NPM3 functions as a lactyltransferase to promote necroptosis in male diabetic cardiomyopathy mice models via FASN transcription modulation

Xu, Hongjiao; Jiang, Xinyu; Wang, Fangrui; Meng, Fufen; Liu, Yue; Huang, Jiechun; Li, Jinbao; Zhu, Minmin

doi:10.1038/s41467-026-70513-0

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Published: 09 March 2026

NPM3 functions as a lactyltransferase to promote necroptosis in male diabetic cardiomyopathy mice models via FASN transcription modulation

Nature Communications volume 17, Article number: 3685 (2026) Cite this article

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Abstract

Diabetic cardiomyopathy (DCM) is characterized by diastolic dysfunction, which progresses to heart failure and abnormal electrophysiological changes in patients with diabetes. Lactate-mediated histone lactylation has been implicated in DCM pathogenesis. We investigated the mechanisms by which lactate-mediated histone-H3-lysine-18 lactylation (H3K18la) and H3K27la promote necroptosis in male DCM mice models. Lactate-mediated H3K18la and H3K27la participate in necroptosis via modulating fatty acid synthase (FASN) transcription in DCM. Moreover, Nucleophosmin/nucleoplasmin-3 (NPM3), identified as a lactyltransferase, regulated H3K18la and H3K27la, thereby activating FASN transcription and triggering necroptosis. Furthermore, dihydroartemisinin (DHA) inhibited NPM3 lactyltransferase activity by competing for binding sites between lactate and NPM3. Importantly, DHA treatment reduced the expression of NPM3, H3K18la, H3K27la, and FASN; alleviated necroptosis and cardiac tissue damage; and resolved diastolic dysfunction and ventricular hypertrophy in DCM models. In conclusion, NPM3 acts as a lactyltransferase to modulate FASN transcription, thereby triggering necroptosis. Moreover, inhibiting NPM3-induced histone lactylation via DHA represents an efficacious therapeutic strategy for DCM.

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Introduction

Diabetes mellitus is a chronic, noninfectious metabolic disorder that has reached pandemic proportions on a global scale^1,2. A substantial epidemiological association exists between diabetes mellitus and cardiovascular disease³. Moreover, diabetes mellitus independently increases the risk of heart failure, irrespective of the presence of hypertension, coronary heart disease, or valvular heart disease³. Persistent hyperglycaemia results in structural and functional alterations in cardiomyocytes that are not attributable to myocardial ischaemia or microvascular atherosclerosis, a condition referred to as diabetic cardiomyopathy (DCM)⁴. Traditionally, DCM is divided into two stages: the early stage is characterized by concentric left ventricular hypertrophy, increased myocardial stiffness, elevated atrial filling pressure, and impaired diastolic function, typically manifesting as an unchanged ejection fraction; the late stage is characterized by increased myocardial fibrosis, further impaired diastolic function, and the emergence of systolic dysfunction⁵. Moreover, patients with type 2 diabetes are at increased risk of developing DCM characterized by diastolic dysfunction and preserved left ventricular ejection fraction⁶. Although various treatment strategies for DCM exist, the primary focus remains controlling blood glucose and lipid levels, with a notable scarcity of effective pharmacological interventions for myocardial damage. Consequently, more than one-third of diabetic patients eventually develop DCM^4,7. Hence, it is imperative to elucidate critical molecular pathways involved in the progression of DCM and to identify potential therapeutic targets that may prevent or mitigate myocardial damage.

Cardiomyocyte death is a pivotal event that occurs because of early molecular changes preceding heart remodelling^8,9. Given that cardiomyocyte death underpins structural alterations in the left ventricular wall that frequently culminate in ventricular dysfunction, targeting cardiomyocyte death pathways may represent a potential therapeutic strategy. Among the various forms of cell death, necroptosis plays a critical role in cardiomyocyte death in DCM¹⁰. Necroptosis is typically initiated by ligands binding to tumour necrosis factor (TNF) family death domain receptors, pattern-recognition receptors, and viral sensors. This process involves the activation of receptor-interacting protein kinase 1 (RIPK1), which recruits RIPK3 and phosphorylates mixed lineage kinase-like (MLKL), thereby forming necrotic vesicles that mediate cell death^11,12,13,14. Previous studies have demonstrated that fatty acid synthase (FASN) plays a significant role in promoting necroptosis to participate in lipodystrophy¹⁵ and ovarian follicular development¹⁶. However, whether FASN contributes to DCM through the activation of necroptosis is not known. Moreover, the transcriptional regulatory mechanism of FASN in DCM has not been fully elucidated. Exploration of the transcriptional mechanism of FASN in DCM may provide potential targets for therapeutic strategies.

A multitude of glucose metabolites, including lactate, play significant roles in the complications related to diabetes^17,18. Lactate is believed to play a role in the pathogenesis of DCM¹⁹. Nonetheless, the exact mechanism by which lactate promotes the progression of DCM remains insufficiently understood. Epigenetics plays a critical role in the complications associated with diabetes²⁰. Our previous research demonstrated that both histone methylation²¹ and histone acetylation²² contribute to the onset and progression of diabetes-related complications. Recently, histone-H3-lysine-18 lactylation (H3K18la) was identified as a tissue-specific active enhancer²³. Furthermore, previous studies have indicated that lactate-mediated histone lactylation, a epigenetic modification, is involved in the onset and progression of diabetes-related complications^24,25. Consistently, lactate-mediated histone lactylation has been implicated in DCM¹⁹. However, the role of histone lactylation in triggering cardiomyocyte necroptosis remains poorly understood. Additionally, the specific lactyltransferases or delactyltransferases involved in DCM have not been well characterized. Filling this knowledge gap by characterizing the relevant lactyltransferases or delactyltransferases represents a vital step toward translational therapies for DCM.

Nucleophosmin/nucleoplasmin 3 (NPM3) is predominantly a nuclear protein belonging to the NPM protein family. Research has demonstrated that the NPM family plays essential roles in DNA replication, histone chaperone activity, ribosome biogenesis, and chromatin remodelling^26,27. In this study, we determined that NPM3 functions as a lactyltransferase, facilitating histone lactylation both in vivo and in vitro. Moreover, our results demonstrated that NPM3 modulates FASN transcription to trigger necroptosis via the regulation of H3K18la and H3K27la in DCM. Importantly, we propose a therapeutically relevant approach for DCM by reducing the lactyltransferase activity of NPM3 through competitive antagonism of the binding between lactate and NPM3 using dihydroartemisinin (DHA). Our work defines a lactylation-dependent pathway in DCM and offers a targeted therapeutic strategy that may prevent or mitigate myocardial damage.

Results

The levels of lactate, lactylation (Kla), H3K18la and H3K27la were increased in DCM models and diabetic patients

To explore the potential mechanisms involved in DCM, we initially constructed a DCM mouse model in which a high-sugar and high-fat diet was combined with multiple low-dose STZ injections. The data for the mice used in this study are shown in Supplementary Table 1. Compared with those of the control animals, the myocardial structure of the DCM model mice changed, characterized by unclear cell edges, disrupted fibre connectivity, and an increased degree of cardiac fibrosis (Fig. 1a). Moreover, the DCM model mice presented diastolic dysfunction and increased ventricular mass (Fig. 1b–g). Lactate has been reported to play a significant role in complications related to diabetes¹⁸. Moreover, lactate dehydrogenase A (LDHA) promotes the conversion of pyruvate to lactate via the glucose pathway. Therefore, we assessed LDHA expression and lactate levels in the hearts of DCM model mice, and our data indicated that LDHA and lactate levels were increased in the hearts of DCM model mice (Fig. 1h–k). A previous study indicated that lactate-induced histone lactylation plays a crucial role in the pathogenesis of DCM¹⁹. Consistently, our data indicated that lactate-mediated Kla expression was increased in the hearts of DCM model mice (Fig. 1j, k). Histone lactylation has been implicated in the regulation of gene transcription under hyperglycaemic conditions²⁸. To determine whether and which histone lactylation plays a major role in DCM, confocal imaging was performed to determine the colocalization between Kla and histone H1 (H1), H2A, H2B, H3 or H4. Our data verified the strong colocalization of Kla with H3 in the hearts of DCM model mice (Fig. 1l). These data indicated that histone lactylation modifications predominantly occurred on H3 in DCM. H3K9la, H3K18la, H3K27la, H4K5la, H4K8la and H4K12la were subsequently assessed in the hearts of DCM model mice. Western blotting and IHC revealed that H3K18la and H3K27la levels were significantly increased in the hearts of mice with DCM, whereas H3K9la, H4K5la, H4K8la and H4K12la expression did not significantly change (Fig. 1j, k).

