L-arginine Attenuates Pressure Overload Induced Cardiac Hypertrophy Via The TGF-Beta/Smad Signaling Pathway

Objective: To investigate the effect of L-arginie on cardiac hypertrophy model induced by pressure overload in rats and elucidates its behavior in regulating the TGF-beta /Smad signal pathway.Material and Methods: The model of pressure overload cardiac hypertrophy in rats was established with proper abdominal aortic constriction,according to the survival rate.The experimental animals were allocated into 4 groups,operation group,sham operation group,operation+L-Arg group and operation +L-Arg+ N-nitro-L-arginine methyl ester(L-NAME)group.(1)Sham operation group: abdominal aorta of rat was dissociated only with no constriction;(2)Operation group: abdominal aorta of rat was partially constricted in accordance with the above method;(3)Operation+L-Arg group: abdominal aorta of rat was partially constricted in accordance with the above method,with that rats were daily fed with L-Arg solution(600mg/kg/day);(4)Operation+L-Arg+L-LAME group: abdominal aorta of rat was partially constricted in accordance with the above method,with that rats were daily fed with L-Arg(600mg/kg/day)and L-LAME(50 mg/kg/day)solution.Six weeks later,all animals were sacrificed.The mean of arterial pressure(MABP),left ventricule mass index(LVMI),left ventricular wall thickness(LVWT),myocardial cell diameter(DCM)were measured.HE and Masson staining for histological evaluation of myocardium tissue were used for cardiomyocte morphological observation and interstitial collagen deposition test to all 4 groups of animal.The myocardial atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)were detected by ELISA,myocardial alpha and beta–myosin heavy chain(MHC)and TGF beta1 and typeⅠ,Ⅲ collagen and their m RNA were measured by Western blotting and Real-time PCR respectively;Cardiomyocytes were harvested by Langerndorf apparatus via isolated perfusion method and adherent cultured in vitro.The nucleus expressions of Smad2/3 were observed by immunofluorescence staining.Results: The survival rate of myocardial hypertrophy induced by modified abdominal aortic constriction approach was higher than traditional approach.Compared with sham-operation group,MABP,LVMI,LVWT and DCM of operation group were significantly increased(p<0.05).By HE staining,the myocardial cell diameter and the nuclei enlarged significantly with obvious nuclear deep staining in operation group and operation+L-Arg+L-NAME group versus sham-operation group.By Masson staining,the myocardial cells were found arranged in disorder,collagen fibers accumulated significantly in operation and operation +L-Arg+L-NAME group compared with the sham-operation and operation+L-Arg group.The concentrations of ANP and BNP in myocardial tissue were detected by ELISA.The level of ANP and BNP in operation group were significantly higher than those in sham operated group;(P<0.05).The ANP and BNP level in operation+L-Arg group were significantly lower than that in the operation group(P<0.05).The level of ANP and BNP in operation+L-Arg+ L-NAME group was higher than that in operation+L-Arg group,but there was no significant difference between two groups(P>0.05).The expression levels of beta-MHC in m RNA or protein were detected by Real-time PCR and Western blotting respectively.These measurements were significantly higher in operation and operation +L-Arg+L-NAME group than those in the other two groups.The expression of type I collagen was not significantly different among these different groups(p>0.05),while there are significant differences in expression of type III collagen in both of m RNA and protein levels.The myocardial collagen I/III ratio decreased significantly in the operation group than that in sham operation group(p<0.05).The myocardial collagen I/III ratio in operation+L-arginine group was significantly increased than that in operation group.The ratio was not significantly different between the operation and the operation +L-Arg+NAME group(p>0.05).The level of TGF-beta1 in myocardial tissue of operation group was significantly higher than that in the sham operation group.With L-arginine administration,TGF-beta1 significantly decreased than that in the operation group(without L-arginine administration).With nitric oxide synthase(NOS)inhibitor L-NAME,the level of TGF-beta1 was higher than that in operation+L-Arg group,but still lower than that in the operation group.Immunofluorescence staining results showed that Smad2/3 nuclear aggregation occurred in the operation and the operation+L-Arg+NAME group,while the Smad2/3 nuclear aggregation were not apparent in the other two groups.Conclusion: The rat model of pressure overload-induced myocardial hypertrophy is successfully established by modified surgical approach,and it has obvious collagen deposition in cardiac tissue.The expressions of ANP,BNP,beta-MHC and TGF-beta1 were increased in its myocardial tissue,accompanied by nuclear translocation of Smad2/3 complex,while L-arginine could partially attenuate these above-mentioned effects.L-NAME,as one NOS inhibitor could partially counteract the beneficial effect of L-arginine on myocardial hypertrophy.The effect of L-arginine on cardiac hypertrophy and fibrosis may be regulated by TGF-beta1/Smad signal pathway,whereas detailed mechanism of this specific effect calls for further investigation in the future.