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Altered mitochondrial structure and function are implicated in the functional decline of skeletal muscle. Numerous cytoskeletal proteins are known to affect mitochondrial homeostasis, but this complex network is still being unraveled. Here, we investigated mitochondrial alterations in mice lacking the cytoskeletal adapter protein, XIN (XIN-/-). XIN-/- and wild-type littermate male and female mice were fed a chow or high-fat diet (HFD; 60% kcal fat) for 8 weeks before analyses of their skeletal muscles were conducted. Immuno-electron microscopy (EM) and immunofluorescence staining revealed XIN in the mitochondria and peri-mitochondrial areas, as well as the myoplasm. Intermyofibrillar mitochondria in chow-fed XIN-/- mice were notably different from wild-type (large, and/or swollen in appearance). Succinate dehydrogenase and Cytochrome Oxidase IV staining indicated greater evidence of mitochondrial enzyme activity in XIN-/- mice. No difference in body mass gains or glucose handling was observed between cohorts with HFD. However, EM revealed significantly greater mitochondrial density with evident structural abnormalities (swelling, reduced cristae density) in XIN-/- mice. Absolute Complex I and II-supported respiration was not different between groups, but relative to mitochondrial density, was significantly lower in XIN-/-. These results provide the first evidence for a role of XIN in maintaining mitochondrial morphology and function., Competing Interests: The authors have nothing to disclose.

Duchenne muscular dystrophy is secondarily accompanied by Ca 2+ excess in muscle fibers. Part of the Ca 2+ accumulates in the mitochondria, contributing to the development of mitochondrial dysfunction and degeneration of muscles. In this work, we assessed the effect of intraperitoneal administration of rhodacyanine MKT077 (5 mg/kg/day), which is able to suppress glucose-regulated protein 75 (GRP75)-mediated Ca 2+ transfer from the sarcoplasmic reticulum (SR) to mitochondria, on the Ca 2+ overload of skeletal muscle mitochondria in dystrophin-deficient mdx mice and the concomitant mitochondrial dysfunction contributing to muscle pathology. MKT077 prevented Ca 2+ overload of quadriceps mitochondria in mdx mice, reduced the intensity of oxidative stress, and improved mitochondrial ultrastructure, but had no effect on impaired oxidative phosphorylation. MKT077 eliminated quadriceps calcification and reduced the intensity of muscle fiber degeneration, fibrosis level, and normalized grip strength in mdx mice. However, we noted a negative effect of MKT077 on wild-type mice, expressed as a decrease in the efficiency of mitochondrial oxidative phosphorylation, SR stress development, ultrastructural disturbances in the quadriceps, and a reduction in animal endurance in the wire-hanging test. This paper discusses the impact of MKT077 modulation of mitochondrial dysfunction on the development of skeletal muscle pathology in mdx mice.

Aim: The present study aimed to investigate the effects of a single bout of resistance exercise on mitophagy in human skeletal muscle (SkM)., Methods: Eight healthy men were recruited to complete an acute bout of one-leg resistance exercise. SkM biopsies were obtained one hour after exercise in the resting leg (Rest-leg) and the contracting leg (Ex-leg). Mitophagy was assessed using protein-related abundance, transmission electron microscopy (TEM), and fluorescence microscopy., Results: Our results show that acute resistance exercise increased pro-fission protein phosphorylation (DRP1 Ser616 ) and decreased mitophagy markers such as PARKIN and BNIP3L/NIX protein abundance in the Ex-leg. Additionally, mitochondrial complex IV decreased in the Ex-leg when compared to the Rest-leg. In the Ex-leg, TEM and immunofluorescence images showed mitochondrial cristae abnormalities, a mitochondrial fission phenotype, and increased mitophagosome-like structures in both subsarcolemmal and intermyofibrillar mitochondria. We also observed increased mitophagosome-like structures on the subsarcolemmal cleft and mitochondria in the extracellular space of SkM in the Ex-leg. We stimulated human primary myotubes with CCCP, which mimics mitophagy induction in the Ex-leg, and found that BNIP3L/NIX protein abundance decreased independently of lysosomal degradation. Finally, in another human cohort, we found a negative association between BNIP3L/NIX protein abundance with both mitophagosome-like structures and mitochondrial cristae density in the SkM., Conclusion: The findings suggest that a single bout of resistance exercise can initiate mitophagy, potentially involving mitochondrial ejection, in human skeletal muscle. BNIP3L/NIX is proposed as a sensitive marker for assessing mitophagy flux in SkM., (© 2024 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Cancer cachexia, the unintentional loss of lean mass, contributes to functional dependency, poor treatment outcomes, and decreased survival. Although its pathogenicity is multifactorial, metabolic dysfunction remains a hallmark of cachexia. However, significant knowledge gaps exist in understanding the role of skeletal muscle lipid metabolism and dynamics in this condition. We examined skeletal muscle metabolic dysfunction, intramyocellular lipid droplet (LD) content, LD morphology and subcellular distribution, and LD-mitochondrial interactions using the Lewis lung carcinoma (LLC) murine model of cachexia. C57/BL6 male mice ( n = 20) were implanted with LLC cells (10 6 ) in the right flank or underwent PBS sham injections. Skeletal muscle was excised for transmission electron microscopy (TEM; soleus), oil red O/lipid staining [tibialis anterior (TA)], and protein (gastrocnemius). LLC mice had a greater number (232%; P = 0.006) and size (130%; P = 0.023) of intramyocellular LDs further supported by increased oil-red O positive (87%; P = 0.0109) and "very high" oil-red O positive (178%; P = 0.0002) fibers compared with controls and this was inversely correlated with fiber size ( R 2 = 0.5294; P < 0.0001). Morphological analyses of LDs show increased elongation and complexity [aspect ratio: intermyofibrillar (IMF) = 9%, P = 0.046) with decreases in circularity [circularity: subsarcolemmal (SS) = 6%, P = 0.042] or roundness (roundness: whole = 10%, P = 0.033; IMF = 8%, P = 0.038) as well as decreased LD-mitochondria touch (-15%; P = 0.006), contact length (-38%; P = 0.036), and relative contact (86%; P = 0.004). Furthermore, dysregulation in lipid metabolism (adiponectin, CPT1b) and LD-associated proteins, perilipin-2 and perilipin-5, in cachectic muscle ( P < 0.05) were observed. Collectively, we provide evidence that skeletal muscle myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in a preclinical model of cancer cachexia. NEW & NOTEWORTHY We sought to advance our understanding of skeletal muscle lipid metabolism and dynamics in cancer cachexia. Cachexia increased the number and size of intramyocellular lipid droplets (LDs). Furthermore, decreases in LD-mitochondrial touch, contact length, and relative contact along with increased LD shape complexity with decreases in circularity and roundness. Dysregulation in lipid metabolism and LD-associated proteins was also documented. Collectively, we show that myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in cancer cachexia.

