The Role of MicroRNA and LncRNA–MicroRNA Interactions in Regulating Ischemic Heart Disease

Abstract

Approximately 2% of the human genome consists of protein-coding regions. Therefore, the majority of transcripts are noncoding RNAs, such as microRNA (miRNA) and long noncoding RNAs (lncRNAs). In ischemic heart disease, the majority of miRNAs are repressors or destabilizers of target messenger RNAs. The lncRNAs are a second class of noncoding RNAs that have recently gained attention for their roles in heart disease and in regulating the functions of miRNA. In this review, we summarize the role of miRNA in pathological cardiac hypertrophy and myocardial infarction. In addition, we discuss the functional interactions of miRNA and lncRNA and its impact on these ischemic heart diseases.

Introduction

More than 98% of the DNA in the human genome consists of sequences defined as noncoding regions, and less than 2% of the genome is translated into proteins. The vast majority of noncoding DNA regions are actively transcribed into RNA but not translated into proteins. These nontranslated proteins are called noncoding RNAs (ncRNAs). ^{1 -3} The ncRNA family is large and heterogeneous, with subgroups defined by function, such as housekeeping RNAs (ribosomal RNA, transfer RNA, telomerase RNA, etc) and regulatory RNAs. The regulatory ncRNAs control the expression of genes involved in various cellular functions. They are broadly classified into 2 groups: short ncRNAs (<200 nucleotides) and long ncRNAs (lncRNAs; >200 nucleotides). ^{4 -9}

Among the short ncRNAs, microRNAs (miRNAs) are well characterized and important regulators of gene expression at the posttranscriptional level under physiological and pathological conditions. ¹⁰ These small (∼21 to 23 nucleotides) nonprotein-coding RNAs efficiently guide a variety of activities, such as transcription rate, initiation and elongation of target messenger RNAs (mRNAs), and the stability of newly synthesized proteins. ^{11 -13} Since the first report in 1993, ^14,15 miRNA generation and miRNA-directed gene regulation have been deeply studied in a wide range of diseases. Those ncRNAs with more than 200 nucleotides are known as lncRNA. Mounting evidence indicates that members of the lncRNA family contribute to intracellular processes by acting as host transcripts for miRNA, regulators of mRNA stability, and cis regulators of gene expression and molecular scaffolds for protein complexes, such as ribonucleoproteins and chromatin-remodeling complexes. ^16,17

Ischemic heart disease is a condition characterized by reduced or insufficient blood supply to the heart. In the heart, blood supply from the coronary arteries is vital for the proper functioning of the heart muscle, and any blockage in the coronary arteries is a potential risk for the development of ischemic heart disease. The main cause of ischemic heart disease is the formation of atherosclerotic plaques in the walls of the coronary arteries, which leads to narrowing/blocking of vessels supplying blood to the heart muscles. Pathological cardiac hypertrophy and myocardial infarction (MI) are the most common ischemia-related diseases that eventually lead to heart failure, a leading cause of hospitalization and death worldwide. In this review, we summarize the role of miRNA and lncRNA in pathological cardiac hypertrophy and MI. Later, we discuss interactions between miRNA and lncRNA and how those interactions affect cardiac disorders.

MicroRNA and lncRNA Functions in Ischemia Heart Diseases

Most of the ncRNAs, including miRNAs and lncRNAs, are located in the nucleus or in the cytoplasm. Cytoplasmic ncRNAs bind with target mRNAs, partly or entirely, by complimentary base pairing to suppress transcription or translation. ^10,16 At the same time, ncRNAs in the nucleus directly bind with gene promoters and regulate (positively or negatively) gene expression and transcription. ^18,19

MicroRNA Functions in Pathological Cardiac Hypertrophy

Cardiac hypertrophy is a compensatory response of the heart to intrinsic or extrinsic stress-induced injury and hemodynamic overload. It can be divided into physiological and pathological cardiac hypertrophies. ²⁰ Both types of hypertrophies exhibit similar histological alterations in heart structure, such as abnormal thickening of the ventricular wall and a decrease in the size of the ventricular chamber. Physiological hypertrophy is related to enhanced heart function. Sustained cardiac hypertrophy is often accompanied by maladaptive repair and cardiac remodeling, which results in heart failure and sudden death. During this process, a variety of intracellular signaling pathways and transcriptional mediators are activated, ^20,21 along with changes in mitochondrial dynamics. Therefore, maladaptive pathological hypertrophy is a therapeutic target for heart failure.

