The recent discovery that thousands of RNAs are transcribed by the cell but are never translated into protein, highlights a significant void in our current understanding of how transcriptional networks regulate cellular function. This is particularly astounding when we consider that over 75% of the human genome is transcribed into RNA, but only approximately 2% of RNA is translated into known proteins. This raises the question as to what function the other so-called ‘non-coding RNAs’ (ncRNAs) are performing in the cell. Over the last decade, an enormous amount of research has identified several classes of ncRNAs, predominantly short ncRNAs (<200 nt) that have been confirmed to have functional significance. Recent advances in sequencing technology and bioinformatics have also allowed for the identification of a novel class of ncRNAs, termed long ncRNA (lncRNA) (>200 nt). Several studies have recently shown that long non-coding RNAs (lncRNAs) are associated with tissue development and disease, particularly in cell types that undergo differentiation such as stem cells, cancer cells and striated muscle (skeletal/cardiac). Therefore, understanding the function of these lncRNAs and designing strategies to detect and manipulate them, may present novel therapeutic and diagnostic opportunities. This review will explore the current literature on lncRNAs in skeletal and cardiac muscle and discuss their recent implication in development and disease. Lastly, we will also explore the possibility of using lncRNAs as therapeutic and diagnostic tools and discuss the opportunities and potential shortcomings to these applications.

INTRODUCTION

It has been predicted that >75% of the entire human genome is transcribed into RNA, however only approximately 2–3% codes for proteins [1]. The remaining RNAs are therefore considered as non-protein coding RNAs (non-coding RNA; ncRNA). Aside from the well described transfer and ribosomal RNAs, the remaining species were previously considered as ‘junk’ or ‘transcriptional artefact’, which had no specific role in cellular function [2]. Over the last decade, an enormous amount of research has identified several classes of ncRNAs, predominantly short ncRNAs (<200 nt) including small nucleolar (sno)RNAs, piwi-interacting (pi)RNA and micro (mi)RNA, that have been confirmed to have functional significance. Recent advances in sequencing technology and bioinformatics has also allowed for the identification of a novel class of ncRNAs, termed long (>200 nt) ncRNA (lncRNA), that often have a similar structure and expression pattern to protein coding messenger RNAs (mRNA) [3].

Skeletal and cardiac muscle development, growth and function are regulated by multiple signalling cascades and core transcriptional pathways. However, previous evidence has demonstrated that in addition to these well accepted pathways, a large amount of regulation occurs outside of protein-centric mechanisms [4,5]. For instance, miRNAs are recognized to be differentially regulated in skeletal and cardiac muscle in settings of disease [6]. Here, we focus on the regulation and potential role of long non-coding RNAs (lncRNAs) in skeletal and cardiac muscle, and discuss their therapeutic and diagnostic potential.

NON-CODING RNAs

ncRNAs are anatomically separated into two classes, short and long, based conveniently on the number of nucleotides (nt) in their final spliced sequence. Small ncRNAs are <200 nt in length and make up a large group of species which include snoRNA, piRNA, siRNA and miRNAs [7]. Many studies have implicated these ncRNAs in cellular regulation, probably none more so than miRNAs of which >2000 have been annotated and partially characterized [2]. Indeed, the discovery of miRNAs and their ability to functionally regulate entire signalling pathways almost exclusively at the RNA level, fundamentally changed the central dogma of transcriptional regulation, and focused the attention of many research groups on ncRNA biology. Finally, the remaining species of ncRNAs are those that are over 200 nt in length, which are called lncRNAs, and have only been identified comparatively more recently.

LONG NON-CODING RNAs

Although the convenient classification of an lncRNA is generally those that are greater than 200 nucleotides, the final spliced form of many lncRNAs can span several kilobases in length [8]. The majority of lncRNAs are very similar in many ways to protein coding mRNA in that they have multiple exons/introns and are transcribed and processed in an almost identical fashion to mRNAs [9] (Figure 1). Furthermore, similar to protein coding mRNAs, lncRNAs also demonstrate classic chromatin modifying marks such as histone methylation and acetylation at their genomic promoters [9]. However as their name suggests, lncRNAs are non-coding and thus are not expected to associate with ribosomes or code for proteins. Intriguingly, this expectation has recently been challenged with a handful of studies demonstrating that transcripts that otherwise qualify as lncRNAs, in fact code for micro-peptides [1012], a discovery which further highlights our lack of understanding and potential mis-classification of some of these transcripts.

Analogous Transcriptional Mechanisms for mRNAs and lncRNAs

Figure 1
Analogous Transcriptional Mechanisms for mRNAs and lncRNAs

Schematic depicting the similar transcriptional mechanisms and nucleotide structure (introns, exons–blue boxes) between mRNA and lncRNA including their polymerization by Pol II, 5′ cap (5’) and poly-A tail. However, by definition lncRNAs do not associate with ribosomes or produce proteins.

Figure 1
Analogous Transcriptional Mechanisms for mRNAs and lncRNAs

Schematic depicting the similar transcriptional mechanisms and nucleotide structure (introns, exons–blue boxes) between mRNA and lncRNA including their polymerization by Pol II, 5′ cap (5’) and poly-A tail. However, by definition lncRNAs do not associate with ribosomes or produce proteins.

