While most studies focus on regeneration of CMs at either fetal or adult stages, the understanding of the transcriptional changes leading to the transition from a fetal to a postnatal CM phenotype is relatively poor [
49]. The postnatal period for a CM is characterized by a change from hyperplastic to hypertrophic growth [
38], a switch from glycolytic to fatty acid oxidation [
50], and sarcomere maturation with adult contractile isoforms [
14,
73]. Together, this leads to cell cycle exit and terminal CM differentiation [
49]. Through a systematic, high-resolution scRNA-seq approach, analyzing mouse CMs around the time of birth, we identified distinct transcriptional profiles of CM subpopulations, and identified multiple master TFs that control numerous cell cycling genes in CMs. Specifically, detailed analysis of ZEB1, a previously unknown factor in CM cell cycling, showed that before birth, ZEB1 works as a key regulator of cell cycle promoting genes and is required for CM proliferation. Yet, after birth ZEB1 mediated CM cell cycling occurs through endoreplication and leads to polyploid CMs. In agreement with the literature [
11,
32,
61], the number of dividing CMs is indeed also very scarce after birth in our data, and it seems likely that CMs also in the mouse already initiates the final round of cell cycling before birth at which point ZEB1 seems to impact CMs. This would be similar to what is observed in humans, where CMs starts the process of terminal differentiation with increased polyploidy already before birth [
18]. Thus, it is possible that ZEB1 represents a mechanism where CM proliferation is sustained by ZEB1 but at some point, mitotic stress is reached forcing the self-limiting DNA damage response to initiate terminal differentiation and avoid cancer. By this, ZEB1 may ensure polyploidy to achieve high production of RNA and proteins required for the high muscle work of fully differentiated CMs in adulthood, and its timed downregulation eventually caused by a self-limiting DNA response then enables p21 mediated CM differentiation. This could be in line with the emerging theory that polyploidy is an essential biological mechanism for tissue differentiation and homeostasis [
19].
Thus, our novel approach has allowed us to acquire valuable new biological information that may be used further to understand the underlying mechanisms of the switch from CM proliferation to polyploidy occurring around the time of birth in mammals. Whether Zeb1 re-expression also underly the cell cycle activity observed in discrete CMs after MI remains to be determined but could provide a target to enable CM proliferation or forced CM polyploidy at this stage to increase CM mass and compensating the CM loss after MI.
Several scRNA-seq studies on in vivo CMs have recently provided new knowledge on heart development by identifying genes that are differentially expressed at different stages of development. Yet, many of the studies are limited in the number of CMs detected [
5,
10,
30,
40] restricting subsequent detailed analysis such as TF binding site enrichment studies. Other studies fail to implement data on ploidy and cell cycle status [
23,
39]. To our knowledge analysis of TFs in CMs based on scRNA-seq has been described in only four settings for CM development, but none used ploidy stratification as performed herein [
10,
27,
30,
39]. However, in a recent study, Yekelchyk et al. showed transcriptional homogeneity by scRNA-seq of adult rod-shaped mono- and multi-nucleated ventricular CMs, although, not performing TF analysis [
77]. With the established protocol, we unravel the uniqueness of in vivo cycling CMs in the G2/M phases around the time of terminal CM differentiation, and besides Zeb1 identified several TFs potentially involved in the process of G2/M phases completion.
Mycn and
Myc were shown to enhance not only S-phase progression, but also G2/M completion, in agreement with recent data [
8,
60]. The more novel players in CM cell cycling:
Arnt,
Zeb1,
Sp1, and
Egr1 specifically promoted S-phase progression herein with high efficiency, yet all four seemed to leave the postnatal CMs in a polyploid state thus favoring karyo-/cytokinesis failure. Whereas
Arnt has been associated with hypoxia [
74],
Egr1 seems to be implicated in several pathologies of the cardiovascular system [
31], and
Sp1 is a well-known TF in cell growth and peripherally related to CM cell cycling [
21].
