Objectives eNOS-derived NO induces acute phase tissue hyperoxia in vivo and hyperoxia induces fibroblast trans-differentiation in vitro. However, little is known about the effect of reperfusion-induced hyperoxia on myocardial infarct healing. The current study is to determine how late phase reperfusion hyperoxia and NO regulate cardiac myofibroblast formation.
Methods C57BL/6 wild-type, eNOS−/− and iNOS−/− mice were subjected to 30-min LAD occlusion followed by 14-days of reperfusion. Myocardial tissue PO2 was monitored with electron paramagnetic resonance oximetry. Protein expression of TGF-beta1, p-Smad2/3, t-Smad2/3, p21 and α-SMA were measured with ELISA and western blot.
Results There was an acute phase overshoot of tissue Po2 in the WT and iNOS−/− but not eNOS−/− mice. After 60 min reperfusion, tissue hyperoxia was observed in all three groups and peaked at day 3 with significantly lower PO2 in the eNOS−/− mice than that in the WT and iNOS−/− mice (22.4±0.8 vs 39.8±1.13 and 26.9±1.3 mm Hg). Protein expression of the total and active TGF-β1, p-Smad2/3 over t-Smad2/3 ratio, p21 and α-SMA were significantly increased after reperfusion in the WT mice. Knockout of eNOS or iNOS further increased the expression of these signals. Immunohistochemical staining indicated the expression of α-SMA in the infarct area. Immunoprecipitation demonstrated the nitration of TGF-β RII. Carbogen (95% O2+5% CO2) treatment increased the expression of p-Smad2/3 over t-Smad2/3 which was inhibited by EUK134 (10006329 EUK 134) and sodium nitroprusside.
Conclusions Late phase reperfusion tissue hyperoxia promoted while eNOS-/iNOS-derived NO/ONOO− inhibited cardiac TGF-β1 signalling and myofibroblast trans-differentiation. These findings may provide new targets to improve myocardial infarct healing and repair.