LncRNAs in Cardiomyocyte Maturation: New Window for Cardiac Regenerative Medicine

Cardiomyocyte (CM) maturation, which is characterized by structural, functional, and metabolic specializations, is the last phase of CM development that prepares the cells for efficient and forceful contraction throughout life. Over the past decades, CM maturation has gained increased attention due to the fact that pluripotent stem cell-derived CMs are structurally, transcriptionally, and functionally immature and embryonic-like, which causes a defect in cell replacement therapy. The current challenge is to discover and understand the molecular mechanisms, which control the CM maturation process. Currently, emerging shreds of evidence emphasize the role of long noncoding RNAs (lncRNAs) in regulating different aspects of CM maturation, including myofibril maturation, electrophysiology, and Ca2+ handling maturation, metabolic maturation and proliferation to hypertrophy transition. Here, we describe the structural and functional characteristics of mature CMs. Furthermore, this review highlights the lncRNAs as crucial regulators of different aspects in CM maturation, which have the potential to be used for mature CM production. With the current advances in oligonucleotide delivery; lncRNAs may serve as putative therapeutic targets to produce highly mature CMs for research and regenerative medicine.


Introduction
Directed differentiation of human pluripotent stem cells (hPSCs) to cardiomyocytes (CMs) has achieved remarkable progress over the past decade. These have provided powerful resources to study human development, translational research to discover disease mechanisms, new drug discovery as well as cell replacement therapy [1]. Despite tremendous achievements in producing the mature PSC-derived cardiomyocytes (PSC-CM) through tissue engineering-based methods, the highly mature CMs have yet to be achieved [2][3][4]. Most protocols generate immature CMs, which lack many attributes of adult CMs. Consequently, the generated cells cannot be used for efficient drug screening, modeling of adult-onset disease, and also as a source for the cell replacement therapy [5]. The current challenge is to discover and understand the molecular mechanisms, which control the CM maturation process. Cardiomyocyte maturation is the last phase of heart development characterized by structural, gene expression, metabolic, and functional specializations. Maturation is a complex trait controlled in different aspects by multiple signaling networks and regulators within the cytoplasm and nucleus [6]. In recent years, the advancement of novel technologies like microarray and next-generation sequencing (NGS) has revealed that 98% of noncoding sequences of the genome are actively transcribed and produce noncoding transcripts, including small and long noncoding RNAs (lncRNAs) [7]. The discovery of lncRNAs has expanded a new layer of regulation to the complexity of transcriptome. The emerging pieces of evidence demonstrate the important role of lncRNAs in CM differentiation and maturation [8]. Interestingly, a large number of lncRNAs are cardiac-specific or dynamically expressed during the differentiation and maturation processes. LncRNAs are the most heterogeneous group of regulators that can way are essential for cardiomyocyte proliferation and can promote the regeneration of cardiac muscle cells [37]. Activation of Notch signaling pathway trigger the BMP10 activity, which in turn promote proliferation of cardiomyocytes through inhibition of the cell cycle inhibitor p57 [38]. It also regulates the transcription of CCND1 and induces cell cycle reentry and progression in neonatal cardiomyocytes [39]. Hippo signaling pathway is also an important regulator of cardiomyocyte proliferation and heart size [37]. The downregulation of cell-cycle-associated genes like Cdk1, Ccnb1 and Aurkb, as well as mentioned proliferation-associated signaling pathways, are the hallmark of this transition [40][41][42][43]. Despite the cell cycle exit, the postnatal heart size also increased more than 30-fold through the maturational hypertrophy [44]. Other aspect of proliferation-to-hypertrophy transition is polyploidization, in which the cardiomyocyte nuclei are polyploid due to DNA endoreplication without karyokinesis ( Figure 1D, top) [45,46]. Cardiomyocytes polyploidization also induces the maturational hypertrophy and cause cell cycle exit in mature cardiomyocytes ( Figure 1D, bottom) [47]. Finally, efficient integration of cardiomyocytes into cardiac tissues require adhesion like structures and costameres to facilitate extracellular matrix (ECM) attachment as well as intercalated discs (ICDs) structures. ICDs contain fascia adherens, desmosomes, and gap junctions, which support well organized cardiomyocytes junctions and synchronized contraction of cardiac cells [48][49][50][51]. Cardiomyocytes undergo myofibril maturation, which is followed by increasing the sarcomere length, assembly and expansion; improvement of sarcomere alignment and M-line formation. (B) Calcium handling maturation is mediated by the development of T-tubules, expression of calcium handling proteins and increased volume of calcium stores of the sarcoplasmic reticulum. (C) Metabolic maturation causes the switch of glycolysis to fatty acid β-oxidation and increases in mitochondria number. The cristae structures in mitochondria are densely organized, and the mitochondria are localized close to the SR and myofibrillar structures to support efficient ATP transition. (D,E) Proliferation-to-hypertrophy transition occurs at the time of maturation. The postnatal heart size is increased through maturational hypertrophy followed by polyploidization (top) cardiomyocyte proliferation rate declines through downregulation of cell-cycle-associated genes (bottom).

Noncoding RNAs
The most recent studies indicate that the developmental complexity of higher eukaryotes is raised from the gene regulation process by a large number of regulators rather than the number of protein-coding genes [52]. The protein-coding genes represent only a small portion of the mammalian genome, while the other majority part produces different classes of noncoding RNAs (ncRNAs). Noncoding RNAs as an important hidden layer of regulation have pivotal roles in almost all biological processes. The ncRNAs can be classified based on their length into small (<200 nucleotides) and long noncoding RNAs (>200 nucleotides) [53][54][55].
LncRNAs are the most diverse and heterogeneous class of noncoding RNAs [56]. Several transcriptional and post-transcriptional processes, including 5 -capping, splicing and polyadenylation, are done to reach a mature form of lncRNAs [57,58]. Generally, lncRNAs have no open reading frame (ORF), while few studies shed light on the existence of lncRNAs with coding peptides [59]. The lncRNAs have a high degree of tissue specificity [60] and are conserved in either expression pattern or transcript structure despite their low sequence conservation [61][62][63]. The localization of lncRNAs is mainly related to their function. Some of them could be located in both the nucleus and the cytoplasm, while some reside only in the cytoplasm or permanently in the nucleus [64,65]. They could be classified according to their genomic position and orientation relative to their adjacent protein-coding gene [66]. The intergenic lncRNAs (lincRNAs), which do not have any overlap with ding genes, are the most defined group across the organisms [67]. Other groups reside inside the introns of protein-coding genes (intronic lncRNAs), or they are transcribed in the opposite direction of adjacent protein-coding genes (divergent lncRNAs). The lncRNAs can be transcribed from either sense or antisense strand of DNA ( Figure 2A) [68][69][70]. They may also be classified based on the chromatin context, in which enhancer-associated (elncRNA) or promoter-associated (plncRNA) lncRNAs are characterized by K4me1 and K4me3 chromatin marks around their transcription start site ( Figure 2B) [63]. The critical functions of lncRNAs belong to their ability to interact with regulatory proteins, DNA elements or even functional RNAs to regulate their interaction. LncRNAs can regulate transcription by recruiting transcription factors and chromatin modifier complexes through different mechanisms, including guide, signal, scaffold, or preventing their interaction through decoy mechanism [71][72][73]. LncRNAs also can facilitate the formation of the enhancer-promoter loop at the site of the target locus through regulating inter and intrachromosomal interaction [74,75]. Furthermore the local formation of R-loops (triple-stranded nucleic acid structures with RNA hybridized to duplex DNA) by some anti-sense lncRNAs recruit transcription cofactors to the promoter of corresponded sense mRNA and regulate its transcription [65,76]. The important function of lncRNAs during heart development, differentiation and disease is well recognized. Some important studies revealed the presence of a large number of lncRNAs, which are indispensable for cardiomyocyte maturation ( Table 1). The sharp alteration of transcriptome, including lncRNAs at the time of maturation reflects the rapid adaptation of cardiomyocytes for metabolism, regeneration capacity as well as functional properties [8,33]. Below, we review the main lncRNAs involved in the cardiomyocyte maturation procedure.

