LCN2-ACOD1 Signalling Affects the Post-Injury Regeneration of Skeletal Muscle Through Mediating Ferroptosis

Abstract

The normal growth and development of skeletal muscle are crucial for the proper function of organisms. During myoblast development, cell death is a fundamental physiological process, and skeletal muscle damage involves various types of cell death, including ferroptosis. However, ferroptosis-related biomarkers in skeletal muscle damage remain unclear. This study aimed to investigate the mechanisms by which lipocalin-2 (LCN2), a key protein of iron metabolism, regulates skeletal muscle regeneration post damage by mediating ferroptosis. When the gastrocnemius muscle (GAS) of mice is acutely injured, LCN2 is significantly upregulated early in the injury. In vitro, LCN2 participates in the inhibition of proliferation and differentiation of C2C12 cells via erastin-induced ferroptosis. Transcriptomic analysis after the overexpression of LCN2 revealed that the one with the most significant difference among all of the differentially expressed genes (DEGs) was aconitate decarboxylase 1 (Acod1). The inhibition of myogenic factors' expression by LCN2 was associated with the activation of the ferroptosis signalling pathway, partly attributed to the mitochondrial dysfunction. The ACOD1 inhibitor attenuated mitochondria-associated ferroptosis induced by LCN2 and alleviated the inhibitory effect of LCN2 on cell viability. These findings highlight the therapeutic potential of targeting the LCN2-ACOD1 signalling to promote myogenesis, providing promising strategies for facilitating the regeneration of skeletal muscle after injury and the treatment of muscle-related diseases.

Keywords

ACOD1 / ferroptosis / LCN2 / mitochondria / skeletal muscle regeneration

References

[1] H. Ju, Y. Yang, A. Sheng, and X. Jiang, “Role of microRNAs in Skeletal Muscle Development and Rhabdomyosarcoma (Review),” Molecular Medicine Reports 11 (2015): 4019–4024.

[2] D. Bertheloot, E. Latz, and B. S. Franklin, “Necroptosis, Pyroptosis and Apoptosis: An Intricate Game of Cell Death,” Cellular & Molecular Immunology 18 (2021): 1106–1121.

[3] C. Sciorati, E. Rigamonti, A. A. Manfredi, and P. Rovere-Querini, “Cell Death, Clearance and Immunity in the Skeletal Muscle,” Cell Death and Differentiation 23 (2016): 927–937.

[4] D. Li, Y. Wang, C. Dong, et al., “CST1 Inhibits Ferroptosis and Promotes Gastric Cancer Metastasis by Regulating GPX4 Protein Stability via OTUB1,” Oncogene 42 (2023): 83–98.

[5] X. H. Ma, J. H. Liu, C. Y. Liu, et al., “ALOX15-Launched PUFA-Phospholipids Peroxidation Increases the Susceptibility of Ferroptosis in Ischemia-Induced Myocardial Damage,” Signal Transduction and Targeted Therapy 7 (2022): 288.

[6] F. Lin, W. Chen, J. Zhou, et al., “Mesenchymal Stem Cells Protect Against Ferroptosis via Exosome-Mediated Stabilization of SLC7A11 in Acute Liver Injury,” Cell Death & Disease 13 (2022): 271.

[7] L. Mahoney-Sánchez, H. Bouchaoui, S. Ayton, D. Devos, J. A. Duce, and J. C. Devedjian, “Ferroptosis and Its Potential Role in the Physiopathology of Parkinson's Disease,” Progress in Neurobiology 196 (2021): 101890.

[8] D. Yuting, Z. Jing, H. Ying, and Z. Jing, “Effect of Ferroptosis on Regeneration After Muscle Injury,” Journal of Shanghai Jiao Tong University 42 (2022): 298.

[9] H. Ding, S. Chen, X. Pan, et al., “Transferrin Receptor 1 Ablation in Satellite Cells Impedes Skeletal Muscle Regeneration Through Activation of Ferroptosis,” Journal of Cachexia, Sarcopenia and Muscle 12 (2021): 746–768.

