Validation of physiological tests in relation to competitive performances in elite male distance cross-country skiing

The purpose of the present study was to establish which physiological test parameters reflects the distance performances in the Swedish National Championships in cross-country skiing (SNC) and the International Ski Federation's ranking points for distance performances (FISdist). The present study also aimed to create multiple regression models to describe skiing performance for the SNC distance races and International Ski Federation's (FIS) ranking. Twelve male, Swedish, national elite, cross-country skiers (maximal oxygen consumption [·VO?max] = 5.34 ± 0.34 L·min?1) volunteered to participate in the study. Their results in the 2008 SNC (15 km race [SNC15] and 30 km race [SNC30]) and FISdist points were used as performance data. On the week preceding the Championship, subjects completed a test battery consisting of 7 physiological tests: isokinetic knee extension peak torque (PT), vertical jumps (VJ), lactate threshold (LT), ·VO?max, and 3 double poling tests of different durations (DP20, DP60, and DP360). Correlations were established using Pearson's correlation analysis, and models to describe skiing performance were created using standard multiple linear regression analysis. Significant correlations were found between the performance parameters and test parameters derived from LT, ·VO?max, and DP60 tests. No correlations with any performance parameter were found for PT, VJ, DP20, and DP360 tests. For FISdist and SNC15, the models explain 81% and 78% of the variance in performance, respectively. No statistically valid regression model was found for SNC30. The results of this study imply that the physiological demands in male elite distance cross-country skiing performances are different in different events. To adequately evaluate a skier's performance ability in distance cross-country skiing, it is necessary to use test parameters and regression models that reflect the specific performance.     

On the regulation of cellular energetics in health and disease

Very recent experimental data, obtained by using the permeabilized cell technique or tissue homogenates for investigation of the mechanisms of regulation of respiration in the cells in vivo, are shortly summarized. In these studies, surprisingly high values of apparent Km for ADP, exceeding that for isolated mitochondria in vitro by more than order of magnitude, were recorded for heart, slow twitch skeletal muscle, hepatocytes, brain tissue homogenates but not for fast twitch skeletal muscle. Mitochondrial swelling in the hypo-osmotic medium resulted in the sharp decrease of the value of Km for ADP in correlation with the degree of rupture of mitochondrial outer membrane, as determined by the cytochrome c test. Very similar effect was observed when trypsin was used for treatment of skinned fibers, permeabilized cells or homogenates. It is concluded that, in many but not all types of cells, the permeability of the mitochondrial outer membrane for ADP is controlled by some cytoplasmic protein factor(s). Since colchicine and taxol were not found to change high values of the apparent Km for ADP, the participation of microtubular system seems to be excluded in this kind of control of respiration but studies of the roles of other cytoskeletal structures seem to be of high interest.In acute ischemia we observed rapid increase of the permeability of the mitochondrial outer membrane for ADP due to mitochondrial swelling and concomitant loss of creatine control of respiration as a result of dissociation of creatine kinase from the inner mitochondrial membrane. The extent of these damages was decreased by use of proper procedures of myocardial protection showing that outer mitochondrial membrane permeability and creatine control of respiration are valuable indices of myocardial preservation. In contrast to acute ischemia, chronic hypoxia seems to improve the cardiac cell energetics as seen from better postischemic recovery of phosphocreatine, and phosphocreatine overshoot after inotropic stimulation.In general, adaptational possibilities and pathophysiological changes in the mitochondrial outer membrane system point to the central role such a system may play in regulation of cellular energetics in vivo.     

Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.     

Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in vivo

In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (K_m = 200-400 M) is much lower than in isolated mitochondria (K_m = 10-20 M). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.     

Increased concentrations of P(i) and lactic acid reduce creatine-stimulated respiration in muscle fibers.

We tested the hypothesis that the respiratory function of skeletal muscle mitochondria is impaired by lactic acidosis and elevated concentrations of P(i). The rate of respiration of chemically skinned fiber bundles from rat soleus muscle was measured at [P(i)] (brackets denote concentration) and pH values similar to those at rest (3 mM P(i), pH 7.0) and high-intensity exercise (20 mM P(i), pH 6.6). Respiration was measured in the absence of ADP and after sequential additions of 0.1 mM ADP, 20 mM creatine (Cr; V(Cr)), and 4 mM ADP. Respiration at 0.1 mM ADP increased after addition of Cr. However, V(Cr) was 23% lower (P < 0.05) during high-intensity conditions than during resting conditions. V(Cr) was also reduced when P(i) or H(+) was increased separately (P < 0.05). Respiration in the absence of ADP and after additions of 0.1 mM ADP and 4 mM ADP was not affected by changes in [P(i)] or [H(+)]. The response was similar, irrespective of when acidosis was induced (i.e., quiescent or actively respiring mitochondria). In conclusion, Cr-stimulated respiration is impaired by increases in [H(+)] and [P(i)] corresponding to those in exercising muscle. Although the reduced Cr-stimulated respiration could be compensated for by increased [ADP], this might have implications for intracellular homeostasis.     

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

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 g/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.     

Calcium-induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells

The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.