Introduction
MicroRNAs (miRNAs) are single-stranded, highly conserved, short non-coding RNAs 22 nucleotides) regulating target gene expression by base pairing with specific sequences of target mRNAs (Ambros, 2004). miRNAs negatively regulate gene expression post-transcriptionally by suppressing translation and/or inducing mRNA degradation. Bioinfomatically, it is estimated that human genome may contain approximately 1000 miRNAs (Bartel, 2004; Berezikov et al., 2005; Griffiths-Jones et al., 2008) and consistently, additional miRNAs are continually being identified (Griffiths-Jones et al., 2006). miRNAs modulate the expression of target proteins in a non-canonical manner by binding to specific sequences regulating functional networks. Consequentially, a single miRNA might target hundreds of distinct genes or alternatively expression of a single coding gene can be regulated by many different miRNAs (Lewis et al., 2005; Miranda et al., 2006). Recent studies show the important role of miRNAs in the regulation of a variety of physiological functions ranging from stem cell differentiation to cardiac muscle development and stress (Krichevsky et al. 2006; Chen et al., 2006; Zhao et al., 2005; Pedersen et al., 2007; Kloosterman et al., 2007; Felli et al., 2005; Tay et al., 2008). Furthermore, aberrant expression of miRNAs has been found in various diseases including cancer, diabetes and cardiac hypertrophy/failure.
The binding specificity of miRNAs depend on complementary base pairing of ~ 7 nucleotide seed sequence region at the 5′ end of the miRNA with the corresponding mRNA target. Another caveat that needs to be considered in the miRNA regulation is the miRNA sequences outside the 7 nucleotide seed region which pairs with the mRNA that may also play a role in determining the strength/efficacy of regulating the target mRNA. The binding of miRNAs to their cognate target mRNAs commonly results in decreased expression of target genes through translational repression or mRNA degradation (Fig. 1). Conversely, decreased expression of miRNAs will lead to increased target gene expression (Gregory et al., 2008).
This realm of knowledge has allowed for studies on miRNAs on their tissue specificity and disease specificity but critically little information is available with regards to temporal or spatial expression profiles of miRNAs in the heart. By and large studies have used micro-array analysis to identify altered miRNAs to define signature of altered miRNAs in a specific cardiac phenotype. As miRNAs target multiple proteins, these signatures have been used to predict the array of molecules altered. Over time sophisticated computational approaches have been developed that has lead to identification of previously unrecognized targets within disease pathways of interest (Ivanovska and Cleary, 2008; Gusev et al., 2007). Among the computational tools the most commonly used target prediction algorithms include DIANA-microT (Kiriakidou et al., 2004), miRanda (Griffiths-Jones et al., 2006), TargetScan (Lewis et al., 2003), TargetScanS (Lewis et al., 2005), PicTar (Krek et al., 2005) and PITA (Kertesz et al., 2007). These algorithms rely on criteria like conservation among species, seed complementarity, thermo-stability of miRNA-mRNA hybrids, delta G of target mRNA binding site, and multiple miRNA binding sites in the 3′UTR (cooperativity) to predict targets (Bartel, 2009; Cacchiarelli et al., 2008; Ivanovska and Cleary, 2008; Gusev et al., 2007). Thus, use of these algorithms provide hundreds of targets indicating that miRNA alteration in expression could have wide ranging effects on molecules belonging to multiple signaling pathways. It is important to note that target prediction with these algorithms remains challenging but these are the tools currently available in field to provide a window into understanding the role of miRNAs. These predicted targets can then be used as a platform for identifying signaling pathways and networks that are altered manifesting in the phenotype. Critically these bioinformatic tools are evolving with the field and are pivotal to understanding the global role of miRNAs in cardiomyopathy. Although miRNA regulation adds another layer of complexity to the already complex etiology, understanding the regulation could provide novel therapeutic strategies due to miRNAs ability to target multiple molecules. In this regard, the focus of our article is to provide an overview of altered miRNAs in cardiac stress and the available tools that could be used to understand their global implications.
miRNA generation
Genome distribution, miRNA processing and nuclear export
miRNAs are encoded by their own genes which are an integral part of cell’s genetic make up and are evolutionarily conserved (Ambros, 2004; Bartel, 2004). miRNAs can be transcribed as polycistronic primary transcripts or as individual transcripts from intergenic regions, exon sequences of non-coding strand or intronic sequences (Kim and Nam, 2006; Altuvia, et al., 2005) (Fig. 1). Intronic miRNAs are generally transcribed coincidentally with the gene and excised by the splicing machinery from the larger gene transcript in which they are embedded (Rodriguez et al., 2004). Indeed, intronic miRNAs may represent a simple way for a protein-coding gene to regulate other protein-coding genes in a non-canonical manner. miRNAs are transcribed by the RNA polymerase II as a primary transcript several kilobases long characterized by stem-loop hairpin structures called pri-miRNAs that are 5′ capped and a poly (A) to stabilize these pre-miRNAs similar to that of the traditional mRNAs (Lee et al., 2004). The generated pre-miRNA is processed in the nucleus and exported out through a regulated process. The stem-loop structures of pre-miRNAs are recognized by Drosha (a double-stranded specific RNase III) and its partner DGCR8 (a double stranded RNA binding protein) that cleave at the hair-pin base to release ~ 70-90 nucleotide stem-loop pri-miRNA precursor (Lee et al., 2003, 2004). In addition to this classical pathway, recent studies have identified alternate pathway wherein intronic pre-miRNA precursors "mirtrons" uses the cellular splicing machinery to bypass Drosha mediated processing (Ruby et al., 2007; Okamura et al., 2007) . The cleaved stem-loop pre-miRNA hairpins are exported into the cytoplasm by the exportin-5 (a Ran-GTP-dependent nuclear transport receptor) (Yi et al., 2003). The interaction of exportin-5 with the pre-miRNA ‘minihelix motif’ (~14 nucleotide stem and a short 30 nucleotide overhang) is thought to stabilize the pre-miRNAs (Yi et al., 2003; Filipowicz, 2005) manifesting in efficient transport.
Fig. 1. MicroRNA (miRNA) genomic organization, biogenesis and function Genomic distribution of miRNA genes. TF: transcription factor. (A) Clusters throughout the genome transcribed as polycistronic primary transcripts and subsequently cleaved into multiple miRNAs; (B) intergenic regions transcribed as independent transcriptional units; (C) intronic sequences of protein-coding or -non-coding transcription units or exonic sequences (black cylinders) of non-coding genes. Primary miRNAs(pri-miRNAs) transiently have a 7- methylguanosine (7mGpppG) cap and a poly(A) tail. The pri-miRNA is processed into a precursor miRNA (pre-miRNA) stem-loop of 60 nucleotides (nt) in length by the nuclear RNase III enzyme Drosha and its partner DiGeorge syndrome critical region gene 8 (DGCR8). Exportin-5 actively transports pre-miRNA into the cytosol, where it is processed by the Dicer RNaselll enzyme, together with its partner TAR (HIV) RNA binding protein (TRBP), into mature, 22 nt-long double strand miRNAs. The RNA strand (in red) is recruited as a single-stranded molecule into the RNA-induced silencing (RISC) effector complex and assembled through processes that are dependent on Dicer and other double strand RNA binding domain proteins, as well as on members of the Argonaute family. Mature miRNAs then guide the RISC complex to the 3′ untranslated regions (3′-UTR) of the complementary messenger RNA (mRNA) targets and repress their expression by several mechanisms: repression of mRNA translation, destabilization of mRNA transcripts through cleavage, de-adenylation, and localization in the processing body (P-body), where the miRNA-targeted mRNA can be sequestered from the translational machinery and degraded or stored for subsequent use. Nuclear localization of mature miRNAs has also been described and is a novel mechanism of action for miRNAs.
