Pellino1 deficiency reprograms cardiomyocytes energy metabolism in lipopolysaccharide-induced myocardial dysfunction

Figures

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  Fig. 1. Silencing Pellino1 alleviates LPS-induced cardiomyocyte apoptosis and inflammation in vitro. a, b Representative western blot of the expression of Pellino1 in the LPS-induced cardiomyocytes. The bar graph shows that the expression of Pellino1 increased gradually with LPS treatment time. β-actin was detected as the loading control. c, d The efficacy of silencing Pellino1 via an adenovirus was assessed by western blotting. β-actin was detected as the loading control. e, f Apoptosis of cardiomyocytes exposed to LPS with or without the Pellino1-silencing adenovirus. The right bar graph shows the results of statistical analysis of the apoptosis ratios. g–i Representative western blot of the expression of Bax, Bcl2, CC3, and C3 in LPS-induced cardiomyocytes with/without si-Pellino1 treatment. The relative protein levels were normalized to the β-actin levels. The data are shown as the mean ± standard error of the mean of triplicates and are representative of three independent experiments performed. j Representative qRT-PCR of mRNA expression of TNF-α, IL-1β, and IL-6 in the LPS-induced cardiomyocytes with/without adenovirus silencing Pellino1. All mRNA expression was normalized to that…

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  Fig. 2. Silencing Pellino1 alleviates the effects of LPS on cardiac mitochondrial function. a–e Representative qRT-PCR of PGC-1α, PGC-1β, PPARα, PPARγ, PPARδ, NRF1, and ERRα mRNA expression following LPS treatment after Pellino1 silencing. f, g qRT-PCR validation of downstream metabolic genes of PGC-1 coactivators, including ATP Synthase β (Atp5b) and Citrate Synthase (Cit syn), involved in mitochondrial function. All mRNA expression was normalized to that of GAPDH. n = 6, results are expressed as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the GFP group

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  Fig. 3. Silencing Pellino1 alleviates the effects of LPS on cardiac fuel metabolism. a–d qRT-PCR validation of downstream metabolic genes of PGC-1 coactivators, including MCPT-1 (Cpt1b), PDK4, MCAD (Acadm), and CD36 involved in fatty acid β-oxidation (FAO). e Representative qRT-PCR of the mRNA expression of GLUT4 (Slc2a4), the PGC-1 target gene encoding the glucose transporter, followed by Pellino1-silencing adenovirus treatment. f, g Representative qRT-PCR of the mRNA expression of PLIN2 and angiopoietin-like 4 (ANGPTL4) involved in the regulation of FAO and myocardial lipid metabolism, respectively, following LPS treatment after Pellino1 silencing. All mRNA expression was normalized to GAPDH. n = 6, results are expressed as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the GFP group

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  Fig. 4. LC–MS-mediated metabolomics analysis of LPS-treated or Pellino1-silenced NRCMs. a Correlation analysis identified good sequencing stability and negative associations among samples. b Different perspectives of two-dimensional principal components (PC) showed the variable reduction outputs analyzed by orthogonal partial least-squares projections to latent structures-discriminant analyses (OPLS-DA) on metabolites from si-Pellino1 and LPS-treated, LPS-treated, and untreated control NRCMs. c, d OPLS-DA score plots of three groups (GFP, GFP + LPS, si-Pellino1 + LPS) according to the differential metabolic profile showed in 2D (c) and 3D (d); each point represents a tested sample. e One-way ANOVA (analysis of variation) of significant levels of all 169 metabolites, P < 0.05

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  Fig. 5. Metabolome view of the top 50 matched metabolic pathways associated with si-Pellino1 treatment. a Impact analysis of metabolites associated with si-Pellino1 treatment relative to the control according to P values (shown in Table 2) from enrichment analysis and impact values from topology analysis. The topological score (X-axis) and the size of each circle represent the importance of different metabolites and their impact values in the enriched pathways, while the color of each circle based on the P value of the enrichment analysis (Y-axis) represents the statistical significance of the overall metabolic changes in the pathways. b Enrichment analysis of metabolites associated with si-Pellino1 treatment relative to the control. The pathway from yellow to red indicates a growing impact

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  Fig. 6. Protein interaction analysis of matched KEGG pathway associations with siPellino1 treatment. a Predicted protein interactions among PGC-1α, PPARα, Slc2a1, CD36, and Cpt1b involved in the adipocytokine signaling pathway. b Predicted protein interactions among PPARγ, PPARα, Angptl4, CD36, Cpt1b, and Acadm involved in the PPAR signaling pathway

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  Fig. 7. Interaction analysis of gene and metabolite associations with si-Pellino1 treatment. Correlation networks of the citrate cycle (TCA cycle); fatty acid metabolism; glyoxylate and dicarboxylate metabolism; propanoate metabolism; valine, leucine, and isoleucine degradation and beta-alanine metabolism showed the inner relationships between metabolites and genes that we previously explored in the main enriched metabolic pathways showing changes