Tibialis Anterior Of Mice Research Articles

UBR2 targets myosin heavy chain IIb and IIx for degradation: Molecular mechanism essential for cancer-induced muscle wasting

Cancer cachexia is a lethal metabolic syndrome featuring muscle wasting with preferential loss of fast-twitching muscle mass through an undefined mechanism. Here, we show that cancer induces muscle wasting by selectively degrading myosin heavy chain (MHC) subtypes IIb and IIx through E3 ligase UBR2-mediated ubiquitylation. Induction of MHC loss and atrophy in C2C12 myotubes and mouse tibialis anterior (TA) by murine cancer cells required UBR2 up-regulation by cancer. Genetic gain or loss of UBR2 function inversely altered MHC level and muscle mass in TA of tumor-free mice. UBR2 selectively interacted with and ubiquitylated MHC-IIb and MHC-IIx through its substrate recognition and catalytic domain, respectively, in C2C12 myotubes. Elevation of UBR2 in muscle of tumor-bearing or free mice caused loss of MHC-IIb and MHC-IIx but not MHC-I and MHC-IIa or other myofibrillar proteins, including α-actin, troponin, tropomyosin, and tropomodulin. Muscle-specific knockout of UBR2 spared KPC tumor-bearing mice from losing MHC-IIb and MHC-IIx, fast-twitching muscle mass, cross-sectional area, and contractile force. The rectus abdominis (RA) muscle of patients with cachexia-prone cancers displayed a selective reduction of MHC-IIx in correlation with higher UBR2 levels. These data suggest that UBR2 is a regulator of MHC-IIb/IIx essential for cancer-induced muscle wasting, and that therapeutic interventions can be designed by blocking UBR2 up-regulation by cancer.

Early-senescent bone marrow mesenchymal stem cells promote C2C12 cell myogenic differentiation by preventing the nuclear translocation of FOXO3

AimsMouse bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells with self-renewal and differentiation abilities. Since the effects of senescent BMSCs on C2C12 cells are not fully clear, the present study aimed to elucidate these effects. Main methodsSenescence-associated β-galactosidase staining and western blotting were performed to confirm the senescence of BMSCs. Immunofluorescence and western blotting were used to assess myoblast differentiation in each group. The role of the AKT/P70 signaling pathway and forkhead box O3 (FOXO3) nuclear translocation was explored by western blotting. BMSC-derived exosomes were injected into the tibialis anterior of mice, and RT-qPCR was used to assess the role of exosomes in promoting muscle differentiation. Key findingsConditioned medium (CM) from early-senescent BMSCs promoted myogenic differentiation in vitro, which was detected as enhanced expression of myosin heavy chain (MHC), myogenin (MYOG), and myogenic differentiation 1 (MyoD). The AKT signaling pathway was found to be regulated by CM, which inhibited FOXO3 nuclear translocation. RT-qPCR analysis results showed that MHC, MyoD, and MYOG mRNA expression increased in the tibialis anterior of mice after exosome injection. SignificanceThe present study demonstrated that early-senescent BMSCs accelerated C2C12 cell myogenic differentiation, and the transcription factor, FOXO3, was the target of senescent cells. Collectively, our results suggest that the AKT/P70 signaling pathway mediates the effect of BMSCs on neighboring cells.

Voluntary Resistance Running as a Model to Induce mTOR Activation in Mouse Skeletal Muscle.

Long-term voluntary resistance running has been shown to be a valid model to induce muscle growth in rodents. Moreover, the mammalian target of rapamycin complex 1 (mTORC1) is a key signaling complex regulating exercise/nutrient-induced alterations in muscle protein synthesis. How acute resistance running affects mTORC1 signaling in muscle and if resistance applied to the wheel can modulate mTORC1 activation has not yet been fully elucidated. Here, we show that both acute resistance running and acute free running activated mTORC1 signaling in the m. gastrocnemius, m. soleus, and m. plantaris, but not in m. tibialis anterior of mice when compared to sedentary controls. Furthermore, only the low threshold oxidative part in the m. gastrocnemius showed increased mTORC1 signaling upon running and acute heavy-load resistance running evoked higher downstream mTORC1 signaling in both m. soleus and m. plantaris than free running without resistance, pointing toward mechanical load as an important independent regulator of mTORC1. Collectively, in this study, we show that voluntary resistance running is an easy-to-use, time-efficient and low stress model to study acute alterations in mTORC1 signaling upon high-load muscular contractions in mice.

