Gut microbiota-derived butyric acid regulates calcific aortic valve disease pathogenesis by modulating GAPDH lactylation and butyrylation

Abstract

The involvement of gut microbiota in calcific aortic valve disease (CAVD) pathogenesis remains underexplored. Here, we provide evidence for a strong association between the gut microbiota and CAVD development. ApoE^−/− mice were stratified into easy- and difficult- to calcify groups using neural network and cluster analyses, and subsequent faecal transplantation and dirty cage sharing experiments demonstrated that the microbiota from difficult-to-calcify mice significantly ameliorated CAVD. 16S rRNA sequencing revealed that reduced abundance of Faecalibacterium prausnitzii (F. prausnitzii) was significantly associated with increased calcification severity. Association analysis identified F. prausnitzii-derived butyric acid as a key anti-calcific metabolite. These findings were validated in a clinical cohort (25 CAVD patients vs. 25 controls), where serum butyric acid levels inversely correlated with disease severity. Functional experiments showed that butyric acid effectively hindered osteogenic differentiation in human aortic valve interstitial cells (hVICs) and attenuated CAVD progression in mice. Isotope labeling and ¹³C flux analyses confirmed that butyric acid produced in the intestine can reach heart tissue, where it reshapes glycolysis by specifically modifying GAPDH. Mechanistically, butyric acid-induced butyrylation (Kbu) at lysine 263 of GAPDH competitively inhibited lactylation (Kla) at the same site, thereby counteracting glycolysis-driven calcification. These findings uncover a novel mechanism through which F. prausnitzii and its metabolite butyric acid contribute to the preservation of valve function in CAVD, highlighting the gut microbiota-metabolite-glycolysis axis as a promising therapeutic target.

Keywords

butyric acid / butyrylation / calcific aortic valve calcification / glycolysis / lactylation

References

[1] Small, Aeron M., Katherine E. Yutzey, Bryce A. Binstadt, Kaitlin Voigts Key, Nabila Bouatia-Naji, David Milan, Elena Aikawa, Catherine M. Otto, and Cynthia St. Hilaire. 2024. “Unraveling the Mechanisms of Valvular Heart Disease to Identify Medical Therapy Targets: A Scientific Statement From the American Heart Association.” Circulation 150: e109-e128. https://doi.org/10.1161/CIR.0000000000001254

[2] Moncla, Louis-Hippolyte Minvielle, Mewen Briend, Yohan Bossé, and Patrick Mathieu. 2023. “Calcific Aortic Valve Disease: Mechanisms, Prevention and Treatment.” Nature Reviews Cardiology 20: 546-559. https://doi.org/10.1038/s41569-023-00845-7

[3] Rutkovskiy, Arkady, Anna Malashicheva, Gareth Sullivan, Maria Bogdanova, Anna Kostareva, Kåre-Olav Stensløkken, Arnt Fiane, and Jarle Vaage. 2017. “Valve Interstitial Cells: The Key to Understanding the Pathophysiology of Heart Valve Calcification.” Journal of the American Heart Association 6: e006339. https://doi.org/10.1161/jaha.117.006339

[4] Huang, Yuming, Chen Jiang, Liang Chen, Juanjuan Han, Ming Liu, Tingwen Zhou, Nianguo Dong, and Kang Xu. 2023. “Gli1 Promotes the Phenotypic Transformation of Valve Interstitial Cells Through Hedgehog Pathway Activation Exacerbating Calcific Aortic Valve Disease.” International Journal of Biological Sciences 19: 2053-2066. https://doi.org/10.7150/ijbs.74123

[5] Wang, Chunli, Yuming Huang, Xianqiong Liu, Lanqing Li, Haiying Xu, Nianguo Dong, and Kang Xu. 2021. “Andrographolide Ameliorates Aortic Valve Calcification by Regulation of Lipid Biosynthesis and Glycerolipid Metabolism Targeting MGLL Expression In vitro and In vivo.” Cell Calcium 100: 102495. https://doi.org/10.1016/j.ceca.2021.102495

[6] Xu, Kang, Shangbo Xie, Yuming Huang, Tingwen Zhou, Ming Liu, Peng Zhu, Chunli Wang, et al. 2020. “Cell-Type Transcriptome Atlas of Human Aortic Valves Reveal Cell Heterogeneity and Endothelial to Mesenchymal Transition Involved in Calcific Aortic Valve Disease.” Arteriosclerosis, Thrombosis, and Vascular Biology 40: 2910-2921. https://doi.org/10.1161/atvbaha.120.314789

