The Role Of Autophagy In Morphine-induced Cardioprotection

Obiective Autophagy plays a role in myocardial ischemia/reperfusion(MI/R),especially in the period of reperfusion. Morphine (Mor) can protect the myocardium fromreperfusion injury. This study investigated the molecular mechanisms of autophagyoccurring during reperfusion through morphological and molecular biology methods,observed whether morphine resist myocardial reperfusion injury by inhibiting autophagy,and explored the molecular mechanisms of autophagy inhibition.Methods This animal model was established on isolated rat hearts which weresubjected to30min ischemia followed by2h of reperfusion using Langendorff system.We examined the myocardial infarct size though Evans blue and TTC double staining todetermine whether the model is successful. Samples were collected from the risk zone atdifferent time points after I/R for Western blot to dectect the expression of autophagymarker protein LC3. Meanwhile, autophagy and autolysosome were observed by electronmicroscopy to futher clarify the specific period autophagy occurs. Morphine was givenfive minutes before reperfusion, infarct size was measured and morphological alterationswere assessed through optical microscopy to confirm the cardioprotection of morphine.Samples were collected at the most significant time point of morphine induced-autophagyfor Western blot. Expression of LC3, beclin1and p-mTOR were analysised.Results1Compared with baseline, the heart rate and coronary flow were decreasedsignificantly during ischemia reperfusion, while increased during early reperfusion period.Myocardial infarct size was too limited to injury the heart severely. With extend hours ofreperfusion, heart rate and coronary flow were decline while infarct size was significantlyincreased to41.27±4.45%. All of these data indicated that the cardiac functionsignificantly reduced with the myocardium infarction after I/R, which also confirmed ouranimal model was established successfully.2Autophagy and autolysosome wrapped withmitochondria were observed by electron microscopy during I/R.3Compared with theSham group, LC3Ⅱ/LC3Ⅰwere increased both at ischemia30min or other time pointsafter reperfusion, it increased the most obviously at reperfusion60min. This resultsuggests that autophagy occurred both during ischemia and reperfusion period, it can besignificantly enhanced by early reperfusion.4Compared with the Sham group， the expression of positive regulatory protein Beclin1has no obvious difference duringdifferent I/R periods. However, the level of phosphorylation of negative regulatoryprotein mTOR decreased during I/R, especially after reperfusion30min and60min.These results suggested that the induction of autophagy during reperfusion is probablydepended on the negative pathway in isolated rat hearts.5Compared to I/R alone,morphine given at reperfusion significantly increased heart rate and coronary flow andreduced myocardial infarct size in isolated rat hearts. Meanwhile HE staining resultshistological changes also show that comparing to the Sham group, I/R cardiomyocytesarranged relatively sloppy, cell gap widened, cell edema, but also the visible part of themuscle fiber rupture; while compared with the I/R group, the extent of damage ofmorphine-treated group was significantly reduced. All the results confirmed thatmorphine postconditioning can resist myocardial ischemia reperfusion injury.6Afterreperfusion60min, LC3Ⅱ/LC3Ⅰratio of I/R group increased significantly comparedwith Sham group, which was reduced by morphine. Meanwhile, morphine did notenhance Beclin1expression, but it increased the phosphorylation of mTOR comparedwith I/R alone. The results suggest that morphine possibly inhibited I/R-inducedautophagy through negative pathway.Conclusions In isolated rat heart, autophagy can be significantly induced byreperfusion injury. Morphine protect the heart from I/R injury probably throughinhibition of I/R-induced autophagy by negative pathway.