Michele Salanova

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 Ca2+-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.

Differential expression of nitric oxide synthases (NOS 1-3) in human skeletal muscle following exercise countermeasure during 12 weeks of bed rest

Adaptive changes of major body systems in astronauts during spaceflight can be simulated by strict anti‐orthostatic head‐down tilt (HDT) bed rest (BR), a ground‐based microgravity (μG) model that provides a meaningful opportunity to study atrophy mechanisms and possible countermeasures under controlled experimental conditions. As nitric oxide (NO) signaling is linked to muscle activity, we investigated altered expression of the three major isoforms of nitric oxide synthase (NOS 1–3) at cellular compartments during prolonged HDT BR without (control group) and with resistance exercise interventions (exercise group) using a flywheel ergometer (FWE). Atrophy detected in mixed (fast–slow) m. vastus lateralis (VL) and slow‐type m. soleus (SOL) myofiber Types I and II (minus 35–40% of myofiber cross‐sectional area) was prevented by FWE training. Concomitant to muscle atrophy, reduced NOS 1 protein and immunostaining was found in VL not in SOL biopsies. In trained VL, NOS 1 protein and immunostaining at myofibers II were significantly increased at the end of BR. Exercise altered NOS 2/caveolin 3 co‐immunostaining patterns of subsarcolemmal focal accumulations in VL or SOL myofibers, which suggests reorganization of sarcolemmal microdomains. In trained VL, increased capillary‐ to‐fiber (C/F) ratio and NOS 3 protein content were documented. Activity‐linked NO signaling may be widespread in skeletal muscle cellular compartments that may be directly or indirectly impacted by adequate exercise countermeasure protocols to offset the negative effects induced by disuse, immobilization, or extended exposure to microgravity.

Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures.

The cellular mechanisms of human skeletal muscle adaptation to disuse are largely unknown. The aim of this study was to determine the morphological and biochemical changes of the lower limb soleus and vastus lateralis muscles following 60 days of head‐down tilt bed rest in women with and without exercise countermeasure using molecular biomarkers monitoring functional cell compartments. Muscle biopsies were taken before (pre) and after bed rest (post) from a bed rest‐only and a bed rest exercise group (n = 8, each). NOS1 and NOS3/PECAM, markers of myofibre ‘activity’ and capillary density, and MuRF1 (E3 ubiquitin‐ligase), a marker of proteolysis, were documented by confocal immunofluorescence and immunoblot analyses. Morphometrical parameters (myofibre cross‐sectional area, type I/II distribution) were largely preserved in muscles from the exercise group with a robust trend for type II hypertrophy in vastus lateralis. In the bed rest‐only group, the relative NOS1 immunostaining intensity was decreased at type I and II myofibre membranes, while the bed rest plus exercise group compensated for this loss particularly in soleus. In the microvascular network, NOS3 expression and the capillary‐to‐fibre ratio were both increased in the exercise group. Elevated MuRF1 immunosignals found in subgroups of atrophic myofibres probably reflected accelerated proteolysis. Immunoblots revealed overexpression of the MuRF1 protein in the soleus of the bed rest‐only group (> 35% vs. pre). We conclude that exercise countermeasure during bed rest affected both NOS/NO signalling and proteolysis in female skeletal muscle. Maintenance of NO signalling mechanisms and normal protein turnover by exercise countermeasure may be crucial steps to attenuate human skeletal muscle atrophy and to maintain cell function following chronic disuse.

Renal Na+-K+-Cl- cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent.

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40-70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na(+)-Cl(-) cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by (86)Rb(+) influx in Xenopus laevis oocytes was markedly reduced by methyl-beta-cyclodextrin (MbetaCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MbetaCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.

Long term bed rest with and without vibration exercise countermeasures: Effects on human muscle protein dysregulation

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2‐D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (∼50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (∼35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of ∼15% in cross‐sectional area and of ∼22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z‐disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.

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 PGC1a 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 ultra‐structural and molecular dysregulation induced by chronic disuse.—Salanova, M., Gelfi, C., Moriggi, M., Vasso, M., Viganò, A., Minafra, L., Bonifacio, G., Schiffl, G., Gutsmann, M., Felsenberg, D., Cerretelli, P., Blottner, D., Disuse deterioration of human skeletal muscle challenged by resistive exercise superimposed with vibration: evidence from structural and proteomic analysis. FASEB J. 28, 4748–4763 (2014). www.fasebj.org

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.—Salanova, M., Bortoloso, E., Schiffl, G., Gutsmann, M., Belavý, D. L., Felsenberg, D., Furlan, S., Volpe, P., Blottner, D. Expression and regulation of Homer in human skeletal muscle during neuromuscular junction adaptation to disuse and exercise. FASEB J. 25, 4312–4325 (2011). www.fasebj.org

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 Ca2+ 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.

Vibration mechanosignals superimposed to resistive exercise result in baseline skeletal muscle transcriptome profiles following chronic disuse in bed rest.

Disuse-induced muscle atrophy is a major concern in aging, in neuromuscular diseases, post-traumatic injury and in microgravity life sciences affecting health and fitness also of crew members in spaceflight. By using a laboratory analogue to body unloading we perform for the first time global gene expression profiling joined to specific proteomic analysis to map molecular adaptations in disused (60 days of bed rest) human soleus muscle (CTR) and in response to a resistive exercise (RE) countermeasure protocol without and with superimposed vibration mechanosignals (RVE). Adopting Affymetrix GeneChip technology we identified 235 differently transcribed genes in the CTR group (end- vs. pre-bed rest). RE comprised 206 differentially expressed genes, whereas only 51 changed gene transcripts were found in RVE. Most gene transcription and proteomic changes were linked to various key metabolic pathways (glycolysis, oxidative phosphorylation, tricarboxylic acid (TCA) cycle, lipid metabolism) and to functional contractile structures. Gene expression profiling in bed rest identified a novel set of genes explicitly responsive to vibration mechanosignals in human soleus. This new finding highlights the efficacy of RVE protocol in reducing key signs of disuse maladaptation and atrophy, and to maintain a close-to-normal skeletal muscle quality outcome following chronic disuse in bed rest.

Homer protein family regulation in skeletal muscle and neuromuscular adaptation

Scaffolding adaptor proteins of the Homer family have recently been implicated in regulation of a large number of physiological processes owing to their remarkable ability to coordinate a complex network of different molecular players within the same signaling pathway. However, because of their unique molecular properties that also allow functional modulation of a plethora of different interacting protein partners, Homers seem to play additional and important roles in the integration of several molecular players belonging to different signaling pathways and thus allowing crosstalk. The role of the Homer protein family has been previously extensively investigated in neuronal tissue where it was first discovered as a new protein family being upregulated in response to brain seizures (Brakeman P.R., et al., Nature 1997, 386, 284–288.). Recently, the role of Homers was also proposed in skeletal muscle physiology. For instance, it has been shown that Homers regulate both the myogenic differentiation program and the open probability (Po) of several ion channels. Furthermore, by knocking out Homer1, one of the three Homer genes, mice carrying such deletion displayed a pronounced skeletal muscle myopathy associated with altered transient receptor potential activity and calcium homeostasis. Homer expression has now been further characterized at the neuromuscular junction in skeletal muscle. Apart from their known role at central synapses, Homers are important physiological determinants in differentiation, development, and adaptation in skeletal muscle and the neuromuscular system and thus integrating motor neuron control, for example, with downstream calcium signaling pathways in muscle fibers.