Vol. 92, Issue 6, 2491-2500, June 2002
Effects of volatile anesthetics on elastic stiffness in
isometrically contracting ferret ventricular myocardium
Anna E.
Bartunek1,2,
Victor
A.
Claes3, and
Philippe R.
Housmans1
1 Department of Anesthesiology, Mayo Foundation,
Rochester, Minnesota 55905; 2 Department of
Cardiothoracic and Vascular Anesthesia and Intensive Care Medicine,
University of Vienna, A-1010 Vienna, Austria; and
3 Laboratory of Human Physiology and
Pathophysiology, University of Antwerp, B-2020 Antwerp,
Belgium
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ABSTRACT |
The effects of
halothane, isoflurane, and sevoflurane on elastic stiffness, which
reflects the degree of cross-bridge attachment, were studied in intact
cardiac muscle. Electrically stimulated (0.25 Hz, 25°C),
isometrically twitching right ventricular ferret papillary muscles
(n = 15) at optimal length
(Lmax) were subjected to sinusoidal length
oscillations (40 Hz, 0.25- 0.50% of Lmax peak to peak). The amplitude and phase relationship with the resulting force oscillations was decomposed into elastic and viscous components of total stiffness in real time. Increasing extracellular
Ca2+ concentration in the presence of anesthetics to
produce peak force equal to control increased elastic stiffness during
relaxation, which suggests a direct effect of halothane and sevoflurane
on cross bridges.
sinusoidal length oscillations; cross bridges; calcium
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INTRODUCTION |
THERE IS EVIDENCE
THAT the volatile anesthetics halothane, isoflurane, and
sevoflurane inhibit actomyosin cross-bridge function at a level
"downstream" from Ca2+ binding to thin filament
regulatory proteins (3, 17, 26, 27, 29, 34). The
contribution of this direct effect on cross-bridge function to the
overall negative inotropic effect of volatile anesthetics appears to be
small, but its relevance in physiological conditions is actually not
known. Information about anesthetics' effects on cross-bridge
performance was obtained mostly in skinned fibers (26, 27, 29,
34), yet results from intact cardiac muscle with intact cell
membranes might reflect more accurately the in vivo physiological
conditions. Instantaneous changes in stiffness on activation arise from
an elastic component associated with each individual attached cross
bridge; stiffness therefore expresses a measure of the number of
attached cross bridges at any one moment (14, 15).
Stiffness measurements were commonly utilized to explore the influence
of different interventions on cross-bridge performance (3, 11,
26, 29, 31, 33, 39). The effects of volatile anesthetics on
active cross bridges were recently studied by detecting changes in
elastic stiffness that occur throughout shortening and lengthening of
isotonic twitches of intact ferret papillary muscle. Halothane and
isoflurane increased elastic stiffness at equal peak shortening and
higher extracellular Ca2+ concentration
([Ca2+]; [Ca2+]o) when compared
with control, an observation that strongly suggests a direct effect of
anesthetics on cross-bridge function (3).
In the present study, stiffness was measured throughout isometric
contraction by imposing sinusoidal length oscillations of small
amplitude and analyzing the amplitude and phase relations of the
resultant force oscillations. Stiffness was instantaneously separated
into its viscous and elastic components, the latter of which is
associated with the elastic elements in the cross bridges.
Instantaneous elastic stiffness measurement in Ca2+
back-titration experiments indicates that halothane and sevoflurane most likely affect cross-bridge performance during isometric relaxation in intact cardiac muscle tissue.
