Springer Science and Business Media LLC

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

Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca2+ release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca2+ handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n′,n′-tetraacetic acid (EGTA) to measure Ca2+ fluxes. Muscles were subjected to both current and voltage clamp conditions. Standard and confocal fluorescence microscopy was used to study Ca2+ release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student’s test with significance set at p < 0.05. We found that the peak value of Ca2+ fluxes elicited by single action potentials was significantly reduced by 15–20 % in C3KO fibers, but the kinetics was unaltered. Ca2+ release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca2+ release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca2+ release in C3KO mice but reduced peak Ca2+ fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca2+ release at all triads consistent with a homogenous reduction of functional voltage activated Ca2+ release sites. Overall, these results suggest that decreased Ca2+ release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

Previous studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle. To gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-β and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle. At 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-β transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers. Our findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy.

A strength of Drosophila as a model system is its utility as a tool to screen for novel regulators of various functional and developmental processes. However, the utility of Drosophila as a screening tool is dependent on the speed and simplicity of the assay used. Here, we use larval locomotion as an assay to identify novel regulators of skeletal muscle function. We combined this assay with muscle-specific depletion of 82 genes to identify genes that impact muscle function by their expression in muscle cells. The data from the screen were supported with characterization of the muscle pattern in embryos and larvae that had disrupted expression of the strongest hit from the screen. With this assay, we showed that 12/82 tested genes regulate muscle function. Intriguingly, the disruption of five genes caused an increase in muscle function, illustrating that mechanisms that reduce muscle function exist and that the larval locomotion assay is sufficiently quantitative to identify conditions that both increase and decrease muscle function. We extended the data from this screen and tested the mechanism by which the strongest hit, fascin, impacted muscle function. Compared to controls, animals in which fascin expression was disrupted with either a mutant allele or muscle-specific expression of RNAi had fewer muscles, smaller muscles, muscles with fewer nuclei, and muscles with disrupted myotendinous junctions. However, expression of RNAi against fascin only after the muscle had finished embryonic development did not recapitulate any of these phenotypes. These data suggest that muscle function is reduced due to impaired myoblast fusion, muscle growth, and muscle attachment. Together, these data demonstrate the utility of Drosophila larval locomotion as an assay for the identification of novel regulators of muscle development and implicate fascin as necessary for embryonic muscle development.

Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation. Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the endogenous Myogenin locus. Expression of tdTOMATO in MyogntdTom mice recapitulated endogenous Myogenin expression during embryonic muscle formation and adult regeneration and enabled the isolation of the MYOGENIN+ cell population. We also show that tdTOMATO fluorescence allows tracking of differentiating myoblasts in vitro and by intravital imaging in vivo. Lastly, we monitored by live imaging the cell division dynamics of differentiating myoblasts in vitro and showed that a fraction of the MYOGENIN+ population can undergo one round of cell division, albeit at a much lower frequency than MYOGENIN− myoblasts. We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts.

Motor unit remodelling involving repeated denervation and re-innervation occurs throughout life. The efficiency of this process declines with age contributing to neuromuscular deficits. This study investigated differentially expressed genes (DEG) in muscle following peroneal nerve crush to model motor unit remodelling in C57BL/6 J mice. Muscle RNA was isolated at 3 days post-crush, RNA libraries were generated using poly-A selection, sequenced and analysed using gene ontology and pathway tools. Three hundred thirty-four DEG were found in quiescent muscle from (26mnth) old compared with (4-6mnth) adult mice and these same DEG were present in muscle from adult mice following nerve crush. Peroneal crush induced 7133 DEG in muscles of adult and 699 DEG in muscles from old mice, although only one DEG (ZCCHC17) was found when directly comparing nerve-crushed muscles from old and adult mice. This analysis revealed key differences in muscle responses which may underlie the diminished ability of old mice to repair following nerve injury.

Mitsugumin 53 (MG53) is a relatively newly identified tripartite motif-containing (TRIM) family muscle-specific E3 ubiquitin ligase that is expressed in skeletal muscle and the heart. It has been postulated to facilitate repair by targeting the site of an injury, and acting as a scaffold for assembly of a repair complex made up of dysferlin, annexin V, caveolin-3, and polymerase I and transcript release factor (PTRF). A recent letter published in Nature by Song et al. proposes an alternate function for MG53: as an E3 ligase that targets the insulin receptor and insulin receptor substrate 1 (IRS1) for degradation, therefore regulating muscle insulin signaling. This work is exciting, as it not only presents a novel role for MG53, but also suggests that muscle insulin signaling has a systemic influence on insulin resistance and the metabolic syndrome.

Measuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation. Here, we developed a new Fiji macro package called ViaFuse (that stands for viability and fusion) to measure skeletal muscle cell viability and differentiation. To test ViaFuse, we utilized immunofluorescence images of differentiated myotubes where the capping actin protein of muscle z-line subunit beta (CAPZB) was depleted in comparison with control cells. We compared the values achieved using the ViaFuse macros first with manual quantification performed by researchers and second with those obtained utilizing the MATLAB muscle-centric software MyoCount. We observed a high degree of correlation between all methods of quantification. ViaFuse can detect the borders of myotubes and identify nuclear clumps which have been limitations of previous muscle-centric imaging software. The ViaFuse macros require little computer power or space to run and user inputs to the ViaFuse macros are minimal, thereby automating the analysis process in a quick, easy, and accurate fashion. Additionally, the ViaFuse macros work with Fiji, an existing imaging software widely used by skeletal muscle researchers. Furthermore, ViaFuse is compatible with many computer systems, has a very intuitive interface, and does not require prior complex mathematical knowledge. Therefore, we propose ViaFuse as a robust and meticulous method to quantify skeletal muscle cell viability and differentiation.

Misexpression of the double homeodomain transcription factor DUX4 results in facioscapulohumeral muscular dystrophy (FSHD). A DNA-binding consensus with two tandem TAAT motifs based on chromatin IP peaks has been discovered; however, the consensus has multiple variations (flavors) of unknown relative activity. In addition, not all peaks have this consensus, and the Pitx1 promoter, the first DUX4 target sequence mooted, has a different TAAT-rich sequence. Furthermore, it is not known whether and to what extent deviations from the consensus affect DNA-binding affinity and transcriptional activation potential. Here, we take both unbiased and consensus sequence-driven approaches to determine the DNA-binding specificity of DUX4 and its tolerance to mismatches at each site within its consensus sequence. We discover that the best binding and the greatest transcriptional activation are observed when the two TAAT motifs are separated by a C residue. The second TAAT motif in the consensus sequence is actually (T/C)AAT. We find that a T is preferred here. DUX4 has no transcriptional activity on “half-sites”, i.e., those bearing only a single TAAT motif. We further find that DUX4 does not bind to the TAATTA motif in the Pitx1 promoter, that Pitx1 sequences have no competitive band shift activity, and that the Pitx1 sequence is transcriptionally inactive, calling into question PITX1 as a DUX4 target gene. Finally, by multimerizing binding sites, we find that DUX4 transcriptional activation demonstrates tremendous synergy and that at low DNA concentrations, at least two motifs are necessary to detect a transcriptional response. These studies illuminate the DNA-binding sequence preferences of DUX4.