Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling

Distinct stressors may induce heart failure. As compensation, β-adrenergic stimulation enhances myocardial contractility by elevating cardiomyocyte intracellular Ca2+ ([Ca2+]i). However, chronic β-adrenergic stimulation promotes adverse cardiac remodelling. Cardiac expression of nuclear receptor Nur77 is enhanced by β-adrenergic stimulation, but its role in cardiac remodelling is still unclear. We show high and rapid Nur77 upregulation in cardiomyocytes stimulated with β-adrenergic agonist isoproterenol. Nur77 knockdown in culture resulted in hypertrophic cardiomyocytes. Ventricular cardiomyocytes from Nur77-deficient (Nur77-KO) mice exhibited elevated diastolic and systolic [Ca2+]i and prolonged action potentials compared to wild type (WT). In vivo, these differences resulted in larger cardiomyocytes, increased expression of hypertrophic genes

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide

Small sample sizes in high-throughput miRNA screens: A common pitfall for the identification of miRNA biomarkers

Since the discovery of microRNAs (miRNAs), circulating miRNAs have been proposed as biomarkers for disease. Consequently, many groups have tried to identify circulating miRNA biomarkers for various types of diseases including cardiovascular disease and cancer. However, the replicability of these experiments has been disappointingly low. In order to identify circulating miRNA candidate biomarkers, in general, first an unbiased high-throughput screen is performed in which a large number of miRNAs is detected and quantified in the circulation. Because these are costly experiments, many of such studies have been performed using a low number of study subjects (small sample size). Due to lack of power in small sample size experiments, true effects are often missed and many of the detected effects are wrong. Therefore, it is important to have a good estimate of the appropriate sample size for a miRNA high-throughput screen. In this review, we discuss the effects of small sample sizes in high-throughput screens for circulating miRNAs. Using data from a miRNA high-throughput experiment on isolated monocytes, we illustrate that the implementation of power calculations in a high-throughput miRNA discovery experiment will avoid unnecessarily large and expensive experiments, while still having enough power to be able to detect clinically important differences. Keywords: MicroRNA, High-throughput screens, Biomarkers, Small sample size error, Array, Methodolog

Increased matrix metalloproteinase-8 and -9 activity in patients with infarct rupture after myocardial infarction

Background: Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an important role in infarct rupture. The present study was designed to study the role of MMP-2, MMP-8, and MMP-9 in human infarct rupture. Methods: Heart samples were obtained from patients who died from infarct rupture and control MI patients. The MMP activity was determined by zymography and quantitative immunocapture activity assay. TIMP-1 levels were measured and immunohistochemistry for MMP-2 and MMP-9 was performed. Results: The amounts of both total and active MMP-8 and MMP-9 were significantly higher in ruptured infarct tissue than in control MI tissue, but no differences in MMP-2 activity were observed. Furthermore, the number of inflammatory cells was significantly higher in the ruptured infarcts than in control infarcts. Conclusions: These data suggest that increased MMP-8 and MMP-9 activity in the infarct area, caused by a more prominent infiltration of inflammatory cells, contribute to infarct rupture in humans. © 2009 Elsevier Inc. All rights reserved

Long noncoding RNAs in cardiac development and ageing.

A large part of the mammalian genome is transcribed into noncoding RNAs. Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators of gene expression. Distinct molecular mechanisms allow lncRNAs either to activate or to repress gene expression, thereby participating in the regulation of cellular and tissue function. LncRNAs, therefore, have important roles in healthy and diseased hearts, and might be targets for therapeutic intervention. In this Review, we summarize the current knowledge of the roles of lncRNAs in cardiac development and ageing. After describing the definition and classification of lncRNAs, we present an overview of the mechanisms by which lncRNAs regulate gene expression. We discuss the multiple roles of lncRNAs in the heart, and focus on the regulation of embryonic stem cell differentiation, cardiac cell fate and development, and cardiac ageing. We emphasize the importance of chromatin remodelling in this regulation. Finally, we discuss the therapeutic and biomarker potential of lncRNAs

Genome-Wide polyadenylation maps reveal dynamic mRNA 3?-End formation in the failing human heart

10.1161/CIRCRESAHA.115.307082Circulation Research1183433-438CIRU

Transcriptome-wide co-expression analysis identifies LRRC2 as a novel mediator of mitochondrial and cardiac function

10.1371/journal.pone.0170458PLoS ONE122e0170458

Variants in the 3&#39; untranslated region of the <em>KCNQ1</em>-encoded K_v7.1 potassium channel modify disease severity in patients with type 1 long QT syndrome in an allele-specific manner.

Aims Heterozygous mutations in KCNQ1 cause type 1 long QT syndrome (LQT1), a disease characterized by prolonged heart rate-corrected QT interval (QTc) and life-threatening arrhythmias. It is unknown why disease penetrance and expressivity is so variable between individuals hosting identical mutations. We aimed to study whether this can be explained by single nucleotide polymorphisms (SNPs) in KCNQ1&#39;s 3&#39; untranslated region (3&#39;UTR). Methods and results This study was performed in 84 LQT1 patients from the Academic Medical Center in Amsterdam and validated in 84 LQT1 patients from the Mayo Clinic in Rochester. All patients were genotyped for SNPs in KCNQ1&#39;s 3&#39;UTR, and six SNPs were found. Single nucleotide polymorphisms rs2519184, rs8234, and rs10798 were associated in an allele-specific manner with QTc and symptom occurrence. Patients with the derived SNP variants on their mutated KCNQ1 allele had shorter QTc and fewer symptoms, while the opposite was also true: patients with the derived SNP variants on their normal KCNQ1 allele had significantly longer QTc and more symptoms. Luciferase reporter assays showed that the expression of KCNQ1&#39;s 3&#39;UTR with the derived SNP variants was lower than the expression of the 3&#39;UTR with the ancestral SNP variants. Conclusion Our data indicate that 3&#39;UTR SNPs potently modify disease severity in LQT1. The allele-specific effects of the SNPs on disease severity and gene expression strongly suggest that they are functional variants that directly alter the expression of the allele on which they reside, and thereby influence the balance between proteins stemming from either the normal or the mutant KCNQ1 allele

Macrophage MicroRNA-155 Promotes Cardiac Hypertrophy and Failure

Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease.Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency.Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure