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Am J Physiol Heart Circ Physiol 285: H1660-H1667, 2003; doi:10.1152/ajpheart.00239.2003
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nNOS gene transfer to RVLM improves baroreflex function in rats with chronic heart failure

Yu Wang,1 Kaushik P. Patel,1 Kurtis G. Cornish,1 Keith M. Channon,2 and Irving H. Zucker1

1Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575; and 2Department of Cardiovascular Medicine, University of Oxford, Oxford OX3 9DU, United Kingdom

Submitted 18 March 2003 ; accepted in final form 17 June 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
We hypothesized that gene transfer of neuronal nitric oxide synthase (nNOS) into the rostral ventrolateral medulla (RVLM) improves baroreflex function in rats with chronic heart failure (CHF). Six to eight weeks after coronary artery ligation, rats showed hemodynamic signs of CHF. A recombinant adenovirus, either Ad.nNOS or Ad.{beta}-Gal, was transfected into the RVLM. nNOS expression in the RVLM was confirmed by Western blot analysis, NADPH-diaphorase, and immunohistochemical staining. We studied baroreflex control of the heart rate (HR) and renal sympathetic nerve activity (RSNA) in the anesthetized state 3 days after gene transfer by intravenous injections of phenylephrine and nitroprusside. Baroreflex sensitivity was depressed for HR and RSNA regulation in CHF rats (2.0 ± 0.3 vs. 0.8 ± 0.2 beats · min–1 · mmHg–1, P < 0.01 and 3.8 ± 0.3 vs. 1.2 ± 0.1% max/mmHg, P < 0.01, respectively). Ad.nNOS transfer into RVLM significantly increased the HR and RSNA ranges (152 ± 19 vs. 94 ± 12 beats/min, P < 0.05 and 130 ± 16 vs. 106 ± 5% max/mmHg, P < 0.05) compared with the Ad.{beta}-Gal in CHF rats. Ad.nNOS also improved the baroreflex gain for the control of HR and RSNA (1.8 ± 0.2 vs. 0.8 ± 0.2 beats · min–1 · mmHg–1, P < 0.01 and 2.6 ± 0.2 vs. 1.2 ± 0.1% max/mmHg, P < 0.01). In sham-operated rats, we found that Ad.nNOS transfer enhanced the HR range compared with Ad.{beta}-Gal gene transfer (188 ± 15 vs. 127 ± 14 beats/min, P < 0.05) but did not alter any other parameter. This study represents the first demonstration of altered baroreflex function following increases in central nNOS in the CHF state. We conclude that delivery of Ad.nNOS into the RVLM improves baroreflex function in rats with CHF.

sympathetic nerve activity; heart rate; nitric oxide; adenovirus

IT HAS BEEN WELL ESTABLISHED that arterial baroreflex function is impaired in patients (8) and animals (48) with chronic heart failure (CHF) and that it is associated with sympathoexcitation (3, 10, 11). This sympathoexcitation, if sustained, clearly aggravates the progression of the CHF state (6). It has been shown that the neuronal isoform of nitric oxide (NO) synthase (nNOS) message and activity are substantially reduced at various central sites in animals with experimental CHF (33). The rostral ventrolateral medulla (RVLM) is the final site of sympathetic processing in the brain and transmits sympathetic excitatory projections to the spinal cord (41). It plays a critical role in the control of arterial pressure, heart rate (HR), and sympathetic outflow from the central nervous system (CNS) (7). As a neuromodulatory substance, NO is believed to elicit effects on several central pathways, including the regulation of sympathetic tone (13). By using immunohistochemistry and NADPH-diaphorase staining, some studies have already demonstrated that nNOS is present in several regions that make up the components of the central neural circuitry mediating baroreflex control of sympathetic outflow, including the RVLM (18) and the nucleus tractus solitarii (NTS) (43).

