| Cardiac hypertrophy is the common pathology of a variety of cardiovascular diseases, which is characterized by the increase in cell volume and protein synthesis and fetal gene expression up-regulation. Although hypertrophy is initially a compensatory mechanism that helps sustain cardiac output, prolonged hypertrophy will inevitably give rise to heart failure and subsequent cardiac death. Therefore, it is very important to prevent or reverse cardiac hypertrophy.Cardiac hypertrophy is the consequence of hypertrophic stimulus-induced changes in gene expression, which is linked by intracellular signal transduction. It is likely, however, that there are"molecular phenotype"differences underlying cardiac hypertrophy triggered by different stimuli, which is caused by the different signal pathways that they initiated. Studying on the signaling pathways in cardiomyocyte hypertrophy contributed to elucidate the cellular and molecular mechanisms of cardiac hypertrophy and might find some new strategies for the prevention of cardiac hypertrophy.RhoA/Rho kinase signaling pathway plays an important role in mediating various cellular functions such as contraction, adhesion,motility, proliferation and apoptosis, and all of which are involved in the pathogenesis of cardiovascular diseases such as hypertension, restenosis, atherosclerosis, pulmonary hypertension, cerebral vasospasm, vascular aneurysms, myocardial ischemia reperfusion injury and cardiac hypertrophy. RhoA/Rho kinase signaling pathway plays an important role in cardiovascular diseases. However, it has not been evaluated the roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by isoproterenol (Iso) and diabetic cardiomyopathy induced by streptozotocin (STZ).In this study, we used Rho kinase inhibitor fasudil (Fas) to investigate: (1) Roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by Iso in rats; (2) Roles of RhoA/Rho kinase signaling pathway in cardiomyocyte hypertrophy induced by phenylephrine (PE), Iso and angiotensin II (Ang II); (3) Roles of RhoA/Rho kinase signaling pathway in diabetic cardiomyopathy induced by STZ in rats.Part 1 Roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by Iso in ratsObjective: To investigate the roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by Iso in rats.Methods: Forty male Wistar rats (200-220 g) were supplied by the Experimental Animal Center of Hebei Medical University. Rats were randomly and equally divided into 5 groups (n=8): control group, Iso model group, low-dose Fas group, high-dose Fas group and captopril (Cap) group. Except for control group, cardiac hypertrophy models rats were induced by injection of Iso (5 mg·kg-1·d-1 ih.) for 7 d. At the same time, Fas group and Cap group were treated with Fas (2, 10 mg·kg-1·d-1 ip.) and Cap (30 mg·kg-1·d-1 ig.) respectively; control group and Iso model group were given an equal volume of onormal saline. Twenty-four hours after the treatment, the following indices were determined: (1) Haemodynamic parameters, including heart rate (HR), maximum ascending and descending rate of left ventricular pressure (±dp/dtmax), left-ventricular systolic pressure (LVSP) and left-ventricular end diastolic pressure (LVEDP), were recorded by using MS4000U-1C biological signal quantitative recording and analyzing system; (2) Biochemical indices, including the contents of Ang II and malondialdehyde (MDA), the activities of superoxide dismutase (SOD), creatine kinase (CK) and lactate dehydrogenase (LDH); (3) Heart weight index (HWI); (4) Histopathological examinations: cardiomyocyte diameter (CMD) and collagen volume fraction (CVF) were analyzed; (5) Expressions of RhoA and Rho kinase mRNA were tested by reverse transcription polymerase chain reaction (RT-PCR).Results: 1 Detection of haemodynamic changes: In this experiment, there was a significant