Wild-type Muscle Research Articles

HIGHLIGHTS ACROSS MYOLOGY: O.13 Altered calcium handling in a zebrafish model of SELENON congenital muscular dystrophy

SELENON congenital muscular dystrophy (previously designated SEPN1 congenital muscular dystrophy) causes muscle weakness, scoliosis, and respiratory insufficiency in infants and children. Targeted treatments for this devastating disease are currently lacking. SELENON encodes the protein Selenoprotein N, a member of the selenoprotein family. Selenoprotein N is expressed at high levels in developing and immature tissues and adopts a ubiquitous, low-level expression pattern in mature tissues. This raises questions about how loss of Selenoprotein N leads to muscle-specific disease. Overlap in clinical and histopathologic characteristics between patients with SELENON congenital muscular dystrophy and certain types of RYR1 myopathy raise the possibility of interactions between Selenoprotein N and the RYR1 calcium channel. This possibility is supported by altered calcium release in SELENON-deficient myotubes in vitro but has not been tested in vivo. Here, calcium release was measured from Selenoprotein N-deficient zebrafish muscle fibers in vivo. Selenoprotein N-deficient zebrafish show decreased amplitude but increased frequency of calcium spikes during myogenesis with subsequent abnormal myofibril alignment in slow-twitch muscle fibers. Further, mature muscle fibers of Selenoprotein N-deficient zebrafish show decreased amplitude and duration of calcium release in response to depolarization compared to wildtype and control muscle fibers. The characteristics of calcium release in Selenoprotein N-deficient zebrafish muscle is suggestive of a leaky RYR1 calcium channel, as is typically seen in the setting of oxidative stress. These data support a role for Selenoprotein N in modulating RYR1 function in vivo, both during muscle development and during excitation-contraction coupling in mature muscle fibers.

Sarcolemma wounding activates dynamin-dependent endocytosis in striated muscle.

Plasma membrane repair is an evolutionarily conserved mechanism by which cells can seal breaches in the plasma membrane. Mutations in several proteins with putative roles in sarcolemma integrity, membrane repair, and membrane transport result in several forms of muscle disease; however, the mechanisms that are activated and responsible for sarcolemma resealing are not well understood. Using the standard assays for membrane repair, which track the uptake of FM 1-43 dye into adult skeletal muscle fibers following laser-induced sarcolemma disruption, we show that labeling of resting fibers by FM1-43 prior to membrane wounding and the induced FM1-43 dye uptake after sarcolemma wounding occurs via dynamin-dependent endocytosis. Dysferlin-deficient muscle fibers show elevated dye uptake following wounding, which is the basis for the assertion that membrane repair is defective in this model. Our data show that dynamin inhibition mitigates the differences in FM1-43 dye uptake between dysferlin-null and wild-type muscle fibers, suggesting that elevated wound-induced FM1-43 uptake in dysferlin-deficient muscle may actually be due to enhanced dynamin-dependent endocytosis following wounding, though dynamin inhibition had no effect on dysferlin trafficking after wounding. By monitoring calcium flux after membrane wounding, we show that reversal of calcium precedes the sustained, slower increase of dynamin-dependent FM1-43 uptake in WT fibers, and that dysferlin-deficient muscle fibers have persistently increased calcium after wounding, consistent with its proposed role in resealing. These data highlight a previously unappreciated role for dynamin-dependent endocytosis in wounded skeletal muscle fibers and identify overactive dynamin-dependent endocytosis following sarcolemma wounding as a potential mechanism or consequence of dysferlin deficiency.

Striated Preferentially Expressed Protein Kinase (SPEG)-Deficient Skeletal Muscles Display Fewer Satellite Cells with Reduced Proliferation and Delayed Differentiation

Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.

A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing.

Loss of the neuromuscular junction (NMJ) is an early and critical hallmark in all forms of ALS. The study design was to develop a functional NMJ disease model by integrating motoneurons (MNs) differentiated from multiple ALS-patients' induced pluripotent stem cells (iPSCs) and primary human muscle into a chambered system. NMJ functionality was tested by recording myotube contractions while stimulating MNs by field electrodes and a set of clinically relevant parameters were defined to characterize the NMJ function. Three ALS lines were analyzed, 2 with SOD1 mutations and 1 with a FUS mutation. The ALS-MNs reproduced pathological phenotypes, including increased axonal varicosities, reduced axonal branching and elongation and increased excitability. These MNs formed functional NMJs with wild type muscle, but with significant deficits in NMJ quantity, fidelity and fatigue index. Furthermore, treatment with the Deana protocol was found to correct the NMJ deficits in all the ALS mutant lines tested. Quantitative analysis also revealed the variations inherent in each mutant lines. This functional NMJ system provides a platform for the study of both fALS and sALS and has the capability of being adapted into subtype-specific or patient-specific models for ALS etiological investigation and patient stratification for drug testing.

