RNA sequencing (RNA-seq) analysis of gene expression provides new insights into hindlimb unloading-induced skeletal muscle atrophy

Abstract

Background: Weightlessness-induced skeletal muscle atrophy, accompanied by complex biochemical and physiological changes, has potentially damaged consequences. However, there is still an insufficient effective strategy to treat skeletal muscle atrophy. Therefore, exploring the molecular mechanisms regulating skeletal muscle atrophy and effective protection is necessary.

Methods: RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in the soleus muscle at 12, 24, 36 hours, three days, and seven days after hindlimb unloading in rats. Pearson correlation heatmaps and principal component analysis (PCA) were applied to analyze DEGs' expression profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for cluster analysis of DEGs. Ingenuity pathway analysis (IPA) was used to analyze specific biological processes further.

Results: At different time points (12, 24, 36 hours, three days, seven days) after hindlimb unloading, the expression levels of 712, 1,109, 1,433, 1,162, and 1,182 genes in rat soleus muscle were upregulated, respectively, whereas the expression levels of 1,186, 1,324, 1,632, 1,446, and 1,596 genes were downregulated, respectively. PCA revealed that rat soleus muscle showed three different transcriptional phases within seven days after hindlimb unloading. KEGG and GO annotation indicated that the first transcriptional phase primarily involved the activation of stress responses, including oxidative stress, and the inhibition of cell proliferation and angiogenesis; the second transcriptional phase primarily involved the activation of proteolytic systems and, to a certain degree, inflammatory responses; and the third transcriptional phase primarily involved extensive activation of the proteolytic system, significant inhibition of energy metabolism, and activation of the aging process and slow-to-fast muscle conversion.

Conclusions: Different physiological processes in rat skeletal muscles were activated sequentially after unloading. From these activated biological processes, the three transcriptional phases after skeletal muscle unloading can be successively defined as the stress response phase, the atrophic initiation phase, and the atrophic phase. Our study not only helps in the understanding of the molecular mechanisms underlying weightlessness-induced muscle atrophy but may also provide an important time window for the treatment and prevention of weightlessness-induced muscle atrophy.

Keywords: RNA sequencing (RNA-seq); Skeletal muscle atrophy; bioinformatics analysis; gene expression; hindlimb unloading.

Figure 1

Three distinct transcriptional phases during hindlimb unloading-induced muscle atrophy. (A) The number of DEGs at different times in SOL muscle post-hindlimb unloading. (B) Pearson correlation heatmaps for different times post-hindlimb unloading, as labeled on lower and right margins. The arrows show two nodal transitions. (C) PCA of DEGs at different times post-hindlimb unloading. The percentage of variance explained by PCs is shown. DEGs, differentially expressed genes; SOL, soleus; PCA, principal component analysis; PCs, principal components.

Figure 2

GO biological process analysis of upregulated genes in SOL muscle after hindlimb unloading. (A) The enriched biological process at different times post-hindlimb unloading. The circle’s size and color show the number and −log10 (P value), respectively, of DEGs, annotated in the specific GO biological process. (B) The average expression profiles for DEGs involved in major biological processes. The y-axis represents the average score of genes in the specific biological process terms. GO, Gene Ontology; SOL, soleus; DEGs, differentially expressed genes.

Figure 3

KEGG pathway analysis of upregulated genes in SOL muscle after hindlimb unloading. (A) The enriched pathways at different times post-hindlimb unloading. The circle’s size and color show the number and −log10 (P value), respectively, of DEGs, annotated in the specific KEGG pathway. (B) The average expression profiles for DEGs involved in major pathways. The y-axis represents the average score of genes in the specific KEGG terms. KEGG, Kyoto Encyclopedia of Genes and Genomes; SOL, soleus; DEGs, differentially expressed genes.

Figure 4

GO biological process analysis of downregulated genes in SOL muscle after hindlimb unloading. (A) The enriched biological process at different times post-hindlimb unloading. The circle’s size and color show the number and −log10 (P value), respectively, of DEGs, annotated in the specific GO biological processes. (B) The average expression profiles for DEGs involved in major biological processes. The y-axis represents the average score of genes in the specific biological process terms. GO, Gene Ontology; SOL, soleus; DEGs, differentially expressed genes.

Figure 5

KEGG pathway analysis of downregulated genes in SOL muscle after hindlimb unloading. (A) The enriched pathways at different times post-hindlimb unloading. The circle’s size and color show the number and −log10 (P value), respectively, of DEGs, annotated in the specific KEGG pathway. (B) The average expression profiles for DEGs involved in major pathways. The y-axis represents the average score of genes in the specific KEGG terms. KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes; SOL, soleus.

Figure 6

Dynamic changes in DEGs associated with aging, angiogenesis, the cell cycle, immune responses, and oxidative stress. Upregulation (red) and downregulation (green) of genes at different time points post-hindlimb unloading; color intensity shows the relative fold-change value. DEGs, differentially expressed genes.

Figure 7

This schematic diagram illustrates a sequence of pathological processes in hindlimb unloading-induced skeletal muscle atrophy. Hindlimb unloading leads to ischemia and hypoxia of SOL muscle, contributing to muscle cell stress. If stress is not controlled, downstream proteolysis and inflammation processes will be activated, contributing to muscle atrophy. This occurs during the atrophic initiation phase and the atrophic phase. SOL, soleus.