| PartⅠ Construction of recombinant adeno-associated virus vector of Ps1 and Ps1-sh RNA and study on transfection effect in vivoObjective:To construct myocardial targeting Ps1 and Ps1-sh RNA recombinant adenoassociated virus vector(r AAV9-Ps1,r AAV9-Ps1-sh RNA)and study transfection effect in vivo.Methods: Three myocardium specific sh RNA fragments for the Ps1 gene were designed.And the r AAV9-Ps1-sh RNA was prepared with r AAV9 as the vector.After the transfection of 293 T cells in vitro,the expression level of Ps1 in cells was detected by Western Blot to pick the most effective fragment out.Myocardium was transfected by tail vein injection of r AAV9-Ps1 and r AAV9-Ps1-sh RNA respectively,and then the transfection effect in vivo was assessed.Results: At the first week after transfection,r AAV9-Ps1 led to a 26% increase of PS1 expression,and the r AAV9-Ps1-sh RNA led to a 63% decrease of PS1 expression as compared to controls.The transfection effect was stable over 1 to 4 weeks.Conclusion: The r AAV9-Ps1 and r AAV9-Ps1-sh RNA constructed in our experiment showed a favorable transfection effect and could be further used for animal experiments in vivo.PartⅡ Effects of Ps1 gene on SR calcium capacity of normal ratsObjective:To study the effect of Ps1 gene on SR calcium capacity of normal rat by changing the expression level of Ps1 gene in myocardium of rats via transfection.Methods:(1)Experimental group and animal models CTRL(n=5): Tail vein injection of stroke-physiological saline solution Ps1-UP(n=8): Tail vein injection of r AAV9-Ps1Ps1-KD(n=8): Tail vein injection of AAV9-Ps1-sh RNA.(2)Test of cardiac function M-type ultrasound system was used to detect cardiac function.(3)Observation of myocardial microstructure and ultrastructure Myocardial tissue of rat was fixed with 4% paraformaldehyde and embedded in paraffin.Changes of myocardial microstructure were observed after massion staining.The other part of the left ventricular myocardial tissue was selected to observe the changes of myocardial ultrastructure by transmission electron microscopy after a conventional sample preparation.(4)Detection of SR calcium capacity in cardiomyocytes The fluorescent intensity of cardiomyocytes isolated by enzymatic hydrolysis were measured by laser confocal microscopy after Fluo5N-AM loaded.The caffeine-induced calcium transients were detected by laser confocal microscopy after Fluo4-AM loaded.Results:(1)Cardiac function index analysis in each group Compared with the CTRL group(n=5),the EF% and FS% of the Ps1-KD group(n=8)were significantly lower(EF%: F=57.215,60.48±1.67 VS 72.28±2.86,P=0.000;FS%: F=97.418,31.70±1.69 VS 42.84±2.30,P=0.000)accompanied with increased LVEDV and LVESV(LVEDV: F=47.647,336.39±9.07 VS 273.40±6.29,P=0.000;LVESV: F=251.901,118.03 ± 4.55 VS 74.50 ± 5.75,P=0.000).There was no significant changes in Ps1-UP group(n=8)(EF%: F=57.215,71.69 ± 2.67 VS 72.28 ± 2.86,P=0.668;FS%: F=97.418,41.21±0.98 VS 42.84±2.30,P=0.096;LVEDV: F=47.647,267.55±22.15 VS 273.40±6.29,P=0.509;LVESV: F=251.901,72.99 ± 3.23 VS 74.50 ± 5.75,P=0.556;HW/BW: F=6.116,3.65±0.089 VS 3.70±0.13,P=0.520).(2)Microscopic and ultrastructural analysis of myocardium Myocardium of Ps1-KD group showed broaden intercalated disc,increased collagenous fiber and mitochondria with high electron density.There was no significant changes in Ps1-UP group.(3)Changes of SR calcium capacity of cardiomyocytes in each group The results showed decreased SR calcium capacity accompanied with induced calcium transient of cardiomyocytes in Ps1-KD group(Fluo5N-AM: ΔF/F0: F=227.131,42.29±2.31 VS 60.61±1.93,P=0.000,n=12;Fluo4-AM: F=106.374,112.23±6.96 VS 136.34±3.11,P=0.000,n=12).There was no changes in Ps1-UP group(Fluo5N-AM: ΔF/F0:F=227.131,60.75±2.97 VS 60.61±1.93,P=0.888,n=12;Fluo4-AM: ΔF/F0: F=106.374,136.30±2.71 VS 136.34±3.11,P=0.985,n=12).Conclusion: Overexpression of Ps1 gene has no significant effect on cardiac function,myocardial structure and SR calcium capacity under normal physiological condition.However,Ps1 silence led to decreased cardiac function,SR calcium capacity as well as changes of the microstructure and ultrastructure.Part Ⅲ Effects of Ps1 gene on calcium homeostasis of heart failure ratsObjective: Model of heart failure was established to study the effect of Ps1 gene on cardiac function of heart failure rats and the calcium mechanism was further explored.Methods:(1)Experimental group and establishment of animal models CTRL group(n=5): The chest was opened and sutured subsequently HF group(n=10): Myocardial infarction surgery(The left anterior descending branch of the heart was ligated)Ps1-UP-HF group(n=10): Myocardial infarction surgery was operated 3 days after the injection of r AAV9-Ps1 Ps1-KD-HF group(n=10): Myocardial infarction surgery was operated 3 days after the injection of r AAV9-Ps1-sh RNA.