Induced Cytokinesis Generates Highly Proliferative Mononuclear Cardiomyocytes at the Expense of Contractility

Nicholas T Lam; Ngoc Uyen Nhi Nguyen; Waleed M Elhelaly; Ching-Cheng Hsu; Ivan Menendez-Montes; Feng Xiao; Shah R Ali; Nelson Vo; Nathan Briard; Lobna El-Feky; Qamar M Omari; Alisson C Cardoso; Yan Liu; Mahmoud Salama Ahmed; Shujuan Li; Suwannee Thet; Chao Xing; Lior Zangi; Hesham A Sadek

doi:10.1161/CIRCULATIONAHA.124.065763

. Author manuscript; available in PMC: 2026 Apr 8.

Published in final edited form as: Circulation. 2025 Feb 6;151(14):1009–1023. doi: 10.1161/CIRCULATIONAHA.124.065763

Induced Cytokinesis Generates Highly Proliferative Mononuclear Cardiomyocytes at the Expense of Contractility

Nicholas T Lam ^1,^2,^3,^#, Ngoc Uyen Nhi Nguyen ¹, Waleed M Elhelaly ^1,², Ching-Cheng Hsu ¹, Ivan Menendez-Montes ^1,², Feng Xiao ¹, Shah R Ali ¹, Nelson Vo ¹, Nathan Briard ¹, Lobna El-Feky ¹, Qamar M Omari ¹, Alisson C Cardoso ¹, Yan Liu ⁴, Mahmoud Salama Ahmed ¹, Shujuan Li ¹, Suwannee Thet ¹, Chao Xing ⁴, Lior Zangi ⁵, Hesham A Sadek ^1,^2,^6,^7,^8,^9,^#

PMCID: PMC12453028 NIHMSID: NIHMS2051079 PMID: 39912233

Abstract

Background:

Cytokinesis is the last step in the eukaryotic cell cycle which physically separates a mitotic cell into 2 daughter cells. A few days after birth in mouse cardiomyocytes, DNA synthesis occurs without cytokinesis leading to the majority of cardiomyocytes becoming binucleated instead of generating 2 daughter cells with one nucleus each. This results in cell cycle arrest of cardiomyocytes and the mouse heart is no longer able to regenerate. A long-standing unanswered question in the field is whether binucleation of cardiomyocytes is a result of cytokinesis failure.

Methods:

To address this, we generated several transgenic mouse models to determine whether forced induction of cardiomyocyte cytokinesis generates mononucleated cardiomyocytes and restores the endogenous regenerative properties of the myocardium. We focused on two complementary regulators of cytokinesis, namely Polo-like kinase 1 (Plk1) and epithelial cell-transformation sequence 2 (Ect2).

Results:

Here we report that cardiomyocyte-specific transgenic overexpression of constitutively active Plk1(T210D) promotes mitosis and cytokinesis in adult hearts, while overexpression of Ect2 alone promotes only cytokinesis. Intriguingly, cardiomyocyte-specific overexpression of both Plk1(T210D) and Ect2 concomitantly (double transgenic) prevents binucleation of cardiomyocytes postnatally and results in widespread cardiomyocyte mitosis, cardiac enlargement, contractile failure and death before two weeks of age. Similarly, doxycycline-inducible cardiomyocyte-specific overexpression of both proteins (inducible double transgenic) in the adult heart results in reversible widespread cardiomyocyte mitosis and contractile failure. Finally, we show that transient induction of both genes in adults improves left ventricular systolic function following myocardial infarction.

Conclusions:

Collectively, these results demonstrate that cytokinesis failure mediates cardiomyocyte multinucleation and cell cycle exit of postnatal cardiomyocytes but may be a protective mechanism to preserve the contractile function of the myocardium.

Keywords: Cardiomyocyte proliferation, cytokinesis, Plk1, Ect2

INTRODUCTION

Heart failure is a costly and deadly disease affecting over 37.7 million patients worldwide, including over 6 million Americans ^1,2. The incessant inability of the adult mammalian heart to regenerate underpins much of the pathology of systolic heart failure, and thus understanding why adult mammals cannot regenerate their hearts is a critical step in the path to development of regenerative therapeutics. In contrast to adult mammals, neonatal mouse hearts have a significant intrinsic regenerative capacity, mediated by proliferation of pre-existing cardiomyocytes, which is lost within the first week of life as cardiomyocytes permanently exit the cell cycle ^3–5. A few days after birth most cardiomyocytes undergo DNA synthesis without cytokinesis leading to the exit of the cell cycle and binucleation in 88–95% of cardiomyocytes in 7 days old to adult mice, and the heart can no longer undergo meaningful regeneration following significant cardiac injury ^3,6. A long-standing unanswered question in the field is whether multinucleation of cardiomyocytes is a result of cytokinesis failure. Specifically, it is unclear why the majority of cardiomyocytes in most mammals are binucleated or multinucleated. For example, proliferative fetal and early postnatal cardiomyocytes in mammals are predominantly mononucleated, while the vast majority of adult cardiomyocytes in the commonly used C57Bl6 mouse strain are binucleated, although that percentage can be lower in other strains which is a phenomenon previously shown to be associated with enhanced endogenous proliferation. Similarly, approximately 50% of human cardiomyocytes are binucleated, with the majority of mononucleated cardiomyocytes displaying polyploidy. Numerous other reports suggest that there is a direct correlation between mononucleation and increased cardiomyocyte turnover ^7,8. Despite this large body of literature, the reason why the majority of cardiomyocytes become binucleated or multinucleated after birth is not known. Theoretically, multinucleation could arise from cytokinesis failure, a scenario which can occur for example if cells fail to pass DNA damage checkpoint, which is a plausible scenario given that spontaneous DNA damage has been previously shown to occur spontaneously in postnatal cardiomyocytes. Understanding the specific sites of cardiomyocyte cell cycle blockages can undoubtedly have important mechanistic and translational implications.

