Cellular Repressor Of E1A-Stimulated Genes Activates Autophagy To Alleviate Myocardial Ischemia Reperfusion Injury

Objective:Cellular Repressor of E1A-stimulated Genes(CREG) is an evolutionarily conserved glycoprotein, the results show that CREG could improve cardiac functions and prevent from cardiac hypertrophy, but there is nothing about the role of CREG in myocardial ischemia reperfusion injury(MIR). CREG, as a lysosomal glycoprotein, may play an important part in the process of autophagy. Thus, we aim to clarify the mechanisms on CREG improving cardiac function in MIR by regulating the autophagic-lysosomal pathway.Methods:(1) Male heterozygous CREG knockdown(CREG+/-) mice were generated at the Southern model animal center and bred at our laboratory.(2) An infusion of recombinantCREG protein(re-CREG)(0.3mg/kg·d) and chloroquine(CQ)(10mg/kg·d).(3)Establishment of MIR models.(4) Establishment the models of overexpression and lowexpression of CREG by using Adenoviral vectors and CREG-si RNA.(5) Detection cardiac function by Echocardiography and area of myocardial infarction by TTC staining.(6) Detection the level of CREG m RNA by RT-PCR.(7) Examination of apoptosis by western blot and Tunel staining.(8) Examination the level of autophagy by western blot and immunofluoresence.Results:The findings were revealed that the level of CREG protein was downregulated when subjected to MIR, especially in CREG+/-mice hearts(P<0.05), but the level of m RNA was not changed.At MIR 28 d, CREG+/-mice had worse cardiac function and larger infarction sizes(P<0.01), but when treated CREG+/+mice with re-CREG protein, the cardiac function was significantly improved( P<0.05). In CREG+/-mice, the number of apoptosis in cardiomyocytes was increased( P<0.01), LC3 A and P62 were accumulated which suggested that autophagy was dysfunctional. However, the upregulation of CREG decreased apoptosis of cardiomyocytes and activated autophagy. Furthermore, we confirmed that the deficiency of CREG protein increased cardiomyocytes apoptosis and inhibited the function of autophagy, the overexpression of CREG protein decreased cardiomyocytes apoptosis and activated autophagy(P<0.05), yet when infusion of CQ(an inhibitor of lysosomal acidification), the overexpression of CREG protein did not play the protection role in vivo. In vitro studies, establishment the models of overexpression(Ad CREG) and lowexpression of CREG(si CREG) by using Adenoviral vectors and CREG-si RNA, and control groups(siscramble and Ad GFP). The si CREG and siscramble were treated with Resveratrol(autophagy agonist) and starvation, we found that there was significantly difference of apoptosis between two groups(P<0.01). The Ad CREG and Ad GFP were treated with CQ and starvation, the results were showed that there was no difference of apoptosis between two groups. These suggest that CREG regulates autophagy to inhibit apoptosis. CREG is a lysosomal protein, we composed that CREG may regulate lysosome to impact autophagy. Thus, the expression of lysosome was detected. The results of Electron microscope revealed that autolysosomes and lysosomes were increased in Ad CREG treated with starvation(P<0.01), but in si CREG treated with starvation, autophagosomes were accumulated and lysosomes were decreased(P<0.01).Then Lysotracker staining revealed that the number of lysosomes were decreased in si CREG,but increased in Ad CREG. Furthermore, using western blot analysis, we found that the lysosome-associated proteins LAMP1/LAMP2/cathepsin D/cathepsin L were decreased in si CREG and increased in Ad CREG. Many studies identified that IGF2 R is the receptor of CREG, moreover, IGF2 R plays the important role in mature of lysosome.We want to know if IGF2 R involves in CREG regulated the development of lysosome.Compared with normal and Ad CREG, the expression of IGF2 R was more in si CREG. The binding efficiency of IGF2 R to LAMP2/cathepsin D was decreased in si CREG, but in Ad CREG the binding efficiency of IGF2 R to LAMP2/cathepsin D was increased. The datas suggest IGF2 R may participate in CREG regulated the development of lysosome.All the results indicate that CREG regulates the development of lysosome, activates autophagy and antagonises cardiomyocyte apoptosis.Conclusion:The results of this study indicate that CREG regulates the development of lysosome,activates autophagy and antagonises cardiomyocyte apoptosis.