β₃-adrenoceptor deficiency blocks nitric oxide–dependent inhibition of myocardial contractility

The cardiac β-adrenergic pathway potently stimulates myocardial performance, thereby providing a mechanism for myocardial contractile reserve. β-Adrenergic activation also increases cardiac nitric oxide (NO) production, which attenuates positive inotropy, suggesting a possible negative feedback mechanism. Recently, in vitro studies suggest that stimulation of the β₃-adrenoceptor results in a negative inotropic effect through NO signaling. In this study, using mice with homozygous β₃-adrenoceptor deletion mutations, we tested the hypothesis that the β₃-adrenoceptor is responsible for β-adrenergic activation of NO. Although resting indices of myocardial contraction were similar, β-adrenergic–stimulated inotropy was increased in β₃^–/– mice, and similar hyper-responsiveness was seen in mice lacking endothelial NO synthase (NOS3). NOS inhibition augmented isoproterenol-stimulated inotropy in wild-type (WT), but not in β₃^–/– mice. Moreover, isoproterenol increased myocardial cGMP in WT, but not β₃^–/–, mice. NOS3 protein abundance was not changed in β₃^–/– mice, and cardiac β₃-adrenoceptor mRNA was detected in both NOS3^–/– and WT mice. These findings indicate that the β₃-adrenergic subtype participates in NO-mediated negative feedback over β-adrenergic stimulation.

Nitric oxide (NO) inhibition of β-adrenergic–stimulated contractility has been demonstrated in vitro (1) and in vivo (2–5). In addition to activating adenylyl cyclase production of cAMP, a cardiostimulatory pathway, β-adrenergic agonists lead to the production of NO. NO stimulates soluble guanylyl cyclase to produce cGMP, a cyclic nucleotide that opposes the cardiostimulatory effects of cAMP. Cardiodepression due to the NO-cGMP pathway likely has pathophysiologic significance because its activity is increased with heart failure (6, 7), sepsis (8), and aging (9, 10).

Whereas β-adrenergic stimulation has been shown to increase NO production (11), the relative contributions of specific β-adrenoceptor subtypes (1, 2, or 3) in this signal-transduction pathway have not been established. The β₃-adrenoceptor, recently appreciated in the mammalian heart (12–14), is widely expressed in adipose, intestinal, and lung tissues (15), where its activation promotes lipolysis and energy expenditure (16–18). β₃-adrenoceptor activation in the human heart produces direct negative inotropic responses (12, 13, 19). Observations that these negative inotropic effects are attenuated by NO synthase (NOS) inhibition suggest that the β₃-adrenoceptor has a role in NO-mediated inhibition of adrenergic stimulation.

The lack of specific β₃-adrenoceptor antagonists has hampered characterization of the precise in vivo role of β₃-NO signaling. To address this issue we measured β-adrenergic contractility in mice with a homozygous deletion mutation of the β₃-adrenoceptor gene (β₃^–/–). Using these animals, we tested the hypothesis that β₃^–/– mice lack the NO negative feedback over β-adrenergic contractility.

Effect of β₃-adrenoceptor and NOS3 deletion on base-line hemodynamic parameters. We used a combined micromanometer-conductance catheter to assess the determinants of cardiovascular performance in WT, β₃^–/–, and NOS3^–/– mice (Table 1). β₃^–/– mice had similar base-line indices of myocardial contractility and afterload compared with WT mice. Both the β₃^–/– and NOS3^–/– mice had smaller chamber sizes (lower EDV) than the WT mice, which was accompanied by impaired relaxation (higher tau) in the β₃^–/– mice. Whereas all three strains of mice had similar body weights, the NOS3^–/– mice had elevated heart weights resulting in increased heart-weight/body-weight ratio. As described previously (23), NOS3^–/– mice had hypertension reflected not only by elevated systemic blood pressure but also by increased Ea, a measure of arterial tone. Also, the NOS3^–/– mice had increased heart rate compared with the WT and β₃^–/– mice.

