Role Of Cardiac Protease Corin In Hypertension

Part I. Plasma Soluble Corin Levels and Corin GeneVariants/Mutations in Patients with HypertensionObjective:Hypertension is a major cardiovascular disease, affecting³0%of adult populationsin the world. Hypertension increases the risk for stroke, heart attack and renal disease.To date, the molecular mechanisms underlying hypertension remain poorly understood. Itis widely believed that the pathogenic factors, including genetic, cardiac, hemodynamic,vascular and neurohormonal factor, may contribute to the disease.Corin is a cardiac membrane protease that activates natriuretic peptides, therebyregulating blood pressure and cardiac function. There are three soluble fragments ofcorin, which are shed from cell membrane with molecular mass of¹80,¹60and¹00kDa respectively. It has been reported that the level of plasma soluble corin was reducedin patients with heart failure, pregnancy-induced hypertension and chronic kidney disease.In this study, we measured the level of plasma soluble corin in patients with hypertensionand examined the correlation between the plasma soluble corin level and hypertension.In previous studies, CORIN gene single nucleotide polymorphisms （SNPs） andmutations were found in patients with hypertension and pregnancy-induced hypertension.In this study, we screened CORIN gene mutations and SNPs in additional patients withhypertension to understand the role of corin in the pathogenesis of hypertension.Methods:1. Blood samples were obtained from480normal individuals and594hypertensionpatients.2. Plasma corin levels were measured by enzyme-linked immunosorbent assay（ELISA）. The data were analysized using statistical software Prism5（GraphPad）.3. Genomic DNAs were isolated from white blood cells using a Qiagen kit. TheDNA samples were used in PCR to amplify CORIN gene exons and intron-exons boundaries. Amplified PCR products were used for direct DNA sequencing.Results:1. We detected soluble corin in plasma samples from hypertension patients andnormal controls. In hypertension patients （n=594）, the plasma soluble corin level was0.6±0.3ng/mL, which was significantly lower than that in normal controls （0.9±0.4ng/mL）（n=480）（p＜0.0001）.2. In hypertension patients, the soluble corin level in females was0.6±0.02ng/mL（n=204）, which was significantly lower than that in males （0.7±0.01ng/mL）（n=390）（p=0.005）. This gender difference also existed in normal controls （0.8±0.03vs.0.9±0.03ng/mL，p=0.005）.3. In each gender group, there were no significant differences among different agegroups in hypertension patients and normal controls.4. We sequenced DNA samples from a Chinese population and identified CORINgene SNPs in patients and normal controls including insA （c.102₁03insA）, C13Y （131A→G）, D95Y （376G→T）, L334W （1094G→T） and H525R （1667A→G）. SNP insAoccurred more frequently in hypertension patients than normal controls （5.7vs.0.9%）.5. CORIN gene mutations were found only in hypertension patients including R349H（1138G→A）, R530S （1683T→G）, P866S （2689T→C） and T924M （2864C→T）.Conclusions:We detected plasma soluble corin levels in hypertension patients and normal controlsand found that the soluble corin level in hypertension patients was significantly lower thanthat in normal controls, indicating that the corin deficiency may contribute to hypertension.Our results suggest that plasma soluble corin level may be used as a biomarker forhypertension.We sequenced the CORIN gene in patients with hypertension and identified ninenovel corin mutations and SNPs. SNP insA appeared in hypertension patients morefrequently than normal controls, whereas mutations R349H, R530S, P866S and T924Mwere found only in hypertension patients. Our results may provide new insights into theimportance of corin-mediated pathway in maintaining normal blood pressure and possiblecontributions of CORIN gene mutation in hypertension. Part II. A Corin Variant in Hypertension Patients with an AlteredCytoplasmic Tail and Reduced Cell Surface Expression and ActivityObjective：Corin is a trypsin-like serine protease identified in the heart, which regulates bloodvolume and pressure by activating the cardiac hormone, atrial natriuretic peptide （ANP）.Corin is a cell membrane-bound protein. Cell surface targeting is essential for corinactivity. Previously, human