Recent Insights into Endogenous Mammalian Cardiac Regeneration Post-Myocardial Infarction

2.1.1. CPCs

CPCs constitute a heterogeneous population of resident cardiac cells distributed throughout the heart [52]. CPCs are immature yet committed myocardial cells capable of proliferation and differentiation into major cardiac cell types, namely CMs, SMCs, and ECs, thereby possibly facilitating the regeneration of damaged cardiac tissue and promoting neovascularisation [53]. Several types of CPCs have been identified in cardiac development and regeneration at various stages that can be classified based on the expression of different surface markers (c-Kit, Sca-1, Mesp1, KDR/Flk-1) and their locations within the heart (mesoderm, epicardium, side population, or cardiosphere-derived) [54].

2.1.2. MSCs

On the other hand, MSCs represent the most undifferentiated stem cells involved in tissue repair across various organs. As stromal cells, they possess a unique capacity for self-renewal and exhibit multilineage differentiation potential [80]. Although there is no concrete evidence regarding their direct intervention in myocardial regeneration through transdifferentiation, researchers have increasingly become interested in MSCs in recent years due to their robust regenerative capabilities and their greater accessibility, especially in the investigation of ischaemic heart disease treatments [81,82]. MSCs secrete a range of bioactive molecules, including GFs, cytokines, chemokines, and EVs, which can modulate the cardiac microenvironment and promote tissue repair processes [83,84,85]. Currently, investigation of MSC-Exosomes (MSC-Exo) and MSC immunomodulatory properties are major research focuses.

2.2.1. Neuregulin-1

Neuregulin-1 (NRG-1) is a growth factor involved in various processes of cardiac development, including CM proliferation and differentiation. Primarily produced and released by vascular ECs, it acts through its binding with the ErbB4 receptor [101]. This binding induces ErbB2-ErbB4 heterodimerization, and the resulting intracellular signal is capable of activating the Ras/ERK, PI3K/Akt, and Src/Fak pathways, which regulate multiple functions of CMs such as growth, proliferation, survival, and structural sarcomeric organisation [102]. The regulatory role of NRG-1 in cardiac development is predominant during the neonatal phase compared to adulthood in mammals, primarily due to the decrease in the cardiac level of ErbB2, which is essential for heterodimerization with ErbB4 [103]. This reduction of ErbB2 was observed in the heart at P7 and further decreased at P28, resulting in the loss of cardiac regenerative capacity. These findings on the role of NRG-1 in cardiac regeneration suggest that combinatorial overexpression strategies of NRG-1 and ErbB2 might be a therapeutic target to initiate cardiac repair after MI.

In mammals, a metabolic switch from glucose to fatty acid metabolism occurs in the first postnatal week and coincides with increased mitochondrial activity and loss of regenerative potential [104]. Although constitutive expression of ErbB2 in the in vitro experiments led to a metabolic shift toward glycolysis, which is beneficial in promoting CM cell cycle re-entry, in the in vivo adult murine model, its constitutive expression induced hypertrophic cardiomyopathy [105,106]. This research group also developed a transgenic mouse model in which the expression of ErbB2 could be transiently induced with Doxycycline. They used a young mouse model (P8) already exited from the regenerative window (MI induced at P7) and a 5-week adult mouse model (MI induced at P42), and subsequently administered Doxycycline for 10 and 21 days, respectively, to achieve transient expression of ErbB2 signalling. This transient expression resulted in a significant improvement in cardiac recovery after MI, with an increase in proliferating CMs (TnT+, Ki67+, and Aurora B+) together with a reduction in scar size in both young and adult models compared to controls [103]. This improvement was particularly pronounced after the cessation of ErbB2 expression, especially in the adult model. Analyses of sarcomeric structures conducted one month after infarction (approximately P70) and ErbB2 induction revealed the presence of robust dedifferentiated cardiac muscle. Moreover, one month after the cessation of ErbB2 expression (approximately P98), both tissue morphology and sarcomeric organisation were restored, indicating the process of CM redifferentiation leading to reduced scar formation and improved cardiac function after MI. These results suggested that a transient expression of ErbB2 in the adult model is sufficient to reopen the regenerative window of CM proliferation. In addition, the analysis of downstream mediators of ErbB2 proved these effects in CMs to be mediated by ERK, AKT, and GSK3β/β-catenin pathway activation [106]. Recent studies have highlighted that in the process of cardiac regeneration mediated by ErbB2 in an adult murine model of MI, an epithelial-mesenchymal transition (EMT)-like process is also involved; thus, the authors suggested that CM migration is an essential element for cardiac repair and scar replacement by new CMs, together with ECM and cytoskeletal remodelling. Furthermore, these authors pointed out the role of YAP, a downstream mediator of the ErbB2 and ERK signalling pathways, the phosphorylation of which is necessary to induce cytoskeletal remodelling and regulate CM proliferation [107]. CM dedifferentiation is associated with an upregulation of foetal gene expression, the disassembly of the sarcomere, and changes in cellular morphology, playing a key role in tissue regeneration [108]. The activation of the EMT process is therefore crucial in promoting the reactivation of efficient regenerative processes in tissues that have lost this capability, such as cardiac tissue. In particular, the transient expression of the ErbB2-YAP signalling axis can lead to the activation of EMT, causing dedifferentiation of adult CMs and their subsequent redifferentiation when ErbB2-YAP expression is stopped. However, only a transient activation is required, while a constitutive activation of ErbB2 can lead to cardiac hypertrophy. This phenomenon has also been confirmed in various murine models following MI [109].

2.2.2. Fibroblast Growth Factors

Fibroblast growth factors (FGFs), comprising a family of 22 members in mammals, play a crucial role in cardiac function, spanning from developmental processes to maintaining homeostasis and contributing to disease progression [110]. Canonical FGFs, such as FGF1-10, FGF16-18, FGF20, and FGF22, are small proteins secreted in the ECM by various cell types, including ECs, FBs, CMs, and MSCs, and function either as paracrine or autocrine GFs [111]. Hormone-like FGFs, such as FGF15, FGF19, FGF21, and FGF23, are released into the blood and regulate different aspects of metabolism. Additionally, there is a subfamily of FGFs comprising four intracellular proteins, namely FGF11-14, which play a role in modulating ion channels [111]. Notably, FGF 1, 2, and 10 mediate a crucial role in the heart by binding to their receptors (FGFRs) [112,113]. The potential of FGF1 has been further underscored through its synergy with NRG-1, an agonist of the epidermal growth factor receptor tyrosine kinase, in both rat and swine models. Both GFs were administered directly into the myocardium, encapsulated within microparticles, four days post-MI. The combination of these growth factors in infarcted hearts led to a significant improvement in cardiac function, with a reduction in necrotic area and appropriate remodelling of the heart compared to the untreated control. Interestingly, a decrease in the number of apoptotic CMs was observed after 3 months of treatment, with an increase in proliferating CMs (cardiac TnT+ Ki67+) in the infarcted zone following treatment [114].

The role of FGF10 in adults is involved in maintaining proper cardiac morphology and, particularly, in preserving the correct thickness of the ventricular wall through the regulation of CM proliferation. Notably, FGF10 controls the CM cell cycle by binding to FGFR2b during embryonic development, while in the adult heart, it exerts this control by binding to FGFR1b [115]. The expression of FGF10 changes during the developmental stages: gene expression analysis in both foetal and postnatal/adult hearts showed that FGF10 expression decreased from 18.5 days of foetal growth (E 18.5) until P10, when CMs exit from the cell cycle. FGF10 expression then increased again at P16, reaching maximal levels at P56 (8 weeks old) before decreasing again at P100 (14 weeks old). These results support a physiologically relevant role for FGF10 in controlling CM proliferation both during foetal development and in the postnatal/adult heart [110,116]. Deletion of FGF10 compromises CM proliferation, as evidenced by the significant reduction in Ki67+ CMs in the right ventricle of FGF10^−/− embryos [117]. During foetal development, FGF10 action involves the phosphorylation of FOXO3 and downregulation of the cyclin inhibitor p27. In the adult transgenic mice model, the analyses of the percentage of CM positive for the proliferative marker Ki67+ and mitotic marker PH3+ (phosphorylated histone 3) reveal that FGF10 overexpression (for 14 days) specifically promotes CM re-entry into the cell cycle. These authors attributed the cell proliferation exclusively to CMs rather than non-myocytic cells, as evidenced by the presence of α-actinin staining. In particular, these smaller and mononucleated new CMs were observed in the infarcted and border zones [117]. The role of FGF10 in cardiac regeneration has also been confirmed using mice with reduced FGF10 expression (FGF10^+/−). Twenty-one days post-MI, endogenous levels of FGF10 increased in CMs, and these FGF10^+/− mice exhibited worsened cardiac performance, including further decreases in ejection fraction and fractional shortening and further increases in left ventricular volume compared to WT. Interestingly, decreased FGF10 levels compromised CM proliferation and impaired fibrosis post-MI. Immunofluorescence staining for Ki67, PH3, and Aurora B revealed significant impairment in CM proliferation in FGF10^+/− mice compared to WT, both at 5 and 21 days after MI. At the same time points, histological analysis with Sirius Red staining showed an increase in fibrotic tissue together with upregulated collagen gene expression in FGF10^+/− mice [117]. These results reinforced the concept that high levels of FGF10 are necessary for cardiac regeneration and the preservation of cardiac function, preventing fibrosis, and reducing post-infarction cardiac remodelling by modulating the gene expression of key regenerative signalling pathways, including the transcription factor Meis1, which controls CMs’ and ECs’ fate, the Hippo signalling pathway, and a pro-glycolytic metabolic switch. Most importantly, in the same study, the pivotal role of FGF10 in cardiac regeneration has also been confirmed in failing human heart biopsies, where FGF10 expression significantly correlated with smaller CM cross-sections and enhanced Ki67+ CM numbers in the border zone, thus reinforcing the conclusion that FGF10 promoted CM renewal [117].

2.2.3. Insulin-like Growth Factors

IGF1 and IGF2 are growth factors that play a central role in activating endocrine, paracrine, and autocrine signalling pathways in cardiac development. While IGF1 is predominantly present in the postnatal period, including adulthood, IGF2 prevails during embryonic development [118].

Indeed, IGF-1 receptor inhibition has been shown to hinder CM proliferation, thereby impeding cardiac development and regeneration in the mouse heart [119]. The main action of IGF1 involves the phosphorylation of MEF2C, resulting in p38-MAPK pathway activation. Additionally, it activates several other signalling pathways, including ERK1/2, PI3K, PKC, PKB, Jak/STAT, and PLC [120].

Overexpression of IGF2 in parthenogenetic stem cells (PSCs) accelerates their differentiation into CMs, resulting in a promising strategy for enhancing cardiac regeneration [121]. Shen et al. recently reported that IGF-2 invalidated the intrinsic regenerative effects in P1-day mice, highlighting the critical role of IGF-2 as a mitogen in neonatal mice [41].