The sorting and assembly machinery (SAM) Complex is responsible for assembling β-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle., (© 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

A diet high in saturated fat leads to skeletal muscle deteriorations including insulin resistance, mitochondrial dysfunction and muscle fiber atrophy. Consumption of long-chain polyunsaturated fatty acids and exercise have shown promise in ameliorating high-fat diet (HFD)-induced oxidative stress and inflammation. However, the impact of extra virgin olive oil (EVOO) on mitochondrial homeostasis in muscle is largely unknown. This study aimed to investigate whether 12 wks of EVOO feeding alone and in conjunction with endurance training could protect against metabolic and mitochondrial dysfunction rat muscle with HFD. Female Sprague-Dawley rats were divided into 4 groups fed a control diet (C), HFD, EVOO diet, and EVOO diet with training (EVOO+T). Mitochondrial enzyme activity and protein content decreased with HFD compared to C, but were restored with EVOO and EVOO+T. EVOO+T elevated muscle cytochrome c and PGC-1α levels. HFD increased muscle proteolytic markers and protein ubiquitination, whereas these effects were not seen in EVOO and EVOO+T. HFD suppressed mitochondrial fusion protein level while increasing fission protein levels, but were restored with EVOO and EVOO+T. Mitophagy marker PINK1 content decreased with HFD, but was unchanged in EVOO and EVOO+T. EVOO+T upregulated autophagy markers, along with decreased phosphorylated/dephosphorylated FoxO3 ratio. Antioxidants enzyme levels were upregulated by EVOO and EVOO+T, and EVOO+T reduced HFD-induced lipid peroxidation. In conclusion, HFD impaired muscle oxidative capacity, promoted protein ubiquitination and mitochondrial fission, and upregulated autophagy markers. Replacement of HFD with EVOO corrected the observed adverse effects, while exercise training in conjunction with EVOO provided additional protection to the muscle., (Copyright © 2021. Published by Elsevier Inc.)

Diabetes mellitus is a systemic metabolic disorder associated with mitochondrial dysfunction, with mitochondrial permeability transition (MPT) pore opening being recognized as one of its pathogenic mechanisms. Alisporivir has been recently identified as a non-immunosuppressive analogue of the MPT pore blocker cyclosporin A and has broad therapeutic potential. The purpose of the present work was to study the effect of alisporivir (2.5 mg/kg/day i.p.) on the ultrastructure and functions of the skeletal muscle mitochondria of mice with diabetes mellitus induced by a high-fat diet combined with streptozotocin injections. The glucose tolerance tests indicated that alisporivir increased the rate of glucose utilization in diabetic mice. An electron microscopy analysis showed that alisporivir prevented diabetes-induced changes in the ultrastructure and content of the mitochondria in myocytes. In diabetes, the ADP-stimulated respiration, respiratory control, and ADP / O ratios and the level of ATP synthase in the mitochondria decreased, whereas alisporivir treatment restored these indicators. Alisporivir eliminated diabetes-induced increases in mitochondrial lipid peroxidation products. Diabetic mice showed decreased mRNA levels of Atp5f1a , Ant1 , and Ppif and increased levels of Ant2 in the skeletal muscles. The skeletal muscle mitochondria of diabetic animals were sensitized to the MPT pore opening. Alisporivir normalized the expression level of Ant2 and mitochondrial susceptibility to the MPT pore opening. In parallel, the levels of Mfn2 and Drp1 also returned to control values, suggesting a normalization of mitochondrial dynamics. These findings suggest that the targeting of the MPT pore opening by alisporivir is a therapeutic approach to prevent the development of mitochondrial dysfunction and associated oxidative stress in the skeletal muscles in diabetes.

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca 2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis., Competing Interests: AG, EC, AG, NC, MA, VR, EG, CJ, CS, LL, CS, FJ, VJ, JT, ML, JC, JG, LS, VG No competing interests declared, (© 2021, Ghasemizadeh et al.)

This study aimed to examine the effects of voluntary wheel running on cancer cachexia-induced mitochondrial alterations in mouse skeletal muscle. Mice bearing colon 26 adenocarcinoma (C26) were used as a model of cancer cachexia. C26 mice showed a lower gastrocnemius and plantaris muscle weight, but 4 weeks of voluntary exercise rescued these changes. Further, voluntary exercise attenuated observed declines in the levels of oxidative phosphorylation proteins and activities of citrate synthase and cytochrome c oxidase in the skeletal muscle of C26 mice. Among mitochondrial morphology regulatory proteins, mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1) were decreased in the skeletal muscle of C26 mice, but exercise resulted in similar improvements as seen in markers of mitochondrial content. In isolated mitochondria, 4-hydroxynonenal and protein carbonyls were elevated in C26 mice, but exercise blunted the increases in these markers of oxidative stress. In addition, electron microscopy revealed that exercise alleviated the observed increase in the percentage of damaged mitochondria in C26 mice. These results suggest that voluntary exercise effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cachexia., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness., Competing Interests: The authors have declared that no competing interests exist.

[Figure: see text].

Treatments are lacking for sarcopenia, a debilitating age-related skeletal muscle wasting syndrome. We identifed increased amounts of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the prostaglandin E 2 (PGE 2 )-degrading enzyme, as a hallmark of aged tissues, including skeletal muscle. The consequent reduction in PGE 2 signaling contributed to muscle atrophy in aged mice and results from 15-PGDH-expressing myofibers and interstitial cells, such as macrophages, within muscle. Overexpression of 15-PGDH in young muscles induced atrophy. Inhibition of 15-PGDH, by targeted genetic depletion or a small-molecule inhibitor, increased aged muscle mass, strength, and exercise performance. These benefits arise from a physiological increase in PGE 2 concentrations, which augmented mitochondrial function and autophagy and decreased transforming growth factor-β signaling and activity of ubiquitin-proteasome pathways. Thus, PGE 2 signaling ameliorates muscle atrophy and rejuvenates muscle function, and 15-PGDH may be a suitable therapeutic target for countering sarcopenia., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mitochondrial dysfunction is a well-characterized consequence of spinal cord injury (SCI). We previously reported that treatment with the FDA-approved β 2 -adrenergic receptor agonist formoterol beginning 8 h post-SCI induces mitochondrial biogenesis (MB) and improves body composition and locomotor recovery in female mice. To determine the time-to-treatment window of formoterol, female mice were subjected to 80 kdyn contusion SCI and daily administration of vehicle or formoterol (0.3 mg/kg) beginning 24 h after injury. This delayed treatment paradigm improved body composition in female mice by 21 DPI, returning body weight to pre-surgery weight and restoring gastrocnemius mass to sham levels; however, there was no effect on locomotor recovery, as measured by the Basso-Mouse Scale (BMS), or lesion volume. To assess the cross-sex potential of formoterol, injured male mice were treated with vehicle or formoterol (0.3 or 1.0 mg/kg) beginning 8 h after SCI. Formoterol also improved body composition post-SCI in male mice, restoring body weight and muscle mass regardless of dose. Interestingly, however, improved BMS scores and decreased lesion volume was observed only in male mice treated with 0.3 mg/kg. Additionally, 0.3 mg/kg formoterol induced MB in the gastrocnemius and injured spinal cord, as evidenced by increased MB protein expression and mitochondrial number. These data indicate that formoterol treatment improves recovery post-SCI in both male and female mice in a dose- and initiation time-dependent manner. Furthermore, formoterol-induced functional recovery post-SCI is not directly associated with peripheral effects, such as muscle mass and body weight., (Published by Elsevier Inc.)

