The Role Of MiRNA103/Cav1.2 Singnal Pathway In The Circadian Rhythm Of Cerebrovascular Function In Simulated Microgravity Rat

BackgroundAstronauts usually show cardiovascular dysfunction characterized by orthostatic intolerance and declined exercise capacity after returning to the ground which affects the astronaut's health. Howerer, the underlined mechanism of cardiovascular dysfunction has not yet been fully elucidated, there has been no effective and satisfactory countermeasures. Previous studies have shown that microgravity causes region specific vascular remodeling. In the vascular beds of lower body, resistance vessels would undergo hypotrophy and decrement in myogenic tone and vasoreactivity. On the contrary, cerebral arteries would undergo hypertrophy and vasoreactivity enhancement due to adaptation to cerebral hypertension. A variety of molecular mechanisms involved in regulating the microgravity induced vascular remodeling such as ion-channel remodeling in vascular smooth muscle cells and vascular NO-NOS and local renin�angiotensin system(L-RAS). Recently, researches on circadian rhythm show that cardiovascular system is closely related to circadian rhythm. Not only the physiological functions of the cardiovascular system exhibite circadian rhythm, but circadian rhythm disorders promote the deterioration of cardiovascular dysfunction. However, whether circadian rhythm disorders contribute to the vascular remodeling induced by microgravity has not been reported.L type calcium channel(Cav1.2) is one of the important pathway of extracellular calcium influx, which plays a key role in the muscle excitation contraction coupling, and closely involved in arterial smooth muscle cell phenotype switch, proliferation and apoptosis regulation.Our previous work has shown that the simulated weightlessness can induce the expression of Cav1.2 in cerebral arteries.Studies have shown that Cav1.2 as an clock controled gene involved in regulating the adaptation of retina to the light changes, and the expression and function of Cav1.2 shows circadian rhythm. However, circadian rhythm of Cav1.2 in the cardiovascular system is not yet fully investigated, so we observed the Cav1.2 circadian expressions in cerebral arteries and the changes induced by simulated microgravity, in order to clarify possible molecular mechanisms of simulated microgravity induced circadian rhythm disorders in cardiovascular functions.Existing research shows that micro RNAs(mi RNA) involve in regulating circadian expressions of Cav1.2. mi RNA is a kind of regulatory non-coding RNA, through combining complementary m RNA induces m RNA degradation or inhibits the protein translations. It has confirmed that there are 8 mi RNA regulating the expression of Cav1.2, and we eventually determined the mi RNA-103 played a role in the circadian expression of Cav1.2 in cerebral arteries, and confered that the disturbed circadian expression of Cav1.2 might be induced by the abnormal expression of mi RNA-103. Purpose 1. To confirm the effect of stimulated microgravity on the circadian rhythm incardiovascular function; 2. To explore the effect of stimulated microgravity on the circadian oscillation of clock genes and the downstream L type calcium channel;3. To confirm the regulation of mi RNA-103 on Cav1.2 circadian expressions.Methods 1. Four weeks tail suspended rats(SUS)were adopted as the animal model to simulate weightlessness. Caudal arterial pressure and heart rate were measured every three hours;2. Middle cerebral artery vessel tone and abdominal aorta vascular contraction were measured at 13:00 and 1:00, respectively;3. Western blot and q PCR was performed to detect the protein and m RNA expression level of clock genes such as Per2(Period2)?Bmal1(Aryl hydrocarbon receptor nuclear translocator � like 1),dbp(D element binding protein) and L-type calcium channel in suprachiasmatic nucleus(SCN) and cerebral artery at different time points(8:00, 12:00, 16:00, 20:00, 24:00, 4:00);4. In vascular smooth muscle cells A7r5, circadian rhythm was induced by serum shock with 50% horse serum treatment for two hours. From the time point of serum shock treatment, every four hours the expression of clock geneswere detected by western blot;5. To screen the candidate mi RNA involved in Cav1.2 expression regulation according to the circadian rhythm and alteration upon stimulated microgravity treatment;6. Vascular smooth muscle cells A7r5 were transfected with mi RNA and followed by serum shock treatment. Then, the circadian expression of Cav1.2 proteins was recorded;7. The software Graph Pad 5 was adopted to statistical analysis. Two groups werecompared using t test and three groups or more were compared using analysis ofvariance(ANOVA) or repeated measurement variance analysis. The results expressedas mean � SEMand P<0.05 represent the difference is significant.Results 1. The circadian rhythm in cardiocascular system is disturbed by simulated microgravity.1) The