The miRNA involvement in hypertrophy (the upper part in Figure 1) was first reported in 2006 by Van Rooij et al. They observed modification in the expression of more than 12 miRNAs in hypertrophic and failing hearts. Among them, upregulation of miR-23a, miR-23b, miR-24, miR-195, and miR-214 provokes a hypertrophic response in cardiomyocytes. ²² The same research group also found that cardiac-specific expressions of miR195²² and miR-208²² are indispensable in cardiomyocyte hypertrophy, pathological cardiac growth, and myocardial fibrosis. In recent years, modern microarray analyses revealed that numerous miRNAs are associated with the hypertrophic response; and some have negative effects, whereas others have positive influences on hypertrophy. The expression of miR-1 and miR-133 is decreased in both physiological and pathological hypertrophy. ²³ Gain- and loss-of-function studies in animal models find that miR-133 protects cardiac contractility and function from pathological hypertrophy by modulating activity of the β1-adrenergic receptor and its signaling cascade components. ²⁴ Similarly, miR-1 protects adult cardiac tissue from pathological hypertrophy by attenuating the calcium-calmodulin signaling-dependent expression of hypertrophic genes. There are also direct targets for miR-1, including the expression of myocyte enhancer factor-2a and Gata4, which are key transcription factors involved in calcium-dependent changes in gene expression. ²⁵ Another member of the miR family, miR-541, remarkably decreases hypertrophic growth in cardiomyocytes. During pathological hypertrophy, miR-541 is downregulated by the microphthalmia-associated transcription factor, a transcription factor involved in β-adrenergic-induced cardiac hypertrophy. Experimental studies in cultured cardiomyocytes and transgenic mice (miR-541 overexpression) suggest that its upregulation in cardiomyocytes can alleviate hypertrophic responses. ²⁶ The antihypertrophic effect of these miRNAs provides a new approach to coping with cardiac hypertrophy.

Figure 1.

The role of miRNA and the regulatory function of lncRNA on miRNAs in ischemic heart diseases. In ischemic heart diseases, such as pathological cardiac hypertrophy and myocardial infarction, miRNAs play multiple roles, including the classical repression of translation or enhancing mRNA degradation. In pathological cardiac hypertrophy, expression levels of miR-208a, miR-195, miR-23a, and miR-34 are increased, whereas miR-1, miR-133, and miR-541 levels are decreased. Myocardial infarction is accompanied by the upregulation of miR-21 and miR-874 and the downregulation of miR-29, miR-499, and miR-484. APF, CARL, MDRL, CHRF, and H19 are recently discovered lncRNAs associated with pathological cardiac hypertrophy or myocardial infarction. These lncRNAs bind miRNAs through partial complementary base pairing and regulate their functions in diseases. APF indicates autophagy-promoting factor; CARL, cardiac apoptosis-related lncRNA; CHRF, cardiac hypertrophy-related factor; lncRNA, long noncoding RNA; MDRL, mitochondrial dynamic-related lncRNA; miRNA, microRNA.

Many stress-inducible miRNAs take part in hypertrophic cascades. In experimental animal models, silencing stress-inducible miR-208a, an miRNA generated from myosin genes, improves cardiac function and survival by preventing cardiac remodeling. ²⁷ Cardiac stress consistently upregulates miR-195, which acts as a regulator in cultured cardiomyocytes or intact hearts. ²⁸ By suppressing the expression of the forkhead family of transcription factor 3a, which retards hypertrophic growth, ²⁹ miR-23a contributes to hypertrophy in a positive manner. The miR-34 family shows increased expression in both pathological cardiac hypertrophy and MI, and they target multiple signaling pathways and downstream factors to regulate cardiomyocyte apoptosis and cardiac contractile function after injury. Therapeutic inhibition of the miR-34 family can protect the heart from pathological hypertrophic remodeling and improve heart function. ³⁰

