Mouse Soleus Research Articles

The importance of muscle activation on the interpretation of muscle mechanical performance.

The work loop technique was developed to assess muscle performance during cyclical length changes with phasic activation, simulating the in vivo conditions of many muscles, particularly during locomotion. To estimate muscle function in vivo, the standard approach involves subjecting a muscle to length trajectories and activation timings derived from in vivo measurements, whilst simultaneously measuring force. However, the stimulation paradigm typically used, supramaximal, 'square-wave' stimulation, does not accurately reflect the graded intensity of activation observed in vivo. While the importance of the timing and duration of stimulation within the cycle on estimates of muscle performance has long been established, the importance of graded muscle activation has not been investigated. In this study, we investigated how the activation pattern affects muscle performance by comparing square-wave, supramaximal activation with a graded in vivo activation pattern. First, we used in vivo electromyography-derived activation patterns and fibre strains from the rabbit digastric muscle during mastication and replayed them in situ. Second, we used Hill-type musculoskeletal model-derived activation patterns and fibre strains in a trotting mouse, replayed ex vivo in the soleus (SOL) and extensor digitorum longus (EDL) muscles. In the rabbit digastric muscle, square-wave activation led to an 8-fold higher estimate of net power, compared with the in vivo graded activation pattern. Similarly, in the mouse SOL and EDL, supramaximal, square-wave activation resulted in significantly greater positive and negative muscle work. These findings highlight that realistic interpretations of in vivo muscle function rely upon more accurate representations of muscle activation intensity.

The 24-hour molecular landscape after exercise in humans reveals MYC is sufficient for muscle growth.

A detailed understanding of molecular responses to a hypertrophic stimulus in skeletal muscle leads to therapeutic advances aimed at promoting muscle mass. To decode the molecular factors regulating skeletal muscle mass, we utilized a 24-h time course of human muscle biopsies after a bout of resistance exercise. Our findings indicate: (1) the DNA methylome response at 30 min corresponds to upregulated genes at 3 h, (2) a burst of translation- and transcription-initiation factor-coding transcripts occurs between 3 and 8 h, (3) changes to global protein-coding gene expression peaks at 8 h, (4) ribosome-related genes dominate the mRNA landscape between 8 and 24 h, (5) methylation-regulated MYC is a highly influential transcription factor throughout recovery. To test whether MYC is sufficient for hypertrophy, we periodically pulse MYC in skeletal muscle over 4 weeks. Transient MYC increases muscle mass and fiber size in the soleus of adult mice. We present a temporally resolved resource for understanding molecular adaptations to resistance exercise in muscle ( http://data.myoanalytics.com ) and suggest that controlled MYC doses influencethe exercise-related hypertrophic transcriptional landscape.

Decreased number of satellite cells-derived myonuclei in both fast- and slow-twitch muscles in HeyL-KO mice during voluntary running exercise

BackgroundSkeletal muscles possess unique abilities known as adaptation or plasticity. When exposed to external stimuli, such as mechanical loading, both myofiber size and myonuclear number increase. Muscle stem cells, also known as muscle satellite cells (MuSCs), play vital roles in these changes. HeyL, a direct target of Notch signaling, is crucial for efficient muscle hypertrophy because it ensures MuSC proliferation in surgically overloaded muscles by inhibiting the premature differentiation. However, it remains unclear whether HeyL is essential for MuSC expansion in physiologically exercised muscles. Additionally, the influence of myofiber type on the requirement for HeyL in MuSCs within exercised muscles remains unclear.MethodsWe used a voluntary wheel running model and HeyL-knockout mice to investigate the impact of HeyL deficiency on MuSC-derived myonuclei, MuSC behavior, muscle weight, myofiber size, and myofiber type in the running mice.ResultsThe number of new MuSC-derived myonuclei was significantly lower in both slow-twitch soleus and fast-twitch plantaris muscles from exercised HeyL-knockout mice than in control mice. However, expect for the frequency of Type IIb myofiber in plantaris muscle, exercised HeyL-knockout mice exhibited similar responses to control mice regarding myofiber size and type.ConclusionsHeyL expression is crucial for MuSC expansion during physiological exercise in both slow and fast muscles. The frequency of Type IIb myofiber in plantaris muscle of HeyL-knockout mice was not significantly reduced compared to that of control mice. However, the absence of HeyL did not affect the increased size and frequency of Type IIa myofiber in plantaris muscles. In this model, no detectable changes in myofiber size or type were observed in the soleus muscles of either control or HeyL-knockout mice. These findings imply that the requirement for MuSCs in the wheel-running model is difficult to observe due to the relatively low degree of hypertrophy compared to surgically overloaded models.

