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1-Adrenoceptor blockade mitigates excessive norepinephrine release into cardiac interstitium in mitral regurgitation in dog
1Auburn University of Veterinary Medicine, Auburn, Alabama; 2Department of Pharmacology, East Tennessee State University, Johnson City, Tennessee; 3Center for Heart Failure Research, Departments of Medicine and Physiology and Biophysics, 4Department of Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama; and 5Department of Pharmacology, University of Montreal, Montreal, Quebec, Canada
Submitted 6 September 2005 ; accepted in final form 25 January 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-adrenergic receptors (ARs). However, activation of neuronal -ARs on cardiac neurons may lead to further catecholamine release, with an attendant risk of functional deterioration. We hypothesize that a beneficial effect of -AR blockade may therefore mitigate excessive catecholamine release from cardiac adrenergic neurons in dogs with MR. We measured the effects of chronic -receptor blockade (-RB) on ISF NE and EP release using in vivo microdialysis in open-chest anesthetized dogs after 4 wk of MR with or without extended release of metoprolol succinate (100 mg/day) as well as in control dogs. Fractional shortening increased by 30% in both MR and MR + -RB dogs after 4 wk of MR. In MR + -RB dogs, stellate-stimulated heart rate change was attenuated compared with control and MR dogs, whereas peak change of LV pressure over time (+dP/dt) increased equally in all groups. Stellate-stimulated ISF NE increased fivefold over baseline in MR versus twofold in control dogs (<0.05), but the NE release was significantly attenuated in MR + -RB dogs. In contrast, stellate-stimulated increases in ISF EP did not differ in control, MR, and MR + -RB dogs. This study demonstrates that -RB attenuates ISF NE release from cardiac neurons and that the LV functional response to MR is not dependent on an excess increase in ISF NE. Thus 1-RB may exert a beneficial effect by attenuating untoward effects of excessive sympathetic efferent neural NE release while sustaining early LV functional adaptation to MR.
interstitial fluid; epinephrine
1-adrenergic receptor (AR) blocker (1-RB) (19). Canine intrathoracic neurons contain both 1- and 2-ARs (25) as well as ANG II receptors that are all involved in eliciting efferent sympathetic responses and catecholamine release in the heart (2, 6).
Using the microdialysis technique in healthy open-chest canines, we have shown that there is compartmentalized NE and EP release into the LV ISF space during electrical stimulation of the stellate ganglion (6, 22). In the present study, we report that excess NE is released into the cardiac interstitium in the early compensated stage of MR (4 wk) and, moreover, that chronic treatment with a 1-AR blocker decreases such excess neurally mediated release of NE but without diminishing the functional LV responses to stellate ganglion stimulation.
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METHODS |
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Mitral valve regurgitation was induced at Auburn University College of Veterinary Medicine in conditioned mongrel dogs of either sex (19–26 kg) by chordal rupture using a fluoroscopic-guided catheterization method previously described in by Dell'Italia and colleagues (4, 17, 21, 22). Dogs were randomly assigned to one of three groups: 1) unoperated controls (n = 6), 2) 4 wk of MR (n = 6), and 3) 4 wk of MR treated with 1-AR blocker (extended release metoprolol succinate, 100 mg orally, once daily; n = 8), starting 24 h after MR induction. In these same animals, our group has previously reported the results of M-mode echocardiography that was performed in the conscious state at baseline and at the time of euthanasia as well as the effects of ANG II infusion in the anesthetized state (22). Dogs were transferred to the University of Alabama at Birmingham for euthanasia. Metoprolol succinate was withheld on the day of the terminal experiments. This study was approved by the Animal Services Committees at the University of Alabama at Birmingham and Auburn University.