The Roles of miRNA in MI

Acute MI is caused by coronary artery occlusion, which remains a leading cause of morbidity and mortality worldwide. Insufficient blood supply to the left ventricular free wall results in the massive death of cardiomyocytes by apoptosis or necrosis and impaired cardiac contractility due to pathological remodeling, such as fibrosis. The pathological consequences of MI are accompanied by dysregulation of several miRNAs. Among them, members of the miR-29 family target mRNAs that encode proteins involved in fibrosis, including collagen and fibronectin. ³¹ However, expression of miR-29 is decreased post-MI; thus, enhancing miR-29 expression could be therapeutically beneficial in preventing post-MI cardiac remodeling and other fibrosis-related disorders. In contrast, miR-21 is upregulated during cardiac stress to promote cardiac fibrosis by enhancing the signaling of extracellular-regulated kinase, a mitogen-activated protein kinase involved in the expression of many profibrotic genes in fibroblasts. ^{32 -35} Similarly, miR-874 expression is substantially increased in response to oxidative stress and promotes necrotic cell death in cardiomyocytes. ³⁶

In recent years, it has been reported that abnormal mitochondrial fission participates in the pathogenesis of MI. Many miRNAs have been reported to regulate mitochondrial integrity and dynamics in a variety of circumstances. A study in cardiomyocytes observed that miR-499 inhibits cardiomyocyte apoptosis by blocking the mitochondrial fission program through the suppression of calcineurin-dependent dephosphorylation of dynamin-related protein 1 (Drp1), an active form of Drp1 required for the initiation of mitochondrial fission. ³⁷ miR-499 downregulates the expression of calcineurin catalytic subunits (CnAα and CnAβ) and inhibits Drp1 activity. However, in an ischemic/anoxic condition, increased levels of p53 downregulate miR-499 expression at the transcription level in cardiomyocytes, which promotes mitochondrial fission and a cardiac hypertrophic response. ³⁷ Likewise, miR-484 protects cardiomyocytes from mitochondrial fission-induced cell death by inhibiting translation of the mitochondrial fission protein 1. ³⁸ Fission protein 1 is an integral protein in the mitochondrial outer membrane, where it forms a complex with Drp1 to initiate mitochondrial fission. Forkhead family of transcription factor 3a acts as an upstream mediator of miR-484 expression. Under anoxic conditions, the expression of miR484 is downregulated, which leads to mitochondrial fragmentation and cardiomyocyte cell death. ³⁸ In addition, several members of the miRNA family, miR-421, miR-361, and miR-30b, are regulators of mitochondrial fission-dependent cardiomyocyte cell death under ischemic conditions. ^{39 -41} Mitochondrial fission plays a crucial role in the activation of cardiac hypertrophy and cardiomyocyte cell death. ⁴² These studies suggest that miRNAs are important partners in maintaining mitochondrial morphology and integrity and that the therapeutic manipulation of their levels could be useful in treating cardiac hypertrophy in clinical settings (the lower part of Figure 1).

The Regulation of lncRNA on Ischemic Heart Diseases

The role of lncRNAs in the pathological response to cardiovascular injury or disease has already been described in detail. ^{43 -45} Here, we discuss what is known about lncRNAs that regulate pathological cardiac hypertrophy. The first known cardio-specific lncRNA is the myosin heavy chain-associated RNA transcript (Mhrt). Mhrt inhibits cardiac hypertrophy and heart failure by reducing the expression of the brahma-related gene 1, which is a chromatin remodeling factor involved in the expression of many prohypertrophic genes. ⁴⁶ Recently, Song et al predicted that approximately 14 lncRNAs regulate hypertrophy-related disease genes. Among them, 3 lncRNAs (SLC26A4-AS1, RP11-344E13.3, and MAGI1-IT1) are highly associated with cardiac hypertrophy gene expression. ⁴⁷

MicroRNA–lncRNA Interaction in Ischemia Heart Diseases

The partial complementary base pairing of miRNA with mRNA inhibits targeted mRNA translation, promotes mRNA degradation, or reduces mRNA stability. Their impact on cardiac hypertrophy and MI confirms that any imbalance/alterations in their levels have detrimental effects on cardiac function. A recent study from our laboratory found that miRNAs are oxidatively modified by Reactive oxygen species (ROS), and oxidized miRNAs recognize and bind new target genes. ⁴⁸ For instance, oxidative stress-induced oxidization of miR-184 leads to its interaction with 3′-untranslated regions of Bcl-xL and Bcl-w. This interaction results in the initiation of apoptosis in cultured cardiomyocytes, as well as in injured (ischemia/reperfusion) cardiac tissue. Interestingly, those 2 molecules are not native targets of miR-184. ⁴⁸ In contrast, oxidative stress induces the generation of many lncRNAs. ⁴⁹ Emerging evidence shows that there is a functional interaction between different classes of ncRNAs; in particular, interactions between miRNAs and lncRNAs are in the spotlight. In fact, they can regulate each other’s activities. The mutual interaction of miRNAs and lncRNAs can be categorized into 4 types depending on the mode and its effects: miRNAs-triggering lncRNA decay, lncRNAs as miRNA sponges/decoys, competing lncRNAs and miRNAs for interaction with mRNAs, and lncRNAs as a precursor for miRNAs. ⁵⁰ These interactions are well defined in tumorigenesis, but few reports are available to define their interactions in cardiovascular diseases. In this section, we discuss what is known about miRNA–lncRNA interactions in cardiac hypertrophy and MI.

