American Journal of Physiology-Cell Physiology

Extracellular vesicles (EVs) released from all cells, are essential to cellular communication, and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle-derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells, pre-differentiation (myoblasts) and post-differentiation (myotubes), as well as from acutely stimulated myotubes (1hr @ 14V, C-Pace EM, IonOptix) using total exosome isolation reagent (TEI, ThermoFisher, referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p<0.001, N=8-10). Two-way ANOVA showed a significant main effect for size distribution of myotube vs. myoblast-EPs (p<0.01, N=10-13). Myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400-600 nm, compared to myotube-EPs that were largely 50-300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than myoblast-EPs, (p<0.001 N=6-9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p=0.07, N=4-6) and had comparable size distribution profiles as EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p<0.05, N=4-6). Myotube-EPs had significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared to myoblast-EPs (p<0.05, N=7-12). Conversely, microvesicle marker ARF6, and lipoprotein marker APO-A1was only found in the myotube-EPs (p<0.05, N=4-12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (N=7), nor on expression of TSG101, ARF6 or CD81 (N=5-6). Myoblasts treated with control or acute stimulation-derived EPs (13 g/well) for 48hrs and 72hrs showed no changes in mitochondrial mass (MitoTracker Red), cell viability or cell count (N=3-4). Myoblasts treated with EP-depleted media (72hrs) had ~90% lower cell counts (p<0.01, N=3). Our data show that EVs differ in size, distribution, protein yield and expression of subtype markers pre- vs. post-skeletal muscle differentiation. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation-derived EPs did not alter mitochondrial mass nor cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle-specific EVs are warranted.

Skeletal muscle wasting and dysfunction is a common characteristic of non-dialysis dependent chronic kidney disease (NDD-CKD). The mechanisms by which this occurs are not clearly understood and one reason for this is a lack of well controlled in-vitro methodologies to simulate NDD-CKD induced muscle wasting for mechanistic investigation at the cellular level. Here we sought to conduct the initial investigations into developing a CKD-induced skeletal muscle model for use as a mechanistic analysis tool as well as a test bed for potential novel therapeutics in this population. Human derived muscle cells (HDMCs) were isolated from n=5 NDD-CKD patients and n=3 matched healthy controls (HC) and taken through proliferation and differentiation phases in cell culture. Upon comparison of the 2 donor types, significantly greater mRNA expression of myogenic markers was noted in the NDD-CKD cultures in comparison to HC cultures, which was carried through to greater mRNA expression of myosin heavy chains (MyHCs) post differentiation. However, this was not carried over to protein expression where Pax7 and MyoD were seen to be expressed to a greater extent in HC cultures. mRNA expression markers of protein degradation were noted to be elevated in NDD-CKD cultures in comparison to HC cultures. In light of our findings, future work should seek to investigate the role of the CKD environment as well as mechanisms implicated in transcription regulation to further advance the current model development as well as the mechanistic understanding of skeletal muscle wasting in CKD.

