PRAVEEN KUMAR DHANDAPANI: Expressing Alternative Oxidase
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PRAVEEN KUMAR DHANDAPANI DHANDAPANI KUMAR PRAVEEN DISSERTATIONES SCHOLAE DOCTORALIS AD SANITATEM INVESTIGANDAM EXPRESSING ALTERNATIVE OXIDASE (AOX) IN MICE: A VALUABLE TOOL TO STUDY MITOCHONDRIAL RESPIRATORY CHAIN DEFECTS RESPIRATORY MITOCHONDRIAL STUDY TO TOOL IN MICE: A VALUABLE (AOX) OXIDASE ALTERNATIVE EXPRESSING UNIVERSITATIS HELSINKIENSIS PRAVEEN KUMAR DHANDAPANI EXPRESSING ALTERNATIVE OXIDASE (AOX) IN MICE: A VALUABLE TOOL TO STUDY MITOCHONDRIAL RESPIRATORY CHAIN DEFECTS ISBN 978-951-51-5998-4 (PRINT) ISBN 978-951-51-5999-1 (ONLINE) ISSN 2342-3161 (PRINT) ISSN 2342-317X (ONLINE) http://ethesis.helsinki.fi HELSINKI 2020 FACULTY OF MEDICINE DOCTORAL PROGRAM IN BIOMEDICINE UNIVERSITY OF HELSINKI 32/2020 Doctoral Programme in Biomedicine Faculty of Medicine University of Helsinki Finland Expressing alternative oxidase (AOX) in mice: A valuable tool to study mitochondrial respiratory chain defects Praveen Kumar Dhandapani ACADEMIC DISSERTATION To be presented, with the permission of Faculty of Medicine of the University of Helsinki, for public examination in room P674, Porthania, Yliopistonkatu 3, Helsinki, on 05.06.2020, at noon. Helsinki 2020 Supervised by Marten Szibor, MD Prof. Howard T. Jacobs, PhD Clinic for Heart and Thorax Surgery Faculty of Medicine and Health Technology Jena University Hospital Tampere University Friedrich-Schiller University of Jena Thesis committee Prof. Katriina Aalto-Setälä, MD, PhD Prof. Klaus-Dieter Schlüter, PhD Faculty of Medicine and Health Physiological Institute Technology Faculty of Medicine Tampere University Justus-Liebig University of Giessen Reviewed by Jaakko Pohjoismäki, PhD Prof. Roberta A. Gottlieb, MD Department of Environmental and Department of Medicine; Department of Biological Sciences Biomedical Sciences University of Eastern Finland Smidt Heart Institute Cedars-Sinai Medical Center Opponent Prof. Anthony Moore, PhD Biochemistry and Biomedicine School of Life Sciences University of Sussex ISBN 978-951-51-5998-4 (paperback) ISBN 978-951-51-5999-1 (PDF) ISSN 2342-3161 (print) ISSN 2342-317X (online) Painosalama Oy Turku 2020 To my mom, dad and mentors We are all born with a divine fire in us. Our efforts should be to give wings to this fire and fill the world with the glow of its goodness. - APJ Abdul Kalam Abstract Mitochondria are vital cellular organelles that produce the majority of the energy required by cells and are therefore represented as "the power house of the cell". Cellular energy, in the form of adenosine triphosphate (ATP), is generated through oxidative phosphorylation (OXPHOS), which is energized by the redox reactions of the electron transport chain (ETC) in mitochondria. Mitochondria are essential for maintaining metabolic homeostasis, fatty-acid metabolism, nucleotide synthesis and cellular redox balance, in addition to producing ATP. Given the multitude of mitochondrial functions, failure of any of them can lead to mitochondrial dysfunction that can have drastic consequences. It can lead to cell loss, organ failure and even death. Mitochondrial disorders are commonly associated with debilitating childhood onset diseases with no known cure. Current therapeutic approaches are largey symptomatic, such as dietary modification for diabetes, anti- convulsant drugs to control seizures, physical exercise for hypotonia, pacemaker implants for cardiac rhythm abnormalities and cochlear implants for sensorineural deafness. However, these are not a permanent solution or cure for severe disorders caused by genetic mutations or irreversible physical damage. Cardiomyopathy, failure of the heart muscle, is one of the most common symptoms of mitochondrial disorders, while cardiomyopathies almost always are associated with mitochondrial dysfunction. In severe forms of cardiomyopathy, the ultimate treatment option of heart transplantation is invasive and carries high risk, and the waiting list for a functional healthy heart is increasingly outstripping availability. I have investigated the use of a mitochondrial enzyme, the alternative oxidase (AOX), which is found in plants and lower eukaryotes, as well as in primitive metazoans but not in mammals as a new approach to better understand the underlying molecular mechanisms of diseases involving mitochondrial dysfunction. On a broader perspective, this would also aid in the development of a possible treatment for diseases associated with mitochondrial dysfunction. If catalytically engaged, AOX branches the mitochondrial ETC by oxidizing ubiquinol, which accumulates in the reduced form if the cytochrome pathway is impaired. By doing so it reduces oxygen directly to water, which is typically carried out by cytochrome oxidase, and thus maintains redox balance by recycling NADH and FADH2, two by-products of the Krebs cycle and upstream metabolism. Focusing on different types of cardiomyopathy, my strategy has been to introduce the AOX gene from the tunicate Ciona intestinalis, a close relative of vertebrates, into mouse models of disease, using genomic manipulation. I have studied and characterized two transgenic mouse models expressing C. intestinalis AOX. The first was designed to express AOX constitutively and ubiquitously. I verified this at the RNA and protein level revealing high-level expression in all tissues tested, including the heart, with the exception of the adult brain. The mice did not show any observable phenotype, making them nearly indistinguishable from their wild-type littermates under non-stressed conditions. Heart function, as measured by ejection fraction and left ventricular mass, treadmill performance and grip strength were all normal in one- year-old AOX-expressing mice. Weight was indistinguishable from that of wild-type littermates on chow or high-fat diet; although on ketogenic diet AOX-expressing mice showed a slightly mitigated weight gain, at least when caged in same-sex groups. A second AOX transgenic line was characterized, in which AOX expression was designed to be activatable by Cre-loxP-mediated removal of a STOP cassette (SNAP coding sequence, pA signal) located downstream of a CAG promoter and upstream of the AOX coding sequence. After verification of the transgenic insertion by PCR and Southern blotting, test activation by breeding to mice ubiquitously expressing Cre recombinase under the control of a β-actin promoter resulted in successful activation, based on ubiquitous expression of AOX and concomitant loss of SNAP expression. This allowed me to test the tissue-specificity of AOX-mediated modification of pathological phenotypes in mouse models of cardiomyopathy. Two different mouse models of cardiomyopathy were investigated. In the first, inflammatory cardiomyopathy was induced by overexpressing monocyte chemo- attractant protein 1 (MCP1) in the cardiomyocytes, which leads to loss of cardiac function commencing during early adulthood (12 weeks of age). Previously published data showed mitochondrial structural damage, decrease in ATP levels; and suggested induced oxidative stress as a pathological mechanism. Although the majority of oxidative stress in the cardiomyocytes of Mcp1 mice is induced by the infiltrating monocytes, via NADPH oxidase pathway, the contribution of mitochondria to the production of reactive oxygen species (ROS) is not well understood. I hypothesized that mitochondrial ROS production, especially via a defective or overloaded cytochrome pathway in the mitochondrial respiratory chain, might play a key role in the underlying molecular mechanisms. Therefore we wanted to test whether AOX could mitigate it, as it has been shown to alleviate oxidative stress under conditions where the cytochrome pathway is dysfunctional. I found that expressing AOX in this model preserved cardiac ejection fraction at 12 weeks of age but not at later time points. At the molecular level, I determined that mitochondrial complex I- linked respiration was severely affected in Mcp1-overexpressing mice irrespective of AOX expression. However, AOX preserved complex II mediated respiration. In theory, this should maintain mitochondrial redox homeostasis and limit oxidative stress and damage within mitochondria, but at the expense of ATP production and mitochondrial-ROS signaling. Ultrastructural analysis revealed mitochondrial damage in the cardiac tissue of 12 week-old Mcp1-overexpressing mice, which was alleviated by AOX expression. Despite this, AOX had no effect on the survival of Mcp1- overexpressing mice, whilst cardiomyocyte-specific AOX expression actually resulted in earlier death than Mcp1 alone. Autophagic markers were mildly elevated in all cases, and metabolic changes consistent with OXPHOS dysfunction were essentially unaffected by AOX expression in the Mcp1 mouse model. Overall, I conclude that AOX is insufficient to block lethal damage to mitochondria and/or other cellular components in this inflammatory cardiomyopathy model, despite transient beneficial effects. Previously, West et al (2011) and Mills et al (2016) reported on the significance of mitochondrial ROS in inducing macrophage activity in a LPS-induced inflammatory mouse model. Additionally, Mills et al (2016) also showed AOX expression prevented LPS-induced lethality in mice, where succinate-dependent ROS production in the ETC was reported to be the underlying cause for the observed severe phenotype. However, since AOX was unable to prevent lethality in Mcp1 mice, it is indicative that the ROS production in this inflammatory model might not be dependent on the ETC. The second model I studied was of Cox-deficient