The role of NO within the RVLM has been investigated by several groups. In anesthetized and conscious rats, studies suggest that NO decreases sympathetic nerve activity and depresses the activity in the RVLM (14, 20, 24, 42, 44).

Because the biological actions of NO are so varied and widespread, many investigators have concentrated on the NOS enzymes as potential targets for gene therapy. An adenoviral vector expressing nNOS (Ad.nNOS) has been used for transferring nNOS into the endothelium of cholesterol-fed rabbits to enhance endothelial function in atherosclerosis (36). Using adenoviral vectors encoding the endothelial NOS isozyme (eNOS), Sakai et al. (37) showed that an increase in NO production in the NTS by Ad.eNOS transfection results in a depressor response, a bradycardia, and a decreased sympathetic nerve activity in conscious rats. But the effect of NO in the RVLM on baroreflex function, especially on impaired baroreflex function in CHF, is still unknown.

It is now becoming clear that the gene transfer technique can be used to uncover basic mechanisms that relate to the role of NO in the pathogenesis of several cardiovascular disease states. The goal of the current study was to investigate the role of NO in the RVLM on baroreflex function in rats with experimental CHF (a sympathoexcitatory state) by using nNOS gene transfer.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The experiments were performed on male Sprague-Dawley rats weighing between 180 and 200 g. Animals were housed in steel cages and maintained at a temperature of 22–24°Cin a controlled room with 12-h/12-h light-dark cycle. Laboratory chow (Purina) and tap water were available ad libitum. All experiments were carried out according to the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings" and approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee.

Coronary artery ligation model of heart failure. Coronary artery ligation in rats was produced by using the technique described elsewhere (35, 40). The rats were assigned randomly to one of two groups: a heart failure group and a sham-operated group. Each rat was anesthetized with an anesthetic cocktail (55 mg/kg ketamine and 10 mg/kg xylazine im). The trachea was intubated, and the rat was placed on a small animal ventilator. A left thoracotomy was performed, and the heart was gently lifted out of the thorax. In the heart failure group, the left coronary artery was ligated with a 6-0 suture between the pulmonary artery outflow tract and the left atrium as it exited the aorta. The sham-operated rats underwent thoracotomy and manipulation of the heart, but the coronary artery was not ligated. After these procedures, the heart was returned, and the thorax was closed. The trachea was extubated after the rat began to recover from the anesthesia. The rats were then maintained on standard chow with water ad libitum for 6–8 wk.

Infarct size determination. At the completion of the experiment, the heart was dissected free of adjacent tissues and the lung. The ventricles were separated from the atria, and the right ventricular free wall was dissected from the septum. The atria and both ventricles were rinsed, blotted, and weighed. The left ventricle was opened with an incision along the septum from base to apex. Incisions were made in the left ventricle so that the tissue could be pressed flat. The circumferences of the left ventricle and the region of infracted tissue were outlined on a clear photograph taken by a digital camera. Infarct size was calculated and expressed as a percentage of left ventricular surface area based on the surface areas measured by the SigmaScan program (SPSS Science; Chicago, IL).

nNOS gene transfer of the RVLM. The nNOS adenovirus originally described by Channon et al. (4) was used in these experiments. To transfect the RVLM, each rat was anesthetized with cocktail (55 mg/kg ketamine and 10 mg/kg xylazine ip) and placed in a stereotaxic frame (David Kopf Instruments; Tujunga, CA). Under sterile conditions, the skull was exposed, and a small burr hole was made on the left side using the following stereotaxic coordinates (34): 12.0 mm posterior to bregma, 2.1 mm lateral to the midline, and 10.0 mm ventral from the dura. A cannula attached to a microsyringe (0.5 µl, model 7000.5, Hamilton microsyringe; Reno, NV) was advanced into the RVLM. A 100-nl solution [final concentration, 1 x 108 plaque-forming units (pfu)/ml, based on our pervious study (28)] of Ad.nNOS or the same adenoviral construct encoding the {beta}-galactosidase (Ad.{beta}-Gal) was injected into the RVLM. All injections were given in artificial cerebral spinal fluid of the following composition (in mmol/l): 124 NaCl, 25 NaHCO3, 3.3 KCl, 0.4 KH2PO4, 1.2 MgSO4, and 2.0 CaCl2; pH 7.4. Injection was given slowly over a 5-min period. After the injection, the wound was sutured, and analgesia (0.05 mg/kg Buprenex sc, Reckitt & Colman Products; Hull, UK) was administered every 12 h for 3 days.

