Transferrin receptor 1 ablation in satellite cells impedes skeletal muscle regeneration through activation of ferroptosis

Abstract

Background: Satellite cells (SCs) are critical to skeletal muscle regeneration. Inactivation of SCs is linked to skeletal muscle loss. Transferrin receptor 1 (Tfr1) is associated with muscular dysfunction as muscle-specific deletion of Tfr1 results in growth retardation, metabolic disorder, and lethality, shedding light on the importance of Tfr1 in muscle physiology. However, its physiological function regarding skeletal muscle ageing and regeneration remains unexplored.

Methods: RNA sequencing is applied to skeletal muscles of different ages to identify Tfr1 associated to skeletal muscle ageing. Mice with conditional SC ablation of Tfr1 were generated. Between Tfr1^SC/WT and Tfr1^SC/KO (n = 6-8 mice per group), cardiotoxin was intramuscularly injected, and transverse abdominal muscle was dissected, weighted, and cryosectioned, followed by immunostaining, haematoxylin and eosin staining, and Masson staining. These phenotypical analyses were followed with functional analysis such as flow cytometry, tread mill, Prussian blue staining, and transmission electron microscopy to identify pathological pathways that contribute to regeneration defects.

Results: By comparing gene expression between young (2 weeks old, n = 3) and aged (80 weeks old, n = 3) mice among four types of muscles, we identified that Tfr1 expression is declined in muscles of aged mice (~80% reduction, P < 0.005), so as to its protein level in SCs of aged mice. From in vivo and ex vivo experiments, Tfr1 deletion in SCs results in an irreversible depletion of SCs (~60% reduction, P < 0.005) and cell-autonomous defect in SC proliferation and differentiation, leading to skeletal muscle regeneration impairment, followed by labile iron accumulation, lipogenesis, and decreased Gpx4 and Nrf2 protein levels leading to reactive oxygen species scavenger defects. These abnormal phenomena including iron accumulation, activation of unsaturated fatty acid biosynthesis, and lipid peroxidation are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis further exacerbates SC proliferation and skeletal muscle regeneration. Ferrostatin-1, a ferroptosis inhibitor, could not rescue ferroptosis. However, intramuscular administration of lentivirus-expressing Tfr1 could partially reduce labile iron accumulation, decrease lipogenesis, and promote skeletal muscle regeneration. Most importantly, declined Tfr1 but increased Slc39a14 protein level on cellular membrane contributes to labile iron accumulation in skeletal muscle of aged rodents (~80 weeks old), leading to activation of ferroptosis in aged skeletal muscle. This is inhibited by ferrostatin-1 to improve running time (P = 0.0257) and distance (P = 0.0248).

Conclusions: Satellite cell-specific deletion of Tfr1 impairs skeletal muscle regeneration with activation of ferroptosis. This phenomenon is recapitulated in skeletal muscle of aged rodents and human sarcopenia. Our study provides mechanistic information for developing novel therapeutic strategies against muscular ageing and diseases.

Keywords: Ferroptosis; Fibro/adipogenic progenitors; Satellite cells; Tfr1.

Figure 1

Identification of Tfr1 as a key biomarker regarding skeletal muscle ageing and satellite cell (SC) activity. (A) Venn diagraph showing overlapped genes between young (2 weeks old) and aged (80 weeks old) mice among four types of muscle [transverse abdominal (TA), extensor digitorum longus (EDL), soleus (Sol), and gastrocnemius (Gas)] (n = 3 per group). (B) Gene ontology (GO: biological process) analysis against down‐regulated genes between 2‐ and 80‐week‐old C57BL/6J mice. (C) Gene Set Enrichment Analysis (GSEA) analysis of down‐regulated pathway in response to the iron homeostasis. (D) Heatmap of cellular iron homeostasis‐related gene expression in TA muscle across five different ages (2, 8, 30, 60, and 80 weeks old). (E) qPCR analysis of Tfr1 expression in four types of skeletal muscles (TA, EDL, Sol, and Gas) between 2‐ and 8‐week‐old C57BL/6J mice (n = 5 per group). (F) Representative western blot image of four types of skeletal muscles (TA, EDL, Sol, and Gas) between 2‐ and 8‐week‐old C57BL/6J mice (n = 5 per group). (G) Representative images of myofibres isolated from 2‐ and 8‐week‐old C57BL/6J mice (n > 50 myofibres from five mice per group). Immunofluorescence of Pax7 (red), Tfr1 (green), Ki67 (pink), and DAPI (blue) staining revealed that Tfr1 is highly expressed in SCs at proliferative state (Ki67⁺) for 2‐week‐old mice but not 8‐week‐old adult mice. (H) Number of Ki67⁺ and Ki67⁻ SCs with different Tfr1 expression level (High, Inter, and Low) per myofibre. (I) Number of Pax7⁺ SCs per myofibre. (J) Number of Ki67⁺ and Ki67⁻ SCs per myofibre. N.S., not significant, **P < 0.01, and ***P < 0.005, by two‐sided Student's t‐test. Data represent the mean ± standard error of the mean.

