| BACKGROUND:Acute myocardial infarction has been concerned due to its high morbidity and mortality,and the most effective treatment is rapid restoration of blood flow.However,further death of myocardial cells is still inevitable in the process of restoring blood flow,and this phenomenon is called myocardial ischemia-reperfusion injury(MIRI).Therefore,MIRI has become an inevitable problem in clinical practice,and more and more studies have focused on this issue.Numerous studies have shown that autophagy in cardiomyocytes is increased in ischemia-reperfusion models and is currently considered as a potential target for the treatment of MIRI,and many autophagy inducers have been shown to protect the myocardium from ischemic injury by promoting autophagy in cardiomyocytes.However,excessive autophagy can also further lead to cell death,so it is important to find drugs that can reasonably promote autophagy for cell survival.As coating drugs for drug-eluting stents,rapamycin,paclitaxel,and everolimus have been shown to be involved in the regulation of autophagy,but whether zotarolimus,a derivative of rapamycin,can be involved in the regulation of autophagy has not been shown.It has been shown that zotarolimus can inhibit mammalian target of rapamycin(mTOR),which is the central point of negative regulation of autophagy,so we speculated that zotarolimus can be involved in the regulation of autophagy.In this study,we mainly simulated the ischemia-reperfusion model in vivo by culturing primary cardiomyocytes and constructing a hypoxia-reoxygenation model to investigate the regulation of autophagy and apoptosis by zotarolimus in the hypoxia-reoxygenation model of primary cardiomyocytes and its mechanism.Part Ⅰ Effects of zotarolimus on hypoxia/reoxygenation injury and its regulation on autophagy in primary cardiomyocytesOBJECTIVE:To investigate the effect of zotarolimus pretreatment on hypoxia-reoxygenation injury in primary cardiomyocytes,as well as its regulation of autophagy.METHOD:We used CCK-8 to examine the effects of different concentrations of zotarolimus on the survival rate of primary cardiomyocytes cultured under normal conditions,and after constructing a hypoxia-reoxygenation model,the effects of different concentrations of zotarolimus on the survival rate of cells under hypoxia-reoxygenation model,and the appropriate concentration range of zotarolimus was selected by these two steps.Next,RT-qPCR was used to detect the expression levels of Bcl-2 and caspase-3 mRNA after pretreatment with each concentration of zotarolimus,so as to select the most appropriate concentration of zotarolimus for the protection of cardiomyocytes injured by hypoxia-reoxygenation.The next experiment was performed at the most appropriate zotarolimus concentration,and the primary cells were first grouped:normal group(control),hypoxia-reoxygenation model group(H/R),and zotarolimus pretreatment group(H/R+zotarolimus).The damage of primary cardiomyocytes in each group was detected by calculating the release of lactate dehydrogenase(LDH)in the cell culture medium.The ultrastructure of primary cardiomyocytes was also observed using transmission electron microscopy.Then different methods were used to detect autophagy in each group of cardiomyocytes:Westernblot was used to detect the expression of autophagy-related proteins LC3-Ⅱ/LC3-Ⅰ,Beclinl and P62;immunofluorescence was used to detect the expression of LC3-II and P62.RESULTS:(1)The results of CCK-8 showed that under normal conditions,different doses of zotarolimus including 0.05 μmol/L,0.1 μmol/L,0.2 μmol/L,0.5 μmol/L,1 μmol/L,5 μmol/L,and 10 μmol/L had no significant effect on the survival rate of primary cardiomyocytes(P>0.05),and the above concentrations of zotarolimus were selected to continue the next experiment.(2)Compared with the normal group,the cell survival rate was significantly lower in the hypoxia-reoxygenation group(P ＜0.01),and compared with the hypoxia-reoxygenation group,except that the concentration of 0.05 μmol/L had no effect on the cardiomyocyte survival rate,the survival rate of 0.01 μmol/L was increased(P ＜0.05),and the survival rate of the 0.2 μmol/L,0.5 μmol/L,1μmol/L,5 μmol/L,and 10 μmol/L groups was significantly increased(P ＜0.01),so the above concentrations of zotarolimus were selected to continue the next experiment.