Zeb1 is mainly described for its enhancing role in epithelial to mesenchymal transition during cancer and embryonic development [
81], but has recently been linked also to Hematopoietic stem cell renewal and asymmetric cell division [
1]. Furthermore, ZEB1 has been suggested to interact directly with the Hippo pathway in cancer cells through YAP [
34]. The functional roles of the TFs, however, were only predicted bioinformatically and not directly examined besides EdU incorporation and determination of ploidy. Herein, we found
Zeb1 to regulate the highest number of genes related to CM cell cycling in our dataset. Knockdown of
Zeb1 in E16.5 CMs led to impaired S-phase progression with reduced expression of the major cell cycle regulators
Ccnd1,
Ccnb1,
Ccnd3, and
Cdk1 as well as a decrease in the level of
Cenpe,
Cenpf,
Aurkb, and
Aurka, which are all genes expressed in the G2/M phases of the cell cycle, confirming our bioinformatic prediction of ZEB1 as a regulator of the cell cycle in CMs before birth.
Ccnb1,
Ccnd1,
Cdk1, and
Cdk4 are known regulators of CM cell cycling and overexpression of these four factors was recently found to promote proliferation of post-mitotic CMs [
46]. Manipulating
Zeb1 by overexpression in postnatal CMs showed that ZEB1 after birth maintains cell cycling in the form of endoreplication specifically governed by Cyclin E while likely inhibiting cell cycle exit through
Cdkn1a (p21) regulation. It is known that p21 binds and inhibits CDK1/Cyclin B1 thereby blocking G1/S and G2/M phase transitions, and that p21 knockout increases CM ploidy significantly [
66], while p21 expression forces CM cell cycle exit [
65]. In agreement, S-phase progression and ploidy was high in ZEB1 expressing CMs. Since, p21 blocks endoreplication [
66] through co-repressing Cyclin E [
24], the major cyclin of endoreplication [
82], it is thus intriguing to speculate that ZEB1 herein also downregulates p21 and hereby increases Cyclin E2 expression resulting in endoreplication of cycling CMs and polyploidy as observed for a fraction of CMs both in vitro and in vivo. In this regard, it is important to note, that Cyclin D1 in parallel was decreased upon ZEB1 overexpression. Whether this in turn inhibits G1/S phase transition and prevents non-cycling CMs from further entering the cell cycle remains elusive but could explain why only a proportion of CMs undergo S-phase progression despite expressing ZEB1. Yet, we did observe that all ZEB1 overexpressing CMs reduced their size in vitro, which therefore likely represents another mechanism. The smaller size of ZEB1 expressing CMs at early timepoints was accompanied by an increase in the major muscle size inhibitors
Mstn,
Ccng2, and
Tead1, and downregulation of the Yap targets
Axl and
Ctgf. All these genes are related to cell size regulation, and recently ZEB1 was demonstrated to inhibit skeletal muscle cell size in mouse [
59]. Thus, the reduced CM size and decrease in Myh6 fit very well with ZEB1 preventing cell cycle exit, while promoting S-phase progression in the early phase. At P8 in vivo, ZEB1-mediated endoreplication then results in polyploid CMs of increased size, which agrees with the literature [
82]. Interestingly, our studies indicate that ZEB1 regulates a distinct set of genes before and after birth, thereby promoting CM proliferation before birth, while favoring polyploidization when reintroduced after birth. To our knowledge this clear molecular switch from proliferation to endoreplication around birth has not previously been shown for one TF. Thus, the decrease in Zeb1 expression occurring around birth may contribute to cell cycle arrest and terminal CM differentiation, as also observed for other TFs [
17]. One example is YAP1, which as part of the Hippo pathway supports CM proliferation during embryonic development in combination with its interaction partner TEAD1 [
43]. Thus, in the postnatal period downregulation of YAP1 and TEAD1 is required for CM cell cycle arrest [
22]. Postnatal upregulation of YAP1 retain CM proliferative capacity after birth causing cardiomegaly and heart failure [
49]. MYC, which was also detected in our TF analysis, induces CM proliferation during development, while expression of MYC in adult mice leads to an increase in polyploid cells [
76]. However, in another study it was suggested that combinational overexpression of MYC and Cyclin T1 induce CM proliferation without any notable change in CM size and nucleation [
8]. Thus, it is intriguing to speculate, whether overexpression of Zeb1 in combination with other genes or TFs also after birth could promote CM proliferation rather than endoreplication, and by this may be a target for therapeutic perspectives in MI patients.
In conclusion, we here provide new knowledge on Zeb1’s cell cycle promoting actions in CMs as well as a comprehensive scRNA-seq of CMs before and after birth, which may be used to further dissect the switch from a proliferative to a terminally differentiated high power beating CM.