Clinical Science (1973) 44, 547-554.
MITOCHONDRIAL VOLUME I N SKELETAL MUSCLE
FROM Y O U N G A N D O L D P H Y S I C A L L Y U N T R A I N E D
A N D T R A I N E D HEALTHY M E N A N D FROM
ALCOHOLICS
K.-H. K I E S S L I N G , L. PILSTRUM, J. KARLSSON"
A N D K A R I N PIEHL"
'
Institute of Zoophysiology, University of Uppsala, Uppsala and
*Department of Physiology, Gymnastik- och Idrottshogskolan, Stockholm, Sweden
(Received 20 December 1972)
SUMMARY
1. Using quantitative electron microscopy, the volume fraction of mitochondria has
been studied in the fibrillar and the perinuclear zones in leg muscle from young and
old healthy men, physically trained and untrained, and from alcoholics with
serious muscle weakness.
2. There is a correlation between the volume fractions of mitochondria in the
fibrillar and perinuclear zones.
3. In young untrained men, the volume fraction of mitochondriain skeletal muscle is
three-quarters of that in old untrained men in both the fibrillar and the perinuclear
zone. In the young men, there are few but comparatively large mitochondria, while
in old men the mitochondria are small but numerous.
4.Training causes a considerable increase in the volume fraction of mitochondria
in young men (approx. 100%) but only a slight increase in old men (approx. 20%).
The increase in the young group is caused by an increase in the number of mitochondria without any change of their volume, but in old men it is due to an increase in size.
5. Alcoholics with serious muscle weakness in their legs have a volume fraction of
mitochondria which is below that of the healthy untrained controls. The decrease is of
the same order as the increase induced by training in the old healthy men.
Key words : alcoholism, exercise, mitochondria, muscle.
Physical training will induce an enlargement of the mitochondrial mass in skeletal muscle in
animals (Gollnick, Ianuzzo & King, 1970) and man (Morgan, Cobb, Short, Ross & Gunn,
1970; Kiessling, Lundquist, Karlsson & Piehl, 1970). The activities of succinate dehydrogenase,
NADH dehydrogenase and cytochrome oxidase increased simultaneously (Morgan et al.,
1970), which indicates that the alterations of the structure are accompanied by corresponding
changes of mitochondrial function.
Correspondence: Professor K.-H. Kiessling, Institute of Zoophysiology, University of Uppsala, Box 560,
S-751 22 Uppsala, Sweden.
547
548
K.-H. Kiessling et al.
Very little is known about the relation between such changes and the age of the experimental
animal or person in training. Our previous study of young men (Kiessling et al., 1970) has
therefore been extended to include trained and untrained middle-aged men. These men were
also compared with a group of alcoholics of the same age suffering from serious muscle
weakness.
Several clinical forms of myopathy of different degrees of severity may be found in alcoholics
(Velez-Garcia, Hardy, Dioso & Perkoff, 1966; Perkoff, Dioso, Bleisch & Klinkerfuss, 1967;
Lafair & Myerson, 1968). In chronic myopathy, muscle weakness and diffuse muscle atrophy
are prominent, especially in the lower extremities. The pathogenesis of the disease is not known.
Regeneration processes have, however, been noted and abstinence results in a significant but
slow improvement (Perkoff et al., 1967; Buge, Autissier, Escourolle, Martin, Rancurel &
Bourdarias, 1967). A possible cause of the muscle weakness may be an inadequate energy
supply. Consequently, the purpose of this part of the study was to determine whether a decrease in the relative mitochondria1 volume occurs in the muscles of alcoholics who suffer from
serious muscle weakness.
MATERIALS AND METHODS
Four groups of male volunteers were studied. The first group included alcoholics suffering
from serious muscle weakness. The average age of the group was 57 years (range 36-73). The
second group included middle-aged healthy men with an average age of 57 years (range 56-59).
This group was divided into two subgroups, depending on the physical status as measured by
the maximum capacity for oxygen uptake, which was 34.0+1.5 ml of 0, min-' (kg body
weight)-' in the untrained group and 53.0k2.4 ml of 0, min-' (kg body weight)-' in the
trained group.
The third group included sedentary men of average age 22 years (range 20-23). These men
participated in a training programme which lasted for 28 weeks. Oxygen uptake was measured
and biopsies were taken at the beginning, in the middle and at the end of this training period.
The oxygen uptakes recorded on these occasions were 47.8 f1.0, 54-65 2 .1 and 55.25 1.0 ml
of 0, min-' (kg body weight)-' respectively.
