Endothelin-1 promotes hypertrophic remodelling of cardiac myocytes by activating sustained signalling and transcription downstream of endothelin type A receptors

Abstract

G-protein coupled receptor (GPCR) mediated activation of the MAPK signalling cascade is a key pathway in the induction of hypertrophic remodelling of the heart – a response to pathological cues including hypertension and myocardial infarction. While levels of pro-hypertrophic hormone agonists of GPCRs increase during periods of greater workload to enhance cardiac output, hypertrophy does not necessarily result. Here we investigated the relationship between the duration of exposure to the pro-hypertrophic GPCR agonist endothelin-1 (ET-1) and the induction of hypertrophic remodelling in neonatal rat ventricular myocytes (NRVM) and in the adult rat heart in vivo. Notably, a 15 min pulse of ET-1 was sufficient to induce markers of hypertrophy that were present when measured at 24 h in vivo and 48 h in vitro. The persistence of ET-1 action was insensitive to ET type A receptor (ETA receptor) antagonism with BQ123. The extended effects of ET-1 were dependent upon sustained MAPK signalling and involved persistent transcription. Inhibitors of endocytosis however conferred sensitivity upon the hypertrophic response to BQ123, suggesting that endocytosis of ETA receptors following ligand binding preserves their active state by protection against antagonist. Contrastingly, α1 adrenergic-induced hypertrophic responses required the continued presence of agonist and were sensitive to antagonist. These studies shed new light on strategies to pharmacologically intervene in the action of different pro-hypertrophic mediators.

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a ‘function follows form’ model of differentiation

“…They examined the temporal profile of ERK phosphorylation and immediate gene activation during hypertrophic remodelling in neonatal myocytes and found them be transient, subsiding after 15 to 30 min. 50 Similar results have been observed for many other signalling pathways in cardiac hypertrophic remodelling. 51 In addition, despite increasing proteome coverage using a fractionation approach, leading to the identification of over 5000 proteins in H9C2 cells, low-abundant proteins such as those related to signal transduction are more difficult to detect compared to those related to morphology or metabolism.…”

MSK-mediated phosphorylation of Histone 3 Ser28 couples MAPK signaling with early gene induction and cardiac hypertrophy

“…Consistent with findings from our laboratory and elsewhere, application of the GPCR agonist endothelin-1 (ET-1) for 24 h stimulated a classical hypertrophic response in neonatal rat ventricular myocytes (NRVMs) that was associated with increases in mRNA levels of Anf/Nppa and Bnp/Nppb, in cell size and in the number of cells positive for perinuclear Anf protein ( Figure 1A-C and S1A). Alongside this hypertrophic response, the expression of IEGs including c-Fos and c-Jun, was rapidly upregulated in ET-1 stimulated NRVMs (Archer et al, 2017) ( Figure 1D, Figure S1B). These effects of ET-1 on both induction of hypertrophy and IEGs were prevented by inhibition of the MAPK pathway with PD184352 (PD, an inhibitor of the direct upstream kinase of ERK1/2, MEK1/2; Figure S1C) (Archer et al, 2017;Heineke and Molkentin, 2006) ( Figure 1A-D).…”

“…Alongside this hypertrophic response, the expression of IEGs including c-Fos and c-Jun, was rapidly upregulated in ET-1 stimulated NRVMs (Archer et al, 2017) ( Figure 1D, Figure S1B). These effects of ET-1 on both induction of hypertrophy and IEGs were prevented by inhibition of the MAPK pathway with PD184352 (PD, an inhibitor of the direct upstream kinase of ERK1/2, MEK1/2; Figure S1C) (Archer et al, 2017;Heineke and Molkentin, 2006) ( Figure 1A-D). The efficacy of PD in preventing ERK1/2 activation (and hence MAPK pathway activation) was confirmed by immunoblotting, which showed a loss of the phosphorylated active form of ERK, pERK1/2, which was elevated in ET-1 stimulated NRVMs ( Figure S1D).…”

“…MAPK signalling pathways, IEG induction and AP-1 transcription factor engagement contribute to the hypertrophic remodelling of cardiomyocytes (Archer et al, 2017). To examine the mechanisms underlying induction of IEG expression, the signalling pathways involved and their relationship to cellular hypertrophic responses were analysed in in vitro and in vivo models following acute exposure to established inducers of pathological hypertrophy.…”

“…Preceding expression of hypertrophic genes, ERK-dependent activation of an immediate early gene (IEG) response is observed in cardiomyocytes. Specifically, IEG expression is rapidly activated in cultured rat neonatal ventricular cardiomyocytes in response to stimulation with hypertrophic agonists, and in vivo following pressure overload (Archer et al, 2017;Iwaki et al, 1990;Izumo et al, 1988). This response is initiated through phosphorylation-dependent activation of the AP-1 family of transcription factors, which promote the induction of IEG expression within minutes of the initiating cue (Balmanno and Cook, 1999;Gille et al, 1992;Karin et al, 1997).…”