Changes in protein fluxes in skeletal muscle during sequential stages of muscle regeneration after acute injury in male mice.

Changes in protein turnover play an important role in dynamic physiological processes, including skeletal muscle regeneration, which occurs as an essential part of tissue repair after injury. The inability of muscle tissue to recapitulate this regenerative process can lead to the manifestation of clinical symptoms in various musculoskeletal diseases, including muscular dystrophies and pathological atrophy. Here, we employed a workflow that couples deuterated water (2H2O) administration with mass spectrometry (MS) to systematically measure in-vivo protein turnover rates across the muscle proteome in 8-week-old male C57BL6/J mice. We compared the turnover kinetics of over 100 proteins in response to cardiotoxin (CTX) induced muscle damage and regeneration at unique sequential stages along the regeneration timeline. This analysis is compared to gene expression data from mRNA-sequencing (mRNA-seq) from the same tissue. The data reveals quantitative protein flux signatures in response to necrotic damage, in addition to sequential differences in cell proliferation, energy metabolism, and contractile gene expression. Interestingly, the mRNA changes correlated poorly with changes in protein synthesis rates, consistent with post-transcriptional control mechanisms. In summary, the experiments described here reveal the signatures and timing of protein flux changes during skeletal muscle regeneration, as well as the inability of mRNA expression measurements to reveal changes in directly measured protein turnover rates. The results of this work described here provide a better understanding of the muscle regeneration process and could help to identify potential biomarkers or therapeutic targets.

Myofibrillar protein lipoxidation in fish induced by linoleic acid and 4-hydroxy-2-nonenal: Insights from LC-MS/MS analysis.

The oxidation of fish lipids and proteins is interconnected. The LOX (lipoxygenase)-catalyzed LA (linoleic acid) oxidation system on MPs (myofibrillar proteins) was established in vitro, to investigate the impact of lipoxidation on the physicochemical properties of fish MPs. By detecting HNE (4-hydroxy-2-nonenal) concentration during LA oxidation, the HNE treatment system was established to investigate the role of HNE in this process. In addition, the site specificity of modification on MPs was detected utilizing LC-MS/MS. Both treatments could induce sidechain modification, increase particle size, and cause loss of nutritional value through the reduction in amino acid content of MPs. The HNE group is more likely to alter the MPs' surface hydrophobicity compared to the LA group. By increasing the exposure of modification sites in MPs, the HNE group has more types and number of modifications compared to the LA group. LA group mainly induced the modification of single oxygen addition on MPs instead, which accounted for over 50 % of all modifications. The LA group induced a more pronounced reduction in the solubility of MPs as compared to the HNE group. In conclusion, HNE binding had a high susceptibility to Lys on MPs. Protein aggregation, peptide chain fragmentation, and decreased solubility occurred in the LA group mainly induced by peroxide generated during lipid oxidation or the unreacted LA instead of HNE. This study fills in the mechanism of lipoxidation on protein oxidation in fish and sheds light on the HNE modification sites of MPs, paving the way for the development of oxidation control technology.

Building, Breaking, and Repairing Neuromuscular Synapses.

A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.

Oleocanthal Protects C2C12 Myotubes against the Pro-Catabolic and Anti-Myogenic Action of Stimuli Able to Induce Muscle Wasting In Vivo.

