Two decades of heart regeneration research: cardiomyocyte proliferation and beyond

Abstract

Interest in vertebrate cardiac regeneration has exploded over the past two decades since the discovery that adult zebrafish are capable of complete heart regeneration, contrasting the limited regenerative potential typically observed in adult mammalian hearts. Undercovering the mechanisms that both support and limit cardiac regeneration across the animal kingdom may provide unique insights in how we may unlock this capacity in adult humans. In this review, we discuss key discoveries in the heart regeneration field over the last twenty years. Initially, seminal findings revealed that pre-existing cardiomyocytes are the major source of regenerated cardiac muscle, drawing interest into the intrinsic mechanisms regulating cardiomyocyte proliferation. Moreover, recent studies have identified the importance of intercellular interactions and physiological adaptations, which highlight the vast complexity of the cardiac regenerative process. Finally, we compare strategies that have been tested to increase the regenerative capacity of the adult mammalian heart.

Graphical Abstract

INTRODUCTION

In 2002, Kenneth Poss and his colleagues published a landmark paper showing that the adult zebrafish heart is fully capable of replenishing lost myocardial tissues without scars within two months after injury (Poss et al., 2002). Although previous researchers provided evidence of significant cardiomyocyte proliferation in adult newts, frogs, and lizards after myocardial injury (Mehdipour et al., 2023), this was the first compelling evidence of complete scarless heart regeneration in any adult vertebrate species. Being genetically tractable, zebrafish have since become a popular model to understand the cellular and molecular mechanisms underlying cardiac regeneration and repair.

There has been a desperate need to understand why our hearts can’t regenerate. It has been well established that the adult mammalian heart does not possess the ability to regenerate itself after injury. Therefore, after a heart attack, humans are often left with a permanent loss of heart muscle cells - cardiomyocytes - while a fibrotic scar eventually forms in place of the lost contractile tissue. This results in deterioration of cardiac contractility and a high risk of life-threatening cardiac arrhythmias. A recent heart disease and stroke statistical analysis reported that 36% of men and 47% of women (45 or older) will die within five years after a heart attack (Virani et al., 2020).

The discovery of the phenomena of heart regeneration in zebrafish has unequivocally kick-started the field of heart regenerative research. Another seminal report that neonatal mouse hearts can fully regenerate further propelled the field (Porrello et al., 2011). It has drawn more investigators to explore organ regeneration in a variety of species across the animal kingdom and at different developmental stages. Such an “evo devo” approach has uncovered conserved mechanisms of heart regeneration and may yield clues to unlock the ability to regenerate the mammalian heart. While early research efforts established the importance of pre-existing cardiomyocytes as a major source for newly regenerated tissue, recent studies revealed key intercellular and physiological control mechanisms required for natural heart regeneration to occur. In this review, we highlight the role of pre-existing cardiomyocytes as the source for newly regenerated cardiac muscle, as well as cell-to-cell interactions and physiological adaptations to oxygen and increased metabolism that regulate cardiomyocyte proliferation and heart regenerative capacity. Furthermore, we compare strategies that have been tested to increase the regenerative capacity of the adult mammalian heart.

PRE-EXISTING CARDIOMYOCYTES ARE THE MAJOR SOURCE OF NEW CARDIOMYOCYTES IN NATURAL HEART REGENERATION

Heart regeneration in fish and amphibians

The adult mammalian heart has limited regenerative capabilities and is generally considered a post-mitotic organ (Jewhurst & McLaughlin, 2015; Kikuchi & Poss, 2012; Rumyantsev, 1977). However, other vertebrates in the animal kingdom are able to robustly regenerate their hearts from birth to adulthood (Kikuchi & Poss, 2012; Vivien et al., 2016). For example, adult salamanders and newts are able to regenerate the myocardium after injury and, unlike their mammalian counterparts, their cardiomyocytes possess the ability to readily enter the cell cycle and successfully divide (Becker et al., 1974; Bettencourt-Dias et al., 2003; Oberpriller & Oberpriller, 1974). Similarly, the Western clawed frog Xenopus tropicalis possesses the ability to regenerate their hearts 30 days after apical resection (Liao et al., 2017). While early studies in amphibians demonstrated cardiomyocyte cell cycle re-entry in response to cardiac injury, the significance of pre-existing cardiomyocytes as the source of newly regenerated cardiac muscle was most notably validated and supported by seminal work using the zebrafish model.

Zebrafish (Danio rerio) are teleosts well known for their exceptional abilities to regenerate various tissues and organs (Gemberling et al., 2013; Jewhurst & McLaughlin, 2015) and are also genetically tractable and amenable to experimental manipulation. Like amphibians, zebrafish possess a remarkable capacity to regenerate their cardiac tissue (Poss et al., 2002). Where do newly generated cardiomyocytes come from? An early study exploited the differential folding rate and protein stability between green fluorescent proteins (GFP) and red fluorescent proteins (RFP) to track cardiomyocyte turnover and generation in zebrafish heart regeneration (Lepilina et al., 2006). This study provided evidence to support that new cardiomyocytes originate from cardiac progenitor cells (Lepilina et al., 2006). However, a technical flaw was found in this study (Kikuchi et al., 2010) and a more rigorous genetic lineage tracing approach was taken by two independent groups, yielding more compelling results that demonstrate pre-existing cardiomyocytes as a major source of regenerated cardiac muscle cells in regenerating zebrafish hearts (Jopling et al., 2010; Kikuchi et al., 2010). These studies established the requirement for the proliferation of pre-existing cardiomyocytes as a fundamental mechanism permitting natural heart regeneration.

Within the last decade, heart regenerative capacity in other fish species has also been evaluated. In a study done by Grivas et al., goldfish (Carassius auratus) with heart tissue damage induced by cauterization were observed to completely regenerate the lost tissue by 30 days post-injury (Grivas et al., 2014). The cardiac regenerative process in goldfish involves the inflammatory response, collagen deposition and neovascularization - all processes shown to occur during zebrafish heart regeneration (Frangogiannis, 2008; Grivas et al., 2014; Tahara et al., 2016). Interestingly, the teleost medaka (Oryzias latipes) exhibits an impaired response to apical resection compared to zebrafish (Ito et al., 2014). Much like the injury response observed in adult mammalian hearts, cardiac injury in medaka resolves with extensive fibrotic scarring without robust cardiomyocyte proliferation (Ito et al., 2014).

