Long Noncoding RNAs in Myocardial Ischemia-Reperfusion Injury

Following an acute myocardial infarction, reperfusion therapy is currently the most effective way to save the ischemic myocardium; however, restoring blood flow may lead to a myocardial ischemia-reperfusion injury (MIRI). Recent studies have confirmed that long-chain noncoding RNAs (LncRNAs) play important roles in the pathophysiology of MIRIs. These LncRNA-mediated roles include cardiomyocyte apoptosis, autophagy, necrosis, oxidative stress, inflammation, mitochondrial dysfunction, and calcium overload, which are regulated through the expression of target genes. Thus, LncRNAs may be used as clinical diagnostic markers and therapeutic targets to treat or prevent MIRI. This review evaluates the research on LncRNAs involved in MIRIs and provides new ideas for preventing and treating this type of injury.


Introduction
An acute myocardial infarction (AMI) is a type of cardiovascular event that seriously endangers human health [1,2]. Timely reperfusion treatment following the onset of an AMI can restore blood flow, salvage viable myocardium, reduce the myocardial infarction size, and preserve left ventricular (LV) systolic function [3][4][5]. However, the process of myocardial reperfusion can further aggravate ischemic myocardium damage, leading to a myocardial ischemia-reperfusion injury (MIRI). Clinical presentations of a MIRI are reperfusion-induced arrhythmias, myocardial stunning, microvascular obstruction, and lethal myocardial reperfusion injury [6][7][8]. Protecting the heart from the detrimental effects of a MIRI is a therapeutic challenge for physicians. Therefore, elucidating the key mechanisms that prevent a MIRI would greatly aid in the development of an effective therapy for AMIs. Recent studies have shown that the mechanisms of a MIRI include cardiomyocyte apoptosis, autophagy, necrosis, oxidative stress, inflammation, mitochondrial dysfunction, and intracellular calcium overload, all of which involve a multitude of signaling pathways and molecular players [6,7,9].
Recently, long noncoding RNAs (LncRNAs) have been shown to modulate key interactions with molecular pathways associated with MIRI progression and potentially serve as novel therapeutic targets. LncRNAs are a class of noncoding RNAs longer than 200 nucleotides. LncRNAs can regulate gene expression at multiple levels, including regulation of epigenetic, transcriptional, and posttranscriptional processes. This review discusses recent studies that have examined the functional role of LncRNAs in MIRIs, their therapeutic potential and aspects that should be further evaluated in the future.

LncRNAs
LncRNAs are a class of noncoding RNAs that are more than 200 nucleotides in length that are typically synthesized via RNA polymerase II. Based on the position of the LncRNA and coding gene, these types of RNAs are divided into five categories: (1) sense LncRNA, (2) antisense LncRNA, (3) intron LncRNA, (4) bidirectional noncoding LncRNA, and (5) intergenic LncRNA. LncRNAs regulate gene expression on both chromatin and DNA at the transcriptional and posttranscriptional level, all of which can affect the occurrence and outcome of various diseases [10]. Some regulatory mechanisms exerted through LncRNAs may involve interfering with the translation of an encoded gene, inhibiting polymerase II activity, facilitating posttranscriptional modification, binding to functional proteins, or serving as a precursor for a small molecule RNA that binds to chromosomes to regulate signaling pathways [11,12].
In addition to being directly involved in regulating gene expression, LncRNAs can also function as a competing endogenous RNA (ceRNA)-i.e., competes with other RNA transcripts for the same microRNA (miRNA), thereby affecting resulting interactions and subsequent signaling cascades. miRNAs are endogenous noncoding RNAs composed of 21 to 25 nucleotides that mainly facilitate the posttranscriptional regulation of target genes through binding to the 3 ′ untranslated region (UTR) of the target mRNA [13]. In a study on the interactions between RNA transcripts, Salmena et al. [14] first proposed a ceRNA-centered hypothesis as a novel model to regulate gene expression. In this model, transcripts such as LncRNA, pseudogene transcripts, or mRNAs can serve as ceRNAs through miRNA response elements (MREs), which can competitively bind to miRNAs with the same MRE to regulate gene expression levels, thereby affecting downstream cellular functions. Currently, functional studies on ceRNAs suggest that LncRNAs and miRNAs are mutually regulated via competitive binding to the corresponding MRE, thereby effectively controlling the subsequent posttranscriptional regulation of the miRNAs [15,16].

