Molecular Mechanism Of Organochlorine Pesticide Pentachloronitrobenzene-Induced Cardiotoxicity

During the development of heart,cardiomyocytes proliferation plays an important role in the morphogenesis of the ventricular wall.Abnormal cardiomyocytes proliferation is closely associated with ventricular noncompaction cardiomyopathy.The organochlorine pesticide Pentachloronitrobenzene(PCNB)is a land fungicide widely used in agricultural production.It has strong bioaccumulation propensity in the ecosystem,which brings serious problems to crop production and food safety.Based on the previous research,this study further explored the effects of PCNB on cardiomyocytes proliferation and the molecular mechanism of PCNB-mediated developmental cardiotoxicity.In this study,pregnant mice(C57BL/6N)were treated with PCNB from E8.5 until the day of delivery.We analyzed the histological appearance of PCNB-exposed embryo heart at E14.5 and E17.5.HE staining analysis showed that heart of embryo treated with PCNB did not display obvious cardiac developmental defects,but exhibited a remarkably thinner compact layer in both ventricles associated with prominent trabeculae,consequently led to a marked increase of ventricle noncompaction-to-compaction ratio,caused ventricular noncompactional-like pathological phenotype.To explore the possible mechanism of PCNB-induced ventricular noncompaction cardiomyopathy,we detected proliferation and apoptosis of cardiomyocytes.Immunofluorescence staining demonstrated that PCNB treatment decreased the level of cell cycle marker Ki67 and mitotic marker phosphohistone H3(pH3)in the compact layer of PCNB-treated hearts.We found that PCNB treatment did not induce significant apoptosis in fetal mice cardiomyocytes via TUNEL staining.These results indicated that PCNB inhibited proliferation and mitosis of fetal mice cardiomyocytes.To explored the toxic effects induced by PCNB treatment,rat cardiomyocytes H9c2 were treated with different concentrations of PCNB.We found that PCNB inhibited cardiomyocytes proliferation and mitosis in dose-dependent manner via EDU and pH3 staining,respectively.The results of flow cytometry demonstrated that PCNB did not induce significant apoptosis of cardiomyocytes.These results suggested that PCNB may cause ventricular noncompaction cardiomyopathy by inducing mitotic abnormalities and inhibiting proliferation of fetal cardiomyocytes.RNA-seq analysis of E17.5 mice heart tissue were used to explore the molecular mechanism of PCNB-induced ventricular noncompaction cardiomyopathy.The results showed that 92 genes were downregulated and 97 genes were upregulated in PCNB-treated mice heart compared with control mice heart.The results of GO analysis showed that differentially expressed genes(DEGs)were mainly related to cell mitosis,chromosome segregation,and kinetochore-microtubule binding.The analysis of DEGs interaction networks demonstrated that the major components of kinetochore(including NDC80 complex members Hec1,Nuf2,Spc25)and regulators of kinetochore-microtubule interaction were located in the center of the network.RT-PCR analysis of candidate gene also verified the results of transcriptome sequencing.As the major event in mitotic,arrangement and separation of chromosomes were coordinated by the dynamic interaction between kinetochore and spindle microtubules.Hee1 constituted the core microtubule-binding site of the kinetochore and played an important role in chromosome separation.We initially chose Hec1 as candidate gene for biological verification.Immunohistochemical staining showed that Hec1 was distributed throughout heart tissue and was highly expressed in the compact layer of ventricular where cardiomyocytes proliferation was highly active.PCNB treatment significantly reduced the expression of Hec1 in the compact layer of ventricular.Western blot analysis demonstrated that PCNB treatment significantly decreased the protein levels of Hec1 in heart tissue.HE staining of heart tissue sections revealed that the percentage of abnormal nuclei was increased after PCNB treatment.Immunofluorescence staining of spindle marker β-tubulin revealed that PCNB treatment caused abnormalities in spindle morphology and chromosome segregation of mitotic cells.The results of cell experiments showed that PCNB treatment downregulated the expression of Hec1,induced abnormalities in the morphology and arrangement of spindle and chromosomes,disturbed cardiomyocytes mitosis and inhibited cell proliferation.We found that endogenous Hec1 mainly localized to the kinetochores of cardiomyocytes.Knockdown endogenous Hec1 in cardiomyocytes by siRNA inhibited cardiomyocytes proliferation and mitosis,increased the percentage of monopolar and multipolar spindles while decreased the percentage of normal bipolar spindles,aggravated the inhibitory effect of PCNB on cardiomyocytes proliferation and mitosis.Overexpression of Hec1 significantly inhibited abnormal spindle morphology caused by PCNB treatment and restored cardiomyocytes proliferation and mitosis activity.These results suggested that PCNB treatment may inhibited cardiomyocytes proliferation and mitosis via downregulating the expression of Hec1,which affected kinetochore-microtubule binding.This study demonstrated that PCNB treatment inhibited cardiomyocytes proliferation and caused ventricular noncompactional-like pathological phenotype in fetal mice heart.PCNB downregulated the expression of kinetochore-associated proteins Hec1,induced abnormalities in spindle morphology and chromosome separation during mitosis,led to mitotic arrest.