Protection against acetaminophen-induced liver injury with MG53: Muscle-liver axis and necroptosis

Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injuryJournal of HepatologyVol. 76Issue 3PreviewDrug-induced liver injury (DILI) remains challenging to treat and is still a leading cause of acute liver failure. MG53 is a muscle-derived tissue-repair protein that circulates in the bloodstream and whose physiological role in protection against DILI has not been examined. Full-Text PDF Reply to: “Protection against acetaminophen-induced liver injury with MG53: Muscle-liver axis and necroptosis”Journal of HepatologyVol. 77Issue 2PreviewWe thank Jaeschke and Umbaugh for their interest in and comments on our study.1 Our findings reveal an important physiological function of MG53, which circulates in the bloodstream, and provide evidence of the effectiveness of exogenously administrated recombinant human MG53 (rhMG53) protein in attenuating acetaminophen (APAP)-induced liver injury.2 We are happy that the RNA-seq data presented by the authors further confirms our finding that hepatocytes and other hepatic non-parenchymal cells do not express endogenous MG53 mRNA before or after exposure to APAP. Full-Text PDF We read with great interest a recent paper by Han et al. showing that the muscle protein Mitsugumin 53 (MG53 or Trim72), is highly effective in attenuating acetaminophen (APAP)-induced liver injury and mortality in a murine model.[1]Han Y. Black S. Gong Z. Chen Z. Ko J.K. Zhou Z. et al.Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.J Hepatol. 2022; 76: 558-567https://doi.org/10.1016/j.jhep.2021.10.017Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar The authors provided evidence for the beneficial effect of MG53 through use of a Mg53-/- mice and injection of human recombinant MG53.[1]Han Y. Black S. Gong Z. Chen Z. Ko J.K. Zhou Z. et al.Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.J Hepatol. 2022; 76: 558-567https://doi.org/10.1016/j.jhep.2021.10.017Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar We want to focus on two fundamental problems with this study. First, the authors hypothesized that MG53 must come from the muscle. Our interrogation of single cell RNA-seq data sets confirmed that neither hepatocytes nor any non-parenchymal cells in the liver express MG53 mRNA in controls or after APAP (Fig. 1). However, according to the human protein atlas (https://www.proteinatlas.org/ENSG00000177238-TRIM72) MG53 is enriched in skeletal muscle cells. This raises the serious question of how APAP metabolized in the liver signals to the muscle to release MG53 and how this, in turn, can affect toxicity in the liver within 3 hours. The authors suggest that H2O2 from hepatocytes may be the signaling molecule that travels through the bloodstream to the muscle. However, this speculation is not supported by established mechanisms of APAP toxicity.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar It is well known that the major oxidant stress and peroxynitrite formation during APAP toxicity occurs inside mitochondria[3]Du K. Ramachandran A. Jaeschke H. Oxidative stress during acetaminophen hepatotoxicity: sources, pathophysiological role and therapeutic potential.Redox Biol. 2016; 10: 148-156https://doi.org/10.1016/j.redox.2016.10.001Crossref PubMed Scopus (286) Google Scholar and that only a very limited amount of superoxide is released towards the cytosol where it functions to activate a mitogen-activated protein kinase cascade.[4]Nguyen N.T. Du K. Akakpo J.Y. Umbaugh D.S. Jaeschke H. Ramachandran A. Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice.Toxicol Lett. 2021; 338: 21-31https://doi.org/10.1016/j.toxlet.2020.12.005Crossref PubMed Scopus (17) Google Scholar There is no evidence that enough cytosolic H2O2 could escape hepatocellular glutathione peroxidase and get into the blood, where it would have to escape erythrocyte glutathione peroxidase while being transported from the hepatic vein through the heart and lung before eventually reaching muscle tissue, entering these cells and triggering the release of MG53. The released MG53 then travels again through the entire circulation to end up in the liver, specifically in centrilobular hepatocytes, to attenuate cell death. Given the strong temporal correlation between the occurrence of the mitochondrial oxidant stress and peroxynitrite formation and cell death,[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar the proposed signaling mechanism between liver and muscle and back through H2O2 and MG53, respectively, is virtually impossible. The fact that Mg53-/- mice show enhanced ALT release from the earliest time point (3 hours), suggests the possibility of off-target effects that are responsible for the aggravating effect in these knockout mice independent of MG53. Thus, the mechanisms underlying the detrimental effects of Mg53-/- might be different to those impacted by treatment with high doses of MG53, which has not been considered or investigated by the authors.The second fundamental problem with this manuscript is the suggested mechanism of cell death, i.e., RIP3K- and MLKL-mediated necroptosis.[1]Han Y. Black S. Gong Z. Chen Z. Ko J.K. Zhou Z. et al.Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.J Hepatol. 2022; 76: 558-567https://doi.org/10.1016/j.jhep.2021.10.017Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar Modes of APAP-induced cell death have been reviewed in detail and it is obvious that APAP causes liver injury through programmed necrosis but not necroptosis.[5]Jaeschke H. Ramachandran A. Chao X. Ding W.X. Emerging and established modes of cell death during acetaminophen-induced liver injury.Arch Toxicol. 2019; 93: 3491-3502https://doi.org/10.1007/s00204-019-02597-1Crossref PubMed Scopus (51) Google Scholar The mechanism of cell death is critically dependent on Cyp2E1-mediated reactive metabolite formation and mitochondrial protein adduct formation, which initiates an oxidant stress that is amplified by c-jun N-terminal kinase activation and mitochondrial translocation, leading to a peroxynitrite-dependent mitochondrial permeability transition pore opening.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar,[5]Jaeschke H. Ramachandran A. Chao X. Ding W.X. Emerging and established modes of cell death during acetaminophen-induced liver injury.Arch Toxicol. 2019; 93: 3491-3502https://doi.org/10.1007/s00204-019-02597-1Crossref PubMed Scopus (51) Google Scholar The severe mitochondrial dysfunction triggers the release of intermembrane proteins such as endonuclease G, which translocate to the nucleus and cause DNA fragmentation.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar,[5]Jaeschke H. Ramachandran A. Chao X. Ding W.X. Emerging and established modes of cell death during acetaminophen-induced liver injury.Arch Toxicol. 2019; 93: 3491-3502https://doi.org/10.1007/s00204-019-02597-1Crossref PubMed Scopus (51) Google Scholar The latter event is considered the point of no-return for cell death.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar Studies of the role of RIP1K and RIP3K in APAP hepatotoxicity have shown inconsistent results. However, reports indicating beneficial effects of RIP1K[6]Dara L. Johnson H. Suda J. Win S. Gaarde W. Han D. et al.Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis.Hepatology. 2015; 62: 1847-1857https://doi.org/10.1002/hep.27939Crossref PubMed Scopus (118) Google Scholar and RIP3K[7]Ramachandran A. McGill M.R. Xie Y. Ni H.M. Ding W.X. Jaeschke H. Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice.Hepatology. 2013; 58: 2099-2108https://doi.org/10.1002/hep.26547Crossref PubMed Scopus (173) Google Scholar have shown reduced mitochondrial dysfunction and necrosis not necroptosis. In addition, Mlkl-/- mice were not protected against APAP toxicity.[6]Dara L. Johnson H. Suda J. Win S. Gaarde W. Han D. et al.Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis.Hepatology. 2015; 62: 1847-1857https://doi.org/10.1002/hep.27939Crossref PubMed Scopus (118) Google Scholar Thus, reactive metabolite formation and mitochondrial protein adducts, mitochondrial oxidant stress, mitochondrial permeability transition pore opening, and nuclear DNA fragmentation are central events in APAP-induced cell death in both the mouse model[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar and in humans.