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MATERIALS AND METHODS |
This study was approved by the Animal Care and Use Committee of
the Mayo Foundation, with protocols completed in accordance with the
National Institutes of Health guidelines and in accordance with the
Guide for the Care and Use of Laboratory Animals (Institute of
Laboratory Animal Resources Commission on Life Sciences, National Research Council). Adult male ferrets (weighing 1,100-1,500 g; 16-19 wk of age) were anesthetized with pentobarbital sodium (100 mg/kg ip), and the heart was quickly removed through a thoracotomy. During generous superfusion with oxygenated physiological solution (see
below), suitable right ventricular papillary muscles were carefully
excised from the beating heart. Papillary muscles were then mounted
vertically in a temperature-controlled muscle chamber containing a
physiological salt solution made up in ultrapure water (Nanopure
Infinity, Barnstead, Dubuque, IA) and with the following composition
(in mM): 137.5 Na+, 5.0 K+, 2.25 Ca2+, 1.0 Mg2+, 127.0 Cl
, 1.0 SO
, 20.0 acetate1, 10.0 glucose, and
5.0 MOPS, pH 7.40, bubbled with 100% O2. Suitable preparations were selected on the basis of the following criteria: length at which twitch active force is maximal
(Lmax) 
3.5 mm, a mean cross-sectional
area 
1.2 mm2, and a ratio of resting to total force
in an isometric twitch at Lmax 0.30. The
tendinous end of each muscle was tied with a thin braided polyester
thread (size 9.0 Deknatel Surgical Suture, Fall River, ME) to the end
of a stiff glass rod (7.5 cm, 40 mg), which was attached to the lever
of a force-length servotransducer. The attachment was sealed with
paraffin. This transducer system (Innovi, Belgium) allows one
simultaneously to 1) measure shortening up to 3 mm
(resolution 0.25 µm), 2) impose loads up to 299 mN, 3) measure force by feedback sensing (resolution <0.1
mN), and 4) impose abrupt changes of load (load clamp) or of
initial length (length clamps: quick release and quick stretch). The
equivalent moving mass was 250 mg, and the static compliance was 0.28 µm/mN. The resonant frequency of the transducer was 250 Hz. The
ventricular end of each muscle was held in a miniature Lucite clip
(Dupont, Wilmington, DE) with a built-in platinum punctate electrode;
two platinum wires were arranged longitudinally, one along each side of
the muscle, and served as anode during punctate stimulation. A Grass
S88D stimulator (Astro-Med, West Warwick, RI) delivered rectangular
pulses of 5-ms duration. Stimuli at 10-20% above threshold, range
5-10 V, were used to minimize the release of endogenous norepinephrine by the driving stimuli. Throughout the stabilization period, the muscles were stimulated and made to contract in alternating series of four isometric and four isotonic twitches for at least 1 h at 0.25 Hz at the temperature of the bathing solution of 30°C. The
solution was changed at least once during this stabilization period.
When muscles had reached steady state, initial muscle length was set at
Lmax. Thereafter the temperature was decreased to 25°C, the definite temperature in which the experiments were carried out. The muscles were then allowed to stabilize for another hour first in the same manner as above and then in isometric
twitches only for muscles contracted isometrically at the preload
of Lmax throughout the experiment. To minimize
effects of release of endogenous catecholamines in ferret cardiac
muscle, 
-adrenoceptor blockade was achieved by the
administration of (±)-bupranolol hydrochloride (107 M).
On-line measurement of elastic stiffness.
When load or length of a muscle is forced to vary in a sinusoidal
manner, the resulting periodic changes are also sinusoidal, but they
will not generally speaking be in phase with the imposed oscillations.
This phase shift is quantified by resolving the sinusoid of the
received signal into two components, one in phase with the imposed
sinusoid and one in quadrature to the imposed sinusoid (i.e., leading
or lagging by 90°). We imposed continuous small rapid length
perturbations on the muscle attached to the electromagnetic lever
system, all of which behaves as a linear second-order mechanical system
(24, 35, 36, 38). The relation of the sinusoidal responses
to the imposed sinusoidal changes can be described mathematically
where m is moving mass, B is viscosity,
Cm is mechanical compliance, v is velocity, f is
force, and t is time. Imposing periodic length
oscillations of certain frequencies induces force oscillations that are
slightly out of phase with length by a phase angle 
(Fig.
1A). The analysis uses force
and velocity vectors that are separated by a phase angle difference of
(90° 
). The force vector is decomposed into a force component
resisting the imposed velocity oscillations (B
:
viscosity) and the vectorial sum of the forces across moving mass
(
m: inertia) and elastic components (/Cm: mechanical compliance). At
resonance, force and velocity oscillations are in phase, and inertial
and elastic forces are equal in magnitude but of opposite signs and
therefore they cancel each other out. Below resonance frequency, as
shown in Fig. 1A, the elastic force is much larger than the
inertial force, yet the opposite is true above resonance frequency.
