CENTRAL ANGIOTENSIN AT₁ RECEPTOR BLOCKADE DECREASES CARDIAC BUT NOT RENAL SYMPATHETIC NERVE ACTIVITY IN HEART FAILURE

Abstract

In heart failure (HF) cardiac sympathetic nerve activity (CSNA) is increased, which has detrimental effects on the heart and promotes arrhythmias and sudden death. There is evidence that the central renin angiotensin system plays an important role in stimulating renal SNA (RSNA) in HF. Since SNA to individual organs is differentially controlled, we have investigated whether central angiotensin receptor blockade decreases CSNA in HF. We simultaneously recorded CSNA and renal SNA (RSNA) in conscious normal sheep and in sheep with HF induced by rapid ventricular pacing (ejection fraction <40%). The effect of blockade of central angiotensin AT₁R by intracerebroventricular infusion of losartan (1 mg/h for 5 hrs) on resting levels and baroreflex control of CSNA and RSNA was determined. In addition, the levels of angiotensin receptors in central autonomic nuclei were determined using autoradiography. Sheep in HF had a large increase in CSNA (43±2 to 88±3 bursts/100 heart beats, P<0.05) and heart rate, with no effect on RSNA. In HF, central infusion of losartan for 5 hours significantly reduced the baseline levels of CSNA (to 69±5 bursts/100 heart beats) and heart rate. Losartan had no effect in normal animals. In HF, angiotensin receptor levels were increased in the paraventricular nucleus and supraoptic nucleus, but reduced in the area postrema and nucleus tractus solitarius. In summary, infusion of losartan reduced the elevated levels of CNSA in an ovine model of HF, indicating that central angiotensin receptors play a critical role in stimulating the increased sympathetic activity to the heart.

Introduction

Heart failure (HF) is a common cause of hospital admission and death in adults over 55 years of age, and with the aging of the population it is becoming a leading cause of death worldwide. Activation of the renin-angiotensin system (RAS) and sympathetic nervous system is a hallmark of HF ^{1, 2}, and inhibition of these systems is a major focus of therapy ^3-5. The sustained and excessive level of sympathetic nerve activity (SNA) has adverse effects that contribute to the progression of HF, and this is particularly the case for the increase in cardiac SNA (CSNA). The high level of norepinephrine release at the heart causes down regulation of cardiac β-adrenoceptors ⁶, has toxic effects on the sympathetic nerve terminals ⁶, induces left ventricular fibrosis and hypertrophy ⁷, and promotes the development of arrhythmias and sudden death ⁸. Underscoring the detrimental effect of the increased sympathetic drive to the heart in HF is the effectiveness of treatment with β-blockers ^{4, 9}, and the finding that cardiac norepinephrine spillover is the strongest prognostic marker in HF patients ⁸.

The mechanisms causing the increase in CSNA in HF are not well defined. The majority of studies examining the increase in SNA in HF have focussed on sympathetic activity to the kidney in animal experiments ^10-13 and to skeletal muscle in patients ^{14, 15}. There is strong evidence from these studies supporting a role for both peripheral and central mechanisms. Regarding central mechanisms, a focus has been the RAS, which acts at multiple brain nuclei to regulate sympathetic outflow ^{16, 17}. Blockade of central angiotensin type 1 receptors (AT₁R) with losartan reduces the elevated renal SNA (RSNA) in rats with HF induced by myocardial infarction ^{10, 18} and restores arterial baroreflex sensitivity in rats and rabbits with HF ^{10, 12}.

The factors that control CSNA are, however, different to those controlling SNA to other beds. For example, we have demonstrated that in the normal animal the resting level of CSNA is set much lower than that of RSNA ¹⁹, that in an ovine pacing-induced model of HF there is a much larger increase in CSNA than RSNA ^{19, 20}, that in this model the inhibition of CSNA in response to volume expansion is abolished whereas the inhibition of RSNA is only attenuated ^{20, 21}, and finally in normal sheep we have shown that ICV infusion of angiotensin II (Ang II) or hypertonic saline have opposite effects on CSNA and RSNA ^{22, 23}. The finding that ICV Ang II increased CSNA, together with findings that central AT₁R blockade reduced RSNA in rodent models of HF, suggested that increased activity of the central RAS could contribute to the cardiac sympathoexcitation in HF.