Transient Receptor Potential Cation Channels and Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia.

Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca dyshomeostasis in a mouse model relevant to malignant hyperthermia. This study examined the mechanisms responsible for Ca dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene-dose-dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia-susceptible muscle, both at rest and during the malignant hyperthermia crisis.

Overexpression of Staufen1 in DM1 mouse skeletal muscle exacerbates dystrophic and atrophic features

In myotonic dystrophy type 1 (DM1), the CUG expansion (CUGexp) in the 3' untranslated region of the dystrophia myotonica protein kinase messenger ribonucleic acid affects the homeostasis of ribonucleic acid-binding proteins, causing the multiple symptoms of DM1. We have previously reported that Staufen1 is increased in skeletal muscles from DM1 mice and patients and that sustained Staufen1 expression in mature mouse muscle causes a progressive myopathy. Here, we hypothesized that the elevated levels of Staufen1 contributes to the myopathic features of the disease. Interestingly, the classic DM1 mouse model human skeletal actin long repeat (HSALR) lacks overt atrophy while expressing CUGexp transcripts and elevated levels of endogenous Staufen1, suggesting a lower sensitivity to atrophic signaling in this model. We report that further overexpression of Staufen1 in the DM1 mouse model HSALR causes a myopathy via inhibition of protein kinase B signaling through an increase in phosphatase tensin homolog, leading to the expression of atrogenes. Interestingly, we also show that Staufen1 regulates the expression of muscleblind-like splicing regulator 1 and CUG-binding protein elav-like family member 1 in wild-type and DM1 skeletal muscle. Together, data obtained from these new DM1 mouse models provide evidence for the role of Staufen1 as an atrophy-associated gene that impacts progressive muscle wasting in DM1. Accordingly, our findings highlight the potential of Staufen1 as a therapeutic target and biomarker.

Mechanisms of altered skeletal muscle action potentials in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) patients suffer from progressive and debilitating motor dysfunction for which only palliative treatment is currently available. Previously, we discovered reduced skeletal muscle Cl- channel (ClC-1) and inwardly rectifying K+ channel (Kir) currents in R6/2 HD transgenic mice. To further investigate the role of ClC-1 and Kir currents in HD skeletal muscle pathology, we measured the effect of reduced ClC-1 and Kir currents on action potential (AP) repetitive firing in R6/2 mice using a two-electrode current clamp. We found that R6/2 APs had a significantly lower peak amplitude, depolarized maximum repolarization, and prolonged decay time compared with wild type (WT). Of these differences, only the maximum repolarization was accounted for by the reduction in ClC-1 and Kir currents, indicating the presence of additional ion channel defects. We found that both KV1.5 and KV3.4 mRNA levels were significantly reduced in R6/2 skeletal muscle compared with WT, which explains the prolonged decay time of R6/2 APs. Overall, we found that APs in WT and R6/2 muscle significantly and progressively change during activity to maintain peak amplitude despite buildup of Na+ channel inactivation. Even with this resilience, the persistently reduced peak amplitude of R6/2 APs is expected to result in earlier fatigue and may help explain the motor impersistence experienced by HD patients. This work lays the foundation to link electrical changes to force generation defects in R6/2 HD mice and to examine the regulatory events controlling APs in WT muscle.

Regulatory T cells suppress novel skeletal muscle macrophages identified by single-cell RNA sequencing

