(A) Schematic figure demonstrating the strategy to establish cell age by ¹⁴C dating. The black curve in all graphs shows the atmospheric levels of ¹⁴C over the last decades since 1930 (data from (14)). The vertical bar indicates the date of birth of the individual. The measured ¹⁴C concentration (1) is related to the atmospheric ¹⁴C level by using the established atmospheric ¹⁴C bomb curve (2). The average birth-date of the population can be inferred by determining where the data point intersects the x-axis (3). ¹⁴C levels in DNA of cells from the left ventricle myocardium in individuals born after (B) or before (C) the nuclear bomb tests correspond to time points substantially after the time of birth, indicating postnatal cell turnover. The vertical bar indicates the date of birth of each individual and the similarly colored dots represent the ¹⁴C data for the same individual. For individuals born before the increase in ¹⁴C levels, it is not possible to directly infer an age as the measured level can be a result of incorporation during the rising and/or falling part of the atmospheric curve, thus the level is indicated by a dotted horizontal line.

(A-C) Flow cytometric analysis of cardiomyocyte nuclei from the left ventricle of the human heart with an isotype control antibody or antibodies to the cardiomyocyte specific antigens cTroponin I or T. Boxes denote the boundaries for the positive and negative sort populations. (D) cTroponin I and T are present in the same subpopulation of heart cell nuclei. (E) Western blot analysis of flow cytometry-isolated nuclei demonstrates close to all detectable cTroponin T (analyzed with two different antibodies) and I protein in the cTroponin T-positive fraction. Brain and heart tissue was used as negative and positive controls, respectively. (F) The cardiac troponin T-positive population is enriched for the cardiomyocyte-specific transcription factors Nkx2.5 and GATA4. Both fractions contain similar amounts of the nuclear protein histone 3 (loading control). (G) Gene expression analysis of flow cytometry-isolated nuclei shows a high expression level of cardiomyocyte specific genes in the cTroponin T-positive fraction (cTroponin I and T, Nkx2.5), while marker genes for endothelial cells (vWF), fibroblasts (vimentin), smooth muscle (ACTA2) and leukocytes (CD45) are highly expressed in the cTroponin T-negative fraction (H). Bars in (G, H) show the average from 3 independent experiments (+−SD).

(A) The ¹⁴C levels in cardiomyocyte DNA from individuals born before the time of the atmospheric radiocarbon increase correspond to time points after the birth of all individuals. The vertical bar indicates year of birth, with the correspondingly colored data point indicating the delta ¹⁴C value. (B) ¹⁴C levels in cardiomyocyte DNA from individuals born after the time of the nuclear bomb test. (C) Average DNA content (2n=100%) per cardiomyocyte nucleus from individuals (without severe heart enlargement, see figure S5) of different ages. Ploidy was measured by flow cytometry. Colored data points identify individuals analyzed for ¹⁴C (n=13). Black data points are from individuals only analyzed with regard to ploidy level (n=23) and white data points are taken from Adler et al. (n=26) (24, 26). The dashed lines indicate the 95% confidence interval for the regression curve. (D) ¹⁴C values corrected for the physiologically occurring polyploidization of cardiomyocytes during childhood for individuals born before and after the bomb spike, calculated based on the individual average DNA content per cardiomyocyte nucleus. The ¹⁴C content is not affected in individuals where the polyploidization occurred before the increase in atmospheric levels.

(A) Individual data fitting assuming a constant turnover (see supporting online text) reveals an almost linear decline of cardiomyocyte turnover with age (R=−0.85; p=0.005). A constant turnover hypothesis might therefore not represent the turnover dynamics accurately. (B) Global fitting of all data points (see supporting online text, SSE=9.4×10³) shows an age-dependent decline of cardiomyocyte turnover. (C) Fraction of cardiomyocytes remaining from birth is depicted as gray area, and the white area is the contribution of new cells. Estimation is from the best global fitting. (D) Cardiomyocyte age estimates from the best global fitting. The dotted line represents the no cell turnover scenario, where the average age of cardiomyocytes equals the age of the individual. The black line shows the best global fitting. Colored diamonds indicate computed data points from ¹⁴C-dated subjects. Error bars in all graphs are calculated for each subject individually showing the interval of possible values fitted with the respective mathematical scenario.