The aim of this work was to characterize quantitatively the arrangement of mitochondria in heart and skeletal muscles. We studied confocal images of mitochondria in nonfixed cardiomyocytes and fibers from soleus and white gastrocnemius muscles of adult rats. The arrangement of intermyofibrillar mitochondria was analyzed by estimating the densities of distribution of mitochondrial centers relative to each other (probability density function). In cardiomyocytes (1,820 mitochondrial centers marked), neighboring mitochondria are aligned along a rectangle, with distance between the centers equal to 1.97 Ϯ 0.43 and 1.43 Ϯ 0.43 m in the longitudinal and transverse directions, respectively. In soleus (1,659 mitochondrial centers marked) and white gastrocnemius (621 pairs of mitochondria marked), mitochondria are mainly organized in pairs at the I-band level. Because of this organization, there are two distances characterizing mitochondrial distribution in the longitudinal direction in these muscles. The distance between mitochondrial centers in the longitudinal direction within the same I band is 0.91 Ϯ 0.11 and 0.61 Ϯ 0.07 m in soleus and white gastrocnemius, respectively. The distance between mitochondrial centers in different I bands is ϳ3.7 and ϳ3.3 m in soleus and gastrocnemius, respectively. In the transverse direction, the mitochondria are packed considerably closer to each other in soleus than in white gastrocnemius, with the distance equal to 0.75 Ϯ 0.22 m in soleus and 1.09 Ϯ 0.41 m in gastrocnemius. Our results show that intermyofibrillar mitochondria are arranged in a highly ordered crystal-like pattern in a muscle-specific manner with relatively small deviation in the distances between neighboring mitochondria. This is consistent with the concept of the unitary nature of the organization of the muscle energy metabolism. confocal microscopy; quantitative analysis; cardiac and skeletal muscles; probability density function; unitary structure of cells RECENT STUDIES HAVE SHOWN the existence of multiple specific functional interactions among mitochondria, sarcoplasmic reticulum (SR), and myofibrils in permeabilized muscle fibers (5,14,30,34). Namely, endogenous ATP has been shown to be more efficient than exogenous ATP in maintaining calcium uptake into SR (14). In addition, kinetic studies have shown a direct supply of endogenous ADP from ATPases to mitochondria (30, 34). Such interaction can be explained by the existence of localized intracellular diffusion restrictions (28, 39). A mild treatment of the fibers with trypsin leads to the removal of these diffusion restrictions, and at the same time, distribution of mitochondria in the fiber is changed from regular arrangement in the control to random distribution after the treatment (28). Similarly, in ischemic hearts, various alterations in mitochondrial function such as the significant decrease in maximal respiration rate and half-saturation constant for ADP were observed in parallel with the changes in structural organization of the cardiac muscle cells (7,1...
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.
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