circadian rhythm in cardiovascular function was disturbed by simulated microgravity. The blood pressure and heart rate in control group was higher at night-time than at day-time which was circadian rhythmic, while the circadian rhythm in SUS was compromised. At daytime, heart rate in suspension rat was higher than that in CON(P < 0.05), and there was no statistical differences of systolic blood pressure and diastolic blood pressure between two groups of rats; At night-time, heart rate of suspension rat was higher than the CON(P < 0.05), while systolic blood pressure and diastolic blood pressure were lower in SUS than CON(P < 0.001, P < 0.05).And the amplitudes of oscillation in heart rate, systolic blood pressure and diastolic blood pressure in SUS were significantly lower than CON(P < 0.001, P < 0.001, P < 0.001), suggesting the circadian rhythm of cardiovascular functions is disturbed by simulated microgravity.2) The difference of abdomen aorta contraction function between day and night was compromised by simulated microgravity. At both day-time and night-time the contraction of abdominal aorta induced by norepinephrine in SUS was significantly lower than CON(P < 0.05, P < 0.001). Furthermore, the difference of abdominal aorta contractive reactivity between day and night was significantly compromised by simulated microgravity(P < 0.001).3) The difference of middle cerebral artery vessel tone between day and night was compromised by simulated microgravity. At both day-time and night-time the middle cerebral artery vessel tone in SUS was significantly higher than CON(P < 0.05, P < 0.05). Furthermore, the difference of middle cerebral artery vessel tone between day and night was significantly compromised by simulated microgravity(P < 0.001).2. The circadian expression of Cav1.2 and clock genes is disturbed by simulated microgravity.1) The circadian expression of CaV1.2 in cerebral arteries was disturbed by Simulated microgravity. The protein expression of Cav1.2 in cerebral arteries from CON was higher at night-time than at day-time which was circadian rhythmic, while the circadian expression of Cav1.2 proteins in cerebral arteries from SUS was compromised. At day-time, Cav1.2 expression in SUS rat is higher than CON(P < 0.01), while the difference of Cav1.2 expression between two groups was vanished at night-time.Furthermore, the amplitude of Cav1.2 protein expression oscillation was compromised in SUS(P < 0.05).In transcription level, the relative expression level of Cav1.2 m RNA in cerebral arteries of both two groups showed no difference between day and night, and the Cav1.2 m RNA were higher in SUS than CON through the day(P < 0.01, P < 0.05).2) The circadian rhythm of clock genes in cerebral arteries was disturbed by simulated microgravity. The protein and m RNA expression level of clock genes Per2, Bmal1, dbp in cerebral arteries showed circadian oscillation in CON, and the oscillation amplitude in SUS was significantly compromised.3) The circadian rhythm of clock genes in suprachiasmatic nucleus(SCN) was disturbed by simulated microgravity. The protein and m RNA expression level of clock genes Per2, Bmal1, dbp in SCN showed circadian oscillation in CON, and the oscillation amplitude in SUS was significantly compromised.3. miRNA-103 is the key molecule in regulating the circadian rhythm of Cav1.21) The circadian expression of clock genes in smooth muscle cell A7r5 could be induced by serum shock.2) Through detecting the expression of mi RNA in cerebral arteries from CON and SUS rats at day-time or night-time, mi RNA-103 was the potential mi RNA which regulated the circadian rhythm of Cav1.2 in cerebral arteries. The expression of mi RNA-103 in cerebral arteries from CON was rhythmic, and simulated microgravity could upregulate the expression and disturb the circadian rhythm of of mi RNA-103 in cerebral arteries.3) Cav1.2 expression in A7r5 cell was reduced after transfection of mi RNA- 103 mimic(P < 0.001), and mi RNA-103 inhibitor transfection could upregulate the expression Cav1.2(P < 0.05).After the transfection with mi RNA-103 mimic, inhibitor into A7r5 cells, the cells were incubated with 50% serum to induce the rhythmicity, and the results showed that transfection with mi RNA-103 mimic would inhibit the expressionof Cav1.2 and narrow the oscillation amplitude, while transfection with mi RNA-103 inhibitor would facilitate Cav1.2 expression and enlarge the oscillation amplitude.Conclusion Our data showed that circadian rhythm of the cardiovascular system could be disturbed by simulated microgravity. The difference between day and night both in blood pressure and heart rate compromised significantly. The circadian thythm disorder in cardiovascular function induced by simulated microgravity was associated with the changes of expression of clock genes and the downstream Cav1.2. In addition, mi RNA-103 was proved to negatively regulate the expression of Cav1.2 and the circadian oscilation.