Figure 4iCSΔ Bmal1 −/− mice have prolonged QTc intervals during the light phase. Activation of the h Kcnh2 reporter is significantly diminished when BMAL1-R91A (B-R91A) was overexpressed with CLOCK (gray bar) or CLOCKΔ19 was overexpressed with BMAL1 (vertical striped bar). Luciferase assay results of transfection experiments using the h Kcnh2 reporter gene in C2C12 myoblasts ( n = 6–10/condition) are presented.ĬLOCK (C) and BMAL1 (B) significantly transactivate the h Kcnh2 reporter gene (solid bars) relative to control transfection (open bars). B: BMAL1 and CLOCK transactivate the human Kcnh2 promoter reporter. Shaded and light regions represent subjective dark and light phases. A: The circadian expression profile for Kcnh2 in the WKY rat heart (n = 4 animals per time point). Figure 3Kcnh2 transcripts follow a circadian pattern of expression in rat hearts, and the human Kcnh2 promoter is transactivated by the coexpression of BMAL1 and CLOCK. C and D: The mean Boltzmann data from the fits to the individual cells for maximally activated I Kr (I MAX, pA/pF), the slope factor (k, mV/e-fold change), and the midpoint potential for half-maximal current activation (V 1/2, mV) are shown for iCSΔ Bmal1 +/+ (solid bars) and iCSΔ Bmal1 −/− (open bars) mice (n = 11–13 cells.
B: The peak inward I Kr measured during the test pulse is plotted as a function of the prepulse potential, and the data are described using a Boltzmann equation (gray line). The peak I Na measured during the initial prepulse is cropped for presentation purposes.
The currents were recorded by prepulsing cells from −80 to 40 mV in 10-mV increments, followed by a test pulse to −80 mV. A: Representative families of currents recorded from iCSΔ Bmal1 +/+ or iCSΔ Bmal1 −/− ventricular cardiomyocytes (isolated from mice after 58 hours in constant darkness). Figure 2Disruption of BMAL1 signaling reduces I Kr. By using JTKCYCLE, the P value for Kcnd2 and Kcnh2 in iCSΔ Bmal1 +/+ mice was estimated as 0.003 and 7.02 × 10 −5, respectively, and the P value for Kcnd2 and Kcnh2 in iCSΔ Bmal1 −/− mice as.0095 and.060, respectively.Ĭhannels with P values. JTKCYCLE statistics were used to determine whether the expression pattern was circadian. The dark and light bars on the graph represent extrapolated subjective day and night as defined by circadian time according to the previous light/dark cycle before released into constant darkness. A: The JTKCYCLE best fit data for the mRNA expression profiles for the K + channel genes Kcnd2 and Kcnh2 from iCSΔ Bmal1 +/+ (solid circles) and iCSΔ Bmal1 −/− (gray squares) mouse hearts (n = 3–4 animals per time point). The molecular data reported will be useful for comparative analysis of gene regulation in the GH/PRL gene family in teleosts.Figure 1The cardiomyocyte molecular clock regulates the circadian expression of Kcnh2 transcripts as well as the expression of several K + channel gene transcripts that are not circadian. From a comparative point of view, this study of PRL gene in Sparus auratus, correlates well with those previously published on tilapia and rainbow trout. Transient expression experiments with 5 ′-delection mutants reveals at least three regulatory regions on the sbPRL gene, two with a stimulatory effect on transcription and one with apparent inhibitory effect. CHO culture cells co-transfected with a sbPRL promoter sequence and a sea bream Pit-1 cDNA expression plasmid showed expression of a linked luciferase reporter gene. Analysis of 1.0 kb of the proximal promoter sequence reveals a consensus TATAA box, up to seven (A/T) 3NCAT consensus motifs for binding of the pituitary-specific factor Pit-1 and putative CREB and GATA binding sites. The gene analyzed comprises 3.5 kb of DNA containing five exons as described previously for other fish PRL genes. A sea bream prolactin (sbPRL) gene was isolated using a prolactin cDNA fragment, generated by PCR as a probe.
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