The limited number of mice that can be housed in the MDS payload together with the unfortunate loss of three mice, represents a critical aspect of the experiment, especially for the reliability of statistical analysis. It was, however, a unique opportunity to study the effects of a long-term exposure to microgravity on several tissues in an animal model and to collect observations that might be relevant for future investigations. The similar levels of OSF1/PTN expression in the skin of the WT and Tg mice and the highly similar gene expression profile found by transcriptomic analysis in the two genotypes within a given experimental group authorized us to group both strains for consolidating the comparisons between the S and G group.

Skin has not yet received much interest in space research although it is the largest organ of the human body. It has multiple functions including thermal regulation, tactile sense, protection against pathogens and deleterious environmental conditions such as radiations and provides a vital barrier against body fluids diffusion and might be the source of health problems, including carcinogenesis, during long term space travels. The three-month duration exposure to weightlessness conditions experienced by mice during the MDS mission is appreciable and provided 20 research teams with organs and tissues collected from these ‘astromice’. The results published at the present time showed a bone loss in the weight-bearing bones,9 atrophy of the antigravity soleus muscle with a concomitant slow-to-fast transition,13 modulation of gene expression in the brain,14 increased lipid peroxidation products, and anti-oxidant defenses in erythrocytes.15 In our study, three major findings concerning different compartments of the skin are reported. Skin is a complex organ made of a stratified keratinizing epithelium, a dermis, and a hypodermis. In mice, it contains a large number of hair follicles, appended sebaceous glands, and arrector pili muscles. Epidermis is separated from the underlying dermis by a dermo-epidermal junction made of a basement membrane supporting the basal layer of germinal keratinocytes. The dermis is made of a scaffold of polymerized fibrillar collagens I, III, and V organized in thick bundles embedded in a highly hydrated gel of hyaluronic acid and proteoglycans. Many adhesive glycoproteins, matricellular proteins, growth factors, and biological mediators are associated with the dermal ECM. This matrix is populated by fibroblasts, which contribute to the maintenance of their own support through a remodeling process which proceeds at a slow rate in healthy adults. Beneath the dermis lies the hypodermis, made of a layer of adipose tissue, and a thin lamina of striated muscle, the panniculus carnosus. Contrasting to muscle and bone cells, dermal fibroblasts are not usually considered as ‘professional’ cells in mechano-sensing and mechano-reaction. We and others have, however, shown in vitro that they are mechano-competent and respond to modification of their mechanical environment by a marked regulation of their metabolic activity,16,17 including in microgravity.18

In the present in vivo study, a significant thinning of the dermis was found in the S mice although it was invaded by growing hair follicles, a process that is known to induce an appreciable increase of its thickness.19 The skin atrophy as shown here is therefore probably largely underestimated. The reduced dermal thickness and the consequent decreased collagen content per unit surface suggest a progressive degeneration of the dermis in agreement with the echographic data obtained in one astronaut.6 Collagen solubilized at low ionic strength at neutral pH represents the newly synthesized procollagen molecules. Its significant increase in S skin suggests an increased turn-over rate. This hypothesis is supported by the upregulation of α1(I) collagen messenger RNA expression and that of two matricellular proteins, CTGF/CCN2 known to stimulate collagen synthesis downstream of TGFβ pathway20 and Cyr61/CCN1, which induces a senescent phenotype associated with an increased collagen degradation.21 Furthermore, one can speculate that the newly formed procollagen molecules are underhydroxylated, as suggested by the observed reduced expression of prolyl-4-hydroxylase, and more prone to lysozomal intracellular degradation, as seen for instance during vitamin C deficiency.22 An impairment of procollagen processing and mature collagen fiber formation might also be involved due to a reduced expression of the enhancer (Pcolce2) of procollagen carboxypeptidase (BMP1). We propose that the dermal atrophy as seen in the S mice might be related to an imbalance between synthesis and degradation with an excessive early degradation of newly formed, perhaps defective, procollagen molecules. Moreover, the reduced expression of hyaluronan synthase, responsible for hyaluronan synthesis coupled to an increased expression of the aggrecanases Adamts 1 and 9 may further participate in the skin atrophy observed in space environment. It is noteworthy that senescent human skin is characterized by a reduction of hyaluronan.

