38, No. 4, April




Mitochondrial of skeletal

in U.,S.A.

enzymatic muscle


to endurance


G. BENZI, P, PANCERI, M. DE BERNARDI, R. VILLA, E. ARCELLI, L. n’ANGEL0, E. ARRIGONI, AND F. BERTh Department of Pharmacology, University of Pavia, 27100 Paviu, Italy

BENZI, G., P. PANCERI, M. DE BERNARDI, R. VILLA, E. ARCELLI, L. D'ANGELO, E. ARRICONI, AND F. BER&. MitochondriaZ enzymatic adaptation of skeletal muscle to endurance training. J. Appl. Physiol. 38(4) : 565-569. 1975.-Some mitochondrial enzymatic activities (succinate dehydrogenase, NADH cytochrome G reductase, cytochrome oxidase) were studied in the gastrocnemius and soleus muscle of the rat. The modifications of the enzyme activity, induced by endurance training, were found to be functions of I) daily work load and 2) total training time. The treatment with an effective dose of vasodilating substances (papaverine, nicergoline, dipyridamole, and bamethan) showed that I) nicergoline, bamethan, and dipyridamole were differently able to shorten the time of appearance of the increase in the enzymatic activities; 2) however, long-term treatments with these drugs did not prove able to modify the plateau level of the enzymatic activity increase, for a given amount of endurance training; .?) the pharmacodynamic effect on enzymatic activities was in no way related to the vasodilating effect of these drugs, since the &ect was not observed with papaverine. The transition from a given level of endurance training to a lower one led to a proportional decrease of the mitochondrial enzymatic activities, thus pointing out the relation between amount of training and enzymatic activity. The drugs studied were unable to modify the decrease of enzymatic activity induced by lower work load. rat skeletal c reductase;

muscle; succinate dehydrogenase; cytochrome oxidase; mitochondria;

NASH drug

cytochrome influence

IN CONNECTION with the problem of enzymatic changes in skeletal muscle related to adaptation to physical training, Hearn and Wainio (6) and Gould and Rawlinson (5, 14) described no changes in succinate dehydrogenase, lactic dehydrogenase, malic dehydrogenase, phosphorylase, adenosine triphosphatase, and creatine phosphokinase in rats exercised by swimming 30 min daily, for six 5-day weeks. Holloszy (7) studied the biochemical adaptation of rats subjected to a strenuous program of treadmill running and found that succinate dehydrogenase, succinate oxidase, INADH cytochrome c reductase, and cytochrorne oxidase activitie.s increased twofold in hindlimb muscles in response to the training. Mitochondria from muscles of exercised animals exhibited a high level of respiratory control and tightly coupled oxidative phosphorylation. Holloszy and co-workers (8) showed that, unlike the constituents of the respiratory chain, the mitochondrial citric acid cycle and citric acid-related enzymes do not increase in parallel during the adaptive response of skeletal muscle to strenuous

exercise. According to Molk and co-workers (lo), also the level of activity of glutamate-pyruvate transaminase showed an 85 % increase in the gastrocnemius muscles of the rats exercised by a strenuous program of treadmill running. Both the mitochondrial and the cytoplasmic forms of glutamate-pyruvate transaminase increased. The succinate dehydrogenase activity was determined by Gollnick and co-workers (4) and by Vihko and coworkers (17) on biopsy samples from the vastus lateralis and deltoid muscles of untrained and trained men. The enzyme activity was highest in the muscles of the groups participating in endurance training, and particularly in the muscles that were extensively engaged in the endurance work, thus suggesting a specific localized training effect. The present study was aimed at investigating a) the influence of both work load and endurance training duration in increasing the activity of some mitochondrial enzymes of skeletal muscle; b) the influence of the work-load decrease on the previously acquired enzymatic modifications due to endurance training; c) the influence of drug administration on these biochemical changes induced by endurance training. The drugs chosen were papaverine, nicergoline, dipyridamole, and bamethan because a) they are well-known peripheral vasodilating agents, able to induce an increase and dipyridamole of muscular blood flow; b) nicergoline increase the cerebral energy charge potential (ECP = 1 (ATP) + 0.5 (ADP) l/l (ATP) + (ADP) + (AMP) 1) previously depressed by hypoxemia in the hypovolemic, hypotensive beagle dog. This action is oxygen-, glucose-, and insulin-dependent and is antagonized by malonate and cocaine (1) ; c) nicergoline, dipyridamole, and bamethan can improve the performance time in mice (11, 18). METHODS

