Effect Of Angiotensin Ⅱ On The Expression Of Clock Genes In Cultured Rattus Cardiac Myocytes

Daily rhythms of mammalian physiology and endocrinology are regulated by circadian pacemakers. A hierarchical multioscillatory system seems to confer precise phase control and stability on the widely distributed physiological systems it regulates. The master circadian pacemaker resides in the suprachiasmatic nucleus (SCN) , which is located in the hypothalamus of the brain, but circadian oscillators also exist in peripheral tissues. For orchestrated circadian timing, the collective SCN synchronizes the timing of slave oscillators, each of which is a multioscillatory entity. Synchronized slave oscillators, in turn, regulate local rhythms in physiology and behavior. Cardiac hypertrophy, an increase in cardiac cell size without cell division, is induced by mechanical overload, neural and humoral factors such as angiotensin II (AngII). AngII, acting by binding with ATI receptor and triggering the second messenger system, can induce protein synthesis and, accordingly, induce cardiac cell hypertrophy. The present study has identified that in rat heart all known clock genes show circadian patterns of expression even when hypertrophic and that the expression of clock output genes [the PAR (rich in proline and acidic amino acid residues) transcription factors dbp, hlf, and tef] are attenuated in the pressure-overloaded hypertrophied heart.The aim of this subject was to study the expression of clock genes in cultured cardiac myocytes and to explore the role of circadian clocks inthe process of .cardiac hypertrophy. This study was composing of three parts:1. Modeling of cultured hypertrophic cardiac myocytes induced by Ang IICardiac myocytes were isolated from the ventricles of the heart by enzymatic dissociation (0.06%trypsin+0.01%collagenase) and were purified using different adhesion. Then the cells were incubated at 37C in 5% CO2. 5' -Brdu was added to the DMEM nutrient medium to inhibit non-cardiac-cells growing. The cells grew to a monolayer adhering to culture flasks 72 hours after inoculating. We changed the nutrient medium and add Ang II to a final concentration at 10-7M once a day for 3days, then cells were collected and the cell surface area of cardiac myocytes was measured under confocal microscopy and the content of protein of the cells was examined. The results showed that Ang II could increase surface area and the content of protein of cardiac myocytes without cell division. A model of cultured hypertrophic cardiac cells was established successfully.2. Establishment of RT-PCR for detecting of clock genes in cultured rattus cardiac myocytesPCR was carried out with 3 primer pairs based on the dbp, bmal 1 and per2 gene of the rattus. The conditions of PCR were optimized and the specificity of amplication was tested, we found that in a volume of 20ul, the optimal PCR mixture of bmal 1 gene contains 0.5u Taq polymerase, 0.006umol dNTP and 0.035umol Mg2+; the annealing temperature being 57C; circle times being 30. In a same volume, the optimal PCR mixture of dbp gene contains 0.5u Taq polymerase, 0.006umol dNTP and 0.03umol Mg2+; the annealing temperature being 58C; circle times being 32. The optimal PCR mixture of per2 gene contains 0.5u Taq polymerase, 0.006umol dNTP and 0.05umol Mg2+; the annealing temperature being 57C; circle times being 30. The PCR method to detect mRNA expression of clock genes in cultured rattus cardiac myocytes was establish successfully.3. Effect of AngII on expression of clock gene in cultured rattus myocytesMethods of cell culturing, hypertrophy inducing and PCR were as part 1 and 2. The expression of clock genes in myocytes coincubated with Ang II (0.1umol/L) for 3 hours and hypertrophic cardiac myocytes induced by Ang II (0.1 u mol/L) coincubated for 72 hours were detected using semi-quantitative RT-PCR. We found that expression of clock genes we studied was lower in hypertrophic cardiac myocytes.We established a method to study clock genes on cell level and found that the expression of clock genes was lower when cardiac myocytes was hypertrophic induced by Ang...