Cytokinesis in mammalian cells is a highly complicated process where a series of coordinated steps result in equal distribution of nuclear and cytoplasmic content, thereby generating two identical daughter cells. Of the multitude of genes involves in cytokinesis, Plk1 and Ect2 play critical and coordinated roles in initiation and completion of cytokinesis ^9,10. Interestingly, previously published RNA-seq data of isolated neonatal and adult mouse cardiomyocytes indicate that Plk1 and Ect2 are the 2 most downregulated cytokinesis genes in adult cardiomyocytes ¹¹. Following chromosome segregation during anaphase, cytokinesis in animal cells is initiated as CDK1 activity declines which leads to microtubule stabilization and mitotic spindle reorganization ^10,12,13. After Cdk1 inactivation, Plk1 promotes accumulation of RhoA at the equatorial plane, assembling F-actin into a contractile ring, and constricts to become the cleavage furrow ¹⁴. Plk1 activity recruits Ect2 to the central spindle, which initiates cytokinesis in human cells ¹⁴. Centralspindlin, a heterotetramer consisting of a kinesin family member 23 (Kif23) dimer and Rac GTPase-activating protein 1 (Racgap1) dimer, participates in most steps of cytokinesis ^15–17. The upregulation of Plk1 activates centralspindlin by phosphorylating Racgap1 which interacts and forms a docking site for Ect2, and Ect2 becomes activated when it docks onto centralspindlin ^14,17,18. Subsequently, Ect2 activates RhoA by transitioning RhoA from the inactive GDP (guanosine diphosphate)-bound to active GTP (guanosine triphosphate)-bound state ^19–21. Activation of RhoA promotes F-actin assembly, and assembles the actin-myosin contractile ring which constricts to form a cleavage furrow that will generate forces to undergo abscission dividing the cell into 2 separate daughter cells ¹⁸. Inhibition of Plk1 or Ect2 causes cytokinesis failure and a multinucleated cell to form, instead of 2 mononucleated daughter cells ^14,19. Furthermore, transient Plk1 expression is essential for adult zebrafish cardiomyocyte proliferation and cardiac regeneration ²². Therefore, although current literature suggests that Plk1 and Ect2 play critical and complementary role in cytokinesis, their roles in cardiomyocyte cytokinesis arrest and multinucleation remains poorly understood. Here we examined the roles of Plk1 and Ect2 in mammalian cardiomyocyte cytokinesis and heart regeneration individually and in combination, using both constitutively active and inducible cardiomyocyte-specific mouse models.

METHODS

Detailed methods are described in the Supplementary Material. Upon request, all data and methods used will be made available. The use of animals in this study was approved by the Institutional Animal Care and Use Committee (IACUC) at the UT Southwestern Medical Center.

Statistical Analyses

Statistical analysis and graphs were generated using GraphPad Prism version 10.2.0 (392) for Windows (GraphPad Software, Boston, MA, USA, www.graphpad.com). Statistical analyses were performed using unpaired Mann-Whitney U test, unpaired Student t test, One-Way ANOVA, Two-Way ANOVA, and Repeated Measures Two-Way ANOVA as specified in the figure legends. For One-Way ANOVA, Two-Way ANOVA or Repeated Measures Two-Way ANOVA, a Tukey post hoc test was applied. All bar graphs and line graphs are represented by mean ± s.e.m. (standard error of the mean), and statistical significance are denoted as P < 0.05. The sample size is described in all figure legends.

RESULTS

Plk1(T210D) is more potent than Plk1 in promoting cardiomyocyte proliferation.

The promotion of mitosis requires Plk1 activation. Plk1 is activated by the phosphorylation of its conserved threonine residue at T210 by aurora kinase A ²³. A Plk1(T210D) phospho-mimicking mutant is an activated Plk1 without the requirement for aurora kinase A. Plk1 is one of the most downregulated genes involved in cytokinesis during the transition from neonatal to adult cardiomyocytes ¹¹. Therefore, we generated and compared 2 transgenic mouse models with a similar expression of Plk1 and Plk1(T210D) under the control of the cardiomyocyte-specific α-myosin heavy chain (αMHC) promoter, namely αMHC-Plk1 and αMHC-Plk1(T210D), respectively (Figure 1A).

Cardiomyocyte-specific overexpression of Plk1 (αMHC-Plk1) (Figure 1B) did not affect the heart weight/body weight (HW/BW) ratio (Figure 1C) or cardiomyocyte cell size (Figure 1D), cardiomyocytes positive for phospho-histone H3 at Ser10 (pH3), a marker associated with mitosis (Figure 1E), BrdU (Bromodeoxyuridine) positive cardiomyocytes from P21–35 (Figure 1F) and Aurora b kinase (Aurkb), a marker associated with cytokinesis was increased in αMHC-Plk1 hearts (Figure 1G). Total isolated ventricular cardiomyocytes from αMHC-Plk1 hearts were similar compared to non-transgenic control hearts (Figure 1H), and the proportion of mono-, bi- and multi-nucleated cardiomyocytes were similar between the 2 groups (Figure 1I). The percentage of mononucleated diploid cardiomyocytes (MNDCMs) was similar between control and αMHC-Plk1 hearts (Figure 1J). Echocardiography of αMHC-Plk1 hearts revealed left ventricular ejection fractions that were mildly decreased after P49 (Figure 1K), but mostly falling within the normal range.

Next, we examined heart from the cardiomyocyte-specific overexpression of the constitutively active Plk1 (αMHC-Plk1(T210D)) mice (Figure 1L). At P35 αMHC-Plk1(T210D) mice did not display a change in heart weight/body weight (HW/BW) ratio (Figure 1M), and cardiomyocyte cell size (Figure 1N). αMHC-Plk1(T210D) hearts exhibited an increase in pH3+ (Figure 1O), BrdU+ (Figure 1P) and Aurkb+ (Figure 1Q) cardiomyocytes (at higher rates compared to αMHC-Plk1 hearts). In addition, in contrast to αMHC-Plk1 hearts (Figure 1H), total isolated ventricular cardiomyocytes from αMHC-Plk1(T210D) hearts were higher than non-transgenic control hearts (Figure 1R). Furthermore, αMHC-Plk1(T210D) hearts showed an increase in the proportion of mononucleated cardiomyocytes and a decrease in the proportion of binucleated cardiomyocytes compared with non-transgenic hearts (Figure 1S). The percentage of MNDCMs was higher in αMHC-Plk1(T210D) than control hearts (Figure 1T). Ejection fraction of αMHC-Plk1(T210D) hearts were lower than non-transgenic control hearts from P21 onwards (Figure 1U). The proportion of mononucleated cardiomyocytes and binucleated cardiomyocytes from P0.5, P3.5, P10 and P56 C57Bl6N hearts (Figure S1A and S1B) were similar to a previous study ⁶, whereby the early neonatal cardiomyocytes are primarily mononucleated and rapidly undergo binucleation leading to the majority of postnatal cardiomyocytes becoming binucleated. These results demonstrate that cardiomyocyte-specific overexpression of Plk1(T210D) phospho-mimetic mutant is more potent than wildtype Plk1 at inducing cardiomyocyte proliferation, and results in an increase in total cardiomyocytes and an increase in the proportion of mononucleated cardiomyocytes and a decrease in binucleated cardiomyocytes.

