Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Centre for Cardiovascular Research (DZHK) Background Cardiovascular diseases such as myocardial infarction (MI) are a leading cause of death worldwide. Since matrix metalloproteinases (MMPs) are essential for the cleavage of collagen as well as for the modification of inflammatory proteins and cytokines, they play a substantial role in remodelling processes after MI. Purpose Previous results of our group revealed, that Mmp13 expression is upregulated post-MI in mice, while it is downregulated after Ischemia/Reperfusion (I/R), indicating an involvement in remodelling processes. In humans, the functional homologue of Mmp13 is MMP1. Single nucleotide polymorphisms (SNPs) in the promotor of MMP1 can lead to alterations in its gene expression level. We analysed the genotype for 3 MMP1 SNPs in a human cohort containing ~2000 patients who presented to the emergency department with suspected MI to identify their associations with development of MI and outcome after MI. Methods The Mmp13 expression in different cardiac cell types was investigated at quiescent stage and under ischaemic conditions, to determine the cellular origin of Mmp13 expression. A MMP13-knockout (KO) mouse model was examined after induction of MI or I/R. Thus, gene expression analysis, histological staining and hemodynamic measurements were conducted to analyse differences between KO and WT as well as between MI and I/R. Out of the human cohort, 2 patient groups (non-MI and MI) were restricted, and Hazard ratios were calculated to evaluate risk for MI and risk for death after MI in dependency of the SNPs. Results The Mmp13 expression in macrophages (6.6-fold to control; p=0.0286) and fibroblasts (4.9-fold; p=0.0079) increased significantly after activation with ischaemic secretome of cardiomyocytes, while Mmp13 expression of leucocytes was unaltered. After stimulation with ischaemic secretome of fibroblasts, Mmp13 expression in macrophages (4.3-fold; p=0.0286) and leukocytes (2.3-fold; p=0.0260) was significantly elevated as well. Comparing MI and I/R, the immune cell infiltration revealed significant differences 1-day post-intervention. About 50% of WT mice but only few KO mice died (p=0.0107) after MI due to cardiac rupture. Moreover, KO mice showed an improved cardiac function compared to WT mice after MI. Risk for death was significantly altered between the investigated genotypes in 2 of 3 investigated SNPs in the BACC cohort. Conclusion Activated macrophages and leucocytes express high levels of Mmp13 in cell culture experiments. The infiltrating immune cell types are different between MI and I/R, which might lead to differences in Mmp13 expression in these models. MMP13 KO mice are protected from cardiac rupture after MI and unveiled improved cardiac function 28 days post-MI. SNPs of the human homologue of Mmp13 – MMP1 – showed an association of MMP1 with remodelling processes after MI.
Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DZHK Ernst und Berta Grimmke Stiftung Background Myocarditis is an inflammatory disease of the myocardium indicated by mononuclear cell infiltration. It is predominantly caused by infectious agents such as coxsackievirus B3 (CVB3). Especially in young adults, myocarditis is a major source of sudden cardiac arrest. However, its clinical course has a broad spectrum of outcomes, ranging from mild symptoms and complete recovery to cardiac dysfunction and dilated cardiomyopathy. G protein-coupled receptor 15 (GPR15) was identified as a T cell homing receptor in the context of inflammatory intestine and skin diseases. We found Gpr15 to be highly upregulated in the left ventricle (LV) 7 days after CVB3 infection in wild type (WT) mice. Purpose GPR15 has not been described in a cardiac context, yet. Our aim was to investigate the role of GPR15 in recruiting immune cell subsets and later in virus elimination during viral myocarditis. Methods Gpr15 deficient (Gpr15gfp/gfp) and WT mice were infected intraperitoneal with CVB3 to investigate the acute (6 & 7 days post infection(p.i.)) and the subacute phase (16 days p.i.) of myocarditis. To study differentially expressed genes, LV tissue was used for TaqMan analysis and RNA-sequencing. Inflammation and fibrosis were evaluated on histological level. For functional characterization, healthy and diseased mice were hemodynamically characterized 16 days p.i.. Furthermore, in vitro migration assays were used to study the interaction between GPR15 and its ligands in vitro. Results Infected Gpr15gfp/gfp mice exhibited higher upregulation of immune response related genes on mRNA level in the acute phase of myocarditis 7 days p.i.. For instance, Cd8a, a cytotoxic T cell marker, and Foxp3, a regulatory T cell marker, were significantly higher in infected Gpr15gfp/gfp compared to infected WT mice. Bulk RNA-sequencing confirmed that the response to virus did not decline from day 6 to 7 in infected GPR15-deficient mice as observed in infected WT mice. Subsequent gene ontology (GO) term analyses reveled enhanced chemotaxis and cytotoxic T cell-related GO terms in GPR15-deficient mice on day 7. Among investigated T cell subsets, GPR15 was highest expressed on CD8+ T cell. Its deficiency abolished chemotaxis of T cells, especially of cytotoxic T cells, towards GPR15 ligand in vitro. In the subacute phase of myocarditis 16 days p.i., viral persistence was observed in more than 85 % of Gpr15gfp/gfp mice. In contrast, more than 70 % of WT mice with verified viremia cleared the virus successfully. Furthermore, Gpr15gfp/gfp mice demonstrated a decreased cardiac function accompanied by increased fibrosis in comparison to WT mice. Conclusion Our findings indicate that despite the prolonged inflammatory response, scant virus elimination was presumably caused by decelerated recruitment of cytotoxic T cells leading to impaired outcome in the GPR15-deficient mice.
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