Abstract/Summary

Background: Multiple diseases, including hypertension, induce stress on the heart, causing cardiac hypertrophy and eventually heart failure. Associated with these disease states are altered protein interactions, which modify cardiac function. Hypertension is estimated to affect 1.28 billion adults worldwide, with heart failure affecting 64 million people. While preclinical models exist and are used in research, they often focus on late-stage hypertension. This thesis presents a series of studies for which a standardised echocardiography protocol was developed to assess the roles of different proteins in the early stages of hypertension-induced cardiac pathology. Aims: To develop a robust, standardised and reproducible in vivo assessment of the effect of early-stage hypertension on the hearts of mouse models. This protocol must adhere to the principles of replacement, reduction, and refinement in animal research. These studies aim to show that it is possible to study the effects of early-stage hypertension on cardiac function and protein signalling. This would provide insights into how alterations in protein signalling contribute to the pathology before significant illness occurs. Methods: An in vivo echocardiography protocol was developed and performed on genetically altered mouse models (e.g., STRN, STRN3, PKN2) and their wild-type (WT) littermates, or through pharmacological inhibition in C57BL/6J mice (e.g. the effect of dabrafenib targeting BRAF). The protocol was used to assess the effect of AngII treatment on cardiac function, cardiac dimensions and arterial blood flow. AngII-induced hypertension causes haemodynamic overload of the heart which induces cardiac hypertrophy. Echocardiography was performed twice before treatment (for baseline normalisation) and sequentially post-AngII administration until the end of the experiment-specific timeline. After the final echocardiography session, tissues were harvested for biochemical and histological analysis. Results: Three papers are presented in this thesis, summarised by the following results. Echocardiography provided insights into the differential roles of STRN, STRN3, and PKN2 genetically altered mice in the cardiac adaptation to AngII-induced hypertension. For the STRN/STRN3 and PKN2 studies, the global heterozygote offspring were successfully produced and appear phenotypically normal. However, homozygote gene deletion was embryonic lethal, suggesting a compensatory mechanism in the heterozygous mice. Consistent with this, the effects of gene deletion or inhibition appeared to have a minimal effect at baseline. However, under stress, each study demonstrated that the genes are required for the heart to adapt. Since the global model affected all cardiac cells other than just cardiomyocytes, it was not clear if these genes were significant in cardiomyocytes themselves. Conditional cardiomyocyte-specific knockouts were successfully generated and applied to demonstrate that these genes play 4 a significant role in cardiomyocyte adaptation to AngII-induced hypertension. Echocardiography also helped identify the cardioprotective effects of RAF inhibition by dabrafenib against AngII-induced hypertrophy. Conclusion: Overall, this thesis contributes to the understanding of protein kinase signalling pathways in cardiac remodelling. It demonstrated that early-stage hypertension-associated cardiac hypertrophy can be assessed, providing insights into potential novel therapeutic interventions. Each chapter demonstrates the importance of echocardiography for the non-invasive assessment of these models. Exploring echocardiography, histology and biochemical analysis further will uncover the full implications of these pathways in the heart.