Research On Inhibition Of Phospholamban Gene Expression With RNA Intrference Therapy For Chronic Heart Failure Rats

[Background] Heart failure (HF),which was called the big last field of cardiopathy, was the end stage of various heart diseases, whose incidence, mortality was increasing in past years. Researches founded Ca²⁺ stablity turbulence not only damaged cardiac function, but played important role in cardiac remodeling as important message transmitter. Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) was responsible for reabsorbing Ca²⁺ from cytoplasma to SR during relax stage. Phospholamban(PLN) was an important regulatory protein of SERCA2a activity. Phosphorylated PLN can increase SERCA2a activity. In heart failuire, PLN decreased little , whereas SERCA2a decreased much more, so PLN /SERCA2a increased and SERCA2a's activity was inhibited and cardiac function worsened.With RNA interference(RNAi), we designed recombinant adeno-associated virus(rAAV) vector expressing PLN short hairpin RNA(shRNA) and transferred to HF rats in order to degrade PLN mRNA and decrease protein expression, add PLN/SERCA2a, increase SERCA2a activity and to improve cardiac function. The research was divided into three parts.[The first part of experiment][Objective] Establishing enrollment criterion of heart failure rats model after abdominal aorta constriction.[Method] 22 rats were randomly divided into 3 months and 6 months group, each group was divided into pseudom-operation subgroup(n=5) and operation subgroup(n=6). At 3 months and 6 months after operation, Echocardiagram, hemodynamics and myocardium pathology examination were done.[Results] At 3 months, echocardiagraph and hemodynamics parameters, such as LVEDD, LVESD, VSD, LVPWD, EF, FS, MBP, LVSP, ±dp/dtmax, HR and LVEDP had no significance compare to those of pseudo-operation group (P>0.05). Pathology examination had myocardium hypertrophy and puntual necrosis, but no pack necrosis;At 6 months, echocardiagraph and hemodynamics parameters significantly exacerbated compare to pseudo-operation group(P<0.05), myocardium pathology examination founded myocardium necrosis and fibrosis;echocardiagraph and ±dp/dtmax had good relevance, the relevent coefficent between EF, FS and +dp/dtmax ,-dp/dtmax were seperatly 0.86 and 0.83, 0.85 and 0.81.[Conclusion] The diagnosis was dependable with echocardiography;Enrollment criterion of heart failure rats model were: 1. 6 months after abdominalaorta constriction, rats emerged less food, feckless, less activity, fur lusterless andpuffed. 2. Echocardiography parameters: LVESD>0.55> LVEDD>0.6, EFO.5,FS<0.25o[The second part of experiment][Objective] To study the therapeutic effects of rAAV2 expressing PLN shRNA on chronic heart failure rats.[Method] we designed two rAAV2 vectors aimed at different site of PLN mRNA (rAAV2-phRil and rAAV2-phRi2). 14 Rats were control group, 56 rats were randomly divided into 4 groups: HF group;rAAV2-enhanced green fluorescence protein(rAAV2-EGFP) group;rAAV2-phRil and rAAV2-phRi2 group.Each group was randomly divided into 10 days and 30 days subgroup(n=7). NS, rAAV2 vectors were respectively injected inito pericardium of HF rats by intrapericardial through trans-diaphragmatic approach. After 10 days and 30 days, hemodynamic parameters, mRNA and proteins expression of PLN and SERCA2a, SERCA2a's activity were measured.The transfer effect was oberserved by pathology examination of rAAV2-EGFP.[Results] Of HF rats, hemodynamics parameters exacerbated, PLN mRNA significantly decreased, SERCA2a mRNA, protein and it's activitydeclined, which had significance compare to control group (P<0.05);after 10 days, of rAAV2-phRil and rAAV2-phRi2 group, hemodynamics parameters had slightly improvement, PLN mRNA and protein droped a little, but no significance compare to HF and rAAV2-EGFP group(P<0.05;.;SERCA2a mRNA, protein and it's activity changed little;After 30 days, Compare to HF group, rAAV2-phRil and rAAV2-phRi2 decreased PLN mRNA by 85.32% and 67.65%, subsequently protein declined by 78.82% and 59.33% respectively, which had significance^^. 