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Carboxylic ionophores are polyether antibiotics used in production animals as feed additives, with a wide range of benefits. However, ionophore toxicosis often occurs as a result of food mixing errors or extra-label use and primarily targets the cardiac and skeletal muscles of livestock. The ultrastructural changes induced by 48 hours of exposure to 0.1 μM monensin, salinomycin, and lasalocid in cardiac (H9c2) and skeletal (L6) myoblasts in vitro were investigated using transmission electron microscopy and scanning electron microscopy. Ionophore exposure resulted in condensed mitochondria, dilated Golgi apparatus, and cytoplasmic vacuolization which appeared as indentations on the myoblast surface. Ultrastructurally, it appears that both apoptotic and necrotic myoblasts were present after exposure to the ionophores. Apoptotic myoblasts contained condensed chromatin and apoptotic bodies budding from their surface. Necrotic myoblasts had disrupted plasma membranes and damaged cytoplasmic organelles. Of the three ionophores, monensin induced the most alterations in myoblasts of both cell lines.

Modulevsky DJ, Tremblay D, Gullekson C, Bukoreshtliev NV, Pelling AE (2012) The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture. PLoS ONE 7(9): e45329. doi:10.1371/journal.pone.0045329 Citation: Modulevsky DJ, Tremblay D, Gullekson C, Bukoreshtliev NV, Pelling AE (2013) Correction: The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture.

Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N -acyl- N -alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N -acyl- N -alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~10 4  M −1  s −1 , comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N -acyl- N -alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery. Chemically modifying proteins is hard to achieve selectively without purifying the target protein. Here, the authors present a method to modify proteins on lysine residues in living cells quicker than via known approaches and show that it can be used to develop protein covalent inhibitors.

Background Skeletal muscle comprises 30–40% of a mammal’s body mass, maintaining its integrity through efficient muscle fiber regeneration, which involves myoblast differentiation into myotubes. Previously, we reported that N-acetylglucosamine (GlcNAc) promotes myogenesis in C2C12 cells, although the underlying processes remained unclear. GlcNAc’s activated form, UDP-GlcNAc, is critical for the biosynthesis of highly branched (N-acetyllactosamine-rich) N-linked oligosaccharides, which are recognized by galectin-3 (Gal-3), a protein that facilitates dynamic cell-cell and cell-matrix interactions and modulating the motility dynamics of membrane-associated proteins. Methods In this study, we used primary myoblasts from both wild-type and Gal-3 null (Gal-3KO) mice, observing myotube formation through long-term live-cell imaging and single-cell tracking to reveal the dynamic process that occurred during the myotube formation. Results We found that GlcNAc enhances myoblast fusion in a dose-dependent manner, and that the addition of Gal-3 with GlcNAc leads to the formation of larger myotubes. Gal-3KO myoblasts exhibited a reduced capacity for myotube formation—a deficiency that was rectified by supplementing with GlcNAc and Gal-3. Our results highlight the role of Gal-3 interaction with oligosaccharides, whose synthesis is promoted by GlcNAc in facilitating myotube formation. Single-cell tracking revealed that GlcNAc and Gal-3 increase myoblast motility, leading to a faster, coordinated, flow-like movement—a collective behavior, along which myotubes form through cell fusion. Interestingly, myoblasts contributing to myotube formation were pre-positioned along the eventual shape of the myotubes before this flow-like movement was fully established. These myoblasts moved along the flow, paused, and even moved against it, suggesting that both coordinated flow and initial spatial positioning contribute to myoblast alignment along the axis of future myotubes. Conclusion Our findings suggest that GlcNAc, in conjunction with Gal-3, enhances myotube formation by fostering an environment conducive to myoblast positioning, establishing optimal coordinated flow-like movement, and facilitating fusion. This suggests potential therapeutic applications of GlcNAc in muscle repair and muscle disorders.

Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD⁺) and its effect on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD⁺ precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the mdx (C57BL/10ScSn-Dmdmdx/J) mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs and melanocyte SCs and increases mouse life span. Strategies that conserve cellular NAD⁺ may reprogram dysfunctional SCs and improve life span in mammals.

Cardiovascular diseases remain the leading cause of global mortality. Cellular senescence has recently been implicated in the pathogenesis of various cardiovascular diseases. Our group has previously shown that the matricellular protein CCN5 is a potent anti-fibrotic molecule capable of inhibiting and reversing cardiac fibrosis. In this study, we investigated whether CCN5 can modulate cellular senescence in the heart utilizing three readouts: western blotting for p53 and p21, staining for senescence-associated β-galactosidase, and microscopic analysis of γH2AX-foci. CCN5 effectively inhibited doxorubicin-induced cellular senescence in both H9c2 cardiac myoblasts and fibroblasts. In addition, CCN5 suppressed cellular senescence in H9c2 cardiac myoblasts induced by the senescence-associated secretory phenotype factors secreted from cardiac fibroblast, and vice versa. CCN5 also restored the apoptotic response of senescent cells. Finally, CCN5 attenuated myocardial infarction-induced cellular senescence in mice. Collectively, our findings provide novel insights into the potential role of CCN5 in the development of anti-senescence therapies.

Skeletal muscle formation occurs through fusion of myoblasts to form multinucleated myofibers. From a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) loss-of-function screen for genes required for myoblast fusion and myogenesis, we discovered an 84–amino acid muscle-specific peptide that we call Myomixer. Myomixer expression coincides with myoblast differentiation and is essential for fusion and skeletal muscle formation during embryogenesis. Myomixer localizes to the plasma membrane, where it promotes myoblast fusion and associates with Myomaker, a fusogenic membrane protein. Myomixer together with Myomaker can also induce fibroblast-fibroblast fusion and fibroblast-myoblast fusion. We conclude that the Myomixer-Myomaker pair controls the critical step in myofiber formation during muscle development.

Stress urinary incontinence commonly arises with aging or following prostatectomy, yet its underlying mechanisms remain unclear. To address this, we investigated the role of metabolic pathways—particularly the tricarboxylic acid (TCA) cycle—in the differentiation of human external urethral sphincter myoblasts. Immortalized sphincter cells (US2-KD) were induced to differentiate over 192 h. Metabolomic profiling using gas chromatography–mass spectrometry, along with pathway enrichment analysis, identified key metabolic changes. Inhibition of mitochondrial pyruvate transport with UK5099 markedly suppressed TCA cycle metabolites, including citrate, α-ketoglutarate, fumarate, and malate. This inhibition also significantly reduced MYH7 expression and intracellular adenosine triphosphate levels throughout the differentiation period. These results demonstrate that the TCA cycle plays a critical role in both energy metabolism and the differentiation of urethral sphincter myoblasts. This study is the first to suggest that impaired TCA cycle activity may contribute to the pathogenesis of Stress urinary incontinence and represents a potential therapeutic target. Our findings offer new insight into age-related metabolic decline associated with Stress urinary incontinence and support the development of therapies that combine metabolic modulation with regenerative approaches.

Muscular dystrophies are a heterogeneous group of genetic diseases, characterized by progressive degeneration of skeletal and cardiac muscle. Despite the intense investigation of different therapeutic options, a definitive treatment has not been developed for this debilitating class of pathologies. Cell-based therapies in muscular dystrophies have been pursued experimentally for the last three decades. Several cell types with different characteristics and tissues of origin, including myogenic stem and progenitor cells, stromal cells, and pluripotent stem cells, have been investigated over the years and have recently entered in the clinical arena with mixed results. In this Review, we do a roundup of the past attempts and describe the updated status of cell-based therapies aimed at counteracting the skeletal and cardiac myopathy present in dystrophic patients. We present current challenges, summarize recent progress, and make recommendations for future research and clinical trials.

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young's modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.