Mhrt-LncRNA
The Myh-associated RNA transcript (Mhrt) is a cardiac-specific lncRNA, which is transcribed within the Myh genomic loci. The Myh genomic locus not only encodes the main cardiac contractile genes, including Myh6 and Myh7 but also miRNAs (miR-208a and miR-208b) and Mhrt lncRNA. Mhrt is transcribed in an antisense direction of Myh6 promoter into the genomic site of Myh7 [77]. The Mhrt expression level is increased gradually from embryonic hearts into mature adult hearts. As a cardiac-specific lncRNA, its expression is limited to the heart with no or minimal expression in other tissues [91]. This lncRNA has a different regulatory function in myofibril formation and hypertrophy at the stage of cardiomyocyte maturation. The lncRNA Mhrt has a tight correlation with the Myh6/Myh7 ratio and regulates their expression during the development through Brg1 inhibition. The BRG1 complex can oppositely regulate the expression of the Myh6 and Myh7 contractile genes through the recruitment of different repressors or activators. It can repress the expression of Myh6 by recruitment of chromatin repressor G9A and DNMT3 on Myh6 promoter and activate Myh7 expression through NFAT1 and AP-1 transcription factor recruitment on the site of Myh7 promoter. Mhrt lncRNA directly binds to the helicase domain of BRG1, which causes BRG1 to be sequestered from its target genomic DNA. This negative regulation of BRG1 by Mhrt is essential for the normal ratio of Myh6/Myh7 during the development and maturation ( Figure 3) [77,116]. On the other side, Mhrt regulates cardiomyocyte hypertrophy through myocardin expression and function regulation. In the heart, myocardin, as a strong trans-activator, binds directly to SRF and activates cardiomyocytes restricted genes containing CarG boxes in their promoter region [117]. The Mhrt upregulation in the mature heart avoids myocardin expression out of control and protects the heart in the development process. Mhrt can raise the Hdac5 level and enrich the HDAC5/myocardin complex, which leads to myocardin deacetylation. Deacetylation loses the activity of myocardin to regulate the cardiac hypertrophy biomarkers expression (

H19-LncRNA
The H19-lncRNA is a highly conserved, imprinted, non-protein-coding gene. The base conservation, along with the highly conserved secondary structure, suggests its pivotal roles in development [96]. H19 has a high-level of expression through cardiomyocyte differentiation, while its expression strongly reduces after birth and through maturation [93]. H19 could regulate the cardiomyocyte maturation process at different steps, including apoptosis, proliferation, hypertrophy and contraction. H19-lncRNA has been demonstrated to regulate hypertrophy in cardiomyocyte cells [80]. H19 physically interacts with polycomb repressive complex 2 (PRC2) to suppress pro-hypertrophic NFAT signaling. Regarding the fact that H19 is downregulated during cardiac development, in the absence of H19, PRC2 hyper-methylates the TESC (tescalin) locus as a main inhibitor of NFAT. This leads to activate NFAT signaling and their corresponding hypertrophy-related target genes ( Figure 4A) [93]. H19 also delicately regulates the function of proteins associate with cardiac contraction through the H19-miR-675 axis. It is shown that calcium/calmodulin-dependent protein kinase II Delta (CamkIIδ) is a direct target of H19-derived miR-675 ( Figure 4B) [80]. CamkIIδ is the predominant cardiac isoform, which can phosphorylate ion channels and critical membrane proteins for cardiac electrical activity and structure like L-type Ca 2+ and voltage-gated Na + and K + channels [118]. The absence of the H19-miR-675 axis in the mature heart increases CamkIIδ expression, which in turn regulates the protein associated with cardiac ca 2+ flux, cardiac contraction and relaxation. Other studies also indicate the pivotal role of the H19-miR-675 axis in the mitochondrial apoptotic pathway. The voltage-dependent anion channel 1 (Vdac1) is a direct target of miR-675, which is a key protein in the process of mitochondria-mediated apoptosis ( Figure 4C) [94]. Moreover, H19-lncRNA regulates proliferation and apoptosis through the miR-19b-Sox6 axis. H19 inhibits miR19b expression, which leads to Sox6 target gene expression upregulation ( Figure 4D). SOX6 is a multifunction transcription factor with critical roles in cell proliferation and apoptosis during the late-stage of cardiomyocyte differentiation [119]. Altogether absence of H19-lncRNA regulates the cardiomyocyte apoptosis, proliferation and protects cardiomyocytes by antiapoptotic effect through either H19-miR-675 or H19-miR-19b axis.

Ahit LncRNA
The antihypertrophic interrelated transcript (Ahit) lncRNA was recently reported that regulate hypertrophy in the postnatal heart. Ahit is highly enriched in the adult heart and is adjacent to Mef2a, which is a pivotal regulator of cardiac development [120]. While the critical function of MEF2 in cardiac development is clear, a novel function of MEF2 transcription factors in the postnatal heart was proposed. The MEF2 expression in the adult heart is at the basal level, and its upregulation to the level seen in the failing heart can promote chamber dilation, mechanical dysfunction, and dilated cardiomyopathy [121,122]. Ahit lncRNA regulates chromatin structure and expression of Mef2a through direct interaction with SUZ12 (suppressor of Zeste 12) and recruiting PRC2 chromatin remodeler at the promoter region of Mef2a locus. PRC2 modulates histone modification and increases H3K27me3 histone marks, which leads to Mef2a transcription repression [120]. Altogether, Ahit regulates the expression of the Mef2 family and prevents their abnormal function from inducing precise hypertrophy in mature adult cardiomyocytes.

