{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/368170"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/368170","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Developing methods for measuring mitochondrial redox status and membrane potential in the isolated perfused heart","abstract":"Mitochondria have been identified as a key player in cardiac ischaemia-reperfusion injury serving as both a source and target of damage. The energetic status of mitochondria critically determines the progression of ischaemia-reperfusion injury by dictating the thermodynamic likelihood of reverse electron transport (RET) at complex I and the production of reactive oxygen species (ROS) and may also influence Ca2+ transport and the opening of the mitochondrial permeability transition pore (MPTP). However, it is not entirely clear how mitochondrial redox and oxygenation status and mitochondrial membrane potential (ΔΨm) respond to ischaemia-reperfusion due to a lack of suitable methods for measuring these bio parameters in *ex vivo* or *in vivo* models in near real-time. Current methods for measuring ΔΨm rely on the uptake and redistribution of exogenous probes and dyes which are then imaged using optical methods or measured by mass spectrometry, ion-selective macroelectrode, or scintillation counter. Transmural absorbance spectroscopy can be used to measure the absorbance of intrinsic chromophores in the isolated perfused heart. The absorbance of mitochondrial cytochromes provides information about reducing equivalent delivery, respiratory flux, tissue oxygenation, and ΔΨm and thus provides a wealth of information about cardiac energetics. The distribution of electrons between *b* haems of complex III depends on ΔΨm and can be determined by absorbance spectroscopy. Thus, I developed a method for measuring ΔΨm in isolated mitochondria and the isolated perfused mouse heart based on the absorbance of intrinsic *b* haems. I validated this approach in isolated perfused mouse hearts under a variety of conditions and then interrogated how ΔΨm changes during global ischaemia-reperfusion. I found that ΔΨm changes dynamically during global ischaemia-reperfusion becoming depolarised during ischaemia and becoming rapidly repolarised during reperfusion. Furthermore, ΔΨm was predictably modulated by targeting glycolytic substrate metabolism or succinate dehydrogenase activity during ischaemia-reperfusion. I also characterised factors contributing to RET including succinate levels, the redox status of the coenzyme Q (CoQ) pool, ATP and ADP levels, and complex I activity during ischaemia-reperfusion with good temporal resolution providing critical insight into the conditions required for RET. Finally, I established a method for measuring H2O2 in the isolated heart using a genetically encoded fluorescent protein which will be used to interrogate the link between mitochondrial-derived ROS and ischaemia-reperfusion injury in the future. Although this work was carried out to address gaps in knowledge surrounding cardiac ischaemia-reperfusion injury, these methods could be applied to a range of research questions related to cardiac physiology in the future.","abstract_html":"Mitochondria have been identified as a key player in cardiac ischaemia-reperfusion injury serving as both a source and target of damage. The energetic status of mitochondria critically determines the progression of ischaemia-reperfusion injury by dictating the thermodynamic likelihood of reverse electron transport (RET) at complex I and the production of reactive oxygen species (ROS) and may also influence Ca<sup>2+</sup> transport and the opening of the mitochondrial permeability transition pore (MPTP). However, it is not entirely clear how mitochondrial redox and oxygenation status and mitochondrial membrane potential (ΔΨ<sub>m</sub>) respond to ischaemia-reperfusion due to a lack of suitable methods for measuring these bio parameters in *ex vivo* or *in vivo* models in near real-time. Current methods for measuring ΔΨ<sub>m</sub> rely on the uptake and redistribution of exogenous probes and dyes which are then imaged using optical methods or measured by mass spectrometry, ion-selective macroelectrode, or scintillation counter. Transmural absorbance spectroscopy can be used to measure the absorbance of intrinsic chromophores in the isolated perfused heart. The absorbance of mitochondrial cytochromes provides information about reducing equivalent delivery, respiratory flux, tissue oxygenation, and ΔΨ<sub>m</sub> and thus provides a wealth of information about cardiac energetics. The distribution of electrons between *b* haems of complex III depends on ΔΨ<sub>m</sub> and can be determined by absorbance spectroscopy. Thus, I developed a method for measuring ΔΨ<sub>m</sub> in isolated mitochondria and the isolated perfused mouse heart based on the absorbance of intrinsic *b* haems. I validated this approach in isolated perfused mouse hearts under a variety of conditions and then interrogated how ΔΨ<sub>m</sub> changes during global ischaemia-reperfusion. I found that ΔΨ<sub>m</sub> changes dynamically during global ischaemia-reperfusion becoming depolarised during ischaemia and becoming rapidly repolarised during reperfusion. Furthermore, ΔΨ<sub>m</sub> was predictably modulated by targeting glycolytic substrate metabolism or succinate dehydrogenase activity during ischaemia-reperfusion. I also characterised factors contributing to RET including succinate levels, the redox status of the coenzyme Q (CoQ) pool, ATP and ADP levels, and complex I activity during ischaemia-reperfusion with good temporal resolution providing critical insight into the conditions required for RET. Finally, I established a method for measuring H<sub>2</sub>O<sub>2</sub> in the isolated heart using a genetically encoded fluorescent protein which will be used to interrogate the link between mitochondrial-derived ROS and ischaemia-reperfusion injury in the future. Although this work was carried out to address gaps in knowledge surrounding cardiac ischaemia-reperfusion injury, these methods could be applied to a range of research questions related to cardiac physiology in the future.","abstract_has_math":false,"creators":["Giles, Abigail"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Murphy, Michael"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-12-29","date_published":"2023-12-29","updated_at":"2026-07-22T22:24:01Z","subjects":["cardiac","membrane potential","mitochondria","optical spectroscopy"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/76cc87b1-31ef-4520-80c1-6b23fa24f015/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.108483","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Murphy, Michael"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["National Institutes of Health"]},{"key":"dc:creator","label":"Author","values":["Giles, Abigail"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-12-29"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/368170"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["cardiac","membrane potential","mitochondria","optical spectroscopy"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/76cc87b1-31ef-4520-80c1-6b23fa24f015/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.108483"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2155f474-3953-4cb1-815e-e09e0b27075a/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Mitochondria have been identified as a key player in cardiac ischaemia-reperfusion injury serving as both a source and target of damage. The energetic status of mitochondria critically determines the progression of ischaemia-reperfusion injury by dictating the thermodynamic likelihood of reverse electron transport (RET) at complex I and the production of reactive oxygen species (ROS) and may also influence Ca2+ transport and the opening of the mitochondrial permeability transition pore (MPTP). However, it is not entirely clear how mitochondrial redox and oxygenation status and mitochondrial membrane potential (ΔΨm) respond to ischaemia-reperfusion due to a lack of suitable methods for measuring these bio parameters in *ex vivo* or *in vivo* models in near real-time. Current methods for measuring ΔΨm rely on the uptake and redistribution of exogenous probes and dyes which are then imaged using optical methods or measured by mass spectrometry, ion-selective macroelectrode, or scintillation counter. Transmural absorbance spectroscopy can be used to measure the absorbance of intrinsic chromophores in the isolated perfused heart. The absorbance of mitochondrial cytochromes provides information about reducing equivalent delivery, respiratory flux, tissue oxygenation, and ΔΨm and thus provides a wealth of information about cardiac energetics. The distribution of electrons between *b* haems of complex III depends on ΔΨm and can be determined by absorbance spectroscopy. Thus, I developed a method for measuring ΔΨm in isolated mitochondria and the isolated perfused mouse heart based on the absorbance of intrinsic *b* haems. I validated this approach in isolated perfused mouse hearts under a variety of conditions and then interrogated how ΔΨm changes during global ischaemia-reperfusion. I found that ΔΨm changes dynamically during global ischaemia-reperfusion becoming depolarised during ischaemia and becoming rapidly repolarised during reperfusion. Furthermore, ΔΨm was predictably modulated by targeting glycolytic substrate metabolism or succinate dehydrogenase activity during ischaemia-reperfusion. I also characterised factors contributing to RET including succinate levels, the redox status of the coenzyme Q (CoQ) pool, ATP and ADP levels, and complex I activity during ischaemia-reperfusion with good temporal resolution providing critical insight into the conditions required for RET. Finally, I established a method for measuring H2O2 in the isolated heart using a genetically encoded fluorescent protein which will be used to interrogate the link between mitochondrial-derived ROS and ischaemia-reperfusion injury in the future. Although this work was carried out to address gaps in knowledge surrounding cardiac ischaemia-reperfusion injury, these methods could be applied to a range of research questions related to cardiac physiology in the future."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["cec87486f61772d97f969a8672446efb","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Developing methods for measuring mitochondrial redox status and membrane potential in the isolated perfused heart"]}]}],"canonical_facts":{"dc:contributor.advisor":["Murphy, Michael"],"dc:contributor.sponsor":["National Institutes of Health"],"dc:creator":["Giles, Abigail"],"dc:date.issued":["2023-12-29"],"dc:description.abstract":["Mitochondria have been identified as a key player in cardiac ischaemia-reperfusion injury serving as both a source and target of damage. 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Transmural absorbance spectroscopy can be used to measure the absorbance of intrinsic chromophores in the isolated perfused heart. The absorbance of mitochondrial cytochromes provides information about reducing equivalent delivery, respiratory flux, tissue oxygenation, and ΔΨm and thus provides a wealth of information about cardiac energetics. The distribution of electrons between *b* haems of complex III depends on ΔΨm and can be determined by absorbance spectroscopy. Thus, I developed a method for measuring ΔΨm in isolated mitochondria and the isolated perfused mouse heart based on the absorbance of intrinsic *b* haems. I validated this approach in isolated perfused mouse hearts under a variety of conditions and then interrogated how ΔΨm changes during global ischaemia-reperfusion. I found that ΔΨm changes dynamically during global ischaemia-reperfusion becoming depolarised during ischaemia and becoming rapidly repolarised during reperfusion. Furthermore, ΔΨm was predictably modulated by targeting glycolytic substrate metabolism or succinate dehydrogenase activity during ischaemia-reperfusion. I also characterised factors contributing to RET including succinate levels, the redox status of the coenzyme Q (CoQ) pool, ATP and ADP levels, and complex I activity during ischaemia-reperfusion with good temporal resolution providing critical insight into the conditions required for RET. Finally, I established a method for measuring H2O2 in the isolated heart using a genetically encoded fluorescent protein which will be used to interrogate the link between mitochondrial-derived ROS and ischaemia-reperfusion injury in the future. Although this work was carried out to address gaps in knowledge surrounding cardiac ischaemia-reperfusion injury, these methods could be applied to a range of research questions related to cardiac physiology in the future."],"dc:format.checksum.md5":["cec87486f61772d97f969a8672446efb","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.108483"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2155f474-3953-4cb1-815e-e09e0b27075a/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/368170"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/76cc87b1-31ef-4520-80c1-6b23fa24f015/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["cardiac","membrane potential","mitochondria","optical spectroscopy"],"dc:title":["Developing methods for measuring mitochondrial redox status and membrane potential in the isolated perfused heart"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:01Z"}