The role of p66ShcA-TLR9 signaling in myocardial remodeling and innate immune responses

Ischemic heart disease and myocardial infarction increase the risk of fatal heart failure via adverse myocardial remodeling. Cardiac cell death during myocardial infarction results in release of pro-inflammatory factors like mitochondrial DNA (mtDNA), leading to innate immunity activation, sterile inflammation, and damage amplification. mtDNA triggers inflammation via Toll-like receptor 9 (TLR9). The p66ShcA is a stress-sensitive protein involved in the regulation of the immune system. This work investigated whether TLR9 and p66ShcA regulate remodeling post-infarction. Cell cultures, p66ShcA knockout mice, an in vivo mouse model of myocardial infarction and tissue samples from patients with ischemic heart disease were used. Cardiac p66ShcA and TLR9 were upregulated, while the p66ShcA knockout mice had improved myocardial healing post-infarction. The p66ShcA was a positive upstream regulator of TLR9 signaling. mtDNA was released into the bloodstream of patients undergoing coronary bypass surgery. The findings suggest that p66ShcA-TLR9 signaling and mtDNA leakage are involved in sterile inflammation and myocardial remodeling post-infarction

Interleukin-17 (IL-17) Expression Is Reduced during Acute Myocardial Infarction: Role on Chemokine Receptor Expression in Monocytes and Their in Vitro Chemotaxis towards Chemokines

The roles of immune cells and their soluble products during myocardial infarction (MI) are not completely understood. Here, we observed that the percentages of IL-17, but not IL-22, producing cells are reduced in mice splenocytes after developing MI. To correlate this finding with the functional activity of IL-17, we sought to determine its effect on monocytes. In particular, we presumed that this cytokine might affect the chemotaxis of monocytes important for cardiac inflammation and remodeling. We observed that IL-17 tends to reduce the expression of two major chemokine receptors involved in monocyte chemotaxis, namely CCR2 and CXCR4. Further analysis showed that monocytes pretreated with IL-17 have reduced in vitro chemotaxis towards the ligand for CCR2, i.e., MCP-1/CCL2, and the ligand for CXCR4, i.e., SDF-1α/CXCL12. Our results support the possibility that IL-17 may be beneficial in MI, and this could be due to its ability to inhibit the migration of monocytes

Inhibiting nucleolin reduces inflammation induced by mitochondrial DNA in cardiomyocytes exposed to hypoxia and reoxygenation

Background and purpose Cellular debris causes sterile inflammation after myocardial infarction. Mitochondria constitute about 30 percent of the human heart. Mitochondrial DNA (mtDNA) is a damage‐associated‐molecular‐pattern that induce injurious sterile inflammation. Little is known about mtDNA's inflammatory signalling pathways in cardiomyocytes and how mtDNA is internalized to associate with its putative receptor, toll‐like receptor 9 (TLR9). Experimental Approach We hypothesized that mtDNA can be internalized in cardiomyocytes and induce an inflammatory response. Adult mouse cardiomyocytes were exposed to hypoxia‐reoxygenation and extracellular DNA. Microscale thermophoresis was used to demonstrate binding between nucleolin and DNA. Key results Expression of the pro‐inflammatory cytokines IL‐1β and TNFα were upregulated by mtDNA, but not by nuclear DNA (nDNA), in cardiomyocytes exposed to hypoxia‐reoxygenation. Blocking the RNA/DNA binding protein nucleolin with midkine reduced expression of IL‐1β/TNFα and the nucleolin inhibitor AS1411 reduced interleukin‐6 release in adult mouse cardiomyocytes. mtDNA bound 10‐fold stronger than nDNA to nucleolin. In HEK293‐NF‐κB reporter cells, mtDNA induced NF‐κB activity in normoxia, while CpG‐DNA and hypoxia‐reoxygenation, synergistically induced TLR9‐dependent NF‐κB activity. Protein expression of nucleolin was found in the plasma membrane of cardiomyocytes and inhibition of nucleolin with midkine inhibited cellular uptake of CpG‐DNA. Inhibition of endocytosis did not reduce CpG‐DNA uptake in cardiomyocytes. Conclusion and implications mtDNA, but not nDNA, induce an inflammatory response in mouse cardiomyocytes during hypoxia‐reoxygenation. In cardiomyocytes, nucleolin is expressed on the membrane and blocking nucleolin reduce inflammation. Nucleolin might be a therapeutic target to prevent uptake of immunogenic DNA and reduce inflammation

Retinoic Acid Signalling Is Activated in the Postischemic Heart and May Influence Remodelling

