【作者】 曲辅政；

【导师】 刘志华；

【作者基本信息】 苏州大学 ， 内科学， 2008， 博士

【摘要】 研究目的:通过观察心衰心肌细胞PLB(受磷蛋白)、SERCA2a(肌浆网钙泵)、CaM(钙调蛋白)、CaMKⅡ(钙调蛋白依赖性蛋白激酶Ⅱ)、PKA(蛋白激酶A)、PP1α(蛋白磷酸酶1α)的表达,PKA、CaMKⅡ的活性和SERCA2a的功能以及缬沙坦(ARB)、辛伐他汀(statins)药物对心衰心肌细胞以上方面的影响,探讨心力衰竭的机制以及ARB、statins药物改善心力衰竭的机制。研究方法:24只新西兰白兔分为4组,第1组为假手术组,2、3、4组联合应用主动脉瓣破坏术及腹主动脉缩窄术建立慢性心力衰竭模型;第2组为心力衰竭组;第3组为缬沙坦干预组,术后给予缬沙坦20mg·kg-1·d-1灌胃,连续7周;第4组为辛伐他汀干预组,术后即给予辛伐他汀10mg·kg-1·d-1灌胃,连续7周。观察开始及结束时进行超声心动图检查,测量左房内径(LAd)、左心室舒张末期内径(LVIDd)、左心室收缩末期内径(LVIDs)、室间隔厚度(IVSd)、左心室后壁厚度(LVPwd)、左心室射血分数(EF)、左心室缩短率(FS)。心导管法记录左心室舒张末压(LVEDP)、左室收缩末压(LVESP)、主动脉收缩压(ASBP)及舒张压(ADBP),同时记录心率(HR)。测量结束后处死动物、取标本。测量心脏重量、左心室重量、心脏重量指数、左心室重量指数;光镜检查心肌组织切片。RT-PCR检测心肌肌浆网钙泵(sarcoplasmic reticulum calcium adenodine triphosphatase,SERCA2a)、受磷蛋白(phospholamban,PLB)mRNA水平; Western blot检测PLB、SERCA2a、钙调蛋白(calmodulin,CaM)、钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinase-Ⅱ,CaMKⅡ)、蛋白激酶A(protein kinase A,PKA)、蛋白磷酸酶1α(protein phosphatase 1 alpha,PP1α)的蛋白表达水平;用γ-32P掺入法测定CaMKⅡ和PKA的活性,无机磷酸根法测定SERCA2a活性,利用光谱荧光检测技术测定肌浆网的摄钙能力。结果:与假手术组比较,心衰组左心室重量、心脏重量指数、左心室重量指数、心率、LVESP、LVEDP显著升高(2.48±0.15g vs 7.15±0.59g,P<0.05;2.29±0.05g/kgvs 5.20±0.25g/kg,P<0.05;1.32±0.06g/kg vs 3.61±0.09g/kg,P<0.05;244.67±9.39bpm vs 270.50±2.88bpm,P<0.05;112.67±3.78mm Hg vs 139.50±3.08mm Hg,P<0.05;-0.50±1.05mm Hg vs 23.00±2.37mm Hg,P<0.05);与心衰组比较,缬沙坦组和辛伐他汀组的左心室重量、心脏重量指数、左心室重量指数、心率、LVESP、LVEDP显著降低(7.15±0.59g vs 4.82±0.21g、5.15±0.24g,P<0.05;5.20±0.25g/kg vs 2.83±0.03g/kg、2.88±0.08g/kg,P<0.05;3.61±0.09g/kg vs 2.07±0.14g/kg、2.17±0.13 g/kg,P<0.05;270.50±2.88bpm vs 252.67±3.50bpm、254.50±2.07 bpm,P<0.05;139.50±3.08mm Hg vs 122.17±0.75mm Hg、123.00±1.09mm Hg , P<0.05 ;23.00±2.37mm Hg vs 2.17±0.72mm Hg、2.33±0.52mm Hg,P<0.05);与假手术组比较,心衰组FS及EF明显降低(37.83±3.58﹪vs 17.38±3.13﹪,P<0.05;71.92±4.56﹪vs 38.50±6.07﹪,P<0.05);缬沙坦干预组和辛伐他汀干预组FS及左室EF明显高于心衰组(17.38±3.13﹪vs 33.83±2.85﹪、33.33±2.21﹪,P<0.05;38.50±6.07﹪vs 64.45±3.66﹪、62.15±3.34﹪,P<0.05)。与假手术组比较,心衰组SERCA2a和PLB mRNA表达下降(SERCA2a/β-actin:1.06±0.16 vs 0.70±0.04,P<0.05;PLB/β-actin:1.12±0.07 vs 0.81±0.04,P<0.05),心衰组SERCA2a、PKA蛋白表达下降(SERCA2a/β-actin:1.02±0.02 vs 0.69±0.04,P<0.05;PKA/β-actin:1.05±0.08 vs 0.61±0.03,P<0.05),心衰组CaMKⅡ、PP1α蛋白表达升高(CaMKⅡ/β-actin:0.89±0.05 vs 1.45±0.13,P<0.05;PP1α/β-actin:0.39±0.07 vs 1.25±0.06,P<0.05),心衰组PKA活性下降(1.85±0.05pmol/min/μg vs 1.09±0.09pmol/min/μg,P<0.05),心衰组CaMKⅡ活性增加(2.18±0.13pmol/min/μg vs 3.54±0.17 pmol/min/μg , P<0.05 ),心衰组SERCA2a活性下降(15.01±1.00μmolPi/mgp/h vs 8.32±0.15μmolPi/mgp/h,P<0.05),心衰组肌浆网摄钙能力下降(肌浆网外液Ca2+浓度下降百分比:95.52±2.12 vs 54.39±7.87,P<0.05)。