Wild-type Muscle Research Articles

Cell transplantation-mediated dystrophin supplementation efficacy in Duchenne muscular dystrophy mouse motor function improvement demonstrated by enhanced skeletal muscle fatigue tolerance

BackgroundDuchenne muscular dystrophy (DMD) is an incurable neuromuscular disease leading to progressive skeletal muscle weakness and fatigue. Cell transplantation in murine models has shown promise in supplementing the lack of the dystrophin protein in DMD muscles. However, the establishment of novel, long-term, relevant methods is needed to assess its efficiency on the DMD motor function. By applying newly developed methods, this study aimed to evaluate the functional and molecular effects of cell therapy-mediated dystrophin supplementation on DMD muscles.MethodsDystrophin was supplemented in the gastrocnemius of a 5-week-old immunodeficient DMD mouse model (Dmd-null/NSG) by intramuscular xenotransplantation of healthy human immortalized myoblasts (Hu5/KD3). A long-term time-course comparative study was conducted between wild-type, untreated DMD, and dystrophin supplemented-DMD mouse muscle functions and histology. A novel GO-ATeam2 transgenic DMD mouse model was also generated to assess in vivo real-time ATP levels in gastrocnemius muscles during repeated contractions.ResultsWe found that 10.6% dystrophin supplementation in DMD muscles was sufficient to prevent low values of gastrocnemius maximal isometric contraction torque (MCT) at rest, while muscle fatigue tolerance, assessed by MCT decline after treadmill running, was fully ameliorated in 21-week-old transplanted mice. None of the dystrophin-supplemented fibers were positive for muscle damage markers after treadmill running, with 85.4% demonstrating the utilization of oxidative metabolism. Furthermore, ATP levels in response to repeated muscle contractions tended to improve, and mitochondrial activity was significantly enhanced in dystrophin supplemented-fibers.ConclusionsCell therapy-mediated dystrophin supplementation efficiently improved DMD muscle functions, as evaluated using newly developed evaluation methods. The enhanced muscle fatigue tolerance in 21-week-old mice was associated with the preferential regeneration of damage-resistant and oxidative fibers, highlighting increased mitochondrial activity, after cell transplantation. These findings significantly contribute to a more in-depth understanding of DMD pathogenesis.

Abstract 58: The 5-HT7 receptor contributes to increased hindquarter blood flow caused by skeletal muscle contraction

Exogenous serotonin (5-hydroxytryptamine, 5-HT) causes a reproducible decrease in blood pressure in normotensive and hypertensive rats. This is associated with dilation of arterioles in skeletal muscle. The 5-HT7 receptor KO rat strain allowed us to test the role of this receptor in both responses as well as the effects of endogenous 5-HT -- acting at 5-HT7 receptors -- on normal circulatory regulation. First, we hypothesized that the 5-HT7 receptor KO rat would have a higher hindquarter (HQ) vascular resistance (HQVR) than wildtype (WT) because of loss of this receptor. In rats anesthetized with isoflurane, instrumented with arterial catheters and flow probes on the terminal aorta, male KO rats had significantly higher resting HQVR [mm Hg/ml/min; KO (16.0±2.0) vs WT (10.8±0.6.0), p = 0.04] and lower HQ blood flow than normal Sprague-Dawley (SD) rats. No differences in resting HQVR or HQ flow were observed in female rats. Intravenous infusion of 5-HT (50 ug/kg/min) reduced systemic blood pressure and HQVR; these parameters were significantly attenuated (~56%) in male KO rats versus WT and SD animals. In females, falls in systemic blood pressure and HQVR were absent in both KO and WT rats. Second, as 5-HT has been implicated in the pathophysiology of heart failure (HF), a left anterior descending artery ligation model of HF was used to investigate the potential role of 5-HT7 regulation of HQ flow in HF-associated exercise intolerance. The ability of hindlimb muscle stimulation to increase blood flow using transdermal neuromuscular electrical stimulation was tested (NMES). M and F KO rats showed a reduced ability to increase HQ flow during muscle contraction vs WT (% over basal: M WT = 118±18 vs KO =14.6±7.1; F WT= 101±12 vs KO = 7.6±6) rats. This was observed in both intact and HF rats (in which flow changes were already impaired). Acute administration of the 5-HT7 receptor antagonist SB269970 abolished the ability of NMES to increase HQ flow in normal SD rats. Importantly, the microcirculatory capacity, visualized by Lectin-594, was not significantly different in the gastrocnemius muscle of WT (arbitrary fluorescence units/area: 946±40) vs KO (914±69). These data support that the 5-HT7 receptor, likely activated by endogenous 5-HT, is an important regulator of skeletal muscle vascular resistance. Thus, 5-HT may have a larger role in normal and pathophysiological regulation of the circulation than has previously been appreciated.

