Research Article

Epigenetic coordination of embryonic heart transcription by dynamically regulated long noncoding RNAs

Scot J. Matkovich, John R. Edwards, Tiffani C. Grossenheider, Cristina de Guzman Strong, and Gerald W. Dorn II
PNAS August 19, 2014 111 (33) 12264-12269; first published July 28, 2014 https://doi.org/10.1073/pnas.1410622111

  1. Edited* by Andrew R. Marks, Columbia University College of Physicians and Surgeons, New York, NY, and approved July 1, 2014 (received for review June 9, 2014)

Significance

The role of noncoding RNAs in mammalian biology is of great interest, especially since the Encyclopedia of DNA Elements results were published. We and others have studied microRNAs in the heart, but little is known about their larger cousins, long noncoding RNAs (lncRNAs). Here, we used genome-wide sequencing and improved bioinformatics to quantify lncRNA expression in mouse hearts, define a subset of cardiac-specific lncRNAs, and measure dynamic lncRNA regulation during the transition between embryo and adult, and in the adult heart after experimental pressure overload (a model resembling human hypertensive cardiomyopathy). We linked specific regulated lncRNAs to cardiac-expressed mRNAs that they target and, through network analyses, discovered a broader role of regulated cardiac lncRNAs as modulators of key cardiac transcriptional pathways.

Abstract

The vast majority of mammalian DNA does not encode for proteins but instead is transcribed into noncoding (nc)RNAs having diverse regulatory functions. The poorly characterized subclass of long ncRNAs (lncRNAs) can epigenetically regulate protein-coding genes by interacting locally in cis or distally in trans. A few reports have implicated specific lncRNAs in cardiac development or failure, but precise details of lncRNAs expressed in hearts and how their expression may be altered during embryonic heart development or by adult heart disease is unknown. Using comprehensive quantitative RNA sequencing data from mouse hearts, livers, and skin cells, we identified 321 lncRNAs present in the heart, 117 of which exhibit a cardiac-enriched pattern of expression. By comparing lncRNA profiles of normal embryonic (∼E14), normal adult, and hypertrophied adult hearts, we defined a distinct fetal lncRNA abundance signature that includes 157 lncRNAs differentially expressed compared with adults (fold-change ≥ 50%, false discovery rate = 0.02) and that was only poorly recapitulated in hypertrophied hearts (17 differentially expressed lncRNAs; 13 of these observed in embryonic hearts). Analysis of protein-coding mRNAs from the same samples identified 22 concordantly and 11 reciprocally regulated mRNAs within 10 kb of dynamically expressed lncRNAs, and reciprocal relationships of lncRNA and mRNA levels were validated for the Mccc1 and Relb genes using in vitro lncRNA knockdown in C2C12 cells. Network analysis suggested a central role for lncRNAs in modulating NFκB- and CREB1-regulated genes during embryonic heart growth and identified multiple mRNAs within these pathways that are also regulated, but independently of lncRNAs.

One of the revelations from sequencing whole genomes and the Encyclopedia of DNA Elements project is the small proportion of the mammalian genome dedicated to protein-coding genes. The majority of genomic DNA encode regulatory noncoding (nc)RNAs, i.e., transcripts that instead of simply acting as templates for protein translation exert their own intrinsic functions. MicroRNAs are the best-studied subclass of ncRNAs, being dynamically regulated small (∼20 nt) single-stranded RNAs that, in the heart, are recognized as central orchestrators of cardiac development and stress adaptation. MicroRNAs control entire biological pathways by targeting multiple mRNAs involved in cell growth, differentiation, and apoptosis by suppressing the translation of central protein effectors (1). By contrast, long ncRNAs (lncRNAs) of 200–2,000 nt or larger are distinguished by a diversity of molecular functioning derived from their ability to fold into complex structures and act as scaffolds for protein-protein interactions and/or chaperones that direct protein complexes to specific RNA or DNA sequences (2). Important roles for some lncRNAs are emerging in heart development (3, 4) and have been suggested in experimental and human heart failure (5, 6). However, interpretation and broader application of these early findings is constrained by uncertainty as to how lncRNAs are regulated in different cardiac developmental and disease states and whether regulated lncRNAs differ between these states. Indeed, it is not yet known with certainty which lncRNAs are expressed in mouse hearts, nor have the identities of lncRNAs exhibiting “cardiac-enriched” expression been defined. To address this deficit, we applied comprehensive next-generation sequencing and advanced computational approaches to identify cardiac-expressed and cardiac-specific lncRNAs, defining cardiac lncRNA expression signatures of late embryonic, normal adult, and hemodynamically stressed adult hearts. Building on this foundation, we used bioinformatic analysis to integrate expression profiles and genomic locations of dynamically regulated lncRNAs and mRNAs, identifying and biologically validating cardiac mRNAs whose expression in the developing embryonic heart appears to be directed in part by regulated lncRNAs.

