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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01159

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Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes

Preeti Ahuja, Evelyne Perriard, Jean-Claude Perriard^* and Elisabeth Ehler

Institute of Cell Biology, Swiss Federal Institute of Technology, ETH Hönggerberg, 8093 Zurich, Switzerland

View larger version (90K): [in a new window]   Fig. 1. Single confocal sections of immunostained cultured embryonic cardiomyocytes at different cell division stages showing the degree of disassembly of the myofibrils with an antibody against the Z-disk protein -actinin (A,D,G,J,M). The spindle apparatus is visualized by staining for tubulin (C,F,I,L,O), while dividing cells can be identified by staining with an antibody that specifically recognizes phosphorylated histone H3 (B,E,H,K,N). In prophase, when the nuclear membrane is still intact, clear cross-striations can be seen for -actinin (A, right hand cell). During metaphase, when the condensed chromosomes are arranged in the middle of the cell, the signal for -actinin becomes diffuse (D) and stays like that during early anaphase (G), when the chromosomes start to be pulled towards the poles, telophase (J) and early cytokinesis (A, left hand cell), when the signal for phosphorylated histone H3 disappears. Reassembly of -actinin to a cross-striated pattern starts in late cytokinesis (M). The arrow in (O) indicates, where the daughter cells are being pinched off. Bar represents 10 µm.

View larger version (32K): [in a new window]   Fig. 2. Single confocal sections showing the comparison of the disassembly level of various sarcomeric proteins present in cultured cardiomyocytes during metaphase (B,D,F,H,J,L,N). While cardiac -actin (E) as well as Z-disk associated epitopes like -actinin (A) and titin T12 (C) display a mostly diffuse staining pattern throughout the entire cytoplasm already at this stage; sarcomeric myosin heavy chain (G), myosin binding protein-C (I) and M-band-associated epitopes, such as myomesin (K) and titin T51 (M), show an intact localization pattern. Bar represents 10 µm.

View larger version (69K): [in a new window]   Fig. 3. Localization pattern of different sarcomeric proteins in cultured cardiomyocytes during later stages of mitosis in single confocal sections. Late metaphase/early anaphase was visualized by staining for phosphorylated histone H3 (D-F), the later stages of mitosis by staining for tubulin (J-L and P-R). Staining for the sarcomeric protein cardiac -actin is shown in the left column, in the middle is myomesin and on the right is sarcomeric myosin heavy chain, as indicated above. While cardiac -actin (A) is completely diffuse at late metaphase, myomesin (B) and sarcomeric myosin heavy chain (C) only start to disassemble at this stage (small arrowheads point at still intact myofibrils). Only when chromosomes are being segregated, cardiac -actin (G), myomesin (H) as well as myosin heavy chain (I) show a diffuse pattern with some remaining aggregates, especially in the case of the latter. During cytokinesis, as identified by tubulin concentration in the midbody (P-R), all sarcomeric proteins start reassembly to myofibrils and display a cross-striated pattern again (M-O). The arrows point at the dividing cells. Bar represents 10 µm.

View larger version (91K): [in a new window]   Fig. 4. Sequential disassembly of different parts of the sarcomere analyzed in single confocal sections. Embryonic rat cardiomyocytes were stained for -actinin (B,E,H,K; red in overlay A,D,G,J) and for MyBP-C (C,F,I,L; green in overlay) as indicated above the columns. The stage of cell division was identified by staining for tubulin (blue in overlay). In the interphase cell, clear cross-striations can be seen for both sarcomeric proteins. At metaphase the localization pattern of -actinin is already diffuse (arrow), while double bands are still visible for MyBP-C (arrowheads), which only redistributes by the anaphase and telophase stage. Bar represents 10 µm.

View larger version (127K): [in a new window]   Fig. 5. Single confocal sections of whole mount preparations of embryonic mouse hearts labelled with antibodies against different sarcomeric proteins (B,E,H,K; red in overlay A,D,G,J) and against phosphorylated histone H3 together with antibodies against beta-catenin to delineate the cell-cell contacts (C,F,I,L; green in overlay). Also in dividing cardiomyocytes in the heart in situ, -actinin (A,B) and titin T12 (D,E), which are Z-disk associated proteins/epitopes, are diffuse at a time when the localization pattern of myomesin (G,H) remains still quite intact (arrowheads delineate the cell borders, small arrows point at intact myofibrils in neighboring cardiomyocytes (D,E; J,K) or in dividing cells (G,H). The staining for titin T51 (J,K), which is an M-band associated titin epitope, becomes diffuse only by anaphase, similar to myomesin (data not shown). Continuous staining for beta-catenin along the plasma membrane also in dividing cardiomyocytes indicates that cardiomyocytes retain their contacts to the neighboring cells during division. Bar represents 10 µm.

View larger version (67K): [in a new window]   Fig. 6. Expression of ubiquitin is upregulated in dividing cardiomyocytes. Cultured cardiomyocytes were stained with antibodies to sarcomeric -actinin (A,D,G) together with antibodies against ubiquitin (B,E,H) and against tubulin (C,F,I) to identify the stage of cell division. Single confocal section reveal that while interphase cardiomyocytes display only little signal for ubiquitin (B); a big increase in the signal for ubiquitin can be detected in cardiomyocytes in metaphase (E) as well as in cytokinesis (H). There is no clear-cut colocalization between -actinin and ubiquitin. Bar represents 10 µm.

View larger version (117K): [in a new window]   Fig. 7. Disassembly of myofibrils is delayed in cardiomyocytes that were treated with MG132 to inhibit proteasome degradation. Single confocal sections of cardiomyocytes stained with monoclonal antibodies to sarcomeric -actinin (B,D; red in A,C) and for tubulin (blue in A,C) as well as for phosphorylated histone (green in A,C) to identify the stage of mitosis. While in control cells at metaphase all the -actinin is localized in a diffuse fashion throughout the entire cell (A,B), cross-striated myofibrils can still be seen in MG132 treated cardiomyocytes (C,D). Bar represents 10 µm.

View larger version (31K): [in a new window]   Fig. 8. Sarcomere disassembly during cell division. Thick (myosin and associated proteins) filaments are represented in dark blue, thin (actin and associated proteins) filaments in yellow and titin filaments in red. The Z-disk is shown in green, the M-band in purple. In metaphase, Z-disk associated proteins are already disassembled (indicated by font in italics), while the thick filaments and the M-band still remain intact, only to be disassembled in late anaphase.