Voltage-gated sodium channels (VGSCs) generate the Na+ current (INa) responsible for the cardiac action potential upstroke largely due to the ‘cardiac’ isoform Nav1.5, but other isoforms are responsible for ˜20% of the upstroke and a component of a sustained sodium current. This sustained component has been attributed with an increasing arrhythmia risk, particularly within the elderly and has been the target of development for new anti-arrhythmic drugs. Our study has investigated the age-associated changes in protein density and localisation of the VGSC alpha-subunits Nav1.5 and Nav1.4 in the rat heart.
Rats at 6, 12 and 28 months of age were sacrificed, their hearts dissected into the regions of left and right ventricle, left and right atria, epicardium and endocardium (n = 5). These regions were analysed by western blot to determine the protein expression of Nav1.5 and Nav1.4 (Alomone, Israel), normalised to the expression of desmin (Dako, UK). Immunocytochemistry of single cardiac myocytes was used to observe the protein distribution and location of Nav1.5 and Nav1.4 conjugated to Alexa 488 (Molecular probes, UK), and cellular membranes were labelled by wheat germ agglutinin conjugated to rhodamine (Vector, UK), viewed by confocal microscopy (Zeiss 710, Germany). Ethical approval was given by the University of Hull. Mean±SEM.
Nav1.5 protein was expressed at the lateral membrane, intercalated disk and t-tubular membrane in single myocytes. When examining the tissues, the relative expression of Nav1.5 was significantly reduced with age (ANOVA, p = 0.01). It was determined Nav1.5 protein expression in the right atria was at 6 months (100 ± 12.3%) and similar at 12 months, however at 24 months there was a significant decline to 50 ± 8.65% (Student T-test, Bonferroni adjustment <p = 0.006). Intense punctate labelling of intercalated disks was observed with Nav1.4, and a similar significant decline in right atrial protein expression observed at 24 months (ANOVA, p = 0.05). We conclude that there is an age-dependent decline in both Nav1.5 and Nav1.4 within the right atria, possibly contributing towards arrhythmia generation in the elderly, however the underlying mechanisms remain unclear.
- Sodium Channel
- western blot
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