GULP1 protects against diabetic cardiomyopathy through IKIP/NF-κB-dependent improvement of mitochondrial function

Figures

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  Fig. 1. GULP1 expression is downregulated in myocardium from patients and mouse models of DCM. A Weighted gene co-expression network analysis (WGCNA) of genes in control and palmitic acid (PA)-treated NMVMs. B Module–trait correlation analysis revealed that the blue module showed the strongest correlation with PA exposure (left). Gene ontology (GO) enrichment analysis of the blue module genes indicated significant enrichment in cytoplasmic translation, lipid localization, and negative regulation of protein metabolism (right). C Representative Western blot images showing GULP1 expression in myocardial tissues from DCM patients and DCM mice. D Densitometric quantification of GULP1 protein levels, normalized to β-actin. n = 6 per group. E Representative immunofluorescence staining of GULP1 (green) in human myocardial samples. α-sarcomeric actin (red) marks cardiomyocyte cytoskeleton; nuclei are counterstained with DAPI (blue). Scale bar = 50 μm. n = 3 per group. F Representative immunofluorescence staining of GULP1 (green) in mouse myocardial samples. α-sarcomeric actin (red) labels cardiomyocyte cytoskeleton; nuclei are counterstained with DAPI (blue). Scale bar = 50 μm. n = 8 per group.…

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  Fig. 2. GULP1 improves cardiac function, mitochondrial morphology, and delays DCM progression. A, B Representative pulse-wave Doppler images and mitral E/A ratios in Gulp1WT and Gulp1KI mice fed either ND or HFD diet. n = 8 per group. C Representative M-mode echocardiographic tracings of the left ventricle. D–G Quantification of LVEF, LVFS, LVESD, and LVEDD. n = 8 per group. H Representative electron microscopy (EM) images of myocardial mitochondria. Scale bar = 1 μm. For each heart, 15–20 random fields (8900×) were analyzed. n = 8 per group. I ATP levels in fresh myocardial tissues, measured spectrophotometrically. n = 8 per group. J Activities of mitochondrial respiratory chain complexes I–V, expressed as percentages of citrate synthase (CS) activity to normalize for mitochondrial content. n = 5 per group. K, L Seahorse XF analysis of complex I-, II-, and IV-dependent oxygen consumption rates (OCR). Representative traces are shown for NADH- and TMPD/ascorbate-induced OCR (K) and succinate/rotenone- and TMPD/ascorbate-induced OCR (L). n = 5 per group. Data are expressed as mean ± SEM. Statistical significance was determined using two-way ANOVA followed by Bonferroni’s multiple…

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  Fig. 3. GULP1 improves mitochondrial morphology and function and reduces oxidative stress and apoptosis in PA-treated cardiomyocytes. A, B Representative Western blot images and densitometric analysis of GULP1 expression in control or PA-treated NMVMs transfected with Ad-Scramble or Ad-Gulp1. β-actin served as a loading control. n = 6 per group. C–E Representative confocal images of MitoTracker Green FM-stained NMVMs and quantification of mitochondrial number per cell and mitochondrial volume. Approximately 90–100 NMVMs from 6 independent experiments were analyzed per group. Bar = 10 μm (ZOOM 5X). F–G Seahorse XF analysis of mitochondrial oxidative capacity, including basal respiration, maximal respiration, ATP-linked respiration, and spare respiratory capacity. n = 5 per group. H–I Representative Western blot images and quantification of MnSOD and GPx4 expression in NMVMs. J–K Representative Western blot images and quantification of apoptosis-related proteins (caspase-3, cleaved caspase-3, Bcl-2, Bax). β-actin served as a loading control. n = 5 per group. Data are presented as mean ± SEM. Statistical significance was determined using two-way ANOVA followed by Bonferroni’s multiple…

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  Fig. 4. GULP1 exerts its cardioprotective effect through the downstream IKIP. A Schematic diagram of NMVMs treatment groups and IP-MS/MS workflow. B Venn diagram of mass spectrometry results, with the protein identifiers of the selected candidate interactors shown in the right box. C Cell lysates from NMVMs were immunoprecipitated with anti-IgG or anti-GULP1 antibodies, followed by immunoblotting for IKIP, GULP1, and β-actin. D Cell lysates from NMVMs were immunoprecipitated with anti-IgG or anti-IKIP antibodies, followed by immunoblotting for IKIP, GULP1, and β-actin. E Cell lysates from NMVMs treated with control or PA were immunoprecipitated with anti-IKIP antibodies, followed by immunoblotting for IKIP, GULP1, and β-actin. F After mutating the ARG7, LYS22, and ASN180 residues of protein GULP1 to Gln, the 3D structures of the mutant proteins were modeled using AlphaFold3. G Cell lysates of H9C2 cells transfected with plasmids Myc-GULP1ARG−7, Myc-GULP1LYS−22, Myc-GULP1ASN−180, and Flag-IKIP were immunoprecipitated with anti-Myc antibodies, and immunoblot assays were performed using Myc, Flag, and β-actin antibodies. H Triple immunofluorescence (IF) staining for GULP1 (Green), IKIP…

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  Fig. 5. GULP1 improves cardiac function and mitochondrial morphology in DCM through IKIP. A Representative pulse-wave Doppler images from HFD-fed Gulp1WT or Gulp1KI mice treated with AAV9-Scramble or AAV9-sg Ikbip. n = 8 per group. B Representative M-mode echocardiographic tracings. n = 8 per group. C Representative electron microscopy (EM) images of cardiac mitochondria. Scale bar = 1 μm. For each heart, 15–20 random fields (8900×) were analyzed. D Representative EM images scored using a 5-grade mitochondrial morphology system: score 4, intact cristae (> 80%); score 3, mildly irregular cristae (60–80%); score 2, major distortions and membrane discontinuities (30–60%); score 1, severely fragmented/swollen cristae (10–30%); score 0, near-complete cristae loss (< 10%). Pie charts indicate grade distribution; bar graphs show mean scores. 150 mitochondria were analyzed per sample. n = 8 per group. E Representative confocal images of MitoTracker Green FM-stained NMVMs transfected with Ad-Scramble or Ad-sg Ikbip, with or without Ad-Gulp1. n = 6 per group. Bar = 10 μm (ZOOM 5X). F Seahorse XF analysis of mitochondrial oxidative capacity in NMVMs following IKIP knockdown, with or without Gulp1…

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  Fig. 6. GULP1/IKIP promotes OPA1 expression via NF-κB signaling, enhancing mitochondrial fatty acid metabolism. A Hierarchical clustering heatmap of differentially expressed genes (baseMean > 5, |log₂FC| > 3, adjusted p < 0.01) between PA and PA + Ad-Gulp1 groups. Expression values (FPKM) were z-score normalized. Red, upregulation; blue, downregulation. B KEGG pathway enrichment (comet plot) of differentially expressed genes. Pathways are ranked by Rich factor; circle color indicates z-score, with warmer colors denoting higher activation. C, D Immunoprecipitation showing IKIP-IKKβ binding in NMVMs under control or PA intervention. E Representative immunoblot analysis of IKIP, IKKβ, NF-κB p65, phosphorylated p65, and OPA1 in NMVMs treated with Ad-Scramble/Ad-Gulp1, with or without Ad-sg Ikbip. F, G Seahorse XF analysis of FAO capacity in AMVMs. Quantification of maximal ΔOCR under PA or BSA conditions, with or without etomoxir (ETO). n = 5 per group. H Fatty acid oxidation (FAO) capacity was measured with 14C-labeled palmitic acid oxidation assays. n = 6 per group. (I) Myocardial triglyceride (TG) content in indicated groups. n = 7 per group. Data are presented as mean ± SEM. Statistical…

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  Fig. 7. Schematic illustration of GULP1-mediated alleviation of DCM via activation of the IKIP/NF-κB Pathway. In the healthy heart, GULP1 binds to IKIP, limiting the interaction between IKIP and IKKβ, thereby facilitating IKKβ-dependent activation of NF-κB and promoting OPA1 expression. This maintains mitochondrial morphology, preserves MRCS, supports oxidative phosphorylation and FAO, and limits oxidative stress and apoptosis. In DCM, cardiomyocyte GULP1 expression of GULP1 is reduced, weakening its interaction with IKIP and favoring IKIP-IKKβ binding. This inhibits IKKβ-mediated activation (phosphorylation) of NF-κB-dependent Opa1 transcription, disrupts mitochondrial morphology, downregulates mitochondrial complexes, impairs oxidative phosphorylation and fatty acid β-oxidation, and enhances oxidative stress, apoptosis, and myocardial lipid accumulation. Restoration of GULP1–IKIP–NF-κB–OPA1 signaling improves mitochondrial structure and function, promotes FAO, reduces lipid overload, and ultimately improves cardiac function and prevents DCM progression. GULP1: PTB-domain engulfment adapter protein 1, IKIP: Inhibitor of nuclear factor kappa-B kinase-interacting, DCM: diabetic…