The bioavailability of IGF is regulated by six members of the IGF-binding protein (IGFBP) family [122].

IGFBP1 regulates cell proliferation, migration, and metabolism by activating integrin-ILK/FAK/PTEN signalling through integrin receptors on the cell membrane [123]. Under hypoxic conditions, IGFBP1 influences myocardial cell apoptosis by regulating Hypoxia-Inducible Factor(HIF)-1α. Its role as a negative regulator of CM proliferation has also been demonstrated in an MI mouse model. Seven days after intramyocardial injection of adenovirus-shRNA-IGFBP1, the animal hearts were subjected to LAD ligation. Silencing IGFBP-1 can reduce infarct size and attenuate apoptosis in CMs, with decreased interstitial fibrosis in the infarcted zone of the shRNA-IGFBP1 group compared to controls. Additionally, immunofluorescence staining showed an increased number of Ki67+ and pH3+ CMs in the border zone of the shRNA-IGFBP1 group compared to the control group, suggesting that knocking down IGFBP-1 promotes cardiac regeneration and reduces fibrosis after MI [124].

Wang et al. identified a regulator of myocardial cell proliferation in the neonatal heart, IGF2BP3 [24]. IGF2BP3 expression is enriched in the regenerative heart following MI and promotes CM division, resulting in a potential target for future cardiac regeneration therapy [24]. IGF2BP3 promoted myocardial regeneration in adult mice by stabilising MMP3 mRNA through the interaction with m6A (N6-methyladenosine) modification. In particular, adeno-associated virus 9 (AAV9)-packaged IGF2BP3 was injected into the myocardium of the left ventricle of P1 mice immediately after MI induction. At 7 and 56 days after IGF2BP3 injection, CM proliferation was markedly increased in P7 and adult mice compared with groups injected with the control vector, as determined by immunostaining of pH3 and Ki67. Furthermore, overexpression of the IGF2BP3 significantly increased the proportion of proliferative CMs in the marginal zone of the MI heart at 14 days [125]. IGF2BP3, along with the cytokine Ccl24, is highly expressed in macrophages of P1 neonatal mice compared to P14. This expression is thought to support cardiac regeneration by promoting postnatal CM proliferation [126].

Taken together, these results highlight the role of IGFs in myocardial regeneration, supporting CM proliferation. Further investigation into their involvement in cardiac repair processes and immune cell regulation could offer new insights for enhancing myocardial regeneration after injury.

2.3.1. Cyclins

Various regulators of cell cycle checkpoints, including cyclins, cyclin-dependent protein kinases (CDKs), CDK-activating kinases (CAKs), and their inhibitors (CKIs), have been identified as key players in regulating the cell cycle activity of CMs during both prenatal and postnatal development [127].

CDKs are activated by complex formation with cyclins, leading to advances in the cell cycle. In mammals, the complexes are Cyclin D-CDK4/6 (G1 phase), Cyclin E-CDK2 (G1/S phase), Cyclin A-CDK2/1 (S/G2 phase), and Cyclin B-CDK1 (M phase) [128]. CDK activities are regulated through interactions with CKIs such as p21, p27, and p57, which are implicated in CM cell cycle arrest. It has been demonstrated that the simultaneous knockdown of p21, p27, and p57 with siRNA induces both neonatal and adult CMs to enter the S-phase and undergo mitosis in vitro [129,130,131]. When complexed with CDK4 or CDK6, D-type Cyclins drive cell cycle re-entry from G0 to G1 phase. Protein levels of D-type Cyclins and activating kinases (i.e., CDK4 and CDK6) have been observed to sharply decline in the early postnatal stages and adulthood [112]. In particular, Cyclin D1 is expressed at very low levels in adult CMs, allowing the maintenance of a quiescent state of these cells. Indeed, specific induction of Cyclin D1 expression in adult mouse CMs leads to an increase in proliferation markers, such as BrdU, Ki-67, and PCNA (Proliferating cell nuclear antigen), as well as an increase in the expression of DNA replication-related proteins, resulting in more than 40% of CMs re-entering in the cell cycle [132]. Recently, it has been demonstrated that miR-301a, enriched in neonatal CMs, is able to induce cell cycle re-entry and enhance cellular proliferation in H9C2 cells and primary CMs by upregulating Cyclin D1. In vitro assays (Ki67, EdU, Aurora B stainings), confirmed that miR-301a overexpression led to increased proliferation and G1/S transition of CMs. AAV9-mediated cardiac delivery of miR-301a promoted cardiac repair and regeneration in an MI murine model, via an increased expression of Cyclin D1 by downregulating PTEN and upregulating phosphorylated AKT and GSK-3β. In the miR-301a-treated mice, 1.5–2.0% of adult CMs were double positive for α-actinin and Ki67, compared to 0.5–1.0% in the control mice, indicating the improved proliferative ability of CMs by miR-301a in vivo [133].

Cyclin B1 interacts with Cyclin-dependent kinase 1 (CDK1) to orchestrate the transition from the G2 phase to mitosis. This complex starts to increase its activity during late G2, through prometaphase until the cell enters metaphase [134]. Scientific evidence in both in vitro and in vivo models demonstrated the importance of both Cyclin D1/CDK4 and Cyclin B1/CDK1 complexes in the cell cycle re-entry of CMs. At first, Tamer et al. proved that a cocktail of Cyclin D1/CDK4 and Cyclin B1/CDK1 efficiently enhances the proliferation of hiPS-CMs, primary mouse neonatal (isolated at P7) and adult rat (isolated at 4 months) CMs. Subsequently, the authors validated these results in an in vivo adult mouse model of coronary ligation with intramyocardial injections of adenoviral vector encoding for CDK1, Cyclin B1, CDK4, and Cyclin D1, showing that adult CMs expressing these four factors underwent stable cell division, resulting in a significant improvement in cardiac function after acute or subacute MI. Interestingly, a similar improvement was obtained even with injection after the formation of scar tissue. Moreover, it has been noted that the use of inhibitors of TGFβ results in an unblocking of adult CM replicative ability by promoting the function of G1 phase cyclins, likely attributable to an indirect suppression of the CDK inhibitor p27 [135].

Cyclin D2 is well known as a key regulator of the cell cycle, particularly in the G1 phase, critical for DNA synthesis and cell proliferation. Specific expression of Cyclin D2 in CMs has a significant impact on their proliferative capacity and cardiac regeneration after MI. Indeed, cardiac-specific expression of Cyclin D2 in both adult mice and pigs leads to increased DNA synthesis up to 150 days post-infarction, increasing CM proliferation and regression of the infarct area. In particular, 7 days after MI induction and treatment, immunofluorescence analysis pointed out a significantly higher CM number expressing Ki67, PH3, and Aurora kinase B in the border zone of mouse hearts treated with CyclinD2-modified mRNA compared to control groups. Similarly, a 5.99-fold increase in Ki67+ and a 4.17-fold increase in PH3+ CM were observed in the border zones of infarcted pig hearts 3 days following intramyocardial administration of Cyclin D2-modified mRNA, compared to control groups [136]. D-Cyclin activity can be modulated by dual-specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1a), which plays a critical role in controlling the progression of the CM cell cycle. Specifically, inhibition of Dyrk1a led to increased expression of D-Cyclins, promoting the activation of the Rb/E2F signalling pathway. The E2F transcription factor family is crucial for CM proliferation. E2F1, 2, and 3 regulate gene expression that influences entry into the cell cycle, especially the G1/S transition. E2F activity is modulated by various proteins, including the retinoblastoma family protein (pRB) [137,138]. Consistent with these findings, DYRK1a can enhance CMcycling and improve heart function in a mouse model of MI. Specifically, when DYRK1a was ablated in mice, there was an upregulation of cell cycle genes and a downregulation of genes related to contractile proteins, compared to the control group [139].

Recently, the role of Cyclin L1 (CCNL1) in CM proliferation has been studied. Gong et al. demonstrated that the presence of CCNL1 increases in the mouse cardiac tissue undergone MI, suggesting a potential interference in the CM proliferation capacity. Gene silencing in murine models confirmed that the absence of CCNL1 promoted CM proliferation and cardiac repair after MI. The negative role of CCNL1 in regulating the CM cell cycle and in post-infarction cardiac repair could probably be attributed to its binding with CDK11 [140,141].

In addition to the cyclins mentioned above, Cyclin A2 also plays a role in the regulation of the cell cycle. It regulates two different stages of the cell cycle; specifically, it causes the entry in the S phase when it combines with CDK2, while it allows the cell to progress to phase M when it binds with CDK1. Indeed, the almost total absence of Cyclin A2 in the adult heart has been correlated with the exit of CMs from the cell cycle in mammals [112]. Several scientific studies in different animal models demonstrated the crucial role of Cyclin A2 in CM cell cycle regulation after cardiac injury. In cardiac tissue, elevated levels of Cyclin A2 are crucial in promoting CM cell cycle re-entry two weeks after acute MI in a rat model [142]. Interestingly, the role of Cyclin A2 in post-MI cardiac regeneration has also been confirmed in a porcine heart model [143]. Researchers induced overexpression with intramyocardial injection of replication-deficient adenoviral vectors containing murine Cyclin A2 one week after MI, observing a significant increase in CM proliferation with improvement in cardiac function compared to control after 6 weeks [143]. Notably, other authors pointed out that the Y-box 1 binding protein (YBX1) can stimulate Cyclin A2 activity. Through the use of a circRNA capable of blocking Nfix (Nuclear factor I X), responsible for the degradation of YBX1, it has been possible to restore the ability of YBX1 to bind and activate the Cyclin A2 promoter, thus inducing CM proliferation and inhibiting apoptosis after MI in a mouse model [144] (Figure 2).

2.3.2. p53

The tumour suppressor protein p53 plays a crucial role in regulating both embryonic and postnatal cardiac development. Under physiological conditions, p53 is functionally active in maintaining cardiac structure and regulating the expression of transcriptomes related to metabolism, mitochondrial biogenesis, cardiac architecture and excitation-contraction coupling. It has been demonstrated that the deletion of p53 triggered murine heart hypertrophy and decreased heart function [145,146].

Qi Xiao et al. developed a tracking system for p53+ CMs in mice, covering various stages of development, from neonatal to adult. Using this system, they observed how p53+ CMs responded to myocardial cryo-injury and contributed to cardiac regeneration during the postnatal age [147]. Conversely, elevated levels of p53 in pathological cardiovascular conditions can lead to the activation of various mechanisms, including CMapoptosis, cell cycle arrest, metabolism alteration, and autophagy [148]. These data have been confirmed by Stanley-Hasnain et al., who demonstrated that simultaneous deletion of both p53 and its inhibitor murine double minute 2 (Mdm2) in the adult mouse heart induces CM cell cycle re-entry through the downregulation of Cdk inhibitors p21 and p27 along with Cyclin E-mediated Cdk2 activation. These results can be explained by the ability of p53 and Mdm2 to regulate the G1-Cyclin-CDK complex, which is responsible for maintaining the quiescence of adult mammalian CMs [149]. p53 is also involved in activating autophagy and inducing CM cell death after ischaemic injury. Its inhibition with long noncoding RNAs (lncRNAs) significantly attenuates the activation of cardiac autophagic flux in the ischaemic heart, resulting in a limitation of myocardial infarct size in an adult murine model [150].

The role of p53 in cardiac regeneration can vary significantly depending on the developmental stage under consideration. Variations in the proliferative capacity of CMs and cellular response to cardiac injury can primarily explain the differences in the p53 role between the neonatal and adult periods.

2.3.3. TBX20

During embryonic development, TBX20 plays a pivotal role in driving CMproliferation by binding to the promoters of key genes such as ccna2, cdc6, Mycn, and ErbB2, thereby triggering their transcription [151,152]. Recent research focused on understanding TBX20’s contribution to cardiac regeneration, in particular its ability to maintain foetal characteristics in the adult heart and thereby stimulate CM proliferation. These investigations highlighted the critical role of TBX20 in cardiac regeneration, suggesting that its overexpression could serve as a promising therapeutic approach for cardiac repair after MI. Chakraborty et al. examined TBX20 functions throughout embryonic and foetal development in a murine model overexpressing this transcription factor [153]. They demonstrated its capacity to drive CM proliferation with the consequent thickening of the ventricle wall. The foetal hearts of transgenic mice overexpressing TBX20 exhibited a significant increase in the number of pHH3+ and MF20+ (myosin 4) CMs compared to the control group [153]. The sustained proliferation of CMs induced by TBX20 was linked to increased N-myc1 transcription, a downstream regulator of the TBX20 pathway, alongside the upregulation of connexin-40 and -43 expression throughout the ventricular wall, without altering the signal propagation [153]. In a subsequent investigation, the same research group confirmed an augmented population of small, mononucleated, proliferating CMs and ventricular wall thickening also in the adult transgenic mice model overexpressing TBX20. Moreover, the authors showed that TBX20 preserved foetal characteristics through the activation of Bmp2/pSmad1/5/8 and PI3K/AKT/GSK3β/β-catenin pathways in adult hearts [154]. The impact of TBX20 was further elucidated in a murine model of MI, where its overexpression in adult hearts enabled CM proliferation without compromising cardiac morphology or function [155]. TBX20 overexpression post-MI led to reduced scar size, diminished cardiac hypertrophy, and increased capillary density in mouse hearts, thus preserving cardiac function and enhancing survival of mice four weeks post-MI [155]. Isolated CMs from TBX20-overexpressing hearts showed, besides a recovery of foetal contractile proteins such as MHC and ssTnI (slow skeletal troponin I), a heightened proliferative activity characterised by increased expression of Cyclin A2 and D1 along with reduced expression of cell cycle inhibitors such as p16, p21, and p27 [155]. Genetic silencing of TBX20 in embryonic murine CMs underscored its importance, resulting in a noticeable reduction in the volume of developing ventricular and atrial chambers along with a decrease in CM proliferation due to arrest in the G1-S phase. TBX20 directly represses the genetic programs of cardiac progenitors in adult CMs [156].

In vitro experiments, human-induced CMs (hiCMs) revealed that TBX20, in conjunction with the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133), induced cardiac reprogramming and activated genes associated with CM contractility and maturation. Notably, CMs induced with MGT+TBX20 exhibited increased beating frequency and enhanced energy metabolism [157].

3.1.1. EPCs

Independent of their origin, it is widely agreed that EPCs display endothelial features and possess a natural affinity for vascular tissues, highlighting the inherent regenerative potential of the vascular system [181]. These EPCs will infiltrate the site of injury, where they can either differentiate into mature ECs or regulate pre-existing ECs via paracrine/juxtacrine signalling [182]. Mobilisation, migration, proliferation, and differentiation of EPCs orchestrate neovascularisation and re-endothelialization by regulating cytokines, receptors, adhesion molecules, proteases, and cellular signalling pathways [183].

The earliest evidence of circulating EPCs dates back to 1997, when researchers isolated CD34+ and Flk-1+ cells from peripheral blood capable of differentiating into ECs [184]. In the years following the study by Asahara and colleagues, numerous types of circulating EPCs have been identified. However, it was only in the last decade that researchers revealed the unique capability of CD34+ EPCs to promote neovasculogenesis during cardiac regeneration [185,186]. Circulating EPCs are recruited from the bone marrow to the ischaemic heart and can differentiate into ECs or SMCs, facilitating the development of new blood vessels [187]. The mobilisation of EPCs from the bone marrow occurs in response to elevated levels of circulating VEGF, which is highly released in response to hypoxia and inflammatory processes in MI [188,189]. In particular, it has been demonstrated that one of the mechanisms by which circulating EPCs induce angiogenesis is mediated through VEGFR-2, and EPCs with elevated VEGFR-2 levels exhibit a greater angiogenic capacity indeed [190]. Moreover, this study highlighted that EPC proliferation, angiogenesis-related gene expression levels, and vessel density were different among donors. Moreover, high concentrations of stem cell-derived factor-1 (SDF-1), also known as CXC motif chemokine 12, both in circulation and within the damaged tissue, contribute to the mobilisation of EPCs from the bone marrow. In particular, SDF-1 favours EPC recruitment to the site of injury, where its levels have been observed to be elevated from the 3rd day post-tissue damage onwards [191]. The onset of tissue regeneration aligns with a reduction in inflammation and an increase in anti-inflammatory cytokines, such as IL-10, which in turn promotes vascular network formation by significantly enhancing the concentration of EPCs at the wound site. Furthermore, an in vitro investigation performed by Short and co-workers revealed that EPC culture treated with an IL-10-conditioned medium effectively promotes endothelial sprouting and network formation [192]. In a murine model, the regenerative effect of exosomes isolated from wild-type EPCs and IL-10 KO EPCs was examined by injecting them into the myocardium immediately post-MI. Exosomes from wild-type EPCs improved neovascularisation and left ventricular cardiac function and significantly reduced CM apoptosis and the extension of infarct size, while EPC exosomes from the IL-10 KO group produced opposite effects [193]. In recent years, among the different types of exosomes derived from EPCs, those containing miRNA-126 play a key role in CM protection, neovascularisation, and tissue repair. Their release is conditioned by various external stimuli, such as hypoxia or inflammatory conditions, which also occur after MI [194].

