Pathways responsible for apoptosis in chick cardiomyocytes
Creators: name: Kong, Jennifer Y.
Publisher: name: University of British Columbia
Year: 2009

description: The mechanisms responsible for apoptosis in the heart are currently being defined. The present study was designed to determine the roles of nuclear enzymes and signal transduction protein kinases in the development of apoptosis in chick embryo cardiomyocytes. Topoisomerase I was chosen as an example of a nuclear enzyme involved in apoptosis. Topoisomerase I is the enzyme responsible for relieving torsional stress in DNA replication and transcription. To determine whether inhibition of topoisomerase I would produce apoptosis in cardiomyocytes, the inhibitor camptothecin was used. Cardiomyocytes, obtained from 7 day old embryonic chick hearts, were treated with camptothecin and examined microscopically or their DNA was examined for fragmentation. Apoptotic cell death was produced by camptothecin as fluorescent microscopy with acridine orange demonstrated cardiomyocytes that were shrunken with cytoplasmic blebs and nuclear fragmentation. In contrast, untreated cells did not manifest these cellular alterations. Apoptosis was further substantiated by Hoescht 33258 dye stained cardiomyocytes that showed a strongly fluorescent nucleus which was undergoing disintegration. Cell death as quantitated by trypan blue exclusion showed that camptothecin, 10 [μM, significantly increased cell death by 25.1±1.4% (+SEM) . Cardiomyocytes were lysed and the DNA isolated and run on a 2% agarose gel. DNA laddering, indicated by fragments of approximately 200 bp or multiples, were found in camptothecin treated cells. DNA fragmentation was also observed quantitatively in camptothecin treated cells, as assessed by an enzyme linked immunosorbent assay (ELISA). Fragmented DNA was isolated from lysed cells and adsorbed onto a microtitre plate. Primary antibody specific for DNA histones was then added and subsequently treated with a horse-radish peroxidase linked-secondary antibody specific for DNA. The colorimetric results were reported relative to control. Camptothecin exposure (10\iM) induced 1.5±0.5 fold more DNA fragmentation than control cells. Alterations in intracellular calcium appeared to be a component of the mechanism of action of camptothecin-induced apoptosis. Ca⁺² levels that can be decreased by the chelator EGTA reduced cell death induced by camptothecin, as demonstrated by membrane bleb formation, DNA fragmentation on agarose gel electrophoresis, and DNA fragmentation on the ELISA. Taurine, a free amino acid in many tissues which affects L-type and T-type Ca⁺² channels, also reduced camptothecin-induced apoptotic morphology and DNA specific fragmentation, determined by ELISA. To further substantiate the role of calcium and to investigate the source of Ca⁺²mediated topoisomerase-induced apoptosis, cardiomyocytes were exposed to thapsigargin, an inhibitor of sarcoplasmic and endoplasmic reticulum Ca⁺²-ATPases which increases intracellular calcium. [Ca⁺²]i increased by thapsigargin exposure yielded greater DNA fragmentation, as assessed by ELISA, than camptothecin alone suggesting that increased [Ca⁺²]i induced apoptosis itself. With the caveat our use of agents that indirectly implicate the mechanism, these data show that apoptosis in cardiomyocytes is under regulatory control by DNA topoisomerase I and intracellular calcium modulates the pathway whereby topoisomerase I inhibition causes apoptosis. To investigate the signal transduction mechanisms responsible for apoptosis, I investigated the role of serine/threonine kinases. Staurosporine, a potent serine/threonine kinase inhibitor, was used to investigate the role of kinase inhibition on the development of apoptosis. Staurosporine induced cell death in a dose and time dependent response to a maximal death of 40. 9±6.3% at 1μM for 6h. DNA fragmentation, 2. 8±1.2 fold that of control, determined by ELISA and electrophoretic separation was observed in staurosporine treated cardiomyocytes. Staurosporine-induced morphology, observed by acridine orange and NBD phallacidin staining, was distinct from usual apoptotic features: staurosporine .induced cytoplasmic condensation resulting in dense vacuoles and a loss in volume. Staurosporine treatment failed to exhibit membrane blebbing and distinct nuclear disintegration. Pre treatment by the Ca⁺² chelator BAPTA blunted the apoptotic response of staurosporine exposure implicating Ca⁺² in staurosporine-induced apoptosis. The activation of protein kinase C (PKC) by the phorbol ester PMA blocked staurosporine-induced cell death, morphology, and DNA fragmentation suggesting that the activation of PKC can reverse staurosporine-induced apoptosis. The addition of trophic factors such as insulin and EGF demonstrated a "rescue" pathway in staurosporine-induced apoptotic cardiomyocytes. In addition, de novo protein synthesis may relate to this rescue pathway. To further investigate the signal transduction mechanisms responsible for apoptosis, the role of PKC was considered. The specific PKC inhibitor chelerythrine chloride was observed to induce cell death of 27.6±7.5% and DNA fragmentation (2.2±0.4 fold that of control) similar to staurosporine. However, chelerythrine exhibited usual apoptotic morphology contrasting staurosporine morphology. In addition, the apoptotic effects of chelerythrine are less potent than staurosporine suggesting that PKC alone is not responsible for staurosporine's apoptotic inducing abilities.