Author + information
- Peter Pfeiffer, MD⁎ (, )
- Patrick Müller, MD,
- Andrey Kazakov, MD,
- Ingrid Kindermann, MD and
- Michael Böhm, MD
- ↵⁎Klinik für Innere Medizin III, Kardiologie, Angiologie, und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, Kirrberger Strasse, 66421 Homburg/Saar, Germany
To the Editor:Recently, the view of the heart as a terminally differentiated organ has been challenged. Cardiomyocytes of recipient origin have been detected in patients who have received gender-mismatched heart (1,2), bone marrow (3–5), and peripheral blood stem cell (4) transplants and have been shown to be able to proliferate (1). However, the numbers of newly formed cells in these studies were extremely variable. One possible explanation was the interval between transplantation and tissue examination (2). We hypothesized that cardiac regeneration might increase rather than decrease over time because after cardiac transplantation and possibly in cardiac disease in general, myocyte loss and regeneration might require some time. To test this hypothesis, we performed a longitudinal chimerism study on gender-mismatched male transplant recipients using immunostaining and fluorescence in situ hybridization (FISH) to detect recipient cells. Moreover, the proliferative capacity of cardiomyocytes in early and later obtained biopsy samples was assessed by Ki67 expression.
Serial right ventricular myocardial biopsy samples (n = 10) were routinely obtained from patients after gender-mismatched cardiac transplantation during normal post-transplantation follow-up. The first biopsy samples were obtained at 6 to 14 days, and other biopsy samples at 3.5 to 13 months after transplantation. Three men with male cardiac allograft transplants as well as 2 women with gender-matched heart transplants were taken as positive and negative controls for the detection of X and Y chromosomes, respectively. Immunosuppressive medication was based on standard protocols. Age at transplantation, interval between transplantation and biopsy examination, and acute rejection according to the International Society for Heart and Lung Transplantation scale are shown in Table 1.All biopsy samples were fixed in 4% buffered formalin, embedded in paraffin, and cut in 6-μm sections.
Immunostaining for cardiomyocytes was performed using antibodies against alpha-sarcomeric actin (Sigma, St. Louis, Missouri) and myoglobin (Dako, Glostrup, Denmark). Coupling between cardiomyocytes was assessed by connexin 43 immunostaining (Sigma). To evaluate proliferative activity in cardiomyocytes, Ki67 (clone MIB-1, Dako) immunostaining was combined with immunostaining for alpha-sarcomeric actin.
For sex chromosome detection, FISH was performed using probes DXZ1 (Oncor, Illkirch, France) for X chromosome and Y3.4 for Y chromosome. Immunostaining and FISH protocols were performed as described previously (2).
Sections were inspected with a Nikon E600 fluorescence microscope (Nikon Europe, Düsseldorf, Germany) and recorded by a Nikon DXM 1200 digital camera. In total, 47,530 nuclei have been examined. The chimerism value was evaluated by dividing the number of Y chromosome-positive cells by the total number of cardiomyocytes. The regeneration rate was obtained by dividing the chimerism value by the hybridization efficiency, which corresponds to the number of Y chromosome-positive cardiomyocytes in the positive controls.
In the positive controls, 52.8% of the cardiomyocytes showed a Y chromosome. Consequently, the regeneration rate was about two times higher than the chimerism rates. In the negative controls, no Y chromosomes were found.
Cardiomyocytes containing Y chromosomes were detected in all biopsy samples obtained from gender-mismatched transplantation patients, in the early as well as in the late cases. Connexin 43 was expressed even in the cardiomyocytes of early biopsy samples (Fig. 1A).The average value of chimeric cardiomyocytes was 0.154 ± 0.043% (mean ± SEM), corresponding well to the value of our previous study (2). Nevertheless, we observed an increase in cardiomyocyte chimerism in the biopsy samples obtained at later time points in every individual patient (Fig. 1B). Taken together, chimerism increased from 0.052 ± 0.01% in the early biopsy samples to 0.256 ± 0.053% in the late biopsy samples (p < 0.02, paired ttest) (Fig. 1C), which corresponds to an increase of from 0.098 ± 0.018% to 0.485 ± 0.101% newly formed cardiomyocytes taken into consideration the hybridization efficiency. Cell fusions were excluded using probes for both sex chromosomes.
Expression of Ki67 was very rare in the early biopsy samples as well as in the late ones. Altogether, only five cardiomyocyte nuclei expressed Ki67 (Figs. 1D and 1F), indicating that 0.031% of all cardiomyocytes showed evidence for proliferation. Only one of five positive nuclei originated from the late biopsy of a patient; the others were derived from the early biopsy of another patient. Eight of 10 biopsy samples did not show any proliferating cardiomyocytes. The chromosomal status of the Ki67-positive nuclei was non-uniform. Three of five nuclei expressed a Y chromosome (Fig. 1E), the others expressed two X chromosomes (Fig. 1G).
The mean size of host- and graft-derived cardiomyocytes in both early and later obtained biopsy samples did not differ significantly. However, Ki67-positive cardiomyocytes had a significant smaller cross-sectional area than Ki67-negative donor- or graft-derived cardiomyocytes (133.68 ± 39.22 μm2vs. 533.69 ± 22.68 μm2; p < 0.001 Mann-Whitney test).
It was suggested that cardiac regeneration initially increases after transplantation, but decreases after several months (2) because resettlement of newly formed cardiomyocytes might be a temporary effect, followed by apoptosis, a well-known mechanism in the pathogenesis of heart failure (6). However, the presented longitudinal study in living patients shows in fact a significant increase in regeneration rates over time, corresponding to another recently published autopsy study that showed a slight amount of chimeric cardiomyocytes after bone marrow transplantation (4). Therefore, cardiac regeneration over time might gradually substitute damaged cardiomyocytes after cardiac transplantation.
Because Ki67-positive cardiomyocytes tend to be smaller than non-proliferating myocytes, they are thought to be young cells that might arise from stem cell differentiation (7). Consequently, if newly formed cardiomyocytes were present in the biopsy samples obtained a few days after transplantation, they would be expected to express Ki67 rather than donor-derived cardiomyocytes, in which the probability to be terminally differentiated is much higher. Furthermore, in cardiac transplants, temporary ischemia could be a stimulus for proliferation. Indeed, 3 of 5 Ki67-positive cardiomyocytes were of recipient origin. However, 8 of 10 biopsy samples did not show any proliferating cardiomyocytes, and overall expression in all biopsy samples was low (0.031%). Size and shape of host-derived cardiomyocytes were also undistinguishable from donor-derived cells. These results indicate that most of the newly formed cardiomyocytes are already terminally differentiated or at least did not proliferate at the time examined. In addition, even newly formed cardiomyocytes in early biopsy samples expressed connexin 43, providing evidence for a functional coupling to adjacent myocytes in the transplanted myocardial tissue. The origin of the newly formed cells might be the bone marrow (3–5). However, because in orthotopic cardiac transplantation the atrial stumps of the donors remain in the thorax, host-derived resident cardiac progenitor cells might also contribute to cardiac regeneration.
Recapitulating, our results illustrate a small but persistent increase in cardiac regeneration. This raises hope that this process could be accelerated and therefore used therapeutically, e.g., by growth factor application or autologous stem cell transplantation. However, the number of regenerated cells is still low, and studies on transplanted myocardium after repetitive rejections or myocardial infarctions will show the biological relevance of cardiac chimerism.
The authors thank Wolfgang Feiden, MD, from the local Institute of Neuropathology, who provided the paraffin sections and histological diagnosis concerning rejection episodes. Julia Michaely supported the authors with excellent technical assistance.
Please note: Supported by a grant of the Ernst und Berta Grimmke-Stiftung, Düsseldorf, Germany. Drs. Pfeiffer and Müller contributed equally to this paper.
- American College of Cardiology Foundation
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