Abstract Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder that results from mutations in the dystrophin gene causing necrosis, inflammation and ultimately fibrosis. In DMD, muscle macrophages are multifaceted effector immune cells that regulate multiple pathogenic processes, including myofiber injury, inflammation and fibrosis, but also promote regeneration. Previously, we demonstrated that regulatory T cells (Tregs) ameliorated the severity of muscular dystrophy by regulating M1-like and M2-like macrophage activation, and more recently, a subset of undefined macrophages. Herein, we used single-cell RNA sequencing (scRNAseq) approaches to define the phenotypic complexity and transcriptional profile of muscle macrophages in healthy and dystrophic muscle. A T-distributed Stochastic Neighbor Embedding (t-SNE) analysis of gene expression data revealed that muscle macrophages clustered into six novel populations defined by unique transcriptional programs. We observed that a population expressing high levels of galectin-3 was absent in wildtype and dystrophic muscle before the onset of disease, but was increased at the onset of acute pathology and further expanded when Tregs were depleted in the mdx mouse model of DMD. In light of emerging evidence in the literature suggesting a role for galectin-3 and Tregs in fibrosis, we performed in vivo functional assays to underscore the role of the Treg-Macrophages axis in the control of fibrosis in DMD. Our data support a new paradigm, in which unique transcriptional programs define novel macrophage populations likely adapted to the diseased muscle milieu, and suggest that therapies focused on augmenting Tregs function in dystrophic muscle may be beneficial to treat fibrosis.

Lower Ca2+ enhances the K+-induced force depression in normal and HyperKPP mouse muscles.

Hyperkalemic periodic paralysis (HyperKPP) manifests as stiffness or subclinical myotonic discharges before or during periods of episodic muscle weakness or paralysis. Ingestion of Ca2+ alleviates HyperKPP symptoms, but the mechanism is unknown because lowering extracellular [Ca2+] ([Ca2+]e) has no effect on force development in normal muscles under normal conditions. Lowering [Ca2+]e, however, is known to increase the inactivation of voltage-gated cation channels, especially when the membrane is depolarized. Two hypotheses were tested: (1) lowering [Ca2+]e depresses force in normal muscles under conditions that depolarize the cell membrane; and (2) HyperKPP muscles have a greater sensitivity to low Ca2+-induced force depression because many fibers are depolarized, even at a normal [K+]e. In wild type muscles, lowering [Ca2+]e from 2.4 to 0.3 mM had little effect on tetanic force and membrane excitability at a normal K+ concentration of 4.7 mM, whereas it significantly enhanced K+-induced depression of force and membrane excitability. In HyperKPP muscles, lowering [Ca2+]e enhanced the K+-induced loss of force and membrane excitability not only at elevated [K+]e but also at 4.7 mM K+. Lowering [Ca2+]e increased the incidence of generating fast and transient contractures and gave rise to a slower increase in unstimulated force, especially in HyperKPP muscles. Lowering [Ca2+]e reduced the efficacy of salbutamol, a β2 adrenergic receptor agonist and a treatment for HyperKPP, to increase force at elevated [K+]e. Replacing Ca2+ by an equivalent concentration of Mg2+ neither fully nor consistently reverses the effects of lowering [Ca2+]e. These results suggest that the greater Ca2+ sensitivity of HyperKPP muscles primarily relates to (1) a greater effect of Ca2+ in depolarized fibers and (2) an increased proportion of depolarized HyperKPP muscle fibers compared with control muscle fibers, even at normal [K+]e.

Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle.

Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.

The Effect of Gas6‐Axl Double Knockout on Satellite Cell Proliferation and Skeletal Muscle Regeneration After Injury

Growth arrest‐specific 6 (Gas6) is an extracellular signaling molecule that is known to play a role in cell proliferation, inflammation and other processes through its interaction with Tyro3, Axl, and Mer receptors, but little is known about its role in skeletal muscle. The purpose of this study was to examine the effects of Gas6‐Axl double knockout (dKO) in skeletal muscle recovery after injury. Muscle injury was produced by injecting BaCl2 into tibialis anterior (TA) muscles of wild type (WT) and dKO mice. Muscle tissue was harvested 3, 7, and 14 days after injury. Gas6 and Axl expression over the injury timecourse was characterized by western blotting and ELISA. Muscle damage and cross‐sectional fiber areas were assessed on hematoxylin and eosin (H&amp;E) stained sections. Muscle satellite cell content and proliferation were assessed by Pax7/Ki‐67 staining. We found that both Gas6 and Axl protein expression increased substantially after injury in WT muscles, peaked at 3 days post injury, then declined toward basal levels thereafter. TA mass was lower for dKO muscles 14 days after injury, but fiber CSA was elevated in dKO muscles, suggesting a deficit in fiber number leading to compensatory hypertrophy of existing fibers. Pax7‐positive satellite cell number was unaffected by genotype in uninjured control muscles, but was ~3X higher in WT vs. dKO muscles at 3 days post injury, suggesting that the rate of satellite cell proliferation was suppressed or delayed by dKO. These results indicate that Gas6‐Axl signaling plays a role in regulating skeletal muscle regeneration after injury. Impaired Gas6 function may be linked to excess hypertrophy and loss of muscle mass after injury.Support or Funding InformationThis work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI) and a BYU Graduate Research Fellowship.

Manipulating Levels of Stress‐Response Proteins in a Drosophila Model of Myosin‐Based Inclusion Body Myopathy 3 Worsens Muscle Dysfunction

Utilizing a Drosophila model of inclusion body myopathy 3 (IBM‐3), we identified a subset of stress‐response proteins essential for skeletal muscle proteostasis in both health and disease. Inclusion body myopathy 3 (IBM‐3) is a rare, dominant skeletal muscle disease caused by an E706K substitution in the SH1 helix of myosin heavy chain IIa. We produced a model of the human disease in the indirect flight muscle (IFM) of Drosophila. As in the human disease, progressive degradation occurs in muscle expressing the IBM‐3 mutant myosin. Homozygotes assemble defective sarcomeres that accumulate thermally unstable myosin and autophagic vesicles. To further investigate the mechanism of pathogenesis in IBM‐3 muscle fibers, we extracted insoluble proteins from IFM of young and old homozygotes and identified aggregated proteins within inclusions. Quantitative iTRAQ proteomics defined 18 proteins with &gt;1.5‐fold difference in relative abundance at both ages in mutants vs. controls. Two of 6 proteins with increased levels in IBM‐3 have small heat shock protein domains (Hsp20, CG7409), while the 12 proteins with decreased levels include Hsp23, Hsp60, and Trim32 (abba/thin), which has ubiquitin‐protein transferase activity. Using the GAL4‐UAS system for over‐expression or knock down (RNAi), we tested the role of these proteins in wild‐type muscle and determined how altering expression affects the IBM‐3 heterozygote phenotype. While knockdown of Hsp20, Hsp23 or CG7409 yielded normal flight in wild type, Hsp23 or CG7409 over‐expression resulted in age‐dependent reduction in flight ability. Knockdown or over‐expression of these chaperones typically exacerbated the IBM‐3/+ phenotype. As expected, Trim32 knockdown eliminated flight in wild‐type or IBM‐3/+ flies and correlated with less organized, thinner myofibrils. Overexpression of Trim32 also eliminated flight and yielded deteriorating myofibrils that hypercontracted by 3 weeks of age. Remarkably, electron microscopy shows the ultrastructure of this phenotype in IBM‐3/+ resembles the dramatic destruction of myofibrils observed in IBM‐3 homozygotes. Overall, each of the stress response proteins assessed is critical for proper muscle development, performance, or maintenance. Altering levels of these key proteins in muscle fibers challenged with the IBM‐3/+ phenotype has detrimental effects. Our findings add to evidence that expression levels of stress response proteins modulate alternative pathways to respond to challenges to proteostasis.Support or Funding InformationThis work was supported by grants from the Muscular Dystrophy Association (MDA217900)and the National Institutes of Health (NIGMS R37 32443) to S.I.B. The authors acknowledgethe Bloomington Drosophila Stock Center (NIH P40OD018537), the Vienna DrosophilaResource Center and Dr. Richard Cripps (SDSU) for Drosophila stocks.

Acute Knockdown of MG29 Alters Skeletal Muscle Cells Differentiation and Leads to Cellular Atrophy