Figure 2

Genetic deletion of Tfr1 in quiescent satellite cells (SCs) abolishes the activation, proliferation, and differentiation. (A) Representative images of myofibres isolated from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice (n > 50 myofibres from five mice per group). Immunofluorescence of Pax7 (red), Tfr1 (green), Ki67 (pink), and DAPI (blue) staining. (B) Representative images of myofibres isolated from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice (n > 50 myofibres from five mice per group). Immunofluorescence of Pax7 (red), Tfr1 (green), MyoD (pink), and DAPI (blue) staining. (C) Number of total, Ki67⁺, and MyoD⁺ SCs per myofibre between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice. (D) Both Tfr1 ^SC/WT and Tfr1 ^SC/KO mice were administrated with tamoxifen (TMX) on the same day. Number of Pax7⁺ SCs per myofibre was calculated at 1, 4, 7, 10, 14, 21, and 30 days after TMX‐induced Tfr1 deletion (n = 5 mice per group per time point). (E) Representative images of SC clusters on myofibre from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice ex vivo cultured for 72 h (n > 50 myofibre). Immunofluorescence of Pax7 (red), Tfr1 (green), MyoD (pink), and DAPI (blue) staining (n > 20 myofibres from seven mice per group). (F) Number of SC clusters per myofibre and number of Pax7⁺ SCs per cluster (n > 50 myofibres from five mice per group). (G) Representative images of differentiated myotubes from SCs on myofibre isolated from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice (n > 10 myofibres from five mice per group). (H) Summary of fusion index of SCs on myofibre differentiated in Dulbecco's modified Eagle's medium supplemented with 2% horse serum (n > 10 myofibres from five mice per group). N.S., not significant, and ***P < 0.005, by two‐sided Student's t‐test. Data represent the mean ± standard error of the mean.

Figure 3

Tfr1 ablation in satellite cells (SCs) delays skeletal muscle regeneration. (A) Timeline characterizing skeletal muscle regeneration upon tamoxifen‐induced Tfr1 ablation in SCs. (B) Representative image of transverse abdominal (TA) muscle upon completion of regeneration between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice at 30 dpi. (C) Summary of body weight, TA muscle weight, and ratio of TA muscle and body weight between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice upon completion of regeneration at 30 dpi (n = 7 per group). (D) Representative images of TA muscle section from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice (n = 6 mice per group). Immunofluorescence of Pax7 revealed a decrease in the number of Pax7⁺ SCs (arrowheads) and number of Pax7⁺ SCs per TA muscle section at 5 dpi (right of immunostaining images). (E) Representative images of TA muscle section from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice (n = 6 mice per group). Immunofluorescence of Pax7 revealed a decrease in the number of Pax7⁺ SCs (arrowheads) and number of Pax7⁺ SCs per TA muscle section at 9 dpi (right of immunostaining images). (F) Immunofluorescence of eMyHC⁺ myotubes after cardiotoxin (CTX) injury (5 dpi) and number of eMyHC⁺ myotubes per TA muscle section area at 5 dpi (right of immunostaining images). (G) Immunofluorescence of eMyHC⁺ myotubes after CTX injury (5 dpi) and number of eMyHC⁺ myotubes per TA muscle section area at 9 dpi (right of immunostaining images). (H) Immunofluorescence of Pax7 revealed a decrease in the number of Pax7⁺ SCs (arrowheads) and number of Pax7⁺ SCs per TA muscle section at 30 dpi (right of immunostaining images, n = 6 mice per group). (I) Representative images of TA muscles from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice with haematoxylin and eosin staining and Masson staining upon completion of CTX‐induced regeneration (30 dpi, n = 6 mice per group). (J) Summary of collagen volume fraction between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice completion of CTX‐induced regeneration at 30 dpi. N.S., not significant, *P < 0.05, **P < 0.01, and ***P < 0.005, by two‐sided Student's t‐test. Data represent the mean ± standard error of the mean.