It is likely for these reasons that lncRNAs have remained unidentified for so many years, confounded by the fact that technology had not advanced sufficiently to distinguish between coding and non-coding RNAs that have similar anatomical features. Even now, the current algorithms and bioinformatics still generate many false positive or negatives, and definitive determinations cannot be made in the absence of laboratory based validations. Although to date over 15000 different lncRNAs have been supposedly annotated by bioinformatics and sequencing programs [8], it is predicted that there may be over 50000 [13] potential lncRNAs in the human genome. Regardless of this astonishing prediction, there is still very little known about lncRNA activity, and indeed until recently no direct evidence existed at all regarding their functional significance. Of those that have been annotated in detail, most lncRNAs have been proposed to act by either modulating chromatin remodelling, altering activity of transcriptional complexes or regulating the activity of other RNA species (sequestration of miRNA activity or mRNA splicing) [9]. Accordingly, their specific involvement in these processes implies that they facilitate broad cellular programs rather than govern them, which might explain in part their wide-ranging ability to influence a vast number of processes including embryonic development, stem cell pluripotency, neurobiology, cancer and cell differentiation.

Perhaps one of the least understood aspects of lncRNAs is how these molecules influence cellular processes. Adding to this lack of understanding is the fact that lncRNAs appear to have very low sequence conservation across evolution compared with other RNA species such as miRNAs and mRNAs [14], making inferences from functional regions of other RNAs difficult to interpret. However, it has been documented that small regions of sequence within lncRNA transcripts demonstrate very high conservation, or that synteny blocks (regions of conserved genomic DNA) that harbour lncRNAs are retained throughout evolution. One such lncRNA called Cyrano was identified in zebrafish. Cyrano has very low homology for the majority of its sequence, however it harbours a ∼100 nt fragment in the 5′ end of exon 3 that demonstrates almost 100% conservation across all vertebrate species, with a further block of conserved sequence towards the 3′ end of exon 3 [15]. The reason for this particular pattern of homology is unknown but may relate to as yet uncharacterized functions of lncRNAs that are determined more on their ability to form common tertiary structures or bind common targets, rather than share highly conserved sequence motifs [16,14].

The two main mechanisms by which lncRNAs are proposed to act relates in part to their ‘cis’ and ‘trans’ activities. ‘Trans-acting’ effects refer to the ability of an RNA transcript to directly interact with other cellular components such as RNA or protein, to modulate cellular signalling, whereas ‘cis-acting’ effects relate to actions that effect transcription of local genes, perhaps merely by the act of transcription of the lncRNA being sufficient to open chromatin and increase transcription of nearby genes [17] (Figure 2). Unfortunately, current knowledge is unable to predict with confidence if a particular lncRNA will act in a cis or trans manner, and thus further experimental exploration is usually required to shed light on a given lncRNAs likely mechanism. These experiments often include determining its genomic location (intergenic/antisense), expression profile (in cis with neighbouring genes) and cellular location (nuclear compared with cytoplasm). These particular functional aspects and genomic attributes are also being used to begin the process of classifying lncRNAs into sub-classes.

“Cis” and “Trans” acting Mechanisms of lncRNAs

Figure 2
“Cis” and “Trans” acting Mechanisms of lncRNAs

Upon transcription of a lncRNA from its gene location, it can function via a number of mechanisms including those which regulate proximal events (cis-acting) or distal events (trans-acting) depending on their specific targets. Targets can include other RNA species (miR sponge), proteins (transcriptional complexes) or genomic DNA (DNA-looping).

Figure 2
“Cis” and “Trans” acting Mechanisms of lncRNAs

Upon transcription of a lncRNA from its gene location, it can function via a number of mechanisms including those which regulate proximal events (cis-acting) or distal events (trans-acting) depending on their specific targets. Targets can include other RNA species (miR sponge), proteins (transcriptional complexes) or genomic DNA (DNA-looping).

The ongoing task of classifying lncRNAs into functional groups is proving to be challenging, particularly due to the large disparity in tissue expression profiles, transcript length and lack of obvious motifs that are shared between members. Nevertheless, as more experimental evidence becomes available some of this information is now starting to appear and indeed, loose classifications are being formed. These classifications are mostly based on the genomic location and transcriptional regulation of the lncRNA itself, rather than on any particular functional annotation relating to their activity. However, some groups are now making attempts to perform these classifications [18,19],

Most previous efforts at classifying lncRNAs either do so by grouping them based on their genomic orientation, or by their proposed function/mode of action such as that performed by Chang and colleagues [20]. However, given very little is still known about most lncRNAs mechanistically, we have chosen to classify them according to three broad sub-classes relating to their genomic orientation, as described briefly below and in Figure 3. 

Varied Genomic Locations of lncRNAs

Figure 3
Varied Genomic Locations of lncRNAs

lncRNAs are dispersed throughout the genome in various configurations in which they fall into three general categories (1) intergenic–coding regions of the lncRNA (white boxes) are found in a genomic location between two known protein coding genes, (2) intragenic–coding regions within introns of known protein coding genes or (3) bi-directional/antisense/multi-coding regions of the lncRNA are either antisense to a known protein coding gene (left) or interspersed across multiple locations/genes in the genome.