The fourth group included volunteers (crack Swedish athletes) with a mean age of 24
years (range 22-28). Their maximum oxygen uptake was 71-4f 1.8 ml of 0, min-' (kg body
weight)-'.
With the aid of a biopsy technique, small pieces of the vastus lateralis muscle were taken
(Bergstrom, 1962). The pieces of tissue were fixed in ice-cold osmium tetroxide by the method
of Caulfield (1957), dehydrated in ethanol and embedded in Epon 812.
Sections approximately 60 nm (600 A) thick were prepared in a LKB Ultrotome, type 1,
stained with uranyl acetate and lead citrate and examined in an electron microscope (Akashi
Tronscope model TRS-50, El).
To obtain a good randomization, no orientations of the sections were made, but micrographs
were taken from longitudinal, oblique and transverse sections. Pictures were taken at random
from each section and the volume of mitochondria per cell volume (volume fraction, V,) was
estimated with the aid of a lattice, as described by Weibel (1969) and Sitte (1967).
Two different parts of the skeletal muscle cell are easily distinguished on the micrographs;
the fibrillar zone and the perinuclear zone. As there are different relative masses of mitochondria in the two zones (see below), they have been studied separately.
Effect of training on mitochondria in muscle
549
The results of the different groups were compared using Student’s t-test.
The application of the biopsy technique in the present investigation was approved by the
ethical committee of the Swedish Medical Research Council.
The formula for the estimation of number of particles per unit volume takes into consideration the mean caliper diameter and the shape coefficient (Weibel, 1969). None of these factors is
known, nor could they easily be estimated for the mitochondria of skeletal muscle, which makes
it impossible to estimate the true number of mitochondria per unit volume. This is also the case
as regards the average size of the mitochondria, as the size distribution is not known.
However, to get an idea of the number and size of the mitochondria in the skeletal muscle of
the different groups, the assumption was made that the distribution of the caliper diameter
and the shape coefficient (p) were equal in the six groups and the ratio K/pwas unity. By
counting the mitochondria1profiles per section area ( N J , the number of mitochondria per unit
were estimated from the equation
cell volume (Nv)
(a
Nv = J ( ~ W V )
The average volume of the mitochondrion in each group was estimated from the volume
fraction (Vv), divided by the number per unit cell volume (Nv).
RESULTS
Table 1 records the relative mitochondrial volumes in muscle from alcoholics and from healthy
trained and untrained men, both young and old. Two zones, the fibrillar and the perinuclear,
were studied.
TABLE
1. The volume fraction of mitochondria in the fibrillar and perinuclear zones of human skeletal muscle
and the maximum capacity for oxygen uptake. Each value denotes a mean value+_SEM. The values in parentheses indicate the number in each group. P1indicates the significance probabilities for the differences in relation
to the alcoholics. Pz denotes the significance probability for the differences between ‘Untrained‘ and ‘Trained’
within the groups ‘Old men’ and ‘Young men’ respectively. N.S., not significant.
Mitochondria1 volume (% of cell volume)
Fibrillar zone
Mean value2 SEM P1 Pz
Alcoh o1ics
Untrained old men
Trained old men
Sedentary young men
Young men after 14 weeks’
training
Young men after 28 weeks’
training
Athletes
C
+
Maximum oxygen
uptake [ml of O2
min-’ (kg body
Mean value+ SEM Pi PZ
weight)- ‘I
Perinuclear zone
-
-
2.40 0.21 (9)
2.82k0.47 (6)
3.3220.36 (6)
2-14+0.21 (15)
N.S.
0.05 N.S.
N.S.
7.850.9 (7)
11.3524 (6)
14.221.7 (6)
8.5 f 0.8 (11)
N.S.
34.0k1.5 (6)
0.01 N.S. 53.022.4 (6)
N.S.
47.8 1.0 (15)
3.1720-20 (15)
0.05 0.01
12.321.4 (11)
0.05 0.05 54622.1 (15)
4.62k0.27 (14)
7.38k0.63 (7)
0.001 0.001 18.0+0-7 (14)
0.001 - 25.6k2.4 (7)
0.001 0.001 55.2k1.9 (14)
0.001 - 71.4k1.8 (7)
+
K.-H. Kiessling et al.