Figure 4

Satellite cell (SC) Tfr1 deletion in transverse abdominal (TA) muscle of Tfr1 ^SC/KO mice results in skeletal muscle dysfunction. (A) Heatmap of mRNA expression profile in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before or after cardiotoxin (CTX)‐induced regeneration at 30 dpi (n = 5 per group). (B) Principal coordinate analysis (PCoA) of transcriptome from TA muscle in adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before or after CTX‐induced regeneration (n = 5 per group). (C) Volcano plot of differentially expressed genes in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced regeneration. (D) Gene ontology (GO) (biological process) analysis of differentially expressed genes (DEGs) for both up‐regulated and down‐regulated genes. (E) Gene Set Enrichment Analysis (GSEA) analysis of macrophage activation pathway between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice upon CTX‐induced regeneration at 30 dpi. (F) Flow cytometry analysis of the percentage of the CD206⁺/CD86⁺ macrophage in total cells obtained from TA muscle of Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi. (G) qPCR analysis of Cd86, Cd163, and Cd206 mRNA expression in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before or after CTX‐induced injury (n = 5 per group). (H) GSEA analysis of collagen‐containing extracellular matrix pathway between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice upon CTX‐induced regeneration at 30 dpi. (I) Heatmap for collagen matrix related gene expression profile between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi (n = 5). (J) qPCR analysis of Col5a3, Col6a1, Col6a3, Col11a2, Col12a1, and Col23a1 mRNA expression in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before or after CTX‐induced injury at 30 dpi (n = 5 per group). (K, L) Tread mill running distance and running time to exhaustion for Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before and after regeneration at 30 dpi. N.S., not significant, *P < 0.05, **P < 0.01, and ***P < 0.005, by two‐sided Student's t‐test. Data represent the mean ± standard error of the mean.

Figure 5

Dysregulation of lipid and iron metabolism activates ferroptosis in injured transverse abdominal (TA) muscle of Tfr1 ^SC/KO mice. (A) Gene Set Enrichment Analysis (GSEA) analysis of adipogenesis pathway between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice upon cardiotoxin (CTX)‐induced regeneration at 30 dpi. (B) qPCR analysis of Adipoq, Fasn, and Cd36 mRNA expression in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before or after CTX‐induced injury (n = 5 per group). (C) Representative images of TA muscle sections from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice upon CTX‐induced injury at 30 dpi (n = 5 per group). Oil Red O (ORO) staining and perilipin (green) and laminin B2 (pink) immunofluorescent staining revealed adipogenesis and lipid accumulation in TA muscle of Tfr1 ^SC/KO mice. (D) qPCR analysis of Pgc1α, Cox7a1, and Cox8b (mitochondrial genes), Tfr1, Slc11a2, Slc40a1, Ftl, and Fth1 (iron homeostasis‐related genes) mRNA expression in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before or after CTX‐induced injury (n = 5 per group). (E) Representative images of TA muscle section with Prussian blue staining (n = 5 per group) and transmission electron microscope images for TA muscle section from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi. (F) Heatmap of ferroptosis‐related gene expression in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi (n = 5 per group). (G) KEGG pathway enrichment analysis of up‐regulated genes in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi. (H) qPCR analysis of Gpx4 and Ptgs2 expression in TA muscle of adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice before or after CTX‐induced injury (n = 5 per group). (I) Representative western blot images of TA muscle between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi. (J) Heatmap of unsaturated fatty acid biosynthesis‐related gene expression in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi (n = 5 per group). (K) GSEA analysis of unsaturated fatty acid biosynthesis pathway between Tfr1 ^SC/WT and Tfr1 ^SC/KO mice upon CTX‐induced regeneration at 30 dpi. (L) qPCR analysis of Fasn, Elvol5, Elvol6, Scd1, Fads1, and Fads2 expression in TA muscle of adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi (n = 5 per group). (M) Saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels (nmol/g) in TA muscle of adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi (n = 4 per group). N.S., not significant, *P < 0.05, **P < 0.01, and ***P < 0.005, by two‐sided Student's t‐test. Data represent the mean ± standard error of the mean.

