Abstract Body: Introduction
All mammals experience a linear decline in heart rate with age. In humans, this decline can become pathological and is the main driver for the implantation of artificial pacemakers. A factor contributing to this slowdown is the reduction in the surface expression of ion channels in pacemaker cells. However, why channel density is reduced remains unknown. Interestingly, these channels are trafficked to and preferentially located within specialized membrane invaginations known as caveolae.
Hypothesis
We hypothesize that aging disrupts the trafficking cycle of membrane proteins and the formation of caveolar microdomains contributing to the reduction of ion channel surface expression.
Aims
We aim to determine age-associated changes in caveolin-3 expression and caveolar integrity in pacemaker cells and evaluate the effect of caveolae disruption on the density of ion channels at the plasma membrane. We also evaluated the effect of age on the density of endosomes and lysosomes.
Methods
Pacemaker tissue or isolated cells were obtained from C57BL mice from two age groups, young (3-6 months) and old (20-30 months). We evaluated the expression of caveolin 3 using qPCR, western blot, and immunocytochemistry. To evaluate changes in vesicular trafficking, we immunolabeled for the early endosomal marker EEA1 and the lysosomal marker LAMP1.
To investigate if caveolae are necessary for the clustering of ion channels, we treated young pacemaker cells with the caveolar disruptor methyl-beta-cyclodextrin (MßCD). Super-resolution microscopy was used to evaluate ion channel cluster size and density at the plasma membrane.
Results
We found a 30% reduction in caveolin-3 expression in old pacemaker tissue (N=6 young and 6 old animals, p = 0.002). MßCD-treatment reduced ion channel cluster density for CaV1.2 (p = 0.007) and CaV1.3 (p = 0.02) (N= 3 young and 3 old animals). Preliminary data suggests an increase in both endosome and lysosome number in old pacemaker cells.
Conclusion
Together, our findings support the hypothesis that aging disrupts the caveolar network and causes an impairment of vesicular trafficking, which can both be linked to the reduction of ion channel surface expression underlying the pacemaker slowdown.