In order to determine whether Plk1(T210D) can induce cardiomyocyte proliferation is impacted by the magnitude of Plk1(T210D) expression, we assessed another αMHC-Plk1(T210D) mouse line [αMHC-Plk1(T210D) High] with a higher level of expression of Plk1(T210D) (Figure S2A and S2B) than αMHC-Plk1(T210D) from Figures 1a and 1l-u,. Compared to non-transgenic control hearts, αMHC-Plk1(T210D) High hearts at P35 exhibit higher HW/BW (Figure S2C), and smaller cardiomyocyte size (Figure S2D). Intriguingly, hearts from this αMHC-Plk1(T210D) High mouse line displayed marked cardiomyocyte proliferation as demonstrated by higher pH3+ cardiomyocytes (Figure S2E), BrdU+ cardiomyocytes from P21–35 (Figure S2F) and Aurkb+ cardiomyocytes (Figure S2G) (several fold higher than the lower expression line). Furthermore, a lineage tracing system to assess completed cytokinesis in cardiomyocytes was used with offspring generated from crosses of cardiomyocyte-specific Myh6^mERcremER (αMHC^mERcremER) and mosaic analysis with double markers (MADM) mice ²⁴ with αMHC-Plk1(T210D) High mice. Tamoxifen was administered daily for 2 weeks from P21 and hearts were harvested at P35. αMHC-Plk1(T210D) High hearts showed an increase in single labelled cardiomyocytes in red or green fluorescent protein confirmed that overexpression of Plk1(T210D) in cardiomyocytes leads to an increase in completed cytokinesis and new cardiomyocyte formation (Figure S2H). Isolated cardiomyocytes from hearts of αMHC-Plk1(T210D) High exhibited an increase in total cardiomyocytes (Figure S2I), as well as an increase in the proportion of mononucleated cardiomyocytes and decreases in the percentage of binucleated and multinucleated cardiomyocytes (Figure S2J). The percentage of MNDCMs increased in αMHC-Plk1(T210D) High compared with control hearts (Figure S2K). Importantly, these hearts exhibited a markedly depressed left ventricular ejection fraction (Figure S2L). These data suggest that the degree of cardiomyocyte proliferation and depression of LVEF are directly related to the magnitude of Plk1(T210D) overexpression.

Ect2 exclusively promotes cytokinesis in cardiomyocytes.

Ect2 expression is high in mammalian cardiomyocytes during the neonatal regenerative window, whereas in adult cardiomyocytes Ect2 is the most downregulated gene involved in cytokinesis ¹¹.

In order to examine the role of Ect2 in cardiomyocyte proliferation, we generated a transgenic mouse model where Ect2 is expressed under the control of the cardiomyocyte-specific αMHC promoter: αMHC-Ect2 (Figure 2A and 2B). αMHC-Ect2 mice at P35 did not display a change in the heart weight/body weight (HW/BW) ratio (Figure 2C), or cardiomyocyte cell size (Figure 2D). Interestingly, αMHC-Ect2 expression did not result in an increase in pH3 positive cardiomyocytes (Figure 2E), and no increase in BrdU+ cardiomyocytes from P21–35 (Figure 2F), but resulted in an increase in cardiomyocytes positive for aurora b kinase (Aurkb) (Figure 2G), suggesting that overexpression of Ect2 in cardiomyocytes exclusively promotes cytokinesis rather than overall mitotic entry. In addition, a lineage tracing system to assess completed cytokinesis in cardiomyocytes was used with offspring generated from crosses of cardiomyocyte-specific Myh6^mERcremER (αMHC^mERcremER); MADM mice ²⁴ with αMHC-Ect2 mice. Tamoxifen was administered daily for 2 weeks from P21 to P35 that resulted in αMHC-Ect2 hearts with increases in single labelled cardiomyocytes in red or green fluorescent protein, confirming that overexpression of Ect2 in cardiomyocytes leads to an increase in completed cytokinesis and new cardiomyocyte formation (Figure 2H). In αMHC-Ect2 hearts, total isolated ventricular cardiomyocytes increased compared with non-transgenic control hearts (Figure 2I). Furthermore, in αMHC-Ect2 hearts the proportion of mononucleated cardiomyocytes were higher, with binucleated and multinucleated cardiomyocytes lower compared with non-transgenic hearts (Figure 2J). The percentage of MNDCMs was significantly higher in αMHC-Ect2 than control hearts (Figure 2K). Echocardiography of αMHC-Ect2 showed a similar left ventricular ejection fraction compared with non-transgenic control hearts until P49 and declines slightly, but still within the normal range (Figure 2L). Overall, these results suggests that Ect2 promotes cytokinesis but not overall mitosis.

Figure 2. — Results for αMHC-Ect2 hearts at P35. A. Schematic of cardiomyocyte-specific Ect2 transgenic mouse line. B. Western blot analysis of Ect2 and β-actin proteins in non-transgenic control versus αMHC-Ect2 hearts (n=3 for each group). C. Heart weight/Body weight of non-transgenic control versus αMHC-Ect2 hearts (n=8 and n=9 for each group, respectively). D. WGA staining and cross-sectional area quantification (n=3 for each group). E. Immunostaining of hearts for cTnT (red) and pH3 (green), and quantification of mitotic cardiomyocytes for non-transgenic control (n=5) and αMHC-Ect2 (n=4) hearts. F. Quantification of BrdU positive cardiomyocytes from P21–35 (n=3 for each group); G. Immunostaining of hearts for cTnT (red) and Aurkb (green), and quantification of cytokinetic cardiomyocytes (n=5 for each group). H. Representative immunofluorescence and quantification of single-labelled (red or green) cardiomyocytes from control (n=3) and αMHC-Ect2 (n=5) mice crossed with MADM and *Myh6*^mERcremER mice. I. Total number of isolated cardiomyocytes per heart (n=5 for each group). J. (**Left panel**) Nucleation quantification (n=5 for each group); (**Right panel**) Representative images of mononuclear, binuclear and multinuclear cardiomyocytes. K. Percentage of MNDCMs (n=5 for each group); L. Left ventricular ejection fraction of non-transgenic control (n=3) and αMHC-Ect2 (n=4) from P21–77. Scale bars = 10μm. Statistical analyses were performed using unpaired Mann-Whitney U test (C-G, I and K), Two-Way ANOVA (H and J), and Repeated Measures Two-Way ANOVA (L).

Concomitant expression of Plk1(T210D) and Ect2 induces robust cardiomyocyte proliferation and prevents binucleation.

