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Am J Physiol Heart Circ Physiol 277: H1609-H1617, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 4, H1609-H1617, October 1999

ACE inhibition improves cardiac NE uptake and attenuates sympathetic nerve terminal abnormalities in heart failure

Hiroya Kawai, Tai-Hwang M. Fan, Erdan Dong, Rizwan A. Siddiqui, Akito Yatani, Suzanne Y. Stevens, and Chang-Seng Liang

Cardiology Unit, Department of Medicine, and Departments of Neurobiology and Anatomy, University of Rochester Medical Center, Rochester, New York 14642


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiac sympathetic nerve terminal dysfunction plays an important role in the downregulation of myocardial beta -adrenoceptors in heart failure. To determine whether chronic angiotensin-converting enzyme (ACE) inhibition improved cardiac sympathetic nerve terminal function and hence increased myocardial beta -adrenergic responsiveness, we administered ACE inhibitors to dogs with chronic right-sided heart failure (RHF) produced by tricuspid avulsion and pulmonary artery constriction. The RHF animals exhibited fluid retention, elevated right heart filling pressures, blunted inotropic response to isoproterenol, and reduced beta -adrenoceptor density. These changes were accompanied by decreases in right ventricular norepinephrine (NE) uptake and neuronal NE histofluorescence and tyrosine hydroxylase immunoreactive profiles. ACE inhibitors had no effect on the production of heart failure but greatly reduced the attenuation of cardiac NE uptake, neuronal NE histofluorescence, and tyrosine hydroxylase immunoreactive profiles. ACE inhibition also improved the inotropic response to isoproterenol and restored myocardial beta -adrenoceptor density. The changes probably are caused by reduction of cardiac NE release by ACE inhibition and may contribute to the beneficial effects of ACE inhibitor therapy in patients with chronic heart failure.

congestive heart failure; sympathetic nerves; angiotensin-converting enzyme inhibition; tyrosine hydroxylase; norepinephrine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SYMPATHETIC NERVE SYSTEM activation has been well demonstrated in congestive heart failure, as evidenced by elevated plasma norepinephrine (NE) (41), increased cardiac NE spillover (20), and increased skeletal muscle sympathetic nerve firing (27). Direct demonstration of elevated cardiac interstitial NE also has been shown in failing myocardium (11). This probably is responsible for the decrease of myocardial beta -adrenoceptor density and abnormal beta -adrenergic responsiveness (6, 11, 15). The increase in interstitial NE probably is caused by both an increase of cardiac neuronal release (13) and a decrease of neuronal uptake (11, 28) of NE. However, in chronic heart failure myocardial NE stores are depleted (8), suggesting that the rate of NE synthesis in the heart is inadequate to compensate for the loss of NE from sympathetic discharge. Impaired NE synthesis is supported by our recent findings that tyrosine hydroxylase immunoreactive profiles are reduced (22). These findings suggest that the changes in sympathetic nerve terminal function in the failing myocardium may increase interstitial NE and cause the beta -adrenoceptor downregulation.

Angiotensin-converting enzyme (ACE) inhibitors, which are commonly used in patients with left ventricular systolic dysfunction, have been shown to reduce mortality and morbidity (9, 34) and improve cardiac function and hemodynamics in patients with congestive heart failure (24). Because angiotensin II is known to enhance sympathetic release of NE (26), part of the beneficial effects of ACE inhibitors may be related to inhibition of the presynaptic effect of angiotensin II on NE release. Early studies have shown that ACE inhibitor therapy reduces plasma NE (5) and attenuates the increased skeletal muscle sympathetic nerve activity in patients with heart failure (18). There is also an improvement in myocardial beta -adrenergic responsiveness in heart failure by chronic ACE inhibition (17, 36). However, direct evidence of effects of ACE inhibitors on sympathetic nerve terminals is lacking. The present study was undertaken to examine the effects of chronic ACE inhibition on cardiac neuronal NE store and on the myocardial beta -adrenoceptor system in congestive heart failure. Studies were performed in dogs with right-sided heart failure (RHF) produced by tricuspid avulsion and progressive pulmonary artery constriction. The animals exhibit a chamber-specific alteration in cardiac sympathetic nerve terminals and changes in myocardial beta -adrenoceptor-coupled adenylyl cyclase system (15, 22, 28), similar to those occurring in human heart failure.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Preparations

Adult mongrel dogs weighing between 19.4 and 27.1 kg were used for the study. RHF was produced in the dogs by performing a modified two-stage surgery (15, 21, 22, 28). The study was approved by the University of Rochester Committee on Animal Resources and conformed to the guiding principles approved by the Council of the American Physiological Society and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports].

