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First published online 19 August 2003
doi: 10.1242/jcs.00698

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Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes

Mónica Bettencourt-Dias1,*,{ddagger}, Sybille Mittnacht2 and Jeremy P. Brockes1

1 Department of Biochemistry, University College London, London WC1E 6BT, UK
2 Centre for Cell and Molecular Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, UK



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  		Fig. 1. Adult ventricular cardiomyocytes in culture. (A) Micrograph of an isolated cardiomyocyte in suspension after dissociation. Note the branched morphology and striated myofibrils. (B,C) Isolated cardiomyocytes after plating. Note the cross striations and expression of the myofibril markers MyHC (green, B and C) and troponin (red, B) or titin (red, C), as visualised by double label indirect immunofluorescence. (D) An interconnected group of cells, at 8 days after plating onto laminin, that beat in synchrony. (E,F) More than 90% of the cells in the culture are cardiomyocytes; expression of the myofibril markers troponin (E) and MyHC (F) at 8 days after plating (DNA stained with Hoechst 33258 in blue). Scale bar: 50 µm.

 


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  		Fig. 2. Cardiomyocytes undergo DNA synthesis and mitosis. Cells were pulsed with BrdU for 9 hours at various times after plating and then fixed and stained for BrdU, MyHC and DNA. Parallel wells were stained for phosphorylated histone H3 (P-H3), MyHC and DNA. (A,B) Labelling of cardiomyocytes with BrdU. Confocal micrograph of two cells stained for BrdU (red), DNA (Hoechst 33258, blue) and MyHC (green). Note that the lower cardiomyocyte has incorporated BrdU. (C) Labelling of cardiomyocytes with an antibody against phosphorylated histone H3 (red) and MyHC (green). Myofibrils are excluded from the central region where mitosis is occurring. (D) Time course of entry into S phase and mitosis. Note that there is a peak of cells undergoing S phase and mitosis at 10 days. The results are the average of duplicate dishes. (E1-E4) Time lapse of a cardiomyocyte undergoing mitosis. Both daughter cells were contracting at 145 minutes (E4). An outline of the cell is seen in the right, lower side of each picture. The time elapsed is shown in the left top side. Note the myofibrils (yellow arrow) and the chromosomes (blue arrow). Red arrow points to the cleavage furrow; N, nuclei. Scale bar: 50 µm (in C and E4).

 


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  		Fig. 3. Analysis of cycle progression by time lapse microscopy. Cells were plated out in a grid, photographed as described in Materials and Methods, then fixed and stained at 18 days. (A,B) Cardiomyocytes may divide more than once. (A) A field where a cardiomyocyte divided twice and another did not divide. The number at the top right of each picture shows the number of days elapsed since the cells were plated. A schematic drawing of the field is shown on the top left part of the figure. Note that the cell on the left at 8 days gives rise to 2 daughter cells at 10 days. Each of the daughter cells divides once more at 12 days. All the progeny cells stain positive for MyHC (lower right), but the staining is less strong and organised (red arrow) than in the non-dividing cell (white arrow). (B) Tree diagrams (one per starting cell) for the proliferation of cardiomyocytes which divided more than once giving rise to mononucleate progeny. Cells that divided only once (n=17) are not represented. Pedigrees were constructed from the time-lapse analysis of three independent cultures and they illustrate the diversity observed. All the cells were followed for 18 days (to simplify the scheme, lines for each cell stop in the last division observed during the 18 days). The arrows indicate clones which continued to divide; using this experimental set-up it was impossible to trace those divisions as accurately as all the others represented. The cross indicates that a cell died. (C) Example of a field where a cardiomyocyte underwent acytokinetic mitosis, thus becoming multinucleate. (D) Same experiment as in B. Pedigrees of cardiomyocytes that gave rise to multinucleate progeny. Cells that underwent only one cycle are not represented. Note that the mitotic division of a multinucleate cell can have several different outcomes. Scale bars: 50 µm.

 


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  		Fig. 4. Fate of single cardiomyocytes. One cell per grid square was labelled with the red fluorescent tracker dye, PKH-26 (see Materials and Methods), and cultured for 24 days prior to fixation and analysis. There were a total of 238 initial cells from three independent experiments. (A,B) Confocal micrographs of squares where two daughter cells (A) or a binucleate cell (B) were observed. Staining for MyHC is in green and DNA (Hoechst 33258) in blue. Scale bar: 50 µm.

 


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  		Fig. 5. Regulation of S phase entry in newt cardiomyocytes. (A) Dependence of entry on serum concentration. Cells were exposed to various concentrations of FBS, as described in Materials and Methods, and pulsed with BrdU 4 days later for 8 hours. Each point is from a separate dish of the same culture, and the peak at approximately 10% FBS was observed in three other comparable experiments. (B,C) Detection of Rb in cardiomyocytes by indirect immunofluorescence with an antibody recognising phosphoserine 608. The cells were stained with anti-MyHC and Hoechst 33258 (DNA; B), anti-phosphoserine 608 (in Rb; C). In control experiments the intensity of nuclear staining was significantly diminished by digestion of fixed cells with lambda phosphatase prior to reaction with the antibody (not shown). Note that most of the nuclei in B are positive for the Rb phosphorylated epitope in C. Scale bar: 50 µm.

 


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  		Fig. 6. Model of cell cycle progression by adult newt cardiomyocytes in culture. This incorporates, into a diagram, the various results described in the text. This figure should be seen only as a model based on the data, since it incorporates data from different experiments (time lapse analysis; dose response assay for FBS; Rb phosphorylation and inhibition of re-entry by p16INK4). Note that all percentages are given with reference to the starting population of cardiomyocytes. Although our data does not rule out the possibility that another member of the pocket-protein family may also mediate the regulation of cell cycle entry, to date no other members of this family have been cloned in amphibia.

 

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© The Company of Biologists Ltd 2003