Figure 1B shows a Bode plot of a muscle at the preload of
Lmax at rest (not stimulated) displaying the
phase angle between force and length oscillations at each of a
large range of frequencies. Resonance frequency ( = 90°) in
this example was 87 Hz. In previous studies, we found that the
resonance frequency of ferret papillary muscle at preload of
Lmax was in the range of 83-154 Hz
(3). During isometric contraction, the resonance frequency
would be higher as muscle stiffness increases with force. We used an
oscillation frequency of 40 Hz in all experiments to minimize the
contribution of inertial forces to the measurement of elastic
stiffness. At frequencies <50% of resonance frequency, the phase
angle was indeed smaller than 15-20° (Fig. 1B),
and the small error introduced by the inertial component can be
ignored. The vectors B and
/Cm m, viscous and elastic components of
total stiffness, respectively, were obtained in real time by
correlating the sine and cosine functions used with amplitude and phase
relations of force and velocity oscillations by means of an electronic
circuit of analog correlators. Henceforth we refer to elastic stiffness
as "stiffness" unless otherwise stated. Viscosity changes were
minimal during contraction and were not pursued in this investigation.
We have previously shown in cat papillary muscle that viscous stiffness
is one order of magnitude smaller than elastic stiffness
(24). The imposed length perturbations, which could be
switched on and off with a toggle switch, were of total amplitude of
0.25 and 0.50% Lmax peak to peak in
Ca2+ back-titration experiments and concentration response
experiments, respectively. Continuous length oscillations resulted in
depression of active force development by 5.1 ± 1.1%
(3.5-6.6%) when oscillation amplitude was 0.50%
Lmax and by 1.7 ± 0.5%
(0.8- 2.4%) when muscles were oscillated with an amplitude of
0.25% Lmax.
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Fig. 1.
A: schematic representation of analysis
methods of the amplitude and phase relationships of length and force
sinusoidal oscillations (left). Force, length, and velocity
vectors are projected on a rotor diagram (right) in which
the vectors rotate counterclockwise, 360° per cycle. The velocity and
force vectors are projected (right) into a
x-y plane in which the force vector  is
decomposed into a resistive component (B, where B is
viscosity and is velocity) and, orthogonal to that,
an [elastic (/Cm) inertial
(m)] component. At frequencies well
below resonant frequency, the inertial component is negligibly small,
and this component represents pure elastic stiffness. Analog
correlators yielded both in-phase and out-of-phase components of total
stiffness from the amplitude and phase relationship between force and
velocity oscillations. B: Bode plot for a representative
muscle at rest at the preload of length at maximal twitch force
(Lmax), illustrating the dependence of the phase
angle on oscillation frequency. The resonance frequency in this
example was 87 Hz. The phase angle between force and length is
small (<15°) and independent of oscillation frequency at
frequencies < 50% of resonance frequency. At resonance, the
phase angle is 90° and force and velocity are in phase with each
other. At frequencies above resonance frequency, force and length tend
to go out of phase. B, viscosity; Cm, mechanical
compliance; f, force; l, length; m, equivalent
moving mass; t, time; v, velocity; , phase
angle; , angular frequency.
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Methods of delivery of volatile anesthetics.
The methods of delivery of anesthetics were the same as previously
described (4, 19). In brief, oxygen flowed through the
calibrated vaporizer for halothane, isoflurane, or sevoflurane and was
allowed to mix with the respective anesthetic in a 3-l reservoir bag.
An occlusive roller pump (Masterflex; Cole-Parmer, Chicago, IL)
delivered a continuous gas flow to the bubbler in the organ bath. The
muscle chamber was covered with a tightly sealing Parafilm (American
Can, Greenwich, CT) except for a narrow slit for the muscle clip and
the glass rod. The concentration of the anesthetic was measured
continuously between the reservoir bag and the roller pump with an
anesthetic agent monitor (Ohmeda 5330, Madison, WI). Gas chromatography
(Hewlett-Packard 5880A, Palo Alto, CA) measurements showed that the
concentrations of halothane, isoflurane, and sevoflurane and their
calculated partial pressure in fluid followed closely imposed changes
of anesthetic vapor concentration in the gas phase. After the
administration of anesthetic was discontinued, anesthetic concentration
was always undetectable at 30 min.
Experimental design.
Two experimental protocols were used to examine the effects of
anesthetics on stiffness in cardiac muscle in which each muscle served
as its own control. Anesthetics were studied in random order. Table
1 summarizes the characteristics of the
muscles used in each of the two experimental series. Muscles twitched isometrically at the preload of Lmax throughout
the experiments. In the first series of experiments, muscles
(n = 7) were exposed to halothane, isoflurane, and
sevoflurane in random order and in concentrations of 0.0, 0.5, 1.0, and
1.5 minimum alveolar concentration (MAC) each. MAC is an anesthetic
half-maximal effective dose as defined by Eger et al.