Oleocanthal (OC) is a monophenol of extra-virgin olive oil (EVOO) endowed with antibiotic, cardioprotective and anticancer effects, among others, mainly in view of its antioxidant and anti-inflammatory properties. OC has been largely investigated in terms of its anticancer activity, in Alzheimer disease and in collagen-induced arthritis; however, the possibility that it can also affect muscle biology has been totally overlooked so far. This study is the first to describe that OC modulates alterations induced in C2C12 myotubes by stimuli known to induce muscle wasting in vivo, namely TNF-α, or in the medium conditioned by the C26 cachexia-inducing tumor (CM-C26). C2C12 myotubes were exposed to CM-C26 or TNF-α in the presence or absence of OC for 24 and 48 h and analyzed by immunofluorescence and Western blotting. In combination with TNF-α or CM-C26, OC was revealed to be able to restore both the myotube's original size and morphology and normal levels of both atrogin-1 and MuRF1. OC seems unable to impinge on the autophagic-lysosomal proteolytic system or protein synthesis. Modulations towards normal levels of the expression of molecules involved in myogenesis, such as Pax7, myogenin and MyHC, were also observed in the myotube cultures exposed to OC and TNF-α or CM-C26. In conclusion, the data presented here show that OC exerts a protective action in C2C12 myotubes exposed to TNF-α or CM-C26, with mechanisms likely involving the downregulation of ubiquitin-proteasome-dependent proteolysis and the partial relief of myogenic differentiation impairment.

Deciphering the Therapeutic Role of Lactate in Combating Disuse-Induced Muscle Atrophy: An NMR-Based Metabolomic Study in Mice.

Disuse muscle atrophy (DMA) is a significant healthcare challenge characterized by progressive loss of muscle mass and function resulting from prolonged inactivity. The development of effective strategies for muscle recovery is essential. In this study, we established a DMA mouse model through hindlimb suspension to evaluate the therapeutic potential of lactate in alleviating the detrimental effects on the gastrocnemius muscle. Using NMR-based metabolomic analysis, we investigated the metabolic changes in DMA-injured gastrocnemius muscles compared to controls and evaluated the beneficial effects of lactate treatment. Our results show that lactate significantly reduced muscle mass loss and improved muscle function by downregulating Murf1 expression, decreasing protein ubiquitination and hydrolysis, and increasing myosin heavy chain levels. Crucially, lactate corrected perturbations in four key metabolic pathways in the DMA gastrocnemius: the biosynthesis of phenylalanine, tyrosine, and tryptophan; phenylalanine metabolism; histidine metabolism; and arginine and proline metabolism. In addition to phenylalanine-related pathways, lactate also plays a role in regulating branched-chain amino acid metabolism and energy metabolism. Notably, lactate treatment normalized the levels of eight essential metabolites in DMA mice, underscoring its potential as a therapeutic agent against the consequences of prolonged inactivity and muscle wasting. This study not only advances our understanding of the therapeutic benefits of lactate but also provides a foundation for novel treatment approaches aimed at metabolic restoration and muscle recovery in conditions of muscle wasting.

MCT4 knockdown by tumor microenvironment-responsive nanoparticles remodels the cytokine profile and eradicates aggressive breast cancer cells.

Breast cancer is a wide-spread threat to the women's health. The drawbacks of conventional treatments necessitate the development of alternative strategies, where gene therapy has regained hope in achieving an efficient eradication of aggressive tumors. Monocarboxylate transporter 4 (MCT4) plays pivotal roles in the growth and survival of various tumors, which offers a promising target for treatment. In the present study, pH-responsive lipid nanoparticles (LNPs) based on the ionizable lipid,1,2-dioleoyl-3-dimethylammonium propane (DODAP), were designed for the delivery of siRNA targeting MCT4 gene to the breast cancer cells. Following multiple steps of characterization and optimization, the anticancer activities of the LNPs were assessed against an aggressive breast cancer cell line, 4T1, in comparison with a normal cell line, LX-2. The selection of the helper phospholipid to be incorporated into the LNPs had a dramatic impact on their gene delivery performance. The optimized LNPs enabled a powerful MCT4 silencing by ∼90 % at low siRNA concentrations, with a subsequent ∼80 % cytotoxicity to 4T1 cells. Meanwhile, the LNPs demonstrated a 5-fold higher affinity to the breast cancer cells versus the normal cells, in which they had a minimum effect. Moreover, the MCT4 knockdown by the treatment remodeled the cytokine profile in 4T1 cells, as evidenced by 90 % and ∼64 % reduction in the levels of TNF-α and IL-6; respectively. The findings of this study are promising for potential clinical applications. Furthermore, the simple and scalable delivery vector developed herein can serve as a breast cancer-targeting platform for the delivery of other RNA therapeutics.

