Mitophagy In Skeletal Muscle Research Articles

Ulk1 phosphorylation at S555 is not required for endurance training-induced improvements in exercise and metabolic capacity in mice.

Endurance exercise training improves exercise capacity as well as skeletal muscle and whole body metabolism, which are hallmarks of high quality-of-life and healthy aging. However, its mechanisms are not yet fully understood. Exercise-induced mitophagy has emerged as an important step in mitochondrial remodeling. Unc-51-like autophagy-activating kinase 1, ULK1, specifically its activation by phosphorylation at serine 555, was discovered as an autophagy driver and to be important for energetic stress-induced mitophagy in skeletal muscle, making it a potential mediator of the beneficial effects of exercise on mitochondrial remodeling. Here, we used CRISPR/Cas9-mediated gene editing and generated knock-in mice with a serine-to-alanine mutation of Ulk1 on serine 555. We now report that these mice displayed normal endurance capacity and cardiac function at baseline with a mild impairment in energy metabolism as indicated by an accelerated increase of respiratory exchange ratio (RER) during acute exercise stress; however, this was completely corrected by 8 wk of voluntary running. Ulk1-S555A mice also retained the exercise-mediated improvements in exercise capacity and metabolic flux. We conclude that Ulk1 phosphorylation at S555 is not required for exercise-mediated improvements of exercise and metabolic capacity in healthy mice.NEW & NOTEWORTHY We have used CRISPR/Cas9-mediated gene editing to generate Ulk1-S555A knock-in mice to show that loss of phosphorylation of Ulk1 at S555 blunted exercise-induced mitophagy and mildly impairs energy metabolism during exercise in healthy mice. However, the knock-in mice retained exercise training-mediated improvements of endurance capacity and energy metabolism during exercise. These findings suggest that exercise-induced mitophagy through Ulk1 activation is not required for the metabolic adaptation and improved exercise capacity in young, healthy mice.

Shenqi Fuzheng injection facilitates skeletal muscle mitophagy mediated by the ubiquitination of HIF-1α to ameliorate cancer-associated fatigue.

Cancer-related fatigue (CRF) significantly impacts the quality of life of cancer patients. This study investigates the therapeutic potential of Shenqi Fuzheng injection (SFI) in managing CRF, focusing on its mechanistic action in skeletal muscle. We utilized a CRF mouse model to examine the effects of SFI on physical endurance, monitoring activity levels, swimming times and rest periods. Proteomic analysis of the gastrocnemius muscle was performed using isobaric tags and liquid chromatography-tandem mass spectrometry to map the muscle proteome changes post-SFI treatment. Mitochondrial function in skeletal muscle was assessed via ATP bioluminescence assay. Furthermore, the regulatory role of the hypoxia inducible factor 1 subunit alpha (HIF-1α) signalling pathway in mediating SFI's effects was explored through western blotting. In CRF-induced C2C12 myoblasts, we evaluated cell viability (CCK-8 assay), apoptosis (flow cytometry) and mitophagy (electron microscopy). The study also employed pulldown, luciferase and chromatin immunoprecipitation assays to elucidate the molecular mechanisms underlying SFI's action, particularly focusing on the transcriptional regulation of PINK1 through HIF-1α binding at the PINK1 promoter region. Our findings reveal that SFI enhances physical mobility, reduces fatigue symptoms and exerts protective effects on skeletal muscles by mitigating mitochondrial damage and augmenting antioxidative responses. SFI promotes cell viability and induces mitophagy while decreasing apoptosis, primarily through the modulation of HIF-1α, PINK1 and p62 proteins. These results underscore SFI's efficacy in enhancing mitochondrial autophagy, thereby offering a promising approach for ameliorating CRF. The study not only provides insight into SFI's potential therapeutic mechanisms but also establishes a foundation for further exploration of SFI interventions in CRF management.

Lack of compensatory mitophagy in skeletal muscles during sepsis.

