Endurance exercise-induced histone methylation modification involved in skeletal muscle fiber type transition and mitochondrial biogenesis.

Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise.

Light-Sheet Microscopy Enables Three-Dimensional Fluorescence Imaging and Live Imaging of Satellite Cells on Skeletal Muscle Fibers.

The ex vivo myofiber culture system has proven to be a useful methodology to explore the biology and behavior of satellite cells within their niche environment. However, a limitation of this system is that myofibers and their associated satellite cells are commonly examined using conventional fluorescence microscopy, which renders a three-dimensional system into two-dimensional imaging, leading to the loss of precious information or misleading interpretation of observations. Here, we report on the use of light-sheet fluorescence microscopy to generate three-dimensional and live imaging of satellite cells on myofibers. Light-sheet microscopy offers high imaging speed and good spatial resolution with minimal photo-bleaching, allowing live imaging and three-dimensional acquisition of skeletal muscle fiber specimen. The potentials of this technology are wide, ranging from the visualization of satellite cell behavior such as cell division and cell migration to imaging the sub-cellular localization of proteins or organelles.

Mitochondrial Adaptation in Skeletal Muscle: Impact of Obesity, Caloric Restriction, and Dietary Compounds.

PURPOSE OF REVIEW: The global obesity epidemic has become a major public health concern, necessitating comprehensive research into its adverse effects on various tissues within the human body. Among these tissues, skeletal muscle has gained attention due to its susceptibility to obesity-related alterations. Mitochondria are primary source of energy production in the skeletal muscle. Healthy skeletal muscle maintains constant mitochondrial content through continuous cycle of synthesis and degradation. However, obesity has been shown to disrupt this intricate balance. This review summarizes recent findings on the impact of obesity on skeletal muscle mitochondria structure and function. In addition, we summarize the molecular mechanism of mitochondrial quality control systems and how obesity impacts these systems. RECENT FINDINGS: Recent findings show various interventions aimed at mitigating mitochondrial dysfunction in obese model, encompassing strategies including caloric restriction and various dietary compounds. Obesity has deleterious effect on skeletal muscle mitochondria by disrupting mitochondrial biogenesis and dynamics. Caloric restriction, omega-3 fatty acids, resveratrol, and other dietary compounds enhance mitochondrial function and present promising therapeutic opportunities.

Regulation of myo-miR-24-3p on the Myogenesis and Fiber Type Transformation of Skeletal Muscle.

Skeletal muscle plays critical roles in providing a protein source and contributing to meat production. It is well known that microRNAs (miRNAs) exert important effects on various biological processes in muscle, including cell fate determination, muscle fiber morphology, and structure development. However, the role of miRNA in skeletal muscle development remains incompletely understood. In this study, we observed a critical miRNA, miR-24-3p, which exhibited higher expression levels in Tongcheng (obese-type) pigs compared to Landrace (lean-type) pigs. Furthermore, we found that miR-24-3p was highly expressed in the dorsal muscle of pigs and the quadriceps muscle of mice. Functionally, miR-24-3p was found to inhibit proliferation and promote differentiation in muscle cells. Additionally, miR-24-3p was shown to facilitate the conversion of slow muscle fibers to fast muscle fibers and influence the expression of GLUT4, a glucose transporter. Moreover, in a mouse model of skeletal muscle injury, we demonstrated that overexpression of miR-24-3p promoted rapid myogenesis and contributed to skeletal muscle regeneration. Furthermore, miR-24-3p was found to regulate the expression of target genes, including Nek4, Pim1, Nlk, Pskh1, and Mapk14. Collectively, our findings provide evidence that miR-24-3p plays a regulatory role in myogenesis and fiber type conversion. These findings contribute to our understanding of human muscle health and have implications for improving meat production traits in livestock.

Dietary short-term supplementation of grape seed proanthocyanidin extract improves pork quality and promotes skeletal muscle fiber type conversion in finishing pigs.

