Download Excitation-contraction Coupling in the Heart and the Negative

䡵 REVIEW ARTICLE
David C. Warltier, M.D., Ph.D., Editor
Anesthesiology 2004; 101:999 –1014
© 2004 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.
Excitation-contraction Coupling in the Heart and the
Negative Inotropic Action of Volatile Anesthetics
Peter J. Hanley, M.B.Ch.B., Ph.D.,* Henk E.D.J. ter Keurs, M.D., Ph.D.,† Mark B. Cannell, Ph.D.‡
IN a comprehensive review in 1987, Rusy and Komai1
discussed the possible mechanisms by which volatile
anesthetics inhibit cardiac contraction. Since that time,
there have been advancements in the understanding of
excitation-contraction coupling, cardiac mechanics, and
the actions of volatile anesthetics. There are essentially
three major factors that determine the force of contraction of heart muscle cells: the magnitude of cytosolic
Ca2⫹ increase after electrical excitation, the responsiveness of the contractile proteins to Ca2⫹, and the sarcomere length (SL) at which the contractile proteins are
activated. Hence, there are two possible ultimate direct
negative inotropic actions of volatile anesthetics: a reduction in Ca2⫹ availability or a decrease in the Ca2⫹responsiveness (Ca2⫹-sensitivity or maximal Ca2⫹-activated force) of the contractile apparatus. The rate of
relaxation of the muscle cells, on the other hand, depends on the rate at which Ca2⫹ is cleared from the
cytosol, which facilitates its dissociation from the regulatory proteins of the contractile system. The control of
calcium cycling and the activity of the contractile proteins consume energy that must be continuously supplied by the mitochondria, another potential site volatile
of anesthetic action.
(pressure) generating myocardial cells within the ventricles. Rapid depolarization in myocytes is mediated by voltage-gated Na⫹ channels, different subtypes of which are
located in the transverse tubules (Nav1.1, Nav1.3, and
Nav1.6) and intercalated disks (Nav1.5).2 The depolarization of the cell leads to the activation of L-type Ca2⫹ channels, which are primarily encoded by the ␣1C gene
(Cav1.2),3 and are the next key element in cardiac excitation-contraction coupling. In addition, other ionic currents,
such as that attributable to Na/Ca exchange, as well as
chloride and potassium currents, all shape the action potential, whose duration is ⵑ300 ms. The action potential
spreads from cell to cell, a process that is facilitated by
Nav1.5 channels (in the intercalated disks) and extensive
gap junctions between cells, each of which is connected to
ⵑ15 of its neighbors.4 In ventricular cells, the action potential passes into transverse tubules (t-tubules) that serve
to minimize delay in excitation throughout the cell.5
Surface membrane depolarization promotes the influx of
Ca2⫹ via the voltage-gated L-type (also called dihydropyridine-sensitive) Ca2⫹ channels and possibly the Na/Ca exchanger (NCX). In the case of the L-type Ca2⫹ channel,
Ca2⫹ influx is limited by both Ca2⫹-dependent inactivation
as well as voltage-dependent and time-dependent inactivation.6 –9 Ca2⫹-dependent inactivation depends on both the
Ca2⫹ that enters the cell via the channel itself (e.g.,
Bechem and Pott10) and Ca2⫹ release from intracellular
stores.11,12 The intracellular mechanism of Ca2⫹-dependent
inactivation appears to involve calmodulin.13,14
Although Ca2⫹ influx via L-type calcium channels will
increase intracellular Ca2⫹ directly by a small amount,
the influx of Ca2⫹ is normally amplified by a larger
release of Ca2⫹ from the sarcoplasmic reticulum (SR) in
a process known as Ca2⫹-induced Ca2⫹ release (CICR).15
This mechanism resides in the Ca2⫹-dependent gating of
ryanodine-sensitive Ca2⫹-release channels in the SR (ryanodine receptors; RyRs) and of the three isoforms
known, the RyR2 isoform predominates in heart (for
review of junctional proteins, see Muller et al.16). Because activation of RyRs depends only on an elevation in
intracellular Ca2⫹, any source of Ca2⫹ could, in principle, activate CICR.
Part I: Excitation-contraction Coupling and
the Contractile Machinery
A diagram of cardiac excitation-contraction coupling is
shown in figure 1. Action potentials, initiated by sinoatrial
nodal cells, are rapidly conducted throughout the heart,
facilitated by the His-Purkinje system, and activate the force
* Research Fellow, Institut für Normale und Pathologische Physiologie, Universität Marburg, Marburg, Germany. † Professor of Medicine, Physiology and
Biophysics, University of Calgary, Calgary, Canada. ‡ Professor of Physiology,
University of Auckland, Auckland, New Zealand.
