Neuraminidase1 Inhibitor Protects Against Doxorubicin-Induced Cardiotoxicity Via Modulating Drp1-dependent Mitophagy

Background and purpose: Anthracyclines,such as doxorubicin(DOX),are among the most effective chemotherapeutic drugs for various malignancies and also serve as headstone for anti-tumor therapy.However,their clinical use is limited by irreversible cardiotoxicity.The underlying mechanisms of DOX-induced cardiotoxicity is still controversial and no consensus has been reached on the best way to prevent DOX-induced cardiotoxicity.As so far,there is no clinical drug that can completely prevent DOX-induced cardiotoxicity.Therefore,it is important to lucubrate and illustrate mechanisms of DOX-induced cardiotoxicity and it is an immediate need for developing novel therapeutic agents on the basis of clinical value.Neuraminidase1(NEU1)is one of members of sialidases and is highly expressed in the heart of mammals,which has been demonstrated to involve in several cardiovascular diseases.However,there are rare researches exploring the relationship between NEU1 and DOX-induced cardiotoxicity so far.In this study,we have three purposes listed as follows: first of all,whether NEU1 is a critical inducer of DOXinduced cardiotoxicity? Second,whether NEU1 inhibitor could improve DOX-induced cardiac dysfunction? Finally,what’s the underlying mechanism of NEU1 inhibitor against DOX-induced cardiotoxicity?Methods: Adult male Sprague–Dawley(SD)rats and H9C2 cells are used in this study.Methods of animal experiments in vivo: Rats were randomized into three groups: control,DOX,and DOX + OSE.Rats in DOX + OSE group received gavage of OSE(20 mg/kg)dissolved in normal saline beginning from 3 days before the first injection of DOX to the end of the experiment.Rats in DOX group and DOX + OSE group received intraperitoneal injection of DOX in a cumulative dose of 15 mg/kg within 2 weeks(2.5 mg/kg for six times).Rats in control group were given equal volume of saline for intraperitoneal injection or gavage.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of NEU1 in rat hearts and plasm;immunoblotting and immunohistochemistry analyses were used to detect expression levels of NEU1 in rat hearts;transthoracic echocardiography was conducted to evaluate cardiac function of rats in different groups;histopathology analysis and biochemical indexes of myocardial injury analysis were conducted to evaluate cardiac injury extent;ELISA assays were used to detect the levels of oxidative stress in adult rat serum;Transmission electron microscope analysis,immunofluorescence analysis,immunoblotting analysis and immunohistochemistry analysis were used to measure the activities of autophagy and mitophagy;Terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining and immunoblotting analysis were used to detect apoptosis of cardiomyocytes.As for cell experiments in vitro,H9C2 cells were used in this study.NEU1 inhibitor OSE and Drp1 inhibitor Mdivi-1 were used as tools for cell experiments to further verify the mechanism of NEU1 inhibitor against DOX-induced myocardial injury.Results: Animal experiments revealed that DOX insult significantly increased the expression and activity of NEU1 in rat hearts,while the increase was dramatically restrained by co-treatment with NEU1 inhibitor OSE.NEU1 inhibitor showed protective role against DOX-induced cardiotoxicity,as evidenced by reduced levels of cardiac injury,reduced myocardial atrophy and fibrosis,reduced oxidative stress and apoptosis,and improved cardiac systolic function.The beneficial effects of NEU1 inhibitor OSE against DOXinduced cardiotoxicity were associated with the suppression of Drp1-dependent mitochondrial fission and mitophagy.Cell experiments further revealed that DOX substantially increased expression of NEU1 in H9C2 cells in a time dependent manner.The elevated NEU1 triggered by DOX further increased the expression of Drp1,which subsequently enhanced mitochondrial fission and PINK1/Parkin pathway-mediated mitophagy,leading to a maladaptive feedback circle towards mitochondrial damage,oxidative stress and myocardial apoptosis.NEU1 inhibitor OSE administration selectively inhibited the increased NEU1 in myocardial cells insulted by DOX,followed by reduction of Drp1 expression,inhibition of PINK1 stabilization on mitochondria,and Parkin translocation to mitochondria,thus alleviating excessive mitochondrial fission and mitophagy,alleviating mitochondrial damage,oxidative stress and subsequent development of cellular apoptotic process.Conclusions: NEU1 inhibitor protects against DOX-induced oxidative stress and myocardial apoptosis through suppressing Drp1-dependent mitophagy,thus alleviating DOX-induced cardiotoxicity.This study confirmed NEU1 as a crucial inducer of DOX cardiotoxicity by enhancing mitochondrial fission and mitophagy,and inhibition of NEU1 by OSE could dramatically improve cardiac dysfunction caused by DOX insult,strongly suggesting the clinical potential of targeting NEU1 as a novel strategy for treatment of chemotherapeutic drugs-induced cardiotoxicity.