The Role of LncRNAs in MIRIs
The relationship between LncRNAs and a MIRI has been reported in several studies. Recently, Wu et al. reported that 2,292 LncRNAs were upregulated and 1,848 LncRNAs were downregulated in patients with a MIRI [17]. Using gene chip analysis in a mouse model of ischemia-reperfusion (I/R), Liu et al. found that 64 LncRNAs were upregulated and 87 LncRNAs were downregulated in the region with the infarction [18]. Similarly, using gene chip analysis in a cell-based study, Huang et al. found that 309 LncRNAs were upregulated and 488 LncRNAs were downregulated following hypoxia/reoxygenation (H/R) in H9c2 cells [19]. Recent studies have provided credible evidence that LncRNAs play a vital role in the initiation and progression of a MIRI through their involvement in apoptosis, autophagy, and necrosis of cardiomyocytes, as well as by affecting oxidative stress, inflammation, mitochondrial dysfunction, and intracellular calcium overload ( Figure 1 and Table 1).

LncRNAs and Apoptosis.
Cardiomyocyte apoptosis is a key pathophysiological mechanism underlying a MIRI. Apoptosis is a type of programmed cell death induced by physiological or pathological factors. The process is characterized by cell shrinkage, chromatin condensation, and systematic DNA cleavage [20]. Apoptosis is a highly regulated cellular process, which can be divided into two classic pathways: the extrinsic or intrinsic pathway. The extrinsic pathway, also known as the death receptor-associated pathway, is activated via the death-inducing signal complex (DISC). The DISC activates caspase-8, which in turn triggers the downstream activation of executioner caspases, leading to apoptosis. The intrinsic pathway, also known as the mitochondrial pathway, is initiated by increased permeability of the outer mitochondrial membrane (OMM), leading to the translocation of proapoptotic molecules such as cytochrome C (cytC) and apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space to the cytosol. This leads to the formation of the apoptosome complex and subsequent downstream activation of the caspase cascade. Members of the Bcl-2 family are major participants in the intrinsic pathway [20,21]. In some circumstances, the intrinsic pathway is also initiated via the endoplasmic reticulum stress-(ERS-) induced apoptotic signaling pathway. It has been demonstrated that prolonged and/or excess ERS may lead to substantial apoptosis and be a major contributor to a MIRI. Herein, we expound on the relationships between different LncRNAs and their role during apoptosis in response to a MIRI [22][23][24][25].
3.1.1. LncRNA MALAT1. The expression of the LncRNA MALAT1 is significantly increased in the myocardial tissue of the rat I/R model. Knockdown of MALAT1 can markedly improve I/R-induced myocardial contractile dysfunction and inhibit cardiomyocyte apoptosis in the rat I/R injury model by upregulating the expression of β-catenin [26]. LncRNA MALAT1 was prominently elevated in oxygen-glucose deprivation/reoxygenation-(OGD/R-) induced H9c2 cells. MALAT1 knockdown inhibited OGD/R-induced cardiomyocyte apoptosis by activating the AKT/GSK-3β/β-catenin signaling pathway via downregulation of miR-122 [27]. LncRNA MALAT1 expression was upregulated in H/Rtreated AC16 cells. Moreover, MALAT1 regulated hypoxiainduced myocardial cell apoptosis by sponging miR-200a-3p to upregulate PDCD4 [28]. The expression of LncRNA MALAT1 and phosphatase and tensin homolog deleted on chromosome 10 (Pten) was upregulated, whereas that of miR-320 was downregulated in myocardial tissues from the AMI mouse model. MALAT1 functions as a ceRNA for miR-320 to regulate Pten in mouse cardiomyocytes. Overexpression of MALAT1 aggravated cardiomyocyte apoptosis in vitro, and knockdown of MALAT1 improved cardiac function, resulting from AMI [29].
3.1.2. LncRNA PFL. The expression level of the LncRNA PFL was markedly improved in the myocardial tissues, whereas the level of HSP-20 was markedly decreased in the myocardium of the I/R rat model. HSP-20 can form a stable complex with Bax and to in turn regulate the ratio of Bax/Bcl-2, thereby maintaining mitochondrial integrity and inhibiting both cytC release and caspase-3 activation. Knocking down PFL can inhibit MIRI-mediated apoptosis by upregulating HSP-20 expression [30].
3.1.3. LncRNA HIF1A-AS1. LncRNA HIF1A-AS1 expression was upregulated in the mouse model of I/R. Interestingly, LncRNA HIF1A-AS1 functions as a ceRNA when it binds with miR-204, thereby suppressing its expression and upregulating suppressor of cytokine2 (SOCS2) expression. Members of the SOCS protein family were initially described as

