STUDY OF REGULATION OF MITOCHONDRIAL RESPIRATION IN-VIVO AN ANALYSIS OF INFLUENCE OF ADP DIFFUSION AND POSSIBLE ROLE OF CYTOSKELETON

The purpose of this work was to investigate the mechanism of regulatio n of mitochondrial respiration in vivo in different muscles of normal mt and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cell s in vivo in the heart, soleus and white gastrocnemius skeletal muscle s of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the ''ghost'' (phantom) cardi omyocytes were produced by extraction of myosin with 800 mM KCI. Use o f confocal immunofluorescent microscopy and anti-desmin antibodies sho wed good preservation of mitochondria and cytoskeletal system in these phantom cells, Kinetics of respiration regulation by ADP was also stu died 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 mus cle the kinetics of mitochondrial respiration regulation by ADP was ch aracterized by very high apparent K-m (low affinity) equal to 300-400 mu M, exceeding that for isolated mitochondria by factor of 25, In ski nned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent K-m for ADP significantly, this excludin g the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative act ivity and by intracellular diffusion problems. However, short treatmen t of fibers with trypsin decreased this constant value to 40-70 mu M, confirming the earlier proposition that mitochondrial sensitivity to A DP in vivo is controlled by some cytoplasmic protein. Phantom cardiomy ocytes which contain mostly mitochondria and cytoskeleton and retain t he normal shape, showed also high apparent K-m values for ADP. Therefo re, 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 kinetic s of respiration regulation by ADP. However, in skinned fibers from th e 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 K-m for ADP and the second one with very low apparent K -m for ADP. Morphological observations by electron microscopy confirme d the existence of two distinct cellular populations in the muscle cel ls of desmin-deficient mice. The results conform to the conclusion tha t the reason for observed high apparent K-m for ADP in regulation of o xidative phosphorylation in heart and slow twitch skeletal muscle cell s in vivo is low permeability of mitochondrial outer membrane porins b ut not diffusion problems of ADP into and inside the cells. Most proba bly, in these cells there is a protein associated with cytoskeleton, w hich 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 c ells with disorganised structure and of altered mitochondrial populati on probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structura l organisation and cytoskeleton in the cells in vivo due to the existe nce of still unidentified protein factor(s). (C) 1997 Elsevier Science B.V.