Sarcopenia is defined as the progressive loss of skeletal muscle mass and development of force/power that occurs during aging. It is estimated that more than 30% of humans over 60 years of age suffer from sarcopenia and age‐related loss of muscle function that contributes to more than half of all chronic conditions in people over 50 and accounts for 1.5% of total U.S. healthcare expenditures (&gt;$40 billion). Mitsugumin 29 (MG29), is a muscle‐specific protein belonging to the synaptophysin and MAL and related proteins for vesicle trafficking and membrane link (MARVEL) families of proteins which are involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the proper formation of the transverse‐tubule (TT) system, maintenance of lipid content of the sarcolemmal membrane, and efficient signaling between RyR1 and the store‐operated Ca2+ entry (SOCE) machinery. MG29 null (mg29−/−) mouse muscle displays abnormalities in the membrane ultrastructure of the triad junction where the TT are swollen and the sarcoplasmic reticulum (SR) networks show a vacuolated and fragmented structure. Clear phenotypic changes are seen in mg29−/− skeletal muscle at young ages (3–6 months) that resemble those seen in aged wildtype muscle. Our previous work clearly demonstrated that SOCE is compromised in an MG29‐associated manner in aged skeletal muscle and that reduced SOCE associates with a drastic decrease in MG29 content (~50%), altered intracellular Ca2+ homeostasis and decreased specific contractile force. But the function of MG29 on muscle differentiation remains unclear. In this study, we knocked down MG29 expression in mouse primary skeletal muscle cells by using MG29 siRNA. Our data shows that 48h after MG29 siRNA (10nM) transfection, the expression of MG29 decreased by ~ 80%, compared to negative control siRNA. After MG29 siRNA treatment, at day 3 of differentiation, thinner, longer myotubes were observed compared to negative controls (diameter of myotubes: negative control = 24.9 ± 2.08μm; MG29 siRNA = 19.1 ± 0.65μm, p &lt; 0.05; Length of myotubes: negative control = 424.9 ± 50.2μm; MG29 siRNA = 580.4 ± 39.9 μm, p &lt; 0.05). Surprisingly, fusion index (FI) was significantly increased compared to control (FI values: negative control= 53.2% ± 6.8; MG29 siRNA = 70.3% ± 7.9, p &lt; 0.05), in agreement with increased mRNA expression of myogenenic markers MyoG and MyoD detected by real time PCR. These results suggest that the acute knock down of MG29 leads in just three days to complex myogenic changes characterized by increased myogenic differentiation but with abnormally thinner and longer muscle cells. These studies further indicate specific roles of MG29 in maintaining normal muscle mass and function, and its importance for cellular homeostasis during aging.Support or Funding InformationThis work was supported by NIH‐National Institutes of Aging R01AG056504 (NW, MB), and the George W. and Hazel M. Jay and Evanston Research Endowments (MB).

Class II phosphatidylinositol 3-kinase \u03b1 and \u03b2 isoforms are required for vascular smooth muscle Rho activation, contraction and blood pressure regulation in mice

Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2β, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2β at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2β KO, but not single KO mice of either PI3K-C2α or PI3K-C2β, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2β but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca2+ mobilization. These data indicate that PI3K-C2α and PI3K-C2β play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.

Targeting RAGE prevents muscle wasting and prolongs survival in cancer cachexia.

BackgroundCachexia, a multifactorial syndrome affecting more than 50% of patients with advanced cancer and responsible for ~20% of cancer‐associated deaths, is still a poorly understood process without a standard cure available. Skeletal muscle atrophy caused by systemic inflammation is a major clinical feature of cachexia, leading to weight loss, dampening patients' quality of life, and reducing patients' response to anticancer therapy. RAGE (receptor for advanced glycation end‐products) is a multiligand receptor of the immunoglobulin superfamily and a mediator of muscle regeneration, inflammation, and cancer.MethodsBy using murine models consisting in the injection of colon 26 murine adenocarcinoma (C26‐ADK) or Lewis lung carcinoma (LLC) cells in BALB/c and C57BL/6 or Ager −/− (RAGE‐null) mice, respectively, we investigated the involvement of RAGE signalling in the main features of cancer cachexia, including the inflammatory state. In vitro experiments were performed using myotubes derived from C2C12 myoblasts or primary myoblasts isolated from C57BL/6 wild type and Ager −/− mice treated with the RAGE ligand, S100B (S100 calcium‐binding protein B), TNF (tumor necrosis factor)α±IFN (interferon) γ, and tumour cell‐ or masses‐conditioned media to analyse hallmarks of muscle atrophy. Finally, muscles of wild type and Ager −/− mice were injected with TNFα/IFNγ or S100B in a tumour‐free environment.ResultsWe demonstrate that RAGE is determinant to activate signalling pathways leading to muscle protein degradation in the presence of proinflammatory cytokines and/or tumour‐derived cachexia‐inducing factors. We identify the RAGE ligand, S100B, as a novel factor able to induce muscle atrophy per se via a p38 MAPK (p38 mitogen‐activated protein kinase)/myogenin axis and STAT3 (signal transducer and activator of transcription 3)‐dependent MyoD (myoblast determination protein 1) degradation. Lastly, we found that in cancer conditions, an increase in serum levels of tumour‐derived S100B and HMGB1 (high mobility group box 1) occurs leading to chronic activation/overexpression of RAGE, which induces hallmarks of cancer cachexia (i.e. muscle wasting, systemic inflammation, and release of tumour‐derived pro‐cachectic factors). Absence of RAGE in mice translates into reduced serum levels of cachexia‐inducing factors, delayed loss of muscle mass and strength, reduced tumour progression, and increased survival.ConclusionsRAGE is a molecular determinant in inducing the hallmarks of cancer cachexia, and molecular targeting of RAGE might represent a therapeutic strategy to prevent or counteract the cachectic syndrome.