As an alternative to apical resection, which cleanly removes part of the heart and does not physiologically model cardiomyocyte death after myocardial infarction, cryoinjury has been developed as a more medically-relevant cardiac injury approach (Chablais et al., 2011; González-Rosa et al., 2011; Schnabel et al., 2011). In this model, a cryoprobe is used to induce necrotic heart lesions, which enables the study of mechanisms involved in clearance of damaged cardiac tissue. Using this approach, a comparative study between zebrafish and medaka cardiac injury responses noted transcriptional differences revealing that the innate immune response in medaka post-cardiac injury is delayed. Further characterization demonstrated blunted macrophage recruitment to the site of injury (Lai et al., 2017). The significance of macrophage recruitment in promoting cardiac regeneration was then further validated by impairing macrophage recruitment in zebrafish, which resulted in reduced regenerative capacity and decreased cardiomyocyte proliferation (Lai et al., 2017). These studies suggest a possible role for the innate immune response in reactivating cardiomyocyte proliferation in response to injury.

The Mexican tetra (Astyanax mexicanus) is another teleost species that exists in two distinct morphotypes: surface- and cave-dwelling (Potts et al., 2021; Stockdale et al., 2018). The two morphotypes are reported to exhibit different degrees of repair after apical resection. Cave-dwelling fish develop a fibrotic scar whereas 50% of surface fish are observed to regenerate their hearts by 64 days post-amputation (Stockdale et al., 2018). Interestingly, significant differences in cardiomyocyte cycling in these distinct populations after injury was not observed (Stockdale et al., 2018). Genomic analyses between these two morphotypes of Astyanax mexicanus further illustrate how environmental factors play a role in the differential gene expression and its effect on metabolism and physiology (Krishnan et al., 2020; Peuß et al., 2020). Therefore, future work in Astyanax mexicanus may provide unique insights as a comparative model to further elucidate how environmental conditions influence cardiac regenerative outcomes (Potts et al., 2021).

Heart regeneration in mammals

The seminal discovery that newborn mice possess a transient but robust capacity for heart regeneration ignited a surge of interest in this experimental model to understand the mechanisms that limit mammalian heart regeneration. Neonatal mice at postnatal day 1 can completely regenerate ventricular tissue after apical resection, while surgeries performed at postnatal day 7 resolve with fibrosis and scarring, similar to that seen in adult mice (Porrello et al., 2011). In response to apical resection, regenerative hearts exhibit widespread dedifferentiation and proliferation throughout the heart, with increased cardiomyocyte cycling at the site of injury (Porrello et al., 2011). It is possible that cardiomyocyte regeneration in this model arises through both compensatory chamber-wide cardiomyocyte proliferation as well as cardiomyocyte cell division at the injury border. It was further demonstrated that heart regeneration similarly occurs in neonates in response to left anterior descending artery (LAD) ligations, a more physiologically relevant injury model for myocardial infarction (Haubner et al., 2012). The observed loss of heart regenerative capacity in neonatal mice after birth suggests that physiological changes occurring during this developmental window inhibit mammalian heart regenerative potential. A similar developmental transition may also be observed in humans (Bergmann et al., 2009).

While the relevance of pre-existing cardiomyocytes as the source of newly regenerated cardiomyocytes had been established in fish and amphibian models of heart regeneration (Becker et al., 1974; Bettencourt-Dias et al., 2003; Jopling et al., 2010; Kikuchi et al., 2010), the progenitors of newly regenerated mammalian cardiomyocytes has historically been a highly debated topic. Progenitor cells from the epicardium have been proposed in the role of cardiomyocyte renewal in adult hearts (Smart et al., 2011; B. Zhou et al., 2008). The origin of de novo cardiomyocytes from nearby bone marrow stem cells has also been implicated (Orlic et al., 2001). Others have suggested the role of stem cell progenitor cells, such as the c-kit+ cardiac stem cells (Bearzi et al., 2007; Beltrami et al., 2003). However, the contribution of c-kit+ cardiac stem cells in cardiomyocyte renewal has been controversial (Bearzi et al., 2007; Bergmann et al., 2009; Elhelaly et al., 2019; Jesty et al., 2012; Khan & Koch, 2016; Kikuchi et al., 2010; Porrello et al., 2013; Senyo et al., 2013; Sultana et al., 2015; van Berlo et al., 2014).

In 2003, Beltrami and colleagues reported a set of cardiac stem cells (CSCs) expressing the stem cell marker c-kit as cardiomyocyte progenitor cells in 20–25 month-old rat hearts in vivo (Beltrami et al., 2003). Furthermore, these c-kit positive CSCs in humans were reported to be clonogenic and possess the ability to differentiate into a variety of cell types including cardiomyocytes in vitro and in vivo (Bearzi et al., 2007). Jesty et al. observed that EGFP-tagged c-kit positive cells could give rise to cardiomyocytes after cryoinjury, but this contribution was most noticeable in neonates with little to no contribution to new cardiomyocytes in adult mice (Jesty et al., 2012). However, more rigorous lineage tracing studies in neonatal mice labeling pre-existing cardiomyocytes through tamoxifen inducible Cre-lox recombination demonstrated that pre-existing cardiomyocytes were the primary source of new cardiomyocytes after injury after LAD ligation (Porrello et al., 2013). Additionally, using a combined stable isotope labeling and multi-isotope imaging mass spectrometry approach to study mammalian cardiomyocyte turnover, Senyo et al. further provided evidence that pre-existing cardiomyocytes are the primary source of new cardiomyocytes in mice (Senyo et al., 2013). Importantly, lineage tracing experiments performed by tamoxifen-inducible labeling of c-kit-positive cells revealed minimal contribution of this cell population to new cardiomyocytes after LAD ligation in mice of a variety of ages (van Berlo et al., 2014). It was then reported that c-kit positive cells do not express myocyte markers after embryonic day 13.5 when using transgenic reporter mice and may actually represent cardiac endothelial cells (Sultana et al., 2015). Therefore, at present, the field is in agreement that c-kit cells are irrelevant as the source of newly regenerated cardiac muscle cells in mammals.

CARDIOMYOCYTE POLYPLOIDIZATION: A BARRIER TO HEART REGENERATION

The cardiomyocytes of adult zebrafish are predominantly mononucleated and diploid (González-Rosa et al., 2018; Wills et al., 2008). Adult newts and salamanders, which retain cardiac regenerative potential, are also observed to have high mononucleated cardiomyocyte frequencies (Becker et al., 1974; Bettencourt-Dias et al., 2003). Additionally, the majority of cardiomyocytes in the newborn mouse heart are initially mononucleated and diploid but then undergo cell cycle arrest and binucleation within the first two weeks of postnatal development (Soonpaa et al., 1996). Therefore, elevated diploid cardiomyocyte frequencies are positively correlated with enhanced cardiac regenerative potential (Figure 1).

Figure 1. Cardiomyocyte polyploidization correlates with the loss of cardiomyocyte proliferative and heart regenerative capacity.

