Effect And Mechanism Of ACE2 Agonist On Left Ventricular Remodeling In Rats Induced By High Salt

Objective:To investigate the effect of angiotensin-converting enzyme2 agonist on high salt-induced left ventricular remodeling and its mechanism in ratsMethods:（1）36 male Sprague-Dawley rats were randomLy divided into Sham group（n=10）,DOCA group[n=13,DOCA50mg/（kg·w）],DIZE group[n=13,DOCA50mg/（kg·w）+DIZE15mg/（kg·d）].Body weight of all rats was measured once a week for eight weeks.Blood pressure of rat tail artery was measured by Softron BP-98A blood pressure meter once a week for 8 weeks.Standard deviation（SD）and coefficient of variation（CV）as indicators of blood pressure variability（BPV）,then,the short-term and long-term BPV was calculated.Left ventricular mass was weighed,left ventricular mass index was calculated.Left ventricular myocardial cell diameter and area were measured by HE staining,and left ventricular fibrosis was measured by Masson staining.ELISA was applied to detect the content of Ang（1-7）in left ventricular.Western blotting was used to dectect relative proteins in left ventricular includingα-smooth muscle actin（α-SMA）,collagenI（ColⅠ）,collagenⅢ（ColⅢ）,angiotensin-converting enzyme（ACE）,angiotensinⅡ（AngⅡ）,angiotensin Ⅱ type1 receptor（AT1R）,angiotensinⅡtype2 receptor（AT2R）,angiotensin-co-nverting enzyme2（ACE2）,Mas receptor（MasR）,Na⁺-K⁺-ATPase（α1,α2）,sodium-hydrogen exchanger1（NHE1）,sodium-calcium exchanger1（NCX1）,transient receptor potential channel1（TRPC1）,transient receptor potential channel 3（TRPC3）,classical transient recep tor potential channel 6（TRPC6）.（2）MTT assay was used to determine the time and concentration of high-salt in H9c2 cardiomyocytes.The experiment was divided into control group（139mM）and high-salt group（150mM,153mM,156mM,159mM,162mM,165mM,168mM,170mM）were treated for 48h respectively.MTT assay was used to detect the concentration of DIZE in H9c2 cardiomyocytes by high-salt intervention.The experim-ent were individed control group,high-salt group and DIZE group（DIZE concentration including 2μg/mL,4μg/mL,6μg/mL,8μg/mL,10μg/mL）.HE staining was used to detect the morphological changes of H9c2 cardiomyocytes in three groups,fluorescence ratio was applied to detect Ca²⁺concentration and pH in H9c2 cardiomyocytes.The Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities of cardiomyocytes in each group were detected by colorimetry.The content of Ang（1-7）was detected by ELISA in the supernatant of H9c2cardiomyocytes.Western blotting was used to evaluate the relative protein expression including BNP,ACE,AngⅡ,AT1R,AT2R,ACE2,MasR,NHE1,NCX1,Na⁺-K⁺-ATPase（α1、α2）,TRPC1,TRPC3and TRPC6 proteins in H9c2 cardiomyocytes.（3）In order to determine the concentration of Ang（1-7）to cardiac fibroblast（CFs）induced by high salt,the experiment was divided into control group（Na⁺139mM）,high-salt group（Na⁺161mM）,high-salt+Ang（1-7）groups（Na⁺161mM+Ang（1-7）0.1μΜ,0.5μΜ,1μΜ,2μΜ,3μΜ,4μM,5μM）.MTT assay was used to determine the intervention concentration of A779 after intervening all groups for 48h respectively.According to the intervention concentration of Ang（1-7）,the experimental groups divided into control group（Na⁺139mM）,high-salt group（Na⁺161mM）,high-salt+Ang（1-7）group,high-salt+Ang（1-7）+A779group（Na⁺161mM+Ang（1-7）+A7790 1μΜ,0.5μΜ,1μΜ,5μΜ,10μΜ,15μΜ）.The contents of ColⅠand Col Ⅲ in the supernatant of CFs cellswere were detected by ELISA.The expressions of ColⅠ,Col Ⅲ and MasR in CFs were detected by Western blotting.RESULTS:（1）Compared with the Sham group,the systolic blood pressure,diastolic blood pressure and mean arterial pressure of the tail artery in the DOCA group increased at the first week of the experiment（P<0.05）,and the pulse pressure increased transiently at the third week and increased again at 5th week until the 8th week（P<0.05）,While the long-term systolic blood pressure variability increased at 0 to 1 week（P<0.05）,long-term diastolic pressure variability increased at 0 to 4 weeks（P<0.05）,long-term mean arterial pressure variability increased from 0 to 2 weeks（P<0.05）,long-term pulse pressure variability increased from 0 to 5 weeks（P<0.05）until the 8th week of the experiment.Compared with the DOCA group,the systolic blood pressure of the tail artery in the DIZE group was decreased at the second week（P<0.05）,the diastolic blood pressure and mean arterial pressure decreased at the 4th week（P<0.05）,and the long-term systolic blood pressure variability was 0² weeks decreased（P<0.05）,long-term mean arterial pressure variability decresd at 0 to 4 weeks（P<0.05）,and continued the 8th week of the experiment.There was no significant difference in short-term BPV between the three groups（P>0.05）.Compared with Sham group,the left ventricular mass,left ventricular mass index,left ventricular myocardial cell diameter,area and left ventricular interstitial fibrosis index in DOCA group were significantly increased,also the expressions