To Study The Role Of Calpain In Cardiac Xenotransplantation Based On Proteomics And Protein Microarray

Objective:Previous studies have shown that inhibition of Calpain activity couldreduce the expression of vascular endothelial cell adhesion molecules,reduce inflammatory cell infiltration of xenogenic heart grafts and prolong their survival time.Howeverthe mechanism of action remains unclear.This study appliedthe mouse to rat cardiac xenotransplantation(CXTx)models to analyse the mechanism of donor endothelial Calpain in xenotransplantation by protein microarray and proteomics.Methods:1.Mouse to rat cardiac xenotransplantation models were established by endothelial cell-specific capnsl knockout(Capnsl-KO)mice and wild type mice through the modified neck cannulation method.On the 5th day after heart transplantation.transplanted hearts were collected and pathological changes were observed by HE staining.2.Serum from recipient rats was collected on the 5th postoperative day.The levels of B7-2b、NGF、CINC-1、CINC-2、IFNg、IL-1a、IL-1b、IL-2、CINC-3、CNTF、Fractalkine、GM-CSF、ICAM-1、IL-4、L-Selectin、MCP-1、PDGF-AA、IL-6、IL-10、LIX、IL-13、Prolactin R、RAGE、TCK-1、TIMP-1、TNF-αandVEGF were detected by rat cytokine protein chip(QAR-CYT-3).3.Serum proteins were identified by Tandem Mass Tag(TMT)technique.Further bioinformatics analysis should be performed.Results:1.HE staining showed that the infiltration of inflammatory cell and thrombosis were inhibited in heart tissue from Capns1-KO mice.2.Cytokine microarray analysis showed that serum IFN-γ and PDGF-AA in Capns1-KO group reduced significantly compared with WT group(P<0.05).3.A total of 401 different proteins were identified by TMT proteomics screening between Capns1-KO group and WT group.209 proteins were up-regulated and 192 proteins were down-regulated in Capns1-KO mice group(P<0.05).The GO function annotation analysis showed that theseproteins were mainly involved in cellular process,biological regulation and metabolic process.It mainly performed molecular functions such as binding and catalytic activity.KEGG pathway enrichment analysis found that differential proteins were enriched to collection tube acid secretion signal pathway,carbohydrate digestion and absorption signal pathway,insulin secretion signal pathway,fat digestion and absorption signal pathway,tumor transcription disturbance signal pathway,calcium signaling pathways;Among them,the lowest P value and the highest proportion of different proteins enriched in calcium signaling pathway suggesting that this signaling pathway was closely related to the mechanism of endothelial calpain in xenotransplantation.Conclusions:1.In the mouse to rat cardiac xenotransplantation models,inhibition of Calpain activity in mouse endothelial cells reduced inflammatory cell infiltration and thrombosis in transplanted hearts by decreasing IFN-γ and PDGF-AA expression in recipient serum.2.Differentially expressed proteins rich in serum calcium signaling pathway(VDAC3,SLC25A5,RyR2,ATP2al,PLC-β4)are closely related to the regulation of donor Calpain enzyme in mouse-rat heart xenotransplantation.It has the impact on cardiac activity and metabolism by participating in the regulation of cell energy metabolism,apoptosis and anti-stress response,thus affecting the state of donor heart transplantation.