Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process.

Long noncoding ribonucleic acids (lncRNAs) are a subclass of regulatory noncoding ribonucleic acids for which expression and function in human endothelial cells and angiogenic processes is not well studied.

So far the pathomechanism of preeclampsia in pregnancy is focussed on increased circulating levels of soluble fms-like tyrosin kinase-1 (sFLT-1) that neutralizes glomerular VEGF-A expression and prevents its signaling at the glomerular endothelium. As a result of changed glomerular VEGF-A levels endotheliosis and podocyte foot process effacement are typical morphological features of preeclampsia. Recently, microRNA-26a-5p (miR-26a-5p) was described to be also upregulated in the preeclamptic placenta. We found that miR-26a-5p targets VEGF-A expression by means of PIK3C2α in cultured human podocytes and that miR-26a-5p overexpression in zebrafish causes proteinuria, edema, glomerular endotheliosis and podocyte foot process effacement. Interestingly, recombinant zebrafish Vegf-Aa protein could rescue glomerular changes induced by miR-26a-5p. In a small pilot study, preeclamptic patients with podocyte damage identified by podocyturia, expressed significantly more urinary miR-26a-5p compared to healthy controls. Thus, functional and ultrastructural glomerular changes after miR-26a-5p overexpression can resemble the findings seen in preeclampsia and indicate a potential pathophysiological role of miR-26a-5p in addition to sFLT-1 in this disease.

Delayed re-endothelialization after balloon angioplasty in patients with coronary or peripheral artery disease impairs vascular healing and leads to neointimal proliferation. In the present study, we examined the effect of RNA-binding motif protein 38 (Rbm38) during re-endothelialization in a murine model of experimental vascular injury.

The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.

Although several risk factors such as infarct size have been identified, the progression of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of post-myocardial infarction (MI), we previously identified 45 proteins differentially modulated during HF by proteomic analysis. This study sought to identify microRNAs (miRNAs) able to regulate these proteins and to test their relevance as biomarkers for HF. In silico bioinformatical analysis selected 13 miRNAs related to the 45 proteins previously identified. These miRNAs were analyzed in the rat and in cohorts of patients phenotyped for left ventricular remodeling (LVR). We identified that 3 miRNAs, miR-21-5p, miR-23a-3p and miR-222-3p, and their target Mn superoxide dismutase (SOD2) were significantly increased in LV and plasma of HF-rats. We found by luciferase activity a direct interaction of miR-222-3p with 3'UTR of SOD2. Transfection of human cardiomyocytes with miR-222-3p mimic or inhibitor induced respectively a decrease and an increase of SOD2 expression. Circulating levels of the 3 miRNAs and their target SOD2 were associated with high LVR post-MI in REVE-2 patients. We demonstrated for the first time the potential of microRNAs regulating SOD2 as new circulating biomarkers of HF.

Hypertrophic cardiomyopathy (HCM) is one of the most common hereditary heart diseases and is associated with a high risk of sudden cardiac death. HCM is characterized by pronounced hypertrophy of cardiomyocytes, fiber disarray and development of fibrosis and can be divided into a non-obstructive (HNCM) and obstructive form (HOCM) therefore requiring personalized therapeutic therapies. In the present study, we investigated the expression patterns of several circulating circular RNAs (circRNAs) as potential biomarkers in patients with HCM. We included 64 patients with HCM and 53 healthy controls to the study and quantitatively measured the expression of a set of circRNAs already known to be associated with cardiac diseases (circDNAJC6) and/or being highly abundant in blood (circTMEM56 and circMBOAT2). Abundancy of circRNAs was then correlated to relevant clinical parameters. Serum expression levels of circRNAs DNAJC6, TMEM56 and MBOAT2 were downregulated in patients with HCM. The inverse association between circRNA levels and HCM remained unchanged even after adjusting for confounding factors. All circRNAs, evaluated separately or in combination, showed a robust discrimination capacity when comparing control subjects with HCM, HNCM or HOCM patients (AUC from 0.722 to 0.949). Two circRNAs, circTMEM56 and circDNAJC6, significantly negatively correlated with echocardiographic parameters for HOCM. Collectively, circulating circRNAs DNAJC6, TMEM56 and MBOAT2 can distinguish between healthy and HCM patients. In addition, circTMEM56 and circDNAJC6 could serve as indicators of disease severity in patients with HOCM. Thus, circRNAs emerge as novel biomarkers for HCM facilitating the clinical decision making in a personalized manner.

