AMPK/PGC1? Signaling Pathway Activation By Melatonin Attenuates Acute Doxorubicin Cardiotoxicity

Objective:Doxorubicin(DOX),also called Adriamycin,is a very effective anticancer anthracycline drug,but its side effect at the level of the heart has limited its widespread clinical application.The mechanisms of DOX-induced cardiotoxicity are attributed,at least in part,to the generation of reactive oxygen species(ROS),mitochondrial damage,impaired calcium homeostasis and induction of cell apoptosis.Melatonin(MEL),an endogenously-produced indolamine,is a documented potent antioxidant and cardioprotective agent,and it is involved in maintaining mitochondrial homeostasis and function.Clinical studies have revealed that MEL enhances the efficacy and reduces the side-effects of chemotherapy,and improves the quality of life of the cancer patients.Therefore,both in vivo and in vitro studies were performed to document melatonin's beneficial actions against DOX-induced acute cardiotoxicity and the detailed underlying mehanisms.Methods:1.The rat cardiomyoblast H9c2 cells were divided into(i)Control group;(ii)MEL group;(iii)DOX group;and(iv)DOX+MEL group.The used dosages of DOX and MEL were 1?M and 100?M,relatively.The CCK-8 cell viability,ROS level,the content of ATP and MDA,TUNEL cell apoptotic rate were respectively investigated.The expression of apoptosis-related signaling(Bax,Bcl2,cleaved caspase 3)were tested by western blot.2.RNA-seq based transcriptome analysis was applied to estimate the transcriptome changes in H9c2 cells among the control group,DOX group and DOX+MEL group,and the changes were further validated by qPCR and western blot analyses.The H9c2 cells were divided into(i)Control group;(ii)AMPK siRNA group;(iii)DOX group;(iv)DOX+MEL group;(v)DOX+MEL+AMPK siRNA group.Western blot was applied to examine the expression of p-AMPK,AMPK,PGC1?,NRF1,TFAM and UCP2.Moreover,we further tested whether AMPK was involved in the melatonin's protection against DOX-induced acute oxidative stress and apoptosis on myocardiocytes through the methods used in part 1.3.C57BL/6 mice were randomly assigned to six groups:(i)Sham group;(ii)MEL group;(iii)DOX group(DOX);(iv)DOX+MEL group;(v)DOX+MEL+Compound C group;(vi)DOX+Compound C group.For constructing the DOX-induce acute cardiotoxicity mice model,DOX was intraperitoneally injected at a total dose of 20 mg/kg.MEL(20 mg/kg/day)or Compound C(20 mg/kg/day)was intraperitoneally injected every day.7 days after the initial injection of DOX,mice were tested by the echocardiography,then all mice were euthanized for further experimental analysis.The myocardial tissues were observed after HE staining,and the ultrastructural morphology of mitochondria in myocardial tissues were checked by transmission electron microscopy.The ROS level,the content of SOD,MDA and GPx,and cell apoptotic rate were respectively investigated.The expression of p-AMPK,PGC1?(NRF1,TFAM,UCP2)cascades and apoptosis-related signaling(Bax,Bc12,cleaved caspase 3)were tested by western blot.4.The H9c2 cells were divided into(i)Control group;(ii)PGC1? siRNA group;(iii)DOX group;(iv)DOX+MEL group;(v)DOX+MEL+PGC1? siRNA group.Moreover,ultrastructural morphology of mitochondria in H9c2 cells were checked by transmission electron microscopy.We then further tested whether PGC1? was involved in the melatonin's protection against DOX-induced acute oxidative stress and apoptosis on myocardiocytes through the methods used in part 1.Results:1.We found 100?M MEL cotreatment could significantly reverse the 1?M DOX-induced downregulation of H9c2 cell viability and ATP content,upregulation of ROS and MDA levels,cell apoptotic rate and Bax and cleaved caspase 3 levels,and decrease of Bcl2 level.2.We found 100 ?M MEL cotreatment could significantly reverse the 1 ?M DOX-induced downregulation of p-AMPK,PGC1?,NRF1,TFAM and UCP2 expression.After the AMPK siRNA treatment,the above-mentioned protective efects of MEL against DOX-induced apoptosis and oxidative stress injury on myocardiocytes were partially reversed.3.In C57BL/6 mice,MEL cotreatment could significantly alleviate the DOX exposure-induced acute cardiac dysfunction,myocardial injury,and mitochondrial dysfunction and morphological disorders.MEL could inhibit DOX-induced oxidative injury,apoptosis and apoptosis-related signaling expression in cardiac tissues.Moreover,MEL cotreatment could promote p-AMPK and PGC1?(NRF1,TFAM,UCP2)cascades levels in DOX-treated hearts.However,inhibiting AMPK activity by Compound C cotreatment significantly reversed the above-mentioned protective effects of MEL on DOX-injured heart.4.After the PGC1? siRNA treatment,the above-mentioned protective effects of MEL against DOX-induced apoptosis and oxidative stress injury on H9c2 were partially reversed.PGC1? knockdown antagonized MEL-exerted upregulating NRF1,TFAM,UCP2 expression,but it was without effect on the AMPK phosphorylation in DOX-treated H9c2 cells.Conclusion:Collectively,our experiments provided in vivo and in vitro mechanistic evidence that melatonin treatment promoted AMPK activation and upregulation of PGC1?(NRF1,TFAM and UCP2)signaling,which contributes to preserving cardiac function and mitochondrial homeostasis,and alleviating oxidative stress and apoptosis in DOX-treated cardiomyocytes,thus alleviating DOX-induced acute cardiotoxicity.Therefore,melatonin could be a promising agent for clinical prevention and treatment of DOX-related acute myocardial injury,and this study will provide theoretical basis for clinical application of melatonin.