Connexin43 Deficiency Leads to Ventricular Arrhythmias by Reprogramming Proline Metabolism

Figures

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  FIGURE 1. GJA1 is identified as one of the crucial genes for ventricular arrhythmias. (A) Venn diagram of the ventricular arrhythmia (VA) putative genes (PVC: premature ventricular contraction; VT: ventricular tachycardia; Vf: ventricular flutter; and VF: ventricular fibrillation) obtained from the OMIM and DisGeNET databases. (B) Visualization of the protein–protein interaction (PPI) network of the obtained genes from Panel A, and top 20 hub genes identified by six algorithms including Stress, MNC, EPC, Degree, Radiality, and Betweenness. The list of genes included in the algorithm is provided in Table S1. (C) Upset plot displaying six VA genes identified by external data (GSE33165) and hub genes. Details of the external patient cohort are provided in Table S2. (D) Bar graph to compare the mRNA expression of GJA1 in left ventricle (LV) between patients with sinus rhythm (SR, n = 5) and VA (n = 8). We averaged the epicardial and endocardial expression values from three nonischemic hearts in the VA group, and applied averaged values as the expression values for the hearts. (E) Bar graph to compare the mRNA expression of GJA1 in left atrium (LA) between patients with SR (n = 5) and VA (n =…

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  FIGURE 2. Connexin43 knockout causes arrhythmias and abnormal action potential properties in induced pluripotent stem cell–derived cardiomyocytes. (A) Schematic diagram of wild‐type (WT) and connexin43 knockout (Cx43‐KO) induced pluripotent stem cells (iPSCs) generated in this study. KO‐1 and KO‐2 denote two different Cx43‐KO iPSC clones. (B) The structure of the GJA1 gene and the location of the guide RNA (gRNA) used for genome editing by CRISPR/Cas9. Sanger sequencing revealed a 14 base‐pair deletion in KO‐1 iPSCs, and a 1 base‐pair insertion in KO‐2 iPSCs, respectively. (C,D) Western blot analysis of Cx43 protein expression in WT and Cx43‐KO iPSC‐derived cardiomyocytes (iPSC‐CMs). n = 4 independently biological repeats. Glyceraldehyde‐3‐Phosphate Dehydrogenase is used as the loading control. (E) Representative action potential waveforms recorded by single‐cell patch clamp from WT and Cx43‐KO iPSC‐CMs. Dash lines indicate 0 mV. Red dots indicate arrhythmias. (F) Bar graph to compare the percentage of cells with arrhythmia between WT and Cx43‐KO iPSC‐CMs. (G,H) Bar graphs to compare the beating rate and action potential duration at 90% of repolarization (APD90) between WT and Cx43‐KO…

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  FIGURE 3. Cx43‐KO leads to metabolic reprogramming in iPSC‐CMs. (A) Volcano plot of differentially expressed genes (DEGs) between WT and Cx43‐KO iPSC‐CMs. (B) Gene ontology (GO) enrichment analysis of upregulated DEGs between WT and Cx43‐KO iPSC‐CMs. (C) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of upregulated DEGs between WT and Cx43‐KO iPSC‐CMs. (D–G) Gene set enrichment analysis (GSEA) demonstrating positively significant enrichment of metabolic‐related pathways including “tricarboxylic acid (TCA) cycle,” “fatty acid β oxidation,” “amino acid metabolism,” and “proline metabolism” in Cx43‐KO iPSC‐CMs. (H) Heatmap demonstrating the gene expression of proline metabolism between WT and Cx43‐KO iPSC‐CMs. (I,J) Volcano plots of differentially expressing metabolites (DEMs) between WT and KO‐1 iPSC‐CMs, as well as between WT and KO‐2 iPSC‐CMs. (K) Venn diagram of DEMs between WT and Cx43‐KO iPSC‐CMs. The DEMs are displayed in a box, and those involved in proline metabolism are highlighted in red. (L,M) Metabolite set enrichment analysis (MSEA) of DEMs between WT and KO‐1 iPSC‐CMs, as well as between WT and KO‐2 iPSC‐CMs. (N) Differential expression of key genes in…

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  FIGURE 4. Cx43‐KO iPSC‐CMs exhibit disrupted mitochondrial dynamics and oxidative stress. (A) Representative graphs of mitochondrial ultrastructure in WT and Cx43‐KO iPSC‐CMs using transmission electron microscopy (TEM). Scale bar, 5 µm. (B,C) Bar graphs to compare the mitochondrial number and mitochondrial cristae number between WT and Cx43‐KO iPSC‐CMs. n = 8 random fields. (D) Representative graphs of mitochondrial morphology labeled with Mito‐Tracker in WT and Cx43‐KO iPSC‐CMs. Scale bar, 100 µm. (E) Representative graphs of mitochondrial membrane potential labeled with a fluorescent dye (tetramethylrhodamine ethyl ester, TMRE) in WT and Cx43‐KO iPSC‐CMs. Scale bar, 100 µm. (F) Bar graph to compare the TMRE fluorescence intensity between WT and Cx43‐KO iPSC‐CMs. n = 8 independently biological repeats. (G) Bar graph to compare the mitochondrial DNA (mtDNA) content between WT and Cx43‐KO iPSC‐CMs. n = 4 independently biological repeats. (H–J) Western blot analysis of the protein expression of total dynamin‐related protein 1 (DRP1) and phosphorylated DRP1 (DRP1‐Ser616) in WT and Cx43‐KO iPSC‐CMs. GAPDH is used as the loading control. n = 4 independently biological repeats. (K) The diagram…

