Expression Of Rho/Rho Kinase In Cardiac Muscle Of Pressure Overload Rats With Chronic Heart Failure And Intervention Of Rho Kinase Inhibitor Fasudil

IntroductionChronic heart failure (CHF) is a complex clinical syndrome , which is the most serious stage of cardiac diseases . CHF is a clinical condition associated with alterations in the normal balance of neurohumoral agents and factors that act on the cardiomyocyte . The mechanisms of heart failure are still not well understood now . Cellular mechanisms inducing changes in intracellular signaling pathway activated by these neurohormonal factors have been intensively investigated . Recently , RhoA - a small , monomeric G - protein , and a member of the Rho subfamily of Ras family of G - proteins has been idenitified as the component of a major pathway for inducing hypertrophy and heart failure . Many of the same receptors that activate the phosphatidylinositol cascade also activate RhoA, and with the help of GEFs , dissociate cytosolic RhoA - GDP from GDI . This allows the exchange of GTP for GDP on RhoA . Active RhoA - GTP activates Rho kinase , which then phosphatase the regulatory subunit of myosin phosphatase and participates in its catalytic activity. The present study is investigated the changes of Rho/Rho kinase signaling pathway in the regulatory mechanisms of heart failure and the effects of Rho kinase inhibitor fasudil on cardiac muscle RhoA , Rho kinase mRNA and intracellular calcium ion concentration ( [ Ca 2+ ] i) in the heart failure rat models subsequent to coarctation of ascending aorta.Materials and methods1. Preparation of animal models (induction of ascending aortic stenosis) Immature female Wistar rats weighting 100 - 150g were anaesthetized andmechanically ventilated . Open the cavity of chest and detach and cerclage the ascending aorta at the initiation and set to an outer diameter of 7 mm . In sham -operated animals , the aortic root was shortly detached without cerclage the ascending aorta . 30 female Wistar heart failure rats were divided randomly into three groups ( n = 10). Heart failure group (natrii chloride , 0. 1ml) ; fasudil group(fasudil , 5mg/Kg) , Bid i. p and Nifedipine group (nifedipine , 10mg/ Kg) ,Tid p. o ,20 weeks after coarctation of ascending aorta operation ,10 age - matched sham operation group as control . Treatment time was 4 weeks.2. Hemodynamics studiesAt terminal experiments , animals were anesthetized and the right carotid artery was isolated and cannulated with a microtip catheter transducer for LV pressure measurement , such as left ventricle diastolic end pressure ( LVDEP) , left ventricle systolic pressure (LVSP) and LV pressure maximal rising and declining velocities ( ± dp/dtmax) were collected to evaluate chamber function .3. [ Ca2+ ] i measurement in cardiomyocyteUsing enzyme , it could obtain isolated cardiomyocytes . Cardiomyocytes were loaded with 5umol/L Fura -2/AM for 30min at 37℃ in a humidified incubator with 95% air /5% CO2. Cells were then washed two times with modified Hanks'buffer (include 0. 2% BSA) . They were centrifuged at 1,000g for 3min. Using HITACHI F-3000 fluorescence spectrophotometer , it provided a measure of intracellular calcium ion.4. The expression level of RhoA and Rho kinase mRNA by reverse transcriptase - polymerase chain reaction ( RT - PCR)At the 24 weeks after surgery , all groups of rats were killed and weigh the body , heart and obtain ratio of LV weight to body weight . The part of left ventricle were fireezed in liguid nitrogen and stored at - 80℃ . Total RNA was extracted from whole hearts using a Quick Prep total RNA extraction kit . At theend of the isolation , RNA samples were dissolved in lml diethylpyrocarbonate (DEPC) -treated water (pH7.5) . The optical density (OD) values of each sample were determined spectrophotometrically using visible spectrophotometer at