Upregulation of p67phox in response to ischemia/reperfusion is cardioprotective by increasing ZIP2 expression via STAT3

While the zinc transporter ZIP2 (Slc39a2) is upregulated via STAT3 as an adaptive response to protect the heart from ischemia/reperfusion (I/R) injury, the precise mechanism underlying its upregulation remains unclear. The purpose of this study was to investigate the role of NADPH oxidase (NOX) isoform NOX2-derived ROS in the regulation of ZIP2 expression, focusing on the role of the NOX2 cytosolic factor p67^phox. Mouse hearts or H9c2 cells were subjected to I/R. Protein expression was detected with Western blotting. Infarct size was measured with TTC staining. The cardiac-specific p67^phox conditional knockout mice (p67^phox cKO) were generated by adopting the CRISPR/Cas9 system. I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression was reversed by the ROS scavenger N-acetylcysteine (NAC) and the NOX inhibitor diphenyleneiodonium (DPI). p67^phox but not NOX2 expression was increased 30 min after the onset of reperfusion, and downregulation of p67^phox by siRNA or cKO invalidated I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression. Both NAC and DPI prevented upregulation of STAT3 phosphorylation and ZIP2 expression induced by overexpression of p67^phox, whereas the STAT3 inhibitor stattic abrogated upregulation ZIP2 expression, indicating that the increase of p67^phox at reperfusion is an upstream signaling event responsible for ZIP2 upregulation via STAT3. Experiments also showed that chelation of Zn²⁺ markedly enhanced p67^phox and ZIP2 expression as well as STAT3 phosphorylation, whereas supplementation of Zn²⁺ had the opposite effects, indicating that cardiac Zn²⁺ loss upon reperfusion triggers p67^phox upregulation. Furthermore, ischemic preconditioning (IPC) upregulated ZIP2 via p67^phox, and cKO of p67^phox aggravated cardiac injury after I/R, indicating that p67^phox upregulation is cardioprotective against I/R injury. In conclusion, an increase of p67^phox expression in response to Zn²⁺ is an intrinsic adaptive response to I/R and leads to cardioprotection against I/R by upregulating ZIP2 via STAT3.