beta(3)-adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility

Abstract

The cardiac beta-adrenergic pathway potently stimulates myocardial performance, thereby providing a mechanism for myocardial contractile reserve. beta-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 beta(3)-adrenoceptor results in a negative inotropic effect through NO signaling. In this study, using mice with homozygous beta(3)-adrenoceptor deletion mutations, we tested the hypothesis that the beta(3)-adrenoceptor is responsible for beta-adrenergic activation of NO. Although resting indices of myocardial contraction were similar, beta-adrenergic-stimulated inotropy was increased in beta(3)(-/-) 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 beta(3)(-/-) mice. Moreover, isoproterenol increased myocardial cGMP in WT, but not beta(3)(-/-), mice. NOS3 protein abundance was not changed in beta(3)(-/-) mice, and cardiac beta(3)-adrenoceptor mRNA was detected in both NOS3(-/-) and WT mice. These findings indicate that the beta(3)-adrenergic subtype participates in NO-mediated negative feedback over beta-adrenergic stimulation.

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.

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.

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.

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.