The efficient production of energy via oxidative phosphorylation is essential to the growth, survival, and reproduction of eukaryotes. The behavior (position of, and communication between, mitochondria) and morphology of mitochondria play key roles in efficient energy production and are influenced by oxidative stressors such as ultraviolet (UV) radiation. We tested the hypothesis that mitochondria change their behavior and morphology to meet energetic demands of responding to changes in oxidative stress. Specifically, we predicted that UV irradiation would increase the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions to influence whole-animal metabolic rate. Using transmission electron microscopy, we found that both three and six hours of UV-A/B irradiation (0.5 W/m 2 ) increased the proportion of inter-mitochondrial junctions (with increasing mitochondrial aspect ratio) and the density of inner mitochondrial membrane in myocytes of Tigriopus californicus copepods. Mitochondrial density increased following both irradiation treatments, but mitochondrial size decreased under the six hour treatment. Metabolic rate was maintained under three hours of irradiation but decreased following six hours of exposure. These observations demonstrate that the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions can play formative roles in maintaining whole-animal metabolic rate, and ultimately organismal performance, under exposure to an oxidative stressor., (Copyright © 2020 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine. Using the linear attenuation coefficient, the mitochondria were identified (0.247 ± 0.04 µm -1 ) presenting average dimensions of 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width. The internal mitochondria structure was successfully identified by reaching up the limit of spatial resolution of 35 nm. The internal mitochondrial membranes invagination (cristae) complexity was calculated by the mitochondrial complexity index (MCI) providing quantitative and morphological information of mitochondria larger than 0.90 mm in length. The segmentation to visualize the cristae invaginations into the mitochondrial matrix was possible in mitochondria with MCI ≥ 7. Altogether, we demonstrated that the MCI is a valuable quantitative morphological parameter to evaluate cristae modelling and can be applied to compare healthy and disease state associated to mitochondria morphology.

Glutamine and glucose are both oxidized in the mitochondria to supply the majority of usable energy for processes of cellular function. Low levels of plasma and skeletal muscle glutamine are associated with severe illness. We hypothesized that glutamine deficiency would disrupt mitochondrial integrity and impair cell function. C2C12 mouse myoblasts were cultured in control media supplemented with 5.6 mmol/L glucose and 2 mmol/L glutamine, glutamine depletion (Gln - ) or glucose depletion (Glc - ) media. We compared mitochondrial morphology and function, as well as cell proliferation, myogenic differentiation, and heat-shock response in these cells. Glc - cells exhibited slightly elongated mitochondrial networks and increased mitochondrial mass, with normal membrane potential (ΔΨm). Mitochondria in Gln - cells became hyperfused and swollen, which were accompanied by severe disruption of cristae and decreases in ΔΨm, mitochondrial mass, the inner mitochondrial membrane remodeling protein OPA1, electron transport chain complex IV protein expression, and markers of mitochondrial biogenesis and bioenergetics. In addition, Gln - increased the autophagy marker LC3B-II on the mitochondrial membrane. Notably, basal mitochondrial respiration was increased in Glc - cells as compared to control cells, whereas maximal respiration remained unchanged. In contrast, basal respiration, maximal respiration and reserve capacity were all decreased in Gln - cells. Consistent with the aforementioned mitochondrial deficits, Gln - cells had lower growth rates and myogenic differentiation, as well as a higher rate of cell death under heat stress conditions than Glc - and control cells. We conclude that glutamine is essential for mitochondrial integrity and function; glutamine depletion impairs myoblast proliferation, differentiation, and the heat-shock response., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Qiangji Jianli Decoction (QJJLD) is an effective Chinese medicine formula for treating Myasthenia gravis (MG) in the clinic. QJJLD has been proven to regulate mitochondrial fusion and fission of skeletal muscle in myasthenia gravis. In this study, we investigated whether QJJLD plays a therapeutic role in regulating mitochondrial biogenesis in MG and explored the underlying mechanism. Rats were experimentally induced to establish autoimmune myasthenia gravis (EAMG) by subcutaneous immunization with R97-116 peptides. The treatment groups were administered three different dosages of QJJLD respectively. After the intervention of QJJLD, the pathological changes of gastrocnemius muscle in MG rats were significantly improved; SOD, GSH-Px, Na + -K + ATPase and Ca 2+ -Mg 2+ ATPase activities were increased; and MDA content was decreased in the gastrocnemius muscle. Moreover, AMPK, p38MAPK, PGC-1α, NRF-1, Tfam and COX IV mRNA and protein expression levels were also reversed by QJJLD. These results implied that QJJLD may provide a potential therapeutic strategy through promoting mitochondrial biogenesis to alleviate MG via activating the AMPK/PGC-1α signaling pathway., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) manifests pathological changes in motor neurons and various other cell types. Compared to motor neurons, the contribution of the other cell types to the ALS phenotypes is understudied. G4C2 repeat expansion in C9ORF72 is the most common genetic cause of ALS along with frontotemporal dementia (C9-ALS/FTD), with increasing evidence supporting repeat-encoded poly(GR) in disease pathogenesis. Here, we show in Drosophila muscle that poly(GR) enters mitochondria and interacts with components of the Mitochondrial Contact Site and Cristae Organizing System (MICOS), altering MICOS dynamics and intra-subunit interactions. This impairs mitochondrial inner membrane structure, ion homeostasis, mitochondrial metabolism, and muscle integrity. Similar mitochondrial defects are observed in patient fibroblasts. Genetic manipulation of MICOS components or pharmacological restoration of ion homeostasis with nigericin effectively rescue the mitochondrial pathology and disease phenotypes in both systems. These results implicate MICOS-regulated ion homeostasis in C9-ALS pathogenesis and suggest potential new therapeutic strategies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The measurement of mitochondrial content is essential for bioenergetic research, as it provides a tool to evaluate whether changes in mitochondrial function are strictly due to changes in content or other mechanisms that influence function. In this perspective, we argue that commonly used biomarkers of mitochondrial content may possess limited utility for capturing changes in content with physiological intervention. Moreover, we argue that they may not provide reliable estimates of content in certain pathological situations. Finally, we discuss potential solutions to overcome issues related to the utilization of biomarkers of mitochondrial content. Shedding light on this important issue will hopefully aid conclusions about the mitochondrial structure-function relationship.

Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca 2+ homeostasis. This work examines the kinetic parameters of Ca 2+ transport and the opening of the Ca 2+ -dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca 2+ uniport, with the kinetics of Na + -dependent Ca 2+ efflux not changing. The data obtained indicate that the decreased rate of Ca 2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca 2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca 2+ homeostasis of skeletal-muscle mitochondria., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Charcot-Marie-Tooth disease (CMT) is a group of inherited neurological disorders of the peripheral nervous system. CMT is subdivided into two main types: a demyelinating form, known as CMT1, and an axonal form, known as CMT2. Nearly 30 genes have been identified as a cause of CMT2. One of these is the 'dehydrogenase E1 and transketolase domain containing 1' (DHTKD1) gene. We previously demonstrated that a nonsense mutation [c.1455 T > G (p.Y485*)] in exon 8 of DHTKD1 is one of the disease-causing mutations in CMT2Q (MIM 615025). The aim of the current study was to investigate whether human disease-causing mutations in the Dhtkd1 gene cause CMT2Q phenotypes in a mouse model in order to investigate the physiological function and pathogenic mechanisms associated with mutations in the Dhtkd1 gene in vivo. Therefore, we generated a knock-in mouse model with the Dhtkd1 Y486* point mutation. We observed that the Dhtkd1 expression level in sciatic nerve of knock-in mice was significantly lower than in wild-type mice. Moreover, a histopathological phenotype was observed, reminiscent of a peripheral neuropathy, including reduced large axon diameter and abnormal myelination in peripheral nerves. The knock-in mice also displayed clear sensory defects, while no abnormalities in the motor performance were observed. In addition, accumulation of mitochondria and an elevated energy metabolic state was observed in the knock-in mice. Taken together, our study indicates that the Dhtkd1 Y486* knock-in mice partially recapitulate the clinical phenotypes of CMT2Q patients and we hypothesize that there might be a compensatory effect from the elevated metabolic state in the knock-in mice that enables them to maintain their normal locomotor function.

