Figure 6.

Fis1^K148R induces mitochondrial fission but does not cause muscle atrophy and preserves mitochondrial function. (A) In vivo imaging of the mitochondrial network was performed in muscles expressing Fis1^K148R. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13-YFP) and Fis1^K148R. Twelve days later muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Mitochondrial network was greatly altered but mitochondrial membrane potential was conserved. (B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (^*P<0.001). (C) hFis^K148R-transfected muscles were collected 12 and 21 days after transfection. Cross-sectional area of transfected fibres, identified by anti-myc immunofluorescence, was quantified (n=550). (D) Fis1-dependent activation of MuRF-1. MuRF-1 promoter was co-transfected into adult TA muscle with or without Fis1 or Fis1^K148R. A renilla-luciferase vector was co-transfected to normalize for transfection efficiency. Eight days later, firefly and renilla-luciferase activity was determined. Each condition represents the average of at least five independent experiments±s.e.m. (^*P<0.05).