Mammals cannot regenerate their hearts despite that some studies have reported that newborn mice can regenerate the heart apex if it is amputated during their first days of life. Also, in some special pathological or physiological conditions adult cardiomyocytes can be forced to re-enter the cell cycle and/ or dedifferentiate. However, these responses cannot sustain the proper regeneration that should take place after, for example, an acute myocardial infarction. In contrast, zebrafish cardiomyocytes retain their ability to dedifferentiate and re-enter the cell cycle throughout their lives, which explain the amazing capacity of these animals to regenerate a large portion of their hearts. Experimentally, regeneration studies in the zebrafish heart are carried out after either amputation or cryoinjury.
Now a recent paper from the laboratory of Anna Jazwinska reports on the spatial and temporal dynamics of cell proliferation and differentiation during cryoinjury-induced heart regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25557620). The authors first used MCM5 as a marker of the G1/S phase of the cell cycle and phospho histone H3 (PH3) to label mitotic cells in G2/M. Moreover, they used these markers on transgenic zebrafish expressing GFP under the control of the cardiac specific promoter cmlc2. During regeneration the initial fibrotic tissue was progressively replaced with new myocardium, which resulted in the decrease of the infarcted area relative to the entire ventricle. As expected, regeneration was characterized by an increase in cardiomyocytes (CM) proliferation, quite evident by 7 dpci (days post-cryoinjury). During the progression phase at 17 dpci the number of proliferating cells declined and at 30 dpci (termination phase) the mitotic levels had declined up to the levels in uninjured fish. However, at this late stage, the number of MCM5-positive CM still remained significantly higher compared to uninjured animals.
Then, the authors determined the spatial distribution of the dividing CMs relative to the injury border. During the initiation phase (7 dpci) there was a graded distribution of PH3 mitotic cells. Thus, 50% of the PH3 cells were within the first 100 mm adjacent to the cryoinjury border; 20% of them were found between 100 and 200 mm from the border, and the remaining 30% were distributed from the 200 to 1000 mm. Thus, most of the mitotic cells were located at the close vicinity of the wounded area. This graded distribution of mitotic cells was not detected during the progression phase (at 17 dpci) when the PH3 cells were nearly evenly spaced within the entire myocardium. It is important to point out that at this later stage the number of PH3 cells was still significantly higher compared to an uninjured heart. Next the authors performed some BrdU labelling and found out, as expected, that the majority (65%) of CMs located in the regenerated myocardium were BrdU-positive, indicating that they derive from newly generated cells. In the adjacent region (0-100 mm from the regenerated myocardium) about 30% of the CMS were BrdU-positive. Beyond this zone and in all the subregions that the authors checked they found an even percentage of BrdU-positive CMs (about 10%), that again was higher compared to uninjured hearts. Overall, these results suggest that in addition to a local rapid and strong proliferative response to cryoinjury there was also a systemic response all throughout the pre-existing heart.
Finally, the authors analysed the formation of immature CMS during regeneration. To do that they took advantage of an antibody raised against a mammalian neonatal muscle and that binds the N-terminal end of slow b/cardiac myosin heavy chain. In zebrafish this antibody seemed to label embryonic undifferentiated CMs. During regeneration (at 7-10 dpci) the authors found a significant number of CMs positive for this antibody at the injury border within a distance up to 100 mm from the post-infarcted tissue. No labelled cells were found in regions beyond this zone. During the progression stage (17 dpci) these positive CMs lost their initial homogenous alignment along the injury border and intermingled with the regenerated mature cardiac tissue. At 30 dpci almost no labelling was detected. Altogether, the authors concluded that during the initial phase of regeneration, undifferentiated CMS appear at the leading edge of the dedifferentiating adult myocardium and would provide a source of new CMs during regeneration.
In summary, the authors propose that upon cryoinjury a local epimorphic regenerative response is triggered within the 100-200 mm adjacent to the injury border and in which a significant increase in immature and proliferating CMs was detected. This initial response in this blastema-like region would be further reinforced by a systemic compensatory regenerative response within the entire pre-existing myocardium.