NADPH-diaphorase histochemistry. Three days after Ad.nNOS infection, the brains were stained for NADPH-diaphorase activity using the protocol previously described by Vincent and Kimura (43) and modified by Zhang et al. (47). Each rat was perfused transcardially with 150 ml heparinized saline followed by 300 ml of freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brain was removed, postfixed at 4°C in paraformaldehyde overnight, and subsequently placed in a 20% sucrose solution for 24 h. The brain was blocked in the coronal plane and sectioned at 30 µm thickness in a cryostat. The sections were collected in 0.1 M phosphate (pH 7.4) containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium, and 1.0 mg/ml {beta}-NADPH. The sections in nitroblue tetrazolium solution were then placed in an incubator at 37°C for 60 min. After the reaction, the sections were rinsed in phosphate buffer (pH 7.4) and mounted onto chrome-alum-coated slides. The slides were air dried overnight, rinsed in distilled water, and dried again. Coverslips were then mounted directly with D.P.X Mounting Medium (Electron Microscopy Sciences; Washington, PA).

Immunocytochemical staining. One section from every five serial sections was prepared for immunohistochemical staining. The sections were rinsed in PBS for 15 min. The sections were then rinsed in acetone-methanol (1:1) for 20 min and PBS for 5 min (1% BSA and 0.2% Triton X-100). Nonspecific staining was blocked by 2% normal goat serum (Jackson Immuno Research Laboratories, West Grove, PA), containing 0.2% Triton X-100, and 0.1% sodium azide for 4 h at room temperature. Sections were incubated with primary antibody of mouse anti-rat nNOS IgG (1:100) (Transduction Laboratories; Lexington, KY) containing 0.2% Triton X-100 overnight at 4°C. These sections were further washed and incubated with goat anti-mouse IgG (1:100) (Molecular Probes; Eugene, OR) and Hoechst 33258 (antinuclei, Molecular Probes) and 0.2% Triton X-100 for 3 h at room temperature. The sections were then rinsed three times in PBS and 0.2% Triton X-100. The sections were mounted and evaluated under epifluorescence in a Leica DMR research microscope (Leica Microsystems Wetzlar; Wetzlar, Germany) equipped with an Optronics Magnafire digital camera (Optronics; Goleta, CA). Sections were digitally photographed. Photomicrographs were arranged using Adobe Photoshop (San Jose, CA) image editing software without any further adjustment to maintain the true nature of the findings.

Histochemical analysis for {beta}-galactosidase gene expression. At the completion of the baroreflex testing, the rats were perfused transcardially with 150 ml heparinized saline, followed by 300 ml of freshly prepared 2% formalin and 0.2% glutaraldehyde in PBS. The brains were removed, and coronal sections of the medulla were cut serially using a cryostat. The sections (30 µm) were evaluated for {beta}-galactosidase expression by X-Gal staining at 37°C overnight.

Western blot analysis of nNOS in the RVLM. Three days after Ad.nNOS infection, the rats were deeply anesthetized with pentobarbital sodium (70 mg/kg ip), and the brains were removed and immediately frozen on dry ice. The brains were blocked in the coronal plane and sectioned at 300 µm thickness in a cryostat. The RVLM was punched according to the method of Palkovits and Brownstein (31) from the virus-injected side and the contralateral control side. The punches were homogenized in ice-cold TRI reagent (Molecular Research Center; Cincinnati, OH) using a sonicator (GraLab 545).

