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Effects of Na+/Ca2+-exchanger Overexpression on Excitation–contraction Coupling in Adult Rabbit Ventricular Myocytes

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Abstract

The Na+/Ca2+-exchanger (NCX) is the main mechanism by which Ca2+ is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation–contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation–contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca2+ could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca2+ entry via the L-type Ca2+ channel. Systolic and diastolic Ca2+ levels were significantly reduced, with peak systolic Ca2+ in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca2+. Both cell relengthening and the decay of the Ca2+ transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca2+ stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca2+ stores and low diastolic Ca2+ levels rather than reduced Ca2+ entry.

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    Please address all correspondence to: Dr Sian E. Harding, Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse StLondon SW3 6LY, UK. Tel: 0044 207 351 8146. Fax: 0044 207 823 3392. E-mail: [email protected]

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