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.

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.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by skeletal muscle degeneration and cardiomyopathy without a cure. This study examined the therapeutic potential of the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) on cardiac function in the dystrophin-deficient mdx mouse model of DMD. Male mice were fed control chow or EMPA-containing chow ([~]25 mg/kg/day), and cardiac function was evaluated longitudinally by four-dimensional ultrasound imaging. EMPA did not alter left ventricular mass or chamber volume but preserved ejection fraction (EF) for 12 weeks, maintained significantly higher EF through 24 weeks, and attenuated global impairment of systolic and diastolic myocardial deformation. These functional improvements were accompanied by reduced cardiomyocyte hypertrophy and decreased expression of cardiac stress genes. EMPA reduced mitochondrial DNA damage, increased mitochondrial DNA copy number, and induced transcriptional signatures consistent with enhanced fatty acid and ketone metabolism, contributing to increased myocardial ATP content. Systemically, EMPA improved body mass trajectory, preserved relative lean mass, enhanced skeletal muscle torque, and did not adversely affect renal function. Together, these findings demonstrate that EMPA improves cardiac performance and mitochondrial integrity while enhancing myocardial energy availability in mdx mice, supporting SGLT2 inhibitors as a promising therapeutic strategy for individuals with DMD.

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.

Interleukin-6 (IL-6), produced by skeletal muscle and extramuscular tissues, regulates skeletal muscle function through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. However, the interaction between intrinsic (locally produced) IL-6 and extrinsic (circulating) IL-6 in skeletal muscle remains unclear. We investigated whether and how intrinsic expression of IL-6 in cultured primary human myoblasts influences their response to extrinsic stimulation with recombinant human IL-6 (rhIL-6). Using gene silencing, we found that suppression of intrinsic IL-6 enhanced rhIL-6-induced phosphorylation of STAT1 and STAT3. Silencing STAT3 also increased rhIL-6-induced STAT1 phosphorylation, but silencing STAT1 had no effect on STAT3 phosphorylation. Pretreatment of myoblasts with neutralising anti-IL-6 antibodies increased phosphorylation of STAT1 and STAT3 induced by 50 ng/mL rhIL-6, whereas pretreatment with 5 ng/mL rhIL-6 reduced this response. Despite increased JAK/STAT signalling, IL-6 silencing decreased glucose and oleic acid uptake and oxidation under both basal and rhIL-6-stimulated conditions. Collectively, our results imply that intrinsic IL-6 restrains activation of the JAK/STAT pathway by extrinsic IL-6, but acts synergistically with it to promote myoblast energy metabolism.

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.

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.

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.

Doxorubicin (DOX), a highly effective and widely used chemotherapeutic agent used to treat various types of cancer. Unfortunately, DOX also has some undesirable and off-target effects, particularly debilitating muscle weakness and fatigue. The mechanism behind this DOX-induced skeletal myotoxicity (DISM) remains unclear. Here, we show that acute DOX exposure, at clinically relevant concentrations, impairs isometric force production and accelerates fatigue in ex vivo murine flexor digitorum brevis (FDB) muscles. Mechanistically, we found that DOX increases the open probability of single RyR1 and disrupts calcium (Ca2+)-dependent inactivation (CDI). This results in a persistent sarcoplasmic reticulum (SR) Ca2+ leak, elevated basal cytosolic Ca2+, and abnormal Ca2+ release during action potentials. This abnormal intracellular Ca2+ handling ultimately leads to increased mitochondrial reactive oxygen species (ROS) production, which, in turn, exacerbates the functional instability of RyR1. Interestingly, the cytosolic basal Ca2+ elevation precedes ROS generation, suggesting that it initiates a destructive cross-talk between Ca2+ dysregulation and oxidative stress. Notably, pharmacological stabilization of the RyR1-FKBP12 complex with novel triazole compounds, MP-001 and MP-034, normalizes RyR1 function, Ca2+ and ROS homeostasis, as well as muscle force and fatigue resistance. Our findings indicate that DISM is initiated by DOX destabilization of the RyR1-FKBP12 complex (abnormal SR Ca2+ leak) and then exacerbated by the Ca-ROS vicious cycle. Limiting RyR1-mediated Ca2+ leak with MP-001 represents a promising therapeutic strategy for anti-DISM, aiming to normalize muscle function in patients undergoing DOX chemotherapy.

Ex vivo culture of isolated muscle fibers can serve as an important model for in vitro research on mature skeletal muscle fibers. Nevertheless, this model has limitations for long-term studies due to structural loss and dedifferentiation following prolonged culture periods. This study aimed to investigate how ex vivo culture affects muscle fiber contraction and to improve the culture system to preserve muscle fiber morphology and sarcomere function. Additionally, we sought to determine which culture-induced changes can negatively affect muscle fiber contraction. We cultured isolated flexor digitorum brevis (FDB) muscle fibers in several conditions for up to 7 days, and investigated viability, morphology, the unloaded sarcomere shortening in intact fibers, along with force generation in permeabilized muscle fibers. In addition, we examined changes to the microtubule network. We found a time-dependent decrease in contractility and viability in muscle fibers cultured for 7 days on a laminin-coated culture dish (2D). Conversely, we found that culturing FDB muscle fibers in a low-serum, fibrin/Geltrex hydrogel (3D) reduces markers of muscle fiber dedifferentiation (i.e. sprouting), improves viability and retains contractility over time. We discovered that the loss of contractility of cultured muscle fibers was not the direct result of reduced sarcomere function but may be related to changes in the microtubule network. Collectively, our findings highlight the importance of providing muscle fibers with a 3D environment during ex vivo culture, particularly when testing pharmacological or genetic interventions to study viability or contractile function. SummaryIn this work, the authors demonstrate that a low-serum, 3D ex vivo culture model preserves muscle fiber viability and contractility while reducing dedifferentiation. This model system can be utilized to conduct prolonged ex vivo pharmacological or genetic interventions to study fundamental muscle fiber processes or disease mechanisms.