In the present studies we have, therefore, investigated whether central administration of the angiotensin AT₁R blocker, losartan, reduces the large increase in CSNA that occurs in conscious sheep with pacing induced HF. As well as investigating the effects of ICV infusion of losartan on CSNA and RSNA in normal and HF sheep, we have compared the levels of angiotensin receptors in central cardiovascular nuclei using autoradiography.

Methods

Experiments were performed on 9 normal and 7 HF adult Merino ewes (30-40 kg) acclimatised to laboratory conditions and housed with other sheep. All experiments were approved by the Animal Experimentation Ethics Committee of the Howard Florey Institute, following guidelines from the National Health and Medical Research Council of Australia.

Surgery

Prior to the studies, sheep underwent three aseptic surgical procedures, separated by recovery periods of at least two weeks (for full details please see http://hyper.ahajournals.org). For all normal animals anesthesia was induced with intravenous sodium thiopental (15mg/kg) and maintained with 1.5-2.0% isoflurane/O₂. For animals in HF, anaesthesia was induced and maintained with isoflurane. In the first operation a carotid arterial loop was constructed, and in HF animals a cardiac pacemaker lead was inserted into the right ventricle. For the second operation, guide tubes were inserted over the lateral cerebral ventricles. The final operation was to implant electrodes in cardiothoracic and renal sympathetic nerves ^{22, 23}. Experiments were started at least 3 days after implantation of electrodes. One day prior to experiments a further cannula was inserted into the carotid artery for measurement of arterial pressure.

Pacing-induced heart failure

Sheep with pacemaking leads underwent left ventricular echocardiograph measurements (Hewlett Packard Sonos 1000) pre-pacing. Sheep were then paced at 200-220 beats/min and echocardiographs were repeated weekly with the pacing off. When ejection fraction was below 40%, electrode placement surgery was performed. The sheep used in this study were paced for 14 ± 3 weeks before nerve recording electrodes were implanted.

Nerve recording

Sympathetic nerve activity was recorded differentially between pairs of electrodes ^{22, 23}. The signal was amplified (× 100,000) and filtered (bandpass 400 to1000 Hz). Sympathetic nerve activity and blood pressure were recorded on computer using a CED micro 1401 interface and Spike 2 software (Cambridge Electronic Design, UK). Details of burst identification are included in the online supplement.

Experimental protocols

After a 5 minute baseline recording, baroreflex responses were generated by measuring CSNA, RSNA, and HR responses to changes in arterial pressure induced by increasing doses of phenylephrine hydrochloride and sodium nitroprusside. Following baseline recordings, the AT₁R antagonist losartan (Merck, New Jersey, USA) was infused ICV (1mg/hour in artificial cerebrospinal fluid (CSF) at 1 mL/h for 5 h), and towards the end of the infusion another baseline recording was made and the baroreflex responses were re-tested. Effectiveness of this dose of losartan has been demonstrated in previous studies in sheep ²². Baroreflex curves were drawn using a four-parameter sigmoidal logistic equation ²⁴. The following control experiments were also conducted: ICV infusion of CSF (1 mL/h for 5h) in normal and HF sheep (n=3/group), IV losartan (1 mg/h for 5 h) in 3 HF sheep, and ICV candesartan (0.25 mg/h for 5 h) in 2 HF sheep.

Autoradiography of angiotensin receptors

To determine changes in the levels of Ang II receptors in heart failure the distribution of angiotensin binding sites was mapped in the brains of normal (n=5) and HF (n=5) sheep. Detailed methods of the autoradiography procedure are available in the online supplement. Briefly, 20 μm coronal slide-mounted sections were incubated using standard solutions containing ¹²⁵I-[Sar¹, Ile⁸] Ang II as radioligand (Prosearch International Australia Ltd, Vic, Australia) and different angiotensin receptor subtype antagonists. After washing, then drying, the sections were apposed to X-ray film (UM-MAC HC, Fujifilm, Tokyo, Japan) and all binding was normalised to total binding in the normal brains.

