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

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

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

Principal Component Analysis (PCA) of gene expression data in <i>soleus</i> and EDL.

<p>PCA analysis of gene expression data in <i>soleus</i> (A) and EDL (B) highlights the high sensitivity of soleus to microgravity exposure compared to EDL.</p

Functional gene clusters differentially regulated in <i>soleus</i> of BION-M1 space flown mice.

<p>Functional gene clusters differentially regulated in <i>soleus</i> of BION-M1 space flown mice.</p

Morphological analysis of mouse <i>soleus</i> and EDL skeletal muscle flown on board the BION-M1 biosatellite for 30 days.

<p>Upper panel, insets show light microscopy images of Haematoxylin Eosin merged to immunofluorescence images of <i>soleus</i> (SOL) and EDL stained for MyHC isoforms (MyHC I: blue, IIa: green, IIb: red) in flown (BF) and control (BG) mice. Lower panel, scatter plots showing the quantification of the myofiber cross sectional area (CSA) and type composition in <i>soleus</i> (n = 2) and EDL (n = 2). Scale bar: 100 μm.</p

Venn diagrams and heat maps showing genes differentially regulated in <i>soleus</i> and EDL of BION-M1 space flown mice.

<p>A, B, Venn diagrams showing the number of genes differentially regulated comparing space flown (BF) with ground controls (BG and FC) in <i>soleus</i> (A) and EDL (B). Comparisons between the three different experimental groups are presented (BF vs. BG, FC vs. BG and BF vs. FC). C, D, hierarchical clustering centred on BF vs. BG in <i>soleus</i> (C, ordered BG-FC-BF) and EDL (D, ordered BF-FC-BG). The differentially regulated genes meeting FDR < 0,05 (<i>soleus</i>), p < 0.05 (EDL) and < -2 & > +2 fold change criteria are included in the heat maps.</p