[8]McGill M.R. Sharpe M.R. Williams C.D. Taha M. Curry S.C. Jaeschke H. The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation.J Clin Invest. 2012; 122: 1574-1583https://doi.org/10.1172/JCI59755Crossref PubMed Scopus (504) Google Scholar Importantly, the current clinical standard of care antidote against APAP overdose, N-acetylcysteine, and fomepizole (4-methylpyrazole), a promising antidote in clinical development, both target these key pathophysiological events.[9]Akakpo J.Y. Ramachandran A. Curry S.C. Rumack B.H. Jaeschke H. Comparing N-acetylcysteine and 4-methylpyrazole as antidotes for acetaminophen overdose.Arch Toxicol. 2022; 96: 453-465https://doi.org/10.1007/s00204-021-03211-zCrossref PubMed Scopus (10) Google Scholar Because MLKL is not a relevant therapeutic target in APAP toxicity and it is highly unlikely to be a relevant target for other drug-induced liver injury models, it would be interesting to investigate how high doses of MG53 could interact with the established mechanisms of APAP-induced cell death and in other models of liver injury. MG53 is certainly a protein that can affect a multitude of intracellular signaling events and positively impact various pathophysiologies.[10]Zhang Y. Wu H.K. Lv F.X. Xiao R.P. MG53 is a double-edged sword for human diseases.Sheng Li Xue Bao. 2016; 68: 505-516https://doi.org/10.13294/j.aps.2016.0037Crossref PubMed Google Scholar However, it also has detrimental effects including negative regulation of myogenesis.[10]Zhang Y. Wu H.K. Lv F.X. Xiao R.P. MG53 is a double-edged sword for human diseases.Sheng Li Xue Bao. 2016; 68: 505-516https://doi.org/10.13294/j.aps.2016.0037Crossref PubMed Google Scholar Thus, a more detailed understanding of the mechanism of protection in various liver disease etiologies but also of the potential adverse effects of MG53 treatment need to be achieved before this compound can be considered for clinical application.Financial supportWork in the authors’ laboratory was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grant R01 DK102142 .Authors’ contributionsHJ: concept, drafting of manuscript. DSU: data acquisition, interpretation of data and critical review of manuscript. All authors read and approved the final manuscript. We read with great interest a recent paper by Han et al. showing that the muscle protein Mitsugumin 53 (MG53 or Trim72), is highly effective in attenuating acetaminophen (APAP)-induced liver injury and mortality in a murine model.[1]Han Y. Black S. Gong Z. Chen Z. Ko J.K. Zhou Z. et al.Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.J Hepatol. 2022; 76: 558-567https://doi.org/10.1016/j.jhep.2021.10.017Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar The authors provided evidence for the beneficial effect of MG53 through use of a Mg53-/- mice and injection of human recombinant MG53.[1]Han Y. Black S. Gong Z. Chen Z. Ko J.K. Zhou Z. et al.Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.J Hepatol. 2022; 76: 558-567https://doi.org/10.1016/j.jhep.2021.10.017Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar We want to focus on two fundamental problems with this study. First, the authors hypothesized that MG53 must come from the muscle. Our interrogation of single cell RNA-seq data sets confirmed that neither hepatocytes nor any non-parenchymal cells in the liver express MG53 mRNA in controls or after APAP (Fig. 1). However, according to the human protein atlas (https://www.proteinatlas.org/ENSG00000177238-TRIM72) MG53 is enriched in skeletal muscle cells. This raises the serious question of how APAP metabolized in the liver signals to the muscle to release MG53 and how this, in turn, can affect toxicity in the liver within 3 hours. The authors suggest that H2O2 from hepatocytes may be the signaling molecule that travels through the bloodstream to the muscle. However, this speculation is not supported by established mechanisms of APAP toxicity.