Received from the Institut für Normale und Pathologische Physiologie, Universität Marburg, Marburg, Germany, the Departments of Medicine, Physiology
and Biophysics, University of Calgary, Calgary, Canada, and the Department of
Physiology, University of Auckland, Auckland, New Zealand. Submitted for publication February 11, 2004. Accepted for publication June 3, 2004. Support was
provided solely from institutional and/or departmental sources.
Address reprint requests to Dr. Hanley: Institut für Normale und Pathologische
Physiologie, Universität Marburg, Deutschhausstrasse 2, 35037 Marburg, Germany.
Address electronic mail to: [email protected]. Individual article reprints
may be purchased through the Journal Web site, www.anesthesiology.org.
Anesthesiology, V 101, No 4, Oct 2004
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HANLEY ET AL.
Fig. 1. Excitation-contraction coupling in
a ventricular myocyte. Excitation of the
sarcolemma and t-tubules by an action
potential leads to activation of L-type
Ca2ⴙ channels and Ca2ⴙ-induced Ca2ⴙ release. The resulting transient elevation of
cytosolic Ca2ⴙ serves to activate the contractile apparatus, producing contraction. Removal of Ca2ⴙ from the cytosol by
various Ca2ⴙ transporters facilitates mechanical relaxation.
Alternative Trigger of CICR
The NCX, which serves as the primary route for Ca2⫹
extrusion at rest,17,18 can be reversed during depolarization as a result of the action potential to bring Ca2⫹ into
the cell. This Na/Ca exchange “reverse mode” Ca2⫹
influx has also been implicated in triggering CICR.19 –21
Although this possibility was first demonstrated under
pathologic conditions of calcium overload, where the
cell is highly sensitive to trigger calcium concentrations,19 subsequent experiments showed that conditions
which would lead to NCX reversal (such as strong depolarization or increased intracellular Na⫹ concentrations) could also trigger CICR.20 –22 It has even been
suggested that the NCX might be a major source of
trigger Ca2⫹ during the normal action potential,21 although this has been debated.23 Blockade of NCX with
an inhibitory peptide suggested that only up to 27% of
the trigger might be attributable to NCX.22 In addition to
the membrane potential, internal Na⫹ concentrations
will also be critical for determining the NCX contribution.24 During the upstroke of the action potential, Na⫹
influx might increase Na⫹ concentrations locally to promote reverse mode exchange20,25 as well modify SR
Ca2⫹ content.26 Some researchers have been unable to
demonstrate potent triggering of CICR at negative potentials by reverse mode exchange when the Na⫹ current is activated,24,27 and it is likely that a major part of
this controversy resides in the difficulty of obtaining
good voltage control during the Na⫹ current while also
trying to prevent all Ca2⫹ influx via L-type Ca2⫹ channels by pharmacological agents.
Anesthesiology, V 101, No 4, Oct 2004
Colocalization of Dihydropyridine Receptors
and RyRs
The reliability of excitation-contraction coupling is
also dependent on the physical relation between the
proteins that provide/regulate the triggers for CICR as
well as the location of the RyRs that respond to the
trigger signal. The membranes of the t-system and the
terminal cisternae of the SR are closely opposed in junctional areas or “dyads,”28 and such structures will increase the reliability of CICR by limiting diffusional loss
of the trigger Ca2⫹ signal within them.29 –31 Although the
dihydropyridine receptors (L-type Ca2⫹ channels) and
the RyRs (Ca2⫹-release channels) are colocalized,32,33 a
combination of immunofluorescence and data deconvolution techniques have led to the suggestion34 that neither voltage-gated Na⫹ channels nor the NCX are located
in the dyad. On the contrary, more recent immunolocalization data from Thomas et al.35 suggest that the NCX is
indeed concentrated in t-tubules.