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in lamin A/C-deficient mice.

Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

Abstract 3028: Diabetic Complication Ischemia Triggers Muscle Mass And Muscle Weight Loss By Reducing Mesenchymal Progenitor Cells Population Proliferation

Introduction: Mesenchymal progenitor cells (MPCs), such as myoblasts and fibroadipogenic progenitors (FAPs), and their communications with each other and with immune and vascular cells are responsible for muscle maintenance and regeneration. We hypothesize that in diabetes, loss of muscle mass after induction of hind limb ischemia is mediated by reduction in myoblast proliferation, thereby impairing muscle regeneration. Methods: Ischemic injury was induced in male diabetic (Akita) and littermate Wild Type (WT) mice via unilateral femoral artery ligation (FAL). Gastrocnemius muscle was collected at baseline, 3 and 7 days after surgery for single-cell RNA sequencing (scRNA seq). We identified the different cell populations in clusters, determined differentially-expressed genes, utilized Reactome Pathway Analysis and analyzed the cell cycle progression status and cell-cell interactions. Results: scRNA seq analysis of diabetic mice muscle, but not WT muscle, showed a reduction in MPCs, myoblasts and fibroadipogenic progenitors (FAPs) at 3 days post-FAL vs. baseline. FAPs were identified as two distinct subclusters; FAPs/Lum+ (associated with cell cycle regulation) and FAPs/Prg4+ (associated with extracellular matrix remodeling). We observed a lack of pathways involved in cell cycle progression in myoblasts at 3 days post- FAL in diabetic vs. WT muscle. At day 7 post-FAL, we observed an increased number of cell cycle pathways vs. day 3 post-FAL in diabetic muscle, implying a delayed response in the myoblasts. Cell cycle progression analysis revealed fewer diabetic myoblasts in S/G2M phase, with lower expression of proliferation markers (Mki67, Top2a, Mcm6 and Pcna), but no significant differences were observed in FAP/Lum+ cells. Cell-cell interaction analysis at day 3 post-FAL revealed fewer FAP-myoblast interactions in diabetic muscle, while interactions between either FAPs/myoblasts and myeloid cells were higher. Conclusion: These findings suggest that understanding the network of inter-cellular communications among MPCs, immune and vascular cells, and their roles in reduced myoblast proliferation after ischemia, is critical to identify new therapies to stimulate regeneration in diabetes and peripheral vascular disease.

Muscle regeneration is not impaired by ablation of UCP-1-expressing cells

INTRODUCTION: Recently, cells expressing uncoupling protein 1 (UCP-1) have been proposed to impact muscle physiology. First, transplant of brown adipose tissue (which has high expression of UCP-1) enhanced regeneration in the injured mouse rotator cuff. Second, isolated UCP-1+ muscle fibroadipogenic progenitors (FAPs) treated to enhance UCP-1 expression improved injured mouse rotator cuff muscle pathology. However, it remains unknown whether endogenous UCP-1-expressing cells contribute to muscle regeneration. Therefore, the objective of this study was to assess whether mice that lack UCP-1-expressing cells demonstrate impaired muscle regeneration following toxin administration. METHODS: Experiments were performed using tibialis anterior (TA) and 5th toe extensor digitorum longus (EDL) muscle of wild type C57BL/6J (WT; n=22), and uncoupling protein 1 diphtheria Toxin A (UCP1-DTA; n=18) mice, which lack all UCP-1 expressing cells. All procedures were performed in accordance with the National Institute of Health’s Guide for the Use and Care of Laboratory Animals and were approved by Washington University School of Medicine IACUC. Muscle injury was induced using intramuscular injection of 50%v/v solution of glycerol. Regeneration was assessed at “early” (7 days post-injury (dpi)), “intermediate” (14 dpi) and “late” (21 dpi) stages. Outcome measures were cell population dynamics by flow cytometry, ex-vivo physiology testing and histological scoring as appropriate for each timepoint. RESULTS: Our preliminary data in early regeneration show that UCP1-DTA mice have significantly greater percentage of FAPs compared to WT mice (29.84 ± 1.24% higher cell population, p<0.0134), however no differences in muscle satellite (stem) cells between genotypes. Physiological and histological outcomes at intermediate and late-stage regeneration find a baseline deficit in muscle mass (23.69 ± 2.3% smaller, p<0.0001), type 2B fiber cross-sectional area (32.56 ± 4.8% smaller, p<0.0001) and normalized muscle active force generation (16.2 ± 3.3% lower force, p<0.0154) in UCP1-DTA compared with WT. However, mass and contractile forces recover to a similar extent between the genotypes following glycerol injection suggesting the phenotypic drivers of the reduced mass and contractile function do not affect muscle regeneration. CONCLUSIONS: Taken together, the data from this study point to a baseline growth deficit in mice lacking UCP-1-expressing cells. However, in two separate timepoints of regeneration (14 and 21dpi), we see that UCP1-DTA mice have similar recovery of mass and normalized contractile force to baseline values suggesting regeneration can proceed normally without UCP-1 expressing cells. However, further data collection will be needed at early-stage regeneration to help discern whether mice lacking UCP-1-expressing cells have initial changes in cell population dynamics given the increased FAP population at 7dpi. ACKNOWLEDGMENTS: This work was supported by R01AR075773 and R21AR071582 to GAM. 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.