GLP-1 and GIP/GLP-1 receptor agonists produce substantial weight loss in clinical trials but significant loss of lean body mass is reported. Whether this reflects a direct pharmacological effect on skeletal muscle or an indirect consequence of caloric restriction and reduced mechanical loading is unknown. Primary myoblasts were isolated from skeletal muscle of older adults with obesity undergoing orthopaedic surgery. GIPR and GLP-1R expression was characterised by RT- qPCR and flow cytometry. Differentiated myotubes were treated with semaglutide or GIP peptide and assessed for atrophy-related gene expression (qPCR), secretome perturbation (Olink Reveal), mitochondrial and glycolytic bioenergetics (Seahorse XF Real-Time ATP Rate Assay, glucose uptake, lactate secretion) and myotube morphology and myogenesis (immunofluorescence). GIPR mRNA was consistently detected across all donors; GLP-1R mRNA was undetectable by PCR, though LUXendin645 flow cytometry identified low-level surface GLP-1R protein in 51-66% of myoblasts. Neither semaglutide nor GIP altered atrophy-related gene expression or the secretome, with no proteins reaching significance. Semaglutide reduced glycolytic and total ATP production rates, accompanied by reduced lactate secretion, suggesting modest suppression of glycolytic flux; mitochondrial parameters were unaffected. Neither treatment impaired myotube thickness or differentiation; GIP increased myotube thickness after 8 days. Direct GLP-1 and GIP receptor activation does not substantively perturb atrophic signalling, myogenesis, or the secretome of primary human skeletal muscle myotubes. These findings suggest that lean mass loss with incretin-based therapies is unlikely to be driven by direct pharmacological action on skeletal muscle - particularly relevant as these agents are increasingly used in older adults at risk of sarcopenia.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent inherited muscular dystrophies, for which there are no disease-modifying therapies. Metabolic perturbation, mitochondrial dysfunction, and oxidative stress are key contributors to FSHD pathology. Here, the effects of the metabolic regulator and anti-diabetic drug Metformin on myogenesis and muscle function in human and murine models of FSHD were investigated. Metformin did not affect the proliferation rate of human control or patient-derived FSHD myoblasts but promoted their myogenic differentiation, increasing myotube formation and maturation. Metformin also enhanced the metabolic health and viability of myotubes. Mechanistic interrogation revealed reduced levels of mitochondrial reactive oxygen species and modified mitochondrial turnover. These cellular investigations were complemented with in vivo functional assessment in a murine model of FSHD, in which Metformin treated mice exhibited significantly improved muscle strength. Collectively, these findings identify metabolic regulation as a therapeutically tractable feature of FSHD and demonstrate that Metformin improves muscle function in multiple models of FSHD via reduction of oxidative stress and augmentation of cellular metabolic fitness. These results provide insight into the therapeutic actions of Metformin and pre-clinical data to support its testing for repurposing in FSHD.

Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes mellitus (T2D). While two-dimensional myotube cultures offer a controlled environment for studying T2D-related metabolic dysfunction, insulin-dependent glucose transporter type 4 (GLUT4) levels are limited and insulin-independent glucose transporter type 1 (GLUT1) expression dominates; reducing physiological relevance. Three-dimensional skeletal muscle microtissue cultures offer a promising alternative, and unlike 2D myotubes, are amenable to repeated contractile stimulation. However, microtissue GLUT1 and GLUT4 glucose transporter profiles remain under-characterized, particularly under physiological glucose and insulin conditions, which is evaluated herein. We report that GLUT1 levels trended [~]3.0-fold lower in microtissues compared with myotubes in 2D culture, although not statistically significant (p = 0.072), while GLUT4 levels were [~]12-fold higher (p < 0.0001), leading to a [~]60-fold increase in the GLUT4:GLUT1 ratio (p = 0.023). Notably, the microtissue GLUT4:GLUT1 profile approached, but did not match that of native human muscle. Microtissues required supraphysiological insulin conditions for the development of maximal contractility, while physiological glucose levels were sufficient. Insulin withdrawal restored insulin responsiveness but impaired microtissue contractile strength (p < 0.0001) and fatigue resistance (p = 0.015). Our findings indicate that the glucose transporter profile of microtissues offers improved physiological relevance. However, their reliance on insulin to maintain contractile function limits their suitability for modeling T2D. The implementation of a robust, insulin-free differentiation protocol would facilitate the development of a microtissue-based T2D model which can be applied to study contraction-mediated increases in insulin sensitivity as a therapeutic approach.