Long-term effects of the chronic administration of doxorubicin on aged skeletal muscle: An exploratory study in mice

The widely used chemotherapeutic drug doxorubicin (DOX) has been associated with adverse effects on the skeletal muscle, which can persist for years after the end of the treatment. These adverse effects may be exacerbated in older patients, whose skeletal muscle might already be impaired by aging. Nonetheless, the mediators responsible for DOX-induced myotoxicity are still largely unidentified, particularly the ones involved in the long-term effects that negatively affect the quality of life of the patients. Therefore, this study aimed to investigate the long-term effects of the chronic administration of DOX on the soleus muscle of aged mice. For that and to mimic the clinical regimen, a dose of 1.5 mg kg−1 of DOX was administered two times per week for three consecutive weeks in a cumulative dose of 9 mg kg−1 to 19-month-old male mice, which were sacrificed two months after the last administration. Body wasting and the atrophy of the soleus muscle, as measured by a decrease in the cross-sectional area of the soleus muscle fibers, were identified as long-term effects of DOX administration. The atrophy observed was correlated with increased reactive oxygen species production and caspase-3 activity. An impaired skeletal muscle regeneration was also suggested due to the correlation between satellite cells activation and the soleus muscle fibers atrophy. Systemic inflammation, skeletal muscle energy metabolism and neuromuscular junction-related markers do not appear to be involved in the long-term DOX-induced skeletal muscle atrophy. The data provided by this study shed light on the mediators involved in the overlooked long-term DOX-induced myotoxicity, paving the way to the improvement of the quality of life and survival rates of older cancer patients.

Low-intensity aerobic training reduces macrophages infiltration and improves morphological characteristics of soleus skeletal muscle from mdx mice

Duchenne Muscular Dystrophy (DMD) is characterized by the absence of the dystrophin protein. The absence of this protein determines recurrent injuries in muscle tissue progressing to necrosis and generalized weakness, leading to the patient’s death due to respiratory and/or cardiac failure. There is no cure for DMD. However, some exercise programs could minimize the disease's progression. Low-intensity training has been used as a rehabilitation program for dystrophic muscles, although the effects are still unclear. This study aimed to analyze the effects of low-intensity aerobic training on the general morphological aspects of the skeletal muscle of mdx mice. Eighteen male mice were divided into three groups with six animals each: (mdx sedentary, mdx trained, and wild-type sedentary). The low-intensity training was performed on a treadmill running during the 37 sessions. After the experiments, the animals were euthanized, and the soleus muscle was excised for histological and immunofluorescence analyses. The training (37 sessions) showed an improvement intoning the morphological aspects of soleus mdx mice and a reduction of macrophage infiltration. The low-intensity training can minimize the inflammatory process and reverse morphological alteration in the soleus muscle of the mdx mice.

Increased Homer Activity and NMJ Localization in the Vestibular Lesion het-/- Mouse soleus Muscle.

We investigated the shuttling of Homer protein isoforms identified in soluble (cytosolic) vs. insoluble (membrane-cytoskeletal) fraction and Homer protein-protein interaction/activation in the deep postural calf soleus (SOL) and non-postural gastrocnemius (GAS) muscles of het-/- mice, i.e., mice with an autosomal recessive variant responsible for a vestibular disorder, in order to further elucidate a) the underlying mechanisms of disrupted vestibular system-derived modulation on skeletal muscle, and b) molecular signaling at respective neuromuscular synapses. Heterozygote mice muscles served as the control (CTR). An increase in Homer cross-linking capacity was present in the SOL muscle of het-/- mice as a compensatory mechanism for the altered vestibule system function. Indeed, in both fractions, different Homer immunoreactive bands were detectable, as were Homer monomers (~43-48 kDa), Homer dimers (~100 kDa), and several other Homer multimer bands (>150 kDA). The het-/- GAS particulate fraction showed no Homer dimers vs. SOL. The het-/- SOL soluble fraction showed a twofold increase (+117%, p ≤ 0.0004) in Homer dimers and multimers. Homer monomers were completely absent from the SOL independent of the animals studied, suggesting muscle-specific changes in Homer monomer vs. dimer expression in the postural SOL vs. the non-postural GAS muscles. A morphological assessment showed an increase (+14%, p ≤ 0.0001) in slow/type-I myofiber cross-sectional area in the SOL of het-/- vs. CTR mice. Homer subcellular immuno-localization at the neuromuscular junction (NMJ) showed an altered expression in the SOL of het-/-mice, whereas only not-significant changes were found for all Homer isoforms, as judged by RT-qPCR analysis. Thus, muscle-specific changes, myofiber properties, and neuromuscular signaling mechanisms share causal relationships, as highlighted by the variable subcellular Homer isoform expression at the instable NMJs of vestibular lesioned het-/- mice.