Plant extracts are commonly used as feed additives to improve pork quality. However, due to their high cost, shortening the duration of supplement use can help reduce production costs. In this study, we aimed to investigate the effects of grape seed proanthocyanidin extract (GSPE) on meat quality and muscle fiber characteristics of finishing pigs during the late stage of fattening, which was 30 days in our experimental design. The results indicated that short-term dietary supplementation of GSPE significantly reduced backfat thickness, but increased loin eye area and improved meat color and tenderness. Moreover, GSPE increased slow myosin heavy chain (MyHC) expression and malate dehydrogenase (MDH) activity, while decreasing fast MyHC expression and lactate dehydrogenase (LDH) activity in the Longissimus thoracis (LT) muscle. Additionally, GSPE increased the expression of Sirt1 and PGC-1α proteins in the LT muscle of finishing pigs and upregulated AMP-activated protein kinase α 1 (AMPKα1), AMPKα2, nuclear respiratory factor 1 (NRF1), and calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) mRNA expression levels. These findings suggest that even during the late stage of fattening, GSPE treatment can regulate skeletal muscle fiber type transformation through the AMPK signaling pathway, thereby affecting the muscle quality of finishing pigs. Therefore, by incorporating GSPE into the diet of pigs during the late stage of fattening, producers can enhance pork quality while reducing production costs.

A mechano- and heat-gated two-pore domain K+ channel controls excitability in adult zebrafish skeletal muscle.

TRAAK channels are mechano-gated two-pore-domain K+ channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K+ ions, of 56 pS unitary conductance in the presence of 5 mM external K+, activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K+ channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K+ channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K+ currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development.

The specific mitochondrial unfolded protein response in fast- and slow-twitch muscles of high-fat diet-induced insulin-resistant rats.

Introduction: Skeletal muscle insulin resistance (IR) plays an important role in the pathogenesis of type 2 diabetes mellitus. Skeletal muscle is a heterogeneous tissue composed of different muscle fiber types that contribute distinctly to IR development. Glucose transport shows more protection in slow-twitch muscles than in fast-twitch muscles during IR development, while the mechanisms involved remain unclear. Therefore, we investigated the role of the mitochondrial unfolded protein response (UPRmt) in the distinct resistance of two types of muscle in IR. Methods: Male Wistar rats were divided into high-fat diet (HFD) feeding and control groups. We measured glucose transport, mitochondrial respiration, UPRmt and histone methylation modification of UPRmt-related proteins to examine the UPRmt in the slow fiber-enriched soleus (Sol) and fast fiber-enriched tibialis anterior (TA) under HFD conditions. Results: Our results indicate that 18 weeks of HFD can cause systemic IR, while the disturbance of Glut4-dependent glucose transport only occurred in fast-twitch muscle. The expression levels of UPRmt markers, including ATF5, HSP60 and ClpP, and the UPRmt-related mitokine MOTS-c were significantly higher in slow-twitch muscle than in fast-twitch muscle under HFD conditions. Mitochondrial respiratory function is maintained only in slow-twitch muscle. Additionally, in the Sol, histone methylation at the ATF5 promoter region was significantly higher than that in the TA after HFD feeding. Conclusion: The expression of proteins involved in glucose transport in slow-twitch muscle remains almost unaltered after HFD intervention, whereas a significant decline of these proteins was observed in fast-twitch muscle. Specific activation of the UPRmt in slow-twitch muscle, accompanied by higher mitochondrial respiratory function and MOTS-c expression, may contribute to the higher resistance to HFD in slow-twitch muscle. Notably, the different histone modifications of UPRmt regulators may underlie the specific activation of the UPRmt in different muscle types. However, future work applying genetic or pharmacological approaches should further uncover the relationship between the UPRmt and insulin resistance.

An Early and Sustained Inflammatory State Induces Muscle Changes and Establishes Obesogenic Characteristics in Wistar Rats Exposed to the MSG-Induced Obesity Model.

