Biologically relevant effects of mRNA amplification on gene expression profiles

BACKGROUND: Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. RESULTS: Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. CONCLUSION: This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways

The long noncoding RNA Wisper controls cardiac fibrosis and remodeling

Long noncoding RNAs (lncRNAs) are emerging as powerful regulators of cardiac development and disease. However, our understanding of the importance of these molecules in cardiac fibrosis is limited. Using an integrated genomic screen, we identified Wisper (Wisp2 super-enhancer–associated RNA) as a cardiac fibroblast–enriched lncRNA that regulates cardiac fibrosis after injury. Wisper expression was correlated with cardiac fibrosis both in a murine model of myocardial infarction (MI) and in heart tissue from human patients suffering from aortic stenosis. Loss-of-function approaches in vitro using modified antisense oligonucleotides (ASOs) demonstrated that Wisper is a specific regulator of cardiac fibroblast proliferation, migration, and survival. Accordingly, ASO-mediated silencing of Wisper in vivo attenuated MI-induced fibrosis and cardiac dysfunction. Functionally, Wisper regulates cardiac fibroblast gene expression programs critical for cell identity, extracellular matrix deposition, proliferation, and survival. In addition, its association with TIA1-related protein allows it to control the expression of a profibrotic form of lysyl hydroxylase 2, implicated in collagen cross-linking and stabilization of the matrix. Together, our findings identify Wisper as a cardiac fibroblast–enriched super-enhancer–associated lncRNA that represents an attractive therapeutic target to reduce the pathological development of cardiac fibrosis in response to MI and prevent adverse remodeling in the damaged heart

Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs

Aim Heart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis. Methods and results We annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodelling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease. Conclusion These findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clini

Dichotomy between the transcriptomic landscape of naturally versus accelerated aged murine hearts

We investigated the transcriptomic landscape of the murine myocardium along the course of natural aging and in three distinct mouse models of premature aging with established aging-related cardiac dysfunction. Genome-wide total RNA-seq was performed and the expression patterns of protein-coding genes and non-coding RNAs were compared between hearts from naturally aging mice, mice with cardiac-specific deficiency of a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity and mice with reduced telomere length. Our results demonstrate that no dramatic changes are evident in the transcriptomes of naturally senescent murine hearts until two years of age, in contrast to the transcriptome of accelerated aged mice. Additionally, these mice displayed model-specific alterations of the expression levels of protein-coding and non-coding genes with hardly any overlap with age-related signatures. Our data demonstrate very limited similarities between the transcriptomes of all our murine aging models and question their reliability to study human cardiovascular senescence

Cartilage intermediate layer protein 1 (CILP1): a novel mediator of cardiac extracellular matrix remodelling

Heart failure is accompanied by extracellular matrix (ECM) remodelling, often leading to cardiac fibrosis. In the present study we explored the significance of cartilage intermediate layer protein 1 (CILP1) as a novel mediator of cardiac ECM remodelling. Whole genome transcriptional analysis of human cardiac tissue samples revealed a strong association of CILP1 with many structural (e.g. COL1A2 r2¿=¿0.83) and non-structural (e.g. TGFB3 r2¿=¿0.75) ECM proteins. Gene enrichment analysis further underscored the involvement of CILP1 in human cardiac ECM remodelling and TGFß signalling. Myocardial CILP1 protein levels were significantly elevated in human infarct tissue and in aortic valve stenosis patients. CILP1 mRNA levels markedly increased in mouse heart after myocardial infarction, transverse aortic constriction, and angiotensin II treatment. Cardiac fibroblasts were found to be the primary source of cardiac CILP1 expression. Recombinant CILP1 inhibited TGFß-induced ¿SMA gene and protein expression in cardiac fibroblasts. In addition, CILP1 overexpression in HEK293 cells strongly (5-fold p¿<¿0.05) inhibited TGFß signalling activity. In conclusion, our study identifies CILP1 as a new cardiac matricellular protein interfering with pro-fibrotic TGFß signalling, and as a novel sensitive marker for cardiac fibrosis

Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass

Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM, we combined expression quantitative trait locus1 and quantitative trait transcript (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of 22,000 transcripts, OGN transcript abundance had the highest correlation with LVM. We further confirmed a role for Ogn in the in vivo regulation of LVM in Ogn knockout mice. Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosi

MicroRNA Involvement in Immune Activation During Heart Failure

Heart failure is one of the common end stages of cardiovascular diseases, the leading cause of death in developed countries. Molecular mechanisms underlying the development of heart failure remain elusive but there is a consistent observation of chronic immune activation and aberrant microRNA (miRNA) expression that is present in failing hearts. This review will focus on the interplay between the immune system and miRNAs as factors that play a role during the development of heart failure. Several studies have shown that heart failure patients can be characterized by a sustained innate immune activation. The role of inflammatory signaling is discussed and TLR4 signaling, IL-1β, TNFα and IL-6 expression appears to coincide with the development of heart failure. Furthermore, we describe the implication of the renin angiotensin aldosteron system in immunity and heart failure. In the past decade microRNAs (miRNAs), small non-coding RNAs that translationally repress protein synthesis by binding to partially complementary sequences of mRNA, have come to light as important regulators of several kinds of cardiovascular diseases including cardiac hypertrophy and heart failure. The involvement of differentially expressed miRNAs in the inflammation that occurs during the development of heart failure is still subject of investigation. Here, we summarize and comment on the first studies in this field and hypothesize on the putative involvement of certain miRNAs in heart failure. MicroRNAs have been shown to be critical regulators of cardiac function and inflammation. Future research will have to point out if dampening the immune response, and the miRNAs associated with it, during the development of heart failure is a therapeutically plausible route to follow

Missing links in cardiology: long non-coding RNAs enter the arena

Heart failure as a consequence of ischemic, hypertensive, infectious, or hereditary heart disease is a major challenge in cardiology and topic of intense research. Recently, new players appeared in this field and promise deeper insights into cardiac development, function, and disease. Long non-coding RNAs are a novel class of transcripts that can regulate gene expression and may have many more functions inside the cell. Here, we present examples on long non-coding RNA (lncRNA) function in cardiac development and give suggestions on how lncRNAs may be involved in cardiomyocyte dysfunction, myocardial fibrosis, and inflammation, three hallmarks of the failing heart. Above that, we point out opportunities as well as challenges that should be considered in the endeavor to investigate cardiac lncRNAs