Skeletal muscle microtissues are valuable in vitro models for studying the stimuli regulating muscle protein synthesis (MPS), the key determinant of changes in muscle mass. Differentiated between opposing posts, microtissues contain aligned, contractile myotubes, providing a controlled system for investigating the responses of skeletal muscle to nutrient and contractile stimulation. However, microtissue MPS responses to these stimuli remain under-characterized. Stable isotope-labeled amino acid tracers deliver sarcoplasmic and myofibrillar fractional synthetic rates (FSR) for MPS in human studies, but have not been implemented in engineered skeletal muscle. We close these gaps by characterizing stimulation-induced MPS, in microtissues and 2D myotubes derived from the same primary myoblast line, using stable isotope tracers and puromycin incorporation. In microtissues, sarcoplasmic FSR increased significantly during the two-hour period following amino acid treatment (p < 0.0001), whereas myofibrillar FSR remained unchanged (p = 0.159). However, both fractions were unresponsive to ketone stimulation and contraction (all p [&ge;] 0.703). 2D myotubes showed significant increases in sarcoplasmic and myofibrillar FSR in response to amino acid treatment (both p = 0.002). Notably, microtissues demonstrated a more stable myofibrillar protein fraction, with a sarcoplasmic-to-myofibrillar FSR ratio of [~]2:1 which closely resembled that of native human muscle. The puromycin-based approach failed to detect MPS responses to any stimulus (all p [&ge;] 0.677), highlighting the superior sensitivity of tracer-based measurements, particularly where longer timescales are needed to capture an effect. These findings support the use of engineered muscle and isotope-derived measurements of MPS in future studies of stimuli regulating skeletal muscle mass. New FindingsO_ST_ABSWhat is the central question of this study?C_ST_ABSStable isotope tracers are emerging as a powerful approach to measure fraction-specific protein synthesis. However, their efficacy relative to conventional puromycin labeling remains unreported, and they have not been applied to engineered skeletal muscle. What is the main finding and its importance?By implementing stable isotope tracers in engineered muscle, we showcase the ability to capture anabolic responses that are undetected by puromycin-based methods. We found that the myofibrillar protein fraction of microtissues is more stable than the sarcoplasmic fraction, a property of native muscle, absent in 2D myotubes. These findings demonstrate the physiological relevance of engineered muscle and support the adoption of isotope-derived measurements in future studies.

Store-operated calcium entry (SOCE) is a ubiquitous calcium (Ca2+) signalling mechanism that replenishes endoplasmic and sarcoplasmic reticulum Ca2+ stores. We previously identified STIM1L, a skeletal muscle-enriched STIM1 splice variant that enables rapid SOCE activation in human myotubes. To determine its physiological role in vivo, we generated a constitutive knockout mouse model that selectively abolishes STIM1L expression while preserving the canonical STIM1 isoform. Stim1l-KO mice developed normally and exhibited no overt abnormalities, but both sexes showed reduced voluntary wheel-running activity, suggesting neuromuscular perturbation. Skeletal muscle deprived of STIM1L did not show any histological alterations or changes in fibre- type and size. Likewise, force-frequency relationships and fatigue resistance measured ex vivo in isolated extensor digitorum longus and soleus muscles were indistinguishable from control muscles. This lack of difference may be explained by the increased expression of STIM1 in several skeletal muscles and suggests that changes in the voluntary running activity of Stim1l-KO mice may arise from STIM1L loss in non-muscular tissues, including the central nervous system. Notwithstanding, primary myotubes derived from Stim1l-KO mice displayed a marked delay in SOCE activation kinetics, whereas SOCE amplitude remained comparable to controls. This was consistent with a compensatory STIM1 upregulation maintaining overall Ca2+ entry capacity. Interestingly, electrically evoked Ca2+ transients in intact Stim1l-KO flexor digitorum brevis fibres exhibited significantly faster decay kinetics than controls. This revealed another role of STIM1L in Ca2+ handling in skeletal muscle that appears independent of its contribution to SOCE activation. Together, these findings establish STIM1L as a regulator of SOCE activation kinetics and indicate an additional role in myofibre Ca2+ clearance, while suggesting that compensatory upregulation of canonical STIM1 may preserve muscle structure and contractile function in its absence in mice.

In Principal cells (PC) of the cortical collecting duct (CCD), the highly regulated and coordinated reabsorption of sodium occurs through the epithelial sodium channel (ENaC) at the apical membrane and Na/K ATPase at the basolateral membrane. However, it is not known how sodium ions (Na+) are transported across the cell. We investigated intracellular transport in mCCDcl1 cells using a fluorescent sodium dye, CoroNa Green AM. Dye uptake was stimulated by aldosterone, blocked by amiloride (an ENaC inhibiter), and basolateral transport was prevented by ouabain (an Na/K ATPase blocker) thus validating the dyes apparently faithful replication of sodium transport. Cells exhibited a consistent pattern of sodium-containing vesicles, of various sizes, surrounded by cytoskeleton and lipid membrane. While the smallest vesicles ([~]0.5m) co-stained with lysotracker, larger vesicles (up to 6.4m) did not co-stain with either lysosomal- or mitochondrial-specific dyes and appeared to have internal structure, suggesting that they were multivesicular bodies. Time-lapse imaging showed a subset of these multivesicular bodies release or take up sodium dye in a controlled manner. Our novel data suggest that intracellular sodium compartmentalisation is highly regulated and offer new insights into intracellular sodium dynamics in the collecting duct, revealing potential new targets for control of sodium homeostasis. New and NoteworthyThe article shows for the first time, to our knowledge, intracellular sodium transport mechanism in mCCDcl1 cells in the form of dynamic vesicular bodies. These structures offer new targets for the regulation of sodium homeostasis and transport in the kidney collecting duct, with wider implications for blood pressure regulation.