increase in LVEDP and significant decrease in LVSP, +dp/dtmax and -dp/dtmax in Iso model group compared with control group. The values of LVEDP increased by 34.3% (P<0.01), LVSP, +dp/dtmax and -dp/dtmax decreased by 16.8% (P<0.01), 16.2% (P<0.01) and 14.4% (P<0.01) respectively in Iso model group compared with control group. Fas (10 mg·kg-1·d-1) and Cap treatment reduced the values of LVEDP by 16.1% (P<0.05) and 28% (P<0.01) respectively from Iso model levels. Fas (10 mg·kg-1·d-1) treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 14.7% (P<0.05), 10.2% (P<0.05) and 9.9% (P<0.05) respectively from Iso model levels. Cap treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 15.8% (P<0.05), 14.4% (P<0.01) and 9.9% (P<0.05) respectively from Iso model levels. Heart rate was not significantly different in all groups (P>0.05).2 Analysis of biochemical indices in blood: The content of Ang II was significantly increased by 34.2% in Iso model group compared with control group (P<0.01). Cap treatment reduced the content of Ang II markedly by 34.8% (P<0.01) from Iso model levels. The content of MDA was significantly increased by 117.0% in Iso model group compared with control group (P<0.01). Fas (2, 10 mg·kg-1·d-1) and Cap treatment reduced the content of MDA by 9.8% (P<0.05), 25.1% (P<0.01) and 28.0% (P<0.01) respectively. The activity of SOD was greatly decreased by 60.4% (P<0.01) in Iso model group compared with control group. Fas (2, 10 mg·kg-1·d-1) and Cap treatment elevated the activity of SOD by 30.7% (P<0.05), 64.8% (P<0.01) and 85.7% (P<0.01) respectively. The activities of LDH and CK were greatly increased by 20.7% (P<0.01), 46.4% (P<0.01) respectively in Iso model group compared with control group. Fas (2, 10 mg·kg-1·d-1) and Cap treatment reduced the activity of LDH by 14.6% (P<0.05), 36.1% (P<0.01) and 42.8% (P<0.01) respectively, and reduced the activity of CK by by 18.3% (P<0.05), 32.2% (P<0.01) and 41.4% (P<0.01) respectively.3 Assessment of HWI:Body weight was not significantly different in all groups at the end of the study (P>0.05). However HWI was significantly increased by 19.2% in Iso model group compared with control group (P<0.01). Fas (2, 10 mg·kg-1·d-1) and Cap treatment reduced HWI by 6.9% (P<0.05), 7.7% (P<0.01) and 9.0% (P<0.01) respectively from Iso model levels.4 Histopathological examinations: The myocardial structure of rats in control group was clearly evident. Myofibrils are the main components of cytoplasm, and they appeared orderly, with bright and dark areas clearly evident. The myocardial structure in Iso model group was characterized by obvious reductions in myofibril content, breaking, and disordered myofilaments. The pathological changes of rats in Fas and Cap groups were slighter than that of Iso model group. CMD and CVF were significantly increased by 16.9% (P<0.01) and 32.9% (P<0.05) in Iso model group compared with control group (P<0.01). Fas (2, 10 mg·kg-1·d-1) and Cap treatment reduced CMD by 8.1% (P<0.05), 12.3% (P<0.01) and 13.5% (P<0.01) respectively from Iso model levels, and reduced CVF by 21.6% (P<0.05) and 26.2% (P<0.05) respectively from Iso model levels.5 Expressions of RhoA and ROCK mRNA:Expression levels of RhoA mRNA and Rho kinase mRNA were both significantly higher in Iso group than that in control group (all P<0.01). Fas (10 mg·kg-1·d-1) and Cap treatment significantly down-regulated expression levels of RhoA mRNA and Rho kinase mRNA (P<0.01 and P<0.05 respectively).Conclusions: RhoA/Rho kinase signaling pathway may play an important role in cardiomyocyte hypertrophy induced byβ-adrenergic receptor stimulation. Rho kinase inhibitor Fas could improve the cardiac dysfunction and the pathological changes in Iso induced cardiac hypertrophy, which is associated with the inhibition of Ang II/Gq/RhoA/Rho kinase signaling