Development of a high-throughput method for real-time assessment of cellular metabolism in intact long skeletal muscle fibre bundles

Abstract

Key points: We developed a method that allows for real-time assessment of cellular metabolism in isolated, intact long skeletal muscle fibre bundles from adult mice. This method can be used to study changes in mitochondrial function and fuel utilisation in live skeletal muscle fibre bundles. Our method enables flexibility in experimental design and high-throughput assessment of mitochondrial parameters in isolated skeletal muscle fibre bundles. Extensor digitorum longus (EDL) fibre bundles obtained from chronic high-fat diet fed mice had lower basal oxygen consumption under FCCP-induced maximal respiration, when compared to control chow-fed mice. EDL fibre bundles obtained from chronic high-fat diet fed mice had enhanced mitochondrial oxidation capacity under FCCP-induced maximal respiration, when compared to control chow-fed mice.

Abstract: Metabolic dysfunction in skeletal muscle contributes to the aetiology and development of muscle diseases and metabolic diseases. As such, assessment of skeletal muscle cellular bioenergetics provides a powerful means to understand the role of skeletal muscle metabolism in disease and to identify possible therapeutic targets. Here, we developed a method that allows for the real-time assessment of cellular respiration in intact skeletal muscle fibre bundles obtained from the extensor digitorum longus (EDL) muscle of adult mice. Using this method, we assessed the contribution of ATP turnover and proton leak to basal mitochondrial oxygen consumption rate (OCR). Our data demonstrate that the mitochondria in EDL fibres are loosely coupled. Moreover, in the presence of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), we show that palmitate exposure induced comparable peak OCR and higher total OCR in EDL fibre bundles when compared to pyruvate exposure, suggesting that fatty acids might be a more sustainable fuel source for skeletal muscle when mitochondria are driven to maximal respiration. Application of this method to EDL fibre bundles obtained from chronic high-fat diet fed mice revealed lower basal OCR and enhanced mitochondrial oxidation capacity in the presence of FCCP when compared to the chow-diet fed control mice. By using a 96-well microplate format, our method provides a flexible and efficient platform to investigate mitochondrial parameters of intact skeletal muscle fibres obtained from adult mice.

Keywords: metabolic flux; method; muscle metabolism; seahorse; skeletal muscle fibre.

Procedural workflow

Figure 1

A, mouse extensor digitorum longus (EDL) muscle fibre isolation and plate seeding (day 1). B, fibre viability normalisation and the Seahorse XF^e96 assay run (day 2). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Validation and optimisation of the muscle fibre seeding procedure

A–D, representative examples of intact single extensor digitorum longus (EDL) muscle fibre (A–C) and small EDL fibre bundles (D). Scale bars: 50 μm. Seeding densities were optimised using single EDL fibres and EDL fibre bundles. E, basal oxygen consumption rate (OCR) readings from single fibres (n = 11), 2–3 single fibres (n = 14), 4–5 single fibres (n = 14) and 7–10 single fibres (n = 15). Wells containing 4–5 single fibres yielded an optimal basal OCR reading between 100 and 200 pmol min^–1. F, basal OCR readings from 4–5 single EDL fibres (n = 14) and small EDL fibre bundles containing 4–5 myofibres (n = 15). The use of small EDL fibre bundles resulted in optimal basal OCR with less variance than multiple single fibres. G, the fluorescence intensity of the alamarBlue cell viability assay was linearly correlated with EDL fibre numbers (total n = 94). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Optimisation of the mitochondrial stress test using extensor digitorum longus (EDL) muscle fibre bundles

A, basal oxygen consumption rate (OCR) readings of EDL fibre bundles remained stable throughout the 80 min of measurement in response to three injections of assay medium (n = 5). B, optimal inhibition of ATP‐coupled OCR in EDL fibre bundles by oligomycin A (Oligo, 0.25‐2.0 μm), followed by injections of carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP, 0.4 μm) and antimycin–rotenone (AA/ROT, 1.0 μm). C, baseline normalised OCR levels at the sixth measurement cycle (M6), after different working concentrations of oligomycin A were injected to inhibit ATP synthesis in EDL fibre bundles. Oligomycin A at 0.25 or 0.5 μm did not decrease the OCR when compared with the basal OCR, while oligomycin A at a working concentration of 1.0 μm or above induced a significant inhibition of OCR in EDL fibre bundles (oligomycin A groups: 0.25 μm, n = 9; 0.5 μm, n = 12; 1.0 μm, n = 10; 1.5 μm, n = 5; 2.0 μm, n = 6; ^* P < 0.05, compared with baseline (B)). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Biogenetic profiles of extensor digitorum longus (EDL) muscle fibre bundles under the basal condition and during FCCP‐induced maximal respiration