Terminal Study: Instrumentation
Animals were maintained at a deep plane of general anesthesia using isoflurane and were mechanically ventilated (Harvard Apparatus, South Natick, MA) as described previously (22). All catheters for LV pressure and coronary sinus and aortic blood samples were inserted as described previously (6, 22). After a median sternotomy, all preganglionic inputs to stellate ganglia were severed and bipolar stimulating electrodes were inserted into both ganglia as described previously (6). Both cervical left and right vagi were sectioned. The heart was suspended in a pericardial cradle. Microdialysis probes (Clirans, Terumo, Tokyo, Japan) were inserted into the LV anterior wall and perfused at 2.5 µl/min as described previously (6, 22). Based on in vitro experiments, 18.8% recovery was used in the calculation of ISF NE and EP values (6, 22).
Experimental Protocol
Dialysate was collected for 10 min before bilateral stellate ganglia stimulation (5 ms, 4 Hz, 8–14 V for 10 min). For each animal, stimulation voltage was set at times two threshold as described previously (6, 22). During stimulation, dialysate was collected separately for the first 3 min, the second 3 min, and the last 4 min. After stimulation, dialysate was collected sequentially for the first and second 5 min. Aortic and coronary sinus blood samples were taken at 8 min of baseline and at 8 min of stellate ganglion stimulation. At the end of the physiological experiments, the heart was arrested with intracardiac KCl and quickly extirpated, placed in phosphate-buffered ice slush, and the coronaries were flushed with oxygenated Krebs solution. The LV was cut into 1-cm cubes and snap frozen in liquid nitrogen for subsequent analysis of catecholamine content and -AR autoradiography.
Biochemical Analyses
ISF and tissue catecholamine levels. ISF NE and EP values at baseline (2 collections) and during stimulation (3 collections) were averaged for one baseline value and one stimulation value. ISF and plasma NE and EP concentrations were determined with the Biotrak catecholamines radioenzymatic assay (Amersham Pharmacia Biotech) as previously described (6, 22).
Tissue catecholamine content. Ventricular tissue NE and EP contents were measured by high-performance liquid chromatography using a Waters 460 electrochemical detector (Milford, MA) as described previously (3).
Receptor Autoradiography
Labeling of 1- and 2-ARs in 20-µm thick sections of unfixed LV myocardium was done by a modification of published protocols (14, 20) using l-[125I]iodocyanopindolol (PerkinElmer Life Sciences; Boston, MA), which is a membrane permeant ligand and measures both extracellular and intracellular receptors. Total binding was determined by incubation of sections for 90 min at 37°C in a Tris-salt buffer containing 0.1 nM [125I]cyanopindolol, 10 µM pargyline, and 10 µM serotonin. Incubation buffers for other sections also contained 3 µM timolol (nonspecific binding) or 0.5 µM CGP-20712A, a 1-AR antagonist. Radioligand binding sites were identified by film autoradiography and quantified by using a microcomputer-assisted imaging device (Imaging Research) as described previously (20). Total -AR levels were calculated by subtracting nonspecific binding from total binding. Nonspecific binding was subtracted from [125I]cyanopindolol binding in the presence of CGP-20712A to determine the abundance of 2-ARs. 1-AR density was calculated by subtraction of 2-AR density from that for total -ARs. Data are presented as femtomoles [125I]cyanopindolol bound per milligram of tissue.
Statistical Analysis
Analysis of NE and EP was compared in control, MR, and MR + 1-RB groups during basal and stellate stimulation separately, using the Kruskal-Wallis test. This statistical test is an analog of ANOVA but does not require normal distribution and constant variance of data. This analysis was required because the data violated assumptions of ANOVA, namely normality and constant variance, which is not unexpected for these physiological variables. If the Kruskal-Wallis test was significant at the 5% level, pairwise comparisons were performed by using Wilcoxon test, which is an analog of the t-test but does not require normal data distribution. Type 1 error was set at 5%. Bonferroni adjustment was utilized to address the issue of inflating type 1 error due to multiple testing in the pairwise comparisons stage. Note that in the comparison of three groups, there are a total of three pairs. Hence, in the pairwise comparisons, only P values <0.01687 (=0.05/3) were considered significant to achieve an overall 5% level of significance.
