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Am J Physiol Heart Circ Physiol 289: H1985-H1992, 2005. First published June 10, 2005; doi:10.1152/ajpheart.00430.2005
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Impaired cardiac and sympathetic autonomic control in rats differing in acetylcholine receptor sensitivity

James R. Padley,1 David H. Overstreet,2 Paul M. Pilowsky,1 and Ann K. Goodchild1

1Hypertension and Stroke Research Laboratories, Department of Physiology, University of Sydney and Department of Neurosurgery, Royal North Shore Hospital, St. Leonards New South Wales, Australia; and 2Skipper Bowles Center for Alcohol Studies, Department of Psychiatry, University of North Carolina, Chapel Hill, North Carolina

Submitted 29 April 2005 ; accepted in final form 6 June 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Acetylcholine receptors (AChR) are important in premotor and efferent control of autonomic function; however, the extent to which cardiovascular function is affected by genetic variations in AChR sensitivity is unknown. We assessed heart rate variability (HRV) and baroreflex sensitivity (BRS) in rats bred for resistance (FRL) or sensitivity (FSL) to cholinergic agents compared with Sprague-Dawley rats (SD), confirmed by using hypothermic responses evoked by the muscarinic agonist oxotremorine (0.2 mg/kg ip) (n ≥ 9 rats/group). Arterial pressure, ECG, and splanchnic sympathetic (SNA) and phrenic (PNA) nerve activities were acquired under anesthesia (urethane 1.3 g/kg ip). HRV was assessed in time and frequency domains from short-term R-R interval data, and spontaneous heart rate BRS was obtained by using a sequence method at rest and after administration of atropine methylnitrate (mATR, 2 mg/kg iv). Heart rate and SNA baroreflex gains were assessed by using conventional pharmacological methods. FRL and FSL were normotensive but displayed elevated heart rates, reduced HRV and HF power, and spontaneous BRS compared with SD. mATR had no effect on these parameters in FRL or FSL, indicating reduced cardiovagal tone. FSL exhibited reduced PNA frequency, longer baroreflex latency, and reduced baroreflex gain of heart rate and SNA compared with FRL and SD, indicating in FSL dual impairment of cardiac and circulatory baroreflexes. These findings show that AChR resistance results in reduced cardiac muscarinic receptor function leading to cardiovagal insufficiency. In contrast, AChR sensitivity results in autonomic and respiratory abnormalities arising from alterations in central muscarinic and or other neurotransmitter receptors.

heart rate variability; baroreflex sensitivity; Flinders Sensitive line; Flinders Resistant line

BAROREFLEX AND RESPIRATORY modulation of heart rate (HR) and arterial pressure are important homeostatic mechanisms and can be severely impaired in cardiac and noncardiac disease (15, 16, 19, 23–25). In the human population, interindividual differences in responses to drugs that influence cardiac autonomic function have been shown to have an inherited basis (18). These differences are likely to be polygenic and may significantly influence susceptibility to cardiac disease (18). Loss of function of ganglionic (13) or smooth muscle (11) acetylcholine receptors (AChR) results in profound impairment of cardiac autonomic function. However, the importance of smaller genetic variations in AChR function is not known.

AChR exist as ligand-gated ion channels (nicotinic, nAChR) or G protein-coupled receptors (muscarinic, mAChR) and are expressed as subtypes differing in subunit composition or G protein signaling, respectively (for review see Ref. 18). AChR subtypes are widely distributed throughout the autonomic nervous system and contribute differentially to the control of sympathetic and vagal outflow. With respect to cardiac autonomic control, nicotinic {alpha}-7 subunits mediate fast synaptic transmission in ganglia supplying the heart (13), and muscarinic M2 receptors in the sinoatrial node are the target of vagal stimulation (11). In the central nervous system nAChR modulate cardiovagal premotor activity via baroreflex- and respiratory-related inputs to the nucleus ambiguus (30, 31, 51). Cholinergic mechanisms (both nicotinic and muscarinic) play a significant role in control of sympathetic vasomotor tone mediated at both central and peripheral sites (2, 14, 20–22, 42).