与心衰组相比,缬沙坦干预组和辛伐他汀干预组SERCA2a、PLBmRNA表达增加(SERCA2a/β-actin:0.70±0.04 vs 0.90±0.04、0.86±0.02,P<0.05;PLB/β-actin:0.81±0.04 vs 1.01±0.06、0.96±0.07,P<0.05),缬沙坦干预组和辛伐他汀干预组SERCA2a、PKA蛋白表达增加(SERCA2a/β-actin:0.69±0.04 vs 0.91±0.02、0.87±0.03,P<0.05;PKA/β-actin:0.61±0.03 vs 0.86±0.06、0.78±0.07,P<0.05),缬沙坦干预组和辛伐他汀干预组CaMKⅡ、PP1α蛋白表达降低(CaMKⅡ/β-actin:1.45±0.13 vs 1.11±0.12、1.16±0.06,P<0.05;PP1α/β-actin:1.25±0.06 vs 0.78±0.12、0.81±0.09 , P<0.05 ),缬沙坦干预组和辛伐他汀干预组PKA活性增加(1.09±0.09pmol/min/μg vs 1.51±0.03pmol/min/μg、1.44±0.04pmol/min/μg,P<0.05),缬沙坦干预组和辛伐他汀干预组CaMKⅡ活性降低(3.54±0.17 pmol/min/μg vs 2.71±0.05 pmol/min/μg、2.83±0.14 pmol/min/μg,P<0.05),缬沙坦干预组和辛伐他汀干预组SERCA2a活性增加(8.32±0.15μmolPi/mgp/h vs 12.46±0.38μmolPi/mgp/h、11.81±0.63μmolPi/mgp/h,P<0.05),缬沙坦干预组和辛伐他汀干预组肌浆网摄钙能力升高(肌浆网外液Ca2+浓度下降百分比:54.39±7.87 vs 81.72±4.86、76.95±3.76,P<0.05)。结论:1.用超容量负荷联合压力负荷,可成功建立兔慢性心力衰竭模型。2.缬沙坦和辛伐他汀有效抑制心室重构,改善心功能。3.心力衰竭机制之一可能是:(1)SERCA2a表达的减少导致其活性下降;(2)PLB的表达减少导致SERCA2a的活性降低;(3)PKA表达、活性降低和PP1α表达增加引起了PLB的去磷酸化程度增加抑制了SERCA2a的活性;(4)CaMKⅡ表达和活性增加加重了心衰。4.缬沙坦和辛伐他汀能够抑制CaMKⅡ、PP1α表达;增加PLB、SERCA2a、PKA表达;增加SERCA2a、PKA活性,抑制CaMKⅡ活性,提高肌浆网的摄钙能力。更多还原

【Abstract】 Objection: This study was to evaluate the effects of valsartan and simvastatin on expressions of PLB、SERCA2a、CaM、CaMKⅡ、PKA、PP1α, CaMKⅡactivity, PKA activity, SERCA2a function, heart function of chronic heart failure and investigate the mechanisms of chronic heart failure and mechanisms of cardioprotective effects of valsartan and simvastatin .Methods: 24 rabbits were divided 4 groups, group I: received sham operation as health control. In other groups, aortic regurgitation and coarctation of ascending aorta were operated in rabbits. Group II was received no drugs. In group III, rabbits were given valsartan 20mg·kg-1·d-1 after the operation. In group IV, rabbits were given simvastatin 10mg·kg-1·d-1 after the operation. At begin and end of treatment period, LAd、LVIDd、LVIDs、IVSd、LVPwd、EF、FS were detected via echocardiography and left ventricular end LVEDP、LVESP、ASBP and ADBP were measured via catheterization. At the end of experiment, heart weight、left ventricular weight、body weight、heart weight index、left ventricular weight index were measured. RT-PCR was used to evaluate( sarcoplasmic reticulum calcium adenodine triphosphatase , SERCA2a) and (phospholamban ,PLB )mRNA expressions.Western blot analysed SERCA2a protein expressions in cardiomyocyte membrane and calmodulin(CaM)、calcium/calmodulin-dependent