Neuronal nitric oxide synthase-CAPON regulation of cardiac sympathetic activity in the development of hypertension

The studies presented in this thesis were undertaken to investigate the cellular and molecular mechanisms responsible for sympathetic hyperactivity that is observed in the Spontaneous Hypertensive Rat (SHR) and whether these abnormalities arise even before the onset of hypertension. Moreover, selected molecular candidates related to oxidative state in cardiac autonomic signalling have been explored for their potential therapeutic effects.
Chapter One is an overview of (i) the relevance of autonomic dysfunction in cardiovascular disease in both human and animal models, (ii) the physiological basis of cardiac sympathetic neurotransmission, (iii) the neuromodulators of peripheral cardiac sympathetic-vagal balance discussed along with how they may be involved in cardiac adrenergic control of neurotransmission and NO-cGMP signalling. This develops the formulation of the specific aims of the thesis.
Chapter Two outlines a detailed rationale for the experimental approach taken to (i) characterise protein expression in the pre-hypertensive animal model with immunohistochemistry and Western blotting, (ii) manipulate selected gene expression to amplify NO-cGMP signalling in vivo and in vitro via viral gene transfer, (iii) investigate calcium handling in cardiac sympathetic stellate neurons with calcium imaging , (iv) measure cardiac noradrenergic neurotransmission from double atria using radioactive-labelled [3H]-noradrenaline.
Chapter Three demonstrated abnormal NO-cGMP signalling in pre-hypertensive SHRs. Endogenous nNOS protein residing in both cardiac parasympathetic and sympathetic neurons was significantly lower in the pre-hypertensive SHR compared to aged-matched WKYs. This was associated with lower cGMP levels. An enhanced depolarization evoked [Ca2+]i transient was observed in cardiac stellate neurons from pre-hypertensive SHR when compared with the WKY, an effect that was reversed by nNOS or sGC inhibition.
Chapter Four investigated the role of nNOS and brain natriuretic peptide (BNP) in cGMP signalling pathways. Gene transfer of nNOS via adenoviral vector in SHR cardiac sympathetic neurons increased cGMP concentration and normalised neuronal calcium handling during depolarization. BNP significantly reduces [3H]- noradrenaline release. Overexpression of PDE2 which facilitates the breakdown of cGMP caused an increase in [3H]- noradrenaline release in response to field stimulation and also prevented the inhibitory action of BNP.
Chapter Five examined the role of the nNOS adaptor protein, CAPON in NO-cGMP signalling. Endogenous CAPON protein is present in cardiac sympathetic neurons in the WKY, and is significantly reduced in pre-hypertensive SHR cardiac neurons. Artificial up-regulation of cardiac sympathetic CAPON via targeted gene transfer directly attenuated neuronal Ca2+ transients, resulting in decreased noradrenaline release in the SHR.
Chapter Six is a concluding discussion summarising the main findings from this thesis, placing them in a physiological context and discussing avenues for further research.

Keywords: nNOS
dysautonomia
Hypertension
CAPON
Advisor: Paterson, D
Herring, N