[7] Liu, Zongtao, Kan Wang, Chen Jiang, Yuqi Chen, Fayuan Liu, Minghui Xie, Wai Yen Yim, et al. 2024. “Morusin Alleviates Aortic Valve Calcification by Inhibiting Valve Interstitial Cell Senescence Through ccnd1/trim25/nrf2 Axis.” Advanced Science 11: e2307319. https://doi.org/10.1002/advs.202307319

[8] Schlotter, Florian, Arda Halu, Shinji Goto, Mark C. Blaser, Simon C. Body, Lang H. Lee, Hideyuki Higashi, et al. 2018. “Spatiotemporal Multi-Omics Mapping Generates a Molecular Atlas of the Aortic Valve and Reveals Networks Driving Disease.” Circulation 138: 377-393. https://doi.org/10.1161/CIRCULATIONAHA.117.032291

[9] Zhu, Zijin, Zhirong Liu, Donghui Zhang, Li Li, Jianqiu Pei, and Lin Cai. 2024. “Models for Calcific Aortic Valve Disease In vivo and In vitro.” Cell Regeneration 13: 6. https://doi.org/10.1186/s13619-024-00189-8

[10] Nemet, Ina, Prasenjit Prasad Saha, Nilaksh Gupta, Weifei Zhu, Kymberleigh A. Romano, Sarah M. Skye, Tomas Cajka, et al. 2020. “A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.” Cell 180: 862-877.e22. https://doi.org/10.1016/j.cell.2020.02.016

[11] Qu, Linghang, Chunli Wang, Haiying Xu, Lanqing Li, Yanju Liu, Qianyun Wan, and Kang Xu. 2022. “Atractylodin Targets GLA to Regulate D-Mannose Metabolism to Inhibit Osteogenic Differentiation of Human Valve Interstitial Cells and Ameliorate Aortic Valve Calcification.” Phytotherapy Research 37: 477-489. https://doi.org/10.1002/ptr.7628

[12] Tang, W. H. Wilson, and Stanley L. Hazen. 2017. “The Gut Microbiome and Its Role in Cardiovascular Diseases.” Circulation 135: 1008-1010. https://doi.org/10.1161/circulationaha.116.024251

[13] Wang, Meng, Xinmin S. Li, Zeneng Wang, Marcia C. de Oliveira Otto, Rozenn N. Lemaitre, Amanda Fretts, Nona Sotoodehnia, et al. 2023. “Trimethylamine N-Oxide Is Associated With Long-Term Mortality Risk: The Multi-Ethnic Study of Atherosclerosis.” European Heart Journal 44: 1608-1618. https://doi.org/10.1093/eurheartj/ehad089

[14] Guo, Hao, Weichun Chou, Yunjia Lai, Kaixin Liang, Jason W. Tam, W. June Brickey, Liang Chen, et al. 2020. “Multi-Omics Analyses of Radiation Survivors Identify Radioprotective Microbes and Metabolites.” Science 370: eaay9097. https://doi.org/10.1126/science.aay9097

[15] Roje, Blanka, Boyao Zhang, Eleonora Mastrorilli, Ana Kovačić, Lana Sušak, Ivica Ljubenkov, Elena Ćosić, et al. 2024. “Gut Microbiota Carcinogen Metabolism Causes Distal Tissue Tumours.” Nature 632: 1137-1144. https://doi.org/10.1038/s41586-024-07754-w

[16] Jie, Zhuye, Huihua Xia, Shilong Zhong, Qiang Feng, Shenghui Li, Suisha Liang, Huanzi Zhong, et al. 2017. “The Gut Microbiome in Atherosclerotic Cardiovascular Disease.” Nature Communications 8: 845. https://doi.org/10.1038/s41467-017-00900-1

[17] Sokol, Harry, Bénédicte Pigneur, Laurie Watterlot, Omar Lakhdari, Luis G. Bermúdez-Humarán, Jean-Jacques Gratadoux, Sébastien Blugeon, et al. 2008. “Faecalibacterium prausnitzii Is an Anti-inflammatory Commensal Bacterium Identified by Gut Microbiota Analysis of Crohn Disease Patients.” Proceedings of the National Academy of Sciences 105: 16731-16736. https://doi.org/10.1073/pnas.0804812105