**Fig. 1: The levels of lactate, lactylation (Kla), H3K18la and H3K27la were increased in diabetic cardiomyopathy (DCM) model mice.**

Next, we constructed hyperglycaemic cellular models using AC-16 cells and mouse primary cardiomyocytes (PCMs). High-glucose treatment increased LDH release in AC-16 cells and PCMs (Supplementary Fig. 1a). Moreover, increases in lactate and LDHA levels were detected in hyperglycaemic cells (Supplementary Fig. 1b–e). Furthermore, the levels of Kla, H3K18la and H3K27la were elevated in hyperglycaemic cells, whereas the level of H3K9la, H4K5la, H4K8la and H4K12l was not significantly changed (Supplementary Fig. 1d).

We subsequently used patient heart tissues to further verify whether the level of histone lactylation increased under hyperglycaemic conditions. HE staining of heart samples from nondiabetic and diabetic patients with valvular heart disease is presented in the present study (Supplementary Fig. 1f). Masson staining revealed increased collagen accumulation and increased interstitial fibrosis in the hearts of diabetic patients (Supplementary Fig. 1f). Moreover, the results of the IHC assay indicated that LDHA, Kla, H3K18la and H3K27la levels were increased in the hearts of diabetic patients, a finding that was similar to the results observed in DCM model mice (Supplementary Fig. 1f). The information regarding the patients included in this study is shown in Supplementary Table 2.

Lactate-mediated H3K18la and H3K27la contributes to DCM in vivo and in vitro

To determine the involvement of lactate-mediated lactylation in DCM, oxamate (OXA) and stiripentol (STP) were administered to DCM model mice and hyperglycaemic cells to inhibit lactate synthesis. Our findings demonstrated that OXA treatment significantly mitigated heart injury and interstitial fibrosis (Fig. 2a); decreased lactylation and H3K18la, H3K27la and LDHA expression (Fig. 2a–c), reduced lactate levels (Fig. 2d); and alleviated hyperglycaemia-mediated diastolic dysfunction and ventricular hypertrophy (Fig. 2e–j) in DCM model mice. Consistently, OXA and STP treatment decreased LDHA, lactylation, and H3K18la and H3K27la expression; reduced lactate levels; and attenuated LDH release in a concentration-dependent manner in hyperglycaemic cells (Supplementary Figs. 2 and 3). Next, we constructed lentiviral plasmids expressing wild-type H3 (H3-WT) and an H3 mutant containing point mutations in which K18/27 is replaced with arginine (H3-K18/27R) to further verify the importance of lactate-mediated expression of H3K18la and H3K27la in DCM. Notably, compared with those in the H3-WT group, the expression of the H3-K18/27R mutant significantly decreased the H3K18la and H3K27la levels and attenuated LDH release following high-glucose treatment (Supplementary Fig. 4). These results suggested that lactate-induced H3K18la and H3K27la expression contributes to the onset and progression of DCM.

**Fig. 2: Lactate-mediated lactylation (Kla) contributed to diabetic cardiomyopathy (DCM) in vivo.**

Lactate-induced H3K18la and H3K27la play crucial roles in the development of DCM by triggering necroptosis

A variety of modes of cell death have been identified in DCM, and cell death plays a crucial role in its pathogenesis^8,9. Given the important role played by H3K18la and H3K27la in DCM, we conducted CUT&Tag experiments using AC-16 cells subjected to high-glucose treatment (Supplementary Data 1 and 2). A joint analysis of H3K18la and H3K27la CUT&Tag data revealed an enrichment of KEGG pathways related to the TNF, FoxO, TGF-beta, p53, and PI3K-Akt signalling pathways (Fig. 3a and Supplementary Data 3). Notably, these pathways play important roles in cell death, including apoptosis and necroptosis^29,30,31. To determine whether apoptosis or necroptosis participated in DCM progression, relevant indicators of apoptosis and necroptosis were examined. Our data indicated that in hyperglycaemic cells, the expression of p-MLKL and p-RIPK1 increased, whereas the expression of BAX, bcl-2 and caspase 3 did not change significantly (Supplementary Fig. 5a). Moreover, the necroptosis inhibitor necrostatin-1 (NSA) inhibited LDH release in hyperglycaemic cells (Supplementary Fig. 5b). However, compared with NSA, the apoptosis inhibitor Z-VAD-FMK (zVAD) had a weaker effect on LDH release in hyperglycaemic cells (Supplementary Fig. 5b). Moreover, the results of the IHC assay indicated that the expression of p-MLKL and p-RIPK1 was increased in the hearts of diabetic patients (Supplementary Fig. 5c). To confirm that necroptosis played a crucial role in DCM, MLKL^−/− mice were used to establish a DCM model (MLKL^−/− + DCM) (Fig. 3b, c). Compared with DCM model mice, MLKL^−/− + DCM model mice presented reduced cardiac injury (Fig. 3b), decreased fibrosis (Fig. 3b), improved diastolic dysfunction and decreased ventricular mass (Fig. 3d–i). In addition, compared with DCM mice, MLKL^−/− + DCM mice presented comparable levels of lactate, H3K18la and H3K27la in cardiac tissues (Supplementary Fig. 5d–f). These findings indicate that necroptosis plays a crucial role in the pathogenesis of DCM but does not affect overall metabolic activity.

**Fig. 3: Lactate-induced H3K18la and H3K27la played crucial roles in the development of diabetic cardiomyopathy (DCM) by triggering necroptosis.**

To determine whether lactate-induced H3K18la and H3K27la participated in DCM by triggering necroptosis, we investigated the effects of OXA on necroptosis in the hearts of DCM model mice. Our data indicated that OXA treatment decreased p-MLKL and p-RIPK1 levels in the hearts of DCM model mice (Fig. 3j, k). Consistent with these findings, OXA and STP treatment decreased p-MLKL and p-RIPK1 expression in hyperglycaemic cells (Supplementary Fig. 5g, h). In addition, overexpression of the H3-K18/27R mutant significantly attenuated p-MLKL and p-RIPK1 expression following high-glucose treatment compared with that in the H3-WT group (Supplementary Fig. 5i). These data showed that lactate-induced H3K18la and H3K27 participated in DCM by triggering necroptosis.

Lactate-mediated H3K18la and H3K27la expression modulates FASN transcription to promote necroptosis in DCM

To elucidate the exact mechanism by which lactate-mediated H3K18la and H3K27la participate in necroptosis in DCM, RNA-seq data from the hearts of diabetic patients (Supplementary Data 4) and DCM model mice (Supplementary Data 5), as well as the top 100 necroptosis-related genes (Supplementary Data 6) obtained from GeneCards (https://www.genecards.org/), were analysed. Our data indicated that FASN might contribute to necroptosis in DCM (Fig. 4a). CUT&Tag data indicated that H3K18la and H3K27la might occupy the promoter region of FASN (Fig. 4b and Supplementary Data 1 and 2), a finding that was confirmed by a ChIP assay (Fig. 4c). Moreover, FASN expression was increased in the hearts of diabetic patients (Supplementary Fig. 6a), DCM model mice (Fig. 4d–f) and hyperglycaemic cells (Supplementary Fig. 6b, c). A previous study indicated that FASN triggers necroptosis via the upregulation of reactive oxygen species (ROS) accumulation¹⁵. Consistent with these findings, both FASN silencing and FASN inhibition (orlistat and C75, inhibitors of FASN) attenuated necroptosis in hyperglycaemic cells via inhibition of ROS accumulation in the present study (Supplementary Figs. 6d–h and 7). In addition, the inhibition of FASN expression by orlistat (Fig. 4d–f) also attenuated necroptosis (Fig. 4d, e), reduced damage to cardiac structure (Fig. 4d), decreased fibrosis (Fig. 4d), alleviated diastolic dysfunction and decreased ventricular mass in DCM model mice (Fig. 4g–l). Furthermore, OXA treatment decreased FASN levels in the hearts of DCM model mice (Supplementary Fig. 8a–c). Similarly, both OXA treatment and STP treatment inhibited FASN expression and ROS accumulation in hyperglycaemic cells (Supplementary Fig. 8d–k). In addition, overexpression of the H3-K18/27R mutant significantly attenuated FASN transcription and ROS accumulation following high-glucose treatment compared with that in the H3-WT group (Supplementary Fig. 9). These data indicated that lactate-mediated H3K18la and H3K27la expression modulated FASN transcription to trigger necroptosis in DCM.

