In heart development, an initial pool of cardiomyocytes is derived by differentiation from early mesodermal progenitors. Once this early process has completed, in every later context in the embryo and throughout postnatal life, essentially all new cardiomyocytes are generated by proliferation of preexisting cardiomyocytes rather than by differentiation from a non-cardiomyocyte progenitor source (Sereti et al., 2018; Li et al., 2019). In mammals, the rate of embryonic cardiomyocyte proliferation is high as growth of the heart keeps pace with overall embryo growth, but then tapers down by the end of gestation (Soonpaa et al., 1996). In normal heart biology, cardiomyocyte proliferation is minimal for the remainder of life; the heart grows considerably in size postnatally, but this is by the hypertrophic enlargement of existing cardiomyocytes rather than by generation of new cardiomyocytes. Nonetheless, in the context of an insufficient number of cardiomyocytes, the mammalian heart at late fetal and very early neonatal stages retains the ability to activate robust cardiomyocyte proliferation and to restore normal cardiomyocyte number. Although there have been some negative observations (Andersen et al., 2016), reactivation of cardiomyocyte proliferation to achieve efficient restoration of heart morphology has been observed in mouse genetic models that cause a non-lethal late-embryonic deficiency of cardiomyocytes (Drenckhahn et al., 2008; Vincentz et al., 2017), and in surgical or congenital injury models that cause a neonatal loss of ventricular myocardium in rats (Robledo, 1956), mice (Sadek et al., 2014), and swine (Mahmoud and Porrello, 2018), and likely also occurs in human (Haubner et al., 2016). In mice, the plasticity to reactivate efficient cardiomyocyte proliferation is mostly lost by postnatal day P7 (Porrello et al., 2011; Alkass et al., 2015), which is understood to mean that most ventricular cardiomyocytes have become postmitotic by this time. Consequently, heart injury in P7 pups or at any later time through adulthood is followed by insufficient cardiomyocyte proliferation and insufficient replacement of lost myocardium, i.e., by a failure to fully regenerate. In animals and humans, failure of regeneration after ventricular injury leads to a permanent diminishment of contractile function with possible further progression to heart failure.

A number of differences distinguish ventricular cardiomyocytes at the beginning and end of the first postnatal week, but one that may determine (rather than correlate with) proliferative ability is their total DNA content (Gan et al., 2020). In mice and perhaps most mammals, essentially all fetal and early neonatal cardiomyocytes have one diploid nucleus (as is the case for most cells). During the first postnatal week (in mice), cardiomyocytes undergo a last round of DNA replication followed by cell cycle arrest either before or after karyokinesis but without cytokinesis, resulting in mononuclear polyploid or multinuclear cardiomyocytes (Soonpaa et al., 1996; Alkass et al., 2015). Typically, only a few percent of ventricular cardiomyocytes thereafter and through the remainder of life are mononuclear diploid, which correlates with the very low level of cellular and functional regeneration usually seen after adult heart injury. Adult zebrafish retain a very high percentage of mononuclear diploid cardiomyocytes throughout adulthood (Patterson et al., 2017; González-Rosa et al., 2018), and heart regeneration is robust in this species (Foglia and Poss, 2016). Thus, from a comparison of multiple species and life stages, there is a precise correlation between the abundance of mononuclear diploid cardiomyocytes and competence to support cardiomyocyte proliferation for embryonic heart growth or post-injury heart regeneration. Recent experimental results in both mouse and zebrafish explicitly demonstrated that polyploid cardiomyocytes are compromised in proliferation and regeneration, and thereby confirmed the relationship between mononuclear diploid cardiomyocytes and adult heart regeneration (Patterson et al., 2017; González-Rosa et al., 2018). Of particular relevance to this paper, we showed (Patterson et al., 2017) that some inbred mouse strains have a much higher percentage of adult ventricular mononuclear diploid cardiomyocytes (e.g., 10% in strain A/J, compared to 2% in C57BL/6J), and that such strains have a correspondingly higher degree of both cellular and functional regeneration after adult heart injury.

