The integrity of the mitochondrial-associated ER membrane is compromised by microgravity in differentiated primary human myotubes. (A) A diagrammatic representation of differentiated human myotubes under normal conditions or exposed to clinorotation. µG; microgravity. (B) Phase contrast images of differentiated primary human myotubes under 1G (upper panel) and µG for 48 hours (left lower panel), for 7 days (right lower panel). Scale bars; 50 µm. (C) Transcription levels of MRF4 (myogenic determination), MYH3 (early differentiation), MuRF1 and FBXO32 (muscle atrophy) in differentiated myotubes with or without microgravity, measured by RT-qPCR and presented relative to transcripts of ribosomal protein RPL13a. (D) Western Blotting analyses of lysates from differentiated myotubes with or without microgravity. Nuclear lysates were analyzed with antibodies against MuRF1 and FBXO32. GAPDH was used as a loading control. (E) Phase contrast images and detectable adjacent mitochondria-associated ER membranes (MAM) by Proximal Ligation Assay in differentiated myotube. Scale bars; 20 µm. (F) The number of MAM in mononuclear myoblasts (mononuclear) or multinucleated fused myotubes (multinuclear) per fixed area. All error bars indicate ±SEM (n=4). P-values are determined by non-parametric Wilcoxon tests or one-way ANOVA and Tukey’s test for comparisons.

Mitochondrial abnormality and the activation of Notch in MFN2-deficient human iPS cells. (A) Phase contrast images (left panels) and MAM visualization (right panels) with or without MFN2 in human iPS cells. Red; MAM (IP3R-VDAC1 by PLA), blue; DAPI, Scale bars; 20 µm. (B) The quantitative analyses of MAM numbers per fixed area in human iPS cells with or without MFN2. (C) Mitochondrial morphology with MitoTracker in human iPS cells with or without MFN2. (Right panels; magnified area outlined in each left panel), Scale bars; 50 µm. (D) Total ATP production (µmol/l) in wild-type or MFN2-deficient human iPS cells. (E) Phase contrast (left images) and Immunostaining for Notch-intercellular domain (NICD; Red) and DAPI (blue) on wild-type or MFN2-deficient human iPS cells (right images). Scale bars; 50 µm. (F) Relative transcription levels of HEY1 and ID3 in wild-type or MFN2-deficient human iPS cells under normal gravity (1G) or microgravity (µG). All error bars indicate ±SEM (n=5). P-values are determined by non-parametric Wilcoxon tests for comparisons.

The improvement of mitochondrial abnormalities in MFN2-deficient human iPS cells treated with gamma-secretase inhibitor DAPT. (A) Mitochondrial morphology visualized by MitoTracker in MFN2-deficient human iPS cells with or without 50µM of DAPT. (Right panels; magnified area outlined in right panels), Scale bars; 50 µm. (B) Relative transcription levels of HES1 and HEY1 in MFN2-deficient human iPS cells with or without DAPT. (C) MAM visualization in MFN2-deficient human iPS cells, with or without DAPT. (Red; MAM (IP3R-VDAC1 PLA), blue; DAPI, Scale bars; 20 µm. (D) The quantitative analyses of MAM numbers in MFN2-deficient human iPS cells with or without DAPT. (E) Total ATP production in MFN2-deficient human iPS cells treated with or without DAPT. All error bars indicate ±SEM (n=5). P-values are determined by non-parametric Wilcoxon tests for comparisons.