Skeletal muscle dysfunction is a major problem in critically ill patients suffering from sepsis. This condition is associated with mitochondrial dysfunction and increased autophagy in skeletal muscles. Autophagy is a proteolytic mechanism involved in eliminating dysfunctional cellular components, including mitochondria. The latter process, referred to as mitophagy, is essential for maintaining mitochondrial quality and skeletal muscle health. Recently, a fluorescent reporter system called mito-QC (i.e. mitochondrial quality control) was developed to specifically quantify mitophagy levels. In the present study, we used mito-QC transgenic mice and confocal microscopy to morphologically monitor mitophagy levels during sepsis. To induce sepsis, Mito-QC mice received Escherichia coli lipopolysaccharide (10mgkg-1 i.p.) or phosphate-buffered saline and skeletal muscles (hindlimb and diaphragm) were excised 48h later. In control groups, there was a negative correlation between the basal mitophagy level and overall muscle mitochondrial content. Sepsis increased general autophagy in both limb muscles and diaphragm but had no effect on mitophagy levels. Sepsis was associated with a downregulation of certain mitophagy receptors (Fundc1, Bcl2L13, Fkbp8 and Phbb2). The present study suggests that general autophagy and mitophagy can be dissociated from one another, and that the characteristic accumulation of damaged mitochondria in skeletal muscles under the condition of sepsis may reflect a failure of adequate compensatory mitophagy. KEY POINTS: There was a negative correlation between the basal level of skeletal muscle mitophagy and the mitochondrial content of individual muscles. Mitophagy levels in limb muscles and the diaphragm were unaffected by lipopolysaccharide (LPS)-induced sepsis. With the exception of BNIP3 in sepsis, LPS administration induced either no change or a downregulation of mitophagy receptors in skeletal muscles.

Maternal prednisone exposure during pregnancy elevates susceptibility to osteoporosis in female offspring: The role of mitophagy/FNDC5 alteration in skeletal muscle

Maternal exposure to glucocorticoids has been associated with adverse outcomes in offspring. However, the consequences and mechanisms of gestational exposure to prednisone on susceptibility to osteoporosis in the offspring remain unclear. Here, we found that gestational prednisone exposure enhanced susceptibility to osteoporosis in adult mouse offspring. In a further exploration of myogenic mechanisms, results showed that gestational prednisone exposure down-regulated FNDC5/irisin protein expression and activation of OPTN-dependent mitophagy in skeletal muscle of adult offspring. Additional experiments elucidated that activated mitophagy significantly inhibited the expression of FNDC5/irisin in skeletal muscle cells. Likewise, we observed delayed fetal bone development, downregulated FNDC5/irisin expression, and activated mitophagy in fetal skeletal muscle upon gestational prednisone exposure. In addition, an elevated total m6A level was observed in fetal skeletal muscle after gestational prednisone exposure. Finally, gestational supplementation with S-adenosylhomocysteine (SAH), an inhibitor of m6A activity, attenuated mitophagy and restored FNDC5/irisin expression in fetal skeletal muscle, which in turn reversed fetal bone development. Overall, these data indicate that gestational prednisone exposure increases m6A modification, activates mitophagy, and decreases FNDC5/irisin expression in skeletal muscle, thus elevating osteoporosis susceptibility in adult offspring. Our results provide a new perspective on the earlier prevention and treatment of fetal-derived osteoporosis.

Cold exposure affects glucose metabolism, lipid droplet deposition and mitophagy in skeletal muscle of newborn goats

Cold exposure is a common stressor for newborn goats. Skeletal muscle plays an important role in maintaining whole-body homeostasis of glucose and lipid metabolism. However, the molecular mechanisms underlying regulation of skeletal muscle of newborn goats by cold exposure remains unclear. In this study, we found a significant increase (P < 0.01) in serum glucagon levels after 24 h of cold exposure (COLD, 6°C), while glucose and insulin concentrations were significantly decreased (P < 0.01) compared to room temperature (RT, 25°C). Additionally, we found that cold exposure reduced glycogen content (P < 0.01) in skeletal muscle. Pathway enrichment analysis revealed that cold exposure activated skeletal muscle glucose metabolism pathways (including insulin resistance and the insulin signaling pathway) and mitophagy-related pathways. Cold exposure up-regulated the expression of genes involved in fatty acid and triglyceride synthesis, promoting skeletal muscle lipid deposition. Notably, cold exposure induced mitophagy in skeletal muscle.