Organ regeneration following injury or tissue loss is generally thought to recapitulate at least some features of embryonic growth, including the redeployment of signaling pathways that had been active earlier in fetal development. The early embryonic heart consists of cardiomyocytes surrounding an inner layer of endocardium (the endothelial lining of the heart). Around embryonic day E10 in mouse, the epicardium, the noncontractile mesothelium of the heart, migrates from a nearby source onto the outer heart surface. In our past work, we showed that the epicardium is the source of secreted mitogenic factors that promote cardiomyocyte proliferation (Chen et al., 2002), and then identified IGF2 (insulin-like growth factor 2) as the major epicardial mitogen responsible for this activity (Li et al., 2011; Shen et al., 2015). Igf2 is expressed in the embryonic epicardium and endocardium (but not myocardium). Mouse embryos deficient in Igf2 expression in all heart mesoderm or specifically in the epicardium lineage were substantially compromised in cardiomyocyte proliferation during the E10-E13 period, which was morphologically manifest in a hypoplastic ventricle. Notably for the present study, conditional Igf2 mutation in the endocardium and endothelium had no phenotypic consequence in the embryonic heart (Shen et al., 2015). Embryos lacking the two signaling-competent IGF2 receptors INSR (insulin receptor) and IGF1R (insulin-like growth factor one receptor) in heart mesoderm or specifically in cardiomyocytes were also compromised in cardiomyocyte proliferation and ventricular morphology (Li et al., 2011), which is predominantly a reflection of the importance of IGF1R rather than INSR in the embryonic heart (Wang et al., 2019). Although the midgestation hypoplastic ventricular phenotypes of conditional Igf2 or IGF receptor mutants were obvious, these were not so severe as to cause embryo lethality. Cardiomyocyte proliferation in mutants then recovered to normal levels at E14 (Li et al., 2011), likely in response to other factors distributed by coronary circulation that begins at that time (Cavallero et al., 2015). Mutant pups were born in normal numbers and of normal size and health, and conditional Igf2 mutants are viable and appear normal throughout a normal lifespan. Because conditional Igf1r/Insr mutants are also normal at birth, the other factors that restore cardiomyocyte proliferation in late gestation in Igf2 mutants seemingly do not signal through IGF1R or INSR and thus are not likely to be IGF1 or insulin.

In this study, we address the reutilization of IGF signaling in neonatal heart regeneration. We find that IGF2 is a required factor for neonatal heart regeneration, although with mechanistic features that are distinct compared to its role in embryonic heart development. After neonatal heart injury, IGF2 is uniquely active during the early part of the first postnatal week when most cardiomyocytes are mononuclear and diploid. A later activity initiates cell cycle entry but is not able to support regeneration, at least under typical circumstances. Most significantly, several independent manipulations that elevate the percentage of mononuclear diploid cardiomyocytes allow rescue of regeneration in Igf2-deficient neonates.

In the mammalian adult heart, myocardial infarction is typically followed by permanently compromised cardiac function and possible progression to heart failure. This outcome is understood to reflect an extremely limited regenerative capacity of the adult heart. In normal biology, hearts are proliferative during embryonic life and can reactivate proliferation following injury during the initial days following birth, but mostly lose regenerative capacity early in the neonatal period. This study adds several new conceptual elements to understanding the regenerative capacity of the heart.

First, our studies support the view that under normal circumstances, the window of heart regenerative competence in mice is limited to only the first few days of the first postnatal week. A similar temporal conclusion was reached by others (Ikenishi et al., 2012; Notari et al., 2018), and is a further refinement of the observation that heart injury at P7 is not followed by effective regeneration (Porrello et al., 2011). In our study, despite robust cell cycle activity at P7 (Figure 5C), Igf2 mutants failed in heart regeneration after P1 injury. Igf2 deficiency therefore unmasks early and late first week cell cycle entry activity and distinguishes the necessity of the former for functional tissue regeneration.

