Effect And Functional Mechanism Of Manganese On Heat Stress-induced Apoptosis In Primary Chicken Embryo Myocardial Cells

Heat stress is a difficult problem that is often encountered in the concentrated feeding of poultry,which can lead to great loss of growth and reproduction.Heat stress may profoundly affect the heart and lead to sudden death in high-density poultry in captivity,especially because of the absence of sweat glands and the covering of feathers in poultry.As the research moves along,it was found that heat stress-induced myocardial cell apoptosis and myocardial injury is one of the causes of poultry sudden death under heat stress.Therefore,inhibition of apoptosis plays an important role in alleviating heat stress injury of cardiomyocytes,maintaining normal cardiac function and reducing heat stress loss.Adding antioxidants into the diet is one of the most studied ways to relieve heat stress.Manganese（Mn）is an essential micronutrient in animals and a component of manganese superoxide dismutase（Mn-SOD）.Studies on the antioxidant activities of Mn are relatively mature,but the current knowledge of the effect of Mn on cell apoptosis induced by heat stress is still very limited.And whether Mn can further regulate apoptosis induced by oxidative stress and the mechanism of action remain unclear.In this study,chicken embryos were used as the test material for the following studies:1)Primary chick embryonic myocardial cells were isolated and cultured,and then a apoptosis model of heat stress was established.2)The regulatory funtion and potential mechanism of Mn in the process of apoptosis induced by heat stress was evaluated by CCK-8,flow cytometry,RT-PCR,western blot,etc.3)Differentially expressed proteins（DEPs）in primary chick embryonic myocardial cells were identified using DIA technology and bioinformatics analysis,followed by the differential expression analysis and functional enrichment analysis.Finally,the key proteins associated with Mn regulated cardiomyocyte apoptosis were screen out.The aim of these studies is to provide a scientific basis for more rational use of Mn and its additives to more accurately regulate cardiac apoptosis induced by heat stress and reduce the adverse effects of heat stress on the production of broilers.The main results of this study were as follows:1.Primary chick embryonic myocardial cells were successfully isolated from chicken embryo and cultured.After heat stress treatment for different time,the activity of cells showed different degree of decreased,and cells apoptosis rate increased significantly in a time-dependent manner.Thus,the time model of heat stress-induced apoptosis was successfully constructed in primary chick embryonic myocardial cells.Caspase activity assaya showed that heat stress-induced cells apoptosis was caspase-dependent,and the apoptosis involved death receptor pathway,mitochondrial pathway and endoplasmic reticulum pathway.After heat stress for 4 h,the cell viability,apoptosis rate and Caspase activity were significantly changed in primary chick embryonic myocardial cells.Therefore,4 h was used as the heat treatment time for subsequent studies.2.To investigate the potential effect of Mn on heat stress-induced apoptosis and mitochondrial function,we examined crucial related factors in the context of heat stress using primary chick embryonic myocardial cells pretreated with Mn for 24 h.The results showed that Mn restored the heat stress-induced decrease in cell viability and reduced the activities of caspase-3（P<0.05）.The anti-apoptotic effect of Mn was different in dose,with the increase of Mn concentration,the anti-apoptotic effect showed a trend of increasing first and then decreasing.And the anti-apoptotic effect was most significant when Mn concentration was 20μM,therefore,20μM is the optimal concentration of Mn to resist heat stress apoptosis.However,there was no significant difference in the effects of the Mn sources（P<0.05）.Compared with the heat stress group,Mn pretreatment prior to heat stress decreased P53 expression and an increased ratio of Bcl-2/Bax（P<0.05）.The repression of theΔψm and intracellular ATP content caused by heat stress was reversed dramatically in the Mn pretreatment group（P<0.05）.Additionally,Mn inhibited heat stress-induced mitochondrial fission,as shown by decreased mitochondrial fission-related protein dynamin-related protein 1（Drp1）expression and increased mitochondrial fusion-related protein opticatrophy 1（Opa1）and mitofusin 1（Mfn1）（P<0.05）in primary chick embryonic myocardial cells.Meanwhile,Mn treatment significantly decreased（P<0.05）the expression levels of the ER stress markers GRP78 and CHOP in primary chick embryonic myocardial cells,which suggested that Mn contributes to relieve endoplasmic reticulum stress induced by heat stress.The results of inhibiting ROS shown that excessive ROS levels caused by heat stress triggered the changes of intracellular Ca²⁺concentration,following by apoptotic signals were activated by endoplasmic reticulum and mitochondrial pathway,which is an important factor leading to apoptosis.Thus,ROS was a key mediator of heat stress-induced apoptosis,and Mn played an anti-apoptotic role by enhancing the expression and activity of Mn-SOD,reducing the intracellular ROS content and the variation range of Ca²⁺concentration.Pearson correlation analysis also showed strong correlation（P<0.05）between ROS and intracellular Ca²⁺concentration,the protein expression of Bax,the protein expression of GRP78,mitochondrial membrane potential（ΔΨm）.The over-expression and down-expression of Opa1 confirmed that down-regulation of Opa1 promoted the release of Cyt-C in mitochondria under heat stress.Mn promoted the expression of Opa1,but there was no significant effect on the release of Cyt-C.Therefore,we believed that Mn exerted anti-apoptotic effects more by regulating mitochondrial dynamic balance,and not by Opa1/Cyt-C pathway.3.Using DIA technique,437 differentially expressed proteins（DEPs）were identified in CON vs HS（161 up-and 276 down-regulated）,93 DEPs were identified in HS vs HS+MnCl₂（30 up-and 63 down-regulated）,121 DEPs were identified in HS vs HS+MN-AA（51 up-and 70 down-regulated）,53 DEPs were identified in HS+Mn-AA vs HS+MnCl₂（20 up-and 33 down-regulated）.Western blot data showed that the proteomic data obtained from DIA were reliable.GO and KEGG analysis showed that DEPs were mainly involved in the biological processes associated with metabolism,biological regulation,stimulus response,localization,development,cell signaling,immunity and so on.Through bioinformatics analysis,five key DEPs related to regulate apoptosis by Mn under heat stress were screened further,namely BAG3,AIFM1,RPA3,DNAJB1 and GLTSCR2.This suggests that Mn maybe by regulating these key genes,and acting on DNA replication,DNA repair and protein synthesis of endoplasmic reticulum,ultimately affects the response of cells to heat stimulation and apoptosis.In conclusion,heat stress-induced apoptosis model was established using chicken embryo primary myocardial cells in vitro,and then a series of experiments have been conducted to confirm that Mn has anti-apoptotic role.Mn helped to alleviate heat stress-induced apoptosis by enhancing the expression and activity of Mn-SOD,reducing the intracellular ROS content and the concentration of Ca²⁺.Base on the DIA technology and bioinformatics analysis,five key DEPs related to regulate apoptosis by Mn under heat stress were screened.The results provide an important reference for further reducing the injury caused by heat stress through mineral nutrition.