Review Article

Proregenerative MicroRNAs to Repair the Damaged Heart

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Abstract

Cardiac regeneration remains a major challenge in clinical medicine. Following MI, up to 25% of cardiomyocytes in the left ventricle can be lost, a key factor contributing to heart failure. In adults, this loss is not compensated by new cardiomyocyte formation. However, in neonatal mammals and some other species, such as fish and amphibians, heart regeneration occurs naturally through the proliferation of the surviving cardiomyocytes. Over the past two decades, substantial progress has been made in understanding the molecular pathways that regulate cardiomyocyte proliferation during development, early neonatal life and in other species. Notably, several human microRNAs, identified through extensive screening for their ability to stimulate cell proliferation, have emerged as potent inducers of cardiomyocyte proliferation and cardiac regeneration when administered therapeutically. This review highlights the gene targets and regenerative effects of the most effective of these microRNAs, including the miR-17-92 and miR-302-367 clusters, miR-199a, miR-1825, miR-590 and miR-33b, and discusses their potential for clinical application in treating MI and heart failure.

Keywords

Cardiac regeneration, microRNA, lipid nanoparticles,

Received:

Accepted:

Published online:

DOI: https://doi.org/10.15420/ecr.2025.34

Disclosure: MG has received grants and consulting fees from and stock options in Heqet Therapeutics and Forcefield Therapeutics, and is named as an inventor in a patent related to some of the microRNAs discussed in this article. CO has no conflicts of interest to declare.

Funding: This review was supported by the European Research Council Advanced Grant HELP-GT (managed by UK Research and Innovation); British Heart Foundation (BHF) Programme Grant RG/F/24/110150; King’s College London BHF Centre of Research Excellence grant RE/24/130035; European Commission Horizon 2020 programme grants 101080897 TiilT and 101080204 GEREMY; Fondation Leducq grant 20CVD04; the MRC/BHF Centre of Research Excellence for Advanced Cardiac Therapeutics; and the National Center for Gene Therapy and Drugs based on RNA Technology of the EU National Recovery and Resilience Plan (PNRR), University of Trieste, Italy. CO is supported by EU Marie Skłodowska-Curie Postdoctoral Fellowship agreement 101205751 (CardioReGen).

Correspondence: Mauro Giacca, The James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK. E: mauro.giacca@kcl.ac.uk

Copyright:

© The Author(s). This work is open access and is licensed under CC-BY-NC 4.0. Users may copy, redistribute and make derivative works for non-commercial purposes, provided the original work is cited correctly.

Heart failure (HF) has reached pandemic proportions, now affecting over 65 million patients and standing as the main cause of morbidity and mortality worldwide.1 Ischaemic heart disease, including MI, plays a major role in the development of HF with reduced ejection fraction (HFrEF), a condition that accounts for most cases of HF. The sudden or chronic loss of functional contractile cardiomyocytes (CMs) is a fundamental factor leading to HFrEF.2 The loss of CMs is not compensated for by the generation of new CMs because of the extremely limited proliferative capacity of these cells during adulthood. The renewal of human CMs was estimated to be as low as ~1% per year at the age of 20 years and to progressively decrease to 0.3% per year in healthy individuals aged 75 years.3

The generation of new myocardial tissue can be achieved through: the transplantation of cardiac progenitor cells, derived from embryonic stem cells or induced pluripotent stem (iPS) cells, or mature CMs; the surgical implantation of ex vivo-generated myocardial tissue patches; or the direct reprogramming of other cell types into CMs.4–9 In essence, all these approaches aim to substitute the lost portion of the myocardium with new contractile cells or tissue generated either ex vivo or directly in vivo (in the case of reprogramming). However, none of these strategies mimics what occurs spontaneously in nature during embryonic and fetal development, after cardiac damage in neonatal mammals and across the entire life span in fish and amphibians: in all these situations, cardiac regeneration occurs spontaneously through the duplication of already differentiated CMs. These cells partially dedifferentiate by dismantling a portion of their sarcomeres, without losing their CM identity, and then re-express cell cycle genes and enter the S phase, culminating in nuclear duplication and, in most instances, cell division.10,11

Numerous studies have identified the relevant molecular mechanisms that control endogenous CM proliferation. At least three main pathways participate in this process, namely the Wnt/β-catenin, Notch and Hippo pathways.12 Although the Wnt/β-catenin and Notch pathways are of relevance for prenatal and early neonatal CM proliferation, CM cell cycle regulation by Hippo also appears to be important and required for postnatal CM replication after cardiac damage. The Hippo pathway negatively controls organ size and growth by inhibiting cell proliferation, promoting apoptosis and limiting cell size.13 The core inhibitory mechanism rotates around a protein kinase cascade that culminates in the inhibitory phosphorylation of the transcriptional coactivator YAP. When not phosphorylated, YAP translocates into the nucleus and associates with the TEAD1–4 transcription factors to stimulate the expression of cell proliferation-related genes.11 Other signal transduction pathways that regulate the CM cell cycle include mitogen-activated protein kinase p38 and the PI3K/AKT pathway.14,15

