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Cell Renewal Demonstrated by Resident Cardiomyocytes

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"Mammalian heart renewal by pre-existing cardiomyocytes"

Controversy reigns in the area of adult heart cell regeneration.   Until recently it was thought there was no adult regenerative capacity, but even though this dogma has now been abolished, the rate at which it occurs and the source of this de novo regeneration is still debated.   Some studies suggest that there is a high level of differentiation of  progenitors to cardiomyocytes (Hosoda et al) and their turnover is high (Kaystura et al), while other studies suggest that new cardiomyocytes are made at a very low level (Soonpaa and Field, Bergmann et al and Walsh et al).   Additional controversy stems from the question of the source of these cardiomyocytes and therefore the plasticity of the heart; do they come from the division of existing myocytes (Kikuchi et al), progenitors residing in the heart (Beltrami et al) or from exogenous niches, such as the bone marrow (Orlic et al)?   To attempt to address these questions, researchers from the laboratory of Richard T. Lee at the Harvard Stem Cell Institute, Cambridge, Massachusetts, USA have used multiple tracking techniques to demonstrate that the genesis of cardiomyocytes indeed occurs at a low rate through the division of pre-existing cardiomyocytes, a phenomenon which is exacerbated by injury (Senyo et al).

The study utilised a multi-isotope imaging mass spectrometry (MIMS) to study cardiomyocyte turnover; this uses non-radioactive stable isotope tracers ([15N]thymidine) allowing the analysis of cardiomyocytes in minute detail (Lechene et al, Steinhauser et al and Zhang et al).   An important consideration in these types of assays is that the tracers do not alter biochemical reactions and are not harmful to the study subject (Klein and Klein).   Nuclear incorporation of [15N] thymidine is evident in cells that have divided during a 1-week labelling period, and initial experiments used this to study age related changes in the cell cycle in C57BL6 mice.   Cardiomyocytes in newborn mice demonstrated high levels of 15N (56%), as expected (Li et al).   While newborn mice exhibited a 1% per day frequency of 15N containing nuclei, this figure was much smaller in young adult mice (0.015%) and smaller again in old adult mice (0.007%).   By extrapolating these figures, DNA synthesis in cardiomyocytes in young adults was 5.5% and 2.6% in old mice, indicative of a low rate of cardiogenesis.   Using [15N] thymidine labelling of double-transgenic MerCreMer/ZEG mice (cardiomyocytes becoming GFP-positive after 4-OH-T treatment) the authors investigated where this cell cycle activity resided; in pre-existing cardiomyocytes or progenitors.   During a chase period, cardiomyocytes which arise from existing cardiomyocytes should be GFP-positive and those generated from progenitors, GFP-negative.    Mice were given 4-OH-T for 2 weeks at 8 weeks of age and then studied for 10 weeks with [15N] thymidine given throughout.   MIMs analysis identified 35 15N-positive cardiomyocytes and, of these, 77% expressed GFP, identical to the surrounding 15N-negative cardiomyocyte population, suggesting that the DNA synthesis observed derived from pre-existing cardiomyocytes and not from stem cell sources.

Multinucleation and polyploidization usually only occur during early postnatal development (Li et al and Soonpaa and Field) but may account for many of the 15N-positive cardiomyocytes observed.  However, while some 15N-positive cardiomyocytes were observed to be polyploid, a higher frequency of diploid nuclei in the 15N-positive fraction was observed relative to the surrounding 15N-negative fraction, consistent with ongoing cell division.   Additionally, 49% of 15N-positive cells were mononucleate, compared to 24% in the surrounding 15N-negative fraction.   Therefore, a great degree of DNA synthesis occurred in polyploid/multinucleate cardiomyocytes, however a combined 17% of cardiomyocytes were diploid and mononucleate, consistent with newly generated cardiomyocytes, and were predominantly GFP-positive, suggesting that they arose from a pre-existing cardiomyocyte.

Next, using the same techniques, the effect of injury was studied with experimental myocardial infarction or sham surgery.   While the number of 15N-positive cardiomyocytes was similar to what was observed previously, the number of 15N-positive cardiomyocytes adjacent to infarcted cardiomyocytes increased to 23%, while GFP analysis suggested that DNA synthesis was primarily occurring in pre-existing cardiomyocytes.   15% of 15N-positive cardiomyocytes were mononucleate and diploid, again suggesting division of pre-existing cardiomyocytes.

Together, collected through the application of multiple techniques for monitoring cell division and DNA synthesis, these data suggest that new cardiomyocytes are formed from pre-existing cardiomyocytes but at a very slow yearly rate (0.76% per year) as observed in young mice, which declines with age but is observed to increase upon myocardial injury, suggesting that cardiac progenitors have a limited role in recovery from injury but no function in myocardial homeostasis.

References

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Bergmann, O. et al.
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Hosoda, T. et al.
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Kajstura, J. et al.
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Kikuchi, K. et al.
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Klein, P. D. & Klein, E. R.
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Soonpaa, M. H. &Field, L. J.
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Steinhauser, M. L. et al.
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Walsh, S.et al.
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Zhang, D. S. et al.
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Study originally appeared in Nature.

Stem CellCorrespondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.