The Role of α7 Nicotinic Acetylcholine Receptor in Modulation of Heart Rate Dynamics in Endotoxemic Rats Roham Mazloom1, Golnar Eftekhari1, Maryam Rahimi2, Vahid Khori3, Sohrab Hajizadeh1, Ahmad R. Dehpour2, Ali R. Mani1* 1 Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran, 2 Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran, 3 Ischemic Disorders Research Center, University of Medical Sciences, Gorgan, Iran
Abstract Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in modulation of heart rate dynamics during systemic inflammation. Citation: Mazloom R, Eftekhari G, Rahimi M, Khori V, Hajizadeh S, et al. (2013) The Role of α7 Nicotinic Acetylcholine Receptor in Modulation of Heart Rate Dynamics in Endotoxemic Rats. PLoS ONE 8(12): e82251. doi:10.1371/journal.pone.0082251 Editor: Charles C Caldwell, University of Cincinnati, United States of America Received July 10, 2013; Accepted October 22, 2013; Published December 10, 2013 Copyright: © 2013 Mazloom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from Tarbiat Modares University and Golestan University of Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected]
Introduction
Experimental studies in animal models have also indicated that endotoxemia is associated with a significant reduction in HRV [6–9]. Likewise, systemic administration of interleukin-6 (IL-6) decreases HRV in mice [10]. The mechanism of increased regularity of cardiac cycle during systemic inflammation in not very well understood. Recent studies have shown that partial uncoupling of cardiac pacemaker cells from cholinergic neural control may play a role in endotoxin-induced loss of HRV in experimental models [9,11]. Moreover, incubation of mouse isolated atria with recombinant IL-6 was associated with a significant reduction in chronotropic response to carbacholine (a cholinergic agonist) in vitro [10]. These reports indicate that activation of inflammatory pathways in cardiac pacemaker cells
Cardiac rhythm displays a complex dynamics in physiological state which is due to non-linear interaction between cardiac pacemaker cells and the autonomic nervous system [1]. A variety of methods have been developed to assess cardiac cycle dynamics in health and disease [2]. These methods have provided evidence to show that heart rate dynamics is altered during systemic inflammation (e.g. in patients with sepsis) [2]. Therefore, heart rate variability (HRV) analysis has been used for non-invasive monitoring of patients with sepsis [3]. These reports have shown that decreased HRV and increased cardiac cycle regularity has diagnostic and prognostic value in patients with systemic inflammatory response syndrome [3–5].
PLOS ONE | www.plosone.org
1
December 2013 | Volume 8 | Issue 12 | e82251
α7nACHR and Heart Rate Dynamics in Endotoxemia
Chemicals
might affect its responsiveness to parasympathetic autonomic nervous control. Recent investigations suggest that activation of vagus nerve can suppress pro-inflammatory cytokine levels in liver and spleen in mice [12]. Furthermore, electrical stimulation of the vagus nerve inhibits tumor necrosis factor-alpha (TNF-α) synthesis in wild-type mice, but fails to inhibit TNF-α synthesis in alpha7-nicotinic acetylcholine receptor (α7nACHR) deficient mice [13]. Since this discovery, the contribution of α7nACHR in modulation of inflammation has been shown in a variety of model systems [14]. α7nACHR is a homopentameric ligandgated ion channel [14] which acts as a calcium channel [15] as well as a ligand-dependent coupler to secondary and tertiary messenger systems [16]. α7nACHR is distributed in several tissues [17], especially in macrophages which are important producer of inflammatory cytokines [14]. Atrial cells receive dense cholinergic innervation and express muscarinic cholinergic receptors [18]. Little is known about the expression of α7nACHR in atrial cells; however previous studies in rats have shown that cardiomyocytes exhibit immunoreactivity for α7nACHR at all embryonic developmental stage, although adult cardiomyocytes immunoreactivity for this receptor is weak [19]. The role of α7nACHR in modulation of cardiac function is not well understood. Deck et al. reported that α7nACHR is not required for parasympathetic control of heart rate in mouse [20]. However the role of this receptor in modulation of inflammatory process in the atria has not been investigated. Sepsis and endotoxemia are associated with decreased HRV and impaired atrial chronotropic responsiveness to muscarinic cholinergic neural control [9]. The underlying mechanism of this phenomenon is uncertain, and the role of anti-inflammatory mediators in modulation of heart rate dynamics during systemic inflammation is not well understood. Fairchild et al. demonstrated that dexamethasone shortened but did not eliminate LPS-induced HRV depression in mice [8]. Likewise, Alvarez et al. reported that administration of a glucocorticoid before endotoxin reduced cytokine levels but did not affect HRV indices in human volunteers [21]. α7nACHR as a key component of cholinergic anti-inflammatory pathway have been shown to modulate local and systemic inflammation in a variety of model systems. However the role of this receptor in sepsis-induced HRV depression is not clear. In present study we investigated the role of α7nACHR in modulation of heart rate dynamics in endotoxemic rats.
