Figure 1.
Novel bioinformatics workflow to identify circadian regulated cardiac genes.
Distinct patterns of circadian gene expression in the heart were visualized using the Circadian Expression Profiles Data Base (CircaDB) and embedded JTK_Cycle algorithm [19]–[22] using the JTK_Cycle parameters p<0.001, period 23 h to 25 h, and phase 16 h to 20 h. The gene list was further enriched for high cardiac mRNA expression levels, using the BioGPS website and GeneAtlas MOE430 gene expression/activity display [28], [29]. Genes were selected if they exhibited a ≥1.5 fold increased expression in heart. An in silico circadian motif search was then performed to detect conserved E-box elements in the gene promoter regions that could be used for CLOCK and BMAL1 transcription, using the University of California Santa Cruz (UCSC) table browser tool [30], the circadian mammalian promoter/enhancer database (PEDB) [32], and the European Bioinformatics Institute pairwise nucleotide alignment tool (EMBOSS) [33]. Circadian regulation of candidate gene Tcap was investigated experimentally using in vivo and in vitro approaches.
Table 1.
Reference Genes.
Table 2.
The 22 Rhythmic Genes Highly Expressed in Heart vs. 96 Murine Tissues.
Table 3.
Heart-enriched rhythmic proteins with human-mouse conserved E-box motifs.
Figure 2.
Diurnal cardiac Tcap mRNA and TCAP protein rhythms.
Hearts were collected every 4 h across the 12∶12 L:D cycle from C57Bl/6N mice, and used for qRTPCR (mRNA) or Western blot (protein) analysis. (A) Tcap mRNA (dotted line) exhibited rhythmic expression (JTK_Cycle, p = 7.41×10−5) with a peak in the light phase at ZT07 (murine sleep time) and trough in the dark phase (n = 3/time point). TCAP protein (solid line) also exhibited a rhythmic profile (JTK_Cycle, p = 0.00139) that peaked in the light and reached a nadir in the dark (n = 3/time point). There was a 4 h phase delay between mRNA expression and protein abundance. (B) Representative Western Blot, illustrating TCAP protein abundance over the 12∶12 LD cycle. The diurnal environment of 12 h dark (black bars, animal’s subjective wake time) and 12 h light (white bars, animal’s subjective sleep time) is illustrated by the bars below the graphs.
Figure 3.
Circadian rhythms in Tcap and Per2 mRNA expression in WT hearts, and blunted expression levels and amplitude in ClockΔ19/Δ19 heart.
Starting at 30 h after transfer into D:D (CT = 18) the mice were euthanized, and hearts collected every 4 h from the ClockΔ19/Δ19 (○) and WT (▪) littermate mice (n = 4/time point). Rhythmic expression of (A) Tcap mRNA (JTK_Cycle, p = 1.09×10−7), and (B) Per2 mRNA (positive control; JTK_Cycle, p = 1.18×10−9) in WT hearts. In contrast in ClockΔ19/Δ19 hearts, gene expression is barely periodic and amplitude is severely blunted consistent with being target genes. The circadian environment of 12 h dark (black bars, animal’s subjective wake time) and 12 h dark (grey bars, animal’s subjective sleep time) is illustrated by the bars below the graphs.
Figure 4.
Tcap is a transcriptional target via binding to E-box motifs.
(A) Chromatin immunoprecipitation (ChIP) assay performed using anti-BMAL1 antibody (n = 3/time point). Rabbit IgG was used as a negative immunoprecipitation control. (B) BMAL1 binding motifs in three E-box consensus sequences in the Tcap promoter, with location relative to the TSS noted. (C) BMAL1 was strongly precipitated by the −274 E-box, by DNA precipitation assays using biotinylated oligonucleotides. (D) BMAL1 increased Tcap promoter activity, whereas deletion of sequences to −257 (relative to the TSS, deleting the −274 motif, represented as the black box) abrogated increases in Tcap promoter activity, by luciferase reporter assay. Mean ± SEM, n = 4, # = p<0.05. (E) Illustration of circadian clock regulation of sarcomere architecture and thus diurnal cardiac structure and function. Left, (∼) = molecular clock mechanism; centre = cardiac sarcomere.