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DISSECTING THE ROLES OF MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) CASCADES IN THE MRNA EXPRESSION RESPONSE TO H2O2 IN CARDIOMYOCYTES
  1. SJ Leonard,
  2. PH Sugden,
  3. A Clerk
  1. School of Biological Sciences, University of Reading, UK

Abstract

Rationale Oxidative stress plays a significant role in the development of heart failure. H2O2 (a physiologically-relevant form of oxidative stress) induces substantial changes in gene expression in cardiomyocytes and modulates apoptosis. It activates three major MAPK cascades [extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), p38-MAPKs] that regulate transcription and gene expression in other cells. The aim is to identify the contribution of each pathway in the gene expression response of cardiomyocytes to H2O2.

Methodology Neonatal cardiomyocytes were exposed to H2O2 (0.2 mM, 2h) in the absence/presence of PD184352 (2 µM; inhibits ERK1/2 signalling), JNK-IN-8 (1 µM; inhibits JNKs) or SB203580 (0.7 µM; inhibits p38-MAPK). Expression of transcription factors or redox enzymes was assessed using qPCR.

Results Upregulation of EGR1 and EGR3 by H2O2 (2.8±0.2 and 3.8±0.7 fold, respectively) was significantly inhibited by PD184352, but not JNK-IN-8 or SB203580. In contrast, upregulation of FosB and Fosl1 (80.9±28.4 and 13.1±2.3 fold; one-way ANOVA with SNK post-test, n=3/4) was unaffected by any inhibitor. Atf3 upregulation (14.1±1.9 fold) was reduced by JNK-IN-8 (to 7.8±1.2 fold; p<0.001), but enhanced by PD184352 (to 23.1±0.8 fold; p<0.001). Interestingly, upregulation of HMOX and GCLC (3.7±0.6 and 3.2±0.7 fold) was reduced by SB203580 (to 2.3±0.29 and 2.3±0.35 fold; p<0.05).

Conclusions The data from even this small subset of genes indicate that the MAPKs play a substantial but differential role in regulating cardiomyocyte gene expression in response to H2O2. To gain global oversight of the relative contributions of each MAPK cascade, future experiments will use microarrays.

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