The model of obesity induced by monosodium glutamate cytotoxicity on the hypothalamic nuclei is widely used in the literature. However, MSG promotes persistent muscle changes and there is a significant lack of studies that seek to elucidate the mechanisms by which damage refractory to reversal is established. This study aimed to investigate the early and chronic effects of MSG induction of obesity upon systemic and muscular parameters of Wistar rats. The animals were exposed to MSG subcutaneously (4 mg·g-1 b.w.) or saline (1.25 mg·g-1 b.w.) daily from PND01 to PND05 (n = 24). Afterwards, in PND15, 12 animals were euthanized to determine the plasma and inflammatory profile and to assess muscle damage. In PND142, the remaining animals were euthanized, and samples for histological and biochemical analyses were obtained. Our results suggest that early exposure to MSG reduced growth, increased adiposity, and inducted hyperinsulinemia and a pro-inflammatory scenario. In adulthood, the following were observed: peripheral insulin resistance, increased fibrosis, oxidative distress, and a reduction in muscle mass, oxidative capacity, and neuromuscular junctions, increased fibrosis, and oxidative distress. Thus, we can conclude that the condition found in adult life and the difficulty restoring in the muscle profile is related to the metabolic damage established early on.

Normal muscle fiber type distribution is recapitulated in aged ephrin-A3-/- mice that previously lacked most slow myofibers.

Individual limb muscles have characteristic representation and spatial distribution of muscle fiber types (one slow and up to three fast isoforms) appropriate to their unique anatomical location and function. This distribution can be altered by physiological stimuli such as training (i.e., for increased endurance or force) or pathological conditions such as aging. Our group previously showed that ephrin-A3 is expressed only on slow myofibers, and that adult mice lacking ephrin-A3 have dramatically reduced numbers of slow myofibers due to postnatal innervation of previously slow myofibers by fast motor neurons. In this study, fiber type composition of hindlimb muscles of aged and denervated/reinnervated C57BL/6 and ephrin-A3-/- mice was analyzed to determine whether the loss of slow myofibers persists across the lifespan. Surprisingly, fiber-type composition of ephrin-A3-/- mouse muscles at two years of age was nearly indistinguishable from age-matched C57BL/6 mice. After challenge with nerve crush, the percentage of IIa and I/IIa hybrid myofibers increased significantly in aged ephrin-A3-/- mice. While EphA8, the receptor for ephrin-A3, is present at all neuromuscular junctions (NMJs) on fast fibers in 3-6 mo old C57BL/6 and ephrin-A3-/- mice, this exclusive localization is lost with aging, with EphA8 expression now found on a subset of NMJs on some slow muscle fibers. This return to appropriate fiber-type distribution given time and under use reinforces the role of activity in determining fiber-type representation and suggests that, rather than being a passive baseline, the developmentally and evolutionarily selected fiber type pattern may instead be actively reinforced by daily living.

Molecular regulation of stretch activation.

Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%-4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development (kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.

Role of PGC-1α in fiber type conversion in the palatopharyngeus muscle of OSA patients.

BACKGROUND: Obstructive sleep apnea (OSA) has a high incidence and is harmful to health. It is characterized by repeated collapse of the upper airway. However, the mechanism underlying upper airway collapse is unclear. METHODS: Patients with OSA and chronic tonsillitis were studied. Pathological changes in palatopharyngeus muscle were detected. The expression of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) and nuclear respiratory factor-1 (NRF-1) in muscles was detected by PCR and Western blotting. Immunofluorescence staining was used to detect the expression of type I and type II myofibril. RESULTS: The structure of the palatopharyngeus muscle was changed, and the expression of PGC-1α and NRF-1 was decreased in the OSA group compared with that in the control group. The expression of PGC-1α, NRF-1, and type I myofibril in C2C12 myoblasts was decreased by intermittent hypoxia exposure. The expression of type I myofibril was decreased when knocking down PGC-1α. CONCLUSION: OSA patients exhibited pathological damage in palatopharyngeus muscle. PGC-1α was involved in the fiber type conversion in palatopharyngeus muscle caused by intermittent hypoxia.

Aerobic exercise training-induced follistatin-like 1 secretion in the skeletal muscle is related to arterial stiffness via arterial NO production in obese rats.

