Research On The Role Of SMYD1 In Myocardial Injury Of Septic Rats

Objective:Sepsis-induced myocardial dysfunction(SIMD)is one of the serious complications of sepsis and a key index affecting the prognosis of sepsis.SMYD1(SET and MYND domain containing 1)gene belongs to SMYD family and contains two functional domains related to chromatin remodeling:MNYD domain and SET domain.Studies have shown that SMYD1 plays a very important role in cardiac development,and SMYD1 overexpression can increase the contractile function of cardiomyocytes,while physical activity after myocardial infarction can increase the expression level of SMYD1.SMYD1 may play a role in restrictive cardiomyopathy and control the size of cardiomyocytes,but its role in SIMD has not been determined.This study explored the role and possible mechanism of SMYD1 in SIMD by establishing lipopolysaccharide(LPS)induced SIMD rat model and LPS induced H9c2 cardiomyocyte model.Methods:Firstly,we used the myocardial microarray data set of patients with septic cardiomyopathy in GEO database to analyze the gene differential expression of SMYD1.In order to verify the role of SMYD 1 in SIMD,we constructed the myocardial injury model of septic rats induced by LPS.SD rats were grouped as LPS treatment group(LPS)and control group(Sham).LPS was injected intraperitoneally into LPS group rats,and equal dosage of normal saline was used to control group.After that,the changes of mean arterial pressure(MAP)were continuously monitored for 6 hours,and the pathological damage of myocardium was detected by HE staining.TUNEL staining was used to detect apoptotic cells.We tested the related protein level of endoplasmic reticulum stress,apoptosis and inflammation in the myocardium of the two groups by Western blot.Then,the expression of SMYD1 in rats’ myocardium was tested through Real-time PCR,Western blot and immunofluorescence staining.The rat H9c2 cardiomyocytes were treated with LPS to construct the rat septic myocardial injury cell model,and the expression of SMYD 1 was detected.Secondly,in order to further study the role of SMYD1,rat H9c2 cardiomyocytes were transfected with SMYD1 overexpression plasmid and empty plasmid,then the cells were treated with LPS.The cells were grouped as follows:blank control group(Control),simple LPS treatment group(LPS),empty plasmid transfection+LPS treatment group(LPS+NC),SMYD1 overexpression plasmid transfection+LPS treatment group(LPS+SMYD1).The level of SMYD1 protein of each group was tested by Western blot method.We adopted MTT assay to detect the viability of the cells.The release amount of LDH and CK-MB in cell supernatant was detected by LDH and CK-MB assay kit.Flow cytometry method and Western blot were adopted to determine and compare the apoptosis of cells in the four groups.Real-time PCR and ELISA methods were selected to test cytokines levels of TNF-α、IL-1β and IL-6 in 4 groups.Finally,we focused on the role of SMYD1 in regulating endoplasmic reticulum stress and inflammatory response in LPS treated H9c2 cells.The positional expressions of SMYD1 and CHOP were determined by immunofluorescence double staining.And the protein levels of endoplasmic reticulum stress related proteins such as p-PERK、p-eIF2α、GRP78、ATF4、CHOP and Cleaved caspase-12 were tested.Western blot was adopted to test the phosphorylation levels of IκBα and NF-κB p65.Results:Part I:Through the analysis of SMYD1 gene difference,it was found that the expression of SMYD1 decreased in myocardial tissue of patients with sepsis induced cardiomyopathy.Two hours after LPS injection,the rats reached shock state(MAP decreased by 25%-30%compared with the basic value).The MAP monitoring values of rats in LPS treatment group decreased remarkably at all time points than control group.HE staining:for control group rats,the myocardial fibers were tightly packed and in good order,and the muscle stria were clearly visible.In LPS group,the myocardial fibers were obviously broken,arranged disorderly,some myocardial cells were necrotic,and some muscle fibers were separated from each other.SMYD1 expression detection:Real-time PCR:the mRNA level of SMYD1 in LPS group was remarkably reduced.Western blot:the expression of SMYD1 protein was down regulated in LPS group,and immunofluorescence staining also demonstrated the expression of SMYD1 protein was reduced in LPS group.Apoptosis detection:through TUNEL staining,there were more positive cells in LPS group.The level of apoptosis promoting protein Cleaved caspase-3 was also increased in LPS group.The endoplasmic reticulum stress protein CHOP and inflammatory index TNF-α and IL-6 expression also higher in LPS group.After LPS treatment,SMYD1 protein level was reduced in H9c2 cells.Part Ⅱ:Real time PCR:the mRNA detection level of SMYD1 in H9c2 cells transfected with SMYD1 overexpression plasmid increased remarkably.Western blot:In the SMYD1 overexpression plasmid transfection group,the level of SMYD1 protein in H9c2 cells upregulated significantly,proving the SMYD1 overexpression H9c2 cell model was well made.After treated with LPS,the level of SMYD1 protein in H9c2 cells decreased remarkably.The expression of SMYD1 in LPS+SMYD1 group increased significantly,indicating that SMYD1 overexpression plasmid restored the expression of SMYD1 in LPS treated H9c2 cells.MTT assay:After LPS treatment,the activity of H9c2 cells decreased,and the activity of LPS+SMYD1 group cells increased compared with LPS+NC group cells.The release amount of LDH and CK-MB of the cells:compared with the control group,the levels of LDH and CK-MB in LPS group increased,and the levels of LDH and CK-MB in LPS+SMYD1 group decreased.Flow cytometry:Apoptosis increased in LPS group,while in contrast with LPS+NC group,that’s decreased in LPS+SMYD1 group.Apoptotic protein detection:the level of Bcl-2 protein in LPS group decreased remarkably than control group,but the level of Bax and Cleaved caspase-3 were on the contrary.Compared with LPS+NC group,the protein level of Bcl-2 was upregulated,while the levels of Bax and Cleaved caspase-3 protein were downregulated in LPS+SMYD1 group.Real time PCR and ELISA detection:the inflammatory cytokines of H9c2 cells increased in LPS treatment group.Contrast with LPS+NC group,the content amount of inflammatory factors in LPS+SMYD1 group reduced.Part Ⅲ:Results of immunofluorescence double staining of SMYD1 and CHOP showed that SMYD1 was mainly located in cytoplasm and cell membrane,CHOP was expressed in nucleus,SMYD1 fluorescence intensity was reduced and CHOP increased in LPS group.Compared with LPS+NC treatment group,SMYD1 fluorescence intensity increased and CHOP fluorescence intensity decreased in LPS+SMYD1 group.Western blot test of endoplasmic reticulum stress related proteins:the levels of them in LPS group were higher in contrast with control group,while the levels of them in LPS+SMYD1 group were less than LPS+NC group.The phosphorylation levels of IκBα and NF-κB p65 were tested through Western blot,levels of them in LPS group were more than that in control group,and that in LPS+SMYD1 group were less than that in LPS+NC group.Conclusion:1.SMYD1 is involved in the process of myocardial injury in LPS induced septic rats.2.The apoptosis levels and inflammatory response of cardiomyocyte were increased in LPS induced septic rats with myocardial injury.3.SMYD1 can alleviate the injury of H9c2 cells induced by LPS.4.SMYD1 may reduce LPS induced apoptosis and inflammatory response of H9c2 cells by inhibiting endoplasmic reticulum stress PERK pathway,which may be the main mechanism of SMYD1 reducing LPS induced cardiomyocyte injury.