The protein was extracted according to the Molecular Research Center published protocol. Protein content in the SDS-supernatant was determined using a bicinchoninic acid protein assay kit (Pierce; Rockford, IL). Protein (6 µg) was mixed with SDS-PAGE buffer containing {beta}-mercaptoethanol and heated at 100°C for 5 min. The protein was then fractionated in a 7.5% polyacrylamide gel along with molecular weight standards, transferred to an Immobilon membrane, and subjected to a Western immunoblotting protocol (28). The membrane was probed with monoclonal anti-nNOS antibody (Transduction Labs) and peroxidase-conjugated goat anti-mouse IgG, and the signal was detected using enhanced chemiluminescence immunoblotting detection system (Pierce). The film was digitized using a Kodak digital camera, and the net intensity was determined using Kodak 1D Image Analysis software.

Acute experiment. On the day of the terminal experiment, the rat was anesthetized with urethane (0.75 g/kg ip) and {alpha}-chloralose (70 mg/kg ip). The temperature was kept between 36 and 37°C. The left femoral vein was cannulated (polyethylene-50) for drug administration. The left femoral artery was cannulated and connected via a pressure transducer (model P23 1D, Gould; Cleveland, OH) to a computer-based data-acquisition system and software (MacLab, AD Instruments; Milford, MA) for recording arterial blood pressure and HR. The right carotid artery was dissected, and a transducer (Millar Instruments, Houston, TX) was advanced to the left ventricle. This was also connected to the MacLab system for recording left ventricular pressure.

The left kidney was exposed through a flank incision. A branch of the renal nerve was isolated from the adipose and connective tissues. The nerve was placed on a stainless steel bipolar electrode. The nerve-electrode junction was insulted electrically from the surrounding tissues with warm mineral oil. The electrical signal from the electrode was amplified with a Grass P55 preamplifier (Grass Instrument; W. Warwick, RI) with high- and low-frequency cutoffs of 1,000 and 100 Hz, respectively. The output from the Grass amplifier was directed to the MacLab system sampling at 1,000 samples/s. The signal was also rectified and integrated. The average rectified signal (RC filtered, time constant, 0.5 s) was then recorded and stored for later analysis. The frequency of nerve discharge was counted by using a window discriminator and ratemeter. The cursor of the window discriminator was set just above the electrical noise. Both frequency and integrated nerve activity were recorded continuously along with the raw nerve activity. The nerve discharge recorded at the end of the experiment after the rat was injected with hexamethonium (30 mg/kg iv) was regarded as background noise. During the experiment, the value of nerve discharge was calculated by subtracting the background noise from the actual recorded value. We determined maximal renal sympathetic nerve activity (RSNA) in each rat by observing its response to a bolus injection of sodium nitroprusside (SNP, 25 µg/kg iv) that lowered arterial pressure to between 40 and 45 mmHg. The baroreflex data were expressed as a percentage of maximum activity.

Arterial baroreflex. Arterial baroreflex curves were constructed as previously described by our laboratory (29, 30). In brief, several points for HR and RSNA were taken during the rise or fall in arterial pressure after the administration of SNP and phenylephrine (PE), respectively. Data points were obtained at ~2-s intervals. The logistic regression curve, as described by Kent et al. (21), was fit to the data points by using the following equation: HR or RSNA = P1/{1 + exp[P2(MAP – P3)]} + P4, where P1 is HR or RSNA range, P2 is the slope coefficient, P3 is the pressure at the midpoint of the range, P4 is minimum HR or RSNA, and MAP is mean arterial pressure. The peak slope (or maximum gain, Gmax) was determined by –P1 x P2/4 of the logistic function curve.

