It is well established that enhanced sympathetic drive contributes to the vicious cycle and poor prognosis in patients with heart failure (HF) (1). Recent large clinical trials have demonstrated that treatment with β-blocker in patients with HF improves their prognosis (2). The enhanced sympathetic drive in HF is also observed in animal models with HF, and there have been many studies examining the mechanism(s) involved (3). Abnormal arterial as well as cardiopulmonary baroreflex control of sympathetic nerve activity is considered to be one of the important mechanisms (3). In particular, afferent abnormalities have been intensively examined (3). However, recent studies have suggested that the central nervous system mechanisms may be significantly involved in the enhanced sympathetic drive in HF (3-6).
There is considerable evidence that nitric oxide (NO) in the brainstem reduces sympathetic nerve activity (7-9). The nucleus tractus solitarii (NTS) and the rostral ventrolateral medulla (RVLM) are important sites for cardiovascular regulation, including the arterial baroreflex control of sympathetic nerve activity (10-12). Nitric oxide in the NTS and the RVLM has been shown to inhibit sympathetic nerve activity (7-9). Recent studies have suggested that NO in the paraventricular nucleus (PVN) of the hypothalamus may play a role in the enhanced sympathetic drive in HF (13). However, there are no studies focusing on examining the expression of both neuronal nitric oxide synthase (nNOS) protein level and mRNA level in the NTS and the RVLM in HF.
Therefore, the aim of this study was to determine the expression level of nNOS protein and mRNA in the NTS and the RVLM of the brainstem in HF. For this purpose, we performed experiments in rats with myocardial infarction as an HF model using Western blot analysis and in situ hybridization techniques.
Male Wistar-Kyoto rats weighing 300-350 g were obtained from an established colony at the Animal Research Institute of Kyushu University Faculty of Medicine (6). This study was reviewed by the Committee on Ethics in Animal Experiments of Kyushu University and was performed in accordance with the Guidelines for Animal Experiments of Kyushu University.
Animal models of HF
Rats were subjected to sham surgery or left coronary artery ligation. Under sodium pentobarbital anesthesia [50 mg/kg, intraperitoneally (i.p.)], myocardial infarction was induced by ligation of the left anterior descending coronary artery, as described elsewhere (4,5,14). Briefly, after intubation of the trachea, and connection to a respirator, the thorax was opened at the fourth intercostal space and the left descending coronary artery was ligated at 2-3 mm from its origin with a 6-0 silk suture. The rats were followed up for 4 to 6 weeks with echocardiographic examination. After confirmation of the enlarged left ventricle (LV) and the reduced left ventricular ejection fraction, rats were used as an HF group. Sham operation was performed without the coronary artery ligation (control group).
Hemodynamic and echocardiographic examination
The rats were anesthetized with sodium pentobarbital (30 mg/kg i.p.), and an echocardiographic examination of the LV diameter and wall motion was performed with a 7.5 MHz probe (SSD-500A, Tokyo, Japan) (6). Short axis views were used to guide the cursor for M-mode images of the LV. Left ventricular diastolic dimensions were measured from the trailing edge of the intraventricular septum to the leading edge of the posterior wall at the point of maximal ventricular diameter. Systolic dimensions were taken at the minimum ventricular diameter of the beats in systole using the same edge definition. Systolic ventricular function was quantified as ejection fraction. For measurement of the LV end-diastolic pressure, a catheter was introduced into the LV from the carotid artery. At the end of the experiments, rats were anesthetized with an overdose of sodium pentobarbital, and organ wet weights and pleural effusions were examined postmortem (6). Urinary norepinephrine excretion was measured by high-performance liquid chromatography in the conscious state, as described elsewhere (15,16).
Western blot analysis
We performed Western blot analysis for nNOS protein from tissue obtained from the NTS or the RVLM anatomically identified by a punch-out technique, as described elsewhere (15,16). For this purpose, we used mouse immunoglobulin G monoclonal antibody to nNOS (1 : 2500, Transduction Laboratories, Lexington, KY, U.S.A.).
In situ hybridization histochemistry for nNOS mRNA
In situ hybridization histochemistry was performed as described previously (17). Frozen 12 μm-thick brainstem sections were cut by cryostat at -20°C, thawed and mounted onto gelatin/chrome alum-coated slides. The sections were fixed, dehydrated and delipidated in 100% chloroform, then partially rehydrated. Hybridization was carried out at 37°C overnight in 45 μl of hybridization buffer with 35S 3′ end-labeled deoxyoligonucleotide probe complementary to transcripts coding for rat nNOS. The specificity of the probe was published elsewhere (17,18). After the sections were washed, hybridized sections of the brainstem were apposed to autoradiography film for 14 days. Slides hybridized to the nNOS probe were dipped in nuclear emulsion (K-5; Ilford, Essex, U.K.) and further exposed for 28 days.
Intracisternal injection ofNG-monomethyl-L-arginine
In order to determine whether the contribution of endogenous NO in the brainstem differed between the HF group and the control group, we examined the effect of intracisternal injection of NG-monomethyl-L-arginine (L-NMMA) (1 μmol) on blood pressure in rats anesthetized with sodium pentobarbital (50 mg/kg i.p. followed by 10-20 mg/kg per hour i.v.), as described elsewhere (15).