Endotoxemia was induced by intraperitoneal (IP) injection of lipopolysaccharide (LPS; Salmonella typhimurium) which was purchased from Sigma-Aldrich (Pool, UK). PHA-543613 hydrochloride (PHA) and methyllycaconitine citrate (MLA) were purchased from Tocris (Bristol, UK) and were used as selective agonist and antagonist for α7nACHR respectively. All of these substances were dissolved in 0.9% saline. All other reagents were purchased from Merck (Darmstadt, Germany) unless stated otherwise.
Animals Male Sprague-Dawley rats were obtained from Razi Institute (Hesarak, Iran). The effect of endotoxin on heart rate dynamics was assessed following IP administration of LPS at two doses of 0.1 and 1 mg/kg. To assess the effect of α7nACHR inhibition on HRV, either saline or LPS (0.1 mg/kg) was injected 30 min after MLA administration (5 mg/kg, IP) [22]. The effect of α7nACHR activation on heart rate dynamics was evaluated by subcutaneous injection of PHA (1 or 4 mg/kg) [23,24] 30 min before IP administration of either control or LPS (1 mg/kg). 6-9 rats were used in each group. Our pilot studies showed that while IP administration of 1 mg/kg of LPS reduced HRV, low dose (0.1 mg/kg) LPS was unable to reduce HRV indices in rats. Based on pilot studies, 1 mg/kg of LPS was the dose that could induce maximum reduction in HRV indices. We observed that higher doses of LPS (e.g. 10 and 20 mg/kg) could not further reduce HRV in rats. We took advantage of this dose-dependent effect of LPS on HRV parameters and chose lower dose of LPS (0.1 mg/kg) for challenge with MLA (α7nACHR antagonist). This enabled us to monitor the effect of MLA on HRV when there is enough capacity for further reduction in HRV indices. Likewise, higher dose of LPS (1 mg/kg) was used to test the effect of PHA (an α7nACHR agonist) on HRV, when there is enough room to restore the loss of HRV parameters after endotoxin challenge.
Telemetric recording of electrocardiogram and body temperature from conscious rats Telemetric recording of electrocardiogram was carried out as described [9,11]. In brief, a dorsally mounted radiofrequency transmitter were implanted subcutaneously (lead I) in animals (body weight 230–250 g) under anesthesia using ketamine (100 mg/kg) and xylazine (10 mg/kg). 14 days after the operation, electrocardiogram (ECG) and body temperature were recorded using a telemetry system (Data Sciences International, St. Paul, Minnesota, USA) connected to a Powerlab data acquisition system (ADInstruments, Sydney, Australia).
Materials and Methods Ethics statement All animal maintenance and procedures were in accordance with recommendations established by the Animal Ethics Committee of Tarbiat Modares University as well as the United States NIH guidelines (publication no. 85-23). The protocol was approved by the Ethics Committee of Tarbiat Modares University. All surgeries were performed under deep anesthesia, and all efforts were made to minimize suffering.
PLOS ONE | www.plosone.org
Data acquisition The R peaks were detected and the R-R interval series were generated using Chart 5 software (ADInstruments). The R-R interval series were visually inspected and 5 minute artifactfree continuous R-R intervals were chosen for analysis.
