Stress signalling to cardiac gene expression and cell death

Background: Cardiovascular diseases such as heart failure and myocardial infarction are associated with increased oxidative stress, the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNFα) and interleukin 1β (IL1β) and increased death of the contractile cardiomyocytes. Oxidative stress (exemplified by H2O2) is a pivotal modulator of the balance between the life and death of cardiomyocytes. H2O2 promotes cardiomyocyte apoptosis, induces substantial changes in gene expression and activates the three principal mitogen-activated protein kinase (MAPK) pathways (ERK1/2, JNKs and p38-MAPKs), which regulate gene expression in other cell types. However, the roles of the MAPK pathways in regulation of cardiomyocyte gene expression in response to H2O2 are yet to be reported. A further pathway that may play important roles cardiac survival vs death is regulated by the protein kinase, RIPK1. In non-cardiac cell types, TNFα signals via RIPK1 to cytoprotection or cell death, depending on the cellular environment. Polyubiquitinylated RIPK1 promotes cytoprotection through activation of NFκB, JNKs and p38-MAPKs while phosphorylation and activation of RIPK1 kinase activity is associated with induction of necroptosis, a novel regulated cell death modality. Hypotheses: The first hypothesis is that ERK1/2, JNKs and p38-MAPKs play substantial roles in regulation of cardiomyocyte RNA expression during cardiomyocyte apoptosis induced by H2O2. The second hypothesis is that RIPK1, which can signal to cytoprotection through NFκB and MAPKs, or to cell death by apoptosis or necroptosis, makes important contributions to mediating the balance between life and death of cardiomyocytes. Results: To dissect the roles of the MAPK pathways in the cardiomyocyte RNA expression response to H2O2, neonatal rat cardiomyocytes were untreated or exposed to H2O2 (0.2 mM, 2 h) with or without pre-treatment (15 min) with PD184352 (2 µM, inhibits ERK1/2 signalling), JNK-IN-8 (1 µM, inhibits JNKs) or SB203580 (0.7 µM, inhibits p38-MAPKα/β) or to the inhibitors alone (2 h 15 min). RNA expression profiles were determined using Affymetrix microarrays and GeneSpring software. PD184352 alone downregulated 92 and upregulated 32 RNAs, indicating that ERK1/2 influence basal gene expression. JNK-IN-8 and SB203580 affected expression of 14 and 6 RNAs, respectively. H2O2 upregulated 295, and downregulated 195 RNAs, of which 43% and 44%, respectively, were unaffected by any inhibitor. MAPK inhibitors affected the upregulation of 37% (PD184352), 25% (JNK-IN-8) or 28% (SB203580) RNAs, and affected the downregulation of 33% (PD184352), 28% (JNK-IN-8) or 35% (SB203580) RNAs. Microarray data for selected genes were validated using qPCR. To examine the roles of cardiac RIPK1, neonatal rat cardiomyocytes were exposed to various pathophysiological stimuli and extracts immunoblotted with antibodies to RIPK1. Proinflammatory cytokines (TNFα or IL1β) induced the appearance of reduced mobility RIPK1 bands within 5 – 15 min, consistent with phosphorylation but not ubiquitinylation. Further evidence of RIPK1 phosphorylation in response to IL1β was obtained using anion-exchange chromatography. Additionally, the p38-MAPKα/β inhibitor SB203580 attenuated the appearance of reduced mobility RIPK1 bands in response to IL1β, suggestive of a potential novel regulatory mechanism of RIPK1. Concentrations of H2O2 that promote apoptosis or necrosis (>0.2 mM) resulted in reduced mobility bands of RIPK1, maximal at 60 min. Reduced mobility bands of RIPK1 were also detected in adult male rat hearts perfused with H2O2 (0.2 mM, 60 min) or subjected to ischaemia-reperfusion. To explore the regulation of RIPK1 by phosphorylation and ubiquitinylation in cardiomyocytes, adenoviruses expressing exogenous FLAG-tagged wild type and mutant RIPK1 were produced. However, the exogenously expressed RIPK1 constructs appeared to undergo cleavage when expressed in cardiomyocytes. Conclusions: The three main MAPK pathways play substantial yet differential roles in the regulation of RNA expression in response to H2O2, with the greatest contribution by ERK1/2 and smaller roles for JNKs and p38-MAPKα/β. Furthermore, RIPK1 in neonatal cardiomyocytes or whole adult hearts exhibits reduced mobility in response to oxidative stress or pro-inflammatory cytokines, likely reflective of phosphorylation and potentially activation. Accordingly, RIPK1 may play important roles in modulating the balance of life vs death of cardiomyocytes. This response may, in part, be mediated by p38-MAPK signalling.