Abstract 251: The Rate of Myocyte Turnover in the Mouse Heart
The accuracy of the estimations of cell turnover in the myocardium is restricted by the difficulty to obtain from histologic samples the information regarding the rate of cell proliferation, i.e., number of cells generated per unit of time. It is easy to identify the fraction of cycling cells, but this value cannot be directly converted into the number of cells generated per day. As a result, the published estimates of cell turnover in the mammalian heart vary dramatically. Here, we propose to use the cell cycle as an internal clock to introduce the time factor in the analysis of population dynamics of cardiac cells. In this novel approach the cells are exposed to two pulses of distinct thymidine analogs, separated by time T. The first pulse is performed with 5-bromo-2’-deoxyuridine (BrdU) and the second pulse with 5-ethynyl-2’-deoxyuridine (EdU). With this protocol, the number of cells that exit the S phase during time T is equal to the number of cells positive for BrdU but negative for EdU. It can be shown that, in non-synchronous population, the number of cells exiting the S phase is equal to the number of cells entering the cell cycle. Importantly, this approach does not require knowledge of the length of the cell cycle. To measure the rate of formation of cells in the myocardium, 2 month-old mice were injected with the nucleotides at 4 hour intervals and sacrified 4 hour later. The hearts were formalin-fixed and histologic sections obtained. The first nucleotide, BrdU, by was detected by immunocytochemistry, and the second, EdU, by the “click” chemistry. Myocytes and non-myocytes were distinguished by α-sarcomeric actin and cardiac troponin I staining and analyzed by confocal microscopy. Spectral analysis was employed to ensure specificity of the detected signals. The enumeration of double positive nuclei, nuclei positive for one nucleotide only, and nuclei negative for both nucleotides indicated that in the entire left ventricle (LV) ~1,250 myocytes are generated per hour. Since the mouse LV contains ~3 x 106 parenchymal cells, these data demonstrate that the entire population of myocytes can be replaced in ~180 days. Thus, our results are consistent with a relatively high turnover rate of myocytes in the mouse heart.
- © 2013 by American Heart Association, Inc.