Distribution of interstitial cells within the mouse esophagus and their spatial relationship to enteric neurons

Background/Objectives: The esophagus is a conduit for the passage of ingested material into the stomach. In humans, it is divided into thirds; the proximal portion is composed of skeletal muscle, the distal is composed of smooth muscle and the middle is a transition zone between skeletal and smooth muscles. In other species such as the mouse, the esophagus has been described to consist entirely of skeletal muscle. Despite this, interstitial cells of Cajal (ICC), cells that regulate gastrointestinal (GI) smooth muscle contractility, have been identified throughout the mouse esophageal body (Rumessen et al., 2001; PMID:11683170). Additional studies in the human esophagus have identified another interstitial cell type, PDGFRa+ cells (Chen et al., 2013; PMID:23734090). However, the distribution of interstitial cells, motor and sensory neurons and their spatial relationship to one another have never been examined. Since interstitial cells modulate contractility of smooth muscle in other GI regions, the presence of these cells leads to the hypothesis that the mouse esophagus also contains smooth muscle cells (SMCs). Therefore, the aims of our study were to 1) determine if SMCs are found within the mouse esophagus, 2) characterize the distribution of ICC and PDGFRa+ cells, and 3) examine the relationship of interstitial cells to enteric motor and sensory neurons. Methods: Wildtype (C57Bl/6) and smooth muscle eGFP (smMHC-eGFP) mouse esophagus was used for dual labeling immunohistochemistry. ICC were labeled with antibodies against Kit or ANO1. PDGFRa+ cells were labeled with antibodies against PDGFRa or SK3. Extrinsic sympathetic neurons were labeled with tyrosine hydroxylase (TH). Motor neurons were labeled with antibodies against NOS, VIP (inhibitory) and vChAT (excitatory). Sensory neurons were labeled with CGRP. Images were taken with a Leica Stellaris 5 confocal microscope and processed using LasX software. Results: SMCs were found throughout the length of the esophagus, although their density declined proximally. Intramuscular ICC expressed ANO1 and were distributed throughout the length of the esophagus with the greatest density in the gastroesophageal junction (GEJ). Submucosal and intramuscular PDGFRa+ cells were distributed throughout the esophagus and GEJ. Intramuscular PDGFRa+ cells expressed SK3 though this expression declined proximally. SMCs, intramuscular ICC and PDGFRa+ cells were closely associated with one another as well as with neurons. Discussion/Conclusions: This study is the first to systematically characterize SMCs, ICC, PDGFRa+ cells and neurons in the mouse esophagus. These findings suggest that the mouse may represent a better translational model for studying esophageal motility than previously assumed. NIH DK129528 and P20GM130459. 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.

Plasticity and structural alterations of mitochondria and sarcoplasmic organelles in muscles of mice deficient in α-dystrobrevin, a component of the dystrophin-glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.

It's more than the amount that counts: implications of collagen organization on passive muscle tissue properties revealed with micromechanical models and experiments.