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar It is well known that the major oxidant stress and peroxynitrite formation during APAP toxicity occurs inside mitochondria[3]Du K. Ramachandran A. Jaeschke H. Oxidative stress during acetaminophen hepatotoxicity: sources, pathophysiological role and therapeutic potential.Redox Biol. 2016; 10: 148-156https://doi.org/10.1016/j.redox.2016.10.001Crossref PubMed Scopus (286) Google Scholar and that only a very limited amount of superoxide is released towards the cytosol where it functions to activate a mitogen-activated protein kinase cascade.[4]Nguyen N.T. Du K. Akakpo J.Y. Umbaugh D.S. Jaeschke H. Ramachandran A. Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice.Toxicol Lett. 2021; 338: 21-31https://doi.org/10.1016/j.toxlet.2020.12.005Crossref PubMed Scopus (17) Google Scholar There is no evidence that enough cytosolic H2O2 could escape hepatocellular glutathione peroxidase and get into the blood, where it would have to escape erythrocyte glutathione peroxidase while being transported from the hepatic vein through the heart and lung before eventually reaching muscle tissue, entering these cells and triggering the release of MG53. The released MG53 then travels again through the entire circulation to end up in the liver, specifically in centrilobular hepatocytes, to attenuate cell death. Given the strong temporal correlation between the occurrence of the mitochondrial oxidant stress and peroxynitrite formation and cell death,[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar the proposed signaling mechanism between liver and muscle and back through H2O2 and MG53, respectively, is virtually impossible. The fact that Mg53-/- mice show enhanced ALT release from the earliest time point (3 hours), suggests the possibility of off-target effects that are responsible for the aggravating effect in these knockout mice independent of MG53. Thus, the mechanisms underlying the detrimental effects of Mg53-/- might be different to those impacted by treatment with high doses of MG53, which has not been considered or investigated by the authors. The second fundamental problem with this manuscript is the suggested mechanism of cell death, i.e., RIP3K- and MLKL-mediated necroptosis.[1]Han Y. Black S. Gong Z. Chen Z. Ko J.K. Zhou Z. et al.Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.J Hepatol. 2022; 76: 558-567https://doi.org/10.1016/j.jhep.2021.10.017Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar Modes of APAP-induced cell death have been reviewed in detail and it is obvious that APAP causes liver injury through programmed necrosis but not necroptosis.[5]Jaeschke H. Ramachandran A. Chao X. Ding W.X. Emerging and established modes of cell death during acetaminophen-induced liver injury.Arch Toxicol. 2019; 93: 3491-3502https://doi.org/10.1007/s00204-019-02597-1Crossref PubMed Scopus (51) Google Scholar The mechanism of cell death is critically dependent on Cyp2E1-mediated reactive metabolite formation and mitochondrial protein adduct formation, which initiates an oxidant stress that is amplified by c-jun N-terminal kinase activation and mitochondrial translocation, leading to a peroxynitrite-dependent mitochondrial permeability transition pore opening.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar,[5]Jaeschke H. Ramachandran A. Chao X. Ding W.X. Emerging and established modes of cell death during acetaminophen-induced liver injury.Arch Toxicol. 2019; 93: 3491-3502https://doi.org/10.1007/s00204-019-02597-1Crossref PubMed Scopus (51) Google Scholar The severe mitochondrial dysfunction triggers the release of intermembrane proteins such as endonuclease G, which translocate to the nucleus and cause DNA fragmentation.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar,[5]Jaeschke H. Ramachandran A. Chao X. Ding W.X. Emerging and established modes of cell death during acetaminophen-induced liver injury.Arch Toxicol. 2019; 93: 3491-3502https://doi.org/10.1007/s00204-019-02597-1Crossref PubMed Scopus (51) Google Scholar The latter event is considered the point of no-return for cell death.[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar Studies of the role of RIP1K and RIP3K in APAP hepatotoxicity have shown inconsistent results. However, reports indicating beneficial effects of RIP1K[6]Dara L. Johnson H. Suda J. Win S. Gaarde W. Han D. et al.Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis.Hepatology. 2015; 62: 1847-1857https://doi.org/10.1002/hep.27939Crossref PubMed Scopus (118) Google Scholar and RIP3K[7]Ramachandran A. McGill M.R. Xie Y. Ni H.M. Ding W.X. Jaeschke H. Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice.Hepatology. 2013; 58: 2099-2108https://doi.org/10.1002/hep.26547Crossref PubMed Scopus (173) Google Scholar have shown reduced mitochondrial dysfunction and necrosis not necroptosis. In addition, Mlkl-/- mice were not protected against APAP toxicity.[6]Dara L. Johnson H. Suda J. Win S. Gaarde W. Han D. et al.Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis.Hepatology. 2015; 62: 1847-1857https://doi.org/10.1002/hep.27939Crossref PubMed Scopus (118) Google Scholar Thus, reactive metabolite formation and mitochondrial protein adducts, mitochondrial oxidant stress, mitochondrial permeability transition pore opening, and nuclear DNA fragmentation are central events in APAP-induced cell death in both the mouse model[2]Ramachandran A. Jaeschke H. Acetaminophen hepatotoxicity.Semin Liver Dis. 2019; 39: 221-234https://doi.org/10.1055/s-0039-1679919Crossref PubMed Scopus (111) Google Scholar and in humans.[8]McGill M.R. Sharpe M.R. Williams C.D. Taha M. Curry S.C. Jaeschke H. The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation.J Clin Invest. 2012; 122: 1574-1583https://doi.org/10.1172/JCI59755Crossref PubMed Scopus (504) Google Scholar Importantly, the current clinical standard of care antidote against APAP overdose, N-acetylcysteine, and fomepizole (4-methylpyrazole), a promising antidote in clinical development, both target these key pathophysiological events.[9]Akakpo J.Y. Ramachandran A. Curry S.C. Rumack B.H. Jaeschke H. Comparing N-acetylcysteine and 4-methylpyrazole as antidotes for acetaminophen overdose.Arch Toxicol. 2022; 96: 453-465https://doi.org/10.1007/s00204-021-03211-zCrossref PubMed Scopus (10) Google Scholar Because MLKL is not a relevant therapeutic target in APAP toxicity and it is highly unlikely to be a relevant target for other drug-induced liver injury models, it would be interesting to investigate how high doses of MG53 could interact with the established mechanisms of APAP-induced cell death and in other models of liver injury. MG53 is certainly a protein that can affect a multitude of intracellular signaling events and positively impact various pathophysiologies.[10]Zhang Y. Wu H.K. Lv F.X. Xiao R.P. MG53 is a double-edged sword for human diseases.Sheng Li Xue Bao. 2016; 68: 505-516https://doi.org/10.13294/j.aps.2016.0037Crossref PubMed Google Scholar However, it also has detrimental effects including negative regulation of myogenesis.[10]Zhang Y. Wu H.K. Lv F.X. Xiao R.P. MG53 is a double-edged sword for human diseases.Sheng Li Xue Bao. 2016; 68: 505-516https://doi.org/10.13294/j.aps.2016.0037Crossref PubMed Google Scholar Thus, a more detailed understanding of the mechanism of protection in various liver disease etiologies but also of the potential adverse effects of MG53 treatment need to be achieved before this compound can be considered for clinical application. Financial supportWork in the authors’ laboratory was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grant R01 DK102142 . Work in the authors’ laboratory was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grant R01 DK102142 . Authors’ contributionsHJ: concept, drafting of manuscript. DSU: data acquisition, interpretation of data and critical review of manuscript. All authors read and approved the final manuscript. HJ: concept, drafting of manuscript. DSU: data acquisition, interpretation of data and critical review of manuscript. All authors read and approved the final manuscript. The authors declare no conflicts of interest that relate to this work. Please refer to the accompanying ICMJE disclosure forms for further details. Supplementary dataThe following are the supplementary data to this article: Download .pdf (.31 MB) Help with pdf files Multimedia component 1 The following are the supplementary data to this article: Download .pdf (.31 MB) Help with pdf files Multimedia component 1