Kuznetsov 2003 Anal Biochem
|Kuznetsov AV, Kunz WS, Saks V, Usson Y, Mazat J-P, Letellier T, Gellerich FN, Margreiter R (2003) Cryopreservation of mitochondria and mitochondrial function in cardiac and skeletal muscle fibers. Anal Biochem 319:296-303.|
Abstract: Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze–thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze–thawing conditions, high rates of adenosine 5′-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5′-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze–thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.
• Keywords: Freeze–thawing, Heart and skeletal muscles, Human biopsies, Mitochondrial oxidative phosphorylation, Permeabilized fibers
Labels: MiParea: Respiration
Stress:Cryopreservation Organism: Human Tissue;cell: Heart, Skeletal muscle Preparation: Permeabilized tissue
Coupling state: OXPHOS Pathway: N, S HRR: Oxygraph-2k