Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans

Akira Higashibata, Toko Hashizume, Kanako Nemoto, Nahoko Higashitani, Timothy Etheridge, Chihiro Mori, Shunsuke Harada, Tomoko Sugimoto, Nathaniel J Szewczyk, Shoji A Baba, Yoshihiro Mogami, Keiji Fukui, Atsushi Higashitani, Akira Higashibata, Toko Hashizume, Kanako Nemoto, Nahoko Higashitani, Timothy Etheridge, Chihiro Mori, Shunsuke Harada, Tomoko Sugimoto, Nathaniel J Szewczyk, Shoji A Baba, Yoshihiro Mogami, Keiji Fukui, Atsushi Higashitani

Abstract

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Quantitative analysis of protein (iTRAQ) and gene expression (microarray) levels in space-flown worms. The levels of certain muscular, cytoskeletal, and mitochondrial proteins and an aspartic protease (ASP-1) were significantly altered in worms cultured under microgravity conditions. *P⩽0.05, **P⩽0.01, Student’s t-test for analyses (details are shown in Supplementary Tables S1 and S2).
Figure 2
Figure 2
Altered gene expression of the sirtuin gene transcript (sir-2.1) and downstream target gene transcripts assessed by quantitative real-time PCR. The expression of sir-2.1 increases in the worms grown in microgravity and SIR-2.1 downstream target genes, abu-6, abu-7, and pqn-5, decrease as expected. Signals were normalized to act-4, which encodes an actin isoform, as a housekeeping gene. **P⩽0.01, Student’s t-test.
Figure 3
Figure 3
Alteration of body length of space-flown C. elegans. (a) Microscopic image of worms grown on the 1 G centrifuge for 4 days. (b) Microscopic image of worms grown in microgravity for 4 days. The worms in both conditions had normal levels of eggs in their bodies. (c) The mean of body length of the worms grown at 1 G was 1.45±0.09 mm, and for microgravity was 1.37±0.05 mm, respectively (n=15, means±s.d.). **P⩽0.01, Welch’s t-test. Scale bars indicate 0.5 mm.
Figure 4
Figure 4
Altered fat storage in microgravity-cultured C. elegans. (a) Images of Sudan Black staining of accumulated fat in C. elegans. Worms grown onboard the 1 G centrifuge (upper image) and in microgravity (lower image) are displayed. Scale bars indicate 100 μm. There was less fat accumulation in microgravity-cultured worms compared with 1 G controls. (b) Density measurements of Sudan Black stained worms (as described in Methods). The mean densities were 135.36±28.12 in microgravity-cultured worms and 176.55±27.61 in 1-G cultured worms (means±s.d.). *P⩽0.05, Student’s t-test. (c) Alteration of fat-related gene expression determined by microarray analysis. P-values as indicated in the panel, Student’s t-test.
Figure 5
Figure 5
Schematic of the TCA cycle and the expression changes under microgravity. The enzymes indicated by blue symbols exhibited lower gene and/or protein expression in space-flown worms. Almost all enzymes (citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase) participated in the TCA cycle showed lower expression.

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