Loss of tet methyl cytosine dioxygenase 3 (TET3) enhances cardiac fibrosis via modulating the DNA damage repair response.

BACKGROUND: Cardiac fibrosis is the hallmark of all forms of chronic heart disease. Activation and proliferation of cardiac fibroblasts are the prime mediators of cardiac fibrosis. Existing studies show that ROS and inflammatory cytokines produced during fibrosis not only signal proliferative stimuli but also contribute to DNA damage. Therefore, as a prerequisite to maintain sustained proliferation in fibroblasts, activation of distinct DNA repair mechanism is essential. RESULT: In this study, we report that TET3, a DNA demethylating enzyme, which has been shown to be reduced in cardiac fibrosis and to exert antifibrotic effects does so not only through its demethylating activity but also through maintaining genomic integrity by facilitating error-free homologous recombination (HR) repair of DNA damage. Using both in vitro and in vivo models of cardiac fibrosis as well as data from human heart tissue, we demonstrate that the loss of TET3 in cardiac fibroblasts leads to spontaneous DNA damage and in the presence of TGF-ß to a shift from HR to the fast but more error-prone non-homologous end joining repair pathway. This shift contributes to increased fibroblast proliferation in a fibrotic environment. In vitro experiments showed TET3's recruitment to H2O2-induced DNA double-strand breaks (DSBs) in mouse cardiac fibroblasts, promoting HR repair. Overexpressing TET3 counteracted TGF-ß-induced fibroblast proliferation and restored HR repair efficiency. Extending these findings to human cardiac fibrosis, we confirmed TET3 expression loss in fibrotic hearts and identified a negative correlation between TET3 levels, fibrosis markers, and DNA repair pathway alteration. CONCLUSION: Collectively, our findings demonstrate TET3's pivotal role in modulating DDR and fibroblast proliferation in cardiac fibrosis and further highlight TET3 as a potential therapeutic target.

Low levels of circulating methylated IRX3 are related to worse outcome after transcatheter aortic valve implantation in patients with severe aortic stenosis.

BACKGROUND: Aortic stenosis (AS) is one of the most common cardiac diseases and major cause of morbidity and mortality in the elderly. Transcatheter aortic valve implantation (TAVI) is performed in such patients with symptomatic severe AS and reduces mortality for the majority of these patients. However, a significant percentage dies within the first two years after TAVI, such that there is an interest to identify parameters, which predict outcome and could guide pre-TAVI patient selection. High levels of cardiac fibrosis have been identified as such independent predictor of cardiovascular mortality after TAVI. Promoter hypermethylation commonly leads to gene downregulation, and the Iroquois homeobox 3 (IRX3) gene was identified in a genome-wide transcriptome and methylome to be hypermethylated and downregulated in AS patients. In a well-described cohort of 100 TAVI patients in which cardiac fibrosis levels were quantified histologically in cardiac biopsies, and which had a follow-up of up to two years, we investigated if circulating methylated DNA of IRX3 in the peripheral blood is associated with cardiac fibrosis and/or mortality in AS patients undergoing TAVI and thus could serve as a biomarker to add information on outcome after TAVI. RESULTS: Patients with high levels of methylation in circulating IRX3 show a significantly increased survival as compared to patients with low levels of IRX3 methylation indicating that high peripheral IRX3 methylation is associated with an improved outcome. In the multivariable setting, peripheral IRX3 methylation acts as an independent predictor of all-cause mortality. While there is no significant correlation of levels of IRX3 methylation with cardiac death, there is a significant but very weak inverse correlation between circulating IRX3 promoter methylation level and the amount of cardiac fibrosis. Higher levels of peripheral IRX3 methylation further correlated with decreased cardiac IRX3 expression and vice versa. CONCLUSIONS: High levels of IRX3 methylation in the blood of AS patients at the time of TAVI are associated with better overall survival after TAVI and at least partially reflect myocardial IRX3 expression. Circulating methylated IRX3 might aid as a potential biomarker to help guide both pre-TAVI patient selection and post-TAVI monitoring.

miR-132-3p and KLF7 as novel regulators of aortic stiffening-associated EndMT in type 2 diabetes mellitus.