[10] H. Zhou, Y. L. Zhou, J. A. Mao, et al., “NCOA4-Mediated Ferritinophagy Is Involved in Ionizing Radiation-Induced Ferroptosis of Intestinal Epithelial Cells,” Redox Biology 55 (2022): 102413.

[11] S. V. Torti and F. M. Torti, “Iron and Cancer: More Ore to Be Mined,” Nature Reviews. Cancer 13 (2013): 342–355.

[12] L. R. Devireddy, C. Gazin, X. Zhu, and M. R. Green, “A Cell-Surface Receptor for Lipocalin 24p3 Selectively Mediates Apoptosis and Iron Uptake,” Cell 123 (2005): 1293–1305.

[13] D. Wang, X. Li, D. Jiao, et al., “LCN2 Secreted by Tissue-Infiltrating Neutrophils Induces the Ferroptosis and Wasting of Adipose and Muscle Tissues in Lung Cancer Cachexia,” Journal of Hematology & Oncology 16 (2023): 30.

[14] N. Chaudhary, B. S. Choudhary, S. G. Shah, et al., “Lipocalin 2 Expression Promotes Tumor Progression and Therapy Resistance by Inhibiting Ferroptosis in Colorectal Cancer,” International Journal of Cancer 149 (2021): 1495–1511.

[15] H. Wang, Z. Wang, Y. Gao, et al., “STZ-Induced Diabetes Exacerbates Neurons Ferroptosis After Ischemic Stroke by Upregulating LCN2 in Neutrophils,” Experimental Neurology 377 (2024): 114797.

[16] I. A. Rebalka, C. M. F. Monaco, N. E. Varah, et al., “Loss of the Adipokine Lipocalin-2 Impairs Satellite Cell Activation and Skeletal Muscle Regeneration,” American Journal of Physiology-Cell Physiology 315 (2018): C714–C721.

[17] K. J. Harber, A. E. Neele, C. P. van Roomen, et al., “Targeting the ACOD1-Itaconate Axis Stabilizes Atherosclerotic Plaques,” Redox Biology 70 (2024): 103054.

[18] C. J. Hall, R. H. Boyle, J. W. Astin, et al., “Immunoresponsive Gene 1 Augments Bactericidal Activity of Macrophage-Lineage Cells by Regulating β-Oxidation-Dependent Mitochondrial ROS Production,” Cell Metabolism 18 (2013): 265–278.

[19] X. Liu, X. P. Wu, X. L. Zhu, T. Li, and Y. Liu, “IRG1 Increases MHC Class I Level in Macrophages Through STAT-TAP1 Axis Depending on NADPH Oxidase Mediated Reactive Oxygen Species,” International Immunopharmacology 48 (2017): 76–83.

[20] Y. Li, P. Zhang, C. Wang, et al., “Immune Responsive Gene 1 (IRG1) Promotes Endotoxin Tolerance by Increasing A20 Expression in Macrophages Through Reactive Oxygen Species,” Journal of Biological Chemistry 288 (2013): 16225–16234.

[21] M. Jamal Uddin, Y. Joe, S. K. Kim, et al., “IRG1 Induced by Heme Oxygenase-1/Carbon Monoxide Inhibits LPS-Mediated Sepsis and Pro-Inflammatory Cytokine Production,” Cellular & Molecular Immunology 13 (2016): 170–179.

[22] B. Wang, Y. Wang, J. Zhang, et al., “ROS-Induced Lipid Peroxidation Modulates Cell Death Outcome: Mechanisms Behind Apoptosis, Autophagy, and Ferroptosis,” Archives of Toxicology 97 (2023): 1439–1451.

[23] X. Wang, W. Zhao, R. M. Ransohoff, and L. Zhou, “Infiltrating Macrophages Are Broadly Activated at the Early Stage to Support Acute Skeletal Muscle Injury Repair,” Journal of Neuroimmunology 317 (2018): 55–66.

[24] X. Wei, J. Wang, Y. Sun, et al., “MiR-222-3p Suppresses C2C12 Myoblast Proliferation and Differentiation via the Inhibition of IRS-1/PI3K/Akt Pathway,” Journal of Cellular Biochemistry 124 (2023): 1379–1390.

