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Abstract

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 microg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon--tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.

Inhibition of the Mitochondrial Permeability Transition by Creatine Kinase Substrates

“…Phosphorylation of these CK substrates occurs in microcompartments formed by mtCK and ANT (see below) (35). Because we did not add external adenine nucleotides, phosphorylation must occur via internally available ATP inside mitochondria, suggesting continuous cycling of internal ADP and ATP between matrix and intermembrane space mediated by ANT, if Cr or CyCr are present.…”

Mechanisms by which opening the mitochondrial ATP- sensitive K+channel protects the ischemic heart

“…Nucleotide permeability is controlled by VDAC, which is normally in a low conductance state that is poorly permeable to ATP (2,34,50), and energy transfers between mitochondria and cytosol are mediated instead by Cr and creatine phosphate (55). Octamers of Mi-CK bridge the IMS between outer membrane VDAC and inner membrane ATP/ADP translocator (62).…”

Heterogeneous effects of old age on human muscle oxidative capacity in vivo: a systematic review and meta-analysis

“…It should be noted that, when mitochondrial function is measured in isolated mitochondria, the organelle is susceptible to structural disruption (Hepple 2014;Picard et al 2011b), which can exaggerate the magnitude of age-related impairments in mitochondrial function compared with saponin-permeabilized fiber bundles (Picard et al 2011c). This latter in vitro approach does not disrupt organelle structure (Saks et al 1998). …”