5 50
In neither of the two zones was a statistically significant difference of the relative mitochondrial volume observed between the middle-aged and young untrained men and the alcoholics,
whereas all the trained groups differ significantly from the alcoholics. However, the difference
between middle-aged, trained and untrained men was not statistically significant (P = 0.40).
The relative mitochondria1 volume in both zones is significantly larger in the trained groups
compared with the alcoholics.
I-
I
0
I
I
I
5.0
I
10.0
I d x V o l u m e fraction of mitochondria in fibrillar zone
FIG.1. Correlation between the volume fractions of mitochondria in the fibrillar zone (abscissa)
and the perinuclear zone (ordinate) of skeletal muscle in six different groups of men. The regression line for the whole material is indicated by the thick line and the regression lines for each
group are indicated by the thin lines. Symbols are as follows: A, old healthy men; A, alcoholics;
0, young sedentary men; 0, young sedentary men after physical exercise for 14 weeks; m, young
sedentary men after physical exercise for 28 weeks; 0 , athletes. The cross (+) represents the
mean coordinates for the whole material. The volume fractions are given in pm3 mitochondria
per pm3 cell volume.
The volume fraction of mitochondria in the perinuclear zone was correlated with that in the
fibrillar zone (Fig. 1). The correlation coefficient (Y) is 0.848, the regression coefficient (b) 3.32
and the residual variance (S,". J 13.26.
The regression coefficients of the different groups (regression lines indicated by the thin lines
in Fig. 1) did not differ significantly from the regression coefficient of the whole material. The
correlation coefficients for the groups were as follows: alcoholics 0.19, middle-aged men 0.83,
sedentary young men 0.29, sedentary young men after training for 14 weeks 0.45, sedentary
young men after training for 28 weeks -0.12, and athletes 0.78.
Alcoholics
Untrained old men
Trained old men
Sedentary young men
Young men after 14
weeks’ training
Young men after 28
weeks’ training
Athletes
1.27f0.15
- 21.1 f3.2
1.35k0.24 N.S.
245k6.3
1.36f0.25 N.S. N.S. 28.7f6.4
0.59+0.08 0.001
37.9f 4.2
41.3f6.9
0-88fO.11 0.05 0.05
1-26+0.10 N.S. 0.001 39.4f3.7
1.23f0.13 N.S. - 62.425-3
15
14
7
N.S.
0.01 N.S.
0.001 -
0.01
N.S.
N.S. N.S.
0.01
-
lo3x Average volume
of mitochondrion @m3)
Mean+SEM PI Pz
9
6
6
15
No. of No. of mitochondria
subjects
per m3
MeanfSEM PI Pz
Fibrillar space
14
7
11
7
6
6
11
4.69k0.48 0.05 0.005 43.8k4.5
4.65f0.46 0.05 - 59.3f7.8
3*70+076 N.S. N.S. 392k5.1
3.33f0.39
- 23*8+1.8
5*11+0*76 N.S.
24-2k6.3
5.62k1.56 N.S. N.S. 32.626.9
266k0.36 N.S.
35.5 f 3.9
u-
0.001 N.S.
0.001 -
N.S.
2
2
33
s
3
- s0
N.S.
N.S. N.S.
0.01
0.01
3
0.3
2-
8.
\
7
2
9
m3) 3
s
No. of No. of mitochondria
lo3x Average volume
subjects
per m3
of mitochondrion
MeanfSEM PI Pz MeanfSEM PI PZ
Perinuclear space
TABLE
2. The number and average volume of mitochondria in the fibrillar and perinuclear zones of human skeletal muscle. The numbers of mitochondria per unit volume (Nv) were estimated from the data of Table 1 by using the formula of Weibel(l969): NV = x ,/(Ni/ Vv)where Na is the number of mitochondria1 profiles per section unit area, Vv is the volume fraction of mitochondria, K is the distribution of the caliper diameter, and /3 is the
shape coefficient. The assumption was made that K//3 = 1 in all groups, which means that the values in the table are not absolute but relative. The
average volume of the mitochondrion was estimated as 7
= Vv/Nv which means that these values also are relative. The values in the table denote
mean values+ SEM. PIindicates the significance probabilities for the difference in relation to alcoholics and PZdenotes the significance probability for
the differences between ‘Untrained’ and ‘Trained’ within the groups ‘Old men’ and ‘Young men’ respectively. N.S., not significant.
552
K.-H. Kiessling et al.
In the young men, moderate training for 28 weeks causes a more than 100% increase of the
mitochondrial volume fraction in both zones (Table 1). This is, however, still only about 60-70
% of the volume fractions observed in the athletes.
