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



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.
School of Biomedical Sciences, University of Queensland, Brisbane, Australia.
Rui
R
Li
School of Biomedical Sciences, University of Queensland, Brisbane, Australia.|University of Queensland Centre for Clinical Research, Brisbane, Australia.
Frederik J
0000-0002-4782-3608
FJ
Steyn
Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA.
Michael B
MB
Stout
School of Biomedical Sciences, University of Queensland, Brisbane, Australia.
Kevin
K
Lee
School of Biomedical Sciences, University of Queensland, Brisbane, Australia.
Tanya R
TR
Cully
Department of Physiology and Biochemistry, Physiology and Biochemistry Research Group-PHYSIS, Faculty of Medicine, University of Antioquia, Medellín, Colombia.
Juan C
JC
Calderón
School of Biomedical Sciences, University of Queensland, Brisbane, Australia.|University of Queensland Centre for Clinical Research, Brisbane, Australia.|Queensland Brain Institute, University of Queensland, Brisbane, Australia.|Department of Neurology, Royal Brisbane and Women's Hospital, Brisbane, Australia.
Shyuan T
ST
Ngo
D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D019069:Cell Respiration / Q000187:drug effects; D059305:Diet, High-Fat; D004734:Energy Metabolism / Q000187:drug effects; D008297:Male; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D019308:Palmitic Acid / Q000494:pharmacology; D019289:Pyruvic Acid / Q000494:pharmacology
D019308:Palmitic Acid; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D019289:Pyruvic Acid
metabolic flux; method; muscle metabolism; seahorse; skeletal muscle fibre