A Krebs cycle intermediate metabolite, itaconate, has gained attention as a potential antimicrobial and autoimmune disease treatment due to its anti-inflammatory effects. While itaconate and its derivatives pose an attractive therapeutic option for the treatment of inflammatory diseases, the effects outside the immune system still remain limited, particularly in the muscle. Therefore, we endeavored to determine if itaconate signaling impacts muscle differentiation. Utilizing the well-established C2C12 model of in vitro myogenesis, we evaluated the effects of itaconate and its derivatives on transcriptional and protein markers of muscle differentiation as well as mitochondrial function. We found itaconate and the derivatives dimethyl itaconate and 4-octyl itaconate disrupt differentiation media-induced myogenesis. A primary biological effect of itaconate is a succinate dehydrogenase (SDH) inhibitor. We find the SDH inhibitors dimethyl malonate and harzianopyridone phenocopie the anti-myogenic effects of itaconate. Furthermore, we find treatment with exogenous succinate results in blunted myogenesis. Together our data indicate itaconate and its derivatives interfere with in vitro myogenesis, potentially through inhibition of SDH and subsequent succinate accumulation. More importantly, our findings suggest the therapeutic potential of itaconate and its derivatives could be limited due to deleterious effects on myogenesis.

Emery-Dreifuss muscular dystrophy 1 (EDMD1) arises from mutations in EMD. Most EDMD1 patients lack detectable emerin expression. They experience symptoms such as skeletal muscle wasting, joint contractures, and cardiac conduction defects. Currently, physicians rely on treating patient symptoms, without addressing the underlying cause - lack of functional emerin protein. Thus, there is a need for therapeutic approaches that restore emerin protein expression to improve patient outcomes. One way would be to deliver emerin mRNA or protein directly to affected tissues to restore tissue homeostasis. Here, we evaluated the utility of lipid nanoparticles (LNPs) to deliver emerin mRNA to diseased cells. LNPs have been studied for decades and have recently been used clinically for vaccination and treatment of myriad of diseases. Here, we show treatment of emerin-null myogenic progenitors with LNPs encapsulating emerin mRNA causes robust emerin protein expression that persists for at least 4 days. Treatment of differentiating emerin-null myogenic progenitors with 2.5 pg/cell emerin LNPs significantly improved their differentiation. Toxicity profiling of emerin mRNA LNP (EMD-LNP) dosing shows little toxicity at the effective dose. These data support the potential use of EMD-LNPs as a viable treatment option and establishes its utility for studying EDMD pathology.

BackgroundEccentric exercise (ECC) is widely recognized as an effective treatment for non-insertional Achilles tendinopathy (NIAT); however, the mechanisms underlying its apparent superiority over concentric exercise (CON) remain poorly understood. This randomized controlled trial aimed to investigate changes in triceps surae motor unit firing properties, pain, function, and AT morpho-mechanical properties following a 6-week intervention involving torque feedback training with isolated ECC and CON contractions in individuals with NIAT. MethodsTwenty-six individuals with NIAT were randomized to ECC or CON training. Motor unit firing properties (mean discharge rate [MDR], recruitment and de-recruitment thresholds, and torque-firing cross-correlation and neuromechanical delay) were assessed in the medial gastrocnemius (MG), lateral gastrocnemius (LG), and soleus (SO) muscles using high-density surface electromyography (HD-sEMG) during isometric plantarflexion at 10%, 40%, and 70% of maximal voluntary contraction (MVC). Pain, function (VISA-A), and tendon properties (ultrasound, elastography) were measured at baseline, week 3, and week 6. ResultsBoth groups showed similar improvements in pain (P < 0.0001) and VISA-A scores (P < 0.001). Tendon stiffness increased in both groups by week 3 but was higher in ECC by week 6 (P = 0.02). Motor unit adaptations differed: CON demonstrated an increase in MG MDR at 40% MVC, while ECC showed a decrease (interaction: P = 0.0008). Only ECC led to increased de-recruitment thresholds in the LG at 70% MVC (P < 0.0001). However, both groups exhibited reduced MDR in the LG at high-force levels (P < 0.05). Additionally, both interventions reduced GL torque-firing relationships (P = 0.025) and decreased SO neuromechanical delay (P = 0.031). ConclusionA 6-week visuo-motor torque feedback training program involving isolated CON or ECC contractions leads to comparable improvements in clinical outcomes. However, contraction-specific and muscle-specific changes in motor unit function and tendon stiffness suggest distinct neuromechanical adaptations. These differences may underlie the observed effects and warrant further investigation in longer-term studies to determine their impact on long-term clinical outcomes. HIGHLIGHTS- Changes in clinical outcomes, tendon morphomechanical properties, and triceps surae motor unit behavior were assessed for the first time following a 6-week rehabilitation protocol for non-insertional Achilles tendinopathy, involving isolated concentric (CON) or eccentric (ECC) contractions. - Both CON and ECC training led to comparable improvements in pain, self-reported outcomes, and perceived function. - Despite these similar clinical improvements, CON and ECC protocols produced distinct adaptations in estimated tendon stiffness and motor unit firing characteristics. These neuromechanical differences may indicate potential long-term divergences between training modalities, which should be explored in studies with extended intervention periods.