Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major determinant of disuse-induced muscle atrophy. Muscle biopsies (thigh vastus lateralis, VL) obtained from healthy male subjects enrolled in the Toulouse Cocktail bedrest (BR) study were used to assess efficacy of an antioxidant cocktail (polyphenols, omega-3, vitamin E, and selenium) to counteract the increased redox homeostasis and enhance the antioxidant defense response by using label-free LC–MS/MS and NITRO-DIGE (nitrosated proteins), qPCR, and laser confocal microscopy. Label-free LC–MS/MS indicated that treatment prevented the redox homeostasis dysregulation and promoted structural remodeling (TPM3, MYH7, MYBPC, MYH1, MYL1, HRC, and LUM), increment of RyR1, myogenesis (CSRP3), and skeletal muscle development (MUSTN1, LMNA, AHNAK). These changes were absent in the Placebo group. Glycolysis, tricarboxylic acid cycle (TCA), oxidative phosphorylation, fatty acid beta-oxidation, and mitochondrial transmembrane transport were normalized in treated subjects. Proteins involved in protein folding were also normalized, whereas protein entailed in ion homeostasis decreased. NITRO-DIGE analysis showed significant protein nitrosylation changes for CAT, CA3, SDHA, and VDAC2 in Treatment vs. Placebo. Similarly, the nuclear factor erythroid 2-related factor 2 (Nrf-2) antioxidant response element (Nrf-2 ARE) signaling pathway showed an enhanced response in the Treatment group. Increased nitrosative redox homeostasis and decreased antioxidant defense response were found in post-BR control (Placebo, n = 10) vs. the antioxidant cocktail treated group (Treatment, n = 10). Taken together, increased nitrosative redox homeostasis and muscle deterioration during BR-driven physical inactivity were prevented, whereas decreased antioxidant nitrosative stress defense response was attenuated by Treatment suggesting positive effects of the nutritional intervention protocol in bedrest.

Limb-girdle muscular dystrophy R3 (LGMDR3) is caused by mutations in the SGCA gene coding for α-sarcoglycan (SG). Together with β- γ- and δ-SG, α-SG forms a tetramer embedded in the dystrophin associated protein complex crucial for protecting the sarcolemma from mechanical stresses elicited by muscle contraction. Most LGMDR3 cases are due to missense mutations, which result in non-properly folded, even though potentially functional α-SG. These mutants are prematurely discarded by the cell quality control. Lacking one subunit, the SG-complex is disrupted. The resulting loss of function leads to sarcolemma instability, muscle fiber damage and progressive limb muscle weakness. LGMDR3 is severely disabling and, unfortunately, still incurable. Here, we propose the use of small molecules, belonging to the class of cystic fibrosis transmembrane regulator (CFTR) correctors, for recovering mutants of α-SG defective in folding and trafficking. Specifically, CFTR corrector C17 successfully rerouted the SG-complex containing the human R98H-α-SG to the sarcolemma of hind-limb muscles of a novel LGMDR3 murine model. Notably, the muscle force of the treated model animals was fully recovered. To our knowledge, this is the first time that a compound designated for cystic fibrosis is successfully tested in a muscular dystrophy and may represent a novel paradigm of treatment for LGMDR3 as well as different other indications in which a potentially functional protein is prematurely discarded as folding-defective. Furthermore, the use of small molecules for recovering the endogenous mutated SG has an evident advantage over complex procedures such as gene or cell transfer.

Long-duration mission (LDM) astronauts from the International Space Station (ISS) (>180 ISS days) revealed a close-to-normal sarcolemmal nitric oxide synthase type-1 (NOS1) immunoexpression in myofibers together with biochemical and quantitative qPCR changes in deep calf soleus muscle. Nitro-DIGE analyses identified functional proteins (structural, metabolic, mitochondrial) that were over-nitrosylated post- vs. preflight. In a short-duration mission (SDM) astronaut (9 ISS days), s-nitrosylation of a nodal protein of the glycolytic flux, specific proteins in tricarboxylic acid (TCA) cycle, respiratory chain, and over-nitrosylation of creatine kinase M-types as signs of impaired ATP production and muscle contraction proteins were seen. S-nitrosylation of serotransferrin (TF) or carbonic anhydrase 3 (CA3b and 3c) represented signs of acute response microgravity muscle maladaptation. LDM nitrosoprofiles reflected recovery of mitochondrial activity, contraction proteins, and iron transporter TF as signs of muscle adaptation to microgravity. Nitrosated antioxidant proteins, alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR), and selenoprotein thioredoxin reductase 1 (TXNRD1) levels indicated signs of altered redox homeostasis and reduced protection from nitrosative stress in spaceflight. This work presents a novel spaceflight-generated dataset on s-nitrosylated muscle protein signatures from astronauts that helps both to better understand the structural and molecular networks associated to muscular nitrosative stress and to design countermeasures to dysfunction and impaired performance control in human spaceflight missions.

The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-form was downregulated in SOL muscle in both models. Homer immunofluorescence intensity analysis at the NMJ of BF and HU animals showed comparable outcomes in SOL but not in the extensor digitorum longus (EDL) muscle. Reduced Homer crosslinking at the NMJ consequent to increased Homer1a and/or reduced Homer2 may contribute to muscle-type specific atrophy resulting from microgravity and HU disuse suggesting mutual mechanisms.

Accelerated postsynaptic remodelling and disturbance of neuromuscular transmission are common features of autoimmune neurodegenerative diseases. Homer protein isoform expression, crosslinking activity and neuromuscular subcellular localisation are studied in mouse hind limb muscles of an experimentally induced autoimmune model of Myasthenia Gravis (EAMG) and correlated to motor end plate integrity. Soleus (SOL), extensor digitorum longus (EDL) and gastrocnemius (GAS) skeletal muscles are investigated. nAChR membrane clusters were studied to monitor neuromuscular junction (NMJ) integrity. Fibre-type cross-sectional area (CSA) analysis is carried out in order to determine the extent of muscle atrophy. Our findings clearly showed that crosslinking activity of Homer long forms (Homer 1b/c and Homer2a/b) are decreased in slow-twitch and increased in fast-twitch muscle of EAMG whereas the short form of Homer that disrupts Homer crosslinking (Homer1a) is upregulated in slow-twitch muscle only. Densitometry analysis showed a 125% increase in Homer protein expression in EDL, and a 45% decrease in SOL of EAMG mice. In contrast, nAChR fluorescence pixel intensity decreased in endplates of EAMG mice, more distinct in type-I dominant SOL muscle. Morphometric CSA of EAMG vs. control (CTR) revealed a significant reduction in EDL but not in GAS and SOL. Taken together, these results indicate that postsynaptic Homer signalling is impaired in slow-twitch SOL muscle from EAMG mice and provide compelling evidence suggesting a functional coupling between Homer and nAChR, underscoring the key role of Homer in skeletal muscle neurophysiology.

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2^-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2^-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2^-/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.