Results

Delineation of Cardiac-Expressed and Cardiac-Enriched lncRNAs.

As a first step to defining mouse cardiac lncRNAs, we interrogated archived raw deep RNA sequencing data from n = 25 normal adult FVB/N mouse hearts (age, 8–16 wk) (7⇓⇓⇓–11) and compared these results to RNA sequencing data from n = 7 mouse livers and n = 6 independent cultures of primary mouse keratinocytes (skin cells). Noncode v3.0 lists ∼37,000 potential lncRNAs in the mouse genome, but these predictions are based largely on unvalidated FANTOM3 cDNAs (12). Therefore, we developed a curated list of 2,997 mouse lncRNAs by combining the annotated lncRNAs from Noncode 2.0, lncRNAdb, Scripture, fRNAdb, Ensembl, RefSeq, and the UCSC Genome database, but eliminated sequences that overlapped with known mRNA exons, leaving 2,140 mouse lncRNAs (SI Methods and Dataset S1). Of these, we detected 736 lncRNAs (∼33% of the annotated list) in at least half of any of the three tissue samples (Dataset S2); lncRNAs comprised 0.3–0.7% of the sequencing reads mapped to transcribed RNAs, which include all defined mRNAs together with the 2,140 defined lncRNAs. Both principal components analysis (Fig. 1A) and unsupervised hierarchical clustering of individual lncRNA sequence read abundance (Fig. 1B and Fig. S1) revealed tissue-specific lncRNA expression profiles, consistent with previous observations that lncRNAs exhibit greater tissue specificity in expression profile than mRNAs (13). A total of 546 lncRNAs were detected in the adult heart samples at levels ranging from 210 RPKM (reads per kilobase of sequence per million reads mapping to transcribed RNAs) for the most abundant lncRNA, n415312, to 0.006 RPKM for lncRNA n411743, one of the longer lncRNAs present at the specified threshold of detection based on sequencing read counts (SI Methods). Approximately 200 lncRNAs detected in hearts were present at very low levels (<0.3 RPKM), likely to be of lesser biological significance (Fig. S1 and Dataset S2). Based on these results and additional RNA sequencing of late embryonic and pressure overloaded hearts (vide infra), we designated 321 lncRNAs as cardiac expressed, i.e., having a mean RPKM of >0.3 in at least one cardiac subgroup (normal embryo, normal adult, adult sham-operated, or pressure overloaded). Of the 321 cardiac-expressed lncRNAs (>0.3 RPKM), 117 were enriched at least threefold in adult hearts compared with liver and skin (Fig. 1 B and C), including the requisite cardiac lncRNA Braveheart (n267831; 16.4 RPKM in adult hearts) (3).

Fig. 1.
Fig. 1.

Tissue selective patterns of mouse lncRNA expression. (A) Principal components analyses of n = 25 adult mouse hearts, n = 7 adult mouse livers, and n = 6 cultured mouse keratinocytes (skin). (B) Heat map display for unsupervised hierarchical clustering of fold-change in expression between the three mouse tissues [reads per million lncRNA aligned reads (RPM)]. (C) Venn diagram revealing patterns of tissue-selective lncRNAs expressed at >0.3 RPKM (reads per kilobase of RNA length per million reads mapped to all transcribed RNA, where all transcribed RNA comprises reads mapped to mRNAs and 2,140 defined lncRNAs).

A total of 152 cardiac lncRNAs were expressed at levels >1 RPKM in normal adult hearts and were designated as abundant cardiac lncRNAs; these comprised at least 90% of the total cardiac lncRNA sequencing reads (Fig. S1 and Dataset S2). Forty-eight of these abundant cardiac lncRNAs, including Braveheart (vide supra), were defined as cardiac enriched. In total, our studies in normal adult mouse hearts identified 48 abundant cardiac-enriched lncRNAs and 104 abundant lncRNAs that are more generally expressed in liver and/or skin, as well as hearts. To determine the cellular fraction(s) to which these abundant cardiac lncRNAs belong, we subjected adult mouse hearts to collagenase digestion (SI Methods) and performed RNA sequencing assays on cardiomyocytes and fibroblasts (Dataset S3). Of the 48 abundant, cardiac-enriched lncRNAs, 32 (67%) were enriched at least threefold in the cardiomyocyte fraction of the heart, whereas only 2 (4.2%) were enriched in the fibroblast fraction (Dataset S3).

Differences in mouse strain can be a source of genetic and transcriptional variation and a confounding factor when comparing experimental results from two or more research groups. We compared lncRNA expression detected by RNA-sequencing on 25 FVB/N hearts and 16 C57BL/6 hearts, two lines with known biological distinctiveness (14⇓⇓–17). Similar lncRNA expression signatures were observed for 546 detectable lncRNAs in these adult mouse hearts (Fig. S2 A and B) with only a few statistically significant differences, most of which involved lncRNAs present at relatively low levels (Fig. S2 C and D and Dataset S4). Thus, lncRNA expression is largely conserved between normal FVB/N and C57BL/6 adult hearts.

lncRNA Expression Profiles Differ in Embryonic vs. Healthy Adult Mouse Hearts.

