Functional Role of microRNAs in Regulating Cardiomyocyte Death

microRNAs (miRNA, miRs) play crucial roles in cardiovascular disease regulating numerous processes, including inflammation, cell proliferation, angiogenesis, and cell death. Herein, we present an updated and comprehensive overview of the functional involvement of miRs in the regulation of cardiomyocyte death, a central event in acute myocardial infarction, ischemia/reperfusion, and heart failure. Specifically, in this systematic review we are focusing on necrosis, apoptosis, and autophagy.


Introduction
MicroRNAs (miRNAs, also known as miRs) are a group of small single-stranded noncoding RNA (ncRNA) with approximately 18-22 nucleotides; miRNAs regulate gene expression by binding to the 3 -untranslated region (3 UTR) of the messenger RNA (mRNA) of a target gene, enhancing its degradation and/or inhibiting protein translation [1][2][3].
Numerous studies have shown that miRNAs play essential roles in cardiovascular diseases regulating a plethora of processes including cell death, cell proliferation, inflammation, and angiogenesis [4]. In this review, we will focus on the involvement of miRNAs in the regulation of cardiomyocyte death.
Cardiomyocyte death is the central event in acute myocardial infarction; additionally, cell death has been shown to contribute significantly to the pathogenesis of ischemia/reperfusion and heart failure [5][6][7].
Various investigations have shown that necroptosis and MPT-dependent necrosis are involved in the pathogenesis of myocardial infarction, ischemia/reperfusion, and heart failure [11,12]. Necroptosis is initiated by the binding of cytokines including tumor necrosis necrosis factor alpha (TNF-α), Fas/CD95, and TRAIL (TNF-related apoptosis-inducing ligand) to death receptors (e.g., TNFR1, TNFR2) and to active receptor interaction protein kinase 1 and 3 (RIP1 and RIP3), causing the formation of the necrosome [13,14].
Another long non-coding RNA, namely necrosis-related factor (NRF), was shown to regulate necrosis in H2O2-treated cardiomyocytes and in mice challenged by ischemia/reperfusion through means of the repression of miR-873; this specific miR reduces necrosis by silencing the translation of RIPK1/RIPK3 [16]. Since NRF is also modulated by p53 [17], NRF overexpression by p53 can markedly aggravate myocardial necrosis [16]. Another study proved that lncRNA E2F1 increases cardiomyocyte necrosis by inhibiting miR-30b and cyclophilin D (CypD); in fact, miR-30b decreases necrosis and infarct size by targeting CypD [18].
miR-874 represents a perfect example of a miR that is mechanistically involved in cardiomyocyte necrosis experimentally induced by H2O2: its expression increases in H2O2treated murine cardiomyocytes and its knockdown reduces necrosis in H2O2-treated cardiomyocytes and in a murine model of ischemia/reperfusion injury; caspase-8, which negatively affects myocardial necrosis by cleaving and inactivating RIP3 [19], was identified as a downstream target of miR-874 [20]; FOXO3a is known to repress miR-874 expression [21] and its overexpression attenuates cardiac necrosis [20].
The downregulation of Adenine nucleotide translocase 1 (ANT1) is another mechanism underlying cardiomyocyte necrosis [25]; by directly targeting ANT1, miR-2861 was shown to induce necrotic cell death in hypoxia-treated murine cardiomyocytes, as well as in mice challenged by ischemia/reperfusion injury [26]; confirming these findings, knocking miR-2861 down preserved cardiomyocytes from necrosis via inhibition of ANT1 degradation [26].

