Identification of Nrf2-responsive microRNA networks as putative mediators of myocardial reductive stress

www.nature.com/scientificreports OPEN Identification of Nrf2‑responsive microRNA networks as putative mediators of myocardial reductive stress Justin M. Quiles1,8, Mark E. Pepin1,2,8, Sini Sunny1, Sandeep B. Shelar1, Anil K. Challa1, Brian Dalley3, John R. Hoidal4,5, Steven M. Pogwizd6, Adam R. Wende1 & Namakkal S. Rajasekaran1,2,4,5,7* Although recent advances in the treatment of acute coronary heart disease have reduced mortality rates, few therapeutic strategies exist to mitigate the progressive loss of cardiac function that manifests as heart failure. Nuclear factor, erythroid 2 like 2 (Nfe2l2, Nrf2) is a transcriptional regulator that is known to confer transient myocardial cytoprotection following acute ischemic insult; however, its sustained activation paradoxically causes a reductive environment characterized by excessive antioxidant activity. We previously identified a subset of 16 microRNAs (miRNA) significantly diminished in Nrf2-ablated (Nrf2−/−) mouse hearts, leading to the hypothesis that increasing levels of Nrf2 activation augments miRNA induction and post-transcriptional dysregulation. Here, we report the identification of distinct miRNA signatures (i.e. “reductomiRs”) associated with Nrf2 overexpression in a cardiac-specific and constitutively active Nrf2 transgenic (caNrf2-Tg) mice expressing low (TgL) and high (TgH) levels. We also found several Nrf2 dose-responsive miRNAs harboring proximal antioxidant response elements (AREs), implicating these “reductomiRs” as putative meditators of Nrf2-dependent post-transcriptional regulation. Analysis of mRNA-sequencing identified a complex network of miRNAs and effector mRNAs encoding known pathological hallmarks of cardiac stress-response. Altogether, these data support Nrf2 as a putative regulator of cardiac miRNA expression and provide novel candidates for future mechanistic investigation to understand the relationship between myocardial reductive stress and cardiac pathophysiology. Abbreviations Nrf2 Nuclear factor, erythroid 2 like 2 CaNrf2-Tg Constitutively active Nrf2 transgenic TgL Low-expressing caNrf2 mouse line TgH High-expressing caNrf2 mouse line ARE Antioxidant response element DEG Differentially-expressed genes DEmiR Differentially-expressed microRNA GSEA Gene set enrichment analysis During ischemia–reperfusion injury, generation of reactive oxygen and nitrogen species results in oxidative stress, which in turn, perturbs cardiac structure and function through calcium mishandling, inflammatory signaling 1 Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, AL, USA. 2Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL, USA. 3Huntsman Cancer Center‑Genomic Core Facility, University of Utah, Salt Lake City, UT, USA. 4Division of Cardiovascular Medicine, Department of Medicine, University of Utah, Salt Lake City, UT, USA. 5Division of Pulmonary Medicine, Department of Medicine, University of Utah, Salt Lake City, UT, USA. 6Comprehensive Cardiovascular Center, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA. 7Division of Molecular and Cellular Pathology, Department of Pathology, Center for Free Radical Biology, The University of Alabama at Birmingham, BMR2 Room 533, 901 19th Street South, Birmingham, AL 35294‑2180, USA. 8These authors contributed equally: Justin M. Quiles and Mark E. Pepin. *email: Scientific Reports | (2021) 11:11977 | https://doi.org/10.1038/s41598-021-90583-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ and extracellular matrix degradation1–3. However, the findings from clinical studies have largely discredited the efficacy of antioxidant therapies4,5. Furthermore, our laboratory has identified the presence of a “reductive stress” wherein aberrant induction of antioxidant response element (ARE)-dependent antioxidant genes produces pathological cardiac hypertrophy and d ysfunction6–8. While the deleterious consequences of reductive 9,10 stress appear highly-conserved , the transcriptional and post-transcriptional mechanisms of the myocardial redox milieu remain unknown. Among the mechanisms known to regulate postnatal heart function, microRNAs (miRNAs) are a class of short (~ 22 nucleotide) RNAs that post-transcriptionally regulate mRNA stability and translational efficiency, often in a tissue-specific m anner11. While miRNAs are necessary for physiologic cardiac function and d evelopment12,13, many have been found to be dysregulated in the failing heart14–16. Specifically, miRNAs have been directly linked to the structural and functional deficits in cardiac function17. Nevertheless, it remains unclear whether ARElinked miRNAs contribute to cardiac pathogenesis. As a transcriptional activator of cis-regulatory AREs18, nuclear factor erythroid 2-related factor 2 (Nfe2l2, a.k.a. Nrf2) plays a critical role in regulating cardiac redox status. Transient Nrf2 signaling is cardioprotective immediately following ischemic insult19, but chronic transactivation of AREs causes reductive stress and cardiac dysfunction20,21. We have recently shown that Nrf2 deficiency (Nrf2−/−) inhibits the expression of several miRNAs in the h eart22, but the genome-wide impact of reductive stress on miRNA expression remains unknown. In this investigation, we identify a miRNA signature for reductive stress to gain insight into potential biomarkers and/or effectors of this novel pathological phenomenon in the heart. The cardiomyocyte-specific and constitutively-active Nrf2 transgenic mouse model (CaNrf2-Tg) was used to conduct a multi-omics analysis of Nrf2-dependent and ARE-bearing miRNAs, which we term “reductomiRs”. Our use of both mRNA-seq and small RNA sequencing (miRNA-seq) in caNrf2 low (TgL) and high-expressing (TgH) mouse lines reveals a distinct signature of transgenic Nrf2 dose-responsive miRNAs linked to a number of suppressed cardiac genes under pro-reductive and reductive stress conditions23. Collectively, this analysis uncovers several novel miRNA candidates for which future mechanistic studies will investigate the interplay between post-transcriptional regulatory responses and redox state in the myocardium of Nrf2-Tg mice. Methods Animals. Our method for establishing the cardiac-specific constitutively active Nrf2 transgenic mouse model (caNrf2-Tg) has been described previously23. Briefly, cDNA encoding a truncated Nrf2 protein lacking the Neh2 domain was ligated into an α myosin heavy chain (αMHC) expression vector, the plasmid backbone was digested, and the αMHC-caNrf2 insert was used for pronuclear injection. Transgenic low (TgL) and transgenic high (TgH) founders were determined using caNrf2 primer sets in real-time qPCR which compared relative transgene expression to endogenous Nrf2 mRNA, and transgenic mice were back-crossed onto the C57BL/6J background for six generations. For expression analyses, sex-matched male and female TgL, TgH and nontransgenic (NT (...truncated)