Fig. 3: AnkB enables fatty acid oxidation to maintain SKM energetics during moderate intensity endurance exercise.

a Mitochondrial content of GC (n = 5 mice/genotype) and SOL (n = 6 mice/genotype), evaluated as mitochondria DNA to nuclear DNA ratio. b Representative images of FDB fibers isolated from 4-mo mice showing ROS levels at baseline and after antimycin A treatment. Scale bar, 20 µm. c quantification of ROS accumulation (MitoSOX signal) normalized to total mitochondria (MitoTracker Green FM) signal at baseline (n = 58 WT, n = 104 AnkB-KO fibers) and after treatment (n = 21 WT, n = 27 AnkB-KO fibers). d Mitochondrial membrane potential of FDB fibers (n = 37 WT, n = 38 AnkB-KO) measured using TMRE. e (left) Simplified schematic of the metabolic pathways that enable utilization of sugars or fatty acids as fuels for mitochondria OXPHOS. Created with elements of BioRender. Voos, K. (2025) https://BioRender.com/y1njcg9. (right) Curve showing the relationship between substrate utilization by SKM mitochondria and maximal oxygen consumption (VO₂max). During low to moderate exercise intensity (VO₂max < 65%) or prolonged exercise, SKM fibers mostly rely on β-oxidation of fatty acids to meet energy requirements. Glucose is the main energy substrate during high-intensity training. Representative high-resolution respirometry traces (e, g) of isolated mitochondria using palmitoylcarnitine (PC) (f) or pyruvate (PYR) (g) as energy substrates. Quantification of O₂ flux after substrate addition (state 2 respiration) (h), ADP-stimulation (state 3 respiration) (i), FCCP addition (maximal-uncoupled respiration) (j), oligomycin inhibition (leak respiration) (k), and antimycin A inhibition (non-mitochondrial respiration) (l) assessed in mitochondria isolated from n = 7 mice/genotype. Data shows mean ± S.D. Statistical differences were determined by one-way ANOVA with Bartlett’s multiple comparison test or by two-tailed multiple unpaired t-test with a significance threshold of p < 0.05.