ADOA-associated Opa1 mutations and Mfn1 silencing inhibit mitochondrial fusion in FDB fibers. (A) Opa1 mutants G300E (GTPase domain) or Δ58 (deletion of the GTPase effector domain) were expressed in FDB muscles together with mtDsRed and mtPA-GFP. Top: examples of the general structure of mitochondria (mtDsRed) before 2P illumination within green boxes, and discrete mitochondrial networks 100 s after photoactivation (mtPA-GFP, white arrowheads), in control and FDB fibers expressing mutant Opa1. Bottom: bar charts show the frequency of fusion events for each condition (2 rats/condition; ***, P < 0.001). (B) Nontargeting (NT) siRNA and Mfn1 or Mfn2 were coexpressed with mtDsRed and mtPA-GFP in FDB muscles. Top: representative images of the mtPA-GFP diffusion (white arrowheads) and fusion events (orange arrowheads). Bar chart displays the fusion event rates (Mfn1siRNA = 3 rats; *, P < 0.05; Mfn2 siRNA = 2 rats). Bottom right: Mfn1 and Mfn2 Western blots of mitochondria isolated from FDB after siRNA treatment.