Physiology - Theses

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    New insights regarding the myocardial-adipose paracrine axis
    Waddell, Helen Moira Munro ( 2021)
    Background: Pericardial adipose accumulation is a major risk factor for atrial fibrillation, independent of body mass index and non-cardiac adipose tissue volumes. Pericardial adipose is associated with detrimental perturbations in atrial electrophysiology, however the cellular mechanisms behind this relationship are poorly understood. Investigations into the pericardial adipose-myocardium paracrine axis have focused on pro-inflammatory/fibrotic factors, without assessing the direct paracrine influence of pericardial adipose on atrial electrophysiology. It is hypothesised that atrial electrophysiology is selectively impaired by pericardial adipose secreted factors compared to non-cardiac adipose, due to differences in the types of secreted proteins. Research Aims: (Relevant chapters in brackets) 1. Optimise and compare electrophysiology recordings of different types of immortalised and primary cardiomyocyte monolayers. (2) 2. Assess whether secreted factors specific to pericardial adipose can influence cardiomyocyte electrical conduction properties. (3) 3. Determine whether greater cardiac adiposity alters pericardial adipose paracrine phenotype and its influence on cardiomyocyte function. (4) 4. Compare and contrast protein release from sheep epicardial, paracardial and subcutaneous adipose tissue samples, to evaluate paracardial adipose as a potential paracrine mediator of cardiac pathology. (5) Methods: In vitro electrophysiology experiments (multi-electrode array) were optimised with neonatal rat ventricular myocytes (NRVM) and compared to immortalised atrial HL-1 cultures. To assess the paracrine influence of pericardial adipose, ovine and murine adipose tissue was incubated to produce a ‘conditioned media’. Multi-electrode mapping of HL-1 cultures was performed in the presence of pericardial adipose conditioned media from normal weight vs obese mice, and murine pericardial vs subcutaneous adipose. Identification of adipose secreted proteins was achieved by explorative proteomics using LC-MS/MS instrumentation. Subsequent gene ontology analysis (Enrichr and PINE software) was applied to proteomic datasets to profile differences between protein subgroups secreted from subcutaneous and pericardial adipose in mice and sheep. Results: Some of the major findings include: 1. Multi-electrode electrophysiological recordings could be comprehensively analysed with HL-1 and NRVM cultures. HL-1 cultures had a slower conduction velocity and shorter field potential repolarisation yet were less variable than NRVMs. 2. In obesity, murine pericardial adipose secreted proteins were substantially different to those secreted from subcutaneous adipose. Slowed electrical propagation was observed in HL-1 cell monolayers that received pericardial adipose conditioned media only. 3. The paracrine influence of pericardial adipose on HL-1 cell electrophysiology, or the types of pericardial adipose secreted proteins, are not altered by cardiac adiposity. Extracellular vesicle and focal adhesion associated proteins were identified in pericardial secretome from both normal and obese mice. 4. Ovine paracardial and epicardial adipose secreted proteins had a high amount of commonality, which included proteins related to inflammation, focal adhesion, and extracellular vesicles. The few points of contrast involved low-density lipoproteins, which may have implications in coronary artery disease. Conclusions: Atrial electrical propagation is selectively slowed by pericardial adipose through a direct paracrine action on cardiomyocytes. The types of proteins secreted by pericardial adipose were highly similar in samples from normal weight mice, obese mice, and normal weight sheep, yet contrasted significantly to subcutaneous secreted factors. The evidence provided herein collectively indicates that the association between obesity and atrial fibrillation is defined by the extent of cardiac adipose accumulation and therefore paracrine potential, and less so by pericardial adipose secretome profile. Therapeutic interventions which limit the release of conduction modulating adipokines from pericardial adipose tissue may provide a novel preventative treatment strategy for re-entrant arrhythmias.
