Atypical expression of circadian clock genes in denervated mouse skeletal muscle

<div><p>The central circadian clock in the suprachiasmatic nucleus of the hypothalamus synchronizes peripheral clocks through neural and humoral signals in most mammalian tissues. Here, we analyzed the effects of unilateral sciatic denervation on the expression of circadian clock- and clock-controlled genes in the gastrocnemius muscles of mice twice per day on days 0, 3, 7, 9, 11 and 14 after denervation and six times on each of days 7 and 28 after denervation to assess the regulation mechanism of the circadian clock in skeletal muscle. Sciatic denervation did not affect systemic circadian rhythms since core body temperature (Day 7), corticosterone secretion (Days 7 and 28), and hepatic clock gene expression remained intact (Days 7 and 28). Expression levels of most circadian clock-related genes such as <i>Arntl, Per1, Rora, Nr1d1</i> and <i>Dbp</i> were reduced in accordance with the extent of muscle atrophy, although circadian <i>Per2</i> expression was significantly augmented (Day 28). Cosinor analysis revealed that the circadian expression of <i>Arntl</i> (Days 7 and 28) and <i>Dbp</i> (Day 28) was phase advanced in denervated muscle. The mRNA expression of <i>Clock</i> was significantly increased in denervated muscle on Day 3 when the severe atrophy was absent, and it was not affected by atrophic progression for 28 days. Sciatic denervation did not affect the expression of these genes in the contralateral muscle (Days 7 and 28), suggesting that humoral changes were not involved in denervation-induced muscle clock disruption. We then analyzed genome-wide gene expression using microarrays to determine the effects of disrupting the molecular clock in muscle on circadian rhythms at Day 7. Among 478 circadian genes, 313 lost rhythmicity in the denervated muscles. These denervation-sensitive genes included the lipid metabolism-related genes, <i>Nrip1, Bbs1, Ptgis, Acot1, Scd2, Hpgd, Insig1, Dhcr24, Ldlr</i> and <i>Mboat1</i>. Our findings revealed that sciatic denervation disrupts the circadian expression of clock and clock-controlled genes either directly or indirectly via muscle atrophy in the gastrocnemius muscles of mice in a gene-specific manner.</p></div

RNA-sequence analysis of <i>Phf2</i> knockout (KO) cells at 2 d post differentiation.

(A) Principal components analysis with RNA-seq analysis of Phf2 control cells and Phf2 KO cells (n = 3 wells in each group in one experiment). (B) Volcano plot of the RNA-seq data obtained from Phf2 KO cells 2 d post differentiation. (C) Heatmap visualizing the expression profiles based on RNA-seq analysis of Phf2 control and Phf2 KO cells (n = 3 wells per group). (D) KEGG pathway analysis of differentially expressed genes (DEGs) in Phf2 KO C2C12 cells. Numbers in parentheses indicate the number of genes associated with each enriched term. (E) BP DIRECT analysis of DEGs in Phf2 KO C2C12 cells. Numbers in parentheses indicate the number of genes associated with each enriched term.</p

The effect of <i>Phf2</i> knockout (KO) on myotube formation at 7 d post differentiation.

(A) Phase-contrast microscopy images of mock and Phf2 KO cells 7 d post differentiation. Scale bar, 100 μm. (B) Myotube diameter in mock and Phf2 KO cells 7 d post differentiation (n = 100 cells per group in one experiment). (C) Immunofluorescence staining of Myosin Heavy Chain (MyHC, red) and nuclei (blue). (D) Total MyHC-expressing area (n = 8, cells per group in one experiment). (E) Myotube fusion index based on the ratio of MyHC-nuclei to total nuclei (n = 10, cells per group in one experiment).</p

<i>Phf2</i> expression and PHF2 localization in C2C12 myotubes.

(A) Phf2 mRNA expression during differentiation in C2C12 myotubes (n = 4 wells per condition in one experiment). (B) Immunofluorescence staining of PHF2 (green) and DAPI (blue) in C2C12 myotubes on days 0 and 7 post differentiation. Scale bar, 100 μm.</p

Expression of genes and recruitment of H3K9me2 related to muscle myogenesis in <i>Phf2</i> KO cells.

(A) mRNA expression of Mef2c, Mybpc2, Myh7, Myh2, Myh1, Myh4, Mylk2, and Tnnt2 in mock and Phf2 KO cells based on qRT-PCR (n = 4 wells per condition in one experiment). (B) CUT&RUN-qPCR analysis of H3K9me2 mark in the promoter region of Mef2c, Mybpc2, and Myh7 using mock and Phf2-KO cells (n = 3 wells per condition in one experiment). The amplicon positions from TSS were indicated in parentheses. *P P P P < 0.0001 compared with mock C2C12 myotubes.</p

The sequence of the oligonucleotides for qPCR.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.</div

Expression levels of genes involved in muscle myogenesis in <i>Phf2</i> KO.

Expression levels of genes involved in muscle myogenesis in Phf2 KO.</p

The primer sequence for qPCR.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.</div

Generation of <i>Phf2</i> knockout (KO) cells in C2C12 myoblasts.

(A) Overview of the generation of Phf2 KO C2C12 cells. (B) Myoblasts expressing ZsGreen were obtained via cell sorting. Sorted untransfected C2C12 cells (without fluorescence) were used as negative controls. Phf2 KO and mock cells were sorted under the same conditions. Sorted cells are indicated in the orange areas. (C) Protein levels of PHF2 were determined using the Simple Western System.</p

Preventive effect of <i>Humulus lupulus</i> on disuse muscle atrophy.

<p>Mice consumed a <i>Humulus lupulus</i>-mixed diet for 14 days, after which denervation was carried out. After 4 days, the weight of the GM was measured. The level of atrophy was calculated to be the ratio of the weight of denervated muscle to the weight of sham muscle in each mouse. Data are the mean ± S.E (n = 3). Asterisks indicate significant differences analyzed by the Student’s <i>t</i>-test (P = 0.0046).</p