Second, IGF2 appears to be the prime if not sole mitogenic factor that directly induces neonatal cardiomyocyte proliferation during the first few days after P1 injury. In Igf2 mutants there was no indication of any further activity that even partially contributes to an increase in cell cycle entry after P1 injury (Figure 5B’). For this reason, effective regeneration does not occur in Igf2 mutants despite robust cell cycle activity at 7dpr. Other secreted factors previously studied in the context of neonatal heart regeneration (Kawagishi et al., 2018; Singh et al., 2018; Das et al., 2019; Wodsedalek et al., 2019) may indirectly influence cardiomyocyte proliferation through alternative cell types or mechanisms, although we cannot formally exclude the possibility that IGF2 and another secreted mitogen are required together to induce proliferation.

Third, it is notable that neonatal heart regeneration relies on paracrine signaling at all. In principle, changes in cardiac output or wall strain after injury might be detected directly by cardiomyocytes, either at myocyte-myocyte junctions or at the cell surface via interaction with extracellular matrix. Such processes (Bassat et al., 2017; Notari et al., 2018) and others (Mahmoud et al., 2015) are clearly relevant but do not substitute for the requirement for IGF2-INSR paracrine signaling. Our conditional manipulation of Igf2 in cardiac endothelium and cardiomyocyte-specific manipulation of Insr establish IGF2 signaling through INSR as the major paracrine pathway that underlies this process.

Fourth, certain circumstances can override the requirement for IGF2 signaling in neonatal regeneration. In this study, we found that mild hypoxia, mCAT expression, and an inbred A/J strain background were each individually sufficient to support efficient regeneration in the absence of IGF2. We interpret these outcomes (see model in Figure 8) to reflect a higher frequency of completed cell cycle events (i.e., proliferation) among the cardiomyocytes that are induced by P1 injury to enter cell cycle during the later part of the first week, when factors other than IGF2 are present (Figure 5). We note a limitation of our analysis is that we have not directly measured proliferation, which for cardiomyocytes is recognized as very difficult to distinguish from polyploidization (Soonpaa et al., 2013; Zebrowski et al., 2016; Leone et al., 2018). Because cardiomyocyte proliferation is necessary for heart regeneration, we infer that rescue of regeneration in Igf2 mutants must have involved proliferation. Our observations also serve as a caution in interpretation of results when strain background or environment are not properly controlled.

Figure 8

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Finally, our results implicate the necessity of proliferation-competent mononuclear diploid cardiomyocytes in order to support neonatal heart regeneration. At P1, most cardiomyocytes are mononuclear diploid, and proliferation and regeneration are robust following injury. By P7, under typical circumstances (such as the strain backgrounds and environmental conditions used in most studies), most cardiomyocytes have become polyploid (Figure 8), and despite the robust cell cycle entry activity that occurs at this time following P1 injury (Figure 5C’), most cardiomyocytes are not able to complete the cell cycle with cytokinesis. By increasing mononuclear diploid cardiomyocyte percentage through P7, regeneration after P1 injury in Igf2 mutants was rescued, presumably via the non-IGF2 activity that is evident at 7dpr (Figure 5C). The presence of this later activity, which is still not defined, was only possible to detect when the earlier and continuing influence of IGF2 was removed genetically. This interpretation for neonatal heart regeneration is consistent with the importance of cardiomyocyte ploidy in adult heart regeneration (Patterson et al., 2017; González-Rosa et al., 2018). We note the possibility of two variant interpretations of our results. First, mild hypoxia, mCAT expression, and the A/J strain background might influence cardiomyocyte proliferation after neonatal heart injury through other mechanisms, with the increase in mononuclear diploid cardiomyocytes associated with these manipulations being a tangential effect not directly related to regeneration. Second, in addition to serving as a mitogenic signal, IGF2-INSR signaling might also modify the components of cardiomyocyte mitosis to promote proliferation rather than polyploidization. If so, then mild hypoxia, mCAT expression, and the A/J strain background must all do the same.

