Age attenuates the T‐type Ca_V3.2‐RyR axis in vascular smooth muscle

Abstract

Caveolae position Ca_V 3.2 (T-type Ca²⁺ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca²⁺ influx to trigger Ca²⁺ sparks and large-conductance Ca²⁺ -activated K⁺ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca²⁺ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Ca_v 3.2 channel inhibition by Ni²⁺ (50 µM) and caveolae disruption by methyl-ß-cyclodextrin or genetic abolition of Eps15 homology domain-containing protein (EHD2) inhibited Ca²⁺ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Ca_v 3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Ca_v 1.2^-/- mice, caffeine (RyR activator) and thapsigargin (Ca²⁺ transport ATPase inhibitor), we found that sufficient SR Ca²⁺ load is a prerequisite for the Ca_V 3.2-RyR axis to generate Ca²⁺ sparks. We identified a fraction of Ca²⁺ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd³⁺ (100 µM), but insensitive to Ca_V 1.2 and Ca_V 3.2 channel blockade. Our data demonstrate that the VSMC Ca_V 3.2-RyR axis is down-regulated by aging. This defective Ca_V 3.2-RyR coupling is counterbalanced by a Gd³⁺ sensitive Ca²⁺ pathway providing compensatory Ca²⁺ influx for triggering Ca²⁺ sparks in aged VSMCs.