| Background: Trauma-induced secondary cardiac injury(TISCI)means that the cardiac dysfunction occurs within a period of time after the body has suffered trauma(such as fall injury,bruise,tissue laceration,etc.)that does not directly affect the heart,which manifested as arrhythmia,ventricular fibrillation,cardiac arrest,etc.The onset of the disease is insidious,and it is easy to be missed.Therefore,it is very important to study the specific pathogenesis of this disease.Studies have shown that myocardial apoptosis is the main reason for the decline of cardiac function caused by secondary cardiac injury.Our research found that among the three cysteine aspartic acid protease(caspase)-induced apoptosis pathways,caspase-12 activation mediating endoplasmic reticulum stress(ERS)apoptotic pathway is the earliest.Caspase-12 is a downstream molecule of ERS,and its activity and expression are regulated by the ERS chaperone Glucose-regulated protein 78(GRP78).Our previous studies have shown that the expression of GRP78 increased after trauma,but the mechanism of regulating the expression and function of GRP78 is still unclear.Therefore,the purpose of this study is: 1)to investigate the changes of protein arginine methyltransferase 1(PRMT1)function and expression in cardiomyocytes at the early stage of trauma;and 2)further to explore the molecular mechanism of its regulation of GRP78 function in cardiomyocytes,and its role in early activation of caspase-12.Objective: 1.To investigate the time course of protein arginine methyltransferase 1(PRMT1)in myocardial tissue of traumatic rats,and explore its relationship with GRP78 expression in vivo.2.To further analyze the effect of PRMT1 on the protein expression and function of glucose-regulated protein 78(GRP78)in post-traumaticcardiomyocytes,and to explore preliminarily the possible mechanism in vitro.Methods: 1.In vivo level: Male SD rats were randomly divided into Sham trauma group and Trauma group(Trauma group included 5 groups,0 h,3 h,6 h,12 h,24 h,n=6 in each group,totaling 36 rats).The traumatic rat model was established by the NobleCollip trauma instrument.(1)The activation level of caspase-12 in myocardial tissue was detected by activity kits.(2)The protein expressions of caspase-12,GRP78 and PRMT1 in myocardial tissue were detected by Western Blot.(3)The m RNA expression of GRP78 in myocardial tissue was detected by Quantitative Real-time PCR(RT-qPCR).2.In vitro level:(1)The H9C2 cell model was induced by fetal bovine serum(FBS)and traumatic rat serum.The FBS group was used as the control group(Sham group),and the traumatic rat serum was used as the experimental group(cultured H9C2 cardiomyocytes for 1 h,3 h,6 h,12 h,24 h,respectively).1)The activation level of caspase-12 in H9C2 cardiomyocytes was detected by activity kits.2)The protein expressions of caspase-12,GRP78 and PRMT1 in H9C2 cardiomyocytes were detected by Western Blot.3)The m RNA expression of GRP78 in H9C2 cardiomyocytes was detected by RT-qPCR.(2)After PRMT1 siRNA was transfected into H9C2 cardiomyocytes,H9C2 cardiomyocytes were divided into Sham group(cultured with FBS),Blank group(cultured with traumatic rat serum),PRMT1 siRNA group and NC RNA group.1)The protein expression of PRMT1 was detected by Western Blot.2)The protein expression of GRP78 was detected by Western Blot.3)The activation level of caspase-12 was detected by activity kits,and the protein expression of caspase-12 was detected by Western Blot.3.The asymmetric dimethylation of arginine(ADMA)of GRP78 was detected by Coimmunoprecipitation(Co-IP).Results: 1.The activity of caspase-12 in myocardial tissue of traumatized rats was enhanced,and the protein expressions of caspase-12,GRP78 and PRMT1 were elevated.1.1 The activation level and protein expression of caspase-12 in myocardial tissue of post-traumatic rats was elevated.The results from caspase-12 activity kits showed that the caspase-12 activity in myocardial tissue of post-traumatic rats was enhanced at 3 h(P<0.05),and peaked at 6 h(P<0.05),compared with the Sham group.The results from Western Blot showed that the protein expression of caspase-12 in myocardial tissue of post-traumatic rats was elevated at 3 h(P<0.05),and peaked at 6 h(P<0.05),compared with the