Upregulation of p67(phox) 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.