| Background Left ventricular hypertrophy (LVH) is an independent risk factor for the morbidity and mortality of essential hypertension, but detailed etiological mechanisms have remained unidentified and there have been no effective prevention and treatment measures so far. It has been proved that cardiac myocytes hypertrophy and phenotype transition from contractile state (adult state) to synthetic state (embryo state) are regarded as one of the important pathological bases of LVH. As characters of hypertrophic myocardium, high quantity oxygen consumption, decreased contractile ability and changes of conduction quality lead to myocardium ischemia, heart failure and arrhythmia as a result. How to prevent and revert cardiac myocytes hypertrophy and LVH has become a heatedly debated global topic. It has been identified by large amount of studies that, except as a potent lipidemia regulator, 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase inhibitor (statins) can not only decrease serum cholesterol and low-density lipoprotein but also inhibit proliferation of vascular smooth muscle cells (VSMCs) and improve endothelium function and its effect is far beyond that of Iipidemia regulation. Statins has been used as a prevention and therapy measure for atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). Recently, it was observed that statins could inhibitcell proliferate and collagen synthesis of cardiac fibroblasts (CFs) and decrease the collagen volume in heart of spontaneously hypertensive rats (SHR), thus, delayed the prosess of cardiac fibosis. It remains unknown whether statins can attenuate cardiac myocytes hyptertrophy and LVH of hypertension, moreover, the underlying precise mechanisms also remain obscure. Phosphoinositide 3-kinase (P13K)/protein kinase B (PKB) pathway has been believed to play an important role in cell proliferation, hypertrophy, survival and apoptosis. Some reports, which based on the fact that the heart volume of PI3K/PKB transgenic mice was much bigger than that of PI3K/PKB(-/-) mice, had revealed that PI3K/PKB pathway may be involved in the prosess of heart development . It has not been reported as far whether PI3K/PKB pathway is involved in the signal transduction of cardiac myocytes hypertrophy modulated by statins. This study was therefore designed to observe the effects of simvastatin on cultured cardiac myocytes hypertrophy in Sprague-Dawley (SD) rats and LVH in SHR, aim to study the possibility of attenuation effects of simvastatin on cardiac myocytes hypertrophy and LVH, investigate the role of PI3K/PKB pathway in the modulation effects of simvastatin on cardiac myocytes hypertrophy, elucidate the cellular and molecular mechanisms of cardiovascular protection effects of simvastatin, and provide new theoretical evidences and prevention and treatment approaches for LVH of hypertension.Methods In this study, SHR and cultured cardiac myocytes of neonatal SD rats were used as experiment models. On the other hand, radioimmunoassay, computerized image analysis, reverse transcription polymcrase chain reaction (RT-PCR) and Western blot were applied to identified: (1) the effects of simvastatin on hypertrophy and phenotype transition of cultured cardiac myocytes induced by animal serum in SD rats; (2) the effects of simvastatin on PKB and phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression of cultured cardiac myocytes; (3) the intervention effects of L-mevelonate(L-MVA) on the modulation effects of simvastatin on cardiac myocytes hypertrophy, phenotype transition, PKB and PTEN expression; (4) the intervention effects of PTEN antisense oligodeoxynucleotides on the biological effects of simvastatin on cardiac myocytes; (5) the effects of simvastatin on LVH and myocytes phenotype transition in SHR; (6) the effects of simvastain on PKB and PTEN expressin of myocardium in SHR.Results (1) After treatment with 15% neonatal bovine serum for 24h, the surface area of cardiac myocytes (1611.16±160.75um2) was much higher than that of serum-free control group (538.04±l 18.60um2) (/><0.01). The surface area of cardiac myocytes was decreased by co-intervention with simvastatin in a concentration dependent manner. They were 799.84±167.70um and 1076.88± 199.28um2 in 10"5 and 10"6mol/L simvastatin groups, respectively, which were both significantly lower than that of serum group (P<0.01). The cell surface areas of 48h group in serum-free control group, 15% serum group and 15% scrum+10"6mol/L simvastatin group were 601.60±130.19um2, 1844.96± 154.68 urn and 1547.44±218.84um , respectively. It was also much lower in simvastatin group than that of serum group after treatment for 48h (P<0.01). (2) [3H]-leucinc incorporation rate of serum group (2360.00±105.59cpm/well) was significantly higher than that of control group (1305.33±91.71cpm/well) (PO.01). They were 1706.67±100.83cpm/well and 1962.00±125.03cpm/well in 10"5 and 10"6mol/L simvastain groups, which were both much lower than that of serum group (P<0.0\). After treatment for 48h, [3H]-lcucine incorporation rate of 10'6mol/L simvastain group (21 13.17±92.17cpm/well) was also lower than that of serum group (2513.67±140.82cpm/well) (PO.Ol). (3) With the increase of simvatatain's concentration, the mRNA expression level of atrial natriuretic peptide (ANP) measured with RT-PCR was decreased gradually compared with that of serum group (0.60±0.03). The levels of mRNA in 10"5 and 10~6mol/L simvastatin groups were 0.29±0.03 and 0.40±0.03, which were both lower than that of serum group(P<0.0\). The mRNA levels of ANP in control group, 15% serum group and 15% serum+10~6mol/L simvastatin group after treatment for 48h were 0.24±0.03, O.85±O.O5 and 0.57±0.04, respectively. It was also much lower in simvastatin treatment group than that of serum group (JP<0.01). (4) ANP protein levels in culured cardiac myocytes supernatant measured with radioimmunoasssy were 854.53±54.94pg/106cells and 358.06±34.38pg/106cells in serum and control groups. It was significantly higher in serum group than that of control group (.P<0.01), while they were much decreased in 10'5 and 10"6mol/L simvastatin groups (405.83±53.48pg/106cells and 489.47±48.35pg/106cells) than that of serum group (P<0.0\). Moreover, the value above in 48h group was also lower in 10~6mol/L simvastatin group (633.86±57.79pg/106cell) than that of serum group (1046.18±74.24pg/106cell) (PO.01). (5) Simvastatin inhibited the expression of PKB mRNA in cardiac myocytes induced by serum in a concentration dependent manner. The mRNA expression level of PKB in serum group was 0.51±0.03, which was much higher than that of control group (0.20±0.02) (PO.01). The mRNA levels in 10"5 and 10"6 mol/L simvastatin groups were 0.28±0.02 and O.36±O.O3, respectively, which were both much lower than that of serum group (P<0.01). The mRNA level of PKB in control group, serum group and serum+ simvastatin group after treatment for 48h were 0.21±0.03, 0.69±0.04 and 0.48±0.04. It was also much lower in simvastatin group than that of serum group (P<0.01). (6) Similarly, simvastatin inhibited the expression of protein of PKB determined with Western blot in cardiac myocytes induced by serum also in a concentration dependent manner as well. Compared with scrum group (60.80±3.49), protein levels of PKB in 10"5 and 10"6 mol/L simvastatin groups (42.41±2.83 and 45.68±3.43) were also both significantly decreased (PO.01), but they were still much higher than that of serum-free control group (35.84±2.54) (PO.01). The protein levels of PKB in control group, 15% serum group and 15% scrum+10~6mol/L simvastatin group after treatment for 48h were 40.12±2.25,67.38±3.43, and 51.31±3.22, respectively. It was also much lower in simvastatin group than that of serum group (PO.01). (7) With the increase of simvastatin's concentration, the mRNA level of PTEN was increased dramaticly. The mRNA levels of PTEN in 10"5, 10"6 and 10~7mol/L simvastatin+15% serum groups were 0.85±0.O5, 0.83±0.04 and 0.38±0.03, respectively, which were all much higher than that of 15% serum group (0.29±0.04) (P<0.01, 0.05). But they were still lower than that of serum-free control group (1.11 ±0.08) (P<0.0\). The mRNA levels of PTEN of 48h groups were 1.04±0.03, 0.21 ±0.04 and 0.67±0.04 in control group, 15% serum group and 15% serum+10"°mol/L simvastatin group, respectively. It was still much higher in simvastain group than that of serum group (P<0.01). (8) Simvastatin could increase PTEN protein level also in a concentration dependent manner. The protein levels of PTEN in 10~\ 10"6 and 10"7mol/L simvastatin+serum group were 47.22±2.39, 46.35±1.78 and 39.25±3.41, all of which were significantly higher than than of serum group (32.21±4.06) (P<0.0\, 0.05). But they were still much lower than that of control group (56.53±4.36) (PO.01). After being treated for 48h, PTEN protein levels of cardiac myocytes in control group, serum group and 10"6mol/L simvastatin group were 56.14±2.76, 25.28±4.63 and 33.33±3.56, respectively. Protein level in simvastatin group was also much higher than that of serum group {P<0.05). (9) The decrease in cell surface area, [3H]-leucine incorporation rate, ANP mRNA and protein levels of cultured cardiac myocytes by 10"6mol/L simvastatin group were almost completely deprived by co-intervention with 10"4mol/L L-MVA for 24h (PO.01). (10) The