Developing methods for measuring mitochondrial redox status and membrane potential in the isolated perfused heart

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 (ΔΨ_m) 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_m 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_m and thus provides a wealth of information about cardiac energetics. The distribution of electrons between *b* haems of complex III depends on ΔΨ_m and can be determined by absorbance spectroscopy. Thus, I developed a method for measuring ΔΨ_m 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_m changes during global ischaemia-reperfusion. I found that ΔΨ_m changes dynamically during global ischaemia-reperfusion becoming depolarised during ischaemia and becoming rapidly repolarised during reperfusion. Furthermore, ΔΨ_m 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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Giles, Abigail

Advisor dc:contributor.advisor

• Murphy, Michael

• cardiac
• membrane potential
• mitochondria
• optical spectroscopy

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/76cc87b1-31ef-4520-80c1-6b23fa24f015/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.108483

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/368170