Mitochondrial-specific autophagy linked to mitochondrial dysfunction following traumatic freeze injury in mice

Tehrani K.F., Latchoumane C.V., Southern W.M., Pendleton E.G., Maslesa A., Karumbaiah L., Call J.A., Mortensen L.J.

Multi-photon scanning microscopy provides a robust tool for optical sectioning, which can be used to capture fast biological events such as blood flow, mitochondrial activity, and neuronal action potentials. For many studies, it is important to visualize several different focal planes at a rate akin to the biological event frequency. Typically, a microscope is equipped with mechanical elements to move either the sample or the objective lens to capture volumetric information, but these strategies are limited due to their slow speeds or inertial artifacts. To overcome this problem, remote focusing methods have been developed to shift the focal plane axially without physical movement of the sample or the microscope. Among these methods is liquid lens technology, which adjusts the focus of the lens by changing the wettability of the liquid and hence its curvature. Liquid lenses are inexpensive active optical elements that have the potential for fast multi-photon volumetric imaging, hence a promising and accessible approach for the study of biological systems with complex dynamics. Although remote focusing using liquid lens technology can be used for volumetric point scanning multi-photon microscopy, optical aberrations and the effects of high energy laser pulses have been concerns in its implementation. In this paper, we characterize a liquid lens and validate its use in relevant biological applications. We measured optical aberrations that are caused by the liquid lens, and calculated its response time, defocus hysteresis, and thermal response to a pulsed laser. We applied this method of remote focusing for imaging and measurement of multiple in-vivo specimens, including mesenchymal stem cell dynamics, mouse tibialis anterior muscle mitochondrial electrical potential fluctuations, and mouse brain neural activity. Our system produces 5 dimensional (x,y,z,λ,t) data sets at the speed of 4.2 volumes per second over volumes as large as 160 x 160 x 35 µm3.

Wosczyna M.N., Rando T.A.

Skeletal muscle has an extraordinary regenerative capacity due to the activity of tissue-specific muscle stem cells. Consequently, these cells have received the most attention in studies investigating the cellular processes of skeletal muscle regeneration. However, efficient capacity to rebuild this tissue also depends on additional cells in the local milieu, as disrupting their normal contributions often leads to incomplete regeneration. Here, we review these additional cells that contribute to the regenerative process. Understanding the complex interactions between and among these cell populations has the potential to lead to therapies that will help promote normal skeletal muscle regeneration under conditions in which this process is suboptimal.

Xie L., Yin A., Nichenko A.S., Beedle A.M., Call J.A., Yin H.

The remarkable regeneration capability of skeletal muscle depends on the coordinated proliferation and differentiation of satellite cells (SCs). The self-renewal of SCs is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in SCs in vivo remains largely unknown. Here, we demonstrate that SCs are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of SCs by maintaining their quiescence, increasing their self-renewal, and blocking their myogenic differentiation. HIF2A stabilization in SCs cultured under normoxia augments their engraftment potential in regenerative muscle. Conversely, HIF2A ablation leads to the depletion of SCs and their consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerates muscle regeneration by increasing SC proliferation and differentiation. Mechanistically, HIF2A induces the quiescence and self-renewal of SCs by binding the promoter of the Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in SCs and may be therapeutically targeted to improve muscle regeneration.

Laker R.C., Drake J.C., Wilson R.J., Lira V.A., Lewellen B.M., Ryall K.A., Fisher C.C., Zhang M., Saucerman J.J., Goodyear L.J., Kundu M., Yan Z.

Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3–12 h and mitophagy at 6 h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer, that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle. Exercise is associated with biogenesis and removal of dysfunctional mitochondria. Here the authors use a mitochondrial reporter gene to demonstrate the occurrence of mitophagy following exercise in mice, and show this is dependent on AMPK and ULK1 signaling.

Ato S., Makanae Y., Kido K., Sase K., Yoshii N., Fujita S.

Previous studies have reported that different modes of muscle contraction (i.e., eccentric or concentric contraction) with similar contraction times can affect muscle proteolytic responses. However, the effect of different contraction modes on muscle proteolytic response under the same force−time integral (FTI: contraction force × time) has not been investigated. The purpose of this study was to investigate the effect of different contraction modes, with the same FTI, on acute proteolytic signaling responses. Eleven-week-old male Sprague–Dawley rats were randomly assigned to eccentric (EC), concentric (CC), or isometric contraction (IC) groups. Different modes of muscle contraction were performed on the right gastrocnemius muscle using electrical stimulation, with the left muscle acting as a control. In order to apply an equivalent FTI, the number of stimulation sets was modified between the groups. Muscle samples were taken immediately and three hours after exercise. Phosphorylation of FoxO3a at Ser253 was significantly increased immediately after exercise compared to controls irrespective of contraction mode. The mRNA levels of the ubiquitin ligases, MuRF1, and MAFbx mRNA were unchanged by contraction mode or time. Phosphorylation of ULK1 at Ser317 (positive regulatory site) and Ser757 (negative regulatory site) was significantly increased compared to controls, immediately or 3 h after exercise, in all contraction modes. The autophagy markers (LC3B-II/I ratio and p62 expression) were unchanged, regardless of contraction mode. These data suggest that differences in contraction mode during resistance exercise with a constant FTI, are not factors in regulating proteolytic signaling in the early phase of skeletal muscle contraction.

Call J.A., Wilson R.J., Laker R.C., Zhang M., Kundu M., Yan Z.

Autophagy is a conserved cellular process for degrading aggregate proteins and dysfunctional organelle. It is still debatable if autophagy and mitophagy (a specific process of autophagy of mitochondria) play important roles in myogenic differentiation and functional regeneration of skeletal muscle. We tested the hypothesis that autophagy is critical for functional regeneration of skeletal muscle. We first observed time-dependent increases (3- to 6-fold) of autophagy-related proteins (Atgs), including Ulk1, Beclin1, and LC3, along with reduced p62 expression during C2C12 differentiation, suggesting increased autophagy capacity and flux during myogenic differentiation. We then used cardiotoxin (Ctx) or ischemia-reperfusion (I/R) to induce muscle injury and regeneration and observed increases in Atgs between days 2 and 7 in adult skeletal muscle followed by increased autophagy flux after day 7. Since Ulk1 has been shown to be essential for mitophagy, we asked if Ulk1 is critical for functional regeneration in skeletal muscle. We subjected skeletal muscle-specific Ulk1 knockout mice (MKO) to Ctx or I/R. MKO mice had significantly impaired recovery of muscle strength and mitochondrial protein content post-Ctx or I/R. Imaging analysis showed that MKO mice have significantly attenuated recovery of mitochondrial network at 7 and 14 days post-Ctx. These findings suggest that increased autophagy protein and flux occur during muscle regeneration and Ulk1-mediated mitophagy is critical for recovery for the mitochondrial network and hence functional regeneration.

Southern W.M., Nichenko A.S., Shill D.D., Spencer C.C., Jenkins N.T., McCully K.K., Call J.A.

We tested the hypothesis that a 6-week regimen of simvastatin would attenuate skeletal muscle adaptation to low-intensity exercise. Male C57BL/6J wildtype mice were subjected to 6-weeks of voluntary wheel running or normal cage activities with or without simvastatin treatment (20 mg/kg/d, n = 7–8 per group). Adaptations in in vivo fatigue resistance were determined by a treadmill running test, and by ankle plantarflexor contractile assessment. The tibialis anterior, gastrocnemius, and plantaris muscles were evaluated for exercised-induced mitochondrial adaptations (i.e., biogenesis, function, autophagy). There was no difference in weekly wheel running distance between control and simvastatin-treated mice (P = 0.51). Trained mice had greater treadmill running distance (296%, P

Le G., Lowe D.A., Kyba M.

Freeze injury is physically induced by exposing skeletal muscle to an extremely cold probe, and results in a robust degenerative and inflammatory response. One unique aspect of freeze injury is that it destroys not only the muscle fiber cells, but also all of the mononuclear cells in the zone of injury. Repair of the muscle is accomplished by satellite cells from outside of the zone of injury, which must migrate in and which may interact with inflammatory cells, hence the length of time before apparent histological recovery of the most damaged zone is typically somewhat longer with freeze injury than with other physical or chemical methods of injury. In this chapter, we present a detailed protocol for the freeze injury of the tibialis anterior (TA) muscle in mouse.

Nichenko A.S., Southern W.M., Atuan M., Luan J., Peissig K.B., Foltz S.J., Beedle A.M., Warren G.L., Call J.A.