The atrophic phenotype of human myotubes under microgravity is alleviated by the gamma-secretase inhibitor DAPT. (A) Phase contrast (left panels) and Immunostaining (right panels) for NOTCH2-intercellular domain (N2ICD; Red) and DAPI (blue) on differentiated myotubes derived from wild-type, or MFN2-deficient human iPS cells by Doxycycline (DOX) treatment. Scale bar; 50 µm. (B) Phase contrast images (left panels) and immunostaining for NOTCH2-intercellular domain (right panels, N2ICD; Red) and DAPI (middle panels, blue) on differentiated myotubes under normal gravity (1G) or microgravity (µG) for 7 days. Scale bar; 50 µm. (C) Relative transcription levels of HES family genes (HES1, HEY1) and ID family genes (ID1, ID3) in differentiated myotubes derived from wild-type or MFN2-deficient human iPS cells after Doxycycline (DOX) treatment. (D) Total ATP production in differentiated human primary myotube under normal gravity (1G) or microgravity (µG) for 7 days. (E) The schematic representation of differentiated human primary myotubes under microgravity (µG) for 7 days with or without DAPT. (F) Relative transcription levels of HES1 or HEY1 in differentiated human primary myotubes under microgravity for 7 days with or without DAPT. (G) Mitochondrial morphology with MitoTracker in differentiated human primary myotubes under normal gravity (1G; left panel) and microgravity with or without DAPT (µG; upper and lower left panels). The average fluorescent intensity of total cells treated with MitoTracker is indicated on the right. Scale bars; 50 µm. (H) Quantitative analyses of MAM numbers in differentiated human myotubes under microgravity with or without DAPT. Scale bar; 50 µm. (I) Total ATP production in differentiated human myotubes under microgravity with or without DAPT. (J) Relative transcription levels of MuRF1 and FBXO32 (muscle atrophy) in differentiated human primary myotubes under normal gravity (1G) and microgravity (µG) with or without DAPT for 7 days. All error bars indicate ±SEM (n=5). P-values are determined by non-parametric Wilcoxon tests or one-way ANOVA and Tukey’s test for comparisons. N.S; not significant.

The regenerative capacity of Mfn2-deficient mouse muscle is reduced and that of Mfn2-deficient muscle stem cells when transplanted into Dystrophic muscle in vivo is improved by DAPT treatment of the muscle. (A) The flowchart to isolate muscle stem cells (MuSCs, SM-C/2.6+) and non-myogenic fibroblasts (FBs) derived from conditionally Mfn2 knockout mice after 4-OH tamoxifen (4-OHT) injection. (B) Immunostaining for Mfn2 (Green), Myogenin (Myog; Red), and DAPI (blue) on differentiated myotubes derived from wildtype or Mfn2-deficient mouse muscle stem cells sorted as SM-C/2.6 positive cells, co-cultured with non-myogenic fibroblasts (FBs). Scale bar; 50 µm. (C) Phase contrast images (left panels) and MAMs visualization (right panels) and quantitative analyses of MAM numbers (right) on cultured muscle stem cells. (Red; MAM (IP3R-VDAC1 PLA), blue; DAPI, Scale bars; 20 µm. (D) Western Blotting analyses of lysates from control and Mfn2-mutant cultured muscle stem cells. Nuclear lysates were analyzed with antibodies against NICD. Histone H3 was used as a loading control. (E) The flowchart for the transplantation into tibialis anterior (TA) muscles of DMD-/y mice (12 weeks old) with Mfn2-deficient muscle stem cells (1.0 x 104 cells) and the treatment with DAPT every 3∼4 days after the transplantation. (F) Transverse sectional images of TA muscles 14 days after the transplantation with the same number of muscle stem cells sorted as SM/C-2.6-positive cells derived from wildtype or conditional Mfn2 Knockout mice. Immunostaining for Dystrophin (Dmd, red as transplanted areas), laminin-a2 (Lama2, white to show the outline of myofibers), and DAPI (blue) on engrafted TA muscle after the transplantation. Scale bars; 50 µm. (G) The quantification of the total number of Dystrophin-positive (Dmd+) regenerated myofibers on the section transplanted with an equivalent number of normal or Mfn2-deficient MuSCs, with or without the treatment of the transplanted muscle with DAPT. (H) The average diameter of Dystrophin-positive (Dmd+) myofibers that are contributed by the transplanted MuSCs, as described in (G). All error bars indicate ±SEM (n=5). P-values are determined by non-parametric Wilcoxon tests or one-way ANOVA and Tukey’s test for comparisons.