INTRODUCTION

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A PREVIOUS MYOCARDIAL infarction (MI) is one of the most common causes of left ventricular dysfunction in humans. Approximately 50% of all deaths after MI have been classified as sudden deaths, the majority of which are caused by ventricular tachyarrhythmias (16). Evidence has been provided that both the baroreflex control of heart rate ("baroreflex sensitivity"; BRS) (3, 27) and the variability of the R-R interval ("heart rate variability"; HRV) during sinus rhythm (16, 20) may be impaired after MI and may identify subgroups of patients with a high susceptibility to malignant ventricular arrhythmias (13, 19). However, available data concerning both evidence and time course of reduction of both BRS and HRV are varying between studies. This may be due to inhomogeneous study populations (e.g., in respect to infarct localization or degree of left ventricular dysfunction) and/or differences in pharmacological and invasive therapy. Therefore, it is desirable to further characterize a small animal model. Experimentally induced chronic ligation of a coronary artery in rats has been shown to provide a MI model that has the advantage of highly reproducible infarct size and localization (24) and thus allows the study of a homogeneous group of experimental animals with chronic left ventricular dysfunction. To date, both BRS and HRV have been studied only at a single time point after MI in rats with pronounced left ventricular dysfunction. A reduced reflex tachycardia but preserved overall BRS (10) and an impaired HRV (30) were observed after MI. It remains unclear whether alterations of BRS and HRV are present only temporarily and whether they are dependent on post-MI hemodynamic changes. It was the aim of the present study to investigate the time course of possible alterations of both reflex and tonic control of heart rate (HR) in rats with MI and only moderately impaired left ventricular function.

Both BRS (7, 12) and HRV (2, 4) are considered as measures of autonomic nervous system activity. Because it is difficult to assess sympathetic and especially vagal tone from direct neural recordings in conscious rats, it was of interest whether the methods used to assess BRS and HRV in this study are suitable to estimate both sympathetic and vagal tone.

	METHODS

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Coronary artery ligation. All experiments of this investigation were approved by the federal authority and conform to the "Guide for the Care and Use of Laboratory Animals" [Department of Health and Human Resources Publication No. (NIH) 85-23, Revised 1985]. A transmural anterior MI was produced in modification of a method previously described (24). Adult male Sprague-Dawley rats (Charles River Wiga, Kisslegg, Germany; 230-275 g) were anesthetized with chloral hydrate (400 mg/kg ip, Riedel de Haen, Seelze, Germany). An oral endotracheal tube was inserted, and mechanical ventilation with room air was instituted. A left-sided thoracotomy was performed, and the proximal left anterior descending coronary artery (LAD) was ligated in situ. Each MI was confirmed by three criteria: 1) inspection immediately after LAD ligation in situ (paleness of left anterior ventricular myocardium); 2) inspection of the heart in situ after thoracotomy at the end of the interventions (groups studied 3 days after ligation, paleness; 28 and 56 days, signs of left ventricular fibrosis); and 3) macroscopic signs of chronic MI at autopsy. All rats with ligation fulfilled these criteria and therefore made up the MI groups. Rats appointed to the sham-operated (SH) groups were subjected to the same procedure except the LAD was not ligated. We did not measure infarct size in the present study. However, according to previous observations in our laboratory, and as described by others (11, 24), an increase of left ventricular end-diastolic pressure (LVEDP) to values of 10-15 mmHg reflects an infarct size of ~30-45%. The perioperative mortality was 45% in MI rats and <1% in SH rats.

Experimental protocol. Different groups of rats were studied 3, 28, and 56 days after SH and MI. On the day before measurements, each rat was anesthetized with chloral hydrate (400 mg/kg ip) to allow for electrocardiogram (ECG) and catheterization of the left femoral artery and vein with heparinized polyethylene tubes (0.58- and 0.4-mm ID, respectively; overall length 50 mm). The free ends of the catheters were subcutaneously led to the back of the neck, exteriorized, and protected using a jacket with a steel tether (Harvard Apparatus, Kent, UK). On the following day (i.e., 3, 28, and 56 days after SH and MI), all experiments were carried out in conscious rats under standardized conditions to avoid possible influences of circadian variation. Via the arterial catheter, 1.5-ml blood samples were withdrawn and replaced by an equal volume of heparinized donor rat blood. The arterial catheter was then connected to a pressure transducer (P231 ID, Gould, Cleveland, OH). The pressure signal was amplified by a manometer (Hugo Sachs, March-Hug-stetten, Germany) and recorded on a two-channel ink writer (Brush 220, Gould). Mean arterial pressure (MAP) was calculated as diastolic pressure plus one-third of the difference between diastolic and systolic pressures. Both R-R interval and HR were derived by beat-to-beat analysis of the peak systolic pressure signal with a HR coupler (Biotachometer; Hugo Sachs).

