Effects Of Fasudil On Cardiac Hypertrophy And Heart Failure In Rats: Roles Of RhoA/ROCK Signaling Pathway

Recent cellular and molecular biology studies have indicated an important role of the RhoA/Rho kinase (ROCK) cascade in many aspects of cardiovascular function such as cardiomyocyte apoptosis, cardiac hypertrophy and ventricular remodeling following myocardial infarction. ROCK, the first Rho effector to be described, is a serine/threonine kinase that is important in fundamental processes of cell migration, cell proliferation and cell survival. There are two isoforms of ROCK, known as ROCKⅠandⅡ. ROCKⅠshows the highest expression level in non-neuronal tissues, whereas ROCKⅡis preferentially expressed in the brain. Inhibition of the RhoA/ROCK signaling pathway may be a suitable target for a number of cardiovascular diseases including hypertension, atherosclerosis, diabetes and hypertrophic heart failure.The knowledge of the involvement of ROCKs in the cardiovascular system came mostly from studies utilizing pharmacological inhibitors. Fasudil, a ROCK inhibitor, was shown to decrease ischemia-reperfusion injury, infarct size and myocardial fibrosis in response to experimental myocardial infarction and in a rat model of chronic hypertension induced congestive heart failure. Moreover, in clinical practice, fasudil treatment significantly ameliorated pacing-induced myocardial ischemia in patients with effort angina, indicating involvement of the ROCK pathway in the pathogenesis of cardiovascular disease in humans. Fasudil did not affect heart rate or blood pressure, and was well tolerated. Based on the above information, we investigated the effect of fasudil on cardiovascular disease and the mechanisms involved, to explore the new potential application of fasudil in the treatment of cardiovascular disease. In this study, we use fasudil investigated:(1) RhoA/ROCK may play an important role in adriamycin (ADR)-induced cardiomyopathy; (2) RhoA/ROCK mediates isoproterenol-induced heart failure in rats via JNK and ERK1/2 pathways; (3) RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.Part 1 RhoA/ROCK signaling pathway may play an important role in adriamycin-induced cardiomyopathyObjective:To investigate the role of RhoA/ROCK signaling pathway in cardiomyopathy induced by ADR in rats and the inhibitory effect of fasudil on this pathway.Methods:60 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. The rats were divided into five groups of twelve animals in each group as follows:control (CONT), ADR alone- (ADR), captopril+ADR (CAP+ADR), low-dose fasudil+ADR (FASL+ADR), high-dose fasudil+ADR (FASH+ADR). All Drugs were given as follows: low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously. Captopril (30 mg/kg/day) were orally administered. Fasudil and captopril were administered over a period of 6 days. ADR (10 mg/kg) was injected intraperitoneally on the 4th day. The CONT animals received an injection of the equivalent volume of saline. The rats given ADR administration were all alive. All evaluations were performed 24 h after the last fasudil and captopril administration.The indices as follow:1 Haemodynamic parameters, including heart rate(HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and the maximum ascending and descending rate of left ventricular pressure (maximum rate of pressure development+dp/dtmax and maximum rate of pressure decrease-dp/dtmax)2 Histopathological examinations, including hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM)3 Measurement of plasma LDH and CPK levels 4 Reverse transcription polymerase chain reaction (RT-PCR):Total RNA was extracted from fresh-frozen myocardium using the Trizol reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.5 