Fig. 3.

Mitochondrial depolarization downstream of H₂O₂ is delayed in single myocytes, but this does not involve altered H₂O₂ scavenging or electron transport chain uncoupling. A: representative images of myocyte areas (×25 μm) containing mitochondria undergoing depolarization with different kinetics (Mfn-1 WT above and Mfn-1 KO below). H₂O₂ was injected at t₀ and imaging began immediately. Images shown were taken at the indicated time points (minutes) following H₂O₂ injection. B: graph showing dissipation of tetramethylrhodamine methylester (TMRM) fluorescence of a selected region of mitochondria after H₂O₂ exposure. On the y-axis, TMRM fluorescence [arbitrary units (AU)] is a quantitative indicator of inner membrane potential (ΔΨ_m) for the selected group of mitochondria. On the x-axis, time after H₂O₂ injection is shown as consecutive slices (time interval: 2.4 s). Channel 1 (blue) shows the course of depolarization in Mfn-1 WT myocytes, and channel 2 (red) shows the depolarization of Mfn-1 KO myocytes at the same conditions. C: TMRM fluorescence at t₀ is quantified in myocytes from each group as a measure of basal membrane potential (ΔΨ_m; P = 0.189). D: capacity of mitochondria to lose TMRM is quantified as the area under the curve from traces such as those shown in B. Confocal microscopy experiment was done using 4 WT and 3 KO mice that were 2.5–5 mo old. Number of myocytes assessed are shown in circles for each group (6 myocytes were Mfn-1^flox without cre, 5 were Mfn-1⁺ with cre, and 15 were homozygous Mfn-1^flox with cre). E: relative levels of mRNAs encoding for proteins with the capacity to alter glutathione levels are quantified in whole-heart extracts. Gss, glutathione synthase; Gpx-1, glutathione peroxidase-1; Gpx-4, glutathione peroxidase-4, mitochondrial variant. F: levels of total glutathione (reduced and oxidized) in whole heart extracts are shown in μg/mg for the 2 experimental groups (2 mice were Mfn-1^flox without cre, 2 mice were Mfn-1⁺ with cre, and 4 mice were homozygous Mfn-1^flox with cre, aged 3.5–4 mo old). G: protein abundance of uncoupling protein-3 (UCP-3) and α-subunit of the F_OF₁-ATPase (F_OF₁-α) in Mfn-1 WT and KO hearts. *P < 0.05, **P < 0.01.