Animals were anesthetized with intravenous pentobarbital sodium (25 mg/kg). They were intubated and ventilated with a respirator (Harvard Apparatus, South Natick, MA). In the first operation, a sterile right thoracotomy was performed through the fifth intercostal space, and an index finger was inserted through a right atriotomy to rupture the anterior chordal tendineae of the tricuspid valves during transient occlusion of the venous return. A heparin-filled Tygon catheter (inner diameter, 1.02 mm; Norton Plastic & Synthetics, Akron, OH) was placed in the right atrium, and the atriotomy was closed. Two weeks later, a second sterile left thoracotomy was performed through the fifth intercostal space. A silicon rubber hydraulic occluder (R. E. Jones, Silver Spring, MD) was placed around the main pulmonary artery, and an implantable micromanometer (Konigsberg Instruments, Pasadena, CA) was inserted into left ventricle via a stab wound at the apex. Tygon catheters were placed in the main pulmonary artery, the left atrium, and the descending thoracic aorta. All catheters and the lead of micromanometer were exteriorized at the nape of the neck, and the chest was closed.

After the dogs were allowed a 2-wk recovery period following the second surgery, the pulmonary artery occluder was progressively inflated. This procedure was repeated at 4- to 7-day intervals for 3 wk to produce a steady increase in mean right atrial pressure to 12-14 mmHg; no further adjustments were made thereafter.

A group of sham-operated animals underwent two surgical procedures identical to those described above, except that neither tricuspid valve avulsion nor the pulmonary artery constriction was included.

Experimental Protocol

Animals were divided randomly to receive either ramipril (Hoechst Marion Roussel, Kansas City, MO) or enalapril (Merck, West Point, PA), two commonly used ACE inhibitors, or no ACE inhibitors. The ACE inhibitors were administered at a dose of 20 mg once a day for 6 consecutive weeks, beginning 2 wk after the second thoracotomy, which corresponded to the start of pulmonary artery constriction in the RHF animals. The doses of ACE inhibitors were chosen to produce significant inhibition of arterial pressor response to intravenous angiotensin I in pilot experiments. To determine the efficacy of ACE inhibitors, we measured the arterial pressor response to intravenous angiotensin I (1.0 µg; Sigma Chemical, St. Louis, MO) before and after 1 and 6 wk of ACE inhibitor treatment. In addition, resting hemodynamics and cardiac inotropic responses to serial doses of isoproterenol (see Hemodynamic Measurements) were measured at the end of 6 wk of pulmonary artery occlusion or 8 wk after the second thoracotomy.

After the final hemodynamic studies, the animals were given a lethal dose (>100 mg/kg) of intravenous pentobarbital sodium. The animals were inspected for ascites. The heart and liver were removed and weighed. The ventricles were separated from the septum and rinsed in ice-cold oxygenated normal saline. The left ventricular weight includes both the septum and left ventricular free wall; the right ventricular weight includes only the free wall. Ventricular muscle blocks were removed quickly from the right and left ventricular free walls 3 cm below the atrioventricular groove. The muscle blocks were trimmed, minced, and homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.4 at 22°C). The homogenate was centrifuged at 40,000 g for 15 min at 4°C. The resultant pellets were stored at -70°C for subsequent radioligand receptor assays. Tissue protein was determined in triplicate (29) with bovine serum albumin used as a standard. Fresh muscle samples were used for measuring myocardial NE uptake activity, whereas other samples were stored in liquid nitrogen for measuring noradrenergic nerve terminal profiles (see Anatomic Studies of Ventricular Sympathetic Nerves).

Hemodynamic Measurements

Animals were acclimatized to the laboratory and personnel and were trained to lie quietly on an experimental table with minimal restraint. For the hemodynamic studies, animals were placed in a lateral decubitus position, and the pulmonary artery occluders were deflated. The previously implanted intravascular catheters were connected to a pressure transducer (Spectramed P23 XL; Spectramed, Oxnard, CA) and an eight-channel recorder (Brush model 480; Gould Instrument Systems, Valleyview, OH) for measuring aortic, right atrial, and left atrial pressures. Left ventricular pressure was measured by the Konigsberg micromanometer connected to the Brush recorder; the first derivative of the left ventricular pressure with respect to time (dP/dt) was obtained using an electronic differentiator. Right ventricular pressure and its dP/dt were measured by a transducer-tipped catheter (Millar Instruments, Houston, TX) inserted through an external jugular vein under local anesthesia. Heart rate was obtained from the electrocardiogram. Cardiac output was determined by injecting indocyanine green (Cardio-Green; Hynson, Westcott & Dunning, Baltimore, MD) into the pulmonary artery and sampling the arterial blood for dye concentration with a cardiac output computer (Honeywell model D-014; Lyons Medical Electronics, Sylmar, CA).

Resting hemodynamic measurements were made in triplicate at 5-min intervals, at least 1 h after the Millar catheter was inserted. Averages of the triplicates were used for statistical analysis. We then administered intravenous isoproterenol (0.05, 0.1, 0.2, and 0.4 µg · kg-1 · min-1) each for 10 min, using an infusion pump (model 600-900; Harvard Apparatus, Dover, MA) at a rate of 0.388 ml/min. The effects of isoproterenol on heart rate, mean aortic pressure, and right ventricular and left ventricular dP/dt were measured at 8-10 min of each infusion.

Myocardial Tissue NE Uptake Activity

Myocardial tissue NE uptake activity was measured in quadruplicate by incubating fresh tissue slices at 37°C for 15 min in 50 nmol/l L-[7-3H(N)]NE (13.8 Ci/mmol; New England Nuclear, Boston, MA) (28). Specific 3H uptake activity, defined as the difference in radioactivity between tissue slices incubated in a [3H]NE-containing solution at 37°C and those at 4°C, is considered to approximate tissue NE uptake activity.

Myocardial beta -Adrenoceptor Density

Myocardial beta -adrenoceptor density was measured by using the radioligand binding technique with a highly specific ligand, [125I]iodocyanopindolol (ICYP, 2,200 Ci/mmol; New England Nuclear) (38). Approximately 20 µg of membrane protein, suspended in 50 mM Tris · HCl buffer (pH 7.4) containing 120 mM NaCl and 5 mM KCl, was incubated in triplicate with eight concentrations of [125I]ICYP (5-250 pM) at 37°C for 60 min in a final volume of 0.25 ml. Nonspecific binding was determined by parallel incubation of samples containing 100 µM propranolol. The reaction was terminated by the addition of ice-cold Tris buffer and immediate filtration through Whatman GF/B filters (Whatman Chemical Separation, Clifton, NJ) on a Brandel cell harvester (Biomedical Research and Development Laboratories, Gaithersburg, MD). The membranes were prepared for quantitation of 125I radioactivity by liquid scintillation spectrometry (Tri-Carb 460 CD; Packard Instrument, Downers Grove, IL). The difference between tissue binding of radioligand in the absence and presence of propranolol was considered to be the specific binding. The maximum number of receptor binding sites and the dissociation constant were calculated with the EBDA computer software program (Elsevier Science, Cambridge, UK) developed by McPherson (30).

Myocardial NE Uptake-1 Carrier Site Density

Myocardial NE uptake site density was measured using the radioligand assay (40) by specific binding of [3H]nisoxetine (New England Nuclear). Nisoxetine is a highly specific ligand. Approximately 80 µg of membrane was incubated in 250 µl of a Tris buffer (50 mM Tris, 300 mM NaCl, 5 mM KCl, pH 7.4) containing 3 nM [3H]nisoxetine and eight increasing concentrations of cold nisoxetine (0.3125 to 10 pM; Sigma Chemical). Incubation was performed in triplicate at room temperature for 90 min. Nonspecific binding was determined in the presence of 10 µM nisoxetine. The reaction was terminated by the addition of an ice-cold Tris buffer. The membrane was rapidly washed three times and filtered through Whatman GF/B filters on a Brandell cell harvester. The filters were counted utilizing a Packard liquid scintillation spectrometer. The difference between binding in the absence and presence of 10 µM nisoxetine was taken as specific binding. The maximal number of receptor binding sites and the dissociation constant were calculated by Scatchard analysis of the binding data as described above under Myocardial beta -Adrenoceptor Density Assay.