Western Blotting, The expressions of bax, bcl-2 andβ-actin protein were determined by Western Blotting.6 A colorimetric non-radioactive NF-κB p65 transcription factor ELISA Assay, was to determine NF-κB activation.Results:1 The effects of fasudil on hemodynamic variables of ADR-induced heart failureTreatments of rats with a single dose of ADR (10 mg/kg) caused significant increases (p<0.05) in HR (from 327.5±30.4 to 408.7±34.8bpm, p<0.05) and LVEDP (from 6.7±1.64 to 18.6±5.15 mmHg, p<0.05) compared to the control values. On the other hand, ADR caused significant decreases in LVSP (from 144.6±26.4 to 99.5±21.7 mmHg,p<0.05) and±dp/dtmax compared to the control values (+dp/dtmax from 6865±1451 to 4509±1524 mmHg/s,p<0.05;-dp/dtmax from 6024±1297 to 3890±813 mmHg/s, p<0.05). LVSP and±dp/dtmax are sensitive to changes in preload and afterload. Haemodynamic parameters changes indicated left ventricular dysfunction in ADR rats. Treatment of ADR-injected rats with fasudil for 6 days markedly normalized the heart function. Both systolic and diastolic function were significantly enhanced, and HR (from 408.7±34.8 to 338.4±45.3 bpm, p<0.05), LVSP (from 99.5±21.7 to 114.8±16.5 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4509±1524 to 5937±1195 mmHg/s, p<0.01;-dp/dtmax from 3890±813 to 5104±1380 mmHg/s, p<0.05) and LVEDP (from 18.6±5.15 to 14.8±3.21 mmHg, p<0.05) were all markedly improved in the FASL+ ADR group compared with the ADR group. HR (from 408.7±34.8 to 336.2±33.2 bpm,p<0.05), LVSP (from 99.5±21.7 to 122.4±21.3 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4509±1524 to 6158±1421 mmHg/s, p<0.05;-dp/dtmax from 3890±813 to 5545±1050 mmHg/s,p<0.05) and LVEDP (from 18.6±5.15 to 12.1±2.82 mmHg, p<0.05) were all markedly improved in the FASH+ADR group compared with the ADR group. 2 The effects of fasudil on heart histopathologyNormal cardiac myocytes were regularly shaped cylinders, comprised the Z lines with diads, sarcomeres, elliptical nucleus, numerous mitochondria and prominent myofilaments as shown in the CONT group. The early manifestations of myocardial necrosis, such as wavy fibers, myocyte vacuolization, mitochondrial swelling, thinning of the Z lines, and loss of myofibrils were detected for rat hearts in the ADR group. The vacuolization of the cytoplasm of the LV was significantly higher in ADR-treated rats (about 31.09%). However, the progressive LV vacuolization was attenuated by fasudil and the effect was in a dose dependent manner (about 24.95% in FASL +ADR and 18.51% in FASH+ADR).3 The effects of fasudil on the serum LDH and CPKThe assessment of serum LDH and CPK activities showed that the administration of ADR significantly increased its activity levels in rat serum compared with control (p<0.01). Treatment of rats with fasudil or captopril, compared to ADR group, markedly lowered the serum LDH and CPK (p< 0.05 or p<0.01). The protective effects obtained by fasudil administration, however, were not complete and did not reach those of the control group4 The effects of fasudil on LV mRNA expressions of ROCKⅠ, c-fos, c-jun, and c-FLIPLAgarose gels with representative PCR products for ROCKⅠ, c-fos, c-jun, and c-FLIPL are depicted. The level of ROCKⅠmRNA showed a significant increase in ADR group compared with CONT group (p<0.01), whereas the mRNA level of ROCKⅠremarkably decreased in CAP+ADR, FASL+ADR and FASH+ADR groups (p<0.01). The c-fos/GAPDH and c-jun/GAPDH mRNA levels in the LV significantly increased in ADR group compared with CONT group (p<0.01), whereas the c-FLIPL/GAPDH mRNA levels were significantly reduced. The level of c-fos mRNA expression in CAP+ADR group was significantly decreased (p<0.01) relative to ADR group whereas there was no statistical difference