The autonomic nervous system is one the most important regulators of the heart. Although much is known about the adrenergic regulation of cardiac function in health and disease, less interest has been dedicated to the cholinergic nervous system. In principle, all regions of the heart are innervated by vagal nerves, although in most species the supraventricular tissues are more densely innervated than ventricles are.1 The release of acetylcholine from parasympathetic nerve terminals activates muscarinic acetylcholine receptors (mAChRs) found in various cardiac tissues including the sinus node, atrium, A-V node, and ventricles.2–5 The principal effects of mAChR stimulation in the heart are slowing or accelerating the heart rate, weakening or strengthening the contractile force, shortening the atrial action potential depolarization, attenuating the atrioventricular nodal conduction velocity, and reducing the cardiomyocyte apoptotic cell death. Low concentration of muscarinic agonists usually causes decreases in heart rate, atrioventricular conduction, and ventricular contraction. Paradoxically, under appropriate conditions, activation of cardiac mAChRs elicits stimulatory effects on the rate and contractile force of the heart.6,7 However, the most common effects of vagal modulation of the heart are to reduce heart rate and A-V node conduction that counterbalances the sympathetic effects of increasing heart rate and contractility.
Molecular studies have identified 5 mAChRs genes that are expressed in multiple tissues, each gene corresponding to the mAChR pharmacological subtypes M1 to M5.8–10 Despite the classic notion that cardiac mAChR is exclusively of the M2 subtype, evidence exists of a possible role of other subtypes, particularly M1 and M3.7,11,12
Baroreceptors (BR) play a pivotal role in modulating the autonomic activity of the cardiovascular system adjusting blood pressure (BP) and heart rate (HR) to maintain homeostasis. The importance of BR in cardiovascular homeostasis has been highlighted by studies after the description of a surgical procedure that disrupted the baro- and peripheral afferent chemoreceptors in the brainstem in the rat,13 known as sinoaortic denervation (SAD). In the acute phase, 6 hours after SAD, an increase in BP and in renal sympathetic activity is observed, accompanied by increased BP lability,14 whereas in the chronic phase, BP and sympathetic nerve activity return to baseline values, but BP lability remains elevated.14,15 Although many studies14–19 have analyzed the role of the sympathetic system on the hemodynamic changes after SAD, little is known about the parasympathetic efferent effect on the heart in this model. Franchini and Krieger20 reported a decrease in bradycardia induced by vagal electrical stimulation and methacholine injection, 6 hours after SAD, a period where the tachycardia seems to be maximal.21 Therefore, the objective of the present study was to analyze the effects of acute (1 day) and chronic (20 days) SAD on the vagal responsiveness and expression of mAChRs in the rat atrium.
Adult male Wistar rats (200–300 g) were housed in the Animal Facility at the Instituto do Coração, Universidade de São Paulo and maintained on a 12-hour light:dark lighting schedule, at 23°C, food and water ad libitum. All procedures were performed according to the Guide for the Care and Use of Laboratory Animals.22 Three groups of animals were used: control rats (CTR), acute sinoaortic denervated rats (animals surgically denervated for 1 day, SADa), and chronic sinoaortic denervated rats (animals surgically denervated for 20 days, SADc).
Sinoaortic denervation was performed using the method described by Krieger.13 Briefly, the animals were anesthetized with sodium pentobarbital (40 mg/kg i.p., Sigma Chemical Company, St. Louis, MO), a midline incision was made, the sternocleidomastoid muscles were reflected laterally, and the neurovascular sheath was exposed. The common carotid arteries and the vagal trunk were isolated and the aortic depressor fibers, either traveling with the sympathetic nerve or as an isolated aortic nerve, were cut. The communication branch of the aortic fibers was resected. The third contingent of aortic baroreceptor fibers traveling with the inferior laryngeal nerve was interrupted by resection of the superior laryngeal nerve after the carotid bifurcation was extensively exposed for carotid stripping. The sinus nerve as well as all carotid branches and the carotid body were resected. Therefore, the BR input to the brain stem was disrupted and its inhibitory action on sympathetic activity and its excitatory influence on vagal activity removed.
Arterial and Venous Catheters
Catheters were implanted in the left femoral vein and artery with rats anesthetized with Ketamine (80 mg. kg−1, Parke-Davis, SP, Brazil) and Xylazine (12 mg.kg−1, Bayer, SP, Brazil) to allow drug infusions and BP recordings, respectively, 1 day before BP monitoring. Catheters were tunneled subcutaneously, exteriorized at the back of the neck, and flushed with heparinized saline solution (100 U/mL) immediately after placement and before the BP signal was recorded.