One or more of these pathways are viably interconnected with the function of a series of individual factors that have been shown to regulate CM proliferation, either positively or negatively. These factors include ErbB2/4, the transcription factors Meis1 and Hand2, PKM2 and a series of stimuli that act outside the CM, such as a few cytokines, including the ErbB2/4 ligand NRG-1, FGF-1 and FSTL-1, and extracellular proteins such as agrin and periostin. The roles of these factors in the regulation of CM proliferation have been reviewed elsewhere.11

The microRNA (miRNA) network also takes part in the regulation of CM proliferation. miRNAs are non-coding RNAs, approximately 21–23 nucleotides (nt) long, that regulate gene expression post-transcriptionally by silencing complementary mRNAs. Most commonly, miRNAs bind a complementary sequence in the 3′ untranslated region (UTR) of their target mRNAs, leading to deanylation and decapping, or inhibiting translation.16 Because target recognition only requires limited pairing between the mRNA 3′UTR and the miRNA 7- to 8-nt seed region, a single miRNA can bind multiple targets and thus exert varied and complex functions, which can be different or even opposite in different organs.17

Two groups of miRNAs with antiproliferative activities were identified in CMs: the miR-15 family, which is involved in the withdrawal of adult CMs from the cell cycle; and the Let-7 family, which takes part in the suppression of the cell cycle during stem cell differentiation.18 The therapeutic inhibition of these miRNAs can induce CM proliferation, but the magnitude of the effect on cardiac regeneration is rather weak.19 More notably, systematic screening has identified several miRNAs that can act as strong inducers of regeneration, even if these miRNAs do not take part in the physiological replicative program of CMs. For example, one of the most effective proproliferative miRNAs in mice and rats is miR-1825 (see below), which has been identified in chimpanzees and humans, but not in rodents (www.mirbase.org).

Three high-throughput screening studies have led to the identification of human miRNAs (hsa-miRNAs) that can stimulate CM proliferation. The first screening study was performed in our laboratory in neonatal rodent CMs, whereas the other two studies were performed in human iPS-derived CMs under normoxic or hypoxic conditions.20–22 The overlap across the results of these screening studies is imperfect in terms of precise miRNA identification, possibly indicating that the stimulation of CM proliferation by miRNAs is largely context dependent. Despite this, the identified proproliferative miRNAs can be broadly classified into three groups: the first group is expressed in embryonic stem cells and is involved in the maintenance of pluripotency (miR-302-367 cluster; miR-290-295 cluster; miR-371-373 cluster and miR-520 family); the second group takes part in cell cycle regulation in different cell types, in particular cancer cells (miR-17-92 and miR-106b-25 clusters); and the third group comprises a series or uncorrelated, individual miRNAs that exert specific functions and do not belong to the other two groups (e.g. miR-590-3p, miR-199a-3p and miR-1825).

Here, we review our knowledge of the activities of the most effective of these proregenerative miRNAs (Table 1). Therapeutic delivery of these miRNAs for cardiac regeneration can be achieved using either viral vectors that selectively express an miRNA duplex in CMs or non-viral methods that deliver a synthetic, chemically modified miRNA mimic. In the former case, the transduced cells also express the complementary miRNA, which has a different sequence and function. In the latter case (miRNA mimics), non-viral transfection also affects other cell types in the heart and elsewhere. Thus, we also consider the effects of the complementary strand of each proproliferative miRNA and the effect of each of the miRNAs on other cell types in the heart.

Table 1: Main miRNAs That Stimulate Cardiac Repair and Regeneration and Their Relevant Target Genes and Potential Mechanism of Action in the Heart

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Table 1: Cont.

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miR-17-92 Cluster

The miR-17-92 cluster is a highly conserved cluster of microRNAs that is essential for heart development and the subsequent maintenance of cardiac function. Changes in the expression of this cluster are involved in different cardiovascular diseases, including HF, arrhythmia and pathological hypertrophy. In the heart, the miR-17-92 cluster is expressed in different cardiac cell types, including CMs, fibroblasts, cardiac progenitor cells and vascular endothelial cells.23,24

The miR-17-92 cluster is located in the lncRNA gene MIR17HG on chromosome 13 in humans and on chromosome 14 in the murine genome. Pri-miR-17-92 is processed into six mature miRNAs: miR-17 (for which both the -3p and -5p strands are generated), miR-18a, miR-19a, miR-19b1, miR-20a and miR-92a1.25 The miR-17-92 cluster has two paralogues, the miR-106b-25 and the miR-106a-363 clusters, located on two different chromosomes in humans. The miR-106b-25 cluster contains three miRNAs (miR-106b, miR-93 and miR-25), whereas the miR-106a-363 cluster contains six miRNAs (miR-106a, miR-18b, miR-20b, miR-19b, miR-92a-2 and miR-363).26 Thus, 15 different miRNAs are collectively expressed from these clusters, several of which are overexpressed in numerous human tumours, as reviewed elsewhere.10,27,28 Due to its high expression in cancer cells, the miR-17-92 cluster, which is activated by the c-Myc oncogene, was also originally known as oncomiR1.29,30