Cardiac-specific patterns of expression for some lncRNAs suggest that they may play a role in heart development. Indeed, the cardiac-enriched lncRNA Braveheart/n267831 reportedly plays a central regulatory role in cardiomyocyte differentiation (3). We determined what other cardiac lncRNAs are differentially expressed in developing embryonic hearts at ∼E13.5 (range, E13–E14.5), a stage when progenitor cell commitment/differentiation is largely complete and myocardial growth through cardiomyocyte proliferation is active. Because we were interested in identifying those lncRNAs with possible roles in cardiac development, we focused our analyses on lncRNAs present at >0.3 RPKM.

Unsupervised clustering of the raw expression (RPM) values of 321 cardiac-expressed lncRNAs perfectly segregated embryonic from adult hearts (Fig. 2A), identifying a distinct lncRNA signature for developing hearts. A total of 157 lncRNAs (approximately half of cardiac-expressed lncRNAs) exhibited significant differences [false discovery rate (FDR) = 0.02, >50% increase or decrease] in expression between embryonic and adult mouse hearts (Fig. 2 B and C and Dataset S5), elucidating a “fetal gene program” for cardiac lncRNAs analogous to that previously described for cardiac mRNAs (18).

Fig. 2.
Fig. 2.

lncRNA expression is regulated during embryonic development but not after TAC. (A–C) Unsupervised cluster analysis of normal adult and ∼E14 C57BL/6 embryonic heart lncRNA expression. (A) Three hundred twenty-one lncRNAs expressed >0.3 RPKM in hearts. (B) Fifty-two cardiac-selective abundant (>1 RPKM) lncRNAs. (C) One hundred five non–cardiac-selective abundant lncRNAs. (D–F) Same lncRNAs as displayed in A–C, showing data from sham-operated vs. pressure overloaded (TAC) adult hearts, 1 wk, and 4 wk.

lncRNA and mRNA Regulation in Early and Late Cardiac Pressure Overload.

We asked if adult cardiac disease induced fetal-like changes in the lncRNA expression signature. RNA sequencing was performed on four pairs of sham-operated hearts and hearts of mice 1 and 4 wk after induction of acute pressure overload by microsurgical subtotal ligation of the transverse aorta [transverse aortic coarctation (TAC)], which evokes changes in cardiac mRNA and microRNA expression also seen in clinical heart failure (8). In accordance with our previous studies (8, 19), we observed hypertrophy without severe deterioration of cardiac function at both of these time points (Fig. S3). Eleven lncRNAs were significantly regulated 1 wk after TAC (Fig. 2 D–F), with similar trends after 4 wk (Fig. 2 D–F and Dataset S6).

Compared with sham-operated controls, only 17 cardiac-expressed lncRNAs were expressed at different levels after 1 or 4 wk of TAC (Fig. 3A and Dataset S6), despite the usual alterations in fetal gene mRNAs (Fig. 3B). Thirteen of these 17 lncRNAs also showed differences in expression between embryonic and adult hearts. Thus, dynamic expression of lncRNAs was more prominent in cardiac growth when transitioning from embryo to adult (157 lncRNAs) than in the hypertrophic response to increased hemodynamic stress (17 lncRNAs). Comparison of the expression signatures for 321 cardiac-expressed lncRNAs (regardless of statistical categorization) across normal E14.5 embryonic, normal adult, sham operated adult, and pressure overloaded adult (TAC) hearts supports the conclusion that lncRNA expression markedly differs between fetal and adult hearts but is similar in healthy and hypertrophied adult hearts (Fig. 3 C and D and Fig. S4).

Fig. 3.
Fig. 3.

Comparison of embryonic and pressure overload heart lncRNA and mRNA signatures. (A) Unsupervised cluster analysis for 17 abundant lncRNAs regulated after 1 or 4 wk TAC. *Opposite regulation by TAC vs. embryonic state; +regulated by TAC only, not by embryonic state. (B) Analysis of fetal gene mRNA expression in the same embryonic (E), adult (A), sham (S), and 1- and 4-wk TAC hearts (1, 4). (C and D) Combined lncRNA analysis across the four study groups; pressure overload responses are shown only for the 4-wk time point. (C) Principal components analysis. (D) Standardized lncRNA expression (fold-change). (E and F) Same as C and D for mRNA expression. Venn diagrams to the right show patterns of overlap between fetal- and TAC-regulated lncRNAs (Upper) and mRNAs (Lower).

Because lncRNAs exert their effects in part via epigenetic regulation of gene expression (2), we asked what mRNA changes occur concomitantly with changes in lncRNA levels. As expected, mRNA sequencing (10) of the same hearts revealed distinct mRNA signatures for embryonic, adult, and hemodynamically stressed hearts (Fig. 3 E and F, Fig. S4, and Dataset S7). Partial overlap of regulated lncRNAs in embryonic and pressure overloaded hearts (Fig. 3D, Venn diagram) resembles the partial recapitulation of embryonic heart mRNA (Fig. 3F, Venn diagram) and microRNA expression signatures in diseased adult hearts (18, 20⇓–22). The minimal overlap of regulated lncRNAs in fetal and pressure overloaded (TAC) hearts suggests a limited role for lncRNAs in genetic reprogramming of hemodynamically stressed adult hearts.

Coregulated cis lncRNA-mRNA Pairs in the Embryonic-to-Adult Heart Transition.

Genomic location is critical to the actions of lncRNAs that regulate neighboring genes in cis and can help uncover functional lncRNA-mRNA relationships (23). To gain insight into the consequences of lncRNAs on cardiac gene expression, we mapped regulated cardiac lncRNAs to the mouse genome, identified coding mRNAs within 10 kb, and assessed their mutual dynamism as a function of cardiac condition.

We identified 33 lncRNA-mRNA cis pairs in which both the lncRNA and mRNA were regulated (FDR < 0.02; >50% increase or decrease) in embryonic hearts (Fig. 4A). Importantly, none of these lncRNAs were significantly regulated in pressure overloaded adult hearts. Twenty-two lncRNA-mRNA pairs (excluding lncRNAs and mRNAs at overlapping genomic loci) exhibited concordant regulation between embryonic and adult hearts (Fig. S5) (24), whereas 11 lncRNA-mRNA pairs showed nonconcordant regulation (Fig. 4A, asterisks), suggesting a suppressive function of these lncRNAs (24).

Fig. 4.
Fig. 4.

Expression of 10-kb lncRNA-mRNA partners. (A) Heat map of standardized (fold-change) expression for 33 coregulated lncRNA-mRNA pairs. lncRNA/mRNA is indicated at the top. Asterisks show instances of nonconcordant regulation. (B) Quantitative expression of the nonconcordantly regulated lncRNA/mRNA pair n413804/Kcnq1 (black is lncRNA; red is mRNA).

The lncRNAs in reciprocally regulated pairs comprised members of natural antisense transcript, intronic, and intergenic families (Fig. S6), including Kcnq1ot1/n413804 that is encoded on the opposite strand of, and has been validated as regulating, the Kcnq1 gene in embryonic hearts (Fig. 4B) (25). As the remaining pairs represented novel relationships, we used gapmeR (antisense-mediated) knockdown of lncRNAs in the C2C12 mouse skeletal myoblast line to validate the dependency of mRNA expression on cis lncRNA levels for two: lncRNA n411949/mRNA Mccc1 and lncRNA n413445/mRNA Relb. Although C2C12 cells are often used in differentiation studies, we performed these experiments in nondifferentiating C2C12 cells to take advantage of muscle-like background gene expression in cells that could nonetheless be readily transfected. lncRNA n411949 is an example of an antisense lncRNA overlapping protein coding exons, whereas lncRNA n413445 represents an intronic lncRNA. Their cognate mRNAs, Mccc1 and Relb, were increased in response to anti-lncRNA gapmeR transfection (compared with a negative control oligonucleotide transfection), using gapmeRs targeted toward two different positions on each lncRNA (Fig. 5). Thus, these data confirm these lncRNA-mRNA regulatory relationships.

Fig. 5.
Fig. 5.

Nonconcordantly regulated 10-kb lncRNA-mRNA partners in embryonic hearts. (A) (Left) Quantitative expression of the nonconcordantly regulated lncRNA/mRNA pair n411949/Mccc1 (black is lncRNA; red is mRNA) in embryonic and adult hearts; expression in sham and 4-wk TAC hearts is shown for comparison (differences with TAC do not meet significance criteria). (Right) Relative positions of lncRNA n411949 and mRNA Mccc1 within the genome; other transcripts within the locus are colored blue. (B) (Left) Positions targeted by anti-lncRNA gapmers, and primer design for lncRNA (arrows) and mRNA (arrows + internal probe) qPCR detection. (Right) lncRNA and mRNA expression in C2C12 cells transfected with anti-lncRNA gapmers, relative to geometric mean of Actb, Gapdh, and Hmbs (mean ± SEM, n = 6; representative of at least two independent experiments). Non, nontransfected cells; Neg, transfected with fluorescein amidite (FAM)-labeled control gapmer; gapmeR-1 and -2, transfected with one of two different gapmeRs against the chosen lncRNA. *P < 0.05 relative to negative control (Neg). (C and D) Same as A and B, but for the nonconcordantly regulated lncRNA/mRNA pair n413445/Relb.

lncRNA Involvement in Regulated Expression of Genes Important for Cardiac Development.

