MicroRNAs (miRNAs) are small, ∼ 22 nucleotide non-coding RNAs that control patterns of gene expression by binding to 3′-untranslated region (3′-UTR) of their targeted mRNAs thereby promoting mRNA degradation or inhibiting mRNA translation (Filipowicz et al., 2008). miRNAs have been demonstrated to play essential roles in proliferation, apoptosis, differentiation and development of many cells and tissues including the heart (Porrello, 2013; Hodgkinson et al., 2015).

MicroRNAs biogenesis is initiated by generation of primary miRNA (pri-miRNA) transcripts. The pri-miRNA are transcribed from DNA sequences by RNA polymerase II and then processed into precursor miRNAs (pre-miRNAs) by the microprocessor complex, consisting of RNase III enzyme Drosha and dsRNA-binding protein DiGeorge Syndrome Critical Region 8 (DGCR8) (Denli et al., 2004). Thereafter, the pre-miRNAs are transported into the cytoplasm via the nuclear export factor exportin 5 (EXP-5)/RanGTP complex and then processed by the RNase III endonuclease Dicer to produce ∼ 22 nucleotide mature miRNA duplexes (Zhang et al., 2004). The mature miRNA duplexes can be loaded into the Argonaute (AGO) family of proteins (AGO 1-4 in humans) in an ATP-dependent manner, and then unwound into the single miRNA guide strand by the RNA-induced silencing complex (miRISC). Then the miRISC complexes specifically bind to the 3′-UTR of target mRNAs and inhibits their translation or leads to their degradation (Bartel, 2004).

Currently, many studies indicate that miRNAs are involved in various specialized biological processes during cardiac development, disease and ultimately cardiac regeneration and repair (Porrello, 2013; Wu et al., 2013; Hodgkinson et al., 2015). A large number of miRNAs have been shown to participate in regulating cardiac regeneration through controlling cardiomyocyte proliferation, cardiomyocyte differentiation, cardiomyocyte reprogramming, and cardiomyocyte survival in vitro and in vivo (Figure 1).

The proliferation capacity of mammalian cardiomyocyte is robust during the fetal period but is switched off early after birth (Gunthel et al., 2018). A number of miRNAs have been identified that induce or inhibit cardiomyocyte proliferation in vitro and in vivo. A high-throughput functional phenotypic screen using a whole-genome miRNA libraryidentifies 204 potential human miRNAs that are able to promote cardiomyocyte proliferation in neonatal rat cardiomyocytes as determined by 5-ethynyl-2′-deoxyuridine (EdU)-incorporation (Eulalio et al., 2012). Among the 204 miRNAs identified, miR-199a, miR-302b, miR-518, miR-590 and miR-1825 are shown to increase EdU-incorporation, Ki-67, and phospho-histone H3 (pHH3) in mouse or rat cardiomyocytes (Eulalio et al., 2012). Moreover, miR-199a and miR-590 were linked to activation of cell cycle induction and progression, and the enhancement of cardiomyocyte proliferation in vitro and in vivo (Eulalio et al., 2012). Furthermore, the induction of cardiomyocyte proliferation induced by these two miRNAs led to improved cardiac function and decrease cardiac fibrosis in response to myocardial infarction (MI) (Eulalio et al., 2012). An independent proliferation screen performed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) identified 96 miRNAs as drivers of DNA synthesis and cell division in cardiomyocytes (Diez-Cunado et al., 2018). Additional chemical screening and computational approaches revealed that 67 of 96 miRNAs stimulated cardiomyocyte proliferation in a yes-associated protein (YAP)-dependent manner in hiPSC-CMs (Diez-Cunado et al., 2018).