Mitochondrial dysfunction has been implicated as a central mechanism in the metabolic myopathy accompanying critical limb ischemia (CLI). However, whether mitochondrial dysfunction is directly related to lower extremity ischemia and the structural and molecular mechanisms underpinning mitochondrial dysfunction in CLI patients is not understood. Here, we aimed to study whether mitochondrial dysfunction is a distinctive characteristic of CLI myopathy by assessing mitochondrial respiration in gastrocnemius muscle from 14 CLI patients (65.3 ± 7.8 y) and 15 matched control patients (CON) with a similar comorbidity risk profile and medication regimen but without peripheral ischemia (67.4 ± 7.4 y). Furthermore, we studied potential structural and molecular mechanisms of mitochondrial dysfunction by measuring total, sub-population, and fiber-type-specific mitochondrial volumetric content and cristae density with transmission electron microscopy and by assessing mitophagy and fission/fusion-related protein expression. Finally, we asked whether commonly used biomarkers of mitochondrial content are valid in patients with cardiovascular disease. CLI patients exhibited inferior mitochondrial respiration compared to CON. This respiratory deficit was not related to lower whole-muscle mitochondrial content or cristae density. However, stratification for fiber types revealed ultrastructural mitochondrial alterations in CLI patients compared to CON. CLI patients exhibited an altered expression of mitophagy-related proteins but not fission/fusion-related proteins compared to CON. Citrate synthase, cytochrome c oxidase subunit IV (COXIV), and 3-hydroxyacyl-CoA dehydrogenase (β-HAD) could not predict mitochondrial content. Mitochondrial dysfunction is a distinctive characteristic of CLI myopathy and is not related to altered organelle content or cristae density. Our results link this intrinsic mitochondrial deficit to dysregulation of the mitochondrial quality control system, which has implications for the development of therapeutic strategies., Competing Interests: The authors declare no conflict of interest.

Background: Canagliflozin (CANA) administration increases the risk of lower limb amputation in the clinic. The present study aimed to investigate whether and how CANA interferes with the intracellular physiological processes of bone marrow derived mesenchymal stem cells (BM-MSCs) and its contribution to ischaemic lower limb., Methods: The in vivo blood flow recovery in ischaemic lower limbs following CANA treatment was evaluated. The cellular function of BM-MSCs after CANA treatment were also assessed in vitro. In silico docking analysis and mutant substitution assay were conducted to confirm the interaction of CANA with glutamate dehydrogenase 1 (GDH1)., Findings: Following CANA treatment, attenuated angiogenesis and hampered blood flow recovery in the ischaemic region were detected in diabetic and non-diabetic mice, and inhibition of the proliferation and migration of BM-MSCs were also observed. CANA was involved in mitochondrial respiratory malfunction in BM-MSCs and the inhibition of ATP production, cytochrome c release and vessel endothelial growth factor A (VEGFA) secretion, which may contribute to reductions in the tissue repair capacity of BM-MSCs. The detrimental effects of CANA on MSCs result from the inhibition of GDH1 by CANA (evidenced by in silico docking analysis and H199A-GDH1/N392A-GDH1 mutant substitution)., Interpretation: Our work highlights that the inhibition of GDH1 activity by CANA interferes with the metabolic activity of the mitochondria, and this interference deteriorates the retention of and VEGFA secretion by MSCs., Funding: National Natural Science Foundation of China, Natural Science Foundation of Zhejiang Province and Wenzhou Science and Technology Bureau Foundation., Competing Interests: Declaration of Competing Interest All of the authors declare no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The main objective of this work was to investigate whether mitochondrial fusion occurs in the skeletal muscle of well-trained athletes in response to high-intensity exercise. Well-trained swimmers ( n = 9) performed a duration-matched sprint interval training (SIT) and high-intensity high-volume training (HIHVT) session on separate days. Muscle samples from triceps brachii were taken before, immediately after, and 3 h after the training sessions. Transmission electron microscopy (TEM) was applied to assess mitochondrial morphology. Moreover, expression of genes coding for regulators of mitochondrial fusion and fission were assessed by real-time quantitative PCR. In addition, mitofusin (MFN)2 and optic atrophy 1 (OPA1) were quantified by Western blot analysis. TEM analyses showed that mitochondrial morphology remained altered for 3 h after HIHVT, whereas SIT-induced changes were only evident immediately after exercise. Only SIT increased MFN1 and MFN2 mRNA expression, whereas SIT and HIHVT both increased MFN2 protein content 3 h after exercise. Notably, only HIHVT increased OPA1 protein content. Mitochondrial morphologic changes that suggest fusion occurs in well-adapted athletes during exercise. However, HIHVT appears as a more robust inducer of mitochondrial fusion events than SIT. Indeed, SIT induces a rapid and transient change in mitochondrial morphology.-Huertas, J. R., Ruiz-Ojeda, F. J., Plaza-Díaz, J., Nordsborg, N. B., Martín-Albo, J., Rueda-Robles, A., Casuso, R. A. Human muscular mitochondrial fusion in athletes during exercise.

Exercise training performed with lowered muscle glycogen stores can amplify adaptations related to oxidative metabolism, but it is not known if this is affected by the "train-low" strategy used (i.e., once-daily versus twice-a-day training). Fifteen healthy men performed 3 wk of an endurance exercise (100-min) followed by a high-intensity interval exercise 2 (twice-a-day group, n = 8) or 14 h (once-daily group, n = 7) later; therefore, the second training session always started with low muscle glycogen in both groups. Mitochondrial efficiency (state 4 respiration) was improved only for the twice-a-day group (group × training interaction, P < 0.05). However, muscle citrate synthase activity, mitochondria, and lipid area in intermyofibrillar and subsarcolemmal regions, and PGC1α, PPARα, and electron transport chain relative protein abundance were not altered with training in either group ( P > 0.05). Markers of aerobic fitness (e.g., peak oxygen uptake) were increased, and plasma lactate, O 2 cost, and rating of perceived exertion during a 100-min exercise task were reduced in both groups, although the reduction in rating of perceived exertion was larger in the twice-a-day group (group × time × training interaction, P < 0.05). These findings suggest similar training adaptations with both training low approaches; however, improvements in mitochondrial efficiency and perceived effort seem to be more pronounced with twice-a-day training. NEW & NOTEWORTHY We assessed, for the first time, the differences between two "train-low" strategies (once-daily and twice-a-day) in terms of training-induced molecular, functional, and morphological adaptations. We found that both strategies had similar molecular and morphological adaptations; however, only the twice-a-day strategy increased mitochondrial efficiency and had a superior reduction in the rating of perceived exertion during a constant-load exercise compared with once-daily training. Our findings provide novel insights into skeletal muscle adaptations using the "train-low" strategy.

