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special communication
Anesthetics can alter subsequent in vitro assessment
of contractility in slow and fast skeletal muscles of rat
BENOÎT M. LAPOINTE AND CLAUDE H. CÔTÉ
Laval University Hospital Research Center, Ste-Foy, Québec, Canada G1V 4G2
muscle relaxant; skeletal muscle; tetanic tension
IN VITRO MEASUREMENT of muscle contractile properties
is a valid and reliable method that allows assessment of
muscle contractility without the possible systemic influences generally associated with in vivo or in situ
protocols. This probably explains its rather wide utilization in different research protocols in which muscle
function per se needs to be evaluated. Measurement of
muscle contractile capacity can also be used as an index
of muscle integrity, as is the case with experiments
dealing with exercise-induced muscle damage. Indeed,
because this type of damage is focal in nature, histological analysis cannot quantitatively account for the totality of the damage as in vitro measurement of muscle
contractile properties does (2).
To obtain valid measurement of muscle maximal
tetanic tension (Po), great care has to be taken to
control several parameters that can potentially influ-
The costs of publication of this article were defrayed in part by the
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ence this variable. However, selecting the appropriate
anesthetic agents is often overlooked, despite the fact
that most commonly used drugs can directly and adversely affect cardiac and skeletal muscle function at
different cellular levels (7, 11, 14). Indeed, the neuromuscular junction, the excitation-contraction coupling
process, and the contractile proteins themselves have
all been shown to be potentially influenced by some of
the anesthetics frequently administered. Furthermore,
when testing muscle function in vivo or in situ, anesthetics can act indirectly on muscle function by altering the
physiology of the cardiovascular and pulmonary systems (13, 15, 19). One would not predict that the
possible impacts of those anesthetics on muscle contractility, previously observed in vivo or in situ, would be
carried over in an isolated in vitro system. In agreement with this line of thinking, we have found no data
on the contractile behavior of skeletal muscles excised
from rats anesthetized with different agents and tested
in vitro.
Ingalls et al. (10) found that pentobarbital sodium
(PS) is the most widely selected anesthetic to study rat
or mouse muscle function, especially in vitro. The use of
a mixture of ketamine and xylazine (KX) is often reported
and has been favored in our institution for induction of
anesthesia in rodents. However, in preliminary experiments in which we measured contractile properties on
in vitro muscles excised from rats anesthetized with PS
and KX, an unexpected level of variation was observed.
We thus decided to conduct an in-depth study aimed at
comparing isometric contractile properties of both fast
[extensor digitorum longus (EDL)]- and slow [soleus
(Sol)]-twitch skeletal muscles obtained from rats anesthetized with PS or KX. To our knowledge, no such
study has yet been done in the rodent. Here we report
that anesthetic administered in vivo can significantly
alter contractility measurements obtained subsequently in vitro, which therefore calls for caution.
MATERIALS AND METHODS
Animals and anesthesia induction. Female Wistar rats
(135–160 g) were used in this study. They had access to
regular rat chow and water ad libitum and were maintained
on a 12-h light-to-dark cycle. All animal care and handling
procedures were approved by the Laval University Research
Center Animal Care and Use Committee and met the guidelines of the American Physiological Society. On injection with
either PS (50 mg/kg ip) or KX (87.5:12.5 mg/kg ip), animals
were left undisturbed under radiant heat to maintain body
0363-6119/99 $5.00 Copyright r 1999 the American Physiological Society
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Lapointe, Benoı̂t M., and Claude H. Côté. Anesthetics
can alter subsequent in vitro assessment of contractility in
slow and fast skeletal muscles of rat. Am. J. Physiol. 277
(Regulatory Integrative Comp. Physiol. 46): R917–R921,
1999.—Anesthetic agents can interfere with measurement of
skeletal muscle contractility in vivo or in situ. Data obtained
in vitro are however believed to be unaffected by such drugs.