pathway and enhancement of antioxidative ability.Part 2 Roles of RhoA/Rho kinase signaling pathway in cardiomyocyte hypertrophy induced by PE, Iso and Ang IIObjective: To investigate the roles of RhoA/Rho kinase signaling pathway in cardiomyocyte hypertrophy induced by PE, Iso and Ang II Methods: Cardiomyocytes were obtained from ventricles of neonatal rats. Bromodeoxyuridine (0.1 mol·L-1) was present to prevent cardiac fibroblast proliferation. Cultured cardiomyocytes were divided into 8 groups: control group, Fas group (Fas 10-5 mol·L-1), Iso group (Iso 10-5 mol·L-1), Iso + Fas group (Iso 10-5 mol·L-1 + Fas 10-5 mol·L-1), Ang II group (Ang II 10-7 mol·L-1), Ang II +Fas group (Ang II 10-7 mol·L-1+Fas 10-5 mol·L-1), PE group(PE 10-5 mol·L-1), PE+Fas group (PE 10 -5mol·L-1+Fas 10-5 mol·L-1).Cell size was obtained by measuring the diameter of trypsinized cell. The protein content, which was measured by the method of Lowry et al, was divided by total cell number to give cell protein concentration (pg/cell). Expressions of RhoA and Rho kinase mRNA were tested by RT-PCR. Expression of c-fos protein was determined with Western blot. Calcineurin (CaN) assessment kit was employed to detect the acitivity of CaN. The acitivity of mitogen-activated protein kinases (MAPK) was measured by method of Tianchang Li.Results:1 Cell size: The diameter of cardiomyocyte was significantly increased in PE, Iso and Ang II groups compared with control group (all P<0.01). Compared with PE group, the diameter of cardiomyocyte was significantly reduced in PE+Fas group (P<0.01). Compared with Ang II group, the diameter of cardiomyocyte was significantly reduced in Ang II+Fas group (P<0.01). There were no significant differences of diameters of cardiomyocyte between Iso and Iso+Fas groups (P>0.05). The single use of Fas had no effect on diameter of cardiomyocyte (P>0.05).2 Cell protein concentration: The cell protein concentration was significantly increased in PE, Iso and Ang II groups compared with control group (all P<0.01). Compared with PE group, the cell protein concentration was significantly reduced in PE+Fas group (P<0.01). Compared with Ang II group, the cell protein concentration was significantly reduced in Ang II+Fas group (P<0.01). There were no significant differences of the cell protein concentration between Iso and Iso+Fas group (P>0.05). The single use of Fas had no effect on the cell protein concentration (P>0.05).3 Expressions of RhoA and Rho kinase mRNA: Expressions of RhoA and Rho kinase mRNA were significantly increased in PE and Ang II groups compared with control group (all P<0.01). Compared with PE group, expressions of RhoA and Rho kinase mRNA were significantly reduced in PE+Fas group (all P<0.01). Compared with Ang II group, expressions of RhoA and Rho kinase mRNA were significantly reduced in Ang II+Fas group (all P<0.01). There were no significant differences among them in control, Iso and Iso+Fas groups (all P>0.05).4 The activity of MAPK: The activity of MAPK was significantly increased in PE, Iso and Ang II groups compared with control group (all P<0.01). There were no significant differences of the activities of MAPK between PE and PE+Fas groups, Iso and Iso+Fas groups, Ang II and Ang II+Fas groups (all P>0.05).5 The activity of CaN: The activity of CaN was significantly increased in PE, Iso and Ang II groups compared with control group (all P<0.01). There were no significant differences of the activities of CaN between PE and PE+Fas groups, Iso and Iso+Fas group, Ang II and Ang II+Fas group (all P>0.05).6 Expression of c-fos protein: Expression of c-fos protein was significantly increased in PE, Iso and Ang II groups compared with control group (all P<0.01). Compared with PE group, expression of c-fos protein was significantly reduced in PE+Fas group (P<0.01). Compared with