[18] Miquel, S., R. Martín, O. Rossi, L. G. Bermúdez-Humarán, J. M. Chatel, H. Sokol, M. Thomas, J. M. Wells, and P. Langella. 2013. “Faecalibacterium prausnitzii and Human Intestinal Health.” Current Opinion in Microbiology 16: 255-261. https://doi.org/10.1016/j.mib.2013.06.003

[19] Machiels, Kathleen, Marie Joossens, João Sabino, Vicky De Preter, Ingrid Arijs, Venessa Eeckhaut, Vera Ballet, et al. 2014. “A Decrease of the Butyrate-Producing Species Roseburia hominis and Faecalibacterium prausnitzii Defines Dysbiosis in Patients With Ulcerative Colitis.” Gut 63: 1275-1283. https://doi.org/10.1136/gutjnl-2013-304833

[20] Fitzgerald, Cormac Brian, Andrey N. Shkoporov, Thomas D. S. Sutton, Andrei V. Chaplin, Vimalkumar Velayudhan, R. Paul Ross, and Colin Hill. 2018. “Comparative Analysis of Faecalibacterium prausnitzii Genomes Shows a High Level of Genome Plasticity and Warrants Separation Into New Species-Level Taxa.” BMC Genomics 19: 931. https://doi.org/10.1186/s12864-018-5313-6

[21] Yang, HaiTao, Zhihui Jiang, Yi Yang, Tingting Wu, Yingying Zheng, Yitong Ma, and Xiang Xie. 2024. “Faecalibacterium prausnitzii as a Potential Antiatherosclerotic Microbe.” Cell Communication and Signaling 22: 54. https://doi.org/10.1186/s12964-023-01464-y

[22] Bo, Tingbei, Xueying Zhang, Jing Wen, Ke Deng, Xiaowei Qin, and Dehua Wang. 2019. “The Microbiota-Gut-Brain Interaction in Regulating Host Metabolic Adaptation to Cold in Male Brandt's Voles (Lasiopodomys brandtii).” The ISME Journal 13: 3037-3053. https://doi.org/10.1038/s41396-019-0492-y

[23] Wang, Jingyue, Xianfeng Zhang, Xinyu Yang, Hang Yu, Mengmeng Bu, Jie Fu, Zhengwei Zhang, et al. 2023. “Revitalizing Myocarditis Treatment Through Gut Microbiota Modulation: Unveiling a Promising Therapeutic Avenue.” Frontiers in Cellular and Infection Microbiology 13: 1191936. https://doi.org/10.3389/fcimb.2023.1191936

[24] Shi, Zhuangzhuang, Min Li, Chen Zhang, Hongwen Li, Yue Zhang, Lei Zhang, Xin Li, et al. 2024. “Butyrate-Producing Faecalibacterium prausnitzii Suppresses Natural Killer/T-Cell Lymphoma by Dampening the JAK-STAT Pathway.” Gut 74: 557-570. https://doi.org/10.1136/gutjnl-2024-333530

[25] Wang, Ruiqing, Xinyu Yang, Jinting Liu, Fang Zhong, Chen Zhang, Yuhong Chen, Tao Sun, Chunyan Ji, and Daoxin Ma. 2022. “Gut Microbiota Regulates Acute Myeloid Leukaemia via Alteration of Intestinal Barrier Function Mediated by Butyrate.” Nature Communications 13: 2522. https://doi.org/10.1038/s41467-022-30240-8

[26] Wu, Haicui, and Jiachi Chiou. 2021. “Potential Benefits of Probiotics and Prebiotics for Coronary Heart Disease and Stroke.” Nutrients 13: 2878. https://doi.org/10.3390/nu13082878

[27] Bridgeman, Stephanie C., Wendy Northrop, Phillip E. Melton, Gaewyn C. Ellison, Philip Newsholme, and Cyril D. S. Mamotte. 2020. “Butyrate Generated by Gut Microbiota and Its Therapeutic Role in Metabolic Syndrome.” Pharmacological Research 160: 105174. https://doi.org/10.1016/j.phrs.2020.105174

[28] Wang, Chunli, Shunshun Wang, Zijun Wang, Juanjuan Han, Nan Jiang, Linghang Qu, and Kang Xu. 2024. “Andrographolide Regulates H3 Histone Lactylation by Interfering With p300 to Alleviate Aortic Valve Calcification.” British Journal of Pharmacology 181: 1843-1856. https://doi.org/10.1111/bph.16332