In Table 2 the apparent number of mitochondria per cell unit volume and their average size
are shown in the different groups. Untrained middle-aged men have about twice as many
mitochondria as sedentary young men. Training causes a considerable rise in the number in
young men but not in middle-aged men. No change in mitochondrial size could be observed in
the fibrillar zone of young men during training and only a slight one in the older group.
The muscles of alcoholics suffering from pronounced muscle weakness contain slightly
smaller mitochondria than those of untrained healthy men of the same age.
DISCUSSION
The aim of the present muscle study was threefold: to establish how the mitochondrial capacity
(expressed as the volume fraction of mitochondria) changes with age, training at different
ages and finally, prolonged alcohol consumption followed by serious muscle weakness.
Two zones of the muscle cell, the fibrillar and the perinuclear, have been studied. A main
function of the mitochondria in the fibrillar zone is obviously to supply energy for contraction
as they are located in direct contact with the contractile elements of the muscle. Physical
exercise induces a larger mitochondrial mass not only in the fibrillar part of the skeletal muscle
but also in the perinuclear part. The mitochondrial masses of the two zones seem to be generally
correlated.
As very little is known about the purpose of the mitochondria in the perinuclear zone, it is
difficult to give an exhaustive explanation of the above results. One possibility is that the
energy produced in the perinuclear zone is used, at least partly, for protein synthesis and other
endergonic processes in this area.
The shapes of the mitochondria are different in the two areas (Fig. 2). In the perinuclear
zone, they are generally round or oval. In the fibrillar zone they are very irregular, often
encircling part of the fibril. This raises the question whether the increase in number of mitochondria per unit cell volume is a true phenomenon or is caused by an irregular increase in
size with the ensuing chance of cutting the same mitochondrion twice.
The round or oval shape of the mitochondria in the perinuclear zone precludes the possibility of cutting the same mitochondrion twice. The values for the number of mitochondria per
unit cell volume in this zone are therefore true values. The possibility of cutting several
mitochondria twice in the fibrillar zone cannot, as stated above, be excluded. The fact, however,
that a rise of the number of mitochondria in the perinuclear zone parallels an equal rise in the
fibrillar zone, strongly suggests that there is an increase in number rather than an increase in
size even in the fibrillar zone.
In middle-aged men this question is of no importance as the relative mitochondrial volume
(Table 1) as well as the number of mitochondria per unit cell volume (Table 2) show no difference between trained and untrained men. In young men, however, both relative volume (Table
1) and number of mitochondria (Table 2) increase markedly during training.
Effect of age
The volume fraction of mitochondria is lower in young men than in middle-aged men in
Efect of training on mitochondria in muscle
FIG.2. Electron micrographs from human skeletal muscle showing irregular-formed mitochondria
in the fibrillar zone (a) and oval-shaped mitochondria in the perinuclear zone (b). Magnification
x 24 000.
(Facing p . 552)
Efect of training on mitochondria in muscle
553
both the fibrillar and the perinuclear zone (Table 1). The differences are not remarkable. If the
volume fraction is taken as a measure of the mitochondrial capacity of the muscle, the results
indicate that this capacity is about the same in the two groups. The results in Table 2, however,
indicate that this volume fraction is maintained in different ways in the two groups. In young
men, there are few but comparatively large mitochondria, whereas middle-aged men have a
larger number of rather small mitochondria. Theoretically, this might mean that enzyme
activities bound to the surface of the mitochondria are more pronounced in middle-aged than
in young men, a hypothesis which will have to be investigated further. Observations which
support this have been obtained from studies of liver mitochondria (Pilstrom & Kiessling,
1972; Reith, 1972).
Effect of training
In Tables 1 and 2, the results from young and middle-aged, untrained and trained, men can
be seen. The two groups are not completely comparable, as in the young groups the same men
were studied before and after a training programme, whereas the older men consist of two
groups, untrained and trained. The maximum oxygen consumption was rather low in the two
untrained groups in comparison with the high values in the trained groups, indicating about
the same work capacity in the two situations.
Training has a very different effect on the volume fraction of mitochondria in the two agegroups. Young muscle responds by increasing the mitochondrial mass by more than 100%
within 28 weeks (Table 1). In older muscle, the corresponding increase is only about 20%. The
results in Table 2 indicate that the considerable increase in the young men is entirely caused by
an increase in the number of the mitochondria, whereas in old men there is an increase in the
size of the mitochondria.
