The stress responses of cardiomyocytes are likely to constitute a significant aspect of the development of cardiac pathologies. Oxidative stress is a common theme in the pathophysiology of ischaemic and non-ischaemic cardiomyopathy. On the other hand, hypertrophic agonists (e.g. endothelin-1, ET-1) are important mediators of the hypertrophic response to hemodynamic overloading, and cardiac pathologies such as heart failure are usually preceded by cardiac hypertrophy. Microarray studies indicate that one of the genes most potently induced by H2O2 (as an oxidative stress) or ET-1 in cardiac myocytes is the transcription factor ATF3. This thesis examines the regulation and role of ATF3 in neonatal rat ventricular cardiomyocytes. The increase in expression of ATF3 mRNA was confirmed in neonatal rat cardiomyocytes exposed to H2O2 or ET-1 using RT-PCR and Q-PCR. Using immunoblotting, it was confirmed that H2O2 or ET-1 increased ATF3 protein expression. ATF3 is an immediate early gene since the upregulation of ATF3 mRNA by ET-1 was not inhibited by cycloheximide (20 μM). Upregulation of ATF3 was inhibited by U0126 (10 μM), suggesting that signalling through ERK1/2 (extracellular signal-regulated kinases 1/2) was required. Other studies suggest that ATF3 downregulates interleukin-6 (IL-6) in human cells. The rat IL-6 promoter possesses an ATF consensus sequence, and chromatin immunoprecipitation (ChIP) analysis indicated that ET-1 or H2O2 increased the association of ATF3 with the IL-6 promoter. IL-6 mRNA and protein expression was transiently upregulated in cardiomyocytes exposed to H2O2 or ET-1, and this was shown to be an immediate early gene response. Following IL-6 mRNA upregulation, its expression was more rapidly downregulated than that of ATF3. The peak of ATF3 protein expression coincided with the return of IL-6 mRNA to basal levels after stimulation with either ET-1 or H2O2. This suggests that ATF3 operates in a negative feedback loop to downregulate IL-6 mRNA expression in cardiomyocytes. This was confirmed by adenoviral-mediated overexpression of full-length ATF3 antisense RNA, which attenuated ATF3 mRNA and protein expression following stimulation with ET-1. Inhibition of ATF3 expression was associated with superinduction of IL-6 mRNA as shown by Q-PCR. Other potential downstream targets of ATF3 were identified from microarray studies using bioinformatics. Those with ATF/CRE consensus sequences in their promoters included epiregulin and leukaemia inhibitory factor (LIF) (also shown to be immediate early genes). Epiregulin and LIF mRNA expression was superinduced by ATF3 antisense RNA. Taken together, these experiments indicate that ATF3 operates in a negative feedback loop by downregulating a cluster of immediate early genes induced by ET-1. Thus, ATF3 may play an important role in the establishment and fine-tuning of an organised and compensatory hypertrophic response in the cardiomyocyte secondary to hypertrophic stimulation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:446028 |
| Date | January 2008 |
| Creators | Giraldo Ramirez, Diego Alejandro |
| Contributors | Sugden, Peter ; Clerk, Angela |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/1300 |
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