Statistics

Results are expressed as mean ± SEM. For baseline data, an unpaired t-test was used to compare normal vs. HF animals. Differences within a group before and after losartan were analysed using 2 way repeated measures ANOVA. A significant result was considered to be p < 0.05.

Results

Resting levels in normal and HF sheep

Ejection fraction (85.2 ± 1.5 to 35.1 ± 3 %; p<0.001) and fractional shortening (53.6 ± 2 to 16.7 ± 1.5 %; p<0.001) decreased in HF sheep over 9-10 weeks of rapid ventricular pacing. In the HF group, HR was significantly increased (100 ± 6 vs. 74 ± 6 beats/min, p<0.05) compared with the normal group, and systolic, diastolic and mean arterial pressures tended to be lower, (95 ± 5 vs. 105 ± 2, 70 ± 3 vs. 74 ± 3 and 78 ± 4 vs. 84 ± 3 mmHg, respectively) and central venous pressure tended to be higher (3.3 ±1.4 vs. 0.4 ± 0.8 mmHg), but the differences did not reach statistical significance (Figure 1). In normal sheep, the resting level of CSNA was significantly lower than that of RSNA (Figure 2). In HF, there was a dramatic increase in resting CSNA (from 43 ± 2 to 88 ± 3 bursts/100 heart beats, P<0.05), whereas the resting levels of RSNA were unchanged (90 ± 4 to 82 ± 5 bursts/100 heart beats) (Figures 1 and 2). Similar changes were seen when total nerve activity, expressed as spikes/second, was analysed (Figure 2).

Figure 1.

Raw traces of arterial pressure (AP), cardiac sympathetic nerve activity (SNA) and renal SNA in a conscious sheep in HF before (panel A) and after (panel B) 5 hours ICV infusion of Losartan (1mg/hr).

Figure 2.

Changes in MAP, HR, CSNA and RSNA over 5 hours ICV infusion of Losartan in conscious normal (solid line) and heart failure (dashed line) sheep. Time 0 shows resting levels before the start of the Losartan infusion. Results are mean ±SEM. † indicates significant decrease in HF animals after 5 hours Losartan. # indicates significant difference between normal and HF animals at the control time point.

Effects of ICV losartan in normal and HF sheep

Infusion of losartan into the cerebral ventricles had no effect on the resting levels of blood pressure in either group, but in the HF group it significantly reduced the raised HR back to normal levels (Figure 2). In the HF group losartan caused a gradual decrease in resting levels of CSNA over the 5 h infusion period towards normal levels (62 ± 10 to 27 ± 7 spikes/s, p<0.05) (Figures 1 and 2). This was due to a significant decrease in the average burst incidence (88 ± 3 to 69 ± 5 bursts/100 heart beats, p<0.05) as well as a decrease in the burst amplitude (40 ± 8 to 29 ± 5 spikes/burst, p<0.05). Losartan infusion tended to decrease in CSNA in the normal animals, but this was not significant. In contrast, ICV losartan had no effect on the resting levels of RSNA in either group (Figures 1 and 2).

Central infusion of candesartan, another AT₁R antagonist, had similar effects to losartan, causing a large fall in CSNA with no effect on RSNA. Intravenous infusion of losartan (1mg/hr), the same dose as given centrally, had no effects in HF sheep and ICV infusion of CSF had no effects on CSNA, RSNA, HR or MAP in either group (data not shown).

Effects of losartan on baroreflex responses in normal and HF sheep

To compare the effects of losartan within each group, arterial baroreflex curves of SNA were constructed from data normalised to the resting activity immediately before starting losartan infusion. In HF animals, the decrease in the resting level of CSNA after 5 hours of losartan infusion was associated with a decrease in the range and upper plateau of the CSNA baroreflex curve (Figure 3, Table 1). In contrast, there was no change in any of these parameters in the normal group. Losartan had no effect on RSNA baroreflex parameters in either group. In HF, there was a decrease in the range and maximum gain of the HR baroreflex compared with normal animals (Figure 3, Table 1). After 5 hours of ICV losartan, the sensitivity of the HR baroreflex curve tended to be increased (−3.26 ± 0.36 to −5.22 ± 0.77 bpm/mmHg, P=0.1).

