IDH2 lactylation promotes angiogenesis in murine diabetic myocardial infarction via blocking Cav1-eNOS interaction

Figures

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  Fig. 1. Increased cardiac lactylation modification following diabetic myocardial infarction (DMI). a Schematic diagram illustrating the construction of the DMI mouse model: Mice were administered streptozotocin (STZ; 40 mg/kg/day) via intraperitoneal injection, followed by left anterior descending coronary artery ligation to induce myocardial infarction (MI), created in BioRender. Zang, G. (2025) https://BioRender.com/snbw805. b Heart vessels were visualized by using Microfil in MI and DMI mice. c Arterial ring experiments were conducted in MI and DMI mice (n = 6). Scale bar = 100 μm. d Representative images and quantification of Masson’s trichrome staining for cardiac fibrosis in the infarct border zone from MI and DMI mice (n = 6). Scale bar = 20 μm. e Representative images and ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs) of cardiac function evaluated by echocardiography (n = 6). f Western blot analysis of Pan Kla levels in the MI border zone tissue of mice after induction of diabetes or MI (n = 6). g Representative Immunofluorescence staining of Pan Kla and endothelial cell…

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  Fig. 2. Proteomic profiling of lysine lactylation following diabetic myocardial infarction (DMI). a Schematic representation of isolating myocardial infarction (MI) border zone tissue from each experimental group for lactylation proteomic analysis, created in BioRender. Zang, G. (2025) https://BioRender.com/95pgpau. b Venn diagrams illustrate the proteins and modification sites undergoing lactylation both in the MI/Sham groups, and in the DMI/MI groups. c Heatmap displays 27 lactylation modification sites in the DMI/MI comparison group, sorted by multiples of differential expressions. d Subcellular localization of lactylated proteins with differential modification between the MI group and DMI group. e GO functional enrichment analysis of differentially lactylated proteins in the DMI/MI comparison group. f KEGG pathway enrichment analysis of differentially lactylated proteins in the DMI/MI comparison group. g Schematic diagram depicting the lactylation modification status of tricarboxylic acid cycle (TCA) rate-limiting enzymes in DMI. Two-sided unpaired student’s t test was performed in (c). Fisher’s exact test was used in (e, f). Source data are provided as a Source data file.

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  Fig. 3. Inhibition of IDH2 lactylation at Lys272 suppresses angiogenesis in high glucose and hypoxia environment. a Western blot analysis of global lactylation modification (Pan Kla) of cardiac microvascular endothelial cells (CMECs) after treatment with normal glucose (NG), high glucose (HG), normal glucose oxygen deprivation (NOD), and high glucose oxygen deprivation (HOD) (n = 6). b, c Western blot analysis of Pan Kla and IDH2 in CMECs with increasing hypoxia duration under high glucose conditions (n = 4). d Coimmunoprecipitation analysis of the lactylation level of IDH2 in the infarct border zone tissue of Sham, Dsham, MI, and DMI mice (immunoprecipitated by Pan Kla antibody). e Coimmunoprecipitation analysis of the lactylation levels of IDH2 in CMECs under NG, HG, NOD, and HOD conditions (immunoprecipitated by Pan Kla antibody). f Western blot analysis of the lactylation level of IDH2-K272 in the infarct border zone tissue of Sham, diabetic Sham (Dsham), myocardial infarction (MI), and diabetic myocardial infarction (DMI) mice (n = 6). g Western blot analysis of the lactylation level of IDH2-K272 in CMECs under NG, HG, NOD, and HOD conditions (n = 6). h Coimmunoprecipitation…

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  Fig. 4. IDH2-K272 mutation in endothelial suppresses angiogenesis and cardiac function after diabetic myocardial infarction (DMI). a Schematic representation of the construction of endothelial cell-specific IDH2-K272R mutant knock-in mice. b Schematic representation of the construction of the DMI model in IDH2EC-K272R mice, created in BioRender. Zang, G. (2025) https://BioRender.com/snbw805. c 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining results of the hearts of IDH2EC-WT and IDH2EC-K272R mice after modeling for MI or DMI (n = 6). Scale bar = 2 mm. d Coimmunoprecipitation analysis of the level of IDH2 lactylation in the infarct border zone tissue of IDH2WT and IDH2EC-K272R mice (immunoprecipitated by Pan Kla antibody). e Western blot analysis of the level of IDH2-K272 lactylation in the infarct border zone tissue of IDH2WT and IDH2EC-K272R mice (n = 6). f Representative Immunofluorescence staining of IDH2-K272la and endothelial cell marker CD31 in heart from IDH2WT and IDH2EC-K272R mice (n = 6). Scale bar = 20 μm. g Arterial ring experiments were conducted in IDH2WT and IDH2EC-K272R mice following induction of MI or DMI (n = 6). Scale bar = 100 μm. h Representative images and…

