Abstract 17314: Direct Determination of the Rate of Cell Turnover in the Mouse Heart
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, severely restricts the accuracy of the estimation of cell turnover in the myocardium. Although it is relatively easy to acquire fractions of cycling cells, these values cannot be directly converted into the number of cells generated per day or any other time interval. 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 in proliferating cells to introduce the time factor in the analysis of population dynamics of cardiac cells. The principle of this novel approach is that 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. Thus, to measure the rate of formation of cells in the myocardium, 3 month old male FVB mice were injected with the nucleotides at 1 hour intervals and sacrified 1 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. 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) 1,000 myocytes and 2,000 non-myocytes are generated per hour. Since the mouse LV contains ∼3 x 106 myocytes and ∼8 x 106 non-myocytes, these data indicate that the entire population of myocytes can be replaced in 125 days, and non-myocytes in ∼170 days. Thus, our results are consistent with a relatively high turnover rate of myocytes in the mammalian heart.
- © 2012 by American Heart Association, Inc.