Collagen accumulation is often used to characterize skeletal muscle fibrosis, but the role of collagen in passive muscle mechanics remains debated. Here we combined finite-element models and experiments to examine how collagen organization contributes to macroscopic muscle tissue properties. Tissue microstructure and mechanical properties were measured from in vitro biaxial experiments and imaging in dystrophin knockout (mdx) and wild-type (WT) diaphragm muscle. Micromechanical models of intramuscular and epimuscular extracellular matrix (ECM) regions were developed to account for complex microstructure and predict bulk properties, and directly calibrated and validated with the experiments. The models predicted that intramuscular collagen fibres align primarily in the cross-muscle fibre direction, with greater cross-muscle fibre alignment in mdx models compared with WT. Higher cross-muscle fibre stiffness was predicted in mdx models compared with WT models and differences between ECM and muscle properties were seen during cross-muscle fibre loading. Analysis of the models revealed that variation in collagen fibre distribution had a much more substantial impact on tissue stiffness than ECM area fraction. Taken together, we conclude that collagen organization explains anisotropic tissue properties observed in the diaphragm muscle and provides an explanation for the lack of correlation between collagen amount and tissue stiffness across experimental studies.

Motor neuron activity enhances the proteomic stress caused by autophagy defects in the target muscle.

Several lines of evidence demonstrate that increased neuronal excitability can enhance proteomic stress. For example, epilepsy can enhance the proteomic stress caused by the expression of certain aggregation-prone proteins implicated in neurodegeneration. However, unanswered questions remain concerning the mechanisms by which increased neuronal excitability accomplishes this enhancement. Here we test whether increasing neuronal excitability at a particular identified glutamatergic synapse, the Drosophila larval neuromuscular junction, can enhance the proteomic stress caused by mutations in the ER fusion/GTPase gene atlastin (atl). It was previously shown that larval muscle from the atl2 null mutant is defective in autophagy and accumulates protein aggregates containing ubiquitin (poly-UB aggregates). To determine if increased neuronal excitability might enhance the increased proteomic stress caused by atl2, we activated the TrpA1-encoded excitability channel within neurons. We found that TrpA1 activation had no effect on poly-UB aggregate accumulation in wildtype muscle, but significantly increased poly-UB aggregate number in atl2 muscle. Previous work has shown that atl loss from either neuron or muscle increases muscle poly-UB aggregate number. We found that neuronal TrpA1 activation enhanced poly-UB aggregate number when atl was removed from muscle, but not from neuron. Neuronal TrpA1 activation enhanced other phenotypes conferred by muscle atl loss, such as decreased pupal size and decreased viability. Taken together, these results indicate that the proteomic stress caused by muscle atl loss is enhanced by increasing neuronal excitability.

Acute exercise modulates Trim63 and Bmal1 in the skeletal muscle of IL-10 knockout mice

The anti-inflammatory role of physical exercise is mediated by interleukin 10 (IL-10), and their release is possibly upregulated in response to IL-6. Previous studies demonstrated that mice lacking IL-6 (IL-6 KO mice) exhibited diminished exercise tolerance, and reduced strength. Rev-erbα, a transcriptional suppressor involved in circadian rhythm, has been discovered to inhibit the expression of genes linked to bodily functions, encompassing inflammation and metabolism. It also plays a significant role in skeletal muscle and exercise performance capacity. Given the potential association between Rev-erbα and the immune system and the fact that both pathways are modulated following acute aerobic exercise, we examined the physical performance of IL-10 KO mice and analyzed the modulation of the atrophy and Rev-erbα pathways in the muscle of wild type (WT) and IL-10 KO mice following one session of acute exercise. For each phenotype, WT and IL-10 KO were divided into two subgroups (Control and Exercise). The acute exercise session started at 6 m/min, followed by 3 m/min increments every 3 min until animal exhaustion. Two hours after the end of the exercise protocol, the gastrocnemius muscle was removed and prepared for the reverse transcription-quantitative polymerase chain reaction (RT-q-PCR) and immunoblotting technique. In summary, compared to WT, the IL-10 KO animals showed lower body weight and grip strength in the baseline. The IL-10 control group presented a lower protein content of BMAL1. After the exercise protocol, the IL-10 KO group had higher mRNA levels of Trim63 (atrophy signaling pathway) and lower mRNA levels of Clock and Bmal1 (Rev-erbα signaling pathway). This is the first study showing the relationship between Rev-erbα and atrophy in IL-10 KO mice. Also, we accessed a public database that analyzed the gastrocnemius of MuRF KO mice submitted to two processes of muscle atrophy, a denervation surgery and dexamethasone (Dexa) injections. Independently of knockout, the denervation demonstrated lower Nr1d1 levels. In conclusion, IL-10 seems to be a determinant in the Rev-erbα pathway and atrophy after acute exercise, with no modulation in the baseline state.

