Expression Of Gelatinases And Their Tissue Inhibitors In A Mechanically Unloaded Rat Heart

Objectives:Left ventricular assist device(LVAD) have emerged as a treatment option to reduce preload and afterload for patients with end-stage heart failure.Studies showed that LVAD can recover the native cardiac function and improve quality of life for patients.However,some groups reported that many patients treated in this fashion have progressed rapidly back to heart failure and aggravate the cardiac function.In addition,there is no consistent results regarding MMPs/TIMPs changes in this process,therefore,in the present work,we perfomed an abdominal heterotopic heart transplantion to achieve pressure and volume unloading (mechanical unloading).Using this model we aimed to investigate the relationship between the expression of MMP-2,MMP-9,TIMP-1 and TIMP-2 and ECM accumulation in rat left ventricle in mechanically unloaded state.Methods:60 male Lewis rats were subjected to abdominal heterotopic heart transplantion to achieve pressure and volume unloading.Some rats were sacrificed at 3^rd d,7^th d and 14^th d after transplantion,and then one part of LV tissue was dissected for protein and total RNA extraction.Another part was paraffin embedded for histological analysis.Age and sex matched in situ heart of Lewis rats were used as control.Total RNA of rats' cardiac tissues were extracted by TRIzol agent.After the first strand of cDNA samples were synthesized by RT-PCR reaction.The relative expression level of MMP-2,MMP-9,TIMP-1 and TIMP-2's in LV were measured by real-time quantitative PCR.LV tissue were homogenated to protein lysate,and concentration of total protein were assayed by BCA method.Protein level of TIMP-1 and TIMP-2 in LV were determined by immunoblotting,and quantified by Image Pro Plus software.MMP-2 and -9 gelatinolytic activity was measured by gelatin-zymography.Picrosirius red staining plus circularly polarized microscopy was performed to analyze collagen volume fraction(CVF) and myocardial cross-sectional area.Results:Myocardial cross-sectional area of transplanted heart was significantly reduced,and accompanied by excessive ECM deposition(CVF 5.22%±1.6%v.s.2.21%±0.9%,P＜0.05) compared to in situ heart.From 3^rd d to 14^th d after transplantion,MMP-2 activity gradually increased,MMP-9 activity was significantly increased at 3^rd d and reached to peak at 7^th d,then gradually decreased. mRNA level of MMP-2 and MMP-9 compared to in situ heart were identical with MMP-2 and MMP-9 activity,mRNA level of TIMP-1 and TIMP-2 increased from 3^rd d to 14^th d,especially for TIMP-1.There's no statistic difference between the mRNA level of TIMP-2 at 3^rd d and control group.TIMP-1 and TIMP-2 protein level were identical with TIMP-1 and TIMP-2 mRNA level.Conclusions:Mechanical unloading of left ventricle will lead to excessive ECM deposition,accompanied by imbalance between MMPs and TIMPs system, especially the upregulation of TIMPs.