in both FASL+ADR and FASH+ADR groups. In CAP+ADR group, c-jun mRNA expression was significantly reduced as well as in both FASL+ADR and FASH+ADR groups (p<0.01). But c-FLIPL mRNA expression in CAP+ADR group was higher than in ADR group. In FASL+ADR and FASH+ADR groups, c-FLIPL mRNA expressions were significantly increased (p<0.05).5 The effects of fasudil on cardiac muscle tissue protein expressions of bax and bcl-2In the LV from ADR group, a dramatic decrease of bcl-2 compared with the CONT group was detected (p<0.01). On the other hand, bax expression in ADR group significantly increased compared with the CONT group (p<0.01). After captopril was administrated, bax expression decreased (p<0.01), but bcl-2 expression showed a significant increase (p<0.05). Meanwhile, fasudil significantly enhanced bcl-2 expressions and reduced bax expressions both in FASL+ADR group (p<0.01) and FASH+ADR group (p<0.01). Because the ratio of bax and bcl-2 expression is the determining factor for the induction of apoptosis, we found that the ratio of bax/bcl-2 protein expression was 7 fold increase in the ADR group (743% of the control, p<0.01), and the ratio of bax/bcl-2 protein expression was dose-dependently decrease after fasudil treatment (p<0.01).6 The effects of fasudil on cardiac muscle tissue NF-κB activityThe CONT group showed a constitutive activation of NF-κB. Treatment with ADR induced a 4 fold increase in NF-κB activation (417% of the CONT group, p<0.01), which was significantly attenuated by captopril, low-dose fasudil or high-dose fasudil (p<0.01).Conclusions:In summary, the present study demonstrated that the RhoA/ROCK signaling pathway plays an important role in the progression of heart failure induced by adriamycin (Fig.6). Furthermore, fasudil is capable of suppressing ADR-induced cardiomyopathy, which is associated with inhibition of bax and NF-κB activation, AP-1 expression and up-regulation of c-FLIP (L) and bcl-2 expressions. These results suggested that fasudil might have therapeutic potential through suppressing RhoA/ROCK dependent cardiocyte apoptosis and preserving LV function in the stressed heart. The study will provide further insight into the molecular mechanisms underlying the beneficial effects of fasudil on heart diseases.Part 2 RhoA/ROCK mediates isoproterenol-induced heart failure in rats via JNK and ERK1/2 pathwaysObjective:To investigate the roles of crosstalk between the ROCK, JNK and ERK1/2 signaling pathways signaling pathway in heart failure induced by ISO in rats, and clarify the possible molecular mechanisms underlying the beneficial effects of fasudil against heart failure.Methods:48 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. The rats were divided into four groups of twelve animals in each group as follows:control (CONT), ISO alone- (ISO), low-dose fasudil treatment+ISO (FASL+ISO) or high-dose fasudil treatment +ISO (FASH+ISO) groups. All Drugs were given as follows:low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously. Fasudil was administered over a period of 7 days. ISO was administered once daily by intraperitoneal injection at 5 mg/kg/day for 7 days. The CONT animals received an injection of the equivalent volume of saline. The rats given ISO administration were all alive after 7 days. All evaluations were performed 24 h after the last ISO administration.The indices as follow:1 Heart weight indexs, including body weight (BW), heart weight (HW), Heart weight to-body weight ratio (HW/BW), left ventricular wet weight (LV WW)-to-body weight ratio (LV WW/BW), left ventricular dry weight (LV DW)-to-body weight ratio (LV DW/BW);2 Histological analysis, including HE staining and masson's trichrome staining protocol;3 Haemodynamic parameters, including