ErbB receptors (ErbB1 - ErbB4) are a subfamily of tyrosine kinase receptors that regulate cell proliferation and differentiation. It has been proposed that the ErbB1 subtype is transactivated by Ang II to mediate cardiac hypertrophy. However, whether other ErbB receptors, in particular the abundant subtype ErbB4, are involved in this process is not known. ErbB4 has four isoforms due to alternative splicing, each of which might play a distinct role in regulating cell activity. However, the role of individual ErbB4 isoforms in hypertrophic signalling has not been investigated. In addition, ErbB4 activation is critical for cardiac development, cardiomyocyte survival in various rodent models of cardiovascular pathologies. However, the physiological role of ErbB4 in the adult heart remains poorly understood. The overall aim of my PhD project is to examine the role of the ErbB4 receptor in mediating hypertrophic growth of cardiomyocytes in vitro, and maintenance of the adult heart in vivo.
To investigate the role of ErbB1, ErbB2 and ErbB4 in mediating hypertrophy, I inhibited individual ErbB receptors in primary neonatal rat ventricular cardiomyocytes using RNA interference or a pharmacological inhibitor (AG1478). Hypertrophy induced with Ang II (100 nM) or NRG1 (10 nM) was assessed by measuring the promoter activity of hypertrophic genes, ERK1/2 activation, and hypertrophic growth. The NRG1-induced hypertrophy was reduced by down-regulation of ErbB4 receptor but not other ErbB receptors. The down-regulation of individual ErbB receptor (ErbB1, ErbB2 and ErbB4) did not affect Ang II-induced hypertrophy. Similar results were observed with the receptor tyrosine kinase inhibitor, AG1478. This suggested that whilst ErbB4 is required for NRG1-induced hypertrophy, none of the individual ErbB receptor subtypes are required for Ang II-induced hypertrophy.
Following the studies above, I investigated whether the different ErbB4 isoforms had any functional differences in the cardiomyocytes. Irrespective of any changes in total ErbB4 mRNA levels, expression of the non-cleavable JM-b isoform was always predominant in adult heart in both physiological and pathological conditions. Although the cleavable isoform JM-a was detectable, it is not cleaved in cardiomyocytes. I replaced the endogenous ErbB4 with exogenous individual isoform in cardiomyocytes and found that all four isoforms of ErbB4 could mediate the NRG1-induced hypertrophic signalling. This suggests that the hypertrophy is triggered by a common feature of the four isoforms (ostensibly the kinase activity), and appears independent of isoform-specific features, such as the cleavable domain.
To investigate the physiological function of ErbB4 in adult heart, we adopted the tamoxifen-inducible αMHC-MerCreMer/loxP system to induce ErbB4 deletion from cardiomyocytes in the adult mouse. The expression of ErbB4 was reduced by ~ 90% at 10 days after tamoxifen treatment. Echocardiography revealed no differences in cardiac function (fractional shortening) between ErbB4-conditional knockout (ErbB4-cKO) and control groups at 3-4 months after deletion of ErbB4. However, the heart weight was increased in ErbB4-cKO animals. Interestingly, there is no change in cardiac structure (cardiomyocyte size and cardiac fibrosis) or the expression of genes associated with pathological hypertrophy. This suggests that the cardiac hypertrophy observed following ErbB4 deletion may be physiological, and raises the question as to how these animals developed cardiac hypertrophy without alteration in cardiomyocyte size or development of fibrosis. One possibility is more cardiac cells generated to cause the cardiac hypertrophy. Indeed, I found that the number of pH3 (phosphorylated histone 3, a marker of proliferation) positive cells was significantly increased in ErbB4-cKO animals. Consistent with this, the expression of NRG1, the ErbB4 selective agonist, was selectively up-regulated following ErbB4 receptor deletion. NRG1 has been suggested to induce cardiomyocyte proliferation and protect the heart under pathological conditions. Thus I proposed that this up-regulation in NRG1 may explain both the cardiac cell proliferation and the lack of cardiac dysfunction in the ErbB4-cKO animals, despite the loss of the ErbB4 receptor. Finally, we examined the long-term effects of cardiac ErbB4 deletion in mice at 7-8 months after tamoxifen treatment. These ErbB4-cKO animals developed milder physiological cardiac hypertrophy than that seen in 3-4 months cohort. Surprisingly, the cardiac ErbB4 expression in the ErbB4-cKO mice was no longer different to the controls, whereas it was reduced by ~67% at 3-4 months and ~90% at 10 days after tamoxifen treatment. The potential reasons for this reversal are not clear, but may explain the unexpected maintenance of cardiac physiology in this model.
In conclusion, the data presented in this thesis demonstrates that activation of the ErbB4 receptor is required for NRG1-induced cardiomyocyte hypertrophy. All of the four isoforms of ErbB4 can mediate this hypertrophic response. Deletion of ErbB4 from cardiomyocytes in adult mice leads to physiological cardiac hypertrophy as well as an up-regulation of NRG1. We speculate that NRG1 might protect the heart from the dysfunction caused by the loss of ErbB4, and promote cell proliferation to cause cardiac hypertrophy.