Transgenic Expression of Nrf2 Induces a Pro-Reductive Stress and Adaptive Cardiac Remodeling in the Mouse

Abstract

Nuclear factor, erythroid 2 like 2 (Nfe2l2 or Nrf2), is a transcription factor that protects cells by maintaining a homeostatic redox state during stress. The constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling. Transgenic and non-transgenic (NTG) mice at 7−8 months of age were used to analyze the myocardial transcriptome, structure, and function. Next generation sequencing (NGS) for RNA profiling and qPCR-based validation of the NGS data, myocardial redox levels, and imaging (echocardiography) were performed. Transcriptomic analysis revealed that out of 14,665 identified mRNAs, 680 were differently expressed (DEG) in TG hearts. Of 680 DEGs, 429 were upregulated and 251 were downregulated significantly (FC > 2.0, p < 0.05). Gene set enrichment analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of the glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 µM/mg protein in mNrf2-TG and CaNrf2-TG vs. NTG). Genes involved in fetal development, hypertrophy, cytoskeletal rearrangement, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG. Non-invasive echocardiography analysis revealed an increase in systolic function (ejection fraction) in mNrf2-TG, suggesting an adaptation, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks). The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e., pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.

Keywords: Nrf2 transgene; RNAseq; constitutively active Nrf2; echocardiography; reductive stress.

Figure 1

Heart specific Nrf2-Transgenic mouse model and transcriptomic changes. (A) Genotyping PCR for mNrf2-TG using αMHC forward and Nrf2 reverse primer (350 bp), (below) FABP (internal control) showed bands at 250 bp. (B) Semi-quantitative PCR for Nrf2 using primer for endogenous Nrf2 showing increased Nrf2 expression in Nrf2-TG compared to NTG mice; acidic ribosomal phosphoprotein (Arbp1/Rplp0) was used as housekeeping. (C) Quantification of semi-quantitative PCR for Nrf2 using image. (D) Three-dimensional principal component analysis for mNrf2-TG and NTG mice. (E) Heatmap representing expression of the 680 most significant (p < 0.05) differentially expressed genes (Log2FC > 1). Some 428 genes were upregulated, and 251 genes were downregulated in mNrf2-TG mice. The heatmap represents normalized count values (RLog) of each DEG in the individual samples, and genes were clustered unsupervised. Fold change > 2 and p-value < 0.05. (F) Volcano plot of DEGs showing log2fold change versus −log10 of (p-value) of transcripts identified by RNASeq analysis between NTG and mNrf2-TG. Significant DEGs expressed in mNrf2-TG (Log2FC ≥ 2 and p < 0.05) are highlighted in red (FDR corrected p-value). Blue dots indicate transcripts with a significant p-value but Log2FC ≤ 2; Grey dots indicate transcripts with a Log2FC ≥ 2 but with an FDR corrected p-value of <0.05; whilst green dots indicate non-significant transcripts with a Log2FC ≤ 2. The total number of genes amounts to 14,665. Error bars represent SEM. ** p-value < 0.01.

Figure 2

Antioxidant transcriptome in enhanced cardiac Nrf2 signalling. (A) GSEA plot of most enriched Nrf2 pathway from Wikipathways. GSEA algorithm calculates an enrichment score for a pathway by walking down the ranked list of genes, increasing a running-sum statistic when a gene is in the gene set and decreasing it when it is not. The magnitude of the increment depends on the correlation of the gene with the phenotype. The ES is the maximum deviation from zero encountered in walking the list. A positive ES indicates gene set enrichment at the top of the ranked list; a negative ES indicates gene set enrichment at the bottom of the ranked list. (B) Heatmap illustrating changes in 103 genes enriched in the Nrf2 pathway. (C) RT-qPCR validation of gene expression of Gstm1, Gsta3, Gstt2 and Gstm3, Nrf2, Txnrd1, Sod3 and Tgfbr2. All RT-qPCR gene expression were normalized to Gapdh. Error bars represent SEM. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Figure 3

Gene expression mapped on the Nrf2 pathway (Wikipathways ID = 4357), cardiac structure and function. (A) Binding with Keap1/Cul3/RBX1 complex allows Nrf2 protein to be continually degraded via ubiquitination/proteosomal degradation. In response to ROS, Keap1 is oxidized or Nrf2 is phosphorylated resulting in translocation to nucleus. Pathway diagram shows changes in mNrf2-TG ARE targets when activated. Colour gradient scaled from blue (negative fold change) to red (positive fold change) was calculated using Log2foldchange, scaled and mapped onto curated network. Nodes (rectangle) represent genes or proteins and edges (or lines) represent interactions and relationships between connected nodes. (B) Validation of protein expression using Western blot, confirming the summary of changes in network. (C) Cardiac structure and function in mNrf2-TG mice. Two-dimensional non-invasive echocardiography (PSLAX axis) was used to determine systolic functions (i.e., ejection fraction and cardiac output), Left ventricle dimensions (i.e., LV mass, intraventricular septum diameter-systole and diastole) and diastolic function, i.e., mitral valve (early, E to late, A ratio) filling (n = 6–8/group).

Figure 4

Survival, redox state, cardiac function, and remodelling in mNrf2 and CaNrf2 mice. (A) Kaplan–Meier survival analysis expressed in terms of percentage survival. (B) Redox scores of mNrf2-TG and CaNrf2 mice compared to controls, mapped on a gradient scale. (C) Redox state and cardiac function of NTG (orange) and mNrf2-TG (green) and CaNrf2 (red) hearts expressed in terms of ejection fraction (%) versus mean redox state (GSH/GSSG). EF and GSH data were paired within experimental groups. (D) Expression of Nogo A and Nogo B detected using Western blot from heart lysates. Membrane was stripped and reprobed for Gapdh. (E) RNAseq mRNA expression heatmap of common cellular hypertrophy indicators in normal, pro-reductive and hyper-reductive hearts. Log normalized data of genes scaled using R scale function shows distinctive changes between control, mNrf2-TG and CaNrf2. Column break for 4 markers (genes) that are downregulated in transgenic models together. Row breaks for groups with n = 3/4. (F) qPCR quantification of mRNA expression for cellular hypertrophy markers normalized to Gapdh. Error bars represent SEM.* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Figure 5

Enhanced Nrf2-Keap1 signalling generates excess Nrf2 at baseline without affecting keap-1 interaction, results in moderate antioxidant upregulation leading to a pro-reductive transcriptome. This leads to an adaptive cardiac remodelling without affecting survival in mNrf2-TG mice. Whereas, affecting the Nrf2-Keap1 signalling renders Nrf2 to be constitutively active. This results in high upregulation of antioxidants that consequentially affect cardiac structure and function resulting in increased mortality rate at 6 months in mice.