LncRNAs and Autophagy.
Autophagy is an intracellular protective mechanism under physiological conditions that can degrade damaged or unnecessary proteins, as well as organelles, to maintain cellular homeostasis. When a MIRI occurs, autophagy is activated in response to oxidative stress, ERS, and energy deprivation. Recent studies have demon-strated that autophagy can be either protective or detrimental to cardiac tissue during a MIRI event [42,43]. Autophagy activated during the initial phase of ischemia has protective effects on cardiomyocytes. For instance, during the myocardial ischemia stage, AMPK is activated in response to the decreasing level of ATP, which occurs when a high ratio of AMP/ATP owing to the deprivation of nutrients and oxygen. Therefore, AMPK is an essential molecule to initiate autophagy in cardiac ischemia. AMPK could launch autophagy through the AMPK/mTORC1/ULK1 signaling pathway or by directly activating ULK1. Autophagy promotes cardiomyocyte survival by generating the amino acids and fatty acids required for maintaining cellular energy levels, as well as activating mitophagy, which can prevent damaged mitochondria from releasing their cytotoxic substances. However, uncontrolled excessive autophagy during the reperfusion stage exerts detrimental effects on cardiomyocytes. During the reperfusion phase, increased ROS generation prompts the overexpression of Beclin-1, which is a crucial mediator of autophagy during this phase. Consequently, overexpressing Beclin-1 can enhance autophagic activity during reperfusion. Excessive autophagy degrades essential proteins or organelles, thereby leading to autophagy-mediated cell death. Additionally, apoptosis is caused by detrimental effects of excessive autophagy in MIRI [44,45].
3.2.1. LncRNA TUG1. LncRNA TUG1 expression was upregulated in I/R-injured heart tissues and H 2 O 2 -challenged 5 Oxidative Medicine and Cellular Longevity cardiomyocytes. TUG1 was identified as a miRNA sponge that targets miR-142-3p, which affects the activity of downstream target molecules, e.g., HMGB1 and Rac1, that contribute to autophagic cell death. Depleting TUG1 could inhibit excessive autophagy-induced cell death and reduce I/R-induced infarction size [46].
3.2.7. LncRNA RMRP. The expression of LncRNA RMRP is increased in hypoxia-treated H9c2 cells as well as in the rat model of I/R. RMRP negatively regulates the expression of miR-206; however, RMRP overexpression can activate the autophagic response and aggravate subsequent cell damage by downregulating miR-206. Moreover, overexpression of miR-206 has a protective effect on hypoxia-induced injuries by targeting the regulation of ATG3 expression. Overexpression of RMRP activates the PI3K/AKT/mTOR signaling pathway, which is reversed following miR-206 overexpression. The RMPR/miR-206/ATG3 axis can mediate cell autophagy by regulating the activation of PI3K/AKT/mTOR signaling in response to a MIRI [51].
3.2.8. LncRNA AK079427. The expression of the LncRNA AK079427, also known as autophagy-promoting factor (APF), was significantly upregulated in A/R-treated cardiomyocytes and the mouse model of I/R. APF specifically sponges miR-188-3p to regulate its activity. Overexpression of miR-188-3p can inhibit both autophagy and myocardial infarction by downregulating its downstream target, ATG7. ATG7 is a key autophagy promoting gene that encodes an E1-like enzyme, a member of the autophagy system. APF knockdown can attenuate cardiomyocyte autophagy and reduce myocardial infarction sizes in response to a I/R injury by targeting the miR-188-3p/ATG7 axis. Thus, the APF/-miR-188-3p/ATG7 axis plays a key role in regulating autophagic cell death in cardiovascular tissues; moreover, it may be a potential target for novel therapeutic strategies for treating myocardial infarctions [52].
3.2.9. LncRNA Galont. LncRNA Galont expression was significantly upregulated in A/R-treated cardiomyocytes. Overexpression of Galont can aggravate cardiomyocyte autophagy and increase cell death in response to A/R stimuli, whereas Galont knockdown had the opposite effect. Galont was confirmed to directly bind with miR-338, a novel suppressor of autophagy. ATG5 is a gene targeted by miR-338, and Galont is positively correlated to ATG5 expression. Thus, the existing evidence suggests that the Galont/miR-338/ATG5 axis plays an important role in promoting A/Rinduced autophagy in cardiomyocytes [53].

3.2.10.
LncRNA 2810403D21Rik/Mirf. LncRNA 2810403D21Rik/Mirf expression was significantly upregulated in cardiomyocytes treated with H 2 O 2 and in the heart tissue from the mouse model of MI. LncRNA 2810403D21Rik/Mirf acts as a ceRNA that competitively binds to and affects the activity of miR-26a. miR-26a also modifies the activity of the downstream target Usp15, which can inhibit mitochondrial autophagy by activating the PINK1/PRKN pathway. Silencing 2810403D21Rik/Mirf and overexpressing miR-26a enhanced cell viability and mitigated myocardial injury by increasing the expression of autophagy-related proteins. Further, overexpression of 2810403D21Rik/Mirf suppresses autophagy and exacerbates myocardial injury by limiting the signaling capacity of the miR-26a/Usp15 axis. Therefore, modulating the impact of the 2810403D21Rik/Mirf-miR-26a/Usp15 axis on autophagy can promote a cardioprotective effect in response to a MIRI [54].
Knockdown of CAIF can aggravate H 2 O 2 -induced cardiomyocyte autophagy, whereas myocardin overexpression augments the size affected from a MIRI. Moreover, CAIF inhibits autophagy and attenuates myocardial injury in the heart by targeting the p53/myocardin-dependent autophagy pathway [55].
3.3. LncRNAs and Necrosis. Cardiomyocyte necrosis is a major pathological event in an AMI. Defining features of necrosis include an increase in cell size from cytoplasmic and mitochondrial swelling, karyolysis, loss of ribosomes from the endoplasmic reticulum, and plasma membrane disruption, all of which initiate inflammation [24,56]. Mitochondria play an important role in the execution of programmed necrotic cell death, especially in cardiomyocytes, through two mechanisms: mitochondrial permeability transition-(MPT-) dependent necrosis and death receptordependent necrosis (necroptosis). Necrosis usually destroys the inner mitochondrial membrane (IMM), thereby inducing the opening of mitochondrial permeability transition pores (MPTPs), which depletes ATP and the plasma membrane loses its integrity [57,58]. Necroptosis can be induced by triggering several molecules, primarily through death receptors-e.g., TNFR, FasR, and TNF-related apoptosisinducing ligand receptor (TRAIL-R)-that are stimulated by cytokines from the TNF family and Toll-like receptors (TLRs) [

LncRNAs and Oxidative Stress.
At the onset of reperfusion, the level of myocardial tissue oxygenation increases after restoring blood flow. However, when in a reduced state, respiratory chain complexes damage the mitochondrial electron transport chain, which generates a high level of ROS and leads to ischemia [61,62]. ROS includes H 2 O 2 , hydroxyl radicals, and superoxide anions. The primary enzyme systems involved in ROS production during a MIRI are xanthine oxi-dase, NADPH oxidase, the mitochondrial electron transport chain, and uncoupled nitric oxide synthase [63]. Oxidative stress occurs when the antioxidant system is overwhelmed by ROS overproduction. ROS-mediated mitochondrial dysfunction and the sequence of biochemical events following reperfusion of the ischemic area are the key pathological features of a MIRI. ROS overproduction promotes the oxidation of DNA and proteins as well as and lipid peroxidation, thereby causing alterations in membrane permeability that structurally and functionally damage cells [62,64,65].
3.4.1. LncRNA Gpr19. The expression of the LncRNA Gpr19 is upregulated in the mouse model of AMI and in neonatal rat ventricular cardiomyocytes (NRCMs) exposed to OGD/R. Gpr19 targets miR-324-5p, a miRNA that negatively regulates Mtfr1 expression at the transcriptional and translational levels. The function of Mtfr1 is primarily related to the IMM. Overexpression of Mtfr1 aggravates oxidative stress in NRCMs induced via OGD/R. Further, suppression of Gpr19 inhibits oxidative stress and attenuates myocardial injury through the miR-324-5p/Mtfr1 axis [66].

