University of Louisville

Poster Title

Development of a Molecular Probe for Measuring Cardiomyocyte Proliferation

Institution

University of Louisville

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 a 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 an innovative approach 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 cytoskeletonassociated protein 2 (CKAP2). CKAP2 remains cytoplasmic during interphase, but translocates to the nucleus following mitotic cell division. Usually, wildtype 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 we show the efficacy of ndCKAP2 as an in vitro cell division probe. NIH 3T3 and HEK 293 cells were transfected with GFP-ndCKAP2. Following transfection, cells were arrested at the G0 phase through serum starvation. Synchronous re-entry into cell cycle was controlled by re-supplying cells with serum-containing media. Proliferation status and localization of ndCKAP2 were studied with epifluorescence and time-lapse microscopy as well as flow cytometry. We also propose an in vivo cardiomyocyte-specific tetracycline-regulated expression system for ndCKAP2. This system will allow for an investigation of 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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Development of a Molecular Probe for Measuring Cardiomyocyte Proliferation

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 a 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 an innovative approach 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 cytoskeletonassociated protein 2 (CKAP2). CKAP2 remains cytoplasmic during interphase, but translocates to the nucleus following mitotic cell division. Usually, wildtype 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 we show the efficacy of ndCKAP2 as an in vitro cell division probe. NIH 3T3 and HEK 293 cells were transfected with GFP-ndCKAP2. Following transfection, cells were arrested at the G0 phase through serum starvation. Synchronous re-entry into cell cycle was controlled by re-supplying cells with serum-containing media. Proliferation status and localization of ndCKAP2 were studied with epifluorescence and time-lapse microscopy as well as flow cytometry. We also propose an in vivo cardiomyocyte-specific tetracycline-regulated expression system for ndCKAP2. This system will allow for an investigation of cardiomyocyte proliferation in vivo under a variety of physiological and pathological conditions, and allow for assessment of various proposed clinical therapies.