**Fig. 4: Lactate-mediated H3K18la and H3K27la modulated FASN transcription to promote necroptosis in DCM.**

NPM3 acts as a lactyltransferase that lactylates H3K18 and H3K27

H3K18la and H3K27la modifications are regulated by writers and erasers. Therefore, determining the regulatory enzymes of H3K18la and H3K27la to further explore the effects of H3K18la and H3K27la on DCM pathogenesis is crucial. First, in cell lysates from high-glucose-exposed cells but not preheated cells (boiled at 95 °C to denature most proteins), lactate markedly increased the lactylation of purified H3 (Fig. 5a). These results suggested that certain proteins in cells exposed to high levels of glucose might utilize lactate to lactylate H3. Recently, p300 and AARS1/2 were shown to serve as lactyltransferases^32,33. To identify potential regulatory enzymes that modulate H3K18la and H3K27la, co-IP coupled with mass spectrometry analysis was conducted. Our data indicated that p300 and AARS1/2 did not interact with either H3K18la or H3K27la (Supplementary Data 7 and 8). However, the NPM family, including NPM1 and NPM3, was found to associate with H3K18la and H3K27la (Fig. 5b, Supplementary Fig. 10a and Supplementary Data 7 and 8). The results of molecular docking experiments and co-IP experiments suggested that NPM1 could directly bind to lactate and H3 (Supplementary Fig. 10b, c). However, an in vitro lactylation assay indicated that NPM1 could not use lactate to lactylate H3 (Supplementary Fig. 10d), indicating that NPM1 might not be a potential lactyltransferase. Next, we initiated experiments to determine whether NPM3 possessed lactyltransferase activity. Crystallographic studies revealed that NPM3 directly bound to H3 (Fig. 5c), which was confirmed by co-IP, GST pulldown and immunofluorescence colocalization assays (Fig. 5d, e and Supplementary Fig. 11a, b). In addition, high-glucose treatment increased the binding between NPM3 and H3K18/27la in a time dependent manner (Supplementary Fig. 11c). Moreover, molecular docking also indicated that NPM3 directly bound to lactate (Fig. 5f), a finding that was confirmed via an isothermal titration calorimetry (ITC) assay (Fig. 5g and Supplementary Fig. 11d). Furthermore, an in vitro lactylation assay indicated that NPM3 used lactate as a substrate to lactylate H3K18 and H3K27 in a concentration-dependent manner (Fig. 5h). Crystallographic studies indicated that ARG:87 and GLN:117 were the binding sites of lactate on NPM3 (Fig. 5f), and we postulated that these two sites played a vital role in the lactyltransferase function of NPM3. Therefore, two mutant plasmids, NPM3^R87G and NPM3^Q117G, and two corresponding purified proteins were constructed. An in vitro lactylation assay indicated that NPM3^Q117G could not lactylate H3K18 or H3K27 (Supplementary Fig. 11e). To verify that NPM3 acted as a lactyltransferase to modulate H3K18la and H3K27la ex vivo, both loss-of-function and gain-of-function experiments were performed. Our data indicated that NPM3 overexpression increased H3K18la and H3K27la levels in cells (Fig. 5i, j and Supplementary Fig. 11f, g). Moreover, only the NPM3^Q117G mutation abolished NPM3-induced H3K18la and H3K27la levels (Fig. 5i, j and Supplementary Fig. 11f, g), confirming the necessity of lactate binding for NPM3-mediated H3K18la and H3K27la. Furthermore, NPM3 silencing decreased high-glucose−induced H3K18la and H3K27la modifications in cells (Fig. 5k, l and Supplementary Fig. 11h, i). In addition, NPM3 overexpression or silencing had no effect on H3K18 acetylation (H3K18ac) and H3K27ac levels (Fig. 5i–l). These data indicated that NPM3 acted as a lactyltransferase and used lactate as a substrate to directly lactylate H3K18 and H3K27.

**Fig. 5: NPM3 acted as a lactyltransferase that modulated H3K18la and H3K27la.**

Next, NPM3 was found to exhibit a high degree of homology across multiple species (Supplementary Fig. 12a). Subsequent examination of the amino acid sequences of NPM3 from various species revealed evolutionarily conserved binding sites for lactate (Supplementary Fig. 12b) and a nuclear localization sequence (NLS) motif (Supplementary Fig. 12c). A previous study indicated that lactate is initially activated by alanyl-tRNA synthetase 1/2 (AARS1/2), a recently confirmed lactyltransferase, through adenosine triphosphate (ATP)-dependent formation of the reaction intermediate lactate-adenosine monophosphate (AMP) to release pyrophosphate (PPi), after which lactate is transferred to lysine residues of target proteins to release AMP³⁰. Therefore, ATP, PPi and AMP levels were assessed in our in vitro lactylation system. Our data indicated that a decrease in ATP and the release of PPi were readily detected after in vitro reactions with lactate, ATP with NPM3 and either with or without the H3 substrate (Supplementary Fig. 12d, e). However, the production of AMP was detected only after the protein substrate H3 was provided (Supplementary Fig. 12f). Together, these results showed that NPM3 catalysed H3 lactylation through a two-step reaction (Supplementary Fig. 12g) similar to that of AARS1/2 reported previously³³.

Next, we assessed the expression of NPM3 in DCM models and diabetic patients. Our data indicated that NPM3 levels were increased in the hearts of DCM model mice (Supplementary Fig. 13a–c) and hyperglycaemic cells (Supplementary Fig. 13d, e), as well as in the hearts of diabetic patients (Supplementary Fig. 13f). These data indicated that NPM3 acted as a lactyltransferase and might play a crucial role in DCM.

NPM3 mediates H3K18la and H3K27la to trigger necroptosis in DCM via the modulation of FASN transcription

Next, we determined whether NPM3-induced H3K18la and H3K27la participated in necroptosis in DCM via the modulation of FASN transcription. Our data showed that NPM3 silencing attenuated FASN levels and inhibited necroptosis in hyperglycaemic cells (Supplementary Fig. 14). In addition, NPM3 overexpression increased FASN expression and promoted necroptosis in cells (Supplementary Fig. 15). Moreover, the effect of NPM3 overexpression on necroptosis was abrogated by FASN silencing (Supplementary Fig. 15). Furthermore, NPM3^Q117G did not affect FASN transcription or necroptosis in cells (Supplementary Fig. 16a–d). In addition, ChIP assays confirmed that NPM3 was enriched in the promoter region of FASN (Supplementary Fig. 16e). However, p300 and AARS1/2 were not enriched in the promoter region of FASN (Supplementary Fig. 16e). Consistently, p300 and AARS1/2 silencing had no effect on FASN transcription (Supplementary Fig. 16f–n). In addition, in AC-16 cells, the regulatory capacity of p300 and AARS1/2 silencing on hyperglycaemia-induced H3K18la and H3K27la expression was weaker than that of NPM3 (Supplementary Fig. 16f–n). Moreover, a luciferase reporter assay indicated that overexpression of NPM3 and NPM3^Q87G enhanced FASN promoter activity, whereas overexpression of NPM3^Q117G had no effect (Supplementary Fig. 16o). Furthermore, the effect of NPM3 silencing was reversed by NPM3 overexpression, while NPM3^Q117G had no effect (Supplementary Fig. 17). These data suggest that the absolute specificity of NPM3 relative to other lactyltransferases lies in its regulation of H3K18la and H3K27la levels, as well as FASN transcription, thereby promoting necroptosis in DCM.

To further confirm that NPM3 mainly exerts its function through modulation of H3K18la and H3K27la in the nucleus, we introduced H3-WT and H3-K18/27R plasmids into the NPM3-overexpressed cells. Our data indicated that compared with that in the H3-WT group, the expression of the H3-K18/27R mutant significantly reduced H3K18la and H3K27la levels and inhibited FASN transcription and necroptosis following NPM3-overexpressed treatment (Supplementary Fig. 18). These data indicated that NPM3 lactylated H3K18 and H3K27 to trigger necroptosis by activating FASN transcription in hyperglycaemic cells.

To confirm that NPM3 modulated H3K18la and H3K27la to trigger necroptosis via the activation of FASN transcription in vivo, we used NPM3^−/− mice. Our data indicated that the inhibition of NPM3 expression attenuated H3K18la, H3K27la and FASN levels (Fig. 6a–d), alleviated necroptosis and cardiac injury (Fig. 6a, b), resolved diastolic dysfunction, and decreased ventricular hypertrophy in DCM model mice (Fig. 6e–j). These data indicated that NPM3 activated FASN transcription to trigger necroptosis via the lactylation of H3K18 and H3K27 in DCM.

