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Review
. 2022 Mar 3;9(3):73.
doi: 10.3390/jcdd9030073.

In Vivo Methods to Monitor Cardiomyocyte Proliferation

Alexander Young  1 , Leigh A Bradley  1 , Matthew J Wolf  1
Affiliations
Review

In Vivo Methods to Monitor Cardiomyocyte Proliferation

Alexander Young et al. J Cardiovasc Dev Dis. .

Abstract

Adult mammalian cardiomyocytes demonstrate scarce cycling and even lower proliferation rates in response to injury. Signals that enhance cardiomyocyte proliferation after injury will be groundbreaking, address unmet clinical needs, and represent new strategies to treat cardiovascular diseases. In vivo methods to monitor cardiomyocyte proliferation are critical to addressing this challenge. Fortunately, advances in transgenic approaches provide sophisticated techniques to quantify cardiomyocyte cycling and proliferation.

Keywords: cardiomyocyte cycling; heart; mouse; regeneration; transgenics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Simplified overview of cardiomyocyte cycling. After G2, failed karyokinesis (nuclear division) produces a single nucleus with >2N genomic DNA content. After mitosis and karyokinesis failed cytokinesis produces a binucleated cell. Further endomitosis of binucleated (on multinucleated) cells produces nuclei with >2N genomic content.
Figure 2
Figure 2
Detecting “Actively cycling” and “Previously cycled” cells. Events represent cycling cells. After an injury, there is an increase and decrease in cycling cells. (A) The “snapshot” approach measures cycling events in blue at a distinct time point after the injury. Typically, cycling cells are detected by the co-localization of markers of cycling (Ki67, PH3, Aurora B, and Anillin) with cardiomyocytes or transgenic mice that report phases of the cell cycle (FUCCI, Ki67-RFP, eGFP-Anillin). (B) The “summation” approach relies on indelibly marking cycling events and measuring total events after an injury. Cycled cells are labeled by incorporating nucleotide analogs into synthesizing genomic DNA (3H-Thymidine, Bromodeoxyuridine, or 5-Ethynyl-2-deoxyuridine) or transgenic mice that mark cells that cycled or underwent mitosis (MADM, Confetti/Brainbow, and αDKRC::RLTG). Figure adapted from Bradley et al., Circ Res 2021, 128, 155–168.
Figure 3
Figure 3
Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) reporters. First-generation FUCCI (A) and FUCCI2 (B). Second-generation Bidirectional R26p-FUCCI2 (C) and polycistronic FUCCI2aR (D). (E) Schematic of cell cycle phase-dependent differential expression of FUCCIs.
Figure 4
Figure 4
Ki67iresCreER mice. Example of Ki67iresCreER::CAG-Lox-STOP-Lox-tdTomato mice. In the presence of tamoxifen, cells undergoing cycling as defined by Ki67 expression have activated Cre recombinase. The activated Cre excises a STOP cassette allowing tdTomato expression under the control of the constitutively active CAG promoter.
Figure 5
Figure 5
CAG-eGFP-Anillin. The subcellular localization of constitutive expression of eGFP-Anillin marks cells that undergo cytokinesis by labeling the mid-body. Figure adapted from Hesse, M. et al., Nat Commun 2012, 3, 1076.
Figure 6
Figure 6
CAG-loxP-STOP-loxP-Confetti (Brainbow). (A) The tamoxifen-induced expression of Cre-dependent causes stochastic recombination of a multi-fluorescent reporter. Identifying of clusters of cells expressing the same fluorescent reporters are used to infer clonal expansion. (B) Schematic of the interpretation of Confetti expression used to identify random stochastic events (left) and clonal expansion consistent with proliferation (right).
Figure 7
Figure 7
Mosaic Analysis with Double Markers (MADM). (A) Schematic of MADM-11GT and MADM-11TG transgenes that harbor spilt GFP and dsRed constructs with intronic LoxP sites. (B) Tamoxifen exposure induces the Cre recombinase expression that catalyzes an inter-chromosomal rearrangement of split fluorescent reporters and labels mitotic events. (C) Recombination during G1 or G1 produces double colored “Yellow” cells. The detection of “Green” or “Red” cells in panel (B) allows the quantification of mitotic events. Figure adapted from Zong, H. et al., Cell 2005, 121, 479–492.
Figure 8
Figure 8
αDKRC::RLTG. (A) The αDKRC transgene. (B) Schematic outlining the expression of GFP in adult cycling cardiomyocytes. Tamoxifen exposure induces the cardiomyocyte-specific Dre-recombinase and subsequent excision of derived poly(A) signal repeats and flanked by two loxP sites from the RLTG reporter, resulting in the expression of tdTomato and RoxedCre to generate catalytically active Cre recombinase under control to the Ki67 cell cycle promoter. Cardiomyocytes reentering the cell cycle, as defined by activation of the Ki67 promoter, express Cre and excise the tdTomato-STOP cassette resulting in the expression of eGFP (enhanced green fluorescent proteins) in cardiomyocytes. A short duration of Tamoxifen exposure activates that reporter system. Schematic of expected changes in tdTomato and eGFP-labeled cardiomyocytes. Figure adapted from Bradley et al., Circ Res 2021, 128, 155–168.
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