Table 1

Base-line conditions

Effect of intravenous isoproterenol on myocardial contractility. To assess the β-adrenergic inotropic response we infused isoproterenol (1, 10, 100 ng/kg/min) to anesthetized mice. As shown in Figure 1, isoproterenol produced an augmented contractile response in β₃^–/– mice (58 ± 7.9% increase in dP/dt-IP at peak isoproterenol response; P < 0.001) compared with WT mice (37 ± 5.9% peak dP/dt-IP increase; P < 0.001 vs. base line, P < 0.01 vs. β₃^–/– mice).

Figure 1

β-Adrenergic concentration-effect curves in β₃^–/– and WT mice. Isoproterenol was administered intravenously at rates of 1, 10, and 100 ng/kg/min to WT mice (FVB, n = 12) and mice with homozygous β₃-adrenoceptor–deletion mutations (β₃^–/–; n = 7). Peak positive dP/dt divided by the instantaneous left-ventricular pressure (dP/dt-IP) was used as an index of contractility and is displayed as a percentage of change from base line. As shown, the inotropic concentration-effect response to isoproterenol was augmented in β₃^–/– mice, relative to FVB. Each concentration-effect relationship was highly significant by one-way ANOVA (P < 0.01). Data are reported as mean ± SEM. ^AP < 0.01, β₃^–/– vs. FVB, by two-way ANOVA.

The effects on contractility, as assessed by dP/dt-IP, were not related to changes in preload or afterload after isoproterenol infusion, because EDP, EDV, Ea, and systolic blood pressure were not changed after isoproterenol infusion in either WT or β₃^–/– mice. Heart rate increased similarly in both the β₃^–/– mice (21.7 ± 3.2%; P < 0.01 vs. base line) and WT mice (29.0 ± 4.3%; P < 0.01 vs. base line, P = not significant [NS] vs. β₃^–/–).

Effect of NOS-inhibition on β-adrenergic stimulation. To determine the role of NO in β-adrenergic contractility we examined the impact of NOS inhibition with L-NMMA on β-adrenergic responses. Mice were administered isoproterenol (5 ng/kg/min) followed by the NOS inhibitor L-NMMA (10 mg/kg/h). At this concentration of isoproterenol, dP/dt-IP increased in both WT (10.1 ± 2.6%; P < 0.05 vs. base line; Figure 2a) and β₃^–/– mice (24.5 ± 3.1%; P < 0.001 vs. base line, P < 0.001 vs. WT; Figure 2a). In WT mice, co-infusion of L-NMMA with isoproterenol resulted in an augmentation of the positive inotropic response (dP/dt-IP increased 28.9 ± 5.6% over base line; P < 0.01 vs. base line, P < 0.05 vs. isoproterenol alone; Figure 2a). Thus, inhibition of NOS augments β-adrenergic–stimulated inotropy, consistent with a NO-related negative-feedback mechanism over cardiac contractile reserve. In contrast to the WT mice, L-NMMA did not augment the already elevated positive inotropic effect in β₃^–/– mice (dP/dt-IP increased 26.4 ± 5.6% over base line; P < 0.001 vs. base line, P = NS vs. isoproterenol alone; Figure 2a). A different NOS inhibitor, L-NAME, was also used in additional experiments. In similar fashion to L-NMMA, L-NAME augmented the isoproterenol inotropic response in WT, but not β₃^–/–, mice (Figure 2b).

Figure 2

(a) Contractile effects of isoproterenol and L-NMMA in WT, β₃^–/–, and NOS3^–/– mice. Isoproterenol was administered intravenously at a rate of 5 ng/kg/min for 4 minutes, followed by a co-infusion with L-NMMA at 10 mg/kg/h for 5 minutes. Contractility was indexed by dP/dt-IP and is shown as a percentage of change from base line. The β₃^–/– mice (n = 10) had greater responses to isoproterenol than did the WT mice (n = 8), but did not show any further augmentation after NOS inhibition with L-NMMA. Similarly, NOS3^–/– mice (n = 15) were hyper-responsive to isoproterenol. L-NMMA augmented the isoproterenol response in WT mice to the level observed in β₃^–/– mice. (b) The effect of an additional NOS inhibitor, L-NAME. L-NAME had an effect similar to L-NMMA, augmenting the response to isoproterenol in WT, but not β₃^–/–, mice. Data are reported as mean ± SEM. ^AP < 0.05 vs. respective base line; ^BP < 0.01 vs. respective base line; ^CP < 0.05 vs. WT isoproterenol response by one-way ANOVA.

