Regulation of Irf2bp2 and Deficiency in Sepsis-Induced QT Prolongation

Abstract

The innate immune system is the body’s first line of defence against viral and bacterial pathogens, and the inflammatory response is crucial in maintaining a healthy cellular environment. Interferon regulatory factor 2 binding protein 2 (IRF2BP2), a zinc finger transcriptional corepressor highly expressed in macrophages, suppresses the interferon response and inflammation. In humans, individuals who carry two copies of a genetic variant (rs3045215) that deletes 9 nucleotides from the long 3’UTR of IRF2BP2 have lower IRF2BP2 protein expression in white blood cells and increased risk of coronary atherosclerosis and calcification. The first objective of this thesis was to determine how expression of IRF2BP2 is regulated by its 3’UTR and how this deletion variant leads to lower protein expression. For this, a combination of cellular and molecular techniques were used. First, we showed that the deletion variant is unable to interact with eukaryotic initiation factor 4H (eIF4H), a translation initiation factor that is required for the unwinding of GC-rich mRNA secondary structures. The 9-nucleotide deletion disrupts a proposed stem-loop structure, which is critical for interaction with eIF4H. siRNA knockdown of eIF4H preferentially disrupts translation of the non-deletion variant of the IRF2BP2 3’UTR, but not the deletion allele, revealing an essential role for the 9-nucleotide sequence in interacting with eIF4H. Previous work from our lab demonstrated that expression of IRF2BP2 is downregulated in response to LPS in bone marrow-derived macrophages (BMDM), attenuating the anti-inflammatory function of IRF2BP2 in the presence of endotoxins. We further uncovered a role for IRF2BP2 protein in binding to its own 3’UTR in the cytoplasm in response to LPS, suggesting an autoregulatory function. This response is lost in the rs3045215 deletion variant, demonstrating the importance of the 9-nnucleotide sequence in the 3’UTR in regulating its own expression. We also confirm that a single nucleotide polymorphism (rs7545855) located in exon 1 of IRF2BP2 that is co-inherited with rs3045215 does not confer any functional differences with respect to the autoregulatory mechanism. Because IRF2BP2 deficiency leads to proinflammatory macrophages, we asked whether mice lacking IRF2BP2 in their bone marrow-derived macrophages would display a heightened response to lipopolysaccharides with respect to their cardiac electrophysiology, which is sensitive to inflammatory signaling. By using LysMCre/Irf2bp2flx/flx mice whose macrophages are prone to inflammation, we revealed that female mice lacking IRF2PB2 in their BMDMs are uniquely protected against severe LPS-induced QT prolongation, an indicator of delayed ventricular repolarization. These female mice displayed an upregulation of growth differentiation factor 3 (GDF3) mRNA and protein, a member of the TGFβ family of signaling molecules that protects mice against LPS-mediated cardiac dysfunction. Our ECG studies revealed that pre-treating male wild type mice with recombinant GDF3 (rmGDF3) 12 hours prior to exposure to LPS similarly protected them from severe LPS-induced QT prolongation. Mechanistically, we discovered that short term exposure to LPS results in the upregulation of the sodium-calcium exchanger (NCX) in the mouse myocardium, which is reduced in mice pre-treated with rmGDF3. Our in vitro studies revealed that rmGDF3 blocks the uptake of toll-like receptor 4 (TLR4) and TRIF signaling in HL-1 cardiomyocytes and neonatal rat ventricular myocytes, providing insight into the possible mechanisms underlying this protection. Together, the data provide valuable insight into the mechanisms governing expression of IRF2BP2 in response to endotoxins. As well, our findings reveal sex-specific differences in the response to IRF2BP2 deficiency, with males showing an increased risk of coronary artery calcification, while females are protected from LPS-induced QT prolongation through the action of increased GDF3. Our results suggest that GDF3 should be further investigated as a potential therapeutic for protecting patients against QT prolongation in the face of inflammatory diseases, who face an increased risk of ventricular arrhythmias and sudden cardiac death.