(2)Detection indicator Changes of cardiac function,myocardial microstructure and ultrastructure,SR calcium capacity and spontaneous calcium release were detected 4 weeks after model establishment.(The specific detection methods were similar to those of the second part);Western Blot was used to detect the expression of Ry R2 and SERCA2 a in myocardium of rats.Results:(1)Analysis of cardiac function index in each group Compared with CTRL group(n=5),the EF% and FS% in HF group(n=9)were significantly decreased(EF%: F=241.562,43.24±1.72 VS 72.28±2.86,P=0.000;FS%: F=226.552,22.32±1.22 VS 42.84±2.30,P=0.000).LVEDV and LVESV were significantly increased(LVEDV: F=793.539,459.81 ± 16.24 VS 273.40 ± 26.76,P=0.000;LVESV: F=601.979,261.78±19.58 VS 72.30±13.36,P=0.000).The EF% and FS% of Ps1-KD-HF group(n=8)were further decreased compared with those of HF group(EF%: F=241.562,25.84±2.76 VS 43.24±1.72,P=0.000;FS%: F=226.552,17.40±2.10 VS 22.32±1.22,P=0.000).LVEDV and LVESV were further increased than those of HF group(LVEDV: F=793.539,709.94±11.29 VS 459.81±16.24,P=0.000;LVESV: F=601.979,544.88±36.59 VS 261.78±19.58,P=0.000).The EF% and FS% of the Ps1-UP-HF group(n = 9)were higher than those of HF group(EF%: F=241.562,51.74±4.28 VS 43.24±1.72,P=0.000;FS%: F=226.552,28.23±1.68 VS 22.32±1.22,P=0.000).And the LVEDV and LVESV concomitantly decreased(LVEDV: F=793.539,356.26±18.93 VS 459.81±16.24,P=0.000;LVESV: F=601.979,163.08±6.53 VS 261.78±19.58,P=0.000).(2)Microscopic and ultrastructural analysis of myocardial tissue in each group Rats of HF group showed increased ventricular volume,weakened movement of the anterior septum,severe fibrosis of the myocardium,loose arrangement of muscle filaments and severely damaged mitochondria and intercalated discs.Rats of Ps1-KD-HF group showed dilated biventricular and severe fibrosis.The anterior septum motion disappeared or even reversed.Part of the muscle filament broke,and even dissolved.Mitochondrial were seriously damaged.Intercalated disc blurred,and even broken.In the Ps1-UP-HF group,myocardial remodeling was significantly alleviated.(3)Changes of SR calcium capacity and spontaneous calcium release in each group The calcium capacity of cardiomyocytes in HF group was significantly lower than that of CTRL group(Fluo5N-AM: F=855.261,29.45±1.71 VS 61.35±2.80,P=0.000,n=12;Fluo4-AM: F=1216.898,60.20±2.37 VS 102.64±4.51,P=0.000,n=12).And spontaneous calcium release increased(F=1161.880,21.50±2.20 VS 3.50±1.00,P=0.000,n=12).Ps1 gene silencing caused further decreased SR calcium capacity(Fluo5N-AM: F=855.261,20.71±2.39 VS 29.45±1.71,P=0.000,n=12;Fluo4-AM: F=1216.898,29.10±2.37 VS 60.20 ± 2.37,P=0.000,n=12)and further increased spontaneous calcium release(F=1161.880,40.83±2.12 VS 21.50±2.20,P=0.000,n=12).Overexpression of Ps1 gene increased the SR calcium capacity of cardiomyocytes in heart failure rats(Fluo5N-AM: F=855.261,39.23±0.82 VS 29.45±1.71,P=0.000,n=12;Fluo4-AM: F=1216.898,78.69±2.37 VS 60.20±2.53,P=0.000,n=12)and reduced the spontaneous calcium release event significantly(F=1161.880,9.25±0.97 VS 21.50±2.20,P=0.000,n=12).(4)Results of molecular biology test Results of molecular biology test showed that the expression of SERCA2 a in myocardial tissue of HF group was significantly decreased(F=370.933,0.525±0.0250 VS 0.908±0.0148,P=0.0662,n=3).Silence of Ps1 gene decreased the expression of Ry R2(F=56.102,0.771±0.0195 VS 0.903±0.0150,P=0.000,n=3).Conclusion:(1)Ry R2 calcium leakage in cardiomyocytes and decreased expression of SERCA2 a may be important factors for the occurrence of heart failure.(2)Ps1 is involved in the pathogenesis of heart failure.Silence of Ps1 aggravated calcium deficiency,myocardial remodeling and reduction of cardiac functionin in heart failure.Ps1over-expression significantly alleviated cardiomyocyte calcium leakage and myocardial remodeling process.And the cardiac function significantly improved at the same time. |
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