LncRNAs and Inflammation.
Inflammation is an important contributor to the pathophysiology of a MIRI. The I/Rinduced injury can activate the inflammatory response by promoting the release of cytokines, chemokines, and other proinflammatory factors [70]. The extent of the inflammatory response to a MIRI can determine the infarct size and subsequent LV remodeling [71,72]. During the postischemic phase, intracellular content released by necrotic cardiomyocytes initiates an intense inflammatory response by activating innate immune responses. During the early reperfusion phase, cardiomyocytes increase the expression of cytokines 7 Oxidative Medicine and Cellular Longevity and adhesion molecules, which prompt the recruitment of neutrophils and other leukocytes to the infarcted myocardium [73]. As the adherent leukocytes transmigrate into these infarcted zones, they disrupt the microvascular barrier and cause hyperpermeability, leading to myocardium edema that increases the diffusion distance for oxygen and nutrients. Neutrophils are the most abundant among infiltrating inflammatory cells during the reperfusion stage. Notably, they impair endothelial function, activate platelets leading to coronary microembolization, and cause the myocardium no-reflow phenomenon. The infiltration of neutrophils induces a reperfusion injury that amplifies the cellular damage initiated by ischemia [71,74].

LncRNA NEAT1.
LncRNA NEAT1 expression is upregulated in H 2 O 2 -treated H9C2 cells and in the mouse model of I/R. NEAT1 was identified as a functional sponge of miR-495-3p that negatively regulates its expression. miR-495-3p blocks the activation of the inflammation process induced via H 2 O 2 treatment by downregulating its target, MAPK6. Thus, the NEAT1/miR-495-3p/MAPK6 axis plays a crucial role in activating inflammation and aggravating cardiomyocyte injury during a MIRI [76].
3.5.3. LncRNA H19. LncRNA H19-a precursor of miR-675 that regulates multifarious target genes posttranslationally and is involved in different biological processes-was upregulated in cardiomyocytes exposed to OGD/R. Silencing H19 reduces the production of proinflammatory cytokines and inhibits apoptosis in cardiomyocytes following exposure to OGD/R by downregulating the levels of p-IκB-α and p-p65. However, the effect of H19 inhibition was partially reversed when miR-675 was overexpressed. Moreover, miR-675 could directly suppress the expression of PPARα, as demonstrated with dual-luciferase reporter assays and software-based analysis. PPARα exerts an anti-inflammatory effect by suppressing the NF-κB signaling pathway. Therefore, H19 inhibition can markedly improve cardiac structure and function in response to a MIRI via the H19/miR-675/PPARα signaling axis [77].
3.5.4. LncRNA Mirt1. Mirt1, a LncRNA mainly expressed in cardiac fibroblasts, was upregulated in both the myocardium of AMI-induced mice and cardiac fibroblasts that were subjected to hypoxia. High Mirt1 levels contribute towards LV dysfunction in AMI-induced mice, whereas its knockdown could decrease the myocardial infarct size, improve cardiac functions, and potentially limit the inflammatory response. Knockdown of Mirt1 inhibits cardiomyocyte apoptosis and reduces the production of inflammatory cytokines by suppressing NF-κB signaling under hypoxic conditions in vitro. These data suggest that Mirt1 knockdown plays a protective role in response to an AMI by inhibiting NF-κB activation [78]. Another study reported that Mirt1 expression levels significantly increased in myocardial tissue of aged diabetic I/R rats. This study also found that the protective effects of Mirt1 inhibition in these rats may result from the inhibition of NF-κB activation by attenuating the inflammatory response, which would normally induce apoptosis and further aggravate the damage to cardiac tissues [79].

