Chowdhury 2015 Biosci Rep
|Chowdhury SK, Djordjevic J, Albensi B, Fernyhough P (2015) Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria. Biosci Rep 36:e00286.|
Abstract: Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of respiratory chain to generate ATP. This study characterized inter-relationship between mtMP, using safranin and TMRM, and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27% and 30-35% in the presence of TMRM and safranin, respectively. Maximal respiration was higher than coupled with Complex I and II linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration in higher concentration of FCCP compared to TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared to pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in the response to mitochondrial substrates were higher in isolated mitochondria compared to tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared to age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.
• Keywords: Cortex, Mitochondrial respiration, Membrane potential, Safranin O, TMRM, Alzheimer’s disease
• O2k-Network Lab: CA Winnipeg Fernyhough P
Labels: MiParea: Respiration, Instruments;methods Pathology: Alzheimer's
Organism: Mouse Tissue;cell: Nervous system Preparation: Homogenate, Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k, O2k-Fluorometer