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Cardiomyocyte cell cycle revealed by FUCCI
Alvarez Jr., Roberto
Sussman, Mark A.
Gustafsson, AsaHampton, RandallWolkowicz, RolandZeller, Robert
Rationale: Pre-existing cardiomyocytes and resident cardiac stem cells are limited in their capacity for substantial regeneration in postnatal endogenous mammalian myocardial repair. Evidence for adult cardiomyocyte proliferation remains inconclusive, relying on proliferation markers that also present in hypertrophy or DNA repair. Unambiguous identification of myocyte cell cycle activity to demarcate de novo cardiomyogenesis within the injured myocardium has not been achieved using traditional markers of cellular proliferation. A powerful tool, Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reporter system utilized to examine dynamic oscillations between G1 and S/G2/M phases of the cell cycle has yet to be exploited in a cardiomyocyte specific fashion. Targeted myocardial expression of FUCCI has the potential to delineate cardiomyocyte cell cycle entry and completion during postnatal development and following pathologic challenge. Objective: Authenticate fidelity of proliferative markers as indicators of de novo cardiomyogenesis and subsequently delineate the post-mitotic status of postnatal cardiomyocytes. Methods and Results: Cardiomyocyte specific FUCCI expression driven by the α- myosin heavy chain (αMHC) promoter was utilized in conjunction with traditional proliferation markers to identify cell cycle status of postnatal murine cardiomyocytes. Cardiomyocyte proliferation rapidly decreased after birth, with cell cycle arrest corresponding to G1/S rather than full mitotic exit at G0. Interestingly, cell cycle activity increased in cardiomyocytes of sham injured hearts, potentially in response to systemic insult. Proliferation markers commonly utilized to ascertain de novo cardiomyocyte formation failed to fully correspond with FUCCI cell cycle activity in injured αMHC-FUCCI hearts. Moreover, rare cKit+ amplifying progenitors identified in early postnatal development confirmed contribution to cardiomyogenesis. Conclusions: Proliferation markers used to identify de novo cardiomyogenesis failed to distinguish cycling cardiomyocytes after injury. Adult cardiomyocytes upregulated cell cycle activity in response to sham injury but failed to complete regenerative proliferation following cell cycle reentry. αMHC-FUCCI elucidated post-mitotic cardiomyocyte arrest at G1/S as opposed to G0 cell cycle exit previously reported in the literature. Finally, αMHC-FUCCI revealed cardiac progenitor cells remain a possible source of neonatal cardiomyogenesis within the myocardium.
Doctor of Philosophy (Ph.D.) University of California, San Diego and San Diego State University, 2018
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