Tesis doctoral de Jisheng Zhang
Apoptosis has been considered as an important factor in controlling the balance between cell proliferation, differentiation and death during development. The regulation of apoptosis is a key aspect to achieve this balance, by eliminating unnecessary or miss-specified cells which, otherwise, may have harmful effects on the whole organism. In the past decade, extensive observations demonstrated that apoptosis plays a vital role in heart morphogenesis. However it is still controversial whether caspases are involved in apoptosis of cardiomyocytes in the adult. Thus, the analysis of apoptosis regulation from a developmental perspective could improve our knowledge about how apoptosis acts in the heart. Therefore, our main objectives were to characterize the regulation of the expression of apoptotic genes during heart development and to identify alternative pathways involved in cardiac damage. expression of proteins involved in the control of caspase-dependent cell death was analyzed in tissues from rats of different ages ranging from the embryo to the adult. The genes involved in the induction of cell death were intensely repressed in the adult heart and, most of them, also in the brain but not in the liver. Exceptions were caspase-2, which was only expressed in embryos, and bak and bax, which were downregulated in all three tissues during postnatal development. The expression of caspase-9 and executioner caspase-3 and -7 was reduced in postnatal heart and brain. With regard to the expression of anti-apoptotic proteins, bcl-2 was silenced in heart and brain during development and undetectable in the liver, whereas bcl-xl was expressed in all tissues until adulthood. Using the semi-quantitative rtpcr method, we found that apoptotic gene silencing involved a reduction of the transcript amount for these genes. During ischemia or staurosporine treatment executioner caspase-3 activation was only observed in embryonic cardiomyocytes, but not in postnatal and adult cardiomyocytes. Furthermore, ischemia induced dna fragmentation that was not inhibited by caspase inhibitors in neonatal cardiomyocytes, which already express very low levels of apoptotic genes. to continue the investigation of cell death in postnatal cardiomyocytes and the switch from caspase-dependent to caspase-independent pathway during heart development, we assessed the potential role of several dnase which may be involved in ischemia-induced dna damage. Knockdown of mitochondrial nuclease gene endonuclease g (endog) decreased the ischemia induced dna damage, which demonstrated that endog plays an important role to the ischemia induced dna fragmentation in this caspase-independent cell death model. Endog release from mitochondria is the key step to dna fragmentation. We have shown that bcl-xl hampers and bnip3 contributes to the release process of endog and, thus, affect in opposite ways to ischemia induced dna damage in differentiated cardiomyocytes. the mechanisms controlling the repression of major apoptotic gene expression have also been investigated. We analyzed two potential candidates, e2f and polypyrimidine tract binding proteins (ptb). In the heart, the expression of e2f and ptb decrease during development, in a similar way to the decrease of apoptotic gene expression. But the analysis of e2f1 and e2f2 single and double knockout mice showed no differences neither in the embryonic expression of these genes nor in their repression during development when compared with their wild type littermates. these results discarded a relevant role of e2f in the regulation of apoptotic gene expression during heart development, contrary to the observations in tumor cell lines. on the other hand, we found that in neonatal cardiomyocytes which express low levels of ptb overexpression of ptb1 and ptb4 induced expression of most of the apoptotic proteins, including apaf-1, caspase-9, caspase-3 and bid. In addition, neonatal cardiomyocytes overexpressing ptbs had more dna damage during ischemia and the dna fragmentation was reduced to the level of wild typecells by caspase inhibitors. This suggested that with recovery of ptb expression level, likely involving the ptb-dependent restoring of caspase-dependent mechanisms, dna damage can go back to the caspase-dependent mechanism in cardiomyocytes. ptb induced an increase in the amount of apoptotic proteins without affecting their transcripts. Luciferase (luc) assays in wild type and ptb overexpressing cultured cardiomyocytes, using bicistronic plasmids in which the 5 utr of caspase-3 and apaf-1 were located between luc renilla and luc firefly, showed an increase of luc firefly activity when preceded by either 5 utr, compared with the control levels. These results suggested that ptbs upregulate apoptotic gene expression at the post transcriptional level via ires-dependent translation.
Datos académicos de la tesis doctoral «Developmental switch on the signaling for cardiomyocyte death: insights into the regulation of apoptotic gene expression and into the control of dna fragmentation«
- Título de la tesis: Developmental switch on the signaling for cardiomyocyte death: insights into the regulation of apoptotic gene expression and into the control of dna fragmentation
- Autor: Jisheng Zhang
- Universidad: Lleida
- Fecha de lectura de la tesis: 12/11/2009
Dirección y tribunal
- Director de la tesis
- Daniel Sanchis Morales
- Tribunal
- Presidente del tribunal: joan xavier Comella carnice
- elisa Cabiscol catal? (vocal)
- almudena Porras gallo (vocal)
- víctor José Yuste mateos (vocal)