Adult zebrafish (A) and neonatal mice (B) retain a high frequency diploid cardiomyocytes that are capable of proliferation, correlates with their robust abilities to regenerate their hearts. Adult mice (C) retain much lower diploid cardiomyocyte frequency. Rather, their hearts contain mostly terminally differentiated polyploid binucleated cardiomyocytes that lack the ability to divide. This correlates with decreased heart regenerative potential.

In contrast, mammalian hearts generally have higher frequencies of polyploid cardiomyocytes (Kikuchi & Poss, 2012; Laflamme & Murry, 2011). The non-regenerating hearts of adult mice are composed of matured mostly polyploid cardiomyocytes that are terminally differentiated and considered postmitotic (Derks et al., 2020; Jewhurst & McLaughlin, 2015; Kikuchi & Poss, 2012; Rumyantsev, 1977; Soonpaa et al., 1996). Polyploidization in rodents occurs within the first two weeks after birth. A peak in cardiomyocyte DNA synthesis is observed around postnatal day 4 accompanied by a gradual rise in binucleated cardiomyocytes, which is largely complete by around postnatal day 10 (F. Li et al., 1996; Soonpaa et al., 1996). Interestingly, in sheep (Ovis aries), cardiomyocyte polyploidization occurs during gestational development (between 65–76 days after conception) and postnatal hearts have reduced proliferative capabilities with observed differences in inflammation and cardiomyocyte proliferation in response to myocardial damage (Herdrich et al., 2010; Jonker et al., 2015; Zgheib et al., 2014). Pigs can also regenerate their hearts within 2 to 3 days after birth and an increase in binucleated and multinucleated cardiomyocyte abundance is observed starting at 15 days after birth (Velayutham et al., 2020; Ye et al., 2018; Zhu et al., 2018). Finally, the human heart has been reported to contain a high frequency of mononucleated CMs, but most are tetraploid and exhibit impaired ability to re-enter and complete the cell cycle (Bergmann et al., 2009, 2015; Kikuchi & Poss, 2012; Laflamme & Murry, 2011; VYa et al., 1994).

The timing of polyploidization described above would suggest that cardiomyocyte endowment, the final number of cardiomyocytes in the heart, is set during the perinatal period. However, it was proposed that a thyroid hormone-dependent burst of cardiomyocyte proliferation after postnatal day 14 - a time when cardiomyocyte polyploidization is already complete - contributes to overall cardiomyocyte endowment (Naqvi et al., 2014). This finding has since been contested by other groups (Alkass et al., 2015; Soonpaa et al., 2015), highlighting the need for improved methods to quantify total cardiomyocyte numbers in the heart.

At the cellular level, cardiomyocyte polyploidization is a result of defective karyokinesis and/or cytokinesis, steps in the cell cycle required for nuclear and cytoplasmic division, respectively (Derks et al., 2020; Engel et al., 2006; Hesse et al., 2018; Leone et al., 2018; Van de Peer et al., 2017; Zebrowski & Engel, 2013). Aberrations and alterations to cytoskeletal components like the mitotic microtubule organizing center as well as formation of a defective cleavage furrow have been suggested to result in cytokinesis failure and give rise to binucleated cardiomyocytes (Leone et al., 2018). Midbody positioning during cytokinesis may also be a determinant of cardiomyocyte binucleation (Hesse et al., 2018). Cardiomyocytes undergoing binucleation exhibit asymmetrical midbody positioning which often resulted in daughter nuclei being in closer proximity to one another, in vitro and in vivo. A larger chromosomal bridge may hinder anaphase and eventual cytokinetic abscission (Engel et al., 2006; Hesse et al., 2018; Hu et al., 2012). Recent work also suggests that homotypic fusion may too be a source of binucleated cardiomyocytes in mice (Ali et al., 2020). However, in zebrafish, the occurrence of cellular fusion does not necessarily give rise to binucleated cells (Kirillova et al., 2021; Sawamiphak et al., 2017). Tetraploidy in human cardiomyocytes arises due to endomitosis, DNA replication in the absence of nuclear division (Edgar et al., 2014; Meckert et al., 2005).

These observations across multiple mammalian species have led to the hypothesis that cardiomyocyte polyploidization may be indicative of cell cycle withdrawal and loss of cardiac regenerative potential (Kikuchi & Poss, 2012; Kirillova et al., 2021; Patterson et al., 2017; Walsh et al., 2010). Supporting this hypothesis, induction of cardiomyocyte polyploidization in zebrafish hearts inhibits cardiac regeneration (González-Rosa et al., 2018) and binucleated cardiomyocytes in adult newts exhibit an impaired ability to properly divide (Matz et al., 1998). Additionally, the frequency of diploid cardiomyocytes in the heart depends on allelic variance (Gan et al., 2019, 2020; Patterson et al., 2017), and performing cardiac injuries in mice strains with higher natural frequencies of diploid cardiomyocytes positively correlates with enhanced regeneration and functional recovery (Patterson et al., 2017). A recent phylogenetic analysis profiling diploid cardiomyocyte frequency recently revealed mammalian species with considerably elevated diploid cardiomyocyte abundances (Hirose et al., 2019). It remains to be tested if these mammals possess enhanced cardiac regenerative potential. For example, the African spiny mouse is quickly gaining popularity as a mammalian model of tissue regeneration (Gaire et al., 2021) and possesses an approximately 2-fold higher frequency in diploid cardiomyocytes relative to that observed in adult mice. Recent reports observe enhanced cardiac repair and recovery in this model (Koopmans et al., 2021; Peng et al., 2021; Qi et al., 2021), but whether or not this involves increased cardiomyocyte cell cycle reactivation and proliferation is not clear.

Contrasting the hypothesis above, some evidence suggests that polyploid cardiomyocytes may retain at least some ability to divide. Postnatal binucleated rat cardiomyocytes stimulated with either serum or a combination of fibroblast growth factor 1 and p38 MAP kinase inhibitor have been observed to re-enter the cell cycle and proliferate through formation of pseudo bi-polar spindles in vitro (Leone & Engel, 2019). Adult binucleated mouse cardiomyocytes have also been reported to dedifferentiate, enter the cell cycle, and divide, when co-cultured with neonatal cardiomyocytes (W. E. Wang et al., 2017). Furthermore, other mammalian cell types are naturally polyploid yet retain the ability to proliferate (Pandit et al., 2013). Polyploid hepatocytes are able to complete their cell divisions through ploidy reversal (Duncan, 2013; M.-J. Wang et al., 2017) and a recent study monitoring the dynamics of cardiomyocyte ploidy through mouse postnatal development suggests that ploidy reversion in cardiomyocytes may be possible as well (Swift et al., 2023). Finally, it is likely that cardiomyocyte polyploidization is not the only barrier limiting cardiac regeneration. Though the hearts of medaka consist primarily of diploid cardiomyocytes (Chowdhury et al., 2022; Ito et al., 2014), heart regeneration still fails in these animals. This suggests that retaining high diploid cardiomyocyte frequency may not be sufficient to support a robust cardiac regenerative response. It is likely that extrinsic signals, possibly from recruited or resident macrophages, are additionally required to stimulate cardiomyocyte proliferation (Chowdhury et al., 2022; Ito et al., 2014; Lai et al., 2017).

