Etruscan shrew muscle: the consequences of being small
Zentrum Physiologie, Medizinische Hochschule, D-30623 Hannover, Germany
* e-mail: Juergens.KlausD{at}MH-Hannover.de
Accepted 13 May 2002
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Summary |
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Key words: Etruscan shrew, Suncus etruscus, skeletal muscle, extensor digitorum longus, soleus, fibre composition, myosin heavy chain, myosin light chain, lactate dehydrogenase, citrate synthase, creatine kinase, myoglobin, Ca2+ transient, contraction, relaxation
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Introduction |
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Because of its large surface-to-volume ratio, the shrew's energy turnover
is extraordinarily high. Under thermoneutral conditions (ambient temperature
35°C), its rate of oxygen consumption is 100 ml kg-1
min-1. At 22°C, a rate of 270 ml kg-1
min-1 has been recorded (Fons
and Sicart, 1976) and a maximal value of 1000 ml kg-1
min-1 (Weibel et al.,
1971
; Jürgens et al.,
1996
) has been estimated. These values are 25, 67 and 250 times
higher than those of humans at rest. Although usually homeothermic, the
species undergoes torpor cycles during times of food restriction and at low
ambient temperatures.
Because muscles play a major role in the capture and chewing of food, in
convective oxygen transport and in the generation of heat, they must be
especially adapted to provide the enormous mass-specific rate of energy
consumption of the body. In earlier studies investigating the properties of
the blood and circulation, it was found that S. etruscus has a large
relative heart muscle mass, 1.2% of its body mass
(Bartels et al., 1979), a value
twice as high as expected from allometry, and a heart rate of up to 1511 beats
min-1 (25 s-1), which exceeds all values reported for
other endotherms (Jürgens et al.,
1996
). The skeletal muscles of S. etruscus are also able
to contract very rapidly: a respiratory rate of up to 900 min-1 (15
s-1) was measured, a stride frequency of 780 min-1 (13
s-1) is estimated from an allometric equation given by Heglund and
Taylor (1988
) and, during cold
tremor, Kleinebeckel et al.
(1994
) observed
electromyographic (EMG) frequencies of up to 3500 min-1 (58
s-1). Although the relative amount of brown adipose tissue is
higher in S. etruscus (on average 9.2% of body mass) than in any
other mammal, shivering has been shown to be important for rapid heat
production and occurs during rewarming from torpor at body temperatures above
17°C (Fons et al.,
1997
).
In a recent study, the contraction parameters, myosin composition and
activity of the metabolic enzymes of the skeletal muscles of S.
etruscus have been investigated and compared with corresponding
properties of larger shrews and other larger mammals
(Peters et al., 1999). In
larger mammals, the soleus and the extensor digitorum longus (EDL) muscles are
typical representatives of slow-twitch and fast-twitch muscles, so they were
chosen to be functionally and structurally investigated in the shrew. In some
cases, other skeletal muscles, such as the diaphragm and gastrocnemius muscle,
were also studied.
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Materials and methods |
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For the contraction measurements, the muscles were mounted in a measuring
chamber and completely submerged in carbogen-equilibrated
KrebsHenseleit solution. To record the twitch contractions, a force
transducer based on a semiconductor sensor device AE 802 (SensoNor a.s.,
Horten, Norway) was developed (Peters et
al., 1999). Ca2+ transients of single twitches of
muscle bundles containing 15-30 fibres were measured fluorometrically using a
microscope photometric apparatus and the fluorescent dye Fura2. The Fura2
technique is described in detail by Wetzel and Gros
(1998
).
Electrophoresis of myosin heavy chain isoforms was carried out for 18 h at
4°C according to the method of Kubis and Gros
(1997) with minor
modifications. Two-dimensional electrophoresis of myosin light chains was
performed using the method of O'Farrell
(1975
). Proteins of muscle
homogenate supernatants were separated by SDSPAGE using slabs with a 5%
stacking gel and a 15% separating gel and visualized by silver staining.
Muscle fibre type identification was performed after the method of Brooke and
Kaiser (1970
).
Immunohistochemical studies with monoclonal and polyclonal antibodies are
described in detail by Peters et al.
(1999
). Lactate dehydrogenase
(LDH) activity was measured according to the method of Bernstein and Everse
(1975
), and citrate synthase
(CS) activity was measured using the method of Bass et al.