Congenital muscular dystrophy with megaconial myopathy (MDCMC) is an autosomal recessive disorder characterized by progressive muscle weakness and wasting. The observation of megamitochondria in skeletal muscle biopsies is exclusive to this type of MD. The disease is caused by loss of function mutations in the choline kinase beta (CHKB) gene which results in dysfunction of the Kennedy pathway for the synthesis of phosphatidylcholine. We have previously reported a rostrocaudal MD (rmd) mouse with a deletion in the Chkb gene resulting in an MDCMC-like phenotype, and we used this mouse to test gene therapy strategies for the rescue and alleviation of the dystrophic phenotype. Introduction of a muscle-specific Chkb transgene completely rescues motor and behavioral function in the rmd mouse model, confirming the cell-autonomous nature of the disease. Intramuscular gene therapy post-disease onset using an adeno-associated viral 6 (AAV6) vector carrying a functional copy of Chkb is also capable of rescuing the dystrophy phenotype. In addition, we examined the ability of choline kinase alpha (Chka), a gene paralog of Chkb, to improve dystrophic phenotypes when upregulated in skeletal muscles of rmd mutant mice using a similar AAV6 vector. The sum of our results in a preclinical model of disease suggest that replacement of the Chkb gene or upregulation of endogenous Chka could serve as potential lines of therapy for MDCMC patients., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Skeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Aberrant proliferation of vascular smooth muscle cells (VSMC) is a critical contributor to the pathogenesis of atherosclerosis (AS). Our previous studies have demonstrated that apelin-13/APJ confers a proliferative response in VSMC, however, its underlying mechanism remains elusive. In this study, we aimed to investigate the role of mitophagy in apelin-13-induced VSMC proliferation and atherosclerotic lesions in apolipoprotein E knockout (ApoE-/-) mice. Apelin-13 enhances human aortic VSMC proliferation and proliferative regulator proliferating cell nuclear antigen expression in dose and time-dependent manner, while is abolished by APJ antagonist F13A. We observe the engulfment of damage mitochondria by autophagosomes (mitophagy) of human aortic VSMC in apelin-13 stimulation. Mechanistically, apelin-13 increases p-AMPKα and promotes mitophagic activity such as the LC3I to LC3II ratio, the increase of Beclin-1 level and the decrease of p62 level. Importantly, the expressions of PINK1, Parkin, VDAC1, and Tom20 are induced by apelin-13. Conversely, blockade of APJ by F13A abolishes these stimulatory effects. Human aortic VSMC transfected with AMPKα, PINK1, or Parkin and subjected to apelin-13 impairs mitophagy and prevents proliferation. Additional, apelin-13 not only increases the expression of Drp1 but also reduces the expressions of Mfn1, Mfn2, and OPA1. Remarkably, the mitochondrial division inhibitor-1(Mdivi-1), the pharmacological inhibition of Drp1, attenuates human aortic VSMC proliferation. Treatment of ApoE-/- mice with apelin-13 accelerates atherosclerotic lesions, increases p-AMPKα and mitophagy in aortic wall in vivo. Finally, PINK1-/- mutant mice with apelin-13 attenuates atherosclerotic lesions along with defective in mitophagy. PINK1/Parkin-mediated mitophagy promotes apelin-13-evoked human aortic VSMC proliferation by activating p-AMPKα and exacerbates the progression of atherosclerotic lesions., (© 2018 Wiley Periodicals, Inc.)

Loss of skeletal muscle mass and function is a hallmark of aging. This phenomenon has been related to a dysregulation of mitochondrial function and proteostasis. Calorie restriction (CR) has been demonstrated to delay aging and preserve function until late in life, particularly in muscle. Recently, we reported the type of dietary fat plays an important role in determining life span extension with 40% CR in male mice. In these conditions, lard fed mice showed an increased longevity compared to mice fed soybean or fish oils. In this article, we analyze the effect of 40% CR on muscle mitochondrial mass, autophagy, and mitochondrial dynamics markers in mice fed these diets. In CR fed animals, lard preserved muscle fibers structure, mitochondrial ultrastructure, and fission/fusion dynamics and autophagy, not only compared to control animals, but also compared with CR mice fed soybean and fish oils as dietary fat. We focus our discussion on dietary fatty acid saturation degree as an essential predictor of life span extension in CR mice., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mitochondria are essential components of eukaryotic cells, carrying out critical physiological processes that include energy production and calcium buffering. Consequently, mitochondrial dysfunction is associated with a range of human diseases. Fundamental to their function is the ability to transition through fission and fusion states, which is regulated by several GTPases. Here, we have developed new methods for the non-subjective quantification of mitochondrial morphology in muscle and neuronal cells of Caenorhabditis elegans. Using these techniques, we uncover surprising tissue-specific differences in mitochondrial morphology when fusion or fission proteins are absent. From ultrastructural analysis, we reveal a novel role for the fusion protein FZO-1/mitofusin 2 in regulating the structure of the inner mitochondrial membrane. Moreover, we have determined the influence of the individual mitochondrial fission (DRP-1/DRP1) and fusion (FZO-1/mitofusin 1,2; EAT-3/OPA1) proteins on animal behaviour and lifespan. We show that loss of these mitochondrial fusion or fission regulators induced age-dependent and progressive deficits in animal movement, as well as in muscle and neuronal function. Our results reveal that disruption of fusion induces more profound defects than lack of fission on animal behaviour and tissue function, and imply that while fusion is required throughout life, fission is more important later in life likely to combat ageing-associated stressors. Furthermore, our data demonstrate that mitochondrial function is not strictly dependent on morphology, with no correlation found between morphological changes and behavioural defects. Surprisingly, we find that disruption of either mitochondrial fission or fusion significantly reduces median lifespan, but maximal lifespan is unchanged, demonstrating that mitochondrial dynamics play an important role in limiting variance in longevity across isogenic populations. Overall, our study provides important new insights into the central role of mitochondrial dynamics in maintaining organismal health.

Objective: To investigate the ultrastructural features of muscle in patients with mitochondrial encephalomyopathy for its diagnosis and differential diagnosis. Methods: The clinical data of 27 mitochondrial encephalomyopathy patients who underwent left or right biceps brachii muscle biopsy at Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University from July 2006 to August 2017 were analyzed retrospectively. The muscle biopsy specimens were examined underlight microscope and transmission electron microscope. Results: There were 27 patients (17 males, 10 females) with an age range of 12 to 62 years (mean 29 years). The age of onset ranged from 3 to 38 years. The course of disease ranged from 1 month to 24 years. Twenty-two cases presented with lactic acidosis and stroke-like episodes (MELAS) syndrome, four with myoclonic epilepsy with ragged red fibers (MERRF) syndrome, and one with chronic progressive paralysis of extraocular muscle (CPEO) syndrome. Skeletal muscle biopsy showed abundant ragged red fibers and strongly SDH-reactive vessel. Genetic studies showed 17 of 22 cases of MELAS syndrome had A3243G mutation, and the other 5 cases had no abnormality. A8344G mutation was found in 3 of 4 cases of MERRF syndrome. No single or multiple mtDNA mutations were found in the single case of CPEO. Transmission electron microscopy of all 27 cases showed diffuse proliferation of mitochondria between the myofibrils and beneath the sarcolemma, with increased spacing between muscle cells. Seven cases showed numerous glycogen and four showed subsarcolemmal lipid droplets, 13 cases showed unusual mitochondrial morphology, including mitochondrial electron-dense substances and paracrystal line inclusions ("parking lot" change)in eight cases. Conclusions: Transmission electron microscopy shows significant differences in ultrastructural pathological changes among different patients with mitochondrial encephalomyopathy. Some patients with mild clinical symptoms have increased mitochondrial number, increased metabolism of glycogen and lipid droplets, while others with severe clinical symptoms have abnormal mitochondrial morphology. Typical crystalloid inclusions are found in mitochondria, which are of great value in the diagnosis of this disease.