CELL-TO-CELL INTERACTIONS REQUIRED FOR HEART REGENERATION

Lineage tracing experiments in both zebrafish and neonatal mice have provided a key insight: pre-existing cardiomyocytes are the main source of newly regenerated cardiac cardiac muscle. This discovery stresses the importance of understanding the intrinsic mechanisms that regulate cardiomyocyte cell cycle control, proliferation, and polyploidization. However, the lack of a robust cardiac regeneration response in other species that retain high frequencies of diploid cardiomyocytes (e.g., medaka), suggest that extrinsic regulators are also required for heart regeneration to occur. In this subsection, we provide an overview of recent work exploring the contributions of other cell types in the heart that may influence the cardiac regenerative response (Figure 2).

Figure 2. Cardiac regeneration requires intercellular communication between other cell types in the heart.

Though the proliferation of pre-existing cardiomyocytes to regenerate new cardiac muscle is required for cardiac regeneration, interactions between immune cells (A), the epicardium (B), and endothelium (C) are necessary to support the reparative process.

Immune cells

As discussed previously, the inability of medaka to regenerate its heart is, at least in part, due to a delayed innate immune response to injury (Ito et al., 2014; Lai et al., 2017). Specifically, the inefficient recruitment of macrophages at the site of injury results in an overall decrease in neutrophil clearance, cardiomyocyte proliferation, neovascularization, and scar resolution The importance of macrophage recruitment was further validated in zebrafish using clodronate liposome-mediated ablation, which resulted in a compromised reparative response. Furthermore, the recruitment of macrophages with Toll-like receptor agonist poly I:C enhanced heart regeneration in medaka (Lai et al., 2017). In adult axolotls, the depletion of macrophages with clodronate liposomes impairs cardiac regeneration after cryoinjury resulting in fibrosis and scarring (Godwin et al., 2013). Interestingly, activation of cardiomyocyte cell cycle markers after injury was still observed despite impaired macrophage recruitment. This may suggest that macrophages do not regulate cardiomyocyte cell cycle entry but rather may assist in cell cycle progression either directly or indirectly through interactions with fibroblasts or other components of the extracellular matrix (Godwin et al., 2013). Macrophage depletion with clodronate liposomes in neonatal mice also causes impaired heart regeneration resulting in fibrotic scars, decreased cardiac function, and decreased angiogenesis (Aurora et al., 2014).

It is important to note that postnatal day 1 and 14 mice exhibit unique age-dependent immune responses to myocardial infarction (Aurora et al., 2014). Activation of the Smad pathway has been shown to regulate phagocytosis and inflammation after myocardial infarction (B. Chen et al., 2019). Smad3 activation in macrophages expressing TGF-β enhanced reparative processes such as initiating phagocytosis of dead cells, regulating anti-inflammatory responses, and activating angiogenesis and fibrosis in the event of myocardial infarction protecting (B. Chen et al., 2019). Macrophages have also been categorized into two subsets. The M1 subset mediates the pro-inflammatory response while M2 populations mediates reparative processes (Nahrendorf et al., 2007). Lavine et al. has also demonstrated that there may be an embryonic-derived lineage of macrophages that promote cardiomyocyte proliferation and coronary angiogenesis while producing little inflammatory response while the more inflammatory monocyte-derived macrophage population exhibits enhanced immune cell recruitment with decreased cardiomyocyte proliferation and angiogenesis (Lavine et al., 2014).

Though evidence supports that macrophage recruitment to damaged heart tissue is generally beneficial for cardiac regeneration, the role of neutrophils in this process is less clear. Whether or not neutrophils promote or inhibit cardiac regeneration may be dependent on the timing and regulation of the inflammatory responses (Frangogiannis, 2008; K. Sun et al., 2021; Timmers et al., 2012). For example, the use of anti-inflammatory glucocorticoids in zebrafish resulted in impaired regenerative capacity of the heart that resulted in excessive collagen deposition after ventricular resection (W.-C. Huang et al., 2013). This provides evidence that the initial inflammatory response may be essential to enable the cardiac regenerative process. In addition, neutrophils have been observed to promote repair of the heart following infarction by helping to polarize macrophages towards the M2 reparative phenotype (Horckmans et al., 2017). Adverse effects associated with the neutrophils and a prolonged inflammatory response have additionally been described (Chia et al., 2009; Guasti et al., 2011; K. Sun et al., 2021; Vinten-Johansen, 2004).

Similar to macrophages and neutrophils, it has been suggested that T-cells may also help to secrete paracrine factors that induce cardiomyocyte proliferation (Dolejsi et al., 2022; J. Li et al., 2019). In fact, there may be age-dependent effects on T-cell mediation of the reparative processes in the heart. For example, adult T-cells producing IFN-5 transplanted into neonatal mice impaired cardiac regeneration after myocardial infarction, suggesting a possible trade-off with T-cell development and maturation with the regenerative capacity of the heart (Dolejsi et al., 2022). This may be correlated to the observed increase in T-helper 1 and 2 cells in patients with severe coronary artery disease and acute myocardial infarction (C. Li et al., 2019). However, it may also be possible that regulation of T helper cells can improve wound healing after myocardial infarction as observed with the knockout of the Dectin-2 receptor resulting in improved wound healing in mice (Yan et al., 2017).

Regulatory T cells (Tregs) also have been reported to aid in the regenerative processes of the heart after injury. Specific depletion of regulatory T cells in mice increased polarization of macrophages to the M1 pro-inflammatory phenotype and deterioration in cardiac function (Weirather et al., 2014). Activation of Tregs improves the healing processes with increased macrophage differentiation into the more reparative and anti-inflammatory M2 phenotype improving survivability of mice after LAD ligation. Additionally, Tregs may have the ability to modulate cardiomyocyte cytokine production and cell cycling. Tang et al. demonstrated Tregs can inhibit the production of inflammatory cytokines from cardiomyocytes effectively protecting the cardiomyocytes from inflammation and improving cardiac function (T.-T. Tang et al., 2011). Tregs have also been observed to decrease apoptosis of cardiomyocytes in a CD39-dependent manner while deficiency in CD39 Tregs resulted in an increase in apoptosis (Fung et al., 2020; Xia et al., 2015). In zebrafish, Tregs have been characterized to secrete growth factors stimulating cardiomyocytes to proliferate (Hui et al., 2017). Similarly, Treg-specific depletion in neonatal mice results in impaired regeneration of the heart after injury and is suggested to signal cardiomyocytes to proliferate through paracrine factors such as CCL24, GAS6 and AREG (J. Li et al., 2019).

