Heart failure (HF) is a complex clinical syndrome with varied pathophysiology that occurs because cardiac output is insufficient to meet the metabolic needs of organs and tissues in the body (1). HF is very common, with an estimated prevalence of 1–3% in the adult population worldwide, and its incidence rises with advancing age (2). Despite substantial therapeutic advances over the past decades, HF remains a major cause of morbidity and mortality worldwide (1). HF survival rates are no better than they were a decade ago, with an ~53% 5-year survival rate from diagnosis (3). Hence, HF is a global public health problem that requires urgent attention to open a window of opportunity for early diagnosis/prognosis, prevention, and treatment (4).

MicroRNAs (miRNAs) are natural, endogenous non-coding single-stranded RNA molecules of around 22 nucleotides that regulate the expression levels of various genes through Watson-Crick base pairing with target messenger RNAs (mRNAs) at the posttranscriptional level, via binding to the 3′ untranslated regions (UTRs) of target mRNAs (5). The role of miRNAs was first described in Caenorhabditis elegans, where silencing of the lin-14 mRNA at various time points during growth resulted in the normal development of a worm from an embryo (6). During the last few decades, increasing numbers of investigations have indicated the regulatory functions of miRNAs in a diverse range of cellular biological processes (7). According to the latest release of miRBase, more than 2500 miRNAs have been reported to exist in the human genome to date (8). Furthermore, bioinformatic analyses have indicated that the expression levels of 30% of human protein-coding genes are regulated by miRNAs through a series of complex signaling pathways (9).

Encyclopedia of DNA Elements (ENCODE) is an international cooperative project that aims to establish a comprehensive database for human genome data research by integrating DNA, RNA, protein, epigenetic modification, and other levels of data (10–12). The ENCODE project is another tremendous achievement of the international scientific community in the field of genomics, following the Human Genome Project, and is an approach that facilitates improved understanding of miRNAs and their effects in disease pathogenesis (13, 14).

In this review, we give an overview of different types of chemical modifications and viral-vector-based delivery systems used for miRNA modulation in HF, as well as summarizing the opportunities and challenges for miRNAs as therapeutic targets for the treatment of HF. Furthermore, we discuss CDR132L, the first miRNA drug used to treat HF, and future prospects for such therapies.

The synthesis of miRNA begins with the transcription of miRNA genes by RNA polymerase II. This transcriptional process, which involves transcription factors in a similar way to protein-coding transcripts, leads to the generation of A-tail-capped long transcripts, referred to as primary microRNA or pri-microRNA (15, 16). In the nucleus, a microprocess complex containing two important constituents, the double-stranded RNase III enzyme, DROSHA, and the cofactor, DiGeorge syndrome critical region 8 (DGCR8), processes pri-miRNA to generate precursor miRNA or pre-miRNA (17, 18). In the cytoplasm, pre-microRNAs are further cleaved by the cytoplasmic nuclease, DICER, to produce double-stranded RNA molecules of 18–22 nucleotides, cut from the stem of the hairpin, similar to the double-stranded structure of small interfering RNA (siRNA) (19). The duplex comprises a guide strand (mature miRNA) and a passenger strand (miRNA^*) (20). While loading on RISC, only the mature miRNA will become active in the silencing procedure, while the miRNA^* is usually non-functional and is degraded (21). A multi-protein RNA-silencing complex composed of RISC and miRNA (22) recognizes target RNA molecules by Watson-Crick base pairing to partially complementary sites, mainly located in the 3′ UTR of target mRNAs, and can inhibit the function of corresponding genes in several ways, as follows: blocking initiation and elongation, forcing premature termination of translation, deadenylation of mRNAs to prevent their reuse, and (most importantly) mRNA degradation (23–27) (Figure 1).

The heart is the first organ to form and function during embryonic development. For normal morphogenesis and function, the development process must be uninterrupted (28). MiRNAs can manipulate cardiac gene expression at the posttranscriptional level (29). The requirement for miRNAs in cardiovascular development and function was initially proven by the deletion of the tissue-specific gene, Dicer1, in mice; Dicer1 encodes an enzyme essential for miRNA processing (30), and its deletion in vascular lineages and myocardial tissue results in embryonic lethality and defective heart morphogenesis (30, 31). Subsequently, numerous miRNAs that contribute to heart morphogenesis have been identified; for example, miR-1 and miR-133 contribute to cardiomyocyte proliferation, as well as the miR-15 family, thereby regulating congenital heart disease development or regeneration (32–35).

Several myomirs, including miR-1, miR-133a, miR-208a, miR-208b, and miR-499, play important roles in normal embryological development of the heart and precise regulation of these myomirs is crucial for normal cardiac development (36). The field of cellular reprogramming and trans-differentiation is rapidly evolving, and cardiac fibroblasts have been demonstrated to directly differentiate into induced cardiomyocytes (iCMs) under the combined influence of the transcription factors GATA4, MEF2C, and TBX5 (GMT) (37). Subsequently, Jayawardena et al. (38) demonstrated that the combination of miR-1, miR-133, miR-208, and miR-499 also function to directly convert fibroblasts to a cardiomyocyte-like phenotype in vitro. Among these miRNAs, miR-133 is considered to mediate the further maturation of transdifferentiated iCMs (39). The importance of miRNA-mediated post-transcriptional regulation in cardiovascular homeostasis, as well as its impact on the pathogenesis, diagnosis, and prognosis of heart disease, is established in the scientific community. Understanding the mechanisms underlying heart development may provide new perspectives relevant to cardiac reprogramming technology, which could, in turn, pave the way for development of miRNA-based approaches for heart disease therapy in the future.

MiRNAs are vulnerable to degradation by nucleases; therefore, chemical modifications to protect miRNAs from nucleases are major solutions that can enhance RNA stability and improve their efficacy (40). Optimal anti-miRNA oligonucleotides (AMOs, or anti-miRs) are designed to be completely complementary with specific mature miRNAs and are chemically modified. Lennox et al. (41) found that chemical modifications of anti-miRs could accelerate their invasion of RISC. At present, the main chemical modification methods used in preclinical studies are phosphorothioate (PS), locked nucleic acid (LNA), and ribose-2'-OH modification (Figure 2).

PS modification, also referred to as oligonucleotide backbone modification, involves replacement of a non-bridging oxygen atom in the phosphodiester bond with a sulfur atom (42). PS can reduce exonuclease- and endonuclease-mediated miRNA degradation; therefore, PS bonds usually are designed near the 5′ and 3′ ends of anti-miRs, to enhance their stability (43). As well as resistance to nucleases, PS-modification can reduce plasma clearance of anti-miRs, increase their stability in serum, and lengthen their half-life, to improve their pharmacokinetic properties, by giving them high affinity with serum albumin (44). Every PS-antimiR substitution reduces the melting temperature of the heteroduplex by 0.5°C (45); however, although the thermal stability of PS-antimiRs is reduced, their increased hybridization specificity and greater nuclease resistance can compensate for this shortcoming. Partial PS oligodeoxynucleotides (ODNs) targeted against the AT1 receptor mRNA are more effective than full PS ODNs in decreasing blood pressure, as well as having lower cost and improved specificity (46). Compared with other modification strategies, PS modification of anti-miRs is more effective in vitro. Two PS modifications of 2'-O-methyl RNA/LNA (2'OMe/LNA) mixtures were reported to be the most efficient of all chemical compounds tested in vitro (43).

LNA, as a modified nucleotide analog, is also termed inaccessible RNA, and uses one of its methylene bridges to fix the ribose ring, by linking an oxygen atom at the 2′ position to a carbon atom at the 4′ position. The methylene bridge is usually in the C3′-endo conformation (47). LNA modifications reduce nuclease degradation by increasing RNA stability and avoid off-target effects through 5′ end modification, which prevents molecules from merging into RISC (48, 49). The high affinity of LNA allows shorter anti-miR sequences to be designed for applications, with similar efficiency (50). Obad et al. (50) first demonstrated that it is feasible for tiny LNAs (including those of only 8 nucleotides) to silence miRNAs with negligible off-target effects. Subsequently, Bernardo et al. (51) showed that the seed-targeting 8-mer LNA-modified antimiR-34 (LNA-antimiR-34) could silence the miR-34 family to prevent pathological cardiac remodeling and improve cardiac function. Another study found that silencing of miR-34a using LNA-antimiR-34a in mice with moderate cardiac pathology reduced atrial dilatation and prevented failure of cardiac function (52). A recent study also found that LNA-antimiR-34 doubled the cardiac progenitor growth rate by inhibiting miR-34 expression (53). The first identified miRNA, miR-1, has a critical role in cardiac development and disease (54); it can significantly inhibit the expression of PKCe and HSP60 to promote cardiac injury. Pan et al. (55) verified that LNA modified antimiR-1 (LNA-antimiR-1), at a dose of 1 mg/kg, down-regulated miR-1 expression by 83% and efficiently reduced cardiac ischemia/reperfusion injury. Wang et al. (56) found that LNA-antimiR-1 remarkably inhibits the production of reactive oxygen species in neonatal Wistar rat cardiomyocytes.