The Influence of Stress and Binge-Patterned Alcohol Drinking on Mouse Skeletal Muscle Protein Synthesis and Degradation Pathways.

Adverse experiences (e.g., acute stress) and alcohol misuse can both impair skeletal muscle homeostasis, resulting in reduced protein synthesis and greater protein breakdown. Exposure to acute stress is a significant risk factor for engaging in alcohol misuse. However, little is known about how these factors together might further affect skeletal muscle health. To that end, this study investigated the effects of acute stress exposure followed by a period of binge-patterned alcohol drinking on signaling factors along mouse skeletal muscle protein synthesis (MPS) and degradation (MPD) pathways. Young adult male C57BL/6J mice participated in the Drinking in the Dark paradigm, where they received 2-4 h of access to 20% ethanol (alcohol group) or water (control group) for four days to establish baseline drinking levels. Three days later, half of the mice in each group were either exposed to a single episode of uncontrollable tail shocks (acute stress) or remained undisturbed in their home cages (no stress). Three days after stress exposure, mice received 4 h of access to 20% ethanol (alcohol) to model binge-patterned alcohol drinking or water for ten consecutive days. Immediately following the final episode of alcohol access, mouse gastrocnemius muscle was extracted to measure changes in relative protein levels along the Akt-mTOR MPS, as well as the ubiquitin-proteasome pathway (UPP) and autophagy MPD pathways via Western blotting. A single exposure to acute stress impaired Akt singling and reduced rates of MPS, independent of alcohol access. This observation was concurrent with a potent increase in heat shock protein seventy expression in the muscle of stressed mice. Alcohol drinking did not exacerbate stress-induced alterations in the MPS and MPD signaling pathways. Instead, changes in the MPS and MPD signaling factors due to alcohol access were primarily observed in non-stressed mice. Taken together, these data suggest that exposure to a stressor of sufficient intensity may cause prolonged disruptions to signaling factors that impact skeletal muscle health and function beyond what could be further induced by periods of alcohol misuse.

Correlation analysis of phosphorylation of myofibrillar protein and muscle quality of tilapia during storage in ice.

In this study, the correlation between protein phosphorylation and deterioration in the quality of tilapia during storage in ice was examined by assessing changes in texture, water-holding capacity (WHC), and biochemical characteristics of myofibrillar protein throughout 7 days of storage. The hardness significantly decreased from 471.50 to 252.17 g, whereas cooking and drip losses significantly increased from 26.5% to 32.6% and 2.9% to 9.1%, respectively (P < 0.05). Myofibril fragmentation increased, while myofibrillar protein sulfhydryl content and Ca2+-ATPase activity decreased from 119.33 to 89.29 µmol/g prot and 0.85 to 0.46 µmolPi/mg prot/h, respectively (P < 0.05). Correlation analysis revealed that the myofibrillar protein phosphorylation level was positively correlated with hardness and Ca2+-ATPase activity but negatively correlated with WHC. Myofibrillar protein phosphorylation affects muscle contraction by influencing the dissociation of actomyosin, thereby regulating hardness and WHC. This study provides novel insights for the establishment of quality control strategies for tilapia storage based on protein phosphorylation.

Macrophage MCT4 inhibition activates reparative genes and protects from atherosclerosis by histone H3 lysine 18 lactylation.