The primary objective of this study was to determine whether alterations in mitochondria affect recovery of skeletal muscle strength and mitochondrial enzyme activity following myotoxic injury. 3-Methyladenine (3-MA) was administered daily (15 mg/kg) to blunt autophagy, and the creatine analog guanidionpropionic acid (β-GPA) was administered daily (1% in chow) to enhance oxidative capacity. Male C57BL/6 mice were randomly assigned to nontreatment (Con, n = 6), 3-MA-treated ( n = 6), and β-GPA-treated ( n = 8) groups for 10 wk. Mice were euthanized at 14 days after myotoxic injury for assessment of mitochondrial remodeling during regeneration and its association with the recovery of muscle strength. Expression of several autophagy-related proteins, e.g., phosphorylated Ulk1 (∼2- to 4-fold, P < 0.049) was greater in injured than uninjured muscles, indicating a relationship between muscle regeneration/remodeling and autophagy. By 14 days postinjury, recovery of muscle strength (18% less, P = 0.03) and mitochondrial enzyme (e.g., citrate synthase) activity (22% less, P = 0.049) were significantly lower in 3-MA-treated than Con mice, suggesting that the autophagy process plays an important role during muscle regeneration. In contrast, muscle regeneration was nearly complete in β-GPA-treated mice, i.e., muscle strength recovered to 93% of baseline vs. 78% for Con mice. Remarkably, 14 days allowed sufficient time for a near-complete recovery of mitochondrial function in β-GPA-treated mice (e.g., no difference in citrate synthase activity between injured and uninjured, P = 0.49), indicating a robust mitochondrial remodeling process during muscle regeneration. In conclusion, autophagy is likely activated following muscle injury and appears to play an important role in functional muscle regeneration.

Zeitler A.F., Gerrer K.H., Haas R., Jiménez-Soto L.F.

Western blots are a commonly used method for protein detection and quantification in biological samples. Compensation of loading variations is achieved by housekeeping protein (HKP) normalization and/or total protein normalization (TPN). However, under infection conditions, HKP normalization, traditionally used in cell biology for quantification of western blots, can be problematic. Binding of microbes to target cells via specific receptors can induce signal transduction events resulting in drastic changes in the level of expression of HKPs. Additionally, samples collected after infection assays will include cellular and microbial proteins altering the analysis with TPN. Here we demonstrate under experimental infection conditions, how a reliable semi-quantitative analysis of proteins in western blots can be achieved using the Stain-Free technology.

Foltz S.J., Luan J., Call J.A., Patel A., Peissig K.B., Fortunato M.J., Beedle A.M.

Secondary dystroglycanopathies are a subset of muscular dystrophy caused by abnormal glycosylation of α-dystroglycan (αDG). Loss of αDG functional glycosylation prevents it from binding to laminin and other extracellular matrix receptors, causing muscular dystrophy. Mutations in a number of genes, including FKTN (fukutin), disrupt αDG glycosylation. We analyzed conditional Fktn knockout (Fktn KO) muscle for levels of mTOR signaling pathway proteins by Western blot. Two cohorts of Myf5-cre/Fktn KO mice were treated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) for 4 weeks and evaluated for changes in functional and histopathological features. Muscle from 17- to 25-week-old fukutin-deficient mice has activated mTOR signaling. However, in tamoxifen-inducible Fktn KO mice, factors related to Akt/mTOR signaling were unchanged before the onset of dystrophic pathology, suggesting that Akt/mTOR signaling pathway abnormalities occur after the onset of disease pathology and are not causative in early dystroglycanopathy development. To determine any pharmacological benefit of targeting mTOR signaling, we administered RAPA daily for 4 weeks to Myf5/Fktn KO mice to inhibit mTORC1. RAPA treatment reduced fibrosis, inflammation, activity-induced damage, and central nucleation, and increased muscle fiber size in Myf5/Fktn KO mice compared to controls. RAPA-treated KO mice also produced significantly higher torque at the conclusion of dosing. These findings validate a misregulation of mTOR signaling in dystrophic dystroglycanopathy skeletal muscle and suggest that such signaling molecules may be relevant targets to delay and/or reduce disease burden in dystrophic patients.

Yang L., Licastro D., Cava E., Veronese N., Spelta F., Rizza W., Bertozzi B., Villareal D., Hotamisligil G., Holloszy J., Fontana L.

García-Prat L., Martínez-Vicente M., Perdiguero E., Ortet L., Rodríguez-Ubreva J., Rebollo E., Ruiz-Bonilla V., Gutarra S., Ballestar E., Serrano A.L., Sandri M., Muñoz-Cánoves P.

