Role Of Angiotensin Ⅱ And Angiotensin Ⅱ Type 1 Receptor Antagonist In Apoptosis In Cultured Cardiomyocytes

Apoptosis is a form of cell death, which removes cells in an orderly or programmed fashion. It plays an essential role in establishing normal embryonic development, maintaining adult tissue homeostasis and contributes to a variety of human diseases including certain pathological processes in the heart. It was first found by Kerr in 1972. During the course of this research, it became clear that in contrast to other classical hormones, the angiotensin is not confined to the blood, but may function as a paracrine and autocrine hormone. Moreover, it participates in a variety of additional processes including cell growth, brain and cardiac fibrosis. Sequence two major AngII receptor subtypes — designated as AT1 and AT2 receptors. We have known that mitochondria plays a critical role in the pathophysiology of myocardial ischemia-reperfusion injury. But the mechanism of Ang II inducing cardiomytcytes apoptosis is not very clear. The purposes of the present study were to determine whether Ang II and Ang II plus Ang II type 1 receptor antagonist (irbesartan )change the expressions of c-fos, PCNA, Bcl-2, iNOS protein and TUNEL -positive nucleis in primary cultured neonatal rat cardiomyocytes. Meanwhile the changes of caspase-9 mRNAand activity of caspase-3 were observed.Material and Methods: 1.The isolated neonatal Wistar rat ventricular cardiomyocytes were cultured and purified with the method of differential attachment. The cultured cells were identified by morphology, spontaneous contraction and specific immunocytochemical stain. 2. Cultured neonatal rat cardiomyocytes were divided into three groups: Cells in control group were exposed to medium free serum for 2h, 6h, 12h, 24h; Cells in Ang II group were exposed to medium free serum but containing with 10~-7mol/L Ang II for 2h, 6h, 12h, 24h; Cells in Ang II plus Ang II type 1 receptor antagonist group were exposed to same medium but containing 10~-7mol/L Ang II plus 10~-5mol/L Irbesartan for 2h, 6h,12h, 24h. The apoptosis cells were detected by TUNEL assay. The c-fos, PCNA, Bcl-2, iNOS protein expression were assessed by Immunohistochemistry stain. Caspase-3 activity was measured by a fluorescent assay kit and caspase-9 mRNA was measured by RT-PCR.Results: l.The shapes of primary cultured cardiomyocytes are spindle? triangle and irregular. Among total cultured cells, the spindle cells are majority of primary cultured cardiomyocytes. Spontaneous contraction of the spindle cell is apparent. There have abundant brownish red filaments in cytoplasm of primary cultured cardiomyocytes with the method of specific immunocytochemical stain. 2.Terminal deoxynuleotidyl transferase-mediated dUTP nick-end labeling assay. Exposure of culture myocytes to 10"7mol/L Ang II increased the number of TUNEL-positive nucleis significantly in time-dependent manner, but exposure of culture myocyte to 10"7mol/L Ang II plus 10"5mol/L Irbesartan reduced the number of TUNEL-positive nucleis significantly. 3.Immunocytochemical stain: (J) Compared with control, the expression of Bcl-2 protein was decreased in time-dependent way in Ang II group, but increased in Ang II plus irbesartan group; ?Compared with control group, a significant increase in expression of c-fos, PCNA protein in group of Ang II reached peak as early as 2 hours and began decreased at 6 hours. Meanwhile expression of c-fos, PCNA protein in group of Angllplus irbesartan reduced significantly at same time point; ?Compared with control group significant increase in expression of iNOS protein in group of Ang II began as early as 2 hours and reached peak at 6 hours. However significant decrease in expression of iNOS protein in group of Ang II plus irbesartan. 4. Fluorescent assay the activity of caspase-3 protease: Compared with control group, a significant increase in activity of caspase-3 in group of Ang II began as early as at 2 hours and reached a plateau at 6 hours and maintained at high level up to 24 hours. However, a significant decrease in activity of caspase-3 in group of Ang II plus irbesartan was observed at same time point. 5.RT-PCR assay the expression of caspase-9 mRNA: A significant increase in caspase-9 mRNA began as early as at 2 hours and reached a plateau at 6 hours and maintained at high level up to 24 hours. However, a significant decrease in expression of caspase-9 mRNA in group of Angllplus irbesartan was observed at same time point.Conclusions: 1.Ang II induced apoptosis in cultured neonatal rat cardiomyocytes in time-dependent manner. 2. Ang II type 1 receptor antagonist irbesartan may be useful for prevention of apoptosis in cultured neonatal rat cardiomyocytes. 3.Ang II induced apoptosis in cultured cardiomyocytes which may be mediated by Bcl-2 protein in time-dependent pathway. 4.Ang II induced apoptosis in cultured cardiomyocytes via caspase-dependentmanner. 5.The expression in instanting early gene c-fos, PCNA protein of cultured cardiomyocyte was significantly increased in induced apoptosis cultured cardiomyocytes by Ang II. 6.The expression in iNOS of cultured cardiomyocyte was significantly increased in induced apoptosis cultured cardiomyocytes by Ang II ,which involved the injury of mitochondria.