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December 2013 | Volume 8 | Issue 12 | e82251
α7nACHR and Heart Rate Dynamics in Endotoxemia
HRV analysis
dissection, a stabilization period of 30 min was allowed before evaluation of the spontaneous electrical activity. The signals were then digitized at the sampling rate of 10 kHz and displayed on a Powerlab system (ADInstruments, Sydney, Australia). Beating rate variability parameters (SDNN, SampEn and DFA) were calculated in 10 min R-R time-series as described above.
The standard deviation of the R-R intervals (SDNN) was calculated on the selected artifact-free trace and used as a measure of total HRV. Non-linear measures of HRV provide information on the structure or complexity of the R-R timeseries. In the present study non-linear measures of HRV were assessed using Poincaré plot, sample entropy (SampEn) as well as detrended fluctuation analysis (DFA). Poincaré plot. The Poincaré plot is a graphical representation of the correlation between consecutive R-R intervals. The standard deviation of the points perpendicular to the line of identity (SD1) describes short-term variability which is mainly related to the effects of respiration on vagal drive [25]. This parameter was calculated as described [26] using the software developed by Niskanen et al. [27]. SampEn. SampEn was developed by Richman and Moorman in 2000 [28] and calculates the probability that epochs of window length m that are similar within a tolerance r remain similar at the next point [28]. A lower value of SampEn reflects a higher degree of regularity, and the higher the entropy value, the more random the time series is. In the present study, the parameter m was fixed to 2, and tolerance level r was 0.2 as described [9]. DFA. DFA quantifies fractal-like correlation properties of RR intervals [29]. In this method, the fluctuation of the integrated and detrended data is measured within observation windows of various sizes and then plotted against window size on a log-log scale. A linear relationship between log (fluctuation) and log (window size) indicates the presence of scaling which serves as a characteristic of a fractal-like time-series [29]. The scaling exponent alpha (α) indicates the slope of this line. An α=0.5 indicates white noise (uncorrelated random data). An α greater than 0.5 and less than or equal to 1.0 indicates persistent longrange power-law correlations in which a large fluctuation is more likely to be followed by another large fluctuation. A special case α=1 corresponds to 1/f noise. When 1
Abstract Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in modulation of heart rate dynamics during systemic inflammation. Citation: Mazloom R, Eftekhari G, Rahimi M, Khori V, Hajizadeh S, et al. (2013) The Role of α7 Nicotinic Acetylcholine Receptor in Modulation of Heart Rate Dynamics in Endotoxemic Rats. PLoS ONE 8(12): e82251. doi:10.1371/journal.pone.0082251 Editor: Charles C Caldwell, University of Cincinnati, United States of America Received July 10, 2013; Accepted October 22, 2013; Published December 10, 2013 Copyright: © 2013 Mazloom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from Tarbiat Modares University and Golestan University of Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected]
Introduction
Experimental studies in animal models have also indicated that endotoxemia is associated with a significant reduction in HRV [6–9]. Likewise, systemic administration of interleukin-6 (IL-6) decreases HRV in mice [10]. The mechanism of increased regularity of cardiac cycle during systemic inflammation in not very well understood. Recent studies have shown that partial uncoupling of cardiac pacemaker cells from cholinergic neural control may play a role in endotoxin-induced loss of HRV in experimental models [9,11]. Moreover, incubation of mouse isolated atria with recombinant IL-6 was associated with a significant reduction in chronotropic response to carbacholine (a cholinergic agonist) in vitro [10]. These reports indicate that activation of inflammatory pathways in cardiac pacemaker cells
Cardiac rhythm displays a complex dynamics in physiological state which is due to non-linear interaction between cardiac pacemaker cells and the autonomic nervous system [1]. A variety of methods have been developed to assess cardiac cycle dynamics in health and disease [2]. These methods have provided evidence to show that heart rate dynamics is altered during systemic inflammation (e.g. in patients with sepsis) [2]. Therefore, heart rate variability (HRV) analysis has been used for non-invasive monitoring of patients with sepsis [3]. These reports have shown that decreased HRV and increased cardiac cycle regularity has diagnostic and prognostic value in patients with systemic inflammatory response syndrome [3–5].