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    Investigating the therapeutic potential of heat shock protein 70 (HSP70) induction for skeletal muscle injury
    Thakur, Savant Singh ( 2020)
    Skeletal muscle has high regenerative capacity due to a resident population of adult stem cells (MuSCs). These MuSCs normally exist in a quiescent state but when a muscle is injured, the MuSCs become activated and re-enter the cell cycle, proliferate, differentiate and undergo fusion to form multinucleated myotubes. During myogenesis, dramatic changes occur in cell size, shape, metabolism and motility, which cause cellular stress and alter muscle proteostasis. Heat shock proteins (HSPs) are molecular chaperones with the potential to maintain proteostasis by regulating protein biosynthesis and folding, facilitating transport of polypeptides across intracellular membranes, and preventing stress-induced protein unfolding/aggregation. HSP70 is the most widely studied HSP relevant to skeletal muscle. How HSPs, particularly HSP70, regulate myogenesis and whether manipulation of HSP expression can enhance muscle repair, remain important unanswered questions. In Chapter 4 of this thesis, HSP expression was characterised in C2C12 cells during proliferation and differentiation. Whole cell lysates prepared from proliferating C2C12 cells and C2C12 cells undergoing myogenic differentiation for 1-4 days (D1-D4), were examined for their expression of various HSPs based on SDS-PAGE and western immunoblotting. HSP25 decreased at D4 of differentiation (P < 0.05). HSP40 expression was high in proliferating myoblasts but decreased at the onset of differentiation (P < 0.05). HSP60 decreased between early and late differentiation (P < 0.001). HSP90 and HSP110 were highly expressed in proliferating myoblasts and decreased during early differentiation. Lastly, HSP70 protein expression peaked during early stages of differentiation, just preceding the expression of myogenin (P < 0.05). These findings imply that many of the HSPs are involved in the regulation of myogenesis at different stages, with HSP70 potentially having a role in myoblast fusion. Based on these findings, the studies in Chapter 5 sought to better understand the roles of Hsp70 in myogenesis. Plasmid DNA encoding GFP alone or a GFP-Hsp70 fusion protein was transiently transfected into proliferating C2C12 myoblasts and effects on cell proliferation, differentiation and fusion were determined. Hsp70 overexpression did not alter either proliferation or early differentiation of C2C12 myoblasts. However, GFP-Hsp70 overexpression resulted in an ~30% increase in both median number of myonuclei per myotube (P < 0.01) and median myotube width (P < 0.0001) relative to GFP, three days after induction of differentiation. Similar increases were observed in median number of myonuclei per myotube (P < 0.001) and median myotube width (P < 0.0001) for GFP-Hsp70 transfected myotubes compared to GFP at D4 post-differentiation. Lastly, in Chapter 6, a preliminary study was conducted in mice that had either a systemic deletion or skeletal muscle-specific overexpression of HSP70 to assess the impact of HSP70 deletion or HSP70 overexpression on muscle regeneration after ischemia-reperfusion (IR) injury. Interestingly, neither HSP70 deletion nor overexpression affected muscle degeneration or regeneration after IR injury. However, muscle mass was significantly reduced at D14 after IR injury in all mouse strains, suggesting that the IR injury model employed had effects on both the blood and the nerve supply to the affected limb. In conclusion, the findings described herein imply that enhanced HSP70 expression promotes myoblast fusion and has the potential for treating muscle injuries and numerous disorders associated with muscle atrophy. Future studies should attempt to uncover the cellular mechanisms and identify fusion-related molecules that interact with HSP70 to drive myoblast fusion.