304-OR: Reduced VDAC1 Limits ROS Release to Restrain Oxidative Damage in Muscle of 3-Year-Old Nonhuman Primates with Early-Life Exposure to Maternal Western Diet or Obesity

Offspring from obese pregnancies have an elevated risk for childhood obesity and chronic metabolic diseases. Using a Japanese macaque model of early-life maternal Western-style diet (mWD) exposure, we previously observed decreased oxidative capacity and elevated markers of mitophagy in skeletal muscle of 3-yr-old offspring. We also found substantial reductions in VDAC1 content, the loss of which significantly correlated with decreased whole-muscle lipid peroxidation in mWD offspring. For this study, we isolated the impact of mWD from mWD-induced obesity by studying offspring from both lean (Ln, body fat &amp;lt;25%) or obese (Ob, body fat &amp;gt;30%) dams fed a WD or lean dams fed a control diet (CD) prior to and throughout pregnancy and lactation. All offspring were weaned to CD and offspring gastrocnemius was collected at 40 months of age. This design created three offspring groups: LnCD, LnWD and ObWD. In offspring muscle, VDAC1, but not VDAC2, was significantly decreased in LnWD, and was further reduced in ObWD. In contrast to whole-tissue, lipid peroxidation in a mitochondrial-enriched fraction was increased in offspring muscle from LnWD and ObWD groups. Markers of mtDNA damage were also elevated in ObWD muscle. Lastly, Sirt3 abundance, a master regulator of mitochondrial antioxidant defense, was reduced in muscle from both LnWD and ObWD. These data suggest that ROS are retained within the mitochondrial compartment due, in part, to VDAC1 suppression, resulting in an overall reduction in cellular oxidant burden. These findings elucidate a long-term adaptation to early life stress caused by mWD and obesity which limits cellular damage at the expense of mitochondrial function. Disclosure K.T.Greyslak: None. C.E.Mccurdy: None. Funding National Institutes of Health (R24DK090964); Eugene Evonuk Memorial Graduate Fellowship

Deciphering the Molecular Mechanism of Yifei-Sanjie Pill in Cancer-Related Fatigue.

The incidence of cancer-related fatigue (CRF) is increasing, but its lack of clear pathogenesis makes its prevention and treatment difficult. Therefore, it is of great significance to clarify the pathogenesis of CRF and find effective methods to treat it. The CRF model was established by intraperitoneal injection of LLC cells in ICR mice to explore the pathogenesis of CRF and verify the therapeutic effect of the Yifei-Sanjie pill (YFSJ). The active components of YFSJ were found by LC/MS, the in vitro inflammatory infiltration model of skeletal muscle was constructed by TNF-α and C2C12 myoblasts, and the results of in vivo experiments were verified by this model. Behavioral analysis results showed that YFSJ alleviated CRF; histological examination results showed that YFSJ could reverse the tumor microenvironment leading to skeletal muscle injury; ELISA and RNA-seq results showed that the occurrence of CRF and the therapeutic effect of YFSJ were closely related to the tumor inflammatory microenvironment; IHC and WB results showed that the occurrence of CRF and the therapeutic effect of YFSJ were closely related to the Stat3-related signaling pathway and autophagy. YFSJ can reduce the level of inflammation in the tumor microenvironment in vivo, inhibit the abnormal activation of the Stat3/HIF-1α/BNIP3 signaling pathway induced by tumor-related inflammation, thereby inhibiting the overactivation of mitophagy in skeletal muscle, and finally alleviate CRF. Quercetin, one of the components of YFSJ, plays an important role in inhibiting the phosphorylation activation of Stat3.