<div><h3>Background</h3><p>All-trans retinoic acid (atRA), an active derivative of vitamin A, regulates cell differentiation, proliferation and cardiac morphogenesis via transcriptional activation of retinoic acid receptors (RARs) acting on retinoic acid response elements (RARE).We hypothesized that the retinoic acid (RA) signalling pathway is activated in myocardial ischemia and postischemic remodelling.</p> <h3>Methods and Findings</h3><p>Myocardial infarction was induced through ligating the left coronary artery in mice. <em>In vivo</em> cardiac activation of the RARs was measured by imaging RARE-luciferase reporter mice, and analysing expression of RAR target genes and proteins by real time RT-PCR and western blot. Endogenous retinoids in postinfarcted hearts were analysed by triple-stage liquid chromatography/tandem mass spectrometry. Cardiomyocytes (CM) and cardiofibroblasts (CF) were isolated from infarcted and sham operated RARE luciferase reporter hearts and monitored for RAR activity and expression of target genes. The effect of atRA on CF proliferation was evaluated by EdU incorporation. Myocardial infarction increased thoracic RAR activity <em>in vivo</em> (p<0.001), which was ascribed to the heart through <em>ex vivo</em> imaging (p = 0.002) with the largest signal 1 week postinfarct. This was accompanied by increased cardiac gene and protein expression of the RAR target genes retinol binding protein 1 (p = 0.01 for RNA, p = 0,006 for protein) and aldehyde dehydrogenase 1A2 (p = 0.04 for RNA, p = 0,014 for protein), while gene expression of cytochrome P450 26B1 was downregulated (p = 0.007). Concomitantly, retinol accumulated in the infarcted zone (p = 0.02). CM and CF isolated from infarcted hearts had higher luminescence than those from sham operated hearts (p = 0.02 and p = 0.008). AtRA inhibited CF proliferation <em>in vitro</em> (p = 0.02).</p> <h3>Conclusion</h3><p>The RA signalling pathway is activated in postischemic hearts and may play a role in regulation of damage and repair during remodelling.</p> </div

Expression of retinoic acid target genes and endogenous metabolites after infarction.

<p>Wild type C57Bl6 mice were subjected to <i>in vivo</i> ligation of the left coronary artery or sham surgery, and hearts were sampled serially for RNA extraction and amplification of retinoic acid target genes with real time PCR, or proteins were extracted for western blotting. Hearts were also sampled from mice without any surgery (preoperative). Gene expression of retinol binding protein 1 (RBP1) (<b>A</b>), aldehyde dehydrogenase 1A2 (ALDH1A2) (<b>B</b>), and cytochrome p45026B1 (CYP26B1) are shown (<b>C</b>). (n = 6–8 in each group at each time point). <b>D)</b> Representative western blot analysis of retinoic acid transporting and metabolizing proteins one week after induction of infarction or sham operation. Ponceau solution was used as protein loading (control). Histograms show the relative density of RBP1 at 16 kDa and ALDH1A2 at 53–57 kDa in infarcted and sham operated hearts. <b>E)</b> Endogenous retinoic acid metabolite concentrations were evaluated by triple-stage liquid chromatography/tandem mass spectrometry one week after induction of myocardial infarction. The infarcted zone of the left ventricle (INF), the periinfarcted zone (PERIINF) and left ventricles from sham operated hearts (SHAM) were investigated (n = 6). Data are shown as mean±SD.</p

<i>In vitro</i> imaging of retinoic acid signaling after myocardial infarction.

<p>Cardiomyocytes and cardiofibroblasts were isolated from left ventricular tissue from RARE-luciferase reporter hearts one week after myocardial infarction or sham operation (SHAM). Cells were isolated from the infarct (INF) and periinfarct zone (PERIINF). After plating for three hours, non-viable cells were removed and luciferin was added for imaging. The upper panel shows a representative image of one experiment (<b>A</b>). The lower panel shows mean±SD of n = 5 experiments in each group (<b>B</b>). Note that the Y-axis labelling is different.</p

Expression of retinoic acid target genes in the infarct zone and effects on cardiofibroblast proliferation.

<p><b>A)</b> Gene expression of retinoic acid target genes in cardiofibroblast (CF) and cardiomyocytes (CM) isolated from left ventricles of infarcted RAR-luciferase reporter hearts 1 week after infarction. RNA was extracted from cells from the infarcted zone and amplified with real time PCR using primers specific for retinol binding protein 1 (RBP1), cytochrome P450 26B1 (CYP26B1), aldehyde dehydrogenase 1A2 (ALDH1A2), and retinoic acid receptors alpha, beta and gamma (RAR α,β,γ). The figure shows test gene expression relative to expression of rpl32, which was similar in both cell types. Data are mean±SD of n = 5 experiments in each group. <b>B)</b> Cardiofibroblasts were isolated from C57BL6 hearts and cultured in medium supplemented with 1 µM all-trans retinoic acid (atRA) and 10 µM EdU, as indicator of cell division. After 96 hours, EdU incorporation was evaluated by flow cytometry. Data are presented as mean±SD of n = 4 in each group.</p