Elementary Ca2⫹ Release Events: Ca2⫹ Sparks
Over the past decade new insights into the basic
events underlying CICR have been obtained by application of confocal microscopy and fluorescent calcium
indicators. Such experiments have shown that the close
opposition of the dihydropyridine receptors and the
RyRs enables local control of Ca2⫹ release into the cytosol as proposed by Stern.36 In this model, it is not the
global cytosolic concentrations of Ca2⫹ (or any other
messenger) that are important but rather the microenvironment around the RyR. The importance of local
rather than global signals is based on the idea that most
NEGATIVE INOTROPIC EFFECTS OF VOLATILE ANESTHETICS
Fig. 2. Elementary Ca2ⴙ release events (Ca2ⴙ sparks) in a ventricular myocyte. The top panel shows confocal images (x,y
scans) of a myocyte obtained using a confocal laser scanning
microscope. The local regions of increased fluo-3 fluorescence
are Ca2ⴙ sparks. The middle panel shows a Ca2ⴙ spark recorded
using the line-scan (x,t) mode of imaging.
biologic processes are inherently nonlinear. Thus, in
terms of cardiac excitation-contraction coupling, we
must consider the microscopic environment produced
by the local activation of trigger mechanisms (such as
L-type Ca2⫹ channels) and the behavior of a local cluster
of RyRs in their vicinity. The discovery of “Ca2⫹ sparks”
or microscopic release events resulting from the local
coactivation of a cluster of RyRs5,37,38 has reinforced this
idea. Ca2⫹ sparks are seen both as spontaneous and
evoked microscopic SR release events inside the cell.
The latter generally require the use of Ca2⫹ channel
antagonists to reduce the probability of spark activation
so that individual sparks can be observed.37,39 From the
site of release (near z-lines) the Ca2⫹ spark diffuses to
cover a region of the cell approximately 2 ␮m in diameter5 with a slightly greater spread in the longitudinal
direction40 (fig. 2). When measured at room temperature, the Ca2⫹ spark reaches a peak in ⵑ10 ms and lasts
Anesthesiology, V 101, No 4, Oct 2004
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ⵑ40 – 80 ms and peak Ca2⫹ during the spark is typically
200 – 400 nM. The decay of the Ca2⫹ spark is a result of
both Ca2⫹ diffusion and SR uptake.41 When sparks are
activated by depolarization, they summate to produce a
larger and slower Ca2⫹ transient, and the reduced rate of
decline of Ca2⫹ can be ascribed to the loss of the diffusive component of spark decay; during the whole-cell
Ca2⫹ transient Ca2⫹ is globally increased so diffusion
cannot serve to reduce Ca2⫹.5
From studying Ca2⫹ sparks we now know that excitation-contraction coupling in the heart is attributable to
the spatio-temporal summation of a very large number of
“elementary” Ca2⫹ sparks.37,42,43 It has been estimated
that during excitation-contraction coupling the spontaneous spark rate is increased by a factor of ⵑ10,000 by
a “local” 100-fold increase in Ca2⫹ resulting from the
trigger Ca2⫹ influx.44 Such a local increase in Ca2⫹ is
consistent with some computer simulations of changes
in Ca2⫹ attributable to L-type Ca2⫹ channels.31 Moreover, the latency for SR Ca2⫹ release is ⬍2 ms,45 consistent with the rate of RyR opening expected from such
high local trigger Ca2⫹ concentrations.30 Although
strictly speaking, Ca2⫹ sparks cannot be elemental if
attributable to the concerted activation of a number of
local RyRs in a cluster,40 the fact that evoked Ca2⫹ sparks
appear to have a modal amplitude distribution at fixed
locations within the cell46 suggests that each spark site
behaves in an “all or none” fashion and are therefore
“elementary” in a functional sense (but see Lipp and
Niggli47). In any case, it is generally agreed that coordinated activation of multiple spark sites gives rise to the
global increase in cytosolic [Ca2⫹] that occurs under
normal conditions. More recently, Wang et al.48 have
shown that the local increase in Ca2⫹ concentration
(“sparklet”) produced by an extended (drug modified)
opening of a single L-type Ca2⫹ channel can trigger a
cluster of about 4 – 6 peripheral RyRs, which then produce a spark. On the other hand, from both experimental and theoretical approaches, both Bridge et al.46 and
Soeller and Cannell49 suggest that sparks may in fact
arise from somewhat larger clusters of RyRs, consisting
of ⬎15 receptors. The number of RyRs underlying a
Ca2⫹ spark is important because it sets limits on the
degree to which different amounts of Ca2⫹ may be
released by modulation of RyR open probability as well
as the “safety factor” inherent in RyR activation.
