| The endogenous rhythm is modulated by the oscillator in the suprachiasmatic nucleus( SCN) in mammalian. Whether the autonomic variations of biological rhythms in different levels of cardiovascular system are modulated by SCN were here investigated .We shall study the modulation mechanism of biological rhythms of thecultured murine myocardiocytes in three stages. First stage, three concentrations: 10-8 mol/L, 10-6 mol/L, 10-4 mol/L Melatonin or/and diazpam interference test were respectively employed. Circadian variabilities in beating frequencies of the cultured myocardiocytes were dynamically measured in 24 h at six time points (local time 00: 00, 04: 00, 08: 00, 12: 00, 16: 00, 20: 00) by a inverted phase-contrast microscope coupled to high speed CCD camera and computer system .Then we analyze the data by single cosinor method. We found that The beating frequencies of the cultured myocardiocytes exist remarkable intrinsic circadian rhythm . Theacrophases of circadian rhythm of beating frequencies which were effected by melatonin were delayed. The acrophases of group in 10-8 mol/L melatonin was delayed significantly than that in 10-6 mol/L(P<0.05) . The biological rhythm of group in 10-4 mol/L Melatonin is disappearance. All of three concentrations diazepam can induce the acrophases of circadian rhythm of beating frequencies of the cultured myocardiocytes delayed , thire acrophases delayed are dose-dependence .Comparing with 10-8 mol/L melatonin group, the acrophasesdelayed of group in 10-8 mol/L melatonin combination with 10-8 mol/L diazepam is not signifinicant, so is the group in 10-8 mol/L melatonin combination with 10-4 mol/L diazepam ; Second stage, melatonin interference test was employed, mperl protein expression in the cultured myocardiocytes was detected by immunohistochemistry . Results indicat that two groups mperl protein expression exist remarkable intrinsic circadian rhythm, the acrophase of mperl protein expression of control group is at 15: 20, melatonin group is at 17: 15. Comparing with control group, the acrophase of melatonin group was delayed about 1. 55 h. It is similar to the acrophase delayed of the beating frequencies of the cultured myocardiocytes which was effected by melatonin; Third stage, Gene expression blocken by ribozyme was employed. We detect mperl mRNA expression and mperl protein expression in the cultured myocardiocytes with RT-PCR, Western blot. Agarose gels electrophoresis shows that PCR products is 640bp in empty vector transfect cultured myocardiocytes group (control group ), it matchs with products we anticipated. The abundance of mperl mRNA shows rhythmic change in six timepoints , it is lowest at 00: 00, and highest at 08: 00; Agarose gels electrophoresis shows that mperl mRNA dose not express in ribozyme transfect cultured myocardiocytes group (ribozyme group). Western blot shows that six bands present at protein marker 120 000, the protein accumulate exist rhythmic change insix timepoints in control group, it is lowest at 04: 00, and highest at 12: 00. mperl protein dose not express in ribozyme group; The beating frequencies of the cultured myocardiocytes exist circadian rhythmic change in control group(empty vector transfect cultured myocardiocytes group ) detected by a inverted phase-contrast microscope coupled to high speed CCD camera and computer system .Its acrophases is at 15: 20. The biological rhythm of ribozyme group is disappearance. From above ,we draw a conclusion: Melatonin and diazepam as 'Zeitgeber' could entrain the circadian rhythm of cultured murine myocardiocytes beatings . The circadian rhythm of mperl gene expression in cultured murine myocardiocytes could be entrained by melatonin ; Ribozym could block mperlmRNA expression therefore the cycle of circadian clock gene was broken and it results in circadian rhythm of beating frequencies of the cultured myocardiocytes disappearing.Based on those results ,circadian rhythm in body dose not only exists in central nervous system ,but it also exists in other organs or tissues ,...
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