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Department of Physiology, Monash University, Victoria 3800, Australia
Submitted 2 January 2003 ; accepted in final form 21 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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20% with Cs+ and nifedipine
(P < 0.05), whereas the heart rate response to norepinephrine was
only reduced with Cs+ (P < 0.05).
Ba2+ attenuated the decrease in heart rate with vagal
stimulation and carbachol by 60% (P < 0.05). These results
are consistent with the idea that sympathetic nerve stimulation modulates
If to increase heart rate in the mouse. Activation of
ICa,L also appears to contribute to the sympathetic heart
rate response. However, the decrease in heart rate with vagal stimulation or
carbachol is likely to result primarily from the activation of
IK,ACh.
sinoatrial node; heart rate; sympathetic; parasympathetic
1-adrenoceptors
(19)] or membrane currents
[e.g., ACh-sensitive K+ current (IK,ACh)
knockout mice (28)] have been
developed. Cardiovascular diseases such as heart failure and hypertension are
associated with abnormal sympathetic and parasympathetic activation of cardiac
autonomic receptors and membrane currents
(11). Genetically altered mice
could therefore be used to investigate the role of the cardiac autonomic
innervation in the development of these cardiovascular diseases. Because the
neuronal control of heart rate has not been examined in mice, the primary aim
of this study was to investigate the effects of cardiac sympathetic and
parasympathetic nerve stimulation on heart rate in spontaneously beating mouse
atrial preparations. Cardiac sympathetic and parasympathetic neurotransmitters are likely to alter heart rate by modulating sinoatrial node ionic currents, such as the hyperpolarization-activated current (If), L-type Ca2+ current (ICa,L), or IK,ACh. Nonmurine studies have produced contradictory results as to the specific sinoatrial node currents that are influenced by cardiac autonomic neurotransmitters. This could be due to the use of different models of autonomic activation (i.e., direct nerve stimulation vs. bath-applied autonomic agonists) or to the use of different preparations (e.g., spontaneously beating isolated atrial preparations, anesthetized animals, or isolated sinoatrial node cells). For example, If and ICa,L are attenuated by bath-applied ACh in isolated cardiac myocytes, whereas the heart rate response to vagal stimulation is not altered, or may even be enhanced in vitro and in vivo, after pharmacological inhibition of these currents (3, 9, 23, 26, 27, 29). Furthermore, ICa,L blockers either have no effect on, or attenuate, the heart rate response to cardiac sympathetic nerve activation in vivo (1, 12, 14, 22). In contrast, pharmacological inhibition of IK,ACh consistently reduces the heart rate response to vagal stimulation in vitro and in vivo, suggesting that the activation of IK,ACh contributes to vagal bradycardia (3, 26). There is little information available concerning the modulation of If by sympathetic nerve activity. A single study (12) reported that the heart rate response to sympathetic nerve stimulation is attenuated after If inhibition in vivo. This result is supported by data from sinoatrial node cells indicating that cAMP enhances If (10).
In the present study, an isolated cardiac preparation was used to investigate the effects of autonomic nerve stimulation on heart rate, because circulating hormones, hemodynamic reflexes, or anesthetics may alter autonomic nerve activity in vivo. To determine whether sinoatrial node currents were likely to be modulated by autonomic nerve stimulation, the effects of pharmacological blockers of If (cesium chloride, Cs+, 1 mM), ICa,L (nifedipine, 200 nM), and IK,ACh (barium chloride, Ba2+, 0.1 mM) were examined on the heart rate responses to nerve stimulation in isolated mouse atria. We found that activation of cardiac sympathetic nerves in isolated mouse atrial preparations is likely to modulate If and ICa,L to produce an increase in heart rate. Furthermore, the activation of IK,ACh appears to contribute to vagal bradycardia. Because both the sympathetic and vagal heart rate responses persisted when If, ICa,L, or IK,ACh were inhibited, they are likely to result from the interplay between several different sinoatrial node currents rather than the action of a single current.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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22°C). The atria, together with either the right stellate ganglion
or right vagus, were dissected free and placed into an organ bath (3 ml
volume) maintained at 37 ± 0.1°C (see
Fig. 1). A silk suture was
placed into the left auricle, and this was attached to an isometric force
transducer (SensoNor). A suture in the right auricle was placed over a hook
attached to the wall of the organ bath. The force response (in mN) was
amplified, and data were acquired (150-Hz sampling rate) with a MacLab
data-acquisition system and Chart software. The software calculated heart rate
(in beats/min) from the rate of atrial contractions. The preparation was left
(45–90 min) to equilibrate until the heart rate did not alter by >10
beats/min for 20 min. The vagus or stellate ganglion was attached to a pair of
platinum electrodes connected to an isolated stimulator. Nerves were
stimulated at 10 V, 1-ms pulse width, at 1–30 Hz for 30 s at 2- to 3-min
intervals; 30 Hz was chosen as the maximum frequency for vagal stimulation
because the preparation usually arrested at or above this stimulation
frequency. A maximum frequency of 10 Hz was selected for sympathetic
stimulation because this produced a submaximal response, and higher
stimulation frequencies often caused the preparations to become
arrhythmic.
