DNA repair in cardiomyocytes is critical for maintaining cardiac function in mice.

Heart failure has reached epidemic proportions in a progressively ageing population. The molecular mechanisms underlying heart failure remain elusive, but evidence indicates that DNA damage is enhanced in failing hearts. Here, we tested the hypothesis that endogenous DNA repair in cardiomyocytes is critical for maintaining normal cardiac function, so that perturbed repair of spontaneous DNA damage drives early onset of heart failure. To increase the burden of spontaneous DNA damage, we knocked out the DNA repair endonucleases xeroderma pigmentosum complementation group G (XPG) and excision repair cross-complementation group 1 (ERCC1), either systemically or cardiomyocyte-restricted, and studied the effects on cardiac function and structure. Loss of DNA repair permitted normal heart development but subsequently caused progressive deterioration of cardiac function, resulting in overt congestive heart failure and premature death within 6 months. Cardiac biopsies revealed increased oxidative stress associated with increased fibrosis and apoptosis. Moreover, gene set enrichment analysis showed enrichment of pathways associated with impaired DNA repair and apoptosis, and identified TP53 as one of the top active upstream transcription regulators. In support of the observed cardiac phenotype in mutant mice, several genetic variants in the ERCC1 and XPG gene in human GWAS data were found to be associated with cardiac remodelling and dysfunction. In conclusion, unrepaired spontaneous DNA damage in differentiated cardiomyocytes drives early onset of cardiac failure. These observations implicate DNA damage as a potential novel therapeutic target and highlight systemic and cardiomyocyte-restricted DNA repair-deficient mouse mutants as bona fide models of heart failure.

C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9.

The proinflammatory alarmins S100A8 and S100A9 are among the most abundant proteins in neutrophils and monocytes but are completely silenced after differentiation to macrophages. The molecular mechanisms of the extraordinarily dynamic transcriptional regulation of S100a8 and S100a9 genes, however, are only barely understood. Using an unbiased genome-wide CRISPR/Cas9 knockout (KO)-based screening approach in immortalized murine monocytes, we identified the transcription factor C/EBPδ as a central regulator of S100a8 and S100a9 expression. We showed that S100A8/A9 expression and thereby neutrophil recruitment and cytokine release were decreased in C/EBPδ KO mice in a mouse model of acute lung inflammation. S100a8 and S100a9 expression was further controlled by the C/EBPδ antagonists ATF3 and FBXW7. We confirmed the clinical relevance of this regulatory network in subpopulations of human monocytes in a clinical cohort of cardiovascular patients. Moreover, we identified specific C/EBPδ-binding sites within S100a8 and S100a9 promoter regions, and demonstrated that C/EBPδ-dependent JMJD3-mediated demethylation of H3K27me3 is indispensable for their expression. Overall, our work uncovered C/EBPδ as a novel regulator of S100a8 and S100a9 expression. Therefore, C/EBPδ represents a promising target for modulation of inflammatory conditions that are characterized by S100a8 and S100a9 overexpression.

Monocyte subpopulation profiling indicates CDK6-derived cell differentiation and identifies subpopulation-specific miRNA expression sets in acute and stable coronary artery disease.

Coronary artery disease (CAD) is a long-lasting inflammatory disease characterized by monocyte migration into the vessel wall leading to clinical events like myocardial infarction (MI). However, the role of monocyte subsets, especially their miRNA-driven differentiation in this scenario is still in its infancy. Here, we characterized monocyte subsets in controls and disease phenotypes of CAD and MI patients using flow cytometry and miRNA and mRNA expression profiling using RNA sequencing. We observed major differences in the miRNA profiles between the classical (CD14++CD16-) and nonclassical (CD14+CD16++) monocyte subsets irrespective of the disease phenotype suggesting the Cyclin-dependent Kinase 6 (CDK6) to be an important player in monocyte maturation. Between control and MI patients, we found a set of miRNAs to be differentially expressed in the nonclassical monocytes and targeting CCND2 (Cyclin D2) that is able to enhance myocardial repair. Interestingly, miRNAs as miR-125b playing a role in vascular calcification were differentially expressed in the classical subset in patients suffering from CAD and not MI in comparison to control samples. In conclusion, our study describes specific peculiarities of monocyte subset miRNA expression in control and diseased samples and provides basis to further functional analysis and to identify new cardiovascular disease treatment targets.

Genomic instability in the naturally and prematurely aged myocardium.