Other studies have implicated additional miRNAs in cardiomyocyte proliferative induction, including the miR-17-92 cluster, miR-25, miR-31a, miR-204, miR-222, miR-294, miR-302-367 cluster, miR-499 and miR-1825. The miR-17-92 cluster, one of the best-characterized miRNA clusters, encodes six mature miRNA members including miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a (Mogilyansky and Rigoutsos, 2013). A recently study has shown that overexpression of the miR-17-92 cluster induces cardiomyocyte proliferation in embryonic, postnatal as well as in adult heart and rescues cardiac function after MI by inhibiting phosphatase and tensin homolog (PTEN) (Chen et al., 2013; Shi et al., 2017). In contrast, inhibition of miR-17 in vivo was shown to attenuate excise-induced cardiac growth and cardiomyocyte proliferation (Shi et al., 2017). The combination of miR-19a and miR-19b led to increased cardiomyocyte proliferation and protected heart against MI in mice (Gao et al., 2019). miR-25, which has the same seed sequences as miR-92a, is also able to stimulate cardiomyocyte proliferation in neonatal rat cardiomyocytes (NRCs) by targeting the Bcl-like protein BCL2 like 11 (Bim) (Qin et al., 2019). Another study has also shown that miR-25 overexpression promotes cardiomyocyte proliferation in hPSC-CMs by targeting F-Box And WD Repeat Domain Containing 7 (FBXW7) (Wang et al., 2020). Overexpressing miR-204 improves cardiomyocyte proliferation via targeting Jarid2, resulted in the upregulation of cell cycle regulator Cyclin A, Cyclin B, cyclin D2, Cyclin E, CDC2 and PCNA in vitro and in vivo (Liang et al., 2015). miR-31a, is significantly upregulated in post-natal day 10 (P10) cardiomyocytes compared to P0, has also been shown to regulate cardiomyocyte cell cycle progression (Xiao et al., 2017). Overexpression of miR-31a in neonatal rat ventricular myocytes (NRVM) and in rat neonates induces cardiomyocyte proliferation by targeting Rho Related BTB Domain Containing 1 (RhoBTB1) (Xiao et al., 2017). In addition, overexpression of miR-222 in mice increased cardiomyocyte proliferation after ischemia/reperfusion (I/R) injury, whereas miR-222 inhibition reduced cardiomyocyte proliferation in response to physical exercise (Liu et al., 2015; Vujic et al., 2018). miR-302-367 cluster is expressed in the embryonic mouse heart and lost in the adult heart, has also been shown to regulate cardiomyocyte proliferation. Loss of miR-302-367 cluster led to decreased cardiomyocyte proliferation in mice, while overexpression of miR-302-367 cluster improved cardiomyocyte proliferation and decreased cardiac fibrosis after MI through repression of the Hippo pathway (Tian et al., 2015). miR-410 and miR-495 both belong to Gtl2-Dio3 miRNAs and have been reported to promote cardiomyocyte proliferation through suppression of CREB binding protein (CBP)/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 (Cited2) as determined by increased EdU incorporation and Ki67 in neonatal cardiomyocytes (Clark and Naya, 2015). miR-294, which is expressed in the heart during development, increased cardiomyocyte proliferation, improved cardiac function and decreased infarct size after MI via suppression of Wee1 in vivo (Borden et al., 2019). miR-499, a myocyte-specific miRNA (myomiR) is expressed within the one of the introns of β-myosin heavy chain (Myh7b) gene, could also promote neonatal cardiomyocyte proliferation by regulating SRY-Box Transcription Factor 6 (Sox6) and cyclin D1 (Li et al., 2013). A recent study demonstrated that miR-1825 increased both DNA synthesis and cytokinesis in adult cardiomyocytes and improve cardiac function after MI (Pandey et al., 2017).

In addition to miRNAs that promote cardiomyocyte proliferation, several miRNAs that are endogenously expressed in cardiomyocytes have been shown to suppress cardiomyocyte proliferation, including let-7i, miR-1, miR-15 family, miR-29a/b, miR-34a, miR-128, miR-128, miR-133 (Braga et al., 2020). Let-7 was one of the first miRNAs isolated and characterized in the nematode C. elegans. Overexpression of let-7i inhibits cardiomyocyte proliferation, whereas the suppression of let-7i induces cardiomyocyte proliferation and improves cardiac function in response to MI by enhancing E2F Transcription Factor 2 (E2F2) and Cyclin D2 (Hu et al., 2019). miR-1 and miR-133 are highly conserved and expressed in cardiac and skeletal muscle and are transcriptionally regulated by myogenic transcription factors MyoD, myocyte enhancer factor-2 (Mef2) and serum response factor (SRF) (Zhao et al., 2007). Overexpression of miR-1 in the developing hearts leads to decreased cardiomyocyte proliferation by targeting the heart and neural crest derivatives-expressed protein 2 (Hand 2) transcription factor (Zhao et al., 2005). Conversely, miR-1 deletion increases cardiomyocyte proliferation in the adult hearts (Zhao et al., 2007). Similar to miR-1, overexpression of miR-133a restricts cardiomyocyte proliferation in zebrafish (Yin et al., 2012), while miR-133a inhibition increases cardiomyocyte proliferation in adult hearts (Liu et al., 2008). The miR-15 family (including miR-15a, miR-15b, miR-16-1, miR-16-2, miR-195 and miR-497), modulates neonatal heart regeneration through inhibition of postnatal cardiomyocyte proliferation and repression of cell cycle genes, thus resulting in postnatal loss of cardiac regenerative capacity (Porrello et al., 2013). Inhibition of miR-15 family from the early postnatal period until adulthood promotes cardiomyocyte proliferation and enhances cardiac function in response to MI (Hullinger et al., 2012; Porrello et al., 2013). Overexpression of miR-195 in the embryonic heart suppresses cardiomyocyte proliferation and impairs the regeneration capability of 1-day-old mouse heart after MI through the repression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1) in vivo (Porrello et al., 2011a, 2013). Inhibition of miR-26a increased proliferation of mouse neonatal cardiomyocytes in vitro and in vivo via regulation of cell cycle inhibitors (Crippa et al., 2016). Overexpression of miR-29a suppresses cardiomyocyte proliferation, while its inhibition enhances cell division by inducing cyclin D2 expression in NRCs (Cao et al., 2013). Similar to miR-29a, inhibition of miR-29b promotes cardiomyocyte proliferation by inactivation of notch receptor 2 (NOTCH2) function in vitro and in vivo (Yang et al., 2020). miR-34a, a regulator of age-associated physiology, also suppresses cardiomyocyte proliferation and its inhibition leads to enhanced cardiomyocyte proliferation and improves cardiac function in response to MI through targeting of silent information regulator factor 2 related enzyme 1 (Sirt1), B-cell lymphoma 2 (Bcl2) and Cyclin D1 (Yang et al., 2015). Inhibition of miR-128 promotes cardiomyocyte proliferation by activating cyclin E- and cyclin dependent kinase 2 (CDK2)-positive cell cycle regulators and improves cardiac function in response to MI (Huang et al., 2018).

In addition to enhancing the proliferative capacity of endogenous cardiomyocytes, transplanting cardiac stem cells or cardiomyocytes derived from iPSCs or ESCs is another therapeutic approach in the field of cardiac regeneration and is considered a promising strategy to heart failure. Pluripotent stem cells provide great promise for regenerative medicine due to their self-renewal potential and ability to differentiate into multiple cell lineages or specific functional cell types. Several studies have shown that miRNAs play a key role in differentiation of stem cells, including miR-1, miR-23, miR-125b, miR-133, miR-199a, miR-203, miR-204, miR-211, miR-222, miR-290, miR-302, miR-499, miR-669.

miR-1 and miR-499 are also highly enriched in cardiomyocytes and upregulated in differentiated cells. Studies have shown that miR-1 promotes cardiac differentiation from ESCs by the downregulation of Notch ligand Delta-like 1 (Chen et al., 2006). Moreover, overexpression of miR-1 itself is sufficient to promote cardiac differentiation mediated by upregulation of the cardiac differentiation genes nk2 homeobox 5 (NKX2.5) and myosin heavy chain beta (β-MHC) in mouse and human ESCs (Ivey et al., 2008). In addition, Sluijter and colleagues have also shown that miR-1 overexpression enhances human cardiomyocyte progenitor cells (hCMPCs) differentiation into cardiomyocytes by repression of Histone Deacetylase 4 (HDAC4) (Sluijter et al., 2010). Furthermore, transplantation of mouse ESCs overexpressing miR-1 (miR-1-ES cells) into the border zone of the infarcted hearts significantly promotes cardiac differentiation and protects against MI-induced apoptosis through activation of PI3K/Akt pathway (Glass and Singla, 2011). Similarly, miR-499 overexpression enhances differentiation of cardiac progenitors into cardiomyocytes in human CMPCs and ESCs by inhibition of Sox6 in human CMPCs (Sluijter et al., 2010). In addition, overexpression of miR-499 significantly enhances cardiac differentiation in human ESC-derived cardiomyocytes by upregulation of cardiac myosin heavy chain genes and cardiac transcription factor MEF2C (Wilson et al., 2010).