HR, LVSP, LVEDP and±dp/dt; 4 Electrocardiography:the leadⅡof a standard body surface electrocardiogram (ECG) is easily reproducible approach by capturing the whole cardiac cycle using a RM6240C Multi-channel physiological signal acquisition and processing system;5 Western blotting, the expressions of myosin phosphatase target subunit-1 (MYPT-1), p-MYPT-1, The extracellular signal-regulated kinases 1/2 (ERK1/2), p-ERK1/2, the the c-jun NH 2-terminal kinase (JNK), p-JNK, c-FLIPL protein were determined by Western Blotting;6 RT-PCR:Total RNA was extracted from fresh-frozen myocardium using the Trizol Reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.Results:1 Effect of fasudil on the heart weight parameters'alterations of ISO-induced heart failureHW/BW was increased by about 41.53% in ISO group as compared to CONT group (ISO:4.26±0.14 versus CONT:3.01±0.04,p<0.01). Moreover, LV WW/BW was increased to 127.60%(ISO:3.19±0.06 versus CONT:2.50±0.28,p<0.05) and LV DW/BW was increased to 138.18%(ISO:0.76±0.01 versus CONT:0.55±0.07,p<0.05). After being administrated with fasudil, HW/BW was significantly reduced in FASL+ISO group (FASL+ISO:3.77±0.10 versus ISO:4.26±0.14, p<0.05) and FASH+ISO group (FASH+ISO: 3.44±0.08 versus ISO:4.26±0.14, p<0.01). WW/BW of LV was significantly reduced by 15.05% in FASL group (FASL+ISO:2.7±0.16 versus ISO:3.19±0.06,p<0.05) and 28.84% in FASH group (FASH+ISO: 2.27±0.02 versus ISO:3.19±0.06, p<0.01) as compared to ISO group. DW/BW of LV showed a similar tendency as LV WW/BW (FASL+ISO:0.63±0.06 versus ISO:0.76±0.01,p<0.05; FASH+ISO:0.60±0.01 versus ISO: 0.76±0.01,p<0.01).2 Effect of fasudil on the morphological alterations of ISO-induced heart failureHE was for morphological analysis and Masson's stain was for fibrosis HE was for morphological analysis and Masson's stain was for fibrosis analysis. Treatment with ISO resulted in marked myocyte loss and increased fibrosis (p<0.01), primarily limited to the subendocardium of the LV free wall and septum. The collagen fraction was determined by measuring the area of blue-stained tissue within a given field with Image-Pro Plus software. The stained area was calculated as a percentage of the total area within an image. The blue-appearing collagen of the LV endocardium was significantly higher in ISO-treated rats (about 84.56%). However, the progressive LV fibrosis was attenuated by fasudil and the effect was in a dose dependent manner (about 43.79% in FASL+ISO and 20.01% in FASH+ISO).3 Effect of fasudil on hemodynamic variables of ISO-induced heart failureTreatments of rats with a single dose of ISO (5 mg/kg/day) caused significant increases in HR (from 343±9.6 to 424±8.2bpm, p<0.01) and LVEDP compared to the control value (from 4.77±0.38 to 15.37±0.49 mmHg, p<0.01), indicating LV haemodynamic overload. On the other hand, ISO caused significant decreases in LVSP (from 143±5.8 to 98±8.2 mmHg, p<0.01) and±dp/dtmax compared to the control values (+dp/dtmax from 8311±437 to 4339±232 mmHg/s,p<0.01;-dp/dtmax from 7116±272 to 3623±598 mmHg/s, p<0.01). LVSP and±dp/dtmax are sensitive to changes in preload and afterload. Haemodynamic parameters changes indicated left ventricular dysfunction in ISO rats. Treatment of ISO-injected rats with fasudil for 7 days markedly normalized the heart function. Both systolic and diastolic function were significantly enhanced, and LVSP (from 98±8.2 to 126±6.7 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4339±232 to 5787±246 mmHg/s,p<0.01;-dp/dtmax from 3623±598 to 5130±1344 mmHg/s, p<0.05) and LVEDP (from 15.37±0.49 to 11.73±1.31 mmHg,p<0.05) were all markedly improved in the FASL+ISO group compared with the ISO group. LVSP (from 98±8.2 to 140±8.0 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4339±232 