Anatomic Studies of Ventricular Sympathetic Nerves

Glyoxylic acid-induced histofluorescence for catecholamines. Histofluorescence staining specific for catecholamines was performed using a modification (4) of the sucrose-potassium phosphate-glyoxylic acid condensation method of de la Torre (10). Tissue blocks from fresh hearts were rapidly frozen and mounted on a cryostat (-20°C) for either longitudinal or cross-section at thickness of 16 µm. Sections were picked up on the glass slides, dipped in sucrose-potassium phosphate-glyoxylic acid solution, heated and dried under oil at 95°C for 2.5 min, coverslipped, and viewed under epifluorescent illumination using a Nikon fluorescence microscope equipped with filters designed for catecholamine fluorescence visualization. All sections were photographed at the same magnification (×50) using 35-mm slide film. The number of stained catecholamine profiles were counted in a 0.221-mm2 (0.003536 mm3) field; the results of five fields were summed to provide an average for each ventricle.

Immunocytochemistry for tyrosine hydroxylase. Ventricular muscle blocks were fixed for 24 h in 4% paraformaldehyde in 0.15 mol/l phosphate buffer (pH 7.4) at 4°C. Blocks were prepared for frozen section at 40 µm. Sections were then incubated in primary anti-tyrosine hydroxylase antibody (1:60,000 dilution in 0.4% Triton X-100 buffer; Chemicon, Temecular, CA) for 24 h at 4°C with gentle agitation. On the following day, sections were rinsed and incubated in the biotinylated secondary antibody (goat anti-rabbit IgG diluted 1:1,000 in buffer plus 0.15% normal goat serum), followed by incubation in avidin-biotin-peroxidase complex (Vector kit; Vector Laboratories, Burlingame, CA) as previously described (22). Sections were finally mounted on gelatin-coated slides, which were viewed and photographed at the same magnification (×50) onto 35-mm slides. The number of tyrosine hydroxylase staining profiles was counted in a 0.00885-mm3 field; the results of five fields were averaged for each ventricle.

Statistical Analysis

The experimental data were analyzed with RS/1 Research System (Bolt, Beranek and Newman Software Products, Cambridge, MA) and SYSTAT (SPSS, Chicago, IL). Two-way analysis of variance (ANOVA) was used to examine the effects of chronic ACE inhibition among the experimental groups. If ANOVA revealed statistical significance of differences between the disease status (sham vs. RHF) or treatment effects (untreated vs. treated with ACE inhibitors), or of disease-treatment interactions, comparisons of individual group means were performed, using post hoc contrast analyses. Pearson correlation was used to determine the significance of correlations between myocardial NE uptake activity and beta -adrenoceptor density and between myocardial NE uptake activity and tyrosine hydroxylase profiles. Data are expressed as means ± SE. A probability value of <= 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dogs were divided into four experimental groups: 1) sham-operated dogs receiving no ACE inhibitors (Sham Control, n = 11); 2) sham-operated dogs receiving ACE inhibitors (Sham + ACE inhibitors, n = 12); 3) RHF dogs receiving no ACE inhibitors (RHF Control, n = 15); and 4) RHF dogs receiving ACE inhibitors (RHF + ACE inhibitors, n = 13). Ramipril was given to six sham-operated and seven RHF dogs. Enalapril was given to six sham-operated and six RHF dogs. Because both ACE inhibitors produced qualitatively similar results, the animals were pooled as one sham and one RHF group treated with ACE inhibitors.

Angiotensin-Converting Enzyme Inhibition

The inhibitory effect of ramipril and enalapril on ACE activity was determined by administrations of intravenous angiotensin I before and after ACE inhibitor treatment. At baseline, angiotensin I produced a 15- to 32-mmHg (23 ± 1 mmHg) increase of mean aortic pressure. Similar increases were produced by angiotensin I 6 wk later in Sham Control and RHF Control dogs. However, the hypertensive effect of angiotensin I was markedly reduced in dogs treated with ACE inhibitors (Fig. 1). Inhibition of the pressor response was present after 1 wk of treatment (4 ± 1 mmHg).