(12). One MAC is the concentration of anesthetic at which
50% of the animals respond to a standardized supramaximal stimulus
with "gross purposeful muscular movements of body or extremities"
(12). It is a measure of anesthetic equipotency. One MAC
halothane, isoflurane, and sevoflurane in the ferret corresponds to 1%
(vol/vol), 1.5% (vol/vol), and 2.7% (vol/vol) of the respective
anesthetic (4, 25). Effects of each anesthetic on
isometric contraction at the preload of Lmax were monitored until a steady state was reached, and this was usually
the case after 10-18 min of equilibration in each concentration. Subsequently, the concentration of the anesthetic was increased. In
control conditions, in each anesthetic concentration, in anesthetic washout, and in Ca2+ back-titration, two records were
taken: one with no oscillations and one when muscle was exposed to
sinusoidal velocity oscillations; the latter was used for
quantification of variables of interest. After the highest
concentration of anesthetic, the anesthetic was turned off, the
reservoir bag was emptied, and the gas-delivering system was flushed
with oxygen. The muscle was allowed to recover for 1 h before new
control measurements were taken and the administration of the next
anesthetic was commenced.
In the second series of experiments (n = 8 muscles), we
determined whether anesthetics change cross-bridge function by a
mechanism different from that exerted by anesthetic-induced
reduced intracellular [Ca2+]. Muscles were stabilized in
the same manner as above. Records, one with no oscillation and one when
muscle was exposed to sinusoidal velocity oscillations, were taken in
control conditions and in one MAC anesthetic, when peak force had
reached steady state. Extracellular [Ca2+] was then
rapidly increased by adding small aliquots of a concentrated CaCl2 solution (0.25 M) to the bathing solution, until the
amplitude of peak force was equal to that in control twitch. In 10 of
24 experiments, peak force slightly exceeded that of the control twitch
after titration with CaCl2. In these instances,
extracellular [Ca2+] was decreased by addition of
20-400 µl of 0.1 M EGTA, pH 7.0, to precisely match peak force
to that in the control twitch. Free Ca2+ in the
Ca2+ back-titrated twitch was calculated by Fabiato's
program (13). After recording the back-titrated twitch,
the anesthetic was turned off, the reservoir bag was emptied, and the
gas-delivering system was flushed with oxygen. The solution was changed
to control physiological salt solution (extracellular
[Ca2+] = 2.25 mM) and replaced at least four times
over a period of 15 min. The muscle was allowed to recover for 1 h
before the same protocol with a different anesthetic was repeated. This
protocol of Ca2+ back-titration allowed us to compare
elastic stiffness in the absence (control) and presence of anesthetic
at equal peak force.
All waveforms of force and elastic stiffness were displayed as a
function of time on a four-channel digital oscilloscope (Nicolet 4094C,
Madison, WI). Waveforms were stored on 5.25-in. floppy disks and
transferred to a desktop computer by software programs written in
WFBASIC (Blue Feather Software, New Glarus, WI), which also measures
all variables of contraction and stiffness. Stiffness trace was
generated by the correlator with a 17-ms delay. Original force trace
with oscillations and filtered force trace with 17-ms delay were stored
(Fig. 1). Maximal amount of developed force (DF), time to DF,
relaxation half time, and the time from DF to half isometric relaxation
were analyzed from filtered and delayed force traces. To obtain
stiffness in the active muscle, resting stiffness was subtracted from
total stiffness. Peak active stiffness (PS), active stiffness at DF,
active stiffness at half isometric relaxation, time to PS, and time
from PS to half active stiffness during relaxation were analyzed from
stiffness traces. Time to DF and time to PS were measured from the
stimulus and corrected for 17-ms delay. Stiffness and force were
normalized for cross-sectional area.
Statistics.
In the concentration-response experiments, measurements during exposure
to different anesthetic concentrations were compared with control
measurements obtained just before exposure to the respective anesthetic
by means of repeated-measures ANOVA, followed by comparisons vs.
control by Bonferroni-corrected paired t-test. Comparisons
between anesthetics of the same clinically effective concentration were performed by repeated-measures ANOVA,
followed by pairwise comparisons with Bonferroni-corrected paired
t-test. In Ca2+ back-titration experiments,
comparisons between measurements in control and in exposure to 1 MAC,
anesthetic and elevated extracellular [Ca2+] were
compared by Student's paired t-test. Differences were
considered significant at the P < 0.05 level. Data are
reported as means ± SD, except in Figs. 3 and 4 where SE are
plotted for reasons of clarity.