Ang II group, expression of c-fos protein was significantly reduced in Ang II+Fas group (all P<0.01). There were no significant differences of c-fos protein expression between Iso and Iso+Fas groups (P>0.05).Conclusions: RhoA/Rho kinase signaling pathway may play an important role in the cardiomyocyte hypertrophy induced by PE and Ang II. Fas could improve the cardiomyocyte hypertrophy induced by PE and Ang II by inhibiting expression of c-fos protein. Part 3 Roles of RhoA/Rho kinase signaling pathway in STZ induced diabetic cardiomyopathyObjective: To investigate the roles of RhoA/Rho kinase signaling pathway in STZ induced diabetic cardiomyopathy and whether administration of Fas, a relatively specific Rho kinase inhibitor, would prevent the diabetic cardiomyopathy as well as the mechanism involved.Methods: Fifty male Wistar rats (200-250 g) were supplied by the Experimental Animal Center of Hebei Medical University. Diabetes was induced in rats fasted for 12 h by a single injection of STZ (60 mg·kg-1, dissolved in fresh 0.1 mol·L-1 citrate buffer, pH 4.5) via the caudal vein of animals. Age-matched rats (n=10) were used as controls and were administered with an equivalent volume of citrate buffer alone via the same route. Induction of diabetes was confirmed by measuring blood glucose levels. Blood sample was taken from the caudal vein 1 week after STZ injection and the blood glucose level exceeded 16.7 mmol·L-1 was considered diabetic. After 8 weeks, diabetic rats in good conditions were randomized into 3 groups, diabetic (n=11), Fas (n=11) and Cap (n=10) groups. Rats of Fas and Cap groups were administered with Fas (5 mg·kg-1 bid, ip.) or Cap (15 mg·kg-1 bid, ig.) respectively, while control and diabetic rats were treated with vehicle (saline, 1 ml·kg-1 bid, ip.) for 4 weeks. Blood glucose levels and body weights were then measured biweekly in both the control and diabetic rats and the general characteristics of all rats were observed throughout the treatment. Twenty-four hours after the treatment, the following indices were determined. (1) Haemodynamic parameters, including heart rate, LVSP, LVEDP and±dp/dtmax, were recorded by using MS4000U-1C biological signal quantitative recording and analyzing system; (2) Biochemical indices, including the contents of Ang II and MDA, the activities of SOD, CK and LDH; (3) Heart weight index (HWI); (4) Histopathological examinations: CMD and CVF were analyzed; (5) Expressions of RhoA and Rho kinase mRNA were tested by RT-PCR; (6) Expressions of Bax and Bcl-2 proteins were quantified by Western blot. Results:1 Detection of haemodynamic changes: In this experiment, there was a significant increase in LVEDP and significant decrease in LVSP, +dp/dtmax and -dp/dtmax in STZ model group compared with control group. The values of LVEDP was increased by 75.9% (P<0.01), LVSP, +dp/dtmax and -dp/dtmax were decreased by 23.9% (P<0.01), 25.9% (P<0.01) and 19.7% (P<0.01) respectively in Iso model group compared with control group. Fas and Cap treatment reduced the values of LVEDP by 20.9% (P<0.05), 27.1% (P<0.01) respectively from STZ model levels. Fas treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 17.9% (P<0.05), 13.6% (P<0.05) and 13.9% (P<0.05) respectively from STZ model levels. Cap treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 24.4% (P<0.01), 19.6% (P<0.01) and 16.6% (P<0.05) respectively from STZ model levels. Heart rate was not significantly different in all groups (P>0.05).2 Analysis of biochemical indices in blood: The content of Ang II was significantly increased by 127.3% in STZ model group compared with control group (P<0.01). Cap treatment reduced the content of Ang II by 38.5% (P<0.01) from STZ model levels. The content of MDA was significantly increased by 95.8% in STZ model group compared with control group (P<0.01). Fas and Cap treatment reduced the content of MDA by 14.8% (P<0.01) and 24.3% (P<0.01) respectively. The activity of SOD was greatly decreased by 51.5% (P<0.01) in STZ model group compared with control group. Fas and Cap treatment increased the activity of SOD by 40.5% (P<0.01) and 62.6% (P<0.01) respectively. The activities of LDH and CK were greatly increased by 149.4% (P<0.01) and 149.5% (P<0.01), respectively in STZ model group compared with control group. Fas and Cap treatment reduced the activity of LDH by 45.1% (P<0.01) and 47.1% (P<0.01) respectively; and reduced the activity of CK by 30.9% (P<0.01) and 43.6% (P<0.01) respectively.3 Assessment of HWI: Compared with control group, body weight was significantly decreased in STZ model, Fas and Cap groups at the end of the study (all P<0.01). However HWI was significantly increased by 13.1% in STZ model group compared with control group (P<0.01). Fas and Cap treatment reduced HWI by 6.3% (P<0.05) and 8.7% (P<0.01) respectively from STZ model levels.4 Histopathological examinations: The myocardial structure of rats in control group was clearly evident. Myofibrils are the main components of cytoplasm, and they appeared orderly, with bright and dark areas clearly evident. The myocardial structure in STZ model group was characterized by obvious reductions in myofibril content, breaking and disordered myofilaments. The pathological changes of rats in Fas and Cap groups were slighter than that of STZ model group. CMD and CVF were significantly increased by 18.3% (P<0.01) and 50.9% (P<0.01) in Iso model group compared with control group. Fas and Cap treatment reduced CMD by 8.7% (P<0.05) and 12.0% (P<0.01), and reduced CVF by 19.8% (P<0.05) and 26.9% (P<0.01) respectively from Iso model levels.5 Determination of RhoA and ROCK mRNA expressions:Expressions of RhoA and Rho kinase mRNA were both significantly higher in STZ group than that in control group (all P<0.01). Fas and Cap treatment down-regulated the expression levels of RhoA mRNA (P<0.01 and P<0.05 respectively) and Rho kinase mRNA (P<0.01 and P<0.05 respectively).Conclusions: RhoA/Rho kinase signaling pathway may play a critical role in the development of diabetic cardiomyopathy. Fas could improve the cardiac dysfunction and the pathological changes in heart of the diabetic animals. This may partly attribute to the suppression of caridac hypertrophy, myocardial fibrosis, and subsequent cardiac remodeling, which is associated with the inhibition of RhoA/Rho kinase signaling pathway, enhancement of antioxidative ability and regulation of expressions of apoptosis-related genes.Conclusions1 RhoA/Rho kinase signaling pathway may play an important role in cardiomyocyte hypertrophy induced byβ-adrenergic receptor stimulation. Rho kinase inhibitor Fas could improve the cardiac dysfunction and the pathological changes in Iso-induced cardiac hypertrophy, which is associated with the inhibition of Ang II/Gq/RhoA/Rho kinase signaling pathway and enhancement of antioxidative ability.2 RhoA/Rho kinase signaling pathway may play an important role in the cardiomyocyte hypertrophy induced by PE and Ang II. Fas could improve the cardiomyocyte hypertrophy induced by PE and Ang II by inhibiting the expression of c-fos protein.3 RhoA/Rho kinase signaling pathway may play a critical role in the development of diabetic cardiomyopathy. Fas could improve the cardiac dysfunction and the pathological changes in heart of the diabetic animals. This may partly attribute to the suppression of caridac hypertrophy, myocardial fibrosis, and subsequent cardiac remodeling, which is associated with the inhibition of Ang II/Gq/RhoA/Rho kinase signaling pathway, enhancement of antioxidative ability, and regulation of expressions of apoptosis-related genes.
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