These various miRNAs exert proproliferative, anti-apoptotic and proangiogenetic effects.25 The main genes targeted by the cluster to activate cell proliferation are the PTEN gene, a tumour suppressor gene that determines cell cycle arrest, and members of the E2F transcription factor family, which play a crucial role in cell cycle progression. miR-19b also directly downregulates CTNNB1 mRNA, a key player in the Wnt signalling pathway that is essential for cardiac progenitor cell differentiation and self-renewal, consistent with the pivotal role of the cluster in cardiac development.31

An additional relevant activity of several miRNAs in the miR-17-92 cluster consists of the regulation of the bone morphogenetic protein (BMP)/transforming growth factor (TGF)-β signalling pathways. miR-17 and miR-20a silence TGFBRII, whereas miR-18a inhibits Smad2 and Smad4 expression.32 Moreover, miR-19a and miR-19b downregulate the cyclin-dependent kinase inhibitor p21, as well as the proapoptotic BIM gene, both of which act as downstream effectors involved in the TGF-β signalling cascade.33 The downregulation of the TGF-β pathway contributes to CM proliferation and reduces collagen deposition.

In addition to CM proliferation, various members of the miR-17-92 cluster have other potentially beneficial actions in the heart. miR-17-3p inhibits fibroblast senescence by suppressing PAR4, thus leading to increased transcription of CEBP and FAK, which promote epithelial-to-mesenchymal transition and cardiac fibroblast renewal, potentially contributing to cardiac damage repair.34 In CM cultures, miR-17-3p also targets TLR4, which modulates inflammation and tissue repair through the regulation of inflammatory cytokines, and the METTL3 gene, which is involved in the maintenance of stemness.35 Finally, miR-18a and miR-19 suppress the expression of the anti-angiogenic factor thrombospondin-1 (TSP-1) and of the connective tissue growth factor (CTGF), thus supporting the generation of new vessels and reducing fibrosis.36

miR-302-367 Cluster

Members of the miR-302-367 cluster also exhibit proproliferative activity in CMs. This is a vertebrate-specific cluster located in an intron of the 4q25 region of chromosome 4. The miRNA precursor generates five miRNA duplexes: miR-302d, miR-302c, miR-302a, miR-302b and the unrelated miR-367.37 The -5p strands of the miR-302 family of miRNAs share the common seed sequence AAGUGCU; remarkably, the miR-17 family of the above-described miR-17-92 cluster also shares the same AAGUGCU sequence, overlapping with its seed sequence at a +1-nt offset position, suggestive of a partially common mechanism of action. Also of note, the mouse genome contains an additional miR-302-related miRNA cluster, namely the miR-290-295 cluster.10 The seed sequences of the -3p strands of three miRNAs of this cluster (miR-291a, miR-294 and miR-294) are the same as those of miR-302, whereas those of the other four miRNAs in the cluster (miR-290a, miR-290b, miR-292a and miR-291b) have only a 1-nt mismatch (in an offset position for miR-290b-3p). In humans, other miRNAs also share this same seed sequence; these are miR-372-3p and miR-373-3p of the miR-371-373 cluster, which is the human homologue of the miR-290-295 cluster in mice, and various members of the miR-520 family, including miR-520e, miR-520a, miR-520b, miR-520c and miR-520d (for a review, see Braga et al.).10 Most of these miRNAs induce neonatal mouse and rat CM proliferation.20

The miRNAs of these families promote cell cycle activation and mitosis in CMs by silencing different effectors of the Hippo pathway, such as the LATS2, MOB1B and Mst1 genes.38 Moreover, the cyclin-dependent kinase inhibitor p21 is also a known target of the cluster; its silencing increases the abundance of the cyclin E/Cdk complex and promotes progression towards the final phases of the cell cycle.37

Members of the miR-302 family also downregulate the left–right determination factors Lefty1 and Lefty2, which are involved in the modulation of the TGF-β/Nodal signalling cascade, thus contributing to balancing cell pluripotency and differentiation.39 The expression of three inhibitors that take part in the BMP signalling pathway, namely TOB2, DAZAP2 and SLAIN1, which control the maintenance of stem cell pluripotency, is also repressed by the cluster.40

