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.
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.
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.
Background
Human skeletal muscle is composed of a functional and metabolic continuum of slow (Type I) and fast fibers (IIa and IIx). Hybrid fibers co‐expressing different myosin heavy chains are also ...present and seem to be more prominent in aging muscle. Their role is debated; hybrid fibers were reported either in a transitional state, between slow and fast fibers, or as fixed individual entities. This study examined the fate of hybrid fibers with an endurance exercise intervention in an elderly sedentary population.
Methods
Twenty‐two sedentary healthy elderly men and women underwent a 16‐week supervised endurance exercise intervention. Eighteen endurance‐trained age‐ and gender‐matched volunteers served as controls. Fiber type distribution was determined by immunohistochemistry on vastus lateralis muscle biopsies pre‐intervention and post‐intervention.
Results
A total of 13840 fibers were analyzed. At baseline, a Type II dominant fiber profile was observed compared with the control group, with more Type IIa (P = 0.0301) and Type IIx fibers (P = 0.0328). Hybrid fibers represented almost 5% of total muscle fibers in both groups. There was no significant difference between groups (I–IIa, P = 0.6719 and IIa–IIx, P = 0.0998). Intervention triggered qualitative dynamics towards an increase in Type I, and decrease in Type II fibers, paralleled by an increase in I–IIa hybrids (P = 0.0301).
Conclusions
The present study is, to our knowledge, the first to examine hybrid muscle fiber type adaptations to an endurance exercise intervention in the elderly. Hybrid fiber proportions did not differ between chronic sedentary state and chronic endurance‐trained state. Exercise intervention increased Type I–IIa hybrid fibers along with shift dynamics in other fiber types suggesting the contribution of hybrid fiber to a fast‐to‐slow fiber type transition, eventually serving as intermediate reservoir from one monomorphic myosin heavy chain expressing fiber type to another. This finding favours the transitional theory regarding hybrid muscle fibers and exercise, crucial to understanding reversible mechanisms of sarcopenia and development of prevention measures.
Intensive-insulin treatment (IIT) strategy for patients with type 1 diabetes mellitus (T1DM) has been associated with sedentary behaviour and the development of insulin resistance. Exercising ...patients with T1DM often utilize a conventional insulin treatment (CIT) strategy leading to increased insulin sensitivity through improved intramyocellular lipid (IMCL) content. It is unclear how these exercise-related metabolic adaptations in response to exercise training relate to individual fibre-type transitions, and whether these alterations are evident between different insulin strategies (CIT vs. IIT).
This study examined glycogen and fat content in skeletal muscle fibres of diabetic rats following exercise-training.
Male Sprague-Dawley rats were divided into four groups: Control-Sedentary, CIT- and IIT-treated diabetic sedentary, and CIT-exercised trained (aerobic/resistance; DARE). After 12 weeks, muscle-fibre lipids and glycogen were compared through immunohistochemical analysis.
The primary findings were that both IIT and DARE led to significant increases in type I fibres when compared to CIT, while DARE led to significantly increased lipid content in type I fibres compared to IIT.
These findings indicate that alterations in lipid content with insulin treatment and DARE are primarily evident in type I fibres, suggesting that muscle lipotoxicity in type 1 diabetes is muscle fibre-type dependant.
Perilipins (PLINs) coat the surface of lipid droplets and are important for the regulation of lipid turnover. Knowledge about the physiological role of the individual PLINs in skeletal muscle is ...limited although lipid metabolism is very important for muscle contraction. To determine the effect of long‐term exercise on PLINs expression, 26 middle‐aged, sedentary men underwent 12 weeks combined endurance and strength training intervention. Muscle biopsies from m. vastus lateralis and subcutaneous adipose tissue were taken before and after the intervention and total gene expression was measured with deep mRNA sequencing. PLIN4 mRNA exhibited the highest expression of all five PLINs in both tissues, and the expression was significantly reduced after long‐term exercise in skeletal muscle. Moreover, PLIN4 mRNA expression levels in muscle correlated with the expression of genes involved in de novo phospholipid biosynthesis, with muscular content of phosphatidylethanolamine and phosphatidylcholine, and with the content of subsarcolemmal lipid droplets. The PLIN4 protein was mainly located at the periphery of skeletal muscle fibers, with higher levels in slow‐twitch as compared to fast‐twitch skeletal muscle fibers. In summary, we report reduced expression of PLIN4 after long‐term physical activity, and preferential slow‐twitch skeletal muscle fibers and plasma membrane‐associated PLIN4 location.
We observed that PLIN4 is an exercise‐responsive PLIN, which is mainly localized on SS LDs in muscle fibers with effects on de novo phospholipid biosynthesis.
