Bedeutung des Transkriptionsfaktors E2F4 für die Regulation der Proliferation von Herzmuskelzellen
Final Report Abstract
The vast majority of mammalian cardiomyocytes (CMs) terminally differentiate shortly after birth – a process punctuated by cytokinesis failure, binucleation, and exit from a proliferative state. Consequently, the adult mammalian heart is incapable of regeneration and rarely forms tumors. It is currently unknown what mechanism underlies terminal differentiation and the strict post-mitotic state of mammalian CMs. On the basis of findings from cell culture experiments, E2F4 is generally considered as an inhibitor of cell proliferation. Thus, we have investigated the role of E2F4 during heart development and in proliferating cardiomyocytes. Nuclear E2F4 expression in cardiomyocytes declined during mouse heart development, which correlates with the loss of proliferative capacity of cardiomyocytes. Re-induction of proliferation in postnatal cardiomyocytes increased nuclear E2F4 expression. E2F4 accumulated in the nucleus at the end of the S phase, remained nuclear during mitosis, and disappeared at the end of cytokinesis. siRNA- mediated inhibition of E2F4 in proliferating postnatal cardiomyocytes resulted in a significant reduction in mitosis, but not in DNA synthesis. Co-staining of E2F4 and Crest revealed that E2F4 co-localizes with kinetochores. Moreover, chromatin immunoprecipitation showed that E2F4 binds to centromeric alpha-satellite DNA during mitosis. Collectively, our data indicate that E2F4 is required for cardiomyocyte proliferation and suggest a function for E2F4 in mitosis. Cell exit upon cardiomyocyte terminal differentiation occurring shortly after birth is associated with down-regulation of Cyclins and CDKs, up-regulation of CDKIs, and hypophosphorylation of pRB. However, genetic manipulation of cell cycle regulators including E2F4 were unable to induce efficient adult cardiomyocyte proliferation. Thus, elucidation of the mechanism of cardiomyocyte terminal differentiation and post-mitotic state requires investigation of novel non-traditional cell cycle regulators. The centrosome and primary cilium have emerged as important mediators of cell cycle progression, division, and differentiation. These structures have yet to be evaluated in CMs. We found the frequency of mammalian CMs containing a primary cilium declined progressively during gestation with only a minor fraction containing a primary cilium postnatally. Onset of terminal differentiation in postnatal mammalian CMs coincided with a relocalization of pericentriolar material protein CDK5RAP2 from the centrosome to the perinuclear space. Concurrently, centrosomes of postnatal mammalian CMs were found to lack centriole cohesion. Collectively, these observations suggest a potential causal relationship between the absence of a primary cilium and loss of centrosome integrity with terminal differentiation and strict governance of a post-mitotic state in mammalian CMs. Understanding these relationships, and underlying mechanisms, should benefit the study of heart regeneration, development, and cancer.
Publications
- Annual meeting of the German Cardiac Society - Heart and Circulation Research. E2F4 is required for cardiomyocyte proliferation. 75. annual meeting (Mannheim, Germany); Clin Res Cardiol 98, Suppl 1, April 2009: V363
an Amerongen MJ, Diehl F, Novoyatleva T, Patra C, Engel FB
- E2F4 is required for cardiomyocyte proliferation. Cardiovasc Res. 86(1):92-102. 2010
van Amerongen MJ, Diehl F, Novoyatleva T, Patra C, Engel FB
- Induction of Cardiomyocyte Proliferation. ed. Engel FB. Heart Regeneration: Stem Cells and Beyond. World Scientific Publishing Company, Inc., Singapore, 2012. ISBN 9-8142-9980-4
van Amerongen MJ and Engel FB
