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First published online 31 October 2006
doi: 10.1242/jcs.03252

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Re-programming of newt cardiomyocytes is induced by tissue regeneration

¹ Institute of Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Hollystrasse 1, 06097 Halle, Germany
² Max-Planck-Institute for Heart and Lung Research, Department of Cardiac Development and Remodelling, Parkstrasse 1, 61231 Bad Nauheim, Germany

View larger version (121K): [in a new window]   Fig. 1. Cardiac injury in newts results in a transient downregulation of contractile proteins. Immunofluorescence staining (green) of sham operated (A,B) and damaged newt hearts (C-F) for MyHC (A,C,E) and cTnT (B,D,F) 1 (C,D) and 14 days (E,F) after mechanical injury. The white dashed lines in C and D indicate the margins of the hearts. Note the massive and widespread downregulation of MyHC and cTnT, which corresponded to the degree of damage exerted to the heart. 14 days after the injury cardiomyocytes in the injured areas re-expressed MyHC and cTnT (E,F). A Hoechst 33342 counterstaining was used to label all nuclei on the sections.

View larger version (40K): [in a new window]   Fig. 2. Transient downregulation of numerous cardiac-specific genes after cardiac injury in newts. RT-PCR of RNA isolated from sham-operated animals (sham), and 7, 14 and 21 days after injury (7d pi, 14d pi, 21d pi). Two separate hearts were analyzed for each time point. Note that hearts shown in lanes 4, 6, and 8 received a more extensive damage than those shown in lanes 5, 7 and 9. The expression of MyHC, cTnT, cTnI, cTnC and cardiac actin was downregulated 1 week after heart injury but returned to levels of sham-operated hearts within three weeks. 40S ribosomal protein S21 was expressed at constant levels during regeneration and served as an internal control. Lane 1, molecular mass marker; lanes 2 and 3, sham operated hearts; lanes 4 and 5, 7 days post injury (pi); lanes 6 and 7, 14 days pi; lanes 8 and 9, 21 days pi.

View larger version (85K): [in a new window]   Fig. 3. Cardiomyocytes maintain the expression of heart-muscle-specific genes after transplantation into intact limbs and under culture conditions. (A,B,D,E,G,H) Stained cryosections. DiI-labeled cardiomyocytes (red) transplanted into intact limb maintained the expression of the cardiac-muscle-specific cTnT (A,B,G,H; green). (C,F,I) Newt cardiomyocytes in culture expressed (C) cTnT and (F,I) MyHC (MF20-staining). Sections and cultures were stained with Hoechst 33342 (blue) to visualize all nuclei.

View larger version (95K): [in a new window]   Fig. 4. Transplantation of newt cardiomyocytes into regenerating limbs results in a rapid downregulation of heart-muscle-specific genes. (A-L) DiI-labeled cardiomyocytes (A-F, red) were transplanted into a regenerating limb 5 days after amputation and staining for desmin (G-I, green) or MyHC (J-L, green) after 1, 7 and 28 days. Newt cardiomyocytes maintained the expression of the intermediary filament protein desmin in the majority of transplanted cells but rapidly lost the expression of MyHC (MF20 staining; note that MF20 detects both skeletal and cardiac MyHC isoforms). After 28 days, transplanted cells re-expressed MyHC. (M-O,P-R) Mergend images. Bars, 100 µm.

View larger version (91K): [in a new window]   Fig. 5. Newt cardiomyocytes rapidly loose the expression of cardiac specific proteins after transplantation in regenerating but not in intact limbs. Western blot analysis of protein extracts prepared from skeletal muscle (M, lane 1), heart (H, lane 2), and cultured cardiomyocytes (C, lane 3) as well as injured and intact limbs (lanes 4-6 and 7-9, respectively) at different time points (2, 14 and 28 days) after transplantation of cardiomyocytes. Note the absence of cTnT after transplantation into injured but not intact limbs.

View larger version (65K): [in a new window]   Fig. 6. Cardiomyocytes that undergo blastema-induced re-programming transiently express blastema cell markers and activate focal adhesion kinase at later stages. (A-L,N-P) Immunofluorescence staining of DiI-labeled cardiomyocytes (red) with vimentin (A,B,F,G,K,L), the blastema cell marker 22/18 (C,D,H,I,N,O) and FAK-Tyr397-P (E,J,P) 1 day and 30 days after transplantation. Bound antibodies on cryosections were detected with FITC-conjugated secondary antibody (green). Hoechst 33342 counterstaining was used to label all nuclei on the sections. Notice that, vimentin and 22/18 are expressed 1 day but not 30 days after transplantation. Activation of FAK occured simultaneously in transplanted and neighboring cells indicating successful integration into the recipient tissue. Bars, 50 µm.

View larger version (89K): [in a new window]   Fig. 7. Newt cardiomyocytes transdifferentiate into skeletal muscle cells in regenerating newt limbs. Immunofluorescence staining of DiI-labeled cardiomyocytes (red) with anti the skeletal-muscle-specific anti-sTnI antibody (A,B,I,J; green) and with the cardiac-muscle-specific anti-cTnT antibody (C,D,K,L; green). Hoechst 33342 counterstaining was used to localize all nuclei on the cryosections. Inlets in C and D, are positive controls for cTNT staining using sections derived from the heart. Note the rapid downregulation of cTnT in transplanted cardiomyocytes and the upregulation of skeletal-muscle-specific proteins in DiI-labeled cells 15 days after transplantation. The formation of cardiomyocyte-derived skeletal myotubes are clearly visible 30 days after transplantation. Bars, 100 µm.

View larger version (55K): [in a new window]   Fig. 8. Newt cardiomyocytes re-enter the cell-cycle in vitro and in vivo and rarely undergo programmed cell death after transplantation. Immunofluorescence staining of phosphorylated histone H3 in cultured cardiomyocytes 24 hours (A,F,K) and 72 hours (B,G,L) after isolation. Cells were labeled with MyHC (red, MF20 staining), anti-phosphorylated histone H3 (green, appears cyan due to color overlay) and Hoechst 33342 to indicate all nuclei. White arrows indicate cells in G2. Immunofluorescent detection of phosphorylated histone H3 in cardiomyocytes 24 hours after transplantation (C,H,M). Cardiomyocytes were DiI-labeled before transplantation and stained with anti-phosphorylated histone H3 (green fluorescence, appears cyan due to color overlay) and Hoechst 33342 to indicate all nuclei. White arrows in (C,H,M) indicate cardiomyocyte-derived cells in G2, yellow arrow indicates a limb-derived cell in G2. TUNEL-labeling of DiI-labeled cardiomyocytes 24 hours after transplantation (D,I,N). Note that transplanted cardiomyocytes only rarely underwent programmed cell death (white arrow). (E,J,O) Cycling cells in regenerating limbs; cells were labeled with MyHC (red, MF20 staining), anti anti-phosphorylated histone H3 (green, appears cyan due to color overlay) and Hoechst 33342 to visualize all nuclei. Note that cell-cycle progression in the regenerating limb occurred both in the skeletal myotubes (red) and in the blastema.

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