Deletion of Tbc1d4/As160 abrogates cardiac glucose uptake and increases myocardial damage after ischemia/reperfusion

Figures

• 

  Fig. 1. Deletion of Tbc1d4 leads to reduced cardiac GLUT4 content and impaired ex vivo glucose uptake. A mRNA copy number, normalized by Tbp expression, of Tbc1d1 and Tbc1d4 in whole heart tissue and isolated left ventricle. B Heart weight/body weight ratio of WT and D4KO animals on standard diet. C Total cardiac glycogen content (n = 7–8); D, E Cardiac protein abundance of TBC1D4, GLUT1 and GLUT4 (n = 7), F Insulin-stimulated [3H]-2-deoxyglucose uptake in isolated cardiac left-ventricular papillary muscle (n = 5–6). Data presented as mean ± SEM. Statistical analysis was performed using unpaired two-tailed student’s t-Test (A-C,E; WT vs. D4KO: *p < 0.05; **p < 0.01; ***p < 0.001) or two-way ANOVA with Bonferroni’s multiple comparison test (F; basal vs. insulin: *p < 0.05; WT vs. D4KO: #p < 0.05); male mice, 36 weeks of age. WT wild type, D4KO Tbc1d4-knockout

• 

  Fig. 2. Deletion of Tbc1d4 leads to impaired in vivo cardiac glucose uptake. A [18F]-fluorodeoxy-D-glucose positron emission tomography (PET) scan technique was applied in order to visualize glucose uptake into the heart (white arrow) for male Tbc1d4-deficient and wild type mice on a Chow-diet. B Quantification of cardiac glucose uptake over time with corresponding (C) area under the curve (AUC) calculation. Data presented as mean ± SEM (n = 4). Statistical analysis was performed using unpaired two-tailed student’s t-Test (C; WT vs. D4KO: *p < 0.05). WT wild type, D4KO Tbc1d4-knockout

• 

  Fig. 3. Tbc1d4-ko and HFD impair heart function and morphology following I/R with unaffected reduced GLUT4 content. Echocardiographic assessment of cardiac A end-systolic and B end-diastolic volume over a time course of 3 weeks (n = 6). C R-wave amplitude at basal state, during ischemia and acute reperfusion phase, normalized to the basal state. Histomorphometrical analysis of WT and Tbc1d4-deficient hearts 3 weeks post-I/R (closed chest) in terms of: D left ventricular wall thickness, E left ventricular lumen, and F infarct size (n = 3). G, H, I Cardiac protein content of H GLUT1 and I GLUT4 three weeks after reperfusion (open chest; n = 6–7). Data presented as mean ± SEM. Statistical analysis was performed using unpaired two-tailed student’s t-Test (A, B, D-F, H, I; WT vs. D4KO: *p < 0.05) or two-way ANOVA with Bonferroni’s multiple comparison test (C; Isch or Rep vs. basal: *p < 0.05, **p < 0.01; WT vs. D4KO: #p < 0.05) (Male mice, 36 weeks of age). WT wild type, D4KO Tbc1d4-knockout, Isch ischemia, Rep Reperfusion

• 

  Fig. 4. Deletion of Tbc1d4 leads to increased extracellular matrix area and impaired Mmp expression profile. Transmission electron microscopy of A WT and B Tbc1d4-deficient cardiac left ventricular tissue and C histomorphometrical analysis of extracellular matrix area. Expression of ECM remodeling markers in terms of D Mmp2, E Mmp9, F Mmp13 and G Mmp2/Timp1 ratio. Data presented as mean ± SEM (n = 3). Statistical analysis was performed using unpaired two-tailed student’s t-Test (C-G; WT vs. D4KO: *p < 0.05; ***p < 0.001). WT wild type, D4KO Tbc1d4-knockout

• 

  Fig. 5. Transcriptome analysis reveals differential cardiac gene expression due to loss of Tbc1d4. Whole heart transcriptome analysis via RNA sequencing and subsequent Ingenuity pathway analysis A Volcano Plot of differentially regulated genes due to the loss of Tbc1d4 displaying the size of differential regulation and size of significance. B Differentially regulated canonical pathways and potential upstream regulators of Ingenuity Pathway Analysis/IPA for differentially regulated genes with adjusted p-value < 0.01. C Differentially regulated genes contained in over-represented Reactome pathways and IPA disease annotations regarding altered metabolic processes. Overlap of genes and corresponding p-value of CPDB over-representation analysis are indicated above the respective heatmap Data presented as Z-Score. Significance threshold was set at p < 0.01 (n = 4). WT wild type, D4KO Tbc1d4-knockout

• 

  Fig. 6. Tbc1d4-ko alters gene expression in insulin signaling and distinctively activates cardiac UPR via eIF2a/ATF4 pathway. A Expression of Tbc1d4-related signaling genes in D4KO and WT hearts. B Atf4 expression and C phosphorylation ratio of phosphorylated eIF2a (Ser51) and total eIF2a protein. D Gene expression of Xbp1 splicing ratio and E phosphorylation ratio of phosphorylated (Thr183/Tyr185) and total SAPK. Data presented as mean ± SEM. Statistical analysis was performed using unpaired two-tailed student’s t-Test (WT vs. D4KO: *p < 0.05; **p < 0.01; ***p < 0.001) n = 6–8. Male mice, 36 weeks of age. WT wild type, D4KO Tbc1d4-knockout