Flinders Sensitive (FSL) and Resistant lines (FRL) were bred for differential sensitivity to the hypothermic effects of cholinesterase inhibition from Sprague-Dawley rats (SD) (33). FSL are more sensitive to the behavioral and physiological effects of muscarinic agonists, whereas responses are attenuated in FRL compared with SD (6, 33–37). FRL and FSL also exhibit differential binding of mAChR ligands in the striatum and hippocampus with some evidence for subtype-specific alterations (6, 33). FRL are otherwise genetically similar to SD (34); thus either the resistant line or outbred SD has been considered an appropriate control for FSL. Muscarinic sensitivity develops early postnatally in FSL (6) and is likely to be polygenic (34). This results in increased sensitivity to nicotine (48, 49), dopaminergic agonists (35, 53), and 5-HT1A receptor agonists (36, 50), occurring via functional interactions with mAChR or from changes in G protein signaling common to specific neurotransmitter receptors (36). At present there are no data demonstrating functional or genetic differences in AChR in cardiovascular brain regions, ganglia, or target organs in FRL and FSL.

From these inherited differences in AChR sensitivity, we hypothesized that vagal and sympathetic control of cardiovascular function would be altered in FRL and FSL compared with SD. To test this hypothesis, we used analysis of HR, HR variability (HRV), and baroreflex sensitivity (BRS) in response to endogenous or induced changes in blood pressure under anesthesia. Analysis of HRV and BRS is used extensively to examine cardiovascular autonomic function in normal subjects and in patients with cardiac and noncardiac disease (7, 15, 16, 23–26).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All animals were bred and housed in a 12-h:12-h light-dark environment with access to food and water ad libitum. Male SD (n = 10), FRL (n = 11), and FSL (n = 9) aged 11–13 wk and weighing 300–450 g were used in the study. All experiments were conducted in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Animal Care and Ethics Committee of Royal North Shore Hospital, University of Technology, Sydney, Australia. After the experimentation, animals were humanely euthanized with an intravenous injection of KCl (1 M) while under deep surgical anesthesia.

Measurement of Sensitivity to Oxotremorine-Induced Hypothermia

Rectal temperature was recorded under brief halothane anesthesia (~1% in O2, Fluothane, Zeneca), and temperatures at baseline were comparable between groups (~37.3°C). Animals were administered sequential intraperitoneal injections of atropine methylnitrate (mATR, 2 mg/kg) and oxotremorine (OXO, 0.2 mg/kg) (6) and allowed to recover, and rectal temperature was recorded 30 min later.

Electrophysiological Recordings

Anesthesia. After 5 days of recovery, animals were briefly anesthetized with halothane (3.5% in O2), and deep surgical anesthesia was achieved with ethyl carbamate (urethane, 1.2–1.5 g/kg ip, Sigma) diluted to 10% (wt/vol) to minimize peritoneal irritation. Depth of anesthesia was assessed regularly using reflex responses to tactile (corneal stroking) and noxious (hind paw pinch) stimuli, and maintenance doses of urethane (0.2 ml iv) were administered as required. Rectal temperature was maintained between 37.0°C and 37.5°C using a thermoregulated heating pad (Harvard Apparatus) and an infrared lamp.

Surgical procedures and electrophysiological recording. The right carotid or femoral artery was cannulated and connected to a pressure transducer (Abbott Critical Care Systems) for measurement of arterial pressure [1401 Plus and Spike 2 version 5.0, Cambridge Electronic Design (CED)]. HR and R-R interval time series were derived from a three-lead ECG attached subcutaneously, sampled at 1,000 Hz, band-pass filtered (0.1–1 kHz), and recorded digitally with Spike 2. The right jugular or femoral vein was cannulated for administration of drugs. HR baroreflexes were examined only in animals that received sodium nitroprusside and phenylephrine via the femoral vein (n = 3 rats/group) to avoid interference with baroreceptive regions or cardiac volume loading. The left phrenic and splanchnic nerves were dissected as described previously (28, 38) and cut distally to eliminate afferent transmission. Nerves were attached to bipolar silver recording electrodes connected to a differential preamplifier (CWE), band-pass filtered (0.05–3 kHz), and amplified (50 000x). All signals were sampled at 1,000 Hz by using an analog-to-digital recorder (1401 Plus, CED) and acquired as waveforms using Spike 2. Animals were placed in a stereotaxic frame and allowed to breathe O2-supplemented room air.