miRNAs and Cardiomyocyte Apoptosis
Apoptosis (from Ancient Greek ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed death [27,28] that plays an important role in the pathogenesis of cardiovascular disorders [29][30][31][32]. In conditions such as myocardial infarction, apoptosis occurs in terminally differentiated cardiomyocytes and inhibition of apoptosis has been shown to protect the heart against ischemia/reperfusion injury [30,33].
The apoptosis machinery is activated by intrinsic mitochondrial pathway and extrinsic cell surface death receptor pathway and is characterized by a series of structural and morphological changes including chromatin condensation, DNA degradation, cell shrinkage, and blebbing of plasma membrane [34,35]. In the extrinsic pathway, extracellular ligands, such as tumor necrosis factor alpha (TNF-α) and FasL bind to the death receptor (e.g., Fas, TNFR) to activate intracellular caspases [36]. πóπτωσις, apóptōsis, 'falling off') is a form of programmed death [27,28] that plays an important role in the pathogenesis of cardiovascular disorders [29][30][31][32]. In conditions such as myocardial infarction, apoptosis occurs in terminally differentiated cardiomyocytes and inhibition of apoptosis has been shown to protect the heart against ischemia/reperfusion injury [30,33].
The apoptosis machinery is activated by intrinsic mitochondrial pathway and extrinsic cell surface death receptor pathway and is characterized by a series of structural and morphological changes including chromatin condensation, DNA degradation, cell shrinkage, and blebbing of plasma membrane [34,35]. In the extrinsic pathway, extracellular ligands, such as tumor necrosis factor alpha (TNF-α) and FasL bind to the death receptor (e.g., Fas, TNFR) to activate intracellular caspases [36].
The upregulation of miR-122 has been reported in rat cardiomyocytes after hypoxiareoxygenation treatment; miR-122 overexpression promotes cell apoptosis via targeting GATA-4 [58]. Liu et al. found in H9c2 cardiomyoblasts that miR-208a triggers apoptosis through silencing activated protein C (APC) in hypoxic conditions; APC reduces apoptosis in hypoxia and knockdown of APC attenuates the inhibitory effects of miR-208a [59]. A recent study revealed that the upregulation of miR-137-3p can aggravate cardiomyocyte apoptosis induced by ischemia/reperfusion through means of the downregulation of the Kruppel-like factor 15 (KLF15). [60]. In vitro, a significant downregulation of miR-7a-5p was detected in H9c2 cardiomyoblasts undergoing hypoxia-reoxygenation; this finding is noteworthy, inasmuch as the overexpression of miR-7a-5p inhibits the expression of cleaved caspase-3 and Bax and promotes the expression of Bcl-2 by targeting voltage-dependent anion channel 1 (VDAC1) [61].
Hypoxia-reoxygenation was shown to reduce miR-24 expression in rat cardiomyocytes, whereas miR-24 mimics increase Bcl-2 protein levels and decrease apoptosis [64]; equally important, Mitogen-Activated Protein Kinase 14 (MAPK14) is a target gene of miR-24 and its expression is negatively regulated by this miR [64]. Notably, miR-135a overexpression is known to decrease cell apoptosis, lactate dehydrogenase levels, Troponin I, and inflammation following isoproterenol treatment [65]; luciferase assay analyses validated Toll-like receptor 4 (TLR4) as the specific target gene of miR-135a [65]. RT-qPCR and immunoblot analyses revealed that oxygen-glucose deprivation/reperfusion (OGD/R) injury significantly enhances miR-210 in primary cardiomyocytes. Decreased caspase-3 activity and cell apoptosis was detected in cells transfected with a miR-210 mimic; furthermore, the transcription factor E2F3, known to trigger cell apoptosis [66], is one of target genes of miR-210 [67].
Emerging evidence indicates a significant downregulation of miR-26a-5p expression in mice undergoing ischemia/reperfusion injury, as well as in cardiomyocytes undergoing hypoxia-reoxygenation. Overexpression of miR-26a-5p significantly inhibits cardiomyocyte apoptosis and improves cardiac function by repressing Phosphatase and tensin homolog (PTEN), thereby activating the PI3K/AKT signaling pathway; thus, miR-26a-5p protects cardiac function via regulating PTEN/PI3K/AKT upon ischemia/reperfusion injury [68]. Wang et al. demonstrated that miR-369 overexpression reduces cell apoptosis, inflammation, and is accompanied by decreased caspase-3 activity, secretion of interleukin (IL)-6, IL-1β, and TNF-α by suppressing Transient Receptor Potential Cation Channel Subfamily V Member 3 (TRPV3) [69]. When overexpressed in rat cardiomyocytes under hypoxiareoxygenation conditions, miR-129-5p decreases cell death by targeting HMGB1 [70]. miR-147 was shown to be downregulated after hypoxia in rat cardiomyocytes and, in vivo, in a rat model of myocardial infarction; overexpression of this miRNA preserves cardiac function by silencing homeodomain interacting protein kinase 2 (HIPK2) [71]. miR-184 depletion in vitro was found to decrease cleaved caspase-3 and Bax and attenuate cell death in cardiomyocytes by targeting F-box protein 28 (FBXO28) under hypoxic conditions [72]. Liu et al. observed that the overexpression of miR-223 reduces cell apoptosis by inhibition of poly (ADP-ribose) polymerase 1 (PARP-1) in rats with myocardial infarction and in hypoxia-treated neonatal rat cardiomyocytes (NRCMs); PARP-1 is a downstream target of miR-223 and these researchers found that silencing PARP-1 can protect cardiomyocytes from hypoxia [73].
Dual-luciferase reporter assay revealed that the secreted protein acidic and rich in cysteine (SPARC) is a direct target of miR-29b-3p [76]. Hypoxia decreases miR-29b-3p expression in H9c2 cardiomyoblasts and increases cell apoptosis. Overexpression of miR-29b-3p can protect H9c2 cells from apoptosis by reducing the TGF-β1/SMAD pathway and SPARC [76].