We further assessed inotropic responses using the relatively load-independent parameter Ees. As shown in Figure 3, isoproterenol-stimulated increases in Ees were augmented by NOS inhibition with L-NMMA in WT, but not in β₃^–/–, mice.

Figure 3

Left ventricular pressure-volume data in WT and β₃^–/– mice. A combined micromanometer-conductance catheter was inserted into the LV through the apex. Transient occlusion of the descending aorta was used to generate the end-systolic pressure-volume relationship (loops not shown). Depicted are (a) example steady-state loops and their respective ESPVR (from which Ees is determined) at base line after receiving isoproterenol (5 ng/kg/min) and after receiving isoproterenol and L-NMMA (10 mg/kg/h). Also shown is (b) pooled data of the augmentation of isoproterenol-stimulated inotropy by L-NMMA in WT (n = 8) and β₃^–/– (n = 10) mice. Isoproterenol-induced increases in Ees were augmented by NOS inhibition in WT, but not in β₃^–/–, mice. Data are reported as mean ± SEM. ^AP < 0.05 vs. base line by paired t test; ^BP < 0.05 vs. WT by unpaired t test.

The contractile effects observed with a co-infusion with L-NMMA were not attributable to changes in preload or afterload (EDV and Ea were not changed). Heart rate increased similarly in the β₃^–/– and WT mice (8.2 ± 2.1% in WT mice and 10.4 ± 3.9% in β₃^–/– mice, P < 0.05 vs. base line for both groups).

Effect of NOS3 deletion on β-adrenergic stimulation. As an additional control, we tested the effects of isoproterenol in NOS3^–/– mice. Isoproterenol (5 ng/kg/min) infusion caused an increase in dP/dt-IP (35.2 ± 6.2%; P < 0.001 vs. base line; Figure 2a) with a magnitude similar to that observed in β₃^–/– mice. This observation provides further confirmation of elevated β-adrenergic contractility in mice lacking NOS3.

Concentration of cGMP. To test the impact of altered NO signaling we assessed concentrations of the NO second messenger cGMP. Basal cGMP was elevated in β₃^–/– (10.7 ± 2 pmol/g, n = 7) vs. WT (2.4 ± 0.4 pmol/g, n = 3; P < 0.05 vs. β₃^–/–). In WT mice, isoproterenol-treated (5 ng/kg/min) hearts had a higher level of cGMP (6.5 ± 0.8 pmol/g, n = 5; P < 0.05) than hearts treated with normal saline. β₃^–/– mice did not exhibit increased cGMP with isoproterenol (10.9 ± 1.7 pmol/g with isoproterenol, n = 6; P = NS vs. normal saline).

NOS3 protein abundance. Western blot analysis of myocardial tissue revealed no change in NOS3 protein levels, relative to p-38 or actin, between WT and β₃^–/– mice (Figure 4a). Protein abundance of NOS3 was similar between WT and β₃^–/– mice (85 ± 5 vs. 87 ± 3 arbitrary units, n = 8 and 9, respectively). Moreover, when corrected for abundance of either p-38 (1.03 ± 0.06 vs. 1.11 ± 0.10_,P = NS) or actin (0.84 ± 0.08 vs. 1.06 ± 0.13, P = NS), NOS3 was similarly unchanged. Whereas NOS3 protein was absent in the NOS3^–/– mice, expression of p-38 and actin was present in similar quantities to the other strains of mice (Figure 4a).

Figure 4

Abundance of NOS3 protein and β₃^–/– mRNA in myocardium. (a) Western blot of NOS3 from mouse heart tissue. Equal amounts of protein extracts were resolved on agarose gels, transferred to nitrocellulose, and exposed to anti-NOS3 Ab or anti–p-38 Ab. The FVB and β₃^–/– mice had similar NOS3 abundance relative to p-38 MAP kinase, and NOS3 was absent in the NOS3^–/– mice. (b) Representative ethidium-stained agarose gel demonstrating expression of β₃-adrenoceptor mRNA in different mice strains: mRNA is expressed in both heart (H) and epididymal fat (F) of NOS3^–/– and FVB control mice, but not β₃^–/– mice. Little or no PCR product is amplified in reactions lacking reverse transcriptase (H-), confirming minimal genomic contamination of mRNA. (c) Autoradiograph of RNase protection assay confirming the expression of β₃-adrenoceptor in the myocardium of FVB and NOS3^–/– mouse hearts. β₃AR P, β₃-adrenoceptor probe; β₃AR PF, β₃-adrenoceptor–protected fragment.