The regenerative properties of muscle stem cells decline with age as the stem cells enter an irreversible state of senescence; a study of mouse muscle stem cells reveals that entry into senescence is an autophagy-dependent process and promoting autophagy in old satellite cells can reverse senescence and restore their regenerative properties in an injury model. During ageing, muscle stem-cell regenerative function declines. At advanced geriatric age, this decline is maximal owing to transition from a normal quiescence into an irreversible senescence state. How satellite cells maintain quiescence and avoid senescence until advanced age remains unknown. Here we report that basal autophagy is essential to maintain the stem-cell quiescent state in mice. Failure of autophagy in physiologically aged satellite cells or genetic impairment of autophagy in young cells causes entry into senescence by loss of proteostasis, increased mitochondrial dysfunction and oxidative stress, resulting in a decline in the function and number of satellite cells. Re-establishment of autophagy reverses senescence and restores regenerative functions in geriatric satellite cells. As autophagy also declines in human geriatric satellite cells, our findings reveal autophagy to be a decisive stem-cell-fate regulator, with implications for fostering muscle regeneration in sarcopenia. The regenerative properties of muscle stem cells decline with age, as they enter an irreversible senescence state. Pura Muñoz-Cánoves and colleagues show that before entering senescence, mouse muscle stem cells preserve their repair properties by returning to a reversible quiescence state in an autophagy-dependent manner. Preventing autophagy in young satellite stem cells promotes their entry into senescence and correlates with an increase in mitochondrial dysfunction and oxidative stress. Conversely, promoting autophagy in old satellite cells reverses senescence and restores their regenerative properties in an injury model.

Klionsky D.J., Abdelmohsen K., Abe A., Abedin M.J., Abeliovich H., Acevedo Arozena A., Adachi H., Adams C.M., Adams P.D., Adeli K., Adhihetty P.J., Adler S.G., Agam G., Agarwal R., Aghi M.K., et. al.

Collins M.A., An J., Peller D., Bowser R.

Cerebrospinal fluid (CSF) has been used to identify biomarkers of neurological disease. CSF protein biomarkers identified by high-throughput methods, however, require further validation. While Western blotting (WB) is well-suited to this task, the lack of a validated loading control for CSF WB limits the method's accuracy.We investigated the use of total protein (TP) as a CSF WB loading control. Using iodine-based reversible membrane staining, we determined the linear range and consistency of the CSF TP signal. We then spiked green fluorescent protein (GFP) into CSF to create defined sample-to-sample differences in GFP levels that were measured by WB before and after TP loading correction. Levels of CSF complement C3 and cystatin C measured by WB with TP loading correction and ELISA in amyotrophic lateral sclerosis and healthy control CSF samples were then compared.CSF WB with the TP loading control accurately detected defined differences in GFP levels and corrected for simulated loading errors. Individual CSF sample Western blot and ELISA measurements of complement C3 and cystatin C were significantly correlated and the methods showed a comparable ability to detect between-groups differences.CSF TP staining has a greater linear dynamic range and sample-to-sample consistency than albumin, a commonly used CSF loading control. The method accurately corrects for simulated errors in loading and improves the sensitivity of CSF WB compared to using no loading control.The TP staining loading control improves the sensitivity and accuracy of CSF WB results.

Acheson J., Joanisse S., Sale C., Hodson N.

Skeletal muscle is a highly plastic tissue that can adapt relatively rapidly to a range of stimuli. In response to novel mechanical loading, e.g. unaccustomed resistance exercise, myofibers are disrupted and undergo a period of ultrastructural remodeling to regain full physiological function, normally within 7 days. The mechanisms that underpin this remodeling are believed to be a combination of cellular processes including ubiquitin-proteasome/calpain-mediated degradation, immune cell infiltration, and satellite cell proliferation/differentiation. A relatively understudied system that has the potential to be a significant contributing mechanism to repair and recovery is the autophagolysosomal system, an intracellular process that degrades damaged and redundant cellular components to provide constituent metabolites for the resynthesis of new organelles and cellular structures. This review summarizes our current understanding of the autophagolysosomal system in the context of skeletal muscle repair and recovery. In addition, we also provide hypothetical models of how this system may interact with other processes involved in skeletal muscle remodeling and provide avenues for future research to improve our understanding of autophagy in human skeletal muscle.

Chu Y., Yuan X., Tao Y., Yang B., Luo J.

Autophagy maintains the stability of eukaryotic cells by degrading unwanted components and recycling nutrients and plays a pivotal role in muscle regeneration by regulating the quiescence, activation, and differentiation of satellite cells. Effective muscle regeneration is vital for maintaining muscle health and homeostasis. However, under certain disease conditions, such as aging, muscle regeneration can fail due to dysfunctional satellite cells. Dysregulated autophagy may limit satellite cell self-renewal, hinder differentiation, and increase susceptibility to apoptosis, thereby impeding muscle regeneration. This review explores the critical role of autophagy in muscle regeneration, emphasizing its interplay with apoptosis and recent advances in autophagy research related to diseases characterized by impaired muscle regeneration. Additionally, we discuss new approaches involving autophagy regulation to promote macrophage polarization, enhancing muscle regeneration. We suggest that utilizing cell therapy and biomaterials to modulate autophagy could be a promising strategy for supporting muscle regeneration. We hope that this review will provide new insights into the treatment of muscle diseases and promote muscle regeneration.