to 7861±324 mmHg/s,p<0.01;-dp/dtmax from 3623±598 to 6660±334 mmHg/s,p<0.01) and LVEDP (from 15.37±0.49 to 7.60±0.33 mmHg, p<0.01) were all markedly improved in the FASH+ISO group compared with the ISO group.4 Effect of fasudil on the ECG alterations of ISO-induced heart failureThe result shows the standard ECGs traces of leadⅡfrom the CONT rats during baseline, ST segment depression with a negative T-wave and typical arrhythmias in the ISO group. Treatment of ISO-injected rats with fasudil for 7 days ameliorated the heart dysfunction.5 Effect of fasudil on ISO-mediated ROCK activationTotal RNA was extracted from the LVs of the CONT, ISO, FASL+ISO and FASH+ISO groups. ISO caused a significant up-regulation of ROCK I mRNA expression compared with the CONT group (p<0.01), and treatment with fasudil resulted in a significant reduction in ROCKⅠmRNA expression (p<0.01). We tested the activation of RhoA/ROCK pathway by the amount of phosphorylated MYPT-1. ISO caused phosphorylation of MYPT-1(p<0.01), which was inhibited by fasudil in a dose-dependent manner (p<0.05 or p<0.01).6 Fasudil reduces ISO-induced activation of ERKWe determined the effect of fasudil on the phosphorylation status of ERK in the nucleus as it may participate in ISO-induced heart failure. ISO increased nuclear translocation of ERK (p<0.01), while fasudil reduced this ISO-induced p-ERK to various degrees (p<0.01).7 Fasudil attenuates ISO-induced JNK activationThe effect of fasudil on ISO-induced JNK activation was observed. ISO increased the activities of JNK phosphorylation to 189.24%(p<0.01). Compared with ISO group, fasudil reduced JNK phosphorylation by about 24.71%(p<0.01) at low dose and 43.37%(p<0.01) at high dose.8 Effect of fasudil on c-fos and c-jun mRNA expressionsWhen the rats were administrated by ISO, the expressions of c-fos and c-jun mRNA quickly increased, reaching about 252.92%(p<0.01) and 247.45%(p<0.01) respectively compared with CONT group. The increases of c-fos and c-jun mRNA expressions by ISO were inhibited by high-dose fasudil by approximately 30.84%(p<0.01) and 41.25%(p<0.01), respectively. There were no significant decreases in the c-fos and c-jun mRNA expressions in low-dose fasudil administrated rats.9 Effect of fasudil on c-FLIPL mRNA and protein expressionsBoth the mRNA and protein expressions of c-FLIPL were determined. The c-FLIPL mRNA expression decreased in the ISO group. By contrast, treatment with fasudil resulted in a significant increase (p<0.01). Simultaneously, the c-FLIPL protein levels changed similarly to that of the c-FLIPL mRNA.Conclusions:RhoA/ROCK is activated by ISO and its activation is essential for heart failure induced by ISO. Furthermore, fasudil is capable of suppressing ISO-induced heart failure, which is associated with inhibition of JNK activation, ERK translocation to the nucleus, subsequent AP-1 (c-fos and c-jun) expression and up-regulation of c-FLIPL expression.Part 3 RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.Objective:To further investigate the mechanism of cardiovascular dysfunction in the hyperthyroid condition, the role of RhoA/ROCK signaling pathway and effects of fasudil were examined in rats treated with L-thyroxine (T4).Methods:48 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. Rats were randomly assigned to one of four groups: control (CONT), thyroxine (T4), low-dose fasudil+T4 (FASL+T4), and high-dose fasudil+T4 (FASH+T4). All Drugs were given as follows: L-Thyroxine (T4) was dissolved in 99% ethanol by adding a small volume (20μl) of 25% NaOH and diluted 33 times by adding 0.9% NaCl to obtain a stock solution of 1 mg/ml. The solution was made in 0.9% NaCl to obtain a concentration of 50μg T4/ml. T4 in daily doses