Figure 3.

Baroreflex relations of % CSNA (a) and % RSNA (b) (both normalised to pre-losartan values in each group) to diastolic blood pressure (dBP), and of HR to systolic blood pressure (sBP) (c). Data are from normal (left panel) and HF animals (right panel) before (solid line) and after (dashed line) ICV infusion of Losartan (1mg/hr for 5 h). Resting points before (filled circle) and after (open circle) Losartan are indicated as mean±SEM. † indicates significant decrease with Losartan, † indicates decreased range with Losartan, *indicates decreased range with HF.

Table 1.

Arterial baroreflex relations in conscious normal and HF sheep before and after 5h ICV infusion of Losartan (1 mg/hr). The % CSNA and % RSNA values during infusion of Losartan are normalised to the resting levels pre-Losartan. Values are mean ± SEM.

Variables	Normal		Heart Failure
	Resting	Losartan (5h)	Resting	Losartan (5h)
dBP and % CSNA
Upper Plateau	363.1 ± 46.8	335.7 ± 31.5	167.7 ± 17.2	112.9 ± 10.7†
Range	372.2 ± 46.2	330.2 ± 29.5	167.6 ± 18.1	105.6 ± 9.3†
Gain	−6.01 ± 1.37	−4.74± 0.75	−4.98 ± 1.12	−3.48 ± 0.9
BP50	73.8 ± 2.8	65.1 ± 4.3	73.8 ± 4.4	69.3 ± 3.2
Threshold (mmHg)	84.6 ± 3.5	79.1 ± 4.7	88.5 ± 5.9	79.5 ± 4.5†
Saturation (mmHg)	49.2 ± 7.7	51.2 ± 4.9	59.1 ± 5.0	59.0 ± 3.7
dBP and % RSNA
Upper Plateau	172.2 ± 16.8	173.5 ± 10.3	201.6 ± 27.5	209.1 ± 40.8
Range	182.7 ± 14.7	185.5 ± 13.4	216.5 ± 29.8	214.8 ± 45.0
Gain	−6.72 ± 1.22	−6.65 ± 0.98	−8.88 ± 2.67	−7.89 ± 1.02
BP50	79.5 ± 3.4	78. ± 3.1	76.1 ± 5.1	73.7 ± 5.5
Threshold (mmHg)	99.3 ± 1.8	97.7 ± 3.3	102.3 ± 12.1	96.8 ± 5.9
Saturation (mmHg)	59.7 ± 6.8	58.3 ± 5.0	50.0 ± 5.4	48.9 ± 7.2
sBP and HR
Upper Plateau (bpm)	167.4 ± 10.7	166 ± 15.3	140.0 ± 3.7	147.9 ± 14.5
Range (bpm)	114.4 ± 8.7	115 ± 12.6	87.0 ± 3.9*	92.1 ± 12.9
Gain (bpm/mmHg)	−4.83 ± 0.54	−5.14 ± 0.86	−3.26 ± 0.36*	−5.22 ± 0.77
BP50 (bpm)	92.8 ± 4.8	93.8 ± 2.8	97.0 ± 5.1	95.8 ± 2.8
Threshold (mmHg)	111.4 ± 5.3	113.7 ± 3.8	118.0 ± 6.5	109.6 ± 4.0
Saturation (mmHg)	74.3 ± 5.0	73.9 ± 4.5	76.0 ± 4.3	82.0 ± 2.8

^*indicates significant effect of HF.

^†indicates significant effect of Losartan.

CSNA – cardiac sympathetic nerve activity, RSNA – renal sympathetic nerve activity, dBP – diastolic blood pressure, sBP – systolic blood pressure, HR – heart rate

Central angiotensin receptor distribution

The distribution of binding sites for ¹²⁵I-[Sar¹, Ile⁸] Ang II was examined in the medulla and the hypothalamus of normal and HF sheep brains. The sites of highest density were in the paraventricular nucleus of the hypothalamus (PVN), the nucleus of the solitary tract (NTS) and the dorsal motor nucleus (DMN) (Figure 4), as has been noted previously in sheep ²⁵. The receptor type was predominantly AT₁R in all regions examined, because co-incubation with losartan decreased the binding intensity to background levels. Comparison of the density of AT₁Rs in regions associated with cardiovascular control showed differential changes in angiotensin receptor binding in the HF group (Figure 4). Of note, there was an increase in the AT₁R levels in the PVN and hypothalamic supraoptic nucleus of HF sheep, and a decrease in AT₁R levels in the area postrema and in the NTS at the level of the area postrema. The AT₁R levels were also examined in the NTS and the DMN at the level of the opening of the central canal and in the OVLT, and there was no significant change in AT₁R levels in these areas.

