Deciphering the crosslink between STAT3 and MAPKs during ischemia/reperfusion and postconditioning

Introduction STAT3 exerts Cardioprotective roles against the ischemia/reperfusion injury (IRI) and during ischemic postconditioning (IPoC). An additional mitochondrial protective role has evolved. In cell line models, STAT3 can be phosphorylated on its tyrosine705 and serine727 residues. The involvement of STAT3 in IPoC, its mitochondrial localization and the specific roles of Y705 and S727 residues are still unclear. Aims We aimed to study the kinetics of STAT3 and ERK phosphorylation following IRI (with and without IPoC) and to decipher the cross link between MAPKs and STAT3 in vitro. Methods In vitro H9C2 cells were treated with LIF, STAT3:STAT1 inhibitor (stattic), MEK/ERK inhibitors (PD98059 & U0126 ethanoate). In vivo C57b male mice were subjected to ischemia followed by different durations of reperfusion, with/without IPoC ( n  = 6). The phosphorylation levels of STAT3 and ERK1/2 MAP kinase were detected. Results In vitro Y705 phosphorylation of STAT3 was increased 4 times with LIF treatment, but decreased 10 times with stattic. The MEK/ERK pathway inhibitors inhibited STAT3 phosphorylation at S727 by 2 times along with a 2-fold increase in the Y705 one. STAT3 was not detected in the mitochondria of cardiac cells. We confirmed a dose-dependent toxicity of STATTIC above 50 μM. In vivo I/R induced ERK phosphorylation, but PoC induced the phosphorylation of S727 STAT3 and ERK1/2 within 15 min of reperfusion. IR or PoC did not affect Tyr705 STAT3 phosphorylation. Conclusions We failed to confirm, using different approaches, the presence of STAT3 in mitochondria. Besides, we found out that STATTIC exerted a toxic effect on cells at concentration above 50 μM. Thus, in any experiment achieved with higher concentrations of STATTIC, the involvement of STAT3 cannot be inferred. Our results also suggest a competitive phosphorylation between STAT3 S727 and Y705 residues. We also found that the former residue is prevalently subjected to phosphorylation during IPoC.