| Background and objective: Myocardial hypertrophy serves as both a common pathway of most forms of cardiovascular diseases and an important structural basis of heart failure, with a similar pathogenesis of increased cell volume and protein systhesis and expression of fetal heart genes upregulated under different stimulations. It was reported recently by Olson that calcineurin (CaN), as a molecule sensitive to intracellular Ca2~, plays an essential role in the development of cardiac bypertrophy, suggesting CaN-NFAT3 pathway be a new signal transduction leading to cardiac hypertrophy, and administration of cyclosporin A(CsA) could be used to relieve its hypertrophy and improve heart failure. As a cross-point of many signal pathways, intracellular Ca2~ concentration([Ca2~]i) of cardiomyocytes has been considered as an underlying second messenger, and regulated by changing Ca2~ inflow and release. Protein kinase C (PKC), mitogen activated protein kinase(MAPK) are also involved in its process. This study based on the primarily cultured cardiomyocytes, stimulating Ca2~ inflow and release by Angiotensin II (AngII) and Ryanodine (RY), is aimed at understanding the effects of Ca2~ from different origins on cell growth, the significance of CaN in the mechanism of myocardial hyerptrophy, the influence of [Ca2~]i on activation or expression of PKC, MAPK, immediate-early genes. Methods: Cardiomyocytes from fetal rat were cultured primarily, and irritated with AngII or RY to cause either Ca2~ inflow or release; 3H-Thymidine (3H-TdR) and 3H-Leucine (3H-Leu) incorporations used to confirm the synthesis velocity of nucleic acid and protein; Fura-2/AM ratio imaging ?III ? analysis applied to detect intracelluar Ca2~ signal, immunocytochemistry for observing the distributions of CaN, NFAT3, C-fos, C-myc; Western blotting for semi-quantification of CaN, NFAT3, GATA4, ERK1I2, a-actin, c-fos, c-myc; RT-PCR for analysis of ~-MHC mRNA; y-32P marked substrate for activity of PKC and MAPK. Results: 1. Ang II caused a concentraion-dependent (1O.s~.~~105 mol/L) increase of [Ca2~]i with marked difference between every experimental group and control group. RY(1 o~?10.6 mol/L) resulted in a similar increase to the former with an exception of its reduce at the concentration of iO~ mol/L. 2. Ang II induced a propotional enhancement of 3H-Leu incorporation of cells in the serum-free medium to stimulation time, depending on AngII concentration ranged from i0~-i0~ mol/L. Similar to AngII, RY(l0~8---- l0~ mol/L) increased its incorporation except for one decrease at the concentration of 1 0~ mol/L. Regarding 3H-TdR incorporation, the results were generally similar to 3H-Leu. CsA, a specific inhibitor of CaN, supressed incorporations of both 3H-TdR and 3H-Leu, with a concentration-dependent manner between the range of 50?00ug/L. 3. CaN expressed mainly in cytoplasm in normal cells, but in cytoplasm and nucli expression both stimulation of AngII and RY for 24 h. CaN expression increased after 24 h of stimulation of AngII and RY, different greatly from control group. 4. NFAT3 normally expressed in cytoplasm and nucli, turned to a predominant nucleus expression after 24 h of stimulation of AngII and RY. It抯 protein expression increased gradually at 24 h and 2 days later. 5. CsA supressed CaN,NFAT3,GATA4 expression induced...
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