FASN-mediated de novo lipogenesis promotes ceramide accumulation and myocardial damage in arrhythmogenic cardiomyopathy

Figures

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  Figure 1. FASN is upregulated in cardiomyocytes of ACM patients (A) TMT-based proteomics heatmap showing upregulation of fatty acid synthesis pathway proteins in ACM (n = 4 per group) versus non-diseased controls (NC). Data are log2-transformed and Z scored. (B) Representative western blots of FASN and GAPDH in left ventricular (LV) and right ventricular (RV) myocardium from ACM patients and NC (n = 3 per group). (C) Densitometric quantification of FASN normalized to GAPDH from (B). Each symbol represents an individual patient (n = 3 per group). Data are mean ± SEM. ∗p < 0.05; ns, not significant (two-way ANOVA with Tukey’s post hoc test). (D) Immunofluorescence staining of human heart sections for α-actinin (red), FASN (green), and DAPI (blue). The white box indicates the region shown at higher magnification in (E). Scale bars, 25 μm. (E) Higher-magnification view of the boxed area in (D), arrow showing FASN (green) localized in the cytoplasm of α-actinin-positive cardiomyocytes (red). Scale bars, 25 μm. (F) Quantification of FASN fluorescence intensity in α-actinin-positive cardiomyocytes (n = 6 patients per group). ∗∗∗∗p < 0.0001 (Student’s t test). (G) Oil red O staining of human ACM…

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  Figure 2. The constructed Dsg2mut/mut transgenic mice exhibit upregulation of FASN (A) Representative B-mode and M-mode echocardiographic images of the left ventricle in WT and Dsg2mut/mut mice at 12 weeks of age. (B) Longitudinal analysis of left ventricular ejection fraction (LVEF, %) from 4 to 12 weeks of age (n = 8 per genotype). ∗p < 0.05, ∗∗p < 0.01 vs. WT (two-way ANOVA with Holm-Šídák test). (C) Right ventricular area (RV area, mm2) over time (n = 8 per genotype). ∗∗p < 0.01 vs. WT. (D) Whole-heart H&E staining at 4, 8, and 12 weeks. Scale bars, 1 mm. (E) Masson’s trichrome staining. Fibrosis is shown in blue. Scale bars, 100 μm. (F) Oil red O staining of mouse hearts at 12 weeks. Scale bars, 50 μm. (G) Quantification of RV thickness (μm) from H&E sections (n = 6 per group). ∗∗∗p < 0.001 vs. WT (Student’s t test). (H) Quantification of inflammation infiltration area (%) from H&E staining (n = 6 per group). ∗∗∗∗p < 0.0001 vs. WT (Student’s t test). (I) Quantification of fibrotic area (%) from Masson’s staining (n = 6 per group). ∗∗p < 0.01, ∗∗∗p < 0.001 vs. WT (Student’s t test). (J) Quantification of oil red O positive area (%) (n = 6 per group). ∗∗∗∗p < 0.0001 vs. WT (Student’s t…

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  Figure 3. JUP synergizes with SREBP1 in the nucleus to activate FASN transcription (A) Schematic of DNA pull-down using biotinylated FASN promoter probes followed by liquid chromatography-mass spectrometry (LC-MS)/MS. (B) MS quantification of JUP binding to FASN probe vs. LacZ control in WT and Dsg2mut/mut hearts. Data are log10 relative quantification (n = 3). (C) Nuclear-cytoplasmic fractionation western blots of JUP, GAPDH, and Histone H3 in WT and Dsg2mut/mut hearts (n = 3). (D) Quantification of JUP in the nuclear fraction (normalized to H3). ∗∗p < 0.01 (Student’s t test). (E) Quantification of JUP in whole-cell lysate (normalized to GAPDH). (Student’s t test). (F) Dual-luciferase reporter assay in HL-1 cells co-transfected with JUP expression vector or empty vector and FASN promoter luciferase construct or basic vector. Relative luciferase activity (firefly/Renilla) is shown (n = 3). ∗∗∗∗p < 0.0001; ns, not significant (one-way ANOVA with Tukey’s test). (G) Predicted molecular docking of JUP and SREBP1 (surface representation). (H) Duolink PLA (proximity ligation assay) in WT and Dsg2mut/mut hearts showing JUP-SREBP1 proximity (orange dots). Scale bars, 50 μm. (I) Quantification of…

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  Figure 4. Isotope tracing indicates de novo lipogenesis in Dsg2mut/mut mouse hearts (A) Schematic representation of in vivo13C-glucose infusion via jugular vein catheterization. (B) Simplified metabolic pathway illustrating the conversion of glucose to pyruvate, acyl-CoA, malonyl-CoA, and downstream fatty acids. (C–E) Mass spectrometry quantification of 13C-labeled (red) and unlabeled (blue) pyruvate (C), acyl-CoA (D), and malonyl-CoA (E) in hearts of wild-type (WT) and Dsg2mut/mut mice (n = 5 per genotype). Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test).

