| Ischemic cardiovascular disease shows trends of increasing morbidity and mortality in the United States and around the world. Current therapeutic options are limited, but the identification of key disease mechanisms and targets will inform novel therapeutic development to help decrease disease burden. One potential target is the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA), a key regulator of Ca2+ homeostasis which plays multiple roles in the cardiovascular system. SERCA catalyzes the hydrolysis of ATP and couples it to the translocation of free cytosolic Ca 2+ into SR/ER stores. SERCA is redox-regulated, and is susceptible to both stimulatory and inhibitory oxidative post-translational modification. For example, oxidation of SERCA by physiological levels of nitric oxide (NO) causes reversible oxidative modification of SERCA cysteine thiols by introducing glutathione adducts. S-glutathiolation enhances SERCA Ca2+ uptake activity, which results in rapid reductions in cytoplasmic Ca2+ levels, and promotes endothelial angiogenic responses in vitro..;S-glutathiolation of SERCA specifically at cysteine-674 (C674) is a key signal regulating SERCA activity under physiological conditions, and a next crucial step is establishing causal relationships between defects in C674 S-glutathiolation and human disease. The following study elucidates the role of redox regulation of the C674 thiol in the mechanisms of vascular disease by employing a SERCA 2 C674S knock-in (SKI) mouse in which the key thiol is lacking in 50% of SERCA, rendering the protein less able to be activated by glutathiolation. Following hind limb ischemia, SKI animals had impaired blood flow recovery, indicating an angiogenic defect. Cultured SKI microvascular endothelial cells showed impaired migration and decreased network formation. Fura-2 Ca2+ signaling studies revealed lower Ca2+ stores and decreased VEGF- and NO-induced Ca2+ influx. Also, hypoxia-induced expression of pro-angiogenic genes (VEGF, VEGF receptor 2 and eNOS) was decreased in SKI endothelial cells. Adenoviral overexpression of calreticulin, a major ER Ca2+ binding protein, enhanced levels of VEGF receptor protein and eNOS phosphorylation. Taken together, these data indicate that impairing normal redox regulation of the C674 thiol via reversible S-glutathiolation interferes with endothelial cell Ca2+ homeostasis and angiogenic gene expression, suggesting mechanisms by which impaired SERCA glutathiolation contributes to worsened angiogenesis during ischemia. |