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Effects of pharmacological inhibition of autonomic function.
Initial experiments examined the effects of the -adrenoceptor antagonist
propranolol (0.1 µM) and the noradrenergic neuron-blocking drug
guanethidine (0.1 µM) on the heart rate response to sympathetic nerve
stimulation. In addition to inhibiting norepinephrine release, guanethidine
also causes the depletion of norepinephrine stores from sympathetic nerve
terminals. The effect of the muscarinic receptor antagonist atropine (0.1
µM) was determined on the heart rate response to vagal stimulation.
Effects of ion channel blockers on heart rate responses to autonomic nerve stimulation and autonomic agonists. After the control heart rate responses to nerve stimulation, the effects of Cs+ (1 mM), nifedipine (200 nM), or Ba2+ (0.1 mM) were determined on these responses. Cs+ at concentrations of up to 2 mM has been reported to specifically block sinoatrial node If, with no effects on sinoatrial node ICa,L or delayed rectifier K+ current [IK (8)]. The concentration of nifedipine (200 nM) was selected because it produced stable bradycardia without arresting the preparation. Previous studies have reported that this concentration of nifedipine inhibits ICa,L (15) and does not affect the transient outward IK (13). There are no specific blockers of IK,ACh; however, 0.1 mM Ba2+ has been reported to significantly inhibit this current, with little or no effects on ICa,L or IK (20). To determine whether Cs+ (1 mM), nifedipine (200 nM), or Ba2+ (0.1 mM) were acting presynaptically to alter neurotransmitter release, their effects were also investigated on the heart rate responses to bath-applied norepinephrine (0.1 µM) and carbachol (0.3 µM).
Drugs. All pharmacological chemicals were obtained from Sigma Aldrich. Stock solutions of the drugs were made up in physiological saline solution on the day of the experiment. Experiments with nifedipine and atropine were performed in the dark because they are light sensitive.
Data analysis and statistics. For all preparations, three heart rate responses to nerve stimulation (or bath-applied autonomic agonist) were obtained at each stimulation frequency (or dose of agonist). The magnitude of these responses was calculated as the difference between the baseline heart rate (averaged over 3 s immediately before autonomic activation) and the maximum response to autonomic activation (averaged over 3 s). The three responses at each stimulation frequency were averaged. Data are presented as means ± SE. Statistical analyses were performed using SPSS (version 11.0) software. ANOVA with repeated measures was used on group data, with the Student-Newman-Keuls test applied for post hoc analysis when significance was obtained. When appropriate, a comparison between two groups was performed by paired Student's t-tests. For all statistical tests, P < 0.05 was accepted as being statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Right vagal stimulation (1–30 Hz) evoked a frequency-dependent decrease in heart rate. At stimulation frequencies at and above 10 Hz, the preparations often arrested. Atropine (0.1 µM) significantly reduced vagal bradycardia without altering the basal heart rate (P < 0.05 by ANOVA; decrease in heart rate with 5-Hz stimulation: control = 89 ± 8 beats/min and atropine = 0 ± 0 beats/min, n = 3). The magnitude of vagal bradycardia did not alter during the time course of the experimental protocols (decrease in heart rate with 3-Hz vagal stimulation: 0 min = 64 ± 6 beats/min, after 20 min = 67 ± 7 beats/min, and after 40 min = 62 ± 2 beats/min, n = 4).
