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Pseudo-bipolar spindle formation and cell division in postnatal binucleated cardiomyocytes

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Abstract

Background

The majority of adult human, mouse and rat cardiomyocytes is not diploid mononucleated. Nevertheless, the current literature on heart regeneration based on cardiomyocyte proliferation focuses mainly on the proliferation capacity of diploid mononucleated cardiomyocytes, instead of the more abundant mononucleated polyploid or binucleated cardiomyocytes. Here, we aimed at a better understanding of the process of mitosis and cell division in postnatal binucleated cardiomyocytes.

Methods and results

Postnatal rat binucleated cardiomyocytes were stimulated to re-enter the cell cycle either by fetal bovine serum or a combination of fibroblast growth factor 1 and p38 MAP kinase inhibitor. Phase-contrast videos revealed that binucleated cardiomyocytes form one metaphase plate and preferentially undergo afterwards cytokinesis failure. The maximum rate of cell division of video-recorded binucleated cardiomyocytes was around 6%. Immunofluorescence analyses of centriole number in mitotic binucleated cardiomyocytes revealed that these cells contain more than four centrioles, which can be paired as well as unpaired. In agreement with multiple and/or unpaired centrioles, multipolar spindle formation was observed in mitotic binucleated cardiomyocytes using fluorescence live imaging of tubulin-GFP. Multipoles were transient and resolved into pseudo-bipolar spindles both in case of cell division and cytokinesis failure. Notably, centrioles were in most cases unevenly distributed among daughter cells.

Conclusions

Our results indicate that postnatal binucleated cardiomyocytes upon stimulation can enter mitosis, cope with their multiple and/or unpaired centrioles by forming pseudo-bipolar spindles, and divide.

Introduction

The prevalence of heart failure is still rising despite significant progress in determining risk factors and attempts to prevent or reduce cardiac injury. Thus, tremendous efforts are invested to reverse cardiac damage. Among diverse strategies, induction of cardiomyocyte proliferation is considered a promising future option [1]. Cardiomyocyte proliferation underlies heart regeneration in zebrafish and newt as well as mammalian fetal heart growth [1]. After birth, mammalian cardiomyocytes acquire a post-mitotic status characterized by loss of centrosome integrity and cell cycle progression results in mononucleated polyploid or binucleated cells due to cytokinesis failure. While the number of diploid mononucleated cardiomyocytes in the adult mouse heart is strain dependent, it is established that the number is <10%. Numbers in human hearts are controversial (<5 to ~30%) [2]. The current literature focuses mainly on the proliferation capacity of diploid mononucleated cardiomyocytes to regenerate the heart. As the polyploid cardiomyocyte population is more abundant, it is of interest to determine whether and how binucleated cardiomyocytes can be induced to divide and how often this process occurs. Despite the importance of these findings for heart regenerative purposes, only few laboratories have attempted to address these questions [[3], [4], [5], [6]]. The absence of clear live cell imaging videos leaves ground to misinterpretations in regards to whether and how binucleated cardiomyocyte divide. As evidence exits that a highly ordered sarcomeric apparatus inhibits cardiomyocyte cell division [7], we chose here rat postnatal day 3 (P3) cardiomyocytes to determine if binucleation by itself, independently from the cardiomyocyte maturation state, represents a terminal cell cycle exit preventing induction of cell division. As shown previously, cardiomyocytes exist as mono- and binucleated cells at P3 whereby mononucleated cardiomyocytes can be stimulated to undergo cell division [4]. Here, we have performed phase-contrast and fluorescence live cell imaging of rat P3 cardiomyocyte cultures stimulated with the pro-proliferative factors fetal bovine serum (FBS) or a combination of fibroblast growth factor 1 (FGF1) and p38 MAP kinase inhibitor (p38i) [8]. Our data indicate that P3 binucleated cardiomyocytes can undergo cell division. However, despite the fact that mitotic binucleated cardiomyocytes cope with their multiple and/or unpaired centrioles by forming pseudo-bipolar spindles, the majority of binucleated cardiomyocyte mitoses resulted in cytokinesis failure.

Section snippets

Methods

Detailed methods are available in the online supplement.

Postnatal binucleated cardiomyocytes can divide forming one metaphase plate

Phase-contrast live cell imaging and subsequent immunofluorescence staining revealed that in Sprague Dawley rat P3 binucleated cardiomyocytes both nuclei enter synchronously into mitosis upon 10% FBS or FGF1/p38i stimulation forming one metaphase plate (Fig. 1A, Supplementary information, Movie S1, Movie S2, Movie S3, Movie S4). These data suggest that mitotic binucleated cardiomyocytes form a single bipolar spindle. Further analysis demonstrated that similar to mononucleated cardiomyocytes [8]

Discussion

Binucleated cardiomyocyte mitosis was previously observed in some cardiomyocyte proliferation studies based on still photos and live cell imaging. Evidence has been provided supporting the idea of two synchronous spindles and two cleavage furrows [4,5] as well as formation of one spindle [3,6]. Our live cell imaging videos not only clearly support and verify the formation of a bipolar spindle, but also allowed to gain novel insights into this process. In fact, we have observed that postnatal

Conclusion

In summary, the data indicate that postnatal binucleated cardiomyocytes can divide, overcoming their supernumerary centrosome/centrioles and/or unpaired centrioles by the formation of intermediate multipolar spindles that resolve into pseudo-bipolar spindles.

The following are the supplementary data related to this article.

Acknowledgments

We thank Jennifer Schmidt for technical assistance, Alisa Shaw and James Bamburg (Colorado State University, USA) for the plasmid GFP-tubulin cloned in ShuttleCMV/pAdEasy1, and Robert Becker and Silvia Vergarajauregui for critical reading of the manuscript. This work was supported by the Emerging Fields Initiative Cell “Cycle in Disease and Regeneration (CYDER)” (Friedrich-Alexander-University Erlangen-Nürnberg, FBE), by the German Research Foundation (DFG, INST 410/91-1 FUGG and EN453/12-1,

Author contributions

ML performed all experiments. ML and FBE designed the experiments and wrote the manuscript. ML and FBE analyzed and interpreted the data.

Disclosures

None.

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