The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is key to Ca2+ homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca2+ uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca2+ studies showed decreased nitric oxide (·NO)-induced 45Ca2+ uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca2+ stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca2+ influx. Adenoviral overexpression of calreticulin, an ER Ca2+ binding protein, increased ionomycin-releasable stores, VEGF-induced Ca2+ influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca2+ homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca2+ stores.

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