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RESULTS |
Table 2 summarizes variables of
isometric contraction and elastic stiffness at the onset of the
experiment. Figure 2, B and C, illustrates force and length traces of an isometrically
twitching muscle in control conditions in a concentration-response
experiment. Length oscillations with amplitude of 0.47%
Lmax (peak to peak) at a frequency of 40 Hz are
visible on the length trace, and the resulting force oscillations are
apparent on the force waveform. Figure 2A shows the
stiffness trace, which is derived from the correlator with a 17-ms
delay, and the force trace (dashed line), which is identical to that in
the Fig. 2B, but filtered and delayed by 17 ms. The length
trace shows a very small amount of shortening during the isometric
contraction (Fig. 2C), which is consistent with a static
compliance of 0.28 µm/mN in the transducer system. In control
conditions of concentration-response experiments, peak stiffness occurs
135 ± 17 ms (114-164 ms) after peak developed force. The
time difference between peak force and peak stiffness decreased with
increasing anesthetic concentrations (not shown) and recovered to
control values when extracellular [Ca2+] was raised until
peak force was the same as in control conditions (not shown). Volatile
anesthetics did not alter resting elastic stiffness measured
immediately before the stimulus (not shown).
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Fig. 2.
Elastic stiffness (solid trace, A) obtained
from sinusoidal length oscillations at 40 Hz (C) and
resulting force oscillations (B) during isometric
contraction in a representative muscle of the concentration-response
experiment series in control conditions. Stiffness and filtered force
trace (dashed trace in A) are delayed by 17 ms compared with
unfiltered force and length trace. Vertical arrow in B
indicates the electric stimulus. Muscle characteristics:
Lmax, 4.29 mm, mean cross-sectional area, 0.42 mm2, ratio of resting to total force (R/T) = 0.14.
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In the first series of experiments, the effects of halothane,
isoflurane, and sevoflurane on force and active stiffness in isometric
twitches were examined in random order in seven muscles in
concentrations of 0.0, 0.5, 1.0, and 1.5 MAC and after 30 min of
anesthetic washout. Figure 3 shows
typical force and stiffness waveforms in control conditions and 1 MAC
anesthetic from a muscle of the first series of experiments. One MAC
sevoflurane, isoflurane, and halothane decreased developed force,
active stiffness, time to peak force, and time to peak active
stiffness. Figure 3C, depicts stiffness plotted against
force. It shows that stiffness at any given force is higher during
relaxation than during contraction. At any given force, anesthetics
increased stiffness during contraction and decreased stiffness during
relaxation resulting in a narrower hysteresis (Fig. 3C).
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Fig. 3.
Force (A) and stiffness (B) as a function of
time in control conditions and during exposure to 1 minimum alveolar
concentration (MAC) isoflurane, sevoflurane, and halothane in a
representative muscle. Stiffness is plotted against force in
C. Vertical arrow in A indicates the electric
stimulus. Muscle characteristics: Lmax, 5.7 mm,
mean cross-sectional area, 0.56 mm2, R/T = 0.13.
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Concentration-dependent and washout effects of volatile
anesthetics on developed force and active stiffness during isometric contraction are displayed in Figs. 4 and
5. Halothane, isoflurane, and sevoflurane
decreased peak force and peak active stiffness in a
concentration-dependent reversible manner (Fig. 4, A and B). Each of the three anesthetics shortened time to peak
force and, to a greater extent, time to peak stiffness (Fig. 4,
C and D). They decreased active stiffness at peak
force (Fig. 5A) and active stiffness at half isometric
relaxation (Fig. 5C). They caused a shortening of time from
peak force to half isometric relaxation (Fig. 5B) and time
from peak stiffness to half active stiffness during relaxation (Fig.
5D). Relaxation half time did not recover to normal
values after 30-min anesthetic washout, and time to peak force did not
recover completely in halothane washout (Figs. 5B and
4C).
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Fig. 4.
Concentration-response effects of halothane, isoflurane, and
sevoflurane on peak developed force (A), peak active
stiffness (B), time to peak force (C), and time
to peak active stiffness (D) in isometric twitches.
* P < 0.05, by repeated-measures ANOVA and
comparisons vs. corresponding control by Bonferroni-corrected paired
t-test. Significant differences, P < 0.05, by repeated-measures ANOVA and pairwise comparisons between equipotent
concentrations of anesthetics by Bonferroni-corrected paired
t-test: #halothane vs. sevoflurane, $isoflurane vs.
sevoflurane, &halothane vs. isoflurane. Values (means ± SE) in
each graph are from the same muscles.
