Physiology - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
ItemCharacterising cellular and molecular mechanisms of cardiac diastolic dysfunction( 2019)Background: Diastolic dysfunction is an important contributor to many cardiac pathologies including diabetic cardiomyopathy and heart failure with preserved ejection fraction. It is characterised by ventricular stiffness, inadequate filling of the ventricles and elevated ventricular pressure. In addition to extracellular influences, evidence suggests that a cardiomyocyte specific intrinsic stiffness may also be an important contributor to diastolic dysfunction, but the mechanisms are not well understood. This might be partly due to the lack of specific animal models available to study underlying mechanisms, in particular in HFpEF. The aim of this thesis was to evaluate cellular and molecular mechanisms of diastolic dysfunction in a model of type 1 diabetes and in a newly characterised model of HFpEF, the Hypertrophic Heart Rat (HHR). Research questions: Q1. Can measurement of in vitro intact cardiomyocyte stiffness be correlated with in vivo diastolic function to determine whether cellular stiffness contributes to cardiac diastolic dysfunction in pathological settings? (Chapter 2) Q2. What are the subcellular mechanisms that contribute to increased stiffness in a pathological model of diastolic dysfunction? (Chapter 3) Q3. Can the Hypertrophic Heart Rat be used as a novel rodent model of HFpEF and what is the underlying cardiomyocyte pathophysiology driving diastolic dysfunction in HFpEF? (Chapter 4) Methods: Type 1 diabetes was induced in Sprague Dawley rats using a single dosage of Streptozotocin. The Hypertrophic Heart Rat (HHR) was characterised and utilised as a model of HFpEF. Echocardiography was used to assess in vivo heart function in diabetic and HFpEF rats. Surface electrocardiogram recordings were performed to assess in vivo electrical activity in HFpEF rats. Cardiomyocytes were isolated by collagenase dissociation. Under loaded conditions glass fibers were attached (MyoTak) at the cell longitudinal surface, and paced cardiomyocytes (1Hz, 2.0mM Ca2+, 37°C) were serially stretched (011.2%, piezomotor). Force development and intracellular Ca2+ transients (Fura-2AM, 5µM) were simultaneously measured (Myostretcher, IonOptix). In the HHR, histological analysis was undertaken to evaluate collagen deposition. Intracellular Ca2+ and contractility was measured in single unloaded cardiomyocytes (4Hz, 2.0mM Ca2+, 37°C). Left ventricular tissue was homogenised and used for Western blot analysis of Ca2+ handling proteins. Results: A1. Validation of in vivo and in vitro methodologies for the measurement of cardiomyocyte and cardiac diastolic function along with confirmation that in vitro cardiomyocyte stiffness directly correlates to in vivo cardiac dysfunction. This verifies the contribution of cellular stiffness to cardiac diastolic dysfunction in the pathological setting. (Chapter 2) A2. Cardiomyocyte stiffness was shown to be an important contributor to diastolic dysfunction in the diabetic heart. The additive contribution of myofilament cooperativity reduction and slowed Ca2+ reuptake were found to be the subcellular mechanisms for the intracellular stiffness. (Chapter 3) A3. A new model of HFpEF was successfully characterised which closely mirrors clinical pathology without surgical or drug intervention. Animals display early mortality, with cardiac diastolic dysfunction, preserved ejection fraction and arrhythmias. The cardiomyocyte pathology was one of hypercontractility and Ca2+ overload, contrasting strongly with what has been reported in systolic failure leading to potential new therapeutic targets for HFpEF treatment. (Chapter 4) Conclusion: This thesis demonstrates that intact cardiomyocyte stiffness contributes directly to cardiac diastolic dysfunction, which was validated in two separate pathological models. Importantly, this is the first evidence that there is an increase in the slope of the end diastolic force length relation in intact diabetic cardiomyocytes indicating increased cellular stiffness. This was linked to changes in Ca2+ reuptake during relaxation and reduced myofilament cooperativity. In addition, a newly characterized model of HFpEF was described, along with cellular and molecular changes that are apparent in this model of diastolic dysfunction, providing new insight and potentially leading to new therapeutic targets to treat HFpEF. Taken together, this thesis advances the mechanistic understanding of the cellular and molecular mechanisms of diastolic dysfunction.
