Figure 8.

Mitochondrial dysfunction by hFis1 is mediated by permeability transition. (A and B) hFis1 induces mitochondrial dysfunction in DKO-SERCA MEFs. DKO-SERCA MEFs cotransfected with GFP, and the indicated plasmid (asterisks in A) were loaded with TMRM, and fluorescence imaging was performed as described in Figure 3. Data represent mean ± SE of three independent experiments. (C and D) Mitochondrial dysfunction induced by hFis1 is inhibited by CsA and NAC. wt MEFs cotransfected with GFP and Myc-hFis1 (asterisks in C) were loaded with TMRM in the presence of 2.5 mM NAC or 1.5 μM CsA when indicated. TMRM fluorescence over mitochondrial regions was imaged, stored, and analyzed as described in Figure 3. Where indicated (arrows in B and D), oligomycin (2.5 μg/ml) and FCCP (4 μM) were added. Data represent mean ± SE of five different experiments. (E) Cell sorting of YFP-expressing wt MEF. Twenty-four hours after transfection with YFP-hFis1 (YFPFis), mtYFP or mtYFP plus hFis1^K148R (Fis^K148R), wt MEFs (10⁸) were washed, harvested, and sorted as described in the Materials and Methods. Dot plot histograms of YFP fluorescence in control (top panel) and transfected (bottom panel) cells are shown. R2 indicates sorted population. Sorted cells were lysed in RIPA buffer, and equal amounts of protein (40 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (F) Representative traces of mitochondrial calcium retention capacity (CRC). Sorted (5 × 10⁶) cells were permeabilized with digitonin (0.001% wt/vol) in experimental buffer, and Ca²⁺ uptake was measured after the fluorescence changes of the Ca²⁺ indicator Ca-Green. Where indicated (arrows), 5 μM Ca²⁺ were added. Final volume, 1 ml, pH 7.4, 37°C. (G) Quantitative analysis of CRC of mitochondria from digitoninpermeabilized cells. CRC of mitochondria of sorted wt MEFs transfected with the indicated plasmids was measured exactly as in F. Data represent mean ± SE of five different experiments.