Brain histology for identification of injection site. At the end of each experiment, the brain was removed and fixed in 10% formalin for at least 24 h. The brain was then frozen, and serial transverse sections (30 µm) were cut using a cryostat (IEC, model CT, International-Harris Cryostat; Minneapolis, MN) at –20°C. The sections were mounted on microscope slides and stained with 1% neutral red. Presence of the needle tract within the RVLM was verified microscopically.

Data analysis. The mean values of each baroreflex curve parameters were used to derive a composite curve for each group of rats.

The data were subjected to one-way repeated-measures ANOVA followed by comparison for individual differences using the Newman-Keuls test. Blood pressure, HR, and the parameters of the baroreflex curve were compared between groups using the unpaired t-test. P < 0.05 was considered to indicate statistical significance. All data are presented as means ± SE.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
NADPH-diaphorase histochemical analysis of nNOS. Figure 1B,a and b, shows an example of the increase (B,a) in the NADPH-diaphorase staining of the RVLM infected with Ad.nNOS at day 3 after gene transfer. The NADPH-diaphorase potitive staining did not differ between Ad.{beta}-Gal-treated RVLM and its contralateral noninjected side (data not shown).



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  		Fig. 1. A: histological section illustrating the rostral ventrolateral medulla (RVLM, the coordinate: 11.90 mm posterior to bregma). x, Microinjection site in a representative animal. B: localization of neuronal nitric oxide synthase (nNOS) expression in RVLM by NADPH-diaphorase staining. a, Ad.nNOS-infected site; b, contralateral uninfected site. C: localization of nNOS expression in RVLM by fluorescent staining. a, Ad.nNOS-infected site; b, contralateral uninfected site. Arrows indicate cells that stain positive for nNOS. D: section through the RVLM with Ad.{beta}-Gal injection.

 

Immunohistochemistry for nNOS and {beta}-galactosidase. We evaluated the efficacy of Ad.nNOS gene transfer in the RVLM by fluorescent staining. We compared the fluorescent staining-positive cells of the RVLM infected with Ad.nNOS with the contralateral RVLM in the same rat at day 3 after gene transfer. Figure 1C,a and b, shows an example of the differences in staining of the infected (C,a) versus noninfected RVLM. Figure 1D shows the staining for {beta}-galactosidase in a section of the RVLM 3 days after gene transfer. Positive staining for {beta}-galactosidase was noted in the infected RVLM. No {beta}-galactosidase labeled cells were seen in the contralateral RVLM.

Western blot analysis of nNOS in the RVLM. We evaluated the efficacy of Ad.nNOS gene transfer in the RVLM by comparing the nNOS protein levels of the viral-injected RVLM with the contralateral noninjected RVLM in the both sham and CHF animals 72 h after gene transfer. In addition to a reduction of nNOS protein levels in CHF animals compared with the sham animals, there was a significant increase in the intensity of the bands for nNOS in the Ad.nNOS-injected RVLM compared with the contralateral noninjected RVLM in both groups of animals (Fig. 2, A and B).



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  		Fig. 2. Western blot analysis of nNOS in the RVLM of contralateral noninjected and Ad.nNOS-injected RVLM of sham and chronic heart failure (CHF) rats. A: representative Western blots showing nNOS protein from the contralateral control (C) and Ad.nNOS (N)-treated RVLM in one individual sham and CHF rat. B: results of densitometric analysis representing means ± SE. *P < 0.05, contralateral noninjected sites vs. Ad.nNOS-injected sites in the same animals.

 

Baseline hemodynamics in sham and CHF rats with or without Ad.nNOS gene transfer. Table 1 shows the values for MAP, HR, maximal pressure change over time (dP/dtmax), and left ventricular end-diastolic pressure (LVEDP) from the four groups studied. All CHF rats exhibited significantly lower dP/dtmax and higher LVEDP than both corresponding sham groups. The CHF rats with Ad.{beta}-Gal gene transfer showed a significantly lower MAP than sham rats with Ad.{beta}-Gal gene transfer, consistent with the hemodynamic changes observed in CHF. MAP in Ad.nNOS sham rats was no different from the Ad.{beta}-Gal-treated counterparts. However, the HR was significantly higher in sham rats treated with Ad.nNOS than the Ad.{beta}-Gal.


