Opposite Regulation of Homer Signal at the NMJ Postsynaptic Micro Domain between Slow- and Fast-Twitch Muscles in an Experimentally Induced Autoimmune Myasthenia Gravis (EAMG) Mouse Model.

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.

Reciprocal Homer1a and Homer2 Isoform Expression Is a Key Mechanism for Muscle Soleus Atrophy in Spaceflown Mice.

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.

Microgravity-Induced Transcriptome Adaptation in Mouse Paraspinal longissimus dorsi Muscle Highlights Insulin Resistance-Linked Genes.

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate longissimus dorsi adaptation to microgravity at both morphological and global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Myofiber cross sectional area and myosin heavy chain subtype patterns were respectively not or slightly altered in longissimus dorsi of BF mice. Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice. Microgravity-induced gene expression changes of lipocalin 2 (Lcn2), sestrin 1(Sesn1), phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 (p85 alpha) (Pik3r1), v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (Mafb), protein kinase C delta (Prkcd), Muscle Atrophy F-box (MAFbx/Atrogin-1/Fbxo32), and Muscle RING Finger 1 (MuRF-1) were further validated by real time qPCR analysis. In conclusion, our study highlighted the regulation of transcripts mainly linked to insulin sensitivity and metabolism in longissimus dorsi following 30 days of microgravity exposure. The apparent absence of robust signs of back muscle atrophy in space-flown mice, despite the overexpression of Atrogin-1 and MuRF-1, opens new questions on the possible role of microgravity-sensitive genes in the regulation of peripheral insulin resistance following unloading and its consequences on paraspinal skeletal muscle physiology.

Gene Expression Profiling in Slow-Type Calf Soleus Muscle of 30 Days Space-Flown Mice.

Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight.

Disuse deterioration of human skeletal muscle challenged by resistive exercise superimposed with vibration: evidence from structural and proteomic analysis.

In the present bed rest (BR) study, 23 volunteers were randomized into 3 subgroups: 60 d BR control (Ctr); BR with resistive exercise (RE; lower-limb load); and resistive vibration exercise (RVE; RE with superimposed vibration). The aim was to analyze by confocal and electron microscopy the effects of vibration on myofibril and filament integrity in soleus (Sol) and vastus lateralis (VL) muscle; differential proteomics of contractile, cytoskeletal, and costameric proteins (TN-C, ROCK1, and FAK); and expression of PGC1α and atrophy-related master genes MuRF1 and MuRF2. RVE (but not RE) preserved myofiber size and phenotype in Sol and VL by overexpressing MYBPC1 (42%, P ≤ 0.01), WDR1 (39%, P ≤ 0.01), sarcosin (84%, P ≤ 0.01), and CKM (20%, P ≤ 0.01) and prevented myofibrillar ultrastructural damage as detectable by MuRF1 expression. In Sol, cytoskeletal and contractile proteins were normalized by RVE, and TN-C increased (59%, P ≤ 0.01); the latter also with RE (108%, P ≤ 0.01). In VL, the outcomes of both RVE (acting on sarcosin and desmin) and RE (by way of troponinT-slow and MYL2) were similar. RVE appears to be a highly efficient countermeasure protocol against muscle atrophy and ultrastructural and molecular dysregulation induced by chronic disuse.

Increased myofiber remodelling and NFATc1-myonuclear translocation in rat postural skeletal muscle after experimental vestibular deafferentation.

BACKGROUND: The vestibular system undergoes considerable modification during spaceflight [5]. This is paralleled by microgravity-induced muscle atrophy [6]. However, the possibility of vestibulo-autonomic regulatory mechanisms affecting skeletal muscle structure and function have not yet been addressed. OBJECTIVE: We hypothesise that the vestibular system affects anti-gravitational skeletal muscle phenotype composition, size and the transcriptional factor called nuclear factor of activated T cells (NFATc1). METHODS: In a laboratory study, we examined the morphological and histochemical properties including intramyocellular NFATc1 changes in slow-type soleus muscle of chemically labyrinthectomized rats (VLx; n=8) compared to a control group (Sham; n=6) after a period of one month. RESULTS AND CONCLUSION: Neurochemical vestibular deafferentation resulted in smaller myofibre sizes, altered myofibre phenotype composition, high yields of hybrid fibre formation, and reduced myonuclear NFATc1 accumulation as signs of slow-type myofibre atrophy, myofibre type remodelling, and altered nuclear transcriptional activity in the postural soleus muscle of rats. We propose that vestibulo-autonomic modification of skeletal muscles occurs during prolonged microgravity. Our findings are likely to have implications for vestibular rehabilitation in clinical settings.

Nitrosative stress in human skeletal muscle attenuated by exercise countermeasure after chronic disuse.

