Abstract 20: Caridomoyocyte Biology Revealed by Fluorescence Ubiquitination-based Cell-cycle Indicators

Abstract

Existing myocyte contribution to new myocyte formation remains an active area of investigation. Novel experimental methodology is needed to faithfully label cardiomyocyte cell-cycle activity after birth and following injury. The Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) system can be used to aid visualization of cell cycle activity and progression by monitoring the inverse oscillation dynamics of fluorescently tagged cell cycle fusion proteins AzG-hGeminin and mKO2-hCdt1. Using this system, we hypothesize that cardiomyocytes retain the capacity to cycle throughout postnatal development and re-enter the cell cycle following acute myocardial infarction injury (MI). A novel cardiac specific FUCCI transgenic mouse model, αMHC-FUCCI, was developed to study cell-cycle dynamics of cardiomyocytes. αMHC-FUCCI hearts were collected throughout postnatal (PN) development to examine cardiomyocyte cell-cycle. Similarly, adult αMHC-FUCCI mice were subjected to MI, injected daily with BrdU and harvested at 3, 7, 10, 14 and 21 days post-MI for further analysis. Peak incidence of single mKO2-hCdt1 (7%, G 1 ) and AzG-hGem (2%, S/G 2 /M) fluorescence in cardiomyocytes occurs at PN7 and decreases over time as as confirmed by colocalization with BrdU and/or mitotic marker phospho-histone 3. Interestingly, continued mitotic activity exists at PN14 as observed by AzG+/pH3+ myocytes and concurrent mKO2+/AzG+ fluorescence is observed in 60% of adult myocardium by at one month. Together, these results indicate cardiac myocytes remain active at least two weeks after birth and transition into a G1/S phase as opposed to a mitotic exit (G0) as adults. Intriguingly, BrdU+ label is only detected in the non-myocyte interstitial population in and around the border zone through the first two weeks post-MI. BrdU+/AzG+ and or/mKO+ myocytes are detectable at 21days post-MI, indicating a lag in cardiomyocyte cell cycle re-entry. These results suggest myocytes retain the ability to re-enter the cell cycle at low levels three weeks post-MI. Future studies will analyze cardiomyocyte cell-cycle biology in response to diffuse injury and will further elucidate the mechanism behind myocardial regeneration.