Study On The Developmental Toxicity Of Chicken Embryo Heart Induced By Perfluorooctanoic Acid Exposure And The Therapeutic Effect Of Levocarnitine

Objective:The aim of this study was to establish perfluorooctanoic acid?PFOA?-induced developmental cardiotoxicity model and lentivirus mediated in ovo gene silencing model with chicken embryo,with which the toxic effects of PFOA exposure at various doses and the underlying molecular mechanisms at animal and cellular level were investigated.Additionally,the potential protective effect of levocarnitine was assessed.This study revealed the mechanism of PFOA-induced developmental cardiotoxicity as well as provided a theoretical basis for the potential use of levocarnitine for heart development protection.Methods:For the animal study,we assigned fertilized eggs into eight groups according to the weight of eggs,including control group?sunflower seed oil?,2 mg/kg?egg weight?PFOA group,2 mg/kg?egg weight?PFOA+100 mg/kg?egg weight?levocarnitine group,control virus group,BMP2 lentivirus silencing group,2 mg/kg?egg weight?PFOA+BMP2lentivirus silencing group,2 mg/kg?egg weight?PFOA+10?g/kg?egg weight?BMP2recombinant protein group,and BMP2 lentivirus silencing+100 mg/kg?egg weight?levocarnitine group.Before hatching,the egg surface was disinfected and exposed to PFOA/levocarnitine via air chamber injection.By the second day of hatching,designated groups were transfected with BMP2-silencing lentivirus or control virus via microinjection;and the PFOA+BMP2 recombinant protein group was injected with BMP2 recombinant protein.By the 15th day of chicken embryo development,some of the embryos were removed from the shell,hearts were dissected out,and subjected to real-time qRT-PCR and western blotting to confirm the silencing efficacy of lentivirus.Remaining eggs were incubated until hatch,heart rates were measured with electrocardiography,and hearts were then subjected to histological assessments for the thickness of right ventricular wall and further BMP2 silencing confirmation.Additionally,the expression levels of PPAR?,BMP2and the activated second messenger of BMP2,pSMAD1 were assessed with western blotting in the hatchling heart tissue samples.At cellular levels,the egg surface was disinfected,and the eggs in the pretreatment groups were exposed to PFOA/levocarnitine via air cell injections,and then put into the incubator.On the second day of incubation,the eggs in the lentivirus silencing groups were microinjected with PPAR?lentivirus and put back into the incubator until the 10th day.The chicken embryos were then removed from the shell,hearts were dissected out,and then the primary chicken embryo cardiomyocytes were extracted.On the other hand,the eggs in the direct exposure groups were incubated without treatment until the 10th day,primary cardiomyocytes were cultured,and then directly exposed to different concentrations of PFOA/levocarnitine for 24/48 hours,and then cytotoxicity was determined with CCK8assay,the intracellular calcium concentration was measured with Fluo-4 AM probe,and the morphology of primary cardiomyocytes was evaluated by hematoxylin&eosin staining.Cardiomyocyte proteins were extracted and the expression levels of Wnt5a and Frizzled2proteins in cardiomyocytes were assessed with western blotting.Results:?1?The results of animal experiments indicated that PFOA exposure significantly increased the heart rate and decreased the thickness of right ventricular wall,which was alleviated by levocarnitine treatment.In the hearts of hatchling chickens,the changes induced by BMP2silencing were very similar to those induced by PFOA exposure.Western blotting results demonstrated that PFOA exposure enhanced the expression of BMP2 but inhibited the expression of pSMAD1 in chicken embryo hearts,while the treatment with levocarnitine could reverse such changes.Comparing with control group and control virus group,BMP2silencing significantly enhanced the expression level of PPAR?protein.?2?The results of cellular study showed that both PFOA pretreatment and direct exposure decreased the viability of primary cardiomyocytes,changed the morphology of cardiomyocytes,and increased the concentration of intracellular Ca²⁺.Levocarnitine treatment effectively abolished such effects.Western blotting results indicated that PFOA pretreatment increased the protein expression levels of Wnt5a and Frizzled2,while direct exposure to PFOA increased the expression level of Frizzled2,but decreased the expression level of Wnt5a.After PPAR?lentivirus silencing,there were no significant difference in the protein expression levels of Wnt5a and Frizzled2 among the pretreatment groups or the direct exposure groups.Conclusion:?1?The developmental toxicity of chicken embryo heart induced by PFOA is associated with BMP2/pSMAD1 signaling pathway,which may be located upstream of PPAR?.Meanwhile,levocarnitine may be a potential therapeutic agent for the developmental toxicity induced by PFOA.?2?Both PFOA pretreatment and direct exposure could decrease the cell viability,increase the intracellular Ca²⁺concentration and change the cell morphology of the primary cardiomyocytes from chicken embryo.These changes are closely associated with the signaling pathway of Wnt5a/Frizzled2.Levocarnitine has a therapeutic effect on cytotoxicity induced by PFOA,potentially through protection against calcium overload and restoration of Wnt5a/Frizzled2 signaling pathway.