The miR-302 family also plays a role in epigenetic modification by silencing both AOF1 and AOF2, MECP1 and MECP2 (the suppression of which destabilises methyltransferase 1) and MBD2 protein (which blocks the reprogramming of somatic cells to iPS cells).15,41 In addition, the miR-302-367 cluster silences the expression of the TGFBR2 and RHOC genes, thus promoting epithelial-to-mesenchymal transition.42

miR-367 has a poor sequence homology with the other members of the cluster and therefore predominantly targets different genes. Its -5p strand represses the COX2 protein, an enzyme that plays a significant role in tissue inflammation by leading to the secretion of cytokines such as interleukin (IL)-1β, IL-6 and tumour necrosis factor-α (TNFα).43 The -3p strand takes part in the development of cardiac fibrosis through the suppression of the cell surface glycoprotein CD69, the earliest T cell activation marker, which leads to a decrease in cytokine levels and increased recruitment of T helper 17 cells.43

Multiple pieces of evidence indicate that the miR-302/miR-290 members maintain the pluripotency of murine embryonic stem cells, with the miR-290 cluster alone constituting approximately 70% of all cellular miRNAs and miR-302-367 being a transcriptional target of the pluripotency transcription factors OCT 3/4, Sox2 and Nanog.44–47 However, contrasting results suggest an antiproliferative role of miR-302 in human iPS cells, exerted by enhancing several G1 phase arrest pathways.41 According to one study, Cdk2, Cdk4 and cyclin D1/D2 (which regulate the balance of the G1-to-S phase transition during the cell cycle), as well as the BMI1 gene, are targeted by members of the miR-302 group, with their suppression ultimately blocking the transition to the S phase of the cell cycle via the Rb/E2F pathway.48 Moreover, the miR-302-367 cluster can also inhibit angiogenic sprouting, in favour of vascular stability, by suppressing ERK1/2 expression and thus increasing the expression of KLF2 and, in turn, S1pr1 and vascular E-cadherin.49 Although this effect is beneficial in limiting tumour growth and progression, the suppression of new vessel formation can be detrimental in the context of cardiac repair.

miR-199a-3p

miR-199a-3p is probably the most investigated cardiac regenerative miRNA. This miRNA was originally identified in a screening study for the stimulation of CM proliferation performed in our laboratory, and it was later studied for cardiac regeneration after MI in both mice and pigs, either expressed using adeno-associated virus (AAV) vectors or transfected as a synthetic miRNA mimic.20,50,51 miR-199a is a vertebrate-specific miRNA, carrying a highly conserved sequence in most species. In humans, the miR-199a gene is duplicated on chromosomes 19 and 1 (miR-199a-1 and miR-199a-2, respectively). miR-199a-1 is located within intron 14 of the dynamin-2 gene and is transcribed in tandem with miR-214. Expression of the miR-199a/214 cluster is controlled by p53.52,53 Both the miR-199a-3p and -5p strands engage in mRNA target recognition, with significantly different effects.

Our original screening study identified different targets of miR-199a-3p in the heart that could mediate its proproliferative activity, among which were Homer1 and Hopx. Homer1 is a widely expressed protein in cardiac muscle, which characterises mature functional CMs and acts as a high-affinity ligand in modulating ryanodine receptor Ca2+ release channels.20,54 Hopx is an important regulator of cardiac development and its expression starts early in cardiogenesis.55 Loss- and gain-of-function murine models have revealed that Hopx operates as a transcription cofactor that modulates cardiac-specific gene programs, and thereby cardiac growth.56

Transcriptional profiling of CMs transfected with miR-199a-3p has identified Cofilin-2, TAOK1 and β-TrCP as additional targets of this miRNA.57 Silencing of each of these genes converges in the activation of the YAP transcriptional cofactor by favouring its translocation into the nucleus through the modulation of different effectors. Cofilin-2 is an actin-regulating protein that mediates the depolymerisation of actin filaments and prevents their reassembly.58 The cytoskeleton is a key upstream regulator of the Hippo/YAP pathway, because F-actin depolymerisation leads to the inhibition of the phosphorylation of Hippo kinases, thus trapping YAP in the cytoplasm. Conversely, polymerised F-actin triggers YAP translocation into the nucleus. TAOK1 is one of the kinases involved in the phosphorylation and inactivation of YAP.59 Therefore, its suppression by miR-199a-3p can independently lead to YAP nuclear translocation. Finally, β-TrCP is an E3 ubiquitin ligase that catalyses the ubiquitination of phosphorylated YAP.60,61 It is of note that the individual downregulation of either TAOK1 or β-TrCP by miR-199a-3p is sufficient to stimulate CM proliferation, indicating the strong impact of this miRNA on these pathways.57

CD151 is an additional functional target of miR-199a-3p.62 The tetraspanin CD151 is a cell surface glycoprotein that induces the expression of MAPK p38, well known to act as a negative regulator of cell proliferation. Suppression of CD151 expression was sufficient to inhibit p38 and promote CM proliferation.62

