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Role of Rho Kinase Inhibitor in Pressure Overload Induced Cardiac Hypertrophy in Rats

Author: WuYanLing
Tutor: ChuLi
School: Hebei Medical University
Course: Pharmacology
Keywords: Rho kinase Fasudil Pressure overload induced cardiac hypertrophy Fibrosis Haemodynamics Apoptosis
CLC: R96
Type: Master's thesis
Year: 2009
Downloads: 78
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Abstract


Objective: To investigate the role of a Rho kinase (ROCK) inhibitor fasudil (Fas) in pressure overload induced cardiac hypertrophy (POH) in rats and the detailed mechanisms involved.Methods:1 Establishment of animal model and drug treatmentIn current investigation, constriction of super-renal abdominal aorta was employed for the generation of POH model in rats. Sixty male Wistar rats weighing 200~220 g were randomly allocated to five groups, sham, vehicle-treated POH, low-dose Fas (L-Fas, 2 mg/kg/d), high-dose Fas (H-Fas, 10 mg/kg/d) groups and Captopril (30 mg/kg/d) group as positive control (n = 12 in each group). Except for sham animals, rats of the other four groups underwent surgery of abdominal aortic constriction. Rats in sham group received surgery without banding the aorta. Treatment commenced 4 weeks after the surgery when the POH model was successfully established and lasted for 4 weeks.2 Measurement of indicesThe general conditions of all rats were observed carefully throughout the experiment. At the completion of the study, all rats were fasted overnight and the following indices were measured: (1) Six rats in each group were randomly chosen for acute study of haemodynamic parameters; (2) The left six rats in each group were decapitated and blood was obtained to analyze the following biochemical indices: activities of superoxide dismutase (SOD) and nitric oxide synthase (NOS) in serum, levels of malondialdehyde (MDA), nitric oxide (NO) in serum and endothelin-1 (ET-1) in plasma; (3) After obtaining the blood samples, hearts were quickly removed en bloc, washed in order to remove peripheral blood contamination, dried, and heart weight (HW) and left ventricular weight (LVW) in milligrams were measured to calculate heart weight index (HWI) and left ventricular weight index (LVWI); (4) Samples were taken from left ventricle once LVW was obtained and placed in 4% paraformaldehyde. After 24 h in 4% paraformaldehyde, tissues were dehydrated in graded alcohol series, embedded in paraffin and cut into slices. After dewaxed, slices were stained with haematoxylin/eosin (HE) or Masson Trichrome respectively. Slices stained by HE were observed under optical microscope for pathological changes of cardiomyocytes and cardiomyocyte diameter (CMD) was measured; Slices stained by Masson Trichrome were observed under optical microscope for the changes of interstitial myocardial collagen and collagen volume fraction (CVF) was analyzed; (5) Hearts of the left six rats were fixed by infusion with 4% paraformaldehyde, then immunohistochemical method was employed to detect the expression levels of apoptosis-related genes Bax, Bcl-2, c-fos and c-myc in left ventricular tissue, furthermore, ratio of Bcl-2/Bax was also caculated; (6) One portion of left ventricular tissue was taken from left ventricle once LVW was obtained and snap-frozened in liquid nitrogen and stored below–70°C for determination of expressions of RhoA mRNA and ROCK mRNA in rats of all groups by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR).Results:1 General characteristics of animalsThere were no significant differences in conditions of rats in sham group before and after surgery. Compared with the state before surgery, rats receiving constriction of abdominal aorta were all in bad mental state, with less activities and intake of food and water within one week after the surgery; after one week, there were not any obvious improvement of the general conditions of POH rats until the end of the experiment compared with those of sham animals. Whereas, the state of Fas-treated or Captopril-treated rats was much better than that of POH rats. Furthermore, the syndromes of heart failure, such as dyspnea, edema and oliguria, were not shown in POH or Fas-treated rats throughout the experiment.2 Detection of haemodynamic changesAt the end of the study, there were significant increase of left ventricular systolic pressure (LVSP) and left ventricular end diastolic pressure (LVEDP) and pronounced reduction of maximum ascending and descending rates of left ventricular pressure (±dp/dtmax) in POH rats in comparison with those of sham group (P<0.01). However, after treatment with L-Fas, H-Fas, or Captopril, LVSP and LVEDP were both markedly decreased (P<0.01), while±dp/dtmax were dramatically elevated (P<0.01).3 Analysis of biochemical indices in bloodCompared with sham animals, concentration of NO and activities of NOS and SOD of POH group were diminished significantly (P<0.01); nevertheless, concentrations of ET-1 and MDA of this group were elevated dramatically (P<0.01). In comparison with those of vehicle-treated POH