Role Of Calcium Homeostasis In Lactate Acid Induced Myocardial Cytoskeleton Injury

Sudden death syndrome of broilers was a nutritional metabolic disease associated with abnormal cardiac function,which had an acute onset and a short course of disease and usually occurs in broilers around 3 weeks of age.The disease was characterized by sudden death in well-developed and rapidly growing broilers.Chickens did not show any specific clinical symptoms or abnormal behavior before the occur of the disease,and suddenly lose balance,ringing,violently flapping their wings,strong muscle contraction and other manic behaviors.The pathogenesis of SDS was associated with abnormalities in many nutritional and environmental factors,but the exact etiology and pathogenesis of the disease are not fully understood.At present,many scholars considered that SDS was caused by heart injury in broilers and was associated with cardiovascular system failure.According to a large number of literature reports and previous studies by our group,SDS showed significantly higher lactate acid levels in the blood than normal chickens.In addition,the occurrence of arrhythmias and sudden death phenomena were induced by intragastric or intravenous administration of lactic acid to broilers.The production of excessive lactic acid was related to the glycolysis of glucose.When the body in stress conditions such as ischemia and hypoxia,it will lead to lactic acid accumulation,metabolic disorders and then damage myocardial cells,causing arrhythmia which ultimately led to the occurrence of SDS.Therefore,we established a lactate poisoning model to simulate sudden death in broilers and explored the mechanism of myocardial cell injury in sudden death broilers in the model.1.Study on the injurious effect of lactate treatment on HL-1 cellsMouse cardiomyocytes（HL-1）were cultured in vitro,and cells were transmitted to the third generation for the assay.After HL-1 cells were treated with 10 or 20 m M lactic acid for 24 h,and then were collected for the following assays.（1）The morphology and number of HL-1 cells were observed using a common light microscope;（2）The effect of lactate acid on the survival rate of HL-1 cells was detected by CCK-8 assay;（3）The cytoskeleton of each test group was stained using phalloidin staining,and the changes of cytoskeleton were observed under a confocal laser scanning microscope;（4）The protein expression levels of RhoA,ROCK1,cofilin,P-cofilin were detected by Western Blot.The results showed that:（1）HL-1 cells treated with lactic acid decreased its density and volume,and the cell attachment became worse;（2）The cell survival rate was significantly decreased in the lactic acid groups;（3）Compare with control groups,the cytoskeleton of cells was damaged in lactic acid groups;（4）The protein expression levels of RhoA,ROCK1,and P-cofilin and the P-cofilin/cofilin ratio was significantly increased in HL-1 cells treated with lactate acid.Conclusion:A model of lactic acid is established,and lactic acid treatment led to HL-1 cell injury and activation of RhoA/ROCK signaling pathway.2.Molecular mechanism of RhoA/ROCK pathway in regulating LA induced myocardial cytoskeletal injuryIn order to further study the molecular mechanism of LA induced cytoskeleton damage in SDS broilers,we selected Y-27632,a specific inhibitor of ROCK kinase,to inhibit the RhoA/ROCK pathway.HL-1 cells were co-treated with 10μM inhibitor Y-27632 with 10 and 20 m M LA for 24 h for the following assays.（1）Cell survival was detected by CCK-8 assay;（2）The expression levels of RhoA and ROCK1 protein were measured by Western Blot in HL-1 cells;（3）Cytoskeleton was stained by phalloidin staining,and cytoskeleton morphology was observed by confocal laser scanning microscopy.The results showed that:（1）Compared with LA groups,the co-treated groups can improve the survival rate of HL-1cells;（2）Y-27632 inhibit the activation of RhoA/ROCK signaling pathway,and the expression levels of RhoA and ROCK1 protein were inhibited;（3）After the addition of Y-27632,the cytoskeleton damage of HL-1 cells induced by LA was alleviated.Conclusion:LA mediates HL-1 cytoskeleton remodeling by activated RhoA/ROCK signaling pathway,which causes cytoskeleton injury.3.Effects of cytosolic calcium overload on myocardial cytoskeleton injury induced by LATo further investigate the relationship between cytoskeleton damage caused by activation of the RhoA/ROCK pathway by LA and cytoplasmic calcium homeostasis,we did the following studies.（1）HL-1 cells were treated with LA in calcium-containing and calcium-free media for different times,and cytoplasmic calcium concentration was detected by a cytoplasmic Ga²⁺specific probe which was measured by fluorescence microplate reader;（2）HL-1 cells were treated with LA for 24 h,and changes in Ca MK II and calpain1 protein expression levels were detected by Western Blot;（3）HL-1 cells were co-treated with10μM 2-APB（2-Aminoethoxydiphenyl,2-APB）and LA,and the survival rate of cells was detected by CCK-8;（4）HL-1 cells were treated with 2-APB and LA for 24 h,and the protein expression levels of RhoA,ROCK 1,Ca MK II and calpain 1 were detected by Western Blot;（5）After co-treatment with2-APB and LA in HL-1 cells for different time,cytoplasmic calcium concentrations were measured with a fluorescence microplate reader;（7）After co-treatment of HL-1 cells with 2-APB and LA for 24 h,F-actin staining were observed by confocal laser scanning microscopy.The results showed that:（1）Under the condition of calcium-containing culture,the concentration of Ga²⁺in the cytoplasm was significantly increased in the LA treated groups compared with the control group;Under the condition of calcium-free culture,the concentration of Ga²⁺in the cytoplasm was still significantly increased in the LA treated groups compared with the control group;（2）Compared with the control group,the expression levels of Ca MK II and Calpain1 protein were significantly increased in LA treatments;（3）Compared with LA treatments,the incubation of 2-APB with LA improved the survival rate of HL-1 cells;（4）Compared with LA groups,the expression levels of RhoA and ROCK 1 were significantly decreased in the co-treated groups;（5）Compared with LA groups,the expression levels of Ca MK II and Calpain 1 protein were significantly decreased in 2-APB and LA co-treatment;（6）Compared with LA groups,the concentration of Ga²⁺was significantly decreased in the cytoplasm in the 2-APB and LA co-treatment groups;The results of F-actin staining showed that the degree of skeleton damage was recovered.Conclusion:LA treatment of HL-1 cells led to cytoplasmic calcium overload and thus activated RhoA/ROCK signaling pathway and further induced cytoskeleton damage.In summary,LA changed the morphology,reduced the survival rate and damaged the cytoskeleton of HL-1 cells,indicated that LA model can be used as an cell model for SDS in vitro.LA activated RhoA/ROCK signaling pathway in HL-1 cells,and then caused cytoskeleton damage.Further studies revealed that cytoplasmic calcium overload is a key factor in activating RhoA/ROCK signaling pathway,and the overloaded Ga²⁺are mainly derived from the endoplasmic reticulum.In this study,we established an cell model to explore the molecular mechanism of SDS cell injury from the correlation between calcium homeostasis and cytoskeleton in vitro,laid a theoretical foundation for the pathogenesis and prevention of SDS.