SR Ca2⫹ Content
The amount of Ca2⫹ released into the cytosol depends
not only on the magnitude of the Ca2⫹-influx current
(the trigger for Ca2⫹ release) but also on the SR Ca2⫹
content.50,51 For a given concentration of trigger Ca2⫹,
the Ca2⫹ release increases as a function of SR Ca2⫹
content. High SR luminal [Ca2⫹] appears to increase the
open probability of the RyRs,52,53 which may explain
why transient inward currents, afterdepolarizations, and
1002
aftercontractions are often seen when the SR contains a
high Ca2⫹ load. Under steady state conditions (constant
heart rate and constant neurohumoral input), the magnitudes of Ca2⫹ influx and efflux across the sarcolemma
are balanced, and thus there is no net change in mean SR
Ca2⫹ content. After ␤1-receptor stimulation, for example, L-type Ca2⫹ current as well as SR Ca2⫹-pump activity
increases, resulting in net Ca2⫹ influx over successive
contraction cycles that loads Ca2⫹ into the SR. The
increase in Ca2⫹ current (trigger Ca2⫹) and SR Ca2⫹
store induced by ␤1-receptor stimulation gives rise to
larger Ca2⫹ transients and therefore more contractile
protein activation. However, it is still unclear whether
this is accompanied by an increase in the number of
Ca2⫹ sparks as well as individual spark amplitude.
Ca2⫹ Transport Systems
The increase in cytosolic [Ca2⫹] and the accompanying force development after electrical activation is transient because Ca2⫹ is rapidly removed from the cytosol
after release. There are four main Ca2⫹ transport systems
that remove Ca2⫹ from the cytosol: the SR Ca2⫹-pump
(sarco-endoplasmic reticular Ca2⫹-adenosine triphosphate [ATP]ase), the sarcolemmal Ca2⫹-pump (Ca2⫹-ATPase), the NCX, and the mitochondrial Ca2⫹-uniporter
(fig. 1). Although the relative contributions of these
systems is species-dependent, the predominant Ca2⫹
transport systems are the SR Ca2⫹-ATPase and the
NCX.50,54,55 The activity of the SR Ca2⫹-ATPase can be
increased by cAMP-dependent phosphorylation of the
endogenous inhibitor phospholamban,56 whereas the
activity of the NCX can be modulated by phosphorylation via protein kinase C.57 The NCX exchanges 3 Na⫹
for 1 Ca2⫹, and therefore it is electrogenic. Hence, its
direction (Ca2⫹-influx versus Ca2⫹-efflux mode) is determined by the prevailing transmembrane gradients for
Na⫹ and Ca2⫹, as well as membrane potential.58,59 Indeed, the NCX may transiently reverse after membrane
depolarization, especially during the peak of the action
potential. It should be reiterated that the magnitude and
direction of the NCX is strongly (in a cubic fashion)
dependent on intracellular [Na⫹], as discussed in detail
by Cooper et al.58 (see also Evans and Cannell24). Hence,
the activity of the Na pump (Na⫹,K⫹-ATPase) plays a
critical role in determining the Gibbs free energy of the
exchanger. It should also be noted that activation of the
exchanger after spontaneous Ca2⫹ release after the Ca2⫹
transient (in Ca2⫹ overloaded cells60 or in nonuniform
cardiac muscle61) will lead to current flow across the
membrane which can lead to after depolarizations sufficiently large to produce action potentials and
extrasystoles.
Activation of the Contractile System
Activation of the contractile system by Ca2⫹ is mediated by its binding to the regulatory protein troponin C
Anesthesiology, V 101, No 4, Oct 2004
HANLEY ET AL.
(TnC).62 The contractile proteins (myofibrils), which
occupy around 60% of the cell volume,63,64 consist of
thick (myosin) and thin (actin) filaments which are arranged between z-lines to form sarcomeres, the repeating units of the myofibrils (fig. 1). The thin filaments are
ⵑ1 ␮m in length and protrude from anchoring points on
the z-line.65,66 Tropomyosin lies in the groove between
the two actin chains of the thin filament and its movements are regulated by the Ca2⫹-sensitive troponin complex.67 Interdigitating with the thin filaments are the
thick filaments, the globular heads of which form the
cross-bridges which interact with the thin filament and
contain actomyosin-ATPase.
The troponin complex has three subunits: TnC (the
Ca2⫹-binding subunit), troponin T (the tropomyosinbinding subunit), and troponin I (the inhibitory subunit).