**Fig. 6: NPM3 mediated H3K18la and H3K27la to trigger necroptosis via the modulation of FASN transcription in diabetic cardiomyopathy (DCM) model mice.**

Lactate-induced H3K18la and H3K27la expression positively regulates NPM3 transcription

Further analysis of the CUT&Tag data revealed that H3K18la and H3K27la were also enriched at the promoter of NPM3 (Supplementary Fig. 19a and Supplementary Data 1 and 2), which was confirmed via a ChIP assay (Supplementary Fig. 19b). Moreover, lactate treatment increased NPM3 promoter activity (Supplementary Fig. 19c). Additionally, after the cells were exposed to 10 mM lactate, the expression of H3K18la, H3K27la and NPM3 subsequently increased (Supplementary Fig. 19d, e). Similarly, OXA treatment decreased high-glucose−induced NPM3 levels in a concentration-dependent manner (Supplementary Fig. 19f, g). Consistent with these findings, OXA treatment reduced NPM3 levels in DCM model mice (Supplementary Fig. 19h–j). In addition, lactate treatment promoted the nuclear translocation of NPM3 in cells (Supplementary Fig. 19k). These results suggested that lactate-induced H3K18la and H3K27la positively regulated NPM3 transcription to form a positive feedback loop and that NPM3 might function as a sensor of intracellular lactate (Supplementary Fig. 19l).

DHA competitively antagonizes NPM3-induced H3K18la and H3K27la expression to inhibit FASN transcription and necroptosis in DCM

Considering that NPM3-induced histone lactylation plays an important role in necroptosis in DCM, the inhibition of NPM3-mediated H3K18la and H3K27la activity may be a potential therapeutic approach for DCM. The results of our previous experiments (Fig. 5f, i and j) confirmed that the ARG:87 and GLN:117 sites played significant roles in the lactylation activity of NPM3. We subsequently conducted molecular docking of this pocket structure with small-molecule compounds in the HY-L022P FDA-Approved Drug Library Plus database and reported that DHA could bind to the same sites (Supplementary Fig. 20a), which was confirmed via ITC (Supplementary Fig. 20b). Next, competitive binding experiments were conducted using ITC to investigate the competitive interaction between lactate and DHA. During the titration of DHA with the NPM3 + lactate mixture, an exothermic event was observed (Supplementary Fig. 20c), indicating that DHA was capable of displacing lactate and forming a binding interaction with NPM3. However, during the titration of lactate with the NPM3 + DHA mixture, no exothermic phenomenon was observed (Supplementary Fig. 20d), suggesting that lactate was unable to displace DHA from NPM3. These data demonstrated that the binding efficiency of DHA with NPM3 was stronger than that of lactate according to ITC assays. A previous study indicated that DHA ameliorated retinal vascular dysfunction in diabetes mellitus via the inhibition of FASN expression³⁴. Nevertheless, the precise mechanism underlying these observations remains to be elucidated. We hypothesize that DHA inhibits the lactyltransferase activity of NPM3 to suppress FASN transcription and necroptosis, thus alleviating DCM. The results of our in vitro lactylation experiment indicated that DHA treatment inhibited NPM3-induced H3K18la and H3K27la expression in a concentration-dependent manner (Supplementary Fig. 20e). These findings suggested that DHA might ameliorated necroptosis in DCM via the suppression of NPM3-mediated lactylation. Thus, in this study, DHA was used to treat a DCM mouse model and hyperglycaemic cells. Our data demonstrated that DHA treatment decreased the expression of NPM3, H3K18la, H3K27la and FASN (Supplementary Fig. 21a–c) and suppressed necroptosis in hyperglycaemic cells (Supplementary Fig. 21d–f). More significantly, DHA treatment also decreased the expression of NPM3, H3K18la, H3K27la and FASN expression (Fig. 7a–d), alleviated necroptosis and cardiac injury (Fig. 7a, b), resolved diastolic dysfunction and decreased ventricular hypertrophy in DCM model mice (Fig. 7e–j). We further verified that DHA mainly exerted its protective function through the inhibition of the lactyltransferase activity of NPM3 by treating NPM3-silenced hyperglycaemic cells with DHA. The results indicated that DHA treatment failed to further inhibit necroptosis in the NPM3-silenced hyperglycaemic cells (Supplementary Fig. 22). Collectively, these observations indicated that targeting the induction of H3K18la and H3K27la induced by NPM3 through DHA treatment might represent an effective therapeutic approach for DCM.

**Fig. 7: Dihydroartemisinin (DHA) competitively antagonized NPM3-induced H3K18la and H3K27la to inhibit FASN transcription and necroptosis in diabetic cardiomyopathy (DCM) model mice.**

Discussion

The primary finding of this study is that the NPM3-mediated lactylation of H3K18 and H3K27 promotes necroptosis by modulating the transcription of FASN, thereby contributing to the initiation and progression of DCM. Furthermore, lactate-induced H3K18la and H3K27la positively regulate NPM3 transcription to form a positive feedback loop, and NPM3 functions as a sensor of intracellular lactate. Notably, the inhibition of NPM3-mediated H3K18la and H3K27la expression by DHA effectively alleviates FASN transcription and necroptosis, thus ameliorating cardiac dysfunction in DCM models.

DCM represents a specific cardiac complication related to diabetes. Researchers have identified multiple factors that may affect the pathogenesis of DCM, such as inflammation, oxidative/nitrosative stress, fibrosis and mitochondrial dysfunction^3,4,7. Nevertheless, the molecular and cellular mechanisms of DCM have not been completely clarified, and current therapeutic strategies have shown limited efficacy. Cardiomyocyte death is a crucial event that occurs as a result of early molecular alterations preceding heart remodelling^8,9. Recently, necroptosis was shown to play a vital role in cardiomyocyte death in DCM¹⁰. Moreover, necroptosis is regarded as a significant and promising target for the treatment of cardiovascular disease³⁵. Our research revealed that the necroptosis inhibitor necrostatin-1 inhibited LDH release in hyperglycaemic cells. Additionally, the hearts of MLKL^−/− + DCM model mice were less impaired, fibrosis was alleviated, and diastolic dysfunction was resolved. These data suggested that inhibiting necroptosis could constitute a therapeutic strategy for improving the prognosis of DCM patients. We subsequently investigated the potential mechanisms through which necroptosis occurs in DCM. By analysing the RNA-seq data, we determined that FASN might be involved in necroptosis in DCM. FASN has been shown to be related to necroptosis and to play a crucial role in necroptosis^15,16. The mechanisms underlying its activity include ROS accumulation¹⁵. Moreover, FASN expression has been reported to be increased in DCM models³⁶. However, whether and how FASN participates in DCM are still not well known. By inhibiting or silencing FASN, we confirmed that FASN played a significant role in necroptosis by increasing ROS accumulation, thus promoting DCM. These findings suggest that FASN serves as both a biomarker and a therapeutic target for DCM. Hence, further exploration of the transcriptional mechanism of FASN in DCM could reveal potential targets for therapeutic strategies.

Epigenetics plays a vital role in the complications associated with diabetes²⁰. Our previous studies revealed that both histone methylation and histone acetylation significantly contribute to the onset and progression of diabetic nephropathy^21,22. Recent studies have revealed that lactate-mediated histone lactylation, a newly identified epigenetic modification, is involved in the onset and progression of diabetes-related complications^24,25. It has been reported that histone lactylation activates PANoptosis to trigger diabetic retinopathy pathogenesis³⁷. Histone lactylation also triggers cellular senescence³⁸ and podocyte epithelial‒mesenchymal transition³⁹ to participate in diabetic kidney disease. Collectively, these findings suggest that lactate-mediated histone lactylation may significantly influence the development and progression of diabetes-related complications. H3K18la and H3K27la were consistently increased in DCM models and diabetic patients. Moreover, both H3K18la and H3K27la were enriched in the promoter of FASN. Additionally, inhibition of lactate-mediated H3K18la and H3K27la expression via OXA decreased FASN expression, thereby suppressing necroptosis in DCM model mice and hyperglycaemic cells. These data indicate that lactate-mediated H3K18la and H3K27la expression modulates FASN transcription to promote necroptosis in DCM.