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  		Fig. 1.   Increases in mean aortic pressure in response to intravenous administration of angiotensin I (1 µg) in four experimental groups of animals. A: sham group; B: right-sided heart failure (RHF). Open bars indicate values obtained at baseline before administration of angiotensin-converting enzyme (ACE) inhibitors (or 2 wk after left thoracotomy). Solid bars indicate values obtained at final hemodynamic study after 6 wk of administration of ACE inhibitors (or 8 wk after left thoracotomy). Bars denote SE. * P < 0.05, compared with baseline (open bars).

Resting Hemodynamics

Table 1 shows the resting hemodynamics obtained at the end of the study in sham-operated and RHF dogs. Ascites was present in dogs with RHF with and without ACE inhibitors. Body weight, right ventricular weight, resting heart rate, and mean right atrial pressure were increased, and cardiac output and peak right and left ventricular dP/dt were decreased in RHF Control dogs compared with Sham Control dogs. Chronic administration of ACE inhibitors did not affect these changes in either sham-operated or RHF dogs. Mean aortic pressure was slightly reduced in RHF Control dogs compared with Sham Control dogs. ACE inhibitors caused a slight decrease of mean aortic pressure in sham-operated dogs but did not affect mean aortic pressure in RHF dogs. Liver weight was increased in RHF dogs with and without ACE inhibitors.

                              
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  		Table 1.   Resting hemodynamics in sham-operated and RHF dogs

Myocardial Tissue NE Uptake Activity and NE Uptake Carrier Site Density

Myocardial tissue NE uptake activity and NE uptake-1 carrier site density were reduced in the right ventricle of RHF dogs compared with sham-operated dogs (Table 2). Chronic ACE inhibition attenuated the reductions of tissue NE uptake activity and NE uptake-1 site density in the right ventricle of RHF dogs, whereas it affected neither parameters in the right ventricle of sham-operated dogs. Furthermore, neither myocardial tissue NE uptake activity nor NE uptake carrier site density differed in the left ventricle between sham-operated and RHF dogs. Neither parameter was affected significantly in the left ventricle by ACE inhibitor treatment.

                              
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  		Table 2.   Myocardial tissue NE uptake activity and NE uptake-1 carrier site density in RHF

Glyoxylic Acid-Induced Histofluorescence and Tyrosine Hydroxylase Immunostained Nerve Profiles

Figure 2 shows that right ventricular NE histofluorescence profile was reduced in RHF Control compared with Sham Control, and that ACE inhibition with enalapril partially restored the NE histofluorescence profile in RHF. Table 3 summarizes the cardiac catecholaminergic histofluorescence and tyrosine hydroxylase immunoreactive profiles in all experimental groups. The catecholaminergic nerve terminal profiles were reduced in both ventricles of RHF dogs compared with sham-operated dogs, but the extent of reduction in the left ventricle was much less than that in the right ventricle. Chronic ACE inhibition did not significantly change the catecholaminergic histofluorescence profiles in sham-operated dogs but attenuated the decrease in the right ventricle of RHF dogs.


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  		Fig. 2.   Representative right ventricular NE histofluorescence pictures in a Sham Control (A), a RHF Control (B), and a RHF + enalapril animal (C).


                              
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  		Table 3.   Myocardial noradrenergic nerve profiles in RHF

Tyrosine hydroxylase-immunostained nerve terminal profiles were reduced in the right ventricles of RHF Control dogs compared with those of Sham Control dogs. Chronic ACE inhibition attenuated this reduction in the right ventricle of RHF dogs, whereas it did not significantly influence tyrosine hydroxylase-immunostained nerve terminal profiles in sham-operated dogs.

Cardiac and Hemodynamic Responses to Isoproterenol

Table 4 shows the changes in heart rate, mean aortic pressure, and peak right and left ventricular dP/dt produced by the largest doses of intravenous isoproterenol. Isoproterenol infusions produced stepwise increases of heart rate and ventricular dP/dt and decreases of mean aortic pressure in all animals. However, increases in heart rate and peak right and left ventricular dP/dt in RHF Control dogs were smaller when compared with Sham Control dogs. ACE inhibition did not affect any of the effects of isoproterenol in sham-operated dogs, but it increased the responses of heart rate and peak right and left ventricular dP/dt to isoproterenol in RHF dogs.

