Fig. 5

Ablation of DRP1 in adulthood modifies mitochondrial shape and reduces RCS assembly, Complex-I-, Complex-II-, and complex-IV-dependent respiration. a SDH staining indicating the presence of bigger mitochondria in KO muscles compared with control. b Representative electron micrographs of EDL muscles of controls and Drp1^−/−. Left panel: in WT mitochondria (pointed by black arrowheads) are placed in proximity of Z lines and usually exhibit an electron-dense matrix (inset). Center panel: in Drp1^−/− mice mitochondria are often larger in size, oriented longitudinally (large arrows), and damaged (white arrowhead and inset). Right panel: a Drp1^−/− fiber presenting spindle-shaped regions (asterisks) which are surrounded by a membrane (inset, white arrow) and that contains vacuoles. c Absence of DRP1 induces mitochondrial depolarization. Isolated adult fibers were loaded with TMRM. Oligomycin and the protonophore FCCP were added at the indicated time points. TMRM staining was monitored in 18 fibers for WT, 52 fibers for KO. Lower panel: representative images of adult myofibers showing altered mitochondrial distribution in DRP1-null muscles. Myofibers were loaded with the potentiometric die TMRM. d Respiratory control ratio (RCR) of muscle isolated mitochondria energized with 5 mM/2.5 mM GLU/MAL or 2 mM rotenone/10 mM succinate or 3 mM ascorbate/10 mM TMPD. The data represent average ± SEM (GLUT/MAL, n = 5 each condition; succinate/rotenone: WT, n = 2; KO, n = 7; ascorbate/TMPD, n = 7 each condition). e The assembly of CIII and CIV in RCS is significantly reduced in mitochondria of DRP1-deficient muscles. Representative Blue Native PAGE analysis showing RCS developed and normalized for individual respiratory chain complexes. CI subunit NDUFB8 (left panel), CIII-Core2 protein 2 (middle panel), and CIV subunit COXI (right panel). f Mitochondrial DNA copy number quantification in controls and KO muscles. mtDNA was amplified by RT-PCR from the total DNA of gastrocnemius muscles of the indicated genotype. The data are normalized to controls and represent the average ± SEM (WT, n = 4; KO, n = 5). g Mitochondrial mass revealed by TOM20 and porin was not affected by DRP1 deletion (n = 4). Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; ***p ≤ 0.001