Figure 6

Ferroptosis in transverse abdominal (TA) muscle of Tfr1 ^SC/KO mice prevents skeletal muscle regeneration. (A) Timeline characterizing the activation of ferroptosis in TA muscle of Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after cardiotoxin (CTX)‐induced injury. (B) qPCR analysis of Gpx4 and Ptgs2 expression in TA muscle from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 5, 9, and 15 dpi. (C) Representative images of TA muscle section with Prussian blue and Oil Red O (ORO) staining from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 5 and 9 dpi. (D) Timeline characterizing the effect of ferrostatin‐1 to inhibit ferroptosis in TA muscle from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice. (E) Representative images of TA muscle section immunostaining for Pax7 (red) and laminin B2 (white). Number of Pax7⁺ SCs per section (right) (n = 6 per group). (F) Representative images of TA muscle section with Prussian blue and ORO staining from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury between saline and ferrostatin‐1 intraperitoneal injection at 30 dpi. (G) Timeline characterizing the effect of lenti‐Tfr1 to inhibit ferroptosis in TA muscle from Tfr1 ^SC/WT and Tfr1 ^SC/KO mice. (H) Representative images of TA muscle section immunostaining for Pax7 (red) and laminin B2 (white) between lenti‐Ctrl and lenti‐Tfr1 intramuscular injection at 30 dpi. Number of Pax7⁺ SCs per section (right) (n = 6 per group). (I) Representative images of TA muscle section with Prussian blue and ORO staining from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury between lenti‐Ctrl and lenti‐Tfr1 intramuscular injection at 15 dpi. (J) Respiratory exchange rate (VCO₂/VO₂) and energy expenditure were monitored over a 48 h period for Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 15 dpi (n = 8 mice per group). (L) Representative transmission electron microscope image of TA muscle samples from adult Tfr1 ^SC/WT and Tfr1 ^SC/KO mice after CTX‐induced injury at 30 dpi. N.S., not significant, *P < 0.05, and ***P < 0.005, by two‐sided Student's t‐test. Data represent the mean ± standard error of the mean.

Figure 7

Slc39a14‐mediated iron absorption and labile iron accumulation induces ferroptosis in aged skeletal muscle. (A) Venn diagraph showing the overlapping genes between aged/young and Tfr1 ^SC/WT/Tfr1 ^SC/KO samples. (B) Heatmap of overlapping gene expression profile for aged/young group (n = 3) and Tfr1 ^SC/WT/Tfr1 ^SC/KO group (n = 5). (C) KEGG pathway enrichment analysis of up‐regulated common genes identified ferroptosis‐related genes highly expressed in transverse abdominal (TA) muscle of aged mice. (D) Serum and total TA muscle non‐haem iron from young (8 weeks old) and aged (80 weeks old) C57BL/6J mice. (E) Representative western blot image of total and membrane protein of TA muscle from young (8 weeks old) and aged (80 weeks old) C57BL/6J mice. (F) Treadmill running distance and running time to exhaustion for aged (80 weeks old) C57BL/6J mice with cardiotoxin (CTX)‐induced injury followed by intraperitoneal injection of either saline or ferrostatin‐1 for 30 days (n = 14). qPCR analysis of iron metabolism‐related (G), adipogenesis‐related (H), and ferroptosis‐related (I) gene expression in skeletal muscle biopsy sample from young and sarcopenia individuals. N.S., not significant, *P < 0.05, and **P < 0.01, by two‐sided Student's t‐test. Data represent the mean ± standard error of the mean.