Ameliorating effect of Trigonella foenum-graecum L. (fenugreek) extract tablet on exhaustive exercise-induced fatigue in rats by suppressing mitophagy in skeletal muscle.

Trigonella foenum-graecum L. (fenugreek) is widely used as a leafy vegetable and spice in China and North Africa. Recent studies have reported that fenugreek can reduce fatigue; however, its antifatigue mechanism remains unclear. Therefore, this study aimed to investigate the potential antifatigue effects of fenugreek extract (FE) on mitophagy and the underlying mechanisms. We evaluated the potential effects of FE tablet on an exhaustive exercise-induced fatigue (EEF) rat model. Oxidative stress indicators and fatigue biomarkers in the serum and skeletal muscle were detected. Mitophagy and mitochondrial morphology were observed using transmission electron microscopy. The expression levels of mitochondrial autophagy-related proteins were detected using western blot and immunofluorescence. Compared with the model group, FE enhanced the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase as well as total antioxidant capacity; however, it decreased the level of malondialdehyde in the serum and skeletal muscle after a 7-day treatment. Moreover, certain indicators of mitochondrial function, such as reactive oxygen species levels, ATP levels, cellular and mitochondrial Ca2+ levels, and ATPase activity, were significantly improved in the FE group compared with the model group. Finally, we found that mitophagy was induced by exhaustive exercise and inhibited by FE. Regarding mitochondrial autophagy-related proteins, the expression levels of LC3B, FUNDC1, PGAM5, PARKIN, and PINK1 in the skeletal muscle tissue were increased in the EEF group compared with the control group. After administration of FE and a positive control drug, a significant reversal in the expression of the above-mentioned proteins was noted. Our findings demonstrate that FE exerted antifatigue effects in the EEF rat model by regulating the mitophagy-related FUNDC1/LC3B signaling pathway rather than the PINK1/PARKIN signaling pathway.

Sensing local energetics to acutely regulate mitophagy in skeletal muscle.

The energetic requirements of skeletal muscle to sustain movement, as during exercise, is met largely by mitochondria, which form an intricate, interconnected reticulum. Maintenance of a healthy mitochondrial reticulum is essential for skeletal muscle function, suggesting quality control pathways are spatially governed. Mitophagy, the process by which damaged and/or dysfunctional regions of the mitochondrial reticulum are removed and degraded, has emerged as an integral part of the molecular response to exercise. Upregulation of mitophagy in response to acute exercise is directly connected to energetic sensing mechanisms through AMPK. In this review, we discuss the connection of mitophagy to muscle energetics and how AMPK may spatially control mitophagy through multiple potential means.

Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1

Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy invivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.

The influence of age, sex, and exercise on autophagy, mitophagy, and lysosome biogenesis in skeletal muscle

BackgroundAging decreases skeletal muscle mass and quality. Maintenance of healthy muscle is regulated by a balance between protein and organellar synthesis and their degradation. The autophagy-lysosome system is responsible for the selective degradation of protein aggregates and organelles, such as mitochondria (i.e., mitophagy). Little data exist on the independent and combined influence of age, biological sex, and exercise on the autophagy system and lysosome biogenesis. The purpose of this study was to characterize sex differences in autophagy and lysosome biogenesis in young and aged muscle and to determine if acute exercise influences these processes.MethodsYoung (4–6 months) and aged (22–24 months) male and female mice were assigned to a sedentary or an acute exercise group. Mitochondrial content, the autophagy-lysosome system, and mitophagy were measured via protein analysis. A TFEB-promoter-construct was utilized to examine Tfeb transcription, and nuclear-cytosolic fractions allowed us to examine TFEB localization in sedentary and exercised muscle with age and sex.ResultsOur results indicate that female mice, both young and old, had more mitochondrial protein than age-matched males. However, mitochondria in the muscle of females had a reduced respiratory capacity. Mitochondrial content was only reduced with age in the male cohort. Young female mice had a greater abundance of autophagy, mitophagy, and lysosome proteins than young males; however, increases were evident with age irrespective of sex. Young sedentary female mice had indices of greater autophagosomal turnover than male counterparts. Exhaustive exercise was able to stimulate autophagic clearance solely in young male mice. Similarly, nuclear TFEB protein was enhanced to a greater extent in young male, compared to young female mice following exercise, but no changes were observed in aged mice. Finally, TFEB-promoter activity was upregulated following exercise in both young and aged muscle.ConclusionsThe present study demonstrates that biological sex influences mitochondrial homeostasis, the autophagy-lysosome system, and mitophagy in skeletal muscle with age. Furthermore, our data suggest that young male mice have a more profound ability to activate these processes with exercise than in the other groups. Ultimately, this may contribute to a greater remodeling of muscle in response to exercise training in males.

A novel PHD2 inhibitor acteoside from Cistanche tubulosa induces skeletal muscle mitophagy to improve cancer-related fatigue.

To study whether ACT exerts anti-fatigue activity against CRF by inducing skeletal muscle mitophagy via suppressing PHD2 to upregulate the HIF-1α/BNIP3 signaling pathway. In this study, the molecular docking virtual screening technique was used to screen active components in Cistanche tubulosa that act as potential PHD2 inhibitors; the preliminary verification was carried out by Surface plasmon resonance (SPR) technology. BALB/c mice were treated with Paclitaxel (PTX, 10mg/kg) and ACT (50, 100mg/kg) alone or in combination for 20 days. Fatigue-related behaviors, energy metabolism and skeletal muscle mitochondria were assessed. Murine C2C12 myoblast was cultured and differentiated; then, a C26 tumor cell-conditioned medium was added to induce cachexia. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential, mitochondrial microstructure and function, autophagy, PHD2/HIF-1 and PINK1/Parkin signal pathway proteins were analyzed. Then, interfering RNA technology was used to silence PHD2 and observe the efficacy of ACT. We demonstrated that ACT exerted good binding activity with PHD2; ACT administration ameliorated PTX-induced muscle fatigue-like behavior via improving muscle quality and mitochondria function, increasing mitophagy, upregulating COXIV, CytoC, PINK1, Parkin, HIF-1α and BNIP3 expression and inhibiting p62, LC3B, PHD2 and Beclin-1 expression. The protective effect of ACT disappeared after transfection with the PHD2 gene knockdown plasmid Egln-1-RNAi. These results suggest that ACT can improve CRF by promoting mitophagy via suppression of PHD2 to remove dysfunctional mitochondria, demonstrating that ACT has huge prospects for clinical application in CRF treatment.

Ambra1 deficiency impairs mitophagy in skeletal muscle.

