The Study On The Relative Mechanism Of Tissue Oxygen Metabolism On Acute Ischemia Reperfusion Injury In Mice Model

Objective:Late phase ischemic preconditioning (LPC) strongly protects the heart against ischemia reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and the related mechanism(s) are unknown. In this study, with an in vivo mouse heart I/R model, we determine whether LPC attenuates post-ischemic tissue hyperoxygenation through preserving mitochondrial function.Methods and Results:Four groups of mice were studied:sham, LPC, I/R, and LPC+I/R mice. Myocardial tissue Po2 was monitored with electron paramagnetic resonance (EPR) oximetry. Upon reperfusion, Po2 rose significantly above the pre-ischemic value in the I/R mice. This hyperoxygenation was attenuated in the LPC+I/R group. Activities of NADH dehydrogenase (NADH-DH), Succinate-Cytochrome c Reductase (SCR) and cytochrome c oxidase (CcO) were increased in the LPC group, reduced in the I/R group, but conserved in the LPC+I/R group compared with sham group. The protein expressions of these enzymes were also increased in the LPC group and LPC+I/R group but no different with I/R group compared to sham group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased in the LPC and LPC+I/R groups compared with sham group. Conclusion:Taken together, LPC attenuates post-ischemic hyperoxygenation and preserves mitochondrial oxygen metabolism through increased SCR and CcO activities and conserved NADH-DH activity due to increased Mn-SOD expression. Objective Ischemic post-conditioning (IPOC) is a powerful method to reduce ischemia and reperfusion injury. Studies also demonstrate that IPOC could be deleterious when the ischemia duration was either too short or too long. Our study is to determine whether there is a salvageable ischemic time window for IPOC and the potential mechanisms involved.Methods C57BL/6 mice underwent 15, 30,45 and 60 min left anterior descending coronary artery (LAD) occlusion followed by reperfusion, w/o IPOC. In vivo tissue oxygenation was monitored with electron paramagnetic resonance (EPR) oximetry. Regional Blood Flow (RBF) was measured by the laser Doppler perfusion monitor. Tissue from the risk area was collected and the activities of mitochondrial were assayed.Results The tissue oximetry data demonstrated that IPOC attenuated tissue hyperoxygenation with 30 and 45 but not 15 or 60 min ischemia. IPOC improved the RBF with 30 and 45 but not 15 or 60 min ischemia. IPOC preserved the activities of mitochondrial enzymes activities at 30 and 45 but not 60 min ischemia. Infarct size measurement indicated that IPOC protected the heart with 30 min and45 min Vint nnt 15 or 60 min ischemia Conclusions The cardioprotection afforded by IPOC is limited to a time window between 30 and 45 min LAD occlusion in mouse. This salvageable ischemic time window is determined by the effect of IPOC on improving the RBF and mitochondrial function. Background eNOS-derived NO induced tissue hyperoxygenation in the acutely reperfused heart in vivo and hyperoxia induced myofibroblast transformation in vitro. However, little is known about the late phase reperfusion tissue oxygenation and its effect on myofibroblast transformation which is critical for the healing and repair of the infarcted myocardium. We hypothesize that eNOS- and iNOS-derived NO induces late phase reperfusion hyperoxygenation which promotes, while NOS/NO itself inhibits, cardiac myofibroblast transformation via TGF-pi/Smad2/3 signaling.Methods and Results C57BL6 wild-type (WT), eNOSA and iNOS'/' mice were subjected to 30 min LAD occlusion followed by 14 days of reperfusion (N=7/group). Myocardial tissue P0₂ was monitored by electron paramagnetic resonance oximetry. Protein expression was detected using ELISA for TGF-pi and Western blot for p-Smad2/3 over total Smad2/3 ratio and p21 at day 3 and a-SMA at day 14 post reperfusion. During 60 min reperfusion, there was a hyperoxygenation state in the WT and iNOS"'" but not eNOS^-/- mice with no difference on the baseline P02 among all the groups (¹1.5 mmHg). However, after 60 min reperfusion, hyperoxygenation was observed in all three groups and peaked at day 3 in the WT (30.3±1.3 mmHg), eNOS"'" (22.4±0.8 mmHg) and iNOS"/" (27.0±1.8 mmHg) mice. Protein expression of the total TGF-pi, p-Smad2/3 over total Smad2/3 ratio, p21 and a-SMA were significantly increased after I/R in the WT (1.43±0.05, 1.60±0.11, 1.55±0.03, 1.68±0.12 fold, p<0.05 vs. WT sham). Knockout of eNOS and iNOS further increased the expression of these proteins (eNOS^-/- mice: 2.19±0.06, 2.37±0.10, 3.39±0.20, 3.34±0.27 fold, p<0.05 vs. WT sham; iNOS"'" mice: 1.69±0.09, 1.98±0.05, 2.10±0.14, 2.81±0.12 fold, p<0.05 vs. WT sham). Immunohistochemical staining at day 14 indicated the expression of a-SMA in the myofibroblasts in the infarct area.Conclusion Thus, eNOS- and iNOS-derived NO induces the late phase reperfusion hyperoxygenation in the post-ischemic heart. Further, the late phase reperfusion hyperoxygenation promotes and NOS/NO inhibits myofibroblast transformation via TGF-pi signaling. These findings are critical to developing new therapeutic interventions to improve infarct healing and repair.