Malleability of skeletal muscle in overcoming limitations: structural elements

Two metabolic systems can supply energy for muscle contraction. The glycolytic system resides in the sarcoplasm and can deliver ATP at high rates but only for short periods of time (Hochachka, 1985). It is thus not able to fuel sustained mechanical activities. The glycolytic system currently eludes a structural analysis as the relevant enzymes in the sarcoplasm are not bound to specific structures which can be estimated by morphometric methods. However, there is some indication that the capillarity of glycolytic muscles might be related to tissue lactate production (Hudlicka, 1985).

The muscle cell’s aerobic system for energy supply mainly consists of mitochondria producing ATP through oxidative phosphorylation. Mitochondria can be well characterized by morphometric methods in terms of their volume density, the surface density of their inner and outer membranes and their distribution within the muscle cells, all of which may be important to mitochondrial energy transduction (Hoppeler et al. 1981). The respiratory system of the muscle cell is supported by capillaries running parallel to the muscle fibres. Capillaries are responsible for an adequate supply of oxygen and of substrates as well as for the removal of ‘waste’ products such as heat.

The highest steady-state energy demand situation an animal can satisfy is assessed by its maximal oxygen uptake capacity ⁠. Under these conditions most of the oxygen taken up by an animal is consumed by its working muscles (Mathieu et al. 1981). In order to appreciate the importance of structural variables on the cellular conductance of oxygen G(c), we have related changes in morphometric estimates of structural determinants of oxygen flow in the muscle cell to experimentally induced changes in maximal steady state oxygen flow rate through the respiratory system (Weibel et al. 1981).

We have exploited two experimental models in which the chronic energy demand of muscle tissue appeared as a variable.

• (a) Endurance exercise and chronic electrical stimulation both lead to metabolic adaptations in muscle cells which allow them to sustain aerobically situations of higher energy demand over prolonged periods of time.

• (b) Small and large animals offer another possibility to compare muscle tissue which greatly differs in its scope of aerobically covering its energy demand, as small animals have to be able to generate ATP at rates several-fold higher than large animals (Weibel et al. 1981).

Taylor et al. (1981) found that the max of a variety of mammals, from pygmy mice (20g) to elands (240kg), scales as body mass to the power 0·809 ⁠, r = 0·987). The slope of this relationship is not significantly different from the slope for resting oxygen consumption versus body mass which scales close to M_b^0·75 (Kleiber, 1932). Thus max is consistently about 10 times resting oxygen consumption. The slope of 0·8 for the allometric relationship of max versus M_b indicates that a mouse consumes some 10 times more oxygen per unit body mass than a cow when both are running at max. We expected mitochondria to be similarly more abundant in mouse than in cow skeletal muscles. In fact the total volume of mitochondria (V_mt) was found to scale close to M_b^0·8 in vastus medialis, semi-tendinosus and diaphragm of a series of African mammals spanning more than three orders of magnitude of body size (Mathieu et al. 1981). The main conclusion of the latter study was that there is a constant (body mass independent) relationship between the maximal rate of aerobic power production in animals and the absolute volume of mitochondria in their muscles. These morphometric results are supported by a study of the scaling of oxidative and glycolytic enzymes in mammals; the scaling factors for oxidation enzymes are similar to those for mitochondrial volumes (Emmett & Hochachka, 1981), while glycolytic enzymes were found to scale as M_b^1·15. This indicates that as animals increase in size, they are endowed with a decreasing scope for aerobic metabolism but with an increasing scope for anaerobic metabolism.

where V_mu is the muscle volume, this means that the volumes of some muscles do not scale linearly to body mass (M_b^1·0). While some muscles scale with an exponent less than one, others must scale with an exponent greater than one, since the total skeletal muscle mass is consistently near 45 % of the total body mass in all mammals irrespective of size (Munro, 1969). For instance, the volume of the diaphragm scales as M_b^0·865 while the volume density of mitochondria in diaphragm scales as M_b^−0·05s. This indicates that the diaphragm is a much smaller fraction of the body mass in large animals than in small ones while the fractional volume of mitochondria decreases only slightly with increasing body size. Perhaps the different scaling of muscle volume and volume density of mitochondria in some muscles might be related to the specific (sizedependent) use of a particular muscle or muscle group. Note that the slope of the allometric regression of the volume density of mitochondria to body size of the remaining three skeletal muscles (semitendinosus, vastus medialis, longissimus dorsi) is not significantly different from the slope of the regression of weight specific max to body mass (Fig. 3). Such a relationship is to be expected if indeed the rate of crossbridge cycling between actin and the myosin heads were to set the energetic cost of muscular contraction in direct proportion to the intrinsic rate of muscle shortening (Hoppeler, Lindstedt & Mathieu, 1980; Heglund, Fedak, Taylor & Cavagna, 1982).