A and B, absolute values (A) and baseline normalised oxygen consumption rate (OCR; B) were determined in EDL fibre bundles at baseline and after FCCP‐induced maximal respiration. OCR levels were inhibited by oligomycin A to 73.94 ± 3.91% of the baseline at the sixth measurement cycle (M6) and surged to 224.05 ± 21.03% of the baseline at M7 following the injection of FCCP. C, key parameters of mitochondrial respiration in EDL fibre bundles determined by the mitochondrial stress assay. Oligomycin A inhibits ATP production‐related mitochondrial respiration, but does not prevent oxygen consumption through proton leak. Mitochondrial electron transport chain (ETC) complex I and III inhibitors antimycin and rotenone completely shut down mitochondrial oxygen consumption, leaving non‐mitochondrial‐related respiration. D, percentage proportions of ATP turnover, proton leak and non‐mitochondrial respiration contributed to total basal respiration and mitochondrial respiration in EDL fibre bundles. E and F, absolute values (E) and baseline normalised extracellular acidification rate (ECAR; F) from EDL fibre bundles. ECAR peaked at M7 following the injection of FCCP, accounting for 220.43 ± 16.36% of baseline respiration. G, matched OCRs and ECARs of EDL fibre bundles at the 3rd, 6th, 7th and 12th measurement, representing the energy phenotype at the basal state (M3) and after the exposure of oligomycin A (M6), FCCP (M7) and antimycin–rotenone (M12). The EDL fibre bundles switched to a high oxidative phosphorylation and high glycolysis phenotype under FCCP‐induced maximal respiration (n = 10). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Differential capacity of extensor digitorum longus (EDL) muscle fibre bundles to utilise pyruvate or palmitate in response to FCCP‐induced maximal respiration

A and B, absolute values of (A) oxygen consumption rate (OCR) and extracellular acidification rate (ECAR; B) at each measurement cycle. EDL fibre bundles utilising palmitate (n = 10) maintained high OCR and ECAR levels for longer than fibres utilising pyruvate (n = 10; ^* P < 0.05, compared with basal measurements). C and D, no difference of basal OCR level or ECAR level was observed between pyruvate and palmitate–BSA treated groups. E and F, EDL fibre bundles exposed to palmitate had higher total OCR and total ECAR (as defined by the area under the curve; AUC) when compared to fibres exposed to pyruvate (^* P < 0.05). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Systemic impairments in glucose tolerance enhance mitochondrial oxidation capacity in extensor digitorum longus (EDL) muscle fibre bundles while lowering the basal respiration

A–D, maintenance of mice on a high‐fat diet (HFD) promotes weight gain (A and B) due to increased fat mass (total, epigonadal (Epi) and inguinal (Ing)) (C and D). E–G, this occurs alongside a worsening of blood glucose clearance (E and F), and an elevation in fasting levels of blood glucose and a rise in plasma insulin levels in the fed state (G). Collectively, these measures are indicative of metabolic syndrome associated with insulin resistance. I, basal OCR (normalised to alamarBlue) of EDL muscle fibre bundles is significantly reduced in HFD mice when compared to mice maintained on a standard chow diet. L, by contrast, basal ECAR (normalised to alamarBlue) does not differ between HFD and chow‐fed mice. H and K, to minimise the effects of different basal respiration rate, baseline normalised OCR (H) and ECAR (K) in EDL muscle fibre bundles of HFD‐fed mice and chow‐fed mice were determined to assess pyruvate or palmitate utilisation capacity during FCCP‐induced maximal respiration. Compared with chow‐fed mice, EDL muscle fibre bundles of HFD‐fed mice exhibited a higher OCR peak when using pyruvate or palmitate as a principal energy substrate. J and M, baseline normalised total OCR (J) and ECAR (M) values in EDL fibre bundles were determined by calculating area under the curve (AUC). When utilising pyruvate as the principal energy source, EDL fibre bundles of HFD‐fed mice had higher total OCR and total ECAR values compared to chow‐fed controls (pyruvate‐treated HFD fibre group: n = 8; palmitate‐treated HFD fibre group: n = 8; pyruvate‐treated chow‐diet fibre group: n = 8; palmitate‐treated chow‐diet fibre group: n = 8; ^* P < 0.05, compared with chow‐fed controls). [Colour figure can be viewed at wileyonlinelibrary.com]