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  Figure 5. Excess ceramides trigger cardiomyocyte death after de novo synthesis of lipids (A) Heatmap of lipidomics data from human ACM and non-diseased control (NC) myocardium (n = 5 per group). Data are log2-transformed and Z scored. (B) Volcano plot of lipid species; upregulated lipids are highlighted in red. (C) Simplified pathway diagram illustrating the conversion of glucose into ceramide. (D) 13C-glucose isotope tracing: relative abundance of 13C-labeled ceramide in Dsg2mut/mut vs. wild-type (WT) hearts (n = 5). ∗p < 0.05 (Student’s t test). (E) qPCR of Sptlc2 in HL-1 sh-NC and sh-Dsg2 cells (n = 3). ∗∗∗∗p < 0.0001 (Student’s t test). (F) Relative ceramide abundance in sh-NC vs. sh-Dsg2 cells measured by LC-MS/MS (n = 3). ∗p < 0.05 (Student’s t test). (G) Experimental design for serum deprivation and siRNA targeting SPTLC1/2. (H) qPCR validation of Sptlc2 knockdown efficiency in indicated groups (n = 3). ∗∗∗∗p < 0.0001 (one-way ANOVA with Tukey’s test). (I) Apoptosis quantified by annexin V/propidium iodide (PI) flow cytometry in the groups shown in (G) (n = 3). ∗∗p < 0.01; ns, not significant (one-way ANOVA with Tukey’s test). (J) Ceramide relative abundance in sh-Dsg2 cells treated…

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  Figure 6. FASN is a potential therapeutic target for ACM (A) Experimental timeline: 6-week-old Dsg2mut/mut mice and littermate wild-type (WT) controls received daily intraperitoneal injections of the FASN inhibitor orlistat or vehicle for 6 weeks. (B) Kaplan-Meier survival curves (n = 10 WT + vehicle, 10 WT + orlistat, 25 Dsg2mut/mut + vehicle, and 22 Dsg2mut/mut + orlistat). ∗∗p < 0.01 (log rank test). (C) Representative B-mode and M-mode echocardiographic images of the left ventricle at 12 weeks. (D) Masson’s trichrome staining of heart sections. Scale bars, 1 mm. (E) Hematoxylin and eosin (H&E) staining of heart sections. Scale bars, 1 mm. (F) Oil red O staining of heart sections. Scale bars, 50 μm. (G) TUNEL staining of heart sections. Scale bars, 50 μm. (H–J) Quantification of left ventricular ejection fraction (LVEF, %) (H), fractional shortening (FS, %) (I), and right ventricular area (RV area, mm2) (J) (n = 15 per group). ∗∗∗∗p < 0.0001 (two-way ANOVA with Holm-Šídák test). (K) Quantification of fibrotic area (%) from Masson’s trichrome staining (n = 6 per group). ∗∗∗p < 0.001 (two-way ANOVA with Holm-Šídák test). (L) Quantification of inflammatory infiltration area (%) from H&E…

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  Figure 7. Pharmacological FASN inhibition ameliorates electrophysiological abnormalities of ACM hearts (A) Representative isochronal activation maps and action potential duration (APD) maps at a pacing cycle length of 100 ms in hearts from WT + vehicle, Dsg2mut/mut + vehicle, and Dsg2mut/mut + orlistat groups. (B) Representative surface electrograms (ECGs) of Dsg2mut/mut + vehicle (upper image) and Dsg2mut/mut + orlistat (lower image). Arrowheads indicate early afterdepolarizations (EADs). (C) Quantification of conduction velocity at a 100-ms pacing cycle length (n = 6 per group). ∗∗p < 0.01 (one-way ANOVA with Tukey’s multiple-comparisons test). (D–F) Quantification of action potential duration at 80% (APD80, D), 50% (APD50, E), and 20% (APD20, F) repolarization at a pacing cycle length of 160 ms (n = 6 per group). ∗p < 0.05, ∗∗p < 0.01 vs. Dsg2mut/mut + vehicle (one-way ANOVA with Tukey’s multiple-comparisons test). Data are mean ± SEM.