Generation of mature miRNA, activation and target recognition
The pre-miRNA is processed into a mature miRNA of ~22 nucleotides long by another double stranded RNase III called the Dicer (Hutvagner et al., 2001). Single stranded RNA is assembled into a RNA-inducing silencing complex (RISC) with the help of Dicer, TAR (HIV) RNA binding protein (TRBP), and dsRNA-binding proteins of the argonaute (AGO) family (Chapman and Carrington, 2007; Filipowicz, 2005; Schwarz et al., 2003; MacRae et al., 2008; Okamura et al., 2004). Additional factors have also been isolated and implicated (Chapman and Carrington, 2007; Filipowicz, 2005; Schwarz et al., 2003; MacRae et al., 2008; Okamura et al., 2004) to be a part of RISC complex bringing about miRNA-mediated silencing of gene expression that could be either a translational repression or degradation of mRNA. miRNAs recognize their target mRNAs through specific interaction of the 5′ end ‘seed’ region (2-8 nt from the 5′ end) and the complementary sequences of conserved target mRNAs (Bartel, 2004). Since only a few miRNAs have perfect complementarity to the target mRNAs leading to degradation, majority of the miRNAs have imperfect match resulting in translational repression (Nilsen, 2007). Another caveat in the miRNA silencing dynamics is the ability of multiple miRNAs to bind to the same mRNA initiating translational repression with different potencies. Repressed mRNAs are sequestered from translational machinery, degraded or stored for subsequent use in large macroscopic cytoplasmic foci, named processing bodies (P-bodies) upon silencing by miRNAs. The P-bodies contain a wide range of enzymes involved in RNA turnover, including de-capping enzymes, de-adenylases and exonucleases (Eulalio et al., 2007). In addition to their cytoplasmic role, miRNAs with nuclear localization sequence have been identified demonstrating their role in transcriptional control of gene expression (Chapman and Carrington, 2007; Volpe et al., 2002; Zilberman, et al., 2003; Aravin et al., 2007; Yu et al., 2008; Hwang et al., 2007; Calin et al., 2007).
miRNAs and cardiac development
miRNA expression in cardiac development
Studies have shown that miRNA mediated fine tuning leads to critical cell lineage commitment and embryonic tissue development (Latronico, et al., 2007; Farh et al., 2005; Ivey et al., 2008). Consistent with the role of miRNA in development, deletion of Dicer leads to embryonic lethality resulting from defects in cardiogenesis due to deficiencies in miRNAs biogenesis (Giraldez et al., 2006; Ebert et al., 2007). In tune with complex process of cardiogenesis many miRNAs are shown to be involved and the selection of miRNAs enriched during the differentiation of mouse embryonic stem cells to cardiomyocytes are detailed in Table 1 (Lakshmipathy et al., 2007; Thum et al., 2007). Significant increase in miRNA expression with development shows that miRNAs play an important role in early embryonic patterning and orchestrating organogenesis (Ivey et al., 2008). Expression pattern of miRNA-1 and -133 show that these two miRNAs play a key role in skeletal muscle proliferation and differentiation (Chen et al., 2006). Specifically, miRNA-1 promotes myogenesis by targeting histone deacetylase (HDAC4), a transcriptional repressor while miRNA-133 enhances myoblast proliferation by repressing serum response factor (SRF) (Zhao et al., 2005, 2007; Kwon et al., 2005; Niu et al., 2007). In this context, loss of function of miRNA-1 in Drosophila results in embryonic/larval lethality due to altered sarcomeric gene expression and increased number of undifferentiated muscle progenitors (Kwon et al., 2005). Where as miRNA-1 gain of function results in embryonic lethality due to insufficient numbers of cardioblasts indicating that cardiogenesis and differentiation is a spatio-temporal process tightly regulated by miRNA-1 mediated by cardiac transcription factor Hand2 (Zhao et al., 2005; Srivastava et al., 1997). Another important component of miRNA expression is the Dicer mediated generation of miRNA during development. Dicer expression during development determines miRNA expression and its regulation. Indeed, Dicer-deficient animals fail to synthesize new miRNAs resulting in embryonic lethality in zebrafish (Giraldez et al., 2005) and mice (Bernstein et al., 2003). More importantly cardiac specific deletion of Dicer results in aberrant cardiac contractile protein expression and severe sarcomere disarray, leading to progressive dilated cardiomyopathy (DCM), failure and postnatal lethality (Chen et al., 2008).
Table 1. Summary of regulated microRNAs (miRNAs) in cardiac development and disease.
