Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFßR3 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 TGFßR3 (TGFß1, TGFß2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFß1 (515), TGFß2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFß1-TGFß2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFß and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFß2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFß2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFßR3. Overall, the genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFßR3 signaling.

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, PLNR9C/+). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs. WT, P = 8 × 10-4) and activation of proinflammatory signaling with notable cardiomyocyte-specific induction of a subset of profibrotic cytokines including TGFß2 and TGFß3. These changes progressed through DCM and HF, resulting in substantial fibrosis (17.6% of left ventricle [LV] vs. WT, P = 6 × 10-33). Cardiomyocytes displayed a marked shift in metabolic gene transcription: downregulation of aerobic respiration and subsequent upregulation of glucose utilization, changes coincident with attenuated expression of PPARα and PPARγ coactivators -1α (PGC1α) and -1ß, and increased expression of the metabolic regulator T-box transcription factor 15 (Tbx15). Comparing DCM transcriptional profiles with those in hypertrophic cardiomyopathy (HCM) revealed similar and distinct molecular mechanisms. Our data suggest that cardiomyocyte-specific cytokine expression, early fibroblast activation, and the shift in metabolic gene expression are hallmarks of cardiomyopathy progression. Notably, key components of these profibrotic and metabolic networks were disease specific and distinguish DCM from HCM.

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. Samples were RNA sequenced and analyzed with the Partek Genomics Suite (Partek Inc., St. Louis, MO) for differential expression. Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA) and Biobase ExPlain (Biobase GmbH, Wolfenbuettel, Germany) systems were used for functional and pathway analyses. RESULTS: Of the 4,098 genes with a mean expression value greater than 5, 90% were down-regulated and 9.1% were up-regulated. Of those, 1,241 were significantly differentially expressed. Gene ontology analysis revealed significant down-regulation in immune inflammatory response and complement activation categories and highly consistent was the down-regulation of intelectin 1, proteoglycan, and secretory leukocyte peptidase inhibitor. Up-regulated genes of interest were FBJ murine osteosarcoma viral oncogene homolog and the hemoglobin genes hemoglobin α1 (HBA1) and hemoglobin ß. In addition, analysis of transcription factor-binding sites revealed interesting targets in factors regulating reactive oxygen species production, apoptosis, immunity, cytokine production, and inflammatory response. CONCLUSIONS: The authors have shown that the human left ventricle exhibits significant changes in gene expression in response to cold cardioplegia-induced ischemia during cardiopulmonary bypass, which provides great insight into the pathophysiology of ventricular ischemia, and thus, may help guide efforts to reduce myocardial damage during surgery.

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). Compared with transcripts from WT mice, 92 genes had altered start-site usage in a mouse model of HCM, including four-and-a-half LIM domains protein 1 (Fhl1). HCM-induced altered transcriptional regulation of Fhl1 resulted in robust myocyte expression of a distinct protein isoform, a response that was conserved in humans with genetic or acquired cardiomyopathies. Genetic ablation of Fhl1 in HCM mice was deleterious, which suggests that Fhl1 transcriptional changes provide salutary effects on stressed myocytes in this disease. Because Fhl1 is a chromosome X-encoded gene, stress-induced changes in its transcription may contribute to gender differences in the clinical severity of HCM. Our findings indicate that 5'RNA-Seq has the potential to identify genome-wide changes in 5' start-site usage that are associated with pathogenic phenotypes.

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. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations.

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. Also described is a custom computational pipeline for analyzing the multiplexed miRNA library sequencing reads generated by Illumina-based technology.

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.A214V/+) ) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, Lrp5(+/+) and Lrp5(p.A214V/+) ) and "insufficient" (Lrp5(-/-) ) diaphyseal bone, and far fewer differentially expressed genes between Lrp5(p.A214V/+) and Lrp5(+/+) diaphyseal bone.

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. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 198 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGFß as a nodal point and identified NF-κB as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-κB, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-κB pathway as regulators of endocardial cell EMT in vitro.

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. METHODS AND RESULTS: Inactivation of the PRC2 subunit Ezh2 by Nkx2-5(Cre) (Ezh2(NK)) caused lethal congenital heart malformations, namely, compact myocardial hypoplasia, hypertrabeculation, and ventricular septal defect. Candidate and genome-wide RNA expression profiling and chromatin immunoprecipitation analyses of Ezh2(NK) heart identified genes directly repressed by EZH2. Among these were the potent cell cycle inhibitors Ink4a/b (inhibitors of cyclin-dependent kinase 4 A and B), the upregulation of which was associated with decreased cardiomyocyte proliferation in Ezh2(NK). EZH2-repressed genes were enriched for transcriptional regulators of noncardiomyocyte expression programs such as Pax6, Isl1, and Six1. EZH2 was also required for proper spatiotemporal regulation of cardiac gene expression, because Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. PRC2 was also required later in heart development, as indicated by cardiomyocyte-restricted TNT-Cre inactivation of the PRC2 subunit Eed. However, Ezh2 inactivation by TNT-Cre did not cause an overt phenotype, likely because of functional redundancy with Ezh1. Thus, early Ezh2 inactivation by Nk2-5(Cre) caused later disruption of cardiomyocyte gene expression and heart development. CONCLUSIONS: Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early in cardiogenesis has sustained disruptive effects at later developmental stages.

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. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number.

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. This normalization is achieved using an approach that was developed for generating expressed sequence tag (EST) libraries that uses the crab duplex-specific nuclease and exploits the kinetics of DNA annealing. That is, double-stranded cDNA is denatured, then allowed to partially re-anneal, and the most abundant species, which re-anneal most rapidly, are digested with crab duplex-specific nuclease. This procedure substantially decreases the proportion of sequence reads from highly expressed RNAs, facilitating assessment of the full spectrum of the sequence and structure of transcriptomes.

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. The expression profiles obtained are highly reproducible, enabling sensitive detection of differences between experimental conditions as well as assessment of the relative transcript abundance of different genes.

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. GDH is known to promote the metabolism of glutamate and glutamine, generating ATP, which promotes insulin secretion. Loss of SIRT4 in insulinoma cells activates GDH, thereby upregulating amino acid-stimulated insulin secretion. A similar effect is observed in pancreatic beta cells from mice deficient in SIRT4 or on the dietary regimen of calorie restriction (CR). Furthermore, GDH from SIRT4-deficient or CR mice is insensitive to phosphodiesterase, an enzyme that cleaves ADP-ribose, suggesting the absence of ADP-ribosylation. These results indicate that SIRT4 functions in beta cell mitochondria to repress the activity of GDH by ADP-ribosylation, thereby downregulating insulin secretion in response to amino acids, effects that are alleviated during CR.

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. Here we identify a previously undescribed mitochondrial isoform of IDE generated by translation at an in-frame initiation codon 123 nucleotides upstream of the canonical translation start site, which results in the addition of a 41-amino-acid N-terminal mitochondrial targeting sequence. Fusion of this sequence to the N-terminus of green fluorescent protein directed this normally cytosolic protein to mitochondria, and full-length IDE constructs containing this sequence were also directed to mitochondria, as revealed by immuno-electron microscopy. Endogenous IDE protein was detected in purified mitochondria, where it was protected from digestion by trypsin and migrated at a size consistent with the predicted removal of the N-terminal targeting sequence upon transport into the mitochondrion. Functionally, we provide evidence that IDE can degrade cleaved mitochondrial targeting sequences. Our results identify new mechanisms regulating the subcellular localization of IDE and suggest previously unrecognized roles for IDE within mitochondria.