Skeletal muscle regulation is responsible for voluntary muscular movement in vertebrates. The genes of two essential proteins, teneurins and latrophilins (LPHN), evolving in ancestors of multicellular animals, form a ligand-receptor pair, and are now shown to be required for skeletal muscle function. Teneurins possess a bioactive peptide, termed the teneurin C-terminal associated peptide (TCAP) that interacts with the LPHNs to regulate skeletal muscle contractility strength and fatigue by an insulin-independent glucose importation mechanism. CRISPR-based knockouts and siRNA-associated knockdowns of LPHN-1 and-3 shows that TCAP stimulates an LPHN-mediated cytosolic Ca2+ signal transduction cascade to increase energy metabolism and enhance skeletal muscle function via increases in type-1 oxidative fiber formation and reduce the fatigue response. Thus, the teneurin/TCAP-LPHN system is presented as a novel mechanism likely to regulate the energy requirements and performance of skeletal muscle.

hERG1 conducts cardiac IKr and is critical for repolarization of the human heart. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden cardiac death. At least two subunits combine to form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG 1a/1b subunit abundance modulates IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue have suggested that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven hiPSC-CM maturation model to investigate how changes in relative hERG 1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immature hiPSC-CMs display a two-fold greater inhibition by extracellular acidosis (pH 6.3) compared to matured hiPSC-CMs. qRT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated, and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared to immature hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by an increased in IKr density. Silencing the impact of hERG1b on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a PAS domain reduced the magnitude of IKr proton inhibition in immature hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and that hERG1 subunit abundance determines IKr sensitivity to protons in hiPSC-CMs.

Skeletal muscle is a dynamic tissue able to regenerate in response to injury. This regeneration relies primarily on the activation of precursor cells, known as muscle stem cells (MuSC). Upon activation, skeletal MuSC re-enter the cell cycle and proliferate as myoblasts, which subsequently either differentiate and fuse to form new fibers or return to quiescence to maintain long-term regenerative capacity. From primary human myoblasts, we generated reserve cells (RCs), quiescent cells similar to MuSC, and investigated their activation after stimulation with a growth medium containing serum. Here, we show that Orai3 calcium channel is primarily expressed in RC, where it promotes RC activation in a calcium-independent manner. We demonstrate that Orai3 does not contribute to store-operated calcium entry (SOCE) or calcium response induced by serum stimulation in RC cells, supporting the hypothesis that Orai3 plays a unique role in RC, distinct from its calcium channel activity. Using a protein proximity assay (BioID), we identified the large scaffold protein AHNAK2 as a potential partner of Orai3. AHNAK2 is expressed at high level in myotubes, and has a similar effect on RC activation than Orai3. Both Orai3 and AHNAK2 are necessary for proper myoblast differentiation and RC activation, while acting through different signaling pathways. Specifically, we show that Orai3 directly influences the RC fate, whereas AHNAK2 facilitates RC activation via signals derived from myotubes. These findings provide new insights into the molecular mechanisms underlying the activation and quiescence of human MuSC.

This study investigates the functional characteristics of induced pluripotent stem cell-derived muscle cells (hiPSC-skMCs) from Duchenne muscular dystrophy (DMD) patients, focusing on their regulation of intracellular calcium concentration. DMD, a progressive muscle degenerative disease, arises from mutations in the dystrophin gene and is characterized by elevated intracellular calcium levels, exacerbating disease progression. This work highlights that DMD hiPSC-skMCs demonstrate unique calcium signatures with increased intracellular calcium compared to healthy counterparts. These cells also exhibit both heightened calcium response when stimulated by electrical fields or acetylcholine and more pronounced constitutive calcium entries. While RNAseq data from these cells reaffirmed known dysregulation mechanisms seen in other dystrophin-deficient models, certain pathways like purinergic or store-operated calcium entries did not show disruption in this DMD model. This discrepancy suggests that not all mechanisms observed in animal models may be equally relevant in human cases, pointing towards specific molecular targets that could be more effective for DMD treatment strategies.