Cardiac diseases are the most frequent causes of death in industrialized countries. Pathological remodeling of the heart muscle is caused by several etiologies such as prolonged hypertension or injuries that can lead to myocardial infarction and in serious cases also the death of the patient. The micro-RNA miR-132 has been identified as a master-switch in the development of cardiac hypertrophy and adverse remodeling. In this study, MALDI-TOF mass spectrometry (MS) was utilized to establish a robust and fast method to sensitively detect and accurately quantify anti-microRNA (antimiR) oligonucleotides in blood plasma. An antimiR oligonucleotide isolation protocol containing an ethanol precipitation step with glycogen as oligonucleotide carrier as well as a robust and reproducible MS-analysis procedure has been established. Proteinase K treatment was crucial for releasing antimiR oligonucleotides from plasma- as well as cellular proteins and reducing background derived from biological matrices. AntimiR oligonucleotide detection was achieved from samples of studies in different animal models such as mouse and pig where locked nucleic acids-(LNA)-modified antimiR oligonucleotides have been used to generate pharmacokinetic data.

Deficient autophagy has been recently implicated as a driver of pulmonary fibrosis, yet bioinformatics approaches to study this cellular process are lacking. Autophagy-related 5 and 7 (ATG5/ATG7) are critical elements of macro-autophagy. However, an alternative ATG5/ATG7-independent macro-autophagy pathway was recently discovered, its regulation being unknown. Using a bioinformatics proteome profiling analysis of ATG7-deficient human fibroblasts, we aimed to identify key microRNA (miR) regulators in autophagy.

Cachexia is associated with poor prognosis in chronic heart failure patients, but the underlying mechanisms of cachexia triggered disease progression remain poorly understood. Here, we investigate whether the dysregulation of myokine expression from wasting skeletal muscle exaggerates heart failure. RNA sequencing from wasting skeletal muscles of mice with heart failure reveals a reduced expression of Ostn, which encodes the secreted myokine Musclin, previously implicated in the enhancement of natriuretic peptide signaling. By generating skeletal muscle specific Ostn knock-out and overexpressing mice, we demonstrate that reduced skeletal muscle Musclin levels exaggerate, while its overexpression in muscle attenuates cardiac dysfunction and myocardial fibrosis during pressure overload. Mechanistically, Musclin enhances the abundance of C-type natriuretic peptide (CNP), thereby promoting cardiomyocyte contractility through protein kinase A and inhibiting fibroblast activation through protein kinase G signaling. Because we also find reduced OSTN expression in skeletal muscle of heart failure patients, augmentation of Musclin might serve as therapeutic strategy.

Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the β-isoform. We previously demonstrated that ∼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively β-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than β-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/β-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted β-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.

Induced pluripotent stem cells (iPSCs) can be generated by overexpression of Oct4, Sox2 and Klf4 in murine fibroblasts. By conducting a microRNA (miRNA) library screen, we identified a set of miRNAs critically regulating iPSC formation. We revealed a new miRNA family (miR-130/301/721) as an important regulator of iPSC induction by targeting the homeobox transcription factor Meox2 (also known as Gax). Meox2-specific silencing mimicked the effects of this miRNA family on reprogramming. Mechanistically, miRNA-resistant Meox2 overexpression abrogated effects of miR-130/301/721 on reprogramming. In conclusion, the miRNA family miR-130/301/721 enhances iPSC generation via repression of Meox2.

Impaired myocardial sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) activity is a hallmark of failing hearts, and SERCA2a gene therapy improves cardiac function in animals and patients with heart failure (HF). Deregulation of microRNAs has been demonstrated in HF pathophysiology. We studied the effects of therapeutic AAV9.SERCA2a gene therapy on cardiac miRNome expression and focused on regulation, expression, and function of miR-1 in reverse remodelled failing hearts.

Pathological growth of cardiomyocytes (hypertrophy) is a major determinant for the development of heart failure, one of the leading medical causes of mortality worldwide. Here we show that the microRNA (miRNA)-212/132 family regulates cardiac hypertrophy and autophagy in cardiomyocytes. Hypertrophic stimuli upregulate cardiomyocyte expression of miR-212 and miR-132, which are both necessary and sufficient to drive the hypertrophic growth of cardiomyocytes. MiR-212/132 null mice are protected from pressure-overload-induced heart failure, whereas cardiomyocyte-specific overexpression of the miR-212/132 family leads to pathological cardiac hypertrophy, heart failure and death in mice. Both miR-212 and miR-132 directly target the anti-hypertrophic and pro-autophagic FoxO3 transcription factor and overexpression of these miRNAs leads to hyperactivation of pro-hypertrophic calcineurin/NFAT signalling and an impaired autophagic response upon starvation. Pharmacological inhibition of miR-132 by antagomir injection rescues cardiac hypertrophy and heart failure in mice, offering a possible therapeutic approach for cardiac failure.

