Abstract
Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) senses oxidative environments and/or stress and initiates a cytoprotective response through transcriptional activation of antioxidant and detoxification genes. Several preclinical studies suggest that Nrf2 combats oxidative stress underlying a variety of pathologies. Despite Nrf2 deficits linked to functional abnormalities in many organ systems, the transcriptional network resulting from Nrf2 deficiency in the heart has remained elusive. Moreover, cross-talk between microRNAs (miRNAs) and cardiac Nrf2 signaling is unknown. Here, we utilized next generation RNA sequencing (RNAseq) to unbiasedly profile basal mRNA and miRNA expression in Nrf2 knockout (Nrf2−/−) hearts. RNAseq of mRNA revealed 152 differentially expressed genes (DEGs) in the Nrf2−/−myocardium, of which 129 were downregulated. Grouping of DEGs based on biological function and real-time qPCR validation indicated that DEGs were enriched for; mitochondrial genome and bioenergetics, oxidoreductase capacity, cardiac development, and chaperone activity. Interestingly, RNAseq analysis uncovered 27 significantly altered miRNAs, of which 11 were upregulated and 16 were downregulated in Nrf2−/− hearts. Expression changes were validated for 12 miRNAs using specific primer assays in real-time and revealed a significant decrease in miR-10b-5p, miR-674-3p, miR-3535, and miR-378c while miR-30b-5p, miR-208a-5p, miR-350-3p, and miR-582-5p, and miR-1249-3p levels were increased. High throughput data were integrated using prediction algorithms, and these in silico analyses discovered potential recognition elements within 39 repressed mRNAs which matched the seed sequence for 4 upregulated miRNAs; miR-30b-5p, miR-208a-5p, miR-350-3p, and miR-582-5p. These high-throughput data reveal transcriptome-wide effects of myocardial Nrf2 deficiency. Further, our results suggest that Nrf2 may directly or indirectly regulate a sub-set of cardiac miRNAs in the basal setting. This comprehensive analysis is the first evidence to demonstrate a plausible regulatory cross-talk among cardiac miRNAs and the Nrf2 transcriptional network, and provides valuable candidates to examine in future mechanistic and preclinical studies.
Highlights
Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) senses oxidative environments and/or stress and initiates a cytoprotective response through transcriptional activation of antioxidant and detoxification genes
Our findings indicate that (i) loss of Nrf2 in the heart results in a modest downregulation of transcripts involved in mitochondrial function, redox homeostasis, metabolism, cardiac pathology, and protein folding, (ii) 27 miRNAs (11 up and 16 downregulated) are significantly altered and differentially regulated in the Nrf2 depleted myocardium, (iii) Nrf2 may either directly or indirectly regulate a sub-set of cardiac miRNAs, and (iv) miR-582-5p, miR-208a-5p, miR-350-3p and miR-30b-5p are likely to contribute to basally downregulated genes in Nrf2−/− hearts
Experimental proof for direct messenger RNA (mRNA) targeting is outside the scope of this study, our results have laid the groundwork for future investigations into the molecular mechanisms of Nrf2 pathway sensitive miRNAs in the heart
Summary
Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) senses oxidative environments and/or stress and initiates a cytoprotective response through transcriptional activation of antioxidant and detoxification genes. Nearly 2 decades of research has delineated beneficial roles for Nrf mediated transcriptional programs in various oxidative stress-related disease models, such as chronic obstructive pulmonary disease (COPD), neurodegeneration, inflammation, carcinogenesis, and pathogenesis associated with environmental toxicant exposure [2,3,4,5,6]. Along these lines, numerous investigations have clearly demonstrated pathological susceptibility of the genetically Nrf ablated (Nrf2−/−) mouse to exogenous oxidative challenges [1]. While basal myocardial antioxidant expression and ROS are comparable between young Nrf2−/− and age matched wild-type (WT) counterparts, knockout hearts are unable to cope with the oxidative burden induced by high intensity exercise and aged Nrf2−/− mice exposed to such stress exhibit pathological atrial hypertrophy [7, 8]
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