All values are expressed as means ± the standard error of the mean. An unpaired t-test was used to compare the values between the two groups. Differences were considered to be significant at the level of p < 0.05.
Hemodynamic and echocardiographic evaluation, organ wet weights, and urinary norepinephrine excretion
Table 1 shows the results of general characteristics of the HF group and the control group. Systolic arterial blood pressure was lower, and the LV end-diastolic pressure was higher in the HF group than in the control group. Echocardiographic examination revealed that both LV end-diastolic diameter and LV end-systolic diameter were enlarged in the HF group compared to the control group. In addition, ejection fraction of the LV was reduced in the HF group compared to the control group. The wet weight of the lungs of the HF group was greater than in the control group. Finally, 24-h urinary norepi- nephrine excretion was greater in the HF group than in the control group.
Western blot analysis for nNOS protein in the NTS and the RVLM
Figure 1 shows the results of Western blot analysis for nNOS protein in the NTS and the RVLM. The expression of nNOS protein levels in the NTS as well as in the RVLM were significantly reduced in the HF group compared to the control group (n = 6 for each group). The group in which echo and hemodynamics was measured was equal to that in which Western blot analysis was performed.
In situ hybridization analysis for nNOS mRNA in the sections of the medulla
The expression of the nNOS gene in the NTS and the RVLM was decreased in the HF group compared to the control group (Fig. 2). Microscopic observation revealed that both the number and the intensity of cells labeled with the nNOS probe tended to be decreased in the NTS and the RVLM in the HF group compared to the control group (n = 3 for each group).
Effect of intracisternal injection of L-NMMA on arterial blood pressure
Figure 3 shows the changes in mean arterial blood pressure evoked by intracisternal injection of L-NMMA; L-NMMA elicited the pressor response in both groups. However, the response to L-NMMA was less in the HF group compared to the control group.
This study has demonstrated that the expression of nNOS protein and mRNA in the NTS and the RVLM was reduced in rats with HF produced by myocardial infarction. In addition, the pressor response elicited by inhibiting NO production with L-NMMA in the brainstem was less in the HF group compared to the control group. These results suggest that reduced production of NO in these nuclei of the brainstem contributes to enhanced sympathetic drive in HF.
The vasodilatory response to acetylcholine or methacholine has been shown to be impaired in HF (19,20). Levels of endogenous endothelial NOS (eNOS) protein and mRNA in peripheral tissues have also been demonstrated to be reduced in HF (21). However, there are a few studies examining the NO system within the central nervous system in HF. Patel et al. showed that the level of expression of nNOS mRNA examined by reverse transcriptase polymerase chain reactions in discrete regions in the brain, particularly the hypothalamus, dorsal pons, and dorsal medulla, is decreased in rats with HF compared to sham-operated rats (22). They also examined the level of expression of the nNOS gene by nicotinamide adenine dinucleotide phosphate-diaphorase staining, and found that the number of NOS-positive neurons in the PVN and supraoptic nucleus of the hypothalamus is decreased in the HF group compared to the sham-operated group (23). Apparently, these authors focused on the hypothalamus, in particular, to examine the role of NO in the PVN in neural control of circulation (24), although they also examined the other areas. In our experiments, we have clearly demonstrated a decreased expression of nNOS protein in the RVLM. In contrast to the earlier findings regarding expression of nNOS mRNA level in the RVLM in the HF group, we found a decrease in nNOS mRNA by in situ hybridization. We do not have a clear answer for the different results between the two studies. We did not quantify nNOS mRNA by in situ hybridization, because the expression of nNOS mRNA was found in many areas in the brainstem other than the RVLM or the NTS, which made it difficult to quantify the expression between the two groups. However, we demonstrated a decrease in nNOS protein in the RVLM in the HF group compared to the control group measured by Western blot analysis. In the NTS, we found that the levels of both mRNA and protein for nNOS were decreased in HF compared with sham-operated controls, which supports earlier results (22).
We evaluated NOS activity by the pressor response evoked by intracisternal injection of L-NMMA. We cannot exclude the possibility that the increased baseline sympathetic nerve activity might affect the results, because sympathetic nerve activity might be close to maximal level in HF. It has been shown that blockade of the endogenous NO by the microinjection of L-NMMA into the PVN increases arterial blood pressure, and renal sympathetic nerve activity in both sham-operated rats and rats with HF (13). However, these responses were significantly reduced in rats with HF as compared to the sham-operated group (13). Importantly, these authors found that sympathetic nerve activity was as increased in an HF model of rats with myocardial infarction as in sham-operated rats, evoked by blockade of the air way (13). This is consistent with our observation that the pressor response evoked by intracisternal injection of L-NMMA was reduced in the HF group compared to the control group.
The mechanism(s) involved in reduced expression of nNOS in HF remains to be elucidated from the results of the present study. Interestingly, recent studies suggest an interaction of NO and angiotensin II (25), and it may be important in the HF state as the angiotensin II level is known to be increased in addition to activation of the sympathetic nervous system (26). In support of this idea, we demonstrated that blockade of angiotensin type-1 receptor in the NTS reduces sympathetic nerve activity in rats with aortocaval shunt (6). Further studies will be needed to clarify these mechanism(s).
In summary, our results suggest that a dysfunction of NO production in the NTS and the RVLM exists in HF, which, in turn, contributes to the increase in sympathetic drive in HF.
Acknowledgement: This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (C11670689).
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