Compared with its skeletal counterpart, the cardiac troponin I isoform has a longer amino terminus containing
two phosphorylation sites that are substrates for cAMPdependent protein kinase A.68 When troponin I is
bisphosphorylated, the Ca2⫹ off-rate of TnC is increased,
which enhances the relaxation rate of a contraction.68,69
Cardiac TnC has three Ca2⫹-binding sites, unlike the
skeletal isoform, which has four.56,70 Two of the sites
bind both Ca2⫹ and Mg2⫹, whereas the other site specifically binds Ca2⫹ and therefore serves as the regulatory site. In resting cardiac muscle, cytosolic [Ca2⫹] is
low (ⵑ70 nM)71 and the Ca2⫹-specific site of TnC (with
a dissociation constant of ⵑ500 nM)55 is unoccupied.72
In this state, tropomyosin, lying in the groove of the thin
filament, prevents interaction of the myosin heads
(cross-bridges) with actin (but see Perry73). When cytosolic [Ca2⫹] increases, after membrane depolarization
and CICR, Ca2⫹ binds to TnC, which causes tropomyosin to move out of the actin groove, thereby allowing
myosin to interact with actin, producing force or shortening (for review, see Perry73).
The mechanism by which myosin interacts with actin
and uses the energy from ATP hydrolysis to produce
mechanical work is known as the “cross-bridge theory of
sliding filaments” and was first proposed in 1957 by
Huxley.74 This mechanism has been extensively reviewed.70,75 As long as Ca2⫹ is bound to TnC, the myosin
head can form cross-bridges with nearby binding sites on
the thin filament. The generation of force or the relative
sliding of the filaments is thought to be brought about by
rotation of the head, the swinging cross-bridge model.76,77 The biochemical steps that drive cross-bridge
cycling (hydrolysis of ATP via actomyosin-ATPase) have
been largely characterized.78 Basically, ATP binds to myosin, causing the globular head domain to detach from
actin, and its subsequent hydrolysis sets the myosin head
into a high energy state. Ishijima et al.79 have simultaneously measured the mechanical events and actomyosin-ATPase activity of a single-headed myosin molecule
interacting with an actin filament. Under these artificial
NEGATIVE INOTROPIC EFFECTS OF VOLATILE ANESTHETICS
1003
Fig. 3. Force-[Ca2ⴙ] relations of trabeculae at various sarcomere
lengths (SLs). Note that there is a shift of the [Ca2ⴙ]-activation
curve to the left, indicating that Ca2ⴙ-sensitivity of the contractile proteins increases as a function of SL. Adapted with permission from Kentish et al.84
conditions, force generation did not always coincide
with the release of adenosine diphosphate from the
myosin head (as previously thought) but could occur
several hundred milliseconds after release. The authors
proposed that the mechanical and biochemical events of
cross-bridge cycling are not tightly coupled in that a
myosin head can undergo several conformational
changes during a single actomyosin-ATPase cycle. However, it should be stressed that the exact nanomechanics
and stoichiometry of cross-bridge cycling still remain to
be elucidated.80
Responsiveness of the Contractile Proteins to Ca2⫹
Although intracellular [Ca2⫹] is the major determinant
of force at any given SL, the development of force is also
dependent on the responsiveness of the contractile proteins to Ca2⫹. Responsiveness refers to the relation between force and intracellular [Ca2⫹] (fig. 3), often expressed as pCa (⫺log10[Ca2⫹]). The pCa-force relation
can be modulated by various factors such as ionic
strength, temperature, pH, [Pi], SL (fig. 3), and phosphorylation state of the contractile apparatus.56 The
[Ca2⫹] at which force is half-maximal provides an index
of ‘Ca2⫹-sensitivity’ of the contractile system whereas
the force obtained at TnC-saturating [Ca2⫹] gives “maximal Ca2⫹-activated force.” Ca2⫹ binding to TnC is cooperative, that is, the binding of one Ca2⫹ ion facilitates
the binding of the next one. This gives rise to a steep
pCa-force relation.
The relation between intracellular [Ca2⫹] and force
has been well characterized using chemically or mechanically “skinned” muscle preparations. Skinning renders
the sarcolemma freely permeable to ions and small molecules, and thus this technique allows the intracellular
environment to be well controlled. However, Marban et
al.81 showed that the relation between intracellular
[Ca2⫹] and force is strikingly steeper in intact cardiac
muscle than is reported for skinned muscle preparations.
This apparent difference was reexamined by Gao et al.,82
who determined the intracellular [Ca2⫹]-force relation
Anesthesiology, V 101, No 4, Oct 2004
Fig. 4. Passive force-SL relations of rat cardiac trabeculae obtained after (A) and before (B) skinning. Note that passive force
at SLs above 2.2 ␮m is reduced after skinning. (Modified from
Kentish et al.84 with permission) At near-resting SL, the perimysial collagen fibers (reconstructed in 3-D by confocal laser scanning microscopy) of cardiac trabeculae are wavy (C), whereas
they become straight at SLs of approximately 2.3 ␮m (D), corresponding to the steep portion of the force-SL relation.