Other genes reported as miR-199a-3p targets are the cell cycle inhibitor Meis2 and the tumour suppressor Rb1, inhibition of which stimulates proliferation, and CABLES1, which encodes an adaptor protein involved in the interaction of Cdk with non-receptor tyrosine kinases, inhibition of which enhances Cdk phosphorylation.17,63 In proliferating cells, CABLES1 links Cdk2 and Wee1, resulting in increased Cdk2 phosphorylation, decreased kinase activity and reduced cell proliferation. The downregulation of CABLES1 by miR-199a-3p regulates the proapoptotic factor p53, promoting cell proliferation and inhibiting apoptosis17

An additional gene targeted by miR-199a is GSK3β, a pro-autophagic and antihypertrophic protein that suppresses mTOR and induces cellular autophagy.64 mTOR activity is also modulated by Hspa5, which is an additional target of both miR-199a-3p and miR-199a-5p.65 Hspa5 is an endoplasmic reticulum stress-associated protein that activates autophagy through mTOR by phosphorylation of AMPK. Therefore, its inhibition by miR-199a suppresses CM autophagy. In addition to the positive regulation of CM proliferation, miR-199a-3p administration can have other positive effects by preventing myocardial fibrosis; for example, in fibroblasts, miR-199a-3p downregulates the serine protease inhibitor SERPINE2, which is involved in the deposition of collagen.66

Of note, although miR-199a-3p is proproliferative for CMs, many studies have shown that the same miRNA has tumour suppressive activity in several human cancers, including papillary carcinoma, ovarian carcinoma, hepatocellular carcinoma, prostate cancer, lung cancer and osteosarcoma.67–75 The targets for this tumour suppressive activity include c-Met, mTOR and CD44.74,76,77

The -5p strand of miR-199a has different effects to miR-199a-3p, which are, in general, detrimental for cardiovascular function, including the development of atherosclerosis and primary hypertension.78 In the heart, miR-199a-5p is upregulated during pathological cardiac hypertrophy, thus negatively correlating with cardiac function.79,80 The selective inhibition of miR-199a-5p can attenuate cardiac hypertrophy and fibrosis after MI.78 miR-199a-5p has various targets, including Sirtuin-1 and HIF-1α, and can regulate contractile function and nitric oxide availability.79,81–83 Given these different and often deleterious functions of miR-199a-5p, when developing a therapeutic strategy involving the use of miR-199a for regenerative purposes, it is important that only the -3p strand is administered or endogenously expressed, rather than the miR-199a duplex.

miR-1825

Another miRNA that exerts a positive effect in sustaining CM proliferation both in vitro and in vivo is miR-1825. The human gene for this miRNA is located in the 3′UTR region of the poFUT1 gene, the upregulation of which leads to cell migration and proliferation.63 miR-1825 and miR-199a may be strictly interconnected, with miR-1825 being an upstream regulator of miR-199a.63 Thus, the direct target genes of miR-199a-3p (in particular Meis2 and Rb1) are also downregulated by miR-1825 treatment.63 In vitro experiments on primary adult CMs transfected with miR-1825 have revealed increased expression of cyclin D1, together with a decrease in p16, a Cdk inhibitor.63

In addition to regulating miR-199a-3p, miR-1825 directly silences additional genes, most of which are involved in mitochondrial oxidative phosphorylation. In particular, miR-1825 reduces mitochondrial mass and function by targeting NDUFA10, which encodes a key protein involved in the electron transport chain.63 Direct inhibition of NDUFA10 promotes re-entry into the cell cycle by suppressing reactive oxygen species production, oxidative stress and DNA damage in adult CMs.63 Reducing the number of mitochondria can have a positive effect on CM proliferation, without hampering the capacity of the cells to generate energy.

In vascular endothelial cells, an additional beneficial activity of miR-1825 is to deregulate the tumour-suppressor protein TSC2-mediated mTOR axis, which is linked to angiogenesis.84 TSC2 acts as a GTPase-activating protein for the GTPase Rheb protein, and the TSC2/TSC1 heterodimer stimulates GTP hydrolysis of Rheb, thus suppressing the mTOR signalling pathway.85 Evidence of this activity was obtained in oral squamous cell carcinoma; however, this is a common pathway and thus it can be expected that part of the beneficial activity exerted by exogenously administered the miR-1825 mimic could be due to the stimulation of revascularisation of the infarcted region, which, in turn, supports cell proliferation and survival.

miR-590-3p

In humans, the miR-590 gene is located on the proximal end of the long arm of chromosome 7. The proregenerative activity of the -3p strand of this miRNA was originally identified during our initial screening in neonatal mouse and rat CMs and later tested in vivo in infarcted mice using an AAV vector expressing the miR-590 pri-miRNA.20 miR-590-3p activates CM replication by silencing different inhibitory components of the Hippo pathway and stimulating actin cytoskeleton remodelling.57 The original screening also identified Homer1 and Hopx as targets of both miR-590-3p and miR-199a-3p; miR-590-3p also downregulates the expression of dimorphic Clic5. Clic5 is present in the endoplasmic reticulum and inner mitochondrial membrane of neonatal CMs and regulates reactive oxygen species production.86