Our objective was to compare in vitro contractile measurements of fast- and slow-twitch muscles dissected from rats
anesthetized with pentobarbital sodium (PS, 50 mg/kg ip) or
with a mixture of ketamine and xylazine (KX, 87.5:12.5 mg/kg
ip). The soleus (Sol) and extensor digitorum longus (EDL)
muscles were precisely dissected 10 and 20 min after induction of anesthesia and equilibrated for 20 min in vitro before
measuring contractile properties. All data obtained from PS
rats were comparable with published values obtained under
similar conditions. In EDL, maximum tetanic tension (Po) in
KX rats was significantly decreased at both times compared
with that in PS muscles. In the Sol, only the muscles exposed
for 20 min to KX showed a decreased Po. These results clearly
emphasize the need for investigators assessing skeletal muscle
contractility in vitro to take into account the type of anesthetics used and the time of in vivo exposition to the drug.
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ANESTHETICS AND IN VITRO MEASUREMENT OF MUSCLE FUNCTION
RESULTS
Fast-twitch EDL muscle. Data for isometric contractile properties of EDL muscles, except Po, are presented in Table 1. After anesthesia with PS, similar
results are obtained with muscles dissected from both
hindlimbs (PS-10 vs. PS-20). All results presented are
in agreement with those previously published and
Table 1. Contractile properties of EDL muscles
from rats anesthetized with PS or KX
PS-10
PS-20
KX-10
KX-20
TPT,
ms
RT1/2 ,
ms
dP/dt,
% Po/ms
Pt,
g
Pt/Po
23.8 ⫾ 0.2
25.1 ⫾ 0.7
22.2 ⫾ 1.4
22.1 ⫾ 0.8
25.4 ⫾ 1.3
25.7 ⫾ 0.2
22.8 ⫾ 1.3
23.8 ⫾ 0.7
2.8 ⫾ 0.2
3.0 ⫾ 0.1
2.6 ⫾ 0.1
2.7 ⫾ 0.3
44.9 ⫾ 0.9
44.5 ⫾ 1.4
34.4 ⫾ 1.0*
36.2 ⫾ 1.2*
0.303 ⫾ 0.005
0.309 ⫾ 0.032
0.285 ⫾ 0.017
0.298 ⫾ 0.014
Values are means ⫾ SE (6–8 muscles in each group). EDL, extensor
digitorum longus; PS, pentobarbital sodium; KX, ketamine-xylazine
mixture; TPT, time to peak tension; RT1/2 , one-half relaxation time;
Pt, maximum twitch tension; Po, maximum tetanic tension. * Significantly different from its respective PS value (P ⬍ 0.05).
Fig. 1. Maximum specific tetanic tension of extensor digitorum
longus (EDL) and soleus (Sol) muscles from rats anesthetized with
pentobarbital sodium (PS) or a ketamine-xylazine mixture (KX).
Maximum tetanic tension (Po) was obtained as described in MATERIALS AND METHODS and normalized for cross-sectional area. Left and
right EDL and Sol muscles from each rat were dissected exactly 10
and 20 min after administration of anesthetic and immediately
installed in the in vitro bathing medium. EDL muscles from PSanesthetized rats had similar values for Po, whereas anesthesia with
KX induced a significant drop in Po already noticeable in KX-10
group. Sol muscles from PS-anesthetized rats had similar values for
Po, whereas anesthesia with KX induced a significant drop in Po, only
noticeable in KX-20 group. aSignificantly different from its respective
PS value; bsignificantly different from KX-10. Values are means ⫾
SE; n ⫽ 6–8 muscles per group, P ⬍ 0.05.
measured under identical temperature. Speed-related
contractile measurements presented in Table 1 were
not influenced by the KX anesthesia as no difference
was seen between both KX hindlimbs and between PS
and KX groups, irrespective of the time of measurement. However, a very significant decrease in maximum twitch tension was seen in both KX groups
compared with PS, but time had no impact on this drop.
Quite surprising results were obtained when looking at
the influence of these drugs over time on Po. A value of
⬃20 N/cm2 was measured for EDL in both PS-10 and
PS-20 groups (Fig. 1), and such a value corresponds to
what is usually published for EDL muscles tested in
vitro at 25°C. However, values for specific Po with rats
anesthetized with KX were significantly lower at both
time points. The deficit was ⬃10% in KX-10 and
reached 20% in KX-20 muscles. The same observation
could be made for values of twitch tension in both KX
groups (Table 1).