Skeletal muscle is composed of different myofiber types that preferentially use glucose or lipids for ATP production. How fuel preference is regulated in these post-mitotic cells is largely unknown, making this issue a key question in the fields of muscle and whole-body metabolism. Here, we show that microRNAs (miRNAs) play a role in defining myofiber metabolic profiles. mRNA and miRNA signatures of all myofiber types obtained at the single-cell level unveiled fiber-specific regulatory networks and identified two master miRNAs that coordinately control myofiber fuel preference and mitochondrial morphology. Our work provides a complete and integrated mouse myofiber type-specific catalog of gene and miRNA expression and establishes miR-27a-3p and miR-142-3p as regulators of lipid use in skeletal muscle., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The QIl1 gene produces a component of the Mitochondrial Contact Site and Cristae Organizing System that forms and stabilizes mitochondrial cristae junctions and is important in cellular energy production. We previously reported a family of Rhodesian Ridgebacks with cardiac arrhythmias and sudden cardiac death. Here, we performed whole genome sequencing on a trio from the family. Variant calling was performed using a standardized bioinformatics approach. Variants were filtered against variants from 247 dogs of 43 different breeds. High impact variants were validated against additional affected and unaffected dogs. A single missense G/A variant in the QIL1 gene was associated with the cardiac arrhythmia ( p < 0.0001). The variant was predicted to change the amino acid from conserved Glycine to Serine and to be deleterious. Ultrastructural analysis of the biceps femoris muscle from an affected dog revealed hyperplastic mitochondria, cristae rearrangement, electron dense inclusions and lipid bodies. We identified a variant in the Q1l1 gene resulting in a mitochondrial cardiomyopathy characterized by cristae abnormalities and cardiac arrhythmias in a canine model. This natural animal model of mitochondrial cardiomyopathy provides a large animal model with which to study the development and progression of disease as well as genotypic phenotypic relationships.

Aim: The subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria in skeletal muscle appear to have distinct biochemical properties affecting metabolism in health and disease. The isolation of mitochondrial subpopulations has been a long-time challenge while the presence of a continuous mitochondrial reticulum challenges the view of distinctive SSM and IFM bioenergetics. Here, a comprehensive approach is developed to identify the best conditions to separate mitochondrial fractions., Methods: The main modifications to the protocol to isolate SSM and IFM from rat skeletal muscle were: (a) decreased dispase content and homogenization speed; (b) trypsin treatment of SSM fractions; (c) recentrifugation of mitochondrial fractions at low speed to remove subcellular components. To identify the conditions preserving mitochondrial function, integrity, and maximizing their recovery, microscopy (light and electron) were used to monitor effectiveness and efficiency in separating mitochondrial subpopulations while respiratory and enzyme activities were employed to evaluate function, recovery, and integrity., Results: With the modifications described, the total mitochondrial yield increased with a recovery of 80% of mitochondria contained in the original skeletal muscle sample. The difference between SSM and IFM oxidative capacity (10%) with complex-I substrate was significant only with a saturated ADP concentration. The inner and outer membrane damage for both subpopulations was <1% and 8%, respectively, while the respiratory control ratio was 16., Conclusion: Using a multidisciplinary approach, conditions were identified to maximize SSM and IFM recovery while preserving mitochondrial integrity, biochemistry, and morphology. High quality and recovery of mitochondrial subpopulations allow to study the relationship between these organelles and disease., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: Skeletal muscle mitochondrial content and function appear to be altered in obesity. Mitochondria in muscle are found in well-defined regions within cells, and they are arranged in a way that form distinct subpopulations of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We sought to investigate differences in the proteomes of SS and IMF mitochondria between lean subjects and subjects with obesity., Methods: We performed comparative proteomic analyses on SS and IMF mitochondria isolated from muscle samples obtained from lean subjects and subjects with obesity. Mitochondria were isolated using differential centrifugation, and proteins were subjected to label-free quantitative tandem mass spectrometry analyses. Collected data were evaluated for abundance of mitochondrial proteins using spectral counting. The Reactome pathway database was used to determine metabolic pathways that are altered in obesity., Results: Among proteins, 73 and 41 proteins showed different (mostly lower) expression in subjects with obesity in the SS and IMF mitochondria, respectively (false discovery rate-adjusted P ≤ 0.05). We specifically found an increase in proteins forming the tricarboxylic acid cycle and electron transport chain (ETC) complex II, but a decrease in proteins forming protein complexes I and III of the ETC and adenosine triphosphate (ATP) synthase in subjects with obesity in the IMF, but not SS, mitochondria. Obesity was associated with differential effects on metabolic pathways linked to protein translation in the SS mitochondria and ATP formation in the IMF mitochondria., Conclusions: Obesity alters the expression of mitochondrial proteins regulating key metabolic processes in skeletal muscle, and these effects are distinct to mitochondrial subpopulations located in different regions of the muscle fibers., Trial Registration: ClinicalTrials.gov (NCT01824173)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The pro-inflammatory cytokine interleukin-15 (IL15) and its receptor α (IL15RA) participate in the regulation of musculoskeletal function and metabolism. Deletion of the Il15ra gene in mice increases spontaneous activity, improves fatigue resistance in the glycolytic extensor digitorum longus (EDL) and protects from diet-induced obesity. In humans, IL15RA single-nucleotide polymorphisms (SNPs) have been linked to muscle strength, metabolism and performance in elite endurance athletes. Taken together, these features suggest a possible role for IL15RA in muscle mitochondrial structure and function. Here, we have investigated the consequences of loss of IL15RA on skeletal muscle fiber-type properties and mitochondrial ultrastructure. Immunostaining of the EDL for myosin heavy chain (MyHC) isoforms revealed no significant changes in fiber type. Electron microscopy (EM) analysis of the EDL indicated an overall higher mitochondria content, and increased cristae density in subsarcolemmal and A-band mitochondrial subpopulations. The higher cristae density in Il15ra -/- mitochondria was associated with higher OPA1 and cardiolipin levels. Overall, these data extend our understanding of the role of IL15RA signaling in muscle oxidative metabolism and adaptation to exercise., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Scope: In this study, we aim to determine the effects of resveratrol (RSV) on muscle atrophy in streptozocin-induced diabetic mice and to explore mitochondrial quality control (MQC) as a possible mechanism., Methods and Results: The experimental mice were fed either a control diet or an identical diet containing 0.04% RSV for 8 weeks. Examinations were subsequently carried out, including the effects of RSV on muscle atrophy and muscle function, as well as on the signaling pathways related to protein degradation and MQC processes. The results show that RSV supplementation improves muscle atrophy and muscle function, attenuates the increase in ubiquitin and muscle RING-finger protein-1 (MuRF-1), and simultaneously attenuates LC3-II and cleaved caspase-3 in the skeletal muscle of diabetic mice. Moreover, RSV treatment of diabetic mice results in an increase in mitochondrial biogenesis and inhibition of the activation of mitophagy in skeletal muscle. RSV also protects skeletal muscle against excess mitochondrial fusion and fission in the diabetic mice., Conclusion: The results suggest that RSV ameliorates diabetes-induced skeletal muscle atrophy by modulating MQC., (© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mitochondria regulate the balance between lipid metabolism and storage in the skeletal muscle. Altered lipid transport, metabolism and storage influence the bioenergetics, redox status and insulin signalling, contributing to cardiac and neurological diseases. Lipid storage disorders (LSDs) are neurological disorders which entail intramuscular lipid accumulation and impaired mitochondrial bioenergetics in the skeletal muscle causing progressive myopathy with muscle weakness. However, the mitochondrial changes including molecular events associated with impaired lipid storage have not been completely understood in the human skeletal muscle. We carried out morphological and biochemical analysis of mitochondrial function in muscle biopsies of human subjects with LSDs (n = 7), compared to controls (n = 10). Routine histology, enzyme histochemistry and ultrastructural analysis indicated altered muscle cell morphology and mitochondrial structure. Protein profiling of the muscle mitochondria from LSD samples (n = 5) (vs. control, n = 5) by high-throughput mass spectrometric analysis revealed that impaired metabolic processes could contribute to mitochondrial dysfunction and ensuing myopathy in LSDs. We propose that impaired fatty acid and respiratory metabolism along with increased membrane permeability, elevated lipolysis and altered cristae entail mitochondrial dysfunction in LSDs. Some of these mechanisms were unique to LSD apart from others that were common to dystrophic and inflammatory muscle pathologies. Many differentially regulated mitochondrial proteins in LSD are linked with other human diseases, indicating that mitochondrial protection via targeted drugs could be a treatment modality in LSD and related metabolic diseases. Cover Image for this Issue: doi: 10.1111/jnc.14177., (© 2018 International Society for Neurochemistry.)