                              
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  		Table 4.   Chronotropic and inotropic response to isoproterenol in dogs

Myocardial beta -Adrenoceptor Density

Myocardial beta -adrenoceptor density was decreased in the right ventricle of RHF Control dogs compared with Sham Control dogs, but it did not differ in the left ventricle between the Sham and RHF animals (Fig. 3). Chronic ACE inhibition attenuated the reduction of myocardial beta -adrenoceptor density that occurred in the failing right ventricle of RHF dogs. There were no significant differences in dissociation constant of the beta -adrenoceptor to ICYP among the experimental groups.


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  		Fig. 3.   beta -Adrenoceptor density in right (A) and left (B) ventricles of four experimental groups. Open bars indicate untreated sham-operated and RHF groups. Solid bars indicate ACE-inhibitor-treated sham-operated and RHF groups. * P < 0.05, compared with untreated sham-operated animals. dagger  P < 0.05, compared with untreated RHF animals.

Correlations Between Myocardial NE Uptake and beta -Adrenoceptor Density

Figure 4 shows a statistically significant positive correlation between right ventricular NE uptake activity and beta -adrenoceptor density in RHF animals with and without ACE inhibitors. A similar correlation exists between right ventricular NE uptake activity and tyrosine hydroxylase profiles in the RHF animals.


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  		Fig. 4.   Correlations between right ventricular beta -adrenoceptor density and tissue NE uptake activity (A) and between right ventricular tyrosine hydroxylase profiles and tissue NE uptake activity (B) in RHF animals. r, Correlation coefficient. Closed circles denote control (untreated) animals; open circles are ACE inhibitor-treated animals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was designed to investigate the effects of chronic ACE inhibition on cardiac sympathetic nerve terminal function in dogs with RHF. We are the first to report that chronic ACE inhibition attenuated the reductions of myocardial NE tissue uptake activity, NE uptake-1 carrier site density, catecholaminergic histofluorescence, and tyrosine hydroxylase immunoreactive profiles in heart failure. The findings suggest that ACE inhibition exerts a protective effect on the sympathetic nerve terminal integrity and function. These effects probably are functionally important because they are linked to improvements of myocardial beta -adrenoceptor density and cardiac inotropic responsiveness to beta -adrenergic stimulation.

RHF produced by tricuspid avulsion and progressive pulmonary artery constriction has been extensively studied by us (15, 22, 28) and other investigators (21). RHF is manifested by ascites, hepatomegaly, weight gain, and elevated right ventricular filling pressures (15, 22). This heart failure model exhibits chamber-specific alterations of the cardiac sympathetic nerve terminal function (22, 28) and downregulation of myocardial beta -adrenoceptors (15, 28, 38). Results of the present study are consistent with our previous reports. The chamber-specific alterations in heart failure suggest that the regulatory mechanisms responsible for modulation of cardiac sympathetic nerve terminal function are under local rather than systemic control. The animals provide an excellent model for studying local effects of enhanced cardiac sympathetic activity in heart failure. Similar chamber specificity of beta -adrenoceptor downregulation was found in humans with pulmonary hypertension-induced RHF (7).

Fluorescent histochemistry is a sensitive and well-developed technique for demonstrating cardiac sympathetic innervation and denervation (14). Formaldehyde (14) and glyoxylic acid (2) have been successfully applied to various tissues for the fluorescent histochemical visualization of catecholamines. An early study in calves with RHF showed the reduction in number and intensity of paraformaldehyde-induced histofluorescence of myocardial noradrenergic nerve terminals (42). We recently employed the sucrose-potassium phosphate-glyoxylic acid-induced histofluorescence technique (28) to assess cardiac intraneuronal NE content. Compared with formaldehyde-induced histofluorescence method, the glyoxylic acid-induced histofluorescence technique gives more NE-specific and highly intensified fluorescence in the varicosities of cardiac sympathetic nerve terminals. Using this technique, we have shown that cardiac neuronal NE content was decreased in the failing myocardium of dogs with both right and left heart failure (22). These findings strongly support the cardiac neuronal NE depletion in congestive heart failure.

