Circular RNA Circ-Cdr1as modulates Macrophage phenotype and Cardiac Reparative Function by Circ-Cdr1as-miR-7-Klf4 pathway

Abstract Background Mechanisms of macrophage switching from pro-inflammatory to anti-inflammatory phenotypes are not well understood. Circular RNAs (circRNAs), a new class of non-coding RNAs, are implicated in immune modulation. We recently identified circ-cdr1as as a regulator of macrophage phenotype in bone marrow derived macrophages (BMDM), however, their role in immunomodulation during cardiovascular injury remains unknown. Methods Cell-specific expression levels of circ-cdr1as was determined in a mouse hearts post-myocardial infarction (MI). Circ-cdr1as was overexpressed in fluorescently labeled BMDMs and injected into the ischemic myocardium immediately following MI. Effect of AAV9-mediated systemic delivery of circ-Cdr1as on post-MI cardiac function and structure was determined. Downstream mechanisms were studied using gain and loss of function strategies. Results Cardiac cell specific expression analysis showed significant downregulation of circ-cdr1as only in macrophages and cardiomyocytes. Overexpression of circ-cdr1as in BMDMs, injected into the ischemic myocardium retained their anti-inflammatory phenotype and significantly improved left ventricular (LV) functions and reduced infarct size. Systemic delivery of AAV9-circ-cdr1as showed similar cardiac reparative activity. Mechanistically, circ-cdr1as directly binds and sponge microRNA-7 and increases the expression of target KLF4. Loss and gain of function studies show that modulation of miR-7 and KLF recapitulates macrophage phenotypic changes. Conclusions Circ-cdr1as plays a crucial role in regulating the anti-inflammatory phenotype of macrophages through modulation of miR-7 and its target gene KLF4. Therefore, circ-cdr1as holds potential as an anti-inflammatory regulator in tissue inflammation post-cardiac injury. Novelty and Significance What is Known? Despite continued research in elucidating mechanisms involved in cardiovascular disease and benefits of approved guideline-based therapies, the leading cause of deaths worldwide continues to be cardiovascular diseases (CVDs) with an increasing incidence of heart failure. Advances in high-throughput RNA sequencing (RNA-seq) allowed the identification of novel transcripts such as microRNAs (miRNA), long non-coding RNAs (lncRNAs), and circular RNAs (circRNA). circular RNAs have recently emerged as promising candidates for targeted therapy due to their circular structure that confers resistance to exonucleases, their capability to regulate gene expression by modulating miRNA activity, sequester proteins by acting as protein sponges, Several studies identified circRNAs to be differentially expressed following myocardial infarction (MI) and to play a role in immunity by contributing to the process of macrophage polarization, appropriate activation of macrophages when exposed to LPS, and inhibition of macrophage biogenesis. However, there are currently no published studies into the role of circular RNAs in the regulation of macrophage plasticity during cardiac injury. What New information Does This Article Contribute? We provide evidence that circ-cdr1as expression is downregulated in the heart 3 days post MI and specifically in cardiomyocytes and macrophages. Our study provides promising evidence that overexpression of circ-cdr1as may be cardioprotective by reducing cardiomyocyte apoptosis, enhancing angiogenesis, limiting infarct size, increasing percentage of anti-inflammatory macrophages, and overall preserving post-MI cardiac function. Mechanistically, we identified a reciprocal relationship between circ-cdr1as and miR-7 at 3 days post-MI and in naïve, pro-, and anti-inflammatory macrophages indicating circ-cdr1as role as a miRNA sponge. This suggests that circ-cdr1as/miR-7/Klf4 play a crucial role in cardiac injury and macrophage phenotype.