The reason for this is difficult to explain. It may be that once a muscle has been induced to
increase its number of mitochondria, this number will not be decreased when training is no
longer maintained. The decrease of the mitochondrial fraction will only be possible by a
decrease of the volume of the individual mitochondria.
Effect of prolonged alcohol consumption
In the alcoholics, who are mainly old men (mean age 56, compared with 57 for the old
healthy men), the mitochondrial volume fraction is slightly lower than in healthy untrained
men of the same age (15% in the fibrillar zone and 31 % in the perinuclear zone). This may seem
negligible, until we compare it with the corresponding differences between the untrained and
the trained men (15% and 28% respectively).
If training, which causes an increase in the maximum oxygen uptake from 34 to 53 ml
min-' kg-l, is the entire cause of the increase in mitochondrial volume fraction, a corresponding decrease of the volume fraction in the alcoholics below that of the untrained men may
very well be the reason for the symptoms of serious muscle weakness observed in these patients
ACKNOWLEDGMENTS
We are indebted to Miss Gun-Britt Jonsson for skilful technical assistance. This work was
supported financially by the Research Council of the Swedish Sports Federation (Grant No.
IFR 71 :37,22), and by the Swedish Medical Research Council (Grant No. B 73-12Y-236406).
554
K.-H. Kiessling et al.
REFERENCES
J. (1962) Muscle electrolytes in man determined by neuron activation analysis on needle biopsy
BERGSTROM,
specimens. Scandinavian Journal of Clinical and Laboratory Investigation, 14, Suppl. 68.
BUGE,A., AUTISSIER,R., BCOUROLLE,
R., MARTIN,M., RANCUREL,
G. & BOURDARIAS,
H. (1967) Myopathie
myoglobinusinque chez un alcoolique. Sociiti Midicale des Hdpitaux de Paris, 118,615-623.
CAULFIELD,
J.B. (1957) Effect of varying the vehicle for Os04 in tissue fixation. Journal of Biophysical and
Biochemical Cytology, 3, 827-830.
GOLLNICK,
P.D., IANUZZO,
C.D. & KING,D.W. (1970) Ultrastructural and enzyme changes in muscles with
exercise. Muscle Metabolism during Exercise, pp. 69-86. Ed. by Pernow, B. & Saltin, B. Plenum Press, New
York and London.
KISSLING,K.-H., LUNDQUIST,C.-G., KARLSSON,
J. & PIEHL,K. (1970) Effect of physical training on ultrastructural features in human skeletal muscle. Muscle Metabolism during Exercise, pp. 97-101. Ed. by
Pernow, B. & Saltin, B. Plenum Press, New York and London.
LAFAIR,J.S. & MYERSON,
R.M. (1968) Alcoholic myopathy with special reference to the significance of creatine
phosphokinase. Archives of Internal Medicine, 122,417-422.
MORGAN,
T.E., COBB,
L.A., SHORT,F.A., Ross, R. & Gu", D.R. (1970) Effects of long-term exercise on human
muscle. Muscle Metabolism during Exercise, pp. 87-96. Ed. by Pernow, B. & Saltin, B. Plenum Press, New
York and London.
PERKOFF,
G.T., DIOSO,M.M., BLEISCH,V. & KLINKERFUSS,
G. (1967) A spectrum of myopathy associated with
alcoholism. I. Clinical and laboratory features. Annals of Internal Medicine, 67,481492.
PILSTR~~M,
L. & KIESSLING,
K.-H. (1972) A possible localization of a-glycerophosphate dehydrogenase to the
inner boundary membrane of mitochondria in livers from rats fed with ethanol. Histochemie, 32,329-334.
REITH, A. (1972) Intramitochondrial localization of glycerol phosphate dehydrogenase. A possible marker
enzyme for the proliferation of mitochondria. Cytobiologie, 5, 384-396.
S m , H. (1967) Morphometrische Untersuchungen an Zellen. Quantitative Methods in Morphology, pp. 167198. Ed. by Weibel, E.R. & Elias, H. Springer, Berlin.
VELEZ-GARC~A,
E., HARDY,P., DIOSO,
M. & PERKOFF,
G.T. (1966) Cystinestimulated serum creatine phosphokinase: Unexpected results. Journal of Laboratory and Clinical Medicine, 68,636-645.
WEIBEL,E.R. (1969) Stereological principles for morphometry in electron microscopic cytology. International
Reviews in Cytology, 26, 235-302.