1432
Exp Physiol 98.10 (2013) pp 1432–1445
Research Paper
Elevated blood pressure, heart rate and body temperature in mice lacking the XLαs protein of the Gnas locus is due to increased sympathetic tone Nicolas Nunn1† , Claire H. Feetham2 , Jennifer Martin1 , Richard Barrett-Jolley2∗ and Antonius Plagge1∗ 1 2
Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK
New Findings
Experimental Physiology
r What is the central question of this study?
Previously, we showed that Gnasxl knock-out mice are lean and hypermetabolic, with increased sympathetic stimulation of adipose tissue. Do these mice also display elevated sympathetic cardiovascular tone? Is the brain glucagon-like peptide-1 system involved? r What is the main finding and its importance? Gnasxl knock-outs have increased blood pressure, heart rate and body temperature. Heart rate variability analysis suggests an elevated sympathetic tone. The sympatholytic reserpine had stronger effects on blood pressure, heart rate and heart rate variability in knock-out compared with wild-type mice. Stimulation of the glucagon-like peptide-1 system inhibited parasympathetic tone to a similar extent in both genotypes, with a stronger associated increase in heart rate in knock-outs. Deficiency of Gnasxl increases sympathetic cardiovascular tone. Imbalances of energy homeostasis are often associated with cardiovascular complications. Previous work has shown that Gnasxl-deficient mice have a lean and hypermetabolic phenotype, with increased sympathetic stimulation of adipose tissue. The Gnasxl transcript from the imprinted Gnas locus encodes the trimeric G-protein subunit XLαs, which is expressed in brain regions that regulate energy homeostasis and sympathetic nervous system (SNS) activity. To determine whether Gnasxl knock-out (KO) mice display additional SNS-related phenotypes, we have now investigated the cardiovascular system. The Gnasxl KO mice were ∼20 mmHg hypertensive in comparison to wild-type (WT) littermates (P ≤ 0.05) and hypersensitive to the sympatholytic drug reserpine. Using telemetry, we detected an increased waking heart rate in conscious KOs (630 ± 10 versus 584 ± 12 beats min−1 , KO versus WT, P ≤ 0.05). Body temperature was also elevated (38.1 ± 0.3 versus 36.9 ± 0.4◦ C, KO versus WT, P ≤ 0.05). To investigate autonomic nervous system influences, we used heart rate variability analyses. We empirically defined frequency power bands using atropine and reserpine and verified high-frequency (HF) power and low-frequency (LF) LF/HF power ratio to be indicators of parasympathetic and sympathetic activity, respectively. The LF/HF power ratio was greater in KOs and more sensitive to reserpine than in WTs, consistent with elevated SNS activity. In contrast, atropine and exendin-4, a centrally acting agonist of the glucagon-like peptide1 receptor, which influences cardiovascular physiology and metabolism, reduced HF power equally in both genotypes. This was associated with a greater increase in heart rate in KOs. Mild stress had a blunted effect on the LF/HF ratio in KOs consistent with elevated basal sympathetic
†Current address: Faculty of Life Sciences, University of Manchester, Manchester, UK ∗ A.P. and R.B.-J. contributed equally to this work. DOI: 10.1113/expphysiol.2013.073064
C 2013 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Exp Physiol 98.10 (2013) pp 1432–1445
Sympathetic cardiovascular stimulation in Gnasxl knock-out mice
1433
activity. We conclude that XLαs is required for the inhibition of sympathetic outflow towards cardiovascular and metabolically relevant tissues. (Received 18 March 2013; accepted after revision 6 June 2013; first published online 7 June 2013) Corresponding author A. Plagge: Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool L69 3BX, UK. Email:
[email protected]. R. Barrett-Jolley: Institute of Ageing and Chronic Disease, 4th floor UCD Building, University of Liverpool, Daulby Str., Liverpool, L69 3GA, UK. Email:
[email protected]
It has been increasingly recognized over recent years that disorders of energy balance and metabolism are often associated with cardiovascular disease. Typically, these symptoms occur jointly when central effects are involved. Specifically, the sympathetic nervous system (SNS) has been recognized as a major regulator of metabolic rate and cardiovascular physiology (Matsumura et al. 2003; Hall et al. 2010; Malpas, 2010). Obesity-related cardiovascular symptoms involve central actions of the adipokine leptin, its downstream effector, the melanocortin system, and subsequently increased sympathetic nerve activity (Hall et al. 2010). Much less is known about cardiovascular complications in disorders of hypermetabolism and leanness. Genetic manipulations of the renin–angiotensin system have been shown to increase metabolic rate and affect blood pressure (BP) through central as well as peripheral mechanisms (Dupont & Brouwers, 2010; Grobe et al. 2010). Another model with elevated sympathetic and cardiovascular tone is the Schlager inbred mouse line, which also displays reduced body weight (Davern et al. 2009). However, the underlying genetic cause for their phenotype is unknown. The anatomical organization of brain regions involved in the control of sympathetic activity is well known, although any differential topographical regulation of SNS outflow towards distinct peripheral tissues is less clear (Sved et al. 2001; Malpas, 2010; Nunn et al. 2011). Retrograde tracing studies from various peripheral tissues have indicated a common set of hierarchically organized brainstem and hypothalamic areas to be involved in the control of SNS outflow to diverse organs (Sved et al. 2001). In this study, we analyse mice deficient for XLαs, a Gprotein α-subunit encoded by the Gnasxl transcript of the complex Gnas locus (Plagge et al. 2008). Gnasxl constitutes an alternative splice variant of Gnas and differs only in the NH2 -terminal exon (Fig. S1). Like Gs α, XLαs links a number of G-protein-coupled receptors in vitro, which results in activation of adenylate cyclase and production of cAMP (Liu et al. 2011). However, the specific receptor(s) that signal through XLαs in vivo remain unknown. Gene expression at the Gnas locus is regulated by the epigenetic mechanism of ‘genomic imprinting’, which leads to the silencing of one allele depending on its parental origin (Plagge et al. 2008). Thus, Gnasxl expression occurs exclusively from the paternal allele. By contrast, Gnas