In addition to miR-1 and miR-499, overexpression of miR-203 improves efficient differentiation and maturation to cardiomyocytes by repression of DNA methyltransferase 3a/b (Dnmt3a/b) in mouse iPSCs (Salazar-Roa et al., 2020). miR-322 and miR-503 clusters have shown that participate in the specification into cardiomyocyte progenitors of the mesodermal cells by the repression of CUGBP Elav-like family member (Celf) family proteins (Shen et al., 2016). miR-204, miR-669 and miR-23 have also been shown to promote cardiac progenitor differentiation (Crippa et al., 2011; Xiao et al., 2012). Furthermore, recently another study shows that a combination of miRNAs (miR-125b, miR-199a, miR-221 and miR-222) promotes the maturation of both mouse and human cardiomyocytes differentiated from ESCs (Lee et al., 2015).

miR-1 and miR-133 are co-transcribed due to their polycistronic clustering on the same chromosome. However, they have antagonistic effects on lineage commitment and terminal differentiation, as miR-133 inhibits cardiomyocyte terminal differentiation and maintains them in a proliferative state (Chen et al., 2006). Overexpression of miR-133 suppresses the expression of cardiac markers in human and mouse ESCs, and inhibits differentiation of ESCs into CMs (Alfar et al., 2018), while miR-133 deletion promotes cardiomyocyte proliferation partly via activation of SRF and G1/S-specific cyclin D2 (Chen et al., 2006; Liu et al., 2008), thus highlighting the inhibitory role of miR-133 in both cardiomyocyte differentiation and proliferation. Recently, a study revealed that inhibition of miR-23 facilitated the transformation of bone marrow mesenchymal stem cells (BMSCs) into myocardial cells and conferred protection against Ischemia/reperfusion (I/R)-induced myocardial damage by activation of Wingless and Int-1 (Wnt) pathway (Lu et al., 2019). miR-302/miR-290 members are involved in the maintenance of pluripotency of murine ES cells, and the members of these families have been shown to suppress cell differentiation (Judson et al., 2009; Melton et al., 2010). In addition, miR-363 negatively regulates in cardiomyocyte specification, miR-363 inhibition leads to an enrichment of left ventricular ESC-derived cardiomyocytes by targeting Hand 1 (Wagh et al., 2014).

Taken together, these studies demonstrate that miRNAs regulate cardiomyocyte differentiation and help balance cardiomyocyte differentiation with proliferation, which could be further translated clinically by using miRNAs to induce transferred ESCs/iPSCs to differentiate into mature cardiomyocytes to replace damaged cardiomyocytes.

There are two methods for cardiomyocyte reprogramming: indirect reprogramming and direct reprogramming. The indirect reprogramming uses an intermediary step where the non-myocytes dedifferentiate into progenitor cells that then could be further programmed into the cardiomyocytes (i.e., iPSC reprogramming). The direct reprogramming directly converts the non-myocytes into cardiomyocytes without passing through the pluripotent state (Farber and Qian, 2020). Over the last decade, direct reprogramming from cardiac fibroblasts of the infarcted area into cardiomyocytes has been successful in the repair of damaged heart tissue, and has great promise for the clinic.

miRNAs are able to directly induce the cellular reprogramming of cardiac fibroblasts into cardiomyocytes in vitro and in vivo (Rao et al., 2006; Jayawardena et al., 2012; Joladarashi et al., 2014). Recently, Jayawardena et al. has identified and evaluated a specific combination of miRNAs (miR-1, miR-133, miR-208 and miR-499; miR combo) which is able to directly reprogram cardiac fibroblasts into cardiomyocyte-like cells in vitro and in vivo (Jayawardena et al., 2012). In vitro, the reprogrammed cardiomyocyte-like cells express cardiomyocyte-specific genes, and exhibit sarcomeric organization and functional properties characteristic of mature cardiomyocytes (Jayawardena et al., 2012). Furthermore, injection of lentivirus miR combo into the border zone of the infarcted heart also induced generation of new cardiomyocyte-like cells from lineage-traced non-cardiac myocytes by 4 weeks post myocardial injury (Jayawardena et al., 2012). In addition, miR-combo significantly decreases cardiac fibrosis and improves cardiac function, as indicated by the improvement of fractional shortening (FS) and ejection fraction (EF) following MI (Jayawardena et al., 2015). These data demonstrate that administration of miRNAs to the peri-infarct area of the infarcted heart in vivo is able to direct reprogramming of cardiac fibroblasts into functional cardiomyocytes.

