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Mccarroll, Douglas (2014) the Effects of Trypanosoma Brucei and Mammalian-Derived Extracellular Cathepsin-L on Myocardial Function

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Mccarroll, Douglas (2014) the Effects of Trypanosoma Brucei and Mammalian-Derived Extracellular Cathepsin-L on Myocardial Function McCarroll, Douglas (2014) The effects of Trypanosoma brucei and mammalian-derived extracellular cathepsin-L on myocardial function. PhD thesis. http://theses.gla.ac.uk/5170/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten:Theses http://theses.gla.ac.uk/ [email protected] The Effects of Trypanosoma brucei and Mammalian-Derived Extracellular Cathepsin-L on Myocardial Function Mr. Douglas McCarroll BVMS (Hons) MSc (VetSci) MRCVS Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy to the Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, U.K. Research conducted at the British Heart Foundation Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, University of Glasgow, U.K. January 2014 2 Abstract African trypanosomiasis is a neglected tropical disease affecting both animals and humans in sub-Saharan Africa. The disease is caused bythe protozoan parasite Trypanosoma brucei, which is transmitted by the tsetse fly (Glossina sp.) vector. In animals, infection leads to severe muscle atrophy and anaemia resulting in significant production and economic losses. In humans, infection leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, and indeed is responsible for the colloquial name “Sleeping Sickness”, the cardiac pathogenesis remains unknown. Previous studies interpreted cardiac dysfunction as being due to immune/inflammatory responses. However, recent work examining the parasite’s interaction with the blood brain barrier, the traversal of which is important for development of neurological signs, has identified direct immune/inflammatory independent mechanisms involving calcium (Ca2+) signalling. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter Ca2+ signalling and cardiac function independent of a systemic immune/inflammatory response. Using a high-throughput method of observing spontaneous contractile activity in isolated cardiomyocytes, we were able to determine that the presence of T. b. brucei parasites resulted in more cardiomyocytes exhibiting spontaneous contractile events. Moreover, when the parasites were removed by careful centrifugation, the culture supernatant had the same effect. Confocal Ca2+ imaging identified an increase in the frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves). Studies utilising specific inhibitors, recombinant protein and RNA interference all demonstrated that this altered SR function was due to cathepsin-L; a cysteine protease produced by T. brucei (TbCatL). Experiments utilising a Langendorff perfusion method revealed that trypanosome culture supernatant could induce ventricular premature contractions in 50% of a cohort of ex vivo whole rat hearts. Mechanistic experiments were performed on single isolated cardiomyocytes stimulated at 1.0 Hz and perfused first with control media followed by 3 trypanosome culture supernatant. The protocol utilised triple caffeine applications: (i) prior to stimulation to empty the SR of Ca2+, (ii) after perfusion with control media and after supernatant to determine the SR Ca2+ content and sarcolemmal extrusion of Ca2+ following each solution. Results were normalised to a parallel set of cardiomyocytes perfused with control media only as time controls. These experiments revealed a 10-15% increase in SR Ca2+ reuptake by the SR Ca2+ ATPase (SERCA) but a reduced SR Ca2+ content suggesting a concomitant increase in SR-mediated Ca2+ leak. This conclusion was supported by the data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide (AIP), highlighting a role for Ca2+/calmodulin kinase II (CaMKII) in the TbCatL action on SR function. When cytosolic diastolic Ca2+ was measured in cardiomyocytes with SR function inhibited by ryanodine and thapsigargin, trypanosome supernatant prevented a decline in cytosolic diastolic Ca2+ that was observed in control media. AIP did not abolish this effect suggesting that TbCatL may raise diastolic Ca2+ that could activate CaMKII leading to the observed effects. These data demonstrated for the first time that African trypanosomes alter cardiac function independent of a systemic immune response via a mechanism involving extracellular cathepsin-L-mediated changes in SR function. Utilising the same (culture adapted and monomorphic) strain of T. brucei as the in vitro experiments, Lister 427, in a rat model of infection we found no significant increase in the arrhythmia frequency as measured by a 15 min electrocardiogram (ECG). However, when hearts were removed and Langendorff perfused with the addition of isoproterenol the arrhythmia frequency was increased. When the pleomorphic strain T. b. brucei TREU 927 was used in rats with continuous ECG recording from biopotential telemetry there was a significant increase in arrhythmia frequency in the infected rats. When hearts were removed and Langendorff perfused with isoproterenol there was a similar increase in arrhythmia frequency as observed with the 427 infected hearts. This suggests that a cardiac dysfunction phenotype is present during trypanosome infections in an animal model providing the basis for future therapeutic work. The relationship between arrhythmogenic SR-mediated Ca2+ release and TbCatL has parallels with endogenous extracellular cathepsin-L (CatL). It has been 4 demonstrated that a basal level of CatL is necessary for normal cardiac function. However, in coronary heart disease (CHD) CatL levels are increased in the serum of patients correlating with the severity of disease. The effects of raised CatL on cardiac function remain unknown. Work in our lab has identified that ex vivo Langendorff perfused hearts that have undergone a 30 min period of ischaemia followed by 90 min reperfusion show greater CatL activity in coronary effluent than hearts perfused without ischaemia. In addition, preliminary data collected in this thesis suggest that human patients that have suffered a myocardial infarction and have undergone reperfusion via percutaneous coronary intervention (PCI) showed higher CatL levels in post-reperfusion serum samples compared to pre-reperfusion serum. When severity of heart function in patients (measured as left ventricular volume at systole and diastole, ejection fraction, infarct size and area at risk) was assessed by magnetic resonance imaging (MRI) in a preliminary study, there was a positive correlation with serum CatL levels. Using recombinant CatL on isolated rat ventricular cardiomyocytes it was found that the SR Ca2+ content and the stimulated Ca2+ transient were significantly reduced in a concentration dependent manner. This suggests a CatL dependent SR dysfunction. This conclusion was supported by an increase in Ca2+ wave frequency measured by confocal Ca2+ imaging in isolated cardiomyocytes. The work in this thesis demonstrates a role for both mammalian-derived and exogenous extracellular cathepsin-L proteases in arrhythmogenic SR-mediated Ca2+ release. 5 Table of Contents The Effects of Trypanosoma brucei and Mammalian-Derived Extracellular Cathepsin-L on Myocardial Function ...................................................... 1 Abstract ....................................................................................... 2 List of Figures ............................................................................... 12 List of Tables ................................................................................ 15 Acknowledgements ......................................................................... 17 Author’s Declaration ....................................................................... 19 Definitions/Abbreviations ................................................................. 20 1 CHAPTER 1 – General Introduction ................................................. 26 1.1 Introduction ....................................................................... 27 1.2 What is Trypanosomiasis? ........................................................ 28 1.2.1 Lifecycle of Trypanosoma spp. ............................................ 29 1.2.2 What are Trypanosomes and How do they Function? .................. 30 1.2.3 How do Trypanosomes Survive in the Host? ............................. 31 1.2.4 What are the Clinical Features of Trypanosomiasis? ................... 32 1.2.5 How is HAT Diagnosed?...................................................... 38 1.2.6 What are the Current Treatment Strategies? ........................... 40 1.2.7 How do Trypanosomes Cross the Blood Brain Barrier .................. 43 1.2.8 Trypanosome Cysteine Proteases ......................................... 49 1.3 Ca2+ and the Heart ...............................................................
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