Currently, the role of cardiac lymphatic vessels with respect to heart regeneration is currently not well characterized, but it has been suggested that these vessels play an important role in cell signaling and transport of macromolecules (Feng et al., 2021). Cardiac lymphatics have been reported to respond to cardiac injury and neo-lymphangiogenesis post-injury may aid in antigen clearance and the resolution of inflammation (Klotz et al., 2015; Vieira et al., 2018). In one study, lymphatic vasculature has also been observed to be fundamental for heart regeneration in zebrafish and mice (Gancz et al., 2019). However, the requirement of lymphatic vessels during heart regeneration isn’t yet substantiated. For example, zebrafish hearts can seemingly mount a complete regenerative response after cryoinjury in mutants that lack lymphatic vasculature, with exception of a small subset that may have enhanced cytokine signaling and inflammation after cryoinjury (Vivien et al., 2019). The cardiac lymphatic vessels may not be required for but may help modulate cardiac injury responses and facilitate the reparative processes of the heart (Feng et al., 2021; Harrison et al., 2019).

Epicardial cells

The epicardium is the layer of mesothelial tissue that envelops the heart, which has been observed to respond to cardiac injury. (Gemberling et al., 2015; González-Rosa et al., 2012; Lepilina et al., 2006; Tahara et al., 2016; Wills et al., 2008). The epicardium has been shown to regulate cardiomyocyte proliferation and heart repair (J. Cao & Poss, 2018; Sucov et al., 2009). It was proposed that epicardial progenitor cells may also contribute to regenerated cardiomyocytes (B. Zhou et al., 2008). A recent study using a Cre-lox reporter system in transgenic salamanders (Pleurodeles waltl) has suggested that epicardial cells can undergo an epicardial cell-to-cardiomyocyte conversion after cryoinjury (Eroglu et al., 2022). However, the fate mapping technique used in this study does not employ genetically encoded reporters to exclusively label epicardial cells. Rather, epicardial cells are targeted by injection of a cell-permeant Cre recombinase into the pericardium to activate a ubiquitously-expressed reporter. Therefore, it remains formally possible that non-epicardial cells may too be labeled with this approach at low frequency, especially in combination with a cardiac injury that would disrupt tissue integrity. The presence of epicardial derived CM progenitor cells has also been debated in the past with lineage tracing studies showing that epicardial cells in zebrafish can differentiate into other cell types, such as myofibroblasts and perivascular cells with no evidence of myocardial cell types (González-Rosa et al., 2012; Kikuchi, Gupta, et al., 2011). The epicardium may also assist in neutrophil recruitment and response after cardiac injury through C/EBP signaling (G. N. Huang et al., 2012). Regardless, after cryoinjury and ventricular resection of the zebrafish heart, the epicardium clearly responds to the injury and helps support cardiac regenerative processes including cardiomyocyte proliferation (González-Rosa et al., 2011; Lepilina et al., 2006; Schnabel et al., 2011).

It is possible that the epicardium serves as a cellular scaffold and an inducer of cardiomyocyte proliferation (González-Rosa et al., 2012; Lepilina et al., 2006; Marín-Juez et al., 2019). For example, the extracellular factor Neuregulin-1 (NRG1) from epicardial perivascular cells has been described as an activator of heart regeneration in zebrafish by acting as a mitogen for zebrafish cardiomyocytes (Gemberling et al., 2015). In addition, there is evidence that paracrine factors secreted from cells derived from the epicardium following cardiac injury promote angiogenesis (B. Zhou et al., 2011). In response to cardiac tissue damage, retinoic acid is another secreted factor of the epicardium that has been shown to stimulate cardiomyocyte proliferation (Kikuchi, Holdway, et al., 2011). Additionally, murine epicardium secreted extracellular vesicles and exosomes have also been reported to promote cardiomyocyte cell cycle activity and proliferation (del Campo et al., 2021).

With specific labeling of endothelial and epicardial cells, it has been observed that endothelial and epicardial cells exhibit a robust injury response after genetic ablation of cardiomyocytes using transgenic Cre-lox mice and diptheria toxin A (DTA) induced by 4-hydroxytamoxifen injection (J. Wang et al., 2011). This suggests that the epicardium has a role in heart regeneration and can facilitate the reparative processes of the heart post injury. Genetic ablation of the epicardium in transgenic zebrafish using nitroreductase to induce the conversion of the non-toxic metronidazole to a cytotoxin was also shown to significantly reduce the reparative processes of the heart after apical resection, suggesting that the epicardium has an important role in heart regeneration (J. Wang et al., 2015). In contrast, a recent report using a selective chemoptogenetic cardiomyocyte ablation strategy in zebrafish resulted in minimal activation of the epicardium and endocardium prior to successful heart regeneration (Missinato et al., 2021). However, epicardial and endocardium marker activation was only evaluated at 3 days post-chemoptogenetic ablation in this study. Therefore, it remains possible that activation of these cellular populations may still occur at later time points during the regenerative process.

The epicardium has also been described to consist of a heterogeneous population of cells distinguishable from one another by expression signatures (J. Cao et al., 2016). Through single cell transcriptome sequencing, it was discovered that all subsets of epicardial cells express the scaffolding protein caveolin 1 (cav1) and its expression facilitates the reparative processes of the heart after apical resection in zebrafish (J. Cao et al., 2016). It was also observed that epicardial cells expressing follistatin-like 1 (Fstl1) are the only epicardial cells capable of promoting regeneration acting as a cardiomyogenic factor after apical resection in mice (Y. Cao & Cao, 2018; Wei et al., 2015). On the other hand, another subset of epicardial cells expressing hapn1 has been reported to play a role in promoting cardiomyocyte expansion and its depletion inhibits regeneration in zebrafish (J. Sun et al., 2022). Though it is not entirely certain whether or not epicardial cells are required for heart regeneration and how the subsets of epicardial cells play a role in heart regeneration, these data further support the idea that there is crosstalk between cardiac tissue and the epicardium during the regenerative process of the heart.

Endothelial cells

Similar to the epicardium, endocardial endothelium also exhibits crosstalk with myocardial tissue after injury to heart tissue. Much like the epicardium, the endothelial cells of the heart have been observed to produce and secrete retinoic acid in response to heart injury which is seen to promote cardiomyocyte proliferation in zebrafish (Kikuchi, Holdway, et al., 2011). Crosstalk between the epicardium and endothelial cells may also indirectly mediate the reparative processes of the heart. For example, epicardial and endothelial cells seem to work together to develop a cellular scaffold that eventually enhances superficial and intra-ventricular coronary artery formation (Marín-Juez et al., 2019). These coronary vessels are suspected to effectively form scaffolds for cardiomyocytes during regeneration and revascularization is essential for cardiac repair (Marín-Juez et al., 2016, 2019). Research with single-cell RNA sequencing of neonatal mice hearts revealed that the expression of the ligand of many endothelial cells, R-spondin 1 (Rspo1), is restricted (Z. Wang et al., 2020). Rspo1 is an activator of the canonical Wnt pathway and suggested to be associated with angiogenesis (Binnerts et al., 2007; Z. Wang et al., 2020). It was observed that adenoviral mediated overexpression of Rspo1 resulted in an enhanced angiogenesis without significant effects on the cardiomyocyte themselves (Z. Wang et al., 2020).