Ribose-2′-OH modification describes the substitution of alternative chemical groups in the 2′ position, primarily 2′-O-methyl (2′-O-Me), 2′-meth-oxyethyl (2′-O-MOE), and 2′-fluoro (2′-F) (57). Ribose-2′-OH modification can enhance nuclease resistance and reduce immunoreactivity (58); however, in most cases, modification is conducted together with LNA or PS modification, or occasionally even both together. Intravenous administration of 2′-O-Me-anti-miRs targeting miR-16, miR-122, miR-192, and miR-194 could decrease the corresponding miRNA levels in mice (59). Similarly, inhibition of miR-133 using an anti-miR with 2′-O-Me modification leads to hypertrophy in vivo (60).

All of the chemical modifications described above have the common advantage of improving nuclease resistance, while PS modification alone also reduces thermal stability and affinity. LNA has become the most commonly used anti-miR chemical modification; however, use of LNA modification alone may decrease RNase H activity. Most anti-miRs tend to be designed with these three chemical modifications in the first and last five nucleotides, referred to as gapmers. These molecules have a good affinity for their target RNAs, effectively improve RNase H activity, and are widely used in many miRNA therapeutics (61, 62).

Overall, AMOs are an effective means of functionalizing miRNAs, both in vivo and in vitro. The differential effects of chemical modifications of AMOs are summarized in Table 1.

When miRNA therapies are applied for heart disease, it is necessary to consider the non-polar and hydrophobic properties of the myocardial cytomembrane, which represent an enormous challenge to miRNA delivery, as miRNAs have a negative charge and are hydrophilic. Therefore, crossing the myocardial cytomembrane is a vital step in transferring miRNAs to their targets successfully and generating effective miRNA therapies. Effective miRNA delivery requires the molecules to exhibit hypocytotoxicity, high transfection efficiency, and specificity. MiRNA delivery vehicles are mainly divided into viral and non-viral vector-based approaches. In this review, we discuss viral-vector-based delivery systems; see refs (79–82) for detailed information on non-viral miRNA vectors. Viral vector delivery aims to reach the target cell using the genome of the virus itself, and has been used extensively for delivery of miRNA therapies. The main viruses used for this purpose are adenoviruses, adeno-associated viruses (AAVs), and lentiviruses.

Adenoviruses, are double-stranded DNA viruses commonly used as viral vectors. As early as 1992, Stratford-Perricaudet et al. (83) found that injecting adenovirus LacZ vectors into neonatal mice resulted in extensive gene transfer in cardiomyocytes. Subsequent studies have shown that adenovirus-mediated miRNA-24 upregulation can enhance myocardial angiogenesis and blood perfusion in myocardial infarction (MI) tissue. While this approach can induce cardiomyocyte and fibroblast apoptosis, overall, it produced a good MI treatment effect (84). Another study using miRNA therapy for heart failure demonstrated that adenovirus vector contributed to up-regulation of AMPKα2, the direct target of miR-195a-3p, helping to relieve cardiac hypertrophy and avoid heart failure (85). Nevertheless, serious flaws have emerged on wide application of adenovirus delivery in clinical studies; for example, use of adenovirus vectors for treatment of malignant intracranial tumors resulted in serious side effects, including headache, change of mental status, and relapsing seizures (86) and such side effects limit the use of adenoviruses for delivery of miRNA therapies.

AAVs are single-stranded, non-enveloped DNA viruses belonging to the “Parvoviridae” family, and are among the most promising vector delivery systems. Unlike other viruses, AAVs are not pathogenic to humans, have minimal immunogenicity, and are low molecular weight (83). AAV2, AAV6, and AAV9 are the most cardiogenic AAVs among AAV1 to AAV9, with AAV6 vector exhibiting the most efficient transduction (87). Bian et al. (88) found that AAV6-mediated over-expression of miR-199a promoted the proliferation of human induced-pluripotent stem cells (hiPSC-CMs), which could improve cardiac function and fibrosis. Further, a recent study demonstrated that myocardial tissue in aorta-constricted mice transfected with miR-124 via AAV9 inhibited the influence of shikonin on sympathetic remodeling, revealing the mechanism underlying the use of shikonin for treatment of chronic heart failure (89). AAV6 and AAV9 have become the main AAVs serotypes used for heart disease therapy. In addition, Gao et al. found that delivery of miR-19a/19b using AAV vectors may reduce the cardiac trauma induced by myocardial infarction and protect heart function (90).

A number of studies have reported the use of AAV vectors for miRNA therapy in heart disease. The latest research showed that varying levels of cardiac miRNA-122 have a crucial effect on the treatment efficiency of AAV vectors regulated by miRNAs (91); however, another report noted severe cardiotoxicity in AAV6 vector-mediated shRNA cardiac gene transfer (92). Notably, AAV antibodies are present in most people, which may influence the effectiveness of vector entry and transgenic expression.