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    Interactions of the enteric nervous system with the gut microbiome in the neuroligin-3 R451C mouse model of autism
    Maduwelthanne Herath Mudiyanselage, Madusani Herath ( 2020)
    Autism patients are four times more likely to be hospitalized due to gastrointestinal (GI) dysfunction compared to the general public. However, the exact cause of GI dysfunction in individuals with autism is currently unknown. Genetic predisposition to autism spectrum disorder (ASD) has been highlighted in various studies and mutations in genes that affect nervous system function can drive both behavioural abnormalities and GI dysfunction in autism. Neuroligin-3 (NLGN3) is a postsynaptic membrane protein and the R451C missense mutation in the NLGN3 gene is associated with ASD. Recent studies revealed that the NLGN3 R451C mutation induces GI dysfunction in autism patients as well as in mice but, the cellular localization and the effects of this mutation on NLGN3 production in the enteric nervous system (ENS) have not been reported to date. The intestinal mucosal barrier is the interface separating the external environment from the interior of the body. Mucosal barrier functions are directly regulated by the enteric nervous system. Therefore, ENS dysfunction can induce mucosal barrier impairments. An impaired intestinal barrier has been reported in autism patients, but neurally-mediated barrier dysfunctions have not been assessed in transgenic autism mouse models with an altered nervous system. The intestinal mucus layer is the outermost layer of the mucosa which separates the intestinal microbiota from the intestinal epithelium. The mucus layer also serves as an energy source for mucus-residing microbes in the intestine. Although the composition of mucus-residing microbiota is altered in a subset of autism patients, the underlying physiological interactions between the host and these microbes are unclear. Identifying the precise spatial location of microbial populations in the gut is essential in order to understand host-microbial interactions but this has not been investigated in Nlgn3R451C mice. In Chapter 3, I developed a method combining RNAScope in situ hybridization and immunohistochemistry technique to localize Nlgn3 mRNA in enteric neuronal subpopulations and glia. Further, a 3-dimensional quantitative image analysis method was developed to measure the Nlgn3 mRNA expression in the ENS. The same protocol was used to determine the effects of the Nlgn3 R451C mutation on Nlgn3 mRNA synthesis in the enteric nervous system. Findings from this study showed that, Nlgn3 mRNA is expressed in most submucosal and myenteric neurons in the ENS. Interestingly, this study revealed for the first time that Nlgn3 mRNA is expressed in enteric glia. In addition, analysis from this study demonstrated that the R451C mutation reduces Nlgn3 mRNA expression in most enteric neurons in mutant mice compared to WT. In Chapter 4, I investigated the effects of the Nlgn3 R451C mutation on intestinal mucosal barrier functions including the paracellular pathway and mucosal secretion in the small intestine. The Ussing chamber technique was used to measure the paracellular permeability and mucosal secretion ex vivo. Since the paracellular pathway is regulated by tight junctions, effects of this mutation on tight junction protein gene expression were measured using real-time (RT) PCR array and droplet digital (dd) PCR approaches. The impact of the Nlgn3 R451C mutation on the neurochemistry of the submucosal plexus was examined using immunocytochemistry. Results from these experiments indicated that the R451C mutation increases paracellular permeability and decreases transepithelial resistance (TER) in the distal ileum. However, ileal tight junction protein gene expression is unchanged in mutant mice compared to WT. Pharmacological stimulation of submucosal ganglia decreased the neurally-evoked mucosal secretion in mutant mice compared to WT. In addition, immunohistochemistry data revealed increased numbers of non-cholinergic and decreased cholinergic neuronal populations in the submucosal plexus in the distal ileum but not in the jejunum in Nlgn3R451C mutant mice. Given that, I identified altered barrier functions in the distal ileum of Nlgn3R451C mutant mice, in chapter 5, I also analysed the spatial distribution of the mucus-residing microbial populations in this region. To determine the spatial distribution of total bacteria, phylum Bacteroidetes, phylum Firmicutes, Akkermansia muciniphila (A. muciniphila) and Bacteroides thetaiotamicron (B. thetaiotamicron) were labelled using fluorescent in situ hybridization. Immunofluorescence for the mucin-2 protein was incorporated to co-stain the mucus and determine the thickness of the mucus layer. Both the spatial pattern of microbial populations and mucus layer thickness were analysed using MATLAB-based BacSpace software. Immunofluorescence experiments revealed that the R451C mutation increases mucus density adjacent to the epithelium. Along with increased mucus density, the total bacterial density was higher in the mucosa in mutant mice. Further, a decreased ratio of Bacteroidetes/Firmicutes, a decreased A. muciniphila density and increased density of B. thetaiotamicron were observed in mutant mice. Overall, findings from this thesis revealed that NLGN3 is expressed in the enteric nervous system and that the R451C mutation reduces Nlgn3 mRNA expression levels in enteric neurons. Furthermore, the Nlgn3 R451C mutation impairs intestinal mucosal barrier integrity. Findings from this study also revealed that this mutation alters mucus density as well as the spatial distribution and composition of the microbial community in the distal ileum in mice. Therefore, these findings highlight that an autism-associated gene mutation that affects nervous system function impairs the mucosal barrier and may contribute to the pathophysiology of GI dysfunction in ASD.