Expression of β₃-adrenoceptor. Expression of β₃-adrenoceptor mRNA was detected by RT-PCR in heart and epididymal fat of both WT and NOS3^–/– mice, but not β₃^–/– animals (Figure 4b). RT-PCR in reverse-transcribed negative reactions revealed little or no PCR product, confirming minimal contamination with genomic DNA. RNase protection assay confirmed the presence of β₃-adrenoceptor mRNA in WT and NOS3^–/– mice (Figure 4c).

In this study we have shown that mice lacking the β₃-adrenoceptor do not exhibit NO-mediated inhibition of β-adrenergic–stimulated inotropic responses. The cardiovascular effects of β₃-adrenoceptor deletion in mice were apparent only during sympathetic stimulation. Under base-line conditions, the β₃^–/– and WT mice exhibited similar heart rates, loading conditions, and contractile states. However, when stimulated with isoproterenol, a nonspecific β-adrenergic agonist, the β₃^–/– mice had a greater positive inotropic response than did the WT mice. In WT mice the inotropic response to isoproterenol was augmented by L-NMMA and L-NAME, NOS inhibitors, to the level seen in β₃^–/– mice with isoproterenol alone. Augmentation by L-NMMA or L-NAME was absent in the β₃^–/– mice. Similarly, mice lacking NOS3 were hyper-responsive to isoproterenol. Taken together, these observations suggest that the β₃-adrenoceptor plays a major role in NO attenuation of β-adrenergic–stimulated positive inotropy.

These present findings agree with those of Gauthier et al. (12, 13, 19) that direct stimulation of β₃-adrenoceptors produces a negative inotropic response due to NO-cGMP signaling. These previous studies were unable to address the impact of inhibiting the β₃-NO pathway because of the lack of availability of β₃-specific antagonists. The present study, by examining the β₃-NO pathway in animals lacking the β₃-adrenoceptor, has established the importance of this receptor in NO-mediated negative feedback over β-adrenergic inotropic responses. In WT mice, NOS inhibition augments β-adrenergic–stimulated positive inotropic effects (2, 3). β₃^–/– mice, on the other hand, are hyper-responsive to isoproterenol infusion, lack augmentation with NOS inhibition, and yet have similar indices at base line. These observations suggest that β₃-adrenoceptor stimulation is coupled to NOS activation, that this coupling offers significant negative feedback over sympathetic activation, and that the β₃-adrenoceptor exerts its influence primarily during sympathetic activation.

β₃^–/– animals had elevated myocardial cGMP levels compared with FVB controls, but only WT animals demonstrated increased cGMP concentrations with exposure to β-adrenergic agonists. The former suggests that the β₃-adrenoceptor negatively modulates basal NO-cGMP production and is consistent with several studies showing that receptors that are coupled to NOS inhibit its activity by a mechanism involving caveolin, the scaffolding protein found in membrane caveolae (see ref. 38 for review and ref. 4). Alternatively, it must be considered that other pathways capable of stimulating cGMP production (e.g., atrial natriuretic peptides; ANP) may be upregulated in these animals. In this regard, Gyurko and colleagues have demonstrated recently increased ANP in NOS3^–/– mice, which results in cGMP levels similar to WT controls (5). The observation that isoproterenol increases cGMP is consistent with β-agonist linkage to NO resulting in cGMP elevation (11). The absence of this response in β₃^–/– is further evidence of a signaling linkage between the β₃-adrenoceptor and NOS.

