Intra-coronary Transfection Of Human Heat Shock70 Gene Protects The Myocardium Against Ischemia/ Reperfusion Injury In Rats

Objectives: To observe the protective effect against myocardial ischemia reperfusion injury after transfer of human HSP70 gene via coronary artery and to investigate the potential mechanisms.Methods:(1) Construction of the replication-deficient recombinant adenovirus: Recombinant plasmids PDC-EGFP (containing enhanced green fluorescence protein EGFP gene), PDC-hsp70 (containing human HSP gene). Recombinant adenovirus Adv-EGFP, Adv-hsp70 were produced by homologous recombinant in 293 cells, amplified also in 293 cells on a large scale, and purified by ultracentrifugation in CsCl step gradient solutions. The concentration of recombinant adenovirus Adv-EGFP purified by ultracentrifugation in CsCl step gradient solutions is 2．0×109Pfu/ml; that of Adv-hsp70 is 2．5×1010Pfu/ml.(2) 50 S-D rats were randomly divided into 5 groups (n=10) . The left thoracotomy was performed and the pericardium opened with the exposure of the heart and the base of pulmonary artery and ascending aorta. Pulmonary artery and ascending aorta were occluded by applying a nontraumatic vascular clamp to achieve complete cessation of outflow, at the same time adenovirus particles (1．0×109pfu) in 0．1 ml of 0．9% saline solution were injected into the left ventricle cavity using a 1-ml syringe with a 27-gauge needle. After 10 seconds of complete outflow occlusion, the clamp was removed. 0．1ml saline solution alone was injected into the rat heart in GroupⅠⅡⅢ. In GroupⅣAdv-EGFP was injected. All of them were closed and recovered, and then stabilized without operative intervention for 4 day.(3) After 4 days, a repeat thoracotomy was performed to expose the anterior surface of the heart. The proximal left anterior descending coronary artery (LAD) was identified and a 6/0 suture (Ethicon) was placed around the artery below the left atrial appendage and the surrouding myocardium. Regional left ventricular ischemia was established for 45 min by ligation of LAD. Ischemia was confirmed by discoloration of myocardium and by changes in cardiac rhythm. At the end of the ischemia period, the ligation was loosened and reperfusion was achieved for 180 min. GroupⅠ(sham-operated group) served as surgical controls and were subjected to the same surgical procedures as the the experimental group, with the exception that the LAD was not ligated. GroupⅢwere subjected to ischemic preconditioning (IPC) before I/R by repeated ischemia and reperfusion through 3 cycles of induced LAD ischemia for 5 min followed by 5 min of reperfusion.(4) To evaluate the effect of 45min ischemia followed by180min reperfusion of the LAD, the area at risk (AAR) and infarct region were determined by Evan's blue dye perfusion and triphenyl tetrazolium chloride (TTC) staining. Aliquots of paraffin section were hematoxylin and eosin stained and examined under the light microscope to measure cardiac inflammation. Determination of Bcl-2, active caspase-3, active NF-κB expression in heart were performed with Western Blotting analysis.Results: (1) The %AAR, determined by the amount of negative staining region with Evan's blue after religation of the LAD, showed no difference among LAD ligated groups (GoupⅡ～Ⅴ). However, a significant reduction of the %infarct size was observed in the Hhsp70 group (27．88±5．3%), and IPC group (27．13±2．7%) compared with either saline control (39．72±4．2%) or adenoviral control (adv-EGFP) (32．73±7．0%) by TTC staining in the regional I/R model.(2) Gene hHSP70 over expression can raise Bcl-2 production,restrain caspase-3 activated.(3) Gene hHSP70 can restrain NF-κB active.(4) The expression of protein in rat heart altered. In Adv-HSP70 group the he expression of HSP70 and Bcl-2 were raised, activated caspase-3 decreased.Conclusions: (1) Transfection of HSP70 inhibits inflammation through NF-κB downregulation. (2) Transfection of HSP70 suppresses apoptosis through increasing Bcl-2 production and decreasing Caspase-3 activation.(3) Transfection of HSP70 demonstrates synergistic effects of myocardial tolerance to I/R injury, which can analogue the protective effect of IPC to a larger degree.