Glycogen synthase kinase 3 (GSK-3) plays an important role in metabolic regulation in skeletal muscles, and both insulin and adrenaline stimulate GKS-3 phosphorylation. The aim of the present study ...was to study the effect of insulin and adrenaline on GSK-3 localisation in skeletal muscles.We characterized subcellular localization of (GSK-3) signal protein in fully differentiated muscle fibre by immunofluorescence and confocal microscopy. We stimulated muscle fibres with insulin and/or adrenaline. Images were analysed by segmentation of single central optical section of the muscle.We found GSK-3 to be localised in clusters. The number of GSK-3 clusters and their average size were increased after stimulation with insulin and/or adrenaline. Average GSK-3 particle size is linearly related to their quantity.We conclude that subcellular GSK-3 in isolated skeletal muscle fibres is localized in clusters and clustering increased after stimulation with insulin and/or adrenaline.
Skeletal muscle is repeatedly exposed to passive stretches due to the activation of antagonist muscles and to external forces. Stretch has multiple effects on muscle mass and function, but the ...initiating mechanisms and intracellular signals that modulate those processes are not well understood. Mechanical stretch applied to some cell types induces production of reactive oxygen species (ROS) and nitric oxide that modulate various cellular signalling pathways. The aim of this study was to assess whether intracellular activities of ROS and nitric oxide were modulated by passive stretches applied to single mature muscle fibres isolated from young and old mice. We developed a novel approach to apply passive stretch to single mature fibres from the flexor digitorum brevis muscle in culture and to monitor the activities of ROS and nitric oxide in situ by fluorescence microscopy. Passive stretch applied to single skeletal muscle fibres from young mice induced an increase in dihydroethidium oxidation (reflecting intracellular superoxide) with no increase in intracellular DAF-FM oxidation (reflecting nitric oxide activity) or CM-DCFH oxidation. In contrast, in fibres isolated from muscles of old mice passive stretch was found to induce an increase in intracellular nitric oxide activities with no change in DHE oxidation.
In the United States, Black Americans are largely descendants of West African slaves; they have a higher relative proportion of obesity and experience a higher prevalence of diabetes than White ...Americans. However, obesity rates alone cannot explain the higher prevalence of type 2 diabetes. Type 2 diabetes is characterized by insulin resistance and beta‐cell dysfunction. We hypothesize that the higher prevalence of type 2 diabetes in African Americans (as compared to White Americans) is facilitated by an inherited higher percentage of skeletal muscle fibre type II and a lower percentage of skeletal muscle fibre type I. Skeletal muscle fibre type II is less oxidative and more glycolytic than skeletal muscle fibre type I. Lower oxidative capacity is associated with lower fat oxidation and a higher disposal of lipids, which are stored as muscular adipose tissue in higher amounts in Black compared to White Americans. In physically active individuals, the influence of muscle fibre composition will not be as detrimental as in physically inactive individuals. This discrepancy is caused by the plasticity in the skeletal muscle fibre characteristics towards a higher activity of oxidative enzymes as a consequence of physical activity. We suggest that a higher percentage of skeletal muscle fibre type II combined with physical inactivity has an impact on insulin sensitivity and high prevalence of type 2 diabetes in Blacks of West African ancestry.
Abstract Accurate and fast measurement of muscle fibre size and evaluation of fibre type proportions in large cross-sectional areas remains challenging as existing methods require extensive manual ...measurements. In this study, we assessed the fibre morphometry of ∼1000 fibres in mouse and human control and diseased muscle cross-sections. We compared fibre size, percentage fibre proportion and percentage fibre surface area results obtained by an automated method using MetaMorph ® with those obtained manually using Image Pro . Data collection using MetaMorph ® software was faster and produced similar results to those obtained using Image Pro . The ability to quickly and accurately measure large numbers of fibres with MetaMorph ® allows the researcher to make a more precise assessment of fibre type and fibre size changes in human muscle biopsies and animal models of muscle disease.
Calcium-dependent signalling pathways are believed to play an important role in skeletal muscle atrophy, but whether intracellular Ca(2+) homeostasis is affected in that situation remains obscure. We ...show here that there is a 20% atrophy of the fast-type flexor digitorum brevis (FDB) muscle in rats hind limb unloaded (HU) for 2 weeks, with no change in fibre type distribution. In voltage-clamp experiments, the amplitude of the slow Ca(2+) current was found similar in fibres from control and HU animals. In fibres loaded with the Ca(2+) dye indo-1, the value for the rate of Ca(2+) decay after the end of 5-100-ms-long voltage-clamp depolarisations from -80 to +10 mV was found to be 30-50% lower in fibres from HU animals. This effect was consistent with a reduced contribution of both saturable and non-saturable components of myoplasmic Ca(2+) removal. However, there was no change in the relative amount of parvalbumin, and type 1 sarco-endoplasmic reticulum Ca(2+)-ATPase was increased by a factor of three in the atrophied muscles. Confocal imaging of mitochondrial membrane potential showed that atrophied FDB fibres had significantly depolarized mitochondria as compared to control fibres. Depolarization of mitochondria in control fibres with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone induced a slowing of the decay of Ca(2+) transients accompanied by an increase in resting Ca(2+) and a reduction of the peak amplitude of the transients. Overall results provide the first functional evidence for severely altered intracellular Ca(2+) removal capabilities in atrophied fast-type muscle fibres and highlight the possible contribution of reduced mitochondrial polarisation.
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