remains expressed biallelically in most cells, with the exception of some tissues where its expression is restricted to the maternal allele, e.g. in the paraventricular nucleus (PVN) of the hypothalamus (Fig. S1; Chen et al. 2009). Loss of XLαs in mice results in a lean and hypermetabolic phenotype, caused by increased sympathetic stimulation of brown and white adipose tissues, and it has been proposed that there may be a systemic increase in SNS outflow (Xie et al. 2006). Expression of XLαs in adult mice is limited almost exclusively to the brain and correlates with SNS control centres, including the intermediolateral layer (IML) of the spinal cord, the ventrolateral medulla, medullary raphe nuclei, the nucleus tractus solitarii (NTS), hypothalamic nuclei (PVN, dorsomedial nucleus, lateral hypothalamic area, arcuate and suprachiasmatic nuclei) and the preoptic area (Pasolli & Huttner, 2001; Krechowec et al. 2012). Its expression in regions such as the PVN and dorsomedial nucleus suggests that it may influence wider sympathetic outflow, including targets within the cardiovascular system (Coote, 2007; Womack et al. 2007). Here, we explore the cardiovascular phenotype of Gnasxl knock-out (KO) mice and its regulation by the autonomic nervous system. The effects of inhibition of the sympathetic and parasympathetic nervous system (PNS), respectively, were examined using reserpine, which blocks the vesicular monoamine transporter at synapses, and atropine, which inhibits muscarinic acetylcholine receptors (Janssen et al. 2000; Young & Davisson, 2011). To begin an investigation into potentially deregulated neuropeptide systems that might be involved in the Gnasxl KO phenotype, we explored responses to activation of the brain glucagon-like peptide-1 (GLP-1) system. Glucagon-like peptide-1, apart from its peripheral role, also acts as a neuropeptide produced in the NTS of the medulla (Llewellyn-Smith et al. 2011). As the GLP1 receptor signals via an α-stimulatory G-protein and cAMP, this pathway could potentially be affected in XLαs KO mice. Activation of the central GLP-1 receptor has dual autonomic effects. It inhibits parasympathetic control of the cardiovascular system, resulting in increased heart rate (HR) and BP (Barragan et al. 1999; Yamamoto et al. 2002; Hayes et al. 2008; Griffioen et al. 2011), while it also stimulates sympathetic outflow towards metabolically relevant tissues (Nogueiras et al. 2009).
C 2013 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.
1434
N. Nunn and others
Furthermore, the c-fos response to the comparatively stable GLP-1 receptor agonist exendin-4 (Ex-4), which elicits identical central effects independent of its route of injection (intraperitoneal or intracerebroventricular), coincides with brain regions that control sympathetic outflow in the hypothalamus, medulla and spinal cord (Yamamoto et al. 2002; Baraboi et al. 2011). In this study, we present BP and HR data obtained from anaesthetized and conscious mice, respectively. Autonomic influences on HR were examined by heart rate variability (HRV) analyses. Heart rate and HRV responses to reserpine, atropine, Ex-4 and handling stress were measured. Additionally, neuronal activation in response to Ex-4 was quantified histologically via c-fos expression analysis in wild-type (WT) and KO mice. Methods
Exp Physiol 98.10 (2013) pp 1432–1445
saline; doses were matched within sibling groups. Body temperature was recorded by rectal probe 3 min after injection of anaesthetic agents and prior to application of external heat. After this, temperature was continuously monitored and maintained at 37 ± 0.5◦ C by heat pad and infra-red lamp. Following loss of reflexes, the trachea was intubated to facilitate breathing, and the carotid artery was cannulated with stretched PP25 tubing filled with heparinized saline, connected to a pressure transducer. The raw blood pressure signal was digitized to a PC with a CED Micro1401 (CED, Cambridge, UK) using Spike2 at 5 kHz. The raw signal was also split, AC coupled and amplified between 10 and 100 times, depending on signal strength, and recorded on Spike2 at 5 kHz. Heartbeats were annotated to the amplified AC-coupled blood pressure signal using Wabp from the Physionet suite of programs (Goldberger et al. 2000). The numbers of animals used are indicated in the Results section.
Ethical approval
All animal work was approved by the Ethical Review Committee of the University of Liverpool and carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 (UK Home Office Project Licences PPL40/3009 and PPL40/3351). All surgery was performed under general anaesthesia as described in detail below. Animals
Gnasxl mutant mice (Plagge et al. 2004) were maintained on a CD1 background. Gnasxl KO offspring (Gnasxl m+/p− , i.e. lacking XLαs expression from the paternally inherited allele) were produced by mating CD1 females with Gnasxl mutation-carrying males. All experiments were performed on young adult (∼4-month-old) male Gnasxl KO and WT siblings. Mice were maintained in the animal facility of the University of Liverpool on a 12 h–12 h light–dark cycle and had unlimited access to water and standard chow diet. Blood pressure recordings and body temperature measurements
For BP recordings via tail volume pressure recording (VPR) plethysmography, which measures systolic and diastolic pressure (CODA VPR system; Kent Scientific, Torrington, CT, USA), mice were lightly anaesthetized (urethane, 1.5 mg kg−1 I.P.; Sigma-Aldrich, UK) and kept in thermoneutral conditions (ambient temperature 30◦ C) throughout recording, which commonly results in lower BP values compared with conscious mice at room temperature (Swoap et al. 2004). A minimum of 10 readings were taken from each mouse; any values with low (