Pharmacological interventions. The influence of -adrenoceptor inhibition on basal HR was studied 3, 28, and 56 days after SH and MI. A bolus intravenous injection of 0.5 mg/kg metoprolol (D,L-metoprolol; Sigma, Munich, Germany; dissolved in 1 ml/kg saline) was administered (followed by 200-µl saline flush) to conscious rats. The effect of muscarinic receptor inhibition with 0.5 mg/kg atropine (atropine methylbromide; Sigma) on basal HR was studied in the same manner in a separate series of rats. The doses of metoprolol and atropine chosen evoked immediate changes of basal HR that remained constant over at least 90 min, but did not affect basal MAP.

Determination of baroreflex sensitivity. To study BRS, intravenous bolus injections of the vasodilator nitroprusside (sodium nitroprusside; Sigma) in four increasing doses (0.2-2.0 µg/kg; each in 100 µl of 0.9% saline followed by 200 µl of 0.9% saline to flush the catheter) were administered with constant monitoring of MAP and R-R interval. Thereafter, four increasing doses (2.0-20.0 µg/kg) of the ₁-adrenoceptor agonist methoxamine (methoxamine hydrochloride; Sigma) were injected in the same manner. Subsequent injections of nitroprusside or methoxamine were only performed when MAP and R-R interval had returned to baseline. The doses of nitroprusside and methoxamine were chosen according to the results of previous experiments (unpublished data) so that the linear range of the relationship between MAP and R-R interval (12, 29) was covered, i.e., changes of basal MAP up to ±25 mmHg. After examination of BRS, each rat was anesthetized with chloral hydrate (100 mg/kg iv) with subsequent tracheotomy and cannulation of the trachea for mechanical ventilation. A transverse thoracotomy was then instituted, and the heart was visually inspected. The left ventricle was directly cannulated at the apex (1.1-mm ID; Venofix S, Braun, Melsungen, Germany) for measurement of LVEDP. The pressure signal was recorded as described above. The influence of metoprolol and atropine on BRS was additionally studied, as described above.

Measurements of HRV. In the studies of HRV, two electrodes for chronic recording of apex-base lead ECG were implanted subcutaneously under anesthesia (chloral hydrate, 400 mg/kg ip). The electrodes were led to the back of the neck, exteriorized, and inserted into a custom-made plug. For ECG recordings, the plug was connected to a rotating swivel, allowing the conscious rat to remain undisturbed. After an accommodation period of 30 min, the ECG was recorded for 3 min under standardized conditions (see above) by modification of a method previously described (28). The signals (0.05-2,500 Hz) were amplified using a one-channel ECG amplifier (Schiller, Bahr, Switzerland) and digitized with a time resolution of 0.1 ms (10-kHz sampling frequency) and 12-bit amplitude resolution by means of a data aquisition board (DT 2812, Data Translation, Marlboro, MA) and a lap-top computer (T 3200, Toshiba, Tokyo, Japan). On a 486-DX50 computer (DSM, Munich, Germany), R-wave recognition and R-R interval tachogram calculation and analysis were carried out. The peak of the R spike served as a reference point for the temporal location of the R wave. Tachograms were checked visually for misdetections and ectopic beats, which were interpolated linearly by taking the mean value of the preceding and following R-R interval. Tachograms containing <800 beats were excluded from processing because of the averaging requirements of the spectral analysis (see below). In the time domain, the mean interval between normal beats ("R-R interval"), its standard deviation, and the coefficient of variance [CV; 100 × standard deviation of mean N-N interval (SDNN)/mean R-R interval] were calculated. For analysis in the frequency domain, the tachogram was divided into segments of 256 intervals overlapping each other by half. After removal of the linear trend and application of the Hanning window, each segment was padded with 256 zeros, submitted to a fast Fourier transform, and magnitude-squared for calculation of the power spectrum according to the periodogram method. The power spectra of all segments were averaged to reduce the variance of fast Fourier transform as spectral estimator. Moreover, the obtained average power spectrum was smoothed using a three-point sliding rectangular window. According to previous HRV studies in rats (4), two regions of interest were defined: low-frequency (LF; >0.5 Hz, <0.8 Hz) and high-frequency bands (HF; 0.8 Hz up to Nyquist frequency, determined by the mean R-R interval of the tachogram, generally <4.5 Hz), expressed as percent of total spectral power. From each rat, ECG signals were recorded both 28 and 56 days after SH and MI. In an additional series of control rats, a venous catheter was inserted as described above. Three days after surgery, ECG signals were recorded in conscious rats both before and 10 min after intravenous injection of atropine (0.5 mg/kg).