Figure 4.

Original x-ray images of Ang II binding in the hypothalamus and brainstem of one normal (Panel A, C) and one HF animal (Panel B, D). The binding was totally displaced after co-incubation with the AT₁R antagonist Losartan. The histogram shows the density of AT₁R binding in major central cardiovascular control nuclei in normal (open bars, n = 5) and heart failure animals (filled bars, n = 5). Receptor binding densities, determined by in vitro autoradiography, are normalized to control values. PVN, paraventricular nucleus of the hypothalamus; SON, Supraoptic nucleus; AP, area postrema; NTS, nucleus of the solitary tract. Results are expressed as means ± SE. *P < 0.05 compared with normals using Student’s t-test.

Discussion

The main findings of this study were that in an ovine model of HF central infusion of losartan significantly decreased the elevated levels of CSNA almost back to control levels. In contrast, RSNA was not increased and treatment with losartan had no effect on RSNA. In HF, losartan infusion decreased the range and threshold of the CSNA arterial baroreflex, but had no effect on the baroreflex control of RSNA. In the HF animals, we observed differential alterations in AT₁R binding densities in specific brain regions, including an increase in AT₁R density in the PVN and a decrease in the NTS. In normal animals, losartan had no significant effects on the resting levels or baroreflex control of CSNA, RSNA, or HR.

Effects of losartan on baseline levels of CSNA and RSNA

Pacing-induced heart failure caused a large increase in baseline levels of CSNA, but no change in the baseline levels of RSNA (Figure 3), confirming our previous findings ^{19, 26}. These findings are similar to those in patients with mild HR where cardiac, but not renal, norepinephrine spillover was increased ²⁷, whereas in more severe HF there was a significant increase in renal norepinephrine spillover ^{28, 29}. The finding that central AT-₁R blockade in HF restored baseline levels of CSNA and heart rate towards normal levels indicates a critical role for central angiotensinergic mechanisms in setting the high level of CSNA in HF. A similarly important role for the central RAS in driving the increased RSNA has been observed in HF models in rats, induced by myocardial infarction, and in rabbits, induced by rapid pacing ^{10, 11, 30}. The lack of a fall in RSNA with losartan in sheep with mild HF is probably because at this level of HF in sheep there was no stimulation of RSNA.

Effects of losartan on baroreflex control of CSNA and RSNA

In this model of HF we found that the sensitivity and range of the HR arterial baroreflex were significantly reduced, which is in accord with findings in humans and other animal models of HF ^{31, 32}. Treatment with ICV losartan did not increase the gain of the HR arterial baroreflex, in contrast to rodent models of HF where inhibition of the central RAS improved HR baroreflex sensitivity ^{11, 18}. As we have reported previously ^{19, 24}, the arterial baroreflex control of CNSA was not altered in this model of HF when the relation between CSNA and diastolic pressure was calculated using CSNA levels normalized to the maximum level obtained during severe hypotension. This finding indicates that altered vagal tone together with cardiac β-adrenoceptor down-regulation are probably the major factors leading to the decreased gain of the arterial baroreflex control of HR in this model of HF.

In the present experiments, the CSNA data used for the arterial baroreflex curves was normalized to the values immediately before losartan infusion, to allow direct comparison of the reflex before and after treatment with losartan. Central infusion of losartan caused a reduction in the upper plateau and the range of the CSNA baroreflex curve in HF, but not normal animals (Figure 3). This effect of losartan on CSNA baroreflex sensitivity in HF is in contrast to the findings in rodent models of HF where the decreased RSNA baroreflex sensitivity was restored by ICV infusion of losartan ¹². The reason for the different responses of the cardiac and renal sympathetic nerves to ICV losartan in HF are unclear, but may relate to the degree of heart failure or different roles of central angiotensin in the control of these two sympathetic outflows, as demonstrated by their opposite responses to central infusion of angiotensin, which increased CSNA and decreased RSNA ^{22, 23}.

