Zinc Oxide Nanoparticles-induced Cardiac Injury And Its Mechanism

Background and Purpose:Zinc oxide nanoparticles(ZnO NPs)are one of the most widely used nanomaterials in a variety of fields such as industrial,pharmaceutical,and household applications.Increasing evidence suggests that ZnO NPs could elicit unignorable harmful effect to the cardiovascular system,and mitochondria are its main targets,but the potential deleterious effects to heart remain to be elucidated.Mitochondrial biogenesis is an important mechanism for regulating mitochondrial function and protecting against cardiac injury.Fox O3 a plays an important regulatory role in maintaining the redox homeostasis of cardiomyocytes,participating in PGC-1?-mediated mitochondrial biogenesis.This study aims to observe the ZnO NPs-induced cardiac injury in vitro and in vivo.The study focuses on perspective of mitochondrial biogenesis and function,revealing the role of Fox O3a-PGC-1?-mediated mitochondrial biogenesis in protecting against cardiac injury.Through this study,we hope to provide new evidence for cardiac safety assessment of ZnO NPs and provide reference strategies and methods for cardiotoxicity testing and safety evaluation of nanomaterials.Methods:1.ZnO NPs were characterized by transmission electron microscopy and dynamic light scattering.2.Cytotoxicity,mitochondrial damage and mitochondrial biogenesis signal pathway were observed in human myocardial cell line AC16.The cytotoxicity-induced by ZnO NPs in AC16 cells was evaluated by determination of cell viability and lactate dehydrogenase release.Cellular reactive oxygen species(ROS)and mitochondrial membrane potential were measured by high-content analysis(HCA).Cellular ATP was measured by luciferase method.Mitochondrial biogenesis was assayed by detection of mt DNA copy number(RT-PCR)and PGC-1? pathway(Western blot).Further,we use transient transfection technology to knock down Fox O3 a,and explore the changes in toxic effects and its potential molecular regulatory mechanism.3.Then,we used a new in vitro model of cardiomyocytes,human induced pluripotent stem cell-derived cardiomyocytes(hi PSC-CMs),which possesses electrophysiological and biochemical properties of human cardiomyocytes.We further confirmed the myocardial ZnO NPs-induced cardiac injury,mainly focus on observing the effect on mitochondrial biogenesis,and changes of cardiac electrophysiological function using microelectrode array technology.4.We used Fox O3a-knockout FVB mice as in vivo model.The mice were intracheally instilled ZnO NPs at dose of 0,0.2,1.0 mg/kg.bw once every two days for one week.Cardiac injury was evaluated by detecting routine blood biochemistry,myocardial injury enzymes,echocardiography,myocardial tissue pathology and ultrastructural changes.SOD activity,GSH and MDA content in heart and Fox O3a-PGC-1? pathway were detected.The possible toxicity mechanism of Fox O3 a on the regulation of oxidative stress and mitochondrial biogenesis function was discussed.Results:1.TEM micrographs demonstrated ZnO NPs were spheroid and rod in shape with mild agglomeration.ZnO NPs could enter AC16 cells and hi PSC-CMs by endocytosis.The hydrodynamic diameter of ZnO NPs in double distilled water and cell culture medium were 214.16 ± 0.31 nm and 266.27 ± 27.36 nm,respectively.2.ZnO NPs concentration-and time-dependently elicited cytotoxicity in AC16 and hi PSC-CMs.ZnO NPs induced cellular ROS accumulation and MMP decline at low cytotoxic exposure doses.Changes in mitochondrial biogenesis were closely related to the exposure concentration of ZnO NPs.Active mitochondrial biogenesis was observed at lower exposure concentration(25,50 ?M 6h),mainly manifested by increased mitochondrial abundance and mt DNA copy number.Conversely,mitochondrial biogenesis impairment was observed at higher exposure concentration.In addition,ZnO NPs at insignificantly cytotoxic concentrations were found to trigger cardiac electrophysiological alterations as evidenced by decreases of beat rate and spike amplitude.3.It was found that Fox O3 a directly regulates PGC-1?-mediated mitochondrial biogenesis in cardiomyocytes.The expressions of PGC-1? and TFAM were up-regulated and then down-regulated,and the inflection point was 50 ?M for 6 h exposure.Fox O3 a knock-down aggravates ZnO NPs induced mitochondrial biogenesis impairment,mitochondria dysfunction and cytotoxicity.4.Cardiac injury was observed in mice after intracheally instilled of ZnO NPs.WBC,Neu%,TG,CK,CK-MB,HBDH and LDH were found increased.Histopathological changes and ultrastructural changes in heart were observed in the high-dose group.Echocardiographic results showed that the anterior and posterior walls of the left ventricle in systole were significantly thickened,and the ejection fraction and fractional shortening were slightly reduced(no significant difference).The heart organ coefficient was significantly increased in exposure group.GSH depletion and MDA accumulation were found in myocardial tissue.5.Compared with wild-type mice,Fox O3 a knockout mice show severer cardiac injury.At the same exposure dose,the content of GSH and MDA,the degree of left ventricular wall thickening,and the level of myocardial injury enzyme were significantly different from those of the wild type.Through pathological sections and TEM observations,severer changes in cardiac structure and mitochondrial damage were observed.6.The Fox O3a-PGC-1? pathway in wild-type mouse was continuously activated after ZnO NPs exposure with activated mitochondrial biogenesis.Fox O3 a knockout mice showed low expression of PGC-1? pathway protein expression,with mitochondrial biogenesis impairment and severer mitochondrial damage.Conclusion:1.ZnO NPs could induce ROS accumulation,oxidative stress and mitochondrial dysfunction,which eventually lead to cardiac injury.2.The Fox O3a-PGC-1? signaling pathway plays an important role in maintaining cellular redox homeostasis and regulating mitochondrial biogenesis.After knocking down(knocking out)of Fox O3 a,ZnO NPs could induce severer cardiac injury.3.Our results showed that the electrophysiological properties of hi PSC-CMs were affected by ZnO NPs at insignificantly cytotoxic doses,and myocardial damage in mice was found at doses below the occupational exposure limit.It is necessary to pay close attention to the cardiovascular damage caused by ZnO NPs at low exposure dose.4.It is recommended that hi PSC-CMs-based HCA and MEA assay will be a stable and reliable strategy for screening of nanomaterial-induced cardiac toxicity screening and safety evaluation.