Experimental Protocol

Cardiorespiratory parameters were acquired at rest for 30–60 min, and then baroreceptor reflexes were tested by ramp changes in mean arterial pressure (MAP) induced by sequential bolus administration of sodium nitroprusside (SNP) and phenylephrine (PE) (10 µg/kg iv) as described previously (40). After a period of recovery (~10 min), a subset of rats (n ≥ 5 rats/group) was administered mATR (2 mg/kg iv), and cardiorespiratory parameters were monitored for an additional 10 min.

Data Analysis

All data were analyzed off-line using Spike 2 (version 5.0) and GraphPad software. For resting cardiorespiratory parameters, analysis was performed on three 5-min segments of data per animal between 3 and 4 h after anesthesia induction. All segments of data for HRV analysis were visually inspected for stationarity and lack of artifacts or ectopic beats.

HR Variability

Time domain analysis. R-R interval time series were constructed in Spike 2 using an algorithm to detect R-wave peaks and the standard deviation of R-R intervals (SDNN) calculated per 5-min segment (Fig. 1A). SDNN provides a reliable estimate of short-term HRV (47). For three separate 5-min segments per animal SDNN was recorded and the mean calculated. After administration of mATR and stabilization of HR (typically 2 min postinjection), SDNN was calculated from a single 5-min epoch.



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  		Fig. 1. Time and frequency domain analysis of heart rate variability and the relationship with mean heart rate in Sprague-Dawley (SD) rats, Flinders Resistant rats (FRL), and Flinders Sensitive rats (FSL) under urethane anesthesia. Representative R-R interval time series (A) and power spectra (B) show that in FRL and FSL fluctuations of R-R interval around the mean were markedly reduced as was total power within the frequency range 0–2.5 Hz, low-frequency power (LF, 0.2–1 Hz), and high-frequency power (HF, 1–3 Hz) compared with SD. C: linear regression (dashed lines) of data from SD (filled squares) indicates that heart rate was inversely correlated with standard deviation of R-R intervals (SDNN) (i) and HF power (ii). In contrast, SDNN (i) and HF power (ii) were consistently clustered at high mean heart rate in FRL (filled circles) and FSL (open circles).

 
Frequency domain analysis. Discrete R-R interval series were interpolated using a weighted sinusoid function and uniformly resampled at 100 Hz (Fig. 1C). Power spectra were calculated by using a fast Fourier transform (FFT)-based nonparametric algorithm (FFT size 256, resolution 0.02 Hz) and viewed in a Hanning window, allowing spectral estimation with frequency measured in hertz and power measured in squared milliseconds (Spike 2). Overall power was divided into three frequency ranges: very low frequency (VLF, ≤0.2 Hz), low frequency (LF, 0.2–1.0 Hz), and high frequency (HF, 1.0–3.0 Hz) and recorded as absolute values or in normalized units as a proportion of combined LF and HF power (excluding VLF component). For each animal, VLF, LF, and HF power from the three separate 5-min spectra was averaged.

Spontaneous BRS. A method for calculation of spontaneous HR BRS using R-R interval time series and the arterial pressure trace was derived for use in rats from the sequence method described in clinical literature (25, 39). The sequence method calculates the average gain, or slope, of HR in response to spontaneous fluctuations in MAP (i.e., when R-R interval and MAP change in the same direction). The slope of each interval sequence was calculated by using an algorithm to detect only sequences of three consecutively lengthening or shortening R-R intervals corresponding to an increase or decrease in MAP of at least 0.5 mmHg, respectively (Spike 2) (25). Spontaneous BRS was calculated as the average slope of R-R interval sequences for all sequences detected during 5-min segments of data (Fig. 2). Separate values of mean BRS were recorded for lengthening (bradycardia) and shortening (tachycardia) interval sequences and only when at least three sequences per 5-min segment were present. The sequence method was applied to both stationary and nonstationary 5-min segments before (at least three per animal) and after (one per animal) administration of mATR.