LncRNAs and Mitochondrial Dysfunction.
Mitochondrial dysfunction is considered as the prominent pathological feature of a MIRI; therefore, it has become a key regulator of its inhibition. Mitochondria synthesize approximately 90% of ATP in cardiomyocytes [81,82]. Mitochondrial dysfunction can cause the imbalance of calcium homeostasis, excess production of ROS, impaired cellular energy metabolism and ATP production, and opening of MPTPs, which can culminate in cell death [83]. Numerous studies have demonstrated that an imbalance between mitochondrial fission and fusion contributes towards mitochondrial dysfunction in response to a MIRI. During the early ischemic period, mitochondrial fusion leads to the generation of a disproportionately large amount of ATP, while sustained ischemia induces excessive mitochondrial fission. During the reperfusion period, excessive ROS and calcium overload exacerbate the activation of mitochondrial fission. Mitochondrial fission generates smaller mitochondrial fragments; however, mitophagy insufficiently eliminates damaged mitochondria. Thus, mitochondrial fission results in a higher susceptibility of opening MPTPs, activating apoptotic pathways by releasing cytC, and activating caspases, all of which result in cell death following myocardial reperfusion [84][85][86].
3.6.1. LncRNA CARL. LncRNA CARL is expressed in both the nucleus and the cytoplasm. The expression of CARL was downregulated in cardiomyocytes following anoxia treatment. CARL acts as an endogenous sponge of miR-539, thereby negatively regulating its levels and activity. miR-539 targets and regulates PHB2 expression, which plays an important role in regulating mitochondrial morphology. Overexpression of CARL attenuates mitochondrial fission and cell death during a MIRI by sponging miR-539 as well as upregulating PHB2 expression in vitro and in vivo. Combined, these data suggest that the CARL/miR-539/PHB2 signaling is crucial in affecting mitochondrial dynamics and cell apoptosis during a MIRI [87]. 8 Oxidative Medicine and Cellular Longevity 3.6.2. LncRNA MDRL. LncRNA MDRL is 1,039 nucleotides and is expressed in both the nucleus and the cytoplasm. MDRL expression was downregulated in A/R-treated cardiomyocytes. The bioinformatics program RNAhybrid revealed that MDRL carries a target site for miR-361. miR-361 promotes mitochondrial fission and apoptosis following A/R treatment by inhibiting the processing of pri-miR-484 by Drosha into pre-miR-484. MDRL acts as an endogenous sponge for miR-361 to regulate mature miR-484 levels. In an animal model, MDRL knockdown attenuated mitochondrial fission, cardiomyocyte death, and myocardial infarction size in response to I/R. These findings suggest that MDRL targets miR-361/miR-484 in the mitochondrial fission cascade and apoptosis during a MIRI, thereby revealing potential targets for treating cardiac disease [88].

LncRNAs and Calcium
Overload. Calcium homeostasis is disturbed during myocardial ischemia-reperfusion. Excessive accumulation and leakage of intracellular calcium lead to calcium overload, which exacerbates a MIRI [89,90]. Cytosolic and mitochondrial calcium overload begins at ischemia and is aggravated during reperfusion. At the onset of ischemia, the Na + /H + exchanger (NHE) is activated and transports H + out of the cytosol in exchange for Na + . During reperfusion, NHE activity is accelerated, leading to additional accumulation of intracellular Na + . As a result, a high Na + concentration activates the Na + /calcium exchanger (NCX), thereby leading to intracellular calcium overload [91]. Alternatively, more calcium influx through L-type calcium channels and the sarcoplasmic reticulum uptake of calcium from the cytoplasm is inhibited, which further exacerbates the calcium overload in response to the I/R [92]. During reperfusion, disruption of the mitochondrial membrane potential accelerates energy depletion, thereby resulting in mitochondrial calcium overload and ROS overproduction. In addition, disrupting the mitochondrial membrane potential also results in ATP synthase behaving as an ATPase, thereby accelerating energy depletion in response to the ischemic insult. Large amounts of calcium accumulated in cells inhibit ATP synthesis in mitochondria, leading to the dysfunctional processing of energy metabolism. These processes can trigger MPTPs to open, leading to the breakdown of respiratory chain coupling, mitochondrial membrane potential disorder, and eventually the induction of either necrotic-or apoptoticmediated cell death [93].
3.7.1. LncRNA ZFAS1. LncRNA ZFAS1, an antisense LncRNA to the 5 ′ end of the protein-coding gene Znfx1, has been identified as an independent predictor of an AMI [94]. Calcium homeostasis is closely related to cardiac contractility, and abnormal intracellular calcium handling might contribute to the impairment of cardiac contractile function caused by ZFAS1 in a MIRI. Sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a) is a key protein involved in the reuptake of calcium into the sarcoplasmic reticulum during diastole, thereby regulating calcium homeostasis in cardiomyocytes. ZFAS1 expression is markedly elevated in the myocardium of mice with an AMI. Knockdown of endogenous ZFAS1 improved cardiac function as the ejection frac-tion (EF), and fractional shortening (FS) resumed to almost normal levels in the mouse model of myocardial infarction. ZFAS1 overexpression in normal mice could impair cardiac function as EF and FS both decreased, thereby promoting an enlarged LV internal dimension at end-diastole (LVIDd) and LV internal dimension at systole (LVIDs), which is similar to the pathological process that occurs in response to a MIRI. Additionally, ZFAS1 conferred a negative effect on SERCA2a by repressing its expression at the transcriptional level. ZFAS1 deleteriously impacted the dynamic influx of calcium, thereby leading to intracellular calcium overload in cardiomyocytes, which might be the underlying mechanism of its proapoptotic property. Moreover, it was found that NFATc2 served as a transactivator to promote ZFAS1 expression [95]. Another study found that ZFAS1 activated the mitochondrial apoptosis pathway in the mouse model of myocardial infarction. Additionally, ZFAS1 knockdown relieved mitochondrial swelling and decreased mitochondrial membrane potential in hypoxia-treated cardiomyocytes. ZFAS1 induced cardiomyocyte apoptosis by inhibiting SER-CA2a, thereby causing intracellular calcium overload. Therefore, knocking down ZFAS1 protects cardiac tissues against MIRI-induced dysfunction and cardiomyocytes against hypoxia treatment-induced apoptosis [96].