(3)The results of RT-qPCR showed that the expression of Bcl-2 mRNA was significantly decreased and the expression level of caspase-3 mRNA was significantly increased in the H/R group compared with the normal group(P ＜0.01),while the expression of Bcl-2 mRNA was significantly increased(P ＜0.01),while the expression level of caspase-3 mRNA was significantly decreased(P ＜0.01)in the zotarolimus groups at each concentration compared with the H/R group.In combination with the above experiments,zotarolimus at a concentration of 0.5 μmol/L was selected for the next experiment.(4)The results of LDH release showed that LDH release was increased in the H/R group compared with the normal group(P ＜0.01),while zotarolimus pretreatment decreased LDH release after H/R treatment(P ＜0.01).(5)Transmission electron microscopy of myocardial cell ultrastructure showed that the cytoplasm of normal myocardial cells was not swollen,the mitochondrial structure was clear,and vacuolar adipocytes and fat droplets were not seen.However,compared with the normal group,the cytoplasm showed moderate swelling and mitochondria showed vacuolization after H/R.Pretreatment with zotarolimus reduced the degree of cytoplasmic swelling as well as mitochondrial vacuolization.(6)Western blot results showed that the expression levels of autophagy-related proteins LC3-Ⅱ/LC3-Ⅰ,Beclinl,and P62 were significantly increased in the H/R group compared with the normal group(P ＜0.01),and pretreatment with zotarolimus further increased the expression of LC3-Ⅱ/LC3-Ⅰand Beclinl(P ＜0.01),while the expression of P62 was significantly decreased(P ＜0.01),and the fluorescence results were consistent with the Western blot results.CONCLUSION:(1)zotarolimus has a protective effect on cardiomyocytes injured by hypoxia-reoxygenation.(2)The protective effect of zotarolimus on cardiomyocytes may be related to its promotion of the patency of cardiomyocyte autophagic flow.Part Ⅱ Study on the protective mechanism of zotarolimus preconditioning on hypoxia-reoxygenation injury in mouse primary cardiomyocytesOBJECTIVE:To investigate whether zotarolimus promotes autophagy and protects cardiomyocytes during hypoxia-reoxygenation by regulating AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)signaling pathway.METHOD:To explore whether zotarolimus acts by regulating the AMPK/mTOR signaling pathway by using the AMPK inhibitor CompoundC,primary cardiomyocytes were first divided into the following four groups:normal group(Control),hypoxia-reoxygenation group(H/R),zotarolimus group(H/R+zotarolimus),and CompoundC group(H/R+zotarolimus+CompoundC).Next,the expression of P-AMPK,AMPK,mTOR,P-mTOR and autophagy-related gene Beclinl protein in primary cardiomyocytes of each group was detected by Westernblot,as well as the expression level of Beclinl mRNA in primary cardiomyocytes of each group by RT-qPCR to verify whether zotarolimus promotes autophagy through the AMPK/mTOR signaling pathway.RT-qPCR was used to detect the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein caspase3 mRNA,and flow cytometry was used to detect the apoptosis rate.LDH release was used to detect the damage of primary cardiomyocytes in each group.RESULTS:(1)Western blot results showed that the ratio of P-AMPK/AMKP in the H/R group was increased compared with the normal group(P ＜0.01),while the ratio of P-mTOR/mTOR was decreased compared with the normal group(P ＜0.01).However,the P-AMPK/AMKP ratio was further increased(P ＜0.01)and the ratio of P-mTOR/mTOR was significantly decreased(P ＜0.01)after zotarolimus pretreatment compared with the H/R group.Use of CompoundC blocks this effect of zotarolimus.(2)Both Western blot and RT-qPCR results showed that the expression of autophagy-related gene Beclinl was increased in the hypoxia-reoxygenation group compared with the normal group(P ＜0.01),and zotarolimus treatment could further increase the expression of Beclinl in cardiomyocytes subsequent to hypoxia-reoxygenation(P ＜0.01),indicating an increase in autophagy levels.Co-treatment of cardiomyocytes with zotarolimus using CompoundC attenuated this effect of tamoxifen.(3)The results of RT-qPCR and flow cytometry as well as LDH release showed that H/R induced apoptosis of cardiomyocytes and increased the release of LDH,a marker of myocardial injury,and zotarolimus pretreatment ameliorated the above damage,while CompoundC attenuated the effect of zotarolimusCONCLUSION:Pretreatment with zotarolimus can induce autophagy in cardiomyocytes after hypoxia-reperfusion by regulating AMPK/mTOR signaling pathway as one of the mechanisms,and regulation of AMPK/mTOR signaling pathway by zotarolimus may be one of the mechanisms by which zotarolimus pretreatment attenuates myocardial injury. |
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