| Cardiac remodeling is manifested clinically as changes in the size, shape, and function of the heart. Histopathologically, it is characterized by a structural rearrangement of components of the normal chamber wall that involves cardiomyocyte hypertrophy, cardiac fibroblast proliferation, fibrosis, and cell death. Cardiac fibrosis, which is a disproportionate accumulation of fibrillar collagen, is an integral feature of the remodeling characteristic of the failing heart. Cardiac fibroblasts are crucially involved in the processes of cardiac fibrosis, producing growth factors and cytokines that act as autocrine and paracrine factors, as well as extracellular matrix proteins and proteinases. Humoral factors that affect the phenotype and function of cardiac fibroblasts include renin-angiotensin-aldosterone system, basic fibroblast growth factor (bFGF), transforming growth factor-β(TGF-β), endothelin, bradykinin, nitric oxide, catecholamines, et al. Aldosterone, which is recognized as vital promoter of myocardial fibrosis, can promote the proliferation of cardiac fibroblast and collagen synthesis, resulting in myocardial fibrosis. Normal cardiac tissue, which has independent aldosterone system, contains abundant, specific, high-affinity mineralocorticoid receptor (MR). Several studies showed that there are increased aldosterone levels found in heart failure patients and high levels of aldosterone is the independent predictor of increased mortality; the MR antagonists can alleviate the progression of cardiac remodeling induced by angiotensinⅡ. So aldosterone plays an important role on heart failure.3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), which are the best way to reduce cholesterol and the risk of heart attack as a lipid-lowering drug, are widely used in the prevention and treatment of cardiovascular disease. In recent years a substantial quantity of data has accumulated showing that statins exert anti-fibrosis effects on myocardium, which are independent of their plasma cholesterol lowering properties. Statins have been shown to inhibit Angiotensin II-mediated cardiac fibrosis as well as to block various intracellular signaling pathways including downregulation of the activity of small GTP-binding proteins. Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis in the liver, which catalyzes the conversion of HMG-CoA to mevalonic acid. In addition to inhibiting cholesterol synthesis, statins also block the synthesis of isoprenoid intermediates such as farnesyl pyrophosphate (FPP) and geranyl- geranyl pyrophosphate (GGPP). Both FPP and GGPP serve as important lipid attachments for the posttranslational modification of a variety of proteins, including heterotrimeric G proteins and small GTP-binding proteins belonging to the family of Ras, Rho GTPases. Isoprenylation is critical for intracellular trafficking and function of small GTP-binding proteins. In general, modification with FPP is necessary for proper localization of Ras family proteins, whereas GGPP is required for Rho family proteins. By inhibiting mevalonate synthesis, statins inhibit the synthesis of isoprenoid intermediates thereby preventing isoprenylation of small GTPases, leading to the inhibition of these signaling molecules, which contributing to the prevention of cardiac fibrosis and heart failure.There are various intracellular signaling pathways participate in the regulation of cell survival and apoptosis, including PI3K/Akt and calcineurin (CaN) pathways. PTEN( phosphatase and tensin homology deleted on chromosome ten), which act as a negative regulator of PI3K/ Akt pathway, is proved have a close relationship with cardiomyocyte hypertrophy. Calcineurin, which is the key link to activated intranuclear hypertrophic gene induced by intra-cellular Ca2+, play the vital role in the development of cardiomyocyte hypertrophy. Recently, sun et al reported that inactivation of PTEN enhances L-type Ca2+ channel (ICa,L) via PI3K-dependent increase in PKB activation and provided powerful evidence for a critical interface between cardiomyocyte Ca2+ signaling and PI3K-regulated pathway. Some domestic studies also showed that cardiac hypertrophy induced by AngⅡcould be blocked by PTEN overexpression via suppressing Ca2+ /Calcineurin pathway. Those data suggested that there may exist some"crosstalking"between PTEN-PI3K/Akt and Ca2+/ Calcineurin pathways.Now, most studies on the role of calcineurin or PTEN are limited to cardiomyocyte hypertrophy, whether calcineurin or PTEN participate in anti-fibrosis effects of statins is still unknown. So we cultured cardiac fibroblasts of neonatal Sprague-Dawley rats induced by aldosterone, treated them with spironolactone, atorvastatin, FPP, GGPP respectively, tested the expression of calcineurin,PTEN mRNA and protein by RT-PCR and Western Blot, to investigate the role of calcineurin and PTEN on the anti-fibrosis effects of atorvastatin and to explore the signal transduction mechanisms.Part 1 The effects of aldosterone on the expression of calcineurin in cultured neonatal cardiac fibroblastsObjective: To investigate the role of calcineurin (CaN) in aldosterone (Ald)–induced proliferation of cardiac fibroblasts and to explore the effects of spironolactone (Spi) on the expression of CaN.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley (S-D) rats, the effects of Ald and Spi on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, CaN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ald and Spi on the proliferation: Compared with the control group, 10-7mol/L,10-8mol/L,10-9mol/L Ald groups significantly increased the proliferation of fibroblasts respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+10-8mol/L,10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group. 