Advanced glycation endproduct-RAGE signaling fosters diabetic cardiomyopathy by enhancing cuproptosis and mitochondrial injury

Abstract

Aim: Diabetic cardiomyopathy is characterized by altered myocardial structure and function, although the underlying mechanism remains unclear. This work evaluated the potential roles of advanced glycation end products (AGEs) and the AGE receptor (RAGE) in diabetes-evoked cardiac defects and the mechanisms involved, with a focus on cuproptosis.

Methods: C57BL/6 mice were induced with diabetes using a high-fat diet and streptozotocin. Diabetic and nondiabetic mice were treated with the RAGE blocker FPS-ZM1, followed by assessment of cardiac geometry, contractile performance, oxidative stress, apoptosis, and cuproptosis.

Results: RNA-seq analysis exhibited differentially expressed cuproptosis-related genes in diabetic hearts, implicating a cuproptosis signature. Levels of AGE and RAGE mRNA were elevated in diabetic hearts. Diabetes triggered cardiac hypertrophy, myocardial tissue fibrosis, impaired fractional shortening, ejection fraction, and cell shortening, alongside mitochondrial ultrastructural and functional defects. Diabetes promoted apoptosis (increased Bax and decreased Bcl2) and cuproptosis [copper transporter 1 (CTR1), ferredoxin 1 (FDX1), dihydrolipoamide S-acetyltransferase (DLAT), and S100 calcium-binding protein A13 (S100A13)], along with dampened Fe-S cluster proteins Aconitase 2 (ACO2) and NADH: ubiquinone oxidoreductase core subunit S8 (NDUFS8). These pathological changes were alleviated by FPS-ZM1, except for CTR1, plasma glucose, and lipid levels. Co-immunoprecipitation and domain mapping favored an interplay between RAGE and the Cu²⁺-binding protein S100A13. In vitro, methylglyoxal-derived AGE (MG-AGE) and high glucose levels prompted Cu²⁺ accumulation and/or compromised cardiomyocyte contractile function, reminiscent of in vivo diabetic cardiomyopathy. Inhibition of RAGE, cuproptosis, and S100A13 using FPS-ZM1, tetrathiomolybdate, and amlexanox, respectively, alleviated MG-AGE or high glucose-evoked Cu²⁺ and cardiomyocyte defects.

Conclusion: These results reveal a role for the RAGE-S100A13-cuproptosis signaling axis in AGE-driven myocardial anomalies in diabetes.

Keywords

Diabetes / advanced glycation endproduct / RAGE / cuproptosis / heart

References

[1] Sun H,Saeedi P,Karuranga S.et al. IDF diabetes atlas: global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045 Diabetes Res Clin Pract. 2022 183 109119 PMC11057359

[2] Li YY,Yu Y,Jing HB.et al. Global epidemiology of diabetes and prediabetes in lean or non-obese patients with NAFLD: a systematic review and meta-analysis Ann Med. 2026 58 2602995 PMC12724282

[3] Jia G,Hill MA,Sowers JR. Diabetic cardiomyopathy: an update of mechanisms contributing to this clinical entity Circ Res. 2018 122 624 38 PMC5819359

[4] Gebretsadik GG,Yang B,Glenn AJ.et al. Dietary manganese, type 2 diabetes, and cardiovascular disease: a UK Biobank cohort study and meta-analysis of over 270,000 individuals J Nutr Health Aging. 2026 30 100754 PMC12756635

[5] Wang M,Zhang SY,Zhai XR.et al. Long-term clinical outcomes in elderly patients with chronic total occlusion and type 2 diabetes: the impact of coronary collateralization following successful recanalization Cardiology Plus. 2024 9 80 90

[6] Tan Y,Zhang Z,Zheng C,Wintergerst KA,Keller BB,Cai L. Mechanisms of diabetic cardiomyopathy and potential therapeutic strategies: preclinical and clinical evidence Nat Rev Cardiol. 2020 17 585 607 PMC7849055

[7] Zhuang Z,Zhu Y,Tao J.et al. UCF101 Rescues against Diabetes-Evoked Cardiac Remodeling and Contractile Anomalies through AMP-activated protein kinase-mediated induction of mitophagy Pharmacology. 2025 110 127 40

[8] Zhang Y,Zou R,Abudureyimu M.et al. Mitochondrial aldehyde dehydrogenase rescues against diabetic cardiomyopathy through GSK3β-mediated preservation of mitochondrial integrity and Parkin-mediated mitophagy J Mol Cell Biol. 2024 15 mjad056 PMC11193060