Adapted with permission from Hanley et al.200
before and after skinning in the same muscle preparation
(rat cardiac trabecula). The [Ca2⫹] required for halfmaximal force was considerably higher after skinning,
suggesting that the skinning procedure may damage or
remove components involved in the regulation of actinmyosin interaction. Hence, data obtained from experiments employing skinned muscle preparations cannot
be readily extrapolated to the intact system.
Length-dependence of Passive and Ca2⫹-activated
Force
One of the major determinants of force is the SL at
which the contractile proteins are activated83,84 (fig. 3).
Typically, cardiac myocytes have a slack (resting) length
of ⵑ1.9 ␮m and can be extended to SLs of up to ⵑ2.3–
2.4 ␮m. In intact cardiac muscle, twitch force (or stress,
which is force/cross-sectional area) increases steeply as a
function of SL (the Frank-Starling mechanism). This
1004
steep increase in force as a function of SL is thought to
be attributable largely to a length-dependent increase in
the sensitivity of the contractile system to Ca2⫹, mediated by increased affinity of Ca2⫹ binding to TnC.62,72 An
increase in overlap of thick and thin myofilaments, a
reduction in interfilament lattice spacing,62,85 and an
increase in intracellular Ca2⫹ release86,87 are thought to
contribute to the increase in force when SL is increased.
The exact mechanism by which an increase in SL
causes an increase in myofilament Ca2⫹ responsiveness
remains elusive. Fuchs and Smith72 argue that the sensitivity of the contractile system to Ca2⫹ is governed by
myofilament lattice spacing rather than muscle length. It
has been shown by radiographic diffraction that interfilament spacing decreases as SL increases in both intact
and skinned cardiac muscle.88 Fitzsimons and Moss85
previously proposed that decreased interfilament spacing increases force development by increasing the probability that myosin will form strong cross-bridges with
actin. Recent work from Konhilas et al.,89 suggests that
changes in lattice spacing may not underlie length-dependent activation. These authors found that osmotic
compression of the myofilament lattice spacing, which
was measured by radiographic diffraction, did not alter
Ca2⫹-sensitivity in skinned cardiac trabeculae. Hence,
the sensor for SL changes probably does not reside in the
interfilament space. The protein titin (also called connectin), which spans the entire sarcomere and interacts
with the thick and thin filaments,90 is in a good position
to sense SL changes, but whether this protein signals
length changes to the regulatory proteins of the thin
filament is not known.91,92
Extension of cardiac muscle beyond a SL that optimizes actin-myosin interaction (a SL of 2.2–2.4 or so,
depending on species), is prevented by the development
of large parallel elastic forces,84,93,94 as shown in figs. 4A
and 4B. The straightening of perimysial collagen fibers,
which are thought to be the dominant contributors to
passive force at SL ⬎2.2 ␮m,95 is presumed to underlie
this end-stop effect (figs. 4C and 4D).
At SLs ⬍ 2.1 ␮m, where passive force is less than 5% of
maximal twitch force, titin overshadows collagen as the
main contributor (⬎90%) to passive force.95 This large
protein, sometimes referred to as the third filament of
the contractile apparatus, is closely associated with the
thick filaments in the A-band region.96,97 The elasticity of
titin resides in its I-band portion, which spans from the
z-line to the tip of the myosin filament.98 This elastic
(I-band) segment of titin is shorter in cardiac muscle than
in skeletal muscle and may explain why titin-based passive force is greater in cardiac than most skeletal muscle
at a given SL. In addition to acting as a molecular spring,
titin may also contribute to the viscoelastic properties of
the sarcomeres by interacting with the thin filament.71,98,99
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HANLEY ET AL.
Fig. 5. Confocal fluorescence image of mitochondria in a living,
intact ventricular myocyte of guinea pig (A) (P. J. Hanley, unpublished data). Mitochondria (and a nucleus) were labeled
with the fluorescent indicator rhod-2. Note the highly ordered
distribution of mitochondria inside the cell. (B) Diagram showing that the mitochondria are spatially arranged within the
intermyofibrillar spaces between the t-tubules.