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Fig. 5.
Concentration-response effects of halothane, isoflurane, and
sevoflurane on active stiffness at peak force (A),
relaxation half time, i.e., time from peak developed force to half
isometric relaxation (B), active stiffness at half isometric
relaxation (C), and stiffness half time, i.e., time from
peak active stiffness to half active stiffness during relaxation
(D). * P < 0.05, by repeated-measures
ANOVA and comparisons vs. corresponding control by Bonferroni-corrected
paired t-test. Significant differences, P < 0.05, by repeated-measures ANOVA and pairwise comparisons between
equipotent concentrations of anesthetics by Bonferroni-corrected paired
t-test: #halothane vs. sevoflurane; $isoflurane vs.
sevoflurane; &halothane vs. isoflurane. Values (means ± SE) in
each graph are from the same muscles.
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Ca2+ back-titration experiment.
To determine whether anesthetics change active stiffness by a mechanism
different from that exerted by anesthetic-induced decrease in
intracellular [Ca2+], Ca2+ back-titration
experiments were performed (Fig. 6).
Force and active stiffness were measured in control and during exposure to anesthetic 1 MAC, both in extracellular [Ca2+] = 2.25 mM. Extracellular [Ca2+] was then rapidly increased
until peak force in anesthetic was the same as in the control twitch.
In two muscles exposed to 1 MAC halothane, force amplitude could not be
raised to control values by increasing extracellular
[Ca2+]. These muscles were excluded from further analysis
regarding the halothane Ca2+ back-titration but were used
for analysis of Ca2+ back-titration in isoflurane and
sevoflurane. In Ca2+ back-titration experiments in the
presence of isoflurane, peak force was minimally higher in each muscle
(2.4-0.6 mN/mm2). This resulted in a significant
different mean peak force (0.64 ± 0.76 mN/mm2).
Therefore, limitations exist in drawing conclusions regarding the
effects of isoflurane. Table 3 summarizes
results of the Ca2+ back-titration experiments to 1 MAC
halothane, isoflurane, and sevoflurane. In Ca2+
back-titration, active stiffness at peak force was slightly higher in
sevoflurane. Halothane and sevoflurane significantly increased active stiffness at half isometric relaxation. In the presence of
anesthetics and elevated Ca2+, time from peak force to half
isometric relaxation and time from peak active stiffness to half active
stiffness during relaxation were significantly abbreviated.
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Fig. 6.
Ca2+ back-titration experiment. Traces of
force and elastic stiffness as a function of time in control
conditions, during exposure to sevoflurane 1 MAC and during sevoflurane
1 MAC and elevated extracellular Ca2+ concentration
([Ca2+]o; 4.07 mM) (dashed trace) are
superimposed in A and B. C: stiffness
plotted vs. force in control conditions, in 1 MAC sevoflurane, and in 1 MAC sevoflurane and elevated [Ca2+]o. In 1 MAC sevoflurane and elevated [Ca2+]o,
stiffness for a given force during the relaxation phase is increased as
indicated by the small vertical arrow at half peak force. Small
vertical arrow in A indicates the electric stimulus. Muscle
characteristics: Lmax, 6.43 mm; mean
cross-sectional area, 0.39 mm2; R/T = 0.18.
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DISCUSSION |
We used continuous small-amplitude sinusoidal length perturbations
to determine the elastic component of stiffness throughout isometric
twitch contractions in the presence of halothane, isoflurane, and
sevoflurane. The three volatile anesthetics decreased peak developed
force and concomitantly active stiffness in a concentration-dependent manner, with halothane being most and sevoflurane least effective, an
observation that is caused mainly by decreased intracellular availability of activating Ca2+ and decreased
Ca2+ sensitivity of the contractile system. When in
Ca2+ back-titration experiments peak force was equal to
that in control conditions, peak active stiffness was increased by
sevoflurane and active stiffness during relaxation was increased by
halothane and sevoflurane, indicating that cross-bridge performance is
affected by a mechanism downstream of Ca2+ binding to
troponin C.
When heart muscle length or load is disturbed sinusoidally, it responds
with sinusoidal force or length alterations like a linear second-order
mechanical system (24, 35, 36, 38). Inertia, viscosity,
and elasticity present in muscle preparations are affected by
sinusoidal forcing functions, and measurement of stiffness therefore
reflects the response of all three components together. Elastic
stiffness is considered to be a static or time-independent property.
