title: Echocardiography for Assessment of Cardiac Hypertrophy in Mice
Creators: name: Cull, Joshua James
Publisher: name: University of Reading
Year: 2024

description: 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 
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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.