Ribonucleotide reductase M2B in the myofibers modulates stem cell fate in skeletal muscle

Abstract

The balance among quiescence, differentiation, and self-renewal of skeletal muscle stem cells (MuSCs) is tightly regulated by their intrinsic and extrinsic properties from the niche. How the niche controls MuSC fate remains unclear. Ribonucleotide reductase M2B (Rrm2b) modulates MuSC quiescence/differentiation in muscle in response to injury. Rrm2b knockout in myofibers, but not in MuSCs, led to weakness of muscles, such as a loss of muscle mass and strength. After muscle injury, damaged myofibers were more efficiently repaired in the Rrm2b myofiber-specific knockout mice than the control mice, but these myofibers were thinner and showed weak functioning. Rrm2b-deleted myofibers released several myokines, which trigger MuSCs to differentiate but not re-enter the quiescent stage to replenish the stem cell pool. Overall, Rrm2b in the myofibers plays a critical role in modulating the MuSC fate by modifying the microenvironment, and it may lead to a possible strategy to treat muscle disorders.

Fig. 1. Rrm2b was upregulated at late stage of regeneration in wild-type skeletal muscle.

a After induction of MuSC differentiation in vitro, the Rrm2b mRNA levels were significantly upregulated at days 3, 5, and 7. b Rrm2b expression levels in the skeletal muscle (gastrocnemius) of 5-month-old F/F mice at days 1, 4, 7, and 14 after damage. The values at day 0 served as the nondamaged controls, and 3–5 mice were in each group. c Rrm2b IHC expression in skeletal muscle (femoris) was higher in the damaged region 14 days after BaCl₂ injection. ND, nondamaged region. D, damaged region showing centrally nucleated fibers. Scale bar, 100 µm. The mice were 3 months old. The results are presented as the mean ± SD. Statistical analysis of differences between the groups was performed by two-tailed, unpaired t-tests, and the P-values were calculated. Asterisks denote statistically significant changes from the control and are defined as *P < 0.05; **P < 0.01.

Fig. 2. Rrm2b deletion in the myofibers leads to severe weakness in skeletal muscles.

a Gene expression levels of Rrm2b and Pax7 were measured in the MuSCs of 3-month-old Rrm2b scKO mice; 3 mice were in each group. b Gene expression levels of Rrm2b were significantly downregulated in the skeletal muscle of the Rrm2b smKO mice; 5 mice were in each group. c Gene expression levels of Rrm2b were not reduced in the MuSCs isolated from the Rrm2b smKO mice; 3 mice were in each group. d Body weight observation of different mouse models from 3 to 24 months old; 10–15 mice were in each group. e Gross view of the skeletal muscles, femoris (F) and gastrocnemius and soleus (G+S) from the control (F/F) and Rrm2b smKO mice. f The muscle weight of the femoris in the Rrm2b smKO mice at all ages; 10–15 mice were in each group. g Grip strength of the Rrm2b smKO mice at 3, 5, 12, and 24 months of age; 6 mice were in each group. The results are presented as the mean ± SD. Statistical analysis of differences between the groups was performed by two-tailed, unpaired t-tests, and the P-values were calculated. Asterisks denote statistically significant changes from the control and are defined as *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. Promotion of the regeneration observed in the Rrm2b smKO mice.

a H&E staining results of skeletal muscle from the Rrm2b smKO mice. Blue arrows indicate the centronucleated muscle fibers. Asterisk indicates adipose accumulation. Scale bar, 100 μm. b The percentage of centronucleated muscle fibers in the Rrm2b smKO mice. At least 6 photos (200×) from different fields were examined for each mouse. All single fibers were counted to determine whether the myonuclei were centrally located; 8 mice were in each group. c Quantification of the adipose area of the Rrm2b smKO mice. Five-month-old Rrm2b smKO mice showed a significant increase in the adipose area. The adipose area within the muscle fibers of the femoris was quantified with ImageJ software. The ratios were calculated by adipose area versus the whole femoris section, with 5 mice in each group. d Muscle fiber diameter of the femoris in the Rrm2b smKO mice. The diameter of 300 myofibers was measured in the femoris muscle of each mouse; 5 mice were in each group. e Immunostaining results of MuSCs (Pax7-positive) on isolated single myofibers in 3-month-old Rrm2b smKO mice; 15–20 myofibers in each mouse and 3 mice in each group were used. f EBD staining of TA muscle sections at 3, 7, and 14 days after injury. Red fluorescence indicates the damaged myofibers. Green fluorescence indicates immunostaining of laminin. Blue fluorescence indicates staining of DAPI. The mice were 5 months old. The results are presented as the mean ± SD. Statistical analysis of differences between the groups was performed by two-tailed, unpaired t-tests, and the P-values were calculated. Asterisks denote statistically significant changes from the control and are defined as *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar, 100 μm.