Changes in central angiotensin receptor levels during heart failure

The ability of centrally administered AT₁R blockers to reduce SNA in HF is likely to be due to their actions to inhibit the effects of both the increase in central angiotensin levels, indicated by the increased CSF levels of angiotensin found in dogs with pacing-induced HF ³³, and the increased density of AT₁R, observed in brain nuclei associated with central sympathetic control in rodent models of HF ^34-36. For example, increased mRNA expression of AT₁R was found in the rostral ventral lateral medulla of rabbits with pacing-induced HF ^{30, 34}, and AT₁R density measured by autoradiography was increased in the subfornical organ, organum vasculosum laminae terminalis, PVN and median preoptic nucleus in rats with HF produced by aortocaval shunt ³⁶.

Our results in sheep with HF indicate decreased AT₁R density in the area postrema and in the NTS at the level of the area postrema (Figure 4). The reasons for the discrepancies regarding changes in the AT₁R levels in the NTS in the sheep and rodent models of HF are not clear. An increase in systemic levels of angiotensin II, as has been observed during HF, can decrease heart rate baroreflex sensitivity ^{16, 37} through actions at the area postrema ³⁸ and the NTS ³⁹. It is possible that the decrease in AT₁R in the area postrema and the NTS may be a secondary response to limit further decreases in baroreflex sensitivity. In addition, our study indicated increased AT₁R density in the PVN and supraoptic nucleus during HF. It is well known that angiotensin II can stimulate vasopressin release through actions at the PVN as well as the supraoptic nucleus ^40-43. Whether the increase in AT₁R in these areas plays an important role in mediating the high levels of vasopressin observed during HF ⁴⁴ remains to be established. In addition to actions on vasopressin release, the increase in AT₁R density in the PVN of HF animals may also play an important role in mediating the increased SNA in HF, but whether it specifically drives the increased CSNA requires further studies examining the responses to PVN microinjections.

We have shown previously that short-term systemic treatment with an angiotensin receptor blocker in the sheep pacing model of HF prevented the reflex increase in CSNA in response to the drug-induced fall in arterial pressure⁴⁵. This intravenous treatment did not, however, reduce the high level of CSNA, probably because, as we demonstrated, it only blocked AT₁R in the periphery, not in the brain. The findings from this and other studies that increased sympathetic outflow to different organs in HF is blocked by central AT₁R blockade suggest that the beneficial effects seen with long-term treatment with this class of drugs in HF patients may result in part from these drugs crossing the blood-brain-barrier and having a central action. For example daily treatment with candesartan decreased CSNA, evaluated by iodine-123 meta-iodobenzylguanidine scintigraphy ⁴⁶. It is important to note, however, that drug induced improvements in hemodynamics are also likely to have acted to reduce CSNA.

Perspectives

Patients with HF die for two main reasons, circulatory insufficiency or sudden death, and since the excessive level of CSNA in HF can induce fibrosis and hypertrophy and promote arrhythmias and sudden death, it is critical to find new mechanisms to decrease CSNA to reduce patient morbidity and mortality. The present findings indicate that central inhibition of AT₁R substantially reduced the elevated CSNA in HF, indicating a primary role for central angiotensin receptors in driving the increased CSNA. These results suggest that oral treatment with high doses of AT₁R blockers that have a greater tendency to cross the blood-brain-barrier may have beneficial actions to reduce the elevated CSNA in HF and the associated detrimental effects.

Non-standard Abbreviations and Acronyms

HF

Heart failure

CSNA

cardiac sympathetic nerve activity

RSNA

renal sympathetic nerve activity

AT₁R

angiotensin receptor type 1

AT₂R

angiotensin receptor type 2

RAS

Renin-angiotensin aldosterone system

Ang II

angiotensin II

ACE

angiotensin converting enzyme