Role And Mechanism Of Rat Sarcoplasmic Reticulum Ca～(2+)-ATPase Promoter In Myocardial Ischemic Preconditioning

Background and Objective: Changes in intracellular calcium ion concentration ([Ca2+]i) is involved in myocardial ischemia-reperfusion injury (IRI) and ischemic preconditioning (IPC). Elevation of [Ca2+]i in the early phase of ischemia is essential for the onset of IPC, and one of the protective mechanisms of IPC is to remain [Ca2+]i homeostasis. Since myocardial sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) play a major role in the control of intracellular calcium homeostasis, the study of transcriptional regulation of SERCA2a in IPC may discover the role and the mechanism of SERCA2a in IPC from the aspect of gene expression regulation. As an important part of the study of IPC, pharmacological preconditioning, possessing evident clinical application perspective is catching more and more people's attention. So the possible mechanism of preconditioning induced by praeruptorin C was also investigated in our study.Methods: Genome DNA was extracted from rat myocardial tissue as a template for polymerase chain reaction (PCR).And the SERCA2a promoter region was obtained by PCR. Then the gene region was ligated into pGEM-T easy vector to construct a plasmid for augmenting through transformation of E.coli Competent Cell JM109 and DNA sequencing. The plasmid was cut into appropriate gene segments of SERCA2a promoter with restriction endonucleases, and the promoter segment was inserted into pGL3-Basic vector to construct a luciferase reporter gene plasmid. Primary culture neonatal rat ventricular myocytes according IRI et al ( CELL: A Laboratory Manual published by Cold Spring Harbor Laboratory Press) had established the cell models of IPC. Cardiomyocytes were transient transfected with reporter gene plasmid and grouped for copying the cell models. The luciferase activity was assayed with SHG-D bio-chemistry luminescence sensor.Results: One of the results was the successful construction of the luciferase reporter gene plasmid. In addition, the result of the luciferase activity assay showed:1. The luciferase activity of the transient ischemic group to be 76.68% of the positive control group2. Preconditioning induced by praeruptorin C group to be 43.67% of the positive control group3. IPC group to be 43.13% of positive control group4. IRI group to be 26.82% of the positive control group...