Single sarcomere contraction dynamics in a whole muscle

The instantaneous sarcomere length (SL) is regarded as an important indicator of the functional properties of striated muscle. Previously, we found greater sarcomere elongations at the distal end compared to the mid-portion in the mouse tibialis anterior (TA) when the muscle was stretched passively. Here, we wanted to see if SL dispersions increase with activation, as has been observed in single myofibrils, and if SL dispersions differ for different locations in a muscle. Sarcomere lengths were measured at a mid- and a distal location of the TA in live mice using second harmonic generation imaging. Muscle force was measured using a tendon force transducer. We found that SL dispersions increased substantially from the passive to the active state, and were the same for the mid- and distal portions of TA. Sarcomere length non-uniformities within a segment of ~30 serial sarcomeres were up to 1.0 µm. We conclude from these findings that passive, mean SLs obtained from a single location are not necessarily representative of the distribution of SL in active muscle, and thus may be misinterpreted when deriving muscle mechanical properties, such as the force-length relationship. In view of these findings, it seems crucial to determine how SL distributions within a muscle relate to the most fundamental properties of muscle, such as the maximal isometric force.

In Vivo Sarcomere Lengths Become More Non-uniform upon Activation in Intact Whole Muscle.

The sarcomere force-length relationship has been extensively used to predict muscle force potential. The common practice is to measure the mean sarcomere length (SL) in a relaxed muscle at a single location and at a given length, and this mean SL is assumed to represent the SLs at other locations across the muscle. However, in a previous study, we found that SLs are highly non-uniform across an intact passive muscle. Moreover, SL non-uniformity increases during activation in single myofibril experiments. Myofibrils lack some structural proteins that comprise an intact muscle, and therefore, the increased SL dispersion upon activation seen in myofibrils may not occur in intact whole muscle. The objectives of the current study were (i) to measure the distribution of SLs in an activated intact muscle; and (ii) to assess the feasibility of using the mean SL measured at a specific location of the muscle to predict muscle force. Using state-of-the-art multi-photon microscopy and a miniature tendon force transducer, in vivo sarcomeres in the mouse tibialis anterior were imaged simultaneously with muscle force during isometric tetanic contractions. We found that in vivo SL dispersion increased substantially during activation and reached average differences of ~1.0 μm. These differences in SL are associated with theoretical force differences of 70–100% of the maximal isometric force. Furthermore, SLs measured at a single location in the passive muscle were poor predictors of active force potential. Although mean SLs in the activated muscle were better predictors of force potential, predicted forces still differed by as much as 35% from the experimentally measured maximal isometric forces.

Sarcomere Length and Passive Sarcomere Lengthening are Location-Dependent in Live Mouse Tibialis Anterior Muscle

Introduction The force produced by muscles depends on sarcomere length (SL). Typically, the SL measured at a single site is taken as representing the SLs of the entire muscle. However, there is little evidence to support that notion. The purpose of this study was to investigate the distribution of in vivo SL along a muscle and to measure sarcomere elongations resulting from passive muscle lengthening. Methods C57BL/6 mice (n=3) were anaesthetized using isoflurane. The skin over the left lower limb was surgically opened to expose the tibialis anterior (TA) muscle. The left knee and the left foot were pinned in a custom-made water chamber to allow imaging of SL using a water-immersion objective. The anisotropic bands (A-band) of the TA were imaged at three anatomical locations (proximal, middle, and distal) by second harmonic generation microscopy with the ankle being fully dorsiflexed (60° relative to tibia) and fully plantarflexed (180°). Results Locally, SL distributions were relatively uniform with a coefficient of variation ranging from 4.2 - 6.6%. However, sarcomere lengths were highly non-uniform along the TA. When fully dorsiflexed, SLs were 2.08±0.09, 2.25±0.08 and 2.16±0.04 µm (mean ± standard deviation) at the proximal, middle and distal sites, respectively. Going to full plantarflexion, the mean sarcomere elongations were also non-uniform. The distal muscle showed the greatest sarcomere elongations (25.1±3.0%), followed by the mid-section (8.6±2.63%) and proximal section (5.6±5.2%). Conclusion We conclude from this study that SLs in mouse TA are location-dependent and that passive elongation of the TA causes further SL non-uniformities along the muscle. Further research is required for active muscles under dynamic conditions to determine to what degree SL varies within a muscle for these conditions.

