Clinical Science (1984) 66, 317-322
317
Experimental mouse muscle damage: the importance of
external calcium
D . A. J O N E S , M . J . JACKSON, G . McPHAIL
AND
R. H. T. EDWARDS
Department of Medicine, University College London, The Rayne Institute, London
(Received 28 Miwchl.5 August 1983; accepted 16 September 1983)
Summary
1. The involvement of extracellular calcium in
experimental muscle damage has been studied in
an isolated mouse soleus muscle preparation.
2. The enzyme efflux and ultrastructural
damage seen after excessive contractile activity
were markedly reduced when the extracellular
calcium was withdrawn. Low extracellular calcium
also protected against the large enzyme efflux
seen after treatment with low concentrations of
detergent.
3. Treatment of the muscle with the calcium
ionophore A 23 187 caused significant release of
enzyme from the muscle.
4. Nifedipine did not prevent the enzyme
release after stimulation and although in some
circumstances verapamil appeared to have some
protective effect this was probably due to a local
anaesthetic action on the muscle and not to any
specific effect on calcium movement.
5. It is concluded that extracellular calcium is
important in mediating at least the two forms of
muscle damage studied here.
Key words: calcium, enzyme efflux, muscle
damage, skeletal muscle.
Introduction
Circumstantial evidence suggests that accumulation of calcium within the muscle fibre may play
an important role in the degeneration seen in
many of the destructive myopathies. Raised
Correspondence: Dr D. A. Jones, Department
of Medicine, University College London, The
Rayne Institute, University
WClE 6JJ.
Street,
London
intracellular levels of calcium have been described
in Duchenne muscular dystrophy [ l , 21 and
experiments with the calcium ionophore A 23187
and caffeine have demonstrated that raised intracellular levels of calcium can lead to disruption of
the internal structure of the muscle fibre [3].
We have recently described an isolated mouse
skeletal muscle preparation that can be used to
examine the factors involved in muscle damage
leading to the release of intracellular enzymes (41.
This system has been used to demonstrate that
enzymes may be released after severe metabolic
stress (caused by contractile activity or metabolic
poisons) or after treatment with detergent. In the
present work we have examined the possible
role of external calcium in mediating the release of
enzyme under these circumstances.
Methods
Muscle preparations
Female white mice, maintained on a standard
laboratory diet (41B, E. Dixon and Sons Ltd,
Herts., U.K.), were killed by an intraperitoneal
injection of pentobarbitone and the soleus muscles
rapidly removed. The muscles were mounted in
special holders and incubated in 2.5 ml of bicarbonate buffered Ringer maintained at 37'C.
The apparatus, experimental conditions and
reagents were all as previously described [4]with
the one modification that all of the holders were
equipped with electrodes, so that all four muscles
could be simultaneously stimulated during the
treatment period. In practice the muscles were
arranged so that, of the two muscles from one
mouse, one was used in the test treatment group
and one in the control, ensuring that the groups
318
D. A . Jones et al.
were of similar composition with respect to muscle
size, age and history of the animal etc.
After an initial 30 min incubation period the
medium was exchanged, and during the treatment
period the muscles were either stimulated (50 Hz
for 0.5 s every 5 s) or agents added to the medium.
After 30 min the medium was exchanged and this
was repeated every 30 min for the next 3 h.
The bicarbonate Ringer solution normally contained 2mmol of calcium chloride/l. In experiments involving calcium free medium the calcium
chloride was simply omitted from the Ringer so
that the calcium was less than 10 pmol/l as
measured by atomic absorption spectrometry.
Just before the change the muscle and incubation
tube were rinsed with 2.5 ml of calcium free
medium, which was removed, discarded and replaced with fresh calcium free medium.
Where the muscles were stimulated under
hypoxic conditions this was achieved by replacing
the gas mixture with N,/C02 (95 : 5, v/v). At all
other times the medium was gassed with Oz/COz
(95 :5, v/v).
Lactate dehydrogenase activity (LDH) was
measured in the incubation medium and in the
muscle homogenates as previously described [4 1.
Muscle morphology
At the end of the experiment some muscles
were Tied in phosphate buffered glutaraldehyde,
post-fmed in osmium tetroxide, and embedded in
Araldite. For light microscopy semi-thin longitudinal sections (0.75 pm) were stained with
0.25% toluidine blue. For electron microscopy
ultra-thin sections (60-90 nm) were stained with
uranyl acetate and lead citrate and examined with
a Phillips E.M. 200 electron microscope.
Reagents and drugs
Verapamil hydrochloride was obtained from
Abbott Laboratories and nifedipine from Bayer
UK Lid. The calcium ionophore A 23187 was
obtained from Sigma Chemical Co., as were
enzymes and cofactors used in the assay of lactate
dehydrogenase. All other reagents were of AnalaR
grade.
Results
Electrical stimulation
Muscles were stimulated under hypoxic conditions during the treatment period, either in
normal medium or in calcium free medium from
No external CaCl,
CaCI. ( 2 mmol/l)l
--
I
-30 0 30 60 90 120 1 5 0 180
Time after end of treatment (min)
No external CaCl,
CaCI, ( 2 m
m-
CaCI, ( 2 mmol/l)
(b1
I
-30
0
30 60 90 120 1 5 0 180
Time after end gf treatment (rnin)
FIG. 1. Enzyme release from stimulated muscles
in the presence and absence of external calcium.
Muscles were stimulated under hypoxic conditions during the treatment period. ( a ) Muscles
were incubated in the presence of CaCl, throughout the experiment ).( or in calcium free medium
from the start of the treatment period (+). ( b )
Muscles were incubated and stimulated as before,
except where the calcium free medium was present
only during the treatment period, returning t o
CaC1, for the remainder of the experiment (+).
Results are given as the means f SEM for four
muscles.
the beginning of the treatment period and for
the remainder of the experiment.
Stimulation under hypoxic conditions gave rise
to a rapid loss of force and development of a
contracture tension (as previously described
[4]). The initial force generated and the subsequent time course were very similar whether the
muscles were in calcium free or calcium containing medium.
Enzyme release as a result of stimulation was
markedly reduced in the absence of extracellular
calcium chloride. The maximum rate of efflux
was less than one-thud of that seen in the presence
of 2 mmol of calcium chloride/l) (Fig. la). In a
separate experiment removal of calcium for the
duration of the treatment period was found to be
just as effective in reducing the enzyme efflux,
even at times when the calcium had been restored
Muscle damage and calcium
to normal (Fig. l b ) . Removing the calcium after
the end of the treatment period had no protective
effect. Although the calcium free medium protected against enzyme release there was no preservation of the contractile properties of the muscle.
At the end of the experiment muscles were
taken for microscopy. Light microscopy examination of frozen cross-sections showed no obvious
abnormalities but when longitudinal semi-thin
sections, stained with toluidine blue, were
examined they showed extensive areas of vacuolation within the fibres in the muscles that had been
stimulated in normal medium but none such were
seen in muscles stimulated in calcium free medium
319
(Fig. 20). On electron microscope examination the
damage in the muscles stimulated in medium of
normal calcium content was seen to display
vesiculation of the sarcoplasmic reticulum,
swelling and disruption of the mitochondria and
loss of Z line material (Fig. 2b).
Detergent treatment
Treatment of the muscle with the detergent
deoxycholate (DOC) at 200 pmol/l without
stimulation resulted in no release of enzyme. At
5M)pmol/l there was a rapid and large release
(Fig. 3) in normal medium. When detergent was
(6)
FIG.2. Ultrastructural changes in muscles stimulated in the presence and absence of
external calcium. Muscles were incubated and treated as in Fig. I(a) and fixed at the
end of the experiment. ( a ) Semi-thin sections stained with toluidene blue: left, muscle
incubated in normal calcium medium throughout; right, muscle in which the medium
was changed to calcium free from the start of the treatment period ( X 480). ( b )
Electron micrographs of ultra-thin sections of the blocks shown above: left, muscle
incubated in normal medium throughout; right, the medium was changed to calcium
free from the start of the treatment period ( X 19 000).
D. A. Jones et al.
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FIG. 5. Enzyme release and the effects of calcium
antagonists. ( a ) Muscles were stimulated under
hypoxic conditions during the treatment period, in
the absence ( 0 ) or in the presence (6)of nifedipine
(25 pmol/l) present from the start of the treatment period and for the rest of the experiment.
Muscle damage and calcium
fact that the verapamil slightly depressed the
contractile activity of the muscle. The initial
tension was reduced to 70% and whereas the
control muscles maintained about 10% of the
initial force after 5 min of fatiguing stimulation,
the verapamil treated muscles were inexcitable.
321
was occurring during the stimulation period and
that this initiated the subsequent release.
Detergent treatment of the muscles released
large quantities of enzyme with a rapid time
course (Fig. 3). We had assumed that the efflux
in these circumstances was due to enzyme passing
though lesions in the surface membrane caused
by the detergent [4]. It was therefore surprising
Discussion
to find that at the lower concentration of DOC
The results presented here show that external
(SOOpmol/l) the efflux was sensitive to the
calcium plays an important role in the release of
presence of external calcium (Fig. 3). The conenzymes from experimentally damaged mouse
clusion must be that at the low concentrations of
muscles. Reducing the external calcium concen- DOC there may be some similarities between the
tration protected against enzyme loss both when
damage caused by contractile activity and dethe damage was initiated by contractile activity tergent. Possibly small lesions in the surface
and after treatment with detergent, suggesting that
membrane allow the inward passage of calcium,
they may share a common mechanism.
which initiates more extensive damage leading to
The protective effect of reducing the external the large and rapid enzyme efflux.
calcium could be because the export of soluble
Experiments with the calcium ionophore
enzyme is a calcium dependent process. It has
A 23 187 have further demonstrated the imporbeen reported that at h g h concentrations of
tance of calcium in initiating muscle damage,
external calcium there is an increased efflux of
although the critical calcium could come from the
creatine kinase from resting skeletal muscle [S],
external medium or be released from the sarcobut it is unlikely that this mechanism was
plasmic reticulum. Duncan and co-workers [3, 61
responsible for the protective effect of low have described both of these changes.
calcium in the present studies. In the work of
The evident importance of external calcium in
Soybell et al. [5] changes in efflux were only
the two forms of muscle damage discussed here
significant when the concentration of calcium was
suggests that an agent that prevented calcium
varied between about 3 and 10 mmol/l; in our
movements might be a valuable means for prework the concentration was never greater than
venting the damage. Although skeletal muscle
2 mmol/l. In addition no changes were seen in the
does not have the same requirements for external
small amount of enzyme efflux from control, un- calcium as smooth and cardiac muscle, it is known
stimulated muscles when the external calcium that there are calcium channels in the surface
concentration was changed from 2 mmol/l to less membrane which may be blocked by nifedipine
than 10 pmolll. More likely is the explanation that
and other calcium antagonists [7, 81. Nifedipine
removing the external calcium prevents the
has been shown to block calcium currents in
damage that leads to enzyme efflux, as witnessed skeletal muscle at a concentration as low as 0.3
by the reduction in ultrastructural changes seen in pmol/l 171, but at 25 pmol/l in the present work
the thin arid ultra-thin sections of the muscle there was no protective action on enzyme release
(Fig. 2).
after stimulation (Fig. 50). There was a suggestion
Despite the significant protection afforded by
that v e r a p d may have some protective effect
the low extracellular calcium with regard to (Fig. 5a) but the interpretation of this result is
enzyme release and ultrastructural damage it is complicated. At the concentration used (50
notable that there was no preservation of the
pmolll) verapamil has a mild anaesthetic action on
contractile properties of the muscle, Low calcium skeletal muscle [9]. Stimulation under hypoxic
conditions minimizes the consequences of this
in the external fluid by itself causes a loss of
action but the metabolic cost of the fatiguing conexcitability over the course of 30 min, probably
by altering the characteristics of the outer mem- tractions may still have been less than that for the
branes rather than depleting the sarcoplasmic control muscles. In other experiments in which the
muscles were poisoned with cyanide and iodostores of calcium (S. J. Mayor & D. A. Jones,
acetate so that the depletion was achieved quite
unpublished observations).
Reducing the extracellular calcium was independently of any electrical activity (see Jones
effective only during the period when the muscle et al. [4]) verapamil was without any protective
was stimulated (Fig. la, lb). This suggests that
effect. On balance it appears that the calcium
although the peak enzyme efflux did not occur
antagonists which are effective with cardiac and
until some 60-90 min after the end of stimula- smooth muscle do not prevent damage to skeletal
tion, some event, probably the entry of calcium, muscle. This may be because in the fatigued
322
D. A . Jones et al.
muscle calcium enters the fibres by a route which
is separate from the normal calcium channel.
We have shown here that two forms of experimental damage, that occurring after exhaustive
contractile activity and after detergent treatment,
are both dependent in some way on the presence
of external calcium. It seems most likely, but is
not yet proven, that in the fatigued muscles
calcium enters the muscle fibres where it initiates
damage leading to the release of cytosolic
enzymes within the fibre. It may be taken up by
mitochondria, overloading them and inhibiting
ATP synthesis, thus exacerbating the metabolic
problems of the fibre [lo]; calcium may activate
the neutral protease [ l l ] or may lead to protein
degradation by a mechanism involving prostaglandin E, [12]. Calcium will also activate the
phospholipase A2 [13], giving rise to an accumulation of free fatty acids and lysophospholipids. All
of these will result in damage to the muscle
fibre.
Calcium entry has been identified as the
common pathway leading to cell death in hepatocytes treated with a number of different toxic
agents [14] and has also been implicated in the
damage caused to individual muscle fibres after
micropuncture lesions [ 151.
Although our work has been with an isolated
animal muscle preparation, the fact that external
calcium has been shown to be important in two
types of damage of quite dissimilar origin does
suggest that this may represent some general
mechanism of damage that may be active in
pathological conditions.
Acknowledgments
We are grateful to Ms C. Forte for excellent
technical help and the Muscular Dystrophy Group
of Great Britain for financial support.
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