Many neurohumoral mechanisms are activated and cause pathophysiologic deterioration in heart failure. Improvement of this deterioration is beneficial for the treatment of chronic heart failure. The activated renin-angiotensin-aldosterone system is one abnormality and has been well studied. The angiotensin-converting enzyme inhibitor has been used to correct this abnormality. The drugs are prescribed almost routinely today for patients with heart failure (1,2).
Another notable phenomenon in patients with chronic heart failure is activation of sympathetic nervous system, which causes in increase of the circulating norepinephrine concentration (3-5). The higher the plasma norepinephrine concentration, the shorter the survival (3,6). Depletion of myocardial norepinephrine also was reported in such a condition (7). The sympathetic nervous system supports the cardiovascular system in maintaining cardiac output and blood pressure. This depletion results in less support for cardiac output when the sympathetic nervous system is activated (8). This is due to a reduced amount of norepinephrine release at nerve endings in response to sympathetic nerve activation. Recovery of cardiac norepinephrine is thought to be one way for the circulatory system to function correctly. Not only enhanced sympathetic nerve tone but also activation of the central nervous system was reported in chronic heart failure (9). Recently the β-blocker has been used for the treatment of heart failure, although its mechanism is controversial. One convincing theory is a neurohumoral mechanism acting at peripheral nerve endings (10). Manipulation of sympathetic nerve activity is thought to be a suitable treatment for patients with chronic heart failure. α2-Adrenoceptor agonists act on the central nervous system and reduce the activity of the sympathetic nervous system (11). These agents have been tried for short-term treatment of chronic heart failure, and improvement in hemodynamics and exercise tolerance have been reported (12-15). Reduction of activated central nervous system tone is one way of correcting the enhanced sympathetic nerve activity and is expected to be a new therapy. In this study, cardiac norepinephrine concentration and tyrosine hydroxylase activity in rats with cardiac hypertrophy recovered after treatment with the adreno-α2-agonist guanabenz.
MATERIALS AND METHODS
Ten-week-old male Wistar rats were obtained from and housed at the Central Animal Laboratory of Hokkaido University. The rats were anesthetized with chloral hydrate and pentobarbital. They were then intubated and mechanically ventilated. After thoracotomy, the ascending aorta was exposed and constricted with a titanium clip to make the same degree of stenosis (∼75%). A sham operation also was performed without placing a clip. After the operation, the rats had free access to normal drinking water and food.
Design of study
Study 1: The norepinephrine concentration in various organs. Four weeks after the operation, the rats were decapitated. Organs such as the heart, adrenal gland, kidney, spleen, liver, and vas deferens were removed and weighed. The organs were then frozen and stored at −70°C until measurement.
Study 2: The appropriate dose of guanabenz. Four weeks after the operation, the clipped rats were randomly treated with either saline (vehicle) or guanabenz (1, 5, or 10 mg/kg). Sham-operated rats received saline. These treatments were performed intraperitoneally every day for 4 weeks. After 4 weeks of treatment, the rats (18 weeks old) were weighed. The heart was removed after decapitation, and the left ventricle was weighed.
Study 3: Cardiac norepinephrine and dihydroxyphenylglycol concentrations after treatment with guanabenz. Four weeks after the operation, the clipped rats were randomly treated with either guanabenz (1 mg/kg) or saline (vehicle). Sham-operated rats also received either guanabenz (1 mg/kg) or saline. These treatments were performed intraperitoneally every day for 4 weeks. After 4 weeks of treatment, the rats (18 weeks old) were decapitated, the heart was removed, and the left ventricle was weighed. The left ventricles were then frozen and stored at −70°C until measurement.
Study 4: Cardiac tyrosine hydroxylase activity after treatment with guanabenz. The same procedure as that in study 3 was performed. After decapitation, the heart was removed, and the left ventricle was weighed and stored on ice. The activity was measured immediately.
Extraction of catecholamines from the tissues and measurement of cardiac tyrosine hydroxylase activity were performed as described by Blank and Pike (16). Assays for catecholamines with high-performance liquid chromatography with electrochemical detection were performed as previously reported (17).
Norepinephrine, dihydroxyphenylglycol, and dihydroxybenzylamine were purchased from Sigma (St. Louis, MO, U.S.A.). Guanabenz was a kind gift from Nippon Shoji (Osaka, Japan).