Spaceflight increases sarcoplasmic reticulum Ca2+ leak and this cannot be counteracted with BuOE treatment

Spending time in a microgravity environment is known to cause significant skeletal muscle atrophy and weakness via muscle unloading, which can be partly attributed to Ca2+ dysregulation. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is responsible for bringing Ca2+ from the cytosol into its storage site, the sarcoplasmic reticulum (SR), at the expense of ATP. We have recently demonstrated that, in the soleus of space-flown mice, the Ca2+ uptake ability of the SERCA pump is severely impaired and this may be attributed to increases in reactive oxygen/nitrogen species (RONS), to which SERCA is highly susceptible. The purpose of this study was therefore to investigate whether treatment with the antioxidant, Manganese(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP5+ (BuOE), could attenuate muscle atrophy and SERCA dysfunction. We received soleus muscles from the rodent research 18 mission which had male mice housed on the international space station for 35 days and treated with either saline or BuOE. Spaceflight significantly reduced the soleus:body mass ratio and significantly increased SERCA’s ionophore ratio, a measure of SR Ca2+ leak, and 4-HNE content (marker of RONS), none of which could be rescued by BuOE treatment. In conclusion, we find that spaceflight induces significant soleus muscle atrophy and SR Ca2+ leak that cannot be counteracted with BuOE treatment. Future work should investigate alternative therapeutics that are specifically aimed at increasing SERCA activation or reducing Ca2+ leak.

Adipose Kiss1 controls aerobic exercise-related adaptive responses in adipose tissue energy homeostasis.

Kisspeptin signaling regulates energy homeostasis. Adiposity is the principal source and receiver of peripheral Kisspeptin, and adipose Kiss1 metastasis suppressor (Kiss1) gene expression is stimulated by exercise. However, whether the adipose Kiss1 gene regulates energy homeostasis and plays a role in adaptive alterations during prolonged exercise remains unknown. Here, we investigated the role of Kiss1 role in mice and adipose tissues and the adaptive changes it induces after exercise, using adipose-specific Kiss1 knockout (Kiss1adipoq-/-) and adeno-associated virus-induced adipose tissue Kiss1-overexpressing (Kiss1adipoq over) mice. We found that adipose-derived kisspeptin signal regulates lipid and glucose homeostasis to maintain systemic energy homeostasis, but in a sex-dependent manner, with more pronounced metabolic changes in female mice. Kiss1 regulated adaptive alterations of genes and proteins in tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OxPhos) pathways in female gWAT following prolonged aerobic exercise. We could further show that adipose Kiss1 deficiency leads to reduced peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α) protein content of soleus muscle and maximum oxygen uptake (VO2 max) of female mice after prolonged exercise. Therefore, adipose Kisspeptin may be a novel adipokine that increases organ sensitivity to glucose, lipids, and oxygen following exercise.

Spatial proteomics of skeletal muscle using thin cryosections reveals metabolic adaptation at the muscle-tendon transition zone

Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.

Effect of high-fat diet on isometric, concentric and eccentric contractile performance of skeletal muscle isolated from female CD-1 mice.

Despite evidence inferring muscle and contractile mode-specific effects of high-fat diet (HFD), no study has yet considered the impact of HFD directly on eccentric muscle function. The present work uniquely examined the effect of 20-week HFD on the isometric, concentric and eccentric muscle function of isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles. CD-1 female mice were randomly split into a control (n=16) or HFD (n=17) group and for 20 weeks consumed standard lab chow or HFD. Following this period, SOL and EDL muscles were isolated and assessments of maximal isometric force and concentric work loop (WL) power were performed. Each muscle was then subjected to either multiple concentric or eccentric WL activations. Post-fatigue recovery, as an indicator of incurred damage, was measured via assessment of concentric WL power. In the EDL, absolute concentric power and concentric power normalised to muscle mass were reduced in the HFD group (P<0.038). HFD resulted in faster concentric fatigue and reduced eccentric activity-induced muscle damage (P<0.05). For the SOL, maximal isometric force was increased, and maximal eccentric power normalised to muscle mass and concentric fatigue were reduced in the HFD group (P<0.05). HFD effects on eccentric muscle function are muscle-specific and have little relationship with changes in isometric or concentric function. HFD has the potential to negatively affect the intrinsic concentric and eccentric power-producing capacity of skeletal muscle, but a lack of a within-muscle uniform response indicates disparate mechanisms of action which require further investigation.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged male mice compared with those in aged female mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