Myers C.

In this chapter, we embark on a journey through the remarkable world of skeletal muscle formation, regeneration, and recovery from injury. Skeletal muscles possess a remarkable ability to regenerate and repair themselves after injury. This chapter delves into the cellular and molecular mechanisms that underpin this ability, focusing on the role of muscle stem cells in the regeneration and repair of skeletal muscles.

Zhang X., Xing T., Zhang L., Zhao L., Gao F.

Abstract Background Wooden breast (WB) myopathy is a common myopathy found in commercial broiler chickens worldwide. Histological examination has revealed that WB myopathy is accompanied by damage to the pectoralis major (PM) muscle. However, the underlying mechanisms responsible for the formation of WB in broilers have not been fully elucidated. This study aimed to investigate the potential role of hypoxia-mediated programmed cell death (PCD) in the formation of WB myopathy. Results Histological examination and biochemical analysis were performed on the PM muscle of the control (CON) and WB groups. A significantly increased thickness of the breast muscle in the top, middle, and bottom portions (P<0.01) was found along with pathological structure damage of myofibers in the WB group. The number of capillaries per fiber in PM muscle, and the levels of pO2 and sO2 in the blood, were significantly decreased (P < 0.01), while the levels of pCO2 and TCO2 in the blood were significantly increased (P < 0.05), suggesting hypoxic conditions in the PM muscle of the WB group. We further evaluated the PCD-related pathways including autophagy, apoptosis, and necroptosis to understand the consequence response to enhanced hypoxic conditions in the PM muscle of birds with WB. The ratio of LC3 II to LC3 I, and the autophagy-related factors HIF-1α, BNIP3, Beclin1, AMPKα, and ULK1 at the mRNA and protein levels, were all significantly upregulated (P < 0.05), showing that autophagy occurred in the PM muscle of the WB group. The apoptotic index, as well as the expressions of Bax, Cytc, caspase 9, and caspase 3, were significantly increased (P < 0.05), whereas Bcl-2 was significantly decreased (P < 0.05) in the WB-affected PM muscle, indicating the occurrence of apoptosis mediated by the mitochondrial pathway. Additionally, the expressions of necroptosis-related factors RIP1, RIP3, and MLKL, as well as NF-κB and the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, were all significantly enhanced (P < 0.05) in the WB-affected PM muscle. Conclusions The WB myopathy reduces blood supply and induces hypoxia in the PM muscle, which is closely related to the occurrence of PCD including apoptosis, autophagy, and necroptosis within myofibers, and finally leads to abnormal muscle damage and the development of WB in broilers.

Pan T., Jiao J., Ye L., Tong X., Wang Q., Ji M.

RESUMO Objetivo: Estudar as alterações temporais dos fatores relacionados à autofagia no músculo esquelético de ratos após exercício exaustivo e trauma contuso. Métodos: Quarenta e dois ratos machos SD foram divididos em 7 grupos com 6 ratos em cada grupo: Grupo de controle silencioso (C), imediatamente após o exercício exaustivo (E0), 24 horas após o exercício exaustivo (E24), 48 horas após o exercício exaustivo (E48), imediatamente após o trauma contuso (D0), 24 horas após o trauma contuso (D24), 48 horas após o trauma contuso (D48). Todos os grupos de ratos foram mortos e rotulados, respectivamente, em diferentes momentos especificados acima, e o músculo gastrocnêmio direito foi retirado, dividido em duas partes, uma para mRNAs de Lamp-2, BNIP3 e NIX por PCR quantitativo fluorescente em tempo real, e a outra para a proteína p62 por imunotransferência. Resultados: (1) Em comparação com o grupo C, os níveis de mRNA de p62, Lamp-2 e NIX no grupo E48 aumentaram significativamente após o exercício exaustivo (P<0,05), sugerindo que a autofagia aumentou em 48 horas após o exercício exaustivo. (2) Em comparação com o grupo C, os níveis de mRNA de p62mRNA e Lamp-2 foram significativamente aumentados imediatamente após o trauma contuso (P<0,05) e diminuíram significativamente em 48 horas após o trauma contuso (P<0,05), sugerindo que a atividade de autofagia foi aumentada imediatamente após o trauma contuso e diminuiu em 48 horas após a lesão. Conclusão: Houve, via de regra, diferenças em cada fase de recuperação entre os modelos de trauma contuso e de exercício exaustivo, sendo que os fatores de autofagia basal e os fatores de autofagia mitocondrial também foram inconsistentes. Os fatores de autofagia basal p62 e Lamp-2 aumentaram significativamente 48 horas após o exercício excêntrico exaustivo e imediatamente após o trauma contuso. O fator de autofagia mitocondrial BNIP3 não aumentou após o exercício exaustivo e o trauma contuso, mas o NIX aumentou somente após o exercício exaustivo. Seu mecanismo molecular precisa ser investigado com mais detalhes. Nível de Evidência III; Estudos Terapêuticos que Investigam os Resultados do Tratamento.