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    Pericardial adiposity and cardiac arrhythmia vulnerability
    Wells, Simon Philip ( 2020)
    Background: The cellular mechanisms that predispose to arrhythmia include heterogeneic conduction slowing and/or changes in repolarisation time (e.g. regional alterations in transmural electrophysiology). Augmented pericardial adiposity is an independent risk factor for atrial and ventricular fibrillation, but the cellular mechanisms are unknown. Very limited data indicate pericardial adipose tissue exhibits paracrine characteristics, secreting factors which modulate cardiac electrophysiology. Recent evidence demonstrates pericardial adipose tissue can synthesise oestrogens which are known to affect cardiomyocyte function. It is hypothesised that augmented pericardial adiposity promotes epicardial conduction slowing and/or repolarisation prolongation through paracrine mechanisms to predispose to arrhythmia. Research aims: (relevant chapters in brackets) 1. Establish that transmural electrophysiology is modulated in the context of elevated cardiac adiposity. (3) 2. Compare electrophysiology of cardiomyocyte cultures from different origins as prelude to examining the paracrine influences of pericardial adipose tissue. (4) 3. Ascertain that obesity and epicardial adiposity associate with cardiac electrophysiological remodelling which may increase arrhythmia vulnerability. (5) 4. Identify that sex steroids can modulate atrial electrophysiology, indicating their potential as paracrine regulators of arrhythmia vulnerability. (6) Methods: Cardiac electrophysiology was assessed in both atrial and ventricular tissues utilising multiple in vitro methodologies. To establish the effects of adiposity on transmural electrophysiology, male rats were fed a high fat diet, then tangential left ventricular slices were electrophysiologically mapped. To optimise cardiomyocyte culture conditions, neonatal rat ventricular myocyte (NRVM) and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) electrophysiology was compared. Fragments of epicardial adipose tissue were co-cultured with hiPSC-CMs to assess the paracrine influence on cardiomyocyte electrophysiology. The effects of obesity on left atrial electrophysiology were determined using male mice fed a Western diet. Left atrial electrophysiology was also assessed in male and female chow-fed mice in the absence/presence of sex steroids. Results: Some of the overall findings of this investigation include: 1. Augmented pericardial adiposity likely disrupts ventricular transmural conduction gradients through putative local actions on the epicardium. 2. hiPSC-CM and NRVM cultures display similar electrophysiology and exhibit good capacity to detect changes in repolarisation via experimental intervention. 3. Obesity associates with prolonged epicardial atrial action potential duration. This is caused by a paracrine influence of pericardial adipose tissue on cardiomyocytes. 4. Oestrogen and testosterone prolong repolarisation and slow conduction in the left atrium, indicating their potential as paracrine regulators of arrhythmia vulnerability. Conclusions: Pericardial adipose tissue has capacity to selectively prolong epicardial activation and repolarisation. This is at least in part, mediated through a paracrine mechanism. Prolonged repolarisation and slowed conduction predispose to triggered and reentrant arrhythmias, respectively. Together, these data indicate that augmented cardiac adiposity has a causative effect on cardiomyocyte electrophysiology to increase arrhythmia likelihood.
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    The effect of early life antibiotic exposure on the development of the gut microbiota and enteric nervous system
    Hung, Lin Yung ( 2019)
    Early postnatal life is a critical stage of microbiota establishment and ENS development. While the initial postnatal stage from birth is fundamental for the development of the gut microbiota and ENS, weaning is another key developmental period where there are major changes in diet, behaviour and physiology, and notably, microbiota. Antibiotics are frequently administered to infants and young children, however, recent studies have identified prospective long-term health consequences of early life antibiotic exposure on the developing gut microbiota. Yet, how antibiotic influences short and long-term ENS development remains unclear. Vancomycin is given as a prophylactic to preterm babies and paediatric patients to treat and prevent infections. It is also one of the most commonly used antibiotics on its own or as part of a cocktail in research to induce dysbiosis in mice. The aim of my PhD was to examine how early life exposure to vancomycin during two critical developmental periods affects microbiota and ENS development and whether changes observed during early postnatal life have long-term repercussions. In chapter 3, I investigated if acute administration of vancomycin, during the early postnatal period, influenced gut microbiota and ENS development. A single regimented dose of either water