The Core Of Circadian Clock Gene BMAL1 Regulates Circadian Rhythmicity Of Cardiac Sodium Calcium Exchanger

Due to the rotation of the earth and illumination,it become a circadian cycle of 24h on earth,which makes the life on the earth evolve the Circadian Clocks systemthat can adapt to these natural environment.Circadian clock gives life the ability to predict changes in time and environment,regulate their behavior and physiologicalactivities,and synchronize the activity of organism with the cycle of external change The mammalian circadian clock can be divided into two categories:central andperipheral circadian clock.The central clock located in the suprachiasmatic nucleus of the hypothalamus(SCN),as a pacemaker,through the acceptance of outside lightstimulation to generate and maintain the daily cycle.At the same time,the peripheral circadian clock is synchronously regulated by signal transmission of the neurohumoral signal to control a series of circadian rhythms of physiological activity,such as heart rate,cardiac output,hormone secretion,energy metabolism and sleep-wake.It has been reported that the incidence of ventricular arrhythmias(VA)andsudden cardiac death(SCD)follows a diurnalvariation with the well-knownmoming peaks.Accumulating evidence has shown that such diurnal variations reflect myocardial stimulus-response coupling which is orchestrated by intrinsic circadianclock.Circadian clocks enable the myocytes to anticipate environmental stimuli,facilitating a timely and appropriate response.Disruption of the diurnal rhythms within cardiomyocytes would directly or indirectly contribute to the etiology of ardiac dysfunction and diseases.Excitation-contraction coupling(E-CC)is the process whereby an action potential triggers myocytes to contract,which is mediated by cyclic changes in intracellular Ca2+.During cardiac cycle,dynamic Ca2+cycling plays an essential role in maintaining the proper E-CC,which is finely tuned via acoordination of Ca2+handling proteins including L-type Ca2+channel,ryanodinereceptor,Ca2+-ATPase and Na+/Ca2+exchangers(NCX1).As one of important Ca2+handling protein that extrudes Ca2+from the cytoplasm,NGX1 participates regulating intracellular Ca2+ homeostasis for cardiomyocytes.In previous study,we observed that NCX1 exhibited Bmall-associated endogenous rhythmic expression in 12:12 light/dark(LD)-entrained mouse heart.In this study,we further define the regulatory role of Bmall on NCX1 with a mousemodel that was kept in constant darkness(DD)to allow for free-running circadianrhythms and the cell model with the circadian oscillation induced by the horse serumshock in the atrial tumor cell derived HL-1 cellsor the primary cultured neonatal ratventricular myocytes(NRVMs).he results were as follows:Under the condition of DD,cardiac NCX1 exihibited intrisinc circadianoscillation characterized as its mRNA and protein expression peaked at CT1 while troughed at CT9,which resembled the patterns of cardiac BMAL1,a component of the core circadian clock gene.2.Adenovirus-mediated overexpression of Bmall in HL-1 cells and primarycultured NRVMs which underwent a stimulation of horse serum shock resulted in the increases in the amplitude of Ncxl circadian oscillation.In contrast,Ad-shRNA-mediated knockdown of Bmall in those cells correspondingly led to decreases in the amplitude of its circadian oscillation.However,no matter the upre-or down-regulation of Bmall had no impact on the phase of Ncxl circadian expression These results implied that cardiac Ncxl is a clock controlled gene.3.The results obtained from ChIP(chromatin immunoprecipitation)assay showed that the core clock gene Bmall was recruited to the upstream sequence of the Ncx1 promoter,where consisted of typical transcriptional regulatory elements(refer to E-box).Furthermore,the reultes of luciferase reporter gene assay confirmed that the binding of Bmall with the proximal 4 E-box sites(CATATG,CATGTG,CACTTG and CAAATG)in upstream(-542bp)sequence of the Ncx1 promoter was required fortransactivation,indicating a direct regulatory role of Bmall on Ncx1.4.The pooled data from NCX1 current(INCX1)recordings in HL-1 cells using whole cell patch clamp technique showed that the overexpression of BMAL1 significantly increased the current density of INCX1 compared with the Ad-GFPcontrol,strongly supporting that the cardiomyocyte molecular clock BMAL1 contributes to the functional regulation of INCX1.Collectively,the present study proved that cardiac Ncx1 exhibited endogenousrhythmicity that was regulated by Bmal1,which was achieved via binding itself on theupstream sequence of Ncx1 promoter containing 4 key transcriptional regulatory elements.