Figure 3
Varied Genomic Locations of lncRNAs

lncRNAs are dispersed throughout the genome in various configurations in which they fall into three general categories (1) intergenic–coding regions of the lncRNA (white boxes) are found in a genomic location between two known protein coding genes, (2) intragenic–coding regions within introns of known protein coding genes or (3) bi-directional/antisense/multi-coding regions of the lncRNA are either antisense to a known protein coding gene (left) or interspersed across multiple locations/genes in the genome.

Intergenic/enhancer-associated lncRNAs

Perhaps the easiest of the lncRNAs to classify and indeed identify by sequence alignments, are the ‘long-intergenic/long-intervening/large-intervening’ non-coding RNAs (lincRNAs) [21]. As their name suggests, these species occupy their own loci in the genome, and apparently do not overlap with other known coding transcripts (Figure 3-1). It is not known whether there is a broad function of these intergenic lincRNAs; however, some have been described to exhibit activity that enhances transcription, thus are sometimes termed enhancer-associated RNAs [22], whereas others act in cis to repress neighbouring gene expression such as HOTAIR, which recruits the polycomb repressive complex 2 (PRC2) to repress HOX gene expression [23].

Intragenic lncRNAs

These lncRNAs are found within introns or exons, or both, of protein coding genes (Figure 3-2), and are generally more difficult to identify due to the significant overlap of their nucleotide sequence with the sequence of the gene within which they reside. As above, there also does not appear to be any broad functional similarities between these lncRNAs. However, given their location it is likely that they often act in cis to fine-tune the signalling networks of their associated protein coding genes and networks [19,24].

Bi-directional, antisense and multi-component lncRNAs

This broad group of lncRNAs is the most complex and difficult to identify. Not only are these lncRNAs found within either promoter regions or exons/introns of protein coding genes, but they can be transcribed in either the sense or antisense direction (Figure 3-3). Furthermore, some of these lncRNAs share sections of nucleotides from protein coding exons of multiple mRNAs, that are stitched together to form a multi-component lncRNA. Accordingly, there are fewer of these lncRNAs identified and indeed, understanding their function through traditional methodologies has proven difficult.

lncRNAs ASSOCIATED WITH SKELETAL MUSCLE DEVELOPMENT AND DISEASE

The molecular control of cell lineage determination and differentiation is incredibly complex, and requires exquisite timing and intimate co-operation between multiple transcriptional pathways. For many cell types these processes have been extensively studied over many years. Subsequently, a great deal of information is available regarding the transcriptional networks involved. Myogenesis for example, is the process of resident stem cell-like progenitors known as satellite cells, giving rise to committed myoblasts that eventually fuse to form multi-nucleated skeletal muscle cells [25]. Indeed, the core transcriptional pathways that initiate and drive this pathway are well described and involve a stage-specific, co-ordinated network of transcriptional regulators that include MyoD, myogenin and myocyte enhancer factors such as Mef2C [26]. However, recent evidence has demonstrated that in addition to these well accepted pathways, a large amount of regulation occurs outside of protein-centric mechanisms [27]. This suggests that many other factors fine-tune the myogenesis process that probably include chromatin remodelling, epigenetic regulation and the action of other undefined transcriptional regulators [26,28]. In support of this is evidence from genome wide association studies which demonstrate that only approximately 7% of disease causing mutations were located within protein coding genes, and that a striking 43% were located in intergenic regions [29,30]. Intergenic regions are highly enriched with ncRNAs and other transcriptional elements, suggesting that many causative genomic alterations may well be located within fundamentally important regulatory regions encoding ncRNAs. Accordingly, it is not surprising that numerous groups over the past decade have demonstrated a substantial role for non-protein coding RNAs in regulating cell function.

Recently, the role of ncRNAs has become an area of focus in skeletal muscle biology and a number of studies have sought to identify ncRNAs, particularly miRNAs and lncRNAs, that are differentially regulated at a genome wide level during skeletal muscle differentiation [3133]. These studies have identified hundreds if not thousands of differentially expressed lncRNAs, almost all of which currently have no known function. Furthermore, most of these have been identified through filtering of RNA-seq data with bioinformatics algorithms and thus, the validation of these transcripts as bona fide lncRNAs has not yet been demonstrated. Nevertheless, using similar investigational approaches a handful of experimentally validated lncRNAs have been systematically demonstrated to participate in, and regulate myogenesis (Table 1 and Figure 4). Of particular note are H19, malat1, MyoD upstream ncRNA (MUNC), lncMyoD, developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA (DUM) and Linc-MD1 [3437], which are all regulated during myogenesis, and likely necessary for efficient differentiation.

ncRNAs with a Demonstrated role in Skeletal and Cardiac Disease

Figure 4
ncRNAs with a Demonstrated role in Skeletal and Cardiac Disease

Overview of several lncRNAs described in the literature in skeletal and cardiac muscle and the role which they have been attributed in each tissue. Many have roles in both development and disease.

Figure 4
ncRNAs with a Demonstrated role in Skeletal and Cardiac Disease

Overview of several lncRNAs described in the literature in skeletal and cardiac muscle and the role which they have been attributed in each tissue. Many have roles in both development and disease.