Work by others has shown that, in human iPS CMs, miR-590-3p targets TSC22D2, a member of the TGF-β family that inhibits cell proliferation through the regulation of PKM2.87 In addition to regulating PKM2, silencing of TSC22D2 upregulates cyclins B1 and D1, contributing to the stimulation of CM proliferation.87 Like miR-199a-5p, miR-590-3p targets HIF-1α, reducing HIF-1α-induced autophagy and apoptosis.88

Another function of miR-590-3p in the heart is to inhibit myocardial inflammation by targeting the p50 subunit of NF-κB.89 NF-κB undergoes nuclear translocation to activate the transcription of a series of proinflammatory genes, including IL-6 and TNFα. In the heart, this initiates a cascade of events sustaining myocarditis. Inhibition of inflammation and direct targeting of the LPL gene in macrophages also explains the anti-atherosclerotic effect of a miR-590-3p agomir in apoE–/– mice.90

In cardiac fibroblasts, an additional role of miR-590-3p is to inhibit proliferation, differentiation, migration and the synthesis of collagen.91 These activities are mediated by the direct targeting of the 3′UTR of ZEB1, a transcription factor involved in the proliferation and invasion of several cell types. Downregulation of ZEB1 decreased levels of α-smooth muscle actin, the main marker of fibroblast-to-myofibroblast differentiation.91 miR-590-3p also promotes the transdifferentiation of pig and human fibroblasts into CMs by repressing the zinc-finger protein Sp1, a ubiquitous transcription factor that binds to GC-rich regions of several promoters.92 Sp1 downregulation potentiates the effect of a cocktail of transcription factors (GMT: Gata4, Mef2c and Tbx5) that was previously shown to transdifferentiate fibroblasts into CMs.8

The -5p strand of miR-590 can also exert an antifibrotic effect by suppressing the TGF-β signalling pathway.93 TGF-β is a multifunctional cytokine that has a critical role in embryonic development, wound healing and tissue homeostasis.94 When activated after injury, TGF-β stimulates extracellular matrix deposition to accelerate wound repair. By targeting TGF-β1 and its receptors TGFBR1 and TGFBR2, miR-590-5p reduces collagen deposition, which could itself be beneficial in infarcted hearts and be important in promoting CM proliferation after MI.93

miR-33b-3p

miR-33b-3p is another miRNA that was originally identified in our screening studies.20 The miR-33 family comprises intronic miRNAs located within the SREBP genes, with which they are co-expressed. Primates (including humans) have two SREBP genes, SREBP-1 and SREBP-2 (known as SREBF1 and SREBF2, respectively), which express three major SREBP proteins. SREBPs are transcription factors that are induced during cellular lipid deficit and upregulate genes involved in cholesterol and fatty acid synthesis and trafficking.95 In humans and other primates, miR-33a and miR-33b are embedded in introns of, and co-transcribed with, SREBF1 (which encodes SREBP-1) for miR-33b and SREBF2 (which encodes SREBP-2) for miR-33a. However, in rodents, a part of miR-33b is missing from a SREBF1 intron (for a review, see Ortega et al.).96

Of note, RNA sequencing information (https://www.mirbase.org) indicates that the -5p strands produced from both human miR-33a and miR-33b dsRNA are by far more prevalent in cells than the -3p strands (the final miRNA strand choice usually occurs at the level of RISC engagement). Thus, by convention, these -5p miRNAs are often referred to simply as miR-33a and miR-33b, respectively. Because rodents and lower species lack miR-33b, miR-33a is often more simply referred to as ‘miR-33’ tout court. However, it has been progressively appreciated that the miR-33b-3p strand (originally indicated as miR-33b*) is also incorporated into RISC.

Human miR-33a-5p and miR-33b-5p have an identical seed sequence and only differ for 3 nt outside of it. In contrast, the -3p strands of these miRNAs are significantly different from one another, including in their seed sequences. Thus, miR-33b-3p and miR-33a-3p target very different cellular mRNAs. As for all miRNAs, the sequences of both -3p strands are completely different from those of their respective -5p strands.