Despite the significant clinical implications of DCM, the roles of lactyltransferases in this condition remain largely unexplored. Therefore, investigating potential lactyltransferases represents a critical step toward developing effective treatments for DCM. Recently, a series of lactyltransferases, including p300 and AARS1/2, were identified^32,33. Nevertheless, our mass spectrometry data failed to detect any indication that the aforementioned lactyltransferase interacted with both H3K18la and H3K27la in hyperglycaemic AC-16 cells. Moreover, ChIP assays revealed that p300 and AARS1/2 were not enriched at the promoter region of FASN, which played a crucial role in necroptosis in DCM. These findings suggested that p300 and AARS1/2 might not play a significant role in the regulatory effect of H3K18la and H3K27la on FASN transcription and necroptosis in DCM. By conducting further in-depth analysis of the mass spectrometry results, we discerned that the members of the NPM family interacted with H3K18la and H3K27la. A subsequent crystallographic study and co-IP, GST pulldown and immunofluorescence colocalization assays confirmed that NPM3 directly interacted with H3K18la and H3K27la. Moreover, molecular docking and ITC assays indicated that NPM3 directly bound to lactate. Furthermore, in vitro lactylation experiments confirmed that NPM3 was a lactyltransferase that mediated the lactylation of H3K18 and H3K27. NPM3 is predominantly a nuclear protein belonging to the NPM protein family. Research has indicated that the NPM family plays essential roles in DNA replication, histone chaperone activity, ribosome biogenesis, and chromatin remodelling^26,27. In the present study, the expression of NPM3 was elevated in hyperglycaemic cells, the hearts of DCM model mice and diabetic patients. Furthermore, the inhibition of NPM3 expression decreased H3K18la, H3K27la and FASN levels; attenuated necroptosis; and alleviated heart injury in both DCM model mice and hyperglycaemic cells. Consistently, the overexpression of NPM3 led to increases in H3K18la, H3K27la and FASN levels, which triggered necroptosis in cells. Notably, NPM3^Q117G, which lacks lactyltransferase activity, did not produce these effects. Additionally, a ChIP assay confirmed that H3K18la, H3K27la and NPM3 were enriched in the promoter region of FASN. Moreover, the induction of necroptosis by NPM3 overexpression was reversed by the introduction of FASN silencing into cells. Collectively, these data suggest that NPM3 acts as a lactyltransferase to lactylate H3K18 and H3K27 to modulate FASN transcription, thereby participating in necroptosis in DCM.

Next, we conducted a comprehensive analysis of the H3K18la and H3K27la CUT&Tag results and confirmed through a ChIP assay that both H3K18la and H3K27la were enriched in the promoter region of NPM3. Furthermore, lactate-induced H3K18la and H3K27la expression positively regulated NPM3 transcription. These results suggest that NPM3 may function as a sensor of intracellular lactate. Given the critical role of NPM3-modulated H3K18la and H3K27la in promoting FASN transcription and triggering necroptosis in DCM, the inhibition of NPM3-mediated lactylation may be a previously uncharacterized strategy for treating DCM. DHA, the active antimalarial metabolite of artemisinin, exhibits antiangiogenic effects in numerous diseases. A previous study indicated that DHA ameliorates retinal vascular dysfunction in diabetes mellitus via the inhibition of FASN expression³⁴. However, the potential mechanism is still not well known. Through molecular docking experiments, we revealed that DHA bound to NPM3 and that the binding site was consistent with that of lactate. Moreover, the ITC findings revealed that the binding strength of DHA to NPM3 was greater than that of lactate to NPM3. Moreover, in vitro lactylation experiments revealed that DHA treatment suppressed NPM3-induced lactylation. Therefore, we administered DHA to hyperglycaemic cells and DCM model mice. Our data indicated that DHA treatment decreased the levels of NPM3, H3K18la and H3K27la, resulting in a decline in FASN transcription; inhibition of necroptosis; reduction in heart injury; and alleviation of cardiac dysfunction in DCM model mice and hyperglycaemic cells. Our findings suggest that DHA represents an effective treatment strategy for DCM through the inhibition of NPM3-induced lactylation.

This study has several limitations. First, the human samples employed in this investigation were not derived from patients with DCM; consequently, the human groupings might be considerably lacking in representativeness to some extent. However, human cardiac samples from patients with and without diabetes have been commonly used in studies of DCM^40,41. Nevertheless, the results of the present study undoubtedly highlight the effects of diabetes on cardiac tissue. Furthermore, the results obtained in humans were in accordance with those obtained from the DCM models. Therefore, we assert that the human results still hold a degree of representativeness. Second, the in vitro lactylation experiments we undertook failed to validate that NPM1 has lactyltransferase activity. Nevertheless, these findings might be correlated with the loss of activity of the purified NPM1 protein. Accordingly, whether NPM1 is also a potential lactyltransferase or a histone lactylation reader may require further exploration. Third, in addition to LDHA, other glycolytic enzymes may be involved in lactate production to participate in DCM; this issue warrants further investigation. Fourth, the results of the present study confirmed that NPM3 functions as a lactyltransferase in the regulation of H3K18la and H3K27la in individuals with DCM; whether other lactyltransferases or delactyltransferases participate in DCM also warrants further exploration. Fifth, previous studies indicated that lactylation¹⁹ and necroptosis¹⁰ are involved in DCM. Moreover, FASN plays a crucial role in necroptosis^15,16. These reported findings limit the innovativeness of this research to some extent. However, the relevant interventions carried out for the above three aspects are still rather limited. In this study, we revealed that NPM3 functions as a previously uncharacterized lactyltransferase to regulate FASN transcription via the modulation of histone lactylation, thus triggering necroptosis in DCM. Moreover, we found that inhibiting NPM3-induced lactylation through DHA may represent an effective therapeutic strategy for DCM. Therefore, interventions targeting potential lactyltransferases may be potential therapeutic targets for treating diabetes-related complications. Sixth, exploring the pharmacokinetic/pharmacodynamic data, toxicity profiles, or optimal dosing windows of DHA in DCM mouse models is highly important. However, the safety of DHA in the treatment of malaria has been fully verified in a wide range of populations, including pregnant women⁴² and children⁴³. Moreover, the core goal of this study was to identify potential molecular targets for the treatment of DCM. We will explore these data in a DCM mouse model in future studies. Seventh, orlistat is an FDA-approved antiobesity drug, and its target was identified as FASN. Therefore, the decrease in mouse body weight in the groups treated with orlistat and DHA alone may have occurred through the inhibition of FASN expression. However, it cannot be completely ruled out that orlistat and DHA may have potentially toxic effects, particularly in the case of DHA. This issue awaits further verification in our subsequent studies. Eighth, although the MV E/A ratio and MV E/E’ ratio are well-established parameters for assessing cardiac diastolic function and the literature supports their ability to reliably reflect diastolic performance, heart rate monitoring remains highly important for the evaluation of diastolic function and warrants inclusion in future studies.

In summary, this study demonstrated that NPM3 functions as a lactyltransferase to lactylate H3K18 and H3K27, thereby activating the transcription of FASN and triggering necroptosis in DCM. Moreover, inhibiting NPM3-induced lactylation through DHA may represent an effective therapeutic strategy for DCM.

Methods

Subjects

This study was conducted in accordance with the Declaration of Helsinki and received approval from the Huashan Hospital of Fudan University Ethics Committee (Ethics number: 2023-1011). Patients with valvular heart disease requiring surgical intervention, regardless of their diabetes status, were enrolled in this study. Informed consent was obtained from all participants. The general clinical characteristics of patients with or without type 2 diabetes in this study are summarized in Supplementary Table 2.

Animal models

This study was approved by the Shanghai First People’s Hospital Clinical Center Laboratory Animal Welfare and Ethics Committee (Ethics Approval Number: 2024AWS227). MLKL-heterozygous mice (MLKL^+/−) and NPM3-heterozygous mice (NPM3^+/−) on a C57BL/6 background were obtained from Jicui Biotechnology Limited (Jiangsu, China). Age-matched male wild-type, MLKL homozygous (MLKL^−/−) and NPM3 homozygous (NPM3^−/−) littermates that we bred were utilized at 3 months of age. The animals were housed in a temperature-controlled environment (ranging from 22 °C to 25 °C) under a 12-h light/dark cycle. Mice that received an intraperitoneal injection of citrate buffer (0.1 M, pH 4.5) for 5 consecutive days were assigned to the control group (Con, n = 8). The mice in the DCM, MLKL^−/− + DCM and NPM3^−/− + DCM groups were fed a high-sugar and high-fat diet for 16 weeks. This dietary regimen was supplemented with an intraperitoneal injection of 50 mg/kg streptozotocin (STZ, HY-13753, MCE, China) for 5 consecutive days. After 7 days, model establishment was considered successful for DCM mice, MLKL^−/− + DCM mice and NPM3^−/− + DCM mice with glucose levels exceeding 16.7 mmol/L. The high-sugar and high-fat diet combined with multiple low-dose STZ-induced diabetes is a well-established approach for inducing type 2 diabetes and subsequent DCM⁴⁴, without causing nonspecific toxicity in animals⁴⁵. DCM model mice that were intraperitoneally injected with 500 mg/kg OXA (HY-W013032A; MCE, China) once a day after the injection of citrate buffer or STZ were designated the OXA group (n = 8) or the DCM + OXA group (n = 8). Mice that were administered orlistat (HY-B0218, MCE, China) by gavage at 240 mg/kg once a day after the injection of citrate buffer or STZ were designated the orlistat group (n = 8) or the DCM+orlistat group (n = 8). Mice that were administered 20 mg/kg DHA (HY-N0176, MCE, China) by gavage once a day³⁴ after the injection of citrate buffer or STZ were designated the DHA group (n = 8) or the DCM + DHA group (n = 8).