Based on our results, Plk1(T210D) is more potent at inducing cardiomyocyte proliferation and Ect2 exclusively promotes cytokinesis. As Plk1 and Ect2 work together to regulate cytokinesis ¹⁴, we therefore generated transgenic mice under the control of a cardiomyocyte-specific αMHC promoter expressing both Plk1(T210D) and Ect2 by the co-injection of αMHC-Plk1(T210D) and αMHC-Ect2 DNA sequences into fertilized mouse eggs (Figure 3A). One founder male mouse survived to adulthood with germline transmission of the transgene and was bred with wildtype C57BL/6N females. Interestingly, all αMHC-Plk1(T210D); αMHC-Ect2 offspring died by ~P15. We therefore, harvested the hearts at P11 prior to the premature death at P15.

We found that hearts from αMHC-Plk1(T210D); αMHC-Ect2 mice (Figure 3A) at P11 had significantly higher Plk1 and Ect2 mRNA expression (Figure 3B and 3C). Cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2 was associated with a marked increase in heart weight/body weight (HW/BW) (Figure 3D) and was associated with a decrease in cardiomyocyte size (Figure 3E). Compared with non-transgenic control hearts, αMHC-Plk1(T210D); αMHC-Ect2 hearts exhibited dilated left ventricular chamber (Figure 3F), but with no change in collagen deposition (Figure 3G). In addition, there was a significant increase in pH3+ (Figure 3H) and Aurkb+ cardiomyocytes (Figure 3I), suggesting an increase in cardiomyocytes undergoing mitosis and cytokinesis, respectively. Isolation of cardiomyocytes showed a marked increase in the total number of cardiomyocytes in αMHC-Plk1(T210D); αMHC-Ect2 hearts (Figure 3J), and there was a substantial increase in the proportion of mononucleated cardiomyocytes and decrease in binucleated cardiomyocytes (Figure 3K) compared with non-transgenic control hearts. Remarkably αMHC-Plk1(T210D); αMHC-Ect2 overexpression resulted in a marked increase in mononucleated cardiomyocytes, approaching 80% compared to approximately 20% mononucleated cardiomyocytes in age matched controls. Furthermore, the percentage of MNDCMs was substantially elevated in αMHC-Plk1(T210D); αMHC-Ect2 than control hearts (Figure 3L). However, αMHC-Plk1(T210D); αMHC-Ect2 overexpression resulted in a marked decrease in left ventricular ejection fraction (Figure 3M). Taken together, this suggests that cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2 are sufficient to induce robust cardiomyocyte mitosis and cytokinesis in neonatal hearts, leading to a substantial increase in total cardiomyocytes and prevention of cardiomyocyte binucleation. However, this occurs at the expense of myocardial contractility, which results in death before 2 weeks of age.

Induction of Plk1(T210D) and Ect2 in adult cardiomyocytes promotes proliferation and cytokinesis.

The continuous overexpression of Plk1(T210D) and Ect2 in cardiomyocytes from birth promotes robust cardiomyocyte proliferation and cytokinesis but decreases left ventricular ejection fraction and premature death in αMHC-Plk1(T210D); αMHC-Ect2 mice. To determine the effect of inducing Plk1(T210D) and Ect2 in mature adult cardiomyocytes (when the majority of cardiomyocytes are already binucleated), an inducible transgenic mice under the control of the tetracycline response element (TRE) promoter ²⁵ with the Plk1(T210D) and Ect2 Open Reading Frames (ORFs) sandwiched by a T2A (thosea asigna virus 2A) sequence were generated as TRE-Plk1(T210D)-T2A-Ect2 (Figure 4A). When TRE-Plk1(T210D)-T2A-Ect2 mice are crossed with an αMHC-rtTA mice ²⁶, the double transgenic offspring (TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA) when given doxycycline (a tetracycline analog) in the drinking water can transiently induce expression of Plk1(T210D) and Ect2 in cardiomyocytes (Figure 4A). The T2A sequence allows this sequence to be transcribed as one sequence, and is cleaved to generate 2 proteins (Plk1(T210D) and Ect2) at similar expression ratios ²⁷. With this model, a transient induction of Plk1(T210D) and Ect2 in the adult heart can address whether adult cardiomyocytes which are mostly binucleated can directly induce cytokinesis and lead to an increase in mononucleated cardiomyocytes.

Figure 4. — A. Schematic of mouse breeding strategy to generate doxycycline inducible cardiomyocyte-specific Plk1(T210D) and Ect2 transgenic mouse line (TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA); B. Western blot of αMHC-rtTA control hearts with normal drinking water, with doxycycline; versus TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts with normal drinking water or doxycycline for 2 weeks; **C-E, G-K.** αMHC-rtTA control and TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA adult mice exposed to doxycycline (0.1g/L) for 2 weeks: C. Schematic; D. Ejection fraction of αMHC-rtTA (n=6) and TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA (n=5) hearts; E. Heart weight/Body weight of αMHC-rtTA control versus TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts (n=6 and n=5 for each group, respectively); F. WGA staining and cross-sectional area quantification of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA with normal (control) versus doxycycline (0.1g/L) drinking water (n=4 and n=5 for each group, respectively); G. Immunostaining of hearts for cTnT (red) and pH3 (green), and quantification of mitotic cardiomyocytes (n=5 for each group); H. Immunostaining of hearts for cTnT (red) and Aurkb (green), and quantification of cytokinetic cardiomyocytes (n=5 for each group); I. Total number of isolated cardiomyocytes per heart (n=4 and n=5 for each group, respectively); J. (**Left panel**) Nucleation quantification (n=4 and n=5 for each group, respectively); (**Right panel**) Representative images of mononuclear, binuclear and multinuclear cardiomyocytes. K. Percentage of MNDCMs (n=4 and n=5 for each group, respectively); **L-O.** αMHC-rtTA control and TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA adult mice exposed to doxycycline for 2 weeks followed by 1 week on normal drinking water: L. Schematic; M. Left ventricular ejection fraction of αMHC-rtTA control and TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts (n=5 for each group); N. Total number of isolated cardiomyocytes per heart (n=5 for each group); O. (**Left panel**) Nucleation quantification (n=5 for each group); (**Right panel**) Representative images of mononuclear, binuclear and multinuclear cardiomyocytes. Scale bars = 10μm. TRE-PTE = TRE-Plk1(T210D)-T2A-Ect2. Statistical analyses were performed using unpaired Mann-Whitney U test (D-I, K and N), Two-Way ANOVA (J and O), and Repeated Measures Two-Way ANOVA (M).

TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice specifically express robust Plk1 and Ect2 protein in the heart only when exposed to doxycycline drinking water (Figure 4B). TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice and αMHC-rtTA mice were exposed to doxycycline drinking water (0.1g/L) for 2 weeks from 8–10 weeks of age and these hearts were studied (Figure 4C). Compared with αMHC-rtTA control hearts, TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts had a lower ejection fraction (Figure 4D) and was associated with a smaller HW/BW (Figure 4E). There was a marked decrease in cardiomyocyte cell size in TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts with doxycycline compared with TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts without doxycycline (Figure 4F). There was a significant increase in pH3+ (Figure 4G) and Aurkb+ cardiomyocytes (Figure 4H), suggesting an increase in cardiomyocytes undergoing mitosis and cytokinesis, respectively. This is supported by the isolation of cardiomyocytes which showed an increase in the total number of cardiomyocytes in TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts on 2 weeks of doxycycline (0.1g/L) drinking water (Figure 4I) and increases in the proportion of mononucleated cardiomyocytes and decreases the proportion of binucleated cardiomyocytes (Figure 4J) and increases the percentage of MNDCMs (Figure 4K).

Transient induction of Plk1(T210D) and Ect2 in adult cardiomyocytes lead to a reversible decrease in ejection fraction and increase in total and mononucleated cardiomyocytes.

Next, to determine if the doxycycline treatment mediated decrease in ejection fraction is reversible, TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA and αMHC-rtTA mice were exposed to doxycycline drinking water (0.1g/L) for 2 weeks from 8–10 weeks of age and exposed to normal drinking water for 1 week from 10–11 weeks of age (Figure 4L). After 2 weeks of doxycycline, the ejection fraction decreases in TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice, but this was restored by 1 week after removing doxycycline water (Figure 4M). After 2 weeks of doxycycline water, followed by 1 week of normal drinking water, isolated cardiomyocytes from these hearts showed that the transient cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2 lead to an increase in total cardiomyocytes (Figure 4N), as well as an increase in the proportion of mononucleated cardiomyocytes and a decrease in binucleated cardiomyocytes (Figure 4O).

Intracardiac injections of modRNA encoding Plk1(T210D) and Ect2 in adult hearts promotes cytokinesis in cardiomyocytes.

To assess the effect of Plk1, Plk1(T210D), and Ect2 on cytokinesis in adult cardiomyocytes (a stage when the majority of cardiomyocytes are already binucleated), intracardiac injections of synthetic modified mRNA (modRNA) ²⁸ encoding Plk1, Plk1(T210D) or Ect2 were performed on the MADM cardiomyocyte lineage tracing mouse model ²⁴.

Tamoxifen was administered daily for 2 weeks in adult (2.5 months) αMHC^mERcremER; MADM mice ²⁴ to induce recombination and prelabeled cardiomyocytes with RFP and GFP (yellow) (Figure S3A). Intracardiac injections of control buffer or modRNA encoding for Plk1, Plk1(T210D) or Ect2 was performed in these prelabeled αMHC^mERcremER; MADM mouse hearts and harvested 1 week later (Figure S3A). Compared with the control group, intracardiac injections of modRNA encoding Plk1(T210D) and Ect2 increases single labelled RFP+ (red fluorescent protein) and GFP+ (green fluorescent protein) cardiomyocytes (Figure S3B and S3C). This suggests that Plk1(T210D) induces cytokinesis in cardiomyocytes more than Plk1, and that Ect2 also induce cytokinesis in adult cardiomyocytes.

Induction of Plk1(T210D) and Ect2 in cardiomyocytes improves ejection fraction following myocardial infarction in adult mice

In TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice, 2 weeks of doxycycline (0.1g/L) treatment leads to a reversible decrease in ejection fraction (Figure 4M). Therefore, we assessed whether 1 week of doxycycline treatment of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice (0.1g/L) can promote cardiomyocyte proliferation without a major decrease in ejection fraction (Figure 5A). TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice exposed to doxycycline (0.1g/L) expressed Plk1 and Ect2 protein in hearts (Figure 5B) The ejection fraction of doxycycline treated TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts were only slightly lower than control hearts (TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA on normal drinking water) (Figure 5C). Compared with control hearts, 1 week of doxycycline treatment of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts was associated with similar HW/BW (Figure 5D), a marked decrease in cardiomyocyte size (Figure 5E), an increase in pH3+ cardiomyocytes (Figure 5F), BrdU+ cardiomyocytes (Figure 5G) and Aurkb+ cardiomyocytes (Figure 5H). To confirm the induction and completion of cytokinesis, TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA; MADM-11^GT/TG; αMHC-MerCreMer transgenic mice were bred (Figure 5I). TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA; MADM-11^GT/TG; αMHC-MerCreMer mice were injected daily from 6–8 weeks of age to prelabel cardiomyocytes with GFP and RFP, and from 9–10 weeks of age these mice were provided with either normal drinking water (control) or doxycycline (0.1g/L) (Figure 5I). Cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2 by doxycycline lead to an increase in single labelled RFP and GFP positive cardiomyocytes (Figure 5J). This suggests that induction of Plk1(T210D) and Ect2 in adult cardiomyocytes (a stage when the majority of cardiomyocytes are binucleated) induces the completion of cytokinesis. Furthermore, 1 week of doxycycline treatment of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts lead to an increase in total cardiomyocytes (Figure 5K) as well as an increase in mononucleated cardiomyocytes and a decrease in binucleated cardiomyocytes (Figure 5L), and increases the percentage of MNDCMs (Figure 5M).