Macrophage activation is a hallmark of atherosclerosis, accompanied by a switch in core metabolism from oxidative phosphorylation to glycolysis. The crosstalk between metabolic rewiring and histone modifications in macrophages is worthy of further investigation. Here, we find that lactate efflux-associated monocarboxylate transporter 4 (MCT4)-mediated histone lactylation is closely related to atherosclerosis. Histone H3 lysine 18 lactylation dependent on MCT4 deficiency activated the transcription of anti-inflammatory genes and tricarboxylic acid cycle genes, resulting in the initiation of local repair and homeostasis. Strikingly, histone lactylation is characteristically involved in the stage-specific local repair process during M1 to M2 transformation, whereas histone methylation and acetylation are not. Gene manipulation and protein hydrolysis-targeted chimerism technology are used to confirm that MCT4 deficiency favors ameliorating atherosclerosis. Therefore, our study shows that macrophage MCT4 deficiency, which links metabolic rewiring and histone modifications, plays a key role in training macrophages to become repair and homeostasis phenotypes.

Human disease-causing mutations result in loss of leiomodin 2 through nonsense-mediated mRNA decay.

The leiomodin (Lmod) family of actin-binding proteins play a critical role in muscle function, highlighted by the fact that mutations in all three family members (LMOD1-3) result in human myopathies. Mutations in the cardiac predominant isoform, LMOD2 lead to severe neonatal dilated cardiomyopathy. Most of the disease-causing mutations in the LMOD gene family are nonsense, or frameshift, mutations predicted to result in expression of truncated proteins. However, in nearly all cases of disease, little to no LMOD protein is expressed. We show here that nonsense-mediated mRNA decay, a cellular mechanism which eliminates mRNAs with premature termination codons, underlies loss of mutant protein from two independent LMOD2 disease-causing mutations. Furthermore, we generated steric-blocking oligonucleotides that obstruct deposition of the exon junction complex, preventing nonsense-mediated mRNA decay of mutant LMOD2 transcripts, thereby restoring mutant protein expression. Our investigation lays the initial groundwork for potential therapeutic intervention in LMOD-linked myopathies.

Myofibrillar protein hydrolysis under hydroxyl radical oxidative stress: Structural changes and their impacts on binding to selected aldehydes.

In this study, a hydroxyl radical oxidation system was established to simulate the oxidation process in fermented meat products. This system was employed to examine the structural changes in myofibrillar proteins (MPs) resulting from tryptic hydrolysis after a hydroxyl radical oxidative regime. The effect of these changes on the ability of MPs to bind selected aldehydes (3-methyl butanal, pentanal, hexanal, and heptanal) was also investigated. Moderate oxidation (H2O2 ≤ 1.0 mM) unfolded the structure of MPs, facilitating trypsin-mediated hydrolysis and increasing their binding capacity for the four selected aldehydes. However, excessive oxidation (H2O2 ≥ 2.5 mM) led to cross-linking and aggregation of MPs, inhibiting trypsin-mediated hydrolysis. The oxidised MPs had the best binding capacity for heptanal. The interaction of the oxidised trypsin-hydrolysed MPs with heptanal was driven by hydrophobic interactions. The binding of heptanal affected the structure of the oxidised trypsin-hydrolysed MPs and reduced their α-helix content.

Proximal tubular FHL2, a novel downstream target of hypoxia inducible factor 1, is a protector against ischemic acute kidney injury.

Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.

Striatin plays a major role in angiotensin II-induced cardiomyocyte and cardiac hypertrophy in mice in vivo.

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with up-regulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN+/- or STRN3+/- male mice (8 weeks) compared with wild-type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN+/- (but not STRN3+/-) mice. Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy.

Structural domain in the Titin N2B-us region binds to FHL2 in a force-activation dependent manner.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2.

Integrated multi-omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex.

