21]. In this regard it is remarkable that in both studies [1,15], overexpression of β3-ARs resulted in reduced heart weight. This suggests that the β3-adrenergic system may be involved in control of cardiac growth. It has been shown that cardiac hypertrophy, fibrosis, and contractile failure in β1-AR transgenic mice can be prevented by inhibition of the Na+–H+ exchanger, the expression of which is upregulated in this animal [25]. In the light of this observation, it would be interesting to know whether β3-adrenergic stimulation can attenuate these processes, including the expression of the Na+–H+ exchanger.

Since the discovery of the existence of a functional β3-system in cardiac cells [2], the potential therapeutic developments aimed to control the function of that system have been suggested [21]. However, the real breakthrough seems to be still far away. One reason for this is the absence of suitable animal models for pharmacological testing. In this regard, the transgenic mice overexpressing human β3-ARs and reproducing the negative inotropic effects observed in humans [1] may become a valuable model in the search for new compounds for human medicine.


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