wavelength 260nm ( λ260) . The amount of RNA in each sample was then determined using the following formula : [ RNA ] = ODλ260 dilution factor 40ng/ ml. OD values of RNA samples were also determined at λ280, and the ODλ260/ λ280 ratio was used as a cursory estimation of RNA quality . Formamide / formaldehyde agarose gels were later used to evaluate RNA quality . We decided to use reverse transcriptase - polymerase chain reaction ( RT - PCR) for detection and quantitation of RhoA , Rho kinase andβ - actin mRNA . Primers which used in PCR reactions for amplification and quantitation of mRNA encodeing RhoA and Rho kinase in rat hearts were : RhoA (244bp) sense : 5' - ACC AGT TCC CAG AGG TGT ATG T - 3', antisense: 5' - TTG GGA CAG AAG TGC TTG ACT TC -3'; Rho kinase (512bp) sense:5' -GAG CAA CTA TGA TGT GCC TGA AAA AT - 3', antisense: 5' - GAT GTC GTT TGA TTT CTT CTA C -3'; β-actin(387bp) sense:5' - CGT AAA GAG CTC TAT GCC AA -3'.antisense: 5' -AGC CAT GCC AAA TGT GTC AT-3'.5. Statistical analysisThe measured values were expressed as means ± SD. The gene expression of Rho and Rho kinase mRNA were compared using F test and Student's t -test. A value of P< 0.05 is considered statistically significant.Results1. Hemodynamics studiesAs summarized in Table 1 and 4, at 24 weeks after surgery ,the heart failure model rats were produced successfully . LVDEP was significantly increased (1.73 ±0.04vs0. 50 ± 0.04, P < 0. 01) and LVSP and ± dp/dtmax were significantly decreased (LVSP: 12. 96 ± 0. 96vsl7. 26 ± 1. 00; + dp/dtmax:658. 11 ± 9 . 05vsl 127. 90 ±9. 99 ; - dp/dtmax :529. 68 ±2. 73vs952. 31 ±4. 75 ;P < 0.01) in heart failure group than in sham group ; LVDEP was significantly decreased (0.80 ±0.09vs1.73 ± 0.04, P < 0.01) and LVSP and ± dp/dtmax weresignificantlyincreased(LVSP:15.27 ±0.39vsl2.96 ±0.96; + dp/dtmax:959. 10 ±9.07vs658.11 ±9.05; - dp/dtmax:824. 34 ±2. 78vs529. 68 ±2. 73 ;P < 0. 01) ;in the fasudil group than in heart failure group ; LVDEP was significantly increased in nifedipine group than in heart failure group (1. 83 ±0.07vs1.73 ±0.04,p<0.01).2. LV weight , body weight and the index of LV hypertrophy (LVDI , the ratio of LV weight to body weight)As summarized in Table 2 and 5 , LVHI was significantly increased in heart failure group than in sham group(4.77 ±0.08 vs2.51 ±0. 12,P <0. 01) ; LVHI was significantly decreased in the fasudil group than in heart failure group (4.05 ±0.08vs4.77 ±0.08,P < 0.01) ; LVHI was not significantly changed in the nifedipine group than in heart failure group (4. 81 ±0.05vs4.77 ±0.08, P >0.05).3. [ Ca2+]; measurementAs summarized in Table 5 , [ Ca2+]i was significantly increased in heart failure group than in sham group (475.93 ±28. 22vs79. 25 ±3. 33 ,P <0. 01) ; [ Ca2+ ]i was not significantly changed in the fasudil group than in heart failure group(462. 78 ± 16. 72vs475. 93 ±28.22,P > 0.05) ; [ Ca2+ ]i was significantly decreased in the nifedipine group than in heart failure group (262. 95 ±12.74 vs475.93±28.22,P<0.01).4. The expression levels of RhoA, Rho kinase mRNATable 6 , Fig. 5 and 6 showed the expression level of RhoA mRNA in cardiac muscle of each group rats . The ratio level of RhoA mRNA and β - actin mRNA was significantly higher in heart failure group than in sham group (1.31 ± 0.07vs0.73 ± 0. 06, P < 0. 01) ; the ratio level of RhoA mRNA andβ - actin mRNA was significantly lower in the fasudil group than in heart failure group (0.82 ±0.03vsl. 31 ±0.07 ,P <0.01) ;the ratio level of RhoA mRNA and $ -actin mRNA was not significantly changed in the nifedipine group than in heart failure group (1.27 ±0.03vsl.31 ±0.07,P>0.05) . Table 6 , Fig. 7 and 8 showed the expression level of Rho kinase mRNA in cardiac muscle of each group rats. The ratio level of Rho kinase mRNA and β - actin mRNA was significantly higher in heart failure group than in sham group (1. 05 ±0.03vs0.46 ±...