max is not only different among animals of different body size, it can also be changed in animals of one particular size class by increasing physical activity. In man, max may vary as much as two-fold between sedentary subjects and extremely well trained endurance athletes such as bicyclists, marathon runners or cross-country skiers (Saltin & Gollnick, 1983). Changes in mitochondrial variables associated with training-induced changes in max have been studied mostly by estimating activities of marker enzymes of aerobic metabolism during training and also during recovery from training. The relative changes in the activities of the most important enzymes of aerobic metabolism invariably exceed the relative changes in max induced by endurance exercise. This topic has been reviewed extensively (Gollnick & Saltin, 1982; Saltin & Gollnick, 1983; Holloszy & Coyle, 1984). Although the changes in structural variables of the mitochondrial compartment have not been documented as well as the biochemical changes, it is commonly assumed that it is the quantity of mitochondria that changes in proportion to the activities of often studied enzymes of the Krebs-cycle such as citrate synthase and succinate dehydrogenase (Hoppeler et al. 1973 ; Davies et al. 1981; Saltin & Gollnick, 1983).

One generally-held view with regard to the disparate adaptation of max and the oxidative capacity of muscle is that mitochondrial adaptations are mostly related to the ability of trained subjects to perform strenuous exercises over prolonged periods of time (Gollnick & Saltin, 1982; Holloszy & Coyle, 1984). A causal relationship between the elevation of max and the activity of oxidative enzymes in skeletal muscle of man in training is refuted on the grounds that these enzyme activities are found to change out of proportion to max (Gollnick & Saltin, 1982). The relationship between central and peripheral determinants of aerobic work performance, and their role, are currently debated. The disproportionate changes in central and peripheral factors of aerobic work performance may not be contradictory, as commonly held, but may well be explained by an inter-related adaptation of the whole cascade of conductances (or resistances) to oxygen flow across the respiratory system of mammals during increased use, as presented elsewhere in this issue (Di Prampero, 1985). By modelling the respiratory system as a cascade of resistances in series, Di Prampero (1985) claims that for two-legged exercise 75 % of max is set by oxygen transport. Hence under these conditions, small changes in max would require large changes in tissue oxidative capacity in selectively trained muscle groups.

Is it possible that there is a constant and predictable relationship between the volume of skeletal muscle mitochondria and tissue oxygen consumption under limiting conditions? A direct link between mitochondrial volumes and oxygen consumption could be established if both of these variables could be determined in identical compartments. An experimental model which allows one to estimate both variables is the isolated, autoperfused cat gracilis and soleus muscle preparation (Bockmann, McKenzie & Ferguson, 1980). By using this model we have calculated oxygen consumption from blood flow obtained by a photoelectric drop-counter inserted in the outflow of surgically isolated muscles and from arteriovenous oxygen content differences estimated by using a Lex-O₂-Con oxygen analyser (Hoppeler, Hudlicka & Uhlmann, 1982). Peak oxygen consumption of the muscles was reached at stimulation frequencies of 6–10 Hz. Volume densities of mitochondria were estimated in muscle samples prepared for electron microscopy by standard stereological procedures. We found peak oxygen consumption to be higher by as much as three-fold in the oxidative soleus as compared to the glycolytic gracilis muscles. Similarly, values for peak oxygen consumption that were three-fold higher than in controls were obtained in gracilis muscles electrically stimulated over periods of up to 28 days. In all muscles analysed, peak oxygen consumption was found to be linearly related to volume density of mitochondria ⁠. Peak oxygen consumption per unit volume of mitochondria can hence be calculated as l·4ml O₂ min⁻¹cm⁻³ (Hoppeler et al. 1982). The highest value for mitochondrial oxygen consumption (2·2ml O₂ min⁻1^cm–3) was found in a gracilis muscle electrically stimulated for 28 days.