BACKGROUND: The prevalence of diabetes mellitus has risen considerably and currently affects more than 422 million people worldwide. Cardiovascular diseases including myocardial infarction and heart failure represent the major cause of death in type 2 diabetes (T2D). Diabetes patients exhibit accelerated aortic stiffening which is an independent predictor of cardiovascular disease and mortality. We recently showed that aortic stiffness precedes hypertension in a mouse model of diabetes (db/db mice), making aortic stiffness an early contributor to cardiovascular disease development. Elucidating how aortic stiffening develops is a pressing need in order to halt the pathophysiological process at an early time point. METHODS: To assess EndMT occurrence, we performed co-immunofluorescence staining of an endothelial marker (CD31) with mesenchymal markers (α-SMA/S100A4) in aortic sections from db/db mice. Moreover, we performed qRT-PCR to analyze mRNA expression of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. To identify the underlying mechanism by which EndMT contributes to aortic stiffening, we used aortas from db/db mice and diabetic patients in combination with high glucose-treated human umbilical vein endothelial cells (HUVECs) as an in vitro model of diabetes-associated EndMT. RESULTS: We demonstrate robust CD31/α-SMA and CD31/S100A4 co-localization in aortic sections of db/db mice which was almost absent in control mice. Moreover, we demonstrate a significant upregulation of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. As underlying regulator, we identified miR-132-3p as the most significantly downregulated miR in the micronome of db/db mice and high glucose-treated HUVECs. Indeed, miR-132-3p was also significantly downregulated in aortic tissue from diabetic patients. We identified Kruppel-like factor 7 (KLF7) as a target of miR-132-3p and show a significant upregulation of KLF7 in aortic sections of db/db mice and diabetic patients as well as in high glucose-treated HUVECs. We further demonstrate that miR-132-3p overexpression and KLF7 downregulation ameliorates EndMT in high glucose-treated HUVECs. CONCLUSIONS: We demonstrate for the first time that EndMT contributes to aortic stiffening in T2D. We identified miR-132-3p and KLF7 as novel EndMT regulators in this context. Altogether, this gives us new insights in the development of aortic stiffening in T2D.

Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1.

Rationale: Cardiac fibrosis is an integral constituent of every form of chronic heart disease, and persistence of fibrosis reduces tissue compliance and accelerates the progression to heart failure. Relaxin-2 is a human hormone, which has various physiological functions such as mediating renal vasodilation in pregnancy. Its recombinant form Serelaxin has recently been tested in clinical trials as a therapy for acute heart failure but did not meet its primary endpoints. The aim of this study is to examine whether Serelaxin has an anti-fibrotic effect in the heart and therefore could be beneficial in chronic heart failure. Methods: We utilized two different cardiac fibrosis mouse models (ascending aortic constriction (AAC) and Angiotensin II (ATII) administration via osmotic minipumps) to assess the anti-fibrotic potential of Serelaxin. Histological analysis, immunofluorescence staining and molecular analysis were performed to assess the fibrosis level and indicate endothelial cells which are undergoing EndMT. In vitro TGFß1-induced endothelial-to-mesenchymal transition (EndMT) assays were performed in human coronary artery endothelial cells and mouse cardiac endothelial cells (MCECs) and were examined using molecular methods. Chromatin immunoprecipitation-qPCR assay was utilized to identify the Serelaxin effect on chromatin remodeling in the Rxfp1 promoter region in MCECs. Results: Our results demonstrate a significant and dose-dependent anti-fibrotic effect of Serelaxin in the heart in both models. We further show that Serelaxin mediates this effect, at least in part, through inhibition of EndMT through the endothelial Relaxin family peptide receptor 1 (RXFP1). We further demonstrate that Serelaxin administration is able to increase its own receptor expression (RXFP1) through epigenetic regulation in form of histone modifications by attenuating TGFß-pSMAD2/3 signaling in endothelial cells. Conclusions: This study is the first to identify that Serelaxin increases the expression of its own receptor RXFP1 and that this mediates the inhibition of EndMT and cardiac fibrosis, suggesting that Serelaxin may have a beneficial effect as anti-fibrotic therapy in chronic heart failure.