Inhibition of Protein Kinase C Signalling On Cardiac Noradrenaline Uptake in the Spontaneously Hypertensive Rat

Abstracts / Autonomic Neuroscience: Basic and Clinical 192 (2015) 56–141

However, there is still no direct evidence to show that in utero exposure to smoking alters the dynamics of cardiac autonomic control in sleeping preterm neonates. Methods and Results: We studied healthy, preterm neonates (postmenstrual age: 36.2 ± 0.9 weeks) born to mothers who had smoked throughout pregnancy but not since birth (n = 16). In uteroexposed neonates were matched with control preterm neonates born to nonsmoking mothers (n = 18). Cardiac autonomic control was monitored as a function of the sleep state by assessing heart rate variability with both linear and non-linear methods. Preterm neonates with in utero exposure to smoking displayed alterations (relative to control neonates) in short-term cardiac autonomic control in all sleep states. These alterations included low vagal activity, elevated sympathetic activity, and low complexity and adaptability in heart rate control dynamics. Conclusion: Our results constitute direct evidence that cardiac autonomic activity and control are altered in preterm infants exposed to smoking in utero. These alterations may place the affected infants at a higher risk of neurological and cardiovascular complications, which could conceivably persist throughout childhood and adulthood. doi:10.1016/j.autneu.2015.07.075

P5.24 Inhibition of Protein Kinase C Signalling On Cardiac Noradrenaline Uptake in the Spontaneously Hypertensive Rat Julia Shanks, Jamie Mahwhinny, David J. Paterson Burdon Sanderson Cardiac Centre Department of Physiology, Anatomy and Genetics, Sherrington Building, Parks Road, Oxford, UK Background: Hypertension is associated with an increase in cardiac sympathetic transmission, although the exact mechanism underlying this is unknown. A decrease in cardiac norepinephrine reuptake transport (NET-1), and therefore the termination of the sympathetic signal has been reported in pre-hypertensive and hypertensive spontaneously hypertensive rats (SHR). NET is known to be a dynamic highly regulated transporter where protein kinase C (PKC) is thought to facilitate its phosphorylation, but also prevent its translocation to the pre-synaptic membrane. Aim: We studied the role of PKC on NET regulation, and tested if its inhibition could rescue the impaired NET activity in the SHR. Method: A novel fluorescent assay of the NET transporter was used on isolated cardiac stellate postganglionic neurons. Results: The PKC inhibitor calphostin C caused a significant dose dependent increase in NET activity (40nM n = 8, 60nM n = 8, 80nM n = 6, and 105nM n = 3) in WKY neurons (105nM n = 5, P b 0.05). Similarly NET activity in SHR neurons was also increased in the presence of calphostin C compared its control (100nM n = 15, P b 0.05). Moreover, there was no difference in NET rate between SHR and WKY neurons after calphostin C. Conclusion: Reduced NET activity in the SHR may be due to increased PKC expression and increased phosphorylation of NET transporters in the cytosol that prevents insertion of the transporter into the presynaptic membrane. Strategies that up regulate NET activity may help rescue the sympathetic hyperactivity observed in this model system. doi:10.1016/j.autneu.2015.07.076

P5.25 Emma Bardsley University of Oxford, UK Background: Spontaneously hypertensive rats (SHRs) and young pre-hypertensive SHRs have enhanced cardiac sympathetic activity; a feature underpinned by impaired cyclic nucleotide signalling linked

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to abnormal calcium-dependent exocytosis. The crosstalk between cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in peripheral stellate neurons is poorly understood. We used Förster Resonance Energy Transfer (FRET) to test whether cAMP and protein kinase A (PKA) activity is greater in sympathetic neurons from SHR and pre-SHR compared with normotensive controls. Method: Cardiac stellate neurons were isolated from male wistar rats and SHR at 4-weeks (pre-hypertensive) or 16-weeks. On the day of culture stellates were infected with either cAMP or PKA reporter adenovirus particles for 24 hours, and FRET experiments were carried out 3-4 days post-isolation. Data were obtained from neurons expressing FRET biosensors using dualemission imaging, where YFP and CFP emission intensities were measured. During imaging, cells were perfused continuously with Tyrode’s, or stimulated with forskolin (0.1-25 μM) and saturating concentrations of 3-isobutyl-1-methylxanthine (IBMX; 100 μM). Results: There was a significant and concentration-dependent increase in cAMP and PKA activity following forskolin administration in the absence and presence of IBMX, which was significantly higher −7 in 16-week SHR vs. normotensive controls (PKA EC50: 1.2x10 M −7 and 5.1x10 M; SHR, wistar respectively). Furthermore, cAMP and PKA hyperactivity was demonstrated in pre-hypertensive SHRs −7 compared with young wistar rats (PKA EC50: 2.8x10 M and −7 5.6x10 M; SHR, wistar respectively). Summary: These results show that cAMP and PKA hyperactivity occurs early in the development of the cardiac sympathetic phenotype in this genetic based model of hypertension.

doi:110.1016/j.autneu.2015.07.077

P6 Cardiac-Heart Rate Variability P6.1 Cardiovascular autonomic regulation in mice overexpressing the vesicular acetylcholine transporter (VAChT) gene G.C.S.V. Tezinia, L.E.V. Silvaa, M. Oliveiraa, S. Guatimosimb, R. Fazan Jr.a, V.F. Pradoc, M.A.M. Pradoc, H.C. Salgadoa a School of Medicine of Ribeirão Preto, University of São Paulo, Brazil b Federal University of Minas Gerais, Brazil c Robarts Research Institute, The University of Western Ontario, Canada Recently, a transgenic animal with overexpression of the vesicular acetylcholine transporter (VAChT) gene, named ChAT–ChR2–EYFP, was developed. This mutant has several extra copies of VAChT exhibiting a three-fold increase in the release of acetylcholine. The consequences of overexpression of VAChT protein for the cardiovascular system were not characterized yet. Mice were assigned as Wild-type (WT, n = 7) and ChAT–ChR2–EYFP (n = 9) and were implanted with probes for arterial pressure (AP) and heart rate (HR) recording by telemetry. Ten days after probe implantation it was assessed the hemodynamics (AP and HR) and autonomic function [Cardiac sympathovagal balance evaluated by HR responses to methylatropine and propranolol; Pulse interval variability (HRV) by linear - time and frequency domain - and nonlinear methods]. As compared to WT, ChAT–ChR2–EYFP mice showed lower HR, similar AP, higher vagal tone and lower intrinsic HR (IHR). Time domain indexes of HRV were similar between groups, while spectral analysis showed lower LF (0.1-1 Hz) and higher HF (1-5 Hz) of HR spectra in ChAT–ChR2–EYFP. Symbolic analysis found lower 0 V and higher 2UV, and multiscale entropy revealed higher entropy at scales 1 to 4 in ChAT–ChR2–EYFP. The overall results suggest that cardiovascular autonomic regulation of ChAT–ChR2–EYFP mice is characterized by