Pan T., Jiao J., Ye L., Tong X., Wang Q., Ji M.

ABSTRACT Objective: To study the temporal changes of autophagy related factors in skeletal muscle of rats after exhaustive exercise and blunt trauma. Methods: Forty-two male SD rats were divided into 7 groups with 6 rats in each group: Quiet control group (C), immediately after exhaustive exercise (E0), 24 hours after exhaustive exercise (E24), 48 hours after exhaustive exercise (E48), immediately after blunt trauma (D0), 24 hours after blunt trauma (D24), 48 hours after blunt trauma (D48). All groups of rats were killed and samped respectively at different time points specified above, and the right gastrocnemius muscle was taken, which was divided into two parts, one for mRNAs of, Lamp-2, BNIP3 and NIX by real-time fluorescent quantitative PCR, and the other for p62 protein by Western blotting. Results: (1) Compared with group C, mRNA levels of p62, Lamp-2 and NIX in group E48 were significantly increased after exhaustive exercise(P<0.05), suggesting that autophagy increased in 48h after exhaustive exercise. (2) Compared with group C, p62mRNA and Lamp-2 mRNA levels were significantly increased immediately after blunt trauma(P<0.05) and decreased significantly in 48h after blunt trauma(P<0.05), suggesting that autophagy activity was enhanced immediately after blunt trauma and decreased in 48h after injury. Conclusions: Generally, there were differences at each recovery phase between blunt trauma and exhausted exercise models, and the basal autophagy factors and mitochondrial autophagy factors were also inconsistent. Basal autophagy factors p62 and Lamp-2 increased significantly 48 hours after eccentric exhaustive exercise and immediately after blunt trauma. Mitochondrial autophagy factor BNIP3 did not increase after exhaustive exercise and blunt trauma, but NIX only increased after exhaustive exercise. Its molecular mechanism needs to be further studied. Level of Evidence III; Therapeutic Studies Investigating the Results of Treatment.

Pendleton E.G., Nichenko A.S., Mcfaline-Figueroa J., Raymond-Pope C.J., Schifino A.G., Pigg T.M., Barrow R.P., Greising S.M., Call J.A., Mortensen L.J.

Hypophosphatasia (HPP) is a rare metabolic bone disorder characterized by low levels of tissue non-specific alkaline phosphatase (TNAP) that causes under-mineralization of the bone, leading to bone deformity and fractures. In addition, patients often present with chronic muscle pain, reduced muscle strength, and an altered gait. In this work, we explored dynamic muscle function in a homozygous TNAP knockout mouse model of severe juvenile onset HPP. We found a reduction in skeletal muscle size and impairment in a range of isolated muscle contractile properties. Using histological methods, we found that the structure of HPP muscles was similar to healthy muscles in fiber size, actin and myosin structures, as well as the α-tubulin and mitochondria networks. However, HPP mice had significantly fewer embryonic and type I fibers than wild type mice, and fewer metabolically active NADH+ muscle fibers. We then used oxygen respirometry to evaluate mitochondrial function and found that complex I and complex II leak respiration were reduced in HPP mice, but that there was no disruption in efficiency of electron transport in complex I or complex II. In summary, the severe HPP mouse model recapitulates the muscle strength impairment phenotypes observed in human patients. Further exploration of the role of alkaline phosphatase in skeletal muscle could provide insight into mechanisms of muscle weakness in HPP.

Rahman F.A., Campbell T., Bloemberg D., Chapman S., Quadrilatero J.

