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. 2021 Oct 5;10:e69207. doi: 10.7554/eLife.69207

Figure 1. GJA1-20k decreases in mitochondrial size.

(A–C) Representative live cell images of mitochondria in HEK293 (A, GST- or GJA1-20k-transfected; B, Control and siGja1) and mouse neonatal cardiomyocytes (C, WT, GJA1-20k-transducted, and Gja1M213L/M213L). The right-most panels are magnified images. (D) and (E) Representative EM images from young mouse hearts (D, WT or Gja1M213L/M213L) and adult mouse hearts (E, GST- or GJA1-20k-injected). (F) Summary of the fold change in the average area of mitochondria. (n = 51 (GST), 67 (GJA1-20k), 52 (Control), or 57 (siGja1) HEK293) cells from five independent experiments; n = 46 (WT), 47 (GJA1-20k), or 48 (Gja1M213L/M213L) cells from four hearts; n = 84 (WT) or 91 (Gja1M213L/M213L) images from six hearts; n = 25 (GST), or 28 (GJA1-20k) images from three hearts. Graphs were expressed as mean ± SD (HEK293) or± SEM (mouse). p values were determined by two-tailed Mann-Whitney U-test or Kruskal-Wallis test with Dunn’s post-hoc test. **p < 0.01, ***p < 0.001. Scale bars, 10 μm and 5 μm in magnified (A–C); 2 μm (D and E). Exact p values and statistical data are provided in the source data.

Figure 1—source data 1. All data points of the mitochondrial size and the statistical data for Figure 1F.

Figure 1.

Figure 1—figure supplement 1. The confirmation of Gja1 knock-down and the mitochondrial morphology rescued by GJA1-20k.

Figure 1—figure supplement 1.

(A) Western blot analysis for Gja1 knock-down by siRNA. Tubulin was used as internal loading control. n = 3 independent experimental repeats. (B) The representative confocal live cell imaging of Gja1 knocked-down HEK293 cells with Cx43-M6L or GJA1-20k transfection. (C) The fold change in the average area of individual mitochondria. n = 52 (Control), 57 (siGja1), 60 (siGja1+ M6 L), or 64 (siGja1+ GJA1-20 k) cells from five independent experiments. The images and the values of Control and siGja1 are also shown in Figure 1. (D) The representative live cell imaging of mitochondria in WT mouse neonatal CMs with adenovirus-mediated GFP induction. The right panel indicates magnified image surrounded by square. (E) The fold change in the average area of individual mitochondria between WT (no virus introduction, the image and the value shown in Figure 1) and GFP-V5 introduction. n = 46 (WT) or 35 (GFP) cells from four hearts. Graphs were expressed as mean ± SD (C) or SEM (E). p values were determined by two-tailed Mann-Whitney U-test or Kruskal-Wallis test with Dunn’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. Scale bars, 10 μm or 5 μm in magnified image. Exact p values and statistical data are provided in the source data.
Figure 1—figure supplement 1—source data 1. All data points of the mitochondrial size and the statistical data for Figure 1—figure supplement 1.
Figure 1—figure supplement 2. The effects of GJA1-20k on membrane potential, ATP production, mitochondrial biogenesis, and mitophagy.

Figure 1—figure supplement 2.

(A) TMRE intensity (mitochondrial membrane potential) in HEK293 cells with GST or GJA1-20k transfection. n = 39 (GST) or 41 (GJA1-20k) cells from three independent experiments. (B) ATP production from HEK293 cells with GST or GJA1-20k transfection. n = 10 replicates. (C) The fold change in mitochondrial DNA copy number in HEK293 or mouse heart with or without the modification of GJA1-20k expression. n = 5 independent experimental repeats (HEK293); n = 5 (WT), or 6 (Gja1M213L/M213L) hearts; or 10 (AAV9-induced-GST or GJA1-20k) hearts. (D) and (F) Western blot analysis for mitochondrial biogenesis and mitophagy marker proteins in HEK293 cells (D) or mouse hearts (F). Tubulin was used as internal loading control. (E) and (G) The fold change of the level of the proteins and ratio of LC3-ii/-i in HEK293 cells (E) or mouse hearts (G). n = 5 independent experimental repeats (HEK293 cells); 4 (LC3) or five hearts from WT or Gja1M213L/M213L mouse. Graphs were expressed as mean ± SD (HEK293) or SEM (mouse). p values were determined by two-tailed Mann-Whitney U-test. *p < 0.05, **p < 0.01; n.s., not significant. Exact p values and statistical data are provided in the source data.
Figure 1—figure supplement 2—source data 1. All data points of the intensity of TMRE, ATP amount, mitochondria DNA copy number, and the protein expression levels and the statistical data for Figure 1—figure supplement 2.