Sham group.1.2 The protein and m RNA expression of GRP78 in myocardial tissue of posttraumatic rats was elevated obviously.The results from Western bot showed that the protein expression of GRP78 in myocardial tissue of post-traumatic rats was significantly elevated at 3 h(P<0.05),peaked at 6 h(P<0.01),and maintained high level of expression at 12 h(P<0.05),compared with the Sham group.The results from RT-qPCR showed that the m RNA expression of GRP78 in myocardial tissue of posttraumatic rats was elevated at 0 h(P<0.05),peaked at 3 h(P<0.01),and maintained high level of expression at 6 h(P<0.05),compared with the Sham group.1.3 The protein expression of PRMT1 in myocardial tissue of post-traumatic rats was elevated obviously.The results from Western bot showed that the protein expression of PRMT1 in myocardial tissue of post-traumatic rats was significantly elevated at 3 h(P<0.05),peaked at 6 h(P<0.01),and maintained high level of expression after 6 h(P<0.05),compared with the Sham group.The protein expression of H4R3me2 a in myocardial tissue of post-traumatic rats was elevated at 3 h(P<0.05),peaked at 6 h(P<0.01),and maintained high level of expression at 6 h(P<0.05),compared with the Sham group.1.4 There was a correlation between the changes of GRP78 and PRMT1 expression in the myocardial tissue of traumatic rats.The results of correlation analysis showed that there was a certain correlation between the changes of GRP78 and PRMT1 protein expressions in the myocardial tissue of post-traumatic rats(r=0.7487,P<0.01).2.Trauma might increase the expression of GRP78 by up-regulating the expression of PRMT1 protein in cardiomyocytes,and further lead to the early activation of caspase-12 protein.2.1 The activation level and protein expression of caspase-12 in H9C2 cardiomyocytes was elevated.The results from caspase-12 activity kits showed that the caspase-12 activity in traumatic H9C2 cardiomyocytes was enhanced at 3 h(P<0.05),and peaked at 6 h(P<0.05),compared with the Sham group.The results from Western Blot showed that the protein expression of caspase-12 in traumatic H9C2 cardiomyocytes was elevated at 3 h(P<0.05),and peaked at 6 h(P<0.05),compared with the Sham group.2.2 The protein expression of GRP78 in H9C2 cardiomyocytes was elevated obviously.The results from Western Blot showed that the protein expression of GRP78 in traumatic H9C2 cardiomyocytes was elevated at 3 h(P<0.05),peaked at 6 h(P<0.01),and maintained high level of expression at 12 h(P<0.05),compared with the Sham group.The results from RT-qPCR showed that the m RNA expression of GRP78 in traumatic H9C2 cardiomyocytes was elevated at 3 h(P<0.05),and maintained high level of expression after 3 h(P<0.05),compared with the Sham group.2.3 The protein expression of PRMT1 in H9C2 cardiomyocytes was elevated obviously.The results from Western Blot showed that the protein expression of PRMT1 in traumatic H9C2 cardiomyocytes was elevated at 3 h(P<0.05),peaked at 6 h(P<0.01),and maintained high level of expression at 12 h(P<0.01),compared with the Sham group.2.4 The protein expression of PRMT1 in H9C2 cardiomyocytes transfected with PRMT1 siRNA was decreased.The protein expression of PRMT1 in traumatic H9C2 cardiomyocytes transfected with PRMT1 siRNA was decreased(P<0.05),compared with the Blank group.2.5 The protein expression of GRP78 in H9C2 cardiomyocytes transfected with PRMT1 siRNA was elevated.The protein expression of GRP78 in traumatic H9C2 cardiomyocytes transfected with PRMT1 siRNA was elevated(P<0.05),compared with the Blank group.2.6 The protein expression of caspase-12 in H9C2 cardiomyocytes transfected with PRMT1 siRNA was decreased.The activity and protein expression of caspase-12 in traumatic H9C2 cardiomyocytes transfected with PRMT1 siRNA was decreased(P<0.05),compared with the Blank group.2.7 GRP78 protein has arginine asymmetric dimethylation modification.The results from Co-IP showed that arginine asymmetric dimethylation modification was found in GRP78.Conclusion: Endoplasmic reticulum stress apoptosis occurs in rat cardiomyocytes in the early stage of trauma,and the possible mechanism is that PRMT1 increases the arginine methylation modification of GRP78. |
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