Determination of plasma concentrations of atrial natriuretic peptide and norepinephrine. For determination of atrial natriuretic peptide (ANP), blood samples were immediately cooled, stabilized by addition of K-EDTA (final concentration 1 mg/ml), and centrifuged at 4,000 revolutions/min for 10 min. The plasma was stored at 20°C until analysis. The plasma samples were extracted as previously described (14). The ANP was determined by radioimmunoassay using a polyclonal antiserum (Peninsula Laboratories, Heidelberg, Germany). Norepinephrine was radioenzymatically assayed (9).

Statistics. Results are expressed as means ± SE. Differences between separate groups were tested by analysis of variance (ANOVA) or by the Mann-Whitney test, where applicable. Intraindividual differences were tested by ANOVA for repeated measures or by the Wilcoxon test, where applicable. Each ANOVA was followed by the Student-Newman-Keuls test. Linear regression analysis was performed by the least-squares method. A P value <0.05 was considered significant.

	RESULTS

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Baseline characteristics. At time of study, neither respiratory distress nor ascites, peripheral edema, or pleural effusion was noted in any animal. Total body weight was not different between respective SH and MI rats studied in different groups 3, 28 and 56 days postoperatively (Table 1). However, total heart weight-to-body weight index was increased after MI as compared with controls (Table 1). The total wet lung weight-to-body weight ratio was not different from SH rats (Table 1). The basal HR was similar in all groups, without major change over time (Table 1). However, basal MAP was lower 28 and 56 days after MI as compared with SH controls (Table 1). In all MI groups, LVEDP was approximately twice as high as in SH rats (Table 1). The plasma concentrations of ANP were approximately three times higher in MI rats, whereas plasma levels of norepinephrine were not different between SH and MI groups (Table 1).

Pharmacological inhibition of the autonomic nervous system. The reduction of basal HR induced by metoprolol (0.5 mg/kg iv bolus) was similar between the respective SH and MI groups 3, 28, and 56 days postoperatively (Table 2). In contrast, the increase of basal HR after application of atropine (0.5 mg/kg iv bolus) was markedly less pronounced in the 3- and 28-day MI groups, as compared with SH controls (Table 2). However, this difference in atropine-induced HR changes between SH and MI was no longer observed 56 days after operation, thus representing a normalization in MI rats. The MAP was not significantly affected by either drug in any group (data not shown).

BRS. The average MAP and R-R interval changes induced by nitroprusside and methoxamine are shown in Fig. 1, A-C. With the individual data pairs, BRS was analyzed for effects of either nitroprusside (causing reflex tachycardia) or methoxamine (causing reflex bradycardia). Reflex tachycardia was not different between any SH and MI group (Table 3; Fig. 2). In contrast, reflex bradycardia was significantly reduced in both the 3- and the 28-day groups as compared with controls (Table 3; Fig. 3). However, reflex bradycardia was normalized 56 days after MI (Table 3; Fig. 3). To examine the relationship between BRS and parameters of left ventricular dysfunction, linear regression analysis was performed with these data. There was no significant correlation between reflex bradycardia and either total heart weight-to-body weight index, basal HR, basal MAP, LVEDP, plasma norepinephrine, or ANP at any of the three time points after MI (data not shown).