Skeletal muscle-derived extracellular vesicles (SkM-EVs) have recently been recognized as novel endocrine factors capable of facilitating inter-organ communication between skeletal muscle and distant organs. These vesicles transport various molecular cargoes, including microRNAs (miRNAs), which are essential regulators of post-transcriptional gene expression. In this study, we characterized the miRNA composition of SkM-EVs using small RNA sequencing and elucidated their putative biological roles via comprehensive bioinformatics analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the predicted miRNA targets revealed that SkM-EV miRNAs are involved in several key pathways, including the FoxO signaling pathway, the insulin signaling pathway, and cancer-related pathways. Our findings suggest that SkM-EV miRNAs may simultaneously promote muscle differentiation and exert protective effects against diabetes and cancer development. These findings provide new insights into the systemic regulatory roles of SkM-EVs and highlight their therapeutic potential for muscular, metabolic, and oncological disorders.

Polycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. Little is known, however, as to whether PC2 contributes to the structure of the primary cilium. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild type LLC-PK1 renal epithelial cells. To identify primary cilia and measure their length, confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated -tubulin antibody. Although primary cilia length measurements did not follow a Normal distribution, data were normalized by Box-Cox transformation rendering statistical difference under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 M) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA also elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length, as compared to their respective scrambled RNA transfected cells. The data indicate that maneuvers that either regulate PC2 function or gene expression, modify the length of primary cilia in renal epithelial cells. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD. Significance StatementPolycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel causing the autosomal dominant polycystic kidney disease (ADPKD). The importance of intact cilia and of fully functional polycystins in the onset of ADPKD cyst formation, point to yet unknown signaling mechanisms occurring within this organelle. We determined that the extracellular Ca2+ concentration, PC2 channel blockers, and PKD2 gene silencing, all contribute to the length of primary cilia in wild type LLC-PK1 renal epithelial cells. The data indicate that proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

The ERG1A K+ channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca2+]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca2+]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca2+]i in C2C12 myotubes, using Fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. We hypothesized that HERG would modulate L-type calcium channel activity, specifically the Cav1.1 channel known to carry signal from the sarcoplasmic membrane of skeletal muscle to the sarcomeres of the myofibrils. However, we find that HERG has no effect on the amplitude of L-type channel current nor does it affect the mRNA levels nor protein abundance of the Cav1.1 channel. Instead we find that, although the rise in [Ca2+]i (induced by depolarization) is greater in myotubes over-expressing HERG relative to controls, the difference between the KCl-stimulated Ca2+ increase in control and HERG over-expressing cells cannot be accounted for by L-type channel mediated Ca2+ influx, which suggests that HERG could modulate excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca2+]i induced by depolarization is blocked by 2-APB, an inhibitor of ECCE (and SOCE). Further, we show data suggesting that HERG also modulates the activity of ryanodine receptors, a component of ECCE, as well as store operated calcium entry (SOCE). Therefore, we investigated the effect of HERG on calsequestrin1, a calcium buffering/binding protein known to modulate ryanodine receptor 1 and store operated Ca2+ entry activities. Indeed, we find that calsequestrin1 mRNA levels are decreased 0.83-fold (p<0.05) and the total protein abundance is lowered 77% (p<0.05) in myotubes over-expressing HERG relative to controls. In summary, the data show that ERG1A overexpression modulates [Ca2+]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1.