Genomic instability, the unresolved accumulation of DNA variants, is hypothesized as one of the contributors to the natural aging process. We assessed the frequency of unresolved DNA damage reaching the transcriptome of the murine myocardium during the course of natural aging and in hearts from four distinct mouse models of premature aging with established aging-related cardiac dysfunctions. RNA sequencing and variant calling based on total RNA sequencing was compared between hearts from naturally aging mice, mice with cardiomyocyte-specific deficiency of Ercc1, a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity, Tert-deficient mice with reduced telomere length, and a mouse model of human Hutchinson-Gilford progeria syndrome (HGPS). Our results demonstrate that no enrichment in variants is evident in the naturally aging murine hearts until 2 y of age from the HGPS mouse model or mice with reduced telomere lengths. In contrast, a dramatic accumulation of variants was evident in Ercc1 cardiomyocyte-specific knockout mice with deficient DNA repair machinery, in mice with reduced mitochondrial antioxidant capacity, and in the intestine, liver, and lung of naturally aging mice. Our data demonstrate that genomic instability does not evidently contribute to naturally aging of the mouse heart in contrast to other organs and support the contention that the endogenous DNA repair machinery is remarkably active to maintain genomic integrity in cardiac cells throughout life.

A genetic variant alters the secondary structure of the lncRNA H19 and is associated with dilated cardiomyopathy.

lncRNAs are at the core of many regulatory processes and have also been recognized to be involved in various complex diseases. They affect gene regulation through direct interactions with RNA, DNA or proteins. Accordingly, lncRNA structure is likely to be essential for their regulatory function. Point mutations, which manifest as SNPs (single nucleotide polymorphisms) in genome screens, can substantially alter their function and, subsequently, the expression of their downstream regulated genes. To test the effect of SNPs on structure, we investigated lncRNAs associated with dilated cardiomyopathy. Among 322 human candidate lncRNAs, we demonstrate first the significant association of an SNP located in lncRNA H19 using data from 1084 diseased and 751 control patients. H19 is generally highly expressed in the heart, with a complex expression pattern during heart development. Next, we used MFE (minimum free energy) folding to demonstrate a significant refolding in the secondary structure of this 861 nt long lncRNA. Since MFE folding may overlook the importance of sub-optimal structures, we showed that this refolding also manifests in the overall Boltzmann structure ensemble. There, the composition of structures is tremendously affected in their thermodynamic probabilities through the genetic variant. Finally, we confirmed these results experimentally, using SHAPE-Seq, corroborating that SNPs affecting such structures may explain hidden genetic variance not accounted for through genome wide association studies. Our results suggest that structural changes in lncRNAs, and lncRNA H19 in particular, affect regulatory processes and represent optimal targets for further in-depth studies probing their molecular interactions.

Interleukin 17 Promotes Expression of Alarmins S100A8 and S100A9 During the Inflammatory Response of Keratinocytes.

Psoriasis is one of the most common immune-mediated inflammatory skin diseases. Expression and secretion of two pro-inflammatory molecules of the S100-alarmin family, S100A8 and S100A9, in keratinocytes is a hallmark of psoriasis, which is also characterized by an altered differentiation of keratinocytes. Dimers of S100A8/S100A9 (calprotectin) bind to Toll-like receptor 4 and induce an inflammatory response in target cells. Targeted deletion of S100A9 reduced the inflammatory phenotype of psoriasis-like inflammation in mice. A role of S100-alarmins in differentiation and activation of keratinocytes was suggested but has been never shown in primary keratinocytes. We now confirm that induction of S100-alarmins in an imiquimod-induced murine model of psoriasis-like skin inflammation was associated with an increased expression of interleukin (IL)-1α, IL-6, IL-17A, or TNFα. This association was confirmed in transcriptome data obtained from controls, lesional and non-lesional skin of psoriasis patients, and a down-regulation of S100-alarmin expression after IL-17 directed therapy. However, analyzing primary S100A9-/- keratinocytes we found that expression of S100A8/S100A9 has no significant role for the maturation and inflammatory response pattern of keratinocytes. Moreover, keratinocytes are no target cells for the pro-inflammatory effects of S100A8/S100A9. However, different cytokines, especially IL-17A and F, highly abundant in psoriasis, strongly induced expression of S100-alarmins preferentially during early maturation stages of keratinocytes. Our data indicate that expression of S100A8 and S100A9 does not primarily influence maturation or activation of keratinocytes but rather represents the inflammatory response of these cells during psoriasis.

LncRNA secondary structure in the cardiovascular system.

Long non-coding RNAs (lncRNAs) have been increasingly studied during the past decade. This led to an immense number of annotated transcripts, out of which many were linked to a diverse range of biological mechanisms and diseases. Due to the variety of their regulatory potential, they are seen as an important link in understanding complex epigenetic mechanisms. Prominent examples of lncRNAs in the cardiovascular system are ANRIL, Braveheart, MALAT1 and HOTAIR which have been excessively studied. But despite the impressive number of described transcripts, only a few examples are characterized functionally. One way to do this is to identify accessible structural domains in the RNA secondary structure which have the ability to bind to DNA, RNA or proteins. Through recent improvements in computational as well as experimental methods, this exploration of secondary structure became not only more efficient than traditional methods like crystallization, but also feasible to investigate whole genome RNA structures. The purpose of this review is to highlight the recent advances in secondary structure probing methods and how these can be applied in order to investigate the functional roles of lncRNAs in the cardiovascular system.