One surprising finding of this study was the presence of a significant proportion of hair follicles in anagen phase in the three mice in Space. These morphological data were validated by the high expression level of a series of keratins known to be specifically expressed in hair follicles during the anagen phase while the interfollicular epidermal keratins were not modified. Follicle cycle, comprising an anagen, catagen, and resting telogen phase, starts soon after birth and is synchronous for the two first cycles before becoming asynchronous. According to the relative duration of hair cycles in C57BL6 mice,11 most hair follicles in these mice, that were 21-week-old at the end of the experiment, should be in resting telogen phase. As the anagen phase normally represents around ¼ of the total cycle duration at this age, it means that the probability to have three mice in anagen stage is 1/64, suggesting that our observations are probably not fortuitous. It might indicate an increase of the relative duration of the anagen phase in microgravity. As hair cycle depends on cyclic activation and silencing of hair stem cells, this suggests that microgravity might alter their differentiation program as observed in several models in microgravity.23

Microgravity affects muscles mass and physical performance in humans and animals.24 In general, slow type contractile proteins were found to be reduced in microgravity concomitantly to an increase of the fast type ones and of proteins involved in glycolytic activity.25 The major finding of the microarray analysis performed in our study highlighted the overexpression in the skin of a high number of structural genes expressed in striated muscles and of genes involved in contraction or encoding transcription factors positively acting in myogenesis or muscle development. As pili arrector muscles and muscle cells lining blood vessels are smooth muscles, the only candidate that could express these specific striated muscle genes is the panniculus carnosus. It is considered as a fast striated muscle as it exhibits fast myosin fiber subtype, is negative for slow myosin and show striations typical of contractile sarcomeres.26 This cutaneous muscle has been reported to be a site of exceptionally rapid wound healing and angiogenesis27 and is believed to participate in thermoregulation. Furthermore, it displays a huge capacity of homing bone marrow-derived stem cells as compared with other muscles, suggesting its high regenerative capacity.26 Increased expression of a number of genes specific of fast type muscle and genes encoding enzymes involved in glycolysis suggests a strengthening of the ‘fast’ phenotype of the panniculus carnosus in microgravity.

Among those genes that are specifically upregulated in the skin of S mice, many encode transcription factors involved in myogenesis such as Myf6, myogenin, MEF2C, and MEF2D, and proteins involved in muscle contraction, neuromuscular junctions, and bioenergetics known or potential targets of MEF2 or myogenin. Together, these data suggest that microgravity increased the expression of a high number of genes in the panniculus carnosus in part through a MEF2C–myogenin pathway.

Muscle atrophy as reported in microgravity may result from both cell death and myofibrillar protein degradation. An increased expression of Trim63 and Fbxo32 genes, two striated muscle-specific proteins associated with the ubiquitin proteasome, and of Serpine1, a gene encoding PAI-1 was observed in a number of models of muscle atrophy, including microgravity and unweighing.28,29 The study performed by Sandona et al13 on the mice muscles in this Tissue Sharing Program showed an increased expression of Trim63 in the soleus, and of Trim63 and Fbxo32 in the extensor digitorum longus. However, muscle atrophy was detected only in the slow postural soleus. Here, we also recorded increased levels of Trim63, Fbxo32, and Serpine1 messenger RNA in the skin of S mice but the thickness of the panniculus carnosus did not seem to be affected by exposure to microgravity, suggesting that it was not atrophied. It is possible that non-postural muscles, such as the panniculus carnosus and extensor digitorum longus, are less sensitive to microgravity.

A recent publication30 showed that many oxidative stress/anti-oxidant defense genes as well as genes encoding ECM structural components were upregulated in the skin of mice flown for 13 days in a space shuttle. None of these reported genes were modulated in our experiment. These discrepancies most probably arise from the very different experimental design, notably in terms of duration of exposure to space environment. Our data may represent a long-term adaptative behavior to microgravity.

Altogether, we have shown that skin may be a target of space flight conditions that lead to dermal atrophy, deregulation of hair cycle, and modulation of the transcriptomic repertoire of the striated muscle panniculus carnosus in mice. This suggests that the skin of astronauts may be affected by pathophysiological alterations that could be detrimental during long trips in space.