Recent studies demonstrate that co-overexpression of the GATA binding protein 4 (Gata4), Mef2c, and T-box transcription factor 5 (Tbx5), termed GMT, leads to the reprogramming of cardiac fibroblasts into cardiomyocytes in vitro and in vivo (Ieda et al., 2010; Qian et al., 2012). Additionally, they observe that the combination of miR-133 and GMT further enhanced cardiac reprogramming of mouse embryonic fibroblasts into cardiomyocytes in terms of kinetics and efficiency by directly binding to the snail family transcriptional repressor 1 (Snail 1) (Jayawardena et al., 2012). Interestingly, a study by Singh and colleagues reveal that miR-590, which is a key miRNA in cardiomyocyte proliferation, could also further enhance GMT-mediated reprogramming in human and pig fibroblasts by suppressing the Sp1 transcription factor (Sp1), that can inhibit the activity of cardiac-specific genes (Singh et al., 2016). In addition, another study shows that the combination of miR-1, miR-133 and Gata4, Mef2c, TBx5, Myocardin could further increase the direct reprogramming efficiency of human fibroblasts to cardiomyocyte-like cells (Nam et al., 2013).

Cardiomyocyte survival is recognized as being a central feature of cardiac remodeling and progression in heart failure (Briasoulis et al., 2016). The underlying triggers of cell survival and development of novel treatment strategies to suppress cardiomyocyte apoptosis is important. Cardiomyocyte apoptosis can be triggered by the activation of two major signaling pathways: the intrinsic and extrinsic pathways. The intrinsic pathway is mediated by mitochondrial membrane permeabilization and the extrinsic pathway involving the activation of surface death receptor by death ligands (Bennett, 2002). Several miRNAs have been shown to pro-apoptotic and anti-apoptotic regulate apoptotic signaling pathways, which may have to effect on survival of cardiomyocytes. miR-21, miR-24, miR-30 family, miR-31a, miR-133, miR-138, miR-199a, miR-181c, and miR-499 are main miRNAs that could inhibit cardiomyocyte apoptosis. The miR-1, miR-29, miR-34a, miR-124, and miR-320 exhibit an pro-apoptotic effect in cardiomyocytes.

Many studies demonstrate that miR-21 promotes cardiac survival and attenuates cardiomyocyte apoptosis in vitro and in vivo. Overexpression of miR-21 protects against hydrogen peroxide (H₂O₂)-induced cardiac apoptosis via inhibition of programmed cell death protein 4 (pDCD4) and activator protein 1 (AP-1) pathway in vitro (Cheng et al., 2009). miR-21 is decreased in the infarct zone after MI, while miR-21 overexpression in rat hearts reduced cardiac fibrosis and decreased cardiomyocyte apoptosis in the infarct and border zone after AMI (Dong et al., 2009; Yan et al., 2015). Furthermore, overexpression of miR-21 alone significantly inhibits autophagic activity and alleviates hypoxic/reoxygenation (H/R)-induced cardiac apoptosis by regulation of PTEN/Akt/mTOR pathway (Huang et al., 2017). In addition, combination of miR-21 and miR-146 could further decrease ischemic/Hypoxic-induced cardiomyocyte apoptosis compared to either miR-21 or miR-146a alone in NRCs (Huang et al., 2016). Furthermore, simultaneous delivery of agomiR-21 and agomiR-146a in mice attenuates cardiomyocyte apoptosis and improves cardiac function following AMI by regulation of p38 MAPK (Huang et al., 2016). In vivo studies show that overexpression of miR-24 protects against cardiomyocyte apoptosis and mediated by repression of BH3-domain-caontaining protein (Bim) in response to MI in mice (Qian et al., 2011). miR-30 family members, including miR-30a, miR-30b and miR-30d, inhibits mitochondrial fission through suppressing the expression of p53 and downstream targets dynamin-related protein-1 (Drp1), leading to decreased cardiomyocytes apoptosis (Li et al., 2010). Furthermore, doxorubicin-induced apoptosis could be rescued by miR-30 expression in rat cardiomyocytes in vivo (Roca-Alonso et al., 2015). miR-31a is upregulated in the failing heart, overexpression of miR-31a protects against the angiotensin II (AngII)-induced apoptosis and caspase-3 activity by targeting Tp53 in the cardiac H9C2 cell line (Yan et al., 2018). Gain-of -function studies demonstrate that miR-133 overexpression inhibits cardiac apoptosis and enhances cardiac function following chronic pressure overload. miR-133 overexpression decreases I/R-induced apoptosis, whereas miR-133 deletion increases cardiac apoptosis mediated by regulating the expression of casp9 (He et al., 2011). miR-199a also plays a crucial role in hypoxia-induced apoptosis (Rane et al., 2009). Replenishing miR-199a during hypoxia decreases cardiomyocytes apoptosis through inhibition of hypoxia-inducible factor (Hif)-1α and its stabilization of p53, while miR-199a inhibition recapitulates hypoxia preconditioning by up-regulating Hif-1α and Sirt1 (Rane et al., 2009). miR-181c is suppressed in the heart tissue of doxorubicin (DOX)-induced heart failure animal model (Li et al., 2020). Study shows that miR-181c overexpression protects heart failure by impeding cardiomyocyte apoptosis through PI3K/Akt pathway (Li et al., 2020). Overexpression or knockdown of miR-499 decreases or increases the cardiomyocyte apoptosis in vitro (Li et al., 2016). In addition, overexpression of miR-499 decreases cardiac apoptosis and reduces myocardial infarct size in the rat AMI models by inhibiting PDCD4 (Li et al., 2016).

In addition to miRNAs that reduce cardiomyocyte apoptosis, there are several miRNAs that exert the opposite effect on cardiomyocyte apoptosis. miR-1, which has been shown to play an essential role in the regulation of cardiac proliferation and differentiation, also plays a critical role in cardiomyocyte apoptosis. Overexpression of miR-1 in mice increases cardiomyocyte apoptosis, while deletion of miR-1 reduces I/R-induced cardiomyocyte apoptosis and attenuates cardiac I/R injury by targeting protein kinase C epsilon (PKCε) (Pan et al., 2012). Overexpression of miR-29 promotes apoptosis, whereas inhibition of miR-29 reduces cardiomyocyte apoptosis and infarct size in hearts subjected to I/R injury by decreasing the expression of pro-apoptotic molecular Bcl-2-associated X protein (Bax) and increasing anti-apoptotic molecular Bcl2 (Ye et al., 2010). miR-34a is induced in the heart during aging in both mice and humans, and its deletion in vivo reduces age-induced cardiomyocyte cell death and fibrosis, and improves cardiac function after AMI by activation of serine/threonine-protein phosphatase 1 regulatory subunit 10 (PNUTS) (Boon et al., 2013). miR-124 is upregulated in a mice model of MI, inhibition of miR-124 decreases MI-induced cardiomyocyte apoptosis and infarct size and improves cardiac function in mice by upregulating signal transducer and activator of transcription 3 (STAT3) (He et al., 2018). miR-320 is significantly decreased in the hearts with I/R injury in vivo and ex vivo, and its overexpression enhances cardiomyocyte apoptosis and death in the hearts on I/R (Ren et al., 2009). Conversely, administration of antagomir-320 reduces I/R-induced cardiac injury and cardiomyocyte apoptosis by targeting heat-shock protein 20 (Hsp20) (Ren et al., 2009).

Together, these studies indicate that miRNAs can effectively stimulate cardiac regeneration by directly modulating target gene expression, and provide promising a therapeutic avenue for heart failure treatment. To date, a large number of miRNAs have been discovered to regulate cardiac regeneration. However, the molecular targets and mechanisms underlying miRNA function needs to be further investigated, and the miRNA-based preclinical and clinical trials need to be better and more stringently assessed for therapeutic efficacy.