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The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice ( p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice ( p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.

New Findings: What is the central question of this study? Does fukutin deficiency in skeletal muscle cause mitochondrial dysfunction, and if so, can AMP-activated protein kinase (AMPK) stimulation via 5-aminoimidazole-4-carboxamide ribonucleotide attenuate this through regulation of mitochondrial biogenesis and autophagy? What is the main finding and its importance? Mitochondrial dysfunction is associated with fukutin deficiency and AMPK stimulation may benefit muscle contractility to a greater extent than mitochondrial function., Abstract: Disruptions in the dystrophin-glycoprotein complex (DGC) are clearly the primary basis underlying various forms of muscular dystrophies and dystroglycanopathies, but the cellular consequences of DGC disruption are still being investigated. Mitochondrial abnormalities are becoming an apparent consequence and contributor to dystrophy disease pathology. Herein, we demonstrate that muscle-specific deletion of the fukutin gene (Myf5/fktn-KO mice (Fktn KO)), a model of secondary dystroglycanopathy, results in ∼30% lower muscle strength (P < 0.001) and 16% lower mitochondrial respiratory function (P = 0.002) compared to healthy littermate controls (LM). We also observed ∼80% lower expression of the gene for peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) (P = 0.004), a primary transcription factor for mitochondrial biogenesis, in Fktn KO mice that likely contributes to the mitochondrial defects. PGC-1α is post-translationally regulated via phosphorylation by AMP-activated protein kinase (AMPK). Treatment with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to rescue mitochondrial deficits in Fktn KO mice (P = 0.458) but did have beneficial (∼30% greater) effects on recovery of muscle contractility following injury in both LM and Fktn KO mice compared to saline treatment (P = 0.006). The beneficial effects of AMPK stimulation via AICAR on muscle contractile function may be partially explained by AMPK's other role of regulating skeletal muscle autophagy, a cellular process critical for clearance of damaged and/or dysfunctional organelles. Two primary conclusions can be drawn from this data: (1) fukutin deletion produces intrinsic muscular metabolic defects that likely contribute to dystroglycanopathy disease pathology, and (2) AICAR treatment accelerates recovery of muscle contractile function following injury suggesting AMPK signalling as a possible target for therapeutic strategies., (© 2020 The Authors. Experimental Physiology © 2020 The Physiological Society.)

The objective of this study was to interrogate the link between mitochondrial dysfunction and mitochondrial-specific autophagy in skeletal muscle. C57BL/6J mice were used to establish a time course of mitochondrial function and autophagy induction after fatigue ( n = 12), eccentric contraction-induced injury ( n = 20), or traumatic freeze injury (FI, n = 28); only FI resulted in a combination of mitochondrial dysfunction, i.e., decreased mitochondrial respiration, and autophagy induction. Moving forward, we tested the hypothesis that mitochondrial-specific autophagy is important for the timely recovery of mitochondrial function after FI. Following FI, there is a significant increase in several mitochondrial-specific autophagy-related protein contents including dynamin-related protein 1 (Drp1), BCL1 interacting protein (BNIP3), Pink1, and Parkin (~2-fold, P < 0.02). Also, mitochondrial-enriched fractions from FI muscles showed microtubule-associated protein light chain B1 (LC3)II colocalization suggesting autophagosome assembly around the damaged mitochondrial. Unc-51 like autophagy activating kinase (Ulk1) is considered necessary for mitochondrial-specific autophagy and herein we utilized a mouse model with Ulk1 deficiency in adult skeletal muscle ( myogenin-Cre ). While Ulk1 knockouts had contractile weakness compared with littermate controls (-27%, P < 0.02), the recovery of mitochondrial function was not different, and this may be due in part to a partial rescue of Ulk1 protein content within the regenerating muscle tissue of knockouts from differentiated satellite cells in which Ulk1 was not genetically altered via myogenin-Cre . Lastly, autophagy flux was significantly less in injured versus uninjured muscles (-26%, P < 0.02) despite the increase in autophagy-related protein content. This suggests autophagy flux is not upregulated to match increases in autophagy machinery after injury and represents a potential bottleneck in the clearance of damaged mitochondria by autophagy.

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., (Copyright © 2017 the American Physiological Society.)

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. [ABSTRACT FROM AUTHOR]

Exercise benefits many organ systems, including having a panacea-like effect on the brain. For example, aerobic exercise improves cognition and attention and reduces the risk of brain-related diseases, such as dementia, stress, and depression. Recent advances suggest that endocrine signaling from peripheral systems, such as skeletal muscle, mediates the effects of exercise on the brain. Consequently, it has been proposed that factors secreted by all organs in response to physical exercise should be more broadly termed the "exerkines." Accumulating findings suggest that exerkines derived from skeletal muscle, liver, and adipose tissues directly impact brain mitochondrial function. Mitochondria play a pivotal role in regulating neuronal energy metabolism, neurotransmission, cell repair, and maintenance in the brain, and therefore exerkines may act via impacting brain mitochondria to improve brain function and disease resistance. Therefore, herein we review studies investigating the impact of muscle-, liver-, and adipose tissue-derived exerkines on brain cognitive and metabolic function via modulating mitochondrial bioenergetics, content, and dynamics under healthy and/or disease conditions. [ABSTRACT FROM AUTHOR]

