Relaxation response induces temporal transcriptome changes in energy metabolism, insulin secretion and inflammatory pathways

Abstract

The relaxation response (RR) is the counterpart of the stress response. Millennia-old practices evoking the RR include meditation, yoga and repetitive prayer. Although RR elicitation is an effective therapeutic intervention that counteracts the adverse clinical effects of stress in disorders including hypertension, anxiety, insomnia and aging, the underlying molecular mechanisms that explain these clinical benefits remain undetermined. To assess rapid time-dependent (temporal) genomic changes during one session of RR practice among healthy practitioners with years of RR practice and also in novices before and after 8 weeks of RR training, we measured the transcriptome in peripheral blood prior to, immediately after, and 15 minutes after listening to an RR-eliciting or a health education CD. Both short-term and long-term practitioners evoked significant temporal gene expression changes with greater significance in the latter as compared to novices. RR practice enhanced expression of genes associated with energy metabolism, mitochondrial function, insulin secretion and telomere maintenance, and reduced expression of genes linked to inflammatory response and stress-related pathways. Interactive network analyses of RR-affected pathways identified mitochondrial ATP synthase and insulin (INS) as top upregulated critical molecules (focus hubs) and NF-κB pathway genes as top downregulated focus hubs. Our results for the first time indicate that RR elicitation, particularly after long-term practice, may evoke its downstream health benefits by improving mitochondrial energy production and utilization and thus promoting mitochondrial resiliency through upregulation of ATPase and insulin function. Mitochondrial resiliency might also be promoted by RR-induced downregulation of NF-κB-associated upstream and downstream targets that mitigates stress.

Figure 1. Individual gene-based differential expression analysis.

A) Differentially expressed genes identified by 3 across-group comparisons (N1 vs. N2, N1 vs. M, and N2 vs. M) at T0, T1 and T2. Venn diagrams depict the overlap of genes identified by these 3 comparisons at each time point. B) Heat map of genes that were significantly differentially expressed comparing N1 vs. N2 and N1 vs. M at T1 and T2 (marked with arrow in Venn diagrams). Gene expression is shown with a pseudocolor scale (−1 to 1) with red color denoting increased and green color denoting decreased fold change in gene expression. The rows represent the genes and columns represent subjects in N1, N2 and M groups at T0, T1 and T2. C) Differentially expressed genes identified by 3 within-group comparisons at different time points (T0 vs. T1, T0 vs. T2 and T1 vs. T2). Venn diagrams depict the overlap of genes identified by the 3 comparisons within each group.

Figure 2. Temporal genomic expression patterns during one session of RR elicitation.

Genes that were differentially expressed either across or within groups comparisons at different time points were used as the seed set of genes for Self-Organizing Map (SOM) analysis. These differentially expressed genes were partitioned to 18 separate maps according to Pearson correlation coefficient based distance metrics (Figure S2). Selected biologically interesting SOM maps were manually clustered into 4 biologically relevant categories based on the gene expression of N1, N2 and M groups at the 3 time points in one session of RR elicitation: Long-term Downregulation; Long-term Upregulation; Progressive Upregulation; and Progressive Downregulation. One representative pattern for each of these 4 biologically relevant categories is shown in the figure. The figure displays the box plot of the gene expression with X-axis representing time points and groups, and Y-axis representing scaled gene expression data from −1 to +1.

Figure 3. Significantly enriched pathways with progressive patterns identified using gene set enrichment analysis.

A) Upregulated Pathways B) Downregulated Pathways. The solid dots indicate significantly affected pathways (False Discovery Rate <25%) identified from across group comparisons (N1 vs. N2, N1 vs. M and N2 vs. M) at a particular time point (T0, T1 and T2). The asterisks represent significance and directionality of enrichment (P value<0.09 *, P value<0.05 **, P value<0.01 ***) identified from within group comparisons at different time points (T0 vs. T1, T0 vs. T2, T1 vs. T2). The red and green color asterisks indicate up- and down-regulated enrichment of pathways respectively. The heatmaps depicting relative expression of selected genes from representative pathways are shown in panels on the right side. Gene expression is shown with a pseudo color scale (−3 to 3) with red and green colors denoting increased and decreased relative expression respectively. Pathways with progressive patterns were enriched (up- or down- regulated) in N2 and M groups with greater significance of enrichments in M group. Furthermore, increasing enrichment over time within one session of RR elicitation was observed in M group.

Figure 4. Significantly enriched pathways with long-term patterns identified using gene set enrichment analysis.

A) Upregulated Pathways B) Downregulated Pathways. The solid dots indicate significantly affected pathways (False Discovery Rate <25%) identified from across group comparisons (N1 vs. N2, N1 vs. M and N2 vs. M) at a particular time point (T0, T1 and T2). The asterisks represent significance and directionality of enrichment (P value<0.09 *, P value<0.05 **, P value<0.01 ***) identified from within group comparisons at different time points (T0 vs. T1, T0 vs. T2, T1 vs. T2). The red and green color asterisks indicate up- and down-regulated enrichment of pathways respectively. The heatmaps depicting relative expression of selected genes from representative pathways are shown in panels on the right side. Gene expression is shown with a pseudo color scale (−3 to 3) with red and green colors denoting increased and decreased relative expression respectively. Pathways with long-term patterns were enriched (up- or down- regulated) only in M group. Furthermore, increasing enrichment over time within one session of RR elicitation was observed in M group.

Figure 5. Interactive network and top focus gene hubs identified from significantly affected pathways.

The figure represents the top focus genes. A) Progressive upregulated Pathways, B) Progressive downregulated Pathways, and C) Integrated network of Long-term and Progressive affected pathways. The top focus hubs were identified from complex interactive networks generated from pathways with progressive and long-term patterns. The focus gene hubs were identified using the bottleneck algorithm for identification of the most interactive molecules with tree like topological structure. The bottleneck algorithm ranks genes on the basis of significance level with smaller rank indicating increasing confidence. The pseudocolor scale from red to green represents the bottleneck ranks from 1 to 20.