PLOS ONE | www.plosone.org
1
December 2013 | Volume 8 | Issue 12 | e82251
α7nACHR and Heart Rate Dynamics in Endotoxemia
Chemicals
might affect its responsiveness to parasympathetic autonomic nervous control. Recent investigations suggest that activation of vagus nerve can suppress pro-inflammatory cytokine levels in liver and spleen in mice [12]. Furthermore, electrical stimulation of the vagus nerve inhibits tumor necrosis factor-alpha (TNF-α) synthesis in wild-type mice, but fails to inhibit TNF-α synthesis in alpha7-nicotinic acetylcholine receptor (α7nACHR) deficient mice [13]. Since this discovery, the contribution of α7nACHR in modulation of inflammation has been shown in a variety of model systems [14]. α7nACHR is a homopentameric ligandgated ion channel [14] which acts as a calcium channel [15] as well as a ligand-dependent coupler to secondary and tertiary messenger systems [16]. α7nACHR is distributed in several tissues [17], especially in macrophages which are important producer of inflammatory cytokines [14]. Atrial cells receive dense cholinergic innervation and express muscarinic cholinergic receptors [18]. Little is known about the expression of α7nACHR in atrial cells; however previous studies in rats have shown that cardiomyocytes exhibit immunoreactivity for α7nACHR at all embryonic developmental stage, although adult cardiomyocytes immunoreactivity for this receptor is weak [19]. The role of α7nACHR in modulation of cardiac function is not well understood. Deck et al. reported that α7nACHR is not required for parasympathetic control of heart rate in mouse [20]. However the role of this receptor in modulation of inflammatory process in the atria has not been investigated. Sepsis and endotoxemia are associated with decreased HRV and impaired atrial chronotropic responsiveness to muscarinic cholinergic neural control [9]. The underlying mechanism of this phenomenon is uncertain, and the role of anti-inflammatory mediators in modulation of heart rate dynamics during systemic inflammation is not well understood. Fairchild et al. demonstrated that dexamethasone shortened but did not eliminate LPS-induced HRV depression in mice [8]. Likewise, Alvarez et al. reported that administration of a glucocorticoid before endotoxin reduced cytokine levels but did not affect HRV indices in human volunteers [21]. α7nACHR as a key component of cholinergic anti-inflammatory pathway have been shown to modulate local and systemic inflammation in a variety of model systems. However the role of this receptor in sepsis-induced HRV depression is not clear. In present study we investigated the role of α7nACHR in modulation of heart rate dynamics in endotoxemic rats.
Endotoxemia was induced by intraperitoneal (IP) injection of lipopolysaccharide (LPS; Salmonella typhimurium) which was purchased from Sigma-Aldrich (Pool, UK). PHA-543613 hydrochloride (PHA) and methyllycaconitine citrate (MLA) were purchased from Tocris (Bristol, UK) and were used as selective agonist and antagonist for α7nACHR respectively. All of these substances were dissolved in 0.9% saline. All other reagents were purchased from Merck (Darmstadt, Germany) unless stated otherwise.
Animals Male Sprague-Dawley rats were obtained from Razi Institute (Hesarak, Iran). The effect of endotoxin on heart rate dynamics was assessed following IP administration of LPS at two doses of 0.1 and 1 mg/kg. To assess the effect of α7nACHR inhibition on HRV, either saline or LPS (0.1 mg/kg) was injected 30 min after MLA administration (5 mg/kg, IP) [22]. The effect of α7nACHR activation on heart rate dynamics was evaluated by subcutaneous injection of PHA (1 or 4 mg/kg) [23,24] 30 min before IP administration of either control or LPS (1 mg/kg). 6-9 rats were used in each group. Our pilot studies showed that while IP administration of 1 mg/kg of LPS reduced HRV, low dose (0.1 mg/kg) LPS was unable to reduce HRV indices in rats. Based on pilot studies, 1 mg/kg of LPS was the dose that could induce maximum reduction in HRV indices. We observed that higher doses of LPS (e.g. 10 and 20 mg/kg) could not further reduce HRV in rats. We took advantage of this dose-dependent effect of LPS on HRV parameters and chose lower dose of LPS (0.1 mg/kg) for challenge with MLA (α7nACHR antagonist). This enabled us to monitor the effect of MLA on HRV when there is enough capacity for further reduction in HRV indices. Likewise, higher dose of LPS (1 mg/kg) was used to test the effect of PHA (an α7nACHR agonist) on HRV, when there is enough room to restore the loss of HRV parameters after endotoxin challenge.
