Cryptochromes Suppress HIF1α in Muscles

Abstract

Muscles preferentially utilize glycolytic or oxidative metabolism depending on the intensity of physical activity. Transcripts required for carbohydrate and lipid metabolism undergo circadian oscillations of expression in muscles, and both exercise capacity and the metabolic response to exercise are influenced by time of day. The circadian repressors CRY1 and CRY2 repress peroxisome proliferator-activated receptor delta (PPARδ), a major driver of oxidative metabolism and exercise endurance. CRY-deficient mice exhibit enhanced PPARδ activation and greater maximum speed when running on a treadmill but no increase in exercise endurance. Here we demonstrate that CRYs limit hypoxia-responsive transcription via repression of HIF1α-BMAL1 heterodimers. Furthermore, CRY2 appeared to be more effective than CRY1 in the reduction of HIF1α protein steady-state levels in primary myotubes and quadriceps in vivo. Finally, CRY-deficient myotubes exhibit metabolic alterations consistent with cryptochrome-dependent suppression of HIF1α, which likely contributes to circadian modulation of muscle metabolism.

Keywords: Cell Biology; Chronobiology.

Graphical abstract

Figure 1

CRYs Suppress HIF1α (A) Accumulation of HIF1α protein detected by immunoblot (IB) in unsynchronized primary myotubes (1°MTs) isolated from WT and dKO mice and treated with vehicle control or 100 μM CoCl₂. Two technical replicates are shown for dKO 1°MTs. (B) Accumulation of HIF1α protein detected by IB in ear fibroblasts (EFs) isolated from WT, Cry1^−/−, Cry2^−/−, and dKO mice and treated with vehicle control or 0–0.2 mM CoCl₂. Faint vertical lines indicate where blot images were spliced to remove samples treated with 50 μM CoCl₂. (C) Expression of the indicated transcripts measured by quantitative PCR (qPCR) in fibroblasts of the indicated genotypes, normalized to Hprt. (D) Accumulation of HIF1α protein detected by IB in EFs isolated from mice of the indicated genotype and infected with virus carrying either empty vector or pBABE-mCry2 plasmid, before treatment with vehicle control (−) or 100 μM CoCl₂ (+). (Note: the faint CRY2 signal in lane 12 reflects a small amount of sample from lane 13 that spilled into the neighboring well). (E) Left, accumulation of HIF1α protein detected by IB in nuclei of cells isolated from quadriceps muscles of mice of the indicated genotype at ZT16. Right, quantitation of three experiments performed as shown at left. (F) Accumulation of HIF1α protein detected by IB in unsynchronized 1°MTs isolated from mice of the indicated genotype upon exposure to 1% O₂ for 0–4 h. (G) Expression of the indicated transcripts measured by qPCR in unsynchronized 1°MTs plated under parallel conditions as those in (F). For (C and E), data represent the mean + SD for 3 samples per condition. ∗p < 0.05 versus WT by one-way ANOVA with Dunn's multiple comparison test. In (G) data represent the range (min to max with mean ± SD shown in box) for 6 samples per condition, each measured in triplicate. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 for a main effect of genotype (G), hypoxia (H), or an interaction between the two, determined by two-way ANOVA. Results of post-hoc analysis are not shown to highlight the main effect results of two-way ANOVA. The position of molecular weight markers (in kDa) are shown to the left of all western blot images. Note that overexpressed epitope-tagged proteins are slightly larger than endogenous ones. See also Figure S1.

Figure 2

HIF1α Interacts with the Core Clock Machinery (A–E) (A–C and E) Proteins detected by immunoblotting (IB) in whole-cell lysates (WCL) or following immunoprecipitation (IP) of the FLAG tag from lysates of HEK293T cells expressing the indicated plasmids and treated with vehicle control (−), 100 μM CoCl₂, or 200 μM DMOG as indicated. (D) Endogenous BMAL1, HIF1α, and β-ACTIN detected in WCL or following IP of HIF1α from lysates of ear fibroblasts of the indicated genotypes treated with vehicle control (−) or 100 μM CoCl₂.

Figure 3

HIF1α Interacts with Clock Proteins via Unique Domains (A) Schematic diagram depicting five residues in the ARNT protein described in Wu et al. (2015) as critical for interaction between ARNT and HIF2α, as well as the location of the corresponding residues in BMAL1 (also known as ARNTL). Gray dashed lines depict interactions between interfaces of each heterodimer pair. Red stars indicate residues found to be critical for interaction between BMAL1 and HIF1α, whereas blue stars indicate residues found to be critical for BMAL1/CRY2 interaction. (B) Sequence conservation between bHLH-PAS family proteins in the 115–343 amino acid region where mutations were introduced. (C–E) Proteins detected by immunoblotting (IB) following immunoprecipitation (IP) of the FLAG (C and D) or hemagglutinin (HA) (E) tags from lysates of HEK293T cells expressing the indicated plasmids and treated with either vehicle control (−) or 100 μM CoCl₂ (+). See also Figure S2.

Figure 4

CRYs Repress BMAL1-Containing Heterodimers (A–F) Luciferase activity in U2OS cells expressing Per2:Luciferase (A–C) or HRE:Luciferase (D–F). Activation of the reporter is achieved by transfection of the indicated plasmids (B, BMAL1; C, CLOCK; H, HIF1α). mCherry was used as a negative control, and luminescence was normalized to Renilla luciferase activity. Data represent the mean + SD superimposed with 3–11 individual replicates per condition from a representative of at least three experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA.

Figure 5

CRYs alter Muscle Metabolic Profile (A–E) Stable isotope tracing analysis and metabolite abundance performed in primary myotubes (1°MTs) from mice of the indicated genotype. Fractional enrichment (A) or mole percent enrichment (MPE) (B–D) in metabolites following addition of indicated tracer for 24 h. For (A–E), data represent the mean ± SEM of 3 replicates per condition, which are representative of 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 from two-tailed Student's t test. (F) Glucose dependency measured in unsynchronized 1°MTs isolated from mice of the indicated genotype and treated with either vehicle control (black) or 100 μM CoCl₂ (blue). ∗∗p ≤ 0.01 versus control by t test using two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. Data in (F) represent the mean + SEM of the results from 4 independent experiments. See also Figure S3.