Consequently, an elastic component responds to a stretch with a change
in tension that is dependent only on the magnitude of the stretch and
not on the rapidity of the stretch. In contrast, viscous and inertial
stiffness both reflect dynamic or time-dependent properties (36,
39). In the present study, the elastic component of stiffness
was separated and recorded instantaneously throughout isometric
contraction. In single activated frog skeletal fibers, elastic
stiffness was thought to reside mainly in attached cross bridges, and
the compliance attributable to cross bridges was found to be
80-90% of the measured instantaneous compliance of the fiber
(14). Thus changes in stiffness reflect changes in the
number of attached cross bridges. It is also generally accepted that
the level of force generated by a muscle fiber is proportional to the
number of cross bridges in the force-generating state
(22). Therefore, if an intervention affects recruitment of
cross bridges during Ca2+ activation, developed force and
active stiffness will change in parallel. Disparate changes in
stiffness and force, as we encountered in our experiments, however, can
be explained by a change in the proportion of force generating to
non-force-generating cross bridges. This might be induced by an
influence on the kinetics of cross-bridge cycle with a modulation in
rate of transition between passive and force-generating cross-bridge
states during activation.
Interpretation of stiffness in isometric twitch contraction.
During isometric contraction in papillary muscle, elastic stiffness
lags behind tension during the tension rise and lags behind during
relaxation (Fig. 2). This results in hysteresis when plotted as
stiffness-tension relation during the isometric contraction cycle, with
stiffness for a given force being higher during relaxation than during
contraction (Fig. 3). This is in agreement with earlier investigations
in intact cat papillary muscle, in which hysteresis of total
(viscoelastic) stiffness-force relation was shown (33, 36)
but not with findings in frog skeletal muscle fiber, in which stiffness
rises earlier than tension during the onset of a tetanus (9,
16) and the plots of force vs. stiffness during development of
force and during relaxation of tetanus were approximately the same
(2). The reason for the discrepancy might be that in
intact papillary muscle a complex composition of intracellular and
extracellular elements forming the entirety of series elasticity induces a nonlinearity of the elastic-stiffness tension relation. Sarcomere compliance residing in the myofilament itself (18, 23,
41), damaged ends of intact muscle preparation
(28), and shearing through Z-lines and intercalated
discs (1) probably contribute to series elasticity.
This might limit to some extent the value of conclusions drawn from
elastic stiffness measurement in intact cardiac muscle on cross-bridge
performance. Yet a change of elastic stiffness attributable to an
intervention's influence on cross-bridge performance can still be
detected and compared, by assuming that the intervention does not
affect passive components in muscle during activation.
The effects of volatile anesthetics on active elastic stiffness.
Evidence exists that contractile proteins undergo changes in the
presence of volatile anesthetics, which result in the modulation of
contractile force (17, 26, 29). Volatile anesthetics' effect on cross-bridge performance became evident in skinned (26, 29) and tetanized (17) cardiac muscle tissue but
not in papillary muscle activated with Ba2+
(31). In intact isotonically twitching papillary muscle,
halothane and isoflurane seemed to exert a direct effect on
cross-bridge performance, as indicated by changes in elastic stiffness
(3). Intact isotonically or isometrically twitching
cardiac muscle tissue might better represent in vivo physiological
conditions than skinned, tetanized, or Ba2+-stimulated
muscle. In isometric Ba2+-stimulated contracture, the
Ca2+-free conditions might impair
Ca2+-dependent protein kinase involved in modulation of
myofilament action. The skinning procedure is likely to change the
microenvironment surrounding the myofilaments and allows the diffusion
out of the cell of low-molecular-weight proteins, which play an
important role in the modulation of muscle contractile function
(43). To explore whether volatile anesthetics'
interference with cross-bridge function is evident in intact
isometrically contracting muscle, extracellular [Ca2+]
was increased during exposure to 1 MAC anesthetic until peak force was
the same as in control conditions. The underlying assumption is that at
equal peak developed force, the Ca2+ binding sites
responsible for Ca2+ regulation of the contractile system
are occupied to an equal extent. The effects observed therefore are
assumed to be caused by effects on contractile proteins downstream to
Ca2+ binding to troponin C. Because peak force in
Ca2+ back-titration in isoflurane experiments was raised
significantly over that in control, we had to exclude isoflurane from
discussion, although data analysis was performed and data are
shown for completeness. In Ca2+ back-titration experiments,
halothane and sevoflurane accelerated isometric relaxation as indicated
by a decreased time from peak force to half isometric relaxation and
increased active stiffness during relaxation as indicated by an
increased stiffness at half isometric relaxation. The rate-limiting
factor in tension relaxation of mammalian muscle is most likely the
rate with which cross bridges detach and not Ca2+ uptake by
the sarcoplasmic reticulum, because the decline of intracellular free
[Ca2+] far precedes the decline of tension
(44). When interpreted in a two-state cross-bridge model,
an increase in the rate constant of cross-bridge detachment
(gapp), an effect which has been shown in skinned rat cardiac muscle preparations under exposure of 2 MAC
halothane but not sevoflurane (29), could explain an
acceleration of isometric relaxation. However, at submaximal levels of
activation as in our experiments, cross-bridge kinetics are primarily
controlled by the kinetics of Ca2+ binding to troponin C
and the related transitions of the regulatory proteins. It is
conceivable, therefore, that volatile anesthetics induce a faster
Ca2+ off-rate from troponin C. Halothane did not alter the
Ca2+ affinity of isolated troponin C (7), but
this could not be shown for intracellular troponin C hitherto. The
elevated active stiffness during relaxation indicates a higher
stiffness-to-force ratio in the presence of halothane and sevoflurane.