Metabolome in Tibialis and Soleus Muscles in Wild-Type and Pin1 Knockout Mice through High-Resolution Magic Angle Spinning 1H Nuclear Magnetic Resonance Spectroscopy.

Skeletal muscles are heterogenous tissues composed of different myofiber types that can be classified as slow oxidative, fast oxidative, and fast glycolytic which are distinguished on the basis of their contractile and metabolic properties. Improving oxidative metabolism in skeletal muscles can prevent metabolic diseases and plays a protective role against muscle wasting in a number of neuromuscular diseases. Therefore, achieving a detailed understanding of the factors that regulate myofiber metabolic properties might provide new therapeutic opportunities for these diseases. Here, we investigated whether peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is involved in the control of myofiber metabolic behaviors. Indeed, PIN1 controls glucose and lipid metabolism in a number of tissues, and it is also abundant in adult skeletal muscles; however, its role in the control of energy homeostasis in this tissue is still to be defined. To start clarifying this topic, we compared the metabolome of the tibialis anterior muscle (mainly glycolytic) and soleus muscle (oxidative) in wild-type and Pin1 knockout mice with High-Resolution Magic Angle Spinning (HR-MAS) NMR on intact tissues. Our analysis reveals a clear demarcation between the metabolomes in the two types of muscles and allows us to decode a signature able to discriminate the glycolytic versus oxidative muscle phenotype. We also detected some changes in Pin1-depleted muscles that suggest a role for PIN1 in regulating the metabolic phenotype of skeletal muscles.

Skeletal muscle myosin heavy chain expression and 3D capillary network changes in streptozotocin-induced diabetic female mice.

It is not well-understood how type 1 diabetes (T1DM) affects skeletal muscle histological phenotype, particularly capillarisation. This study aimed to analyze skeletal muscle myosin heavy chain (MyHC) fibre type changes and 3D capillary network characteristics in experimental T1DM mice. Female C57BL/6J-OlaHsd mice were categorized into streptozotocin (STZ)-induced diabetic (n = 12) and age-matched non-diabetic controls (n =12). The muscle fibre phenotype of the soleus, gluteus maximus, and gastrocnemius muscles were characterized based on the expression of MyHC isoforms, while capillaries of the gluteus maximus were assessed with immunofluorescence staining, confocal laser microscopy and 3D image analysis. STZ-induced diabetic mice exhibited elevated glucose levels, reduced body weight, and prolonged thermal latency, verifying the T1DM phenotype. In both T1DM and non-diabetic mice, the gluteus maximus and gastrocnemius muscles predominantly expressed fast-twitch type 2b fibers, with no significant differences noted. However, the soleus muscle in non-diabetic mice had a greater proportion of type 2a fibers and comparable type 1 fiber densities (26.2 ± 14.6% vs 21.9 ± 13.5%) relative to diabetic mice. T1DM mice showed reduced fiber diameters (P = 0.026), and the 3D capillary network analysis indicated a higher capillary length per muscle volume in the gluteus maximus of diabetic mice compared to controls (P < 0.05). Overall, T1DM induced significant changes in the skeletal muscle, including shifts in MyHC fibre types, decreased fibre diameters, and increased relative capillarisation, possibly due to muscle fibre atrophy. Our findings emphasize the superior detail provided by the 3D analytical method for characterizing skeletal muscle capillary architecture, highlighting caution in interpreting 2D data for capillary changes in T1DM.

