The Protective Effect Of NMN Against Doxorubicin-Induced Cardiotoxicity And Its Related Mechanisms

Doxorubicin is a representative of anthracycline antitumor drugs,which has a broad antitumor spectrum and definite efficacy,and is widely used in the chemotherapy of many kinds of tumors in adults and children.However,the cardiotoxicity of doxorubicin is prominent,with high incidence and poor prognosis,which hinders the clinical application of doxorubicin and quality of life of patients.Oxidative stress is a canonical mechanism of doxorubicin cardiotoxicity,and myocardial mitochondria damaged by doxorubicin are important sources of reactive oxygen species.Since the antioxidant system of cardiomyocytes is relatively weak,autophagy serves as an important defense mechanism against oxidative stress.Recent studies have found that lysosomal dysfunction and the resultant autophagy disturbance can exacerbate doxorubicin-related cardiac injury.However,the pathogenesis of doxorubicin cardiotoxicity remains incompletely understood,and targeted prevention and treatment methods are very limited and may weaken the antitumor effect of doxorubicin.Therefore,it is of great importance and urgency to further explore the molecular mechanism of doxorubicin cardiotoxicity and to develop new therapeutics with high efficacy and low toxicity.Depletion of nicotinamide adenine dinucleotide(NAD+)in the myocardium contributes to the development of doxorubicin cardiotoxicity.NAD+ is important in the maintenance of cellular redox homeostasis,DNA damage repair,protein deacetylation and other processes,which is crucial for maintaining the normal function of mitochondria,lysosome and other organelles.Previous studies have shown that boosting NAD+is a promising therapeutic strategy for a variety of heart diseases,including doxorubicin myocardial injury.As an NAD+ precursor,nicotinamide mononucleotide(NMN)protects the heart from injuries related to aging,ischemia/reperfusion,obesity,etc.However,the protective effect of NMN against doxorubicin-related myocardial injury still needs further investigation,and its effect on the antitumor efficacy of doxorubicin is unclear.In addition,the mechanisms of action of NMN against doxorubicin cardiotoxicity remain elusive.The present study investigated the ameliorative effects of NMN on doxorubicin related myocardial injury in vivo and in vitro,and determined whether NMN had any effect on the anti-tumor effect of doxorubicin.The present study also explored the mechanisms of NMN protecting against doxorubicin myocardial injury by focusing on improving mitochondrial and lysosomal function.Objectives:1.To evaluate whether NMN improves doxorubicin-related myocardial injury without affecting its anti-tumor efficacy.2.To examine the effect of NMN on mitochondrial oxidative stress of cardiomyocytes induced by doxorubicin and its effect on ameliorating myocardial injury induced by doxorubicin.3.To investigate the effect of NMN on doxorubicin-induced lysosomal injury of cardiomyocytes.4.To determine whether NMN alleviates doxorubicin-induced lysosomal dysfunction of cardiomyocytes by reducing mitochondrial reactive oxygen generation.Methods:1.Male C57BL/6 mice were intraperitonealy injected with a single dose of doxorubicin to establish an acute myocardial injury model.Meanwhile,intraperitoneal injection of NMN was used for intervention.Five days later,cardiac systolic function was detected by echocardiography,serum Troponin I and CK-MB levels were detected by ELISA.The structure of heart tissue,myocardial cell death and collagen deposition were observed by HE staining,Evans blue staining and Sirus red staining,respectively.Myocardial mitochondria were isolated,and the level of NAD+and reactive oxygen species generation were detected.The levels of protein carbonylation and lipid oxidative damage in myocardial tissue were detected by colorimetry,and the expression of Bnip3 was detected by Western Blot and qPCR.2.The cardiomyocyte injury model induced by doxorubicin was established in rat embryonic cardiomyocyte line H9c2 cells and neonatal mouse cardiomyocytes,and NMN was used