This is consistent with findings in skinned rat cardiac fibers, in
which halothane, enflurane, and isoflurane increase the
stiffness-to-force ratio (26). We interpret the disparate
change in stiffness and force during exposure to halothane and
sevoflurane as an effect on cross-bridge cycling kinetics. A modulation
in rate of transition between passive and force-generating cross-bridge
states could influence the proportion of force-generating to
non-force-generating cross bridges. The molecular levels at which
volatile anesthetics modulate cross-bridge performance cannot be
detected by the technique used in this study. Anesthetics might modify
rate constants of transitions between various states in the
cross-bridge cycle by affecting contractile proteins themselves or by
influencing second messenger cascades. There is growing evidence that
volatile anesthetics activate protein kinase C, a mechanism that seems
to play a role in myocardial protection exerted by volatile anesthetics
(10, 40). Combined with the observation that protein
kinase C inhibits cross-bridge cycling rate by phosphorylation of
troponin I (32), this second messenger pathway might be
considered to be involved in the effects observed.
The present findings must be interpreted within the constraints
of several possible limitations. Although ferret papillary muscle is
stable for many hours (3-5, 17, 19-21), we
experienced a significant decrease of developed force from 55.5 mN/mm2 in the first control measurement to 50.0 mN/mm2 in the washout measurement of the last anesthetic
~5 h later. Because anesthetics were studied in random order, this
should not markedly confound the results.
Second, as mentioned earlier, in the presence of a high amount of
series and parallel elastic elements and nonuniformities in intact
muscle preparations, limitations might exist to deduct cross-bridge
mechanical properties from whole muscle measurements (8).
An alternative to the intact papillary muscle is the use of skinned
fiber preparations (26, 29, 42), in which effects on
myofilaments, practically anatomically isolated, can be tested. The
studies by Prakash et al. (29) and Murat et al.
(26) support our findings that volatile anesthetics affect
cross-bridge kinetics.
A third limitation of our study is that we did not determine frequency
dependence of stiffness. Dynamic transfer function of stiffness
(42) and frequency at which stiffness is minimal (6,
30, 31) would give valuable information about dynamics and
cycling rates of cross bridges. But those methods can only be used in
muscle preparations in which steady-state activation of the contractile
system is carried out, either in skinned fiber preparations or in
intact tetanized or Ba2+-activated muscle.
In conclusion, volatile anesthetics exert a direct effect on
cross-bridge performance, which becomes apparent during isometric relaxation (halothane and sevoflurane) and as shown recently during isotonic contraction (halothane and isoflurane) in intact cardiac muscle tissue, an effect that might contribute significantly to their
depressant action on the heart.
| |
ACKNOWLEDGEMENTS |
We thank Laurel Wanek and Jonathan Nesbitt for outstanding
technical support.
| |
FOOTNOTES |
This work was supported by grant GM-36365 (to P. R. Housmans) from
the National Institutes of General Medical Sciences, Bethesda, MD, and
by the Mayo Foundation, Rochester, MN.
Address for reprint requests and other correspondence:
P. R. Housmans, Dept. of Anesthesiology, 2-752 MB, Mayo
Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail:
housmans.philippe{at}mayo.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 15, 2002;10.1152/japplphysiol.00841.2001
Received 10 August 2001; accepted in final form 9 February 2002.
| |
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ABSTRACT
INTRODUCTION