BackgroundMaintaining healthy mitochondria is mandatory for muscle viability and function. An essential surveillance mechanism targeting defective and harmful mitochondria to degradation is the selective form of autophagy called mitophagy. Ambra1 is a multifaceted protein with well‐known autophagic and mitophagic functions. However, the study of its role in adult tissues has been extremely limited due to the embryonic lethality caused by full‐body Ambra1 deficiency.MethodsTo establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivo overexpression of a mitochondria‐targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle‐specific Ambra1 knockout (Ambra1 fl/fl:Mlc1f‐Cre) mice. Mitochondria‐enriched fractions were obtained from muscles of fed and starved animals to investigate the dynamics of the mitophagic flux.ResultsOur data show that Ambra1 has a critical role in the mitophagic flux of adult murine skeletal muscle and that its genetic inactivation leads to mitochondria alterations and myofibre remodelling. Ambra1 overexpression in wild‐type muscles is sufficient to enhance mitochondria clearance through the autophagy‐lysosome system. Consistently with this, Ambra1‐deficient muscles display an abnormal accumulation of the mitochondrial marker TOMM20 by +76% (n = 6–7; P < 0.05), a higher presence of myofibres with swollen mitochondria by +173% (n = 4; P < 0.05), and an alteration in the maintenance of the mitochondrial membrane potential and a 34% reduction in the mitochondrial respiratory complex I activity (n = 4; P < 0.05). Lack of Ambra1 in skeletal muscle leads to impaired mitophagic flux, without affecting the bulk autophagic process. This is due to a significantly decreased recruitment of DRP1 (n = 6–7 mice; P < 0.01) and Parkin (n = 6–7 mice; P < 0.05) to the mitochondrial compartment, when compared with controls. Ambra1‐deficient muscles also show a marked dysregulation of the endolysosome compartment, as the incidence of myofibres with lysosomal accumulation is 20 times higher than wild‐type muscles (n = 4; P < 0.05). Histologically, Ambra1‐deficient muscles of both 3‐ and 6‐month‐old animals display a significant decrease of myofibre cross‐sectional area and a 52% reduction in oxidative fibres (n = 6–7; P < 0.05), thus highlighting a role for Ambra1 in the proper structure and activity of skeletal muscle.ConclusionsOur study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

TGFβ Induces Changes in Mitochondrial Homeostasis in Skeletal Muscle of Mice with Bone Metastases

Cancer cachexia is a common neoplastic syndrome of advanced cancers characterized by a loss of skeletal muscle mass and decreased function. Patients with cancer cachexia have higher chances of morbidity and mortality. We have shown that mice inoculated with human MDA‐MB‐231 breast cancer cells, which metastasize to bone, have skeletal muscle weakness, cachexia, and altered skeletal muscle metabolism. Mice with bone metastases have increased serum levels of transforming growth factor beta (TGFβ). Serum TGFβ decreased after treatment with the bone anti‐resorptive agent, zoledronic acid (ZA), suggesting TGFβ is, in part, bone‐derived. Additionally, mice with bone metastases treated with ZA had increased skeletal muscle function. TGFβ has also been shown to decrease the master regulator of mitochondrial biogenesis, peroxisome‐activated receptor γ coactivator 1α (PGC1α), in C2C12 myotubes. Mitochondria provide muscle with energy (ATP) via oxidative phosphorylation, so we measured oxygen consumption and energy production using indirect calorimetry in mice with bone metastases. Oxygen consumption and energy production were both decreased in these mice suggesting a decrease in mitochondrial function. We then examined skeletal muscle mitochondria using transmission electron microscopy and western blotting. We found highly dysmorphic mitochondria and altered protein markers of mitochondrial biogenesis and dynamics. Additionally, LC3B protein was increased in skeletal muscle mitochondria demonstrating increased mitophagy in skeletal muscle of mice with bone metastases. From our previous knowledge and data of TGFβ and its role in mice with bone metastases, we hypothesize that TGFβ causes mitochondrial dysfunction in skeletal muscle of mice with bone metastases. We have shown that a 48‐hour treatment with TGFβ decreases protein expression in markers of mitochondrial biogenesis and dynamics in C2C12 myotubes. This data suggests that TGFβ could be causing the mitochondrial dysfunction and dysmorphia we see in our mouse model, but further experiments are needed to prove this hypothesis. Our data demonstrates the importance of mitochondrial function in skeletal muscle cells and skeletal muscle of mice with bone metastases. Understanding the role TGFβ plays in changes of skeletal muscle mitochondria in mice with bone metastases will help contribute to defining treatments for patients with cancer cachexia.

Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control.

BackgroundSarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified that a mitochondrial‐targeted furazano[3,4‐b]pyrazine named BAM15 is orally available and selectively lowers respiratory coupling efficiency and protects against diet‐induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity.MethodsEighty‐week‐old male C57BL/6J mice with obesity were randomized to 10 weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body weight and food intake were measured weekly. Body composition, muscle function, energy expenditure, locomotor activity, and glucose tolerance were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signalling, and mitochondrial structure and function.ResultsBAM15 decreased body weight (54.0 ± 2.0 vs. 42.3 ± 1.3 g, P < 0.001) which was attributable to increased energy expenditure (10.1 ± 0.1 vs. 11.3 ± 0.4 kcal/day, P < 0.001). BAM15 increased muscle mass (52.7 ± 0.4 vs. 59.4 ± 1.0%, P < 0.001), strength (91.1 ± 1.3 vs. 124.9 ± 1.2 g, P < 0.0001), and locomotor activity (347.0 ± 14.4 vs. 432.7 ± 32.0 m, P < 0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (interleukin 6 and gp130, both P < 0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis. Specifically, BAM15 activated mitochondrial quality control (PINK1‐ubiquitin binding and LC3II, P < 0.01), increased mitochondrial activity (citrate synthase and complex II activity, all P < 0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P < 0.0001) while limiting ER stress (decreased PERK signalling, P < 0.0001), apoptotic signalling (decreased cytochrome C release and Caspase‐3/9 activation, all P < 0.001), and muscle protein degradation (decreased 14‐kDa actin fragment insoluble/soluble ratio, P < 0.001).ConclusionsMitochondrial uncoupling by agents such as BAM15 may mitigate age‐related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity.

The role of Sirt3 in the changes of skeletal muscle mitophagy induced by hypoxic training.

We aimed to explore the role of Sirt3 in the regulation of skeletal muscle mitophagy with hypoxic training. C57BL/6J mice were randomly divided into four groups: C group (control), HT group (mice performed a hypoxic training of living in an environment with an oxygen concentration of 13.8% and treadmill exercise under normoxia for 6 weeks), T group (mice were subjected to an intraperitoneal (i.p.) injection of the Sirt3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) 50 mg/kg three times per week for 6 weeks) and THT group (the hypoxic training of HT group with i.p. injection of 3-TYP in T group). The results showed that 6 weeks of hypoxic training could improve ATP synthesis in skeletal muscle. After the combined intervention of 3-TYP injection and hypoxic training, Sirt3, FOXO3a, and SOD2 protein contents were still lower than those in hypoxic training group. Hypoxic training cannot improve the negative effect of Sirt3 inhibition on muscle PINK1/Parkin signal. This study demonstrated that Sirt3 plays a key role in mediating skeletal muscle mitophagy by hypoxic training. The results of our study also provided the first evidence that mitophagy caused by hypoxic training might be transduced through the Sirt3-FOXO3a signaling pathway.

The role of Sirt3 in the changes of skeletal muscle mitophagy induced by hypoxic training

The role of Sirt3 in the changes of skeletal muscle mitophagy induced by hypoxic training

P53 regulates skeletal muscle mitophagy and mitochondrial quality control following denervation-induced muscle disuse

Persistent inactivity promotes skeletal muscle atrophy, marked by mitochondrial aberrations that affect strength, mobility, and metabolic health leading to the advancement of disease. Mitochondrial quality control (MQC) pathways include biogenesis (synthesis), mitophagy/lysosomal turnover, and the mitochondrial unfolded protein response, which serve to maintain an optimal organelle network. Tumor suppressor p53 has been implicated in regulating muscle mitochondria in response to cellular stress; however, its role in the context of muscle disuse has yet to be explored, and whether p53 is necessary for MQC remains unclear. To address this, we subjected p53 muscle-specific KO (mKO) and WT mice to unilateral denervation. Transcriptomic and pathway analyses revealed dysregulation of pathways pertaining to mitochondrial function, and especially turnover, in mKO muscle following denervation. Protein and mRNA data of the MQC pathways indicated activation of the mitochondrial unfolded protein response and mitophagy–lysosome systems along with reductions in mitochondrial biogenesis and content in WT and mKO tissue following chronic denervation. However, p53 ablation also attenuated the expression of autophagy–mitophagy machinery, reduced autophagic flux, and enhanced lysosomal dysfunction. While similar reductions in mitochondrial biogenesis and content were observed between genotypes, MQC dysregulation exacerbated mitochondrial dysfunction in mKO fibers, evidenced by elevated reactive oxygen species. Moreover, acute experiments indicate that p53 mediates the expression of transcriptional regulators of MQC pathways as early as 1 day following denervation. Together, our data illustrate exacerbated mitochondrial dysregulation with denervation stress in p53 mKO tissue, thus indicating that p53 contributes to organellar maintenance via regulation of MQC pathways during muscle atrophy.

