Figure 6.

Selective Disruption of Mitochondrial Thiol Redox State Enhances Mitochondrial ROS Production, Mitochondrial Fragmentation, and Retrograde Signaling

(A) MitoCDNB enhances mitochondrial H₂O₂ production. Heart mitochondria (70 μg protein) were incubated with MitoCDNB (10 μM) or vehicle for 10 min then MitoPQ (5 μM) or vehicle was added and H₂O₂ production measured for 5 min. Data are means ± SEM, N = 4. ***p < 0.001 relative to control; ^###p < 0.001 relative to control + MitoPQ.

(B) MitoCDNB effect on mitochondrial ROS production in cells by confocal microscopy. Representative maximum projections of ROS production measured by MitoSOX fluorescence in C2C12 myoblasts at 0 or 30 min after 5 μM MitoPQ addition. Myoblasts were incubated with MitoSOX (5 μM) and either 0.1% ethanol (control) or MitoCDNB (10 μM) for 10 min prior to MitoPQ addition. Red is oxidized MitoSOX and blue is DAPI nuclear staining. Scale bar, 20 μm. The graph is fold change relative to control. Data are means ± SEM, N = 3. *p < 0.05, **p < 0.01.

(C) MitoCDNB effect on mitochondrial ROS production in cells assessed by flow cytometry. C2C12 cells were incubated with MitoSOX Red and treated with 0.1% ethanol (control), or 10 μM of MitoCDNB, TPMP or CDNB. Data are means ±SEM, N = 5. ***p < 0.001.

(D) Representative maximum projection images of C2C12 myoblasts (upper panels). Cells were incubated with 1, 5, and 10 μM MitoCDNB, TPMP, or FCCP for 4 hr, and analyzed by confocal microscopy to visualize mitochondrial fragmentation. The lower panels expand the indicated sections of the upper panels. Images are representative of 3 independent experiments. Scale bars, 20 μm.

(E) Quantification of mitochondrial morphology in C2C12 myoblasts assessed as in (D) after incubation with 1, 5, or 10 μM of MitoCDNB, TPMP, or FCCP. Mitochondrial morphology was assigned as tubular, intermediate or fragmented and presented as mean % of all cells ± SEM. Data are mean ± SEM from 3 independent experiments, 100 cells were counted for each condition. ***p < 0.001.

(F and G) Effect of MitoCDNB on the mouse transcriptome. Mice (six in each condition) were administered MitoCDNB (5 mg/kg) or carrier and then the liver transcriptomes were analyzed by RNA sequencing 1 and 4 hr later. (F) Volcano plot of the significance of the transcriptional changes caused by MitoCDNB 1 hr after injection compared with control. (G) Heatmap of changes in expression for the 20 most upregulated and most downregulated genes 1 hr after MitoCDNB injection.

See also Figure S6.