Molecular and Cellular Biochemistry

, Volume 184, Issue 1–2, pp 81–100 | Cite as

Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vitro

  • Vladimir I. Veksler
  • Andrei V. Kuznetsov
  • Laurence Kay
  • Peeter Sikk
  • Toomas Tiivel
  • Leone Tranqui
  • Jose Olivares
  • Kirstin Winkler
  • Falk Wiedemann
  • Wolfram S. Kunz

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 μg/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.

mitochondrial respiration skinned fibers permeabilized cell heart skeletal muscle regulation cytoskeleton myopathies 

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Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Vladimir I. Veksler
    • 1
  • Andrei V. Kuznetsov
    • 2
  • Laurence Kay
    • 3
  • Peeter Sikk
    • 1
  • Toomas Tiivel
    • 1
  • Leone Tranqui
    • 1
  • Jose Olivares
    • 1
  • Kirstin Winkler
    • 1
  • Falk Wiedemann
    • 3
  • Wolfram S. Kunz
    • 3
  1. 1.Laboratories of Bioenergetics, Institute of Chemical Physics and Biophysics, TallinnEstonia and J. Fourier UniversityGrenobleFrance
  2. 2.INSERM U446Chatenay-MalabryFrance
  3. 3.Neurobiochemisches Labor, Klinik für NeurologieUniversitätsklinikum MagdeburgGermany

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