Regulation of miRNA transcription
Understanding transcriptional regulation of miRNAs is critical as expression of miRNAs is a major determinant of miRNA dependent regulatory mechanisms. As miRNAs are transcribed like other genes, they are regulated by transcription factors and expression of transcription factors determines the miRNA expression. SRF is a cardiac enriched transcription factor that regulates sarcomere organization in the heart and SRF expression follows a restrictive pattern during development (Olson and Schneider, 2003; Niu et al., 2007; Barron et al., 2005). SRF expression is very important as multiple SRF binding sites have been identified in promoters of genes regulating contractility, cell movement, and growth signaling (Sun et al., 2006; Zhang et al., 2005). Consistent with the role of SRF in cardiac development, several miRNAs have been identified to contain SRF binding sites in their promoter including miRNA-1-1, -1-2, -21, -206, -214, -133 and others (Niu et al., 2007). In addition, studies have unequivocally have shown that miRNA-1-1, -1-2 and -133 are regulated by SRF transcription factor alone or in conjunction with co-factors like GATA5, MyoD, Nkx 2.5 or MEF2 (Fig. 2) (Zhao et al., 2005; Chen et al., 2006; Niu et al., 2007; Rao et al., 2006; Xiao et al., 2007). It is important to note that the outlined co-factors of SRF by themselves can act as transcriptional regulators in their own right increasing complexity of regulation. For example, MEF2 along with MyoD is known to regulate miRNA-1-2/133a-1 in myotomes during embryogenesis and all skeletal fibers in adulthood (Liu et al., 2007).
miRNA expression patterns
miRNA expression is greatly enriched in a tissue/cell-specific manner indicating unique signature patterns for each type. This enrichment and signature pattern suggests that miRNAs play a critical role in regulating and maintaining the specific cellular phenotype which is of essence in an organ with diverse cell/tissue types contributing to effective functioning. In this regard, heart as an organ contains many "non-cardiomyocyte" cell types like endothelial cells, smooth muscle cells and fibroblasts and each of which have distinct function in the heart. Consistently, differential enrichment of miRNAs are observed in cardiomyocyte versus cardiac fibroblasts indicating important role in cellular specificity (Landgraf et al., 2007; Kuehbacher et al., 2007; Harris et al., 2008). Although specific miRNA enrichments are being found in different cell types (Kuehbacher et al., 2007; Harris et al., 2008), lot more work needs to be done to determine contribution of miRNAs towards regulation of global networking pathways that defines specific fingerprints for each cell type. The current studies have all been focused on one or two miRNAs (Chen et al., 2006; Gregory et al., 2008; Harris et al., 2008) which by themselves may not be sufficient to determine a cellular phenotype indicating requirement of more comprehensive studies on specific miRNAs signature for cellular phenotype.
miRNAs and cardiac disease
The heart is responsive to physiological stimuli or pathological stress and accordingly undergoes remodeling to meet the demand (Catalucci et al., 2008). Following stress, the heart undergoes extensive remodeling in the form of physiological or pathological hypertrophy defined as an augmentation of ventricular mass due to increased cardiomyocyte size. Cardiac hypertrophy is characterized by initial compensatory mechanisms that adapt the heart towards sustaining the cardiac output. However, this process is only an initial ‘adaptive’ response and chronic exposure to stress eventually leads to impaired function that, in many cases, progresses to failure. This maladaptive change is accompanied by alterations in the underlying molecular map including a switch in the gene expression program leading to reexpression of fetal genes (Catalucci et al., 2008; Thum et al., 2007). The involvement of miRNAs in this pathological process has been recognized and is thought to be integral ‘switch’ in the gene expression program. Intense efforts have been put into identifying miRNAs altered in pathology and evolving signature of deregulated miRNAs identified cardiac disease is detailed in Table 2.
Myocardial hypertrophy, remodeling, and heart failure
Cardiac remodeling is characterized by structural alterations of myocardial tissue, modification of the extracellular matrix, and reshaping of left ventricle geometry and performance (Catalucci et al., 2008; Dash et al., 2001). The presence of chronic stress results in deleterious remodeling.
Multiple studies have been carried out to reveal important roles of miRNAs in cardiac hypertrophy and heart failure. Studies have found that a unique set of miRNAs are upregulated, downregulated or unaltered during the adaptive response of the heart to stress stimuli (Latronico et al., 2007). Furthermore, unique subset of miRNAs are known to be altered within the various etiologies of heart failure indicating significant role of miRNAs in these disease states (Sucharo et al., 2008). Consistent with the reexpression of fetal gene program, a high degree of similarity has been found between the miRNA expression pattern occurring in failing human hearts and those observed in the 12- 14 week-old hearts (Thum et al., 2007). Approximately, 80% of the analyzed miRNAs are similarly altered in failing adult and fetal human hearts compared to non-failing hearts. Multiple miRNAs have been implicated in cardiomyocyte hypertrophy and studies have consistently found upregulation of miRNA-21, -23a, -23b, -24, -195, -199a and miR-214 and downregulation of miRNA-1, -7, -133 and 378 (Naga Prasad and Karnik, 2010). Many of these miRNAs have been tested for hypertrophic response in neonatal cardiomyocytes. Concordant data from human and mice samples indicate that miRNAs may be involved in common pathway mediating hypertrophic response (Thum et al., 2007; van Rooij et al., 2006; Chen et al., 2008; Ikeda et al., 2007; Tatsuguchi et al., 2007; Sayed et al., 2007; Cheng et al., 2007) .
