Abstract 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.

Aims Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell-specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry, and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19. Methods and results In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). Massive analysis of cDNA ends (MACE)-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset ‘Heart Cell Atlas’ and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis. MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The Gene Ontology (GO) term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response. Conclusion This study reveals that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.

Rationale: 3′,5′-cAMP is a ubiquitous second messenger which, upon β-AR (β-adrenergic receptor) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac RyR2 (ryanodine receptor type 2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum often occurring in heart failure. Objective: We sought to establish a real-time live-cell imaging approach capable of directly visualizing cAMP in the vicinity of mouse and human RyR2 and to analyze its pathological changes in failing cardiomyocytes under β-AR stimulation. Methods and Results: We generated a novel targeted fluorescent biosensor Epac1 (exchange protein directly activated by cAMP 1)-JNC (junctin) for RyR2-associated cAMP and expressed it in transgenic mouse hearts as well in human ventricular myocytes using adenoviral gene transfer. In healthy cardiomyocytes, β 1 -AR but not β 2 -AR stimulation strongly increased local RyR2-associated cAMP levels. However, already in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of PDEs (phosphodiesterases) 2, 3, and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurable β 2 -AR/AMP signals in the vicinity of RyR2 in failing mouse and human myocytes, increased β 2 -AR–dependent RyR2 phosphorylation, sarcoplasmic reticulum calcium leak, and arrhythmia susceptibility. Conclusions: Our new imaging approach could visualize cAMP levels in the direct vicinity of cardiac RyR2. Unexpectedly, in mouse and human failing myocytes, it could uncover functionally relevant local arrhythmogenic β 2 -AR/cAMP signals which might be an interesting antiarrhythmic target for heart failure.

1 Abstract Background Analyses in hospitalized patients and small autopsy series suggest that severe SARS-CoV-2 infection may affect the heart. We investigated heart tissue by in situ hybridization, immunohistochemistry and RNA sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19. Methods Left ventricular tissue from 95 deceased with diagnosed SARS-CoV-2 infection undergoing autopsy was analyzed and clinical data were collected. RNA was isolated to examine virus load of SARS- CoV-2 and its replication in the heart. A virus load >1000 copies per µg RNA was defined as relevant. Viral RNA and inflammatory cells were assessed using histology. RNA sequencing and gene ontology (GO) enrichment were performed in 10 cases with high cardiac virus load and 10 age-matched cases without cardiac infection. Results A relevant SARS-CoV-2 virus load was detected in 41 out of 95 deceased (43%). The median cardiac virus load was 7952 copies per µg RNA (IQR 2507, 32 005). In situ hybridization revealed SARS- CoV-2 RNA primarily in the interstitium or interstitial cells. Virus detection was not associated with increased inflammatory cells. Relevant cardiac infection was associated with increased expression of the entry factor TMPRSS2 . Cardiac virus replication was found in 14/95 hearts (15%). Remarkably, cardiac virus replication was associated with shorter time between diagnosis and death. RNA sequencing revealed clear activation of immune response pathways to virus infection and destruction of cardiomyocytes. Hearts with high virus load showed activation of the GO term “extracellular exosomes”. Conclusion SARS-CoV-2 infection including virus replication and distinct transcriptomic alterations without signs of myocarditis demonstrate a cardiac involvement. In this autopsy series, cardiac replication of SARS-CoV-2 was associated with early death.