Purpose Acute lower extremity ischemia is still a main cause of mortality and morbidity in orthopedic traumatology and reconstructive surgery. In acute lower extremity ischemia, the skeletal muscles are the tissues that are the most vulnerable to ischemia. The aim of this study was to evaluate the effects of iloprost (IL) therapy on skeletal muscle contractile impairment and mitochondrial degeneration in an acute lower extremity ischemia-reperfusion rat model. Main Methods Forty Wistar albino rats were randomly divided into a control group and four experimental groups. Experimental groups were either subjected to 2 h of lower extremity ischemia followed by a 4-h reperfusion period or to 4 h of ischemia followed by an 8-h reperfusion period. Except for the animals in the control group, all animals received IL (1 ng/kg/min) or saline (1 ml/kg) by intraperitoneal infusion for 10 min immediately before reperfusion. At the end of the recording of skeletal muscle electrical activity and contractility, all rats were sacrificed by decapitation and muscle samples of lower extremity were immediately harvested for histopathologic analyses. Results After ischemia-reperfusion, a breakdown in the force-frequency curves of extensor digitorum longus muscle was observed, showing the diminished muscle contractility. However, IL significantly improved muscle contractility following injury induced by 2 h of ischemia followed by a 4-h reperfusion period. In addition, IL partially ameliorated mitochondrial degeneration in the muscle cells of ischemia groups. Conclusion This study indicates that immediate IL therapy repairs muscle damage especially after 2 h of ischemia and 4 h of reperfusion and therefore that IL improves contractile function.

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic β cyto - or γ cyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific β cyto - and γ cyto -actin KO mice. We found β cyto - and γ cyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking β cyto - and/or γ cyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific β cyto - and γ cyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old β cyto - and γ cyto -actin muscle-specific KO mice. Our data suggest that impaired Ca 2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy., (© 2017 Federation of European Biochemical Societies.)

Sarcopenia is the degenerative loss of muscle mass and strength with aging. Although a role of mitochondrial metabolism in muscle function and in the development of many diseases has been described, the role of mitochondrial topology and dynamics in the process of muscle aging is not fully understood. This work shows a time line of changes in both mitochondrial distribution and skeletal muscle function during mice lifespan. We isolated muscle fibers from flexor digitorum brevis of mice of different ages. A fusion-like phenotype of mitochondria, together with a change in orientation perpendicular to the fiber axis was evident in the Adult group compared to Juvenile and Older groups. Moreover, an increase in the contact area between sarcoplasmic reticulum and mitochondria was evident in the same group. Together with the morphological changes, mitochondrial Ca 2+ resting levels were reduced at age 10-14 months and significantly increased in the Older group. This was consistent with a reduced number of mitochondria-to-jSR pairs in the Older group compared to the Juvenile. Our results support the idea of several age-dependent changes in mitochondria that are accentuated in midlife prior to a complete sarcopenic phenotype.

The spatial density of mitochondria was studied by thin-section electron microscopy in smooth muscles of bladder, iris and gut in mice, rats, guinea-pigs and sheep. Morphometric data included areas of muscle cell profiles (~6,000 muscle cells were measured) and areas of their mitochondria (more than three times as many). The visual method delivers accurate estimates of the extent of the chondrioma (the ensemble of mitochondria in a cell), measuring all and only the mitochondria in each muscle cell and no other cells. The digital records obtained can be used again for checks and new searches. Spatial density of mitochondria varies between about 2 and 10% in different muscles in different species. In contrast, there is consistency of mitochondrial density within a given muscle in a given species. For each muscle in each species there is a characteristic mitochondrial density with modest variation between experiments. On the basis of data from serial sections in the rat detrusor muscle, mitochondrial density varies very little between the muscle cells, each cell having a value close to that for the whole muscle. Mitochondrial density is different in a given muscle, e.g., ileal circular muscle, from the four mammalian species, with highest values in mouse and lowest in sheep; in mice the mitochondrial density is nearly three time higher that in sheep. In a given species there are characteristic variations between different muscles. For example, the bladder detrusor muscle has markedly fewer mitochondria than the ileum, and the iris has markedly more.

Introduction: Botulinum neurotoxin A (BoNTA) has long been used as a therapeutic agent and has been widely accepted as a cosmetic agent in recent years. It can inhibit function and induce structural changes in skeletal muscle., Methods: Specimens of fresh dissected human masseter muscle were used to observe the ultrastructural changes that occurred at 6 and 12 months following BoNTA injection., Results: The findings observed were muscle fiber distortion, sarcomere shortening, mitochondrial vacuolar degeneration, glycogen accumulation, and H and M band disruption in the triad of tubules. At 12 months after injection, there was still evidence of degenerative changes in muscle ultrastructure, whereas most organelles exhibited a normal structure., Discussion: Profound ultrastructural and organelle disfiguring changes were observed after BoNTA injection into human masseter muscle. Most changes were transient, however, and were resolved by 12 months after injection. Muscle Nerve 57: 96-99, 2018., (© 2017 Wiley Periodicals, Inc.)