rats, content of NO in L-Fas group was elevated while content of ET-1 of this group was diminished dramatically (P<0.01 and P<0.05, respectively); activities of SOD and NOS were raised up to some extent, and concentration of MDA of L-Fas treated rats was reduced to some extent, but there were no significant differences in comparison with those of POH rats; meanwhile, after treated with H-Fas or Captopril, activities of NOS and SOD were also increased, and concentration of NO was increased significantly, furthermore, levels of MDA and ET-1 were reduced dramatically, and they were all restored toward to the values of sham animals (P<0.01).4 Assessments of HWI and LVWIHW, HWI, LVW and LVWI of vehicle-treated POH rats were increased significantly in comparison with those of rats receiving sham operation (P<0.01). However, after treatment with L-Fas, H-Fas or Captoptil, HW was reduced at different degrees (P<0.05, P<0.01, and P<0.01, respectively). Furthermore, LVW, HWI and LVWI of Fas-treated rats and rats of positive control group showed significant reductions in comparison with those of POH rats (P<0.01).5 Histopathological examinationsSections of left ventricle samples stained by HE observed under light microscope exhibited a clear structure of cardiomyocyte and no obvious pathological changes was shown in sham group. However, hypertrophic cardiomyocytes disarranged were shown in POH rats, with significantly thicked myofilament and larger and more round nucleus, compared with sham group. The pathological changes of rats in L-Fas, H-Fas and Captopril group were much slighter than those of POH group. Sections of heart samples stained by Masson Trichrome showed that, the degree of collagen deposition in POH group was much severe than that of sham rats, and enhanced fibrosis was observed in that group, compared with sham animals. While the degrees of interstitial myocardial fibrosis in Fas-treated or Captopril-treated rats were much better than those of POH animals.Compared with those of sham animals, there were pronounced elevation of CMD and CVF in POH rats (P<0.01). However, after L-Fas, H-Fas or Captopril treatment, CMD was reduced at different degrees compared with that of the POH rats (P<0.05, P<0.01 and P<0.01, respectively). Furthermore, CVF of H-Fas and positive control groups were much lower than that of POH rats (P<0.01).6 Expressions of apoptosis-related genesCompared with sham animals, expressions of Bax, c-fos and c-myc in cardiomyocytes were significantly up-regulated in POH rats (P<0.01), while expression of Bcl-2 was down-regulated markedly in that group (P<0.01), with the ratio of Bcl-2/Bax reduced dramatically (P<0.01). Compared with POH group, up-regulated expressions of c-fos was suppressed after L-Fas treatment (P<0.05), furthermore, expression levels of Bax and c-myc in L-Fas group were suppressed and expression of Bcl-2 of that group was enhanced, but there were no significant differences. Expressions of Bax, c-fos and c-myc were down-regulated in H-Fas group and positive control group (P<0.01), and Bcl-2 in the two groups was much higher than that of POH rats (P<0.01), with the ratio of Bcl-2/Bax up-regualated in Fas-treated and Captopril groups at different degrees, comparing to that of POH rats (P<0.05, P<0.01 and P<0.01, respectively).7 Determination of RhoA mRNA and ROCK mRNA expressionsExpression levels of RhoA mRNA and ROCK mRNA were both significantly higher in POH rats than that of sham animals (P<0.01). Nevertheless, expression levels of RhoA mRNA and ROCK mRNA were down-regulated in heart tissues of L-Fas and H-Fas groups at different degrees in comparison with that of POH rats (P<0.05 and P<0.01, respectively). Expression levels of RhoA mRNA and ROCK mRNA of Captopril-treated rats were also dramatically decreased (P<0.01).Conclusion: RhoA/ROCK signaling pathway may play a critical role in the pathogenesis and development of POH, and ROCK inhibitor Fas could effectively prevent the development of POH, and protect the cardiac function of POH rats. This may attribute to:(1) Attenuation of cardiac fibrosis and cardiac remodeling by up-regulation of activity of NOS and content of NO as well as down-regulation of a potential growth factor for cardiomyocytes and hypertrophic factor ET-1;(2) Prevention of development of cardiac hypertrophy by regulation of antioxidative ability of cardiomyocytes of POH rats, resulting from increased activity of SOD with decreased content of MDA followed;(3) Decrease of HWI and LVWI, inhibition of cardiac hypertrophy and collagen synthesis, with subsequent improvement of cardiac fibrosis and cardiac remodeling;(4) Suppression of expressions of apoptosis promotor, such as Bax, c-fos and c-myc, and enhancement of expression of Bcl-2, an apoptosis inhibitor, with elevated ratio of Bcl-2/Bax followed, resulting in the inhibition of cellular apoptosis and regulations of signaling pathway transducted by these proto-oncogenes, both of which would prevent the development of cardiac hypertrophy and cardiac fibrosis.

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