Mitochondrial Respiration and H2O2 Emission in Saponin‐permeabilized Murine Diaphragm Fibers: Optimization of Fiber Separation and Comparison to Limb Muscle

Abstract

Chronic diseases such as cancer, COPD, and heart failure impair diaphragm metabolism and mitochondrial function. Mice are used extensively to replicate disease conditions and offer the advantage of studying genetically modified animals. Mouse diaphragm contains a high abundance of connective tissue, with relatively thin (15–25 μm diameter) and fragile fibers. In preliminary studies, we discovered that standard fiber separation approaches utilized for limb muscles are unsuitable for the diaphragm. Thus, the purpose of this study was to optimize a protocol for diaphragm (Dia) fiber bundle preparation to evaluate the mitochondrial respiration and reactive oxygen species emission. We used the red gastrocnemius (Gast) muscle as a ‘standard’ for comparison.Michaelis‐Menten kinetics of ADP‐stimulated O2 consumption with complex I substrates (JO2 in pmol/s/mg wet weight) showed 3‐fold higher Vmax in Dia than Gast (Gast 69 ± 18, Dia 211 ± 20; p < 0.05, n = 9 mice), whereas Km was not significantly different. Maximal JO2 in Dia was 3‐fold higher with complex I + II substrates (Gast 148 ± 25, Dia 387 ± 45; p < 0.05), and 2‐fold higher with palmitoyl‐CoA + carnitine (Gast 7 ± 0, Dia 15 ± 2, p < 0.05) compared to Gast. Baseline JH2O2 (pmol/min/mg wet weight) was higher in Dia (Gast 0.8 ± 0.2, Dia 1.7 ± 0.3; p < 0.05), but succinate‐induced JH2O2 was not different between muscles. Citrate synthase activity was 3‐fold higher in Dia (μmol/min/mg protein: Gast 43 ± 3, Dia 118 ± 4; p < 0.05). The protein abundance of electron transport chain complexes I‐V were 2–4 fold higher in the Dia than Gast (p < 0.05). When we normalized maximal JO2 by citrate synthase activity, there were no differences between Dia and Gast for complex I and complex I + II substrates. JH2O2 normalized to citrate synthase activity was higher for Gast at baseline (pmol/min/mg wet weight/U CS: Gast 2.1 ± 0.3, Dia 0.3 ± 0.1; p < 0.05), but there was no difference in succinate‐induced JH2O2 normalized to citrate synthase activity.The values for maximal diaphragm mitochondria JO2 in our study are 2–3 fold higher than reported in the literature using the standard ‘limb muscle’ approach for the diaphragm fiber preparation. The similar maximal JO2 normalized to citrate synthase activity between diaphragm and red gastrocnemius muscles suggest that our new approach is valid for the assessment of intact diaphragm mitochondrial function in permeabilized fiber bundles.Support or Funding InformationFunding source: NIH 1R01HL130318‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.