-
ItemAdipose, sex steroids and atrial arrhythmia vulnerability( 2018)Background: Pericardial adipose deposition occurs in ageing and obesity, and independently contributes to the development of atrial fibrillation. The mechanisms underlying this association are not yet understood. Investigations to date have focused on physical conduction block posed by infiltrating adipose and the secretion of pro-inflammatory/pro-fibrotic paracrine factors into the atria. Though not yet investigated in the pericardial adipose, white adipose depots are established sites of oestrogen synthesis. Considering the reported actions of oestrogens on the heart, it is hypothesised that pericardial adipose may represent an important source of local oestrogen synthesis, exerting paracrine actions on the myocardium. Research questions: 1. Do myocardial and pericardial adipose tissues express aromatase, and do locally-derived oestrogens affect the vulnerability to atrial arrhythmia? (Chapter 2) 2. Does disruption of aromatase activity in aged and obese mice influence basal cardiac electrophysiology and the susceptibility to atrial arrhythmia? (Chapter 3) 3. Can liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) sensitive methodology be used to quantify androgens and oestrogens in human and mouse myocardium and pericardial adipose tissues? (Chapter 4) Methods: Aromatase expression in human and rodent myocardium and pericardial adipose was measured by Western immunoblotting. Arrhythmia vulnerability was assessed in isolated hearts from male C57BL/6 mice (‘young’, ‘aged’ or ‘aged’ + high fat diet). Hearts were perfused with a hypokalaemic solution (2 mmol [K+]) and subjected to programmed electrical stimulation to provoke arrhythmias. In addition, hearts were perfused with acute perfusion with 17β-oestradiol (or vehicle) and arrhythmic provocation repeated. Aromatase knockout and wild type mice (male and female) were fed a control or high fat diet for 40 weeks. Mice were subjected to electrocardiographic and echocardiographic assessment prior to isolated heart atrial arrhythmia provocation experiments. Human and mouse myocardium and adipose tissues were homogenised, derivatised with dansyl chloride and subjected to LC-MS/MS for sex steroid quantification. Mass spectrometric technique was developed using the aromatase knockout as a positive control for androgens and a negative control for oestrogens. Results: 1. Aromatase is expressed in human/rodent myocardium and pericardial adipose, conferring the capacity for local androgen to oestrogen synthesis. Pericardial adipose capacity to synthesise oestrogens increased by 30-50x in aged hearts, which were significantly more vulnerable to atrial arrhythmias. (Chapter 2) 2. The aromatase knockout model of oestrogen depletion and androgen excess revealed a sex-differential phenotype in the susceptibility to atrial arrhythmia. Left atrial action potential duration was prolonged and arrhythmia vulnerability greater in female aromatase knockout mice compared to all other groups. The combined influence of extensive pericardial adipose deposition and a highly androgenic/oestrogen-depleted environment was unique to the female aromatase knockout mice and may have been decisive in driving the exacerbated vulnerability to atrial arrhythmias. (Chapter 3) 3. LC-MS/MS methodologies were optimised for the detection and quantification of sex steroids in human/mouse myocardium and adipose. Successful quantification of testosterone and progesterone was achievable, but concentrations of oestrogens in tissues were below the technical limits of detection. (Chapter 4) Conclusions: This thesis identifies that pericardial adipose expresses aromatase and indicates a probable capacity for oestrogen synthesis, hence supporting the presence of a local cardiac androgen-oestrogen system. Pericardial adipose derived oestrogens (and androgens) are recognised as probable paracrine mediators capable of altering atrial arrhythmic vulnerability. In addition, the data support the clinically observed correlation between pericardial adipose accumulation and atrial fibrillation. Mass spectrometric methodology is capable of quantifying tissue testosterone and progesterone concentrations, but tissue oestrogens are below the limits of detection. Taken together, this thesis advances the mechanistic understanding of the link between pericardial adipose accumulation and greater atrial arrhythmia vulnerability.
-
ItemFructose and the heart: myocardial remodelling and functional responses( 2011)Context: Large population studies have demonstrated that high dietary sugar is associated with increased risk for type 2 diabetes and cardiovascular disease independent of body mass index. Specifically, fructose intake has been linked with the onset of insulin resistance and evidence is emerging that dietary fructose induces a specific cardiac pathology. Metabolism of fructose bypasses the phosphofructokinase regulatory step of glycolysis and high throughput may lead to distinct cellular disturbances. But whether fructose can have direct effects on cardiomyocytes is unknown. The heart may be especially vulnerable to the indirect (i.e. systemic insulin resistance) and direct (i.e. cardiomyocyte metabolic dysregulation via phosphofructokinase bypass) effects of fructose and requires investigation. Aims: This thesis aimed to investigate the cardiac-specific effects of high dietary fructose, specifically assessing whole heart morphology and signalling, and cardiomyocyte performance. Evaluation of cardiomyocyte capacity for fructose transport and utilisation was undertaken to assess the potential for high fructose intake to have direct effects on the heart. Specific questions of cardiac pathophysiological importance were addressed: 1. Do cardiomyocytes have the capacity to transport and utilise fructose? [Chp3]2. How does dietary fructose affect cardiac morphology and cell survival signalling? [Chp4]3. How does dietary fructose affect cardiomyocyte contractile function and Ca2+ handling? [Chp5]4. Does cardiac angiotensin II (AngII) upregulation interact with dietary fructose-induced cardiac signalling alterations? [Chp6] Methods: Detailed in vitro studies manipulating glucose/fructose substrate were used to functionally demonstrate that rodent cardiomyocytes have the capacity to utilise fructose. Dietary fructose-induced cardiac pathology was evaluated in mice with histological, biochemical, molecular, and cellular techniques. Assessment of dietary fructose cardiac effects in the presence of an underlying predisposition for renin-angiotensin system upregulation utilised the cardiac-specific angiotensinogen overexpressing mouse model. Results: The major overall findings of this thesis are: 1. The fructose-specific transporter, GLUT5, is expressed in adult rat ventricular cardiomyocytes, and functional cardiomyocyte fructose utilisation is evident. Fructose reversed the 2-deoxyglucose(2DG)-induced slowing of the Ca2+ transient time to peak: 2DG: 29.0±2.1ms vs. glucose: 23.6±1.1ms vs. fructose + 2DG: 23.7±1.0ms; p<0.05). 2. A high fructose diet induces a specific cardiac pathology associated with systemic insulin resistance. This cardiac pathology is characterised by: • myocardial autophagy activation (46% increase in LC3BII:I ratio; 47% increase in p62) but no evidence of apoptosis induction • cardiac remodelling (28% increased collagen deposition with no change in heart size)