The Effects Of Fasudil On Cardiac ACE-Ang Ⅱ And ACE2-Ang(1-7)in Pressure-overload Rats

BACKGROUNDCardiac remodeling is the pathophysiology basis of heart diseases such as heart failure, hypertensive, myocardial infarction and cardiomyopathy and so on. Cardiac fibrosis, a key part of the cardiac remodeling, is tissue excessive deposition of collagen fibers, significant increase of collagen concentration and collagen volume fraction, disorders of the proportion of collagen and disordered arrangement, and is closely related with arrhythmia, cardiac dysfunction and even sudden cardiac death. These changes lead to derangement of cardiac structure, ventricular wall stiffness, resulting in cardiac systolic or diastolic dysfunction and arrhythmias.Renin-angiotensin system (RAS) plays an important role on the development of pathological ventricular remodeling. Intervention targets RAS has become the fundamental treatment about remodeling related diseases. Heart tissue can synthetize almost all of the components of RAS, which is independent from circulating RAS. There are evidences that tissue RAS rather than circulating RAS is a key stimulus of myocardial fibrosis. Angiotensin Ⅱ (Ang Ⅱ) is the major effect factor of the RAS, and angiotensin-converting enzyme (ACE) is a key enzyme of Ang Ⅱ generation. Angiotensin-converting enzyme2(ACE2) is an isozyme of ACE. There is about42%homology between the two in catalytic domain. The major role of ACE2is hydrolysis the octapeptide AngⅡ to the heptapeptide Angiotensin(1-7)[Ang(1-7)]. Ang(1-7), the main effectors of ACE2, through its specific receptor Mas activate or inhibite multiple intracellular signaling pathways, to antagonize the effect of AngⅡ at the level of intracellular signaling molecules. So, ACE2-Ang-(1-7)-Mas axis is the endogenous antagonistic system of ACE-Ang II-AT1axis.The cytoskeleton is an important structure of the eukaryotic cell to maintain its basic form. It provide attachment bracket for intracellular proteins involved in cell division, growth, material transport, migration, contraction, and many other important life activities. Rho kinase, the major downstream effector of the small G protein RhoA, is widely expressed in the organization, and was proved to be important factors regulating the cytoskeleton reorganization. Recent studies have demonstrated that Rho kinase plays an important role in heart disease. Rho kinase activity is positively related with left ventricular remodeling and cardiac pumping function disability in patients with heart failure. Animal experiments showed that Fasudil, a Rho kinase specific inhibitor, can significantly reduce pathological stimulus such as pressure-overload, damage, neuroendocrine ligands and diabetes induced cardiomyocyte hypertrophy, interstitial fibrosis and inflammatory infiltration, and inprove the systolic or diastolic dysfunction.Based on the above background, Rho-kinase is a potential and effective target for treatment of ventricular remodeling, but its mechanism is not yet clearly elucidated. Fasudil is the only specific inhibitor of Rho kinase listed for clinical. This study treated pressure overload rats to observe the effects of Fasudil on cardiac ACE-Ang Ⅱ and ACE2-Ang(1-7), and provide more evidence about Rho kinase as a new clinical target for anti-ventricular remodeling.OBJECTIVETo observe the effects of Fasudil, an inhibitor of Rho kinase, on cardiac ACE-AngⅡ and ACE2-Ang(1-7) in pressure-overload rats, and to explore the role of Rho kinase in myocardial fibrosis.METHODS50male SD rats weighing (180±20) g. Select8rats as sham group according to the random table. The remaining42rats were preparated with abdominal aorta banding, following the methods used by Anversa, etc. Abdominal aortas of sham group were separated out following the procedure described above, but not clipping. All rats were fed under standard condition.4weeks after operation,36rats survived and14dead, there was no dead in sham group. These rats were divided randomly into4groups:Sham (n=8, NS2ml/kg-d i.p.), Model (n=10, NS2ml/kg-d i.p.), Fasudil High-dose group (FH, n=9,30mg/kg-d i.p.) and Fasudil low-dose