A premise of our interpretations is that the conditional mutant mice used in this study are normal prior to neonatal injury; if this were not true, then their failure to regenerate after injury could reflect a preexisting deficiency rather than an injury-induced requirement for IGF2-INSR signaling. The fact that Nkx2.5-Cre/Igf2 mutants have a transient embryonic hypoplastic heart phenotype (Li et al., 2011) perhaps supports this scenario. We counter this possibility with several observations. First, the embryonic role for Igf2 is a manifestation of expression in the epicardium, yet epicardium-specific Tbx18-Cre/Igf2 mutants with the exact same embryonic heart phenotype as Nkx2.5-Cre/Igf2 mutants (Shen et al., 2015) were able to regenerate after P1 injury while Nkx2.5-Cre/Igf2 mutants were not. Second, both Tek-Cre/Igf2 and Nfatc1-Cre/Igf2 mutants have no embryonic heart phenotype (Shen et al., 2015) and yet were unable to regenerate after P1 injury. Third, the INSR has no observable role in heart development (Wang et al., 2019) and yet is required for neonatal heart regeneration. These results clearly segregate the consequences of IGF2 signaling in embryonic heart development from its independent role in postnatal heart regeneration. We do note that there is a low level of INSR phosphorylation in uninjured neonatal heart (Figure 4), which may have some role or consequence. We evaluated several aspects of postnatal heart biology in uninjured Nkx2.5-Cre/Igf2 mutants and all were found to be normal (Figure 2), with the possible exception of a small difference in the percentage of mononuclear cardiomyocytes at P7 (Figure 2G). As commented above, we suspect this to be more likely related to the exact circumstances of the pups that were used for this analysis (their exact ages and number of pups per litter; both parameters have a prominent impact on polyploidy [Brodsky et al., 1985]), rather than an indication of a biologically meaningful difference at P7, although we have not formally disproven the latter. Clearly though, the observation that IGF2 signaling through the INSR is typically required for heart regeneration, and that this requirement can be circumvented by several different manipulations (Figure 5) that are unlikely to be relevant to embryonic heart development, supports the interpretation of a unique program of IGF2 signaling that is activated by early postnatal heart injury to promote heart regeneration.

IGF2 is involved in midgestation mouse heart development, so perhaps it is not surprising that it is also involved in neonatal heart regeneration. Nonetheless, with closer examination, there are several features related to its role in neonatal heart regeneration that indicate this to be a unique process and not simply a reiteration of developmental events. First, the lineage sources of IGF2 in heart development and neonatal regeneration are distinct: as shown by genetic manipulation, IGF2 originates from the epicardium (or epicardium-derived mesenchymal cells) in embryonic heart growth, whereas in neonatal heart regeneration, IGF2 derives from the cardiac endothelium (which could include endocardium, coronary endothelium, hematopoietic cells derived from hemogenic endocardium, or endocardium-derived mesenchymal cells). Igf2 is expressed in both epicardium and endocardium in the embryo and neonate, so the completely opposite lineage source of IGF2 in each context implies unique posttranscriptional modes of signaling regulation. Second, the receptors that mediate IGF2 signaling also differ in the embryo and injured neonate: embryonic development requires IGF1R, whereas neonatal regeneration exclusively uses INSR. The mechanisms that account for this distinction in receptor activity are unknown. Clearly though, when looking at IGF signaling in the embryonic and neonatal heart, the oft-stated paradigm that regeneration is a recapitulation of development is superficially true, but each also has unique features that indicate a deeper complexity and subtlety.

