A study of excitation contraction coupling in rabbit cardiomyocytes

Miller, Stewart L.W (2004) A study of excitation contraction coupling in rabbit cardiomyocytes. PhD thesis, University of Glasgow.

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Abstract

Excitation-Contraction coupling is the process that links the depolarisation of the myocardium to its contraction. We have utilised an adenoviral- mediated strategy to overexpress sorcin, calsequestrin (CSQ), FKBP 12.6 and the sodium/calcium exchanger (NCX) in isolated rabbit cardiomyocytes over a period of 24hrs in cell culture. The effects of overexpression of the above-mentioned proteins on excitation-contraction coupling were subsequently investigated using whole cell voltage clamp. The loading of cardiomyocytes with FURA-2 enabled simultaneous measurement of intracellular calcium concentration. Cardiomyocytes transfected with an adenovirus that caused overexpression of the enzyme beta-galactosidase were employed as a control. Sorcin overexpression was found to significantly reduce both the amplitude of the intracellular Ca2+ transient and the size of the SR Ca2+ load under conditions of whole cell voltage clamp when compared to control. It had no effect on the L-type Ca2+ current but significantly enhanced the activity of the sodium/calcium exchanger. Subsequently, overexpression of NCX was shown to affect excitation contraction coupling in a similar manner to sorcin overexpression. CSQ overexpression was shown to significantly increase the size of L-type Ca2+ current, the intracellular Ca2+ transient amplitude and the SR Ca2+ load under conditions of whole cell voltage clamp when compared to control. When the L-type Ca2+ current in the CSQ overexpressing cardiomyocytes was normalised using nifedipine the SR Ca2+ load remained increased while the amplitude of the intracellular Ca2+ transient was similar to that observed in control cardiomyocytes. Finally, FKBP 12.6 overexpression significantly increased both the amplitude of the intracellular Ca2+ transient and the size of the SR Ca2+ load under conditions of whole cell voltage clamp when compared to control. It had no effect on the L-type Ca2+ current. Over expression of an FKBP 12.6 mutant that lacked a calcineurin binding site had little effect on the size of the intracellular Ca2+ transient but still significantly increased the SR Ca2+ load under conditions of whole cell voltage clamp when compared to control. Experiments previously carried out in the laboratory using confocal microscopy have demonstrated that both sorcin and FKBP 12.6 overexpression alter the occurrence of spontaneous Ca2+ sparks in permeabilised cardiomyocytes. Conversely, CSQ overexpression was shown to have no effect on the occurrence of Ca2+ sparks.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Godfrey Smith
Keywords: Physiology
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-70988
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 09 May 2019 14:28
Last Modified: 09 May 2019 14:28
URI: http://theses.gla.ac.uk/id/eprint/70988

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