Minuzzi 2019 MiPschool Coimbra

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A pilot study on mitochondrial function of mouse skin using high-resolution respirometry.

Link: MitoEAGLE

Minuzzi LM, Santos D, Leal E, Carvalho E (2019)

Event: MiPschool Coimbra 2019

COST Action MitoEAGLE

Diabetic wounds are a serious complication of diabetes and a cause of non-traumatic amputation of the lower limbs. Thus, understanding the mechanisms underlying development of chronic ulcer is important to improve treatment strategies and the quality of life of patients. We aimed to evaluate mitochondrial respiration on mouse skin in the presence or absence of diabetes and 3 days post wounding. Skin samples were collected from diabetic (DM) and nondiabetic (CTL) C57/BL6 mice at day0 and day3 post wounding. Small skin explants were finely minced and permeabilized with saponin (5mg/ml). Mitochondrial respiration was assessed using 2 different high-resolution respirometry protocols (Oxygraph-2k; Oroboros, AU), as described by Chicco et al. [1]. Briefly, one protocol focused on fatty acid (FAO)-linked respiration with sequentially added NADH-linked substrates, followed by uncoupler titrations. The other protocol focused on coupling control with primarily NADH-linked and FADH2-linked substrate sources, followed by inhibitors titrations without a fatty acid. These protocols measure the respiratory capacity and substrate control during oxidative phosphorylation (OXPHOS), and the respiratory “leak” (LEAK), as well as the capacity of the electron transfer system (ETS). Basal respiratory flow and leak respiration were similar between the groups. Mitochondrial OXPHOS and FAO in the permeabilized skin at day0 was similar between CTL and DM mice. The ETS capacity, evaluated after FCCP titration, was higher in skin of CTL mice at day0 compared to the day3 post wounding. In addition, the OXPHOS capacity was higher in CTL mice at day0 compared to the day3 and no differences were observed in the DM group. Moreover, rat skin showed a higher Complex I- and Complex II-linked OXPHOS capacity than CTL mouse skin. The optimization of protocols showed robust and stable oxygen fluxes in both protocols. This technique proves useful for assessing mitochondrial respiration in the skin in 15 mg amount of tissue, with possible application in the identification of mitochondrial dysfunction in skin in different conditions in diabetes.


Bioblast editor: Plangger M


Labels: MiParea: Respiration  Pathology: Diabetes 

Organism: Mouse  Tissue;cell: Endothelial;epithelial;mesothelial cell  Preparation: Permeabilized tissue 


Coupling state: LEAK, OXPHOS, ET  Pathway: F, N, S  HRR: Oxygraph-2k 


Affiliations

Center Neuroscience Cell Biology, Univ Coimbra, Portugal. - lucielegm@gmail.com

References

  1. Chicco AJ, Le CH, Schlater A, Nguyen A, Kaye S, Beals JW, Scalzo RL, Bell C, Gnaiger E, Costa DP, Crocker DE, Kanatous SB (2014) High fatty acid oxidation capacity and phosphorylation control despite elevated leak and reduced respiratory capacity in northern elephant seal muscle mitochondria. J Exp Biol 217:2947-55.