The results are expressed as mean ± SEM. Differences among groups were examined by using the unpaired t test for study 1, one-way analysis of variance (ANOVA) for study 2, and two-way ANOVA for studies 3 and 4.
All of the rats that recovered from the operation survived through the 4- or 8-week study period. None of the animals had ascites at death. Stenosis of about three fourths of the ascending aorta was confirmed at decapitation in study 1.
There were 12 rats in the clipped (C) group and eight in the sham (S) group. Left ventricular weight in C (2.23 ± 0.04 mg/g body weight) was significantly (p < 0.01) greater than that in S (1.57 ± 0.05 mg/g body weight). Left ventricular norepinephrine concentration in C (273 ± 13 ng/g heart weight) was significantly (p < 0.01) lower than that in S (445 ± 33 ng/g heart weight). Right ventricular norepinephrine concentration in C (333 ± 16 ng/g heart weight) was also depleted significantly (p < 0.01) compared with that in S (490 ± 31 ng/g heart weight). However, there were no significant intergroup differences in norepinephrine concentration in the adrenal gland (62.4 ± 5.0 μg/g in C vs. 75.0 ± 7.6 μg/g in S), kidney (155 ± 11 ng/g in C vs. 174 ± 17 ng/g in S), spleen (317 ± 24 ng/g in C vs. 369 ± 48 ng/g in S), liver (49.1 ± 3.9 ng/g in C vs. 51.2 ± 6.2 ng/g in S), and vas deferens (7.73 ± 0.67 μg/g in C vs. 8.00 ± 0.70 μg/g in S).
Left ventricular weight and body weight of each of the seven rats after the treatment are shown in Table 1. Although left ventricular mass was significantly regressed after treatment with guanabenz, body weight also was remarkably reduced at the higher dose of the drug. The most suitable dose of guanabenz was therefore considered to be 1 mg/kg body weight.
Body weight, left ventricular weight, and norepinephrine and dihydroxyphenylglycol concentrations in the left ventricle after treatment with guanabenz in each of the seven rats are shown in Table 2. Body weight was not significantly influenced by clipping or guanabenz. Left ventricular mass was significantly increased by clipping (p < 0.001) and regressed significantly after treatment with guanabenz (p < 0.001). Left ventricular mass to body weight ratio was significantly increased by clipping (p < 0.001) and reduced significantly after treatment with guanabenz (p < 0.01). Norepinephrine concentration in the left ventricle was significantly decreased by clipping (p < 0.001) and recovered significantly after treatment with guanabenz (p < 0.05). Dihydroxyphenylglycol concentration in the left ventricle was significantly decreased by clipping (p < 0.01) but did not change significantly after treatment with guanabenz (NS). There were also no significant differences in the dihydroxyphenylglycol to norepinephrine concentration ratio in the left ventricle among the four groups.
Tyrosine hydroxylase activity in the left ventricle of each of the eight rats after treatment with guanabenz is shown in Table 3. The activity in the left ventricle was significantly decreased by clipping (p < 0.001) and significantly recovered after treatment with guanabenz (p < 0.01).
It is well known that clipping the ascending aorta induces cardiac hypertrophy and a reduction in the norepinephrine concentration in the heart. Cardiac norepinephrine concentration is influenced by various factors such as cardiac sympathetic nerve activity, distribution of this nerve, uptake of norepinephrine, and metabolism of norepinephrine (5,7,18-20). In this study, depletion of norepinephrine was detected only in the heart. This nonuniform reduction of norepinephrine concentration is very interesting. An increased cardiac sympathetic nerve drive was reported in human heart failure before generalized sympathetic activation occurred (20). Enhanced sympathetic nerve activity to the heart, which means nonuniform sympathetic nerve activation, was thought to trigger the myocardial norepinephrine concentration reduction, although other mechanisms might be involved.