Igf2 has been studied in the context of zebrafish heart development and adult heart regeneration, via the use of heat shock-regulated ubiquitous expression of an igf1r dominant negative construct and by treatment with an igf1r-specific antagonist (Choi et al., 2013; Huang et al., 2013). These studies do not provide insight on the lineage sources of IGF2 in the fish heart, but do implicate igf1r as the major receptor in both contexts. At the moment, it is not possible to reconcile the exclusive use of INSR in neonatal mouse heart regeneration with the apparent use of igf1r in zebrafish adult heart regeneration, and it is possible that regeneration in zebrafish has different genetic controls compared to the mouse. Nonetheless, the conservation of reliance on IGF signaling in heart development and regeneration in both species is striking.

It is intriguing that the IGF2-INSR signaling axis is inactive in the uninjured neonatal heart and only becomes active following injury. Igf2 expression at the RNA and protein levels, and Insr expression at the RNA level, are all unchanged by injury, suggesting a posttranslational block to signaling in the normal heart and an alleviation of this block following injury. It is currently unknown what accounts for this block; possible mechanisms could include storage of IGF2 protein in vesicles prior to exocytosis from their source, sequestration after exocytosis by IGF binding proteins or by other components of the extracellular matrix, or some inhibitory feature in uninjured cardiomyocytes that prevents INSR from responding to IGF2. It is interesting that INSR activation (Figure 4B, Figure 4—figure supplements 1 and 2), cardiomyocyte cell cycle activation (Figure 5) and cardiomyocyte proliferation (Porrello et al., 2011) are all induced across the entire ventricle following apex resection; this suggests that the alleviation of IGF2 signaling after injury involves a broadly distributed mechanism rather than one localized to the site of injury. How this occurs is unknown, although because this involves IGF2-INSR, there are now molecular targets through which this process can be explored.

A major reason for interest in the very efficient process of neonatal heart regeneration is its contrast with the very limited regeneration seen in the adult heart after injury. Mouse cardiomyocytes transition from being mostly proliferative to mostly nonproliferative during the first postnatal week, although a small subset of cardiomyocytes remain mononuclear diploid and proliferative. Our analysis indicates that regeneration in the neonate requires both a mitogenic stimulus and an adequate population of proliferation-competent cardiomyocytes. It is notable that although Igf2 is not basally expressed in the adult mouse heart, it is reexpressed following injury, albeit in an inactive manner (Figure 7), and possibly inactive through the same mechanisms that prevent IGF2 signaling in the neonate prior to injury. This suggests the possibility that derepressing IGF2 signaling in the adult heart might promote adult heart regeneration, at least among the population of proliferation-competent cardiomyocytes that are present.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Hua Shen

   Department of Stem Cell Biology and Regenerative Medicine, University of Southern California Keck School of Medicine, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

2. Peiheng Gan

   1. Department of Stem Cell Biology and Regenerative Medicine, University of Southern California Keck School of Medicine, Los Angeles, United States
   2. Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, United States
   3. Department of Medicine Division of Cardiology, Medical University of South Carolina, Charleston, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Kristy Wang

   Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Ali Darehzereshki

   Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

5. Kai Wang

   Department of Cardiovascular Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. S Ram Kumar

   Department of Surgery, University of Southern California Keck School of Medicine, Los Angeles, United States

   Contribution

   Conceptualization

   Competing interests

   No competing interests declared

7. Ching-Ling Lien

   Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, United States

   Contribution

   Conceptualization, Supervision

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5100-9780

8. Michaela Patterson

   Department of Cell Biology, Neurobiology and Anatomy, and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3805-4181

9. Ge Tao

   Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, United States

   Contribution

   Conceptualization, Formal analysis, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

10. Henry M Sucov

    1. Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, United States
    2. Department of Medicine Division of Cardiology, Medical University of South Carolina, Charleston, United States

    Contribution

    Conceptualization, Supervision, Funding acquisition, Project administration

    For correspondence

    sucov@musc.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-3792-3795

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