Follistatin-like 1 (FSTL1), which is mainly secreted from skeletal muscle and myocardium, upregulates protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) phosphorylation in vascular endothelial cells. It is unclear whether skeletal muscle- and myocardium-derived FSTL1 secretion induced by aerobic exercise training is involved in the reduction of arterial stiffness via arterial NO production in obese rats. This study aimed to clarify whether aerobic exercise training-induced FSTL1 secretion in myocardium and skeletal muscle is associated with a reduction in arterial stiffness via arterial Akt-eNOS signaling pathway in obese rats. Sixteen Otsuka Long-Evans Tokushima Fatty (OLETF) obese rats were randomly divided into two groups: sedentary control (OLETF-CON) and eight-week aerobic exercise training (treadmill for 60min at 25m/min, 5days/week, OLETF-AT). Eight Long-Evans Tokushima Otsuka (LETO) rats were used as a healthy sedentary control group. In OLETF-CON, serum FSTL1, arterial Akt and eNOS phosphorylation, and arterial nitrite/nitrate (NOx) levels were significantly lower, and carotid-femoral pulse wave velocity (cfPWV) was significantly greater than those in LETO. These parameters were improved in the OLETF-AT compared to the OLETF-CON. In the OLETF-AT, FSTL1 levels in slow-twitch fiber-rich soleus muscle were significantly greater than those in the OLETF-CON, but not in myocardium, fast-twitch fiber-rich tibialis anterior muscle, and adipose tissue. Serum FSTL1 levels were positively correlated with soleus FSTL1, arterial eNOS phosphorylation, and NOx levels and negatively correlated with cfPWV. Thus, aerobic exercise training-induced FSTL1 secretion in slow-twitch fiber-rich muscles may be associated with a reduction in arterial stiffness via arterial NO production in obese rats.

2,3',4,4',5-Pentachlorobiphenyl attenuated fast-twitch fibers and fiber size of skeletal muscle via disturbing thyroid hormone signaling and mitochondrial dynamics.

Polychlorinated biphenyls (PCBs) affect multiple organs, and some of the effects are mediated by interfering with thyroid hormone (TH) signaling that regulates physiological processes in mammals. It remains unclear how PCBs affect skeletal muscle (SM). In our study, wistar rats were injected 2,3',4,4',5-pentachlorobiphenyl (PCB118) intraperitoneally at 0, 10, 100, and 1000 µg/kg/day for 13 weeks, and C2C12 myoblasts were treated PCB118 (0, 0.25, 25, and 50 nM) for 24 h or 48 h. We found that myocyte cross-sectional area (MCSA) was reduced, MyHC IIa and MyHC IIb mRNA levels significantly decreased, and muscle strength was weakened in PCB118-exposed rats. TH receptor α (TRα) and iodothyronine deiodinase type 2 (DIO2) were upregulated after PCB118 exposure both in vivo and in vitro. Transmission electron microscopy showed significant mitochondrial abnormalities in PCB118-treated rats, and the expression of mitochondrial regulators such as PTEN-induced kinase 1 (PINK1) and GTPase dynamin-related protein 1 (DRP1) were altered after PCB118 exposure. These results suggest that PCB118 could weaken muscle strength and attenuate fast-twitch fibers and fiber size of SM in rats. TH signaling, mitochondrial dynamics and mitophagy were also disturbed by PCB118, which may contribute to the alternations of SM structure and function.

VEGF alleviates lower limb ischemia in diabetic mice by altering muscle fiber types.