Telemetric recording of electrocardiogram and body temperature from conscious rats Telemetric recording of electrocardiogram was carried out as described [9,11]. In brief, a dorsally mounted radiofrequency transmitter were implanted subcutaneously (lead I) in animals (body weight 230–250 g) under anesthesia using ketamine (100 mg/kg) and xylazine (10 mg/kg). 14 days after the operation, electrocardiogram (ECG) and body temperature were recorded using a telemetry system (Data Sciences International, St. Paul, Minnesota, USA) connected to a Powerlab data acquisition system (ADInstruments, Sydney, Australia).
Materials and Methods Ethics statement All animal maintenance and procedures were in accordance with recommendations established by the Animal Ethics Committee of Tarbiat Modares University as well as the United States NIH guidelines (publication no. 85-23). The protocol was approved by the Ethics Committee of Tarbiat Modares University. All surgeries were performed under deep anesthesia, and all efforts were made to minimize suffering.
PLOS ONE | www.plosone.org
Data acquisition The R peaks were detected and the R-R interval series were generated using Chart 5 software (ADInstruments). The R-R interval series were visually inspected and 5 minute artifactfree continuous R-R intervals were chosen for analysis.
2
December 2013 | Volume 8 | Issue 12 | e82251
α7nACHR and Heart Rate Dynamics in Endotoxemia
HRV analysis
dissection, a stabilization period of 30 min was allowed before evaluation of the spontaneous electrical activity. The signals were then digitized at the sampling rate of 10 kHz and displayed on a Powerlab system (ADInstruments, Sydney, Australia). Beating rate variability parameters (SDNN, SampEn and DFA) were calculated in 10 min R-R time-series as described above.
The standard deviation of the R-R intervals (SDNN) was calculated on the selected artifact-free trace and used as a measure of total HRV. Non-linear measures of HRV provide information on the structure or complexity of the R-R timeseries. In the present study non-linear measures of HRV were assessed using Poincaré plot, sample entropy (SampEn) as well as detrended fluctuation analysis (DFA). Poincaré plot. The Poincaré plot is a graphical representation of the correlation between consecutive R-R intervals. The standard deviation of the points perpendicular to the line of identity (SD1) describes short-term variability which is mainly related to the effects of respiration on vagal drive [25]. This parameter was calculated as described [26] using the software developed by Niskanen et al. [27]. SampEn. SampEn was developed by Richman and Moorman in 2000 [28] and calculates the probability that epochs of window length m that are similar within a tolerance r remain similar at the next point [28]. A lower value of SampEn reflects a higher degree of regularity, and the higher the entropy value, the more random the time series is. In the present study, the parameter m was fixed to 2, and tolerance level r was 0.2 as described [9]. DFA. DFA quantifies fractal-like correlation properties of RR intervals [29]. In this method, the fluctuation of the integrated and detrended data is measured within observation windows of various sizes and then plotted against window size on a log-log scale. A linear relationship between log (fluctuation) and log (window size) indicates the presence of scaling which serves as a characteristic of a fractal-like time-series [29]. The scaling exponent alpha (α) indicates the slope of this line. An α=0.5 indicates white noise (uncorrelated random data). An α greater than 0.5 and less than or equal to 1.0 indicates persistent longrange power-law correlations in which a large fluctuation is more likely to be followed by another large fluctuation. A special case α=1 corresponds to 1/f noise. When 1
