Investigation into the effects of viral-mediated Ang-(1-9) delivery on cardiac function and remodelling in a mouse model of myocardial infarction

Fattah, Caroline (2015) Investigation into the effects of viral-mediated Ang-(1-9) delivery on cardiac function and remodelling in a mouse model of myocardial infarction. PhD thesis, University of Glasgow.

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Coronary heart disease (CHD) leading to myocardial infarction (MI) is the primary cause of morbidity and mortality globally. Following an MI, a number of structural and functional changes to the myocardium occur, known as cardiac remodelling. Initially, these changes are adaptive. Inflammation and deposition of extracellular matrix (ECM) components and collagen occurs in order to form the scar tissue, and in response to the rise in wall stress cardiomyocytes undergo adaptive hypertrophy in order to maintain contractile performance of the heart. There are also alterations in the electrical properties of the heart, including dysregulation of a variety of Ca2+-handling proteins. In the long term these processes become maladaptive. Reactive fibrosis stiffens the ventricular wall, eccentric hypertrophy contributes to expansion of the left ventricle (LV) and the electrical changes lead to reduced contraction and increased propensity to arrhythmia, all of which can contribute to the development of heart failure (HF) and the possibility of sudden cardiac death (SCD). Dysregulation of the renin-angiotensin system (RAS) is one of the factors responsible for driving these adaptive and maladaptive remodelling processes. The main effector peptide of the RAS, Angiotensin II (Ang II), acting via the Angiotensin type 1 receptor (AT1R) mediates the majority of the maladaptive changes which occur post-MI. The counter-regulatory axis of the RAS has been found to counteract many of deleterious effects associated with Ang II signalling. The peptide Ang-(1-7), signalling via Mas, has been found to exert anti-fibrotic and anti-hypertrophic effects and improve LV function post-MI. Less is known about the peptide Ang-(1-9), however there is evidence that it too is able to exert anti-hypertrophic and anti-fibrotic effects post-MI, however improvements on cardiac function have not been previously demonstrated. Therefore, the main aim of this thesis was to investigate the therapeutic potential of Ang-(1-9) via a gene transfer approach on adverse remodelling in a mouse model of MI, with a focus on cardiac functional parameters.

First, the mouse model of MI was established and characterised for adverse structural and functional remodelling parameters. Haemodynamic and functional measurements using echocardiography and pressure-volume (PV) loops demonstrated a reduction in contractility and ejection fraction (EF) following MI. This was also associated with an increase in concentric cardiomyocyte hypertrophy, fibrosis and collagen deposition. Moreover, alterations in expression of cardiac AT1R, Angiotensin type 2 receptor (AT2R), Angiotensin converting enzyme (ACE) and Angiotensin converting enzyme 2 (ACE2) were detected. Following characterisation, this model was utilised to assess the effects of viral-mediated Ang-(1-9) delivery.

Initially, an adenoviral vector (Ad) expressing a biological peptide pump enabling the synthetic production of Ang-(1-9) [RAdAng-(1-9)] was utilised in order to assess the effects of the peptide following MI. Efficient transduction of the healthy myocardium following MI was demonstrated using direct intramyocardial Ad injection. Initially, it was found that administration of RAdAng-(1-9) reduced the mortality rate associated with the MI procedure, with a reduction in deaths from unknown causes and cardiac rupture. Functional cardiac parameters were monitored using echocardiography for a 4 week period, with RAdAng-(1-9) administration found to be associated with increased LV fractional shortening (FS) compared to MI controls from 1 to 4 wks. PV loop measurements confirmed this improved function, with increased end systolic pressure (ESP) and normalised EF found in RAdAng-(1-9) administered animals. Post-mortem analysis and histology demonstrated an anti-hypertrophic effect of RAdAng-(1-9) delivery, with reduced heart weight and cardiomyocyte thickness in MI animals over-expressing Ang-(1-9). An anti-fibrotic effects was also evident, with a reduction in total LV fibrosis demonstrated, primarily due to reduced collagen I expression.

Next, an adeno-associated virus serotype 9 (AAV9) vector expressing the Ang-(1-9) fusion protein expression cassette [AAVAng-(1-9)] was utilised in the same mouse MI model in order to assess the effects of Ang-(1-9) 8 wks post-MI. Global transduction of the healthy myocardium in MI hearts using a single tail vein injection of AAV9 was demonstrated, with transgene expression detectable as early as 1 wk post-MI. Similarly to the previous study, AAVAng-(1-9) delivery demonstrated a reduction in the incidence of cardiac rupture following MI. Echocardiography also demonstrated improvements in cardiac contractility, with increased FS evident from 1 to 8 wks post-MI in AAVAng-(1-9) transduced animals. Again, PV loop measurements found that AAVAng-(1-9) increased ESP and normalised EF. Moreover, CO was significantly elevated in Ang-(1-9) expressing animals. Due to the more advanced 8 wk time-point, a reduction in LV stiffness was detectable in AAVAng-(1-9) animals compared to controls as measured by the end diastolic pressure-volume relationship (EDPVR). In contrast to RAdAng-(1-9) administration at 4 wks post-MI, no anti-hypertrophic effect was detectable, with an increase in heart weight and cardiomyocyte thickness found in AAVAng-(1-9) animals equivalent to control MI groups. However, AAVAng-(1-9) administration was associated with a reduction in total fibrosis in MI animals, which was attributable to reduced collagen I expression. Moreover, gene expression analysis in MI animals found AT2R expression was elevated in the myocardium of AAVAng-(1-9) administered animals, which had not previously been identified.

Finally, in order to attempt to elucidate the mechanism of action of Ang-(1-9), single cardiomyocyte Ca2+-handling measurements were utilised in order to assess its effects on Ca2+-handling protein function. Pre-treatment of isolated mouse cardiomyocytes with 1 µM Ang-(1-9) increased cardiomyocyte Ca2+-transient amplitude and shortening compared to control, equivalent to the effects seen using 1 µM of Ang II. However, only Ang II induced an increase in spontaneous rises in intracellular Ca2+. Ang-(1-9) pre-treatment was also associated with increased sarcoplasmic reticulum (SR) Ca2+ content.

Overall the findings from this thesis have demonstrated for the first time that Ang-(1-9) exerts beneficial effects on cardiac function post-MI, which may in part be due to modulation of cardiomyocyte Ca2+-handling. These findings provide the impetus to further investigate the potential of Ang-(1-9) as a possible therapeutic agent to prevent progression of adverse cardiac remodelling and HF post-MI.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Supported by the British Heart Foundation.
Keywords: Ang-(1-9), counter-regulatory renin-angiotensin system, cardiac function, MI.
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health > Cardiovascular & Metabolic Health
Supervisor's Name: Nicklin, Dr. Stuart A and Loughrey, Dr. Christopher M
Date of Award: 2015
Depositing User: Miss Caroline Fattah
Unique ID: glathesis:2015-6874
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 16 Nov 2015 15:43
Last Modified: 05 Nov 2018 10:37
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