Effect of Cs+ on autonomic control of heart rate. The If blocker Cs+ (1 mM, n = 33, combined values for all Cs+ experiments) decreased heart rate from 339 ± 9 to 270 ± 15 beats/min. The heart rate returned to control levels when Cs+ was washed out with fresh physiological saline solution [washout = 305 ± 9 beats/min, P < 0.05 (by ANOVA) for Cs+ vs. control and washout]. In addition to slowing the heart rate, Cs+ significantly attenuated the positive chronotropic responses to sympathetic nerve stimulation (1–10 Hz, n = 10; see Fig. 2, A and B) and 0.1 µM norepinephrine [n = 10, heart rate response to norepinephrine: control = 177 ± 13 beats/min, Cs+ = 148 ± 12 beats/min, and washout = 186 ± 14 beats/min; P < 0.05 (by ANOVA) for Cs+ vs. control and washout; see Fig. 2C]. The decreases in heart rate with vagal stimulation (1–30 Hz, n = 8, see Fig. 2D) and carbachol (0.3 µM, n = 5) were not altered by Cs+ (heart rate response with carbachol: control = –158 ± 22 beats/min and Cs+ = –161 ± 19 beats/min).
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To determine whether the slowed baseline heart rate with Cs+
contributed to the attenuated sympathetic and norepinephrine heart rate
responses, the effect of reducing the baseline heart rate with carbachol was
determined on these -adrenoceptor-mediated responses. Carbachol (0.1
µM) produced a similar decrease in heart rate (n = 15, heart rate
decreased by 85 ± 15 beats/min) to 1 mM Cs+ (n =
33, heart rate decreased by 63 ± 14 beats/min). As shown in
Fig. 3, in contrast to the
inhibitory effects of Cs+, carbachol did not alter the heart rate
responses to sympathetic nerve stimulation (n = 7) and norepinephrine
(n = 8).
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Effect of nifedipine on autonomic control of heart rate. Nifedipine (200 nM) slowed the baseline heart rate from 319 ± 6 to 267 ± 12 beats/min (n = 28, combined values for all nifedipine experiments; P < 0.05 by paired t-test). It also significantly decreased in the magnitude of the heart rate response to sympathetic nerve stimulation (1–10 Hz, n = 6; see Fig. 4, A and B). Nifedipine had no affect on the heart rate responses to vagal stimulation (n = 6; see Fig. 4C), 0.1 µM norepinephrine (heart rate response: control = 170 ± 11 beats/min and nifedipine = 156 ± 11 beats/min, n = 10) or 0.3 µM carbachol (heart rate response: control = –174 ± 19 beats/min and nifedipine = –166 ± 18 beats/min, n = 6).
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Effect of Ba2+ on autonomic control of
heart rate. Vagal bradycardia (1–30 Hz, n = 6; see
Fig. 5, A–C) and
the heart rate response to carbachol (0.3 µM, n = 5) were
significantly reduced (by 60%) when IK,ACh was
inhibited with Ba2+ (0.1 mM). This effect was reversed
when Ba2+ was washed out with fresh physiological saline
solution (heart rate response to carbachol: control = –171 ± 22
beats/min, Ba2+ = –72 ± 22 beats/min, and
washout = –140 ± 18 beats/min; P < 0.05 by ANOVA).
Ba2+ had no affect on the heart rate responses to
sympathetic nerve stimulation (n = 7; see
Fig. 5D) and
bath-applied norepinephrine (heart rate response to norepinephrine: control =
177 ± 28 beats/min and Ba2+ = 141 ± 19
beats/min, n = 5).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Sympathetic and vagal control of heart rate in an isolated mouse atrial
preparation. In this study, the normal physiology of the autonomic
control of heart rate was mimicked by the use of an isolated double-atrial
preparation with intact cardiac sympathetic or parasympathetic postganglionic
innervation. This is the first time that the effects of cardiac sympathetic
and parasympathetic nerve activation on heart rate have been specifically
investigated in an isolated mouse cardiac preparation. In a recent study
(6), our laboratory examined
the vagal heart rate responses in mouse atria from normal and neuronal nitric
oxide synthase knockout mice. Other studies have indirectly examined neuronal
heart rate responses in the mouse by using field stimulation of the atria or
by activation of cardiac nerves in vivo via the baroreflex (for example, see
Refs. 7 and
28). Consistent with other
mammalian species, we found that in mouse atria, right stellate ganglion
stimulation caused the release of neurotransmitter(s), which interacted with
-adrenoceptors to produce an increase in heart rate, and right vagal
stimulation caused a decrease in heart rate that resulted from the activation
of muscarinic receptors.