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  Fig. 5. IDH2-K272 lactylation triggers eNOS activation by coupling with Cav1. a, b Western blot analysis of the protein expression of eNOS and p-eNOS after the IDH2-K272R mutation both in vivo and in vitro experiments (n = 6). c NO content of cardiac microvascular endothelial cells (CMECs) after transfected with WT or K272R plasmid under normal glucose oxygen deprivation (NOD) and high glucose oxygen deprivation (HOD) stimulation was quantified by NO fluorescent probe (n = 6). Scale bar = 100 μm. d IP-MS analysis of CMECs treated with Flag-IDH2-WT and Flag-IDH2-K272R to screen for proteins interacting with IDH2, created in BioRender. Zang, G. (2025) https://BioRender.com/th0j3wz. e ZDOCK SERVER simulated the protein binding spatial conformation of IDH2 and Cav1, where yellow represents IDH2 and blue represents Cav1. f Coimmunoprecipitation experiment assessing the binding capacity of Cav1 with IDH2 and eNOS in CMECs after IDH2-K272R mutation under NOD and HOD stimulation (immunoprecipitated by Cav1 antibody, n = 3). g Schematic representation illustrating the effect of IDH2-Cav1 binding on the interaction between Cav1 and eNOS, created in BioRender. Zang, G. (2025)…

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  Fig. 6. ACAT1 is the lactyltransferase for IDH2-K272. a Immunoprecipitation coupled with mass spectrometry (IP-MS) identified ACAT1 as a protein interacting with IDH2 possessing acyltransferase activity. b ZDOCK SERVER simulated the protein binding spatial conformation of IDH2 and ACAT1, where yellow represents IDH2 and green represents ACAT1. c Coimmunoprecipitation experiments assessed the altered interaction ability between ACAT1 and IDH2, as well as IDH2-K272la, under normal glucose oxygen deprivation (NOD) and high glucose oxygen deprivation (HOD) stimulation (immunoprecipitated by ACAT1 antibody, n = 3). d Western blot analysis of the expression levels of ACAT1 and IDH2-K272la in CMECs treated with the ACAT1 inhibitor Avasimibe (20 µM) under NOD and HOD stimulation (n = 6). e Coimmunoprecipitation experiment assessing the binding capacity of Cav1 with IDH2 and eNOS in CMECs treated with the ACAT1 inhibitor Avasimibe under NOD and HOD stimulation (immunoprecipitated by Cav1 antibody, n = 3). f Schematic representation of ACAT1 regulating IDH2 lactylation influencing Cav1-eNOS interaction, created in BioRender. Zang, G. (2025) https://BioRender.com/1kizpt1. g Evaluation of tube…

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  Fig. 7. Empagliflozin (EMPA) induces angiogenesis and ventricular remodeling by augmenting IDH2-K272 lactylation following diabetic myocardial infarction (DMI). a Western blot analysis of IDH2-K272 lactylation levels in infarct border zone tissues of IDH2WT and IDH2EC-K272R mice following induction of myocardial infarction (MI) or DMI with gavage administration of EMPA (i.g. 30 mg/kg/day, 7 days; n = 6). b Representative Immunofluorescence staining of IDH2-K272la in heart from IDH2WT and IDH2EC-K272R mice following induction of MI or DMI with gavage administration of empagliflozin. CD31 is an endothelial cell marker (n = 6). Scale bar = 20 μm. c Coimmunoprecipitation experiment assessing the binding capacity of Cav1 with IDH2 and eNOS in infarct border zone tissues of IDH2WT and IDH2EC-K272R mice following induction of MI or DMI with gavage administration of empagliflozin (immunoprecipitated by Cav1 antibody). d Arterial ring experiments were conducted in IDH2WT and IDH2EC-K272R mice following induction of MI or DMI with gavage administration of empagliflozin (n = 6). Scale bar = 100 μm. e Representative images and quantification of Masson’s trichrome staining for cardiac fibrosis in the…