BackgroundPeri-synaptic astrocyte processes (PAPs) play a fundamental role in synapse formation and function. Central afferent synapse loss and astrocyte dysfunction greatly impede sensory-motor circuitry in spinal muscular atrophy (SMA) disease progression, however mechanisms underpinning tripartite synapse dysfunction remains to be fully elucidated. The aims of this study were to further define PAP and motor neuron synaptic defects in human SMA disease pathology and implement a therapeutic intervention strategy to improve motor neuron function. MethodsWe derived astrocyte monocultures and motor neuron astrocyte co-cultures from healthy and SMA patient induced pluripotent stem cell (iPSC) lines to assess intrinsic astrocyte filopodia defects and phenotypes occurring at the synapse-PAP interface, respectively, using cell surface capture mass spectrometry proteomics, confocal and super resolution microscopy, synaptogliosome isolation, and electrophysiology. ResultsSMA astrocytes demonstrated intrinsic filopodia actin defects featuring low abundance of actin-associated cell surface N-glycoproteins, and decreased filopodia density and CDC42-GTP levels after actin remodeling stimulation. This phenotype is likely driven by the significant reduction of CD44 and phosphorylated ezrin, radixin and moesin ERM proteins (pERM) within SMA astrocyte filopodia. The dual combination of SMN1 gene therapy and forskolin treatment, an adenylyl cyclase activator leading to increased cyclic adenosine monophosphate (cAMP) levels and actin signaling pathway stimulation, led to extensive branching and increased filopodia density of SMA astrocytes during actin remodeling. SMA patient-derived motor neuron and astrocyte co-cultures, particularly samples derived from male patient iPSC lines, demonstrated a significant decrease in synapse number, actin-associated pre-synaptic neurotransmitter release protein, synapsin I (SYN1), and PAP-associated expression of pERM and glutamate transporter, EAAT1. Our astrocyte-targeted SMN1 augmentation and forskolin treatment paradigm restored SYN1 protein levels within the SMA synaptogliosome, resulting in significant increases in motor neuron synapse formation and function, but did not fully restore PAP-associated proteins levels at the synapse. ConclusionsSMA astrocytes demonstrate intrinsic actin-associated defects within filopodia, which correlates with decreased pERM levels at tripartite motor neuron synapses. We also define a SMN- and cAMP-targeted treatment paradigm that significantly increases pre-synaptic neurotransmitter release protein levels to improved SMA motor neuron synapse formation and function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/714618v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1257ab8org.highwire.dtl.DTLVardef@19c0010org.highwire.dtl.DTLVardef@c84552org.highwire.dtl.DTLVardef@3f1e62_HPS_FORMAT_FIGEXP M_FIG C_FIG

Defects in ATGL-mediated myocellular LD lipolysis results in mitochondrial dysfunction of unknown origin, which can be rescued by PPAR agonists. Here we examine whether ATGL-mediated lipolysis is required to maintain mitochondrial network connectivity and function. Moreover, we explored if the functional implications of ATGL deficiency for mitochondrial network dynamics and function can be alleviated by promoting PPAR and/or PPAR{delta} transcriptional activity. To this end, we cultured human primary myotubes from patients with neutral lipid storage disease with myopathy (NLSDM), a rare metabolic disorder caused by a mutation in the PNPLA2 gene. These myotubes possess dysfunctional ATGL and compromised LD lipolysis. In addition, mitochondria-LD contacts, mitochondrial network dynamics, and TMRM intensity were abrogated. Using a humanized ATGL inhibitor in myotubes cultured form healthy donors, revealed similar results. Upon stimulating PPAR{delta} transcriptional activity, mitochondrial respiration improved by more than 50% in human primary myotubes from healthy lean individuals. This increase in respiration was dampened in myotubes with dysfunctional ATGL. Stimulation of PPAR{delta} transcriptional activity had no effect on mitochondria-LD contacts, mitochondrial network dynamics, and TMRM intensity. Our results demonstrate that dysfunctional ATGL results in compromised mitochondrial-LD contacts and mitochondrial dynamics, and that functional ATGL is required to improve mitochondrial respiratory capacity upon stimulation of PPAR{delta} transcriptional activity.

Heart failure is a major source of mortality in Duchenne muscular dystrophy (DMD). DMD arises from mutations that ablate expression of the protein dystrophin, which render the plasma membrane unusually fragile and prone to disruption. In DMD patients, repeated mechanical stress leads to membrane damage and cardiomyocyte loss. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study specific mutations in the context of a human cell, but these models can be improved by adding physiologic stressors. We modeled the primary defect underlying DMD by applying equibiaxial mechanical strain to DMD iPSC-CMs. DMD iPSC-CMs demonstrated an increased susceptibility to equibiaxial strain after 2 hours at 10% strain relative to healthy control cells, measured as increased lactate dehydrogenase (LDH) release. After 24 hours, both DMD and healthy control iPSC-CMs showed evidence of injury with release of LDH and cardiac troponin T. We exposed iPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced LDH and troponin release in DMD and control iPSC-CMs that had been subjected to 24 hour strain at 10%. We used aptamer protein profiling of media collected from DMD and control iPSC-CMs and compared these results to serum protein profiling from DMD patients. We found a strong correlation between the proteins in DMD patient serum and media from DMD iPSC-CMs subjected to mechanical stress. By developing an injury assay that specifically targets an underlying mechanism of injury seen in DMD-related cardiomyopathy, we demonstrated the potential therapeutic efficacy of the protein membrane resealer, recombinant annexin A6, for the treatment of DMD-related cardiomyopathy and general cardiac injury.