protein kinase-Ⅱ(CaMKⅡ)、protein kinase A(PKA) and protein phosphatase 1 alpha(PP1α) expressions in cardiomyocytes cytosol. CaMKⅡand PKA activity was determined by [γ-32P] ATP-binding assays. SERCA2a activity was examined by inorganic phosphate. Calcium uptake by sarcoplasmic reticulum was determined with fluorescence spectroscopy.Results: Compared with sham-operated rabbits, heart weight、heart weight index、left ventricular weight index、HR、LVESP、LVEDP of congestive heart failure (CHF) rabbits were significantly increased ((2.48±0.15g vs 7.15±0.59g,P<0.05; 2.29±0.05g/kg vs 5.20±0.25g/kg,P<0.05;1.32±0.06g/kg vs 3.61±0.09 g/kg,P<0.05;244.67±9.39bpm vs 270.50±2.88bpm,P<0.05;112.67±3.78mm Hg vs 139.50±3.08mm Hg,P<0.05;-0.50±1.05mm Hg vs 23.00±2.37mm Hg,P<0.05),compared with CHF rabbits, in rabbits received treatment of valsartan and simvastatin, heart weight、heart weight index、left ventricular weight index、HR、LVESP、LVEDP were significantly decreased ((7.15±0.59g vs 4.82±0.21g、5.15±0.24g,P<0.05;5.20±0.25g/kg vs 2.83±0.03g/kg、2.88±0.08g/kg,P<0.05;3.61±0.09g/kg vs 2.07±0.14g/kg、2.17±0.13 g/kg,P<0.05;270.50±2.88bpm vs 252.67±3.50bpm、254.50±2.07 bpm,P<0.05;139.50±3.08mm Hg vs 122.17±0.75mm Hg、123.00±1.09mm Hg,P<0.05;23.00±2.37mm Hg vs 2.17±0.72mm Hg、2.33±0.52 mm Hg,P<0.05);compared with sham-operated rabbits, the EF and FS of CHF rabbits were significantly decreased (37.83±3.58﹪vs 17.38±3.13﹪,P<0.05;71.92±4.56﹪vs 38.50±6.07﹪,P<0.05),compared with CHF rabbits, in rabbits received treatment of valsartan and simvastatin, EF and FS were significantly increased (17.38±3.13﹪vs 33.83±2.85﹪、33.33±2.21﹪,P<0.05;38.50±6.07﹪vs 64.45±3.66﹪、62.15±3.34﹪,P<0.05).Compared with sham-operated rabbits, the gene expressions of SERCA2a、PLB of CHF rabbits were significantly decreased (SERCA2a/β-actin:1.06±0.16 vs 0.70±0.04,P<0.05;PLB/β-actin:1.12±0.07 vs 0.81±0.04,P<0.05),and protein expressions of SERCA2a、PKA of CHF rabbits were significantly decreased(SERCA2a/β-actin :1.02±0.02 vs 0.69±0.04,P<0.05;PKA/β-actin:1.05±0.08 vs 0.61±0.03,P<0.05), protein expressions of CaMKⅡ、PP1αof CHF rabbits were significantly increased(CaMKⅡ/β-actin:0.89±0.05 vs 1.45±0.13,P<0.05;PP1α/β-actin:0.39±0.07 vs 1.25±0.06,P<0.05),the activity of PKA of CHF rabbits was significantly decreased(1.85±0.05pmol/min/μg vs 1.09±0.09pmol/min/μg,P<0.05),activity of CaMKⅡof CHF rabbits was significantly increased(2.18±0.13pmol/min/μg vs 3.54±0.17 pmol/min/μg , P<0.05) , activity