Aims: (i) To determine whether exercise-induced increases in muscle mitochondrial volume density (Mito VD ) are related to enlargement of existing mitochondria or de novo biogenesis and (ii) to establish whether measures of mitochondrial-specific enzymatic activities are valid biomarkers for exercise-induced increases in Mito VD ., Method: Skeletal muscle samples were collected from 21 healthy males prior to and following 6 weeks of endurance training. Transmission electron microscopy was used for the estimation of mitochondrial densities and profiles. Biochemical assays, western blotting and high-resolution respirometry were applied to detect changes in specific mitochondrial functions., Result: Mito VD increased with 55 ± 9% (P < 0.001), whereas the number of mitochondrial profiles per area of skeletal muscle remained unchanged following training. Citrate synthase activity (CS) increased (44 ± 12%, P < 0.001); however, there were no functional changes in oxidative phosphorylation capacity (OXPHOS, CI+II P ) or cytochrome c oxidase (COX) activity. Correlations were found between Mito VD and CS (P = 0.01; r = 0.58), OXPHOS, CI+CIIP (P = 0.01; R = 0.58) and COX (P = 0.02; R = 0.52) before training; after training, a correlation was found between Mito VD and CS activity only (P = 0.04; R = 0.49). Intrinsic respiratory capacities decreased (P < 0.05) with training when respiration was normalized to Mito VD. This was not the case when normalized to CS activity although the percentage change was comparable . CONCLUSIONS: Mito VD was increased by inducing mitochondrial enlargement rather than de novo biogenesis. CS activity may be appropriate to track training-induced changes in Mito VD., (© 2017 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Serial-section electron tomography may be adapted to study any cellular structure in three-dimensions.

Key Points: Obesity during pregnancy and childbirth is associated with labour dystocia leading to instrumental or operative delivery, but the underlying pathophysiological mechanisms remain unclear and insufficient uterine contractility has been suggested. This study examined whether reduced myometrial mitochondrial capacity or quantity could contribute as a pathophysiological mechanism to labour dystocia. Data did not support reduced myometrial mitochondrial capacity or quantity in the myometrium at term in obese women, but a reduced myocyte density with increased triglyceride content was demonstrated, which could lead to poorer uterine contractility. These results add to the understanding of systemic effects of obesity, placing also the myometrium at term as an affected non-adipose tissue., Abstract: Obesity is known to increase the risk of labour dystocia and insufficient energy supply, due to reduced mitochondrial capacity or quantity, could be a possible mechanism leading to reduced efficiency of uterine contractility during labour. In the present study of 36 women having an elective Caesarean section at term, obesity did not change mitochondrial phenotype in the myometrial myocyte obtained from uterine biopsies taken at delivery. Respiration rates in isolated mitochondria were unaffected by obesity. No indication of reduced content, investigated by quantification of the complexes of the respiratory chain, or altered regulation, examined by myometrial mRNA levels of genes related to mitochondrial biogenesis and inflammation, was detected. Yet we found increased myometrial triglyceride content in the obese group (2.39 ± 0.26 vs. 1.56 ± 0.20 mm, P = 0.024), while protein content and citrate synthase activity per gram wet weight myometrium were significantly lower in the obese (109.2 ± 7.2 vs. 139.4 ± 5.6 mg g -1 , P = 0.002, and 24.8 ± 1.0 vs. 29.6 ± 1.4 U g -1 wet wt, P = 0.008, respectively). These differences were substantiated by our histological findings where staining for nuclei, cytoplasm, glycogen and collagen supported the idea of a smaller muscle content in the myometrium in obese women. In conclusion no indication of myometrial mitochondrial dysfunction in the isolated state was found, but the observed increase of lipid content might play a role in the pathophysiological mechanisms behind labour dystocia in obese women., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Background: Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle wasting disorder caused by the absence of dystrophin, a large cytoskeletal muscle protein. Increasing the levels of the dystrophin-related-protein utrophin is a highly promising therapy for DMD and has been shown to improve pathology in dystrophin-deficient mice. One contributing factor to muscle wasting in DMD is mitochondrial pathology that contributes to oxidative stress and propagates muscle damage. The purpose of this study was to assess whether utrophin could attenuate mitochondria pathology and oxidative stress., Methods: Skeletal muscles from wildtype C57BL/10, dystrophin-deficient mdx, dystrophin/utrophin double knockout (dko) and dystrophin-deficient mdx/utrophin over-expressing mdx-Fiona transgenic mice were assessed for markers of mitochondrial damage., Results: Using transmission electron microscopy, we show that high levels of utrophin ameliorate the aberrant structure and localisation of mitochondria in mdx mice whereas absence of utrophin worsened these features in dko mice. Elevated utrophin also reverts markers of protein oxidation and oxidative stress, elevated in mdx and dko mice, to wildtype levels. These changes were observed independently of a shift in oxidative phenotype., Conclusion: These findings show that utrophin levels influence mitochondrial pathology and oxidative stress. While utrophin deficiency worsens the pathology, utrophin over-expression in dystrophic muscle benefits mitochondria and attenuates the downstream pathology associated with aberrant mitochondrial function.

The effects of mitofusin 2 (MFN2) deficiency, on mitochondrial morphology and the mitochondria-junctional sarcoplasmic reticulum (jSR) complex in the adult heart, have been previously investigated using 2D electron microscopy, an approach which is unable to provide a 3D spatial assessment of these imaging parameters. Here, we use 3D electron tomography to show that MFN2-deficient mitochondria are larger in volume, more elongated, and less rounded; have fewer mitochondria-jSR contacts, and an increase in the distance between mitochondria and jSR, when compared to WT mitochondria. In comparison to 2D electron microscopy, 3D electron tomography can provide further insights into mitochondrial morphology and the mitochondria-jSR complex in the adult heart., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: Skeletal muscle is central to locomotion and metabolic homeostasis. The laboratory worm Caenorhabditis elegans has been developed into a genomic model for assessing the genes and signals that regulate muscle development and protein degradation. Past work has identified a receptor tyrosine kinase signalling network that combinatorially controls autophagy, nerve signal to muscle to oppose proteasome-based degradation, and extracellular matrix-based signals that control calpain and caspase activation. The last two discoveries were enabled by following up results from a functional genomic screen of known regulators of muscle. Recently, a screen of the kinome requirement for muscle homeostasis identified roughly 40% of kinases as required for C. elegans muscle health; 80 have identified human orthologues and 53 are known to be expressed in skeletal muscle. To complement this kinome screen, here, we screen most of the phosphatases in C. elegans., Methods: RNA interference was used to knockdown phosphatase-encoding genes. Knockdown was first conducted during development with positive results also knocked down only in fully developed adult muscle. Protein homeostasis, mitochondrial structure, and sarcomere structure were assessed using transgenic reporter proteins. Genes identified as being required to prevent protein degradation were also knocked down in conditions that blocked proteasome or autophagic degradation. Genes identified as being required to prevent autophagic degradation were also assessed for autophagic vesicle accumulation using another transgenic reporter. Lastly, bioinformatics were used to look for overlap between kinases and phosphatases required for muscle homeostasis, and the prediction that one phosphatase was required to prevent mitogen-activated protein kinase activation was assessed by western blot., Results: A little over half of all phosphatases are each required to prevent abnormal development or maintenance of muscle. Eighty-six of these phosphatases have known human orthologues, 57 of which are known to be expressed in human skeletal muscle. Of the phosphatases required to prevent abnormal muscle protein degradation, roughly half are required to prevent increased autophagy., Conclusions: A significant portion of both the kinome and phosphatome are required for establishing and maintaining C. elegans muscle health. Autophagy appears to be the most commonly triggered form of protein degradation in response to disruption of phosphorylation-based signalling. The results from these screens provide measurable phenotypes for analysing the combined contribution of kinases and phosphatases in a multi-cellular organism and suggest new potential regulators of human skeletal muscle for further analysis., (© 2017 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)