Two-thirds of the lncRNA-associated mRNAs in the 33 coregulated lncRNA-mRNA pairs could be assigned to functional classes relating to tissue growth and development, including DNA replication and transcription, mRNA processing and translation, and protein synthesis and transport (Table S1). Network analysis of these lncRNA-modulated mRNAs uncovered involvement in major cardiac development and metabolic pathways and also pointed to central roles for the transcription factors CREB1 and NFκB (of which RelB is a critical subunit). Other regulated mRNAs in these pathways are apparently modulated through conventional transcriptional mechanisms rather than by dynamically expressed cardiac lncRNAs (Fig. 6).

Fig. 6.
Fig. 6.

Signaling networks of mRNAs involved in lncRNA-mRNA relationships. MetaCore analysis of functional networks involving regulated cardiac mRNAs linked to regulated cardiac lncRNAs. Green arrows, up-regulation; red arrows, down-regulation; gray lines, context-dependent interaction. Blue circles are mRNAs directly linked to regulated lncRNAs, green circles are mRNAs also regulated, but not connected to lncRNAs described in our study. Note central nodes for NFκB and CREB1.

Discussion

There is an explosion of interest in regulatory noncoding RNAs, especially lncRNAs. We and others have been frustrated by the absence of basic general information on lncRNAs in the fully differentiated mammalian heart and especially by the lack of rigorously annotated, quantitative expression data in adult mouse models (26⇓–28). Here, we identified hundreds of cardiac-expressed and dozens of cardiac-enriched (not cardiac-specific) lncRNAs, providing a foundation on which we can advance our understanding of lncRNA biology in the heart. Our studies delineate greater than 100 dynamically regulated lncRNAs in embryonic hearts but relatively few in reactive adult cardiac hypertrophy. In fact, lncRNAs regulated in hypertrophy thus exhibit only limited recapitulation of fetal lncRNA expression. This observation is consistent with the notion, supported by functional classification and network analysis of regulated lncRNAs and their co- or reciprocally regulated mRNA partners, that a dominant function of cardiac lncRNAs is epigenetic modulation of developmental gene expression.

lncRNAs use diverse molecular regulatory mechanisms to regulate their target genes, including acting as antisense transcripts that directly bind mRNA or acting as chaperones that recruit macromolecular protein complexes to specific sequence-specified locations in the genome (29). A common theme for the latter mechanism is chromatin remodeling that evokes long-term changes in transcriptional activity (27). Such remodeling represents chronic, rather than acute, regulation of gene expression, consistent with our observation that dynamic regulation of lncRNAs and their partner mRNAs is more evident during embryonic heart development than in the adult cardiac stress response. The abundance of lncRNAs that are an order of magnitude less than that of their validated and putative partner mRNAs is also consistent with the notion that at least some lncRNAs act in a nonlinear manner via recruitment of chromatin remodeling protein complexes to regulate mRNA transcription.

Little is known about overall lncRNA function in the heart, but three cardiac-expressed lncRNAs have been studied in detail. Kcnq1ot1 (n413804) mediates transcriptional silencing through histone methylation of the overlapping Kcnq1/Kv7.1 potassium channel gene and other genes at the same genomic locus (30⇓–32). In our studies, Kcnq1ot1 is expressed at high levels in embryonic hearts, but decreases by fivefold in adult hearts. Its mRNA partner, Kcnq1, encodes the Kv7.1 slow delayed rectifying potassium channel that is essential for normal cardiomyocyte repolarization that terminates the action potential and cardiomyocyte contraction. Coinciding with decreased Kcnq1ot1 expression, levels of Kcnq1 mRNA increase, consistent with the need for enhanced pump function during the transition between developing embryonic and fully functioning adult hearts.

Fendrr is expressed specifically in embryonic lateral mesoderm where it regulates heart development, likely by modifying the chromatin signatures of genes encoding transcription factors that direct cardiomyocyte differentiation (4). We did not detect Fendrr in hearts of ∼E14.5 mouse embryos or of adult mice, consistent with a transient role in the specification and differentiation of mesoderm to cardiomyocytes, which is largely complete and supplanted by cardiomyocyte proliferation at E14.5.

Braveheart (Bvht) is another cardiac-expressed lncRNA that epigenetically regulates cardiomyocyte differentiation (3). In our studies, Braveheart (annotated as n267831) was cardiac-enriched approximately threefold compared with other tissues, but expressed in the heart at similar levels in E14.5 embryos and adults; Braveheart is also not regulated late after hemodynamic stress. Constitutive cardiac expression of Braveheart suggests that it may have “housekeeping” roles in adult hearts in addition to its canonical role upstream of MesP1 to stimulate and maintain cardiomyocyte fate.