The study conducted by Huang et al. underscored the significance of predominantly anti-inflammatory signalling in facilitating EPC-mediated myocardial revascularisation. These authors engineered exosomes from adipose tissue-derived stem cells (ADSCs) to overexpress Sirtuin 1 (SIRT1) and treated circulating EPCs isolated from MI patients, thus enhancing their cell migratory capacity. Moreover, in vivo administration of these exosomes in a mouse MI model led to increased angiogenesis within the infarcted tissue and improved cardiac function 28 days post-MI [195].

Two distinct subsets of EPCs have emerged for their roles in post-MI neovascularisation: myeloid angiogenic cells (MACs) and endothelial colony-forming cells (ECFCs). MACs are characterised by a relatively low proliferation rate, elevated CD34 levels, and diminished expression of stem cell-related markers CD133 and c-Kit [196]. Conversely, ECFCs exhibit robust proliferation, clonogenic potential, and substantial postnatal vascularization capacity also in the infarcted cardiac tissue [197]. Moreover, besides being CD34+, ECFCs express late endothelial differentiation genes including CD105, CD146, CD31, and VE-cadherin [198,199]. However, ECFCs have demonstrated diminished vascular regenerative capabilities in ischaemic settings, while enhanced angiogenesis has been observed when ECFCs were utilised in post-MI regenerative therapy in conjunction with MSCs [200].

In contrast, another recent study involving a limited participant cohort asserts that circulating EPCs responsible for cardiac neovascularisation did not originate from bone marrow but from the vessel wall [201]. The study enrolled male patients who received allogeneic bone marrow transplants from female donors, allowing the researchers to distinguish EPCs based on their genotype. The authors observed that ECs derived from the vascular wall were capable of proliferating in culture and forming a monolayer of CD31+ cells with an XY genotype, thus excluding that these cells originated from the bone marrow [201]. In line with these authors, there is also evidence suggesting the presence of resident EPCs in the heart. By using an EC-specific multispectral lineage-tracing mouse model (Pdgfb-iCreERT2-R26R-Brainbow2.1) combined with single-cell RNA sequencing, Li and colleagues demonstrated that a specific subset of resident EPCs actively contributes to the formation of new blood vessels in the adult mouse heart 7 days after MI [202]. Additionally, this study identified plasmalemma vesicle-associated protein (Plvap) as a novel endothelial-specific marker of cardiac neovasculogenesis, which was also identified in cardiac samples from patients with ischaemic heart disease [202]. The repair of damaged vessels by EPCs can occur through direct differentiation into ECs and integration into the injured vessels. However, an emerging feature of EPCs is their ability to act on cells and blood vessels through paracrine activity. EPCs release soluble pro-angiogenic factors, which directly or indirectly support the angiogenesis process by influencing migration, differentiation, mesenchymal to endothelial transition (MET), and integration of the differentiated cells as part of the intrinsic repair process [203]. EPCs also release EVs with a specific payload of pro-angiogenic miRNAs that regulate the molecular signalling pathways involved in neovascularisation [204].

The positive effects of an EPC-EV-based treatment were demonstrated in a rat model of MI [205]. The study highlighted the effectiveness of using a shear-thinning hydrogel to enhance EV delivery and retention in the ischaemic region. LAD occlusion was performed in rats, and then EPCs and EPC-EVs were injected into the border zone of the infarcted area. The results showed that both EPC and EPC-EV injections significantly improved vascular structure, cell proliferation, and haemodynamic function compared to the control group [205].

3.1.2. CPCs

There is compelling evidence suggesting that c-Kit+ CPCs possess the ability to augment angiogenesis and attenuate myocardial fibrosis in rat models post-MI [206].