group (FL, n=9,10mg/kg-d i.p.). At8weeks after surgery, the Heart/Weight Index (HWI) and the Left Ventricle/Weight Index (LVWI) were calculated, and the content of cardiac Hydroxyproline (HYP) was measured using alkaline hydrolysis. Study the myocardial histopathology and interstitial fibrosis with HE and Masson staining. Immunohistochemical analysis was used to measure ACE, ACE2, TGF-β1and CTGF protein levels. RT-PCR was used to detect the expression of ACE2mRNA. Cardiac and plasma AngⅡ and Angiotensin(1-7)[Ang(1-7)] concentration were detected with ELISA.Statistical analysis:The data was analyzed by SPSS13.0statistic software. Values were presented as means±SD (x±s). If variance to be equalized when two sets compared with each other, data were analyzed with independent sampler t test. If variance to be not equalized, data were analyzed with Satterthwaite t test. Comparisons among groups were performed by one way ANOVA analysis. If the variance to be equalized, two sets data were analyzed with LSD test, But if not, it were analyzed with Dunnett’s T3test. p≤0.05was considered significant. RESULTS1. Comparison of cardiac fibrosis in each groupCompared with sham group, the HWI and LVWI in model group were significantly increased, and cardiac HYP content were elevated (p<0.01), suggesting cardiac fibrosis inrats. Compared with Model group, HWI, LVWI and the cardiac HYP content in the FH and FL group have different levels of reduction, the difference was statistically significant (p<0.01or p<0.05).2. Myocardial tissue HE and Masson staining in each group.In sham group, the cardiomyocytes were arranged in neat rows, and cytoplasmic dyeing uniformity, and no focal necrosis. Interstitial collagen fiber hyperplasia is not obvious. Compared with the sham group, the cardiomyocytes in model group were enlargement, disorder, multiple focal necrosis, and inflammatory cells infiltration. In perivascular and interstitium, there was much collagen deposition. Compared with Model group, cardiomyocytes of FH group, similar with sham group, improved significantly. The extent of fibrosis significantly reduced, and had much less collagen deposition; Cardiomyocytes of FL group were also enlargement, disorder, but had few focal necrosis, and small amount of inflammatory cell infiltration. In perivascular and interstitium, there still was collagen deposition, but much less than Model group.3. Comparison of levels of cardiac p-MYPT1in each group.MYPT1is a specificity substrate of Rho kinase. Its phosphorylation level may reflect the activity of Rho kinase. The immunohistochemical staining brown prompted positive expression of p-MYPTl. Brown areas of sham group were little and scattered, and overall lightly stained. Brown areas of model group were significantly more, according with an increase in picture deep dye. Brown areas of FH group significantly reduced, the overall coloring was similar with Sham group. Brown areas of FL group were smaller than the Model group, but overall staining was slightly darker than Sham group. The semi-quantitative analysis showed that, compared with the Sham group, MOD of p-MYPT1in the Model group significantly increased (p<0.01). Compared with Model group, MOD of FH and FL group were significantly lower (p<0.01).4. Comparison of levels of cardiac TGF-β1, CTGF in each group.TGF-β1immunostaining in the Sham group was shallow, and few brown areas. Model group staining was significantly deeper than the Sham group, and brown areas were much more. The FH group had less brown areas than Model group, but overall slightly darker than Sham group. Brown areas and color of FL group were fall in between Model group and FH group. Semi-quantitative analysis showed that the MOD of TGF-β1in Model group was significantly higher than Sham group (p<0.01). MOD of FH and FL group were significantly lower (p<0.01,p<0.05).CTGF staining of model group had more brown areas than Sham group, and overall color significantly deepened; FH group was more lightly stained than in the Model group, and brown areas in FH group were little and scattered. In FL group, size and color of brown areas were between