Abstract 18159: Myocyte Turnover in the Mouse Heart
Despite the identification of cardiac stem cells and their ability to differentiate in the various cardiac cell lineages, the controversy on the turnover rate of myocytes in the adult heart continues. Two independent but complementary protocols were employed to define myocyte renewal in the mouse heart. Mice 2-3 months of age were injected every 12 hours with BrdU (50mg/kg bw) for 6 days prior to sacrifice. A second group of mice received one injection of 14C-thymidine (300 pCi) and the animals were sacrificed from 1 to 90 days later. The BrdU method allowed us to measure the number of myocytes formed in 6 days. The 14C-thymidine strategy provided us with an evaluation of myocyte regeneration as a function of time.
In assay 1, BrdU localization in myocytes was determined by immunolabeling and confocal microscopy. After 12 injections of BrdU, 3.1±0.4% or 100,000±13,000 (n=8) left ventricular (LV) myocyte nuclei were labeled, indicating that a significant number of parenchymal cells was added to the LV in 6 days. Given that the number of LV myocytes is constant in the adult mouse heart, myocyte apoptosis and necrosis were measured by hairpin 1 and hairpin 2 in situ ligation, respectively. Myocyte necrosis was also assessed by the incorporation of circulating vitronectin that reflects an irreversible loss of integrity of the plasma-membrane. Apoptosis comprised 186±55 myocyte nuclei/106 (n=6) and cell necrosis 316±71/106 nuclei. Similarly, vitronectin-positive myocytes were 280±103/106 cell profiles (n=4). Since apoptosis last at most 4 hours and necrosis 20 hours, there was a 2.9±0.4% drop out of myocytes in 6 days.
In assay 2, LV myocytes were isolated, purified by differential centrifugation, and the DNA extracted for the measurement of the kinetics of 14C concentration by Accelerator Mass Spectrometry. 14C concentration in myocyte DNA increased progressively from 1 to 10 days, and remained essentially constant up to 90 days. These findings indicate that a significant number of new myocytes was formed in 10 days. The regenerated myocytes had a lifespan of at least 90 days as demonstrated by the lack of changes in 14C from 10 to 90 days. This value is consistent with the rate of myocyte formation and death measured above. Thus, myocyte turnover is high in the adult mouse heart.
- © 2013 by American Heart Association, Inc.