Effect of Cs+ on autonomic control of heart
rate. Mangoni and Nargeot
(18) recently recorded
If in isolated mouse sinoatrial node cells. We found that
inhibition of If with Cs+ in isolated mouse
atria reduced their spontaneous beating rate, indicating that this current is
likely to be involved in the generation of sinoatrial node pacemaker action
potentials in this species. In addition, Cs+ attenuated the
positive chronotropic responses to right stellate ganglion stimulation and
bath-applied norepinephrine, suggesting that -adrenoceptor activation
enhances the activation of If to cause an increase in
heart rate. It is unlikely that the sympathetic heart rate response was
attenuated in the presence of Cs+ due to the associated decrease in
baseline heart rate, because a similar slowing of heart rate with carbachol
failed to attenuate the sympathetic heart rate response.
In the autonomically decentralized heart of the anesthetized dog,
zatebradine, a potent inhibitor of sinoatrial node If,
also attenuated the increase in atrial beating rate evoked by cardiac
sympathetic nerve activation
(12). Furthermore,
-adrenoceptor agonists increase the amplitude of If
in isolated rabbit sinoatrial node pacemaker cells by shifting the activation
curve 11 mV in the positive direction, an effect that would enhance
If, increase the slope of the diastolic depolarization,
and elevate the heart rate
(10). More recently, Mangoni
and Nargeot (18) reported that
norepinephrine caused an increase in the amplitude of If
recorded from isolated mouse sinoatrial node cells. Therefore, norepinephrine
released from sympathetic nerve terminals innervating the mouse sinoatrial
node could increase the activation of If and thus increase
the heart rate. This agrees with our data from mouse atria, which indicate
that when If is inhibited the heart rate response to
sympathetic activation is significantly attenuated.
A hyperpolarization-activated current, Ih, which is similar to sinoatrial node If (also called Ih) has been recorded in isolated sympathetic postganglionic neurons (16). It is therefore possible that Cs+ inhibited Ih in both sinoatrial node myocytes and sympathetic neurons in our preparation. Inhibition of Ih with Cs+ in sympathetic neurons causes hyperpolarization of the resting potential, implying that If normally contributes an inward current to the resting potential of the neurons (16).
Modulation of If is unlikely to contribute to the cholinergic control of heart rate in the mouse because Cs+ did not alter the drop in heart rate with vagal stimulation or bath-applied carbachol. The magnitude of the vagal bradycardia was also unchanged with If inhibition in isolated guinea pig atria and the anesthetized dog (23, 26). However, 5 µM carbachol inhibited If by 37% in isolated mouse sinoatrial node cells (18). It is possible that high concentrations of ACh inhibit If in the mouse sinoatrial node. However, in the current study, concentrations of carbachol above 1 µM arrested the preparations. This suggests that these high concentrations of bath-applied carbachol are unlikely to be physiological.
Effect of nifedipine on autonomic control of heart rate. A recent study (25) using isolated rabbit sinoatrial node cells indicates that ICa,L contributes to pacemaker diastolic depolarization. It has been established that ICa,L is also present in murine sinoatrial node myocytes (2). Furthermore, knockout mice deficient in voltage-gated L-type Ca2+ channels exhibit bradycardia in vivo relative to their wild-type controls (21). Similarly, in the present study, partial inhibition of ICa,L with a low concentration of nifedipine (200 nM) reduced the beating rate of spontaneously beating mouse atria. Therefore, modulation of ICa,L by autonomic neurotransmitters could alter heart rate in the mouse.
Inhibition of ICa,L with nifedipine significantly attenuated the increase in heart rate evoked by activation of the right stellate ganglion. Nifedipine has also been reported to attenuate the increase in heart rate with cardiac sympathetic nerve stimulation in the dog and cat in vivo (1, 14, 22). It is possible that the attenuated sympathetic heart rate response with nifedipine could be at least partly due to the inhibition of neuronal L-type Ca2+ channels. The membranes of postganglionic sympathetic neurons possess several types of voltage-dependent Ca2+ channels, including P-, Q-, N-, and L-type Ca2+ channels (5). The contribution of L-type Ca2+ channels in sympathetic postganglionic neurons to calcium entry, and therefore transmitter release, is unclear. In isolated somata from the rat superior cervical ganglion, 80% of the current passed through N-type channels and only 5% through L-type channels (24). In the present study, nifedipine attenuated the positive chronotopic response to sympathetic activation but had no significant effect on a similar increase in heart rate caused by bath-applied norepinephrine. These results are consistent with the idea that ICa,L contributes to calcium entry in mouse cardiac sympathetic neurons and thus to neurotransmitter release and the postsynaptic heart rate response.
Effect of Ba2+ on autonomic control of heart rate. Because Ba2+ significantly attenuated the decrease in heart rate with vagal stimulation and carbachol in isolated mouse atria, these responses are likely to be primarily due to the activation of IK,ACh. Similarly, Wickman et al. (28) found that mice genetically deficient for IK,ACh had a 50% reduction in the vagal modulation of heart rate (assessed by bradycardia in response to baroreflex activation in conscious mice). Previous studies (3, 4, 26) in isolated rabbit and toad atria, as well as in the anesthetized dog, have also indicated a substantial role for IK,ACh in vagal bradycardia. In all of these studies, inhibition of IK,ACh never completely abolished vagal bradycardia. This indicates that other sinoatrial node channels are likely to be modulated by the parasympathetic neurotransmitter ACh, or by a parasympathetic cotransmitter such as nitric oxide (6), to cause alter heart rate. Our data indicate that If and ICa,L are unlikely to be involved in vagal bradycardia in isolated mouse atria. However, parasympathetic modulation of IK may contribute to the vagal heart rate response because carbachol has been reported to inhibit this current in isolated rabbit sinoatrial node cells (17).
Perspectives. This study used ion channel blockers to examine the sinoatrial node pacemaker currents that are likely to be modulated by autonomic activation. We understand that the autonomic modulation of ionic currents can only be directly examined in isolated sinoatrial node cells. However, because the aim of this study was to investigate the neuronal control of heart rate in the mouse, a multicellular preparation with intact innervation was required. We attempted to determine the effects of autonomic nerve stimulation on intracellular recordings of sinoatrial node action potentials in isolated mouse atria. However, it was impossible to maintain impalements with the high beating rates in this preparation. As mentioned in the DISCUSSION, it is possible that the pharmacological agents used in this study may affect the autonomic neurons as well as cardiac myocytes. Taken together with the results of this study, the development of genetically manipulated mice with alterations in cardiac membrane currents [e.g., IK,ACh knockout mice (28)] will further our understanding of how autonomic neurotransmitters modulate ionic currents to alter heart rate.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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1-adrenoceptors. Am J
Physiol Heart Circ Physiol 271:
H1465–H1472, 1996.This article has been cited by other articles:
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  M. E. Mangoni and J. Nargeot Genesis and Regulation of the Heart Automaticity Physiol Rev, July 1, 2008; 88(3): 919 - 982. [Abstract] [Full Text] [PDF]
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 J. K. Choate, S. M. Murphy, R. Feldman, and C. R. Anderson Sympathetic control of heart rate in nNOS knockout mice Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H354 - H361. [Abstract] [Full Text] [PDF]
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H. Kiriazis, X.-J. Du, X. Feng, E. Hotchkin, T. Marshall, S. Finch, X.-M. Gao, G. Lambert, J. K. Choate, and D. M. Kaye Preserved left ventricular structure and function in mice with cardiac sympathetic hyperinnervation Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1359 - H1365. [Abstract] [Full Text] [PDF]
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A. Nygren, A. E. Lomax, and W. R. Giles Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2634 - H2643. [Abstract] [Full Text] [PDF]
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R. B. Robinson, M. Baruscotti, D. DiFrancesco, J. K. Choate, and R. R. Feldman Autonomic Modulation of Heart Rate: Pitfalls of Nonselective Channel Blockade Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2865 - H2866. [Full Text] [PDF]
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