CPT1B K321 crotonylation contributes to cardiac dysfunction in endotoxic shock

www.nature.com/emm ARTICLE OPEN CPT1B K321 crotonylation contributes to cardiac dysfunction in endotoxic shock Ni Yang1,5, Jingjing Yang2,5, Yang-Fan Xu1,5, Xin-Mei Huang3, Ying Gu4, Ri Wen1, Yu-Hang Yang1, Peng-Hui Hao1, Xin-Ru Yang1, ✉ ✉ Chun-Feng Liu1, Wanshan Ning4 and Tie-Ning Zhang 1 © The Author(s) 2026 1234567890();,: Lysine crotonylation (Kcr) is a novel posttranslational modification that has been proven to have evolutionary conservation. However, the role of protein Kcr in the pathogenesis of endotoxic shock-induced secondary cardiomyopathy is elusive. Here a classic rat model of endotoxic shock was induced by intraperitoneal lipopolysaccharide (LPS) injection. Crotonylproteomic analysis was subsequently performed on myocardial tissues to profile the Kcr modifications that occurred during endotoxic shock, and we found that the Kcr of carnitine palmitoyltransferase 1B (CPT1B) at the K321 site was significantly upregulated in LPS-treated rats. These findings were also confirmed in rat primary cardiomyocytes and H9C2 cells exposed to LPS. Furthermore, we demonstrated that the K321cr of CPT1B impaired CPT1B activity. Moreover, we found that the mutation of K321 to R prevented the mutant CPT1B protein from being crotonylated by LPS, thus alleviating LPS-induced lipid droplet deposition and mitochondrial dysfunction, as reflected by the recovery of ATP generation and mitochondrial membrane potential. Consistently, in vivo cardiac-specific overexpression of CPT1B via an AAV9 vector with a cardiomyocyte-specific promoter (cTnT) also confirmed that the K321-R mutation of CPT1B protected against endotoxic shock-induced secondary cardiomyopathy. Further liquid chromatography–tandem mass spectrometry analysis revealed that the crotonyl-transferases P300 and CBP might be involved in the Kcr of CPT1B. Mechanistically, LPS led to the dissociation of CBP from CPT1B, which promoted the binding of P300 to CPT1B, thereby increasing the level of CPT1B K321cr in H9C2 cells. Taken together, the results of our study revealed the regulatory axis of CPT1B K321 cr/CBP/ P300 in LPS-induced endotoxic shock. Experimental & Molecular Medicine (2026) 58:1674–1687; https://doi.org/10.1038/s12276-026-01730-2 Graphical Abstract INTRODUCTION Septic shock is a state of acute circulatory failure with a high burden of organ dysfunction and profound cellular metabolic abnormalities during severe infection1,2. As a specific etiological subset of septic shock, endotoxic shock is caused primarily by Gram-negative bacterial lipopolysaccharide (LPS) and is characterized by systemic hypotension and reduced myocardial contractility3,4. Despite promising preclinical findings, clinical interventions for endotoxic shock, including antibiotics, intravenous immunoglobulin and detoxifying agents such as alkaline phosphatase, have shown limited efficacy5–7. Therefore, elucidating the signaling mechanisms that drive endotoxic shock progression is critical for developing effective therapies against this condition and septic shock. 1 Department of Pediatrics, PICU, Shengjing Hospital of China Medical University, Shenyang, China. 2Department of Pulmonary and Critical Care Medicine, the First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China. 3Department of Endocrinology, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, China. 4Institute for Clinical Medical Research, the First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China. 5These authors contributed equally: Ni Yang, Jingjing Yang, Yang-Fan Xu. ✉email: ; Received: 2 July 2025 Revised: 25 February 2026 Accepted: 1 March 2026 Published online: 28 May 2026 N. Yang et al. 1675 Lysine crotonylation (Kcr) was initially described in 2011 as a protein posttranslational modification (PTM) on the lysine residues of histones8. More recent studies have identified Kcr among a large number of nonhistone proteins9,10, which are essential for multiple cellular processes, such as remodeling, RNA splicing and metabolism11. On the one hand, the Kcr process can be regulated by the cellular concentration of crotonyl-CoA12. On the other hand, similar to lysine acetylation (Kac), Kcr is catalyzed mainly by three types of protein kinase13,14: writers, which are acyltransferases that are responsible for installing crotonyl groups from modified histones or nonhistones, such as acetyltransferase P300, CREB-binding protein (CBP) and general control nonderepressible 5 (GCN5); erasers, which are deacylases that catalyze the removal of acyl groups from amino acid residues, such as NAD+-dependent enzymes class III HDACs (Sirt1-3); and readers, which are the specific proteins responsible for reading the acylation marks, with unknown mechanisms. Recently, an increasing number of studies have attempted to explore the relationships between Kcr and multiple diseases, including infectious diseases15, metabolic diseases16, neurological diseases17, cancer, and embryonic and reproductive diseases14 in animal and cell models. Notably, Kcr has also been shown to be involved in the significant physiological processes of cardiovascular diseases18,19. Chen et al.20 reported that the Kcr of SERCA2a at Lys120 was significantly increased after cardiac-specific knockout of Sirt1 (Eraser) in mice, which induced cardiac hypertrophy and abnormal energy metabolism in the heart, ultimately leading to arrhythmias. Short-chain enoyl-CoA hydratase (encoded by ECHS1), a hydratase that hydrolyzes crotonyl-CoA, mediates histone crotonylation21,22. Mutation of the ECHS1 gene leads to hypertrophic cardiomyopathy21. The overexpression of ECHS1 significantly inhibited H3K18cr and H2BK12cr, thereby suppressing Ang II-induced cardiac hypertrophy18. In a mouse model of ischemia‒reperfusion, increased general Kcr via sodium crotonate provision or upregulation of the site-specific Kcr of selected mitochondrial protein IDH3a and the cytoskeletal protein TPM1 could protect cardiomyocytes from apoptosis and fibrosis19. However, the role of protein Kcr in endotoxic shock-induced secondary cardiomyopathy is poorly understood. In this study, to identify new therapeutic targets or strategies for prognostic interventions in the context of endotoxic shock, crotonyl-proteomics analysis was performed on myocardial tissues to profile the Kcr modifications that occurred following LPSinduced endotoxic shock. Accordingly, we identified upregulated crotonylation of carnitine palmitoyltransferase 1B (CPT1B) and provided evidence that crotonylation at the K321 site of CPT1B might be crucial to cardiac dysfunction in endotoxic shock. MATERIALS AND METHODS Cardiomyocyte-specific AAV9 vectors Cardiomyocyte-specific adeno-associated virus serotype 9 (AAV9) vectors were designed, constructed, and packaged by General Biol (Anhui) Co., Ltd. (China). The vectors utilized the cardiac troponin T (cTnT) promoter to ensure heart-restricted transgene expression. All the co (...truncated)