Our studies identified possible functional interactions between 10 further lncRNAs and neighboring mRNAs that were reciprocally regulated between embryonic and adult hearts; we validated two of these using lncRNA knockdown in mouse C2C12 skeletal myoblasts. The importance of one of these lncRNA-regulated mRNAs, Relb, was captured in our network analysis pointing to a central role for NFκB in modulating altered gene expression during cardiac growth and transition to the adult heart. lncRNA n413445 expression is high in the embryonic heart but is quite low in adult hearts. As its levels decline, levels of Relb mRNA increase, providing increased expression of the RelB transcription factor that is an essential component of the NFκB pathway (33). An increase of the lncRNA n411949-regulated mRNA Mccc1 during fetal cardiac growth may be of key importance for sensing free leucine levels and thus the availability of branched-chain amino acids for anabolic signaling in muscle (34, 35). Mccc1 is one among several enzymes that metabolize leucine; interestingly, the mRNA levels of at least three others in the same catabolic pathway (Ivd, Mccc2, and Auh) increase dramatically during the transition to the adult heart (Dataset S7).

Study Limitations.

Our studies of embryonic hearts were designed to be compared with pressure overloaded adult hearts and therefore focused on a relatively late time during development when cardiomyocyte growth was dominant and mesodermal specification and differentiation were largely complete. A detailed time course of lncRNA expression in earlier stage embryos will be required to understand the role of lncRNA regulation in cardiomyocyte differentiation. Likewise, studies in postnatal hearts during the transition from cardiomyocyte proliferation to cardiomyocyte hypertrophy are needed to define the roles of lncRNAs during that important transition. We studied pressure overloaded hearts at 1 and 4 wk after surgical transverse banding, i.e., early cardiomyocyte hypertrophy (1 wk) (8) and late hypertrophy with mild deterioration of heart function (4 wk) (19). We selected these time points to capture lncRNA regulation in either cardiac growth or the transition to cardiac failure and found relatively few alterations in lncRNAs. Nonetheless, analyses performed even earlier after surgical modeling or late in overt heart failure may reveal additional regulated lncRNAs that our studies did not identify. Finally, we used conventional RNA sequencing that does not discriminate between mRNA and lncRNA exons encoded on opposite strands at the same genomic location. Future studies could use strand-specific RNA sequencing from nuclear and cytoplasmic fractions to expand the universe of constitutively expressed and dynamically regulated cardiac lncRNAs described here.

On a positive note, our studies of anti-lncRNA knockdown and assessment of neighboring mRNA expression demonstrated functional relationships between cis lncRNA and mRNA pairs. This approach should be useful in future studies designed to further elucidate cardiac lncRNA function.

Methods

Definition of lncRNA and mRNA Sequences.

lncRNA sequences were obtained from annotated lncRNA entries from Noncode 2.0, lncRNAdb, Scripture, fRNAdb, Ensembl, RefSeq, and the UCSC Genome database. Annotation details and genomic locations (using the NCBIM37/mm9 release of the mouse genome) for 2,140 lncRNAs can be found in Dataset S1. mRNA sequences were obtained from the gtf supplied with the Ensembl iGenomes 2011 (NCBIM37) index.

RNA Sequencing Library Preparation, Read Generation, and Mapping.

Mouse heart, liver, and primary keratinocyte (skin) RNA sequencing libraries were prepared from polyA-selected RNA as previously described (19, 36). Alignment with Tophat before differential expression analysis with DESeq was performed as previously described (37).

Heart libraries were sequenced to a depth of 6.7 ± 0.8 × 106 (mean ± SEM) reads mapped to mRNAs and 3.0 ± 0.3 × 104 reads mapped unequivocally to lncRNAs within the same libraries. Similar sequencing depths were obtained for liver and skin libraries. Overall, lncRNAs comprised ∼0.4–0.7% of the total reads assigned to transcribed RNA.

RNA Quantitation and Differential Expression.

RNA count data in heatmaps are presented as read counts normalized for total read number only, e.g., RPM (lncRNA reads per million reads mapped exclusively to lncRNAs). Alternatively, RPM input data were standardized (mean of 0, SD of 1) to better display the extent of variation among sample groups for a given lncRNA. Use of raw or standardized heatmaps is denoted in figure panels. All unsupervised clustering was performed with Euclidean distance and average linkage.

RNA expression is presented in the text, tables, and datasets as RPKM. Partek Genomics Suite 6.6 (Partek) was used for principal components analyses, standardization, and heatmap generation.

For differential expression analyses, RNA read count data were not normalized or standardized before input into the DESeq package (38). Cutoffs were established a priori at a fold-change ≥ 50% (FDR < 0.02). Output data in the tables and datasets are presented as RPKM for consistency with the remainder of the text, together with fold-change and adjusted P values (FDRs) computed by DESeq.

Further details are given in SI Methods.

Acknowledgments

This work was supported by National Institutes of Health Grants R01 HL108943-02 (to G.W.D.) and UL1 TR000448 (to the Washington University Institute of Clinical and Translational Sciences from the National Center for Advancing Translational Sciences).