Among the miRNAs altered in various cardiac etiologies, some of them have been studied indepth and these include miRNA-1, -21, -133 and -208. It is well known that miRNA-1 is downregulated with a week of transverse aortic banding and its expression is inversely correlated with cardiac hypertrophy (Table 2) (Sayed et al., 2007; Catalucci et al., 2008; Ikeda et al., 2009, Naga Prasad et al., 2009 after al., 2009)). Similarly, Care et al., observed impaired expression of both miR-1 and miR-133 in patients with hypertrophic cardiomyopathy and atrial dilatation as well as in 3 different murine models of cardiac hypertrophy (Catalucci et al., 2008). In vitro cellular overexpression of miRNA-133 resulted in suppression of protein synthesis and block in hypertrophic response. Contrastingly, utilization of a decoy for miRNA-133 resulted in cellular hypertrophy and in vivo administration resulted in significant myocardial hypertrophy associated with reexpression of the fetal gene program (Catalucci et al., 2008). Some of targets of miRNA-133 have been validated and many are still being validated to provide evidence of miRNA-133 targeting multiple molecules to bring about hypertrophic response. miRNA-133 is encoded by 133a-1 and -2 and deletion of individual miRNA have no obvious cardiac abnormalities but combined deletion results in severe cardiac malformations with embryonic and post-natal lethality (Care et al., 2007). In contrast, overexpression of miRNA-133a results in embryonic lethality (E 15.5) caused by ventricular septal defected and impaired cardiomyocyte proliferation resulting in thinning of ventricular walls unable to meet hemodynamic needs (Care et al., 2007). These studies reveal that miRNA-133 plays a key role in myocardial development, hypertrophy and function. miRNA-208 is unique as it is a cardiac-specific miRNA encoded within the intron of a-myosin heavy chain (a-MHC) gene. miR-208 knockout mice are viable and do not show any obvious cardiac phenotype, but they fail to undergo stress-induced cardiac remodeling, hypertrophic growth, and a-MHC upregulation following transverse aortic constriction (Table 2) (van Rooij et al., 2007). It is believed that miRNA-208 regulation of this process involves a-MHC alterations balancing a-MHC.
While miRNA-208 mediates cardiac function by cardiomyocyte specific expression, miRNA-21 regulates cardiac function by its expression in both myocytes as well as cardiac fibroblasts. Recent studies (Thum et al., 2008) have shown progressive upregulation of miR-21 during late stages of heart failure, with an expression profile restricted exclusively to cardiac fibroblasts (Table 2). Upregulation of miR-21 was shown to be responsible for increased extracellular signal-regulated kinase (ERK) signaling through inhibition of its target, spry1 (sprouty 1), an inhibitor of the ERK/ extracellular signal-regulated kinase pathway. These studies suggest that miRNA-21 expression results in increased fibroblast survival and reduced interstitial fibrosis independent of cardiomyocyte loss that may provide protective effects (Thum et al., 2008). Likewise, it has been (van Rooij et al., 2008) recently demonstrated that downregulation of the fibroblast-enriched miRNA-29 family in fibrotic areas surrounding a cardiac infarct is responsible for the regulation of mRNAs that encode a multitude of proteins involved in fibrosis such as collagens, fibrillins, and elastins. In addition to these miRNAs, we have recently shown that 8 miRNAs are differentially expressed in human dilated cardiomyopathy (DCM) (Naga Prasad et al., 2009). The miRNA-1, -29b, -7, and -378 were significantly down-regulated in the DCM samples compared with non-failing controls. In contrast, miRNA-214, -342, -125b and -181b were significantly upregulated in DCM compared with non-failing controls. These studies identified miRNA-7 and -378 as novel miRNAs which are significantly downregulated during end stage cardiac dysfunction whose role in cardiac pathology remains to be determined.