Animal care and exercise program. Wistar strain male rats, weighing 120-140 g, were maintained under standard conditions (temp: 23 & 1 “C ; relative humidity: 55-60 Yo) and fed a standard diet and water. The rats were trained to run on a rotarod treadmill (Basile, Milan), When a rat fell off its cylinder section onto the plate below, the corresponding electronic counter was tripped, thereby recording the animal’s endurance time in seconds. In a preliminary experiment, the running speed was progressively increased over a 2-wk period from 2.5 to 10 m/min. The animals were distributed according to 565

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their performance time at 10 m/min. For the experiments, those animals (weighing ZOO-220 g) were chosen whose performance times were closest to the average value of the whole population examined. In the first experiment, the selected rats were divided at random into exercising groups (A and B) and sedentary groups (S). The exercising groups A were trained to run continuously for 60 min at 20 m/min (group Al), or for 120 min at 25 m/min (group AZ), or for 180 min at 30 m/min (group A3). Th e exercising groups B were trained to run continuously for 60 min at 20 m/min (group Sl), or for 180 min at 30 m/min (group B2) both with sprints at 40-45 m/min, each lasting 15-30 s, interspersed at IO-min intervals through the workout. All the groups were trained 6 days a week. The work load was progressively increased to reach the indicated levels within a maximum of 30 days (groups Al and Sl), of 45 days (group AZ), or of 60 days (groups A3 and BZ). The animals which were not able to attain this working capacity within the respective times assigned to them, were discarded. The biochemical determinations were carried out after 15, 30, 60, and 120 days of training; consequently, the enzymatic recordings at the 15th day (for all groups) and at the 30th day (for groups AZ, A3, and B2) were carried out on the assumption that the animals examined would acwithin the tuallv be able to execute their performance established time. The second experiment was devised to establish whether the enzymatic changes due to training were affected by exogenous factors such as the administration of drugs. The rats selected were divided into groups of 1) trained controls (AC); 2) trained-treated with drugs (groups AS, AD, AB, and Al’); 3) sedentary controls (SC) and sedentarytreated \vith drugs (groups ZX, W, SB, and Sr). The pharmacological treatments were carried out by intrawith Q) 1,6-dimethyl-8P-(5-bromoperitoneal injection nicotinoylhydroxymethyl)lOa-methoxyergoiine tartrate or nicergolinc (groups AS and &‘X) ; 6) 2, &bis(diethanolamino)4,8-dipiperidinopyrixnido 1 5,4-d 1 pyrimidine or dipyridamole (groups AlI and Sr>) ; c) 1-(p-hydroxyphenyl)-2butylarninocthanol or bamethan (groups AB and SB) ; papaverine d) 6,7-dimethoxy1 -veratryl-isoquinoline or (groubs AI’ and Sr). The rats were injected daily with 0.5 ml/kg of a 4 X 1W3 M solution of the substances studied. The kind of training carried out both in treated and in nontreated animals leas 180 mm/day at 30 m/min. The third experiment was aimed at studying the bchavior of training-induced changes of enzymatic activity some in connection with time. After 120 days of training, to groups A2 and A3 were subof the animals belonging jetted for 2 mo to a training of lower intensity and duration. In fact, after 120 days of training some animals of group A3 were submitted to the work load of group A2 (group A3 -+ AZ), while some rats of gro@ A2 were submitted to the work load of group Al (group A2 -+ Al). The fourth experiment was planned in order to establish whether the modifications of muscular enzymatic activities, induced by changes in the daily work load, lvere to any extent affected by an exogenous factor such as pharmacological treatlnent. The procedure of the fourth experiment \yas the same as for the third: however. some rats of



