cTnIR193H restrictive cardiomyopathy mice satisfy high-energy metabolic demands through regulating glucose metabolism

Figures

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  Figure 1. Cardiac function of 4-month-old mice. (A) Representative M-mode echocardiography of short axis and representative PW-mode echocardiography of four-chamber section. (B) Left ventricular ejection fraction (EF%). (C) Left ventricular fractional shortening (FS%). (D) Left ventricular isovolumic relaxation time (IVRT). (E) E peak. (F) A peak. (G) E/A. n = 4; ∗P < 0.05; ∗∗P < 0.01.

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  Figure 2. Cardiac energy metabolism. (A) ATP concentration (n = 3) and ATPase activity (n = 4) in the hearts of 4-month-old WT and cTnI193His-M mice. (B) Electron micrographs of sections from 4-month-old WT and cTnI193His-M mouse hearts (n = 3; 12,000×). (C) Statistical analysis of mitochondrial number under an electron microscope. ∗P < 0.05; ∗∗P < 0.01.

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  Figure 3. GLUT4 and CD36 expression levels. (A) GLUT4 and CD36 mRNA expression levels (WT, n = 4; cTnI193His-M, n = 3). (B) Immunohistochemical staining of GLUT4 and CD36 in heart tissue (n = 3). Photographs were taken at 400× using an electron microscope. (C) CD36 and GLUT4 protein levels (n = 3). (D) Statistical analysis of western blotting results for the detection of protein expression. (E) GLUT4 immunofluorescence staining on the cell membrane (n = 3). (F) CD36 immunofluorescence staining on the cell membrane (n = 3). Photographs were captured at 400× using a confocal laser. ∗P < 0.05; ∗∗P < 0.01.

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  Figure 4. Proteomic analysis and glucose metabolism in the heart. (A) Proteomics protein identification and quantitative results statistics. (B) A Venn diagram showed the overlapping differentially expressed proteins between the RCM group and the WT group. (C) The principal component analysis of all samples. (D) GO annotation statistics of differentially expressed proteins in the RCM_vs_WT group. The ordinate represented the secondary GO function annotation information, including biological processes, molecular functions, and cell components, which were successively distinguished by blue, red, and orange. The abscissa indicated the number of differentially expressed proteins in each functional category. (E) KEGG enrichment analysis, top20 KEGG TopMapStat sorted by the number of proteins enriched on the KEGG pathway, and up-down-regulation analysis of KEGG enrichment pathway. The blue represents down-regulation and the red represents up-regulation. (F) Glucose content in heart tissue (n = 3). (G) Lactic acid content in heart tissue (WT, n = 5; cTnI193His-M, n = 4). ∗∗P < 0.01.

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  Figure 5. Phosphorylation omics analysis and expression of PI3K/AKT pathway-related proteins in the heart. (A) Phosphorylation omics identification and quantitative results statistics. (B) Venn diagram of phosphopeptides identified between all sample groups and Venn diagram of phosphoproteins identified between all sample groups. (C) Distribution ratio diagram of S/T/Y phosphorylation modification sites. (D) GO annotation statistics of differentially expressed phosphoproteins corresponding to differentially expressed phosphopeptides in the RCM and WT groups. The ordinate represents the secondary GO function annotation information, including biological processes, molecular functions, and cell components, which were successively distinguished by blue, red, and orange. The abscissa represents the number of differentially expressed proteins in each functional category. (E) KEGG enrichment analysis, top20 KEGG TopMapStat sorted by the number of proteins enriched on the KEGG pathway, and up-down-regulation analysis of KEGG enrichment pathway. The blue represents down-regulation and the red represents up-regulation. (F) PI3K protein levels. (G) Statistical chart of PI3K protein expression levels…

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  Figure 6. Cellular ATP concentration, glucose metabolism, and oxidative stress. (A) ATP concentration of primary cardiomyocytes 72 h after transfection of virus (n = 3). (B) Glucose concentration in cell culture medium, 72 (Control, NC-AV, n = 4, cTnIR193H-AV, n = 3), 96 (n = 5), and 108 (n = 4) h after transfection of virus. (C) GLUT4 protein levels. (D) Statistical chart of GLUT4 protein expression levels (n = 4). (E) PI3K protein levels. (F) Statistical chart of PI3K protein expression levels (n = 5). (G) AKT and p-AKT protein levels. (H) Statistical chart of p-AKT/Akt protein expression ratio (n = 4). (I) PI3K protein levels. (J) Statistical chart of PI3K protein expression levels (n = 5). (K) AKT and p-AKT protein levels. (L) Statistical chart of p-AKT/Akt protein expression ratio (n = 4). (M) Glucose concentration in cell culture medium, 72 (n = 6), 96 (n = 6), and 108 (n = 5) h after transfection of virus. (N) Malondialdehyde (MDA) concentration (n = 4) and superoxide dismutase (SOD) activity (n = 3) in primary cardiomyocytes 130 h after transfection of virus. ∗P < 0.05 versus control; ∗∗∗P < 0.01 versus control; #P < 0.05 versus NC-AV; ##P < 0.01, ###P < 0.01, and ####P < 0.01…