Effects Of High Fat On The Expression Of Circadian Genes In Mouse Cardiomyocytes

The circadian clock is involved in regulating behavior and many physiological functions. The rhythms are entrained to the 24h day by the light-dark (LD) cycle to adapt the natural conditions. Circadian rhythms are generated by the transcription-translation feedback loops of the core circadian genes. In mammals, the master circadian clock is located in suprachiasmatic nucleus (SCN) of the hypothalamus. Apart from SCN, it is shown that peripheral tissues (such as heart, liver, kidney, etc) have circadian oscillators. By regulating the clock controlled genes (CCGs), clock genes regulate many physical functions. Clock genes also can be expressed rhythmically in cultured cells after induced by two-hour serum shock.Many researches show that the circadian clock plays important roles in several pathological processes such as tumor and cardiovascular diseases besides physiological functions. In cardiovascular disease, the occurrence of stroke and acute myocardial infarction showed a circadian rhythm with the peak in the morning hours. Although the precise mechanism underlying the phenomenon is still not clear, circadian clock may involve in the process. Our previous research demonstrated that the expression amplitude and rhythmicity of all the target clock genes and clock controlled genes which controlled apoptosis, blood clotting, vasoconstriction and lipid metabolism was changed in apoE-/-mice, respectively. Besides, it started to be paid attention to the relationship of the clock genes and metabolic disturbance of lipid. However, no report showed the exact day when the clock genes started to show rhythmicity in mouse. We would ascertain the exact day when the clock genes expressed with circadian rhythms in the mouse SCN and heart, then observed the effect of high fat on the expression of circadian genes in mouse cardiomyocytes and approach the relationship of the clock genes and metabolic disturbance of lipid. Part I The time when clock genes expressed circadian rhythms in C57BL/6J mouse heart and central tissuePurpose The aim was to ascertain the exact day when the clock genes expressed with circadian rhythms in mouse heart and central tissue.Design Female C57BL/6J mice were maintained separately on a 12L:12D light-dark cycle. Day of delivery was designated the postnatal day 0 (PO). For postnatal studies of PI, P3 and P5, infants were kept with their mother through the experiment. According to Zeitgeber time (ZT), three pups of mixed sexes were sampled at different time points including ZTO, ZT4, ZT8, ZT12, ZT16 and ZT20. Hearts were harvested, frozen quickly in liquid nitrogen. Brains were taken away from the skull. And SCN was dissected grossly, quick-frozen in liquid nitrogen and stored at-80℃until RNA isolation. The daily expression of four clock genes mRNA (Bmall, Per2, Cryl and Rev-erb alpha) in mouse SCN and heart was measured at P1, P3 and P5 by real-time PCR.Results:All the studied mice clock genes began to express in a circadian rhythms manner in heart and SCN at P3 and P5, respectively. Interestingly, the daily rhythmic phase of some clock genes shifted during the postnatal days. Moreover, the expressions of clock genes in heart were not synchronized with those in SCN until at P5.PartⅡEffects of high fat on the expression of circadian genes in mouse cardiomyocytesPurpose The goal was to detect the impacts of triglycerides (TGs) on the expression of circadian genes in the cultured mouse cardiomyocytes.Design Hearts of C57BL/6J mice were harvested at postnatal day 3. Cardiomyocytes were isolated from the hearts. After cultured on plates for 48 hours, the cardiomyocytes were treated with 50%horse serum for 2 hours (serum shock).Clock genes could be expressed rhythmically except mClock. Serial concentration (0.5,1,2.5,5,10mmol/L) of TGs was given to the cardiomyocytes in different petri dishes after serum treatment, and the Ommol/L TGs group was the control. According to Zeitgeber time (ZT), the time when to give the TGs was ZTO. Then the clock genes mRNA expression level was detected by real-time PCR every 4 hours in the next 60 hours and stored at-80℃until RNA isolation. There are 5 core circadian genes in mouse such as mBmall (brain and arylhydrocarbon receptor nuclear translocator-like protein 1), mRev-erba (reverse strand of the c-erba), mPer2 (period 2), mCry (Cryptochrome) and mClock which were measured by real-time PCR.Results We found that the mRNA expressions in the cells started to show 2 circles of daily rhythms from ZT12 to ZT60 after serum treatment, and the peak of the second circle was lower than the first. The mRNA expressions of circadian genes in cardiomyocytes treated with 2.5mmol/L TGs were equal with TGs-free group. However, the expressions of circadian genes in cardiomyocytes were decreased with lower concentration of TGs (0.5, 1mmol/L), and were dramatically increased with higher level of TGs (5,10mmol/L).Conclusion1. In mouse, clock genes began to express in a circadian rhythms manner in heart and SCN at P3 and P5 respectively, and the central clock synchronized the peripheral clock as early as P5.2. The mRNA expressions in the cells started to show 2 circles of daily rhythms from ZT12 to ZT60 after serum treatment, and the peak of the second circle was lower than the first.3. The mRNA expression of clock genes could be affected by the TGs and the tendency was bidirectional according to the concentration of TGs.