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RESULTS |
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In comparison with the control group, peak change of LV pressure over time (+dP/dt) was significantly lower at baseline in both the MR and MR + 1-RB, which was in parallel with significantly lower baseline mean arterial pressure (Fig. 1). Stellate-mediated increases in +dP/dt did not differ from control in both the MR and MR + 1-RB groups. The chronotropic responses were similar in the control and MR group, whereas the increase in heart rate was significantly blunted in the MR + 1-RB dogs (last dose, 36 h before experiment).
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Baseline transcardiac plasma NE levels measured in MR dogs with and without 1-RB were not statistically different from controls (Table 1). During stellate ganglion stimulation, significant transcardiac NE differences of a similar magnitude (aortic-coronary sinus levels 
1,000 pg/ml) were detected in all three groups (data not shown). ISF NE levels increased over baseline in all groups. However, the peak ISF NE level achieved during stelllate ganglion stimulation in the 4-wk MR group was significantly greater than in the control group, whereas peak ISF NE levels in the MR + 1-RB group were not different from controls (Fig. 2). It is noteworthy that the stimulated ISF NE increments were preserved or increased (in comparison to the control group) despite a significant 45% decrease in the tissue NE stores in MR dogs with and without 1-RB (Table 1).
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Similar baseline transcardiac plasma EP levels and similar tissue EP levels were measured in all three groups (Table 1). In response to stellate ganglion stimulation, EP ISF levels increased significantly in all three groups without differences among them (Fig. 3).
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-Adrenoreceptor Density
When compared with the density of the control group, LV 1-AR density was reduced by 50% at 4-wk MR in the two groups with or without +1-RB, whereas 2-AR values were not significantly different among all three groups (Fig. 4).
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DISCUSSION |
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The current investigation also demonstrates that 1-RB mitigates excessive sympathetic neuronal NE release in response to electrical stimulation of the stellate ganglia in the early phase of the volume overload of MR. These results suggest that, in addition to the direct effects mediated on cardiomyocyte -ARs, a major target for 1-RB in MR may be the -ARs on cardiac neurons (25). This mechanism of action thus provides an added benefit of decreasing the potentially long-term toxic effects of elevated NE in the interstium of the heart. Work performed in the tachycardia-induced heart failure model indicates that chronic sympathetic efferent neural activation induces a state of oxidative stress in the cardiac ISF (10), which can directly activate matrix metalloproteinases (18, 24). The canine MR model is marked by activation of matrix metalloproteinases and a 50% decrease in extracellular matrix as early as 2 wk that persists for 6 mo (21). We have shown that 1-AR blockade significantly attenuates extracellular matrix dissolution in the MR model at 4 wk (22).
We postulate that increased ISF NE levels in the conscious dog are reflected by our neural-stimulated levels in the anesthetized state, thereby accounting for the decrease in 1-AR density. Although chronic 1-RB in MR dogs attenuated the rise in neuronal NE release in response to stellate stimulation, downregulation of the 1-AR was not reversed by 1-RB. This suggests that suppression of sympathetic drive was not complete, as evidenced by our persistent decrease in tissue NE levels. Nevertheless, 1-RB did result in a decrease in stellate-stimulated heart rate compared with control dogs. It should be noted that at this early stage of MR, 1-RB failed to attenuate the small (15%) increase in LV end-diastolic dimension in our MR dogs at 4 wk (22). This adaptive increase in LV end-diastolic dimension in the early stage of MR could also be a stimulus for 1-AR downregulation, despite the attenuation of excessive ISF NE release after 1-AR blockade.
In dogs with MR and MR + 1-RB treatment, the increase in stellate-evoked +dP/dtmax was similar to control at this early compensated stage of MR. However, the positive chronotropic response was slightly decreased in MR + 1-RB compared with controls but without differing significantly from untreated MR animals. This could be due to a greater sensitivity of pacemaker mechanisms to the reduced NE release into cardiac ISF in the MR + 1-RB group, whereas the preservation of the ISF EP response could be a contributing factor to the increase in stellate evoked increase in dP/dtmax. In the present study, ISF EP release was equivalent in MR and MR + 1-RB dogs, despite a tendency for lower LV EP tissue levels in the untreated MR group. This could be an indication that most of the EP present in adrenergic nerve terminals is readily available for release even when the stores are reduced in this model. It is noteworthy that there was also a tendency for higher circulating levels of EP and lower LV EP levels among the untreated MR canines. This may be related to the previously reported downregulation of the cardiac renin-angiotensin system and reduction of ANG II-mediated plasma and ISF EP release after chronic 1-RB in this model (22).
