title: The role of the signalling protein XLalphas in cardiovascular control in mice
Creators: name: Nunn, Nicolas
Publisher: name: University of Liverpool Repository
Year: 2013

description: Loss of the signalling protein XLαs in mice leads to a lean phenotype characterised by increased energy expenditure due to elevated sympathetic nervous system (SNS) stimulation of brown adipose tissue. XLαs is the protein produced from the Gnasxl transcript of the imprinted Gnas locus, and has a restricted expression pattern that includes a number of brain regions essential for SNS control of both energy expenditure and the cardiovascular system. However, it is unknown to what degree XLαs influences overall sympathetic tone, or how XLαs signalling in the brain causes these phenotypic changes. Using arterial cannulation, anaesthetised Gnasxl knockout mice had elevated blood pressure, shown to be caused by increased SNS stimulation by a greater blood pressure response to the sympatholytic reserpine in knockouts. Using electrocardiogram (ECG) telemetry, conscious Gnasxl knockout mice had elevated heart rate at night, as well as a significant heart rate response to both reserpine and the parasympatholytic atropine. This supported the previous results showing elevated SNS stimulation of the cardiovascular system, but paradoxically also suggested elevated parasympathetic stimulation. Therefore, autonomic control of the cardiovascular system was investigated in further detail by analysing heart rate variability (HRV). A number of HRV analyses were experimentally validated in wildtype mice. The most reliable method was the fast Fourier transform (FFT); high frequency (HF) power was used as a measure of parasympathetic activity, and low frequency (LF)/HF ratio was used as a measure of sympathetic activity. Gnasxl knockouts had a greater LF/HF response to reserpine, but an equivalent HF response to atropine, suggesting the mice had elevated SNS activity only. Additionally, knockouts had elevated LF/HF ratio at night, suggesting consistently elevated SNS output. Neuronal signalling pathways that may be deregulated in Gnasxl knockouts were investigated by injection of MTII and Exendin-4, agonists to the melanocortin 3/4 and GLP-1 receptors, respectively. Gnasxl knockouts had a hypersensitive heart rate response both to centrally injected MTII in anaesthetised mice and peripherally injected Exendin-4 in conscious mice. The hypersensitivity to Exendin-4 was investigated further by HRV analysis, which showed that Exendin-4 had no effect on the SNS, but caused a potent reduction in parasympathetic activity in both wildtypes and knockouts. Neuronal signalling changes in response to Exendin-4 were investigated by antibody staining for the early response gene c-fos. No significant differences were seen in overall numbers of activated neurones between wildtypes and knockouts in a number of brain regions including the nucleus of the solitary tract (NTS). Interestingly, neurones expressing XLαs showed no c-fos response to Exendin-4, except in the area postrema. In summary, loss of XLαs in mice resulted in elevated SNS stimulation of the cardiovascular system, as well as hypersensitivity to Exendin-4 that was unlikely to be caused by increased activation of XLαs-deficient neurones.