Unraveling the Interplay of p21, Autophagy, and Mitophagy in Mitigating Skeletal Muscle Dysfunction during Aging and Obesity

Introduction: The accumulation of senescent cells accelerates aging, a process further exacerbated by obesity. The decline of muscle function is a hallmark of this progression due to muscle atrophy stemming from dysregulated autophagy and mitochondrial function in skeletal muscles. p21 is one of the major regulators, which accumulates in tissues with aging and obesity. Previous studies have demonstrated that the elimination of p21-highly-expressing ( p21high) cells improves muscle function. However, the connection between p21 and its influence on muscle function remains unclear. One possible explanation is associated with alternations in autophagy and mitophagy through mitochondria dysfunction and increased reactive oxygen species. Therefore, this study investigated the link between autophagy, mitophagy, and p21 in the context of aging and obesity. Methods: Four 18-month-old male p21-Cre/+; +/+ (P) and p21-Cre/+; DTA/+ (PD) mice, fed by high-fat diet for 15 months were administered with tamoxifen for twice. We previously observed the P group with accumulation of p21high cells, which were effectively eliminated in PD group. Soleus muscles were collected for subsequent immunoblotting analysis, employing primary antibodies targeting key proteins involved in autophagy and mitophagy cellular processes. Results: Our study elucidates dynamic changes driven by eliminating p21high cells in autophagy and mitophagy marker levels within the soleus muscles of mice exposed to a high-fat diet for 15 months. There was no significant difference in the body weight of P and PD mice (65.1±2.58 g vs. 64.3±2.22 g). In P mice, an increase in the upstream autophagy initiator, Beclin1, was observed, along with elevated levels of ATG4A, ATG7, and the ATG12-ATG5 complex, indicating elongation of autophagosome formation. Despite increased autophagosome marker p62, the LC3-II/LC3-I ratio remained unchanged. Lysosomal markers, LAMP1 and LAMP2, were both elevated in P mice. Conversely, PD mice exhibited heightened mitochondrial damage elimination markers, Parkin, VDAC1, and DRP1. Although PINK1 levels were not significantly changed, they showed an increasing trend. Conclusion: Our results suggest that eliminating p21high cells may be the potential therapeutic approach to enhance skeletal muscle function in the context of obesity and aging, possibly through autophagy and mitophagy. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effects of muscle starting length on work loop power output of isolated mouse soleus and extensor digitorum longus muscle.

Force-length relationships derived from isometric activations may not directly apply to muscle force production during dynamic contractions. As such, different muscle starting lengths between isometric and dynamic conditions could be required to achieve maximal force and power. Therefore, this study examined the effects of starting length [±5-10% of length corresponding to maximal twitch force (L0)] on work loop (WL) power output (PO), across a range of cycle frequencies, of the soleus (SOL) and extensor digitorum longus muscle (EDL; N=8-10) isolated from ∼8week old C57 mice. Furthermore, passive work was examined at a fixed cycle frequency to determine the association of passive work and active net work. Starting length affected maximal WL PO of the SOL and EDL across evaluated cycle frequencies (P<0.030, ηp2>0.494). For the SOL, PO produced at -5% L0 was greater than that at most starting lengths (P<0.015, Cohen's d>0.6), except -10% L0 (P=0.135, d<0.4). However, PO produced at -10% L0 versus L0 did not differ (P=0.138, d=0.35-0.49), indicating -5% L0 is optimal for maximal SOL WL PO. For the EDL, WL PO produced at -10% L0 was lower than that at most starting lengths (P<0.032, d>1.08), except versus -5% L0 (P=0.124, d<0.97). PO produced at other starting lengths did not differ (P>0.163, d<1.04). For the SOL, higher passive work was associated with reduced PO (Spearman's r=0.709, P<0.001), but no relationship was observed between passive work and PO of the EDL (Pearson's r=0.191, r2=0.04, P=0.184). This study suggests that starting length should be optimised for both static and dynamic contractions and confirms that the force-length curve during dynamic contractions is muscle specific.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

Plasminogen deficiency exacerbates skeletal muscle loss during mechanical unloading in developing mice.