Cardiac Energy Metabolism and Mitochondrial
Function
Cardiac myocytes, the functional unit of the heart
(accounting for ⬎90% of heart volume), require a continual supply of free energy to perform mechanical
work, synthesize various molecules, and do electrochemical work (maintain ion gradients across membranes). This free energy comes from the oxidation of
metabolic substrates such as long-chain fatty acids and,
to a lesser extent, glucose and lactate.100 –102 Mitochondria occupy ⵑ30% of the myocyte volume4,63,64 and
have a highly ordered distribution in the living myocyte,
lying in the interfibrillar space between z-lines (fig. 5).
Oxygen is the ultimate acceptor of electrons when metabolic substrate is oxidized. Electrons are transferred to
oxygen via the electron transport chain, a series of
proton pumps and electron carriers located in the mitochondrial inner membrane. A proton gradient is established across the mitochondrial inner membrane, giving
rise to a potential of ⵑ200 mV, as electrons are transferred along the various complexes of the electron transport chain. The energy stored in this electrochemical
NEGATIVE INOTROPIC EFFECTS OF VOLATILE ANESTHETICS
gradient is used to drive the synthesis of the energy carrier
molecule ATP. Mitochondrial matrix ATP is then tranported via the ATP-adenosine diphosphate translocase to
the cytosol where, via the creatine-creatine phosphate
shuttle system,103 it serves as a readily accessible source of
free energy for mechanical and electrochemical work.
Ultimately, all the energy used by the myocyte to
maintain its structure and function is degraded to heat.
From studies in which the rate of heat production of
cardiac trabeculae of guinea pig was measured under
various conditions, Schramm et al.104 deduced that actomyosin-ATPase, Ca2⫹-ATPase, and Na⫹,K⫹-ATPase accounted for 76%, 15%, and 9%, respectively, of the overall rate of ATP turnover. In accord with these results,
Ebus and Stienen105 measured the rate of ATP hydrolysis
in saponin-skinned trabeculae of rat and showed that
approximately 15% of maximal Ca2⫹-activated ATPase was
membrane-bound, around two thirds of which was attributable to the sarco-endoplasmic reticular Ca2⫹-ATPase. The
source of the heart’s extraordinarily high basal rate of
metabolism, which accounts for 25–30% of the energy
expenditure of the beating heart, remains unknown (for
review, see Gibbs and Loiselle106).
Mitochondrial Ca2⫹ Uptake
Mitochondrial Ca2⫹ influx occurs via a ruthenium-red
sensitive Ca2⫹-uniporter107 (fig. 1), whereas its efflux is
mediated by a Na/Ca exchange mechanism (and, indirectly, Na⫹-H⫹ exchange), the stoichiometry of which
has not been established.108 Ca2⫹ influx into the mitochondria is thought to regulate metabolism because an
increase in mitochondrial [Ca2⫹] stimulates ATP synthase109 and the activity of pyruvate dehydrogenase and
␣-ketoglutarate dehydrogenase, enzymes linked with the
tricarboxylic acid cycle.110
Recent evidence suggests that cytosolic Ca2⫹ signals
are probably communicated directly to the mitochondria. After selectively loading the fluorescent Ca2⫹ indicator rhod-2 into the mitochondria of electrically stimulated ventricular myocytes of rabbit, Trollinger et al.111
were able to demonstrate mitochondrial Ca2⫹ transients
using confocal microscopy. In accord, in work with
patch-clamped ferret or cat ventricular myocytes, Zhou
et al.112 were also able to detect mitochondrial Ca2⫹
transients during a twitch, albeit only when the cytosolic
resting [Ca2⫹] exceeded physiologic concentrations.
Compared with the cytosolic Ca2⫹ transient, the kinetics
of the mitochondrial Ca2⫹ transient were much slower.
Griffiths,113 using cardiac myocytes and techniques to
load indo-1 (a fluorescent Ca2⫹ indicator) selectively into
mitochondria, found that mitochondrial transients accompanied twitches in guinea pig but not rat, suggesting
that there may be species-differences in mitochondrial
Ca2⫹ cycling. It should be noted that the surface area of
the mitochondrial inner membrane is 10-fold higher than
the area of the sarcolemma and t-tubule system.56 Hence,
Anesthesiology, V 101, No 4, Oct 2004
1005
small changes in mitochondrial Ca2⫹ permeability could
greatly influence Ca2⫹ distribution in the cell.