The detected actions of miR-33b-3p include a series of variegate effects broadly related to cell survival. miR-33b-3p was reported to downregulate: CDKN1A (p21), and enhance the survival of lung cancer cells in response to cisplatin treatment; ULK1, and inhibit autophagy; DNMT3A, and inhibit chondrocyte apoptosis and cartilage extracellular matrix degradation in osteoarthritis; IRAK3, and relieve chondrocyte inflammation and apoptosis in osteoarthritis; DOCK4, and act as a tumour suppressor in prostate cancer; and fibronectin-1, and regulate extracellular matrix deposition.97–104

In contrast, the effects of miR-33a-5p (in humans and rodents) and miR-33b-5p (in humans) are quite variegated and include at least four different categories. Category 1 includes metabolic effects. These are exerted by targeting: ABCA1 (and ABCG1 in rodents) to reduce cellular cholesterol efflux, thus collaborating with SREBPs to elevate cellular HDL levels;105,106 PCK1 and G6PC, thus cooperating with SREBP1 in reducing glucose production in liver cells; CROT, CPT1A, HADHB and AMPKa to reduce fatty acid β oxidation and degradation;107 and IRS2 and SIRT6 to reduce insulin signalling.108

Category 2 effects of miR-33b-5p are related to the regulation of atherosclerosis. Inhibition of miR-33b was shown to reduce atherosclerosis in mice by regulating several genes involved in lipid metabolism and macrophage function.108,109 Category 3 effects include a series of tumour-suppressive effects. The -5p miRNAs were shown to inhibit tumour cell proliferation by targeting Cdk6 and CCND1 in mice, as well as the proto-oncogene PIM-1.110,111 Finally, category 4 effects include the regulation of abdominal aortic aneurysm formation, also via targeting ABCA1.112,113

Of note, only miR-33b-3p, and not miR-33a-3p (which, as reported above, has a very different seed sequence), is effective at stimulating CM proliferation; in contrast, miR-33b-5p exerts an opposite, antiproliferative effect in these cells, in line with the broader tumour suppressive activity of this -5p miRNA.20

Path to Clinical Translation

As is evident from the above descriptions, each of these proregenerative miRNAs targets a variety of different genes and thus exerts pleiotropic effects in both CMs and other cell types (Figure 1). Such pleiotropy can be a desirable property in the case of CMs, because the proliferation of these cells is a complex process that demands a variety of molecular events, ranging from sarcomere disassembly to the reactivation of the cell cycle genes, and thus requires the regulation of multiple genes. However, the effect of each of these proproliferative miRNAs on other cell types and organs poses important specificity and safety hurdles to clinical translation. The therapeutic use of miRNAs for cardiac regeneration, not different from other applications, deserves careful consideration in at least three areas: the choice of miRNA, the vehicle for cardiac delivery and the route of administration. These three areas are discussed below.

Figure 1: Main Processes and Pathways Modulated by Proproliferative miRNAs

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Choice of miRNA

An ideal miRNA should stimulate S phase entry and completion of the cell cycle, including cytokinesis, without targeting other relevant genes (in particular those regulating the calcium cycle and propagation of the action potential), in addition to having no effect in other cell types. No such ideal miRNA exists. Most of the miRNAs described above were selected for their ability to stimulate CM proliferation and they are effective at this task. Several of them were later verified for efficacy in mice, and some in pigs, thus showing that they exert no major deleterious effect in terms of electrophysiological dysfunction. Thus, the choice of the most translatable miRNAs for clinical applications largely depends on their extracardiac effects. Because selected and controlled transfection or transduction of CMs is difficult to achieve (see below), a main problem is to minimise the risk of carcinogenesis. In this respect, members of miR-17-92 cluster, which is tumour-associated, can be considered less favourably than other miRNAs, such as miR-199a-3p or miR-33b-3p, which are known to exert tumour suppressive effects in other organs. A most effective refinement in the choice of an optimal miRNA for regeneration, or even the identification of additional and more effective miRNAs by screening of genome-wide human miRNA libraries, can now take advantage of the current availability of more sophisticated, 3D tissue models, such as human cardiac organoids, engineered heart tissue or living myocardial slices.114,115

Vehicle for Cardiac Delivery

miRNAs are dsRNA molecules that are relatively large, positively charged, hydrophilic and sensitive to nucleases; thus, their cellular uptake needs to be facilitated by viral or non-viral methods. To date, the most effective mean of expressing an miRNA specifically in CMs is by cloning its pri-miRNA gene into the genome of AAVs under the control of a CM-specific promoter and then packaging this vector using a cardiotropic AAV serotype (e.g. AAV9 for in vivo and AAV6 for ex vivo applications in mice).116 This ensures CM-specific delivery and expression of the miRNA. There are, however, two fundamental drawbacks to this approach. First, because the miRNA duplex is generated inside the cells, it is not possible to choose the desired miRNA strand that is eventually incorporated into RISC, unless resorting to complex molecular biology methods for selective passenger strand inactivation.117 As is apparent from the information reported above on the individual miRNAs, the two strands of the same miRNA duplex often exert opposite effects, which can lead to unwanted outcomes. For example, our own work with miR-199a pri-miRNA has shown that, in pig hearts, both the -5p (which leads to pathological hypertrophy and fibrosis) and the -3p strand (which stimulates regeneration) are expressed at comparable levels.51 Similar considerations would hold for miR-33b, for which the -5p strand exerts a variety of undesirable metabolic, proatherogenic and antiproliferative effects, whereas the -3p strand stimulates CM proliferation. The second drawback relates to the fact that, to date, all the RNA Pol II promoters that are used in gene therapy are constitutively active over time. Prolonged expression of a proproliferative miRNA can lead to hyperproliferation and arrhythmia in the long term, as we have shown in pigs.51 The long-awaited generation of pharmacologically inducible or deactivatable promoters would solve this issue in the future.

