TECRL deficiency results in aberrant mitochondrial function in cardiomyocytes

Figures

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  Fig. 1. Generation of Tecrl KO mice using CRISPR/Cas9. a The general structure of Tecrl gene. Two sgRNA sequences and a schematic diagram of two sgRNA target sites located in exon 3 and exon 4 of the mice Tecrl locus. Target sites of two sgRNA sequences (Tecrl-T1 and Tecrl-T2) are highlighted in red. b Representative of sgRNA images. c Representative of the PCR images. Only one band at position 537 represented the Tecrl KO mice, one band at position 455 represented the WT mice, while one at position 537 and one at position 455 represented the heterozygote mice. d Quantification of the Tecrl mRNA level in the WT and Tecrl KO mice. e Representative of the immunoblotting images and quantification of the expression of Tecrl in the WT and Tecrl KO mice. The DNA ladder (DL2000). Values are means ± SE. P < 0.05 was considered significant.

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  Fig. 2. Tecrl deficiency-induced cardiac dysfunction. a Representative echocardiography images of WT and Tecrl KO mice. b Quantification of LVEF, LVFS, LVIDd, LVIDs, LVEDV, and LVESV in WT and Tecrl KO mice (8 weeks) (n = 8). c–f Representative ECG recordings and percent of VT episode (%) in WT and Tecrl KO mice before and after epi/caffeine stimulation (n = 10). g Quantification of VT (n = 10). h Quantification of mice heart rate (n = 8). Values are mean ± SE. P < 0.05 was considered significant.

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  Fig. 3. Visualization of the differentially regulated mRNAs in cardiac tissues of WT and Tecrl KO mice. a Representative volcano plots of differentially regulated mRNAs in the WT and Tecrl KO mice cardiac tissues. Those with P < 0.05 and |fold change | >1.5 are highlighted. b GO analyses of differentially expressed mRNAs. c KEGG analyses of differentially expressed mRNAs (n = 6).

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  Fig. 4. Tecrl deficiency significantly suppressed mitochondrial respiration. a, b The cardiac ultrastructure of the mice was examined by TEM. Representative image and quantitative analysis of cardiac ultrastructure of the WT and Tecrl KO mice at the age of 8 weeks. Magnification is 6700 x, scale bar = 1 μm (n = 4). c, d Measurements of OCR and respective quantitative analysis of cardiomyocytes isolated from WT and Tecrl KO mice (WT, n = 34, Tecrl KO, n = 32). e, f Representative image and quantitative analysis of DHE staining. The WT and Tecrl KO mice tissues were both measured at the age of 8 weeks (n = 6). g, h Representative image and quantitative analysis of DHE staining in primary neonatal cardiomyocytes (n = 7). i, j Representative image and quantitative analysis of Mitosox staining in primary neonatal cardiomyocytes (n = 4). FCCP, trifluoromethoxy carbonyl cyanide phenylhydrazone. Values are mean ± SE. P < 0.05 was considered significant.

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  Fig. 5. Tecrl regulates mitochondrial function through PI3K/Akt signaling and mitochondrial fusion protein MFN2. a–d Representative immunoblotting images and quantification of the expression of p-Akt, Mfn1, Mfn2, Nrf2, Fas, and Tecrl in WT and Tecrl KO mouse hearts (n = 6). Values are mean ± SE. P < 0.05 was considered significant.

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  Fig. 6. Tecrl regulates PI3K/Akt signaling and mitochondrial fusion protein in hiPSC-CMs. a Representative immunofluorescence images of OCT4 in the hiPSCs (n = 3). b Representative immunofluorescence images of α-actinin and cTnT in the hiPSC-CMs (n = 6). c Representative images and quantification of the velocity of contraction and relaxation of hiPSC-CMs. d–g Representative immunoblotting images and quantification of the expression of p-AKT, NRF2, MFN2, FAS, and TECRL in hiPSC-CMs following TECRL knockdown. Values are mean ± SE. P < 0.05 was considered significant.

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  Fig. 7. Overexpression of TECRL can induce mitochondrial respiration in hiPSC-CMs. a, b Representative immunoblotting images and quantification of the expression of FAS, MFN2, and TECRL of hiPSC-CMs following TECRL overexpression (n = 6). c Representative images of co-immunoprecipitation of TECRL and ATPB (n = 3). d, e Measurements of OCR and respective quantitative analysis in hiPSC-CMs (n = 12). f Representative images of Cyc and AIF colocalization with Tom 20 in hiPSC-CMs (n = 6). Scale bar = 10 μm. g Cyc and AIF released from the mitochondria into the cytoplasm were analyzed by western blotting. VDAC1 was used as a loading control for mitochondria gradients. GAPDH was used for cytosolic gradient (n = 4). Values are mean ± SE. P < 0.05 was considered significant.