Apart from the autoperfused single muscle preparation in the cat, there are a few more studies in several animal species in which one can at least approximate ‘maximal’ oxygen consumption of a unit volume of skeletal muscle mitochondria (Table 1).

• (1) In man, Saltin (1985) elegantly demonstrated the maximal oxygen consumption of the functionally isolated extensor muscle group of the knee joint to be 30–36 ml O₂ min⁻¹100 g⁻¹ of muscle tissue. By assuming the mitochondrial volume density of all extensor muscles to be similar to that of vastus lateralis (approx. 5 % of the fibre volume in untrained subjects; Hoppeler et al. 1973) the mitochondrial oxygen consumption can be calculated as 5 ml O₂min⁻¹ cm⁻³.

• (2) In the mouse, a novel sampling technique has made it possible to determine the total skeletal muscle mitochondrial volume (Hoppeler et al. 1984). We were thus able to measure maximal running and cold exposure oxygen consumption, as well as whole body skeletal muscle mitochondrial volumes in the same animals (European woodmice). Mitochondrial oxygen consumption under both conditions was found to be 4·9 ml O₂min⁻¹ cm⁻³. This value indicates a lower limit of mitochondrial oxygen consumption in this species as (a) not the whole muscle mass is activated simultaneously in running and (b) not all the oxygen taken up by the body is used by the skeletal muscles.

• (3) In a series of African mammals, Mathieu et al. (1981) have demonstrated a strong and constant relationship between the mitochondrial volume of individual muscles and whole animal max. As mammals are all nearly 45 % muscle (Munro, 1969) it must then hold that the total volume of all skeletal muscle mitochondria likewise scales parallel to max. By assuming the mean volume density of the leg muscles to be representative for the volume density of muscle mitochondria of the whole body (Hoppeler et al. 1984), we can calculate a lower limit for mitochondrial oxygen consumption at max for the series of African mammals to be 5·3 ml O₂min⁻¹ cm⁻³.

• (4) In birds, Pennycuick & Rezende (1984) analysed the sustained power requirements of flight muscles by means of a simplified model of muscle function in which they also measured the volume density of mitochondria in sparrow and quail pectoralis muscle. They proposed that the specific power output of flight muscles required 1 ml of mitochondria to sustain a mechanical power output of 1–2W. Converting Watts into units of oxygen consumption this would indicate that mitochondrial oxygen consumption would minimally have to be 3–6 ml O₂ min⁻¹ cm⁻³.

In summary, we have several lines of evidence which suggest that there is a rather constant volume of oxygen metabolized under limiting conditions of max per unit volume of skeletal muscle mitochondria. This holds despite a more than five-fold difference in weight specific max among the species analysed (not including birds). This might support Pennycuick & Rezende’s (1984) hypothesis that the potential for aerobic power production of skeletal muscles can be characterized by estimating their volume density of mitochondria. Such a prediction critically depends on the composition of the mitochondria and the efficiency of the muscular energy conversion being constant or known for one set of experiments.

The quantitative structural composition of skeletal muscle tissue shows a wide range of variability among different species of animals and in any one species among muscles with a different function. Moreover, experimental manipulations such as exercise training or chronic electrical stimulation can dramatically change the ultrastructural appearance of the muscles involved. Both in endurance exercise and in chronic electrical stimulation the volume density of mitochondria can be increased greatly (by more than three-fold in the stimulation experiments). This happens without an apparent change of the internal architecture of the mitochondria, since the surface density of the inner mitochondrial membranes remains constant. In situations where both the mitochondrial volume and the maximal rate of oxygen consumption of the muscle tissue are known, these two variables are found to be linearly related. It can be calculated that the ‘maximal’ oxygen consumption of a unit volume of mitochondria in muscle is close to 5 ml O₂ min⁻¹ cm⁻³ under comparable conditions in man, mouse and a series of African mammals. It is hypothesized that there is a constant volume of oxygen metabolized per unit volume of mitochondria and unit time under limiting conditions in working skeletal muscle tissue. Given the efficiency of muscular energy conversion, this would allow an estimate of the potential for aerobic power production of a muscle from measurement of its volume density of mitochondria.

The authors wish to express their sincere gratitude to Ms H. Claassen, Ms E. Uhlmann, Ms M. Schweizer and Mr K. Babl for excellent technical assistance and to Ms R. M. Fankhauser for typing the manuscript. This work was supported by Grant 3.128.81 from the Swiss National Science Foundation.