Data Collection and Analysis
Blood pressure was recorded from the left femoral artery for 90 minutes in conscious rats by using pressure transducers (P23Db, Gould-Statham, Oxnard, CA) connected to an amplifier (Stemtech, Milwaukee, WI). Before the analog to digital conversion, blood pressure was low-pass filtered (fc=50 Hz, Butterworth, 4th order-Bioengineering Division, InCor, São Paulo, Brazil) for high-frequency noise removal, and recorded on a microcomputer (Gateway 2000, 4DX2-66 V) with commercially available software (Codas, Dataq, Akron, OH), with a 2kHz sampling frequency. Pulse intervals (PI) were measured considering intervals between consecutive diastoles and measured in milliseconds (ms). HR was calculated as the inverse of PI and measured in beats per minute (bpm).
The first 45-minute period of recording was assigned to animal adaptation to laboratory environment, equipment, and for signal quality adjustments. The following 20-minute period was used for HR and BP time domain analysis. Blood pressure signals were analyzed with a specific semi-automatic monitor (MATLAB 6.0, Mathworks, Inc., Natick, MA). Systolic and diastolic values were detected after parabolic interpolation and signal artifacts were visually identified and removed. Time-domain analysis consisted of calculating mean PI, systolic BP (SBP), diastolic BP (DBP), and SBP variability as the standard deviation from its respective time series. HR was calculated as the inverse of PI periods. To confirm denervation effectiveness, baroreflex sensitivity was calculated after bolus infusions of phenylephrine (Sigma Chemical Company, St. Louis, MO) enough to elevate BP at least 20 mm Hg. Reflex bradycardia was lower in SADa (−0.24 bpm/mm Hg) and lower in SADc (−0.03 bpm/mm Hg) rats compared with that in CTR rats (CTR: −1.54 bpm/mm Hg).
Vagal Nerve Stimulation
After BP recording, rats were anesthetized with sodium pentobarbital (30 mg.kg−1 e.v., Sigma Chemical Company, St. Louis, MO), the right vagus was isolated from the carotid artery and the sympathetic trunk, and its proximal portion was tied and cut. Platinum electrodes were positioned for electrical stimulation by using increasing frequencies of 2 Hz to 16 Hz, with pulse energy of 5 V, duration of 2 milliseconds for 7 seconds, with an electrical stimulus generator (Divisão de Bioengenharia, InCor, USP, São Paulo, Brazil). After stabilization of BP and HR, vagal electrical stimulation was carried out. For each stimulation frequency, bradycardia was calculated as the difference between mean resting HR values and minimal HR value during electrical stimulation. The relative (percentage) bradycardia was calculated for each stimulation frequency over a stimulation frequency range from 2 Hz to 16 Hz. Linear regression analysis was applied to data and individual regression lines obtained. For each group, a common regression line was calculated.23
Muscarinic Acetylcholine Receptor (mAChR) Binding Assays
The right and left atria obtained from 12 to 15 animals were used for each experiment. Tissues were prepared as described by Maróstica et al.24 Protein concentration of membrane preparations was determined with a protein reagent assay (Bio Rad Laboratories, Hercules, CA).
Membrane Binding Assay
In preliminary studies, the appropriate conditions for binding assays of [3H] N-methylscopolamine ([3H]NMS, specific activity 82.0–84.3 Ci.mmol−1, New England Nuclear, Boston, MA), a muscarinic acetylcholine receptor nonselective antagonist, were determined. According to the results, all subsequent binding studies were performed with membrane protein concentration of 100 μg/mL and incubation time of 90 minutes at 30°C.
Saturation binding experiments were performed as described by Maróstica et al.24 Briefly, atrial membrane preparations (100 μg protein/mL) were incubated with 0.05 nM to 16 nM [3H]NMS in the absence (total binding) and presence (nonspecific binding) of atropine sulfate (Sigma Chemical Company, St. Louis, MO) for 90 minutes at 30°C. Specific binding was calculated as the difference between total and nonspecific binding. Nonspecific binding, near the KD value, was below 20% of the [3H]NMS total binding.
Saturation binding data were analyzed using a weighted nonlinear least-square interactive curve-fitting program called GraphPad Prism (GraphPad Prism Software Inc, San Diego, CA). A mathematical model for one or two sites was applied. The dissociation constant (KD) and the maximum number of binding sites (Bmax) were determined from a Scatchard plot.25
Differences among CTR, SADa, and SADc groups for hemodynamic parameters, mAChR density, and affinity were tested through one-way analysis of variance with a Bonferroni multiple comparisons post hoc test. Differences among regression lines calculated for bradycardia were tested by analysis of variance with the Bonferroni post hoc correction test.23P values <0.05 were accepted as significant.
