Oxidation of SERCA2 at cysteine 674 increases ER stress during hind limb ischemia in mice

Reversible glutathione adducts on the cysteine 674 (C674) thiol of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) stimulate Ca 2+ uptake activity, endothelial cell (EC) angiogenic signaling, and migration. This study was to determine if oxidation of SERCA C674 and endoplasmic reticulum (ER) stress affects angiogenesis during hind limb ischemia. SERCA2 C674 serine mutant heterozygote knock in (SKI) mice, lacking one allele for the redox‐regulated C674 thiol, had impaired blood flow recovery after femoral artery ligation. Immunostaining demonstrated increased SERCA C674 thiol oxidation in microvascular endothelium of ischemic limbs, and double staining revealed increased BIP expression co‐localized with the EC marker isolectin B4. Staining was greater in SKI compared with WT, indicating thiol oxidation or mutation in ischemia is associated with ER stress activation. Western blotting showed ER stress markers p‐PERK and CHOP were greater in both non‐ischemic and ischemic SKI limbs compared to WT. Cultured SKI microvascular ECs showed smaller Ca 2+ stores, and higher basal and hypoxia‐induced levels of BIP and CHOP. Collectively, these data suggest that lack of the SERCA C674 thiol caused by oxidation or mutation increases EC ER stress. Thus, oxidation of SERCA2 C674 and associated Ca 2+ store depletion and ER stress during hind limb ischemia may contribute to impaired angiogenesis. Funding NIH RO1 HL31607