[29] Xie, Fei, Juanjuan Han, Dashuai Wang, Peng Liu, Chao Liu, Fuqiang Sun, and Kang Xu. 2022. “Disturbing Effect of Cepharanthine on Valve Interstitial Cells Calcification via Regulating Glycolytic Metabolism Pathways.” Frontiers in Pharmacology 13: 1070922. https://doi.org/10.3389/fphar.2022.1070922

[30] Feng, Fan, Jiaqin Wu, Qingjia Chi, Shunshun Wang, Wanqian Liu, Li Yang, Guanbin Song, et al. 2024. “Lactylome Analysis Unveils Lactylation-Dependent Mechanisms of Stemness Remodeling in the Liver Cancer Stem Cells.” Advanced Science 11: e2405975. https://doi.org/10.1002/advs.202405975

[31] Pan, Lianhong, Fan Feng, Jiaqin Wu, Shibing Fan, Juanjuan Han, Shunxi Wang, Li Yang, et al. 2022. “Demethylzeylasteral Targets Lactate by Inhibiting Histone Lactylation to Suppress the Tumorigenicity of Liver Cancer Stem Cells.” Pharmacological Research 181: 106270. https://doi.org/10.1016/j.phrs.2022.106270

[32] Xu, Haiying, Lanqing Li, Shunshun Wang, Zijun Wang, Linghang Qu, Chunli Wang, and Kang Xu. 2023. “Royal Jelly Acid Suppresses Hepatocellular Carcinoma Tumorigenicity by Inhibiting H3 Histone Lactylation at H3K9la and H3K14la Sites.” Phytomedicine 118: 154940. https://doi.org/10.1016/j.phymed.2023.154940

[33] Zhang, Di, Zhanyun Tang, He Huang, Guolin Zhou, Chang Cui, Yejing Weng, Wenchao Liu, et al. 2019. “Metabolic Regulation of Gene Expression by Histone Lactylation.” Nature 574: 575-580. https://doi.org/10.1038/s41586-019-1678-1

[34] Uemura, Masato, Stefan Blankvoort, Sean Shui Liang Tok, Li Yuan, Luis Fernando Cobar, Kwok Keung Lit, and Ayumu Tashiro. 2021. “A Neurogenic Microenvironment Defined by Excitatory-Inhibitory Neuronal Circuits in Adult Dentate Gyrus.” Cell Reports 36: 109324. https://doi.org/10.1016/j.celrep.2021.109324

[35] Kang, Sang-Ho, Ramesh Prasad Pandey, Chang-Muk Lee, Joon-Soo Sim, Jin-Tae Jeong, Beom-Soon Choi, Myunghee Jung, et al. 2020. “Genome-Enabled Discovery of Anthraquinone Biosynthesis in Senna tora.” Nature Communications 11: 5875. https://doi.org/10.1038/s41467-020-19681-1

[36] Nshanian, Michael, Joshua J. Gruber, Benjamin S. Geller, Faye Chleilat, Samuel M. Lancaster, Shannon M. White, Ludmila Alexandrova, et al. 2025. “Short-Chain Fatty Acid Metabolites Propionate and Butyrate Are Unique Epigenetic Regulatory Elements Linking Diet, Metabolism and Gene Expression.” Nature Metabolism 7: 196-211. https://doi.org/10.1038/s42255-024-01191-9

[37] Huang, Yuming, Chunli Wang, Tingwen Zhou, Fei Xie, Zongtao Liu, Haiying Xu, Ming Liu, et al. 2024. “Lumican Promotes Calcific Aortic Valve Disease Through H3 Histone Lactylation.” European Heart Journal 45: 3871-3885. https://doi.org/10.1093/eurheartj/ehae407

[38] Callahan, Benjamin J., Paul J. McMurdie, Michael J. Rosen, Andrew W. Han, Amy Jo A Johnson, and Susan P. Holmes. 2016. “DADA2: High-Resolution Sample Inference From Illumina Amplicon Data.” Nature Methods 13: 581-583. https://doi.org/10.1038/nmeth.3869

[39] Bolyen, Evan, Jai Ram Rideout, Matthew R. Dillon, Nicholas A. Bokulich, Christian C. Abnet, Gabriel A. Al-Ghalith, Harriet Alexander, et al. 2019. “Reproducible, Interactive, Scalable and Extensible Microbiome Data Science Using QIIME 2.” Nature Biotechnology 37: 852-857. https://doi.org/10.1038/s41587-019-0209-9

RIGHTS & PERMISSIONS

2025 The Author(s). iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.