Gene set enrichment analysis (GSEA)
To dissect genetic processes specific for each phase of atrophy-recovery process, we conducted analogous Gene Set Enrichment Analysis (GSEA) for DEGs unique in each phase (D1-3-7 and R1-3-7). (refer to DEGs on each phase and PANTHER results in SM). In agreement with DEGs data in each phase, we observed significant differences in GSEs for unique differentially expressed genes between slow and fast muscles. The “regulation of adaptive immune response” and “related T cell immunity” were the only enriched terms in the last disuse phase (day 7 of unloading experiment) in slow muscle, while the regulation of adaptive immune response and ATP, nucleotides metabolic processes were presented in the enrichment set at days 1 and 3 of the disuse in slow muscle, correspondingly. GSEs of the unique DEGs for recovery were distinct between slow and fast muscles too. Thus, in R1, the unique signatures in fast muscle were enriched with cell proliferation terms (See ST4), while the unique signatures in slow muscle were enriched with terms related to metabolism (See ST5). Notably, gene set enrichments in the slow muscle are much widely represented in each recovery phase and demonstrate consistent functional pattern during recovery in this type of the skeletal muscle: involvement of signaling (via G-protein-coupled receptor and phosphorylation cascades of intermediate and target proteins) and metabolic processes (cellular respiration, nucleotides biosynthesis, actin filament organization) in early phase R1, follow-up activation of translational complex machinery in R3 and cell development and differentiation in R7 phase.
Unlike fast muscle, differentially expressed genes in slow muscle, which were common to phase-control and time-course comparisons, formed four clusters, the two largest of which were enriched with features of metabolism regulation through MAP kinase activity and actin biosynthesis, other two belonged to nucleosome assembly coordinated with muscle contraction and metabolism (See ST6). According to the heatmap, MAP kinase regulation activated only on the first day of recovery (R1 phase) and actin biosynthesis lasted during the whole recovery period, being the most expressed in R1. Notably, members of nucleosome assembly pathways were active only in disuse and metabolism features were downregulated in the initial stages of recovery (Figure 2A).
Figure 2A. Clusterization of differentially expressed peaks (both genic and intergenic) common to both phase-control and time-course comparisons. FDR threshold 5e-04. Clusters of the metabolism regulation through MAP kinase activity, actin biosynthesis, nucleosome assembly, and lipid metabolism are shown in black, green, blue, and red, respectively (See SF).
To take a wider look on the changes specific for atrophy-associated changes, we further performed the similar analysis for each phase of the experiment as compared to control animals. In the slow muscle, most of the enriched terms were related to parent classes of muscle tissue remodelling and metabolism. Expectantly, enriched features of actin cytoskeleton (sarcomere) reorganization were up-regulated in recovery phases (R1 - R7), and down-regulated mostly in disuse phases (D1 - D7). Notably, a vast number of sarcomere features were downregulated in the early stages of recovery [2]. Surprisingly, downregulated signatures of R1 and R3 phases were enriched with terms of muscle cell differentiation and energy derivation (except for receptor signalling pathways), that is likely to be interpreted as a signature of strong impact of stress associated with atrophic process on cell homeostasis.