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  		Table 1. Baseline values for mean arterial pressure, HR, dP/dtmax, and LVEDP in each rat group

 

Effects of overexpression of nNOS in the RVLM on the arterial baroreflex control of HR and RSNA in CHF rats. Composite arterial baroreflex and gain curves for the control of HR in the sham and CHF rats are shown in Fig. 3. The data for the curve parameters are shown in Table 2. As can be seen, rats with CHF exhibited a depressed baroreflex control of HR. This depression was due primarily to a reduction in the HR range. nNOS gene transfer did not significantly alter the baroreflex sensitivity (Gmax) in sham rats (Fig. 3A). However, there was an increase in the HR range. In CHF rats, Ad.nNOS gene transfer normalized baroreflex control of HR by increasing the peak slope and the HR range (Fig. 3B).



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  		Fig. 3. Arterial baroreflex curves and their respective slopes (insets) for control of heart rate [HR in beats/min (bpm)] in sham (A) and CHF (B) rats with Ad.{beta}-Gal or Ad.nNOS gene transfer. MAP, mean arterial pressure. *P < 0.01, Ad.{beta}-Gal/CHF vs. Ad.nNOS/CHF.

 

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  		Table 2. Logistic parameters of baroreflex control of HR in each rat group

 

The baroreflex control of RSNA (Fig. 4 and Table 3) was depressed in CHF rats with Ad.{beta}-Gal gene transfer compared with sham rats with Ad.{beta}-Gal. For the baroreflex control of RSNA, Ad.nNOS normalized the sensitivity by increasing the RSNA range in CHF group (Fig. 4B) compared with the Ad.{beta}-Gal gene transfer group.



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  		Fig. 4. Arterial baroreflex curves and their respective slopes (insets) for control of RSNA in sham (A) and CHF (B) rats with Ad.{beta}-Gal or Ad.nNOS gene transfer. *P < 0.01, Ad.{beta}-Gal/CHF vs Ad.nNOS/CHF.

 

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  		Table 3. Logistic parameters of baroreflex control of RSNA in each rat group

 

Histological verification of microinjection sites. Figure 1A indicates the microinjection site in a representative animal. The needle tract can also be observed in Fig. 1D.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
In the present study, we used Ad.nNOS gene transfer to induce nNOS overexpression unilaterally in the RVLM. The restriction to unilateral injection may explain why nNOS overexpression did not significantly alter baseline blood pressure in either CHF or sham rats. However, we found nNOS overexpression normalized the impaired baroreflex function in CHF, suggesting that the lack of NO is involved in this dysfunction.

The dose and time period of gene transfer are based on previous studies using the same recombinant virus (5, 28). It has been shown that the dose of 1 x 108 pfu/ml and the time course of 3 days yield maximal transgene expression without apparent neuronal or vascular injury. Furthermore, we found Ad.{beta}-Gal gene transfer did not alter basal arterial blood pressure, HR, and RSNA compared with untreated control rats (data not shown). These results suggest that the changes in arterial baroreflex function with Ad.nNOS gene transfer in the RVLM were not mediated by inflammation or cytotoxicity but primarily mediated by the augmentation of NO production. From our immunohistochemical staining, we noticed that there are various cell types transfected. Whereas it is difficult to discern specific cell types (e.g., various types of glial cells), the NADPH-diaphorase staining suggests that at least neurons appeared to be transfected. From our prelimary study, which transfected the same virus to the fibroblasts in culture, we found that the nitrite or nitrate levels increased in the medium in proportion to the viral titer. This suggests the NO production is augmented no matter which cell types are being transfected. Because some studies suggest that the adenovirus can be taken up by nerve terminals and retrogradely transported, we examined the retrograde transportation to caudal ventrolateral medullar (CVLM) and NTS by NADPH-diaphorase staining 3 days after administering Ad.nNOS to RVLM. We did not find a significant increase in the number of diaphorase-positive cells in the CVLM as well as NTS compared with nontransfected brains.