Lentiviruses are a subgroup of retroviruses, and lentivirus vectors are often used for miRNA delivery into myocardial cells. Yang et al. (93) demonstrated that downregulation of miR-322 via lentiviral transduction could further prevent cardiomyocyte apoptosis induced by hypoxia. Similarly, Wang et al. (94) showed that reduction of miR-137 levels, mediated by lentivirus vectors, decreased cardiomyocyte apoptosis. Although lentivirus-mediated miRNA therapy has been applied for the treatment of heart disease, the disadvantages of insertional mutagenesis may limit its use (95, 96).

MiRNA-related therapy based on viral vectors has certain advantages. For example, it is easy to generate vectors and they exhibit high transduction efficiency. Moreover, the long-term and stable gene expression mediated by these vectors makes them potentially ideal in this context; however, safety issues represent a huge obstacle that requires further research.

Cardiac remodeling, a basic mechanism underlying HF, is the process of generating changes in the size, shape, and function of the heart, and responds to internal and external cardiovascular injury or risk factors (97). Pathological ventricular remodeling has three main characteristics: extensive fibrosis, pathological cardiomyocyte hypertrophy, and myocardial cell apoptosis (98). In this review, we summarize the regulatory effects of newly-discovered miRNAs in cardiac remodeling, particularly cardiac hypertrophy (Figure 3), fibrosis (Figure 4), and apoptosis (Figure 5).

Progress over the past three decades has significantly improved the development of nucleic acid therapeutics. The first-in-human study of an miRNA-based therapy was MRX34, a liposome-based miR-34a mimic, for the treatment of advanced solid tumors in April 2013 (132, 133). This provided valuable insights into the potential for application of new oligonucleotide-based drugs in oncology (134). To date, several RNA-targeted drugs have been approved for commercial use, while others are in the final phases of clinical trials (135–137).

MiRNA-132-3p (miR-132) is a non-coding RNA whose cardiac expression is up-regulated in patients under cardiomyocyte stress. High expression of miR-132 in heart tissue leads to progressive cardiac remodeling, and thereby HF events. In addition, preclinical animal experiments show that miR-132 can affect signaling pathways related to cardiomyocyte growth, autophagy, calcium handling and contraction and, more significantly, can down-regulate FOXO3 levels and inhibit the expression of genes related to intracellular calcium handling and contraction, which can lead to cardiac remodeling (138). Hence, miR-132 has attracted the attention of clinicians as a promising molecular target for HF treatment.

CDR132L, a synthetic lead-optimized oligonucleotide targeting miR-132, was the first miRNA-132 inhibitor. In related preclinical studies, the application of CDR132L significantly improved cardiac function, and can attenuate, or even reverse, HF (139).

Based on these findings, Täubel et al. published the first in-human clinical trial of CDR132L in patients with HF. The study was a randomized, double-blind, placebo-controlled, dose-escalation clinical trial. This Phase 1b clinical trial confirmed for the first time that CDR132L is safe and well-tolerated in humans, with no obvious toxicity. Simultaneously, CDR132L can significantly reduce the level of NT-proBNP, a biomarker of heart failure and narrow QRS complex, and lead to positive trends in myocardial fibrosis markers (140). This represents a huge transformation in treatment for patients with heart disease, from symptomatic and supportive therapies, to etiological treatments (141). As clinical researchers and medical workers, while maintaining a cautious attitude toward data generated from small sample sizes, we should also actively recognize the “encouraging” pharmacodynamic performance of CDR132L. Larger-scale clinical studies are needed in the future to further confirm the positive role of CDR132L in the treatment of heart failure. More broadly, this research can inspire more studies investigating RNA-based therapeutics for cardiovascular disease.

As miRNAs can target a wide range of genes (mRNAs), and an individual gene can be regulated by multiple miRNAs, the complex communication networks between these two molecule types indicate that miRNAs can regulate numerous different biological phenomena, ranging from hypertrophy and fibrosis to angiogenesis, among other processes. Therefore, miRNAs have significant potential for preventing, alleviating, and even restoring cardiac dysfunction, such as adverse ventricular remodeling. Further, therapeutic cocktails including miRNA inhibitors and treatment strategies targeting multiple genes involved in disease development may produce remarkable results; however, the broad clinical application of miRNA-related therapy must first overcome several obstacles, including targeted delivery, off-target effects, and hepatic/renal toxicity. Thus, further attempts to promote clinical application of miRNA-related therapeutics are urgently required.

HZ drafted the manuscript. CF designed the study. WT, JY, JP, and JG revised the manuscript. HZ and WT were responsible for the collection of data or analysis. All authors read and approved the final manuscript.

This work was supported by the Key Project of Science and Technology of Hunan Province (No. 2020SK53420 to JY). Hunan Province Outstanding Postdoctoral Innovative Talent Project (2021RC2106 to CF).

JG and CF was employed by the company, Hunan Fangsheng Pharmaceutical Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.