The Role Of Cardiac And Renal Corin In The Regulation Of Cardiovascular Homeostasis

Objective:Atrial natriuretic peptide(ANP)is an important endocrine hormone that plays an important role in maintaining cardiovascular homeostasis and normal blood pressure.Corin is a transmembrane serine protease found in the heart.It can activate atrial natriuretic peptide precursor(pro-atrial natriuretic peptide,pro-ANP)and exert its biological function of natriuretic and diuretic.Corin deficiency can lead to major cardiovascular diseases such as hypertension,heart disease and preeclampsia.For a long time,it has been considered that ANP is a cardiogenic endocrine hormone,which reaches the kidney through blood circulation,promotes sodium excretion and diuresis,and regulates body fluid balanceIn addition to the heart,Corin is also expressed in the pregnant uterus and kidney.Our team found that the expression of Corin,ANP and their receptors in various segments of the renal tubule overlaps,suggesting that the kidney may also be the producing organ of ANP,and the Corin-ANP expressed in the kidney may play a role in local autocrine.The role of this autocrine function in maintaining cardiovascular homeostasis may be different from the Corin-ANP endocrine function of the heart.In this study,we will construct Corin systemic and tissue-specific gene knockout mice to clarify the special role of heart and kidney Corin-ANP pathway in the regulation of cardiac function,water-sodium balance and vascular homeostasis,and to understand the mechanism of Corin-ANP disorders in different tissues.Methods:(1)The vector containing two loxP sites on both sides of exon 4 of CORIN gene was constructed and inserted into mouse CORIN gene by homologous recombination to obtain Cor+/flox mice.To generate Corin KO mice,Cor+/flox mice were crossed with the CMV-Cre mice(B6.C-Tg(CMV-cre)1Cgn/J)which expressed a ubiquitously Cre gene.(2)To generate corin heart cKO mice,Cor+/flox mice were crossed with B6.FVB(129)-Tg(Myh6-cre/Esr*)1 Jmk/J mice expressing Cre in cardiomyocytes under the myosin heavy chain promoter to disrupt the Corin gene specifically in the heart.(3)To generate corin kidney cKO mice,Cor+/floxx mice were mated with Tg(Ggt1-cre)M3Egn/J mice expressing Cre recombinase in the proximal and distal tubules and collecting ducts of the kidney.(4)The offspring was screened by PCR to identify.Corin mRNA and protein expression in Corin KO,heart and kidney cKO mice were verified by RT-PCR and Western blotting analysis.(5)A computerized non-invasive photoelectric tail-cuff system(Visitech Systems,BP-2000)was used to measure blood pressure in mice.(6)Transthoracic echocardiograms were performed using Visual Sonic Vevo 2100 Imaging System(VisualSonic Inc.).Briefly,mice were anesthetized with 1.5%inhaled isoflurane with oxygen.Two-dimensional and M-mode images of the heart and vasculature were obtained from the parasternal long-axis and short axis acoustic windows.Analysis was blindly completed post-recording using VevoLAB software.Heart function and morphometrics were measured directly or calculated using standard equations within the software.(7)Heart tissues were was collected from 15-months old mice,the ratio of heart weight to body weight was calculated.The cardiac tissue sections were stained with H&E,Wheat germ agglutinin immunofluorescence staining and Sirius red staining.(8)In order to evaluate the renal function of mice,urine samples were collect from 12 months-old mice.(9)To exam the effect of salt diet on blood pressure,we examined blood pressure in mice on normal(0.3%NaCl)and high-salt(4%NaCl)diets.(10)Metabolic cage was used to study the metabolism of mice.Mice were feed with normal(0.3%NaCl)and high-salt(4%NaCl)diet.The food intake and drinking water were measured every day.Urine was collected and 24 h urine volume was recorded.Results:(1)After genomic DNA,RT-PCR and Western blotting identification,homozygous Corin KO,Myh6-cKO and Ggt1-cKO mice were obtained.(2)The blood pressure of Corin KO,Myh6-cKO and Ggt1-cKO mice was significantly higher than that of WT mice(p<0.01).During the experiment,no increase in blood pressure was observed with age.(3)Echocardiography showed that there was no significant difference in cardiac function at 3,6 and 9 month-old in Corin KO,Myh6-cKO,Ggt1-cKO mice and WT mice.At the age of 12 and 15 months,the LV mass of Corin KO,Myh6-cKO and Ggt1-cKO mice was significantly higher than that of WT mice(p<0.05),and the left ventricular ejection fraction and short axis shortening rate were significantly lower than those of WT mice(p<0.01),and left ventricular systolic and end-diastolic volume increased significantly(p<0.05).It is suggested that cardiac function is impaired in:Corin KO,heart and kidney cKO mice.(4)At 15 months of age,the ratio of heart weight to body weight(HW/BW)and the ratio of heart weight to tibia length(HW/TL)in Corin KO,Myh6-cKO and Ggt1-cKO mice were significantly higher than WT Mice(p<0.05)(5)Myocardial cell hypertrophy were found in Corin KO,Myh6-cKO,and Ggt1-cKO mice.Myocardial cell diameter in Corin KO,Myh6-cKO,and Ggt1-cKO mice was significantly higher than WT Mice(p<0.001).The degree of myocardial fibrosis in Corin KO,Myh6-cKO and Ggt1-cKO mice was significantly deepened(p<0.01).(6)Urinary protein levels in Corin KO,Myh6-cKO and Ggt1-cKO male mice increased,compared to those in WT male mice,but the difference was not statistically significant.(p>0.05).In female mice,urine protein levels in female Corin KO,Myh6-cKO and Ggt1-cKO was similar to that of WT female mice(p>0.05).(7)Under high-salt diet,SBP in WT mice did not change significantly,SBP in Corin KO and kidney cKO mice were increased significantly(p<0.05),while cardiac cKO(Myh6-cKO)mice showed no significant changes.(8)Under normal diet(0.3%NaCl),there was no significant difference in water and food intake between mice in each group.Under high-salt diet(4%NaCl),food intake of WT,Corin KO,Myh6-cKO,and Ggt1-cKO mice did not show significant changes,but water intake and urine output increased significantly.Conclusions:We obtained Corin KO,Myh6-cKO,and Ggt1-cKO mice,and found hypertension,impaired cardiac function,and myocardial hypertrophy in these mice.Our study demonstrated that both cardiac and renal corin-ANP pathway are important in regulating cardiac function,salt-water balance and vascular homeostasis.However,the Corin-ANP autocrine function of the kidney is different from the Corin-ANP endocrine function of the heart.Our results may also help to understand how defects of the corin-ANP pathway in different organs contribute to cardiovascular and kidney diseases.