[9] Jiang MY,Man WR,Zhang XB.et al. Adipsin inhibits Irak2 mitochondrial translocation and improves fatty acid β-oxidation to alleviate diabetic cardiomyopathy Mil Med Res. 2023 10 63 PMC10712050

[10] Dandamudi S,Slusser J,Mahoney DW,Redfield MM,Rodeheffer RJ,Chen HH. The prevalence of diabetic cardiomyopathy: a population-based study in Olmsted County, Minnesota J Card Fail. 2014 20 304 9 PMC4076144

[11] Segar MW,Khan MS,Patel KV.et al. Prevalence and prognostic implications of diabetes with cardiomyopathy in community-dwelling adults J Am Coll Cardiol. 2021 78 1587 98

[12] Schalkwijk CG,Stehouwer CDA. Methylglyoxal, a highly reactive dicarbonyl compound, in diabetes, its vascular complications, and other age-related diseases Physiol Rev. 2020 100 407 61

[13] Berdowska I,Matusiewicz M,Fecka I. Methylglyoxal in cardiometabolic disorders: routes leading to pathology counterbalanced by treatment strategies Molecules. 2023 28 7742 PMC10708463

[14] Lai SWT,Lopez Gonzalez EJ,Zoukari T,Ki P,Shuck SC. Methylglyoxal and its adducts: induction, repair, and association with disease Chem Res Toxicol. 2022 35 1720 46 PMC9580021

[15] Mota KO,de Vasconcelos CML,Kirshenbaum LA,Dhalla NS. The role of advanced glycation end-products in the pathophysiology and pharmacotherapy of cardiovascular disease Int J Mol Sci. 2025 26 7311 PMC12347567

[16] Vianello E,Beltrami AP,Aleksova A.et al. The advanced glycation end-products (AGE)-receptor for age system (RAGE): an inflammatory pathway linking obesity and cardiovascular diseases Int J Mol Sci. 2025 26 3707 PMC12028226

[17] Wang B,Jiang T,Qi Y.et al. AGE-RAGE axis and cardiovascular diseases: pathophysiologic mechanisms and prospects for clinical applications Cardiovasc Drugs Ther. 2025 39 1489 506 PMC12717123

[18] Zhang L,Huang D,Shen D.et al. Inhibition of protein kinase C βII isoform ameliorates methylglyoxal advanced glycation endproduct-induced cardiomyocyte contractile dysfunction Life Sci. 2014 94 83 91

[19] Yang L,Yang P,Lip GYH,Ren J. Copper homeostasis and cuproptosis in cardiovascular disease therapeutics Trends Pharmacol Sci. 2023 44 573 85

[20] Liu Y,Shen M,Zhu S.et al. Metallothionein rescues doxorubicin cardiomyopathy via mitigation of cuproptosis Life Sci. 2025 363 123379

[21] Li P,Li Y,Meng Q,Wang J,Wang K,Yang S. Copper dyshomeostasis and cardiovascular disease: Molecular mechanisms and new strategies for targeted intervention with cuproptosis (Review) Int J Mol Med. 2026 57 19 PMC12634067

[22] Tsvetkov P,Coy S,Petrova B.et al. Copper induces cell death by targeting lipoylated TCA cycle proteins Science. 2022 375 1254 61 PMC9273333

[23] Saha PP,Kumar SKP,Srivastava S,Sinha D,Pareek G,D’silva P. The presence of multiple cellular defects associated with a novel G50E iron-sulfur cluster scaffold protein (ISCU) mutation leads to development of mitochondrial myopathy J Biol Chem. 2014 289 10359 77 PMC4036159

[24] Wang Z,Wu C,Li R.et al. Cuproptosis and cardiovascular diseases: mechanisms, pathophysiology, and therapeutic strategies - a narrative review Rev Cardiovasc Med. 2025 26 38833 PMC12516759

[25] Wold LE,Ceylan-Isik AF,Ren J. Oxidative stress and stress signaling: menace of diabetic cardiomyopathy Acta Pharmacol Sin. 2005 26 908 17

[26] Liu C,Zhang H,Guan R.et al. Inhibition of the HMGB1-RAGE axis attenuates microglial inflammation and ameliorates hypoxia-induced cognitive impairment Int J Mol Sci. 2025 26 8782 PMC12469424

[27] Chen L,Yin Z,Qin X.et al. CD74 ablation rescues type 2 diabetes mellitus-induced cardiac remodeling and contractile dysfunction through pyroptosis-evoked regulation of ferroptosis Pharmacol Res. 2022 176 106086