Impaired muscle spindle function in murine models of muscular dystrophy.

Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin- and dysferlin-deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin-deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy. Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin- and dysferlin-deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin-deficient mice. Single-unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp-and-hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy.

It's well and truly time to stop stating that AMPK regulates glucose uptake and fat oxidation during exercise.

None for Perspective.

Mechanical factors tune the sensitivity of mdx muscle to eccentric strength loss and its protection by antioxidant and calcium modulators

BackgroundDystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10–95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC.MethodsWe completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin).ResultsThe magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss.ConclusionsThese results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

Histological features of masticatory muscles after botulinum toxin A injection into the right masseter muscle of dystrophin deficient (mdx-) mice

Objective/backgroundThe most frequently used animal model for human DMD (Duchenne muscular dystrophy) research is the mdx mouse. In both species, characteristic histological changes like inflammation, muscle fiber degeneration and fibrosis are the same, but in contrast to humans, in mdx mice, phases of muscle fiber degeneration are compensated by regeneration processes. AimTherefore, the interest of this study was to evaluate histological features in masticatory muscles after BTX-A injection into the right masseter muscle of wild type and dystrophic (mdx) mice, illustrating de- and regeneration processes induced by this substance. Material and methodsThe right masseter muscle of 100 days old healthy and mdx mice were selectively paralyzed by a single intramuscular BTX-A injection. Masseter as well as temporal muscle of injection and non-injection side were carefully dissected 21 days and 42 days after injection, respectively, and fiber diameter, cell nuclei position, necrosis and collagen content were analyzed histomorphologically in order to evaluate de- and regeneration processes in these muscles. Statistical analysis was performed using SigmaStat Software and Mann Whitney U-test (significance level: p < 0.05). ResultsAt both investigation periods and in both mouse strains fiber diameter was significantly reduced and collagen content was significantly increased in the right injected masseter muscle whereas fiber diameters in mdx mice were much smaller, and these differences were even more apparent at the second investigation period. Necrosis and central located nuclei could generally be found in all mdx mice muscles investigated with an amount of centronucleation exceeding 60%, and a significant increase of necrosis six weeks after injection. In wild type mice central located nuclei could primarily be found in the treated masseter muscle with a portion of 2.7%, and this portion decreased after six weeks, whereas in mdx mice a decrease could also be seen in the non-injected muscles. In contrast, in wild type mice necrosis was not apparent at any time and in all muscles investigated. ConclusionFrom our results it can be concluded that in mdx mice masticatory muscles de- and regeneration processes were extended, triggered by a selective BTX-A injection, or mdx mice at this age, independently of BTX-A treatment, went through another cycle of de- and regeneration as a characteristic of this disease.

Effects of a titin mutation on force enhancement and force depression in mouse soleus muscles.

The active isometric force produced by muscles varies with muscle length in accordance with the force-length relationship. Compared with isometric contractions at the same final length, force increases after active lengthening (force enhancement) and decreases after active shortening (force depression). In addition to cross-bridges, titin has been suggested to contribute to force enhancement and depression. Although titin is too compliant in passive muscles to contribute to active tension at short sarcomere lengths on the ascending limb and plateau of the force-length relationship, recent evidence suggests that activation increases titin stiffness. To test the hypothesis that titin plays a role in force enhancement and depression, we investigated isovelocity stretching and shortening in active and passive wild-type and mdm (muscular dystrophy with myositis) soleus muscles. Skeletal muscles from mdm mice have a small deletion in the N2A region of titin and show no increase in titin stiffness during active stretch. We found that: (1) force enhancement and depression were reduced in mdm soleus compared with wild-type muscles relative to passive force after stretch or shortening to the same final length; (2) force enhancement and force depression increased with amplitude of stretch across all activation levels in wild-type muscles; and (3) maximum shortening velocity of wild-type and mdm muscles estimated from isovelocity experiments was similar, although active stress was reduced in mdm compared with wild-type muscles. The results of this study suggest a role for titin in force enhancement and depression, which contribute importantly to muscle force during natural movements.