ofα-SMA,ColⅠ,ColⅢ were significantly higher than Sham group（P<0.05）,but the left ventricular mass,left ventricular mass index,left ventricular myocardial cell diameter and area and left ventricular interstitial fibrosis index and the expressions ofα-SMA,ColⅠ decreased in DIZE group（P<0.05）.Compared with Sham group,ACE,AngⅡ,AT1R,NHE1,NCX1,TRPC3 and TRPC6 in left ventricleofDOCAgroupweresignificantlyincreased（P<0.05）,but ACE2,Na⁺-K⁺-ATPase（α2）was significantly decreased（P<0.05）,while the expression of TRPC1,AT2R and Na⁺-K⁺-ATPase（α1）was not changed,and the content of Ang（1-7）was significantly decreased（P<0.05）.Compared with the DOCA group,the expressions of ACE,AngⅡ,AT1R,NHE1,NCX1,TRPC3 and TRPC6 in the left ventricle of the DIZE group were significantly decreased（P<0.05）,however the expression of proteins inculding ACE2,Na⁺-K⁺-ATPase（α2）was significantly increased（P<0.05）,but TRPC1,AT2R was not significantly changed.（2）MTT assay showed that Na⁺162mmol/L interfered with H9c2cardiomyocytes for 48h promote cell proliferation.ACE2 agonist DIZE concentration was6μg/mL,which significantly inhibited H9c2 cardiomyocytes proliferation by high salt（Na⁺162mM）.Compared with the control group,the cell diameter and area of the high-salt group were increased significantly（P<0.01）,but the cell diameter and area of the cardiomyocytes in DIZE group were significantly decreased（P<0.05）,compared with the high-salt group.The intracellular pH and Ca²⁺concentration of the high-salt group higher than control group,but Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities decreased（P<0.05）.Compared with the high-salt group,the intracellular pH and Ca²⁺concentration of the DIZE group were decreased,but Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities increased（P<0.05）.Compared with the control group,the expressions of BNP,ACE,AT1R,NHE1,NCX1,TRPC3 and TRPC6 proteins in the high-salt group were significantly increased（P<0.05）,ACE2,MasR,Na⁺-K⁺-ATPase（α1）,Na⁺-K⁺-ATPase（α2）was significantly decreased（P<0.05）,the expression of TRPC1 and AT2R was not significantly changed,and the content of Ang（1-7）was significantly decreased（P<0.05）.Compared with the high-salt group,the expression of BNP,ACE,AT1R,NHE1,NCX1,TRPC3,and TRPC6 protein in DIZE group was significantly decreased（P<0.05）,but protein the expression of ACE2,MasR,Na⁺-K⁺-ATPase（α1）,Na⁺-K⁺-ATPase（α2）was significantly increased（P<0.05）,the protein expression of TRPC1 and AT2R was not significantly changed,the content of Ang（1-7）was significantly increased（P<0.01）,and the protein expression of TRPC1 and AT2R was not changed.（3）MTT assay showed that concentration of Ang（1-7）1μM could inhibit the proliferation of CFs induced by high salt（P<0.05）,and concentration of MasR blocker A779 1μM could prevent the action of Ang（1-7）（P<0.05）.The results of EdU showed that the proliferation rate of high-salt group was higher than control group（P<0.05）.Compared with the high-salt group,the proliferation rate of high-salt+Ang（1-7）group was decreased,the cell proliferation rate of high-salt+Ang（1-7）+A779 group was higher than high-salt+Ang（1-7）group（P<0.05）.Cell cycle results showed that the percentage of S phase and proliferation index of CFs in high-salt group were higher than the control group（P<0.05）,and the percentage of S phase and proliferation index in high-salt+Ang（1-7）group were lower than high-salt group（P<0.05）,the percentage of S phase in the high-salt+Ang（1-7）+A779 group and proliferation index were higher than high-salt+Ang（1-7）group（P<0.05）.Compared with the control group,the secretion and protein expression of ColⅠand Col Ⅲ of CFs were increased,but the expression of MasR protein in high-salt group was decreased（P<0.05）.Compared with the high-salt group,the high-salt+Ang（1-7）group the protein expression of ColⅠ and Col Ⅲ were decreased,the expression of MasR protein was increased（P<0.05）,the secretion and protein expression of ColⅠ and ColⅢ were increased in high-salt+Ang（1-7）+A779group,but the expression of MasR protein was decreased（P<0.05）.Conclusion:Ⅰ.High-salt increases the blood pressure and long-term blood pressure variability in DOCA-salt sensitive hypertensive rats,while induces left ventricular remodeling,myocardial cell hypertrophy,the proliferation of CFs and the secretion and synthesis of ColⅠ and ColⅢ.Ⅱ.High-salt up-regulates the expression of Ca²⁺-related proteins including TRPC3、TRPC6 and NCX1 by activating cardiac local RAS,promotes Ca²⁺influx,contribute to left ventricular remodeling and cardiomyocyte hypertrophy.Ang（1-7）inhibits the proliferation of CFs and decreases the secretion and synthesis of ColⅠ and ColⅢ in CFs by MasR.Ⅲ.The ACE2 agonist negatively regulates myocardial local RAS,down-regulates the expression of Ca²⁺-related proteins including TRPC3、TRPC6 and NCX1,then reduces Ca²⁺influx,reduce left ventricular remodeling and cardiomyocytes cell hypertrophy induced by high-salt.