(1969
). The specific activities
of these enzymes are presented per milligram of cytoplasmic protein instead of
per milligram wet mass of tissue because the tissue mass of the tiny muscles
changes critically depending on the amount of water adhered to the muscle
surface, either from the rinsing solution (fresh muscles) or from condensed
water vapour (frozen muscles). The concentration of myoglobin was measured
according to the method of Reynafarje
(1963
).
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Results and Discussion |
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Time to peak force is not significantly shorter in the EDL than in the
soleus muscle, whereas the times to 50%, 75% and 90% relaxation are all
significantly shorter in the EDL than in the soleus muscle
(Table 1). This difference was
confirmed by force measurements on fibre bundles from these muscles, which
were performed together with recordings of the Ca2+ transients
(Wetzel and Gros, 1998). Here,
both time to peak force and times to 50% and 75% relaxation were significantly
shorter in the EDL than in the soleus muscle, and the rise and decay of the
cytosolic Ca2+ concentration were significantly faster in EDL
muscle (Table 2).
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To reveal the reasons for the functional difference between these two muscles, we investigated their fibre composition, their composition of myosin heavy and light chains and the activity of two enzymes important for ATP generation, lactate dehydrogenase and citrate synthase.
Myosin chain composition
Histochemical staining for myosin ATPases revealed identical fibre type
patterns in the EDL and soleus muscle. All fibres showed the same degree and
pattern of staining, indicating the presence of only alkaline-resistant myosin
ATPase, which is typical of type II fibres. Immunochemistry using monoclonal
antibodies against type I and type II myosin heavy chains also revealed only
the presence of heavy chain type II in all leg muscles. In addition,
SDSPAGE and two-dimensional electrophoresis of EDL and soleus muscles
showed no difference in the pattern of myosin heavy chains and myosin light
chains. Only one type of heavy chain is found in both muscles, and the same
holds for the diaphragm muscle of S. etruscus. This heavy chain type
was identified as the isoform HCIId by comparison with myosin electrophoresis
from rabbit muscles (Fig. 2).
The same two types of fast light chains, fLC1 and fLC2, with molecular masses
of 24 and 21.5 kDa, were detected in the EDL and soleus muscle
(Fig. 3). Moreover, in a
mixture of leg muscles from the Etruscan shrew, only these fast light chains
were detected.
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In contrast to larger mammals, no slow-twitch type I fibres are present in the EDL and soleus muscle of S. etruscus. As shown in Table 3, the number of type I fibres in soleus muscles decreases and the percentage of fast-twitch fibres increases with decreasing body mass in a range of mammals. Shrew muscles, in general, seem to be composed of type II fibres only but, within this family, there is a correlation between body mass and HCII-subtype composition (Table 4). The number of type IIA fibres decreases and the number of IID fibres increases with decreasing body mass, leading to the unique situation that all the muscles of the smallest mammals are probably composed solely of type IID fibres. Nevertheless, as shown by the contraction measurements (Tables 1, 2), there are functional differences between different muscles.
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Activity of metabolic enzymes
Can the functional difference between the EDL and soleus muscles of S.
etruscus be attributed to different activities of the glycolytic and the
oxidative metabolic pathway? Measurements of the activities of lactate
dehydrogenase (LDH) and citrate synthase (CS) revealed no significant
difference in activities between the two muscles
(Table 5), suggesting that the
functional difference between the EDL and soleus muscles cannot be explained
by different activities of CS and LDH. The gastrocnemius muscle also did not
differ from the EDL and soleus with respect to the activities of these
enzymes. Moreover, the specific activity of CS turned out be higher in the
muscles of S. etruscus than in those of other mammals, whereas the
activity of LDH was much lower than in larger species. The ratio of LDH/CS
activity, given in Fig. 4 as a
function of body mass, is almost 1 in the smallest mammal, a value that is
smaller than in any other mammal.
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The measured enzyme activities indicate that the function of the type IID
fibres depends almost entirely on oxidative ATP production; the activity of
the glycolytic pathway appears to be negligible. The importance of oxidative
metabolism is also underlined by the finding of a very high capillary density
of up to 2800 mm-2 in the soleus muscle of the Etruscan shrew
(Pietschmann et al., 1982) and
a very high volume fraction of mitochondria (0.23 in leg muscles, 0.35 in
diaphragm) (Hoppeler et al.,
1981
). Moreover, because of the foldings, the surface area of the
inner mitochondrial membranes is considerably larger in the shrew than, for
instance, in man (Bartels,
1980
). All Etruscan shrew muscles studied to date are also uniform
with respect to their myoglobin content, which is approximately 150 µmol
l-1.
Specialization of Etruscan shrew muscles
From the results of the myosin studies and a consideration of energy
metabolism, it can be concluded that oxidative fast-type IID fibres are most
appropriate to meet the demands of the smallest mammal. The lighter the
species, the less important are slow-twitch types of fibre, which constitute
muscle specialized for static contractions to maintain posture. Gravitational
forces, which are proportional to body mass, decrease with decreasing body
size, and the locomotory behaviour of the smallest mammals reveals that moving
is much more important for them than standing.
Type IID fibres combine a high shortening velocity which, according to
studies in rats is highest in type IIB fibres, a little lower in type IID
fibres and considerably lower in type IIA and I fibres, and a low fibre
diameter, which is lowest in type I and IIA fibres, a little larger in type
IID fibres and largest in type IIB fibres
(Galler et al., 1994).
Provided that the fibre properties found in the rat also hold for shrews, type
IID fibres seem to be an appropriate compromise between a high ATP turnover,
which is required not only for physical performance but also for heat
production, and a small fibre diameter which, together with a high capillary
density, enables a high oxygen flux into the fibre and, hence, a high rate of
ATP production, as a result of the short diffusion distance for oxygen between
the blood and the mitochondria.
The importance of the oxidative metabolic pathway is supported by the
capacity of the shrew's type IID fibres to store oxygen bound to myoglobin.
The myoglobin concentration is intermediate: type I and IIA fibres are known
to have a myoglobin concentration higher than 150 µmol 1-1, and
type IIB fibres are free of myoglobin in all mammals studied so far. The
activity of metabolic enzymes leads to the conclusion that aerobic metabolism
may meet any level of energy demand by the animal so that glycolytic
metabolism is not necessarily required for ATP production. The glycolytic
metabolic pathway may be underdeveloped because, compared with the enormous
mass-specific energy requirements of the organism (its rate of oxygen
consumption can rise to 1000 ml O2 kg-1
min-1) (Jürgens et al.,
1996), the amount of ATP that can be produced glycolytically
during an oxygen debt would be almost negligible even at significantly higher
levels of LDH activity.
Reasons for the functional differences between the EDL and soleus
muscles of Suncus etruscus
Despite the uniformity of Etruscan shrew muscles with regard to their
myosin composition and their metabolic enzyme activities, differences in
contraction times and the kinetics of Ca2+ transients have been
observed. The rate at which Ca2+ is released from the sarcoplasmic
reticulum and subsequently resequestered is significantly higher in the EDL
than in the soleus muscle of S. etruscus
(Table 2). A shorter relaxation
time could be the result of a higher concentration of parvalbumin in the EDL,
but immunohistochemical investigations have provided no evidence for the
presence of parvalbumin in shrew muscles
(Peters et al., 1999). A
larger volume fraction of sarcoplasmic reticulum and, hence, greater numbers
of Ca2+ channels and higher Ca2+-ATPase activity in the
EDL could also be responsible, but these have not been measured.
The results might be explained, at least in part, by an effect of creatine
kinase (CK) activity on Ca2+-ATPase activity. From electrophoretic
studies of cytoplasmic proteins (Fig.
5) and subsequent immunoblotting with anti-CK antibodies, it has
been shown that cytosolic CK is present in remarkable amounts in shrew muscles
and that its concentration in the EDL muscle is three times higher than in the
soleus muscle. The presence of CK close to the Ca2+-ATPase of the
sarcoplasmic reticulum has been shown to enhance Ca2+ release and
uptake rates in the muscles of mice
(Steeghs et al., 1997). CK
activity is functionally coupled to the activity of the Ca2+-ATPase
because CK controls the local [ATP]/[ADP] ratio at the sarcoplasmic reticulum
and, thus, determines the ATP concentration available for the
Ca2+-ATPase and, therefore, the velocity of muscle relaxation
(Minajeva et al., 1996
).
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Acknowledgments |
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