expression levels of PKB mRNA and protein in 10'6mol/L simvastatin+10"4moI/L L-MVA group (0.52±0.03 and 58.68±3.10) were both significantly higher than those of simvastatin single treatment group (0.33±0.02 and 46.01 ±3.77) (P<0.01), but, there were no significant differences between simvastatin+L-MVA group and serum group (O.5O±O.O3 and 60.52±4.00)(/3>0.05). (11) The mRNA and protein levels of PTEN in simvastatin+L-MVA group(0.31 ±0.03 and 33.28±4.79) were both much lower than those of simvastatin single treatment group (0.80±0.04 and 45.08±2.10) (PO.01). But there were no significant differences between simvastatin+L-MVA group and serum group (0.28±0.03 and 31.71 ±5.92) (P>0.05). (12) Cell surface area, [3H]-leucine incorporation rate, ANP mRNA and protein levels in 10"6mol/L simvastatin+ 5x10"'moI/L PTEN antisense oligodeoxynucleotides group were significantly higher than those of simvastatin single treatment group (P<0.01), however, they were still lower than those of serum group (P<0.01). On the contrary, there were no significant differences between PTEN sense or mismatch oligodeoxynucleotides group and simvastatin group (Z^O.05). (13) The mRNA expression levels of PTEN in simvastatin group, simvastain+PTEN antisense oligodeoxynucteoti-des group, simvastatin+sense group, simvastatin+mismatch group were 0.80±0.04, 0.22±0.03, 0.81 ±0.04 and 0.83±0.03, respectively. The protein levels of PTEN in these groups above were 45.08±2.10, 23.22±5.10, 41.13±4.61 and 40.08±2.60. The mRNA and protein levels in PTEN antisense oligodeoxynucleotides group were significantly lower than those of simvastatin group (P<0.0\), however, there were no significant differences between sense or mismatch group and simvastatin group (Z^O.05). (14) After ten-week-treatment, systolic blood pressures of SHR control group and simvastatin treatment group were 220.75±9.85mmHg and 217.25±8.48mmHg. Systolic blood pressure of Wistar-Kyoto (WKY) rats in normal control group (126.00±5.78mmHg) was much lower than those of SHR groups (P<0.0\). Systolic blood pressure of treatment group was a little lower than that of control group of SHR, but it was not significant (P>0.05). (15) The ratios of left ventricular mass to body weight (LVM/BW) of SHR control group and WKY normal control group were 4.10±0.13mg/g and 3.04±0.12mg/g, respectively. It was significantly higher in SHR control group than that of normal control group (PO.01). However, LVM/BW of SHR treatment group (3.73±0.08mg/g) was much lower than that of SHR control group (P<0.01). (16)The mRNA expression levels of ANP in SHR control group and normal control group were 0.44±0.03 and 0.17±0.03. It was much higher in SHR control group than that of normal control group (P<0.0\). However, it was significantly decreased in simvastatin treatment group (0.27±0.03) than that of control group of SHR (P<0.01). (17) The mRNA expression level of PKB in SHR control group was 0.45±0.05. The mRNA level of PKB in normal control group were 0.19±0.02, which were much lower than that of SHR control group (P<0.01). The mRNA level was significantly decreased in simvastatin treatment group (0.32±0.03) than that of SHR control group (/><0.01). (18) The protein levels of PKB measured with Western blot in WKY normal control group, SHR control group and simvastatin treatment group were 43.31±3.86, 62.34±3.47 and 50.85±3.20, respectively. PKB level of SHR control group was much higher than that of WKY normal control group (P<0.0\), while simvastatin group was significantly lower than that of SHR control group (PO.01). (19) The mRNA level of PTEN was significantly decreasd in SHR control group (0.36±0.04) than that of WKY control group (0.87±0.05) (P<0.01), however, it was significantly increased in simvastatin treatment group (0.60±0.05) than that of SHR control group (PO.01). (20) The expression level of PTEN protein in SHR control group (24.65±3.89) was much lower than that of WKY control group (50.53±2.92) (P<0.01), while it was significantly increased in simvastatin treatment group (40.32±4.04) than that of SHR control group (P<0.01).Conclusion Simvastatin had dramatic inhibition effects on hypertrophy and phenotype transition of cultured cardiac myocytes in SD rats in a concentration dependent manner, moreover, it could decrease PKB expression and increase PTEN expression also in a dose dependent manner. The biological effects of simvastatin on cardiac myocytes were almost completely abrogated by addition of low dose of L-MVA, while they could be partly inhibited by intervention with PTEN antisense oligodeoxynucleotides. Furthermore, simvastatin could...
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