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  FIGURE 5. ROS‐mediated abnormal calcium handling confers the arrhythmic phenotype of Cx43‐KO iPSC‐CMs. (A) Representative calcium (Ca2+) transient waveforms recorded by ratiometric Fura‐2 imaging from WT and Cx43‐KO iPSC‐CMs. (B–H) Bar graphs to compare key parameters of calcium transients between WT and KO iPSC‐CMs, including diastolic calcium, calcium amplitude, peak calcium, maximal rising rate, maximal decay rate, time to peak, and transient duration 90. n = 40 cells. (I) Mitochondrial calcium analysis through Rhod‐2 staining in WT and Cx43‐KO iPSC‐CMs with or without ISO (100 nm, 2 h) stimulation, and with or without treatment of either Mito‐TEMPO (MT, 5 µm, 72 h) or N‐acetyl‐l‐cysteine (NAC, 5 mm, 4 h). n = 4–6 independently biological repeats. (J) Representative action potential waveforms recorded by single‐cell patch clamp from Cx43‐KO iPSC‐CMs treated with phosphate‐buffered saline (PBS, vehicle), NAC (5 mm, 4 h) or proline (Pro, 100 nm, 24 h). Dash lines indicate 0 mV. Red dots indicate arrhythmias. (K) Bar graph to compare the percentage of cells with arrhythmia among different groups in Panel J. n = 31–65 cells. (L,M) Bar graphs to compare the beating rate and APD90 among…

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  FIGURE 6. Downregulation of the Cx43‐interacting protein SNAT2 leads to disorders of proline metabolism and disturbances in redox balance of Cx43‐KO iPSC‐CMs. (A) Bar graph to compare the mRNA expression of the proline transporters (SLC36A1, SLC36A2, SLC38A1, and SLC38A2) between WT and Cx43‐KO iPSC‐CMs. n = 4 independently biological repeats. (B–D) Western blot analysis of the protein expression of SNAT2 (sodium‐coupled neutral amino acid transporter) and PRODH (proline dehydrogenase) in WT and Cx43‐KO iPSC‐CMs. GAPDH is used as the loading control. n = 3 independently biological repeats. (E) Co‐immunoprecipitation (co‐IP) assay showing that SNAT2 was detected in anti‐Cx43 immunoprecipitates in WT iPSC‐CMs. (F) Molecular docking simulation using AutoDockTools and GRAMM. Cx43 and SNAT2 are represented as slate and cyan cartoon models, respectively. (G) Measurement of proline contents through a proline assay kit in Cx43‐KO iPSC‐CMs transfected with green fluorescent protein (GFP) only (negative control, NC) (KO + NC) or SNAT2 protein labeled with GFP (SNAT2‐OE) (KO + SNAT2‐OE). OE, overexpressing. n = 5 independently biological repeats. (H) Bar graph to compare the mitochondrial ROS level…

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  FIGURE 7. Exogenous proline supplementation alleviates the proline metabolic dysfunction and suppresses the stress‐induced arrhythmias in heterozygous cardiac‐specific conditional Cx43‐KO mice. (A–C) Western blot analysis of the protein expression of membrane and cytosol Cx43 and SNAT2 in ventricular tissues isolated from 2‐month‐old Myh6‐Cre−‐Gja1 flox/+ (WT) and heterozygous (HET) Myh6‐Cre+‐Gja1 flox/+ cardiac‐specific conditional Cx43‐KO (Cx43‐cKO) mice. Na+/K+‐ATPase and GAPDH are used as the membrane and cytosol loading controls, respectively. n = 4 mice. (D) Schematic representation of animal experiments. 2‐month‐old WT and HET Cx43‐cKO mice were used in the study. Normal group was received daily oral gavage of normal saline (NS); proline supplemented group was fed on normal diet and received daily oral gavage of NS supplemented with 5% proline for 14 days; proline deprived group was fed on proline‑deprived diet and received daily oral gavage of NS for 14 days. The mice were euthanized with carbon dioxide after the experiments. (E) Bar graph to compare the proline content in ventricle among 2‐month‐old WT and HET Cx43‐cKO mice with or without proline supplementation. n = 4 mice. (F)…