Zfas1 and Dach1 LncRNAs
Intracellular Ca 2+ homeostasis is a key factor to regulate cardiomyocyte contraction as well as cell viability through regulating mitochondrial-mediated apoptosis. This process is tightly regulated by an array of protein and nonprotein-coding genes. SERCA2a (SR Ca 2+ -ATPase 2a) is the main protein, which is indispensable for the normal intracellular Ca 2+ handling process through regulating the Ca 2+ reuptake into SR in cardiac muscles [123]. The more recent studies indicate that Serca2 expression and function are delicately regulated by lncRNAs. Zfas1 (zinc finger antisense 1), an antisense lncRNA to the 5 end of the proteincoding gene Znfx1, is a cardiac-related lncRNA, which regulates cardiac apoptosis as well as contraction activity [4]. Zfas1 negatively regulates the Serca2a through either its expression repression or functional restriction ( Figure 5A). This aims to decrease Ca 2+ reuptake to the SR, which leads to cytosolic Ca 2+ overload [81]. The studies indicate that Ca 2+ overloads cause cardiac dysfunction [81] and induce mitochondria-mediated apoptosis [97].
More recently, a highly conserved lncRNA dachshund homolog 1 (Dach1) was also reported by the same group to have a main role in regulating the mature cardiomyocyte proliferation and function. Dach1 expression was gradually upregulated after birth in a mature heart. It has a hallmark role in the blockage of mitosis and proliferation in the mature heart through the Hippo signaling pathway ( Figure 5A) [98]. It is well-known that the Hippo signaling pathway regulates the mitosis and proliferation of cardiomyocytes [124]. Dach1 directly interacts with protein phosphatase 1 alpha (PP1A) and reduces its dephosphorylation activity. This leads to improving yes-associated protein 1 (YAP1) phosphorylation, reduces its nuclear localization, which in turn inhibits cardiomyocyte proliferation in the mature heart [98]. Dach1 lncRNA also regulates the function of mature cardiomyocytes through regulating Ca 2+ homeostasis in the cells. Dach1 directly interacts with SERCA2a protein, promote its ubiquitination and proteasome degradation ( Figure 5A) [82]. All together, Zfas1 and Dach1 manipulate the cardiac function by regulating Ca 2+ homeostasis, proliferation and apoptosis in mature cardiomyocytes.

Carl and Mdrl LncRNAs
Metabolic maturation, which is occurred at the level of prenatal to postnatal transition, is an important event in cardiomyocyte maturation. The increase in myofibril density and contractile components require more ATP production during postnatal hypertrophic growth. Mitochondria are highly dynamic, and their fusion and fission are crucial for maintaining mitochondrial function and fidelity [125]. The mitochondrial biogenesis is tightly regulated by a different network of proteins, miRNAs and lncRNAs. The cardiac apoptosis-related lncRNA (Carl), which is highly expressed at the adult heart, suppresses the cardiomyocytes fission and apoptosis by targeting miR-539 and Prohibitin 2 (PHB2) [88]. PHB2 has main roles in the functional and structural integrity of mitochondria, the cristae morphology and the formation of a super respiratory complex in mitochondria [126]. Carl lncRNA sponge miR-539 in the cardiomyocytes, which in turn upregulates the expression of Phb2 as its target gene ( Figure 5B). PHB2 inhibits the fission in the mitochondria and suppresses apoptosis [88].
The mitochondrial fission and apoptosis are also regulated by other cardiomyocytes enriched lncRNA called mitochondrial dynamic related lncRNA (Mdrl). The studies indicate that nucleus enriched miR-361 inhibits prito pre-miR-484 processing through direct interaction with a primary transcript of miR-484. Mdrl lncRNA has direct interaction with miR-361 and downregulates its expression levels, which in turn promotes the processing of pri-miR-484 [89]. Upregulation of miR-484 represses Fis1 expression that is involved in mitochondrial fission and apoptosis ( Figure 5B) [127]. Carl and Mdrl lncRNAs regulate mitochondrial fission and apoptosis through regulating the combination of miRNAs and transcription factors in adult cardiomyocytes.

Cpr and Sarrah LncRNAs
Most recent studies indicate the important function of lncRNA Cpr (cardiomyocyte proliferation regulator) in mature cardiomyocyte proliferation. The expression level of Cpr lncRNA is increased in postnatal and adult hearts and is enriched in the nucleus of cardiomyocytes cell type. This lncRNA attenuates the cardiomyocyte proliferation in postnatal and adult hearts. Cpr lncRNA directly interacts and recruits DNMT3A protein to enhance CpG methylation of minichromosomal maintenance 3 (Mcm3) promoter ( Figure 5C) [78]. MCM3 belongs to the family of proteins involved in the initiation of eukaryotic genome replication and cell cycle progression [128]. The Mcm3 downregulation in mature cardiomyocytes inhibits cardiomyocyte proliferation. In addition, Liu et al. indicate that Cpr remarkably causes a hypertrophic response in mature cardiomyocytes, which has been shown by sarcomere organization, increased cell surface area, and elevated hypertrophy markers like Anf and Myh6 [78].
The association between lncRNAs and cardiomyocyte survival and function is coming to light more by other reports. The lncRNA Sarrah (short for SCOT1-antisense RNA regulated during aging in the Heart) was reported to have antiapoptotic and pro-survival effects in adult cardiomyocytes. Sarrah directly binds to the promoter region of the Nrf2 gene through RNA-DNA triple helix formation and recruits CRIP2 and p300 transcription factors ( Figure 5C) [99]. The Nrf2 pathway promotes cell survival and is well-known for its antiapoptotic and cardio-protective effects [129]. The evolutionary conserved lncRNA Sarrah, as a regulator of cardiomyocyte survival, also enhance the contractile capacity of adult cardiomyocytes [99].

Therapeutic Target of LncRNAs in Cardiac Remodeling
The biological relevance of lncRNAs to different stages of cardiomyocyte maturation has triggered tremendous interest in their application as a therapeutic option. Nevertheless, there are several important issues that need to be considered before lncRNAs can be used as potential therapeutic targets in regeneration medicine. First, many lncRNAs are not conserved at the sequence level, and this makes the identification of human lncRNAs and their clinical testing challenging [130]. Second, as we described in this review, lncRNAs regulate gene expression through different mechanisms, including chromatin remodeling, miRNA regulation or binding to different proteins, RNA and even DNA elements. This phenomenon brings about potential off-target effects for lncRNAs expression, which should be considered. Consequently, the most promising lncRNAs as therapeutic targets are the ones, which their mechanism of action is well described. Finally, the delivery method of oligonucleotides and their duration of action in vivo should be considered before manipulating the candidate lncRNAs [131]. On the other hand, lncRNAs have high tissue specificity, which makes conserved lncRNAs a promising target for drug development, and this also helps to have less remote off-target effects [132]. In spite of the obstacles mentioned above, the role of CHROME [133] and H19 [134] lncRNAs in cardiovascular disease have been investigated in animal models. Of interest, H19 lncRNA with a pivotal role in the cardiomyocyte maturation process was studied in both mouse and mini-pig models. Li et al. reported that H19 lncRNA is a novel regulator of smooth muscle cell survival in abdominal aortic aneurysm development and progression [134]. All together, further works should be done to focus and clarify the lncRNAs regulatory network, investigate their role in known beneficial/harmful signaling pathways and optimize the delivery technology in animal models to promote the rapid clinical transformation of lncRNAs. In summary, based on the available in vitro and in vivo animal model, current studies with human cells and tissue and optimized delivery system, lncRNAs has potential to be considered as a novel therapeutic option in different filed of cardiovascular development specially maturation process.

Conclusions
Over the past decades, several differentiation protocols have been optimized to produce functional cardiomyocytes, which still are structurally, transcriptionally and functionally immature and somehow embryonic-like. In spite of the fact that differences between the immature and mature cardiomyocytes have been explained with details, underlying molecular mechanisms and corresponded regulators that mediate this transition still remain less elaborated. Critical regulators with combinational effects are needed to regulate and optimize cardiomyocyte maturation as a complex process. As previously discussed, several lncRNAs are expressed during the heart development and of interest cardiomyocyte maturation. LncRNAs are defined to regulate the maturation in different aspects, including myofibril maturation, electrophysiology and Ca 2+ handling maturation, metabolic maturation and proliferation to hypertrophy transition. LncRNAs like Mhrt, H19, Zfas1 and Carl could act as important participants in promoting the maturation. As a result, they enhance a wide range of phenotypic changes, which promote the fetal to adult cardiomyocytes transition. This review highlights lncRNAs as the multi-effect regulators, which have the potential to promote mature cardiomyocyte production. Targeting the lncRNAs may be a novel approach to producing effective adult-like cardiomyocytes. However, we must consider several challenges, including the lack of sequence conservation, delivery strategy and off-target effect. With the current advances in oligonucleotide delivery and gene therapy, the studies in the next few years may demonstrate the possible role of lncRNAs as a therapeutic target to produce highly mature cardiomyocytes in cell replacement or other clinical therapies.