Fig. 4. Myofiber-specific Rrm2b deletion weakens the regenerative capacity.

a Donor and recipient mouse preparation timetable for muscle homogenate and MuSCs transplantation. b Quantification of engrafted myofiber size from homogenate transplantation between Rrm2b smKO and F/F mice. Each spot indicates one engrafted myofiber (myofibers with green fluorescence). c Representative images of engrafted myofibers in old recipient TA muscle. 14 days after MuSCs transplantation, activated MuSCs from young donors engrafted old recipient mice and differentiated into green myofibers with central nuclei. Scale bar, 100 μm. d Quantification of engrafted myofiber size from MuSCs transplantation between Rrm2b smKO and F/F mice. Each spot indicates one engrafted myofiber (myofibers with green fluorescence). e Histology of the gastrocnemius muscle from ROSA^mT/mG, ROSA^mT/mG;Pax7^CreERT2, and Rrm2bF/F;ROSA^mT/mG;Pax7^CreERT2 mice at 14 days after damage. The mice were 5 months old, with 3 mice in each group. Scale bar, 100 μm. The results are presented as the mean ± SD. Statistical analysis of differences between the groups was performed by two-tailed, unpaired t-tests, and the P-values were calculated. Asterisks denote statistically significant changes from the control and are defined as **P < 0.01; ***P < 0.001.

Fig. 5. Rrm2b deletion drives MuSCs toward differentiation but not proliferation.

a Quantitative data of mRNA expression of myogenic genes related to quiescence exit, proliferation, and differentiation in skeletal muscles (gastrocnemius) of the Rrm2b smKO mice at ages 3, 5, 12, and 24 months old using RT-qPCR analysis. Five mice were in each group. The data are presented as the mean ± SD. *P < 0.05; **P < 0.01. b The IPA analysis of RNA sequencing data. The red bar indicates the pathways related to cell differentiation. The blue bar indicates the pathways related to cell proliferation (cell cycle, cell growth and proliferation, and growth factor signaling). The selected pathways had a P-value < 0.05 and Z-score > 1 or <−1. c Enrichr analysis, including KEGG_2019_Mouse, WikiPathways_2019_Mouse, BioPlanet_2019, and MSigDB_Hallmark_2020. The top pathways were selected. The results were compared to those of the control group.

Fig. 6. Myofibers modulate stem cell function by myokine secretion.

a RNA sequencing data showed the expression of myokines in the gastrocnemius muscle of the Rrm2b smKO, scKO and F/F mice. b mRNA expression levels of Fgf21 were verified in muscle of the Rrm2b smKO mice. c Fgf21 levels in circulation were determined by ELISAs. d mRNA expression levels of Gdf15 and Mthfd2 were detected in muscle of the Rrm2b smKO mice. e mRNA expression levels of mTOR and Atf4 were detected in muscle of the Rrm2b smKO mice. f Gene expression levels of ERRγ were measured in muscle of the Rrm2b smKO mice at 3, 5, 12, and 24 months of age. g mRNA expression levels of PGC1α were detected in muscle of the Rrm2b smKO mice. In b–g, six mice were in each group. h mRNA expression levels of mTOR and Il6 were detected in MuSCs isolated from the Rrm2b smKO mice. In h, eight mice were in each group. The results are presented as the mean ± SD. Statistical analysis of differences between the groups was performed by two-tailed, unpaired t-tests, and the P-values were calculated. Asterisks denote statistically significant changes from the control and are defined as *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7. Decreases in mitochondria in skeletal muscles of the Rrm2b smKO mice.

a mtDNA copy number was detected in the gastrocnemius muscle from 5-month-old Rrm2b smKO mice. Six mice were in each group. b Long-range PCR (13.6 kb) of mtDNA and detection of D-17 deletion (851 bp) using genomic DNA isolated from skeletal muscles. Genomic DNA was used for DNA input in PCR. c RNA sequencing data showed the expression of mitochondrial genomic DNA-encoded genes in the gastrocnemius myofibers of the Rrm2b smKO mice at 5 months of age. d Subsarcolemmal accumulation of mitochondria was observed in the type I muscle of the Rrm2b smKO mice at 5 months of age by transmission electron microscopy. e RNA sequencing data showed the expression of mitochondrial genomic DNA-encoded genes and mitochondrial metabolism-related genes in the type I myofibers of the Rrm2b smKO mice at 5 months of age. The results are presented as the mean ± SD. Statistical analysis of differences between the groups was performed by two-tailed, unpaired t-tests, and the P-values were calculated. Asterisks denote statistically significant changes from the control and are defined as *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 8. Rrm2b deletion in the myofibers promotes differentiation and arrests quiescence of stem cells when skeletal muscle is injured.

a Under muscle damage, quiescent MuSCs enter the cell cycle, proliferate, and are then activated. Activated MuSCs either differentiate to form new myofibers or self-renew to re-enter the quiescent stage. b In the Rrm2b knockout myofibers, increased myokines continuously drive spontaneous MuSC activation and differentiation but inhibit self-renewal, resulting in a low number of MuSCs and myofiber weakness. c Our working hypothesis revealed that Atf4 and Errγ through PGC1α-independent signaling pathways and mitochondrial defects, upregulated expressions of Fgf21, Gdf15, Mthfd2 and other factors, which associated myogenesis in the Rrm2b-deleted myofibers.