MicroRNAs (miRs) are small non- coding RNA molecules controlling a plethora of biological processes such as development, cellular survival and senescence. We here determined miRs differentially regulated during cardiac postnatal development and aging. Cardiac function, morphology and miR expression profiles were determined in neonatal, 4 weeks, 6 months and 19 months old normotensive male healthy C57/Bl6N mice. MiR-22 was most prominently upregulated during cardiac aging. Cardiac expression of its bioinformatically predicted target mimecan (osteoglycin, OGN) was gradually decreased with advanced age. Luciferase reporter assays validated mimecan as a bona fide miR-22 target. Both, miR-22 and its target mimecan were co- expressed in cardiac fibroblasts and smooth muscle cells. Functionally, miR-22 overexpression induced cellular senescence and promoted migratory activity of cardiac fibroblasts. Small interference RNA-mediated silencing of mimecan in cardiac fibroblasts mimicked the miR-22-mediated effects. Rescue experiments revealed that the effects of miR-22 on cardiac fibroblasts were only partially mediated by mimecan. In conclusion, miR-22 upregulation in the aging heart contributed at least partly to accelerated cardiac fibroblast senescence and increased migratory activity. Our results suggest an involvement of miR-22 in age-associated cardiac changes, such as cardiac fibrosis.

Viperid snake venoms contain a unique family of cytotoxic proteins, the Lys49 PLA2 homologs, which are devoid of enzymatic activity but disrupt the integrity of cell membranes. They are known to induce skeletal muscle damage and are therefore named 'myotoxins'. Single intact and skinned (devoid of membranes and cytoplasm but with intact sarcomeric proteins) rat cardiomyocytes were used to analyze the cytotoxic action of a myotoxin, from the venom of Bothrops asper. The toxin induced rapid hypercontraction of intact cardiomyocytes, associated with an increase in the cytosolic concentration of calcium and with cell membrane disruption. Hypercontraction of intact cardiomyocytes was abrogated by the myosin inhibitor para-aminoblebbistatin (AmBleb). No toxin-induced changes of key parameters of force development were observed in skinned cardiomyocytes. Thus, although myosin is a key effector of the observed hypercontraction, a direct effect of the toxin on the sarcomeric proteins -including the actomyosin complex- is not part of the mechanism of cytotoxicity. Owing to the sensitivity of intact cardiomyocytes to the cytotoxic action of myotoxin, this ex vivo model is a valuable tool to explore in further detail the mechanism of action of this group of snake venom toxins.

Cardiac fibroblasts constitute the major cell type of the murine and human heart. Once activated, they contribute to an excessive deposition of extracellular matrix (ECM) leading to cardiac fibrosis and subsequently organ dysfunction. With the exception of the pulmonary drugs, nintedanib and pirfenidone, drugs specifically targeting anti-fibrotic pathways are scarce. We recently performed large library screenings of natural occurring compounds and identified first lead structures with anti-fibrotic properties in vitro and in vivo. In line, we now aimed to improve efficacy of these anti-fibrotic lead structures by combining in vitro validation studies and in silico prediction. Next to this combined approach, we performed large OMICs-multi-panel-based mechanistic studies. Applying human cardiac fibroblasts (HCF), we analysed 26 similars of the initially identified anti-fibrotic lead molecules bufalin and lycorine and determined anti-proliferative activity and potential toxicity in an array of in vitro and ex vivo studies. Of note, even at lower concentrations, certain similars were more effective at inhibiting HCF proliferation than nintedanib and pirfenidone. Additionally, selected similars showed low cytotoxicity on human iPS-derived cardiomyocytes and anti-fibrotic gene regulation in human ex vivo living myocardial slices. Further, array and RNA sequencing studies of coding and non-coding RNAs in treated HCFs revealed strong anti-fibrotic properties, especially with the lycorine similar lyco-s (also known as homoharringtonine), that led to a nearly complete shutdown of ECM production at concentrations 100-fold lower than the previously identified anti-fibrotic compound lycorine without inducing cellular toxicity. We thus identified a new natural compound similar with strong anti-fibrotic properties in human cardiac fibroblasts and human living heart tissue potentially opening new anti-fibrotic treatment strategies.

Long non-coding RNAs (lncRNAs) have potential as novel therapeutic targets in cardiovascular diseases, but detailed information about the intercellular lncRNA shuttling mechanisms in the heart is lacking. Here, we report an important novel crosstalk between cardiomyocytes and fibroblasts mediated by the transfer of lncRNA-enriched extracellular vesicles (EVs) in the context of cardiac ischemia. lncRNA profiling identified two hypoxia-sensitive lncRNAs: ENSMUST00000122745 was predominantly found in small EVs, whereas lncRNA Neat1 was enriched in large EVs in vitro and in vivo. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. In addition, lncRNA Neat1 was transcriptionally regulated by P53 under basal conditions and by HIF2A during hypoxia. The function of Neat1 was further elucidated in vitro and in vivo. Silencing of Neat1 in vitro revealed that Neat1 was indispensable for fibroblast and cardiomyocyte survival and affected fibroblast functions (reduced migration capacity, stalled cell cycle, and decreased expression of fibrotic genes). Of translational importance, genetic loss of Neat1 in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance.

Peripheral artery disease (PAD) is a significant burden, particularly among patients with severe disease requiring invasive treatment. We applied a general Machine Learning (ML) workflow and investigated if a multi-dimensional marker set of standard clinical parameters can identify patients in need of vascular intervention without specialized intra-hospital diagnostics.

Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and in the absence of culprit coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS.