| Background: In all mammals, behavioral and physiological processes display 24-hour rhythms that are regulated by circadian pacemakers. The circadian clock is a self-sustaining oscillator with a period of about 24 hours that includes input, central oscillator and output element. In doing so, circadian clocks confer the selective advantage of anticipation, conditioning the cell to changes in its environment before they occur. In mammals, the central circadian pacemaker is located in the suprachiasmatic nucleus of the brain, whose phase is directly light-entrained by the retina. Recent experiments have shown that peripheral tissues like heart, liver and kidneys also have oscillatory systems that contain a transcriptional - translational feedback loop of clock genes, which involve three homologs of the Drosophila gene period (mPerl, roPer2, and roPer3), two cryptochrome genes (mCryl and mCry2), and the transcriptional activator genes Clock and Bmall. The heart also contains fully functional internal clocks, and several cardiac genes exhibit circadian oscillation in their expression. Given the universal appreciation for diumal variations in both physiological and pathophysiological cardiovascular events, the recent exposure of molecular mechanism within the individual cells of the cardiovascular system that has the potential of modulating an array of cellular processes has sparked increasing interest.Myocardial hypertrophy, an increase in cardiac cell size without cell division, is induced by multiple stimulators, such as mechanical overload, neural stress and humoral factors. Among the neurohormenes, AngiotensinⅡ(AngⅡ) is an important regulator of blood pressure, fluid, electrolyte balance. As a mitogen activating factor, AngⅡseems to be involved in the pathogenesis of cardiovascular disorders such as hypertension and pressure overload cardiac hypertrophy. AngⅡhas been shown to activate various protein kinase pathways through the AngⅡtype 1 receptor. Especially, it activates ERK, JNK, P38, which are critical protein kinases in cells for their growth, apoptosis and gene expression.Objective: We investigated whether the circadian expression of clock genes will change, and whether there is a circadian expression and activation of ERK in AngⅡ-induced hypertrophic cardiomyocytes. Furthermore, we try to elucidate the relationship between ERK and clock genes.Method: (1) Myocytes were prepared from cardiac ventricles of 3-day-old neonatal Sprague-Dawley rats. The cells were cultured at 37℃ in 5% CO2 for 72 hours before our treatment. (2) The cells were divided into 3 groups: AngⅡ, Telmisartan (Tel), Control. For AngⅡgroup, cells were treated by AngⅡwith a final concentration of 10-7 mol/L. While for the Tel group, telmisartan was used with a final concentration of 10-7 mol/L for 30 minutes before AngⅡtreating. For each group, we did these once a day at the same time (6:00 PM) for 3 days. (3) We then detected the gene expression of At1, At2, Bmall and Per2 of three groups respectively. (4) We detected the expression of AT1, AT2, ERK, phospho-ERK, BMAL 1 and PER2 protein of three groups respectively.Results: (1) The results of measuring the cell surface area of cardiomyocytes and total amount of protein suggest that the model of cultured hypertrophic cardiac cells was established successfully. (2) According to the results of RT-PCR, expression of At1 in AngⅡgroup significantly increased, compared with the control group. In contrast, the expression of At2 in all groups had no conspicuous differences. In three groups, the gene expression of Bmall had a peak at CT16, a trough at CT8. Per2 has an antiphase rhythm, with a peak at CT4 and a trough at CT16. Treatment of cardiomyocytes with angiotensinⅡ(10-7mol/L, 3 days) resulted in a significant increase in rhythmic expression of Bmall and Per2. This effect was completely abolished by preincubation with telmisartan. (4) The results of Western Blot suggested that the protein expressions of AT1, ERK and phospho-ERK in the AngⅡgroup significantly increased. In contrast, the protein expression of AT2 in all the three groups had no conspicuous differences. The expression of BMAL 1 and PER2 protein displays rhythmicity in all the three groups. Unremit treatment of cardiomyocytes with angiotensinⅡresulted in a significant increase in rhythmic expression of BMAL 1 and PER2, which was completely abolished by preincubation with telmistan.Discussion: The cultured cardiomyocytes possess a circadian oscillation system which is comparable to that of in vivo intact heart. AngⅡ, as a potential zeitgeber, upregulates circadian gene expression in cultured hypertrophic cardiomyocytes which is trigged by increasing activation of ERK pathway.
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