or vancomycin was administered daily to Wnt1-Cre;R26R-GCaMP3 mouse pups from postnatal (P) day 0 to P10/11. These mice contain a genetically-encoded fluorescent Ca2+ indicator in all enteric neurons and glia. At P10/11, vancomycin-fed pups showed significant dysbiosis, reduced myenteric neuron density and altered nNOS and calbindin neuronal subtype proportions compared to water-fed littermates. Using Ca2+ imaging, I showed that vancomycin-fed pups had more neurons responding to electrical stimulation applied to interganglionic connectives and larger amplitudes of train-evoked [Ca2+]i transients. These changes in the ENS contributed to dysmotility of the colon of vancomycin-fed pups. In contrast to the colon, the structure of the ENS and motility patterns of the duodenum were not affected by vancomycin, ruling out drug toxicity effects. P10/11 vancomycin-fed pups also had lower numbers of serotonin (5-HT) positive cells in the colonic mucosa. Altered 5-HT metabolism in these animals were confirmed by performing mass spectrometry on 5-HT biosynthesis intermediates, showing reduced concentrations of the 5-HT metabolite, 5-HIAA and droplet digital PCR (ddPCR) revealing increased gene expression of the 5-HT transporter, SERT. Bypassing tryptophan hydroxylase, by supplementing vancomycin-fed pups with 5-HTP, restored 5-HIAA levels in the colonic mucosa and prevented some of the vancomycin-induced effects on myenteric neurons, colonic motility and gut microbiota. Therefore, vancomycin exposure during the neonatal period induced significant developmental changes to both the gut microbiota and ENS. Some of these changes could be mediated by altered mucosal serotonergic signalling. In Chapter 4, I examined if vancomycin-induced changes on the gut microbiota and ENS observed at P10 were long-lasting. Newborn mouse pups were only treated with water and vancomycin till P10, then pups were left to grow to adulthood. 6-week-old mice given neonatal vancomycin had enlarged caeca, which is an indication of dysbiosis. This suggests that the gut microbiota of vancomycin-fed mice was not fully recovered despite cessation of antibiotic treatment. Adult mice treated with neonatal vancomycin had sustained reduction in myenteric neuron density. However, alterations in the proportions of nNOS+ and calbindin+ neurons observed during the neonatal periods was now restored. In contrast to the heightened [Ca2+]i activity at P10s, adult mice given neonatal vancomycin had lower numbers of neurons responding to electrical stimulation and no change in the amplitudes of electrically-evoked [Ca2+]i transients in their myenteric neurons compared to water-fed controls. Furthermore, there were no treatment-induced changes in colonic motility. Interestingly, faecal water content, which was unaffected in vancomycin-fed pups at P10, was lower in adult mice given neonatal vancomycin compared to controls. These findings indicate that although vancomycin treatment is terminated, the gut microbiota is not fully recovered and significant re-modelling of the ENS occurs, some of which are distinct to changes observed during the neonatal period. In Chapter 5, I explored the effects of vancomycin exposure between weaning and adulthood. From the day of weaning, mice were administered vancomycin or sterile water in their drinking bottles for three weeks. At 6-weeks of age, vancomycin-treated mice had dysbiosis accompanied with enlarged caeca. Similar to vancomycin-treated neonates in Chapter 3, increased synaptic activity exhibited by enteric neurons were mainly observed by larger amplitudes of train-evoked [Ca2+]i transients and increased number of neurons responding to electrical stimulation. However, in contrast to antibiotic exposure during the neonatal period, vancomycin-treated mice displayed significantly slower colonic motility, increased faecal water content and a decrease in the proportions of ChAT+ cholinergic neurons including calbindin and neurofilament-M subtypes in the myenteric plexus of the colon. Moreover, vancomycin treatment between weaning and adulthood had no effects on the serotonergic system in the colonic mucosa. Collectively, these findings suggest that vancomycin exposure from weaning had differential effects on the gut microbiota and ENS compared to administration of the antibiotic during the neonatal period. Together, my study is the first to identify and compare effects of antibiotic exposure on the gut microbiota and ENS during two critical stages of development. While vancomycin did not deplete bacterial diversity and abundance, it caused profound shifts in microbial composition in both developmental periods. Additionally, acute vancomycin exposure in both periods, resulted in dysmotility and alterations of the neuronal circuitry. Although the effects on colonic motility for mice given neonatal antibiotic treatment did not appear to be long-lasting, changes in the ENS and disrupted faecal and caeca weights, which manifested only in adulthood, suggests that early life exposure to antibiotics can have other long-term consequences on microbiota and host gut physiology.
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    Characterising cellular and molecular mechanisms of cardiac diastolic dysfunction
    Raaijmakers, Antonia Johanna Adriana ( 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 (011.2%, 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.
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    Synaptic mechanisms and function in the mouse enteric nervous system
    Swaminathan, Mathusi ( 2018)
    Virtually all functions of the enteric nervous system (ENS) rely on synaptic transmission, which occurs at specialised sites referred to as synapses. Molecular mechanisms behind synaptic transmission at central synapses have been extensively characterised, studies accordingly show that pre- and post-synaptic proteins localized to these synapses regulate transmission. However, little is known about the synaptic machinery involved in regulating excitatory transmission at enteric synapses. There is growing evidence to suggest that patients with synaptic protein associated neurodevelopmental and neurodegenerative diseases, such as Parkinson’s Disease, also display abnormalities in gastrointestinal function. This suggests there is a commonality between the central nervous system (CNS) and the ENS. Excitatory transmission within the ENS is primarily mediated by acetylcholine (ACh) acting on nicotinic receptors, there are also many other putative excitatory neurotransmitters in the system whose roles remain elusive. Therefore, the aim of my PhD thesis was to elucidate molecular and pharmacological mechanisms underlying excitatory transmission in the ENS. In Chapter 2, I localized synaptic vesicle proteins synaptophysin, synaptotagmin-1 and vesicular acetylcholine transporter (vAChT) to enteric varicosities. I developed two high-throughput analysis methodologies to quantify co-expression in varicosities and their close contact with enteric neurons. Using these analysis tools, I found that synaptic vesicle proteins synaptophysin and synaptotagmin-1, described to be ubiquitous in pre-synaptic terminals, are not found in all cholinergic varicosities (vAChT+) in the myenteric plexus. I found that in the submucosal plexus, all cholinergic varicosities contained synaptophysin, but some lacked synaptotagmin-1. This highlights the sensitivity of the analysis tool developed and the disparity in synaptic protein localization at cholinergic varicosities between the two plexuses. Additionally, using 3D rendering I examined close contacts between varicosities expressing synaptophysin and vAChT on neuronal nitric oxide synthase (nNOS+) neurons. I found that nNOS+ neurons receive three distinct classes of input. This includes varicosities that either contain vAChT, synaptophysin or both. Overall, my findings demonstrate that there is molecular heterogeneity in cholinergic varicosities within the ENS, which will likely transpire into distinct modes of cholinergic transmission or ACh release at enteric synapses. Moreover, this study highlights the use of advanced image analysis tools to examine connectivity and mechanisms of transmission within the ENS. In Chapter 3, I described the expression of post-synaptic density protein PSD93 in the ENS using immunohistochemical methods. I found that most myenteric neurons, including subpopulations of cholinergic and nitrergic neurons express PSD93. The wide spread expression of PSD93 in the cytoplasm and axons of enteric neurons indicates that it is an unsuitable marker for identifying excitatory post-synaptic densities in the myenteric plexus. Instead, PSD93 is likely to be involved in other cytosolic processes in addition to any role as a post-synaptic density protein at excitatory synapses. In Chapter 4, I demonstrate importance of α-synuclein (α-Syn) in cholinergic function within the ENS. α-Syn is a synaptic vesicle protein pathologically linked to neurodegenerative diseases. I show that α-Syn is expressed in varicosities and some neuronal somata within the mouse colon, a result described previously in other species. Using the quantitative method described in Chapter 2, I found that most cholinergic varicosities (vAChT+) contained α-Syn. I also investigated the implications of α-Syn deletion for ENS function using α-Syn knock out (KO) mice. α-Syn KO mice have increased proportions of cholinergic neurons in the myenteric plexus. Additionally, cross-sections of mouse colon preparations also show that α-Syn KO mice have increased cholinergic innervation to the circular muscle. Calcium (Ca2+) imaging studies reveal that fast synaptic transmission mediated by nicotinic receptors is increased in α-Syn KO mice. However, I found that α-Syn KO mice have a reduced incidence of spontaneous circular muscle contractility, suggesting that there are changes in the circuitry underlying motor patterns. Collectively, these findings suggest that there are