Succinate–GPR91 signaling promotes cardiomyocyte metabolic reprogramming and NAD+ production to alleviate HFpEF

Figures

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  Fig. 1. Reduced succinate levels and downregulation of its receptor GPR91 in HFpEF. A, Succinate levels in cardiac tissue of HFpEF patients, based on targeted metabolomics data from a published study. B, Succinate levels in cardiac tissue of WT regular chow (RC) and HFpEF mice, measured using a colorimetric assay. n = 6 per group. C, The mRNA expression levels of Gpr91 in cardiac tissues of WT RC and HFpEF mice. n = 6 in WT RC, n = 9 in WT HFpEF. D, Western blot analysis and quantification for GPR91 expression in the cardiac tissues of control mice compared with db/db or HFpEF mice. n = 6 per group. E, Western blot analysis and quantification of GPR91 expression in AC16 cardiomyocytes cultured under control or HG + PA conditions for 48 h (n = 3 per group). F, Western blot analysis and quantification of GPR91 expression in human iPSC-derived cardiac organoids exposed to control or HFpEF-like comorbidity conditions (n = 3 per group). G, Representative images of immunofluorescence staining of GPR91 (Red) in the heart of RC and HFpEF mice, and the corresponding quantification of GPR91 fluorescence intensity. n = 7 per group. All data are presented as the mean ± SEM. P < 0.05 was considered…

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  Fig. 2. Supplementation with succinate mitigates diastolic dysfunction and metabolic remodeling. C57BL/6J male mice (8-week-old) were fed with regular chow or high-fat diet (HFD) + Nω-nitro-L-arginine methyl ester (L-NAME), and received either control or 1.5% succinate-supplemented drinking water for 12 weeks. A, Experimental design. B, Body weight. n = 6 in RC and RC + SUC; n = 13 in HFpEF and HFpEF + SUC. C, representative images of echocardiography. D, E/A ratio. E, E/E′ ratio. F, Representative image of myocardial strain analysis using VevoStrain. G, Left ventricular global longitudinal strain (GLS). H, Tei index. I, Percentage of LVEF. n = 4 in RC and RC + SUC; n = 7–10 in HFpEF and HFpEF + SUC. J, Rotarod-determined running time during the exercise exhaustion test. n = 6 in RC and RC + SUC; n = 8 in HFpEF and HFpEF + SUC. K and L, Representative heart images, heart weight (mg) and tibia length(mm) ratio (HW/TL). n = 5–6 in RC and RC + SUC; n = 7–8 in HFpEF and HFpEF + SUC. M, Representative images of H&E, Masson, WGA and Oil Red-stained sections. N, Quantification of interstitial fibrosis (%) based on Masson’s trichrome staining, and cardiomyocyte cross-sectional area based on WGA…

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  Fig. 3. Succinate Fails to Confer Cardiometabolic Protection in Gpr91 Knockout HFpEF Mice. Global Gpr91 knockout (Gpr91-/-) and wild-type (WT) C57BL/6J male mice (8 weeks old) were fed with either regular chow or HFD + L-NAME, and received either control or 1.5% succinate-supplemented drinking water for 12 weeks. A, Experimental design. B, Body weight. n = 7–10. C, Representative echocardiographic images. D, E/A ratio. E, E/E′ ratio. F, Representative myocardial strain analysis using VevoStrain. G, Tei index. H, Left ventricular global longitudinal strain (GLS). I, Left ventricular ejection fraction (LVEF, %). n = 6–12 per group. J, Rotarod-determined running time during the exercise exhaustion test. n = 7–8 per group. K, L, Representative images of hearts and heart weight-tibia length ratio (HW/TL). n = 9–10 per group. M, Representative histological sections stained with H&E, Masson’s trichrome, wheat germ agglutinin (WGA), and Oil Red O. N, Quantification of interstitial fibrosis (%) based on Masson’s staining, and cardiomyocyte cross-sectional area based on WGA staining. n = 6 per group. O, Relative mRNA expression levels of Nppa, Nppb, and Myh7, Col1a1, Col3a1, and Acta2 in cardiac…