the A3 + A2 group were treated with nicergoline (group A3 -+ A2 + N) or with dipyridamole (group A3 -+ A2 + D), while some rats of the A2 --) Al group were treated with bamethan (group A2 + AI + B). The drugs were injected as described in the case of the third experiment Preparation of mitochondriu and assay methods. According to Molk and co-workers (IO), the rats were not exercised for 72 h prior to sacrifice to avoid any acute effects of exercise. The gastrocnemius and soleus muscles were dissected out, trimmed of fat and connective tissue, weighed, and minced. Preparation of skeletal muscle mitochondria by homogenization and differential centrifugation was made according to Ernster and Nordenbrand (3). This method is known to result in the recovery of only a part of the mitochondria contained in the muscle. Furthermore, a loss of soluble and Ioosely bound mitochondrial enzymes into the supernatant “cytoplasmic” fraction is observed. In the present study, enzymatic activities are referred to micromoles of substrate utilized per minute by mitochondrial protein, per gram of fresh tissue. Spectrophotometric assays were performed in a PerkinElmer 124/56 double-beam spectrophotometer with a thermostated cell compartment, in l-ml cuvettes of l-cm light path, at 30°C. Initial reaction rates were determined from a segment of the linear portion of the change in absorbance and corrected for the rates of any nonspecif% activity. The assays were performed only under conditions where the reaction rate was proportional to enzyme concentration. Enzyme activities were evaluated on material simultaneously taken from the soleus and gastrocnernius muscles. Succinate dehydrogenase activity was measured by the method of Bonner (2). NADH cytochrome c reductase activity was measured by the method of Mason and Vasington (12). Rotenone sensitivity of KADH cytochrome c reductase was assayed as described by Sottocasa and co-workers (15). Cytochrome oxidase activity \\ras measured in whole homogenates according to the method of Potter (13). For the sake of a more immediate interpretation, all the data reported under RESULTS and concerning each individual enzymatic activity tested, were plotted by assuming the activity at lime 0 of the sedentary controls equal to 1. During the observation period (on the 120th and on the 180th day) no enzyme activity changes were observed in the muscle of sedentary controls, as compared to initial values at time 0. Protein kvas determined by the method of Lowry and co-workers (9). was performed according to the Statistical analysis Student t-test (significance: 1’ < 0.05). RESULTS

As Figs. 1 and 2 show, training leas responsible for rnodifications in the activity of succinatc dehydrogenase and cytochrome oxidase. These changes are strictly related to 1) the amount of daily work load: indeed, only group A3 which had a high work load (30 m/min for 3 h) and, to a lower extent, group A2 which had a moderate work load (25 m/min for 2 h) exhibited a significant modification of the enzyn~atic activities assayed; 2) total training time, in ,proub A2 the succinate dehvdrogcnasc activity e-e.,

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2.6 c 2 5

” F “r >g w

2.4 2.2

2.0 1.8 1.6 1.4 I.2







FIG. 1. Succinate dehydrogenase gastrocnemius and soleus muscles training time (abscissa) and of



relative activity (ordinate) of rats, expressed as a function daily work load (for symbols,

of of see

Enzymatic activity was expressed by assuming the acof sedentary controls (at time 0) equal to 1 (& standard error). point represents the mean of 5 rats, except for base values at


tivity Each

0, where


n =

could modify the pattern of biochemical adaptation to endurance training was checked by means of a daily intraperitoneal treatment with papaverine, nicergoline, dipyridamole, and bamethan. As for succinate dehydrogenase, it can be observed from Fig. 4 that nicergoline and, in order, bamethan and dipyridamole, favored the increase of enzymatic activities. However, the differences between trained and trained-and-treated lots became statistically significant only at the 30th day; at any rate, at the 120th day the values of the succinate dehydrogenase activity were the same in the two groups (trained and trained-andtreated with the drugs mentioned above). The chronic treatment with papaverine did never increase the values of succinate dehydrogenase activity. The mitochondrial enzymatic activity of the muscles of sedentary animals treated with nicergoline, dipyridamole, bamethan, or papaverine was also unchanged as compared to sedentary controls. The decrease, due to lower daily work load, of the mitochondrial enzymatic activities acquired through intense endurance training is shown in Fig. 5. At the end of the 4-mo standard training, the animals of grou;bs A3 and A2