In summary, there are attendant risks to excessive sympathetic efferent neural activation, including receptor desensitization, eventually leading to diminished functional responses to the sympathetics (9). In addition to its effect on 1-AR signaling, NE itself generates toxic oxidative metabolites in the cardiac ISF space that can reduce cardiac nerve and myocyte viability (11) and promote extracellular matrix degradation (18, 24). Dell’Italia and colleagues (21) have previously reported that extracellular matrix degradation appears to be important in the pathophysiology of adverse LV remodeling in the dog with experimentally induced MR. Taken together with the previous report, this study suggests that the 1-RB attenuates the risk of potentially untoward effects from excessive sympathetic efferent neural release of NE into the ISF as well as from ANG II-stimulated NE release in the volume overload of MR.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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-adrenoceptor down-regulation by norepinephrine is linked to reduced norepinephrine uptake activity. Eur J Pharmacol 384: 17–24, 1999.[CrossRef][Web of Science][Medline]-adrenoceptor density in pacing-induced heart failure. Am J Physiol Heart Circ Physiol 278: H1708–H1716, 2000.-adrenergic signaling in heart failure? Circ Res 93: 896–906, 2003.-adrenergic receptor subtypes in the canine conduction system. Circ Res 71: 51–57, 1992.-adrenergic support for left ventricular dysfunction in experimental mitral regurgitation normalizes indexes of pump and contractile function. Circulation 89: 818–826, 1994.1-adrenergic receptor blockade attenuates angiotensin II-mediated catecholamine release into the cardiac interstitium in mitral regurgitation. Circulation 108: 225–230, 2003.-adrenergic blockade on the left ventricular and cardiocyte abnormalities of chronic canine mitral regurgitation. J Clin Invest 93: 2639–2648, 1994.[Web of Science][Medline]1- and 2-Adrenoreceptor subtypes in canine intrathoracic efferent sympathetic nervous system regulating the heart. Am J Physiol Regul Integr Comp Physiol 261: R1269–R1275, 1991.This article has been cited by other articles:
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  B. Pat, C. Killingsworth, T. Denney, J. Zheng, P. Powell, M. Tillson, A. R. Dillon, and L. J. Dell'Italia Dissociation between cardiomyocyte function and remodeling with {beta}-adrenergic receptor blockade in isolated canine mitral regurgitation Am J Physiol Heart Circ Physiol, December 1, 2008; 295(6): H2321 - H2327. [Abstract] [Full Text] [PDF]
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 R. A.H. Stewart, O. C. Raffel, A. J. Kerr, R. Gabriel, I. Zeng, A. A. Young, and B. R. Cowan Pilot Study to Assess the Influence of {beta}-Blockade on Mitral Regurgitant Volume and Left Ventricular Work in Degenerative Mitral Valve Disease Circulation, September 2, 2008; 118(10): 1041 - 1046. [Abstract] [Full Text] [PDF]
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 A. Sabri, K. Rafiq, R. Seqqat, M. A. Kolpakov, R. Dillon, and L. J. Dell'italia Sympathetic Activation Causes Focal Adhesion Signaling Alteration in Early Compensated Volume Overload Attributable to Isolated Mitral Regurgitation in the Dog Circ. Res., May 9, 2008; 102(9): 1127 - 1136. [Abstract] [Full Text] [PDF]
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), mitral regurgitation (MR; 
) and MR + 
). Each point is recorded every 2 min. +P < 0.05 vs. baseline; #P < 0.05 vs. control; *P < 0.05 MR and MR + 
P < 0.05 vs. control stimulation.