Given these intrinsic problems related to the use of AAV vectors, an appealing alternative is to administer synthetic miRNA mimics, namely chemically modified molecules that selectively mimic the activity of a specific miRNA strand, using non-viral delivery. Of note, non-viral delivery would solve the issues of both miRNA strand specificity and duration of effect, because duration can be modulated by introducing sets of different chemical modifications in the mimic molecule. We have recently reviewed the properties of the different available non-viral methods for sncRNA delivery elsewhere.10 A progressively appreciated non-viral method is based on lipid nanoparticles obtained using the stable nucleic acid lipid particle (SNALP) technology, which was originally developed over 20 years ago and has become very popular with the approval of the first siRNA therapeutics (patisiran) in 2018 and of two COVID-19 vaccines in 2020.118–121 However, the development of CM-specific SNALPs is not as straightforward as would be desirable. Systemically injected SNALPs home to the liver and, to a considerably lesser extent, to the spleen and lung, with only a non-significant amount found in the heart.122 Specific CM transfection through the inclusion of targeting ligands is an appealing possibility that is currently the research focus of several laboratories. Our own experience indicates that most current SNALP formulations, when injected into the myocardium, are largely ineffective at specifically transfecting CMs. Cell type specificity can be modulated by changing the lipid formulation. SNALPs usually comprise four lipids: one is cholesterol, the second is commonly a phosphatidylcholine-containing lipid, the third contains a polyethylene glycol-conjugated lipid and the fourth, and most important, is an ionisable lipid that is cationic in acidic conditions and becomes neutral at physiological pH. The field is eagerly waiting for new lipid nanoparticle formulations that can deliver RNA specifically to CMs.

Route of Administration

Direct cardiac catheterisation can be used to overcome the inefficacy of systemic non-viral miRNA complex administration or to improve myocardial uptake of AAV vectors. Direct cardiac delivery would also minimise the potential extra-cardiac effects of the miRNA payload. Treatments can be administered by direct intramyocardial injection, through either surgical access (e.g. mini thoracotomy or during open-chest surgery) or by intraventricular injection using a percutaneous catheter. Even though the surgical approach may be more invasive, it has the advantage of permitting precise delivery in multiple locations of the infarct border zone. This is also possible, even if more complicated, with percutaneous transendocardial delivery, which is, however, safer and less invasive.123 A simpler approach is intracoronary administration, by infusion either after complete balloon occlusion of the coronary flow or during a sub- or non-occlusive procedure.18 However, for this approach to be successful, the treatment needs to cross the endothelial cell barrier, which is normally impermeable to particles larger than 20 nm in diameter.123 Of note, after MI, inflammation leads to the generation of large fenestration between endothelial cells, an effect known as enhanced vascular permeability and retention (EPR). EPR offers an advantage for the extravasation of particles from the vascular network during a narrow time window (approximately 48 hours) from the infarction, which could be sufficient for the therapeutic administration of miRNA complexes.12 As an alternative to anterograde coronary infusion, retrograde administration route via the coronary sinus can be used, which may be more effective but can be associated with a risk of coronary sinus rupture. Finally, percutaneous pericardial administration can also be considered; this method is safe but restricts delivery to the epicardial surface and runs the risk of removal of the therapeutic agent by the lymphatic system.

Conclusion

The possibility of exploiting miRNAs to stimulate the proliferation of endogenous CMs offers an unprecedented clinical possibility to achieve cardiac regeneration. Systematic research on miRNAs now indicates that the proliferation of CMs is a process that can be therapeutically stimulated in adulthood. In addition, the proregenerative effect of miRNAs could reasonably be enhanced by additional interventions, including, for example, their application concomitant with left ventricle unloading or the simultaneous administration of metabolic modulators or treatments that alter the epigenetic state of the cells, all of which could favour CM proliferation.124–126

Should miRNA therapeutics be developed for cardiac regeneration, these would offer tremendous advantages over other approaches to achieve remuscularisation of the myocardium, most notably those based on the transplantation of ex vivo-generated CMs or engineered cardiac tissue. miRNA-based products could be administered with relative ease by interventional cardiologists or, should cardiac targeting be achieved soon, even more simply injected systemically. As indicated by the successful COVID-19 vaccination story, RNA-based nanomedicines are relatively easy to produce, store and distribute, all of which are of paramount importance for a condition such as HFrEF, which has now reached pandemic proportions and for which most patients live in developing countries.

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