2. Effects of Ald and Spi on the hydroxyproline concentration: Compared with the control group, 10-7mol/L,10-8mol/L,10-9mol/L Ald groups significantly increased the hydroxyproline concentration respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7mol/L Ald+10-8 mol/L,10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group.3. Effects of Ald on the expression of CaN mRNA: Compared with the control group, the expression of CaN mRNA in 10-7mol/L,10-8mol/L,10-9mol /L Ald groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Spi on the expression of CaN mRNA: Compared with 10-7mol/L Ald group, the expression of CaN mRNA in 10-7mol/L Ald+10-8 mol/L,10-9mol/L Spi groups were significantly decreased respectively (P<0.05), which were no difference with the control group.5. Effects of Ald on the expression of CaN protein: Compared with the control group, the expression of CaN protein in 10-7mol/L,10-8mol/L,10-9mol /L Ald groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).6. Effects of Spi on the expression of CaN protein: Compared with 10-7mol/L Ald group, the expression of CaN protein in 10-7mol/L Ald+10-8 mol/L,10-9mol/L Spi groups were significantly decreased respectively (P<0.05), which were no difference with the control group.Conclusions: Exogenous aldosterone stimulated cardiac fibroblasts proliferation,collagen synthesis and increased the expression of CaN mRNA,protein. The effects of Ald on calcineurin were realized by genomic way.Part 2 The effects of aldosterone on the expression of PTEN in cultured neonatal cardiac fibroblastsObjective: To investigate the role of PTEN in aldosterone (Ald)–induced proliferation of cardiac fibroblasts and to explore the effects of spironolactone (Spi) on the expression of PTEN.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley (S-D) rats, the effects of Ald and Spi on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, PTEN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ald and Spi on the proliferation: Compared with the control group, 10-7mol/L,10-8mol/L,10-9mol/L Ald groups significantly increased the proliferation of fibroblasts respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+10-8mol/L,10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group.2. Effects of Ald and Spi on the hydroxyproline concentration: Compared with the control group, 10-7mol/L,10-8mol/L,10-9mol/L Ald groups significantly increased the hydroxyproline concentration respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7mol/L Ald+10-8 mol/L,10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group.3. Effects of Ald on the expression of PTEN mRNA: Compared with the control group, the expression of PTEN mRNA in 10-7mol/L,10-8mol/L,10-9 mol/L Ald groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Spi on the expression of PTEN mRNA: Compared with 10-7mol/L Ald group, the expression of PTEN mRNA in 10-7mol/L Ald+10-8 mol/L,10-9mol/L Spi groups were significantly increased respectively (P<0.05), which were no difference with the control group.5. Effects of Ald on the expression of PTEN protein: Compared with the control group, the expression of PTEN protein in 10-7mol/L,10-8mol/L,10-9 mol/L Ald groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).6. Effects of Spi on the expression of PTEN protein: Compared with 10-7mol/L Ald group, the expression of PTEN protein in 10-7mol/L Ald+10-8 mol/L,10-9mol/L Spi groups were significantly increased respectively (P<0.05), which were no difference with the control group.Conclusions: Exogenous aldosterone stimulated cardiac fibroblasts proliferation,collagen synthesis and decreased the expression of PTEN mRNA,protein. The effects of Ald on PTEN were realized by genomic way.Part 3 The effects of atorvastatin on the expression of calcineurin in aldosterone–induced cardiac fibroblastsObjective: To investigate the effects of atorvastatin(Ato)on calcineurin in aldosterone–induced cardiac fibroblasts.Method: In aldosterone–induced cardiac fibroblasts of neonatal Sprague- Dawley(S-D) rats, the effects of Ato,FPP,GGPP on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, CaN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ato on the proliferation in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7mol/L Ald group significantly increased the proliferation of fibroblasts(P<0.05). Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+ 10-7,10-6,10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.2. Effects of Ato on the hydroxyproline concentration in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7 mol/L Ald group significantly increased the hydroxyproline concentration (P<0.05). Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7mol/L Ald+10-7,10-6,10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.3. Effects of Ato on the expression of CaN mRNA in aldosterone–induced cardiac fibroblasts: Compared with the control group, the expression of CaN mRNA in 10-7mol/L Ald group was significantly increased (P<0.05). Compared with 10-7mol/L Ald group, the expression of CaN mRNA in 10-7 mol/L Ald+10-7,10-6,10-5mol/L Ato groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Ato on the expression of CaN protein in aldosterone–induced cardiac fibroblasts: Compared with the control group, the expression of CaN protein in 10-7mol/L Ald group was significantly increased (P<0.05). Compared with 10-7mol/L Ald group, the expression of CaN mRNA in 10-7 mol/L Ald+10-7,10-6,10-5mol/L Ato groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).5. Effects of FPP,GGPP on the expression of CaN mRNA in aldosterone–induced cardiac fibroblasts: The expression of CaN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group was significantly increased compared to 10-7mol/L Ald+10-5mol/L Ato group (P<0.05), while the expression of CaN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group was no difference with 10-7mol/L Ald+ 10-5mol/L Ato group.6. Effects of FPP,GGPP on the expression of CaN protein in aldosterone–induced cardiac fibroblasts: The