Endothelial cells secrete angiocrines which can regulate cardiomyocyte compaction and proliferation (Rhee et al., 2021). The findings that secreted factors from endothelial cells can regulate cardiomyocyte proliferation and compaction supports the idea that these cells help with organization and maturation of cardiomyocytes in the heart. Endothelial-cardiomyocyte crosstalk through paracrine signaling may protect the myocardial tissue from injury such as pharmacological damage (Leucker et al., 2011). Interestingly, rat and mice cardiomyocytes overexpressing vascular endothelial growth factor B (VEGF-B) have also been observed to promote angiogenesis and enhance endothelial proliferation (Räsänen et al., 2021). These findings may suggest that though the epicardium and endocardium has been observed to enhance and aid in the reparative process of the heart, the myocardium may interact with these tissues to regulate cardiac regeneration as well.

METABOLIC ADAPTATIONS

Beyond our appreciation for the intercellular mechanisms that may be required to support vertebrate cardiac regeneration, recent findings provide evidence that physiological adaptations to support the increasing metabolic demands of an animal may negatively impact its ability to regenerate its heart (Figure 3).

Figure 3. The shift towards fatty acid oxidation to support increasing metabolic demand may inversely correlate with cardiac regenerative potential.

(A) After birth, the hearts of neonatal mice shift from utilizing glycolysis to fatty acid oxidation. (B) Animal metabolic needs increase both during mammalian postnatal development and during the evolution of endothermic mammals. (C) The shift to fatty acid metabolism and the increase in animal metabolic needs inversely correlate with the loss of heart regenerative capacity.

Lipid metabolism

One suggested barrier to mammalian heart regeneration is the maturation process of adult mammalian cardiomyocytes, which may be driven by a hypoxic-to-hyperoxic environmental transition after birth (Puente et al., 2014). The development of endothermy and the observed shift from glycolysis to fatty acid oxidation have also been hypothesized as “evolutionary trade-offs” that drive cardiomyocyte cell cycle arrest in adult mammals (Ross et al., 2022; Sakaguchi et al., 2020). The transition to the hyperoxic postnatal environment has been proposed to increase mitochondrial reactive oxygen species production, which activates the DNA damage response and inhibits cardiomyocyte cell cycle progression (Puente et al., 2014). Furthermore, the utilization of fatty acids in the mitochondria has been shown to increase chromatin oxidative stress in cardiomyocytes (Menendez-Montes et al., 2021). Inhibition of the DNA damage response increased the postnatal window of cardiomyocyte proliferation proliferation (Puente et al., 2014). This transition seems to be correlated with a metabolic shift in cardiomyocytes from glycolysis to fatty acid oxidation (Cardoso et al., 2020). Inhibition of pyruvate dehydrogenase kinase 4 (PDK4) enhances pyruvate dehydrogenase abundance and activity and shifts cardiomyocytes toward glycolytic metabolism. This resulted in increased cardiomyocyte proliferation and reduction in DNA damage response markers (Cardoso et al., 2020). Zebrafish embryos treated with long chain acyl carnitines, fatty acid intermediates, exhibited impairment to cardiac function and mitochondrial function (Park et al., 2021). On the other hand, the stimulation of glycolysis in zebrafish has also been observed to promote CM proliferation after injury (Fukuda et al., 2020). Furthermore, malonate has also been observed to induce CM proliferation and enhance cardiac regeneration through succinate dehydrogenase inhibition in turn decreasing production of reactive oxygen species (Bae et al., 2021). These findings indicate that metabolic substrate utilization, mitochondrial reactive oxygen species, the DNA damage response associated with the cardiac metabolic shift to fatty acid oxidation may play a key role in restricting mammalian heart regenerative capacity.

In contrast to the above work performed in the mouse model, the timing of the hypoxic-to-hyperoxic environmental shift in sheep does not correlate with the loss of cardiomyocyte proliferation and regenerative (Lock et al., 2019). Sheep cardiomyocytes can enter the cell cycle after injury during a proliferative window between 65–76 days of gestation but lose their proliferative potential after birth (Herdrich et al., 2010; Zgheib et al., 2014). Therefore, sheep seem to lose the ability to regenerate their hearts before birth, which could be due to the precocial nature of this animal where fetal maturation occurs rapidly within the intrauterine environment (Lock et al., 2019). Consistent with this, differences in growth and maturation have been observed between fetal sheep and rodent cardiomyocytes (Drake et al., 2022; Jonker et al., 2018).

Interestingly, in 3-month-old mice, it has also been suggested that hypoxic environments can induce cardiomyocyte proliferation. One study observed a decrease in ROS and oxidative DNA damage in hypoxic mice after 2 weeks of being in a hypoxic environment (7% O₂) (Nakada et al., 2017). With chronic hypoxia, 3-month-old mice also were observed to exhibit a higher number of cardiomyocytes with a higher percentage of mononucleated cardiomyocytes and an increase to heart weight in comparison to normoxic mice with similar effects observed with mild hypoxia (10% O₂) (Nakada et al., 2017). Similarly, one study using an EdU mini pump implanted in 3-month-old mice under hypoxic conditions also observed an increase in cardiomyocyte proliferation according to EdU incorporation further supporting the aforementioned study done by Nakada and colleagues (Johnson et al., 2023)

Acquisition of Endothermy

The acquisition of endothermy has also been proposed to be an evolutionary adaptation that is associated with the loss of regenerative capacity of the heart (Amram et al., 2021; Hirose et al., 2019; Ross et al., 2022). In support of this, there may be an inverse correlation with the development of endothermy and the regenerative capacity of the heart (Hirose et al., 2019). Many animals that possess robust heart regenerative capacity, such as fish, amphibians, and reptiles, also happen to be ectothermic (Hirose et al., 2019). Thyroid hormones have been hypothesized to drive the shift towards the development of endothermy. Consistent with this hypothesis, inhibition of thyroid hormone production during postnatal development decreases thermogenesis (Little & Seebacher, 2014; Payumo et al., 2021). Furthermore, it has been observed that exposure to thyroid hormones inhibits cardiomyocyte proliferation in fetal sheep and zebrafish in vitro while deficiency in thyroid hormone signaling in adult mice increases regenerative potential of the heart (Chattergoon et al., 2006; Hirose et al., 2019).