Skeletal muscle is a complex tissue comprising multinucleated and post-mitotic cells (i.e., myofibers). Given this, skeletal muscle must maintain a fine balance between growth and degradative signals. A major system regulating the remodeling of skeletal muscle is autophagy, where cellular quality control is mediated by the degradation of damaged cellular components. The accumulation of damaged cellular material can result in elevated apoptotic signaling, which is particularly relevant in skeletal muscle given its post-mitotic nature. Luckily, skeletal muscle possesses the unique ability to regenerate in response to injury. It is unknown whether a relationship between autophagy and apoptotic signaling exists in injured skeletal muscle and how autophagy deficiency influences myofiber apoptosis and regeneration. In the present study, we demonstrate that an initial inducible muscle-specific autophagy deficiency does not alter apoptotic signaling following cardiotoxin injury. This finding is presumably due to the re-establishment of ATG7 levels following injury, which may be attributed to the contribution of a functional Atg7 gene from satellite cells. Furthermore, the re-expression of ATG7 resulted in virtually identical regenerative potential. Overall, our data demonstrate that catastrophic injury may “reset” muscle gene expression via the incorporation of nuclei from satellite cells.

Picca A., Guerra F., Calvani R., Romano R., Coelho-Junior H.J., Bucci C., Leeuwenburgh C., Marzetti E.

Mitochondrial remodeling is crucial to meet the bioenergetic demand to support muscle contractile activity during daily tasks and muscle regeneration following injury. A set of mitochondrial quality control (MQC) processes, including mitochondrial biogenesis, dynamics, and mitophagy, are in place to maintain a well-functioning mitochondrial network and support muscle regeneration. Alterations in any of these pathways compromises mitochondrial quality and may potentially lead to impaired myogenesis, defective muscle regeneration, and ultimately loss of muscle function. Among MQC processes, mitophagy has gained special attention for its implication in the clearance of dysfunctional mitochondria via crosstalk with the endo-lysosomal system, a major cell degradative route. Along this pathway, additional opportunities for mitochondrial disposal have been identified that may also signal at the systemic level. This communication occurs via inclusion of mitochondrial components within membranous shuttles named mitochondrial-derived vesicles (MDVs). Here, we discuss MDV generation and release as a mitophagy-complementing route for the maintenance of mitochondrial homeostasis in skeletal myocytes. We also illustrate the possible role of muscle-derived MDVs in immune signaling during muscle remodeling and adaptation.

Alway S.E., Paez H.G., Pitzer C.R.

Musculoskeletal health is directly linked to independence and longevity, but disease and aging impairs muscle mass and health. Complete repair after a pathological or physiological muscle injury is critical for maintaining muscle function, yet muscle repair is compromised after disuse, or in conditions such as metabolic diseases, cancer, and aging. Regeneration of damaged tissue is critically dependent upon achieving the optimal function of satellite cells (muscle stem cells, MSCs). MSC remodeling in muscle repair is highly dependent upon its microenvironment, and metabolic health of MSCs, which is dependent on the functional capacity of their mitochondria. Muscle repair is energy demanding and mitochondria provide the primary source for energy production during regeneration. However, disease and aging induce mitochondrial dysfunction, which limits energy production during muscle regeneration. Nevertheless, the role of mitochondria in muscle repair likely extends beyond the production of ATP and mitochondria could provide potentially important regulatory signaling to MSCs during repair from injury. The scope of current research in muscle regeneration extends from molecules to exosomes, largely with the goal of understanding ways to improve MSC function. This review focuses on the role of mitochondria in skeletal muscle myogenesis/regeneration and repair. A therapeutic strategy for improving muscle mitochondrial number and health will be discussed as a means for enhancing muscle regeneration. Highlights: (a). Mitochondrial dysfunction limits muscle regeneration; (b). Muscle stem cell (MSC) function can be modulated by mitochondria; (c). Enhancing mitochondria in MSCs may provide a strategy for improving muscle regeneration after an injury.

Chatzinikita E., Maridaki M., Palikaras K., Koutsilieris M., Philippou A.

Mitochondria are cellular organelles that play an essential role in generating the chemical energy needed for the biochemical reactions in cells. Mitochondrial biogenesis, i.e., de novo mitochondria formation, results in enhanced cellular respiration, metabolic processes, and ATP generation, while autophagic clearance of mitochondria (mitophagy) is required to remove damaged or useless mitochondria. The balance between the opposing processes of mitochondrial biogenesis and mitophagy is highly regulated and crucial for the maintenance of the number and function of mitochondria as well as for the cellular homeostasis and adaptations to metabolic demands and extracellular stimuli. In skeletal muscle, mitochondria are essential for maintaining energy homeostasis, and the mitochondrial network exhibits complex behaviors and undergoes dynamic remodeling in response to various conditions and pathologies characterized by changes in muscle cell structure and metabolism, such as exercise, muscle damage, and myopathies. In particular, the involvement of mitochondrial remodeling in mediating skeletal muscle regeneration following damage has received increased attention, as modifications in mitophagy-related signals arise from exercise, while variations in mitochondrial restructuring pathways can lead to partial regeneration and impaired muscle function. Muscle regeneration (through myogenesis) following exercise-induced damage is characterized by a highly regulated, rapid turnover of poor-functioning mitochondria, permitting the synthesis of better-functioning mitochondria to occur. Nevertheless, essential aspects of mitochondrial remodeling during muscle regeneration remain poorly understood and warrant further characterization. In this review, we focus on the critical role of mitophagy for proper muscle cell regeneration following damage, highlighting the molecular mechanisms of the mitophagy-associated mitochondrial dynamics and network reformation.

