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Mitochondrial Respiration and H 2 O 2 Emission in Saponin‐permeabilized Murine Diaphragm Fibers: Optimization of Fiber Separation and Comparison to Limb Muscle
Author(s) -
Hahn Dongwoo,
Kumar Ravi A,
Ryan Terence E,
Ferreira Leonardo F
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.543.7
Subject(s) - citrate synthase , chemistry , respiration , mitochondrion , medicine , skeletal muscle , endocrinology , anatomy , biochemistry , biology , enzyme
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 O 2 consumption with complex I substrates ( J O 2 in pmol/s/mg wet weight) showed 3‐fold higher V max in Dia than Gast (Gast 69 ± 18, Dia 211 ± 20; p < 0.05, n = 9 mice), whereas K m was not significantly different. Maximal J O 2 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 J H 2 O 2 (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 J H 2 O 2 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 J O 2 by citrate synthase activity, there were no differences between Dia and Gast for complex I and complex I + II substrates. J H 2 O 2 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 J H 2 O 2 normalized to citrate synthase activity. The values for maximal diaphragm mitochondria J O 2 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 J O 2 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 Information Funding source: NIH 1R01HL130318‐01 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .