Physiology - Theses
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ItemNew insights regarding the myocardial-adipose paracrine axis( 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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ItemInvestigating the therapeutic potential of heat shock protein 70 (HSP70) induction for skeletal muscle injury( 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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ItemInteractions of the enteric nervous system with the gut microbiome in the neuroligin-3 R451C mouse model of autism( 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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ItemPericardial adiposity and cardiac arrhythmia vulnerability( 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.