Footnotes

  • ↵1To whom correspondence may be addressed. Email: smatkovi{at}dom.wustl.edu or gdorn{at}dom.wustl.edu.

  • Author contributions: S.J.M. and G.W.D. designed research; S.J.M., T.C.G., and C.d.G.S. performed research; J.R.E. contributed new reagents/analytic tools; S.J.M. and G.W.D. analyzed data; and S.J.M. and G.W.D. wrote the paper.

  • The authors declare no conflict of interest.

  • ↵*This Direct Submission article had a prearranged editor.

  • Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession nos. GSE58453, GSE58455, and GSE56890) and Sequence Read Archive (SRA) database (accession no. SRP026654).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1410622111/-/DCSupplemental.

References

  1. ↵
    1. Mendell JT,
    2. Olson EN
    (2012) MicroRNAs in stress signaling and human disease. Cell 148(6):1172–1187.
    .
    OpenUrlCrossRefPubMed
  2. ↵
    1. Batista PJ,
    2. Chang HY
    (2013) Long noncoding RNAs: Cellular address codes in development and disease. Cell 152(6):1298–1307.
    .
    OpenUrlCrossRefPubMed
  3. ↵
    1. Klattenhoff CA,
    2. et al.
    (2013) Braveheart, a long noncoding RNA required for cardiovascular lineage commitment. Cell 152(3):570–583.
    .
    OpenUrlCrossRefPubMed
  4. ↵
    1. Grote P,
    2. et al.
    (2013) The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse. Dev Cell 24(2):206–214.
    .
    OpenUrlCrossRefPubMed
  5. ↵
    1. Lee JH,
    2. et al.
    (2011) Analysis of transcriptome complexity through RNA sequencing in normal and failing murine hearts. Circ Res 109(12):1332–1341.
    .
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Yang KC,
    2. et al.
    (2014) Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support. Circulation 129(9):1009–1021.
    .
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Dorn GW 2nd
    (2012) Decoding the cardiac message: The 2011 Thomas W. Smith Memorial Lecture. Circ Res 110(5):755–763.
    .
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Hu Y,
    2. et al.
    (2012) Epitranscriptional orchestration of genetic reprogramming is an emergent property of stress-regulated cardiac microRNAs. Proc Natl Acad Sci USA 109(48):19864–19869.
    .
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Matkovich SJ,
    2. Van Booven DJ,
    3. Eschenbacher WH,
    4. Dorn GW 2nd
    (2011) RISC RNA sequencing for context-specific identification of in vivo microRNA targets. Circ Res 108(1):18–26.
    .
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Matkovich SJ,
    2. et al.
    (2010) MicroRNA-133a protects against myocardial fibrosis and modulates electrical repolarization without affecting hypertrophy in pressure-overloaded adult hearts. Circ Res 106(1):166–175.
    .
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Chen Y,
    2. et al.
    (2013) A nucleus-targeted alternately spliced Nix/Bnip3L protein isoform modifies nuclear factor κB (NFκB)-mediated cardiac transcription. J Biol Chem 288(22):15455–15465.
    .
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Maeda N,
    2. et al.
    (2006) Transcript annotation in FANTOM3: Mouse gene catalog based on physical cDNAs. PLoS Genet 2(4):e62.
    .
    OpenUrlCrossRefPubMed
  13. ↵
    1. Cabili MN,
    2. et al.
    (2011) Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev 25(18):1915–1927.
    .
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Dansky HM,
    2. et al.
    (1999) Genetic background determines the extent of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 19(8):1960–1968.
    .
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Jo J,
    2. et al.
    (2009) Hypertrophy and/or hyperplasia: Dynamics of adipose tissue growth. PLOS Comput Biol 5(3):e1000324.
    .
    OpenUrlCrossRefPubMed
  16. ↵
    1. Li TT,
    2. et al.
    (2004) Genetic variation responsible for mouse strain differences in integrin alpha 2 expression is associated with altered platelet responses to collagen. Blood 103(9):3396–3402.
    .
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Kim DH,
    2. Gutierrez-Aguilar R,
    3. Kim HJ,
    4. Woods SC,
    5. Seeley RJ
    (2013) Increased adipose tissue hypoxia and capacity for angiogenesis and inflammation in young diet-sensitive C57 mice compared with diet-resistant FVB mice. Int J Obes (Lond) 37(6):853–860.
    .
    OpenUrlCrossRefPubMed
  18. ↵
    1. Izumo S,
    2. Nadal-Ginard B,
    3. Mahdavi V
    (1988) Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc Natl Acad Sci USA 85(2):339–343.
    .
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Matkovich SJ,
    2. Hu Y,