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  Fig. 4. Succinate-Mediated Improvement of Diastolic Dysfunction in HFpEF Depends on Cardiomyocyte GPR91. Cardiomyocyte-specific GPR91 knockout mice and their littermate controls C57BL/6J male mice (8 weeks old) were fed with either regular chow or HFD + L-NAME, and received either control or 1.5% succinate-supplemented drinking water for 12 weeks. A, Experimental design. B, E/A and C, E/E′ ratio. D,Representative echocardiographic images. E, Left ventricular global longitudinal strain (GLS). F, Tei index. G, Representative myocardial strain analysis using VevoStrain. H, Left ventricular ejection fraction (LVEF, %). n = 6–12 in each group. I, Rotarod-determined running time during the exercise exhaustion test. n = 6–9 in each group. J, Representative images of hearts and heart weight/tibia length ratio (HW/TL). n = 8–10 in each group. K, L, Quantification of interstitial fibrosis (%) based on Masson’s staining, and cardiomyocyte cross-sectional area based on WGA staining. n = 7 in each group. M, Representative histological sections stained with H&E, Masson’s trichrome, wheat germ agglutinin (WGA), and Oil Red O. N, Representative electron microscopy image shows the lipid droplets (yellow)…

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  Fig. 5. Succinate–GPR91 Activates AMPK to Improve Energy Metabolism in HFpEF Mice. A, Venn diagram and table showing shared GO biological processes among groups. (Upregulated Terms in Gpr91fl/fl HFpEF + SUC vs. Gpr91fl/fl HFpEF and downregulated Terms in Gpr91ΔCM HFpEF + SUC vs. Gpr91fl/fl HFpEF + SUC) B, Venn diagram and table showing shared KEGG-enriched pathways among groups. Groups are the same as in (A). C, Heart NAD+ levels in Gpr91fl/fl and Gpr91ΔCM HFpEF or HFpEF + SUC groups. n = 6 in each group. D, Heart succinate levels in four groups measured by colorimetric assays. n = 7–8 in each group. E, Heatmap depicting relative expression of significantly altered AMPK downstream target genes in the hearts. n = 3 in each group. F, qPCR analysis of relative expression levels of AMPK downstream target genes. n = 6 in each group. G, Western blotting analysis of AMPK and phosphorylated AMPK (p-AMPK) levels in heart tissue. n = 5 in each group. Data are expressed as mean ± SEM. P < 0.05 was considered statistically significant, and exact P-values are indicated in the respective figure panels. Data were analyzed using one-way ANOVA (D-G), followed by Tukey’s multiple comparisons test

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  Fig. 6. Gq-Dependent AMPK Activation Mediates Succinate–GPR91 Metabolic Effects. AC16 were treated with high glucose (33mM) plus palmitate (200µM) for 48 h in a sustained medium to mimic HFpEF-like conditions in vitro. A, Representative immunoblot images and densitometry analysis of GPR91 in AC16 cells treated with succinate (400µM) 24 h, under normal or HG + PA conditions 48h. n = 3 in each group. B, Representative immunoblot images and densitometry analysis of p-AMPK/AMPK in primary neonatal ventricular cardiomyocytes isolated from WT and Gpr91−/− mice. n = 3 in each group. C, AC16 cells were treated with high glucose (33mM) plus palmitate (200µM) for 48 h, then 1µM Gqi pretreat 1 h, succinate treat 5 min, Representative blot images of p-AMPK and AMPK. n = 3 in each group. D, qPCR analysis of AMPK downstream target genes in AC16 cells under indicated treatments. n = 4 in each group. E, Intracellular NAD+ levels in AC16 cells treated with succinate and/or Gqi (1µM) in normal and HG + PA environments. n = 5–6 in each group. F, NAD+ levels following treatment with succinate and/or AMPK inhibitor (Compound C 10µM) in normal and HG + PA environments. n = 3 in each group. Data are expressed…

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  Fig. 7. NAM supplementation partially restores cardiac function and NAD+ levels in Gpr91−/−HFpEF mice. WT mice were fed with RC diet. Gpr91−/− mice receiving HFD + L-NAME were treated with or without 40 mM nicotinamide in drinking water for 12 weeks.A, Experimental design. B, Body weight. n = 6–10. C, Representative echocardiographic images. D, E/A ratio. E, E/E′ ratio. F, Tei index. G, Left ventricular global longitudinal strain (GLS). H, Representative myocardial strain analysis using VevoStrain. I, Left ventricular ejection fraction (LVEF, %). n = 6–10 in each group. J, Rotarod-determined running time during the exercise exhaustion test. n = 6–10 in each group. K, L, Representative images of hearts and heart weight/tibia length ratio (HW/TL). n = 7–8 in each group. M, Representative histological sections stained with H&E, Masson’s trichrome, wheat germ agglutinin (WGA), and Oil Red O. N, Quantification of interstitial fibrosis (%) based on Masson’s staining, and cardiomyocyte cross-sectional area based on WGA staining. n = 6 in each group. O, Relative mRNA expression levels of Nppa, Nppb, and Myh7, Col1a1, Col3a1, and Acta2 in cardiac tissue from each group. n = 6 per group. P, Heart…