Since complete cardiac vascular regeneration cannot be achieved only through the direct differentiation of progenitor cells into mature cells, it has been demonstrated that these cells exert their regenerative potential through a paracrine mechanism mediated by EVs present in their secretome [51]. CPC-EVs are taken up by ECs, thus promoting angiogenesis through the delivery of both associated and co-isolated proteins [207]. These vesicles contain proangiogenic factors, including VEGF and FGF, as well as matrix proteins and integrins. CPC-EVs interact with ECs via endocytosis or membrane fusion, activating key signalling pathways such as PI3K/Akt and MAPK that enhance EC proliferation, migration, and survival. This results in improved vascular regeneration after myocardial injury by creating a favourable microenvironment for tissue repair [207]. Recent research has revealed that adult CPCs, however, exhibit a reduced regenerative capacity compared to neonatal CPCs [208]. For instance, adult CPCs lack the expression of YAP1, which is crucial for activating pro-regenerative and pro-survival pathways. This limitation was highlighted in a recent work where 72-h exposure of adult CPC clones to EVs derived from neonatal CPCs fosters the AKT signalling pathway, reflecting the well-established interplay between AKT and YAP1 in promoting cell proliferation and survival [208]. Vrijsen and colleagues pinpointed the role of CD147, also known as Extracellular Matrix Metalloproteinase Inducer (EMMPRIN), present in EVs of adult human CPC in mediating in vivo vascular regeneration through the stimulation of angiogenesis, as evidenced by the increase in CD31+ and alfa-SMA+ cells along with their co-localisation, typical of mature vessels [209]. In another study, human CPC-Exo were enriched in miR-210 and miR-132, which play important roles in angiogenesis and vascular remodelling. In a preclinical rat model of MI, CPC-Exo were able to promote angiogenesis and inhibit CM apoptosis, along with the improvement of cardiac function, through miR-132 and miR-210 [210]. miR-132 stimulates EC proliferation and migration by activating pro-angiogenic signalling pathways, thereby contributing to the formation of new blood vessels. On the other hand, miR-210 enhances cell survival under hypoxic conditions and regulates the cellular response to ischaemic stress while also promoting EC differentiation and angiogenesis [210]. Furthermore, CPC-Exo can also be engineered to deliver key molecules aimed at promoting cardiac neovascularization. An increase in capillary density has been observed in the infarcted area of the myocardium in mice following the administration of exosomes isolated from CPCs and bioengineered with a pro-angiogenic miR-322, delivered through the caudal vein [211].

3.1.3. MSCs

One of the key mechanisms contributing to the beneficial outcomes of MSCs in cardiac vascular regeneration, akin to EPCs, is their paracrine activity [40,212]. The secretome of MSCs is believed to play a central role in both the initial phases of response to MI-induced damage, facilitating and sustaining the inflammatory process, and in the subsequent stages, aimed at attenuating inflammation and fostering an anti-inflammatory milieu (for an in-depth review, refer to Wagner et al., [213]). MSCs secrete a range of bioactive molecules, such as VEGF, HGF, and IGF1, and are shown to promote angiogenesis and cell survival, as well as reduce inflammation and inhibit apoptosis [214].

Among the recent evidence, Klopsch et al. demonstrated that CD45− CD44+ DDR2+ MSCs participate in the early stages of cardiac regeneration after MI [215]. Indeed, following the ischaemic event, both oxygen tension and paracrine activity of infiltrated polarised macrophages can regulate the MSC niche, promoting the expression of CD44 and DDR2 antigens. This study successfully identified tissue-specific ischaemia-responsive cardiac MSCs that possibly could take part in the regeneration of the ischaemic heart [215]. Subsequently, the same group demonstrated that co-culturing HUVECs with MSCs (CD45−CD44+DDR2+) significantly enhanced angiogenesis, leading to a roughly fourfold increase in the formation of branched and junctional networks, while unstimulated HUVECs exhibited only minimal angiogenic activity [216].

Moreover, MMPs are implicated in tissue revascularisation, regulating capillary diameter, and possibly stabilising nascent vessels after MI, highlighting the potential role of MMPs in angiogenesis after MI [80]. Other authors have demonstrated in a murine model of MI that the promotion of EC-induced microvascular regeneration is mediated by the MSC-derived SDF-1-enriched exosome, which stimulated MMP-2 and -9 protein levels [217]. Co-culturing human MSCs with ECs has led to the secretion of pro-angiogenic growth factors and matrix remodelling through the activation of MMP-2. MSC-EC crosstalk resulted in the formation of a rich vascular network with intercapillary distances similar to those observed in native myocardium [218]. In fact, it has been seen that the angiogenic potential of ECs could be enhanced by EMMPRIN, MM9, and VEGF contained in MSC-derived EVs, the key enzyme involved in promoting ECM remodelling and consequently supporting tissue regeneration in the myocardium [209]. The in vitro EMMPRIN-knockdown MSC-derived exosomes significantly reduced EC migration capacity with consequent almost complete inhibition of vessel formation, thus confirming the crucial role of EMMPRIN. These studies strengthen that MSCs can promote angiogenesis by modulating protease expression [209]. Endogenous c-Kit+ MSCs play a crucial role in enhancing capillary neovascularisation and improving cardiac function in mice with MI lesions. Specifically, Set domain-containing protein 4 (Setd4) epigenetically modulates the quiescence of c-Kit+ cells. In Setd4 knockout models, activated c-Kit+ cells have been observed, leading to improved cardiac function in mice with MI through capillary neovascularisation [180].

Transcription factors are pivotal molecules that govern gene expression, and, in the adult heart, GATA4 has a central role in progenitor cell signalling in cardiac repair and regeneration [219]. The analysis of mouse cardiac tissue at two, three, and four days after MI revealed that intravenous transplantation of GATA-4-overexpressing MSCs significantly increased the number of blood vessels and c-Kit+ cardiac cells [220]. The role of GATA-4 in promoting MSC-mediated cardiac vascular regeneration appears to be closely associated with the release of EVs [221]. In particular, exosomes derived from GATA-4-modified MSCs contain significant levels of the Let-7 miRNA family, which is known for its pro-angiogenic properties. Let-7 miRNAs regulate the expression of genes critical for EC proliferation, migration, and survival processes that are essential for new blood vessel formation [221]. The pro-angiogenic role of GATA-4-overexpressing MSC-derived exosomes has been further confirmed in a mouse MI model. A significant enhancement in vessel density was observed in the hearts of mice treated with GATA-4 MSC-derived exosomes 48 h after MI induction, compared to those treated with control MSC exosomes or the untreated MI groups [222].