A recent study by Kishi et al. (24) demonstrated that overexpression of eNOS in the RVLM of the conscious Wistar-Kyoto rat produced hypotension and bradycardia 7 days after transfection. In our study we observed that the sham rats with Ad.nNOS gene transfer exhibited insignificantly lower MAP compared with sham rats with Ad.{beta}-Gal gene transfer. HR was significantly higher in the sham Ad.nNOS group compared the Ad.{beta}-Gal gene transfer group. Because we conducted the study in urethane-anesthetized rats, alteration of blood pressure and HR by anesthesia may account for these differences. Alternatively, the production of NO by eNOS versus nNOS may be different. All CHF rats exhibited significantly lower dP/dtmax and higher LVEDP than either sham group. All CHF rats also exhibited a lower baseline MAP. Ad.nNOS gene transfer in the CHF group resulted in a decrease in dP/dtmax compared with the Ad.{beta}-Gal gene transfer CHF group. The mechanism(s) for these findings is not clear; however, it may be due to a sympathoinhibitory response of central NO in the RVLM (44). These results are consistent with the finding of Kishi et al. (24) that the urinary norepinephrine excretion was decreased on day 7 after the Ad.eNOS gene transfection in conscious rats. In addition, another study by Kishi et al. (23) demonstrated that an increase in NO production in the RVLM by Ad.eNOS transfection improved the impaired baroreflex control of HR in stroke-prone spontaneously hypertensive rats. This suggests that NO plays an important sympathoinhibitory role in the RVLM of stroke-prone spontaneously hypertensive rats as well as in the CHF state.

In the Ad.{beta}-Gal CHF group, baroreflex sensitivity for the control of HR and RSNA was, as we expected, depressed compared with the sham group. The BP50 (an index of resetting) of the baroreflex control of HR in the Ad.{beta}-Gal CHF group was lower than that in the sham group, which corresponds to a lower baseline MAP. In the CHF group, Ad.nNOS gene transfer normalized arterial baroreflex sensitivities by increasing the HR and RSNA range compared with the Ad.{beta}-Gal gene transfer group. However, Ad.nNOS gene transfer did not alter the arterial baroreflex sensitivities in the sham groups.

It has been well accepted that sympathetic nerve activity is elevated in patients and animals with CHF (3, 10, 11). This sympathoexcitation, if sustained, is clearly deleterious in terms of mortality and progression of the CHF state (6). It is well established that arterial baroreflex function is impaired in patients and animals with CHF (8, 48). Although the mechanisms for sympathoexcitation are not completely understood, therapeutic targeting of sympathetic outflow, such as {beta}-blocker therapy and exercise training, has proved to be beneficial for patients and animals with CHF (12, 29). Enhanced baroreflex sensitivity may provide for a more stable arterial pressure and increased HR variability (25). In this study, we showed the augmentation of nNOS expression in the RVLM. Although we could not differentiate this effect between neurons and glia or other kinds of cells, we believe the mechanism of normalization of baroreflex in CHF after Ad.nNOS gene transfer is due to the sympathoinhibitory effect of NO in the RVLM (20, 24, 44).

