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Abstract
The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) 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). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) 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 Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores.
Highlights
The sarco/endoplasmic reticulum Ca2ϩ ATPase (SERCA) is key to Ca2ϩ homeostasis and is redox-regulated
Ischemia-induced oxidant production initiates signaling cascades essential for vascular repair [20], and our work here shows that redox regulation of SERCA 2 cysteine 674 (Cys-674) via reversible S-glutathiolation modification plays a significant role in ischemia-induced angiogenic responses in vivo
We provide new evidence that SERCA Cys-674 is post-translationally modified by GSH during ischemia and hypoxia, and that redox regulation by SERCA 2 of Ca2ϩ stores is essential for normal endothelial responses to vascular endothelial growth factor (VEGF) and hypoxia
Summary
The sarco/endoplasmic reticulum Ca2ϩ ATPase (SERCA) is key to Ca2ϩ homeostasis and is redox-regulated. JULY 18, 2014 VOLUME 289 NUMBER 29 thelial 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. In cardiac microvascular endothelial cells isolated from the SKI mouse, ER Ca2ϩ stores were depleted, and endothelial angiogenic behavior was impaired, but these defects could be rescued by directly increasing Ca2ϩ stores by overexpressing calreticulin, an ER Ca2ϩ store-binding protein These studies indicate that redox-sensitive Ca2ϩ store maintenance via S-glutathione adducts on the key SERCA 2 Cys-674 thiol is required for normal angiogenic endothelial cell function and blood flow recovery after hind limb ischemia
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