Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: The single-cell RNA-sequencing (scRNA-seq) was conducted in T2DM hearts. a The t-SNE plot showed different cell clusters in control (Ctrl) and T2DM hearts, colored by experimental groups. b Volcano plot displayed the differential expression of deubiquitinating enzymes (DUBs) in heart tissues between control and T2DM mice. NS no significance. c Dot plot illustrated the expression levels of Otud1 in control and T2DM hearts. d The mRNA levels of Otud1 in heart tissues. e Representative western blot images and quantification of protein levels of OTUD1 in heart tissues. f The t-SNE plot identified seven main cell types, including fibroblasts (FBs), cardiomyocytes (CMs), endothelial cells (ECs), smooth muscle cells (SMCs), macrophages (MPs), T cells, and B cells in scRNA-seq analysis. g A biaxial scatter plot showing the expression pattern of Otud1 across these cell clusters. h Dot plot indicated the relative expression of Otud1 in the CMs. i Representative images of immunofluorescence staining for OTUD1 (green), DAPI (blue), α-actinin (red), or vimentin(red) in heart sections from control and T2DM mice. j OTUD1 protein expression in neonatal rat primary cardiomyocytes (NRPCs) treated with high glucose (HG; 33 mM) and palmitic acid (PA; 100 μM) for indicated times (0, 12, 24, and 48 h). For i , scale bar = 50 μm. Source data are provided as a file. Data are presented as mean ± SEM ( a – c , f – h : n = 3; d , e : n = 6 for each group; i , j : n = 3 independent experiments); For ( d , e ), P values were determined by two-tailed unpaired t -test; For ( j ), P values were determined by one-way ANOVA followed by Tukey post hoc tests.

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: RNA Sequencing, Control, Quantitative Proteomics, Expressing, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a Schematic representation of the experimental design. Otud1 fl/fl and Otud1 cardiomyocyte-specific knockout mice were induced to T2DM by low-dose streptozotocin (STZ, 35 mg/kg) administration for 3 days combining high-fat diet (HFD) feeding for 16 weeks. After 16 weeks, the cardiac function of the mice was evaluated, and blood samples along with heart tissues were harvested. b , c Blood glucose levels and body weight of sham and T2DM mice in both Otud1 fl/fl and Otud1 conditional knockout (CKO) groups. d , e Serum levels of lactate dehydrogenase (LDH) and atrial natriuretic peptide (ANP) in different groups of mice. f , g Echocardiography was performed to assess ejection fraction (EF%) and fractional shortening (FS%). h , i Representative echocardiographic images and gross heart morphology from each group. j Wheat germ agglutinin (WGA) staining and quantitative analysis of cardiomyocyte area in heart tissues. k , l The mRNA levels of hypertrophic genes ( Nppa , Nppb , Myh7 , and Acta1 ) and fibrotic genes ( Col1a1 and Tgfb1 ) were quantified by RT-qPCR in heart tissues. m Masson’s trichrome staining and quantification in heart tissues. For ( j , m ), scale bar = 50 μm. Source data are provided as a file. Data are presented as mean ± SEM ( n = 6 for each group); For ( d, f , g , j – l ), P values were determined by one-way ANOVA followed by Tukey post hoc tests; For ( e , k ) ( Bnp ), and ( m ), P values were determined by Kruskal–Wallis test with Dunn post hoc tests.

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Knock-Out, Staining, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a Schematic of the experimental design. Otud1 fl/fl and Otud1 conditional knockout (CKO) mice were induced to T1DM by a high dose of streptozotocin (STZ, 120 mg/kg) injection. Sixteen weeks later, cardiac function was assessed, and blood and heart tissue samples were collected. b , c Blood glucose levels and body weight were measured in sham and T1DM mice in both Otud1 fl/fl and Otud1 conditional knockout (CKO) groups for 16 weeks. d , e Serum lactate dehydrogenase (LDH) and atrial natriuretic peptide (ANP) levels in different groups of mice. f , g Statistical graphs of ejection fraction (EF%) and fractional shortening (FS%) in mice. h , i Representative images of echocardiographic and gross heart. j Wheat germ agglutinin (WGA) staining and quantitative analysis of heart sections. k , l The mRNA expression levels of hypertrophic genes ( Nppa , Nppb , Myh7 , Acta1 ) and fibrotic genes ( Col1a1 , Tgfb1 ) in heart tissues. m Masson’s trichrome staining and quantification in heart tissues from different groups. For ( j , m ), scale bar = 50 μm. Source data are provided as a file. Data are presented as mean ± SEM ( n = 6 for each group); For ( d , f , g , j – m ), P values were determined by one-way ANOVA followed by Tukey post hoc tests; For ( e ), P values were determined by Kruskal–Wallis test with Dunn post hoc tests.