Skeletal muscle mitochondria are highly adaptable, highly dynamic organelles that maintain the functional integrity of the muscle fiber by providing ATP for contraction and cellular homeostasis (e.g., Naþ/Kþ ATPase). Emerging as early modulators of inflammation, mitochondria sense and respond to cellular stress. Mitochondria communicate with the environment, in part, by release of physical signals called mitochondrial-derived damage-associated molecular patterns (mito-DAMPs) and deviation from routine function (e.g., reduced ATP production, Ca2þ overload). When skeletal muscle is compromised, mitochondria contribute to an acute inflammatory response necessary for myofibril regeneration; however, exhaustive signaling associated with altered or reduced mitochondrial function can be detrimental to muscle outcomes. Here, we describe changes in mitochondrial content, structure, and function following skeletal muscle injury and disuse and highlight the influence of mitochondria-cytokine crosstalk on muscle regeneration and recovery. Although the appropriate therapeutic modulation following muscle stressors remains unknown, retrospective gene expression analysis reveals that interleukin-6 (IL-6), interleukin-1b (IL-1b), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1) are significantly upregulated following three unique muscle injuries. These cytokines modulate mitochondrial function and execute bona fide pleiotropic roles that can aid functional recovery of muscle, however, when aberrant, chronically disrupt healing partly by exacerbating mitochondrial dysfunction. Multidisciplinary efforts to delineate the opposing regulatory roles of inflammatory cytokines in the muscle mitochondrial environment are required to modulate regenerative behavior following skeletal muscle injury or disuse. Future therapeutic directions to consider include quenching or limited release of mito-DAMPs and cytokines present in cytosol or circulation. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy induction, i.e., the formation of autophagosomes, is robust following many forms of muscle injury. Autophagy inhibition studies strongly indicate that autophagy is necessary for successful muscle fiber recovery. Now, there are accumulating pieces of evidence indicating that autophagosome clearance, i.e., autophagy flux, does not increase to match the burden of accumulating damaged proteins and organelles after muscle fiber damage, creating a bottleneck effect. Some potential consequences of the bottleneck effect are reduced regenerative capacity marked by the inadequate activation of muscle stem cells (i.e., satellite cells) and a lesser commitment toward differentiation due to a deficiency in energetic substrates and/or molecular signaling pathways. These findings highlight an emerging area of investigation for both autophagy and muscle regeneration fields. The identification of the molecular mechanisms governing autophagy and autophagy flux may serve as targets for future therapies to enhance the recovery of its function in healthy and diseased muscle. BNIP3: BCL2/adenovirus E1B interacting protein 3; CQ: chloroquine; DMD: Duchenne muscular dystrophy; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ULK1: unc-51 like kinase 1. [ABSTRACT FROM AUTHOR]

—Ischemia-reperfusion (IR) due to temporary restriction of blood flow causes tissue/organ damages under various disease conditions, including stroke, myocardial infarction, trauma, and orthopedic surgery. In the limbs, IR injury to motor nerves and muscle fibers causes reduced mobility and quality of life. Endurance exercise training has been shown to increase tissue resistance to numerous pathological insults. To elucidate the impact of endurance exercise training on IR injury in skeletal muscle, sedentary and exercise-trained mice (5 wk of voluntary running) were subjected to ischemia by unilateral application of a rubber band tourniquet above the femur for 1 h, followed by reperfusion. IR caused significant muscle injury and denervation at neuromuscular junction (NMJ) as early as 3 h after tourniquet release as well as depressed muscle strength and neuromuscular transmission in sedentary mice. Despite similar degrees of muscle atrophy and oxidative stress, exercise-trained mice had significantly reduced muscle injury and denervation at NMJ with improved regeneration and functional recovery following IR. Together, these data suggest that endurance exercise training preserves motor nerve and myofiber structure and function from IR injury and promote functional regeneration. NEW & NOTEWORTHY This work provides the first evidence that preemptive voluntary wheel running reduces neuromuscular dysfunction following ischemia-reperfusion injury in skeletal muscle. These findings may alter clinical practices in which a tourniquet is used to modulate blood flow. [ABSTRACT FROM AUTHOR]

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<0.001), and ankle plantarflexor contractile fatigue resistance (9%, P<0.05) compared to sedentary mice, independent of simvastatin treatment. At the cellular level, trained mice had greater mitochondrial biogenesis (e.g., ~2-fold greater PGC1α expression, P<0.05) and mitochondrial content (e.g., 25% greater citrate synthase activity, P<0.05), independent of simvastatin treatment. Mitochondrial autophagy-related protein contents were greater in trained mice (e.g., 40% greater Bnip3, P<0.05), independent of simvastatin treatment. However, Drp1, a marker of mitochondrial fission, was less in simvastatin treated mice, independent of exercise training, and there was a significant interaction between training and statin treatment (P<0.022) for LC3-II protein content, a marker of autophagy flux. These data indicate that whole body and skeletal muscle adaptations to endurance exercise training are attainable with simvastatin treatment, but simvastatin may have side effects on muscle mitochondrial maintenance via autophagy, which could have long-term implications on muscle health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of nitric oxide-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF.We demonstrated that systemic administration of endogenous nitric oxide donor S-nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, as well as the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis (muscle creatine kinase [MCK]-EcSOD) in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF (α-myosin heavy chain-calsequestrin), MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced HF. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria, and vascular rarefaction in skeletal muscle.EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF. [ABSTRACT FROM AUTHOR]

The article reports on research which was conducted to investigate whether low-intensity exercise in mdx mice improved plantar flexor muscle contractile function, resistance to fatigue, and mitochondrial adaptations without exacerbating muscular dystrophy. Researchers evaluated mice who participated in 12 weeks of voluntary low-resistance wheel running or normal cage activity which was followed by plantar flexor torque generation and fatigue resistance or running capacity on a treadmill. They found that the exercise resulted in skeletal muscle adaptation, led to improved contractile function and reduced fatigability.