9 1.8 2 F 1.6 N =

?j 1.4







1.2 15




1.0 >w



5 0.8 der 0.6






G rcductase of rats.


relative as in Fig.


of gas-


0.4 15


oxidase Cytochrome and soleus muscles of rats. Legend FIG.


relative activity as in Fig. 1.







significantly departed from normal values only after the 60th day of training. The kinetics of succinate dehydrogenase activity as a function of total training time \yas markedly different from that of cytochrome oxidase, probably because of the different techniques used for their determination. The inclusion of interspersed sprints in the endurance training (groups BI and B2) did not modify the increase in enzymatic activities as compared to the lots lvhere no sprints were included in the endurance training (groups Al and A3). The relative activity of the NADH cytochrome c reductase followed a pattern remarkably different from that of both succinate dehydrogenase and cytochrome oxidase. As Fig. 3 shows, only in the lots with a high lf\rork load (A3 and SZ) and only at the 120th day of training there was a statistically significant difference as compared to control values, the increase in the enzymatic activity being rotenone sensitive. The hypothesis that some pharmacological treatments



5 F



2 2.0





d =

0.6 DAYS I





1 30

1 60

dehydrogenase relative activity of gastrocnemius of rats. Legend as in Fig. 1. AC = control trained rats; A,;V, AD, AR, and AP = trained rats treated daily with 0.5 ml/kg IP of a 4 X 10s3 hlI solution of nicergoline, dipyridamole, bamethan, and papaverine, respectively. SN, SI), SB, and SP = sedentary rats treated daily with 0.5 ml/kg IP of a 4 X lOA” hl solution of nicergoline, dipyridamole, bamethan, and papaverine, reFIG.


4. Succinate





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2.6 z 3 F 2

2.4 2.2










FIG. 5. Succinate dehydrogenase relative activity of gastrocnemius and soleus muscles of rats. Legend as in Fig. 1. Numbers reported in abscissa indicate the days of training with reduced work load. Numbers in parentheses in abscissa indicate the days from the beginning

of training.



see text



were subjected to a lower daily work load, respectively corresponding to that of groups A2 and A I (group A3 -+ A2 and group A2 -+ Al of Fig. 5). In about 40 days, the groz#s A3 + A2 and A2 + AI showed the same succinate dehydrogenase levels as those reached by groups A2 and Al durini the 4-mo standard training. The decrease of enzymatic activity induced by a lower work load was statistically significant. The possible ability of some pharmacological treatments to counteract to any extent the tendency of mitochondriaJ. enzymatic activity to decrease as a consequence of a lower work load represents another factor to be verified. Figure 5 showTs that the chronic intraperitoneal treatment with nicergoline or dipyridamole, which was started at the end of the 120-day standard training in the rats of group A3 = group A3 -+ A2 + IV; dipyridamole = grouf (niccrgoline A3 + A2 + 11) did not significantly modify the decrease of the succinate dehydrogenase activity induced by lower work load. Similar results were obtained for the intraperitoneal chronic treatment with bamethan, started at the end of the 120-day standard training in the rats of groupA2 (groupA2 -+A1 + BofFig. 5).

The data reported in this paper show that the training described here leads to an improvement in performance. The enzymatic changes can be merely a byproduct of the training, or they can contribute i.n some way to the perforrnance . The ra ts imDrove thei .r performance in the first 15 days of training before homogeneous changes in the enzymatic activities studies are noted. On the other hand, the groups with a low daily work load continue their performance for 120 days without changes in the enzymatic activity. As a con sequence, the increase in the succinate dehydrogenase and cytochrome oxidase activities induced