[25] X. Cao, L. Xue, X. Yu, et al., “Myogenic Exosome miR-140-5p Modulates Skeletal Muscle Regeneration and Injury Repair by Regulating Muscle Satellite Cells,” Aging (Albany NY) 16 (2024): 4609–4630.

[26] S. Wang, X. Zhao, Q. Liu, Y. Wang, S. Li, and S. Xu, “Selenoprotein K Protects Skeletal Muscle From Damage and Is Required for Satellite Cells-Mediated Myogenic Differentiation,” Redox Biology 50 (2022): 102255.

[27] F. J. Bock and S. W. G. Tait, “Mitochondria as Multifaceted Regulators of Cell Death,” Nature Reviews. Molecular Cell Biology 21 (2020): 85–100.

[28] Q. Huang, Y. Ru, Y. Luo, et al., “Identification of a Targeted ACSL4 Inhibitor to Treat Ferroptosis-Related Diseases,” Science Advances 10 (2024): eadk1200.

[29] P. Grubwieser, N. Brigo, M. Seifert, et al., “Quantification of Macrophage Cellular Ferrous Iron (Fe(2+)) Content Using a Highly Specific Fluorescent Probe in a Plate Reader,” Bio-Protocol 14 (2024): e4929.

[30] Y. Bi, S. Liu, X. Qin, et al., “FUNDC1 Interacts With GPx4 to Govern Hepatic Ferroptosis and Fibrotic Injury Through a Mitophagy-Dependent Manner,” Journal of Advanced Research 55 (2024): 45–60.

[31] K. Long, X. Li, D. Su, et al., “Exploring High-Resolution Chromatin Interaction Changes and Functional Enhancers of Myogenic Marker Genes During Myogenic Differentiation,” Journal of Biological Chemistry 298 (2022): 102149.

[32] X. Li, T. X. Wang, X. Huang, et al., “Targeting Ferroptosis Alleviates Methionine-Choline Deficient (MCD)-Diet Induced NASH by Suppressing Liver Lipotoxicity,” Liver International 40 (2020): 1378–1394.

[33] J. P. Friedmann Angeli, M. Schneider, B. Proneth, et al., “Inactivation of the Ferroptosis Regulator Gpx4 Triggers Acute Renal Failure in Mice,” Nature Cell Biology 16 (2014): 1180–1191.

[34] S. Javadov, “Mitochondria and Ferroptosis,” Current Opinion in Physiology 25 (2022): 100483.

[35] A. M. Battaglia, R. Chirillo, I. Aversa, A. Sacco, F. Costanzo, and F. Biamonte, “Ferroptosis and Cancer: Mitochondria Meet the Iron Maiden Cell Death,” Cells 9 (2020): 1505.

[36] P. Luo, Q. Zhang, S. Shen, et al., “Mechanistic Engineering of Celastrol Liposomes Induces Ferroptosis and Apoptosis by Directly Targeting VDAC2 in Hepatocellular Carcinoma,” Asian Journal of Pharmaceutical Sciences 18 (2023): 100874.

[37] Y. Wang, R. Yu, L. Wu, and G. Yang, “Hydrogen Sulfide Guards Myoblasts From Ferroptosis by Inhibiting ALOX12 Acetylation,” Cellular Signalling 78 (2021): 109870.

[38] Y. Yoshimoto, M. Ikemoto-Uezumi, K. Hitachi, S. I. Fukada, and A. Uezumi, “Methods for Accurate Assessment of Myofiber Maturity During Skeletal Muscle Regeneration,” Frontiers in Cell and Developmental Biology 8 (2020): 267.

[39] Y. Zhang, H. Tan, J. D. Daniels, et al., “Imidazole Ketone Erastin Induces Ferroptosis and Slows Tumor Growth in a Mouse Lymphoma Model,” Cell Chemical Biology 26 (2019): 623–633.e9.

[40] S. Codenotti, M. Poli, M. Asperti, D. Zizioli, F. Marampon, and A. Fanzani, “Cell Growth Potential Drives Ferroptosis Susceptibility in Rhabdomyosarcoma and Myoblast Cell Lines,” Journal of Cancer Research and Clinical Oncology 144 (2018): 1717–1730.

[41] M. A. Badgley, D. M. Kremer, H. C. Maurer, et al., “Cysteine Depletion Induces Pancreatic Tumor Ferroptosis in Mice,” Science 368 (2020): 85–89.

[42] B. R. Stockwell, “Ferroptosis Turns 10: Emerging Mechanisms, Physiological Functions, and Therapeutic Applications,” Cell 185 (2022): 2401–2421.

[43] Y. Huang, B. Wu, D. Shen, J. Chen, Z. Yu, and C. Chen, “Ferroptosis in a Sarcopenia Model of Senescence Accelerated Mouse Prone 8 (SAMP8),” International Journal of Biological Sciences 17 (2021): 151–162.

[44] X. Xiao, B. S. Yeoh, and M. Vijay-Kumar, “Lipocalin 2: An Emerging Player in Iron Homeostasis and Inflammation,” Annual Review of Nutrition 37 (2017): 103–130.

[45] Y. Chi, J. Remsik, V. Kiseliovas, et al., “Cancer Cells Deploy Lipocalin-2 to Collect Limiting Iron in Leptomeningeal Metastasis,” Science 369 (2020): 276–282.

[46] L. Luo, L. Deng, Y. Chen, R. Ding, and X. Li, “Identification of Lipocalin 2 as a Ferroptosis-Related Key Gene Associated With Hypoxic-Ischemic Brain Damage via STAT3/NF-κB Signaling Pathway,” Antioxidants 12 (2023): 12.

[47] J. Liu, X. Song, F. Kuang, et al., “NUPR1 Is a Critical Repressor of Ferroptosis,” Nature Communications 12 (2021): 647.

[48] F. Yao, Y. Deng, Y. Zhao, et al., “A Targetable LIFR-NF-κB-LCN2 Axis Controls Liver Tumorigenesis and Vulnerability to Ferroptosis,” Nature Communications 12 (2021): 7333.

[49] J. Naujoks, C. Tabeling, B. D. Dill, et al., “IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection via IRG1-Derived Itaconic Acid,” PLoS Pathogens 12 (2016): e1005408.

[50] J. Domínguez-Andrés, B. Novakovic, Y. Li, et al., “The Itaconate Pathway Is a Central Regulatory Node Linking Innate Immune Tolerance and Trained Immunity,” Cell Metabolism 29 (2019): 211–220.

[51] J. Pan, X. Zhao, C. Lin, et al., “Immune Responsive Gene 1, a Novel Oncogene, Increases the Growth and Tumorigenicity of Glioma,” Oncology Reports 32 (2014): 1957–1966.

[52] Y. P. Cheon, X. Xu, M. K. Bagchi, and I. C. Bagchi, “Immune-Responsive Gene 1 Is a Novel Target of Progesterone Receptor and Plays a Critical Role During Implantation in the Mouse,” Endocrinology 144 (2003): 5623–5630.

[53] M. S. Kwun and D. G. Lee, “Ferroptosis-Like Death in Microorganisms: A Novel Programmed Cell Death Following Lipid Peroxidation,” Journal of Microbiology and Biotechnology 33 (2023): 992–997.

[54] Y. Zhao, Z. Liu, G. Liu, et al., “Neutrophils Resist Ferroptosis and Promote Breast Cancer Metastasis Through Aconitate Decarboxylase 1,” Cell Metabolism 35 (2023): 1688–1703.

[55] R. Wu, R. Kang, and D. Tang, “Mitochondrial ACOD1/IRG1 in Infection and Sterile Inflammation,” Journal of Intensive Medicine 2 (2022): 78–88.

[56] L. Galluzzi, O. Kepp, C. Trojel-Hansen, and G. Kroemer, “Mitochondrial Control of Cellular Life, Stress, and Death,” Circulation Research 111 (2012): 1198–1207.

[57] M. Gao, J. Yi, J. Zhu, et al., “Role of Mitochondria in Ferroptosis,” Molecular Cell 73 (2019): 354–363.

[58] S. Naghdi and G. Hajnóczky, “VDAC2-Specific Cellular Functions and the Underlying Structure,” Biochimica et Biophysica Acta 1863 (2016): 2503–2514.

RIGHTS & PERMISSIONS

2025 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.