Fig 2: Depolarization of the mitochondrial membrane by midivi-1 associated with L-OPA1 proteolytic degradation. (A, B) Representative Western blot showing changes in expression of OPA1, MFN2, and Drp1 in CCCP (10 µmol/L for 1 or 3 h) or vehicle-treated cardiomyocytes. GAPDH was used as a loading control (n = 4). (B) Representative Western blot showing changes in expression of OPA1, MFN2, and Drp1 in Antimycin A (AA) (10 µmol/L for 1 or 3 h) or vehicle-treated cardiomyocytes. GAPDH used as a loading control (n = 4). (C) Representative Western blot and densitometric quantification showing changes in expression levels of OPA1, LC3BII, and p62 in cardiomyocytes treated with mdivi-1 (100 µmol/L for 24 h) followed by CCCP (10 µmol/L for 3 h) or AA (10 µmol/L for 3 h). GAPDH was used as a loading control. (D) Representative Western blot and densitometric quantification showing temporal changes in expression levels of OMA1 and YME1L1 (data from Fig. 8 used as a control) as well as cardiomyocytes treated with mdivi-1 (100 µmol/L, 24 h) along with CCCP (10 µmol/L, 3 h) or AA (10 µmol/L, 3 h). Ponceau S protein staining of the membrane after transfer was used as a loading control to confirm approximately equal loading across the gel (n = 4). Bars represent mean ± SEM. P values were determined by one-way ANOVA followed by Tukey's post hoc test. NS = non-significant.

Fig 3: HG induces cardiomyocyte hypertrophy with abnormalities in mitochondrial dynamics and calcium handling.A WGA staining (red) of the surface membrane of cardiomyocytes, and DAPI staining (blue) of nuclei. Scale bars = 50 µm. To quantify cardiomyocyte size, HG induced an increase in cardiomyocyte size (n = 45 cells). B Western blotting images and summarized data showing ß-MHC (n = 5) and ANP (n = 11) levels in NRCMs. C Confocal images of mitochondrial morphology in NRCMs. RGB color, scale bars = 50 µm; 8-bit color, scale bars = 3 µm. There was a main effect of HG on the number of individuals and networks, and the size of the mitochondrial footprint. Overall, HG decreased all mitochondrial network parameters (n = 140 cells). D JC-1 staining of NRCMs. Red fluorescence is from JC-1 aggregates in healthy mitochondria with polarized inner mitochondrial membranes, while green fluorescence is emitted by cytosolic JC-1 monomers and indicates MMP dissipation. Merged images indicate the co-localization of JC-1 aggregates and monomers. Scale bar = 50 µm. MMP of cardiomyocytes for each group was calculated as the fluorescence ratio of red to green. HG decreased the MMP (n = 390 cells). E Western blotting images and summarized data showing Drp1 protein level and phosphorylation at S616 (n = 6) or S637 (n = 8) in NRCMs. F Western blotting images and summarized data showing Opa1 (n = 4) and Mfn2 (n = 3) protein levels in NRCMs. G Western blotting images and summarized data showing Orai1 protein levels in NRCMs (n = 4). H Representative traces of Ca2+ influx in NRCMs are shown. Fluorescence intensity measurements of Fluo4-AM revealed the intracellular Ca2+ concentration in NRCMs following CaCl2 stimulation (n = 76 cells). F1 fluorescence intensity, F0 baseline fluorescence. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s. no significant statistical difference.

Fig 4: Altered expression of mitochondrial dynamics and OXPHOS regulatory protein in the hearts of chronic Dox-cardiomyopathy mice. (A) Representative Western blot of the whole cell fraction showing expression of mitochondrial dynamic regulatory proteins in the acute Dox-treated hearts: Drp1, OPA1, and MFN2. Ponceau S protein staining of the transfer membrane confirmed approximately equal loading across the gel. (B) Representative Western blot showing expression of Complex I, Complex II, Complex III, Complex V and PDH complex protein derived from the whole cell fraction isolated from acute Dox-treated hearts at different time points. Ponceau S protein stain of the transfer membrane was used to confirm approximately equal loading. (C) Densitometric quantification of the temporal changes in Drp1, OPA1, and MFN2 protein expression in Dox-treated hearts. (D) Densitometric quantification of OXPHOS complex and PDH complex protein. Bars represent mean ± SEM. n = 4 mice per group at each time point. P values were determined by Tukey’s post-hoc test. NS = not significant.

Fig 5: GDAP1 KD reduces F-actin fibers at mitochondrial surfaces and restricts access of DRP1 to the mitochondria resulting in less DRP1 and MFF.a Live cell imaging of GFP-F-tractin transfected cells. Mitochondria were stained with MitoTracker and images analyzed for contact area using Imaris. b Immunoblot of DRP1 in cytosolic and mitochondrial fractions demonstrating a reduced DRP1 abundance in mitochondrial fractions. TOM20 and actin served as loading controls, size is indicated. c, d Live cell imaging of GFP-DRP1 transfected cells. Mitochondria were stained with MitoTracker and images analyzed for (c) colocalization and (d) area. e Immunoblot of total DRP1 and MFF demonstrating a reduced abundance of both proteins. Actin served as loading control, size is indicated. f Scheme depicting our findings. Cof1, Cofilin-1. In the absence of GDAP1 mitochondrial F-actin is reduced resulting in an increased distance between ER and mitochondria, reduced mitochondrial Ca2+ levels, and a reduced presence of DRP1 at mitochondrial constriction sites. Data from (a) are from n = 19 cells per cell line from three independent experiments performed in triplicates. Data from (c) are from n = 10 and (d) from n = 16 cells per cell line from three independent experiments. Data points corresponding to the examples are highlighted. Statistical variation is shown as scatter plots with the indication of mean ± SEM and significance calculated using the non-parametric Mann–Whitney test, *p < 0.05.