by endurance training indicate that two of the factors determining such increase are I) the amount of daily work load (intensity X duration); 2) the overall training time. The discrepancy in the results of Hearn and Wainio (6), Gould and Rawlinson (5, 14) and Holloszy and co-workers (7, 8, 10) is therefore only apparent when one considers that only a heavy work load over a prolonged period of time is able to induce an increase in the activity of mitochondrial enzymes. The higher working ability early during training, constantly observed in the animals with a low work load, should be ascribed to extraenzymatic factors. At first sight, NADH cytochrome c reductase would seem to behave differently from both succinate dehydrogenase and cytochrome oxidase, since its activity increase is quite delayed and much lower than that of the other two enzymes. However, when one takes into account the fact that this increase is rotenone sensitive, then it becomes clear that the discrepancy is more apparent than real. Indeed, according to Sottocasa and co-workers (16), the icrespiratory chain-linked” rotenone-sensitive NADH cytochrome G reductase is concentrated in the heavy subfraction, which represents the inner membrane system, with some of the matrix contents retained. On the contrary, linked” rotenone-insensitive the “respiratory-chain-not NADH cytochrorne G reductase activity is concentrated in the light subfraction which consists of vesiculated derivatives of the outer membrane. The relative specific activity of rotenone-insensitive NADH cytochrome c reductase is much higher than the specific activity of the rotenonesensitive one. The latter activity is affected by training, since the increase observed at the 120th day is rotenone sensitive. The overall increase of the NADH cytochrome c reductase activity before the 120th day is negligible or nonexistant since the increase in the rotenone-sensitive activity was largely masked because it makes up only a little part of the total activity- On the whole, these observations seem to indicate that a high level of endurance training affects those portions of mitochondrial enzymatic activities which are bound to the respiratory chain. Other investigators (8, 10) have reported increases also in various mitochondrial matrix enzymes and in some extramitochondrial enzymes. Obviously, the evaluation of a few, or of a number, of enzymatic activities related to the energy transduction system is not very meaningful. The methods of determination of these activities totally disregard the regulative interactions which render the cell a functionally integrated system. Only a comprehensive study of biochemical correlations at a subcellular level could offer a complete and reliable picture. This being however quite a diiscult task, one should therefore try to get a dynamic view of the whole process starting from very specific situations. At any rate, in man (4, 17) an increase in succinic dehydrogenase specific of the athletes’ muscles subjected to a high work load was also observed. From this standpoint, the relations between the level and duration of endurance training on one side and the modifications of mitochondrial enzymatic activities on the other can be useful in establishing correlations which, though being far from absolute ones, can lay the foundations for a more detailed biochemical study of these processes.

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The chronic pharmacological treatment emphasizes at least four basic points. I) In the animals treated with some drugs (i.e., nicergoline, bamethan, dipyridamole) shorter training times are suf5cient to reach the values of mitochondrial enzymatic activities which are reached later in untreated animals. 2) Such pharmacodynamic action is not exclusively due to the peripheral vasodilating effect; for example, all tested drugs possess a vasodilating power, but papaverine proves totally unable to interfere with biochemical changes due to endurance training. 3) The pharmacological treatment does not modify the maximal level of activity increase due to a certain amount of work (e.g., 30 m/min for 180 min/day), performed over a suitable number of days (e-g., 120 days of training). 4) The drugs exert their action only if the enzymatic svsterns are activated by training, as shown by the fact that ‘they have no effect whatsoever on sedentary animals. The transition from a certain level of daily work load to a lower one leads to a decrease of the mitochondrial

enzymatic activities which had previously been stimulated by endurance training. This resul t emphasizes the relation between amount of training .X enzymatic and II litochondria activities. The modifications of mitochondrial enzymatic activities, induced by training, are therefore reversible. In the absence of continuous intense training these enzymatic xtivities drop to a lower level, thus adjusting to the new lower work load. This decrease is not inhibited by chronic treatment with the drugs tested. It is therefore evident that pharmacological factors can only partially and rather marginally influence the phenomena I;hich are at the basis of the relation between amount of training and enzymatic activity. The


UniversitA throughout Received


di Trieste-Italia) the period for





for his of experimental 13 May




helpful suggestions work.




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Mitochondrial enzymatic adaptation of skeletal muscle to endurance training.

JOURNAL Vol, OF APPLIED 38, No. 4, April ~'I~YS~OEOGY 1975. Printed Mitochondrial of skeletal in U.,S.A. enzymatic muscle adaptation to end...
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