Biomarkers.
Due to the high mortality rate of patients with an AMI, early detection and treatment can considerably improve the prognosis of these patients. Finding a myocardial biomarker with high specificity and sensitivity has always been a hotspot in the cardiovascular research field.
LncRNAs have a high degree of tissue specificity and are relatively stable secondary structures present within samples of plasma, serum, urine, and other body fluids. Thus, LncRNAs can be novel biomarkers to diagnose and predict the prognosis of several diseases [97,98] (Table 2). An increasing number of clinical studies have measured changes in circulating LncRNAs of AMI patients and have investigated their roles as novel diagnostic biomarkers for AMIs. Lu et al. discovered seven LncRNAs by analyzing LncRNA expression in genomes from 52 acute coronary syndrome patients; moreover, they compared the LncRNA expression pattern between patients with a myocardial infarction or an unstable angina. The overall classification exhibited 90.38% accuracy, 100% sensitivity, and 68.75% specificity. These results suggest that these novel, functional LncRNAs may be candidate diagnostic biomarkers and potential therapeutic targets [99]. Li et al. reported LncRNA expression patterns using two microarray datasets from patients with AMI and healthy samples from the Gene Expression Omnibus (GEO) database. They found that 11 differentially expressed LncRNAs had a diagnostic value in AMI patients and may be used as potential biomarkers for early diagnosis of AMIs [100]. Gao et al. found that HOTAIR expression levels in the serum of AMI patients were significantly decreased compared with that of healthy controls. Thus, the LncRNA HOTAIR may serve as a sensitive predictor for AMI 9 Oxidative Medicine and Cellular Longevity diagnosis [101]. Li et al. found that the LncRNA LIPCAR may serve as a potential biomarker for ST-segment elevation myocardial infarction. The extent of LncRNA elevation also could reflect the severity of a coronary stenosis. Multivariate Cox regression analysis revealed that the Gensini score and LIPCAR levels were independent predictors of major adverse cardiac events following an AMI [102]. Yan et al. found that LncRNA UCA1 levels decreased in plasma samples from patients with early stage AMI and increased in 72-96 h following AMI onset. They proposed that the circulating concentration of LncRNA UCA1 may serve as a potential biomarker for diagnosing AMI [103]. Vausort et al. reported that LncRNAs ANRIL and KCNQ1OT1 within the peripheral blood of patients with AMI can be used as predictors of LV dysfunction following a MIRI [104]. Wang et al. found that LncRNA CHAST expression was upregulated in patients with AMI at 24 h but substantially decreased 3 d following AMI onset, indicating that CHAST may serve as a potential biomarker for diagnosing AMI. Moreover, circulating CHAST levels within 24 h can be used to predict cardiac contractile function in patients with AMI independently [105]. Wang et al. found that the LncRNAs H19, MIAT, and MALAT1 indicated an adverse outcome from an AMI. Peripheral blood mononuclear cell-(PBMC-) derived levels of the LncRNAs H19, MIAT, and MALAT1 were increased in AMI patients suggesting that they may be a useful diagnostic biomarker for AMI [106]. Zhang et al. found that LncRNA ZFAS1 expression was downregulated, but LncRNA CDR1AS expression was upregulated in the peripheral blood of patients with AMI. They also found that either 10 Oxidative Medicine and Cellular Longevity ZFAS1 or CDR1AS correlated with an AMI; however, the combination of the two or their reciprocal changes elicited greater sensitivity and specificity to accurately predict, thereby indicating that they are superior biomarkers for AMI [94].
In addition, there are several studies demonstrating that LncRNAs also serve as novel diagnostic biomarkers of coronary artery disease (CAD). Zhang et al. found that increased plasma levels of H19 and LIPCAR were associated with an increased risk of CAD and may be considered as novel biomarkers for diagnosing CAD [107]. Interestingly, the polymorphisms of H19 are associated with the risk and severity of CAD in a Chinese population, which might provide new markers for the prevention and early intervention of CAD [108]. Li et al. identified aberrantly expressed LncRNAs in CAD patients by analyzing LncRNA expression in the transcriptomes of 93 CAD patients and 48 healthy controls. The authors identified ENST00000444488.1 and uc010yfd.1 as novel LncRNA biomarkers for diagnosing CAD [109]. A recent study found that the LncRNA OTTHUMT00000387022 in PBMCs can be recognized as a novel biomarker due to its high sensitivity and specificity for diagnosing CAD [110].
Although the prospect of using LncRNAs as new diagnostic and prognostic biomarkers in cardiovascular disease is advancing, there are some limitations. For example, some LncRNAs need to be revalidated in a multicenter clinical study with a large sample size to ensure their diagnostic potential as a biomarker. Additionally, some previous clinical studies and samples related lack follow-up information; therefore, the predictive value of the LncRNA cannot be fully assessed. Recently, an in-depth study on the epigenetic mechanisms of differentially expressed LncRNAs in response to a MIRI is in progress; however, the standardized protocol of the test operation has not been confirmed. For instance, from acquiring biological fluids to recovering a small amount of nucleic acids, the quantification of these samples also presents a challenging technical query that should be further explored.

