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The electrophysiological and molecular effects of chronic beta-adrenoceptor antagonist therapy on human atrium

Marshall, Gillian Elizabeth (2008) The electrophysiological and molecular effects of chronic beta-adrenoceptor antagonist therapy on human atrium. PhD thesis, University of Glasgow.

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Abstract

The chronic treatment of patients with a β-adrenoceptor antagonist is associated with prolongation of the atrial cell action potential duration (APD), potentially contributing to the ability of these drugs to prevent atrial fibrillation (AF). The mechanisms underlying this APD prolongation are not fully understood but may involve pharmacological remodelling of atrial K+ currents and underlying ion channel subunits. This project aimed to test the hypothesis that various characteristics of human atrial K+ currents, including voltage, time and rate dependency, differ between patients treated and not treated with a β-blocker as a result of altered expression of ion channel pore-forming and accessory subunits. Human atrial myocytes were isolated enzymatically from right atrial appendage tissue obtained from consenting patients, in sinus rhythm, undergoing cardiac surgery. Using whole cell patch clamping, K+ currents were recorded at physiological temperature. Treatment of patients with β-blockers for a minimum of 4 weeks duration was associated with a significant, 34% reduction in the transient outward K+ current (ITO) density but no change in the sustained outward current (IKSUS). There was a reduction in the Ba2+-sensitive, inwardly rectifying K+ current (IK1) but only at -120 mV and the physiological significance of this is unclear. The reduction in ITO density was not secondary to changes in the voltage dependency of the current, as determined by Boltzmann curve fits. There was no difference in the time dependent inactivation or re-activation of ITO between cells from non β-blocked and β-blocked patients, indicating these current characteristics were not contributing to β-blocker induced APD prolongation. The density of ITO decreased significantly with increasing stimulation rate in cells from both patient groups but remained significantly reduced in β-blocked patients at all rates studied. To determine a possible mechanism underlying the reduction in ITO density, the expression of Kv4.3 mRNA, the pore-forming subunit responsible for this current, was compared in right atrial appendage tissue from non β-blocked and β-blocked patients using real-time RT-PCR. mRNA levels were normalised to the expression of both 28S, a marker of total RNA, and the housekeeping gene GAPDH. The levels of mRNA for the accessory subunits KChIP2, KChAP, Kvβ1 and 2 and Frequenin, which modify Kv4.3 expression and function, were also measured. No change was found in the relative mRNA levels of any of these ion channel subunits in association with chronic β-blockade. mRNA for the pore-forming subunits Kir 2.1 and 2.2 and Kv1.5 which are responsible for IK1 and IKSUS respectively, in addition to mRNA for the pore-forming subunits underlying the L-type calcium current and sodium-calcium exchanger were also measured. Again, no significant changes in expression were found in association with chronic β-blockade. The possibility of ion channel remodelling at a translational level was investigated by measuring Kv4.3 protein levels using Western blotting with a monoclonal anti-Kv4.3 antibody. Kv4.3 protein levels were normalised to GAPDH which was used as a loading control. Chronic β-blockade did not change the ratio of the level of Kv4.3 protein relative to GAPDH. In conclusion, chronic treatment of patients with a β-blocker is associated with a reduction in atrial ITO density which may contribute to the APD prolongation reported in cells from these patients. However, this cannot be explained by changes in the expression of Kv4.3 or by changes in the expression of its regulatory accessory subunit genes.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: chronic beta-blockade, human atrium, atrial fibrillation, electrophysiology, pharmacological remodelling, patch clamp, repolarising potassium currents, ion channels, pore forming subunits, accessory subunits, western blotting, RT-PCR
Subjects: Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Supervisor's Name: Workman, Dr Anthony J and Rankin, Prof Andrew C and Kane, Prof Kathleen A
Date of Award: December 2008
Depositing User: Dr Gillian E Marshall
Unique ID: glathesis:2008-12-406
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 Nov 2008
Last Modified: 10 Dec 2012 13:18
URI: http://theses.gla.ac.uk/id/eprint/406

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