miRNAs and Cardiomyocyte Autophagy
Autophagy (from the Ancient Greek α 3p can preserve cardiomyocytes under hypoxia from apoptosis by inhibiting HMGB1 and Rac1 [75]. Dual-luciferase reporter assay revealed that the secreted protein acidic and rich in cysteine (SPARC) is a direct target of miR-29b-3p [76]. Hypoxia decreases miR-29b-3p expression in H9c2 cardiomyoblasts and increases cell apoptosis. Overexpression of miR-29b-3p can protect H9c2 cells from apoptosis by reducing the TGF-β1/SMAD pathway and SPARC [76].
The main miRs involved in the regulation of cardiomyocyte apoptosis, necrosis, and autophagy are reported in Table 1.
Ex vivo models, including engineered heart tissue (EHTs) and living myocardial slices derived from human cells or tissues, are used as a bridge between in vitro and in vivo studies. Differentiated cardiomyocytes from human induced pluripotent stem cells (hiPSC) do not fully recapitulate the complex intercellular interactions observed in the whole human heart. Instead, several studies have shown that EHTs can recapitulate chronic heart disease phenotypes and miRNA-based drug development [135][136][137].
In an in vivo study carried out in mice, miR-92a was found to be overexpressed following cardiac ischemic injury; strikingly, administering antimiR-92a encapsulated in bioabsorbable and biocompatible microspheres via intracoronary injections in a swine model of myocardial infarction substantially improved angiogenesis [138]. A miR-92a inhibitor (Drug: MRG-110) intending to promote angiogenesis is currently under investigation in phase I clinical trials (Clinical Trial Identifiers: NCT03603431 and NCT03494712).
Another miRNA that is undergoing a Phase Ib clinical trial is miR-132 (Clinical Trial Identifiers: NCT04045405). Foinquinos et al. reported strong evidence for therapeutic efficacy of a locked nucleic acid based antisense inhibitor of miR-132 (antimiR-132) in a swine model of heart failure [101,139]. A miR-132 inhibitor (known as CDR132L) significantly preserves cardiac function and reverses cardiac remodeling in heart failure patients [140].

Conclusions
The overview on miRs modulating cardiomyocyte death presented here underlies the active research in this area and embodies a useful guide for the investigators in this field. Considering that targeting miRs and other non-coding RNAs represents a specific strategy to counteract cardiomyocyte death, we anticipate substantial research in this direction in the next years.

Conflicts of Interest:
The authors declare no conflict of interest.