High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis.

Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes.

Growth differentiation factor-15 is associated with muscle mass in chronic obstructive pulmonary disease and promotes muscle wasting in vivo.

BackgroundLoss of muscle mass is a co‐morbidity common to a range of chronic diseases including chronic obstructive pulmonary disease (COPD). Several systemic features of COPD including increased inflammatory signalling, oxidative stress, and hypoxia are known to increase the expression of growth differentiation factor‐15 (GDF‐15), a protein associated with muscle wasting in other diseases. We therefore hypothesized that GDF‐15 may contribute to muscle wasting in COPD.MethodsWe determined the expression of GDF‐15 in the serum and muscle of patients with COPD and analysed the association of GDF‐15 expression with muscle mass and exercise performance. To determine whether GDF‐15 had a direct effect on muscle, we also determined the effect of increased GDF‐15 expression on the tibialis anterior of mice by electroporation.ResultsGrowth differentiation factor‐15 was increased in the circulation and muscle of COPD patients compared with controls. Circulating GDF‐15 was inversely correlated with rectus femoris cross‐sectional area (P < 0.001) and exercise capacity (P < 0.001) in two separate cohorts of patients but was not associated with body mass index. GDF‐15 levels were associated with 8‐oxo‐dG in the circulation of patients consistent with a role for oxidative stress in the production of this protein. Local over‐expression of GDF‐15 in mice caused wasting of the tibialis anterior muscle that expressed it but not in the contralateral muscle suggesting a direct effect of GDF‐15 on muscle mass (P < 0.001).ConclusionsTogether, the data suggest that GDF‐15 contributes to the loss of muscle mass in COPD.

Carbonic Anhydrase III Contributes to Fatigue Tolerance and Recovery of Skeletal Muscle

Carbonic anhydrase III (CA3) is a metabolic enzyme with a potential role in regulating intracellular pH. CA3 is highly expressed in slow twitch skeletal muscles. Here we demonstrated that mouse tibialis anterior (TA), a fast twitch muscle, also expresses a high level of CA3 while its myofilament protein contents were similar to that of CA3-negative extensor digitorum longus (EDL) muscle. To investigate the function of CA3 in muscle contractility and tolerance to fatigue, we studied skeletal muscles of CA3 knockout (Car3-/-) mice. Sciatic nerve stimulation-generated in situ contractility of EDL and TA muscles were examined in comparison with wild type controls. The results of isometric twitch and tetanic contractions showed no significant difference between TA and EDL muscle of wild type mice or between Car3-/- and wild type TA muscles. Nonetheless, intermittent fatigue treatment revealed faster fatigue and slower recovery of wild type TA muscle than that of wild type EDL muscle. Car3-/- TA muscle exhibited slower and less fatigue but also slower recovery than that of wild type TA muscle, whereas the ultimate level of force recovery was unchanged. It is suggested that CA3 increases the sensitivity of muscle to fatigue, which might serve as an acute physiological protection. In the meantime, Western-blot detected a low molecular weight fast troponin T (TnT) variant specifically in TA muscle of adult Car3-/- mice, suggesting a chronically adaptive response through alternative RNA splicing. The role of CA3 in fatigue tolerance and recovery of skeletal muscle suggests a molecular therapeutic target for functional enhancement.

Growth‐related signaling increases with duty cycle following high force contractions in mouse tibialis anterior

High force contractions (HFC) activate multiple cascades related to protein synthesis. Although some studies have suggested force‐dependent activation of various growth‐related kinases, we found no correlation between force magnitude and several members of these signaling cascades when metabolic stress was minimized. HFC impose both mechanical and metabolic stresses, and we hypothesized that MAPK phosphorylation during high force isometric activation would be increased when the work‐to‐rest ratio (duty cycle, DC) and metabolic cost are increased. Mouse tibialis anterior muscles were subjected to high (H, 15%) or low (L, 1.5%) DC stimulation protocols, using long or short duration activations to further manipulate ATP depletion. Force‐time integrals were ~4X greater in HDC than LDC and fatigue was evident in all groups except LDC, short activations. ACC phosphorylation, indicative of AMPK activity, was greater in HDC protocols indicating greater energetic stress. FAK and p38 phosphorylation were also greater in HDC than LDC, which could reflect either force or energy‐depletion dependent signaling. Akt phosphorylation decreased most in LDC with long activations, but ERK was similarly phosphorylated by stimulation in all groups. Combined with our previous observation of force‐independent signaling, we conclude that metabolic stress is a major contributor to high force‐related signaling. DC05017