Cell culture and treatment

AC-16 human cardiomyocytes (AC-16 cells) were obtained from COWELDGEN SCIENTIFIC (Shanghai, China). PCMs were isolated from neonatal C57BL/6 mouse hearts strictly in accordance with a previously established protocol⁴⁶. The cells were cultivated in DMEM (CM-H061; CM-0109; Procell, China) for serial subcultivation. The AC-16 cells and PCMs cultured in DMEM supplemented with 5 mM glucose for 3 days were designated the control group (Con). Cells cultured in DMEM supplemented with 25 mM glucose for 3 days were designated the high-glucose (HG) group. This concentration of glucose has been shown to be reliably used in a previous study⁴⁷ and our prior reports^48,49. Glucose (5 mM) plus mannitol (20 mM) was used as an osmotic control (mannitol) to mitigate the effects of elevated osmotic pressure resulting from the high-glucose treatment of cells^48,49. The LDHA inhibitor OXA (HY-W013032A; MCE, China) was used in the cellular experiments. The cells were incubated with different concentrations of OXA (0.5, 1, 5 or 10 mM) for 3 days. The optimal concentration for the significant inhibitory effects of OXA on 25 mM glucose-induced LDH release was determined. Another LDHA inhibitor, STP (HY-103392; MCE, China), was applied at different concentrations (125, 250 or 500 nM) for 3 days. The optimal concentration for the significant inhibitory effects of STP on 25 mM glucose-induced LDH release was determined. The FASN inhibitor orlistat (HY-B0218; MCE, China) was used in the cellular experiments. The cells were incubated with different concentrations of orlistat (125, 250 or 500 nM) for 3 days. The optimal concentration for achieving the significant inhibitory effects of orlistat on FASN expression induced by 25 mM glucose was determined. Another FASN inhibitor, C75 (HY-12364; MCE, China), was used in the cellular experiments. The cells were incubated with different concentrations of C75 (2.5, 5, or 10 μM) for 3 days. The optimal concentration for the significant inhibitory effects of C75 on 25 mM glucose-induced FASN expression was determined. DHA (HY-N0176; MCE, China) was used to inhibit NPM3-induced H3K18la and H3K27la. The cells were incubated with different concentrations of DHA (2.5, 5 or 10 μM) for 3 days. The optimal concentration for the significant inhibitory effects of DHA on 25 mM glucose-induced H3K18la and H3K27la expression was determined.

LDH release assay

An LDH release kit (C0016; Beyotime, China) was used in the present study to assess the degree of cellular damage. In brief, an appropriate number of cells were seeded into 96-well cell culture plates. The culture medium was aspirated, and the cells were washed once with PBS. Following the addition of LDH release reagent, the cells were incubated in a cell culture incubator for 3 h. The supernatant from each well was subsequently collected, and the absorbance of the samples was measured at 490 nm.

shRNA, siRNA and plasmid treatments

After the cells were seeded and reached 70–80% confluence, they were transfected with plasmids, siRNA or shRNA using Lipofectamine 3000 (L3000015; Thermo Fisher, USA). As specified in the user guide, 2500 ng of plasmid or shRNA was initially mixed thoroughly with the P3000 Reagent. Next, this mixture was combined with Lipofectamine™ 3000. Finally, the resulting solution was added to the cell culture medium. The sequences of the shRNAs and siRNAs used in this study are shown in Supplementary Table 3.

Western blotting analysis

After being washed with cold phosphate-buffered saline (PBS), the cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (PC101; Epizyme Biotech, Shanghai, China) supplemented with phenylmethylsulphonyl fluoride (PMSF, ST2573-5g; Beyotime Biotechnology, Shanghai, China). The protein concentration was measured using a BCA protein assay kit (P0009; Beyotime, Shanghai, China). Equal amounts of protein (20 μg) from each group were separated by sodium dodecyl sulphate‒polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (ISEQ00010; Millipore, Billerica, USA). The primary antibodies used in this study were as follows: monoclonal antibodies against β-actin (66009-1-Ig; ProteinTech, Wuhan, China), Kla (PTM-1401RM; PTM Biolabs, China), H3K9la (PTM-1414RM; PTM Biolabs, China), H3K18la (PTM-1427RM; PTM Biolabs, China), H4K5la (PTM-1407RM; PTM Biolabs, China), H4K12la (PTM-1411RM; PTM Biolabs, China), phospho-MLKL (ab187091; Abcam, USA), MLKL (66675-1-Ig; ProteinTech, Wuhan, China) and phospho-RIPK1 (66854-1-Ig; ProteinTech, Wuhan, China) and polyclonal antibodies against histone-H3 (17168-1-AP; ProteinTech, Wuhan, China), histone-H4 (16047-1-AP; ProteinTech, Wuhan, China), H3K27la (PTM-1428; PTM Biolabs, China), H4K8la (PTM-1415; PTM Biolabs, China), FASN (10624-2-AP; ProteinTech, Wuhan, China), RIPK1 (17519-1-AP; ProteinTech, Wuhan, China), NPM3 (11960-1-AP; ProteinTech, Wuhan, China), AARS1 (17394-1-AP, ProteinTechWuhan, China), AARS2 (22696-1-AP, ProteinTechWuhan, China) and p300 (20695-1-AP, ProteinTechWuhan, China).

Quantitative real-time PCR (qPCR)

Total RNA and cDNA from cells or mouse heart tissues were obtained using a Universal RNA Purification Kit (EZB-RN4; EZbioscience, USA) with HyperScript IlI RT SuperMix (R202-02; EnzyArtisan, Shanghai, China). Universal SYBR qPCR Mix (EnzyArtisan, Shanghai, China) was used to perform real-time PCR in an ABI7500 real-time PCR system (Applied Biosystems). All transcript levels were normalized to β-actin levels. The primers used in the present study are listed in Supplementary Table 4.

In vitro lactylation and GST pulldown assay

In vitro lactylation assays were performed in the present study as described previously³³. In brief, purified GST-NPM3 protein (Ag2564; Proteintech, Shanghai, China) was incubated with recombinant human His-histone H3 (Ag10644; Proteintech, Wuhan, China) and lactate (5 mM; HY-B2227; MCE, China) in incubation buffer (50 mM HEPES, 25 mM KCl, 2 mM MgCl2, and 4 mM ATP; pH 7.5) for 3 h at 37 °C. The samples were subsequently denatured at 95 °C for 5 min and analysed via Western blotting.

GST pulldown assays were performed with a GST pulldown kit (FI8804, FITGENE, Guangzhou, China). In brief, GST-NPM3 (Ag2564; Proteintech, Shanghai, China) and His-histone H3 (Ag10644; Proteintech, Wuhan, China) fusion proteins were used for the GST pulldown assays. Equal amounts of the fusion proteins were incubated together for 12 h at 4 °C in GST binding buffer (50 mM HEPES, pH 7.6; 50 mM NaCl; 0.1% Nonidet P-40; 5 mM EDTA; and 10% glycerol). Anti-GST beads were mixed with the fusion protein mixture for another 4 h. The beads were subsequently washed three times with wash buffer (200 mM Tris–Cl (pH 8.0), 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton ×-100, and 0.4 mM PMSF), and target proteins were assessed by Western blotting.

Isothermal titration calorimetry (ITC) assay

ITC assays were performed using a MicroCal PEAQ-ITC calorimeter with an automated system (Malvern) at 25 °C. For calorimetric measurements, purified NPM3 was loaded into the ITC cell at a concentration of 5 µM, and 50 µM lactate or 50 µM DHA in 50 mM Tris HCl and 150 mM NaCl were autoloaded into a syringe. In parallel experiments, the ITC cell was loaded with a mixture of 5 µM NPM3 and 5 µM LA, while the syringe was filled with 50 µM DHA; alternatively, the cell contained a mixture of 5 µM NPM3 and 5 µM DHA, with the syringe loaded with 50 µM LA. Each titration included a single 1 µL injection followed by 19 sequential injections of 2 µL aliquots, with a spacing of 300 s between the injections, and stirring at 500 rpm. The data were analysed using ORIGIN data analysis software (MicroCal Software).