of 0.25mg/kg body weight was given subcutaneously once daily for 14 days. This treatment results in a long-term moderate hyperthyroidism. The control rats were treated with subcutaneous injections of normal saline given once daily for 14 days. Low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously at the same time. The rats given T4 administration were all alive. All evaluations were performed 24 h after the last fasudil and T4 administration.The indices as follow:1 Measurement of cardiac hypertrophyCardiac hypertrophy was assessed by the measurement of HW (in mg), BW (in g) and HW/BW (in mg/g).2 Hormone concentrationsRats were anaesthetized, and then the blood samples (4 ml) were collected for the analysis of plasma T4 levels. Following centrifugation (3500 g), plasma was separated and stored at -70℃. Total plasma T4 levels were determined by radioimmunoassay according to procedures of the kits.3 Measurement of mechanical functionA miniature pressure transducer (Millar Micro-Tip) was inserted into the LV via the right carotid artery. The HR, LVEDP, LVSP, and±dp/dtmax were monitored by using MS4000U-1C biological signal quantitative recording and analyzing system.4 Histology analysisThe LV was removed and fixed, and routinely embedded. Paraffin sections were cut at 5μm thickness and mounted on glass slides. Slides were routinely stained with hematoxylin and eosin for histopathologic review.5 TUNEL assaysTUNEL assays were performed with the use of 3,3'-diaminobenzidine (DAB). Photographs were taken with the use of a microscope. The numbers of TUNEL-positive cardiac myocyte nuclei were quantified using Image Pro Plus software (Media Cybernetics, Silver Spring, MD). The chosen fields of view represented areas of heaviest TUNEL staining.6 Western blotting The expressions of bax, bcl-2 and p-MYPT-1 protein were determined by Western Blotting.7 RNA extraction and RT-PCR analysisTotal RNA was extracted from fresh-frozen myocardium using the Trizol Reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.Results:1 Heart weightsT4 induced a significantly increased HW/BW ratio (T4:4.33±0.13 mg/g vs. CONT:3.12±0.15 mg/g, n=6 each; p<0.01) accompanied by a significantly decreased body weight than that seen in the CONT (T4:191.2±3 g vs. CONT:229.4±5 g, n= 6 each; p<0.01). Combined treatment by T4 and low-dose fasudil (4.07±0.15 mg/g vs.4.33±0.13 mg/g, n=6;p<0.05), T4 and high-dose fasudil (3.92±0.09 mg/g vs. 4.33±0.13 mg/g, n=6;p<0.01) showed significantly lower HW/BW ratios than that of T4。2 Thyroid state of T4-treated animalsRats treated with T4 for 14 days had a serum T4 level 4 times higher than that seen in the control (euthyroid) rats (120±13 nmol/l vs. 38±3 nmol/l, n=6 each; p<0.01). Combined treatment by T4 and low-dose fasudil (118±14 nmol/l vs. 120±13 nmol/l, n= 6; p<0.05), T4 and high-dose fasudil (124±17 nmol/l vs. 120±13 nmol/l, n= 6; p<0.01) resulted in T4 concentrations not differing from T4 treated group values.3 Measurement of mechanical functionT4 depressed left ventricular±dp/dtmax by～1/3 (from 6856±524 to 4489±371 and 6019±357 to 3890±312 mmHg/s for +dp/dtmax and -dp/dtmax, respectively) and LVSP by 28%(from 142.2±22.4 to 102.8±21.4 mmHg). T4 increased HR by 14%(from 357.5±10.4 to 408.7±34.8 bpm) and LVEDP by nearly 3 times (from 6.7±0.58 to 17.6±1.15). FASL+T4 and FASH+T4 groups demonstrated return toward normal HR, LVEDP, LVSP,+dp/dtmax and-dp/dtmax (for FASL+T4 and FASH+T4 groups, HR decreased to 338.4±43.8 and 330.2±32.2 bpm, LVEDP decreased to 14.7±2.01 and 12.1±1.68 mmHg, LVSP increased to 109.4±10.3 and 127.2±19.7 mmHg,+dp/dtmax increased to 5887±393 and 6162±553 mmHg/s,-dp/dtmax increased to 5104±380 and 5545±437 mmHg, respectively).4 Myocardial histology analysisMyocardial