Activity-induced nitric oxide (NO) imbalance and "nitrosative stress" are proposed mechanisms of disrupted Ca(2+) homeostasis in atrophic skeletal muscle. We thus mapped S-nitrosylated (SNO) functional muscle proteins in healthy male subjects in a long-term bed rest study (BBR2-2 Study) without and with exercise as countermeasure in order to assess (i) the negative effects of chronic muscle disuse by nitrosative stress, (ii) to test for possible attenuation by exercise countermeasure in bed rest and (iii) to identify new NO target proteins. Muscle biopsies from calf soleus and hip vastus lateralis were harvested at start (Pre) and at end (End) from a bed rest disuse control group (CTR, n=9) and two bed rest resistive exercise groups either without (RE, n=7) or with superimposed vibration stimuli (RVE, n=7). At subcellular compartments, strong anti-SNO-Cys immunofluorescence patterns in control muscle fibers after bed rest returned to baseline following vibration exercise. Total SNO-protein levels, Nrf-2 gene expression and nucleocytoplasmic shuttling were changed to varying degrees in all groups. Excess SNO-protein levels of specific calcium release/uptake proteins (SNO-RyR1, -SERCA1 and -PMCA) and of contractile myosin heavy chains seen in biopsy samples of chronically disused skeletal muscle were largely reduced by vibration exercise. We also identified NOS1 as a novel NO target in human skeletal muscle controlled by activity driven auto-nitrosylation mechanisms. Our findings suggest that aberrant levels of functional SNO-proteins represent signatures of uncontrolled nitrosative stress management in disused human skeletal muscle that can be offset by exercise as countermeasure.

Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission.

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.

Expression and regulation of Homer in human skeletal muscle during neuromuscular junction adaptation to disuse and exercise.

Protein calcium sensors of the Homer family have been proposed to modulate the activity of various ion channels and nuclear factor of activated T cells (NFAT), the transcription factor modulating skeletal muscle differentiation. We monitored Homer expression and subcellular localization in human skeletal muscle biopsies following 60 d of bedrest [Second Berlin Bedrest Study (BBR2-2)]. Soleus (SOL) and vastus lateralis (VL) biopsies were taken at start (pre) and at end (end) of bedrest from healthy male volunteers of a control group without exercise (CTR; n=9), a resistive-only exercise group (RE; n=7), and a combined resistive/vibration exercise group (RVE; n=7). Confocal analysis showed Homer immunoreactivity at the postsynaptic microdomain of the neuromuscular junction (NMJ) at bedrest start. After bedrest, Homer immunoreactivity decreased (CTR), remained unchanged (RE), or increased (RVE) at the NMJ. Homer2 mRNA and protein were differently regulated in a muscle-specific way. Activated NFATc1 translocates from cytoplasm to nucleus; increased amounts of NFATc1-immunopositive slow-type myonuclei were found in RVE myofibers of both muscles. Pulldown assays identified NFATc1 and Homer as molecular partners in skeletal muscle. A direct motor nerve control of Homer2 was confirmed in rat NMJs by in vivo denervation. Homer2 is localized at the NMJ and is part of the calcineurin-NFATc1 signaling pathway. RVE has additional benefit over RE as countermeasure preventing disuse-induced neuromuscular maladaptation during bedrest.

Localization of NOS-1 in the sarcolemma region of a subpopulation of atrial cardiomyocytes including myoendocrine cells and NOS-3 in vascular and endocardial endothelial cells of the rat heart.

Cellular localization patterns of NOS isoforms 1 and 3 (nNOS and eNOS, respectively) in the mammalian heart under basal (non-stimulated) working conditions are still a matter of discussion. Therefore, this issue was reinvestigated in rats using RT-PCR, Western blotting, catalytic histochemistry, immunohistochemistry and image analysis. Tongue and extensor digitorum longus muscles served as positive controls for NOS-1 and NOS-3. RT-PCR revealed NOS-1 mRNA and NOS-3 mRNA in atria and ventricles. Western blotting showed NOS-1 protein in atria and NOS-3 protein in the walls of both heart chambers. Localization of the activity of urea-resistant (and therefore specific) NADPH diaphorase (NADPH-D) and NOS-1 immunohistochemistry showed that NOS-1 is present in the sarcolemma region of a subpopulation of atrial cardiomyocytes but not in working and impulse-conducting cardiomyocytes of atria and ventricles. Atrial natriuretic peptide (ANP) immunohistochemistry revealed that a minority of the NOS-1-expressing atrial cardiomyocytes are myoendocrine cells. eNOS immunostaining was present in endothelial cells of capillaries of the conducting and working myocardium and endocardial cells. Image analysis of the activity of urea-resistant NOS diaphorase showed that NOS-1 activity is lower in the sarcolemma region of atrial cardiomyocytes than in that of tongue and extensor digitorum longus myofibers. These data suggest that, in the non-stimulated rat heart. NOS-1 is expressed in a subpopulation of atrial cardiomyocytes including myoendocrine cells, and that NOS-3 is expressed in the vascular and endocardial endothelium.