Therapy.
With the recent advancing research on MIRI mechanisms, new therapeutic strategies have been developed to help limit the myocardium injury following an AMI. To reduce the infarct size, preserve the LV function, and improve clinical outcomes of AMI patients, LncRNAs have emerged as important regulatory molecules in the pathological progression of MIRIs [111]. Recent studies have reported that upregulating or downregulating the expression level of LncRNA can relieve a MIRI in mice. These trials also indicated multiple LncRNAs are likely to emerge as novel therapeutic candidates to target and treat AMI ( Table 2).
In the rat I/R model, HOTAIR overexpression could suppress inflammation and myocardial cell apoptosis, as well as reduce myocardial infarction sizes [113]. Additionally, inhibiting either Mirt1 [79] or ZFAS1 [114] suppressed myocardial cell apoptosis, reduced myocardial infarction sizes, and improved cardiac function following an I/R-induced injury.
Although the results of using LncRNA therapeutics in animal experimental studies are promising, several limitations persist. Interestingly, owing to their therapeutic potential in preventing and treating a MIRI, gene therapy has become a promising approach to effectively target LncRNAs. However, our present understanding is limited to the animal models, and the safety and effectiveness of its clinical application require further research.

Conclusions and Perspectives
We have begun the era of "noncoding." We look forward to seeing more reports of noncoding RNAs, particularly LncRNAs, that regulate various basic biological processes including cardiovascular biology and related diseases. LncRNAs have the potential to be biomarkers for clinical diagnoses and targets for gene therapy. However, LncRNAbased research in MIRIs is still in its infancy, and further investigation is required to fully elucidate the role of LncRNAs in MIRIs. For example, the majority of available evidence linking LncRNAs and MIRIs is limited in vitro data; moreover, nonconservative sequences and limitations of some experimental methods hinder the progress of in vivo experiments. Whether modifying LncRNAs could fully regulate the cellular response to a MIRI, there are relatively few studies on LncRNAs in this field. These hurdles must be overcome before LncRNAs can be used in clinical trials. With the innovation and advancement of LncRNA-based research, their mechanistic role in MIRIs is expected to be elucidated. Therefore, LncRNAs would be ideal and novel targets for the diagnosis and treatment of patients with AMI.

Conflicts of Interest
The authors declare that they have no conflict of interests.

Authors' Contributions
Zhuo Zhao and Wei Sun contributed equally to this work.