Effects of spaceflight on murine skeletal muscle gene expression

Abstract

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.

Fig. 1.

mRNAs identified as differentially expressed in spaceflight samples and in response to ground-based unloading models. A: expression patterns of the 272 mRNAs differentially expressed in mouse gastrocnemius in response to spaceflight. Hierarchical clustering was performed for the 272 mRNAs affected by spaceflight using expression data for spaceflight (SF) and the animal enclosure module (AEM) controls as well as for hindlimb suspension (Sus), hindlimb suspension plus reloading (Sus+Rel), and control (Con). The bracket at right highlights mRNAs that generally indicate a pattern in which hindlimb suspension mimics the effects of spaceflight and for which reloading alleviates the effect of hindlimb suspension. B and C: relative expression patterns of mRNAs regulated by spaceflight and by suspension in a consistent manner. B shows expression patterns for 113 mRNAs upregulated by spaceflight and by suspension; and C shows expression patterns for 92 mRNAs downregulated by spaceflight and by suspension. D: Venn diagram schematically depicting the numbers and associations of mRNAs found to be significantly affected by suspension, reloading, suspension plus reloading, and/or spaceflight stimuli.

Fig. 2.

Quantitative RT-PCR analysis of myostatin, ActRIIb, and FSTL3 in ground control (AEM) and SF gastrocnemius muscle. Bars represent means ± SE for n = 4 mice per group. All values were normalized to a β-actin duplexed internal normalizing control. A: myostatin mRNA levels. B: ActRIIb mRNA levels. C: FSTL3 mRNA levels. Myostatin increased and ActRIIb and FSTL3 mRNA levels decreased in SF gastrocnemius, but only the changes in ActRIIb mRNA levels were statistically significant. *P < 0.05 compared with AEM control.

Fig. 3.

TaqMan analysis of microRNA (miRNA) expression in ground control (AEM) and SF gastrocnemius muscle for miR-1 (A), miR-133a (B), miR-206 (C), and the ratio of miR-1 to miR-133a (D). Total RNA was subjected to reverse transcription using gene-specific primers miR-1, miR-133a, miR-206, and small nuclear RNA (snRNA) U6 and then quantified using quantitative real-time PCR. Fold change was determined using the ΔΔCt relative quantitation method with snRNA U6 as the endogenous control gene. *P < 0.05 compared with AEM control.