Transcriptomic Validation of the Protective Effects of Aqueous Bark Extract of Terminalia arjuna (Roxb.) on Isoproterenol-Induced Cardiac Hypertrophy in Rats

Abstract

Aqueous extract of the bark of Terminalia arjuna (TA) is used by a large population in the Indian subcontinent for treating various cardiovascular conditions. Animal experiments have shown its anti-atherogenic, anti-hypertensive, and anti-inflammatory effects. It has several bioactive ingredients with hemodynamic, ROS scavenging, and anti-inflammatory properties. Earlier we have done limited proteomic and transcriptomic analysis to show its efficacy in ameliorating cardiac hypertrophy induced by isoproterenol (ISO) in rats. In the present study we have used high-throughput sequencing of the mRNA from control and treated rat heart to further establish its efficacy. ISO (5 mg/kg/day s.c.) was administered in male adult rats for 14 days to induce cardiac hypertrophy. Standardized aqueous extract TA bark extract was administered orally. Total RNA were isolated from control, ISO, ISO + TA, and TA treated rat hearts and subjected to high throughput sequence analysis. The modulations of the transcript levels were then subjected to bio-informatics analyses using established software. Treatment with ISO downregulated 1,129 genes and upregulated 204 others. Pre-treatment with the TA bark extracts markedly restored that expression pattern with only 97 genes upregulated and 85 genes downregulated. The TA alone group had only 88 upregulated and 26 downregulated genes. The overall profile of expression in ISO + TA and TA alone groups closely matched with the control group. The genes that were modulated included those involved in metabolism, activation of receptors and cell signaling, and cardiovascular and other diseases. Networks associated with those genes included those involved in angiogenesis, extracellular matrix organization, integrin binding, inflammation, drug metabolism, redox metabolism, oxidative phosphorylation, and organization of myofibril. Overlaying of the networks in ISO and ISO_TA group showed that those activated in ISO group were mostly absent in ISO_TA and TA group, suggesting a global effect of the TA extracts. This study for the first time reveals that TA partially or completely restores the gene regulatory network perturbed by ISO treatment in rat heart; signifying its efficacy in checking ISO-induced cardiac hypertrophy.

Keywords: Terminalia arjuna; biological network; cardiac hypertrophy; heart failure; transcriptomics.

Figure 1

(A) Scheme of data mining workflow for analyzing the transcriptome data. (B) The bar graph indicates the number of upregulated and downregulated gene in each condition. (C) Principal component analysis (PCA) of gene expression data sets from all three groups represented as two-dimensional plot of PC1 (Component 1) and PC2 (Component 2). (D) Venn Diagram representing overlap of differentially expressed Genes in ISO, ISO_TA, and TA groups. (Groups: ISO = ISO treated, ISO_TA = ISO + T. arjuna treated, TA, T. arjuna treated).

Figure 2

KEGG pathways significantly enriched after multiple testing adjustments (p < 0.1) (A) Bar graph indicates KEGG pathway upregulated and downregulated after ISO treatment. Pathways under the same group (carbohydrate metabolism, fatty acid metabolism, etc.) are clustered together and marked by the same color. The number of unique genes modulated in each group are shown in parenthesis next to the respective group names. The total number of genes modulated in various pathways under each group is generally larger than the total number of unique genes mentioned in parenthesis. It is due to the occurrence of same genes in multiple pathways and accordingly, those were counted more than once. The individual pathways given in the bar graph (A-O1) is tabulated in Table 1 . (B) Bar graph representing genes upregulated and downregulated by ISO + TA is shown. The total number of unique genes that were downregulated is 14. The total number of genes modulated in various pathways under each group is more than 14 as the same genes might be associated with multiple pathways, hence counted more than once. (C) Bar graph representing three different pathways involving 13 unique genes upregulated by TA alone is shown. The individual pathways given in the bar graphs in A and B is tabulated in Table 1 . (D) Heatmap of significantly regulated genes in the ISO group compared to ISO_TA group along with expression value. (E) Heatmap of significantly regulated genes in the ISO _TA group compared to ISO group along with expression value. The genes, which are not significantly regulated, are indicated as ns (non-significant). A detail of expression value of heatmap along with p-value is tabulated in Supplementary Table S5 .

Figure 3

Comparison of enriched non-redundant biological processes represented as “degree sorted circular view” networks of upregulated genes in ISO group extracted from “GeneMANIA” (Cytoscape plugins) along with ISO_TA and TA group overlaid network. Upregulated, downregulated, and undifferentiated gene represented as red node, green node, and gray node respectively.

Figure 4

Comparison of enriched non-redundant biological processes represented as “degree sorted circular view” networks of downregulated genes in ISO group extracted from "GeneMANIA" (Cytoscape plugins) along with ISO_TA and TA group overlaid network. Upregulated, downregulated, and undifferentiated gene represented as red node, green node, and gray node respectively.