4.3.1. Wnt/β-Catenin

In addition to the widely studied classical pathways, Wnt/β-catenin plays a role in both embryonic development and adult tissue homeostasis regeneration. This pathway can be classified into two main types: canonical, known as Wnt/β-catenin, and non-canonical. In the canonical way, β-catenin translocates into the nucleus, leading to the activation of TCF/LEF transcription factors, primarily regulating cell proliferation. The non-canonical way operates independently of β-catenin and regulates cell polarity and migration [251]. The Wnt/β-catenin pathway is an important regulator of cardiac development and growth, and its activity in healthy adult hearts is low, essential for maintaining normal heart function. Acute activation of the Wnt/β-catenin pathway is thought to play a cardioprotective role after infarction through the upregulation of pro-survival genes and metabolic reprogramming [252]. Through the use of a reporter system in murine and rat models, the precise localisation of Wnt pathway activation following cardiac injury was identified specifically within the border zone of the infarcted region [253]. It has been reported that this pathway is gradually activated 24 h after MI and subsequently downregulated three weeks post-MI [254]. Recently, it has also been shown the direct activity of β-catenin in inducing mature CM proliferation after MI [255]. Reactivation of Wnt/β-catenin signalling enhances CM proliferation by increasing the nuclear import of β-catenin, which in turn activates the transcription of key target genes (such as Wnt1, Wnt2, Wnt6, Axin2, and Lef1). This process induces CM cytokinesis in both the infarct and border zones after MI, ultimately reducing infarct size and improving cardiac function [255]. Despite these findings demonstrating that the reactivation of the Wnt/β-catenin pathway leads to cardiac regeneration, as far back as 2008, indications emphasised that the silencing of β-catenin in KO mice resulted in increased differentiation of resident cardiac progenitor cells, suggesting that the downregulation of β-catenin was directly involved in endogenous cardiac regeneration [256]. These results were also recently confirmed by Hodgkinson et al., which demonstrated that inhibition of β-catenin induces the differentiation of c-Kit+ resident cells into CMs in a murine model of MI [257]. Therefore, the role of the Wnt/β-catenin pathway in inducing CM proliferation remains controversial.

Wnt signalling is triggered after MI injury and is implicated in cardiac regeneration, but it is also related to inflammation and angiogenesis [258]. Mice treated with the GSK-3β inhibitor (responsible for β-catenin degradation) displayed a robust activation of the Wnt/β-catenin signalling pathway with enhanced expression of VEGF. This overexpression led to a significant increase in CD31+ positive cells 7 and 14 days after MI, resulting in augmented capillary density and tissue healing [259]. High β-catenin expression could promote EMT, which might be an important source of angiogenesis and muscle fibre cells and play a significant role in tissue repair [260]. In effect, Wnt family protein levels (Wnt2, Wnt4, Wnt10b, and Wnt11) are increased 5 days following MI in murine actin+ perivascular SMCs and subepicardial ECs, apparently through EMT mechanism [254]. In addition, genetically enhanced Wnt10b expression in transgenic mouse CMs improved arterial formation and attenuated fibrosis in cardiac tissue after injury [261].

4.3.2. JAK-STAT

The JAK-STAT signalling pathway plays a crucial role in maintaining cardiac homeostasis. Considerable attention has been directed towards understanding the biological and pathophysiological significance of the JAK-STAT signal in cardiac myocytes. Collecting evidence suggests that this signalling pathway is activated in cardiac myocytes by various cytokines, including IL-6, granulocyte colony-stimulating factor (G-CSF), leptin, and erythropoietin, among others. Activation of the JAK-STAT pathway in the heart promotes CM survival and enhances myocardial angiogenesis, thereby protecting the myocardium from pathological stresses [262]. There are four members in the JAK family (JAK1, JAK2, JAK3, and TYK2) and seven members in STAT (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6) [263]. The first studies regarding the role of the JAK/STAT pathway in the cardiac tissue have primarily focused on the investigation of STAT1 and STAT3 [264]. For instance, the supernatant from necrotic primary CMs activates the JAK1-STAT1 pathway and promotes the nuclear translocation of c-Fos and NFκB p65 when simulating the MI microenvironment, reducing CM apoptosis under hypoxic conditions. However, silencing STAT1 diminishes the apoptosis induced by the necrotic supernatant [265]. Conversely to STAT1’s pro-apoptotic role, STAT3 demonstrates an anti-apoptotic effect [266]. These findings suggest a close association between the JAK/STAT pathway and the apoptotic response following MI, with STAT1 and STAT3 exhibiting opposite effects.

The involvement of the JAK/STAT pathway in neovascularisation during MI was investigated with STAT3, which plays a central role in the formation of blood vessels. Its activity has been investigated in STAT3 cardiac-specific KO mice that displayed decreased myocardial capillary density and increased susceptibility to injury [267]. Similarly, the administration of hyaluronic acid oligosaccharides into MI mice promoted neovessel formation and improved cardiac function as assessed 28 days post-intervention. These beneficial effects were associated with the activation of chemokines expression implicated in macrophage polarisation and the stimulation of MAPK and JAK/STAT signalling pathways for myocardial function reconstruction, as revealed by transcriptomic analyses [268]. These studies suggest that the JAK/STAT signalling pathway has a role in cardiac remodelling after cardiac infarction by controlling angiogenesis [269].