Mechanical-unloading-induced skeletal muscle atrophy results in physical frailty and disability. Elucidating its mechanism is required to establish effective countermeasures for this muscle adaptation. First, we analyzed the proteome profile in the gastrocnemius (Gast) and soleus muscles of space-flown mice raised under microgravity or artificial 1-g for 30 days, and found that the expression levels of fibrinolysis-related proteins were significantly elevated in the mechanical-unloaded muscles. Next, we investigated the roles of the fibrinolytic system in skeletal muscle atrophy induced by mechanical unloading on the ground. Eight-week-old male mice with plasminogen gene deficiency (Plg-/-) and their wild-type littermates were divided into control and hindlimb-suspended groups and were raised for 21 days. Plasminogen deficiency significantly enhanced the decrease in muscle mass at the lower limbs of mice following hindlimb unloading, and the Gast muscle atrophy was more prominent in Plg-/- mice. In addition, plasminogen deficiency significantly increased the expression of autophagy-related markers, beclin1 mRNA and LC3B protein, in the mechanical-unloaded Gast muscles, but did not affect the increase in the gene expression of ubiquitin ligases, atrogin-1 and MuRF1. Neither plasminogen deficiency nor hindlimb unloading affected the Akt/mechanistic target of rapamycin pathway in the Gast muscles. These results suggested that plasminogen deficiency might accelerate protein breakdown via the autophagy-lysosome, but not the ubiquitin-proteasome, system in the mechanical-unloaded Gast muscles. In conclusion, we first showed that plasminogen deficiency exacerbated the Gast muscle atrophy in hindlimb-unloaded mice. Plasminogen and the fibrinolysis system might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.NEW & NOTEWORTHY The expression levels of fibrinolysis-related proteins, including plasminogen, were significantly elevated in the gastrocnemius (Gast) and soleus muscles of mice following 30-day microgravity exposure. Plasminogen deficiency exacerbated atrophy of the Gast, but not the soleus, muscles in mice following 21-day hindlimb suspension. It was also suggested that protein breakdown via the autophagy-lysosome system was accelerated in the Gast muscles. Plasminogen might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.

Transcriptional dysregulation of autophagy in the muscle of a mouse model of Duchenne muscular dystrophy

It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.

The MuSK-BMP pathway maintains myofiber size in slow muscle through regulation of Akt-mTOR signaling

Myofiber size regulation is critical in health, disease, and aging. MuSK (muscle-specific kinase) is a BMP (bone morphogenetic protein) co-receptor that promotes and shapes BMP signaling. MuSK is expressed at all neuromuscular junctions and is also present extrasynaptically in the mouse soleus, whose predominantly oxidative fiber composition is akin to that of human muscle. To investigate the role of the MuSK-BMP pathway in vivo, we generated mice lacking the BMP-binding MuSK Ig3 domain. These ∆Ig3-MuSK mice are viable and fertile with innervation levels comparable to wild type. In 3-month-old mice, myofibers are smaller in the slow soleus, but not in the fast tibialis anterior (TA). Transcriptomic analysis revealed soleus-selective decreases in RNA metabolism and protein synthesis pathways as well as dysregulation of IGF1-Akt-mTOR pathway components. Biochemical analysis showed that Akt-mTOR signaling is reduced in soleus but not TA. We propose that the MuSK-BMP pathway acts extrasynaptically to maintain myofiber size in slow muscle by promoting protein synthetic pathways including IGF1-Akt-mTOR signaling. These results reveal a novel mechanism for regulating myofiber size in slow muscle and introduce the MuSK-BMP pathway as a target for promoting muscle growth and combatting atrophy.

Effects of plasminogen activator inhibitor-1 deficiency on bone disorders and sarcopenia caused by adenine-induced renal dysfunction in mice.

Chronic kidney disease (CKD) is a significant global health issue and often involves CKD-mineral and bone disorder (MBD) and sarcopenia. Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis. PAI-1 has been implicated in the pathogenesis of osteoporosis and muscle wasting induced by inflammatory conditions. However, the roles of PAI-1 in CKD-MBD and sarcopenia remain unknown. Therefore, the present study investigated the roles of PAI-1 in bone loss and muscle wasting induced by adenine in PAI-1-deficient mice. CKD was induced in PAI-1+/+ and PAI-1-/- mice by administration of adenine for ten weeks. Muscle wasting was assessed by grip strength test, quantitative computed tomography (CT) analysis and muscle weight measurement. Osteoporosis was assessed by micro-CT analysis of femoral microstructural parameters. PAI-1 deficiency did not affect adenine-induced decreases in body weight and food intake or renal dysfunction in male or female mice. PAI-1 deficiency also did not affect adenine-induced decreases in grip strength, muscle mass in the lower limbs, or the tissue weights of the gastrocnemius, soleus, and tibialis anterior muscles in male or female mice. PAI-1 deficiency aggravated trabecular bone loss in CKD-induced male mice, but significantly increased trabecular bone in CKD-induced female mice. On the other hand, PAI-1 deficiency did not affect cortical bone loss in CKD-induced mice. In conclusion, PAI-1 is not critical for the pathophysiology of CKD-MBD or CKD-induced sarcopenia in mice. However, PAI-1 may be partly related to bone metabolism in trabecular bone in the CKD state with sex differences.