Timon-Gomez 2023 EUROMIT2023 Bologna
Event: EUROMIT2023 Bologna IT
Ischemia-reperfusion damage occurs when there is a disruption and restoration of blood supply to an organ and it underlies many pathologies, mostly heart attack and stroke. Warm ischemia is defined as the time interval after the interruption of blood flow while the tissue remains at body temperature. The evaluation of mitochondrial damage caused by ischemia-reperfusion is essential to assess treatments for protecting or rescuing the tissue from an injury.
We studied brain homogenate and isolated cardiac mitochondria from C57BL/6N mice, and HEK 293T cells by high-resolution respirometry. Warm ischemia (WI) was induced by incubating brains and hearts in preservation buffer BIOPS for 1 h at 37 °C, followed by homogenization or mitochondria isolation. HEK 293T cells were exposed to anoxia in the O2k chamber for 1 h. For OXPHOS analysis, we used two substrate-uncoupler-inhibitor titration (SUIT) reference protocols, RP1 and RP2, interrogating 20 respiratory states. These include fatty acid oxidation F-, NADH N-, succinate S-, glycerol phosphate Gp- electron transfer (ET) pathways. Fluxes were normalized by tissue or protein mass, or by cell count. Respirometric OXPHOS analysis does not focus on single enzymes but provides a broad diagnostic spectrum on multiple pathways and coupling control.
In all models, OXPHOS and ET capacities were decreased in all pathways (F, N, S) and their combinations (NS, FNS, FNSGp, SGp) after 1 h warm ischemia, compared to controls. In cardiac mitochondria, flux control ratios showed a specific defect in OXPHOS and ET capacities of fatty acid oxidation, and a significant cytochrome c effect revealed an injury of the outer mitochondrial membrane. In contrast, in brain tissue and HEK 293T cells, there was no cytochrome c effect and flux control ratios indicated a major damage in N-linked respiration after warm ischemia.
Our results show that warm ischemia produces tissue-specific mitochondrial damages. In carbohydrate-dependent tissues, such as brain, the damage is highest in the N-pathway, similarly as in HEK 293T cells. Whereas the F-pathway is mainly affected in tissues that are more dependent on fatty acid oxidation, such as heart. SUIT protocols provide a broad scope to detect specific OXPHOS defects, offering a more sensitive diagnostic approach compared to respiratory protocols with non-permeabilized living cells.
• Bioblast editor: Plangger M
- Oroboros Instruments GmbH, Oroboros Ecosystem, Innsbruck, Austria.
List of abbreviations, terms and definitions - MitoPedia
Labels: MiParea: Respiration
Stress:Ischemia-reperfusion Organism: Human, Mouse Tissue;cell: Heart, Nervous system, HEK Preparation: Homogenate, Isolated mitochondria, Intact cells
Coupling state: OXPHOS, ET Pathway: F, N, S, Gp HRR: Oxygraph-2k