Figure 5. — TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA adult mice exposed to doxycycline (0.1g/L) or normal drinking water (no Dox) for 1 week: A. Schematic of doxycycline inducible cardiomyocyte-specific Plk1(T210D) and Ect2 transgenic mouse line; B. Western blot of hearts without doxycycline and with doxycycline (n=3 for each group). C. Ejection fraction of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts on normal drinking water (n=4) and doxycycline (0.1g/L) drinking water (n=5) for 1 week; D. Heart weight/Body weight of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts on normal drinking water versus Doxycycline drinking water (n=4 and n=5 for each group, respectively); E. WGA staining and cross-sectional area quantification of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA on normal versus doxycycline drinking water (n=3 and n=5 for each group, respectively); F. Immunostaining of hearts for cTnT (red) and pH3 (green), and quantification of mitotic cardiomyocytes of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA on normal versus doxycycline drinking water (n=3 and n=5 for each group, respectively); G. Quantification of BrdU+ cardiomyocytes from adult TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice injected with BrdU daily and drinking water with and without doxycycline (0.1g/L) from 9–10 week of age (n=4 for each group); H. Immunostaining of hearts for cTnT (red) and Aurkb (green), and quantification of cytokinetic cardiomyocytes of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA on normal versus doxycycline drinking water (n=3 and n=5 for each group, respectively); **I-J.** TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA; MADM-11^GT/TG; αMHC-MerCreMer transgenic mice prelabelled with RFP and GFP by daily injections of tamoxifen from 6–8 weeks of age, and from 9–10 weeks of age the mice receive normal drinking water or doxycycline (0.1g/L) water: I. schematic and J. quantification of single-labelled RFP+ and GFP+ cardiomyocytes (n=3 for each group); K. Total number of isolated cardiomyocytes per heart (n=5 and n=6 for each group, respectively); L. (**Left panel**) Nucleation quantification of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA on normal versus doxycycline drinking water (n=5 and n=6 for each group, respectively); (**Right panel**) Representative images of mononuclear, binuclear and multinuclear cardiomyocytes. M. Percentage of MNDCMs (n=5 for each group); **N-O.** TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA 2-month-old adult mice underwent MI, and exposed to normal (control) or 0.1g/L doxycycline drinking water from 1–2 weeks post MI: N. Schematic; O. (**Left panel**) Left ventricular ejection fraction of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice exposed to normal (control) or doxycycline drinking water from 1–2 weeks post MI (n=5 and n=6 for each group, respectively). (**Right panel**) Representative short axis M-mode echocardiography image of TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mouse hearts at 1 week and 8 weeks post MI treated with normal (control) and doxycycline drinking water for 1–2 weeks post MI. Scale bars = 10μm. TRE-PTE = TRE-Plk1(T210D)-T2A-Ect2. Statistical analyses were performed using unpaired Mann-Whitney U test (C-H, K and M), Two-Way ANOVA (J and L), and Repeated Measures Two-Way ANOVA (O).

To identify transcriptional pathways involved with the induction of Plk1(T210D) and Ect2, RNA sequencing was performed on adult TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts treated with doxycycline (0.1g/L) versus controls hearts on normal drinking water (Figure S4A). Based on P<0.05, 1377 genes were upregulated and 1375 genes were downregulated. The gene ontology terms for Plk1(T210D) and Ect2 overexpression promoted pathways in telomerase, cell cycle and glycolytic process, and downregulated pathways in fatty acid metabolism (Figure S4B). During the transient overexpression of Plk1(T210D) and Ect2 in adult cardiomyocytes [TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA model], some of the enrichment of genes in the glycolytic pathways upregulated include Adpgk, Gapdh, Gapdhs, Gck, Ier3, Myc, and Prkag3. Furthermore, the enrichment of genes in the fatty acid biosynthetic pathway that are downregulated include Abcd2, Abcd3, Abhd1, Acacb, Acsm5, Alox5, Oxsm, Pecr, Prkab1, Prkag2, and Ptgds.

To determine if the Plk1(T210D) and Ect2-mediated cardiomyocyte proliferation can contribute to cardiac regeneration following injury, Plk1(T210) and Ect2 were induced transiently after myocardial infarction, with doxycycline drinking water administered from 1–2 weeks post myocardial infarction (Figure 5N). Myocardial infarction (MI) was surgically induced in TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice by ligation of the left anterior descending (LAD) coronary artery. At 1 week post MI, mice were treated with either normal (control) or 0.1g/L doxycycline (Dox) drinking water to induce cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2, from 1 to 2 weeks post MI (Figure 5N). The transient overexpression of Plk1(T210D) and Ect2 in cardiomyocytes in TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts after MI lead to a progressive increase of ejection fraction from 8 weeks post MI onwards compared with 1 week post MI, when doxycycline treatment began (Figure 5O). In contrast, the ejection fraction did not improve in TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA hearts after MI with no doxycycline, which is consistent with post-infarction remodeling (Figure 5O). Importantly, transient induction of Plk1(T210D) and Ect2 from 1–2 weeks post MI results in a higher ejection fraction compared to control hearts from 4 weeks post MI onwards (Figure 5O). These results suggest that transient overexpression of Plk1(T210D) and Ect2 can induce myocardial regeneration and improve left ventricular systolic function following injury.

DISCUSSION

One of the enduring mysteries in the cardiac regeneration field is the mechanism of mammalian cardiomyocytes binucleate in the early postnatal period, which coincides with their permanent cell cycle arrest. A number of theories have been put forth, including a leading untested hypothesis which states that postnatal cardiomyocyte binucleation is a result of cytokinesis failure. Although a large number of cardiomyocyte cell cycle regulators have been identified to date, the role of cytokinesis failure in mammalian cardiomyocyte binucleation and cell cycle arrest is unclear.

Cardiomyocyte-specific knockout of Ect2 at embryonic and early neonatal stages leads to increases in binucleated cardiomyocytes ^29,30. Nkx2.5^Cre; Ect2^flox/flox mice died by E10.5 and primarily composed of binucleated cardiomyocytes (with control hearts mostly mononucleated cardiomyocytes) ²⁹. Furthermore, Mlc2v^Cre; Ect2^flox/flox mice at P3 with Ect2 knocked down of ventricular cardiomyocytes results in an increase in binucleated cardiomyocytes ²⁹. Mlc2v^Cre; Ect2^flox/flox mice underwent MI at P1 exhibited higher cardiac fibrosis compared with control mice at P8, demonstrating that lack of Ect2 impairs cardiac regeneration. αMHC^Cre; Ect2^flox/flox mice increased binucleated cardiomyocytes by 3.2 fold at P1 and had half as many total cardiomyocytes and all pups died by P2 ³⁰. Plk1 knockout mice are embryonic lethal by the 8-cell stage ³¹, however the effect of Plk1 inhibitor (Volasertib) on human pluripotent stem cell derived cardiomyocytes resulted in increased binucleation and maturation ³². Data extracted from publicly available RNA-sequencing data of isolated cardiomyocytes from neonatal and adult mice ¹¹, shows that Plk1 and Ect2 mRNA were the most downregulated genes involved in cytokinesis. Interestingly, the significant decline in Plk1 and Ect2 expression in cardiomyocytes coincides with binucleation of cardiomyocytes during the neonatal period ⁶. Our data show that αMHC-Plk1(T210D); αMHC-Ect2 mouse hearts exhibit a significant reduction in binucleation leading to the majority of cardiomyocyte remaining mononucleated. Collectively taken together, the natural decline of Plk1 and Ect2 in cardiomyocytes increases cytokinesis failure and plays a role for why cardiomyocytes become binucleated, so that cardiomyocytes mature to support an increasing cardiac workload.

