Trimetazidine Alleviates Cardiomyocytes Hypoxia/reoxygenation Injury By Promoting AMPK-dependent Autophagic Flux

ObjectivesTo verify the protective effect of Trimetazidine(TMZ)pre-treatment against cardiomyocytes Hypoxia/reoxygenation(H/R)injury and investigate whether TMZ pre-treatment regulate autophagy via AMP-activated protein kinase(AMPK)signaling during cardiomyocytes H/R.Methods1.To clarify the protective effect of TMZ during H/R injury and the most effective concentrationCardiomyocytes,isolated from newly born rats,were randomly assigned into 5 groups:Control,H/R,H/R+TMZ(10?M),H/R+TMZ(50?M),H/R+TMZ(100?M).Cell viability was examined by MTS.Culture solution was collected to evaluate the level of LDH which reflected the cell injury.The most effective concentration was verified via comparing the efficacy of each concentration.2.To explore the effect of TMZ on autophagy during cardiomyocytes H/R with or without autopagy inhibitionChloroquine(Cq)was used to inhibit autophagic flux.Cardiomyocytes wer e randomly divided into 7 groups:Control,Control+TMZ,Control+ Cq,H/R,H/R+TMZ,H/R+Cq,H/R+TMZ+Cq.Cell viability was examined by MTS.Cell injury was detected by LDH measurements.Western blot was performed to d etect the level of autophagy-related proteins,including Beclin-1,LC3 and p62.mRFP-GFP-LC3 were transfected to evaluate the distribution of autophagy-rela ted proteins via confocal microscopy.The quantity of autophagosome each gro up can be directly evaluated via Transmission electron microscope.TUNEL sta ining was used to detect cardiomyocyte apoptosis.3.To investigate the effect of AMPK intervention on TMZ-regulated autophagy during cardiomyocytes H/R.Compound C(com C)was used to inhibit AMPK signaling.The experiments were performed on the following groups:Control,Control+com C,H/R,H/R+TMZ,H/R+com C,H/R+TMZ+ com C.Cell viability was examined by MTS.LDH release was detected through kit.Western blot was performed to detect autophagy-related protein(LC3 and p62)and AMPK signaling protein(p-AMPK/AMPK and p-mTOR/mTOR).Results1.TMZ pre-treatment protected cardiomyocytes against H/R injuryMTS assay were performed to examine cell viability.Compared with Control group,cell viability was lower in the H/R group(P<0.05).Interestingly,TMZ pre-treatment significantly increased cell viability,especially for the 50 ?M(P<0.05).LDH measurements were performed to assess cell injury during H/R.Our results show that LDH was higher in the H/R group than in the Control group(P<0.05).Compared with H/R group,The 3 TMZ pre-treatment groups have lower LDH realease(P<0.05).The LDH in the H/R+TMZ(50?M)group was lower than in the H/R+TMZ(10?M)group,but there was no significant difference between H/R+TMZ(50?M)group and H/R+TMZ(100?M)group(P>0.05).Thus,50?M was the most effective concentration.2.TMZ pre-treatment promoted autophagic flux during cardiomyocytes H/RWestern blot was performed to detect the expression of Beclin-1,LC3 and p62.Our results showed that H/R treatment increased all of the autophagy-related proteins(P<0.05,in Control vs.H/R).However,TMZ pre-treatment markedly increased the ratio of LC3-?/LC3-? and Beclin-1,and decreased p62(P<0.05,in H/R vs.H/R+TMZ),which suggested that TMZ pre-treatment could induce autophagy.Under the transmission electron microscope,autophagosomes are small vesicles with a double membrane surrounding cytoplasmic material.In Control group cell membrane,nuclear membrane,mitochondria and endoplasmic reticulum were all integrate,with a few autophagosomes in the cytoplasm.Compared with Control group,there was more autophagosomes in H/R group(P<0.05).Compared with H/R group,H/R+TMZ group has more autophagosomes(P<0.05).In agreement with Western blot,it can be concluded that TMZ pre-treatment could induce autophagy during cardiomyocytes H/R.To further assess the effect of TMZ on autophagic flux,we transfected cardiomyocytes with mRFP-GFP-LC3.Our results revealed that there was no evident LC3 gathering in Control group.H/R treatment increased autolysosomes(P<0.05,in Control vs.H/R).Compared with H/R group,H/R+TMZ group has higher autolysosomes(P<0.05),which indicated that TMZ pre-treatment could upregulated autophagy with intact process of autophagic flux.3.Cq pre-treatment abolished the cardioprotection of TMZWestern blot indicated that Cq significantly increased the ratio of LC3-?/LC3-I and p62(P<0.05,in H/R+TMZ vs.H/R+TMZ+Cq).Under the confocal microscopy,Cq markedly decreased autolysosomes(P<0.05,in H/R+TMZ vs.H/R+TMZ+Cq).Thus,TMZ could promoted autophagic flux,which could be abolished by Cq.MTS assay indicated that the cell viability in H/R+TMZ group was higher than in H/R+TMZ+Cq group.In addition,LDH measurement indicated that Cq markedly increased the level of LDH(P<0.05,in H/R+TMZ vs.H/R+TMZ+Cq),which suggested that TMZ might protect against H/R injury via promoting autophagic flux.4.TMZ pre-treatment attenuated apoptosisTUNEL assay was used to examine the level of apoptosis.Compared with Control group,H/R increased apoptotic cells(P<0.05).TMZ pretreatment significantly reduced apoptotic cells(P<0.05,in H/R vs.H/R+TMZ).However,co-administering TMZ with Cq reversed that protection with increased apoptotic cells(P<0.05).These results suggested that TMZ might attenuated apoptosis through promoting autophagy.5.TMZ pre-treatment regulated autophagy trough the AMPK-mTOR pathway during H/RWestern blot was performed to detect the expression of p-AMPK,AMPK,p-mTOR and mTOR.Our results showed that TMZ pre-treatment significantly increased the ratio of p-AMPK/AMPK and reduced the ratio of p-mTOR/mTOR(P<0.05,in H/R vs.H/R+TMZ),which indicated that TMZ increased the phosphorylation of AMPK and decreased the phosphorylation of mTOR.Compared with H/R+TMZ group,com C reduced the ratio of p-AMPK/AMPK and increased the ratio of p-mTOR/mTOR(P<0.05).Furthermore,com C decreased the ratio of LC3-?/LC3-? and increased p62,which suggested that com C could block TMZ-induced autophagy through the AMPK-mTOR pathway.MTS assay and LDH measurement both indicated that com C treatment decreased cell viability and increased LDH release((P<0.05,in H/R+TMZ+com C vs.H/R+TMZ),which suggested com C partly counteract the protective effect of TMZ against H/R injury.Thus,AMPK-dependent autophagy was involved in TMZ-induced cytoprotection during H/R.ConclusionsThis study provided the evidences that TMZ pre-treatment alleviated H/R injury through activating the AMPK-mTOR pathway and hence promoting autophagic flux.