the Model group and the FH group. The semi-quantitative analysis showed that, compared with Sham group, MOD of CTGF were elevated in the Model group (p<0.01). The FH group were similar with the Sham group, and was significantly lower compared with Model group (p<0.01). MOD of FL group were also lower than the Model group (p<0.05).5. Comparison of levels of cardiac ACE in each group.ACE immunostaining in the Sham group was shallow andhad small brown areas. Staining of Model group significantly deepen, and had a significant increase of the brown areas. Brown areas of FH group were significantly reduced compared with the Model group, but the overall color is slightly darker than Sham group. Brown areas and color of the FL group were between the Model group and the FH group. The semi-quantitative analysis showed that, cardiac ACE protein levels in the Model group were significantly higher than Sham group (p<0.01). Compared with the Model group, ACE protein levels in FH group approaching normal levels, while the FL group decreased by35%(p<0.05).6. Comparison of levels of cardiac ACE2in each group.Under the light200times, positive cells were stained brow. There were scattered brown areas in Sham group, and the cells Contour is clear. Compared with Sham group, brown areas of Model group is rare, the slices were lightly stained. Brown areas of the FH group increased significantly compared with the Model group. There were pieces of positive cells, and the slices deeply stained. Brown areas of FL group were also significantly increased compared with the Model group, but the slices stained slightly lighter than the FH group. The semi-quantitative analysis showed that, ACE2protein levels of the Model group were significantly reduced compared with Sham group. Compared with the Model group, Fasudil significantly elevated cardiac ACE2protein levels. Levels of ACE2in the fh group increased by197%, and FL group increased by136%(p<0.01).7. Comparison of cardiac expression of ACE2mRNA in each group.Compared with Sham group, ACE2mRNA expression of the Model group was significantly reduced (p<0.01). Compared with the Model group, Fasudil increased ACE2mRNA expression by181%in FH group and by119%in FL group (p<0.01).8. Comparison of cardiac and plasma Ang Ⅱ, Ang(1-7) in each group.In rat myocardium, Ang Ⅱ levels of Model group were increased, while Ang(1-7) levels were significantly lower (p<0.01), and Ang Ⅱ/Ang(1-7) ratio remarkably increased. Ang Ⅱ levels in FH group were reduced to the normal, while Ang(1-7) levels increased by213%compared with the Model group (p<0.01), and the AngⅡ/Ang (1-7) ratio was reversed and<1. Compared with the Model group, AngⅡ levels in FL group decreased by35%(p<0.05), Ang(1-7) levels raised by76%(p<0.01), and Ang Ⅱ/Ang (1-7) is reduced to the level of Sham group.In rats plasma, plasma Ang II of Model group were significantly higher than Sham group (p<0.01), but the change of plasma Ang(1-7) was not statistically significant, Ang Ⅱ/Ang (1-7) ratio also remarkably increased. Compared with the Model group, Fasudil reduced Ang II levels in FH group by49%, increased Ang(1-7) by106%, and AngⅡ/Ang(1-7) ratio was significantly lower (p<0.01). Compared with the Model group, Ang II in FL group decreased by33%(p<0.01), Ang(1-7) increased43%(p<0.01), while AngⅡ/Ang(1-7) was reduced to the level of Sham group.CONCLUSION1. Pressure overload induced by abdominal aortic coarctation resulted in significant myocardial fibrosis8weeks after the surgery, associated with increase of cardiac ACE and Ang Ⅱ levels, and decrease of ACE2and Ang(1-7).2. Rho kinase was involved in pressure overload-mediated cardiac fibrosis. The activity of Rho kinase was consistent with the levels of Ang Ⅱ, TGF-β1and CTGF.3. Inhibition of Rho kinase activity by Fasudil can significantly improve the pressure overload induced myocardial fibrosis, associated with reduction of cardiac ACE, Ang Ⅱ, TGF-β1and CTGF levels, and increase of cardiac ACE2and Ang(l-7).4. The improvement of cardiac RAS and the reduction of cardiac TGF-β1may contribute to the improvements of pressure overload-induced cardiac fibrosis by Fasudil.