Effects of SAD on Hemodynamic Parameters
The effects of SAD on the hemodynamic parameters are summarized in Table 1. Figure 1 is representative of HR and SBP behavior of 20-minute periods of continuous recording in CTR, SADa, and SADc rats. As expected, acute SAD caused an increase in HR, systolic, diastolic mean values, and increased BP lability. However, these parameters were similar to control values after 20 days of SAD, except for BP lability that remained elevated.
Vagal Electrical Stimulation
Reductions in HR due to vagal electrical stimulation were observed in a frequency-dependent fashion in all 3 groups of rats. Bradycardia was greater from SADa and SADc rats compared with that in CTR rats. Figure 2 is representative of HR and SBP responses during vagal electrical stimulation set at 4 Hz. Regression lines obtained from SADa and SADc rats were significantly different from regression lines from CTR rats, showing that vagal electrical stimulation after SAD induces more intense bradycardia (Fig. 3).
Effects of SAD on [3H]NMS Binding in Rat Atrial Membranes
The binding of [3H]NMS to rat atrial membranes was specific and saturable (Fig. 4). Scatchard analysis of the specific binding of [3H]NMS indicated the presence of a single class of [3H]NMS high-affinity sites in membranes derived from CTR, SADa, and SADc (Fig. 4, right panel). An analysis of 4 experiments performed in duplicate yielded a dissociation constant (KD) and maximum number of binding sites (Bmax), summarized in Table 2. The comparison of binding parameters indicated that sinoaortic denervation for 20 days induced an increase in affinity and receptor density when compared with that in CTR or SADa (P<0.05).
The SAD procedure has shown the importance of the arterial baroreflex that tonically modulates the sympathetic and parasympathetic efferent to the heart and vessels.13 In the SADa rats, hypertension, tachycardia, and an increase in BP lability were observed in the acute phase, whereas in the chronic phase only BP lability is usually observed.13–15,21,26,27 Franchini and Krieger26 showed that the normalization of BP after chronic SAD is in part due to denervation of the chemoreceptors that exerted tonic influence on the sympathetic system. Acute SAD is known to produce adrenergic hyperactivity and Franchini and Krieger20 showed that 6 hours after SAD the bradycardic responses to vagal electrical stimulation or injections of methacholine had decreased. The mechanisms underlying these changes are not completely understood, but adrenergic and muscarinic receptor responsiveness to neurotransmitters might be involved in the heart. Indeed, it was shown that SAD after 7 days induced downregulation19 and desensitization of atrial beta-1 adrenoreceptors and reduction in the sympathetic21 and parasympathetic28 tone. Although vagal tone is reduced in SAD rats,21,28 the present study showed that the parasympathetic efferent pathway is still responsive to acetylcholine released by electrical stimulus and may respond in an even greater magnitude than in rats with the intact baroreflex arc. Similarly, vagal hyperresponsiveness was also observed in aged29 and spontaneous hypertensive rats.30
Chronic sinoaortic denervation induced an increase in mAChR affinity and density. These modifications were accompanied by augmented vagal responsiveness. These observations are in agreement with the higher bradycardia shown by SADc rats during vagal electric stimulation and may represent a peripheral adjustment to a central decrease in the vagal tonus. Indeed, chronic treatment with muscarinic antagonists like atropine for up to 14 days induces a dose-dependent increase in mAChR number in several brain regions, the heart, and airways.31–35 This increase in receptor number may be due in part to the inverse agonist activity of muscarinic antagonist, which increases the proportion of receptors in the inactive state at the expense of the active state,36 thereby attenuating receptor downregulation. Antagonist treatment may also prevent endogenously released acetylcholine from binding to the receptor and thereby agonist-induced receptor downregulation. In general, the increases in receptor number in response to chronic atropine or scopolamine administration are accompanied by augmented mAChR responsiveness.35 Similar mechanisms may occur with chronic sinoaortic denervation.
In contrast to that observed in SADc rats, a relationship between the regulation of mAChRs and increased vagal responsiveness was not shown in acute SAD, indicating that different mechanisms may be involved in this process. In the acute phase of SAD, no significant changes in the number and affinity of mAChRs were observed, suggesting that other mechanisms might be involved in the increased bradycardia in SADa rats during vagal stimulation. Considering the increase in sympathetic tonus to the heart at this time, a probable interaction between sympathetic and parasympathetic action37 may be associated with this observed “accentuated antagonism.”38
The present results provide evidence of the effects of SAD on mAChRs and parasympathetic function involved in regulating cardiac function. The functional data showing an adaptive increase in peripheral parasympathetic function of the heart associated with the increase in the number and affinity of mAChRs may represent a new possibility of management in pathophysiological states associated with baroreflex sensitivity attenuation like heart failure and hypertension in which a clearly parasympathetic impairment may occur.
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