hypertrophy with large nuclei, severe myofiber disorganization, and interstitial fibrosis was found in T4 group. T4 induced cardiomyocyte hypertrophy was significantly attenuated by fasudil even though cardiomyocyte areas from the FASL+T4 group remained significantly greater that those of CONT. Cardiomyocyte hypertrophy was significantly attenuated in FASH+T4 group.5 Apoptotic changes in the LVsBecause HW/BWs were observed to significantly increase in the T4 group compared to the CONT group, the possible involvement of apoptosis was examined. Apoptosis was evaluated within the LV tissue by the TUNEL method. The number of TUNEL-positive cells per microscopic field was significantly increased in the T4 group compared in the CONT group. Fasudil caused a decrease in apoptosis in the cardiac tissue, more significantly at the higher dose, compared to the T4 group6 ROCK activationIn left ventricle mRNAs of ROCKⅠand GAPDH were detected by RT-PCR analysis. The expression of ROCKⅠmRNA was significantly up-regulated by nearly 2 times in T4 group compared with CONT group (p<0.01). Compared with T4 group, the mRNA level of ROCKⅠremarkably decreased in FASL+T4 and FASH+T4 groups (p<0.01). Consistent with RT-PCR observations, p-MYPT-1 expression was significantly higher in T4-treated rats and remarkably decreased in FASL+T4 and FASH+T4 groups.7 The effects of fasudil on LV mRNA expressions of c-fos, c-jun, and c-FLIPLAgarose gels with representative PCR products for c-fos, c-jun, and c-FLIPL are depicted. The c-fos/GAPDH and c-jun/GAPDH mRNA levels in the LV significantly increased in T4 group compared with CONT (p<0.01), whereas the c-FLIPL/GAPDH mRNA levels significantly reduced (p<0.01). The level of c-fos/c-jun mRNA expressions in FASL+T4 and FASH+T4 groups significantly decreased (p<0.01) relative to T4 group. c-FLIPL mRNA expressions in FASL+T4 and FASH+T4 groups significantly increased (p<0.01).8 Bax and bcl-2 expressionsThe left ventricle bax expression of T4 group was 5 times higher than the CONT group. Bcl-2 expression in T4 group significantly decreased compared with the CONT group (p<0.01). Accompany with fasudil administrated, bax expression significantly decreased (p<0.01) on dose-dependent manner. Meanwhile, high-dose fasudil increased the expression of bcl-2 (p<0.01). Low-dose fasudil treated group showed no significant vary on bcl-2 expression.Conclusions:this study demonstrated that T4 enhanced cardiac hypertrophy by concomitant enhancement of ROCK and AP1 activity and reduction of c-FLIPL expression. It also confirmed that this response can be inhibited by fasudil in dose dependent manner.1 RhoA/ROCK is activated by ADR and its activation is essential for ADR-induced cardiomyopathy. Furthermore, fasudil is capable of suppressing ADR-induced cardiomyopathy, which is associated with inhibition of bax and NF-κB activations, subsequent AP-1(c-fos and c-jun) expression and up-regulation of c-FLIPL expression.2 RhoA/ROCK is activated by ISO and its activation is essential for heart failure induced by ISO. Furthermore, fasudil is capable of suppressing ISO-induced heart failure, which is associated with inhibition of JNK activation, ERK translocation to the nucleus, subsequent AP-1(c-fos and c-jun) expression and up-regulation of c-FLIPL expression.3 T4 enhanced cardiac hypertrophy by concomitant enhancement of ROCK and API activity and reduction of c-FLIPL expression. It also confirmed that this response can be inhibited by fasudil.In summary, the present studies demonstrated that RhoA/ROCK is activated by heart failure and cardiac hypertrophy in different experimental model of rats, fasudil has beneficial effects on hypertrophic heart failure by suppressing the pathological processes.