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Angiotensinergic Neurons in the Neonatal Rat Heart and in Atrial Cell Culture

Mosimann, Stefanie Lea (2020). Angiotensinergic Neurons in the Neonatal Rat Heart and in Atrial Cell Culture. (Thesis). Universität Bern, Bern

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Abstract

The renin-angiotensin system (RAS) and its main effector peptide angiotensin II (Ang II) are known to play a pivotal role in the regulation of heart function, development of cardiac remodeling, arrhythmia, and heart failure. Recently, a novel population of sympathetic neuronal cells containing Ang II was described in the adult rat heart. The aim of the current study was to investigate the presence of Ang II positive neurons in the neonatal rat heart and to develop methods to maintain these neurons in primary atrial cell cultures. The immunohistochemical characterization of angiotensinergic neurons in whole rat hearts and in primary cell culture preparations was based on colocalization studies of Ang II with other components of the RAS and known neuronal cell markers. The survival of Ang II positive neurons in primary cell culture was studied under different culture conditions. Single cell electrophysiological studies were performed using patch clamp recording on isolated neurons in cell culture. Viral transduction using a neuron specific promoter served to investigate the neuronal identity of angiotensinergic cells. In the atria of whole neonatal rat heart preparations, neuronal cell bodies and fibers of intracardiac ganglia were found to contain Ang II that colocalized with neuron specific beta III tubulin (NM III), synaptophysin and calcitonin gene related peptide (CGRP). Ang II and the vesicular acetylcholine transporter (VAChT) displayed an intriguing staining pattern with VAChT positive structures encircling angiotensinergic ganglion cells, which may indicate a functional link between the parasympathetic nervous system and the angiotensinergic system. Labeling of the sympathetic nervous system with antibodies against dopamine-β-hydroxylase (DβH) resulted in strong staining of neuronal cell processes while cell bodies were only weakly stained. Neither renin nor cathepsin D could be detected immunohistochemically. In primary atrial cell cultures, neuron-like cells with long and slender processes exhibiting colocalization of Ang II with NM III were found. Similar to intact tissue, these neurons were renin negative. The finding that VAChT staining was unspecific and DβH was negative suggests that neither sympathetic nor parasympathetic neuronal cells were present in cell culture. The quantity of angiotensinergic neurons in primary culture was unaffected by addition of nerve growth factor, Ang II or aliskiren to the cell culture medium. Adding pepstatin A at high concentration reduced the number of Ang II positive neurons relative to control conditions. Patch clamp recordings of Ang II positive neurons revealed these cells to be excitable. Resting membrane potentials averaged -64.45 mV ± 2.83 mV (n=6). The threshold potential for eliciting action potentials was -47.43 mV ± 6.38 mV (n=6). Action potential duration was ~5 ms and the maximal spike frequency observed was ~20 Hz. Viral transduction experiments showed expression of the enhanced yellow fluorescent protein (eYFP) under the calcium/calmodulin-dependent protein kinase IIa (CaMKIIa) promoter in Ang II positive cells thereby underlining the neuronal identity of the angiotensinergic cells under investigation. Overall, the results show that rat hearts contain an early developed intracardiac angiotensinergic nervous system. When isolated and kept under appropriate culture conditions, Ang II positive cells show the typical electrophysiological characteristics of neurons. Further investigation will be needed to elucidate the specific physiological role of angiotensinergic neurons in heart function.

Item Type: Thesis
Dissertation Type: Single
Date of Defense: 2 September 2020
Subjects: 600 Technology > 610 Medicine & health
Institute / Center: 04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Physiology
Depositing User: Hammer Igor
Date Deposited: 19 Oct 2020 08:04
Last Modified: 02 Sep 2021 00:30
URI: https://boristheses.unibe.ch/id/eprint/2264

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