Age-related Differences In Post-infarct Cardiac Rupture And Remodeling And Targeted Inhibition Of NF-κB Signal Pathway In Aged Mice

Objective:The research is aimed at mechanisms of higher rate of cardiac rupture, more severe cardiac remodeling in old mice by study of expressions of NF-κB signal pathway post infarction, meanwhile the study is to investigate if targeted inhibition of NF-κB can attenuate post-infarct left ventricular rupture and remodeling in aged mice by ribozyme gene transfer with adeno-associated virus serotypes 9. The present study focuses on the following issues:1) To establish the acute myocardial infarction mouse model, study differential expression of p65, p50, intercelluar adhesion molecule (ICAM)-l and vascular celluar adhesion molecule (VCAM)-1 in different age groups post-infarction.2) To evaluate the transfection efficiency of recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein (rAAV9-eGFP) to mouse heart in vivo and the impact on cardiac function.3) To investigate if targeted inhibition of NF-κB can attenuate post-infarct left ventricular rupture and remodeling in aged mice by ribozyme gene transfer with adeno-associated virus serotypes 9. Methods: Part one:The part is about the expression of NF-κB signal pathway in mice with different age post myocardial infarction.1) The acute myocardial infarction model of mouse has been established by ligation of left coronary artery in 160 adult C57BL/6 mice (80 3-month-old,80 18-month-old) in which 20 were sham operation group (SH).Compare infarct size, rate of cardiac rupture and cardiac remodeling between different age-group. 2) Choose another 96 mice:48 3-month-old, other 48 18-month-old, set up AMI model by ligation of left coronary artery. They were randomly divided into four different groups according to time of postinfarction:SH, MI 3d,7d and 14d. The mice were sacrified at the time postinfarction according to group division and hearts were kept in liquid nitrogen then moved to-80℃refrigerator or in 4% paraformaldehyde. Expression of p65, p50 were detected by immunohistochemistry method and Western Blot, while expression of ICAM-1, VACM-1 were detected by Western Blot.Part two:1) 16 C57BL/6 mice were transfected rAAV9-eGFP by tail injection. EGFP expression in the heart, liver, lung, kidney and brain cryosections was observed under inverted fluorescence microscope 7, 14,21,28 days after the injection of rAAV9-eGFP and eGFP was quantitated by Western Blot.2) 20 C57BL/6 mice were divided into control group and rAAV9-eGFP group randomly, and were received with saline or rAAV9-eGFP. The echocardiography and hemodynamics were performed 28 days after the injection of saline or rAAV9-EGFP. Part three:1) We used 160 old mice (18-month-old) to set up acute myocardial infarction model.75 were given AAV9-R65 by tail vein injection 16 days before operation (MI+R65).75 and 10 mice were divided into myocardial infarction group (MI) and sham operation group (SH). Echocardiography was carried on 7 days post-operaion. Compare infarct size, rate of cardiac rupture and cardiac remodeling between these two groups.2) Compare activities of matrix metalloproteinases 2,9, expression of TNF-α, IL-1β, ICAM-1, VCAM-1 and content of collagen to discuss mechanisms. Results:Four results have been obtained through the study,1) There was no difference in infarct size between old and young group. Rate of cardiac rupture is higher in old group than that in young group(33.9% vs 15.6%, P=0.017). Dilation of left ventricle (LV) and contractile dysfunction were more severe in old group.2) It is observed that p65, p50 positive cells are much higher in old mice than in young mice by immunohistochemistry methods (p65 400 vs 154, p50 179 vs 83, P<0.05). Expression of p65 in SH, MI 3d,7d group in old mice is higher than in young mice, which means higher activities of NF-κB in old animals post infarction. There were no statistics differences in expression of ICAM-1 between old and young. Expression of VCAM-1 is higher in old than in young in SH and MI 14d group (P=0.001,0.034 respectively).3) eGFP expression in the heart reached the maximum at day 21, at the point of which the transduction efficiency of rAAV9-eGFP in myocardium was 32%. The other tissues had a little or no eGFP expression. The cardiac function did not reveal significant difference between rAAV9-eGFP group and the control group after transfection (P>0.05).4) Rate of cardiac rupture decreased greatly in MI+R65 group (32.8% vs 15.2%, P=0.018). Diameter of LV decrease, ventricular wall thickness, and fraction shortening increased in MI+R65 group by echocardiaography (P <0.05). Expression of MMP-9 and MMP-2, TNF-αand early VCAM-1 decreased in MI+R65 group (P<0.05). While CVF also decreased in MI+R65 group (P<0.05). But there were no changes of expression of IL-1βand ICAM-1in MI+R65 group. Inhibition of NF-κB leads to lower rate of cardiac rupture and lower expression of cytokines which identified action of NF-κB signal pathway in cardiac rupture. And age-related change in NF-κB signal pathway maybe associated with age-related cardiac rupture. Conclusions: The conclusion of the research can be drawn from the following four aspects,1) NF-κB signal pathway was activitied postinfarction and activities in old group is higher than that in young group. This result identified age-related expression differences of NF-κB signal pathway in old mice postinfarction.2) rAAV9-eGFP gene can be stably and efficiently expressed in mouse heart, and has no toxic effect on cardiac function.3) Rate of cardiac rupture and cardiac remodeling decreased greatly in old mice by ribozyme gene transfer with adeno-associated virus serotypes 9. It maybe caused by decreased collagen as the result of decreased MMP-9, MMP-2, TNF-αand early VCAM-1 which proved that NF-κB signal pathway may be associated with age-related cardiac rupture and cardiac remodeling.