corin cytoplasmic tail was found important for corin surfacetargeting and zymogen activation.The goal of this study is to test the hypothesis that naturally occurring variantsaltering the corin cytoplasmic tail may exist and that such variants may impair corinactivity, thereby contributing to hypertension.Methods:1. pcDNA3.1-based plasmids （Invitrogen） were used to express human corinwild-type （WT） and mutants.2. The plasmids were transfected into human embryonic kidney293（HEK293） cells.Conditioned medium was collected after48h and the transfected cells were lysed.Immunoprecipitation and Western analysis of corin proteins in the conditioned mediumand cell lysate were performed. Pro-ANP processing was analyzed byimmunoprecipitation, SDS-PAGE and Western blotting.3. To examine corin expression on the cell surface, HEK293cells expressing corinproteins were incubated with membrane impermeable Sulfo-NHS-SS-biotin （ThermoScientific）. Biotin-labeled proteins were precipitated with avidin-agarose beads andanalyzed by Western blotting. Flow cytometric analysis also was used to examine corinexpression on the cell surface.4. We examined the intracellular distribution of the corin variant by immunostainingand confocal microscopy.Results:1. We sequenced DNA samples from a Chinese population and identified a novelvariant of a single adenine insertion （c.102₁03insA） in exon1of the CORIN gene.Unlike WT corin, which starts at Met-1with a cytoplasmic tail of45amino acids, the insAvariant is expected to have a shorter cytoplasmic tail of16amino acids. The insA variantappeared to occur more frequently in patients with hypertension than normal individuals. 2. Western analysis showed that the insA variant was expressed in transfectedHEK293cells, and that the expression level of the insA variant was similar to that of WTand mutants R801A and S985A, indicating that the variant did not alter corin expression intransfected HEK293cells. In a pro-ANP processing assay, variant had41.3±8.5%ofactivity compared with that of WT （p＜0.01）.3. We analyzed the zymogen activation of the insA variant by Western blotting.An⁴0kDa band was detected in WT corin. The intensity of the⁴0kDa band in theinsA variant was markedly reduced, indicating that the insertion did not prevent the variantexpression but impaired its zymogen activation in the transfected cells.4. We examined the cell surface expression of corin by biotin-labeling followed byWestern blotting. Cell surface expression of the insA variant was much less abundantthan that of WT and mutants R801A and S985A. In controls, levels of total corinexpression in cell lysate were similar for all corin proteins. We verified these results byflow cytometry. In HEK293cells transfected with plasmid expressing WT corin,32.7±4.9%cells were corin-positive on the surface. In contrast, only7.9±2.1%of cells werecorin-positive on the surface when plasmids expressing the insA variant was transfected.5. By immunostaining and confocal microscopy, we found that the insA variantprotein was partially retained in the Golgi.6. We examined the insA variant and WT corin shedding in transfected HEK293cells. Three distinct fragments of¹80,¹60and¹00kDa were found in theconditioned medium from cells expressing WT corin. In cells expressing the insA variant,the¹80kDa fragment was less abundant and the¹00kDa fragment was hardly detected.There was no significant difference of¹60kDa fragment between insA and WT.7. Plasma corin levels in25individuals with insA corin allele were0.6±0.1ng/mL,which were significantly lower than that in215normal controls （0.9±0.02ng/mL,p<0.01）.Conclusions:In this study, we identified a new CORIN variant allele in a Chinese population that ispresent preferentially in hypertension patients. The corin protein encoded by this variantallele is predicted to have a shortened cytoplasmic tail. In functional studies, the variantwas found to have poor intracellular trafficking, reduced cell surface expression, andimpaired zymogen activation, which may explain low plasma corin levels in hypertension patients who carried the variant allele. Together, our data provide new insights into thestructural basis for corin biosynthesis and activity. Our data also indicate that corindeficiency may represent an important molecular mechanism underlying hypertension.