Sympathetic activation of the β-adrenergic pathway in the heart produces positive inotropic, chronotropic, and lusitropic responses. These responses are mediated through the activation of the β₁- and β₂-adrenoceptors coupled to a stimulatory G protein (G_s) (39), which in turn stimulates cAMP production by adenylyl cyclase (AC) (39). cAMP, through cAMP-dependent protein kinase-A (PKA), directly augments the L-type Ca⁺⁺ current (I_Ca), which enhances excitation-contraction coupling (40). The best-understood signaling pathway opposing β-adrenergic cardiac activation is mediated by muscarinic-cholinergic stimulation of the heart (41, 42). Muscarinic receptors are coupled to an inhibitory G protein (G_iα) that both inhibits AC production of cAMP and stimulates NO production via NOS3 (42). NO, which stimulates the production of cGMP, can have both cGMP-dependent (43–47) and independent (48–50) inotropic effects. Thus, the traditional view of cardiac inotropic regulation by the autonomic nervous system is that of two limbs of the autonomic nervous system acting through separate receptor/signaling pathways, exerting opposing forces on myocardial regulation.

Description of β₃-adrenoceptors in the heart (13) activated by traditional β-agonists, leading to negative inotropic effects, has raised the possibility that there may be negative feedback over contractility within the sympathetic pathway itself. Initially, β₃-adrenoceptors were found to be expressed in adipose tissues, where their activation promotes lipolysis and energy expenditure, and in gastrointestinal tissues (16–18), where they regulate motility. Recently, unique β₃-selective agonists have been shown to cause negative inotropic effects in the heart (12, 13, 19, 51) that are accompanied by increases in NO and production of cGMP (12). Inhibition of NO attenuates the negative inotropic effects of β₃-selective agonists (12), suggesting that β₃-adrenoceptor activation is coupled to NOS stimulation. Activation of NOS by the β₃-adrenergic pathway may be through a G_iα protein, similar to the muscarinic system (42), since the negative inotropic effects of β₃-adrenergic stimulation are attenuated by pertussis-toxin treatment (12, 13). Unlike the muscarinic-cholinergic pathway, the β₃ pathway may serve as negative feedback over sympathetic activation as β₃ receptors are activated by the same agonists that activate β_1,2-adrenoceptors. These findings suggest a role for the β₃-adrenoceptor in inhibition of β_1,2-adrenergic–stimulated positive inotropic effects in a pathway that is NO mediated.

Recent studies demonstrating that the β₃-adrenoceptor is upregulated in humans (52) and animals (53) with heart failure (52) suggest that the β₃-NO pathway has implications in the pathophysiology of heart failure. The failing heart is characterized by downregulation of both the β₁- and β₂-adrenoceptors (54), through protein kinase A or β-adrenoceptor kinase (55), and subsequent attenuation of catecholamine induced positive inotropy. Unlike the β_1,2 subtypes, the β₃-adrenoceptor is unlikely to be downregulated by these mechanisms (56). Given the high sympathetic tone associated with heart failure, the increased ratio of β₃- to β_1,2-adrenoceptors and their respective inotropic effects could have profound consequences important for myocardial function in the failing heart.

This study uses a miniature combined manometer-conductance catheter to acquire pressure-volume loops in mice, thus enabling integrated measurement of cardiovascular performance. This method allows for the simultaneous determination of load-independent measures of contractility (Ees, for example), preload, afterload, and heart rate. The combined conductance-manometer catheter permits accurate determination of cardiovascular performance and has been used to characterize cardiomyopathy in transgenic mice (24, 57). Use of this technique has also shown that β₃^–/– and NOS3^–/– mice have smaller chamber sizes than WT mice. The latter can be attributed to myocardial hypertrophy (increased heart-weight/body-weight ratio), whereas the former appears to be related to diastolic filling abnormalities (increased tau). The pressure-volume data illustrate that these differences do not influence the contractile parameters reported here (i.e., Ees and dP/dt-IP are independent of chamber size). Diastolic abnormalities mediated by the β₃-NO pathway are under active investigation by our laboratory.

In conclusion, we have established a physiologic role for the β₃-adrenoceptor in the heart. Using transgenic mice lacking the β₃-adrenoceptor, we have shown that the β₃-adrenoceptor is responsible for NO-mediated negative feedback over β-adrenergic–stimulated positive inotropy.

This work was supported by NIH grant K08 HL-03238 and a Grant-in-Aid from the American Heart Association, Mid-Atlantic affiliate.

Data from this paper have been presented at the 72nd Scientific Sessions of the American Heart Association in Atlanta, Georgia, November 7–10, 1999.

Paul Varghese and Robert W. Harrison contributed equally to this work.

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