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  		Fig. 2. Spontaneous baroreflex sensitivity (BRS) measured using the sequence method and its relationship with mean heart rate in SD, FRL, and FSL under anesthesia (n ≥ 8 rats/group). A: linear regression (dashed lines) of data from SD (filled squares) shows that heart rate was inversely correlated with the slope of lengthening and shortening sequences. The slope of lengthening and shortening sequences was reduced in FRL (filled circles) and FSL (open circles), and despite comparably high heart rates, values for baroreflex sensitivity were reduced in FSL compared with FRL. B: slope of lengthening sequences detected using the sequence method was significantly reduced (*P < 0.05) after treatment with methylatropine (2 mg/kg iv) in SD (filled squares), whereas it had little effect on the low values for slope obtained in FSL (open circles). After methylatropine administration, the slope of lengthening sequences was reduced in FSL compared with FRL (filled circles) and SD, whereas slope was similar in FRL and SD.

 
BRS Derived From Induced Changes in Arterial Pressure

Ramp baroreflex method. Baroreflex gain was estimated using ramp changes in arterial pressure induced by intravenous administration of SNP and PE (10 µg/kg iv) (Fig. 3). The absolute change in arterial pressure evoked by SNP and PE was not different among FRL, FSL, and SD (see RESULTS). However, peak HR responses took significantly longer to occur in FSL compared with FRL and SD (see RESULTS); thus a linear rather than a logistic approach was used to estimate HR baroreflex gain.



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  		Fig. 3. A: changes in heart rate and splanchnic nerve activity (SNA) evoked by sequential intravenous administration of sodium nitroprusside (SNP) and phenylephrine (PE) are illustrated in a representative trace from an SD rat. B: splanchnic logistic function curves in SD (solid line, filled square), FRL (dashed line, filled circle), and FSL (dotted line, open circle) (n ≥ 6 rats/group) were in general similar, although gain was selectively reduced in FSL. C: heart rate BRS was reduced in FSL compared with FRL (P < 0.05) and SD (P < 0.01) using the slope of lengthening (sequence L) or shortening intervals (sequence S) or gain of R-R interval responses to ramp increases (linear PE) or decreases (linear SNP) in arterial pressure (P < 0.05 vs. SD). Resting [i.e., at mean arterial pressure (MAP)] and maximal splanchnic nerve baroreflex gain was also selectively reduced in FSL compared with FRL and SD (P < 0.05) (C).

 
HR baroreflex gain. R-R interval and MAP were averaged with a time constant of 0.2 s, and linear regression analysis was applied to R-R interval and MAP data over a defined change in MAP (40 mmHg). Only animals in which drugs were administered through the femoral vein (n = 3 rats/group) were included to avoid interference with baroreceptorial regions or cardiac volume loading after intrajugular drug administration. Separate values for BRS with SNP and BRS with PE were recorded for each animal.

Splanchnic sympathetic baroreflex gain. Arterial pressure and splanchnic nerve activity (SNA) were averaged with a time constant of 1 s, and SNA was normalized by setting maximum SNA inhibition following PE as 0% and resting SNA as 100%. Normalized values (nu) of SNA versus MAP were fitted to a four-parameter sigmoid logistic function (Eq. 1) by nonlinear regression (GraphPad) over the duration of the baroreflex stimulus (43). Goodness of fit was shown by r2 values ≥0.95.

(1)
where y is SNA, x is MAP, P1 is the range of SNA response, P2 is the slope coefficient, P3 is MAP at 50% SNA range (MAP50), and P4 is the lower plateau. The first derivative of the logistic function was used to calculate maximal gain (Gmax; gain at MAP50) and gain at resting MAP (GMAP).

Statistical Analysis

All data are presented as individual data points or means ± SE. Data were analyzed using one-way analysis of variance (ANOVA) and using a post hoc test for significance performed with Bonferroni's correction. A Student’s t-test was used to compare individual means or effects of mATR.

Drugs

OXO and mATR (Sigma Aldrich) were stored at 4°C or room temperature, respectively. All drugs were dissolved in saline (0.9% NaCl, pH 7.4) on each experimental day and stored at 4°C until use. All agents were administered in a volume of 0.4 ml/kg at the required concentration, the exception being PE at 0.8 ml/kg.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Sensitivity to OXO-Induced Hypothermia

Administration of OXO (0.2 mg/kg ip) following mATR (2 mg/kg ip) produced a fall in rectal temperature in FSL (–2.5 ± 0.1°C, n = 9) that was significantly greater (P < 0.001) and nonoverlapping compared with FRL (–0.7 ± 0.1°C, n = 11) and SD (–0.6 ± 0.2°C, n = 10). Changes in rectal temperature in FRL compared with SD were not significantly different.

