Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC163
How does ageing modulate intracellular Ca homeostasis in the sheep atria?
J. D. Clarke1, J. L. Caldwell1, D. A. Eisner1, A. W. Trafford1, K. M. Dibb1
1. Unit of Cardiac Physiology, Univ Manchester, Manchester, United Kingdom.
Ageing is an important risk factor for the development of the most common cardiac arrhythmia, atrial fibrillation (AF). The prevalence of AF is set to increase as society ages. Despite the fact that AF may arise due to perturbations in Ca handling, little is known regarding how intracellular Ca is affected by ageing. We have previously shown altered Ca handling in the aged atria. Here we have investigated how changes in expression levels and functional properties of key proteins could account for these changes. Young adult (18 months) and old sheep (>8 years) were euthanized (200 mg/kg intravenous pentobarbitone) and myocytes isolated from the left atrium by enzymatic digestion. Atrial myocytes were stimulated at 0.5Hz under voltage clamp control using the perforated patch clamp technique at 37oC. Changes in intracellular Ca were measured using Fluo-5F AM. Data are presented as mean ± SEM for n cells and statistical analysis was performed using a t-test or a Mann Whitney Rank Sum Test. Ageing decreased the peak of the L-type Ca current (ICa-L) by 19% (2.26 ± 0.14 vs. 1.84 ± 0.11 pA/pF, p<0.05, n = 47-67) which is likely to play a role in the 24% decrease in Ca transient amplitude. An age associated reduction in SERCA function was accompanied by a reduction in SERCA protein levels with no change in phospholamban as shown by western blotting (p<0.001). Surprisingly sarcoplasmic reticulum (SR) Ca content was increased (83.4 ± 3.48 vs. 101.9 ± 4.40μmol/l, n=35-50 cells, p<0.01). We sought to investigate SR function by plotting the relationship between Ca transient amplitude and SR Ca content (Trafford et al., 2001;Kettlewell et al., 2005). This was achieved by depleting the SR with caffeine and refilling to different levels by altering the number of recovery pulses before assessing SR Ca content again by caffeine application. This protocol was repeated until Ca transient amplitude had returned to steady state. A 3 parameter power function was fitted to the relationship between Ca transient amplitude and SR Ca content. Ageing resulted in a rightward shift of this relationship (p<0.001) suggesting a decrease in the sensitivity of Ca release. To investigate potential roles for decreased ICa-L vs. decreased ryanodine receptor (RyR) Ca release capacity we divided Ca transient amplitude by peak ICa-L and expressed this as a function of SR content. Ageing still resulted in a rightward shift of this relationship suggesting roles for both ICa-L and RyR. In summary, decreased ICa-L may underlie the age associated reduction in Ca transient amplitude however it remains to be determined if reduced ICa-L can fully account for reduced Ca transient amplitude in the presence of increased SR Ca content. A decreased Ca release capacity of RyR may offset decreased SERCA function in determining SR Ca content in the aged atria.
Where applicable, experiments conform with Society ethical requirements