The Role Of SIRT1 In Doxorubicin-induced Myocardial Toxicity And Mitochondrial Dysfunction

ObjectiveThe mitochondrial biogenesis dysfunction and the consequent mitochondrial structure collapsion are considered to be one of the most important mechanisms of cardiotoxicity of Doxorubicin.Mitochondrial biogenesis was regulated by both nuclear genome and mitochondrial.The key adjuster of mitochodnrial biogeneisis,peroxisome proliferator-activated receptor gamma coactivator-1 alpha(PGC-1α),is modified by its upstream factor silent mating type information regulation 2 homolog 1(SIRT1)by deacetylation.The SIRT1/PGC-1α pathway has been proved to be the vital signal routing that help maintaining the normal function of mitochondrion and regulating cell metabolism.The SIRT1 agonist can protect mitochondrial function by upregulating expression and activity of SIRT1 and subsequently improve the transcription of PGC-1α and its target genes.This study intends to explore for the role that SIRT1 palys in DOX induced cardiac mitochondrial toxicity and to look for the new toxicology mechanism for the prevention and therapy of side effects of DOX.Methods1.Role of SIRT1 in RSV’s protection against DOX induced mitochondrial toxicity.AC16 human cardiomyocytes were either pretreated with RSV to activate SIRT1 or transfected with siRNA to knock down SIRT1 and then were exposed to DOX.The changes of parameters such as cell viability,intracellular general ROS production,mitochondrial membrane potential,activity of citroyl synthetase and ATP content demonstrated the great damage to mitochondrial structure,antioxidative capacity and respiration function.The mtDNA copy number,mitochondrial content and expression of PGC-1α pathway related proteins are tested for the supervision of mtiochonrial biogenesis.The activity and expression of SIRT1,as well as the acetylation of PGC-1α were tested,too.The SIRT1 deficient cells were measured with cell viability,mtDNA copy number,general ROS production,ATP content,mitochondrial membrane potential and proteins that related to mitochondrial biogenesis.2.The mechanism of EPO’s protection agains DOX induced cardiotoxicity: the role of SIRT1 in mitochondrial function.The aim of this chapter was to find whether and how did SIRT1 take part in the protection of EPO’s against DOX induced cardiotoxicity.The AC16 human cardiomyocytes were expposued to DOX with or without pretreatment of EPO.Cell viability,mitochondrial superoxide,mitochondrial membrane potential,ATP content,proteins expression of superoxide dismutase SOD2 and the key enzyme of Kerb’s cycle,the citroyl synthetase,were all measured for evaluating the mitochondrial structures and function of oxidation resistance and ability of energy production.The parameters such as mt DNA copy number,proteins expression of VDAC and COXIV,as well as the molecule that related to PGC-1α mediated mitochondrial biogenesis pathway,were all tested to find any changes that EPO made to mitochondrial biogenesis.SIRT1 activity and expression,together with PGC-1α acetylation were tested for the measurement of SIRT1’s participation during the whole protection of EPO against DOX exposure.Again,the SIRT1 deficient AC16 cells were used to test the parameters related to mitochondrial biogenesis and functions.3.Bechmark dose approach and western blot were used to evaluate the different toxic effects between sigle and repeated dose of DOX exposures,with the aim of confirming existence of probability of different molecular events that might have been involved.Results1.SIRT1 plays a key role in RSV protection against DOX induced cardiotoxicity.DOX was found to decrease the cell viability,SIRT1 activity,mitochondrial function and biogenesis in both 12 h and 24 h exposures of DOX.Both RSV and EPO pretreatment were found to upregulate SIRT1 activity and protein expression to reverse DOX-induced acetylation of PGC-1α and suppression of a suite of PGC-1α-activated genes involved in mitochondrial function and biogenesis.Silencing of SIRT1 via small RNA interference sensitized AC16 cells to DOX-induced cytotoxicity and reduction in mitochondrial function and biogenesis.The toxic effects of mtDNA copy number,MMP,ATP content,ROS and proteins expression of PGC-1α pathway were all deteriorated by SIRT1 knocking down.The fact that SIRT1 deficiency eliminated the protection of RSV and EPO suggested that the protection of the both agents were SIRT1 depentent.2.The relationship between SIRT1 and EPO’s protection against DOX induced toxicity.After being approved to be related to SIRT1,EPO was expoused as a protective agent against DOX induced toxicity with unknown mechanism.EPO greatly attenuated DOX induced ctytotoxicity,mitochondrial biogenesis dysfunction such as decrease of mtDNA copy number,protein expression of PGC-1α pathway related molecules,as well as mitochondrial dysfunction such as oxidative stress,decrease of CS and SOD expression and MMP.SIRT1 defecient cells were more vulnerable to cytotoxicity,oxidative stress and demonstrated lower mtDNA copy number,ATP and MMP.The results demonstrated that SIRT1 played an important role in maintaining the normal function of mitochondrion and cell and further proved that EPO protected against DOX in a SIRT1 dependent manner,so as to imply the important role of SIRT1 in both protection and maintenance of normal mitochondrial structures and functions.3.Difference between single dose and repeated dose exposures.The tolerance to DOX of cells that pretreated with non-toxic concentration of repeated DOX exposure for 3 days were higher than that of the single dose;yet the pretreatment with non-toxic concentration of repeated DOX for 6 days oppositely made the cells more vulnerable than that of the single dose.The transformations of the tolerance of cells were evaluated by IC50 and BMDL.In another repeated exposure schedule,the cells were exposued to DOX with same accumulative dosage yet with different frequency.The western blots showed there were significant differences in the protein expressions of SIRT1 and PGC-1α pathway related molecule between single and repeated exposures.Conclusion1.SIRT1 was the key toxicity target of DOX and as well as the therapeutic target of protection agents.It is SIRT1 activity suppression caused by DOX that induced the perturbation of mitochondrial biogenesis pathway,which in hence leads to the dysfunction of mitochondrial biogenesis and normal physiological function of mitochondrion.In the other hand,activation of SIRT1 can demonstrate the remarkable protection against DOX induced cardiotoxicity and mitotoxicity.The agent that can activate SIRT1 such as RSV and EPO is proved to have the prospect of becoming the candidate that could play important role in protection against DOX side effects in cilinic.SIRT1,as one of the key molecules of mitochondrial toxicity pathway and important post-translational modification enzyme,plays a vital role in matianing the normal function of cardiomyocytes.These findings are of great significance to exploration of mechanism of DOX cardiotoxicity and also to finding the novel approach for treatment of DOX induced side effect in chemotherapy.2.Difference of viability and protein expression between single and repeated dose of DOX exposure.Repeated dose of DOX exposure lead to variation of IC50 and BMDL of cell viability to single dose.SIRT1/PGC-1α pathway expressions were also demonstrated a difference.It implied there maybe different molecular mechanism which was involved in single and repeated dose exposures.