of SERCA2a of CHF rabbits was significantly decreased(15.01±1.00μmolPi/mgp/h vs 8.32±0.15μmolPi/mgp/h,P<0.05),calcium uptake by sarcoplasmic reticulum of CHF rabbits was significantly decreased(percent of downregulation of calcium concentration out of SR:95.52±2.12 vs 54.39±7.87,P<0.05).In rabbits treated by valsartan and simvastatin, the gene expressions of SERCA2a and PLB were upregulated(SERCA2a/β-actin:0.70±0.04 vs 0.90±0.04、0.86±0.02,P<0.05;PLB/β-actin:0.81±0.04 vs 1.01±0.06、0.96±0.07,P<0.05);valsartan and simvastatin significantly promoted protein expressions of SERCA2a、PKA (SERCA2a/β-actin:0.69±0.04 vs 0.91±0.02、0.87±0.03,P<0.05;PKA/β-actin:0.61±0.03 vs 0.86±0.06、0.78±0.07,P<0.05),and decreased protein expressions of CaMKⅡ、PP1α(CaMKⅡ/β-actin:1.45±0.13 vs 1.11±0.12、1.16±0.06,P<0.05;PP1α/β-actin:1.25±0.06 vs 0.78±0.12、0.81±0.09,P<0.05);valsartan and simvastatin significantly diminished activity of CaMKⅡ(3.54±0.17 pmol/min/μg vs 2.71±0.13 pmol/min/μg、2.83±0.14 pmol/min/μg,P<0.05)and increased activity of SERCA2a (8.32±0.15μmolPi/mgp/h vs 12.46±0.38μmolPi/mgp/h、11.81±0.63μmolPi/mgp/h,P<0.05)and PKA(1.09±0.09pmol/min/μg vs 1.51±0.03 pmol/min/μg、1.44±0.04pmol/min/μg , P<0.05). Valsartan and simvastatin significantly increased calcium uptake by sarcoplasmic reticulum(percent of downregulation of calcium concentration out of SR:54.39±7.87 vs 81.72±4.86、76.95±3.76,P<0.05).Conclusion: 1.Animal model of heart failure were established by volume overload plus pressure overload in rabbits successfully.2.Given valsartan and simvastatin in chronic heart failure rabbit models could prevent heart enlarge and inhibite the development of cardiac hypertrophy and improve cardiac function. 3.The mechanisms of heart failure include: (1) Downregulation of protein expression of SERCA2a led to downregulation of SERCA2a activity; (2) Downregulation of expression of PLB decreased SERCA2a activity; (3) Downregulation of protein expression of PKA and PKA activity、upregulation of protein expression of PP1αincreased phosphorylation of PLB,which inhibited SERCA2a activity;(4) Upregulation of protein expression of CaMKⅡand CaMKⅡactivity aggravated heart failure. 4.Valsartan and simvastatin inhibited the protein expressions of CaMKⅡ、PP1αand CaMKⅡactivity in cardiomyocytes cytosol, promoted gene expressions of SERCA2a and PLB, increased protein expressions and activity of SERCA2a、PKA , increased calcium uptake by sarcoplasmic reticulum.更多还原