Mitochondrial respiration and H2O2emission in saponin-permeabilized murine diaphragm fibers: optimization of fiber separation and comparison to limb muscle

Abstract

Diaphragm abnormalities in aging or chronic diseases include impaired mitochondrial respiration and H2O2emission, which can be measured using saponin-permeabilized muscle fibers. Mouse diaphragm presents a challenge for isolation of fibers due to relatively high abundance of connective tissue in healthy muscle that is exacerbated in disease states. We tested a new approach to process mouse diaphragm for assessment of intact mitochondria respiration and ROS emission in saponin-permeabilized fibers. We used the red gastrocnemius (RG) as “standard” limb muscle. Markers of mitochondrial content were two– to fourfold higher in diaphragm (Dia) than in RG ( P < 0.05). Maximal O2consumption ( JO2: pmol·s−1·mg−1) in Dia was higher with glutamate, malate, and succinate (Dia 399 ± 127, RG 148 ± 60; P < 0.05) and palmitoyl-CoA + carnitine (Dia 15 ± 5, RG 7 ± 1; P < 0.05) than in RG, but not different between muscles when JO2was normalized to citrate synthase activity. Absolute JO2for Dia was two– to fourfold higher than reported in previous studies. Mitochondrial JH2O2was higher in Dia than in RG ( P < 0.05), but lower in Dia than in RG when JH2O2was normalized to citrate synthase activity. Our findings are consistent with an optimized diaphragm preparation for assessment of intact mitochondria in permeabilized fiber bundles. The data also suggest that higher mitochondrial content potentially makes the diaphragm more susceptible to “mitochondrial onset” myopathy. Overall, the new approach will facilitate testing and understanding of diaphragm mitochondrial function in mouse models that are used to advance biomedical research and human health.