Metabolism-related gene PDK1 regulates myocardial cell remodeling and its mechanism

Figures

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  Fig. 1. PDK1−/−hiPSC-CMs showed myocardial remodeling phenotype. (A) Cell apoptosis was detected by TUNEL staining. The rate of apoptotic cells significantly increased in KO-CM’s, Tunnel (green) and DAPI (blue) (Scale bar, 200 μm) and quantification. (B) Expression of Atrial Natriuretic Peptide (ANP), (C) B-type Natriuretic Peptide (BNP), (D) B-MHC detected by Real time PCR in cardiomyocytes. (E) Cell viability determined by CCK8 assay in KO-CM’s.(F) Expression of cleaved caspase-3 was assessed by real time PCR. (G) Western Blot confirmed the expression levels of apoptosis-related protein (Cleaved Caspase-3) and quantification. Full-length blots are presented in Supplementary file 5, Fig. 7. Data are analyzed with two-sample t test and shown as means ± SD. **P <.01, ***P <.001

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  Fig. 2. KO hiPSC-CMs showed abnormal mitochondrial metabolism and elevated oxidative stress level. (A) Schematic depicting the glycolysis and the citric acid cycle. Significantly upregulated metabolites highited by *. (B) Heatmap of all metabolites in both groups. (C) Immunostaining of ROS (red) and DAPI (blue) shows increase ROS production in KO CMs (Scale bar, 200 μm (D) The OCR curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the aerobic respiration level of KO hiPSC-CMs was reduced. (E) The ECAR curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the level of glycolysis is reduced in KO hiPSC-CMs. (F) ATP level has been determined by enhanced ATP assay kit (beyotime). Result shows highly significant difference between the groups. (G, H) Quantitative statistical analysis of basal and maximal respiration levels in WT and KO hiPSC-CMs indicated that basal and maximal respiration levels were decreased in KO hiPSC-CMs Data are analyzed with two-sample t-test and shown as means ± SD. ***P <.001

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  Fig. 3. PDK1 knockout cardiomyocytes are more sensitive to hypoxia (18 h). (A) Hypoxia significantly increased the number of apoptotic cells (Scale bar, 100 μm) and quantification. (B) Immunostaining of ROS (red) and DAPI (blue) shows increase ROS production in KO CMs (Scale bar, 100 μm). (C) Hypoxia treatment significantly increased cleaved caspase 3 activity confirmed by real-time PCR. (D) Cell viability determined by CCK8 assay in KO-CM’s. Result shows highly significant difference between the groups under 18 h hypoxia. (E) The expression level of cleaved caspase-3 protein was detected by Western Blot and quantification. Full-length blots are presented in Supplementary file 5, Fig. 2. (F) The OCR curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the aerobic respiration level of KO hiPSC-CMs was reduced as compared to WT group under 18 h hypoxia. (G) ECAR. curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the level of glycolysis is reduced in KO hiPSC-CMs (18 h Hypoxia). (H) ATP level determined in both groups (18 h Hypoxia). (I) Quantitative statistical analysis of basal and maximal respiration levels in WT and KO…

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  Fig. 4. Transcriptomes are different between pyruvate dehydrogenase kinase 1-deficient and wild-type CMs. (A) Gene expression profile was examined by RNA-seq. The volcano plot shows differentially expressed genes between two groups (n = 3, respectively). Red denotes up-regulated genes, whereas green represents down-regulated genes. (B) GO analyses include three categories, cell components, molecular function and biological process are shown here. (C) Sequencing analysis show second highest gene ratio in ischemia and myocardial infarction. (D) Significantly enriched KEGG (Kyoto Encyclopedia Genes and Genomes) pathways in differential genes. The PI3-AKT pathway enriched the most differential genes with high reliability

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  Fig. 5. Apoptosis of mitochondrial pathway was activated in PDK1 knockout CM’s. (A-C) Expression of P-AKT, GSK3- β and Cyto-C detected by qPCR and suggesting that level of P-AKT and GSK3-β upregulated in knockout cardiomyocytes and Cyto-C level decreases. (D) Expression of P-AKT and Cyto-C verified by the WB results that P-AKT and Cyto-C level downregulated in knockout CM’s. Full-length blots are presented in Supplementary file 5, Fig. 7. (E) The quantification data of the WB result. (F) Cytochrome-C level determined by ELISA. Cytochrome-C release was significantly decreases in knockout cardiomyocytes under basal conditions. This effect is more pronounced under hypoxia. (G) As expected, flow cytometry results showed that PDK1-KO cardiomyocytes induce the opening of mPTP. Hence, mPTP served as a downstream process of Akt-GSK3β pathway. This effect is more sensitive under hypoxia. *P <.05, **P <.01, ***P <.001

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  Fig. 6. Improving metabolism rescues the myocardial remodeling phenotype of PDK1-KO hiPSC‐CMs by lentivirus transfection. (A) Western blot results confirm the overexpression of PDK1 in PDK1 deficient cells. P-AKT level increased in OE cells as compared to KO. (B) Quantification. Full-length blots are presented in Supplementary file 5, Fig. 4. (C) Tunel staining for apoptosis. Hypoxia significantly increased the number of apoptotic cells as compared to Normoxia (Scale bar, 100 μm). Overexpression partially rescue the cells and quantification. (D) ROS levels in WT, KO, and PDK1-KO-OE groups were detected, and the results showed that ROS levels in KO hiPSC-CMs were reduced to varying degrees after overexpression under both conditions. (E) The OCR curves of WT, KO, and KO-OE hiPSC-CMs were detected by Seahorse experiments, the results showed that oxidative phosphorylation was restored in KO hiPSC-CMs after PDK1 overexpression (18 h hypoxia). (F) The ECAR curves of WT, KO, and KO-OE hiPSC-CMs were detected by Seahorse experiments, the results showed that aerobic glycolysis was restored in KO hiPSC-CMs after PDK1 overexpression (18 h hypoxia). (G) The ATP content of WT, KO and PDK1-KO-OE, was…

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  Fig. 7. Schematic diagram of the proposed mechanism. Mitochondrial oxidative damage via the PDK1/Akt- GSK3-β signaling pathway to exacerbate cardiomyocytes from apoptosis