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The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).

The kinetics of in vivo regulation of mitochondrial respiration by ADP was studied in rat heart, slow-twitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius, plantaris, quadriceps and tibialis anterior) by means of saponin-skinned fibres. Mitochondrial respiratory parameters were determined in the absence and presence of creatine (20 mM), and the effect of proteolytic enzymes (trypsin, chymotrypsin or elastase) on these parameters was investigated in detail. The results of these experiments confirm the observation of Veksler et al. [Veksler, V.I., Kuznetsov, A. V., Anflous, K., Mateo, P., van Deursen, J., Wieringa, B. & Ventura-Clapier, R. (1995) J. Biol. Chem. 270, 19921-19929], who studied muscle fibres from normal and transgenic mice, that the kinetics of respiration regulation in muscle cells is tissue specific. We found that in rat cardiac and soleus muscle fibres the apparent K(m) for respiration regulation was 300-400 microM and decreased to 50-80 microM in the presence of creatine. In contrast, in skinned fibres from gastrocnemius, plantaris, tibialis anterior and quadriceps muscles, this value was initially very low, 10-20 microM, i.e. the same as that is in isolated muscle mitochondria, and the effect of creatine was not observable under these experimental conditions. Treatment of the fibres with trypsin, chymotrypsin or elastase (0.125 micrograms/ml) for 15 min decreased the apparent K(m) for ADP in cardiac and soleus muscle fibres to 40-98 microM without significant alteration of Vmax or the intactness of outer mitochondrial membrane, as assessed by the cytochrome c test. In fibres from gastrocnemius, trypsin increased the apparent K(m) for ADP transiently. The effects of trypsin and chymotrypsin were studied in detail and found to be concentration dependent and time dependent. The effects were characterised by saturation phenomenon with respect to the proteolytic enzyme concentration, saturation being observed above 1 microM enzyme. These results are taken to show that in cardiac and slow-twitch skeletal muscle, the permeability of the outer mitochondrial membrane to adenine nucleotides is low and controlled by a cytoplasmic protein that is sensitive to trypsin and chymotrypsin. This protein may participate in feedback signal transduction by a mechanism of vectorial-ligand conduction. This protein factor is not expressed in fast-twitch skeletal muscle, in which cellular mechanism of regulation of respiration is probably very different from that of slow-twitch muscles.