for intervention.The intracellular NAD+level was detected by colorimetry.Myocardial cell survival rate and LDH leakage were determined to evaluate cell damage.Caspase-3 activity,caspase-3 cleavage and DNA fragmentation were detected to observe the apoptosis level.MitoSOXTM staining was performed to detect mitochondrial reactive oxygen species,ATP production was detected by bioluminescence,and Bnip3 expression was detected by Western Blot.LysoID? Red staining was used to detect lysosomal perturbation,LysoSensorTM Yellow/Blue DND-160 staining was used to detect lysosome pH,and Western Blot was used to detect cathepsin B expression and autophagic flux.On this basis,the effect of combined usage of bafilomycin A1,an inhibitor of lysosomal acidification,on the protective effect of NMN against doxorubicin cardiotoxicity was observed to explore whether NMN could counteract doxorubicin cardiomyopathy by improving lysosomal function and regulating autophagy.3.In doxorubicin-injured H9c2 cells,the effects of mitochondrial targeted reactive oxygen scavengers mito-Tempo on myocardial cell apoptosis,lysosome perturbation,lysosome pH,cathepsin B maturation and autophagic flux were observed to explore whether blocking mitochondrial reactive oxygen species could improve lysosome dysfunction.Furthermore,the effects of NMN and mito-Tempo on the oxidation of ATP6V0D1 were observed by means of immunoprecipitation combined with DNP derivation.4.Breast cancer cells were cultured in vitro,and a tumor-bearing nude mouse model was established.The effects of NMN alone or in combination with doxorubicin on tumor growth were observed.Results:1.In cultured cardiomyocytes,NMN boosted intracellular NAD+level,alleviated cell damage caused by doxorubicin,and inhibited necroptosis and apoptosis.In mouse model of doxorubicin-induced myocardial injury,NMN alleviated myocardial injury,improved left ventricular systolic function and myocardial structure,and reduced cell death.2.NMN did not promote the proliferation of cultured breast cancer cells,nor did it weaken the inhibitory effect of doxorubicin on the proliferation of breast cancer cells.In nude mice bearing breast cancer xenografts,NMN did not promote the growth of breast cancer subcutaneous grafts,nor did it affect the in vivo antitumor effect of doxorubicin.3.In the mouse model,NMN increased the level of NAD+in myocardial mitochondria of doxorubicin-exposed mice,reduced the generation of mitochondrial reactive oxygen species,and alleviated oxidative damage of myocardial tissue.In cultured cardiomyocytes,NMN inhibited the generation of mitochondrial reactive oxygen species and increased ATP productionin doxorubicin-damaged cardiomyocytes.4.NMN alleviated doxorubicin-induced lysosomal damage,improved lysosomal acidification and function,and improved autophagy flux in cardiomyocytes.Inhibition of lysosomal acidification weakened the protective effect of NMN on doxorubicin-damaged cardiomyocytes.5.Mito-Tempo,a mitochondria-targeted reactive oxygen scavenger,could alleviate doxorubicin-induced myocardial cell damage and reduce apoptosis.Mito-Tempo alleviated lysosomal damage,improved lysosomal acidification and function,and improved autophagy flux in doxorubicin-induced cardiomyocytes.6.Both NMN and mito-Tempo inhibited oxidative modification of ATP6V0d1 induced by doxorubicin.Conclusions:1.NMN alleviates doxorubicin-related myocardial injury without hindering the anti-breast tumor activity of doxorubicin.2.The protective effect of NMN against doxorubicin-related cardiac injury is related to the inhibition of mitochondrial reactive oxygen generation.3.NMN reduces doxorubicin myocardial injury by improving lysosomal function.4.NMN alleviates doxorubicin-induced lysosomal damage of cardiomyocytes by reducing mitochondrial reactive oxygen generation.Significance:This study has provided significant insights into the therapeutic effects of NMN on doxorubicin cardiotoxicity.The findings may inform clinical trials for alleviating doxorubicin cardiotoxicity with NMN,which may provide a therapeutic option to protect the heart and hopefully,minimize mortality.