    3. Eschenbacher WH,
    4. Dorn LE,
    5. Dorn GW 2nd
    (2012) Direct and indirect involvement of microRNA-499 in clinical and experimental cardiomyopathy. Circ Res 111(5):521–531.
    .
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Frey N,
    2. Olson EN
    (2003) Cardiac hypertrophy: The good, the bad, and the ugly. Annu Rev Physiol 65:45–79.
    .
    OpenUrlCrossRefPubMed
  21. ↵
    1. Rajabi M,
    2. Kassiotis C,
    3. Razeghi P,
    4. Taegtmeyer H
    (2007) Return to the fetal gene program protects the stressed heart: A strong hypothesis. Heart Fail Rev 12(3-4):331–343.
    .
    OpenUrlCrossRefPubMed
  22. ↵
    1. Thum T,
    2. et al.
    (2007) MicroRNAs in the human heart: A clue to fetal gene reprogramming in heart failure. Circulation 116(3):258–267.
    .
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Derrien T,
    2. Guigó R,
    3. Johnson R
    (2011) The long non-coding RNAs: A new (p)layer in the “dark matter” Front Genet 2:107.
    .
    OpenUrlPubMed
  24. ↵
    1. Katayama S,
    2. et al.,
    3. RIKEN Genome Exploration Research Group,
    4. Genome Science Group (Genome Network Project Core Group),
    5. FANTOM Consortium
    (2005) Antisense transcription in the mammalian transcriptome. Science 309(5740):1564–1566.
    .
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Korostowski L,
    2. Sedlak N,
    3. Engel N
    (2012) The Kcnq1ot1 long non-coding RNA affects chromatin conformation and expression of Kcnq1, but does not regulate its imprinting in the developing heart. PLoS Genet 8(9):e1002956.
    .
    OpenUrlCrossRefPubMed
  26. ↵
    1. Ounzain S,
    2. Crippa S,
    3. Pedrazzini T
    (2013) Small and long non-coding RNAs in cardiac homeostasis and regeneration. Biochim Biophys Acta 1833(4):923–933.
    .
    OpenUrl
  27. ↵
    1. Papait R,
    2. Kunderfranco P,
    3. Stirparo GG,
    4. Latronico MV,
    5. Condorelli G
    (2013) Long noncoding RNA: A new player of heart failure? J Cardiovasc Transl Res 6(6):876–883.
    .
    OpenUrlCrossRefPubMed
  28. ↵
    1. Kataoka M,
    2. Huang ZP,
    3. Wang DZ
    (2013) Build a braveheart: The missing linc (RNA). Circ Res 112(12):1532–1534.
    .
    OpenUrlFREE Full Text
  29. ↵
    1. Wang KC,
    2. Chang HY
    (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43(6):904–914.
    .
    OpenUrlCrossRefPubMed
  30. ↵
    1. Mohammad F,
    2. Mondal T,
    3. Guseva N,
    4. Pandey GK,
    5. Kanduri C
    (2010) Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1. Development 137(15):2493–2499.
    .
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Mohammad F,
    2. et al.
    (2008) Kcnq1ot1/Lit1 noncoding RNA mediates transcriptional silencing by targeting to the perinucleolar region. Mol Cell Biol 28(11):3713–3728.
    .
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Pandey RR,
    2. et al.
    (2008) Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell 32(2):232–246.
    .
    OpenUrlCrossRefPubMed
  33. ↵
    1. Perkins ND
    (2007) Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8(1):49–62.
    .
    OpenUrlCrossRefPubMed
  34. ↵
    1. Norton LE,
    2. Layman DK
    (2006) Leucine regulates translation initiation of protein synthesis in skeletal muscle after exercise. J Nutr 136(2):533S–537S.
    .
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Suryawan A,
    2. et al.
    (2012) Enteral leucine supplementation increases protein synthesis in skeletal and cardiac muscles and visceral tissues of neonatal pigs through mTORC1-dependent pathways. Pediatr Res 71(4 Pt 1):324–331.
    .
    OpenUrlCrossRefPubMed
  36. ↵
    1. Matkovich SJ,
    2. Zhang Y,
    3. Van Booven DJ,
    4. Dorn GW 2nd
    (2010) Deep mRNA sequencing for in vivo functional analysis of cardiac transcriptional regulators: Application to Galphaq. Circ Res 106(9):1459–1467.
    .
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Matkovich SJ,
    2. Hu Y,
    3. Dorn GW 2nd
    (2013) Regulation of cardiac microRNAs by cardiac microRNAs. Circ Res 113(1):62–71.
    .
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Anders S,
    2. Huber W
    (2010) Differential expression analysis for sequence count data. Genome Biol 11(10):R106.
    .
    OpenUrlCrossRefPubMed
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lncRNAs in the mouse heart
Scot J. Matkovich, John R. Edwards, Tiffani C. Grossenheider, Cristina de Guzman Strong, Gerald W. Dorn
Proceedings of the National Academy of Sciences Aug 2014, 111 (33) 12264-12269; DOI: 10.1073/pnas.1410622111
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