ERK1/2 Signaling Dominates Over RhoA Signaling in Regulating Early Changes in RNA Expression Induced by Endothelin-1 in Neonatal Rat Cardiomyocytes

Abstract

BackgroundCardiomyocyte hypertrophy is associated with changes in gene expression. Extracellular signal-regulated kinases 1/2 (ERK1/2) and RhoA [activated by hypertrophic agonists (e.g. endothelin-1)] regulate gene expression and are implicated in the response, but their relative significance in regulating the cardiomyocyte transcriptome is unknown. Our aim was to establish the significance of ERK1/2 and/or RhoA in the early cardiomyocyte transcriptomic response to endothelin-1.Methods/Principal FindingsCardiomyocytes were exposed to endothelin-1 (1 h) with/without PD184352 (to inhibit ERK1/2) or C3 transferase (C3T, to inhibit RhoA). RNA expression was analyzed using microarrays and qPCR. ERK1/2 signaling positively regulated ∼65% of the early gene expression response to ET-1 with a small (∼2%) negative effect, whereas RhoA signaling positively regulated ∼10% of the early gene expression response to ET-1 with a greater (∼14%) negative contribution. Of RNAs non-responsive to endothelin-1, 66 or 448 were regulated by PD184352 or C3T, respectively, indicating that RhoA had a more significant effect on baseline RNA expression. mRNAs upregulated by endothelin-1 encoded a number of receptor ligands (e.g. Ereg, Areg, Hbegf) and transcription factors (e.g. Abra/Srf) that potentially propagate the response.Conclusions/SignificanceERK1/2 dominates over RhoA in the early transcriptomic response to endothelin-1. RhoA plays a major role in maintaining baseline RNA expression but, with upregulation of Abra/Srf by endothelin-1, RhoA may regulate changes in RNA expression over longer times. Our data identify ERK1/2 as a more significant node than RhoA in regulating the early stages of cardiomyocyte hypertrophy.

Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active

“…Primary cultures of ventricular cardiac myocytes were prepared from 1- to 2-day-old Sprague–Dawley rats by an adaptation of the method of Iwaki et al [11] as described elsewhere [12] . Hearts were removed, ventricles were dissected away from atria, and ventricular myocytes were dissociated by serial digestion with 0.4 mg/ml collagenase and 0.6 mg/ml pancreatin in sterile digestion buffer (116 mM NaCl, 20 mM HEPES, 0.8 mM Na 2 HPO 4 , 5.6 mM glucose, 5.4 mM KCl and 0.8 mM MgSO 4 , pH 7.35).…”

Small G Proteins in the Cardiovascular System: Physiological and Pathological Aspects

“…In neonatal ventricular myocytes, pharmacological inhibition of Rho-kinase (fasudil or Y27632) or transfection of dominant negative forms of Rho-kinase prevents morphological and cytoskeleton changes, protein synthesis, and ANF expression associated with response induced by hypertrophic factors such as PhE, endothelin-1, or leptin (181,245,576). In neonatal ventricular myocytes, Ras activation by hypertrophic factors is also essential to their hypertrophic effects (see above), suggesting that Ras/ MAPK and RhoA induce a hypertrophic response by separate, complementary, and synergistic pathways (58,292). In fact, RhoA/Rho-kinase-mediated actin cytoskeleton remodeling has been shown to be necessary for the translocation of ERK to the nucleus, providing a mechanism for the synergy between RhoA and Ras signaling pathways regulating gene transcription and inducing hypertrophy.…”

p90 Ribosomal S6 kinases play a significant role in early gene regulation in the cardiomyocyte response to Gq-protein-coupled receptor stimuli, endothelin-1 and α1-adrenergic receptor agonists

“…ERK1/2 phosphorylate DNA-binding transcription factors, including GATA4 (GATA-binding protein 4) and Elk1 [5], consistent with a role in direct transcriptional regulation [2]. Small-molecule inhibitors of ERK1/2 signalling inhibit the increases in expression of >70% of the mRNAs up-regulated by ET-1 [13–15]. Presumably, activation of other signalling pathways by ET-1 (e.g.…”

“…Presumably, activation of other signalling pathways by ET-1 (e.g. c-Jun N-terminal kinases and p38 MAPKs [2]) contributes to the transcriptional changes, but the data suggest that the ERK1/2 cascade plays a major role in regulating cardiomyocyte gene expression [2,5,12–15]. ET-1 is particularly potent at activating ERK1/2.…”