Autophagy-Promoting Factor

Autophagy is an intracellular degradative process that is essential for the removal of damaged or excessive organelles, proteins, and lipid aggregates. Autophagy-promoting factor (APF) is a recently identified lncRNA that regulates autophagic cell death in cardiomyocytes by influencing the expression of autophagy-related protein 7 (ATG7) in vivo and in vitro. Autophagy-promoting factor contains a complementary site for miR-188-3p, which actively suppresses ATG7 to prevent autophagic cell death. Under ischemia/reperfusion conditions, increased levels of APF lead to direct binding with miR-188-3p, which inhibits miR-188-3p activity and results in enhanced cardiac autophagy and consequently worsening MI injury. ⁵¹ The RNA–RNA binding energy affects target accessibility and is therefore crucial for determining lncRNA–miRNA interactions. An RNA hybrid analysis found that the minimum free energy of APF-miR-188-3p binding is −26.2 kcal/mol, which indicates that this interaction is stronger in cases of MI injury. ⁵¹ The APF lncRNA-miR-188-3p-ATG7 pathway is a typical example of how lncRNAs act as miRNA sponges/decoys and provides a model of the molecular regulation of the autophagic program in MI. Thus, the identification and targeting of lncRNAs with similar functions could yield novel therapeutic candidates to control cardiac disease.

Cardiac Apoptosis-Related lncRNA

Cardiac apoptosis-related lncRNA (CARL) is an lncRNA that is important in the regulation of mitochondrial dynamics, cardiac apoptosis, and the resulting cardiac dysfunction. Prohibitin 2 (PHB2) is a subunit of the prohibitin complex, which is an estrogen receptor-binding protein that represses the activity of many transcription factors. Prohibitin 2 can inhibit mitochondrial fission and apoptosis. However, miR-539 binds PHB2 mRNA and inhibits its activity. Recently, Wang et al observed that CARL acts as a competing endogenous RNA to “sponge up” miR-539, thereby upregulating the expression of PHB2 and suppressing mitochondrial fission and apoptosis. ⁵² Adenosine Triphosphate (ATP)-generating pathways are required to maintain heart function, which is why cardiomyocytes are rich in mitochondria. These studies show that lncRNAs can regulate mitochondrial dynamics by modulating miRNA activity/expression. These findings provide a new pathway to regulate the energy supply in cardiomyocytes under normal or pathological circumstances.

Mitochondrial Dynamic-Related lncRNA

The name mitochondrial dynamic-related lncRNA (MDRL) indicates an lncRNA that regulates mitochondrial dynamics. For example, miR-484 is required for the inhibition of mitochondrial fission-mediated apoptotic cell death. However, nuclear miR-361 can directly bind with the primary transcript of miR-484 and prevent its maturation. A recent report from our laboratory demonstrated that MDRL acts as an endogenous sponge by binding to miR-361, downregulating its expression and inhibiting mitochondrial fission and apoptosis in cardiomyocytes. ⁵³ Thus, the contribution of MDRL to the maturation of miR-484 illustrates another novel model of interactions between ncRNAs in cardiac pathophysiology.

Cardiac Hypertrophy-Related Factor

Cardiac hypertrophy-related factor (CHRF) is an lncRNA that is upregulated in response to cardiac hypertrophy. Cardiac hypertrophy-related factor is also elevated in the failed heart tissue of humans. Like MDRL, CHRF acts as an endogenous “miR sponge” to downregulate miR-489 expression. Studies show that miR-489 targets myeloid differentiation primary response gene 88 (Myd88) to inhibit hypertrophy in vivo and in vitro. Cardiac hypertrophy-related factor interferes with miR-489 expression and activity by promoting the expression of hypertrophic genes such as Myd88. ⁵⁴ Therefore, modulating the expression of ncRNAs could be helpful to tackle cardiac hypertrophy.