CARDIOMYOCYTE CELL-CYCLE REACTIVATION: CHALLENGES AND ALTERNATIVE STRATEGIES

While much progress has been made to understand the mechanisms that support or limit heart regeneration, how to best apply this knowledge to therapeutically reactivate a cardiac regenerative response in adult mammals is not clear. In this subsection, we compare existing approaches to reactivate the regenerative capacity of the mammalian heart (Figure 4).

Figure 4. Strategies to replace lost cardiomyocytes after injury.

(A) Reactivation of cell cycle activity and proliferation of mature adult cardiomyocytes. (B) Heterocellular reprogramming and transdifferentiation of other cell types in the heart (e.g., cardiac fibroblasts) towards a cardiomyocyte-like fate. (C) Derivation of cardiomyocytes from patient-derived induced pluripotent stems (iPSCs).

Reactivation of cell cycle in adult cardiomyocytes

Genetic and epigenetic repression of embryonic cardiomyocyte gene expression during development and maturation may also contribute to the loss of cardiac regenerative capacity (Cui et al., 2018; Kraus, 2022). For example, the transcription factor Meis1 inhibits cardiomyocyte cycling during cardiogenesis by activating the expression of cyclin dependent kinase (CDK) inhibitors p15, p16, and p21 potentially contributing to cell cycle arrest in cardiomyocytes in mice (Mahmoud et al., 2013). The targeted expression of cyclin D2 and deregulation of D-cyclin-mediated Rb/E2f-signaling has also been suggested to affect cardiomyocyte proliferative potential (Hille et al., 2016; Pasumarthi et al., 2005). E2F-dependent genes have been reported to become silenced in adult mouse cardiomyocytes and results in epigenetic changes in heterochromatin associated with retinoblastoma (Rb) family and p130 recruiting heterochromatin protein 1 (HP1) proteins (Sdek et al., 2011). Depletion of both Rb and p130 increased proliferation in adult cardiomyocytes. Similarly, inhibition of HP1-γ resulted in an increase to genes associated with cardiomyocyte proliferation supporting that epigenetic changes have a role in cell cycle arrest (Sdek et al., 2011). Histone acetylation and methylation also regulates DNA synthesis and differentiation in cardiomyocytes (Kou et al., 2010). .

Key efforts in the field have focused on reactivating the cell cycle in terminally differentiated adult cardiomyocytes. For example, the glycogen synthase kinase-3 (GSK3) inhibitor 6-bromoindirubin-3’-oxime (BIO) has been observed to induce proliferation in both neonatal and adult rat CMs (Tseng et al., 2006). The use of GSK3 inhibitors to induce cell cycle entry in adult cardiomyocytes in vitro shows that it may be possible that terminally differentiated cardiomyocytes possess developmental plasticity and can be reprogrammed to divide. However, cardiomyocyte-specific deletion of GSK3 in vivo results in mitotic catastrophe through dysregulation of cell cycle checkpoints and severe DNA damage (J. Zhou et al., 2016).

Modulation of the Hippo signaling pathway has been another route researchers have investigated to improve cardiomyocyte proliferation and function (Heallen et al., 2011, 2013; Leach et al., 2017; Xin et al., 2013). Interestingly, the Hippo effector YAP has been characterized to play a role in crosstalk with canonical Wnt signaling by negatively regulating β-catenin by nuclear interactions and inhibiting CM proliferation (Heallen et al., 2011). This is generally supported by other work that shows deficiency or downregulation of Hippo signaling results in an increase in CM proliferation. There is additional evidence that Hippo signaling is upregulated in cases of human heart failure and deficiency in Hippo signaling can increase CM proliferation and may enhance the reparative process of the heart (Leach et al., 2017).

Similarly, stimulation of adult cardiomyocyte proliferation and cardiac regeneration has been observed with the modulation of NRG1 and its tyrosine kinase receptor ERBB2 (D’Uva et al., 2015). The study done by D’Uva et al., suggests that induction of ERBB2 can dedifferentiate adult cardiomyocytes and increase cardiomyocyte proliferation (D’Uva et al., 2015). These results further suggest that adult cardiomyocytes may be able to re-enter and potentially complete the cell cycle under permissive systemic conditions. Supporting the idea that cell cycle progression and completion could be regulated in adult cardiomyocytes, overexpression of cell-cycle regulators associated with stable cytokinesis cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 was observed to induce cell-cycle re-entry, successful division, and proliferation (Mohamed et al., 2018). Other pathways and environmental factors have also been considered to play a role in regulating adult cardiomyocyte proliferation ranging from Notch signaling to regulation of the extracellular matrix (Gong et al., 2021).

Heterocellular reprogramming

The first known case of heterocellular reprogramming was done by Qian et al. using cardiac fibroblasts (Qian et al., 2012). In the study, retroviral administration of Gata4, Mef2x, and Tbx5 (GMT) was done by direct intramyocardial injection in murine cardiac fibroblasts. With the retroviral-mediated gene transfer of GMT into cardiac non-myocytes, transdifferentiation efficiency was estimated to be about 10–15% for both in vivo and in vitro induced cardiomyocytes (iCMs). iCMs in vivo were reported to resemble endogenous cardiomyocytes more than their in vitro counterparts which could indicate the microenvironment may enhance the transdifferentiation of cardiac non-myocytes to iCMs (Qian et al., 2012).

Since the development of GMT-derived iCMs, many have tried to further optimize the transdifferentiation protocol and conditions. The addition of other transcription factors associated with cardiomyocyte function and maturation has been a common approach to further optimize the transdifferentiation process (Addis et al., 2013; Singh et al., 2021; Song et al., 2012; Y. Tang et al., 2022). For example, the Hippo pathway effector transcriptional enhance associate domain 1 (Tead1) was observed to further increase transdifferentiation efficiency and can replace Tbx5 in the GMT cocktail (Singh et al., 2021). Though a general increase in cardiomyocyte associated structures and functionality was observed with cultures transdifferentiation by Gata4, Mef2c, and Tead1 (GMTd) compared to GMT treated cultures, human iCM cultures exhibited to increase slightly (~4% to ~8% cTnT positive cells, ~2% to ~6% ɑ-actinin positive cells at 2 weeks, and ~3% to ~12% ɑ-actinin positive cells at 4 weeks). Similar results are generally seen with the incorporation of other factors along with the GMT cocktail, such as Hand2, Nkx2.5, and TBX20, but there is general trend of these iCMs still being a small percentage of cells present (Addis et al., 2013; Muraoka et al., 2014; Y. Tang et al., 2022).