05;. Of rAAV2-phRil and rAAV2-phRi2 group, SERCA2a mRNA, protein had no change, so PLN/SERCA2a decreased and SERCA2a's activity was 2.04 and 1.74 times of HF group. Hemodynamics parameters improved significantly compare to HF group(P<0.05);SERCA2a's activity had good relevance with PLN/SERCA2a and absolute value of ±dp/dtmax. Absolute value of ±dp/dtmax of rAAV2-phRil was more than rAAV2-phRi2;In myocardium pathology examination , green fluorescence of 10 days after transfer was weak than that of 30 days.[Conclusion] Gene transfection by intrapericardial through trans-diaphragmatic was effective;Two rAAV2-phRi can effectively inhibit the expression of PLN mRNA and protein, decreased PLN/SERCA2a, increase SERCA2a's activity and improve cardiac function. The effects of rAAV2-phRilwas superior to those of rAAV2-phRi2. [The third part of experiment][Objective] To study the medium- and long-term therapeutic effects and safety of rAAV2/l expressing PLN shRNA on chronic HF rats.[Method] we designed adeno-associated virus2/l vectors expressing PLN shRNA (rAAV2/l-phRil). 14 Rats were control group* 42 rats were randomly divided into 3 groups: HF group, rAAV2/l-phRil low and high dose(lxlOnvg and 2xl0nvg) group.Each group was randomly divided into 30days and 60days subgroup(n=7). rAAV2 vectors(carring EGFP gene, rAAV2/l-phRi) were respectively injected inito pericardium of HF rats by intrapericardial through trans-diaphragmatic approach. After 30days and 60days, hemodynamic parameters, the genes and proteins expression of PLN, SERCA2a and SERCA2a 's activity were measured.At the same time, side effects were observed, i.g gene expression outside of heart, myocardium oxygen consumption(MVO2), arrhythmia, liver and kindy function and systemic and local inflammation.[Results] Of HF group rats, hemodynamics parameters exacerbated, PLN mRNA decreased, SERCA2a mRNA and protein and it's activity significantly declined, which had significance compare to control group (P<0.05);after 30 days, in rAAV2/l-phRil high and low dose groups, PLN mRNA decreased by85.62% and 79.36%, subsequently protein declined by 78.41% and 71.29% respectively, which had significance(P<0.05,). SERCA2a mRNA, protein had no change, PLN/SERC A2a decreased, so it's activity was 1.96 and 1.70 times of HF group rats'. Hemodynamics parameters improved significantly compare to HF group (P<0.05). After 60 days, The inhibitory efficency was slightly droped. Compare to HF group, rAAV2/l-phRil high and low dose group decreased PLN mRNA by 80.18% and 65.74% respectively, subsequently protein declined by 72.27% and 60.26% respectively, which had significance compare to HF rats(P<0.05). SERCA2a mRNA, protein had no change, PLN/SERCA2a decreased, SERCA2a's activity was 1.93 and 1.63 times of HF group rats'. Hemodynamics parameters improved significantly compare to those of HF group(P<0.05,). absolute value of±dp/dtmax in high dose group rats were more than those of low dose group(P<0.05,);SERCA2a's activity had good relevance with PLN/SERCA2a and absolute value of ±dp/dtmax. In side effects, green fluorescence was only seen in myocardium,which implied there was no gene expression outside of heart;Compare to control and HF rats, MVO2 of rAAV2/l-phRil rats didn't increaesd(P>0.05,);There only existed occasional ventricular premature beat;There was not obvious local and systemic inflammation, C-reaction protein(CRP) and creatine kinase(CK) had nosignificence among groups(P>0.05,). There was not inflammation and damage in pathology examination;The liver and kindy function of rAAV2/l-phRi group rats had no significance compare to control and HF rats,even high dose group(P> 0.05). green fluorescence was similar at 30days and 60days.[Conclusion] rAAV2/l-phRil can effectively inhibit the expression of PLN mRNA by RNAi and protein, decreased PLN/SERCA2a, increase SERCA2a's activity and improve cardiac function. The long-term effects of high dose was superior to those of low dose. There was no obvious side-effects.