Date on Master's Thesis/Doctoral Dissertation

4-2014

Document Type

Dissertation/Thesis

Department

Chemistry

Author's Keywords

Cardiac; Cardiomyocyte; Proliferation; Regenerative medicine; Molecular probe; Mitosis; CKAP2

Abstract

Currently, one of the biggest controversies in the field of cardiac regenerative medicine revolves around the ability of cardiomyocytes to proliferate. In contrast to the long-held hypothesis that the heart is terminally differentiated, post-mitotic organ, some studies have suggested that the heart is capable of undergoing limited regeneration following injury. Others have reported induction of cardiomyocyte proliferation following various treatments, mostly in vitro. Conventional tools such as BrdU labeling fail to distinguish between mitotic events and other phenomena such as endoreduplication or poly-nucleation, thus making it difficult to assess cardiomyocyte proliferation. The present study presents a novel and innovative way to unambiguously study cardiomyocyte proliferation by use of a cell division probe to identify cells that undergo mitosis. The system utilizes a mutant form of the mitotic regulator cytoskeleton-associated protein 2 (CKAP2). CKAP2 remains cytoplasmic during interphase, but translocates to the nucleus following mitotic cell division. Usually, wild-type CKAP2 is degraded via the ubiquitin-proteosome pathway following translocation to the nucleus; however, by mutation of a destruction motif, the protein persists in the daughter nuclei following cell division. Thus, this non-degradable mutant of CKAP2 (ndCKAP2) can be used to track mitotic events - ndCKAP2 should remain cytoplasmic in quiescent cells but appear nuclear in cells that have undergone mitosis. Here, the efficacy of ndCKAP2 as a cell division probe is demonstrated; in particular, ndCKAP2 is demonstrated to be easily visualizable, unambiguously track mitotic events, and present a relatively long half-life (and is therefore integrative in output). It is expected that future studios will utilize this molecular probe system to investigate cardiomyocyte proliferation in vivo under a variety of physiological and pathological conditions, and allow for assessment of various proposed clinical therapies.

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