It has been known that the presympathetic neurons in the RVLM project to the intermediolateral cell column of the spinal cord (IML). Thus the RVLM acts as the major source of sympathoexcitatory activitity from the CNS. In the RVLM, a number of neurotransmitters influence the presympathetic neuronal activities through both excitatory and inhibitory effects. GABA is the primary inhibitory neurotransmitter in RVLM. A major source of the GABAergic inhibition arises from the CVLM. Previous studies by Kishi et al. (24) suggested that the GABA levels in the RVLM are significantly increased in the Ad.eNOS-transfected rats. This may explain their observation that blood pressure, HR, and urinary norepinephrine excretion decreased after overexpression of eNOS in the RVLM. A study by Zhang et al. (46) suggests that the inhibitory effect of endogenous NO within the paraventricular nucleus (PVN) is mediated by GABA (for a review, see Ref. 32). Interestingly, their recent study (45) showed that endogenous GABA effects in the PVN of rats with CHF were reduced. These studies suggest that GABA may be responsible for the sympathoinhibition when NO production is augmented.

Glutamate elicits an excitatory effect on presympathetic neurons in the RVLM (38). This may be another mechanism by which NO reduces sympathetic tone and augments baroreflex function in the RVLM. It has been shown that central NO inhibits N-methyl-D-aspartic acid (NMDA)-mediated increases in RSNA in PVN (27). It has also been shown that NO in the PVN increased both excitatory and inhibitory amino acids, although blood pressure decreased after administration of NO (16). Kishi et al. (24) also showed that both GABA and glutamate levels were significantly increased in the RVLM in Ad.eNOS-transfected rats. They considered that in the resting condition GABAergic input for inhibitory sympathetic nerve activity may be greater than glutamatergic input. But glutamate might be primarily involved in the hypotensive response evoked by NO. This notion is consistent with the present study, which showed that during hypotension in the CHF rats, the RSNA increased to a higher level in the Ad.nNOS-treated group. A balance between excitatory and inhibitory inputs may exist in the RVLM. In some disease states, such as hypertension, this balance is shifted toward the excitatory pathway (19). In CHF, we believe this balance is similarly shifted toward sympathoexcitation because the resting RSNA is generally elevated (10, 11, 49). nNOS gene transfer by increasing inhibitory inputs to the RVLM results in normalization of baroreflex sensitivity in CHF.

Another neurotransmitter, ANG II, has been investigated in the RVLM recently. Microinjection of ANG II into the RVLM results in an increase in the release of glutamate in the IML (17), as well as an increased in arterial pressure and sympathetic vasomotor activity (1, 15, 39). Immunofluorescence combined with confocal microscopy demonstrated that most of the glutamatergic and GABAergic neurons in the RVLM were double labeled with the AT1 receptors (17). This suggests that AT1 receptors are closely related to glutamatergic and GABAergic pathways in RVLM. The relationship between NO and ANG II in the RVLM is still unclear. Recent studies showed that the endogenous angiotensin receptors in the RVLM were involved in the maintenance of hypertension in NG-nitro-L-arginine methyl ester-treated rats (2) and the angiotensin-converting enzyme mRNA level was increased in the brain stem in N{omega}-nitro-L-arginine methyl ester-treated rats (9). High levels of ANG II may act to depress NOS gene expression (22) or reduce the bioavailability of NO by an increase in endogenous superoxide production (26).

In conclusion, we developed a model for nNOS gene transfer to the RVLM in rats with CHF. This study demonstrated that in vivo gene transfer of nNOS in RVLM of rats with CHF increased baroreflex sensitivity. These data implicate a loss of NO production in the etiology of sympathoexcitation in the CHF state. Our results provide a novel approach to restore central nNOS in experimental disease states, such as heart failure and hypertension.


   DISCLOSURES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute grant PO-1 HL-62222. Y. Wang was supported by a predoctoral fellowship from the American Heart Association, Heartland Affiliate.


   ACKNOWLEDGMENTS
 
The authors thank Johnnie F. Hackley and Pamela Curry for expert technical assistance. We also thank Yi-Fan Li for help with the immunofluorescence and Western blots.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Zucker, Dept. of Physiology and Biophysics, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: izucker{at}unmc.edu).

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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