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.