Resting Cardiovascular Parameters

MAP was similar in SD, FRL, and FSL (Table 1). In contrast, resting HR was significantly elevated (P < 0.01) in FRL and FSL compared with SD (Table 1). Mean HR in SD exhibited significantly greater variance (P < 0.001) compared with FRL and FSL (Table 1, Fig. 1C).


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  		Table 1. Resting cardiorespiratory parameters in SD, FRL and FSL at rest under urethane anesthesia

 
In the presence of mATR (2 mg/kg) there was no significant change in MAP, HR, or respiratory frequency in FRL and FSL. mATR had a significant effect (P < 0.01) on HR in SD producing a sustained tachycardia ({Delta}95 ± 12 beats/min). In the presence of mATR, MAP and HR were similar among SD, FRL, and FSL (Table 1).

Resting Respiratory Parameters

The frequency of phrenic nerve discharge was significantly reduced in FSL compared with FRL (P < 0.01) and was not different between FRL and SD (Table 1). In the presence of mATR, respiratory frequency was unchanged in all groups and remained significantly lower in FSL compared with FRL (P < 0.05) and SD (P < 0.01) (Table 1).

HR Variability

Time domain analysis. HRV in the time domain (SDNN) was markedly reduced in FRL and FSL compared with SD (P < 0.01) (Table 2, Fig. 1A). SDNN in SD showed greater variance (P < 0.001) compared with that in FRL and FSL. In the presence of mATR (2 mg/kg), SDNN was unchanged in FSL and FRL but significantly reduced (P < 0.01) in SD. In the presence of mATR SDNN was similar in all three groups (Table 2).


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  		Table 2. Time and frequency domain analysis of heart rate variability in SD, FSL, and FRL under urethane anesthesia

 
Frequency domain analysis. Spectral analysis of HRV showed distinct VLF, LF, and HF components in SD with central frequencies around 0.1, 0.6, and 2 Hz, respectively (Fig. 1B). HF was synchronous with mean phrenic nerve frequency during the 5-min segment, consistent with work showing coupling of the HF band and respiratory sinus arrhythmia (41). HRV spectra from FRL and FSL showed a reduction in spectral power over all frequencies (Fig. 1B). Total spectral power, LF, and HF power were significantly reduced (P < 0.01) in FSL and FRL compared with SD (Table 2). Normalized LF power was also reduced, whereas normalized HF power was increased in FSL and FRL compared with SD (Table 2). HF predominance was also evident as a reduction in LF-to-HF ratio (LF/HF) in FSL and FRL compared with SD (Table 2).

HR dependency of HRV parameters. In SD there was a significant inverse correlation between mean HR and SDNN (P < 0.01, Fig. 1C, i), HF power (P < 0.01, Fig. 1C, ii), and LF power (P < 0.01, data not shown) and a nonsignificant positive correlation between mean HR and LF/HF (data not shown). HRV parameters were clustered at high mean HR in FRL and FSL, and there was no correlation with HR in these animals (Fig. 1C, i, C, ii). In all three groups, high mean HR was associated with low SDNN and reduced LF and HF power.

Spontaneous HR BRS

Spontaneous BRS for lengthening and shortening interval sequences combined was significantly reduced (P < 0.01) in FRL (0.41 ± 0.04 ms/mmHg) and FSL (0.17 ± 0.02 ms/mmHg) compared with SD (1.52 ± 0.19 ms/mmHg). In SD mean HR was inversely correlated with spontaneous BRS for both lengthening and shortening interval sequences (P < 0.01, Fig. 2A). Spontaneous BRS was clustered at high mean HR in FRL and FSL; however, BRS was significantly reduced (P < 0.01) in FSL compared with FRL (Fig. 2A).

Administration of mATR significantly reduced (P < 0.05) spontaneous BRS in SD but not in FRL or FSL (Fig. 2B). In the presence of mATR, spontaneous BRS of lengthening interval sequences was compared between groups and found to be significantly reduced (P < 0.05) in FSL (0.16 ± 0.05 ms/mmHg) compared with FRL (0.62 ± 0.19 ms/mmHg) and SD (41 ± 0.10 ms/mmHg) (Fig. 2B).