Lower limb ischemia caused by diabetic foot (DF) is one of the most serious complications of diabetes. The therapeutic role of VEGF in DF is well documented. However, the mechanism for action of VEGF is still not clear. The present study aimed to explore the effects of VEGF-mediated skeletal muscle fiber type switch in angiogenesis and the treatment of DF. C57BL/6 mice housed in cages equipped with a voluntary running wheel were used to access VEGF protein level and citrate synthase activity (by ELISA) as well as muscle fiber type changes (by immunofluorescence) in the gastrocnemius muscle. C57BL/6 mice were fed on a high-fat diet for 6 weeks and then injected with streptozocin to induce diabetic lower limb ischemia model. Control adenovirus (Ad-GFP) or Ad-VEGF-GFP were then injected into the left gastrocnemius of the ischemic diabetic mice. Blood flow perfusion was examined by laser Doppler imaging at 1, 3, 7 and 14 days after adenovirus transduction. On day 14, all mice were anesthetized and sacrificed. VEGF expression levels, citrate synthase activity and muscle fiber type changes in the gastrocnemius muscle were assayed by ELISA and immunofluorescence analysis of myosin heavy chain IIa (MHCIIa) expression, respectively. Transwell assays were performed to determine whether VEGF-treated C2C12 myotubes played a role on tubule formation and migration of HUVECs. It was found that VEGF levels and citrate synthase activity were upregulated after voluntary exercise, along with the increased frequency of oxidized muscle fibers. Notably, adenovirus-mediated VEGF overexpression in the muscle also increased the frequency of oxidized (MHCIIa-positive) muscle fibers, enhanced citrate synthase activity and ameliorated lower limb ischemia in diabetic mice. VEGF treatment enhanced the phosphorylation of PI3K, Akt and AMPK (assayed by western blotting), as well as glucose consumption and metabolism (assayed by western blotting and glucose uptake assay), in the C2C12 myotubes. Interestingly, VEGF-treated C2C12 myotubes promoted the migration and tubule formation of HUVEC cells. The present findings suggest that skeletal muscle fiber conversion might be a potential approach for VEGF-mediated angiogenesis and disease treatment, which may provide new options for the prevention and treatment of DF.

Single skeletal muscle fiber mechanical properties: a muscle quality biomarker of human aging.

Skeletal muscle strength, mass, and function should be carefully monitored for signs of decline with advanced adult age. An understanding of the pathophysiology and severity of sarcopenia can be improved with the exploration of changes in muscle fiber properties. Furthermore, although functional decline with increase age is a well-known phenomenon, the mechanisms underlying this decline, and the features that characterize it, are complex and variable. The age-related decline of muscle function is a result of not only a decrease of muscle mass but also a decline in the intrinsic properties of muscle fibers that are independent of size. We believe it is important to understand changes in muscle quality (force adjusted for size), and not to focus solely on muscle mass, because muscle quality is closely related to measurements of function and could potentially predict clinical outcomes such as morbidity, disability, and mortality. Neurological and metabolic mechanisms contribute to muscle quality, but the intrinsic properties of muscle cells are central to the maintenance of force-generating capacity. Muscle quality can be evaluated with the assessment of morphological, physiological, and mechanical properties in single permeabilized or skinned fibers. This approach excludes the influence of the nervous system, tendons, and the extracellular matrix. In this review, we summarized the changes in active and passive mechanical properties at the single muscle cell level in older skeletal muscles. We argue that intrinsic mechanical changes in human single muscle fibers are useful biomarkers and indicators of muscle quality.

Dietary lycopene supplementation improves meat quality, antioxidant capacity and skeletal muscle fiber type transformation in finishing pigs.

This study aimed to investigate effects of dietary lycopene supplementation on meat quality, antioxidant ability and muscle fiber type transformation in finishing pigs. In a 70-day experiment, 18 Duroc × Landrace × Yorkshire barrows were randomly allocated to 3 dietary treatments including a basal diet supplemented with 0, 100 and 200 mg/kg lycopene, respectively. Each dietary treatment had 6 replicates with one pig each. Results showed that dietary 200 mg/kg lycopene supplementation increased muscle redness a∗ value, intramuscular fat and crude protein contents, and decreased muscle lightness L∗ and yellowness b∗ values (P < 0.05), suggesting that addition of 200 mg/kg lycopene to the diet of finishing pigs improved color, nutritional value and juiciness of pork after slaughter. Results also showed that dietary lycopene supplementation enhanced antioxidant capacity of finishing pigs (P < 0.05). Moreover, dietary supplementation of 200 mg/kg lycopene significantly increased slow myosin heavy chain (MyHC) protein level and slow-twitch fiber percentage, and decreased fast MyHC protein level and fast-twitch fiber percentage (P < 0.05), suggesting that the addition of 200 mg/kg lycopene to the diet of finishing pigs promoted muscle fiber type conversion from fast-twitch to slow-twitch. Together, we provide the first evidence that dietary 200 mg/kg lycopene supplementation improves meat quality, enhances antioxidant capacity and promotes muscle fiber type transformation from fast-twitch to slow-twitch in finishing pigs.