ATP, PPi and AMP detection assays

ATP, PPi and AMP levels in the in vitro lactylation system were measured using an Enhanced ATP Assay Kit (S0027; Beyotime, China), a Pyrophosphate Assay Kit (ab234040; Abcam, Cambridge, UK), and an AMP Assay Kit (ab273275; Abcam, Cambridge, UK) in accordance with the manufacturers’ instructions. A multiscan spectrum (SpectraMax Mini, Molecular Devices, USA) was used for detection.

Coimmunoprecipitation (co-IP) assay

A classic magnetic bead immunoprecipitation kit (88804; Thermo Scientific, USA) was used to perform co-IP assays. AC-16 cells were seeded in 100-mm dishes, cultured in 25 mM DMEM for 3 days, harvested and extracted with cell lysis buffer (G2038; Servicebio, Wuhan, China) containing a protease phosphatase inhibitor (P1051; Beyotime Biotechnology, Shanghai) to obtain protein lysates. A portion of the lysate supernatant (30 μL) was reserved as the input sample. The remaining supernatant was incubated with the corresponding primary antibodies and A/G Dynabeads at 4 °C overnight to perform endogenous IP. Western blotting was performed to analyse the input, IgG and IP fractions.

Chromatin immunoprecipitation (ChIP)

ChIP analysis was performed using an EZ ChIP Kit (17–371; MERCK, USA). The cells were subsequently washed with PBS and incubated at room temperature with 1% formaldehyde for 10 min to cross-link the DNA and proteins. Next, 2.5 M glycine was added to terminate the reaction. The chromatin was sonicated for 10 cycles (each consisting of 7 s of sonication followed by a 7-s pause) to fragment it into suitable lengths. The samples were then divided and incubated overnight at 4 °C with specific primary antibodies or IgG. The following day, the immunoprecipitated complexes were captured using agarose beads and washed, and the DNA‒protein cross-links were reversed by incubation in a metal bath at 65 °C for 5‒6 h. The enriched DNA sequences were subsequently amplified by PCR using specific primers and analysed by agarose gel electrophoresis. The oligonucleotide primer sequences are provided in Supplementary Table 5.

Echocardiography

Echocardiography was carried out using a small animal ultrasound imaging platform (Vevo3100, Fuji Visual Sonics, Japan). The examination was executed under general anaesthesia. The mice were placed in a 10-cm-diameter glass chamber and positioned on an electric blanket maintained at 37 °C. Oxygen was supplied at a flow rate of 2 L/min, and 3% sevoflurane was administered via inhalation for the induction of anaesthesia. Upon loss of consciousness, a face mask was placed on the mice to maintain anaesthesia, with the oxygen flow rate remaining at 2 L/min. The animals were then secured on the imaging platform and underwent echocardiographic examination. When the echocardiographic plane was satisfactory, the probe position was fixed using a fixed frame. Cardiac images were continuously monitored for 5 min, during which stable images were captured. Following the examination, the mice were placed in a recovery cage equipped with an electric blanket and returned to their breeding cages upon full recovery. The diastolic function of the mouse heart was evaluated by the MV E/A ratio and MV E/E’ ratio, while systolic function was assessed by the left ventricular ejection fraction (LVEF). The LVEF, MV E/A ratio and MV E/E’ ratio were calculated in accordance with previously established methods⁵⁰.

Haematoxylin and eosin (HE) staining, Masson staining and immunohistochemistry (IHC)

HE staining was performed as described previously²¹. Paraffin sections were incubated in an oven at 60 °C for 1 to 2 h and subsequently subjected to dewaxing with xylene (10023418; National Pharmaceutical Group, Beijing, China) and ethanol. Haematoxylin (H3136; Sigma‒Aldrich, St. Louis, USA) was used to stain the nuclei for approximately 10 min, after which eosin (E4009; Sigma‒Aldrich, St. Louis, USA) was used to stain the cytoplasm for 30 s. Finally, the sections were mounted with neutral balsam (10004160; National Pharmaceutical Group, Beijing, China), dried at room temperature, and examined under an optical microscope (Nikon Eclipse Ci-L; Nikon, Tokyo, Japan). Myocardial injury was assessed using a semi-quantitative morphometric (SQM) scoring system that considers cardiomyocyte morphology, myofibre arrangement, average transverse diameter, and interstitial inflammatory cell infiltration. The total SQM score was calculated as the sum of individual component scores. Cardiomyocyte morphology was scored on a scale from 1 (normal) to 4 (myofibrillar lysis or necrosis), incorporating features such as cloudy swelling and vacuolar degeneration. Myofibre arrangement was graded from 1 (well-organized and aligned) to 4 (severely disorganized with loss of structural integrity). The transverse diameter of cardiomyocytes was classified from 1 (normal: <18 μm in humans or <14 μm in mice) to 4 ( > twofold increase over normal, indicative of severe pathological hypertrophy). Interstitial inflammation was scored from 1 (absent) to 4 (moderate to diffuse inflammatory infiltration). Masson’s trichrome staining and IHC analysis were performed as described previously²¹. The primary antibodies used for the IHC analysis are described in detail in the preceding sections.

Immunofluorescence (IF) staining

For mouse heart tissue sections: Paraffin-embedded sections were dewaxed and rehydrated. Endogenous peroxidases were blocked by incubation with 100 μl of hydrogen peroxide blocking solution for 10 min at room temperature. The sections were subsequently subjected to heat-induced antigen retrieval using 1 mmol/L Tris-EDTA buffer (Tris-base, 648310; Sigma, USA; EDTA, E9884; Sigma, USA). Thereafter, the sections were blocked with 5% bovine serum albumin (BSA; B2064; Sigma, USA) and incubated overnight at 4 °C with primary antibodies histone H1 (18201-1-AP, ProteinTech, Wuhan, China), histone H2A (16441-1-AP, ProteinTech, Wuhan, China), histone H2B (15857-1-AP, ProteinTech, Wuhan, China), histone H3 (17168-1-AP, ProteinTech, Wuhan, China), or histone H4 (16047-1-AP, ProteinTech, Wuhan, China). The following day, the sections were incubated with a labelled secondary antibody, goat anti-rabbit IgG H&L (HRP) (ab205718, Abcam, USA) at 37 °C for 30 min. Following the addition of the try-488 tyrosine conversion reagent (Bry-try488, Runnerbio, China). After a second round of heat-induced antigen retrieval and blocking, the sections were incubated with an primary antibody against Kla (PTM-1401RM; PTM Biolabs, China). The sections were subsequently incubated with a labelled secondary antibody and try-Cy3 tyrosine conversion reagent (Bry-Trycy3; Runnerbio, China) in the same manner. Finally, the sections were mounted with DAPI-containing anti-fluorescence quenching mounting medium (P0131; Beyotime, China) and sealed. Quantitative co-localization analysis was analyzed using Fiji software. Colocalization between histone and Kla signals was assessed with the Just Another Colocalization Plugin (JACoP).

For PCM and AC-16 cells: Immunofluorescence was also performed as previously described22. The cells were fixed, permeabilized, and blocked before they were incubated with the following primary antibodies: NPM3 Mouse mAb (64015, Signalway Antibody) and either H3K18la rabbit mAb (PTM-1427RM, PTM Biolabs) or H3K27la rabbit pAb (PTM-1428, PTM Biolabs). In parallel experiments, the membrane translocation of p-MLKL was detected using a rabbit monoclonal antibody (ab187091; Abcam, USA) after using a membrane staining kit with DiI (C1991, Beyotime, China). Following primary antibody incubation, the cells were thoroughly washed and subsequently incubated with the following species-specific secondary antibodies: Alexa Fluor 594-conjugated IgG (red) and Alexa Fluor 488-conjugated IgG (green). The nuclei were counterstained with DAPI. All fluorescence images were acquired using a confocal microscope (Leica Microsystems, Germany).

Dual-luciferase assay

The effects of lactate on the activity of the NPM3 promoter and the effects of the NPM3 or NPM3 mutation on the activity of the FASN promoter were assessed using a Firefly & Renilla-Light Luciferase Reporter Assay Kit (MA0520; Meilun Bio, Dalian, China). The FASN and NPM3 promoters were amplified and ligated into the pGL3-basic vector to generate the pGL3-FASN and pGL3-NPM3 constructs. The pGL3-FASN or pGL3-NPM3 plasmid was transfected along with a Renilla luciferase vector into AC-16 cells.

