| Objective Diabetes mellitus(DM) is a group of metabolic disease, characterized by chronic elevated blood glucose levels, and is caused by insulin secretion and/or founction deficiency. With the increasing prevalence of diabetes, more attention has been paid to diabetic cardiomyopathy(DCM), which is one of diabetes mellitus complications. DCM is a common cardiovascular complication of DM, and is the leading cause of heart failure and sudden death in patients with DM. The typical clinical symptoms of DCM are cardiac dilatation, heart failure, even occurring congestive heart failure more easily. The pathogenesis of DCM is complex, and has not yet been clarified. Recently researches showed that Corin, as a cardiac serine protease, is mainly expressed in the heart, and can convert pro-atrial natriuretic peptide(pro-ANP) to biologically active atrial natriuretic peptide(ANP) specifically. ANP is a polypeptide hormone, which is mainly synthesized and released by atrial myocytes. It has a strong function of promoting natriuresis, diuresis, vasodilatation. Meanwhile, ANP is positively associated with the development of heart failure and hypertension. It has been discovered that Corin gene deletion or down-regulated expression is closely related to the mechanisms of serious cardiovascular diseases, such as heart failure, cardiac hypertrophy and hypertension. However, it is not clear that whether Corin-mediated processing of pro-ANP plays a role in DCM. Thus, we used the STZ-induced DCM rat model, and high ambient glucose-induced neonatal rat cardiomyocytes and H9c2 cardiomyocytes in vivo and in vitro, to explore the relationship between Corin-mediated processing of pro-ANP and DCM at the whole body, cellular and molecular levels.Methods 1. We established the animal model of DM SD rats with STZ intraperitoneally; 2. Echocardiography, hemodynamics were performed, and heart weight/body weight(HW/BW) was detected in the animal model of DCM SD rats; 3. Transmission electron microscopy observed the ultrastructural changes of cardiomyocytes in DCM rats; 4. RT PCR and Real Time PCR measured Corin and ANP m RNA expression in the myocardial tissue of DCM rats; 5. Western Blot and immunohistochemistry measured Corin and ANP protein expression in the myocardial tissue of DCM rats; 6. Western Blot measured Corin and ANP protein expression in the myocardial tissue of DCM rats at 12, 16, 20 and 33 weeks; 7. ELISA measured the concentration of NT-pro ANP in the plasma of DCM rats; 8. Real Time PCR detected Corin and ANP m RNA expression in high ambient glucose-induced neonatal rat cardiomyocytes; 9. Western Blot and immunofluorescent measured Corin and ANP protein expression in high ambient glucose-induced neonatal rat cardiomyocytes; 10. Western Blot measured Corin and ANP protein expression in high ambient glucose-induced neonatal rat cardiomyocytes at 36 h, 48 h and 60 h; 11. Western Blot measured Corin and ANP protein expression in high ambient glucose-induced H9c2 cardiomyocytes at 36 h, 48 h and 60 h.Results 1. We established the animal model of diabetes mellitus SD rats successfully: One week after STZ injection, the random blood glucose of DCM rats was more than 16.7 mmol/L, urine glucose was positive, and DCM rats showed polydipsia, polyphagia, polyuria symptoms. Compared with Ctrl group, the random blood glucose level was significantly increased in DCM group(P < 0.01), the body weight was significantly decreased in DCM group(P < 0.01); 2. We established the animal model of diabetes cardiomyopathy SD rats successfully: Echocardiography showed, compared with Ctrl group, the IVSd, LVPWd and LVIDd of DCM group were decreased(P < 0.05), the LVIDs of DCM group was significantly increased(P < 0.01), the FS % and EF % of DCM group were significantly decreased(P < 0.01); the hemodynamic showed, the + dp/dt and- dp/dt of DCM group were significantly lower than that in Ctrl group(P < 0.01), MABP of DCM group had no significant difference with Ctrl group (P > 0.05); HW/BW in DCM group was significantly higher than that in Ctrl group(P < 0.01); 3. The transmission electron microscope showed, in Ctrl group, the cardiac muscle fibers were arranged in order, the morphology of cardiomyocytes was normal, the ultrastructure was intact, the mitochondrial membrane was intact, and the distribution of mitochondrial cristae was uniform; but in DCM group, the cardiac muscle fibers were arranged in disorder, the morphology and ultrastructure of cardiomyocytes were abnormal, and the mitochondrial membrane was not clear and damaged, the distribution of mitochondrial cristae was not uniform, the spacing of mitochondrial cristae was increased, the mitochondrial burst and glycogen deposition could also be seen; 4. RT-PCR and Real-Time PCR showed that Corin and ANP m RNA expression in the myocardial tissue of DCM group were significantly lower than that in Ctrl group(P < 0.05); 5. Western Blot and immunohistochemistry results showed that Corin and ANP protein expression in the myocardial tissue of DCM group were significantly lower than that in Ctrl group(P < 0.05); 6. Western Blot showed that Corin and ANP protein expression in the myocardial tissue of DCM rats were gradually decreased in time dependent manner; 7. ELISA showed that the concentration of NT-pro ANP in the plasma of DCM rats was significantly higher than that in Ctrl group(P < 0.01); 8. Real Time PCR showed that Corin and ANP m RNA expression in high ambient glucose-induced neonatal rat cardiomyocytes were significantly lower than that in Ctrl group(P < 0.01); 9. Western Blot and immunofluorescent showed that Corin and ANP protein expression in high ambient glucose-induced neonatal rat cardiomyocytes were significantly lower than that in Ctrl group(P < 0.05); 10. Western Blot showed that Corin and ANP protein expression in high ambient glucose- induced neonatal rat cardiomyocytes were gradually decreased in time dependent manner; 11. Western Blot showed that Corin and ANP protein expression in high ambient glucose-induced H9c2 cardiomyocytes were gradually decreased in time dependent manner.Conclusions 1. The animal model of diabetes mellitus SD rats was established successfully; 2. The animal model of diabetic cardiomyopathy SD rats was established successfully; 3. Corin and ANP m RNA expression were decreased in diabetic cardiomyopathy; 4. Corin and ANP protein expression were decreased in diabetic cardiomyopathy; 5. Corin-mediated processing of pro-ANP was involved in the pathogenesis of diabetic cardiomyopathy, and was closely related to the course of diabetic cardiomyopathy. |
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