Heart failure is a consequence of various cardiovascular diseases and associated with poor prognosis. Despite progress in the treatment of heart failure in the past decades, prevalence and hospitalisation rates are still increasing. Heart failure is typically associated with cardiac remodelling. Here, inflammation and fibrosis are thought to play crucial roles. During cardiac inflammation, immune cells invade the cardiac tissue and modulate tissue-damaging responses. Cardiac fibrosis, however, is characterised by an increased amount and a disrupted composition of extracellular matrix proteins. As evidence exists that cardiac inflammation and fibrosis are potentially reversible in experimental and clinical set ups, they are interesting targets for innovative heart failure treatments. In this context, animal models are important as they mimic clinical conditions of heart failure patients. The advantages of mice in this respect are short generation times and genetic modifications. As numerous murine models of heart failure exist, the selection of a proper disease model for a distinct research question is demanding. To facilitate this selection, this review aims to provide an overview about the current understanding of the pathogenesis of cardiac inflammation and fibrosis in six frequently used murine models of heart failure. Hence, it compares the models of myocardial infarction with or without reperfusion, transverse aortic constriction, chronic subjection to angiotensin II or deoxycorticosterone acetate, and coxsackievirus B3-induced viral myocarditis in this context. It furthermore provides information about the clinical relevance and the limitations of each model, and, if applicable, about the recent advancements in their methodological proceedings.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine known to play a major role in inflammatory diseases such as myocardial infarction (MI), where its expression increases. Cardio protective functions of MIF during ischemia have been reported. Recently, the structurally related MIF-2 was identified and similar effects are assumed. We wanted to further investigate the role of MIF and MIF-2 on inflammatory processes during MI. Therefore, we subjected mice to experimentally induced MI by coronary occlusion for one and five days. During the acute phase of MI, the gene expression of Mif was upregulated in the infarct zone, whereas Mif-2 was downregulated, suggesting a minor role of MIF-2. Simulating ischemic conditions or mechanical stress in vitro, we demonstrated that Mif expression was induced in resident cardiac cells. To investigate possible auto /paracrine effects, cardiomyocytes and cardiac fibroblasts were individually treated with recombinant murine MIF, which in turn induced Mif expression and the expression of pro-inflammatory genes in cardiac fibroblasts. Cardiomyocytes did not respond to recombinant MIF with pro-inflammatory gene expression. While MIF stimulation alone did not change the expression of pro-fibrotic genes in cardiac fibroblasts, ischemia reduced their expression. Mimicking the increased MIF levels during MI, we exposed cardiac fibroblasts to simulated ischemia in the presence of MIF, which led to further reduced expression of pro-fibrotic genes. The presented data show that MIF was expressed by resident cardiac cells during MI. In vitro, Mif expression was induced by different external stimuli in cardiomyocytes and cardiac fibroblasts. Addition of recombinant MIF protein increased the expression of pro-inflammatory genes in cardiac fibroblasts including Mif expression itself. Thereby, cardiac fibroblasts may amplify Mif expression during ischemia promoting cardiomyocyte survival.

Rationale Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which, upon β-adrenergic receptor (β-AR) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating the cAMP-dependent protein kinase (PKA) phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac ryanodine receptor type 2 (RyR2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum (SR) often occurring upon RyR2 hyperphosphorylation by PKA and calcium/calmodulin-dependent kinase. Objective We sought to establish a real time approach capable of directly visualizing cAMP and its pathological changes in the vicinity of RyR2 by generating a proper targeted biosensor and transgenic mouse model to express it in adult cardiomyocytes. Methods and Results We generated transgenic mice expressing a novel targeted fluorescent biosensor for RyR2-associated cAMP in adult mouse cardiomyocytes. In healthy cardiomyocytes, β 1 -AR but not β 2 -AR stimulation strongly increased local RyR2-associated cAMP levels. However, in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of phosphodiesterases (PDEs) 2, 3 and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurable β 2 -AR-induced cAMP signals, increased SR calcium leak and arrhythmia susceptibility. Conclusions Our new targeted biosensor expressed in transgenic mice can visualize cAMP levels in the vicinity of cardiac RyR2 in healthy and diseased cardiomyocytes. In the future, this novel biosensor can be used to better understand alterations of RyR2-associated cAMP in cardiovascular diseases and local actions of new therapies.

Increased adrenomedullin (ADM) levels are associated with various cardiac diseases such as myocardial infarction (MI). ADM is cleaved off from the full-length precursor protein proadrenomedullin (ProADM) during its posttranslational processing. To date, no biological effect of ProADM is reported, while ADM infusion leads to antiapoptotic effects and improved cardiac function. Using an MI mouse model, we found an induction of ProADM gene as well as protein expression during the early phase of MI. This was accompanied by apoptosis and increasing inflammation, which substantially influence the post-MI remodeling processes. Simulating ischemia in vitro, we demonstrate that ProADM expression was increased in cardiomyocytes and cardiac fibroblasts. Subsequently, we treated ischemic cardiomyocytes with either ProADM or ADM and found that both proteins increased survival. This effect was diminishable by blocking the ADM1 receptor. To investigate whether ProADM and ADM play a role in the regulation of cardiac inflammation, we analyzed chemokine expression after treatment of cells with both proteins. While ProADM induced an expression of proinflammatory cytokines, thus promoting inflammation, ADM reduced chemokine expression. On leukocytes, both proteins repressed chemokine expression, revealing antiinflammatory effects. However, ProADM but not ADM dampened concurrent activation of leukocytes. Our data show that the full-length precursor ProADM is biologically active by reducing apoptosis to a similar extent as ADM. We further assume that ProADM induces local inflammation in affected cardiac tissue but attenuates exaggerated inflammation, whereas ADM has low impact. Our data suggest that both proteins are beneficial during MI by influencing apoptosis and inflammation.