Differentiation of skeletal muscle is associated with increased mitochondrial biogenesis and reliance of oxidative phosphorylation (OXPHOS). The terminal enzyme complex in the electron transport chain, cytochrome c oxidase (COX), requires copper for its assembly and activity, and copper delivery to mitochondria is essential for OXPHOS. However, when mitochondrial copper becomes essential during skeletal myoblast differentiation is not known. Here, we show that genetic deficiency of the mitochondrial copper and phosphate carrier SLC25A3 induced prior to myoblast differentiation leads to the formation of smaller myotubes, but SLC25A3 deficiency induced in mature myotubes leads to cell death and detachment. Both phenotypes are recapitulated upon genetic knockdown of COX17, a critical assembly protein for both COX copper cofactors, or by chemical inhibition of COX. Importantly, myotube death caused by SLC25A3 deficiency is rescued by copper supplementation or expression of an SLC25A3 variant that transports copper but not phosphate. Taken together these data support a model wherein copper transport by SLC25A3 and copper delivery to COX is critical for survival in mature myotubes.

Background: The neuromuscular junction (NMJ) is the site of communication between myofibers and a-motor neurons. Cellular and molecular mechanisms that determine, maintain, and remodel the neuromuscular synapse are poorly understood. Coactivator-associated arginine methyltransferase 1 (CARM1) post-translationally modifies target proteins by methylating arginine residues and has emerged as a key determinant of skeletal muscle biology. Methylarginine signalling is required for the maintenance and repair of the NMJ, but the direct role of CARM1 on the NMJ in health and disease remains unexplored, particularly in humans. Methods: We generated Carm1 skeletal muscle-specific knockout-out (mKO) mice to gain a basic understanding for the role of the enzyme in NMJ biology under homeostatic and denervated conditions. Additionally, we investigated CARM1 activity in severe mouse models of neuromuscular disorders (NMDs) including D2.mdx and Smn2B/- mice, which replicate Duchenne's muscular dystrophy (DMD) and spinal muscular atrophy (SMA), respectively, and exhibit chronic remodelling of the NMJ. Lastly, to evaluate if methylarginine signalling is implicated during NMJ instability in human skeletal muscle, we obtained samples from healthy volunteers before and after 14 days of single leg immobilization as well as from patients with myotonic dystrophy type 1. Results: Our results demonstrated that Carm1 mRNA expression and activity are elevated (P<0.05) in NMJ-enriched regions of healthy murine skeletal muscle. Carm1 muscle-specific deletion reduced NMJ compactness (-9.3%; P<0.05), increased fragmentation (+33%; P<0.05), and disrupted the expression of synapse-specific transcripts basally and following sciatic nerve transection. In skeletal muscle from pre-clinical models of NMDs, we observed a compensatory upregulation in CARM1-dependent arginine methylation as evident by +54% and +71 increases (P<0.05) in asymmetric dimethylarginine (ADMA)-marked CARM1 substrates in DMD and SMA mice, respectively. Similarly, muscle CARM1 was hyperactive with increased NMJ instability during neuromuscular disuse (+22%; P<0.05), and disease (+30%; P<0.05), in humans. In a cohort of muscular dystrophy patients and healthy volunteers, elevated CARM1 signalling was negatively correlated with clinical metrics of skeletal muscle health including grip strength (r = -0.583; P<0.05) as well as positively correlated with mRNA expression of NMJ machinery such as CHRNA1 (r = 0.578; P<0.05). Conclusion: In summary, we highlight that muscle-specific CARM1 is required for maintaining NMJ morphology and transcriptional regulation. Insults to NMJ stability during muscle disuse or in myopathic conditions were associated with enhanced CARM1-mediated methylarginine signalling in mice and humans. Collectively, our findings demonstrate CARM1 as a key mediator of NMJ biology and plasticity in health and disease.