Biomarkers for Duchenne muscular dystrophy progression: impact of age in the mdx tongue spared muscle

BackgroundDuchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation, and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-β, are potential biomarkers for dystrophic muscle characterization.MethodsTo investigate disease progression and aging, we utilized young (1 month old) and old (21–25 months old) mdx and wild-type tongue muscles. Metabolite changes were analyzed using 1H nuclear magnetic resonance, while TNF-α and TGF-β were assessed using Western blotting to examine inflammation and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups.ResultsThe histological analysis of the mid-belly tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild-type or mdx whole tongues of the same age. The metabolites alanine, methionine, and 3-methylhistidine were higher, and taurine and glycerol were lower in young tongues in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine, and the proteins TNF-α and TGF-β had no difference in the analysis between groups (p > 0.05).ConclusionsSurprisingly, histological, metabolite, and protein analysis reveal that the tongue of old mdx remains partially spared from the severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes in the tongue muscle. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-β do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.

Combining electromyographic and electrical impedance data sets through machine learning: A study in D2-mdx and wild-type mice.

Needle impedance-electromyography (iEMG) assesses the active and passive electrical properties of muscles concurrently by using a novel needle with six electrodes, two for EMG and four for electrical impedance myography (EIM). Here, we assessed an approach for combining multifrequency EMG and EIM data via machine learning (ML) to discriminate D2-mdx muscular dystrophy and wild-type (WT) mouse skeletal muscle. iEMG data were obtained from quadriceps of D2-mdx mice, a muscular dystrophy model, and WT animals. EIM data were collected with the animals under deep anesthesia and EMG data collected under light anesthesia, allowing for limited spontaneous movement. Fourier transformation was performed on the EMG data to provide power spectra that were sampled across the frequency range using three different approaches. Random forest-based, nested ML was applied to the EIM and EMG data sets separately and then together to assess healthy versus disease category classification using a nested cross-validation procedure. Data from 20 D2-mdx and 20 WT limbs were analyzed. EIM data fared better than EMG data in differentiating healthy from disease mice with 93.1% versus 75.6% accuracy, respectively. Combining EIM and EMG data sets yielded similar performance as EIM data alone with 92.2% accuracy. We have demonstrated an ML-based approach for combining EIM and EMG data obtained with an iEMG needle. While EIM-EMG in combination fared no better than EIM alone with this data set, the approach used here demonstrates a novel method of combining the two techniques to characterize the full electrical properties of skeletal muscle.

REPTOR and CREBRF encode key regulators of muscle energy metabolism

Metabolic flexibility of muscle tissue describes the adaptive capacity to use different energy substrates according to their availability. The disruption of this ability associates with metabolic disease. Here, using a Drosophila model of systemic metabolic dysfunction triggered by yorkie-induced gut tumors, we show that the transcription factor REPTOR is an important regulator of energy metabolism in muscles. We present evidence that REPTOR is activated in muscles of adult flies with gut yorkie-tumors, where it modulates glucose metabolism. Further, in vivo studies indicate that sustained activity of REPTOR is sufficient in wildtype muscles to repress glycolysis and increase tricarboxylic acid (TCA) cycle metabolites. Consistent with the fly studies, higher levels of CREBRF, the mammalian ortholog of REPTOR, reduce glycolysis in mouse myotubes while promoting oxidative metabolism. Altogether, our results define a conserved function for REPTOR and CREBRF as key regulators of muscle energy metabolism.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of β-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle

ObjectiveThe AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. MethodsA recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. ResultsSTO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. ConclusionsWe demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Skeletal muscle overexpression of sAnk1.5 in transgenic mice does not predispose to type 2 diabetes