Slow-twitch Sol muscle. Table 2 presents data for
contractile properties (except Po) of the slow-twitch Sol
muscle. No difference could be observed between
muscles from PS-10 and PS-20 groups, and all values
presented agree very well with data previously published for contractile properties of rat Sol muscle
obtained in vitro. Comparison between groups of
muscles dissected from KX-anesthetized animals reveals the same absence of influence of time on these
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temperature. Doses given were all near values reported in the
literature (10). Ten and twenty minutes after administration
of the anesthetic, muscles (EDL or Sol) were carefully dissected free and immediately transferred to the in vitro muscle
bath. One group of animals was used for each muscle tested.
PS-10 and PS-20, for example, indicate that muscles were
precisely dissected 10 and 20 min after administration of PS.
Measurement of contractile properties. In vitro measurement of muscle contractility was performed as described
previously (6, 9). Briefly, muscles were incubated at 25°C in a
buffered solution (Krebs-Ringer) supplemented with glucose
and bubbled with carbogen. One tendon was attached to a
rigid support at the bottom of the bath while the other one
was connected to an isometric force transducer (Grass FT-03).
Muscles were then adjusted to their optimal length, defined
as the length at which isometric twitch tension was maximal.
After a 20-min equilibration period, a single-twitch contraction was generated to obtain values for maximum twitch
tension, time to peak tension, and one-half relaxation time
(RT½ ). Muscles were then stimulated once every 60 s for 400
ms (EDL) or 1 s (Sol) at various frequencies ranging between
5 and 150 Hz to obtain the force-frequency relationship and
the Po. Stimulation consisted of 25-V square pulses of 0.2-ms
duration delivered through platinum field electrodes. After
measurement of Po, muscles were weighted to permit calculation of maximum specific tetanic tension (specific Po), which
is force production normalized for the 1/1,000 s crosssectional area as described elsewhere (16). The value used for
muscle density was 1.062 g/cm3, and the ratios of fiber length
to muscle length used were 0.40 for EDL and 0.62 for Sol (16).
When the total length of the protocol was taken into account,
including dissection, values for Po of both muscles tested
were obtained 45 and 55 min after induction of anesthesia in
PS-10 and KX-10 and in PS-20 and KX-20, respectively.
Statistical analysis. All data are expressed as means ⫾ SE.
Data on measurement of contractile properties for a single
anesthetic agent were analyzed by a one-way analysis of
variance for repeated measures followed by the Tukey’s post
hoc test on detection of significance. A two-way analysis of
variance was used when comparisons between anesthetics
were performed. In all cases, the level of significance was set
at P ⬍ 0.05.
ANESTHETICS AND IN VITRO MEASUREMENT OF MUSCLE FUNCTION
Table 2. Contractile properties of Sol muscles from rats
anesthetized with PS or KX
TPT,
ms
PS-10
PS-20
KX-10
KX-20
RT1/2 ,
ms
65.5 ⫾ 1.9 85.1 ⫾ 7
66.3 ⫾ 3
88.5 ⫾ 3.8
70.5 ⫾ 2.1 100.8 ⫾ 5.4*
72.7 ⫾ 3.4 105.4 ⫾ 6.1*
dP/dt,
% Po/ms
Pt,
g
Pt/Po
0.71 ⫾ 0.07
0.66 ⫾ 0.08
0.66 ⫾ 0.07
0.65 ⫾ 0.07
15.4 ⫾ 0.9
15.7 ⫾ 1.7
16.8 ⫾ 1.5
16.0 ⫾ 1.4
0.214 ⫾ 0.006
0.217 ⫾ 0.010
0.215 ⫾ 0.009
0.229 ⫾ 0.015
Values are means ⫾ SE (6–8 muscles in each group). Sol, soleus
muscle. *Significantly different from its respective PS value (P⬍0.05).