Activation of the dopamine 1 and dopamine 5 receptors increase skeletal muscle mass and force production under non-atrophying and atrophying conditions

BackgroundControl of skeletal muscle mass and force production is a complex physiological process involving numerous regulatory systems. Agents that increase skeletal muscle cAMP levels have been shown to modulate skeletal muscle mass and force production. The dopamine 1 receptor and its closely related homolog, the dopamine 5 receptor, are G-protein coupled receptors that are expressed in skeletal muscle and increase cAMP levels when activated. Thus we hypothesize that activation of the dopamine 1 and/or 5 receptor will increase skeletal muscle cAMP levels thereby modulating skeletal muscle mass and force production.MethodsWe treated isolated mouse tibialis anterior (TA) and medial gastrocnemius (MG) muscles in tissue bath with the selective dopamine 1 receptor and dopamine 5 receptor agonist SKF 81297 to determine if activation of skeletal muscle dopamine 1 and dopamine 5 receptors will increase cAMP. We dosed wild-type mice, dopamine 1 receptor knockout mice and dopamine 5 receptor knockout mice undergoing casting-induced disuse atrophy with SKF 81297 to determine if activation of the dopamine 1 and dopamine 5 receptors results in hypertrophy of non-atrophying skeletal muscle and preservation of atrophying skeletal muscle mass and force production.ResultsIn tissue bath, isolated mouse TA and MG muscles responded to SKF 81297 treatment with increased cAMP levels. Treating wild-type mice with SKF 81297 reduced casting-induced TA and MG muscle mass loss in addition to increasing the mass of non-atrophying TA and MG muscles. In dopamine 1 receptor knockout mice, extensor digitorum longus (EDL) and soleus muscle mass and force was not preserved during casting with SKF 81297 treatment, in contrast to significant preservation of casted wild-type mouse EDL and soleus mass and EDL force with SKF 81297 treatment. Dosing dopamine 5 receptor knockout mice with SKF 81297 did not significantly preserve EDL and soleus muscle mass and force although wild-type mouse EDL mass and force was significantly preserved SKF 81297 treatment.ConclusionsThese data demonstrate for the first time that treatment with a dopamine 1/5 receptor agonist results in (1) significant preservation of EDL, TA, MG and soleus muscle mass and EDL muscle force production during periods of atrophy and (2) hypertrophy of TA and MG muscle. These effects appear to be mainly mediated by both the dopamine 1 and dopamine 5 receptors.

P85, Optison microbubbles and ultrasound cooperate in mediating plasmid DNA transfection in mouse skeletal muscles in vivo

Pluronic block copolymers, a kind of non-ionic surfactant, also known as poloxamers, and ultrasound-targeted microbubble destruction have been respectively investigated as vectors for gene delivery in vitro and in vivo. However, they are limited for clinical application due to the relatively low transfer efficiency of each individual vector. In the present study, we explored if the combination of P85, a pluronic block copolymer, Optison, a microbubble contrast agent and ultrasound enhances the transfection of plasmid DNA in vivo using mouse skeletal muscle models. Plasmid encoding green fluorescent protein (GFP) was respectively conjugated with 0.05%P85, 10%Optison, or 0.05%P85 plus 10%Optison, and injected into mouse tibialis anterior (TA) muscles with or without ultrasound irradiation (1 MHz, 1 W/cm 2, 2 min and 20% duty cycle). Mice were sacrificed 1 week after injection. The TA muscles were collected and cryo-sectioned into a series of 7 μm slices. To assess the efficiency of plasmid DNA transfection, tissue sections were counterstained with DAPI and scored by counting the number of GFP-positive fibers. Meanwhile the area of damaged muscles was measured based on the tissues stained with hematoxylin and eosin. Both P85 and Optison significantly enhanced the delivery of plasmid DNA in mouse TA skeletal muscles ( P < 0.01 and P < 0.05 respectively, compared to saline control). In combination with Ultrasound irradiation, P85 ( P < 0.01, compared to P85 alone) but not Optison ( P > 0.05, compared to Optison alone) exerted a more pronounced effect on the transfection efficiency. Furthermore P85-induced gene delivery was higher than that by Optison regardless of the presence of ultrasound ( P < 0.01). The highest transfection efficiency was observed when P85, Optison and ultrasound irradiation were administrated together ( P < 0.01, compared to any other treatment in this study). The area of damaged muscles was enlarged by ultrasound irradiation in the presence of Optison microbubbles ( P < 0.01, compared to those groups without ultrasound irradiation). These results suggest that P85, microbubbles and ultrasound irradiation synergistically enhance plasmid DNA delivery in mouse skeletal muscles in vivo.