Our present study shows that the sympathetic neuronal NE content in the failing ventricle was increased by chronic ACE inhibition treatment. The findings are supported by clinical studies using the NE analog [123I]metaiodobenzylguanidine that enalapril treatment increased cardiac NE content in patients with heart failure (35, 39). This increase in neuronal NE was accompanied by similar increases of myocardial tissue NE uptake activity and tyrosine hydroxylase immunostained nerve profiles. The findings suggest that normalization of NE uptake mechanism is an integral component of the beneficial effect of ACE inhibition in heart failure. Early studies have shown that chronic ACE inhibition reduces plasma NE (5) and attenuates the increased skeletal muscle sympathetic nerve activity in patients with severe heart failure (18). However, in mild-to-moderate heart failure, a selective increase in cardiac NE spillover appears to precede the augmented sympathetic outflow to the skeletal muscle, which is usually found in advanced heart failure (33). The preservation of cardiac NE in the ACE inhibitor-treated RHF may suggest a reduction of cardiac neuronal NE release in heart failure.

Alternatively, the preserved cardiac NE content could be explained by an improvement in the NE uptake mechanism. As we have shown previously, myocardial tissue NE uptake activity and NE uptake-1 carrier site density were reduced in the failing right ventricle of RHF animals. Chronic ACE inhibition increased myocardial NE uptake activity in the right ventricle of RHF dogs compared with that in the untreated dogs. These changes of NE uptake activity were accompanied by quantitatively similar changes of myocardial NE uptake-1 carrier site density. Angiotensin II has been shown to enhance sympathetic nerve activity by several actions (23, 26). Facilitation of NE release from nerve terminals by angiotensin II has been well demonstrated (26), whereas inhibition of neuronal NE uptake mechanism by angiotensin II has been shown in some (23) but not in other studies (37). Results of the present study support that excess angiotensin II may lead to reductions of cardiac NE uptake-1 carrier site density and tissue NE uptake activity, and that chronic ACE inhibition may exert its beneficial effect by blocking the inhibitory effect of angiotensin II on cardiac neuronal NE uptake.

Cardiac neuronal NE uptake is the predominant mechanism for terminating the action of released NE on myocardial beta -adrenoceptors. Reduced myocardial NE uptake in heart failure is one of the crucial mechanisms for increased synaptic NE concentration (11, 28). Thus, given the evidence of improved NE uptake, we could expect that chronic ACE inhibition would have reduced the level of interstitial NE in heart failure, which in turn may be responsible for the less reduction of myocardial beta -adrenoceptor density in the present study.

Tyrosine hydroxylase-immunostained nerve terminal profiles are reduced in the failing myocardium. The changes probably are responsible for the reduced cardiac tyrosine hydroxylase activity in heart failure (8). Because tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis (31), reduced biosynthesis of NE may contribute to neuronal depletion of NE. Because both enzyme activity (1) and protein level of tyrosine hydroxylase (22) could be inhibited by NE through the end-product feedback inhibition, the elevated synaptic NE level as seen in congestive heart failure (11) may contribute to a reduced cardiac neuronal tyrosine hydroxylase activity. Chronic ACE inhibition improved the reduced tyrosine hydroxylase immunostained nerve terminal profiles in the failing myocardium in the present study. The improvement of tyrosine hydroxylase protein level might be related to a decreased synaptic NE concentration by chronic ACE inhibition and contribute to restoration of cardiac neuronal NE.

Our present study demonstrates significant positive correlations of the relationships of myocardial NE uptake activity with both beta -adrenoceptor density and tyrosine hydroxylase-immunostained profiles. However, correlations of determination (r2) indicate that the changes in NE uptake activity explained only 22-40% of changes in myocardial beta -adrenoceptor density and tyrosine hydroxylase-immunostained profiles. On the other hand, there was a much closer correlation (r = 0.848, P < 0.001) between left ventricular interstitial NE concentration and beta -receptor density in pacing-induced cardiomyopathy (11). The findings suggest that the interstitial NE concentration is affected not only by NE uptake capacity, but also by other factors such as cardiac sympathetic nerve activity and circulating plasma NE concentration. A decrease in myocardial NE uptake activity, which is pathophysiologically important in heart failure (15, 22), would not necessarily cause changes in the adrenergic terminal endings or myocyte beta -adrenoceptor density unless there is a concomitant increase in NE release into the myocardium.