BACKGROUND: Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions. METHODS: Skeletal muscles from 2-month-old SPEG-deficient (Speg-CKO) and wild-type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg-CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg-CKO mice. Based on the multi-omics results, we performed quantitative real-time PCR, co-immunoprecipitation and immunoblot to verify the findings. RESULTS: We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 [P < 0.05] and 18 ± 3% for FSD2 [P < 0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg-CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90 ± 0.24-fold], S162 [1.61 ± 0.37-fold] and S165 [1.66 ± 0.13-fold]; decreased phosphorylation at S228 and S231 [39 ± 6%], S234 [50 ± 12%], S593 [48 ± 3%] and S613 [66 ± 10%]; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction (P < 1e-15) and peroxisome proliferator-activated receptor signalling (P < 9e-14). CONCLUSIONS: We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG-interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi-omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.

Intramuscular injection of mesenchymal stem cells augments basal muscle protein synthesis after bouts of resistance exercise in male mice.

Skeletal muscle mass is critical for activities of daily living. Resistance training maintains or increases muscle mass, and various strategies maximize the training adaptation. Mesenchymal stem cells (MSCs) are multipotent cells with differential potency in skeletal muscle cells and the capacity to secrete growth factors. However, little is known regarding the effect of intramuscular injection of MSCs on basal muscle protein synthesis and catabolic systems after resistance training. Here, we measured changes in basal muscle protein synthesis, the ubiquitin-proteasome system, and autophagy-lysosome system-related factors after bouts of resistance exercise by intramuscular injection of MSCs. Mice performed three bouts of resistance exercise (each consisting of 50 maximal isometric contractions elicited by electrical stimulation) on the right gastrocnemius muscle every 48 h, and immediately after the first bout, mice were intramuscularly injected with either MSCs (2.0 × 106 cells) labeled with green fluorescence protein (GFP) or vehicle only placebo. Seventy-two hours after the third exercise bout, GFP was detected only in the muscle injected with MSCs with concomitant elevation of muscle protein synthesis. The injection of MSCs also increased protein ubiquitination. These results suggest that the intramuscular injection of MSCs augmented muscle protein turnover at the basal state after consecutive resistance exercise.

APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

Beef peptides mitigate skeletal muscle atrophy in C2C12 myotubes through protein degradation, protein synthesis, and the oxidative stress pathway.

This study aimed to investigate the potential of beef peptides (BPs) in mitigating muscle atrophy induced by dexamethasone (DEX) with underlying three mechanisms in vitro (protein degradation, protein synthesis, and the oxidative stress pathway). Finally, the anti-atrophic effect of BPs was enhanced through purification and isolation. BPs were generated using beef loin hydrolyzed with alcalase/ProteAX/trypsin, each at a concentration of 0.67%, followed by ultrafiltration through a 3 kDa cut-off. BPs (10-100 µg mL-1) dose-dependently counteracted the DEX-induced reductions in myotube diameters, differentiation, fusion, and maturation indices (p < 0.05). Additionally, BPs significantly reduced FoxO1 protein dephosphorylation, thereby suppressing muscle-specific E3 ubiquitin ligases such as muscle RING-finger containing protein-1 and muscle atrophy F-box protein in C2C12 myotubes at concentrations exceeding 25 µg mL-1 (p < 0.05). BPs also enhanced the phosphorylation of protein synthesis markers, including mTOR, 4E-BP1, and p70S6K1, in a dose-dependent manner (p < 0.05) and increased the mRNA expression of antioxidant enzymes. Fractionated peptides derived from BPs, through size exclusion and polarity-based fractionation, also demonstrated enhanced anti-atrophic effects compared to BPs. These peptides downregulated the mRNA expression of primary muscle atrophy markers while upregulated that of antioxidant enzymes. Specifically, peptides GAGAAGAPAGGA (MW 924.5) and AFRSSTKK (MW 826.4) were identified from fractionated peptides of BPs. These findings suggest that BPs, specifically the peptide fractions GAGAAGAPAGGA and AFRSSTKK, could be a potential strategy to mitigate glucocorticoid-induced skeletal muscle atrophy by reducing the E3 ubiquitin ligase activity.

Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity.

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable-and easily accessible-tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.