Repairing process in the transected muscle fibers of the mouse tibialis anterior.

Repairing process at the injury site in the transected muscle fibers of the mouse tibialis anterior was studied by light and electron microscopy. Immediately after the transection, the cut end (approximately 10 µm) was filled with dense network of disorganized myofilaments, in which disrupted membranous structures and degraded mitochondria were scattered. In the portion next to the portion exhibiting sudden necrotic changes, morphological features of the myofilaments, mitochondria and membranous structures appeared to be almost normal. The degradation of disorganized myofilaments at the cut end began within 1 hour after the transection, and at 1hour after the transection, the degenerating areas were noted in most of muscle fibers up to 150-250 µm from the cut end. Following the degradation, accumulation of mitochondria occurred between the necrotic and myofilament-predominant living portions, and several transverse tubules (T-tubules) and sarcoplasmic reticula were found between the mitochondria-accumulated and myofilament-predominant areas. In most cases, demarcation membrane formed between the mitochondria-accumulated and myofilament-predominant areas, and the fusion of T-tubules and sarcoplasmic reticula was encountered in these areas, suggesting that at least some parts of the demarcation membranes formed through fusion of T-tubules and sarcoplasmic reticula. This repairing process was completed in a number of muscle fibers within 6 hours after the transection. Macrophages were first found in the injured portions at 6 hours after the transection, increased in number with time, and several macrophages were distributed at 1 to 3 days after the transection. Some spindle-shaped cells were first found in the degenerating portions of the muscle fibers at 1 day after the transection. Since they were located along the basal lamina of the muscle fiber, and had a long oval pale nucleus and relatively abundant cytoplasm, they can be regarded as activated satellite cells. They gradually increased in number with time, and became larger and longer. On and after 5 days, thin regenerating muscle fibers exhibiting centrally located nuclei were observed, and they became gradually thicker with time. These findings indicate that the muscle regeneration was actively occurring during these periods. The repairing process is followed by the invasion of macrophages, and then the occurrence of muscle regeneration in the sequential order. These findings suggest that there might be close chronological relationship among these events.

Mutated fukutin-related protein (FKRP) localises as wild type in differentiated muscle cells

The mechanism of disease in forms of congenital and limb girdle muscular dystrophy linked to mutations in the gene encoding for Fukutin-related protein (FKRP) has previously been associated with the mis-localisation of FKRP from the Golgi apparatus. In the present report, we have transfected V5-tagged Fukutin-related protein expression constructs into differentiated C2C12 myotubes and the tibialis anterior of normal mice. The transfection of either wild type (WT) or several mutant constructs (P448L, C318Y, L276I) into myotubes consistently showed clear co-localisation with GM130, a Golgi marker. In contrast, whilst WT and the L276I localised to the Golgi of Cos-7 cells, the P448L and C318Y was mis-localised in the majority of these undifferentiated cells. The injection of the same constructs into the tibialis anterior of mice resulted in similar localisation of both the WT and all the mutants. Immunolabelling of FKRP in the muscle of MDC1C and LGMD2I patients was found to be indistinguishable from normal controls. Overall, these data suggest that retention in the endoplasmic reticulum of FKRP is not the main mechanism of disease but that this may instead relate to a disruption of the functional activity of this putative enzyme with its substrate(s) in the Golgi.