Genome-wide association studies (GWAS) and cis-expression quantitative trait locus (cis-eQTL) analyses indicated an association of the rs508419 single nucleotide polymorphism (SNP) with type 2 diabetes (T2D). rs508419 is localized in the muscle-specific internal promoter (P2) of the ANK1 gene, which drives the expression of the sAnk1.5 isoform. Functional studies showed that the rs508419 C/C variant results in increased transcriptional activity of the P2 promoter, leading to higher levels of sAnk1.5 mRNA and protein in skeletal muscle biopsies of individuals carrying the C/C genotype. To investigate whether sAnk1.5 overexpression in skeletal muscle might predispose to T2D development, we generated transgenic mice (TgsAnk1.5/+) in which the sAnk1.5 coding sequence was selectively overexpressed in skeletal muscle tissue. TgsAnk1.5/+ mice expressed up to 50% as much sAnk1.5 protein as wild-type (WT) muscles, mirroring the difference reported between individuals with the C/C or T/T genotype at rs508419. However, fasting glucose levels, glucose tolerance, insulin levels and insulin response in TgsAnk1.5/+ mice did not differ from those of age-matched WT mice monitored over a 12-month period. Even when fed a high-fat diet, TgsAnk1.5/+ mice only presented increased caloric intake, but glucose disposal, insulin tolerance and weight gain were comparable to those of WT mice fed a similar diet. Altogether, these data indicate that sAnk1.5 overexpression in skeletal muscle does not predispose mice to T2D susceptibility.

Plasticity in airway smooth muscle differentiation during mouse lung development.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlinopathy.

Dysferlin-null A/J myofibers generate abnormal Ca2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J Physiol., 2017). Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM; however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was also ineffective when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling; GCaMP6fu alone or as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft. Supported by the Jain Foundation, and NIH (RO1 AR064268).

Eccentric contraction-induced strength loss in dystrophin-deficient muscle: Preparations, protocols, and mechanisms.

The absence of dystrophin hypersensitizes skeletal muscle of lower and higher vertebrates to eccentric contraction (ECC)-induced strength loss. Loss of strength can be accompanied by transient and reversible alterations to sarcolemmal excitability and disruption, triad dysfunction, and aberrations in calcium kinetics and reactive oxygen species production. The degree of ECC-induced strength loss, however, appears dependent on several extrinsic and intrinsic factors such as vertebrate model, skeletal muscle preparation (in vivo, in situ, or ex vivo), skeletal muscle hierarchy (single fiber versus whole muscle and permeabilized versus intact), strength production, fiber branching, age, and genetic background, among others. Consistent findings across research groups show that dystrophin-deficient fast(er)-twitch muscle is hypersensitive to ECCs relative to wildtype muscle, but because preparations are highly variable and sensitivity to ECCs are used repeatedly to determine efficacy of many preclinical treatments, it is critical to evaluate the impact of skeletal muscle preparations on sensitivity to ECC-induced strength loss in dystrophin-deficient skeletal muscle. Here, we review and discuss variations in skeletal muscle preparations to evaluate the factors responsible for variations and discrepancies between research groups. We further highlight that dystrophin-deficiency, or loss of the dystrophin-glycoprotein complex in skeletal muscle, is not a prerequisite for accelerated strength loss-induced by ECCs.

Carboxyl-terminal modulator protein (CTMP) deficiency mitigates denervation-induced skeletal muscle atrophy

Denervated skeletal muscles show decreased Akt activity and phosphorylation, resulting in atrophy. Akt inhibits downstream transcription of atrophy-associated ubiquitin ligases like muscle ring-finger protein 1 (MuRF-1). In addition, reduced Akt signaling contributes to aberrant protein synthesis in muscles. In ALS mice, we recently found that carboxyl-terminator modulator protein (CTMP) expression is increased and correlated with reduced Akt signaling in atrophic skeletal muscle. CTMP has also been implicated in promoting muscle degeneration and catabolism in an in vitro muscle atrophy model. The present study examined whether sciatic nerve injury (SNI) stimulated CTMP expression in denervated skeletal muscle during muscle atrophy. We hypothesized that CTMP deficiency would reduce neurogenic atrophy and reverse Akt signaling downregulation. Compared to the unaffected contralateral muscle, wild-type (WT) gastrocnemius muscle had a significant increase in CTMP (p < 0.05). Furthermore, denervated CTMP knockout (CTMP-KO) gastrocnemius weighed more than WT muscle (p < 0.05). Denervated CTMP-KO gastrocnemius also showed higher Akt and downstream glycogen synthase kinase 3β (GSK3β) phosphorylation compared to WT muscle (p < 0.05) as well as ribosomal proteins S6 and 4E-BP1 phosphorylation (p < 0.001 and p < 0.05, respectively). Moreover, CTMP-KO mice showed significantly lower levels of E3 ubiquitin ligase MuRF-1 and myostatin than WT muscle (p < 0.05). Our findings suggest that CTMP is essential to muscle atrophy after denervation and it may act by reducing Akt signaling, protein synthesis, and increasing myocellular catabolism.