Li J., Wu D., Wang Z., Wang X., Ke Z., Wang R.

High mobility group box-1 protein (HMGB1) is an evolutionarily ancient protein, which, as an important non-histone chromosome-binding protein in organism cells, is involved in a variety of important biological processes, including DNA repair, gene transcription, cellular inflammatory response, and autophagy. In this study, we established an eccentric exercise model to observe the effect of HMGB1 on skeletal muscle autophagy and to investigate the underlying molecular mechanisms. Forty-eight male 8-week-old SD rats were randomly divided into control group (C) and exercise group (E). Group E was subjected to a bout of eccentric exercise on a treadmill and sampled soleus at 0 h, 12 h, 24 h, 48 h, and 72 h post-exercise. The speed of the exercise protocol in this study was 16 m/min, the slope was −16°, and the time was 90 min. The ultrastructural changes of skeletal muscle were observed by transmission electron microscopy. The protein expressions of HMGB1, Beclin1, and LC3 were detected by Western Blot. The co-localizations of Beclin1/Bcl-2, Beclin1/HMGB1, and Beclin1/Vps34 were measured by immunofluorescence. The results show that eccentric exercise leads to abnormal changes in the ultrastructure of skeletal muscle, and the protein levels of Beclin1, LC3-II/LC3-I, and the content of HMGB1 in nuclear and cytoplasm were significantly increased at 24 h post-exercise (P < 0.05). The co-localization of Beclin1/Bcl-2 and Beclin1/HMGB1 were increased significantly at 0 h post-exercise and then decreased, while the co-localization of Beclin1/Vps34 showed the highest level at 24 h post-exercise. In conclusion, HMGB1 facilitates the separation of Beclin1 from Bcl-2 and promotes Beclin1 binding to Vps34, which may play an important role in eccentric exercise-induced skeletal muscle autophagy.

Chen W., Chen Y., Liu Y., Wang X.

Chen M., Li Y., Deng S., Zhao Y., Lian Z., Yu K.

Skeletal muscle fibers contain a large number of mitochondria, which produce ATP through oxidative phosphorylation (OXPHOS) and provide energy for muscle contraction. In this process, mitochondria also produce several types of “reactive species” as side product, such as reactive oxygen species and reactive nitrogen species which have attracted interest. Mitochondria have been proven to have an essential role in the production of skeletal muscle reactive oxygen/nitrogen species (RONS). Traditionally, the elevation in RONS production is related to oxidative stress, leading to impaired skeletal muscle contractility and muscle atrophy. However, recent studies have shown that the optimal RONS level under the action of antioxidants is a critical physiological signal in skeletal muscle. Here, we will review the origin and physiological functions of RONS, mitochondrial structure and function, mitochondrial dynamics, and the coupling between RONS and mitochondrial oxidative stress. The crosstalk mechanism between mitochondrial function and RONS in skeletal muscle and its regulation of muscle stem cell fate and myogenesis will also be discussed. In all, this review aims to describe a comprehensive and systematic network for the interaction between skeletal muscle mitochondrial function and RONS.

Li Y., Zhang F., Yu L., Mu J., Yang Y., Yu J., Yang X.

PINK1, also known as PARK6, is a PTEN-induced putative kinase 1 that is encoded by nuclear genes. PINK1 is ubiquitously expressed and regulates mitochondrial function and mitophagy in a range of cell types. The dysregulation of PINK1 is associated with the pathogenesis and development of mitochondrial-associated disorders. Many natural products could regulate PINK1 to relieve PINK1-associated diseases. Here, we review the structure and function of PINK1, its relationship to human diseases, and the regulation of natural products to PINK1. We further highlight that the discovery of natural PINK1 regulators represents an attractive strategy for the treatment of PINK1-related diseases, including liver and heart diseases, cancer, and Parkinson’s disease. Moreover, investigating PINK1 regulation of natural products can enhance the in-depth comprehension of the mechanism of action of natural products.