Fig. 2. Force-frequency curves for Sol muscles dissected from rats
anesthetized with PS or KX. Tetanic contractions at various stimulation frequencies were triggered at 60-s intervals in 3 groups of
muscles shown on graph. For clarity, curve for PS-10 muscles was
deleted because it exactly superimposed on curves for PS-20 and
KX-10 groups. Sol muscles from KX-20 group not only have a
significant deficit in maximal absolute or normalized force production, but they also show decreased force production at submaximal
level of stimulation. a All points on the curve for KX-20 are significantly different from the two other curves except at 10 Hz. Values are
means ⫾ SE; n ⫽ 6–8 muscles per group, P ⬍ 0.05.
group are significantly lower than in all other groups.
Also, when the same curve was plotted with force
expressed as a percentage of Po to isolate significant
right or left shift of the curve, a small but statistically
significant left shift was seen at the midfrequencies of
stimulation with the KX-20 muscles (data not shown)
as Sol muscles from PS-20 stimulated at 35 Hz developed 90% of their Po compared with 94% in KX-20
groups.
DISCUSSION
In various experimental paradigms, assessment of
skeletal muscle function is the outcome measure of
choice. Contractile measurements can be obtained under in vivo, in situ, or in vitro conditions depending on
the specific goals pursued. Anesthetic agents have been
shown to alter directly skeletal muscle contractility by
acting at different points in the molecular cascade of
events that leads to contraction, these being scattered
between the neuromuscular junction and the contractile proteins (7, 11, 14). The barbiturate compound PS,
at doses usually given, is known to potentiate twitch
tension, whereas a decrease in Po is seen with higher
concentrations of PS (17, 18). Ketamine is a dissociative
anesthetic, whereas xylazine is a nonnarcotic sedative
and analgesic agent with muscle relaxing properties.
This cocktail can influence the muscle acetylcholinesterase activity (1) and the excitation-contraction coupling
process probably by interfering with calcium homeostasis (14). When used for in situ or in vivo studies, PS and
KX can also influence contractile performance through
systemic actions, notably at the cardiovascular and
respiratory levels, and those are usually taken into
consideration in such protocols.
In the field of exercise-induced muscle damage, in
vitro measurement of isometric contractile properties
is a widely used method to evaluate muscle damage and
the time course and extent of recovery. Curiously, it
seems that the potential deleterious effect(s) of anesthetic agents administered in vivo on muscle function
measured in vitro has never been of any concern. In this
study, all results obtained with PS are in agreement
with data previously published by various groups for
contractile measurements obtained in vitro at 25°C on
muscles dissected from PS-anesthetized animals (3–6,
9, 16). This makes this group a solid basis for comparison in the absence of what could be called a true control
group, as no muscles were obtained on unanesthetized
or killed animals, because our goal was not to determine whether PS had an effect on contractility in vitro.
Such an experiment is basically unfeasable at the
moment for various ethical and physiological reasons.
However, comparisons of data for specific Po (N/cm2 )
obtained with muscles dissected from PS-anesthetized
mammals in general with various data published for
maximum voluntary force obtained in vivo in humans
suggest that PS has no negative effect on normalized
force production (8).
The most important finding is that the determination
of muscle maximum isometric tension in vitro can be
influenced by the anesthetic administered before the
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contractile variables as was seen in the EDL muscle.
However, compared with PS muscles at 10 or 20 min,
the value RT1/2 significantly longer when KX is used.
Values for maximum specific tension of Sol obtained
after anesthesia with PS and KX are shown in Fig. 1.
Muscles dissected from the PS-10 and KX-10 hindlimbs
had similar values; however, Sol muscles that were in
contact for 20 min with KX showed a significant 15%
decrease in Po compared with both the PS-10 and
PS-20 rats. It is worth noting that in the KX-20 group
the capacity to generate force was significantly decreased even at submaximal levels, as shown by the
force-frequency curve depicted in Fig. 2 in which,
except for tension at 10 Hz, all points for the KX-20
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R920
ANESTHETICS AND IN VITRO MEASUREMENT OF MUSCLE FUNCTION
cally, KX should be avoided because of its inherent
negative influence on force production.
Perspectives
The use of laboratory animals for experimental purposes is governed by constantly evolving guidelines and
considerations. Surgical and anesthetic procedures are
two areas in which animal care committees have low
tolerance. In the near future, new anesthetic or analgesic regimens, presently prescribed for human or veterinary use, will certainly be made available to laboratory
investigators. Inevitably, the influence of these new
drugs on physiological systems affecting measurements made in situ or in vivo will have been characterized already. However, the present experiments suggest that muscles, or any other tissue, dissected from
anesthetized animals for in vitro experiments can still
be influenced by the drugs administered. Furthermore,
one cannot assume that the effects observed in situ or
in vivo will be directly transposed in vitro, which means
that every new anesthetic regimen should be tested for
its potential influence on data recorded subsequently in
vitro.
We thank Julie Lefebvre for technical assistance and Dr. P.
Frémont for fruitful discussions.
This work was supported by grants from the Fonds de la Recherche en Santé du Québec (FRSQ) and from the Rehabilitation Research Provincial Network (REPAR) to C. H. Côté. B. M. Lapointe
was the recipient of scholarships from FRSQ and REPAR.
Address for reprint requests and other correspondence: C. H. Côté,
Laval Univ. Hospital Research Center, Rm. 9500, 2705 Blvd. Laurier,
Ste-Foy, Québec, Canada G1V 4G2 (E-mail: claude.h.cote@crchul.
ulaval.ca).
Received 17 December 1998; accepted in final form 20 May 1999.
REFERENCES
1. Arora, R. C., and H. Y. Meltzer. Muscle cholinesterase: effect of
phencyclidine and ketamine on rat and human muscle cholinesterase activity. Exp. Neurol. 67: 1–10, 1980.
2. Brooks, S. Rapid recovery following contraction-induced injury
to in situ skeletal muscles in mdx mice. J. Muscle Res. Cell Motil.
19: 179–187, 1998.
3. Close, R. Dynamic properties of fast and slow skeletal muscles of
the rat. J. Physiol. (Lond.) 173: 74–95, 1964.
4. Close, R. I. Dynamic properties of mammalian skeletal muscles.
Physiol. Rev. 52: 129–197, 1972.
5. Côté, C., T. P. White, and J. A. Faulkner. Intramuscular
substrate depletion and fatigability of soleus grafts in rats.
Can. J. Physiol. Pharmacol. 66: 829–832, 1988.
6. Côté, C. H., G. Perreault, and J. Frenette. Carbohydrate
utilization in rat soleus muscle is influenced by carbonic anhydrase III activity. Am. J. Physiol. 273 (Regulatory Integrative
Comp. Physiol. 42): R1211–R1218, 1997.
7. Davies, A. E., and J. L. McCans. Effects of barbiturate
anesthetics and ketamine on the force-frequency relation of
cardiac muscle. Eur. J. Pharmacol. 59: 65–73, 1979.
8. Edgerton, V. R., P. Apor, and R. R. Roy. Specific tension of
human elbow flexor muscles. Acta Physiol. Hung. 75: 205–216,
1990.
9. Frémont, P., H. Riverin, J. Frenette, P. A. Rogers, and C.
Côté. Fatigue and recovery of rat soleus muscle are influenced by
inhibition of an intracellular carbonic anhydrase isoform. Am. J.
Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R615–
R621, 1991.
10. Ingalls, C. P., G. L. Warren, D. A. Lowe, D. B. Boorstein, and
R. B. Armstrong. Differential effects of anesthetics on in vivo
skeletal muscle contractile function in the mouse. J. Appl.
Physiol. 80: 332–340, 1996.
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.247 on October 6, 2016
dissection. In both slow- and fast-twitch muscles tested,
Po is significantly decreased when KX is used instead of
PS. In the case of the EDL, this effect is already
noticeable in the KX-10 muscles and is time dependent
as the second muscle dissected showed a larger decrease in Po. This negative influence on maximum force
production takes more time to appear in the Sol as a
significant drop in Po was seen only in the muscles
dissected after 20 min of exposition to KX. These
observations were rather unexpected as we were unable to find in the literature any evidence that the
influence of KX could still be present once the muscles
are incubated in a Krebs-Ringer physiological solution.
Maximum twitch tension of the EDL was also significantly depressed in KX animals compared with the PS
groups, whereas all speed-related twitch contractile
parameters in this same group showed a tendency to be
faster (or shorter) than the values for PS animals.
Overall, these observations suggest that administration of KX at doses efficient for anesthesia can interfere
with calcium fluxes in fast-twitch muscle, an observation in agreement with previously published results
(14). We also observed that the value for RT½ in Sol was
significantly prolonged when KX was used, which also
supports an effect on calcium fluxes. More specifically,
it suggests that these drugs could decrease the rate of
calcium reuptake by the sarcoplasmic reticulum in
slow-twitch muscle, an influence previously noted with
cardiac muscle (12).
A thorough comparison of our results with previous
reports on the effects of PS and KX on muscle contractility is almost impossible to do as experimental protocols
used in most of these studies were very different and
quite often very distant from the real physiological
situation faced during experiments aimed at measuring muscle contractility in situ or in vitro. For instance,
Taylor et al. (17) dissected muscles from a dead animal,
and muscles were then incubated in vitro with the
anesthetic for the duration of the experiment as it was
added to the bath. In other cases, single fibers or entire
muscles from frogs were incubated in vitro with concentrations of anesthetics not necessarily mimicking the
real situation (11). These earlier studies are important
in that they gave mechanistic clues on how anesthetic
agents can influence muscle contractility but are not
entirely relevant to our situation. In our case, muscles
were in contact with the anesthetic for ⬃10 or 20 min
through the vasculature of the animal. They were then
incubated in vitro for a total duration of ⬃35 min before
measurement of Po in 150 ml of Krebs-Ringer solution,
which should further dilute the anesthetic present in
the muscles. In summary, these results have indubitable consequences on experimental design in which
conclusions are drawn from in vitro measurement of
maximum force production, a marker of muscle integrity. The fact that the influence of anesthetic agents
administered in vivo can be transferred in vitro and
that its extent may vary depending on the time of
exposition to the drugs should always be kept in mind
when designing an experimental protocol. More specifi-
ANESTHETICS AND IN VITRO MEASUREMENT OF MUSCLE FUNCTION
11. Khan, A. R. Mechanism of action of pentobarbital on the
contractile system of isolated frog muscle fibres. Acta Physiol.
Scand. 108: 405–409, 1980.
12. Lain, R. F., M. L. Hess, E. W. Gertz, and F. N. Briggs. Calcium
uptake activity of canine myocardial sarcoplasmic reticulum in
the presence of anesthetic agents. Circ. Res. 23: 597–604, 1968.
13. Lang, R. M., R. H. Marcus, A. Neumann, D. Janzen, A. M.
Hansen, A. M. Fujii, and K. M. Borow. A time-course study of
the effects of pentobarbital, fentanyl, and morphine chloralose on
myocardial mechanics. J. Appl. Physiol. 73: 143–150, 1992.
14. Marwaha, J. Some mechanisms underlying actions of ketamine
on electromechanical coupling in skeletal muscle. J. Neurosci.
Res. 5: 43–50, 1980.
15. Namba, T., M. Takaki, J. Araki, K. Ishioka, and H. Suga.
Energetics of the negative and positive inotropism of pentobarbital
16.
17.
18.
19.
R921
sodium in canine left ventricle. Cardiovasc. Res. 28: 557–564,
1994.
Segal, S. S., and J. A. Faulkner. Temperature-dependent
physiological stability of rat skeletal muscle in vitro. Am. J.
Physiol. 248 (Cell Physiol. 17): C265–C270, 1985.
Taylor, R. G., R. T. Abresch, J. S. Lieberman, and W. M.
Fowler, Jr. Fast and slow skeletal muscles: effect of secobarbital
on contractility of muscles from mice. Arch. Phys. Med. Rehabil.
61: 160–166, 1980.
Taylor, R. G., R. T. Abresch, J. S. Lieberman, W. M. Fowler,
and M. M. Portwood. Effect of pentobarbital on contractility of
mouse skeletal muscle. Exp. Neurol. 83: 254–263, 1984.
Urthaler, F., A. A. Walker, and T. N. James. Comparison of
the inotropic action of morphine and ketamine studied in canine
cardiac muscle. J. Thorac. Cardiovasc. Surg. 72: 142–149, 1986.
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.247 on October 6, 2016