alterations in the enteric neural circuitry of α-Syn KO mice and that α-Syn is important for cholinergic transmission. In Chapter 5, I used Ca2+ imaging and high-resolution microscopy to elucidate the mechanisms behind glutamatergic transmission within the ENS. Thus far there is conflicting evidence to suggest the involvement of ionotropic receptors and metabotropic glutamate receptors (mGluRs) in synaptic transmission. I show that many myenteric varicosities that contain vesicular glutamate transporter 2 (vGluT2) are non-cholinergic and express synaptic vesicle proteins synaptophysin using tools I developed in Chapter 2. Using 3D rendering I showed that calbindin (calb+) neurons receive more vGluT2 varicosities than nNOS+ neurons. Exogenous application of glutamate predominantly excites calb+ neurons in the myenteric plexus. Calb+ neurons also receive slow synaptic transmission mediated by endogenous release of glutamate excited by a train of electrical stimuli. Using ionotropic and group I metabotropic glutamate receptor (mGluR) antagonists, I found that group I mGluRs are involved in mediating slow synaptic transmission. This study demonstrates a role for glutamate in mediating excitability of myenteric calb+ neurons. Overall, I have developed powerful methodologies that will provide valuable tools to contribute to understanding mechanisms underlying excitatory transmission within the ENS. The molecular heterogeneity of cholinergic varicosities identified in this thesis, provides a foundation for elucidating ACh release at enteric synapses. I have also shown that post-synaptic density markers that identify excitatory synapses in the autonomic nervous system (ANS) are unsuitable for labelling excitatory synapses in the ENS. This indicates that mechanisms underlying excitatory transmission could differ between the ANS and ENS. I have highlighted the difficulty in establishing a marker for post-synaptic densities within the ENS, which is necessary for a detailed understanding of excitatory transmission. Moreover, I have shown that α-Syn is associated with cholinergic synapses and the deletion of the synaptic vesicle protein has consequential effects on cholinergic transmission and function, thus implicating α-Syn in gastrointestinal pathophysiology. I have also identified a role for group I mGluRs in mediating excitatory slow synaptic transmission, indicating that glutamate is an excitatory neurotransmitter within the ENS. These findings provide a foundation for future analyses of synaptic function in the ENS and point to key questions for further investigation of this understudied nervous system.
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    The metabolic microenvironment regulates myogenic fate decisions through altered histone acetylation
    Ly, Chi ( 2018)
    1. Skeletal muscle has an extensive capacity for regeneration, a property conferred on this tissue by a resident population of skeletal muscle stem cells (MuSCs). Any impairment to this population of stem cells can lead to increased morbidity and mortality. MuSCs normally exist in a quiescent state marked by the paired box transcription factor Pax7. In response to an activating signal, MuSCs rapidly undergo a process of activation and cell-cycle re-entry. At this early stage, MuSCs begin to express the myogenic determination factor MyoD and initiate the process of commitment to the myogenic lineage. Importantly, a sub-population of MuSCs will return to quiescence so as to prevent depletion of the stem cell pool. This decision for MuSCs to either undergo commitment or self-renewal remains ill defined. Studies in cancer, and developmental and stem cell biology has identified cellular metabolism as playing a key role in directing changes associated with stem cell self-renewal, lineage specification and the processes of proliferation and differentiation. Therefore, the aim of this study was to investigate this link between metabolism and the processes of self-renewal and myogenic commitment in MuSCs and to identify how metabolism may regulate these processes. 2. To efficiently isolate a large number of primary MuSCs I utilized Pax7creERT2xROSA26eYFP transgenic mice which allows for the fluorescent labelling of MuSCs and subsequent isolation via fluorescence activated cell sorting. To examine the link between innate cell metabolism and MuSC heterogeneity, single cell RNA sequencing (scRNAseq) was performed on either freshly isolated MuSCs or MuSCs that had been cultured ex vivo for 96 hrs. The scRNAseq results revealed that while freshly isolated MuSCs are largely homogeneous, cultured MuSCs exhibited significant heterogeneity with divergent metabolic signatures. These metabolic signatures marked cells either undergoing myogenic commitment or self-renewal. 