Myocardial contractile response to beta -adrenergic agonists is reduced in both right and left ventricles, although myocardial beta -adrenoceptor density is reduced only in the right ventricle. We have further shown that the stimulatory guanine nucleotide-binding regulatory protein (Gs) is decreased in both ventricles (25). The results suggest that the beta -adrenergic desensitization in the failing right ventricle is due to a combination of beta -adrenoceptor downregulation and reduced Gs, whereas in the unloaded left ventricle it is mainly due to reduced Gs. The present study has shown that chronic ACE inhibition was accompanied by the restoration of myocardial beta -adrenoceptor density in the right ventricle. Furthermore, the beta -adrenergic desensitization in both ventricles was attenuated, suggesting the possible beneficial effect of chronic ACE inhibition on the impaired postreceptor signal transduction including Gs.

The exact mechanism by which chronic ACE inhibition attenuates the cardiac sympathetic nerve terminal dysfunction in the failing ventricle is not known. However, because neither body weight nor resting hemodynamics differed between the RHF dogs with and without ACE inhibitors, it appears unlikely that the improved cardiac sympathetic nerve terminal function is causally related to changes in fluid retention or resting hemodynamics. ACE inhibitors probably exert the effects on the sympathetic nerve endings by inhibiting the formation of angiotensin II, which enhances the release of NE after sympathetic nerve stimulation (26). Angiotensin II has also been shown to attenuate baroreflex control of sympathetic nerve outflow (19), resulting in increased sympathetic excitation. Another possible mechanism is the accumulation of bradykinin after ACE inhibition, which has a sympathoinhibitory effect (16). Further studies are needed to determine the relative contributions of the angiotensin II and bradykinin.

This present study shows that ACE inhibitors produced only partial restoration of the sympathetic nerve terminal function in RHF. Because we administered only one dose of the ACE inhibitors, we cannot state with certainty whether fuller restoration would occur if larger doses of the drugs were administered. We used two different ACE inhibitors in the study. Ramipril is known to have greater tissue affinity than enalapril (3). Results of our study indicate that there were no qualitative differences between the two kinds of ACE inhibitors. Furthermore, because angiotensin I can be converted to angiotensin II by chymase (12), ACE inhibitors may not have eliminated the formation of angiotensin II in RHF and thus caused only partial restoration of the angiotensin II-induced abnormalities. Alternatively, the sympathetic nerve activation may occur independently of the renin-angiotensin system activation and contributes to the sympathetic nerve terminal dysfunction. Further studies with an angiotensin II receptor blocker or a specific chymase inhibitor may help elucidate the relative roles of ACE and chymase in heart failure.

In conclusion, chronic ACE inhibition in dogs is associated with attenuation of reduced cardiac NE uptake activity and NE uptake carrier site density and prevention of myocardial beta -receptor downregulation and beta -adrenergic subsensitivity in heart failure. These findings strongly support that chronic ACE inhibition in heart failure is associated with the improvement of cardiac sympathetic nerve terminal function. These actions of ACE inhibitors on cardiac sympathetic nerve endings may contribute to the beneficial effects of ACE inhibitors on cardiac function and mortality in patients with congestive heart failure.


    ACKNOWLEDGEMENTS

The authors thank Amy M. Mohan and Sherry D. Steinmetz for excellent technical support and Barbara C. Entz for secretarial assistance.


    FOOTNOTES

An unrestricted grant support from the Merck Research Laboratories is gratefully acknowledged. The study was supported in part by National Heart, Lung, and Blood Institute Grants HL-07229 and HL-39260 and by a Paul N. Yu Fellowship.

The study was presented in part before the 1996 Scientific Sessions of the American Heart Association at Anaheim, California, on November 12, 1996.

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. §1734 solely to indicate this fact.

Address for reprint requests: C-s. Liang, Cardiology Unit, Box 679, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: Chang-seng_Liang{at}urmc.rochester.edu).

Received 2 February 1999; accepted in final form 26 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 277(4):H1609-H1617
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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