In the current study, we examined whether Plk1 and Ect2 mediate the failure of cytokinesis that occurs postnatally in mammals, resulting in generation of binucleated, cell cycle arrested cardiomyocytes. We determined whether the induction of Plk1(T210D) and Ect2 in adult mouse cardiomyocytes in vivo, alone or in combination (using genetic mouse models), can induce cytokinesis and generate mononucleated proliferative cardiomyocytes, and we applied these genetic models in an adult myocardial infarction model.

First, we generated a number of cardiomyocyte-specific (αMHC-driven) transgenic mouse lines to overexpress Plk1, constitutively active Plk1(T210D) or Ect2. As Plk1 is involved in both mitosis and cytokinesis ³³, αMHC-Plk1(T210D) hearts exhibited more cardiomyocytes undergoing mitosis and cytokinesis compared with control hearts. Normally, Aurora kinase A (Aurka) phosphorylates Plk1 at Thr210 ²³, however the expression of Aurka is extremely low in mouse adult cardiomyocytes ¹¹, and thus activation of Plk1 is unlikely to occur. As expected, we found that cardiomyocyte-specific overexpression of Plk1(T210D), a phospho-mimic mutant is more potent than Plk1 in induction of mitosis and cytokinesis, and resulted in an increase in total cardiomyocyte number, with an increase in mononucleated cardiomyocytes and a decrease in binucleated cardiomyocytes. Interestingly, the ejection fraction of αMHC-Plk1(T210D) hearts were mildly decreased compared to the non phospho-mimetic αMHC-Plk1, possibly linking enhanced cardiomyocyte cytokinesis to decreased myocardial systolic function. In contrast, we found that overexpression of Ect2 alone in the αMHC-Ect2 hearts did not increase the number of cardiomyocytes undergoing mitosis (pH3 positive) but increased the number of cardiomyocytes undergoing cytokinesis (Aurkb-kinase positive and MADM cardiomyocyte lineage tracing), which resulted in an increase in total cardiomyocytes with an increase in the proportion of mononucleated cardiomyocytes and a decrease in binucleated and multinucleated cardiomyocytes. This is perhaps not surprising given that Plk1 is involved in both mitosis and cytokinesis, whereas Ect2 is involved only in cytokinesis.

Next, we tested whether the complementary roles of Plk1 and Ect2 would force cardiomyocyte cytokinesis by generating a double transgenic mouse overexpressing Plk1(T210D) and Ect2 in cardiomyocytes from birth (αMHC-Plk1(T210D); αMHC-Ect2). We found that the αMHC-Plk1(T210D); αMHC-Ect2 hearts were larger, with smaller cardiomyocytes which exhibited markedly higher degrees of mitosis and cytokinesis. Remarkably, the majority of cardiomyocytes in these hearts were mononucleated, which supports the notion that postnatal binucleation is a result of cytokinesis failure. However, these hearts exhibited markedly impaired systolic function, which resulted in death within approximately 2 weeks after birth.

An important question in the field is whether cell cycle arrest and binucleation of postnatal cardiomyocyte are permanent. Therefore, we generated transgenic mice where cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2 occurs only upon administration of doxycycline (TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA). Upon administration of large doses of doxycycline, these mice exhibited widespread induction of cardiomyocyte mitosis and cytokinesis resulting in partial reversal of cardiomyocyte binucleation where a significant proportion of cardiomyocytes became mononucleated (23.4%) compared to control cardiomyocytes (12.8%). Importantly, this phenotype was also associated with a significant reduction in left ventricular systolic function, which was readily reversible upon discontinuation of doxycycline. Collectively, these results suggest that forced induction of cytokinesis markedly enhances cardiomyocyte proliferation and can prevent as well as reverse binucleation, but this occurs at the expense of left ventricular systolic function.

Finally, to determine whether modest induction of cytokinesis can induce myocardial regeneration, TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice underwent experimental myocardial infarction, which was followed by administration of a lower dose of doxycycline in drinking water transiently post MI (to avoid continuous induction of cytokinesis). Although low dose doxycycline administration resulted in a modest decline in LVEF, this was transient, and the infarcted TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA mice showed rapid improvement of systolic function, which surpassed their post-infarction LVEF, as well as that of the control infarcted hearts. Interestingly, Plk1 is essential for adult zebrafish cardiomyocyte proliferation and cardiac regeneration ²², and Ect2 is also upregulated ³⁴. Our study in mice shows that cardiomyocyte-specific induction of Plk1(T210D) and Ect2 after myocardial infarction leads to an improvement in ejection fraction.

Our results highlight several points of physiological and translational significance. First, the results suggest that the postnatal loss of cardiomyocyte cytokinesis may be a protective effect to maintain normal myocardial contractility. This is supported by results in both early postnatal as well as adult mice, where forced induction of cytokinesis by concomitant expression of Ect2 and Plk1 results in a rapid decline in LVEF, which is readily reversible upon cessation of cytokinesis gene expression. Second, our findings also support the notion that postnatal binucleation of cardiomyocytes is, in part, due to cytokinesis failure. However, this does not completely negate other potential hypotheses of mechanisms and potential roles of binucleation in postnatal cardiomyocytes, such as providing a larger transcriptional machinery for a cell the size of adult mammalian cardiomyocytes, which can exceed 200 μM in length. Third, these results suggest that transient and focal induction of cytokinesis, as we have achieved using intramyocardial injection of modRNA, can induce cardiomyocyte cytokinesis and may be a viable therapeutic strategy. However, generalized, and persistent induction of cytokinesis is associated with significant contractile dysfunction and should not be considered as a safe strategy for myocardial regeneration.

In summary, our study shows that forced cytokinesis using cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2 can both prevent and partially reverse mammalian cardiomyocyte binucleation. However, this occurs at the expense of myocardial contractility, which suggests that the postnatal cytokinesis failure and binucleation of cardiomyocytes might be a physiological phenomenon designed to maintain normal myocardial contractility. Importantly, we show that transient controlled induction of cardiomyocyte cytokinesis can rapidly regenerate the adult infarcted myocardium. These results indicate that regulation of cardiomyocyte cytokinesis is critical for maintenance of normal myocardial function and provides important insights into the potential role of cytokinesis in myocardial regeneration.

Supplementary Material

Supplemental Publication Material

NIHMS2051079-supplement-Supplemental_Publication_Material.pdf^{(48.1MB, pdf)}

Uncroppoed Gel Blots

NIHMS2051079-supplement-Uncroppoed_Gel_Blots.pdf^{(1.7MB, pdf)}

CLINICAL PERSPECTIVE.

What Is New?

In contrast to the natural postnatal decline of Plk1 and Ect2 expression coinciding with binucleation of most cardiomyocytes and cell cycle exit, constitutively active Plk1(T210D) and Ect2 overexpression from birth prevents binucleation of cardiomyocytes with most cardiomyocytes remaining mononucleated.
Reexpression of Plk1(T210D) and Ect2 in mature multinucleated cardiomyocytes promotes mitosis and induces cytokinesis and increases mononucleated and total cardiomyocytes.
After myocardial infarction, transient cardiomyocyte-specific overexpression of Plk1(T210D) and Ect2 progressively improves left ventricular ejection fraction.
Transient induction of cytokinesis in multinucleated cardiomyocytes could be a novel strategy for generating new cardiomyocytes for regenerating the heart after injury.

What Are the Clinical Implications?

A significant proportion of human cardiomyocytes are multinucleated, and induction of multinucleated cardiomyocytes could be a potential strategy to increase cardiomyocytes after a myocardial infarction to prevent the onset of heart failure or to reverse it to restore heart function.
Transient overexpression of Plk1(T210D) and Ect2 could offer a potential therapy to improve left ventricular ejection fraction after myocardial infarction.

Acknowledgements

We acknowledge the core facilities at UT Southwestern including Transgenic Core (Robert Hammer, John Ritter and Mylinh Nguyen) and Mouse Genome Engineering Facility (Hao Zhu) for microinjections to generate transgenic mice; Molecular Pathology Core (John Shelton) for histology support; and Sequencing Facility for RNA-sequencing (Jian Xu and Yoon Jung Kim).

Sources of Funding

H.A.S. is supported by grants from the NIH (1R01HL115275, 5R01H2131778, 1P01HL160476-01A1, R35HL166563-01 and P01HL160488), Hamon Center for Regenerative Science and Medicine, and Leducq Foundation (Redox Regulation of Cardiomyocyte Renewal).

N.T.L. was supported by a Haberecht Wildhare-Idea Research Grant, an American Australian Association ‘Sir Keith Murdoch’ Australia to US Fellowship and an American Heart Association’s Second Century Early Faculty Independence Award (24SCEFIA1252824).

Nonstandard Abbreviations and Acronyms

αMHC: α-myosin heavy chain
ANOVA: Analysis of variance
Aurka: Aurora kinase A
Aurkb: Aurora b kinase
BrdU: Bromodeoxyuridine
Dox: doxycycline
Ect2: epithelial cell-transformation sequence 2
GAPDH: Glyceraldehyde 3-phosphate dehydrogenase
GDP: guanosine diphosphate
GFP: Green fluorescent protein
GTP: guanosine triphosphate
HW/BW: heart weight/body weight
IACUC: Institutional Animal Care and Use Committee
Kif23: kinesin family member 23
MADM: mosaic analysis with double markers
MI: Myocardial infarction
MNDCMs: mononuclear diploid cardiomyocytes
modRNA: modified mRNA
ORFs: Open Reading Frames
pH3: phospho-histone H3 at Ser10
Plk1: polo-like kinase 1
Racgap1: Rac GTPase-activating protein 1
RFP: Red fluorescent protein
s.e.m.: standard error of the mean
T2A: thosea asigna virus 2A
TRE: tetracycline response element
WGA: Wheat germ agglutinin

Footnotes

Disclosures

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Publication Material

NIHMS2051079-supplement-Supplemental_Publication_Material.pdf^{(48.1MB, pdf)}

Uncroppoed Gel Blots

NIHMS2051079-supplement-Uncroppoed_Gel_Blots.pdf^{(1.7MB, pdf)}

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PERMALINK

Induced Cytokinesis Generates Highly Proliferative Mononuclear Cardiomyocytes at the Expense of Contractility

Nicholas T Lam, PhD

Ngoc Uyen Nhi Nguyen, PhD

Waleed M Elhelaly, MD, PhD

Ching-Cheng Hsu, PhD

Ivan Menendez-Montes, PhD

Feng Xiao, PhD

Shah R Ali, MD

Nelson Vo

Nathan Briard

Lobna El-Feky

Qamar M Omari

Alisson C Cardoso, PhD

Yan Liu, PhD

Mahmoud Salama Ahmed, PhD

Shujuan Li, MD

Suwannee Thet, MS

Chao Xing, PhD

Lior Zangi, PhD

Hesham A Sadek, MD, PhD

Abstract

Background:

Methods:

Results:

Conclusions:

INTRODUCTION

METHODS

Statistical Analyses

RESULTS

Plk1(T210D) is more potent than Plk1 in promoting cardiomyocyte proliferation.

Figure 1. Plk1(T210D) is more potent than Plk1 in promoting cardiomyocyte proliferation.

Ect2 exclusively promotes cytokinesis in cardiomyocytes.

Figure 2. Ect2 exclusively promotes cytokinesis in cardiomyocytes.

Concomitant expression of Plk1(T210D) and Ect2 induces robust cardiomyocyte proliferation and prevents binucleation.

Figure 3. Concomitant expression of Plk1(T210D) and Ect2 induces robust cardiomyocyte proliferation and prevents binucleation.

Induction of Plk1(T210D) and Ect2 in adult cardiomyocytes promotes proliferation and cytokinesis.

Figure 4. Transient induction of Plk1(T210D) and Ect2 in adult cardiomyocytes promotes proliferation and leads to a reversible decrease in ejection fraction and increases total and mononuclear cardiomyocytes.

Transient induction of Plk1(T210D) and Ect2 in adult cardiomyocytes lead to a reversible decrease in ejection fraction and increase in total and mononucleated cardiomyocytes.

Intracardiac injections of modRNA encoding Plk1(T210D) and Ect2 in adult hearts promotes cytokinesis in cardiomyocytes.

Induction of Plk1(T210D) and Ect2 in cardiomyocytes improves ejection fraction following myocardial infarction in adult mice

Figure 5. Transient induction of Plk1(T210D) and Ect2 in adult cardiomyocytes promotes cardiomyocyte proliferation and cytokinesis, and improves ejection fraction after myocardial infarction.

DISCUSSION

Supplementary Material

CLINICAL PERSPECTIVE.

What Is New?

What Are the Clinical Implications?

Acknowledgements

Sources of Funding

Nonstandard Abbreviations and Acronyms

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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