Background Diabetic cardiomyopathy (DCM), a fatal cardiovascular complication of diabetes mellitus,

Background Diabetic cardiomyopathy (DCM), a fatal cardiovascular complication of diabetes mellitus, often leads to progressive heart failure, however its pathogenesis remains unclear. The mRNA and protein levels of corin and ANP in rat hearts and cardiomyocytes were determined by quantitative real-time PCR, Tepoxalin IC50 western blotting and immunohistochemical staining, respectively. H9c2 cardiomyoblasts proliferation was detected by MTT colorimetric assay and viable cell counting with trypan blue. The effect of H9c2 cardiomyoblasts on EA.hy926 cells migration was measured by the wound healing scratch assay. Results The corin and ANP expression in mRNA and protein levels was decreased in DCM rat hearts. Corin and ANP levels of neonatal rat cardiomyocytes and H9c2 cardiomyoblasts Tepoxalin IC50 treated with high glucose were significantly lower than that of normal glucose treated. Precisely, corin and ANP levels decreased in DCM rats at 12, 16, 20 and 33?weeks; neonatal cardiomyocytes and H9c2 cardiomyoblasts treated with high glucose at 36, 48 and 60?h demonstrated significant reduction in corin and ANP levels. H9c2 cardiomyoblasts showed decreased proliferation. Culture supernatants of H9c2 cardiomyoblasts prevented endothelial cell line EA.hy926 migration in the wound healing scratch assay. Furthermore, iso-lectin expression in arteriole and capillary endothelium was down-regulated in DCM rats. Conclusions Our results indicate that corin plays an important role in cardioprotection by activating pro-atrial natriuretic peptide pathway in DCM. Corin deficiency leads to endothelial dysfunction and vascular remodeling. Electronic supplementary material The online version of this article (doi:10.1186/s12933-015-0298-9) contains supplementary material, which is available to authorized users. H9c2 cardiomyoblasts culture supernatants on EA.hy926 cells migration by wound healing scratch assay. Our results indicated that corin exerted cardioprotective action via pro-ANP activating pathway in DCM, meanwhile, corin deficiency was associated with endothelial dysfunction and vascular remodeling. Methods Induction of the diabetes model Forty-five male SpragueCDawley rats (180C220?g) were purchased from the experimental animal center of Academy of Military Medical Sciences (Beijing, China). The animals were housed at 22??2?C with 12?h lightCdark cycles. All care and experimental procedures of animals were in accordance with the guidelines for the Care and Use of Laboratory Animals published by the National Institute of Health and approved by the Animal Care & Welfare Committee of Tianjin Medical University. The rats were randomly divided into two groups: control group and diabetes group. Diabetes group was induced by Tepoxalin IC50 a single intraperitoneal injection of STZ (Sigma; 65?mg/kg dissolved in 0.1?mol/L citrate buffer, pH 4.5). The control group received the same dose of citrate buffer alone. The two groups received normal chow. Blood glucose levels were measured on day 3 and 7 after STZ or citrate buffer administration by a hand-held glucometer (UltraEasy, Johnson, USA). Rats with random blood glucose (RBG) >16.7?mM in two consecutive examinations were considered as diabetic model. We monitored body weight, blood glucose, and urine glucose every week. The two groups were sacrificed under deep anesthesia (a single intraperitoneal injection of 3?% sodium pentobarbital at the dose of 50?mg/kg body weight) by exsanguinations. Echocardiography and hemodynamic measurements Transthoracic echocardiography was performed by the vivid 3 pro imaging system (GE, USA) in both groups at 4, 8, 12, 16, 20?weeks. Images were obtained from two-dimensional, M-mode, pulsed-wave Doppler imaging. All measurements were the average of six consecutive cardiac cycles and performed by the same operator. Briefly, male SD rats were lightly anaesthetized with 3? % inhaled isoflurane and set in a supine position. The hemithorax of each rat was carefully shaved. Diastolic interventricular septal wall thickness (IVSd), left ventricular posterior wall thickness in diastole (LVPWd), left ventricular internal dimension in diastole (LVIDd), left ventricular internal dimension in systole (LVIDs), fractional shortening (FS %) and left ventricular ejection fraction (EF %) were measured. Mean arterial blood pressure (MABP), maximal rate of rise in LV pressure (+dP/dt), and maximal rate of decline in LV pressure (?dP/dt) of DCM and Ctrl rats at 20?weeks were measured with a manometer-tipped catheter (SPR-320NR, Millar, USA) and recorded by an MP150 system (Biopac Systems, USA). Histology and immunohistochemistry Paraformaldehyde (4?%)-fixed hearts were embedded in paraffin, and cut into 5?m sections. The extent of myocyte hypertrophy was evaluated by hematoxylin-eosin staining. Interstitial and perivascular fibrosis Rabbit polyclonal to DUSP10 were evaluated by Massons trichrome staining. For immunohistochemical staining, sections were incubated with anti-corin (sc-67179, Santa Cruz Biotechnology Inc.) or anti-ANP (sc-18811, Santa Cruz Biotechnology Inc.) antibody and a secondary Tepoxalin IC50 antibody conjugated with HRP (horseradish peroxidase). Nuclei were counterstained with haematoxylin. Immuno-reactivity was exhibited by 3, 3-diaminobenzidine (DAB, BOSHIDE). Data were collected from at least five rats per study group. Immunofluorescent studies were carried out in.