BRS Derived From Ramp Changes in Arterial Pressure

Ramp decreases and increases in MAP and corresponding reflex changes in HR and splanchnic nerve activity produced by intravenous injection of SNP or PE (10 µg/kg) are illustrated in Fig. 3A. SNP evoked very similar reductions in MAP in all groups (SD, 43 ± 3 mmHg; FRL, 38 ± 2 mmHg; FSL, 41 ± 2 mmHg; n ≥ 8 rats/group). Similarly, increases in MAP evoked by PE were not significantly different between groups (SD, 50 ± 3 mmHg; FRL, 46 ± 2 mmHg; FSL, PE, 54 ± 3 mmHg; n ≥ 8 rats/group).

HR baroreflex latency. The time delay to peak change in HR following peak change in MAP evoked by SNP or PE was significantly increased in FSL (SNP; 24 ± 4 s, PE; 2.6 ± 0.3 s) compared with FRL (SNP; 11 ± 2 s, PE; 1.0 ± 0.4 s) and SD (SNP; 5 ± 1 s, PE; 0.7 ± 0.2 s) (P < 0.05).

HR BRS. HR baroreflex gain, assessed as the slope of R-R interval and MAP following SNP or PE, was reduced in FSL (SNP 0.14 ± 0.03; PE; 0.42 ± 0.10 ms/mmHg) compared with FRL (SNP 0.36 ± 0.09; PE 0.69 ± 0.08 ms/mmHg) and SD (SNP 0.49 ± 0.10; PE 0.80 ± 0.17 ms/mmHg); however, reduced gain in FSL only reached significance compared with SD (P < 0.05, n = 3 rats/group) (Fig. 3C).

Splanchnic sympathetic BRS. The range and midpoint of average baroreflex function curves for splanchnic nerve activity were not significantly different among SD, FRL, and FSL (Fig. 3B, n ≥ 6 rats/group). Resting MAP was close to threshold (MAP50) in all groups. However, GMAP was reduced in FSL (2.27 ± 0.15% nu/mmHg) compared with FRL (3.21 ± 0.51% nu/mmHg) and was significantly reduced (P < 0.05) compared with SD (3.98 ± 0.36% nu/mmHg). Gmax was significantly reduced (P < 0.05) in FSL (2.54 ± 0.10% nu/mmHg) compared with both SD (4.59 ± 0.45% nu/mmHg) and FRL (4.04 ± 0.45% nu/mmHg) (Fig. 3C).

Comparison of BRS Measurements

FRL and FSL showed a reduction in spontaneous BRS compared with SD (Figs. 2A and 3C); however, spontaneous BRS was significantly reduced in FSL compared with FRL also (Figs. 2A and 3C). Considering the rate dependency of spontaneous BRS (Fig. 2A), higher mean HR was not a factor skewing BRS to lower values in FSL versus FRL. HR baroreflex gain following ramp changes in pressure was significantly reduced in FSL compared with SD (Fig. 3C), and splanchnic nerve baroreflex gain obtained using similar methods was significantly reduced in FSL compared with both FRL and SD (Fig. 3C). There was general agreement in values of HR BRS obtained using lengthening or shortening interval sequences or using pharmacological methods. A notable exception was that in three SD, spontaneous BRS was greater than BRS obtained using ramp changes in MAP (Fig. 3C).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We show that in anesthetized FRL and FSL, normal blood pressure is maintained but cardiovagal tone and spontaneous baroreflex control of HR are diminished. Additionally, in FSL, baroreflex control of sympathetic activity and respiratory frequency is reduced compared with FRL and SD. These findings indicate that selective breeding for reduced or increased AChR sensitivity results in specific cardiorespiratory deficits. FRL have reduced muscarinic sensitivity and may exhibit a reduction in functional cardiac mAChR required for vagal modulation of sinus rhythm (11, 13). In contrast, FSL show a number of central changes in ACh (nicotinic and muscarinic) and aminergic receptors (34–37, 53) that accompany increased muscarinic sensitivity, and these may be linked to the vagal, baroreflex, and respiratory abnormalities described.

In the present work, we characterized HRV and BRS (both spontaneous and pharmacological gains) in SD compared with FRL and FSL under anesthesia. These methods indicate predominantly reflex vagal and sympathetic modulation of sinus rhythm, and where applicable, arterial pressure, but do not directly reflect levels of autonomic tone. Reduced HRV and BRS are powerful predictive markers of prognosis and cardiac mortality in clinical settings (23, 24) and have been interpreted as indicators of sympathovagal balance as it pertains to cardiac autonomic modulation (10, 26).

Cardiac vagal activity was reduced in FRL and FSL compared with SD, indicated by reduced HRV in the time domain and reduced HF power. HF oscillations in HR originate from vagal modulation because they are synchronous with vagally mediated respiratory sinus arrhythmia and are abolished after atropine (12, 41). Cardiac vagal tone was markedly diminished in both lines, indicated by elevated resting HR unresponsive to mATR. Under these conditions, it is likely that sympathetic modulation of HR predominates and risk of ventricular arrhythmia is increased (10, 26).

The reduction in vagal activity was the clearest difference between FRL and their parent strain SD. Vagal efferent modulation of HR is mediated entirely by muscarinic M2 receptors in the sinoatrial node (11). It is likely that a reduction in functional cardiac M2 receptors accounts for reduced vagal activity in FRL compared with SD, particularly since they are otherwise genetically and phenotypically similar (34, 35, 53). Furthermore, apart from their resistance to mAChR agonists, multiple chemical sensitivities are unaltered in FRL (34, 35, 53). However, opposite effects on vagal activity were not seen in FSL, suggesting they do not show increased cardiac muscarinic sensitivity. Peripheral muscarinic sensitivity is evident in FSL in some tissues; for example, methacholine-induced intestinal and bronchial constriction were enhanced in FSL compared with FRL (8, 9). Because M3 receptors predominantly mediate the contractile response in these tissues (27), FSL may show enhanced M3 but not M2 receptor-mediated effects in the periphery. Subtype-selective differences in muscarinic sensitivity in FSL have also been observed in the striatum, where M1 but not M2 receptor binding was found to be elevated (6).

The diminution of vagal activity seen in FSL may result from changes in central pathways regulating vagal premotor output. This could also potentially mask any change in muscarinic sensitivity at the level of the heart. Furthermore, it is possible that muscarinic receptors are not involved as FSL exhibit increased nicotinic (48, 49) and 5-HT1A (36, 50) receptor sensitivities that could contribute to vagal inhibition. For example, nicotinic {alpha}4{beta}2-subunits (31, 51) and 5-HT1A receptors (45) strongly modulate the tonic and reflex inhibition of cardiovagal premotor neurons in the nucleus ambiguus. Nicotinic {alpha}4{beta}2-subunit binding is increased in the brains of FSL (48), and chronic nicotine exposure fails to evoke receptor desensitization in FSL compared with FRL (49). However, the precise mechanisms underlying reduced vagal activity in FSL remain to be elucidated.

FSL exhibited a significantly slower rate of phrenic nerve discharge, indicating a reduction in central respiratory drive compared with FRL and SD. Although blood gas analysis was not performed, phrenic activity exhibited a typical augmenting pattern in all cases indicating normocapnic conditions (28). Muscarinic M1 and M3 receptors are important in the neural control of breathing where they strongly modulate activity of ventral medullary cell groups controlling respiratory rhythmogenesis (29, 44). This may indicate abnormal rhythmogenic control of breathing by mAChR in FSL. Respiratory patterns also influence cardiovagal activity; however, whether or not reduced respiratory frequency in FSL contributes to diminished cardiac vagal nerve traffic is unclear (10, 41).

Normal blood pressure appeared to be maintained in FRL and FSL compared with SD under anesthesia, indicating an absence of cholinergic tone in blood pressure regulation. Descending cholinergic input to the ventrolateral medulla may influence long-term levels of blood pressure because local mAChR activation evokes sustained increased in sympathetic vasomotor activity (14). This pathway does not appear to be tonically activity under normotensive conditions (2, 14, 52), but it is strongly activated in animals with spontaneous (20) or deoxycorticosterone acetate-salt hypertension (21). Our findings indicate that increased AChR sensitivity alone is not sufficient to produce hypertension in FSL, at least at the ages tested. However, because sympathetic tone was not directly assessed, it is possible that compensatory mechanisms (renal or neurohumoral) act to maintain normal blood pressure.

Baroreflex control of HR and arterial pressure were markedly and differentially affected in FRL and FSL. Diminished cardiovagal activity in both lines is likely to account for the observed reduction in spontaneous (HR) BRS compared with SD, because in the latter group BRS was sensitive to mATR and was correlated with mean HR. After ramp changes in arterial pressure, FSL alone showed reduced HR baroreflex gain and longer latency to peak changes in HR. Sympathetic and vagal activities contribute relatively equally to HR gain under these latter conditions (17), suggesting that FSL show a disturbed sympathetic component. This was also apparent as a selective reduction in the gain of splanchnic SNA following baroreflex activation in FSL compared with FRL and SD. Furthermore, SNP and PE elicited comparable blood pressure changes in SD, FRL, and FSL, indicating no differences in vascular {alpha}-adrenoreceptor sensitivity. Because responses to peripheral {beta}-adrenergic agonists are also similar in FRL and FSL (53), the sympathetic effects seen in FSL appear to be generated centrally. Reduced sympathetic baroreflex gain may result from changes in AChR (3, 22), 5-HT1A (45), or other aminergic receptors (35, 53) involved in the central integration and modulation of the baroreflex. For example, baroreflex activation evokes release of ACh in the rat rostral ventrolateral medulla (22). Muscarinic M2 and 5-HT1A receptors also strongly modulate activity of presympathetic neurons in this region (14, 28).

Abnormal modulation of sympathetic outflow also underlies exaggerated hypothermic responses to muscarinic and 5-HT1A receptor agonists in FSL (4, 46). The interaction between muscarinic and 5-HT1A receptors may therefore play a key role in abnormal sympathetic control of cardiovascular and thermoregulatory targets in FSL. M2 and 5-HT1A receptors influence a common pool of Gi proteins leading to inhibition of adenylyl cyclase (32), suggesting that phenotypic abnormalities may result from alteration of G proteins or second messengers in FSL (36). Future studies are required to test this hypothesis.

Anesthesia is an important consideration in this study, first, because urethane anesthesia affects cardiorespiratory function in general and respiratory sinus arrhythmia in particular (5). Furthermore, FRL and FSL may be more or less sensitive to the cardiovascular effects of anesthesia. Confirmation of the present observations will require similar studies in conscious animals. An advantage of the use of anesthesia here is to enable comparison of autonomic regulation in the absence of locomotor activity, higher order inputs, and stress reactivity that are known to differ between FRL and FSL (1, 37, 53).

In conclusion, the present study shows that autonomic control of the heart and circulation is impaired in rats differing in AChR sensitivity. FRL may exhibit a functional reduction in cardiac mAChR resulting in reduced vagal control of HR. Given that FRL are otherwise genetically and phenotypically similar to their parent strain SD, FRL may provide a novel model of innate vagal insufficiency. Furthermore, this indicates that care should be taken in future studies using FRL as a control for FSL. In contrast, FSL show a number of central changes in ACh (nicotinic and muscarinic) and aminergic receptors that may be linked to concomitant alteration of vagal, sympathetic baroreflex, and respiratory functions observed here. FRL and FSL may have an increased susceptibility to arrhythmia and myocardial damage and may provide a model for investigating an inherited basis of increased cardiac risk. FRL and FSL may be important also for evaluating the cardioprotective effects of agents that differentially influence cardiac vagal and sympathetic reflex function.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Work in the authors' laboratory is supported by grants from the National Health and Medical Research Council of Australia (211023, 211196) Northern Sydney Area Health Service 2005:27 and Garnett Passe and Rodney Williams Memorial Foundation. J. R. Padley is supported by an Australian Postgraduate Award and a Northern Sydney Health Centenary Foundation scholarship. A. K. Goodchild is a Fellow of the High Blood Pressure Research Foundation of Australia.


   ACKNOWLEDGMENTS
 
The authors thank the staff at Gore Hill Research Laboratories for careful breeding of the Flinders lines, Dr. Richard Piper for critical review of the paper, Graham Nisbet for technical recommendations, and David Crick and Greg Smith for writing the first version of the script for sequence analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. K. Goodchild, Hypertension and Stroke Research Laboratories, Ground Floor Block 3, Dept. of Neurosurgery, Royal North Shore Hospital, St. Leonards, NSW Australia 2065 (e-mail: anng{at}physiol.usyd.edu.au)

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
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 DISCUSSION
 GRANTS
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