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Title: Neuronal control of cardiac excitability in pro-hypertensive states
Author: Larsen, Hege Ekeberg
ISNI:       0000 0004 6496 4976
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Hypertension is associated with marked cardiac sympathetic over-activity and end organ hyper-responsiveness. The sympathetic dysfunction is caused by aberrant calcium (Ca2+) handling resulting in enhanced neurotransmission. However, it remains unclear whether the sympathetic neuron or the myocytes is the primary driver behind the initiation and maintenance of the autonomic phenotype. The work in this thesis characterises the Ca2+ dysfunction and regulation at the membrane level. Further, it employs physiologically coupled sympathetic neurons and ventricular myocytes to determine the cellular driver of cardiac dysautonomia in the pro-hypertensive state. Chapter 1 provides a general overview of the field of autonomic hypertension with a specific focus on the sympathetic control of cardiac excitability. In particular, the role of Ca2+ and cyclic nucleotides in the facilitation of neurotransmission are explored. Chapter 2 details the methods used in this thesis. It provides rationale for the approaches taken to record membrane Ca2+ currents, cyclic adenosine monophosphate (cAMP) levels and cAMP-activated protein kinase (PKA) activity, and the development and uses of a co-culture of coupled sympathetic neurons and ventricular myocytes. Chapter 3 describes the successful development of an effective voltage clamp method to isolate whole cell Ca2+ currents in sympathetic neurons. It details the issue of space clamp problem when using this technique on peripheral neurons and provides experimental guidance on how to quantify and limit theses issues. Chapter 4 identifies that the pro-hypertensive four-week old neurons from the spontaneously hypertensive rat (SHR) have significantly larger whole cell Ca2+ currents when compared to normotensive (Wistar Kyoto-WKY) neurons, that are largely N-type in nature. Restoring the cGMP cyclic nucleotide dysfunction seen in these cells, rescues the ion channel phenotype and bring the Ca2+ down to levels seen in the normotensive WKY neuron. Further, it identifies that phosphodiesterase (PDE) 2A inhibition differentially affects the currents in the WKY and SHR, further supporting the notion of PDE2A dominance. Chapter 5 identifies the presence and functional relevance of cGMP cross-talk with the cAMP-PKA pathway in sympathetic neurons. This cross talk is significantly altered in the pro-hypertensive state, via the differential involvement of PDEs. It functionally identifies the presence of PDE3 and PDE2A and provides further evidence that these enzymes could be dysregulated in pro-hypertensive neurons. Chapter 6 describes the use of a co-culture model of ventricular myocytes and sympathetic neurons. Physiological stimulation of the sympathetic neuron with nicotine whilst monitoring cAMP levels in the myocytes confirms that the cellular phenotypes seen in the individual cells are functionally present in the co-culture. Using cross-cultures, it identifies the neuron as the principal driver behind the cardiac sympathetic responses observed in pro-hypertension. The results provide evidence for a dominant role played by the neuron in driving the adrenergic phenotype seen in cardiovascular disease and highlights the potential of using healthy neurons to turn down the gain of neurotransmission, akin to a smart pre-synaptic β-blocker. Chapter 7 forms the concluding discussion that summarises the main findings of this thesis and attempt to place it in a clinical context, and highlights avenues of further research. In particular, the possibility of using a cell therapeutic approach to treat sympathetic hyperactivity.
Supervisor: Larsen, Hege Ekeberg ; Paterson, David J. Sponsor: Wellcome Trust ; Physiological Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Neuro-Cardiac excitability ; Autonomic dysfunction ; Hypertension ; Sympathetic nervous system ; Cardiac neurobiology ; Cardiac-neuronal cell culture ; Cardiac excitability ; co-culture