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Major interest surrounds how angiotensin II triggers cardiac hypertrophy via epidermal growth factor receptor transactivation. G protein-mediated transduction, angiotensin type 1 receptor phosphorylation at tyrosine 319, and β-arrestin-dependent scaffolding have been suggested, yet the mechanism remains controversial. We examined these pathways in the most reductionist model of cardiomyocyte growth, neonatal ventricular cardiomyocytes. Analysis with [(32)P]-labeled cardiomyocytes, wild-type and [Y319A] angiotensin type 1 receptor immunoprecipitation and phosphorimaging, phosphopeptide analysis, and antiphosphotyrosine blotting provided no evidence for tyrosine phosphorylation at Y319 or indeed of the receptor, and mutation of Y319 (to A/F) did not prevent either epidermal growth factor receptor transactivation in COS-7 cells or cardiomyocyte hypertrophy. Instead, we demonstrate that transactivation and cardiomyocyte hypertrophy are completely abrogated by loss of G-protein coupling, whereas a constitutively active angiotensin type 1 receptor mutant was sufficient to trigger transactivation and growth in the absence of ligand. These results were supported by the failure of the β-arrestin-biased ligand SII angiotensin II to transactivate epidermal growth factor receptor or promote hypertrophy, whereas a β-arrestin-uncoupled receptor retained these properties. We also found angiotensin II-mediated cardiomyocyte hypertrophy to be attenuated by a disintegrin and metalloprotease inhibition. Thus, G-protein coupling, and not Y319 phosphorylation or β-arrestin scaffolding, is required for epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy via the angiotensin type 1 receptor. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy is characterized by stimulation of hypertrophic signalling pathways representing the reactivation of the ‘foetal gene program’ (hypertrophic genes) and activation of the MAPK pathway. Angiotensin II (AngII) stimulates cardiac hypertrophy, a process which may involve the transactivation of epidermal growth factor receptors (ErbBs). Three subtypes of ErbBs are present in the postnatal heart (ErbB1, 2, 4), yet their precise roles are poorly understood. We have shown that the ErbB4-selective ligand neuregulin 1 beta-1 (NRG1-β1) potently stimulates cardiomyocyte hypertrophy. We now aim to determine if AngII activates hypertrophic signalling in cardiomyocytes by transactivation of ErbBs, particularly ErbB4.Ventricular cardiomyocytes from neonatal rats were treated with AngII (100 nM) or NRG1-β1 (10 nM) to induce hypertrophic signalling. ErbB1, 2, 4 were downregulated using shRNA and siRNA. Reporter assays were used to measure promoter activity of the hypertrophic genes myosin light chain 2v (MLC-2V), atrial natriuretic peptide (ANP) and cyclin D. ERK1/2 activation was measured via Western blot.NRG caused robust activation of the hypertrophic gene MLC-2V (11.3 fold increase versus untreated control) that was significantly reduced to 4.3-fold by silencing of ErbB4 (n = 5, P < 0.01), but not ErbB1 or ErbB2, confirming that ErbB4 activation contributes to hypertrophic signalling. However, knockdown of ErbB1, 2 or 4 did not affect AngII-induced activation of MLC-2V (5.8 fold increase versus untreated control). Similar results were seen for ANP and cyclin D. Similarly, ErbB4 knockdown reduced NRG1-β1-induced MAPK activation from 21.0-fold to 13.5 fold (n=4, p<0.05), whilst Ang II-induced MAPK activation was unaffected.Cardiomyocyte hypertrophy can be promoted by activation of AT1 and ErbB4 receptors. AngII-induced hypertrophic signalling (measured by promoter activity of MLC-2V, ANP and cyclin D and MAPK activation) may not involve transactivation of ErbB receptors. We are now extending these findings in vivo; we have established a model of cardiac-specific ErbB4 deletion using Cre/Lox recombination, and are undertaking echocardiographic analysis to measure cardiac hypertrophy and function. [ABSTRACT FROM AUTHOR]