Mechanisms Of Abnormal Sarcoplasmic Reticulum Ca²⁺-ATPase Of Chronic Heart Failure Myocytes And Effects Of Valsartan And Simvastatin

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.