Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight

Abstract

Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLab^TM payloads on the ISS.

Fig. 1. Experimental design, implementation, and RNA analysis of the skeletal muscle MPS payload for the international space station.

A Human cell banks. Skeletal muscle biopsies were obtained from the vastus lateralis from volunteers (AdventHealth, Orlando). Isolated muscle precursor cultures were enriched for CD56+ (myogenic) cells. B Experimental flight timeline. Photos are numbered in the order of the process. 1. Tissue chips were seeded with myoblasts, 2. pre-differentiated using a syringe pump, 3. loaded into the CubeLab™ and 4. launched to the ISS on SpaceX CRS-21. Crew members installed the PAUL on the EXPRESS rack locker and the on-orbit experiment was initiated after plug-in. 5. Images of the myobundles were captured on orbit by the optics system installed into the lid of the CubeLab^TM. Scalebar = 500 μm. 6. The Optics system automatically moved in the x,y,z axes to collect images. 7. Ten days post launch, crew members moved the payload to cold stowage following experiment termination with RNALater. C RNA-Seq volcano plots. YA flight vs ground; OS flight vs ground; Ground OS vs YA and Flight OS vs YA. Colored points are differentially expressed between comparisons. Blue: significantly down-regulated genes. Red: significantly up-regulated genes. Significance is determined according to log2 fold change with threshold set at ±2 and -log10 p-value ≤ 0.05. Vertical dotted lines are positioned at log2 fold-change of ±2 and horizontal dotted lines are positioned at the -log10 p-value = 0.05. RNA-Seq analysis measured three distinct chips each containing one myobundle derived from five male donor cells pooled in equal ratios.

Fig. 2. Transcriptional responses of muscle myobundles during flight and ground culturing conditions.

A Comparative analysis of differential gene expression changes between flight and ground controls of genes involved in muscle myogenesis and contraction YA (orange bars) and OS (blue bars). B Panther classification system of the 59 shared genes shown in the Venn diagram for overlapping and distinct DEGs among Flight YA vs GC and Flight OS vs GC datasets. The horizontal bar chart identifies the four protein classes and relative number of DEGs of each functional class by applying a cut-off threshold of 10 genes. C Significantly enriched KEGG pathways in response to flight either in YA- or OS-derived myobundles relative to their respective ground controls. Significantly affected “hallmark” gene sets from the Molecular Signature Database for (D). Ground OS vs YA and (E). Flight OS vs YA (MSigDB). C, D, E The box and whisker plots on the left summarize the distribution of all the differentially expressed genes in the two pathways. The box represents the 25–75 quartile with a median line, the whiskers show the top and bottom 25% of the data, while the outliers are represented by doted lines. RNA-Seq analysis measured distinct three individual chips each containing one myobundle derived from 5 male donor cells pooled in equal ratios.