Fig 2: Loss of skeletal muscle Bmal1 alters the transcriptional response to acute exercise. (A) Experiment 1. Vehicle and iMSBmal1KO mice completed a 30-minute, work-matched 15° inclined treadmill bout and gastrocnemius muscles were collected 1-hour post. (B) Exercise bout work was matched between genotypes. (C) RNA-sequencing was used to assess the number of DEGs 1-hour post exercise in vehicle and iMSBmal1KO mice. (D) Venn overlap of vehicle and iMSBmal1KO mice exercise responses. (E) Pathway analysis of shared exercise response genes. (F) Z-scores of exercise responsive clock factors and common exercise response genes shown between genotypes. Associated pathway databases are indicated by (R) Reactome, (K) KEGG, and (BP) Biological Processes. Pathway analysis of vehicle exercise responsive genes (G) and iMSBmal1KO exercise responsive genes (H).

Fig 3: Loss of skeletal muscle BMAL1 indirectly disrupts transcriptional homeostasis of peripheral tissues. (A) Upset plot of the differentially expressed genes affected by loss of skeletal muscle BMAL1 across tissues. Set size indicates the total number of differentially expressed genes regulated by loss of BMAL1 in skeletal muscle in each tissue. Numbers above bars indicate the number of differentially expressed genes. Overlapping differentially expressed genes which are common between tissues are indicated by the connected points below bars. In summary when comparing vehicle control and iMSBmal1KO controls we identified 684 (425 upregulated, 259 downregulated) differentially expressed genes in inguinal white adipose tissue (iWAT), 199 (86 upregulated, 114 downregulated) in liver, 1177 (665 upregulated, 512 downregulated) in the heart and 110 (82 upregulated, 28 downregulated) in lung tissue where identified. Pathway analysis for differentially expressed genes in inguinal white adipose tissue (B), liver tissue (C), heart tissue (D) and lung tissue (E). Red segments of bars indicate downregulation with loss of BMAL1, blue segments of bars indicate upregulation with loss of BMAL1. Associated pathway databases are indicated by (R) Reactome, (K) KEGG and (BP) Biological Processes.

Fig 4: Disruption of circadian rhythm and BMAL1 expression in the skeletal system in senile osteoporosis(A) PET scans of 2-month-old and 20-month-old C57BL/6J mice after 18F-NaF tail vein injection at the indicated times. ZT0 is defined as the time the light was turned on (8 a.m.). (B) PET analysis of maximum and mean SUV. (C and D) Western blot analysis of the levels of the core rhythm components BMAL1 and CLOCK in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (E and F) HE staining (scale bar, 100 μm) of bone specimens from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (G and H) Immunofluorescence staining (scale bar, 100 μm) showed Bmal1 and Clock expression in the Ocn+ osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (I-J) Immunofluorescence staining (scale bar, 100 μm) showed BMAL1 and CLOCK expression in the OCN+ osteoblast lineage in young patients and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

Fig 5: Diurnal phosphoproteome of mouse liver. a Numbers of phosphosites, phosphopeptides, and phosphoproteins detected in our diurnal phosphoproteome of mouse liver. The distribution of diurnal rhythmic phosphoproteins in the total phosphoproteins. Hierarchical clustering of the cycling phosphoproteins ordered by the phase of the oscillation. Values for each phosphoprotein at all analyzed samples (columns) are color code based on the intensities, low (blue) and high (yellow) z-scored normalized iBAQ. The upper white to black bar indicates the 2 days’ cycle. Daytime is shown in white, while nighttime is shown in black. b Temporal abundance of DNA-binding activity of Bmal1, Clock, Dbp, and Nr1d1/Rev-erbα and their correlated abundance of their phosphorylated forms. X axis represents the sampled time points, Y axis represents the z-scored abundance. c Time-resolved map of signaling cascades regulated by diurnal changed phosphoproteins. Proteins (kinases, phosphoproteins, TFs) were colored based on their peak time, daytime-peaked proteins are yellow (detected in this study) or orange (detected in the study by Robles et al.10), while nighttime-peaked proteins were blue (detected in the study by Robles et al.10) or green (detected in this study). Diurnal rhythmic proteins (this study: JTK_CYCLE p < 0.1, Robles et al.10: q (adjusted p) < 0.1) were shown with red border. Box plots show the path from substrates to pathway-specific TFs were shorter than the path from substrates to all detected TFs (pair tailed Student’s t test p < 0.05). For the box plot, the bottom and top of the box are the first and third quartiles, and the band inside the box is the median of the paths between TFs and substrates