H19

H19 is an lncRNA highly expressed in the heart under physiological conditions. ⁵³ A recent study in our laboratory revealed that H19, which is required to attenuate necrotic responses, interacts with the binding of miR-103/107 to the Fas-associated protein with death domain (FADD). The FADD is a component of the apoptotic cell death inducing a signaling complex, which inhibits certain types of necrosis by negatively regulating receptor-interacting serine/threonine-protein kinases 1 and 3. A member of the miR family, miR-103/107, directly binds FADD to block its inhibitory action on necrosis. However, the miR-103/107-FADD pathway does not participate in tumor necrosis factor–dependent necrosis, which is a common extrinsic pathway of cell death. H19 lncRNA can act as a sponge to block miR-103/107 expression. However, H19 expression is downregulated under ischemic conditions, which could be associated with myocardial necrosis. ⁵⁵ This newly identified regulatory pathway strengthens our understanding of the molecular mechanisms of cardiomyocyte necrotic cell death.

Conclusion

Long ncRNAs and miRNAs can affect all aspects of cellular function by regulating gene expression at transcriptional, posttranscriptional, and posttranslational levels. Both miRNAs and lncRNAs have significant impacts on the pathogenesis of ischemic heart diseases, such as cardiac hypertrophy and MI. Emerging evidence highlights interactions between these 2 groups of ncRNAs that regulate several molecular events under pathological circumstances (Table 1). In most instances, lncRNAs absorb miRNAs, which is the only regulatory mechanism well recognized in ischemic heart disease. However, it is possible that in ischemic heart disease, lncRNAs affect miRNA expression/activity by mechanisms other than miRNA absorption. Similar mechanisms may occur in other diseases, such as cancer. ⁵⁰ For example, lncRNAs can act as miRNA precursors or compete with miRNAs to bind mRNA in tumor cells. ⁵⁰ Recently, a muscle-specific lncRNA was reported to encode a 34-amino acid peptide that induces heart failure. ⁵⁶ This suggests that lncRNAs can act as transcription factors that regulate miRNA expression. However, more detailed studies are required to elucidate the complex interactions of lncRNA–miRNA in cardiac diseases. Numerous experimental studies show that ncRNAs, miRNAs in particular, play a crucial role in normal and pathological circumstances. Many have been successfully tested in experimental animal models for efficiency in treating various disorders, including cardiac hypertrophy and MI. Currently, considerable effort has been invested in translating miRNA-based therapy from experimental settings to clinical situations. The role of lncRNAs in regulating miRNAs has provided important clues for designing therapeutics that target lncRNAs (Table 1). Given that both miRNAs and lncRNAs influence miRNA function in cardiac diseases, a clear understanding of how lncRNAs modify miRNA expression and activity may provide an efficient and powerful tool for the effective treatment of pathological hypertrophy or MI in the future.

Table 1.

Examples of Direct Interaction Between lncRNAs and miRNAs in Cardiac Ischemia Diseases.

Disease	lncRNA	miRNA	Interaction	Target	Consequence	Reference
MI	APF	miR-188-3p	miRNA sponges	ATG7	Enhanced cardiac autophagy and worsen MI injury	51
	CARL	miR-539	miRNA sponges	PHB2	Suppressed mitochondrial fission and apoptosis	52
I/R	MDRL	miR-361 pri-miR-484	miRNA sponges	miR-484	Inhibit mitochondrial fission and apoptosis	53
Hypertrophy	CHRF	miR-489	miRNA sponges	Myd88	Inhibit hypertrophy	54
Necrosis	H19	miR-103/107	miRNA sponges	FADD	Inhibit myocardial necrosis	55

Abbreviations: APF, autophagy-promoting factor; ATG7, autophagy-related protein 7; CARL, cardiac apoptosis-related lncRNA; CHRF, cardiac hypertrophy-related factor; FADD, Fas-associated protein with death domain; lncRNA, long noncoding RNA; I/R, Ischemia/Reperfusion; MDRL, mitochondrial dynamic-related lncRNA; MI, myocardial infarction; miRNA, microRNA; Myd88, myeloid differentiation primary response gene 88; PHB2, prohibitin 2.