ROS assay

Intracellular ROS levels were measured using a ROS Assay Kit (S0034S; Beyotime, China) according to the manufacturer’s instructions. In brief, after the experimental treatments were performed, the cells were washed with PBS and subsequently incubated with 10 μM DCFH-DA diluted in serum-free medium at 37 °C for 30 min in the dark. Following incubation, the cells were washed three times with PBS to remove excess probe. The fluorescence intensity was immediately measured using a fluorescence microplate reader (SpectraMax Mini, MOLECULAR, China) with excitation and emission wavelengths set at 488 nm and 525 nm, respectively.

Low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), glycated albumin (GA), lactate and triglyceride (TG) levels in mouse serum

LDL-C levels in mouse serum were measured using an LDL-C assay kit (ADS-W-D012; AIDISHENG, China). TC levels in mouse serum were measured using a TC assay kit (ADS-W-ZF014; AIDISHENG, China). HDL-C levels in mouse serum were measured using an HDL-C assay kit (ADS-W-D011; AIDISHENG, China). GA levels in mouse serum were measured using an enzyme-linked immunosorbent assay kit (TW7949; Tongwei Biotech, Shanghai, China), lactate levels in mouse serum were measured using a lactate assay kit (ADS-W-T009-96; AIDISHENG, China), and TG levels in mouse serum were measured using a TG assay kit (ADS-W-ZF013; AIDISHENG, China).

Prediction of potential inhibitors of NPM3 and virtual screening

The 3D structure of human NPM3 (AlphaFold ID: AF-O75607-F1) was downloaded from the AlphaFold website (https://AlphaFold.ebi.ac.uk/entry/O75607). The data from the virtual screening workflow module indicated that ARG:87 and GLN:117 were the binding sites of lactate on NPM3. Next, the Receptor Grid Generation module was used to create a grid file for NPM3, centred on the amino acid residues ARG:87 and GLN:117, with the box size set to 20 × 20 × 20 Å. Finally, the 2D format of the HY-L022P FDA-Approved Drug Library Plus database (containing 3400 compounds) was processed through the Schrödinger software LigPrep Module for hydrogen addition, energy optimization, etc., and the 3D structures were output for virtual screening.

Pathway enrichment analysis

The Kyoto Encyclopaedia of Genes and Genomes (KEGG) database was used to identify enriched pathways, and a two-tailed Fisher’s exact test was used to determine the enrichment of differentially expressed genes. These pathways were classified into hierarchical categories according to the KEGG website.

Statistics and reproducibility

For human myocardial samples, continuous variables are presented as mean ± SD and compared using an paired two-tailed t‑test. Categorical variables are expressed as number (n) with percentage (%) and analyzed with the nonparametric Mann–Whitney U test. Statistical analyses for human samples were performed using SPSS 20.0. In animal experiments, a sample size of n = 8 biologically independent mice was used. For cell‑based in vitro assays, n = 5 biologically independent replicates were included; cell‑free in vitro experiments were repeated three times. Each experiment was independently repeated three times. Analyses were conducted with GraphPad Prism 6. Data are shown as mean ± SD and were compared by unpaired two-tailed t‑tests. A P value < 0.05 was considered statistically significant. In figures, significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data, including exact P values are provided as a Source Data file.

Ethics approval

This study was conducted in accordance with the Declaration of Helsinki and received approval from the Huashan Hospital of Fudan University Ethics Committee (Ethics number: 2023-1011). Informed consent was obtained from all participants. This study was approved by the Shanghai First People’s Hospital Clinical Center Laboratory Animal Welfare and Ethics Committee (Ethics Approval Number: 2024AWS227). All methods were performed in accordance with the relevant guidelines and regulations in the present study.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq, CUT&Tag, and mass spectrometry (proteomics) data generated in this study have been deposited in the China National Center for Bioinformation (CNCB) database under BioProject accession code PRJCA049021 (https://ngdc.cncb.ac.cn). These data are available under restricted access to ensure compliance with China’s regulations on human genetic resource management and participant confidentiality agreements. Access can be obtained by submitting a formal request outlining the academic, non-commercial research purpose to the corresponding author, Prof. Minmin Zhu (zhu_mm@126.com), for review and approval by the institutional data access committee. The numerical data underlying all graphs and the uncropped scans of all blots and gels presented in this study are provided in the Source Data file. Source data are provided with this paper. All Supplementary Figures/Tables and their source data are in the Supplementary Information file. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Special Talent Training Program—Shanghai Leading Talent 2023—Shanghai General Hospital (1,000,000 CNY to Minmin Zhu); the Hospital-level Discipline Construction Project of Pudong Hospital Affiliated with Fudan University (NO. Yjfcxk2025-03, 1 200,000 CNY to Minmin Zhu); The Medical Discipline Construction Program of Shanghai Pudong New Area Health Commission (the Key Weak Disciplines Program, NO. PWZbr2025-01, 300,000 CNY to Minmin Zhu); and The Healthcare Talents Elite Program of Shanghai Pudong New Area (NO. 2026PDWSYCBJ-05, 800,000 CNY to Minmin Zhu).

Author information

These authors contributed equally: Hongjiao Xu, Xinyu Jiang, Fangrui Wang.

Authors and Affiliations

Department of Anesthesiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Hongjiao Xu, Xinyu Jiang & Jinbao Li
Department of Cardiothoracic Surgery, Huashan Hospital of Fudan University, Shanghai, P. R. China
Fangrui Wang & Jiechun Huang
Department of Anesthesiology, Xinjiang Medical University Affiliated Cancer Hospital, Urumqi, Xinjiang, China
Fufen Meng
School of Pharmacy, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, Yinchuan, China
Yue Liu
Department of Anesthesiology, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, China
Minmin Zhu

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Hongjiao Xu
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Xinyu Jiang
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Fangrui Wang
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Fufen Meng
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Yue Liu
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Jiechun Huang
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Contributions

M.M.Z., J.B.L., J.C.H., H.J.X., X.Y.J. and F.R.W. had full access to all the data in the study and accepted responsibility for the integrity of the data and the accuracy of the data analyses. M.M.Z. designed the study. M.M.Z., J.B.L., J.C.H., H.J.X., X.Y.J. and F.R.W. organized and conducted the experiments. H.J.X., X.Y.J., F.R.W. and M.M.Z. conducted quality control and analysed the data. M.M.Z., J.B.L., J.C.H., H.J.X., X.Y.J., F.F.M., Y.L. and F.R.W. wrote the first draft of the manuscript. All the authors participated in discussions and approved the final manuscript.

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Correspondence to Jiechun Huang, Jinbao Li or Minmin Zhu.

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Nature Communications thanks Jun Ren, Lu Cai, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Xu, H., Jiang, X., Wang, F. et al. NPM3 functions as a lactyltransferase to promote necroptosis in male diabetic cardiomyopathy mice models via FASN transcription modulation. Nat Commun 17, 3685 (2026). https://doi.org/10.1038/s41467-026-70513-0

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Received: 12 March 2025
Accepted: 27 February 2026
Published: 09 March 2026
Version of record: 22 April 2026
DOI: https://doi.org/10.1038/s41467-026-70513-0

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Abstract

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Introduction

Results

The levels of lactate, lactylation (Kla), H3K18la and H3K27la were increased in DCM models and diabetic patients

Lactate-mediated H3K18la and H3K27la contributes to DCM in vivo and in vitro

Lactate-induced H3K18la and H3K27la play crucial roles in the development of DCM by triggering necroptosis

Lactate-mediated H3K18la and H3K27la expression modulates FASN transcription to promote necroptosis in DCM

NPM3 acts as a lactyltransferase that lactylates H3K18 and H3K27

NPM3 mediates H3K18la and H3K27la to trigger necroptosis in DCM via the modulation of FASN transcription

Lactate-induced H3K18la and H3K27la expression positively regulates NPM3 transcription

DHA competitively antagonizes NPM3-induced H3K18la and H3K27la expression to inhibit FASN transcription and necroptosis in DCM

Discussion

Methods

Subjects

Animal models

Cell culture and treatment

LDH release assay

shRNA, siRNA and plasmid treatments

Western blotting analysis

Quantitative real-time PCR (qPCR)

In vitro lactylation and GST pulldown assay

Isothermal titration calorimetry (ITC) assay

ATP, PPi and AMP detection assays

Coimmunoprecipitation (co-IP) assay

Chromatin immunoprecipitation (ChIP)

Echocardiography

Haematoxylin and eosin (HE) staining, Masson staining and immunohistochemistry (IHC)

Immunofluorescence (IF) staining

Dual-luciferase assay

ROS assay

Low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), glycated albumin (GA), lactate and triglyceride (TG) levels in mouse serum

Prediction of potential inhibitors of NPM3 and virtual screening

Pathway enrichment analysis

Statistics and reproducibility

Ethics approval

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding authors

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Peer review

Peer review information

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Supplementary information

Source data

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