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Knock-Out, Injection, Staining, Expressing

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a , c Pathway enrichment analysis related to AMPK pathway and mitochondrial homeostasis by GSEA in heart tissues of T2DM-challenged Otud1 fl/fl and Otud1 conditional knockout (CKO) mice. b Western blot analysis and quantification of protein levels of phosphorylated AMPK (p T172 -AMPK), AMPK, p-ACC, ACC, and HK-1 in heart tissues from sham and T2DM mice in both Otud1 fl/fl and Otud1 CKO groups. d Representative images of MitoSOX staining showed mitochondrial reactive oxygen species levels in heart sections. e Representative images of transmission electron microscopy illustrated mitochondrial morphology in heart tissues from each group. Red arrows show abnormal mitochondria. f Quantitative analysis of mitochondrial morphology scores. g Measurement of ATP content in heart tissues from all mice. h Western blot analysis and quantification of mitochondrial respiratory chain complex proteins in heart tissues. For ( d ), scale bar = 50 μm; for ( e ), Scale bar = 1 μm (upper) and 2 μm (lower). Source data are provided as a file. Data are presented as mean ± SEM ( a , c : n = 4 for Otud1 fl/fl group, and n = 3 for Otud1 CKO group; b and d – h : n = 4 for each group); For ( b , h ), P values were determined by one-way ANOVA followed by Tukey post hoc tests; For ( g , h (CIII)), P values were determined by Kruskal–Wallis test with Dunn post hoc tests.

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Knock-Out, Western Blot, Staining, Transmission Assay, Electron Microscopy

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a Neonatal rat primary cardiomyocytes (NRPCs) transfected with siRNA targeting OTUD1 (siOTUD1), scrambled negative control (NC), OTUD1 plasmids (OTUD1 OE ), or empty vector (EV) for 24 h, and were subsequently stimulated with high glucose (HG; 33 mM) and palmitic acid (PA; 100 μM) for 2 or 24 h. The expressions of OTUD1, p T172 -AMPK, AMPK, p-ACC, ACC, and HK-1 were detected. b NRPCs transfected with siOTUD1 or NC were stimulated with HG, combining PA for 1 h. Flow cytometry analysis of the mitochondrial reactive oxygen species levels in NRPCs. c After 24 h of HG + PA stimulation, the ATP content was detected in NRPCs. d Western blot analysis of mitochondrial respiratory chain complexes (Grim19, SDHA, UQCRC2, MTCO1, and ATP5A) in NRPCs. e , f Analysis of oxygen consumption rate (OCR) in NRPCs treated as indicated. Oligo oligomycin, FCCP carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, Rot/AA rotenone/antimycin A. g , i Representative images of rhodamine phalloidin staining for cardiomyocyte area measurement. h , j RT-qPCR analysis of hypertrophic genes ( Nppa , Nppb , Myh7 , Acta1 ) in NRPCs with HG + PA stimulation for 24 h. k RT-qPCR analysis of hypertrophic genes in AMPKα2 knockout H9C2 cells ( gAMPKα2 ) or control ( gctrl ) cells challenged with HG and PA for 24 h. For ( g , i ), scale bar = 100 μm. Source data are provided as a file. Data are presented as mean ± SEM ( a , b , d – k : n = 3 independent experiments; c : n = 6 independent experiments); For ( c ), P values were determined by two-tailed unpaired t -test; For ( f – k ), P values were determined by one-way ANOVA followed by Tukey post hoc tests; For ( f ) (ATP Production), P values were determined by Kruskal–Wallis test with Dunn post hoc tests.

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Transfection, Negative Control, Plasmid Preparation, Flow Cytometry, Western Blot, Staining, Quantitative RT-PCR, Knock-Out, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a Schematic illustration of a quantitative proteomic screen to identify proteins binding to OTUD1. b Venn diagram represents potential OTUD1-binding proteins associating with DCM, AMPK pathway, and mitochondrial energy metabolism. c , d Co-immunoprecipitation (Co-IP) of the interaction between OTUD1 and AMPKα2 in heart tissues of T2DM mice and in high glucose (HG; 33 mM) + palmitic acid (PA; 100 μM)-treated neonatal rat primary cardiomyocytes (NRPCs). e Immunofluorescence staining revealed the colocalization of OTUD1 and AMPKα2 in NRPCs treated with HG + PA for 12 h. f Co-IP analysis confirmed the interaction between OTUD1 and AMPKα2 in Hek293T cells co-transfected with plasmids expressing Flag-OTUD1 and HA-AMPKα2. g Bio-layer interferometry (BLI) assay demonstrated the interaction between OTUD1 and AMPK protein, with AMPK added at different concentrations ranging from 18.75 to 300 nM, and KD values were calculated. h Co-IP of OTUD1 and different subunits of AMPK in Hek293T cells. i Schematic diagram of truncated mutants of AMPKα2. KD kinase domain, AID autoinhibitory sequence domain, CTD carboxyl-terminal domain. j Co-IP of OTUD1 and either wild-type AMPKα2 (HA-AMPKα2) or mutants-AMPKα2 in Hek-293T cells. k Schematic diagram of truncated mutants of OTUD1. OTU motif named as ovarian tumor, UIM ubiquitin-interacting motif. l Co-IP of AMPKα2 and either wild-type OTUD1 or mutants-OTUD1. For ( e ), scale bar = 200 μm. Source data are provided as a file. Data are presented as mean ± SEM ( n = 3 independent experiments).

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Transfection, Expressing, Sequencing, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a Hek-293T cells were co-transfected with HA-AMPKα2, Flag-OTUD1, and MYC-ubiquitin (MYC-Ub), followed by treatment with 10 μM MG132 for 6 h. Ubiquitinated AMPKα2 was detected via immunoblotting. b Western blot analysis of total AMPK (T-AMPK) and AMPKα2 in Hek-293T cells (upper), transfected with Flag-OTUD1 plasmids, as well as in NRPCs (lower). c Co-immunoprecipitation (Co-IP) assay demonstrated the ubiquitination of AMPKα2 by K48-Ub or K63-Ub in Hek-293T cells. d Co-IP assay was conducted in Hek-293T cells co-transfected with HA-AMPKα2 (WT) or mutants carrying lysine-to-arginine substitutions at two sites (K60R and K379R) or four sites (K60R, K379R, K391R, and K470R), and MYC-Ub plasmids to determine AMPKα2 ubiquitination levels. e Ubiquitination levels of AMPKα2 were assessed in Hek-293T cells co-transfected with WT HA-AMPKα2, HA-AMPKα2-K60R, or HA-AMPKα2-K379R, and MYC-Ub plasmids. f Western blot analysis was performed to evaluate phosphorylated AMPK (p T172 -AMPK) and total AMPK in NRPCs transfected with WT, K60R, or K379R AMPKα2 under high glucose (HG; 33 mM) and palmitic acid (PA; 100 μM) stimulation for 2 h. g Co-IP assay was used to analyze ubiquitinated AMPKα2 in Hek-293T cells co-transfected with WT-OTUD1 or the 320 cysteine mutant to alanine (C320A; OTUD1-Mut), and MYC-Ub plasmids. h Western blot analysis of p T172 -AMPK and T-AMPK in NRPCs overexpressing WT-OTUD1 or C320A-OTUD1 under HG and PA stimulation for 2 h. i RT-qPCR analysis of hypertrophic genes in NRPCs challenged with HG + PA for 24 h. j Co-IP of CAMKK2, LKB1, and p T172 -AMPK in Hek-293T cells treated as indicated. k CAMKK2 and p T172 -AMPK levels were analyzed in Hek-293T cells overexpressing WT-OTUD1, Mut-OTUD1, and MYC-Ub. Source data are provided as a file. Data are presented as mean ± SEM ( n = 3 independent experiments); For ( i ), P values were determined by one-way ANOVA followed by Tukey post hoc tests.

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Transfection, Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 and inducing mitochondrial dysfunction

doi: 10.1038/s41467-025-61901-z

Figure Lengend Snippet: a Adeno-associated virus 9 (AAV9) carrying shRNAs targeting Otud1 (AAV9-sh Otud1 ) or Ampka2 (AAV9-sh Ampka2 ) with the cardiomyocyte-specific promoter cTNT was administered to db/db mice via tail vein injection. b Western blot analysis of OTUD1 and p T172 -AMPK in heart tissues. c The levels of serum atrial natriuretic peptide (ANP) in different groups. d , e Echocardiographic analysis of ejection fraction (EF%) and fractional shortening (FS%) in different groups. f , g Representative images of echocardiographic and gross heart morphology in each group. h Wheat germ agglutinin (WGA) staining and quantitative analysis in heart sections. i RT-qPCR analysis of hypertrophic genes in heart tissues. j Images of transmission electron microscopy showed mitochondrial morphology in heart tissues from different groups. Red arrows show abnormal mitochondria. k Quantitative analysis of mitochondrial morphology scores. l Western blot analysis of mitochondrial respiratory chain complex proteins (SDHA, UQCRC2, MTCO1) in heart tissues. For ( h ), scale bar = 50 μm; for ( j ), scale bar = 1 μm. Source data are provided as a file. Data are presented as mean ± SEM ( n = 6 for each group); For ( c – e , i ), P values were determined by one-way ANOVA followed by Tukey post hoc tests; For ( h ), P values were determined by Kruskal–Wallis test with Dunn post hoc tests.

Article Snippet: The primary antibodies utilized were: OTUD1, p T172 -AMPK (50081, Cell Signaling Technology, MA, USA), p-ACC (3661S, Cell Signaling Technology), ACC (3662S, Cell Signaling Technology), HK-1 (19662-1-AP, Proteintech), AMPK (2532S, Cell Signaling Technology), Grim19 (sc-365978, Santa, USA), SDHA (ab14715, Abcam), UQCRC2 (ab14745, Abcam), MTCO1 (ab14705, Abcam), ATP5A (ab14748, Abcam), CAMkk2 (11549-1-AP, Proteintech), LKB1(10746-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech).

Techniques: Virus, Injection, Western Blot, Staining, Quantitative RT-PCR, Transmission Assay, Electron Microscopy