trans 10, cis 12, but Not cis 9, trans 11 Conjugated Linoleic Acid Isomer Enhances Exercise Endurance by Increasing Oxidative Skeletal Muscle Fiber Type via Toll-like Receptor 4 Signaling in Mice.

Conjugated linoleic acid (CLA) has been implicated in regulating muscle fiber. However, which isomer elicits this effect and the underlying mechanisms remain unclear. Here, male C57BL6/J mice and C2C12 cells were treated with two CLA isomers, and the exercise endurance, skeletal muscle fiber type, and involvement of Toll-like receptor 4 (TLR4) signaling were assessed. The results demonstrated that dietary t10, c12, but not c9, t11-CLA isomer enhanced exercise endurance of mice (from 115.88 ± 11.21 to 130.00 ± 15.84 min, P < 0.05) and promoted the formation of oxidative muscle fiber type of gastrocnemius muscle (from 0.15 ± 0.04 to 0.24 ± 0.05, P < 0.05). Consistently, t10, c12-CLA isomer increased the mRNA expression of oxidative muscle fiber type in C2C12 myotubes (from 1.00 ± 0.08 to 2.65 ± 1.77, P < 0.05). In addition, t10, c12-CLA isomer increased TLR4 signaling expression in skeletal muscle and C2C12 myotubes. More importantly, knockdown of TLR4 eliminated the t10, c12-CLA isomer-induced enhancement of exercise endurance in mice and elevation of oxidative muscle fiber type in C2C12 myotubes and gastrocnemius muscle. Together, these findings showed that t10, c12, but not c9, t11-CLA isomer enhances exercise endurance by increasing oxidative skeletal muscle fiber type via TLR4 signaling.

Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells.

Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded biosensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO2 must be controlled to avoid pH fluctuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle.

Skeletal muscle type-specific mitochondrial adaptation to high-fat diet relies on differential autophagy modulation.

In obesity, skeletal muscle mitochondrial activity changes to cope with increased nutrient availability. Autophagy has been proposed as an essential mechanism involved in the regulation of mitochondrial metabolism. Still, the contribution of autophagy to mitochondrial adaptations in skeletal muscle during obesity is unknown. Here, we show that in response to high-fat diet (HFD) feeding, distinct skeletal muscles in mice exhibit differentially regulated autophagy that may modulate mitochondrial activity. We observed that after 4 and 40 weeks of high-fat diet feeding, OXPHOS subunits and mitochondrial DNA content increased in the oxidative soleus muscle. However, in gastrocnemius muscle, which has a mixed fiber-type composition, the mitochondrial mass increased only after 40 weeks of HFD feeding. Interestingly, fatty acid-supported mitochondrial respiration was enhanced in gastrocnemius, but not in soleus muscle after a 4-week HFD feeding. This increased metabolic profile in gastrocnemius was paralleled by preserving autophagy flux, while autophagy flux in soleus was reduced. To determine the role of autophagy in this differential response, we used an autophagy-deficient mouse model with partial deletion of Atg7 specifically in skeletal muscle (SkM-Atg7+/- mice). We observed that Atg7 reduction resulted in diminished autophagic flux in skeletal muscle, alongside blunting the HFD-induced increase in fatty acid-supported mitochondrial respiration observed in gastrocnemius. Remarkably, SkM-Atg7+/- mice did not present increased mitochondria accumulation. Altogether, our results show that HFD triggers specific mitochondrial adaptations in skeletal muscles with different fiber type compositions, and that Atg7-mediated autophagy modulates mitochondrial respiratory capacity but not its content in response to an obesogenic diet.