Reduction of the accelerated cardiac sympathetic nerve activity is considered to be one way to restore the norepinephrine concentration in the heart if increase of the activity to the heart is the reason for the reduction. Reserpine is a sympatholytic drug. This agent reduces sympathetic nerve activity through kicking off the storage of norepinephrine at the peripheral nerve ending. Administration of this drug to patients with cardiac disease reduced the myocardial norepinephrine concentration (21). Guanabenz is also used as a sympatholytic drug. In this study, guanabenz (1 mg/kg, i.p.) restored the level of myocardial norepinephrine concentration. Determining the suitable dose of guanabenz is important for treatment. A high dose of guanabenz (e.g., 60 mg/kg, p.o.) caused no weight gain in young rats (22). We therefore investigated the suitable dose of this drug. The pharmacologic mechanism of this drug is a reduction in sympathetic nerve activity by stimulation of adreno-α2-receptors in the central nervous system and also suppression of norepinephrine release from nerve endings by activation of α2-receptors. This mechanism works favorably for the nerve and results in an improvement in norepinephrine concentration. Guanabenz also reduced the increase in norepinephrine turnover. Norepinephrine is one of the most stimulating cardiotrophic agents. The reduction of the increase in norepinephrine turnover caused less stimulation to the myocardium. Therefore a reduction of cardiac hypertrophy was obtained by this drug with the recovery of norepinephrine concentration.
Recovery of cardiac tyrosine hydroxylase activity is another important reason for the improvement in cardiac norepinephrine concentration. This enzyme is required for norepinephrine biosynthesis and is a rate-limiting enzyme for the synthesis. A reduction of its activity was reported in heart failure, and this was considered to be one of the reasons for the reduction of norepinephrine (18). Thus recovery of cardiac tyrosine hydroxylase activity is beneficial for norepinephrine synthesis. It is not known why cardiac tyrosine hydroxylase activity was recovered by guanabenz. Previous studies showed that the activity of ganglionic tyrosine hydroxylase was induced by drugs. The activity of tyrosine hydroxylase could be increased by the peripheral effect of drugs or by the central effect of drugs. An increase in cyclic adenosine monophosphate (cAMP) concentration by drugs could increase the tyrosine hydroxylase activity (23). This enzyme activity was also induced by reserpine without an increase in cAMP concentration, and this increase could be prevented by decentralization (24). In our study, activation of the cardiac sympathetic nervous system by narrowing of the aorta caused a reduction in the enzyme activity along with a reduction in the norepinephrine concentration. Suppression of the activated sympathetic nerve restored the enzyme activity. The activity of the nerve was thought to be a key factor. Moreover, the tyrosine hydroxylase activity was higher in the sham/guanabenz group than in the sham/saline group. Therefore we considered that the recovery of the enzyme activity by guanabenz is due to the peripheral as well as the central effect of the drug.
It was reported that the dihydroxyphenylglycol/norepinephrine ratio in the myocardium increased after the development of heart failure as a result of left ventricular infarction (25). This is not compatible with our results. Dihydroxyphenylglycol is the major metabolite of norepinephrine (25,26). This metabolite is formed within sympathetic neurons by monoamine oxidase after taking up norepinephrine into the neurons (uptake-1). In normal subjects, cardiac dihydroxyphenylglycol spillover into the bloodstream had a positive relation with that of norepinephrine during sympathetic nerve activation (26). In our study, the metabolite concentration was reduced in parallel with the level of norepinephrine. The ratio of dihydroxyphenylglycol and norepinephrine in the myocardium was the same in each group. In heart failure, neuronal uptake of norepinephrine (uptake-1) is remarkably reduced (5,19). This reduction is mimicked by the effects of uptake-blocking agents in the nonfailing heart. This reduced pump activity takes up less norepinephrine into the neurons. The synthesis ratio of dihydroxyphenylglycol from norepinephrine might not change in heart failure because monoamine oxidase activity did not change (27). A small amount of available norepinephrine in neurons is metabolized into a small amount of dihydroxyphenylglycol. Moreover, dihydroxyphenylglycol removal from the heart increased in heart failure patients (5). This might be due to sympathetic nerve overfilling. These phenomena might explain why the level of dihydroxyphenylglycol in the heart with an activated sympathetic nervous system decreases.
Adreno-α2-agonists were reported to have a beneficial effect on patients with enhanced sympathetic nerve activity (28-30). These agents reduced energy demand at the heart and decreased cardiac ischemia. These agents also improved arrhythmogenicity and increased ventricular-fibrillation thresholds. These abnormalities also are seen in heart failure. We consider adreno-α2-agonists to be useful for treatment of heart failure, along with the improvement of these abnormal pathophysiologic conditions.
In conclusion, a reduction in overactivated sympathetic nerve tone by guanabenz resulted in regression of cardiac hypertrophy, recovery of tyrosine hydroxylase activity, and restoration of cardiac norepinephrine concentration. Modulation of this activity by the central α2-adrenoceptor stimulator could be a new approach to the treatment of heart failure.
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