Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFβR3 In Vitro.

The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFβR3 (TGFβ1, TGFβ2, BMP2), for 72 hours were harvested for RNA-seq analysis.

Molecular profiling of dilated cardiomyopathy that progresses to heart failure.

Dilated cardiomyopathy (DCM) is defined by progressive functional and structural changes. We performed RNA-seq at different stages of disease to define molecular signaling in the progression from pre-DCM hearts to DCM and overt heart failure (HF) using a genetic model of DCM (phospholamban missense mutation, PLN). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs.

Using next-generation RNA sequencing to examine ischemic changes induced by cold blood cardioplegia on the human left ventricular myocardium transcriptome.

Background: The exact mechanisms that underlie the pathological processes of myocardial ischemia in humans are unclear. Cardiopulmonary bypass with cardioplegic arrest allows the authors to examine the whole transcriptional profile of human left ventricular myocardium at baseline and after exposure to cold cardioplegia-induced ischemia as a human ischemia model.

Methods: The authors obtained biopsies from 45 patients undergoing aortic valve replacement surgery at baseline and after an average of 79 min of cold cardioplegic arrest.

5'RNA-Seq identifies Fhl1 as a genetic modifier in cardiomyopathy.

The transcriptome is subject to multiple changes during pathogenesis, including the use of alternate 5' start-sites that can affect transcription levels and output. Current RNA sequencing techniques can assess mRNA levels, but do not robustly detect changes in 5' start-site use. Here, we developed a transcriptome sequencing strategy that detects genome-wide changes in start-site usage (5'RNA-Seq) and applied this methodology to identify regulatory events that occur in hypertrophic cardiomyopathy (HCM).

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice.

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery-Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis.

Quantification of microRNA expression with next-generation sequencing.

Rapid advancement of next-generation sequencing technologies has made it possible to study expression profiles of microRNAs (miRNAs) comprehensively and efficiently. Multiplexing miRNA libraries by barcoding can significantly reduce sequencing cost per sample without compromising library quality. This unit provides a step-by-step protocol for isolating miRNAs and constructing multiplexed miRNA libraries.

Spatial transcriptional profile of the chick and mouse endocardial cushions identify novel regulators of endocardial EMT in vitro.

Valvular Interstitial Cells (VICs) are a common substrate for congenital and adult heart disease yet the signaling mechanisms governing their formation during early valvulogenesis are incompletely understood. We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs.

An RNA-seq protocol to identify mRNA expression changes in mouse diaphyseal bone: applications in mice with bone property altering Lrp5 mutations.

Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5(+/+) ), knockout (Lrp5(-/-) ), and high bone mass (HBM)-causing (Lrp5(p.

Polycomb repressive complex 2 regulates normal development of the mouse heart.

Rationale: Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, which establishes H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs.

Objective: We studied the function of PRC2 in murine heart development using a tissue-restricted conditional inactivation strategy.

Barcoding bias in high-throughput multiplex sequencing of miRNA.

Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode.

Construction of normalized RNA-seq libraries for next-generation sequencing using the crab duplex-specific nuclease.

RNA-seq is a method for studying the transcriptome of cells or tissues by massively parallel sequencing of tens of millions of short DNA fragments. However, the broad dynamic range of gene expression levels, which span more than five orders of magnitude, necessitates considerable over-sequencing to characterize low-abundance RNAs at sufficient depth. Here, we describe a method that enables efficient sequencing of low-abundance RNAs by normalizing or reducing the range spanned by the most abundant RNA species to the least abundant RNA species.

Quantification of gene transcripts with deep sequencing analysis of gene expression (DSAGE) using 1 to 2 µg total RNA.

Deep sequencing analysis of gene expression (DSAGE) measures global gene transcript levels from only 1 to 2 µg total RNA by massively parallel sequencing of cDNA tags. This unit describes the construction of 21-bp cDNA tag libraries appropriate for massively parallel sequencing and analysis of the resulting sequence data. The adapter oligonucleotides used are optimized for sequencing with current Illumina massively parallel sequencers, and a step-by-step implementation of the analysis protocol is described.

SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells.

Sir2 is an NAD-dependent deacetylase that connects metabolism with longevity in yeast, flies, and worms. Mammals have seven Sir2 homologs (SIRT1-7). We show that SIRT4 is a mitochondrial enzyme that uses NAD to ADP-ribosylate and downregulate glutamate dehydrogenase (GDH) activity.

Alternative translation initiation generates a novel isoform of insulin-degrading enzyme targeted to mitochondria.

IDE (insulin-degrading enzyme) is a widely expressed zinc-metallopeptidase that has been shown to regulate both cerebral amyloid beta-peptide and plasma insulin levels in vivo. Genetic linkage and allelic association have been reported between the IDE gene locus and both late-onset Alzheimer's disease and Type II diabetes mellitus, suggesting that altered IDE function may contribute to some cases of these highly prevalent disorders. Despite the potentially great importance of this peptidase to health and disease, many fundamental aspects of IDE biology remain unresolved.