View larger version (23K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Fig. 1.   Average changes () of mean arterial pressure (MAP) and R-R interval induced by bolus injections of nitroprusside or methoxamine (each in 4 increasing doses) 3 (A), 28 (B), and 56 days (C) after sham operation and after myocardial infarction. Bold dashed lines were calculated by linear regression of average R-R interval vs. MAP (separately for nitroprusside and for methoxamine). Values are means ± SE.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Baroreflex sensitivity (BRS; reflex tachycardia, induced by nitroprusside) and effect of pretreatment with metoprolol (0.5 mg/kg iv bolus) 3, 28, and 56 days after sham operation (A) and myocardial infarction (B). BRS is expressed as slope of regression line of individual data pairs of R-R interval vs. MAP. Values are means ± SE. + P < 0.05 vs. corresponding group without pretreatment of same time point. There were no significant differences between any sham operation and myocardial infarction group without pretreatment. For data, see Table 3.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   BRS (reflex bradycardia, induced by methoxamine) and effect of pretreatment with atropine (0.5 mg/kg iv bolus) 3, 28, and 56 days after sham operation (A) and myocardial infarction (B). BRS is expressed as slope of regression line of individual data pairs of R-R interval vs. MAP. Values are means ± SE. * P < 0.05 vs. sham-operated group without pretreatment of same time point. + P < 0.05 vs. corresponding group without pretreatment of same time point. For data, see Table 3.

HRV. To validate the effect of muscarinic receptor inhibition on HRV, ECG signals were recorded both before and 10 min after intravenous injection of atropine (0.5 mg/kg) in control rats without thoracotomy (n = 6). In addition, a mean reduction of the R-R interval of 29 ± 5 ms (P < 0.05 between control and treatment) and a decrease of SDNN of 2.3 ± 0.9 ms (P < 0.05) in the time domain was induced by atropine. In the frequency domain, LF and HF power was markedly reduced (LF, 1.4 ± 0.4%; HF, 9.9 ± 2.9%, i.e., differences in percent of total spectral power; P < 0.05 both).

	DISCUSSION

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Severity of left ventricular dysfunction. In the present study, several parameters were determined to assess the severity of left ventricular dysfunction after MI. In all MI groups, the LVEDP, one of the most sensitive criteria of decreased left ventricular function (24), was approximately doubled as compared with SH groups. An increase of LVEDP to values of 10-15 mmHg reflects an infarct size of 30-45% (11, 24). The interindividual variance of LVEDP was very low in all SH controls as well as within each MI group, thus indicating not only a high reproducibility of the technique used for measurement of LVEDP, but also a high homogeneity with respect to left ventricular dysfunction within MI groups. In agreement with previous investigations, several other characteristics of moderately impaired left ventricular systolic function were observed; the plasma concentrations of ANP were approximately threefold higher after MI as compared with SH controls (14). Moreover, the total heart weight-to-body weight index was increased in all MI groups. However, there was no evidence of severe heart failure, since the plasma levels of norepinephrine were not elevated, the lung weight-to-body weight index and total body weight remained unchanged, and neither pleural effusion nor ascites was observed. Furthermore, basal HR remained unchanged, and only a slight decrease in MAP was found 4 and 8 wk after MI. In summary, these observations clearly demonstrate the induction of moderate left ventricular dysfunction associated with compensated heart failure early after MI without major changes over the first 8 wk of the post-MI period.

Effect of pharmacological autonomic inhibition on basal heart rate. Even though basal HR remained unchanged in MI rats, a latent imbalance of autonomic control of HR might occur in these rats. Therefore, the effect of pharmacological inhibition of -adrenoceptors (by metoprolol) and muscarinic acetylcholine receptors (by atropine) on basal HR was investigated. The dose of either drug was chosen so that MAP remained unchanged to avoid interferences with the baroreflex component of HR control. The decrease of basal HR by metoprolol was not different between MI and SH rats, which may indicate that the tonic sympathetic influence on HR is preserved in this model. In contrast, a reduced increase of basal HR by atropine was observed 3 and 28 days after MI. However, the effect of atropine on basal HR was normalized by the 56th day of the post-MI period. The fact that atropine had a reduced effect on basal HR 3 and 28 days after MI even though these rats had no change in basal HR and no difference in the effect of metoprolol on basal HR might be explained by a change of intrinsic HR. However, the intrinsic HR was not evaluated (by combined administration of metoprolol and atropine) in the present study. In summary, these data indicate that a transient post-MI attenuation of tonic vagal activity is uncovered after autonomic blockade but appears to be masked under control conditions. To further analyze autonomic control of HR in MI rats, BRS and HRV were assessed.

BRS. One major finding of this study is the significant impairment of BRS as early as 3 days after MI. No further change in BRS was observed 28 days after MI. Therefore, it may be concluded that BRS is rapidly and persistently reduced in rats during the first 4 wk of the post-MI period. However, 8 wk after MI, BRS was completely recovered, thus demonstrating the transient character of BRS depression. The reduction of BRS, observed 3 and 28 days after MI, was due to a decrease in reflex bradycardia, whereas reflex tachycardia was not different between MI and SH groups. Likewise, the recovery of BRS observed 56 days after MI was due to a normalized reflex bradycardia.

HRV. In contrast to the depression of BRS, no significant changes of HRV parameters were observed, thus underlining the independence (13, 18) of the two methods used to assess autonomic HR control. The analysis of beat-to-beat HRV has been previously shown to provide an estimation of autonomic control of HR in humans (16, 20, 28) as well as in dogs (1) and rats (2, 17). In the time domain, a reduction of SDNN is considered to reflect a decrease in vagal tone (13). In the frequency domain, an impaired vagal tone has been attributed to a reduced power of the HF band, whereas the LF band is thought to be modulated by both sympathetic and vagal tone (2, 17). The very-low-frequency band was not analyzed in the present study because it is less suitable to detect sympathetic or vagal activity (4). Because R-R intervals in rats are much smaller than in humans and dogs, we developed a novel data analysis software to allow for adequate resolution of ECG signals. To validate whether the resolution of the method used is sufficient to evaluate vagal tone, the effect of atropine on HRV was examined in healthy control rats. Both SDNN and LF and HF power were reduced by atropine. However, both 28 and 56 days after MI, all HRV parameters determined in the time and frequency domain were unchanged. As previously described for HRV in rats, a relatively high interindividual variance of HRV parameters in the frequency but not in the time domain (17) was found. Because a transient post-MI reduction of resting vagal tone could be unmasked after autonomic blockade (see above) but could not be detected by the assessment of HRV without autonomic blockade, it may be concluded that HRV in rats is less dependent on the absolute vagal tone than on relative influences of vagal and sympathetic tone. Furthermore, both the threshold and the time course of infarction-induced changes in HRV may be different from the other estimates of vagal activity. In more severe states of heart failure in rats, HRV (studied in the time domain only) was found to be reduced 56 days after MI (30). In dogs, a reduction of HRV was observed at 5 but not at 10-30 days after MI (1); however, these HRV parameters remained decreased in a subgroup of MI dogs susceptible to ventricular fibrillation during the whole observation period. Clinical trials revealed HRV depression in patients 2 wk after MI with recovery 6 mo later (20). In summary, the present data indicate a normal HRV in rats with moderate left ventricular dysfunction 28 and 56 days after MI when analyzed as a whole group. The study design did not allow us to differentiate in regard to subgroups at increased risk for mortality. Moreover, the effects of atropine and -adrenoceptor antagonists on HRV in MI rats have not yet been characterized. Likewise, it remains to be determined whether arterial blood pressure variability is altered in this model.

Conclusions. The present study indicates that BRS is transiently depressed in conscious rats with moderate left ventricular dysfunction after MI, whereas HRV remains unchanged. The BRS changes do not appear as a direct consequence of the hemodynamic alterations after MI. The methods used allow the evaluation of vagal activity and discrimination between its tonic and reflex components. It is concluded that MI rats exhibit temporarily reduced vagal activity; however, the attenuation of its tonic component is unmasked only after autonomic blockade. Because of the discrepancies in HRV data between rats and humans described above, MI rats may have only a limited value as a model for studies of tonic vagal control of HR. However, both the time course and the pathophysiological pattern of BRS depression and recovery in rats parallel the BRS changes observed in humans after MI. There is evidence of a causal relationship between reduction of BRS and impaired hemodynamic tolerability of both general cardiovascular stress and sustained ventricular tachycardia (8, 18, 31). Therefore, therapeutic interventions improving BRS after MI are desirable. The MI rat may be considered as a small animal model to further investigate both pathophysiological mechanisms and pharmacological modulation of reflex control of heart rate.