Protease corin expression and activity in failing hearts

Abstract

Atrial and brain natriuretic peptides (ANP and BNP) regulate blood pressure and cardiac function. In patients with heart failure (HF), plasma levels of pro-ANP and pro-BNP, the precursor forms of ANP and BNP, are highly elevated, but the mechanism underlying the apparent deficiency in natriuretic peptide processing is unclear. Corin is a cardiac protease that activates natriuretic peptides. In this study, we examined corin protein expression and activity in mouse and human failing hearts. Tissue samples were obtained from a mouse model of HF induced by myotrophin overexpression and from human nonfailing, hypertrophic, and failing hearts. Corin protein levels in the membrane fraction and tissue lysate were measured by Western blotting and ELISA. Corin catalytic and biological activities were measured by fluorescent substrate and pro-ANP processing assays. In mice, corin protein levels did not change with age in normal hearts but increased significantly in failing hearts. In humans, corin protein levels were similar in the atrium from nonfailing and failing hearts but were increased in the ventricle in failing hearts compared with those in nonfailing or hypertrophic hearts. Unlike the protein level, however, corin activity did not increase in failing hearts, as measured by fluorogenic substrate and pro-ANP processing assays. Our results indicate that corin activation is a rate-limiting step in failing hearts. Insufficient corin activation is expected to prevent natriuretic peptide processing and may contribute to body fluid retention and impaired cardiac function in patients with HF.

Fig. 1.

Corin protein expression in mouse hearts. A: heart membranes were from wild-type (WT) and transgenic (Tg) mice of the indicated ages (w, weeks). Corin protein was analyzed by Western blotting. Samples from parental HEK 293 cells (negative) or HEK 293 cells expressing human corin (positive) were used as controls. Blots were reprobed with an anti-GAPDH antibody, as an internal control. B: corin protein levels were quantified by densitometric analysis of Western blots. The data were from 3 independent experiments. *P < 0.05 vs. WT of the same age group; n.s., not statistically significant.

Fig. 2.

Corin mRNA and protein expression in human nonfailing hearts. A: total RNAs from atria and ventricles were analyzed by quantitative RT-PCR. Corin mRNA levels were higher in the atrium than that in the ventricle. *P < 0.005; n = 8. B: tissue lysates from atria and ventricles were analyzed by ELISA. Corin protein levels were higher in the atrium than in the ventricle. *P < 0.0001; n = 10.

Fig. 3.

Corin protein levels in human failing hearts. Tissue lysate was from left and right atria (LA and RA, top) and ventricles (LV and RV, bottom) from nonfailing and failing hearts. Corin protein levels were measured by ELISA. Samples numbers in each group were ≥5. *P < 0.05 vs. nonfailing hearts of the same group.

Fig. 4.

Corin protein levels in human nonfailing, hypertrophic, and failing hearts. Tissue lysate was prepared with ventricular samples from nonfailing, hypertrophic, and failing hearts. Corin expression levels were measured by ELISA. At least 9 samples were included in each group. **P < 0.05 vs. nonfailing or hypertrophic.

Fig. 5.

Serine protease activity in human nonfailing and failing hearts. Serine protease activity in membrane fractions from left ventricles of nonfailing and failing hearts was measured by a fluorescent substrate assay. The reaction was monitored by a plate reader using a kinetic mode. Hirudin and benzamidine were used to determine assay specificity. The data were from 3 independent experiments, each of which included 4 samples. n.s., Not statistically significant vs. nonfailing hearts.

Fig. 6.

Pro-atrial natriuretic peptide (ANP) processing activity in human nonfailing and failing hearts. Membrane fractions from left ventricles of nonfailing and failing hearts with indicated corin concentrations were incubated with human pro-ANP. Conversion of pro-ANP to ANP was analyzed by immunoprecipitation and Western blotting (top). Data from 3 independent experiments were quantified by densitometry (bottom).