The electrophysiological and mechanical effects of gap junction uncoupling in cardiac muscle

Kettlewell, Sarah (2002) The electrophysiological and mechanical effects of gap junction uncoupling in cardiac muscle. PhD thesis, University of Glasgow.

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Abstract

The aim of this study was to study the electrophysiological and mechanical effects of the gap junction uncoupler 1-heptanol in the left ventricle (epicardial surface) of the artificially perfused Langendorff rabbit heart. Specifically, electrical restitution and the dispersion of repolarisation were studied.

Methods. Using a single monophasic action potential (MAP) electrode, in the healthy and failing (coronary ligated) heart, the effect of 1-heptanol was studied on rate dependent changes in action potential duration. Dispersion of repolarisation was measured sequentially.

A 32 MAP electrode array was developed to simultaneously record dispersion of repolarisation from the epicardial surface of the left ventricle of healthy hearts. Restitution was studied using an extrastimulus protocol that involved electrically stimulating the heart with 16 S1 stimuli (350ms intervals), and an extrastimulus S2. S1-S2 interval was increased progressively from 70 to 600ms. S1-S2 changes of 5ms were made between 70 and 150ms, 10ms between 150 and 350ms, and 50ms between 350 and 600ms. Protocols were run at 37°C, initially in Tyrode's solution, then after addition of 0.3mM 1-heptanol.

Results. The single catheter study showed that failure significantly (P<0.05) prolongs MAP duration between cycle lengths of 250ms and 650ms. No base to apex changes, changes in dispersion of repolarisation or ventricular fibrillation thresholds were observed. 1-Heptanol, at cycle lengths above 350ms, significantly (P<0.05) decreased MAP duration in failing and healthy hearts. 1-Heptanol however did not alter the dispersion of repolarisation or ventricular fibrillation threshold in healthy and failing hearts.

The last SI MAP in the 16 beat train and the S2 MAP obtained using the 32 electrode array were analysed at 90% repolarisation (MAPD90). S2 MAPD90 increased with S I-S2 interval up to -180ms but decreased at longer intervals. 0.3mM l-heptanol exacerbated this negative slope in the restitution curve from (mean±SEM) -0.031±O.004 in Tyrode's compared to -O.063±O.005 in O.3mM 1-heptanol (P<O.OOI). Dispersion of repolarisation was increased (p>O.05) in the presence of O.3mM l-heptanol. Conduction delay was increased from (mean±SEM) 44.2±0.82ms to 49.2±O.87ms (P<O.OOI).

The possibility of an effect on the single cell being the mechanism behind the exacerbation of the negative slope by l-heptanol was investigated in a single cell study. The effect of l-heptanol on single cell fractional shortening and Ca2+ handling was examined. At 1 and 3Hz l-heptanol decreases fractional cell shortening from (mean±SEM): at 1Hz 9.3±0.8% in Krebs to 5.7±O.7% (O.03mM 1-heptanol), 5.6±O.9% (O.lmM l-heptanol) and 3.2±0.8% (O.3mM lheptanol) (P<O.Ol); at 3Hz lO.6±O.8% in Krebs to 6.S±1.4% (O.03mM l-heptanol), 7.7±1.4% (O.lmM l-heptanol) and 4.7±l.S% (O.3mM l-heptanol) (P<O.Ol). The negative inotropic (Le. the reduction in contractility) effect of I-heptanol indicated that this agent is not a specific gap junction uncoupler. SR ci+ release was reduced at 3Hz only by (mean±SEM) 7.8±O.04% (O.03mM l-heptanol), 5.0±0.03% (O.lmM 1-heptanol) and 7.9±O.02% (P<O.OS),indicating an effect on the L-type Ca2+ channel or ryanodine receptor.

The effect on the L-type Ca2+ channel was investigated by the use of nifedipine firstly in single cells then in the whole heart. Nifedipine decreased single cell fractional shortening at 1Hz from (mean±SEM) 8.9±O.7% in Krebs to S.8±O.7% (O.lJ,tM nifedipine), 3.4±O.9% (O.l5J,tM nifedipine, P<O.OOI)and O.8±O.2% (O.2JlM nifedipine, P<O.OOl). SR Ca2+ release was also reduced from (mean±SEM) O.75±O.017% in Krebs to O.72±O.024% (O.IJlM nifedipine), O.73±0.024% (O.l5JlM nifedipine), O.71±O.016% (O.2J,tM nifedipine) (P<O.OS). In the whole heart nifedipine did not induce a negative slope in the restitution curve indicating no role of the L-type Ca2+ channel in this phenomenon.

Carbenoxolone, a novel specific gap junction uncoupler failed to induce a negative slope in the electrical restitution curve of the whole heart but did increase dispersion of repolarisation (P>0.05) and caused a significant conduction slowing from (mean±SEM) 45.50±2.I2ms in Tyrode to 55.11±2.82ms in carbenoxolone (P<0.05). Carbenoxolone has an inconsistent effect on single cell fractional shortening and Ca2+ handling.

Conclusions. The biphasic relationship and the increased dispersion of repolarisation in the presence of 0.3mM I-heptanol may have implications for the development of alternans and/or arrhythmias (Gilmour and Chialvo, 2000). The cause of the negative slope is as yet unknown. but it is likely that it is an effect on the single cell rather than gap junction uncoupling.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: R Medicine > RM Therapeutics. Pharmacology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Funder's Name: UNSPECIFIED
Supervisor's Name: Supervisor, not known
Date of Award: 2002
Depositing User: Ms Mary Anne Meyering
Unique ID: glathesis:2002-6223
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Mar 2015 12:19
Last Modified: 27 Mar 2015 12:21
URI: http://theses.gla.ac.uk/id/eprint/6223

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