Table 1
lncRNAs demonstrated to vary during and/or regulate skeletal muscle development in humans or mice and their corresponding citation
lncRNASetting/roleSpeciesReference
H19 miR sponge, epigenetic regulation/regeneration Human/mouse [39
Linc-MD1 miR sponge/Duchenne muscular dystrophy Human/mouse [34
Malat1 Epigenetic regulation, miR sponge/myogenesis Mouse [37
Gtl2/Meg3 Epigenetic regulation Mouse [86
Neat1 Paraspeckle functionality Mouse [87
Nctc1 Genomic looping, promoter enhancer Mouse [88,89
lncMyoD Transcriptional co-regulator/myogenesis Mouse [41
MUNC Transcriptional co-regulator/myogenesis Mouse [36
DBE-T Transcriptional co-regulator/FSHD Mouse/human [45
DUM Transcriptional co-regulator/myogenesis Mouse [42
lncRNASetting/roleSpeciesReference
H19 miR sponge, epigenetic regulation/regeneration Human/mouse [39
Linc-MD1 miR sponge/Duchenne muscular dystrophy Human/mouse [34
Malat1 Epigenetic regulation, miR sponge/myogenesis Mouse [37
Gtl2/Meg3 Epigenetic regulation Mouse [86
Neat1 Paraspeckle functionality Mouse [87
Nctc1 Genomic looping, promoter enhancer Mouse [88,89
lncMyoD Transcriptional co-regulator/myogenesis Mouse [41
MUNC Transcriptional co-regulator/myogenesis Mouse [36
DBE-T Transcriptional co-regulator/FSHD Mouse/human [45
DUM Transcriptional co-regulator/myogenesis Mouse [42

H19 is an imprinted lncRNA that is highly expressed in fetal tissues and adult muscle. Accordingly, it has been associated with a range of genetically inherited disorders and particularly studied in the setting of cancer. Among other functions, H19 was recently shown to harbour miR-675 sites within its sequence that regulate myogenesis [35], and also led to functional depletion of mRNAs in a miRNA-mediated fashion limiting rhabdomyosarcoma growth [38]. Furthermore, in skeletal muscle Kallen et al. [39] demonstrated that it also has a developmental role in skeletal muscle myogenesis, by sponging the Let-7 family of miRNAs from within canonical sites in its sequence. This mechanism of molecular sponging appears to be a common mode of action of lncRNAs, as Linc-MD1 and malat1 [34,40] also act via this sponging or ‘competing endogenous’ mechanism. Indeed, both Linc-MD1 and malat1 have been shown to sponge miR-133, a critical miRNA shown to potently regulate the mRNA abundance of important myogenic transcription factors including serum response factor (SRF), master-mind-like-1 (MAML1) and Mef2C [34,40]. Hence, the presence of these lncRNAs at particular times throughout myogenesis can have significant impacts on the differentiation process.

Not all lncRNAs in skeletal muscle have been shown to act via sponging, and indeed as described previously lncRNAs can act by cis or trans mechanisms. For example, lncMyoD is expressed distally from the MyoD locus and was shown to directly bind to the protein IMP2 (IGF2-mRNA-binding protein 2), an important protein that controls expression of genes necessary to promote cell cycle arrest. Accordingly, siRNA mediated depletion of lncMyoD led to augmented activity of IMP2, failure to exit the cell cycle and therefore impaired myogenesis [41]. Interestingly, another lncRNA is also expressed from the MyoD locus, albeit 5 kb upstream, known as the MyoD upstream ncRNA (MUNC) [36]. This 5 kb region upstream of MyoD is an important regulatory site for MyoD expression known as the distal regulatory region (DRR), and is a site that is necessary for the feed-forward enhancement of MyoD expression. Thus, MUNC is also known as DRR-enhancer RNA (DRR-eRNA). Although modulating MUNC expression was sufficient to affect MyoD activity and its ability to enhance its own expression, MUNC also affects the expression of other non-MyoD regulated genes, and thus is not exclusively a MyoD enhancer RNA. The most recent lncRNA shown to regulate myogenesis is known as Dppa2 Upstream binding Muscle lncRNA or DUM [42]. DUM expression was shown to be significantly regulated during myogenesis both in vitro and in vivo. Mechanistic studies demonstrated that DUM is expressed from a region near the gene Dppa2, and that it acts directly in cis to repress its neighbouring gene by recruiting members of the DNA methyl-transferase (Dnmt) family to the locus.

Collectively, lncRNAs that have been mechanistically characterized in skeletal muscle myogenesis display a diverse range of functions that affect the differentiation program. Of note, is the observation that the lncRNAs in skeletal muscle often regulate broad cellular mechanisms such as transcription factor activity or methylation programs, and thus provide further evidence towards a role for lncRNAs in fine tuning the activity of core transcriptional programs. Accordingly, it is not surprising that some of these lncRNAs have also been implicated in skeletal muscle dysfunction and thus, also possess potential therapeutic utility.

Strong evidence suggests that lncRNAs are likely to impact significantly on disease. Firstly, the fact that lncRNAs are major regulators of developmental programs is defining, especially given that in muscle tissues many developmental programs are often re-activated in the setting of disease [43]. Furthermore as mentioned above, a recent analysis of several genome wide association studies revealed that of the SNPs associated with disease, ∼43% were found in intergenic regions that are enriched with ncRNAs. Indeed, direct evidence for a role for lncRNAs in muscle dysfunction has been described in a number of studies. Clearly, one of the most studied disorders in this area is the genetically inherited muscular dystrophies. Several lncRNAs have been shown to be alternatively expressed in the setting of Duchenne muscular dystrophy (DMD) [17], although direct evidence for a functional role is still not definitively described. However, one lncRNA, Linc-MD1 [34,44], has been shown to be reduced in DMD cells and further demonstrated to result in improved differentiation of DMD myotubes following ectopic re-introduction of Linc-MD1.

Another form of dystrophy with evidence of a role for lncRNAs is facioscapulohumeral muscular dystrophy (FSHD). FSHD was known to be associated with a reduction in the copy number of the D4Z4 repeat mapping to 4q35, by a previously unknown mechanism. However Cabianca et al. [45] identified the lncRNA DBE-T to be one of the regulatory factors that coordinates the de-repression of 4q35 genes. Finally, a recent study which identified the lncRNA DUM as a regulator of myogenesis, also showed that overexpression of DUM in the setting of injury-induced muscle regeneration, led to a greater ability to regenerate muscle mass [42].

Collectively, although still limited in number, these studies provide significant evidence towards a plausible role for lncRNAs in mediating a ‘fine-tuning’ effect in the overall myogenesis pathway. Furthermore, these findings may also help explain in part, the varied presentation of disease phenotypes in many muscle disorders that are unexplained by our current technologies and understanding. Thus, even if a given lncRNA does not demonstrate a significant therapeutic effect, it may indeed serve as an important diagnostic marker to guide future treatment regimes.

lncRNAs ASSOCIATED WITH CARDIAC MUSCLE DEVELOPMENT AND DISEASE

The molecular regulation of cardiac commitment and development has been an intense area of research for many years. As above with skeletal muscle, the discovery of lncRNAs has pioneered a new area of investigation in cardiac biology. Several groups have identified numerous cardiac specific lncRNAs that are involved in almost all stages of cardiac commitment, development and dysfunction (Table 2 and Figure 4).

Table 2
lncRNAs demonstrated to vary during and/or regulate cardiac development and disease in humans or mice and their corresponding citation
lncRNASetting/roleSpeciesReference
APF Cardiac hypertrophy Human [67
ANRIL Myocardial infarction Human [62,90
MIAT Myocardial infarction Human [62
LIPCAR Myocardial infarction Human [53
Chast Cardiac hypertrophy Human/mouse [69
Bvht Ventricle function/lineage commitment Mouse [46
Mhrt Cardiac hypertrophy Mouse [50
Fendrr Cardiac development Mouse [48
Chrf Cardiac hypertrophy/miR sponge Mouse/human [51
Carl Mitochondria/miR sponge Mouse [65
Novlnc6 Lineage commitment Mouse [61
lncRNASetting/roleSpeciesReference
APF Cardiac hypertrophy Human [67
ANRIL Myocardial infarction Human [62,90
MIAT Myocardial infarction Human [62
LIPCAR Myocardial infarction Human [53
Chast Cardiac hypertrophy Human/mouse [69
Bvht Ventricle function/lineage commitment Mouse [46
Mhrt Cardiac hypertrophy Mouse [50
Fendrr Cardiac development Mouse [48
Chrf Cardiac hypertrophy/miR sponge Mouse/human [51
Carl Mitochondria/miR sponge Mouse [65
Novlnc6 Lineage commitment Mouse [61

Following the first detailed description of a disease linked, cardiac specific lncRNA called Braveheart (bvht) in 2013 [46], several lncRNAs have since been identified and implicated in cardiac commitment, most of which have been shown to affect the activity of lineage specific transcriptional pathways [4749]. Furthermore, the expression of some lncRNAs has also been strongly associated with cardiovascular disease [5053], particularly heart failure and pathological cardiac hypertrophy.

However, although there have been a handful of lncRNAs that are shown to be regulated by or associated with cardiac function, it is still not known if there is a primary fundamental process in which they participate. Interestingly, many of the lncRNAs that are differentially expressed in a pathological setting in the heart appear to be developmental in origin and have been demonstrated to participate in cell lineage commitment or speciation. The expression of embryonic genes is not uncommon in cardiac tissue and indeed it has been known for several decades that hypertrophy and heart failure reactivates fetal gene programs including the expression of brain natriuretic peptide (BNP) and beta myosin heavy chains (βMHC) [54]. A simple answer to these observations would be that in the period following damage, the cardiac tissue activates regenerative pathways and subsequent expansion of stem cell populations, which are closer to embryonic lineages and would account for fetal transcriptional signals. However, this is unlikely to be the case because the capacity of the adult heart to regenerate and replace damaged cardiomyocytes is very poor in comparison with other striated muscles such as skeletal muscle [55]. In fact, the annual rate of turnover of cardiomyocytes in a normal adult heart is less than 1% [55] compared with skeletal muscle which is much higher. Moreover, although pathological injury to the heart such as myocardial infarction (MI) has been shown to elevate the rate of cardiac regeneration, this rate of regeneration is considerably inadequate and thus the tissue mostly resolves injury in the long term with fibrotic scar tissue, rather than functional myocardium [56]. On the other hand, skeletal muscle injury can induce almost complete regeneration within a few weeks. Elegant studies using the snake venom cardiotoxin in murine mouse models, demonstrate complete depolarization and loss of mature muscle fibres within approximately 24–48 h, followed by classic stem cell activation/mobilization and complete regeneration to fully functioning skeletal muscle within 3 weeks [57].

Why this discrepancy exists between two tissues that are quite similar in morphology and function is still not well characterized, and it is certainly of significant interest to the field. Indeed, numerous studies have begun investigating why the adult heart is unable to efficiently regenerate, with some seminal work demonstrating that murine neonatal hearts retain the ability to regenerate for up to 1 week of age [58]. This program was shown to be strongly influenced by miRNAs, a program that is specifically lost in adult hearts [59]. Thus, these findings suggest that cardiac regeneration and recovery following injury maybe modulated by ncRNAs, highlighting further the recent interest in what function lncRNAs might be playing in regulating these pathways.

Several studies including those from the Pedrazzini, Thum and Dorn groups have utilized screening and discovery platforms to aid in identification and cataloguing of lncRNAs associated with cardiac commitment, or those that are differentially expressed in cardiac tissue under pathological conditions [22,48,60]. This has led to the discovery of several lncRNAs whose expression is shown to be associated with either lineage commitment and/or altered in the setting of disease (see Table 2 for a selected list). The first detailed description of a disease-associated lncRNA was called BraveHeart [46] which prevents cardiac stem cell differentiation into cardiomyocytes by regulating the lineage commitment factor MesP1. Since then, other lncRNAs shown to be involved in lineage commitment include Fendrr [48] and Novlnc6 [61], whose expression were also shown to be associated with ventricular defects and dilated cardiomyopathy respectively.

Perhaps of most interest in the clinical arena are lncRNAs that demonstrate diagnostic or prognostic potential. Indeed, a significant amount of effort has been invested into identifying lncRNAs that associate with pathological cardiac conditions either in the heart muscle itself [53,62,63], or potentially more diagnostically relevant in the plasma. Along these lines, one study has shown that the abundance of several lncRNAs was differentially detected in whole blood following an acute MI, which the authors speculate might serve as prognostic markers for left ventricular (LV) remodelling [62]. Furthermore, another study also detected a significant increase in plasma levels of the lncRNA LIPCAR, which they also suggested might provide diagnostic value in monitoring LV remodelling [53]. These are exciting studies and provide strong rationale for further work; however, the obvious self-reported limitations of the studies include the inability to demonstrate whether the source is specifically cardiac derived, or whether the lncRNAs were being expressed or just released into plasma following death of cardiomyocytes.

Heart failure is another condition that is being investigated in detail with regards to lncRNA expression and function. In particular, dilated cardiomyopathy and pathological cardiac hypertrophy are both associated with significant reductions in heart function and are known to activate fetal gene expression. A group that has contributed significantly to this area is the Pedrazzini laboratory. In several elegant studies this group has defined experimental conditions and methodologies that are optimal for identifying lncRNAs in cardiac tissue, and in the process have described several lncRNAs that associate with cardiac hypertrophy and ischemic heart disease. In particular, the studies by Ounzain et al. [64] use stringent criteria to define novel lncRNAs that are expressed primarily in cardiomyocytes, and have shown that lncRNAs including novlnc6 and others, demonstrate cell and context specific expression that are likely to participate in chromatin modifying processes.

Another group that has made substantial contributions to this area is the Li group, who have published a number of papers on ncRNAs that both associate with and regulate severity of cardiac hypertrophy. In particular, they have identified several lncRNAs whose expression is altered in this setting, including cardiac hypertrophy related factor (CHRF) [51], cardiac apoptosis regulated lncRNA (CARL) [65], mitochondrial dynamic related lncRNA (MDRL) [66] and autophagy promoting factor (APF) [67], and further shown in a number of conditions that modulating expression of these lncRNAs can alter heart function. Separately, a study by Yang et al. [68] performed an lncRNA screen that identified ∼18000 lncRNAs expressed in the human heart, of which approximately 600 were differentially expressed in heart failure, some of which were rescued upon patients being placed on LV assist devices.

Finally, two recent studies and probably the most in-depth to date have provided very strong evidence for lncRNAs being bona fide targets for therapeutic intervention in heart failure. The first of these identified a group of lncRNAs named Myheart (mhrt), which are expressed from the Myh7 locus in a normal heart, but repressed in the setting of stress and subsequent heart failure. This study went on to demonstrate that if mhrt is re-expressed during stress conditions, it was sufficient to prevent subsequent hypertrophy and heart failure, potentially by binding and inactivating Brg1 [50]. The second study used stringent bioinformatics criteria to identify a lncRNA named CHAST, which is elevated in both murine and human cardiac hypertrophy. Excitingly, this study also demonstrated for the first time that antisense (GAPmeR)-mediated depletion of a lncRNA in vivo, was able to both prevent and regress disease in a mouse model of pathological cardiac hypertrophy [69]. Thus, these two studies demonstrate that therapeutic manipulation of specific lncRNAs in the heart can improve cardiac function in the setting of cardiovascular disease. Accordingly, such findings not only confirm the relevance of lncRNAs in cardiac disease, but also highlight the therapeutic potential of targeting these molecules.

In summary, work over the past five years has made significant advancements in our understanding of lncRNAs in the heart and the role they play in facilitating or preventing disease pathways. Importantly, with continuing advances in small molecule design and antisense technology, studies are now beginning to demonstrate that targeting specific lncRNAs in cardiac tissue can have functional effects on cardiovascular function. Nevertheless, a number of hurdles still remain before these therapies can be taken to the clinic.

CURRENT LIMITATIONS TO lncRNAs BEING THERAPEUTIC TARGETS AND DIAGNOSTICS

Clearly, experimental data are the key to continuing to build a substantial body of evidence that supports a role for lncRNAs being independently sufficient to modulate pathological pathways. Although several lncRNAs have demonstrated promise in this capacity, there are still some obstacles that need to be overcome before their validation as bona fide targets for therapeutic or diagnostic applications.

Lack of homology

Perhaps the most significant translational issue at present with lncRNAs is their apparent lack of conservation in nucleotide sequences between species, including mice and humans [14]. Identifying orthologues of murine protein coding transcripts (e.g. mRNAs) in the human genome is relatively simple in the post genome sequencing era, particularly because of the conserved nature of many of these transcripts. However, lncRNAs have far less sequence similarity than mRNAs. Unfortunately, it is also not as simple as mapping the same chromosome and regions of both genomes, because although there is >80% homology in coding nucleotide sequence between mice and humans, the chromosome location and gene arrangement is often very different [70]. However, because of reasons that are now becoming more apparent, large blocks of chromosomal DNA known as synteny blocks are passed on to offspring presumably as a means of keeping clusters of genes together, possibly due to their co-dependence for efficient biological activity. This often includes dozens of mRNAs and sometimes several lncRNAs. For example, if an identified lncRNA is in an antisense direction at the 5′ end of a given gene in mice, it is common that the same region in humans may also harbour a similar lncRNA, even if sequence similarity is low or it is found on a different chromosome. RNA-sequencing can subsequently identify if a transcript is expressed from that region in humans and the transcript be investigated for functionality. Accordingly, agents can then be designed to specifically modulate that lncRNA in humans.

Physical location of expressed lncRNAs

Further to the issue of sequence homology, another significant problem that requires consideration before therapeutic targeting can be contemplated is the cellular location of a given lncRNA. Similar to other ncRNA species such as miRNAs, lncRNAs have been shown to reside in different cellular locations including the nucleus, mitochondria and the cytoplasm [71]. This is in line with the numerous functions described for lncRNAs including their cis- and trans-acting capabilities. Indeed, several lncRNAs have also been detected in extracellular locations and in the plasma, suggesting the possibility that they may be secreted or exported in vesicles [53,62]. What governs the location of a given lncRNA is still not well described, although its function is undoubtedly a strong determinant of its location, or vice versa. The cellular location is an important consideration in the design of any potential therapeutic, particularly as some pharmacological agents do not equivalently penetrate all intracellular locations. Therefore, before employing strategies for modulating a given lncRNA, it is necessary to determine its cellular location in order to determine the best potential therapeutic approach.

POTENTIAL APPROACHES FOR lncRNA TARGETING

Given that lncRNAs are mostly single-stranded RNA transcripts, classic modes of therapeutic modulation that are otherwise suitable for proteins or protein coding transcripts, do not necessarily have application for lncRNAs. Nevertheless, several studies have had success in modulating lncRNAs using viral approaches including adenovirus [7274] and lentivirus [21,7577] to either overexpress or deplete lncRNAs in the setting of disease. However, given the slight but obvious risks associated with therapies involving viruses, these are still not considered a viable long-term therapeutic option. This being said, a newer type of viral delivery approach, adeno-associated viruses (AAVs), has recently shown promise in delivering protein coding genes in pre-clinical models and several human clinical trials have been undertaken without side-effects [7880]. However, the use of AAVs for the delivery of lncRNA therapeutics has yet to be rigorously investigated.

Outside of viral delivery methods, two other broad approaches have been trialled in their capacity to modulate lncRNA function, small molecule inhibitors and antisense oligonucleotide (ASOs) based depletion strategies (Figure 5). A recent study described a platform that tested hundreds of small molecule inhibitors to disrupt the interaction between the lncRNA HOTAIR and its binding protein partner enhancer of zeste homologue 2 (EZH2) [81]. In this study, they identified several compounds that significantly inhibited the interaction. However, none of these molecules were shown to be specific for the interaction. Nevertheless, they demonstrated the capacity for small molecules to bind and interfere with lncRNA functionality.

Potential Therapeutic Approaches for Modulating lncRNAs in vivo

Figure 5
Potential Therapeutic Approaches for Modulating lncRNAs in vivo

Schematic outlining potential approaches to therapeutically increase/activate lncRNAs (left box) or decrease/inactivate lncRNAs (right box). Several of these approaches have already been successfully employed for other ncRNA species, but are yet to be determined for lncRNAs.

Figure 5
Potential Therapeutic Approaches for Modulating lncRNAs in vivo

Schematic outlining potential approaches to therapeutically increase/activate lncRNAs (left box) or decrease/inactivate lncRNAs (right box). Several of these approaches have already been successfully employed for other ncRNA species, but are yet to be determined for lncRNAs.

Undoubtedly, the most promising and tested approach to lncRNA therapeutic intervention are the oligonucleotide-based approaches which include both ASOs and shRNA interference. In fact, ASOs have already been used to target numerous miRNAs in preclinical models of heart disease [82]. Both approaches result in degradation of target RNAs by generating small RNA molecules that directly bind target RNA and promote their degradation through either an RNase H or complementary RNA cleavage pathway respectively. ASOs are small single stranded oligonucleotides that are directly introduced as chemically modified molecules that do not require processing by the cell [83]. shRNA therapies are usually introduced as longer double stranded RNA molecules that are processed by the endogenous Dicer pathway to generate siRNAs [84]. Accordingly, because ASOs do not require processing, they are effective at silencing RNAs in the nucleus, whereas shRNA therapies are more effective at silencing cytoplasmic RNAs due to their requirement for processing by Dicer. These technologies propose an exciting approach to modulating lncRNAs in a therapeutic manner, and have been shown in a handful of cell and pre-clinical models to be efficacious in models of skeletal and cardiac disease.

EVIDENCE FOR IN VIVO lncRNA TARGETING IN SKELETAL AND CARDIAC MUSCLE

Given the fact that lncRNAs have only recently emerged as modifiers of pathological processes, coupled with the very recent advances in ASO technology, it is not surprising that only two studies have been published in the setting of muscle disease demonstrating in vivo evidence for systematically administered therapeutics that manipulate lncRNA activity.

The first was a study by Wheeler et al. [85] that identified that the expanded CUG containing RNA responsible for promoting the hereditary degenerative disease myotonic dystrophy type 1 displayed an unusually high sensitivity to ASO mediated depletion. Subsequently, these authors demonstrated that systemic administration of targeted ASOs mediated the depletion of this RNA for up to a year after ASO administration, with measureable effects on disease prevention. Independently, this study also demonstrated successful depletion of the lncRNA Malat1 in skeletal muscle using systemic ASO administration, a lncRNA implicated in skeletal muscle myogenesis.

Secondly, a more recent study mentioned earlier in this article by Viereck et al. [69] demonstrated that systemic administration of an ASO mediated the depletion of a cardiac lncRNA known as cardiac hypertrophy-associated transcript (CHAST). CHAST was discovered as an lncRNA that was increased in the setting of pathological cardiac hypertrophy, thus systemic ASO administration and depletion of CHAST promoted significant improvements in cardiac function in a trans-aortic constriction model of heart failure.

Excitingly, these two studies provide important experimental evidence that lncRNAs may be legitimate targets for therapeutic intervention in both skeletal and cardiac diseases. However, whether these results translate into clinical outcomes, or ASOs become viable options for therapeutic manipulation of muscle diseases in humans, remains to be determined.

CONCLUSIONS

Collectively, these studies highlight lncRNAs as an emerging class of regulators that may significantly affect fundamental cellular processes important to human disease. In particular, the identification of lncRNAs that are functionally important in skeletal and cardiac muscle development and diseases thereof, highlights an exciting therapeutic avenue to pursue in a setting where there are currently very few effective treatments. With the advancements in gene therapy and oligonucleotide delivery, studies over the next few years will undoubtedly demonstrate whether lncRNAs are viable therapeutic targets, and whether they yield efficacy in a clinical setting.

FUNDING

B.G.D. would like to thank the following for their support; NH&MRC Australia, Diabetes Australia Research Program (DARP), The Mason Foundation, The Miller Foundation, The CASS Foundation, BakerIDI Metabolism Program and Monash University Central Clinical School. J.R.M. is a National Health and Medical Research Council Senior Research Fellow [grant number 1078985].

Abbreviations

     
  • AAV

    adeno-associated virus

  •  
  • APF

    autophagy promoting factor

  •  
  • ASO

    antisense oligonucleotide

  •  
  • CARL

    cardiac apoptosis regulated lncRNA

  •  
  • CHAST

    cardiac hypertrophy-associated transcript

  •  
  • CHRF

    cardiac hypertrophy related factor

  •  
  • DRR

    distal regulatory region

  •  
  • DRR-eRNA

    DRR-enhancer RNA

  •  
  • DMD

    Duchenne muscular dystrophy

  •  
  • Dppa2

    developmental pluripotency-associated 2

  •  
  • DUM

    Dppa2 upstream binding muscle lncRNA

  •  
  • FSHD

    facioscapulohumeral muscular dystrophy

  •  
  • IMP2

    IGF2-mRNA-binding protein 2

  •  
  • lncRNA

    long non-coding RNA

  •  
  • LV

    left ventricular

  •  
  • MDRL

    mitochondrial dynamic related lncRNA

  •  
  • mRNA

    messenger RNA

  •  
  • nt

    nucleotides

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