[28] Shen C,Ma Y,Zeng Z.et al. RAGE-specific inhibitor FPS-ZM1 attenuates AGEs-induced neuroinflammation and oxidative stress in rat primary microglia Neurochem Res. 2017 42 2902 11

[29] Shen L,Zhang T,Yang Y,Lu D,Xu A,Li K. FPS-ZM1 alleviates neuroinflammation in focal cerebral ischemia rats via blocking ligand/RAGE/DIAPH1 pathway ACS Chem Neurosci. 2021 12 63 78

[30] Gautier L,Cope L,Bolstad BM,Irizarry RA. Affy - analysis of Affymetrix GeneChip data at the probe level Bioinformatics. 2004 20 307 15

[31] Ashburner M,Ball CA,Blake JA.et al. Gene Ontology: tool for the unification of biology. The Gene Ontology Consortium Nat Genet. 2000 25 25 9 PMC3037419

[32] Kanehisa M,Goto S,Hattori M.et al. From genomics to chemical genomics: new developments in KEGG Nucleic Acids Res. 2006 34 D354 7 PMC1347464

[33] Subramanian A,Tamayo P,Mootha VK.et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles Proc Natl Acad Sci U S A. 2005 102 15545 50 PMC1239896

[34] Peng H,Fu S,Wang S.et al. Ablation of FUNDC1-dependent mitophagy renders myocardium resistant to paraquat-induced ferroptosis and contractile dysfunction Biochim Biophys Acta Mol Basis Dis. 2022 1868 166448

[35] Chen Y,Zhao J,Ye H.et al. Beneficial impact of cardiac heavy metal scavenger metallothionein in sepsis-provoked cardiac anomalies dependent upon regulation of endoplasmic reticulum stress and ferroptosis but not autophagy Life Sci. 2024 336 122291

[36] Ren J,Dominguez LJ,Sowers JR,Davidoff AJ. Metformin but not glyburide prevents high glucose-induced abnormalities in relaxation and intracellular Ca2+ transients in adult rat ventricular myocytes Diabetes. 1999 48 2059 65

[37] Quan MY,Song XJ,Liu HJ.et al. Amlexanox attenuates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and reprogramming effector and regulatory T cell responses J Neuroinflammation. 2019 16 52 PMC6396467

[38] Rani SG,Mohan SK,Yu C. Molecular level interactions of S100A13 with amlexanox: inhibitor for formation of the multiprotein complex in the nonclassical pathway of acidic fibroblast growth factor Biochemistry. 2010 49 2585 92

[39] Ren J,Sun M,Zhou H.et al. FUNDC1 interacts with FBXL2 to govern mitochondrial integrity and cardiac function through an IP3R3-dependent manner in obesity Sci Adv. 2020 6 eabc8561 PMC7494344

[40] Blackburn NJR,Vulesevic B,Mcneill B.et al. Methylglyoxal-derived advanced glycation end products contribute to negative cardiac remodeling and dysfunction post-myocardial infarction Basic Res Cardiol. 2017 112 57

[41] Li F,Hu H,Wu L.et al. Ablation of mitophagy receptor FUNDC1 accentuates septic cardiomyopathy through ACSL4-dependent regulation of ferroptosis and mitochondrial integrity Free Radic Biol Med. 2024 225 75 86

[42] Chen N,Guo L,Wang L,Dai S,Zhu X,Wang E. Sleep fragmentation exacerbates myocardial ischemia-reperfusion injury by promoting copper overload in cardiomyocytes Nat Commun. 2024 15 3834 PMC11076509

[43] Huo S,Wang Q,Shi W.et al. ATF3/SPI1/SLC31A1 signaling promotes cuproptosis induced by advanced glycosylation end products in diabetic myocardial injury Int J Mol Sci. 2023 24 1667 PMC9862315

[44] Abramson J,Adler J,Dunger J.et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3 Nature. 2024 630 493 500 PMC11168924

[45] Sivaraja V,Kumar TK,Rajalingam D,Graziani I,Prudovsky I,Yu C. Copper binding affinity of S100A13, a Key Component of the FGF-1 nonclassical copper-dependent release complex Biophys J. 2006 91 1832 43 PMC1544301

[46] Landriscina M,Prudovsky I,Mouta Carreira C,Soldi R,Tarantini F,Maciag T. Amlexanox reversibly inhibits cell migration and proliferation and induces the Src-dependent disassembly of actin stress fibers in vitro J Biol Chem. 2000 275 32753 62