3. To examine the role of the metabolic microenvironment in regulating MuSC lineage specification whole skeletal muscle fibres, isolated primary MuSCs or C2C12 cells were cultured in media containing different carbohydrate conditions; high glucose (25 mM glucose, HG), low glucose (5 mM glucose, LG) or galactose (10 mM galactose, GAL). Following culture, the downstream effects on metabolism, including measurements of mitochondrial DNA, mitochondrial abundance, key electron transport chain proteins and cellular bioenergetics was assessed. Myogenic specification was assessed via examination of key myogenic regulatory factors by PCR, western immunoblotting, immunofluorescence, whole transcriptome sequencing and single cell sequencing (scRNAseq). Finally, to link alterations to metabolism to changes in gene transcription, global histone acetylation was examined. Extracellular carbohydrate availability directly regulates both innate cellular metabolism and gene expression via acetyl-CoA availability and histone acetylation. Importantly, use of several pharmacological modulators of metabolism confirm a central role of carbohydrate metabolism in histone acetylation. Combining both whole transcriptome sequencing and scRNAseq, extracellular carbohydrate availability was shown to directly influence lineage fate decisions, with reduced carbohydrate availability linked to a reduction in the proportion of cells undergoing myogenic commitment. The scRNAseq dataset presented provides entirely new information of subpopulations of cells; including true MuSCs, primed MuSCs, early committed muscle progenitors (CMPs) and late CMPs and show that the extracellular metabolic environment directly influences the proportion of cells in each of these subpopulations. Finally, single fibre experiments showed that reduced carbohydrate availability was linked to increased rates of asymmetric division and self-renewal. 4. These results provide the first evidence that the extracellular metabolic microenvironment is able to directly alter MuSC lineage commitment and self-renewal with reduced carbohydrate availability leading to a maintenance of the true MuSC population a result of an increased proportions of asymmetric divisions. Finally, metabolic remodelling can be used to enhance the efficiency of MuSC transplantation.
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    Adipose, sex steroids and atrial arrhythmia vulnerability
    Bernasochi, Gabriel Brian ( 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.
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    Chronic ephedrine administration decreases brown adipose tissue activity in a randomised controlled human trial: implications for obesity
    Pajtak, Renata ( 2018)
    Aim Activation of Brown Adipose Tissue (BAT) may have therapeutic potential to combat obesity. Acute treatment of mice with sympathomimetic drugs activates BAT thermogenesis, and chronic treatment increases BAT thermogenic capacity. It has previously been demonstrated that human BAT is acutely responsive to oral administration of the sympathomimetic ephedrine. This study aimed to determine whether chronic treatment with ephedrine could mimic adaptive thermogenesis in humans. Methods Twenty-three healthy young men were recruited via general advertisement from Melbourne, Australia to participate in a randomised, placebo-controlled, parallel group trial. Recruited individuals were unmedicated, non-smokers, physically inactive and had no prior history of either cardiovascular disease, insulin resistance or diabetes. They were allocated to either a placebo (n=11; 22±2 years, 23±2 kg/m2) or 1.5 mg/kg/day ephedrine (active group; n=12, age 23±1 years, BMI 24±1 kg/m2) treatment group for twenty-eight days. Body composition was measured before and after the intervention by dual energy x-ray absorptiometry. BAT activity, measured before and after the twenty-eight day intervention period, via 18F-fluorodeoxyglucose positron emission tomography computed-tomography (18F-FDG PET/CT) in response to a single dose of 2.5mg/kg ephedrine, was the primary outcome measure. Results After twenty-eight days of treatment, the active treatment lost significantly more total body fat (placebo 1.1± 0.3 kg, ephedrine -0.9 ± 0.5kg; p<0.01) and visceral adipose tissue (placebo 6.4 ± 19.1g, ephedrine -134 ± 43g; p<0.01), with no change in lean mass or bone mineral content, compared with the placebo group. In response to acute ephedrine, BAT activity (change in mean standardised uptake value: placebo -3 ± 7 %, ephedrine -22 ± 6%) and the increase in systolic blood pressure were significantly reduced (p<0.05) in the active group compared with placebo. Conclusion Chronic ephedrine treatment reduced body fat content, however, it was independent of an increase in BAT activity. Rather, chronic ephedrine treatment suppressed BAT glucose disposal, suggesting that chronic ephedrine treatment decreased, rather than increased BAT activity.