Mitochondrial Dynamics and Mitophagy in Skeletal Muscle Health and Aging.

The maintenance of mitochondrial integrity is critical for muscle health. Mitochondria, indeed, play vital roles in a wide range of cellular processes, including energy supply, Ca2+ homeostasis, retrograde signaling, cell death, and many others. All mitochondria-containing cells, including skeletal muscle cells, dispose of several pathways to maintain mitochondrial health, including mitochondrial biogenesis, mitochondrial-derived vesicles, mitochondrial dynamics (fusion and fission process shaping mitochondrial morphology), and mitophagy—the process in charge of the removal of mitochondria though autophagy. The loss of skeletal muscle mass (atrophy) is a major health problem worldwide, especially in older people. Currently, there is no treatment to counteract the progressive decline in skeletal muscle mass and strength that occurs with aging, a process termed sarcopenia. There is increasing data, including our own, suggesting that accumulation of dysfunctional mitochondria contributes to the development of sarcopenia. Impairments in mitochondrial dynamics and mitophagy were recently proposed to contribute to sarcopenia. This review summarizes the current state of knowledge on the role played by mitochondrial dynamics and mitophagy in skeletal muscle health and in the development of sarcopenia. We also highlight recent studies showing that enhancing mitophagy in skeletal muscle is a promising therapeutic target to prevent or even treat skeletal muscle dysfunction in the elderly.

Short-Term High-Fat Feeding Does Not Alter Mitochondrial Lipid Respiratory Capacity but Triggers Mitophagy Response in Skeletal Muscle of Mice.

Lipid overload of the mitochondria is linked to the development of insulin resistance in skeletal muscle which may be a contributing factor to the progression of type 2 diabetes during obesity. The targeted degradation of mitochondria through autophagy, termed mitophagy, contributes to the mitochondrial adaptive response to changes in dietary fat. Our previous work demonstrates long-term (2-4 months) consumption of a high-fat diet increases mitochondrial lipid oxidation capacity but does not alter markers of mitophagy in mice. The purpose of this study was to investigate initial stages of mitochondrial respiratory adaptations to high-fat diet and the activation of mitophagy. C57BL/6J mice consumed either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 3 or 7 days. We measured skeletal muscle mitochondrial respiration and protein markers of mitophagy in a mitochondrial-enriched fraction of skeletal muscle. After 3 days of HFD, mice had lower lipid-supported oxidative phosphorylation alongside greater electron leak compared with the LFD group. After 7 days, there were no differences in mitochondrial respiration between diet groups. HFD mice had greater autophagosome formation potential (Beclin-1) and greater activation of mitochondrial autophagy receptors (Bnip3, p62) in isolated mitochondria, but no difference in downstream autophagosome (LC3II) or lysosome (Lamp1) abundance after both 3 and 7 days compared with the LFD groups. In cultured myotubes, palmitate treatment decreased mitochondrial membrane potential and hydrogen peroxide treatment increased accumulation of upstream mitophagy markers. We conclude that several days of high-fat feeding stimulated upstream activation of skeletal muscle mitophagy, potentially through lipid-induced oxidative stress, without downstream changes in respiration.