In addition to the use of lenti-viral transduction to overexpress specific transcription factors to aid in the transdifferentiation of cardiac non-myocytes into iCMs, some have investigated the potential of utilizing short hairpin RNA to silence specific factors to enhance the generation of iCMs (Patel et al., 2018; Pinnamaneni et al., 2022). Downregulation of p63 further enhances the generation efficiency and functionality of iCMs and epigenetically regulates iPSC reprogramming (Patel et al., 2018; Pinnamaneni et al., 2022). Others have reportedly enhanced the generation of iCMs with the addition of small-molecule cocktails with the GMT cocktail supplemented with other transcription factors and entirely with small-molecule cocktails alone (N. Cao et al., 2016; Singh et al., 2020). Optimization of the iCM culturing conditions by altering the microenvironments has also been done to yield higher transdifferentiation efficiency as well where the use of 3D microenvironments increase reprogramming efficiency (Jin et al., 2022.).

Though all of these instances represent progress within the field, as mentioned in Qian et al., the transdifferentiation of cardiac fibroblast behavior may also have unforeseen effects on heart function as fibroblasts and other cardiac non-myocytes contribute to the effects of scar formation and heart function (Qian et al., 2012).

iPS cell-derived cardiomyocytes

The discovery and development of induced pluripotent stem cells has been instrumental in developing the field of stem cell research as it stands today. With the discovery of the Yamanaka factors, Oct3/4, Sox2, c-Myc, and Klf4, somatic cells have been able to be reprogrammed into a state of pluripotency; referred to as induced pluripotent stem cells (iPSCs) today (Takahashi & Yamanaka, 2006). With the relative success of this technique, many have taken advantage of this discovery and investigated how to differentiate iPSCs into a variety of cell types such as CMs, developing multiple potential models for basic research and regenerative medicine (Yu et al., 2007; Zhang et al., 2009).

Though iPSCs can serve as useful models due to availability and protocol reproducibility, iPSC-derived cardiomyocytes are generally considered immature compared to those in adult mammals (Patterson et al., 2012; van den Berg et al., 2015). In particular, iPSC-derived cardiomyocytes (iPSC-CMs) are immature with respect to their cell morphology, transcriptome, electrophysiology, metabolic activity, and contractile force production (Gherghiceanu et al., 2011; Karbassi et al., 2020; van den Berg et al., 2015; Wickramasinghe et al., 2022). As a result, different conditions have been tested over the years to determine possible approaches to generate iPSC-CMs more comparable to mature cardiomyocytes including culturing long-term, stimulating cultures with electrical currents, utilizing mechanical stress, and the use of biochemical induction (Wu et al., 2021). Some have attempted to further optimize known differentiation protocols by investigating the effects of pathways associated with cardiomyocyte maturation such as the peroxisome-proliferator-associated receptor (PPAR) pathway due to its relation to fatty acid oxidation pathways (Wickramasinghe et al., 2022). With the PPAR pathway, the PPAR-delta (PPARd) isoform along with long-chain fatty acid supplementation was observed to increase binucleation of iPSC-CMs, enhance myofibril organization and contractility while also increasing mitochondrial content and activity along with gene regulatory networks associated with FAO (Wickramasinghe et al., 2022). This data suggests there are signaling pathways that may signal for maturation based on activation and or the downregulation of important pathways associated with maturing CMs such as the metabolic switch from glycolysis to fatty acid oxidation. These results also further support previous findings that fatty acid supplementation can enhance iPSC-CM maturation (Horikoshi et al., 2019; Yang et al., 2019). Though PPARd activation seems to enhance iPSC-CM maturation, much of the increases observed are still within several percentile of the control and differentiation efficiency may be seen as another problem being observed in less than 60% of the cells in culture.

Optimization and adjustments to culturing microenvironments have also been done. For example, with the critical role of the extracellular matrix, combinational polymer matrices were used to determine the potential effect on iPSC-CM maturation (Chun et al., 2015). Utilizing a variety of copolymer matrices, iPSC-CMs cultured with 4% polyethylene glycol (PEG) - 95% poly-ε-caprolacton (PCL) matrices ultimately exhibited increased expression of cardiac troponin I, integrin alpha-7, and increased levels of contractility along with mitochondrial activity (Chun et al., 2015). These results suggest that the microenvironment may play a vital role in iPSC-CM differentiation to which the substrate type can inadvertently affect mechanosensory transduction pathways that may play a role with intermediate filament regulation in iPS-CMs.

With the variety of developments made over the years further unraveling the mysteries of iPSC-CM maturation, it is also important to appreciate the heterogeneity of cardiomyocytes in the heart. In fact, a variety of cardiomyocyte subtype-specific differentiation protocols exist (Grassam-Rowe et al., 2020; Lyra-Leite et al., 2022). Additionally, it is worth mentioning that with PPARd activation, iPSC-CM transcriptomes were still observed to vary from one another depending on the culture formats from 2D monolayers to 3D engineered heart tissues; yet another reminder of the complexities and caveats associated with different model systems (Wickramasinghe et al., 2022).

CONCLUSION

The work and progress in the field of heart regeneration over the last two decades has been immense. From the discovery (and debate) of the origin of newly regenerated cardiomyocytes to the appreciation of intercellular interactions and the significance of metabolic adaptations, it is clear that cardiac regeneration is complex. The proliferation of pre-existing cardiomyocytes alone is likely not sufficient to support the cardiac regenerative response. Therefore, efforts in determining the extrinsic mechanisms that permit cardiomyocytes to divide will likely be a major area of research in future. How loss of cardiac regenerative potential in mammals may be linked to the development of endothermy and the subsequent metabolic shift from glycolysis to fatty acid metabolism may help us further understand the evolutionary framework for how heart regenerative capacity is lost in mammals.

However, with the plethora of knowledge obtained over the years regarding the mechanisms that both support and limit heart regeneration, there is still much left to be desired from a therapeutic standpoint. Some have tested modulation of different signaling pathways to initiate cell cycle re-entry and stimulate cardiomyocyte proliferation. Others have approached the issue with attempts at derivation of cardiomyocytes from sources other than terminally differentiated cardiomyocytes. For example, iPSCs have been a popular model and potential source of new cells in many different fields. Heterocellular reprogramming is another approach where surrounding fibroblasts are modified with the modulation of different molecular effectors to induce cellular reprogramming to generate new cardiomyocytes in vivo. Unfortunately, as of the time of writing, these two general approaches to generate new cardiomyocytes are still burdened with the difficulties of generating developmentally mature cardiomyocytes at high enough efficiencies to be therapeutically relevant. The use of surrounding cardiac fibroblasts to generate new cardiomyocytes in vivo could be a viable therapeutic system, but like many other techniques, it is worth noting that this approach may alter the composition of the heart tissue itself. All existing strategies have their own limitations and caveats. Regardless, the work in the field over the past two decades has given hope that there is a possibility to enhance the regenerative potential of the adult human heart.