expression of CaN protein in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group was significantly increased compared to 10-7mol/L Ald+10-5mol/L Ato group (P<0.05), while the expression of CaN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group was no difference with 10-7mol/L Ald+10-5mol/L Ato group.Conclusions: Atorvastatin could decrease the expression of calcineurin by inhibiting Ras activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.Part 4 The effects of atorvastatin on the expression of PTEN in aldosterone–induced cardiac fibroblasts Objective: To investigate the effects of atorvastatin(Ato)on PTEN in aldosterone–induced cardiac fibroblasts.Method: In aldosterone–induced cardiac fibroblasts of neonatal Sprague- Dawley(S-D) rats, the effects of Ato,FPP,GGPP on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, PTEN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ato on the proliferation in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7mol/L Ald group significantly increased the proliferation of fibroblasts(P<0.05). Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+ 10-7,10-6,10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.2. Effects of Ato on the hydroxyproline concentration in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7 mol/L Ald group significantly increased the hydroxyproline concentration (P<0.05). Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7 mol/L Ald+10-7,10-6,10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.3. Effects of Ato on the expression of PTEN mRNA in aldosterone–induced cardiac fibroblasts: Compared with the control group, the expression of PTEN mRNA in 10-7mol/L Ald group was significantly decreased (P<0.05). Compared with 10-7mol/L Ald group, the expression of PTEN mRNA in 10-7mol/L Ald+10-7,10-6,10-5mol/L Ato groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Ato on the expression of PTEN protein in aldosterone– induced cardiac fibroblasts: Compared with the control group, the expression of PTEN protein in 10-7mol/L Ald group was significantly decreased (P<0.05). Compared with 10-7mol/L Ald group, the expression of PTEN mRNA in 10-7mol/L Ald+10-7,10-6,10-5mol/L Ato groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).5. Effects of FPP,GGPP on the expression of PTEN mRNA in aldosterone–induced cardiac fibroblasts: Compared with 10-7mol/L Ald+10-5 mol/L Ato group, the expression of PTEN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group and 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group were significantly decreased respectively (P<0.05).6. Effects of FPP,GGPP on the expression of PTEN protein in aldosterone–induced cardiac fibroblasts: Compared with 10-7mol/L Ald+10-5 mol/L Ato group, the expression of PTEN protein in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group and 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group were significantly decreased respectively (P<0.05).Conclusions: Atorvastatin could increase the expression of PTEN by inhibiting Ras and Rho activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.Part 5 The Correlation between the expression of calcineurin and PTEN in aldosterone–induced and atorvastatin-interfered cardiac fibroblastsObjective: To investigate the correlation between the expression of calcineurin and PTEN in aldosterone–induced and atorvastatin-interfered cardiac fibroblasts.Method: In aldosterone–induced and atorvastatin-interfered cardiac fibroblasts of neonatal Sprague-Dawley rats, the effects of Ald,Ato on the expression of calcineurin and PTEN were tested by RT-PCR and Western Blot, respectively, the correlation between the expression of CaN and PTEN were analyzed by Spearman rank correlation test.Results: 1. The correlation between the expression of CaN and PTEN mRNA in aldosterone–induced cardiac fibroblasts: The expression of CaN mRNA were negatively correlated with the expression of PTEN mRNA (r=﹣0.860, P<0.01).2. The correlation between the expression of CaN and PTEN protein in aldosterone–induced cardiac fibroblasts: The expression of CaN protein were negatively correlated with the expression of PTEN protein (r=﹣0.868, P<0.01).3. The correlation between the expression of CaN and PTEN mRNA in atorvastatin-interfered cardiac fibroblasts: The expression of CaN mRNA were negatively correlated with the expression of PTEN mRNA (r=﹣0.808, P<0.01).4. The correlation between the expression of CaN and PTEN protein in atorvastatin-interfered cardiac fibroblasts: The expression of CaN protein were negatively correlated with the expression of PTEN protein (r=﹣0.799, P<0.01).Conclusions: In aldosterone–induced and atorvastatin-interfered cardiac fibroblasts, the expression of CaN mRNA,protein were negatively correlated with the expression of PTEN mRNA,protein respectively, suggested there were a close relation between the expression of CaN and PTEN.Conclusions1. Exogenous aldosterone stimulated cardiac fibroblasts proliferation,collagen synthesis and increased the expression of CaN mRNA,protein. The effects of Ald on calcineurin were realized by genomic way.2. Exogenous aldosterone stimulated cardiac fibroblasts proliferation,collagen synthesis and decreased the expression of PTEN mRNA,protein. The effects of Ald on PTEN were realized by genomic way.3. Atorvastatin could decrease the expression of calcineurin by inhibiting Ras activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.4. Atorvastatin could increase the expression of PTEN by inhibiting Ras and Rho activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.5. In aldosterone–induced and atorvastatin-interfered cardiac fibroblasts, the expression of CaN mRNA,protein were negatively correlated with the expression of PTEN mRNA,protein respectively, suggested there were a close relation between the expression of CaN and PTEN. |
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