Neurohumoral Regulation of Contraction
Although neurohumoral regulation is outside the
scope of this review (see, for example, Morris and
Malbon114 or Xiao115), a brief overview of the major
signaling pathways in the myocyte follows. Each myocyte has three major intracellular signaling cascades that
are modulated via G protein-coupled surface receptors,
each of which contains a conserved structure of seven
transmembrane ␣-helices.116,117 The G protein-coupled
receptors regulate the activity of various membranebound enzymes including adenylate cyclase, guanylate
cyclase, and phospholipase C, which produce, respectively, the secondary messengers cAMP, cGMP, and
DAG.118 In each case, the secondary messengers activate
protein kinases (protein kinase A, protein kinase G, and
protein kinase C, respectively) that phosphorylate specific amino acid residues on contractile proteins, ion
channels, and pumps. The adrenergic receptors and the
muscarinic cholinergic receptors are the most important
G protein-coupled receptors in the heart. At least nine
subtypes of adrenergic receptors have been cloned:
three ␣1-receptors, three ␣2-receptors, and three ␤-receptors. When agonist is bound to the receptor the G
proteins dissociate into effector subunits that modulate
the activity of various membrane-bound targets such as
adenylate cyclases, phospholipases, and ion channels.
For example, when agonists bind to ␤1-receptors, the
G␣s subunit dissociates and stimulates adenylate cyclase,
which produces the secondary messenger cAMP, a protein kinase A activator. Protein kinase A phosphorylates
the following: L-type Ca2⫹ channels (which increases
Ca2⫹ influx), phospholamban (which enhances SR Ca2⫹uptake, thereby increasing the rate of relaxation), RyRs
(which may facilitate Ca2⫹ release, a controversial issue),119 troponin I (which reduces filament sensitivity to
Ca2⫹ ions and by itself decreases force but accelerates
relaxation), and myosin binding protein C. The net effect
is positive inotropy and positive lusitropy (increased rate
of relaxation). Stimulation of muscarinic receptors yields
G␣i, which inhibits adenylate cyclase, producing the
opposite effect to adrenergic receptor stimulation.
Part II: Mechanisms of Negative Inotropy
Induced by Volatile Anesthetics
Volatile anesthetics have been shown to inhibit or
stimulate various cellular components such as ion channels, pumps, exchangers, enzymes, gap junctions, and
components of the contractile system, as schematically
illustrated in figure 6. Most studies have shown effects at
moderately high concentrations, suggesting that volatile
anesthetics may perturb lipid bilayers, which would ex-
1006
Fig. 6. Sites of action of volatile anesthetics in a ventricular
myocyte. The red spots indicate inhibitory actions whereas the
green spots indicate stimulatory actions.
plain why these lipophilic agents affect diverse molecular targets. However, the fundamental molecular mechanisms by which these agents inhibit or stimulate various
membrane-bound proteins and the contractile apparatus
remains unclear. For a recent discussion of the possible
mechanisms by which molecules of volatile anesthetics
interact with protein structures, lipid bilayers and various molecular interfaces, see a review by Urban.120
Anesthetic concentrations are usually expressed as
mM, volume%, or their equivalent MAC (minimal alveolar [anesthetic] concentration) value, where 1 MAC is
defined as the minimal alveolar (anesthetic) concentration at one atmospheric ambient pressure required to
prevent movement in response to a noxious stimulus in
50% of animals.121 Anesthetic concentrations corresponding to 1 MAC (in rat) at 37°C, for example, are as
follows: halothane (0.27 mM), isoflurane (0.31 mM), and
sevoflurane (0.35 mM).122 When comparing studies performed at different temperatures, it is important to note
that MAC expressed as volume% varies considerably
with temperature, whereas the equivalent liquid-phase
concentration, typically expressed in mM, changes
little.122
Ultimately, for volatile anesthetics to inhibit cardiac
contractility they must reduce Ca2⫹-availability or decrease the Ca2⫹-responsiveness of the contractile proteins. Although direct actions at the level of the contractile apparatus or components of excitation-contraction
coupling are likely to be more important, volatile anesthetics could also inhibit contractile function indirectly
by impairing mitochondrial energy supply.
Anesthetic Effects on Ca2⫹ Availability
In 1986, Bosnjak and Kampine123 showed that halothane decreased the Ca2⫹ transient in cat papillary musAnesthesiology, V 101, No 4, Oct 2004
HANLEY ET AL.
Fig. 7. Fura-2 fluorescence (top trace), an index of [Ca2ⴙ], and
cell length (bottom trace), an index of contraction, were measured simultaneously in an electrically stimulated rat ventricular myocyte. Application of halothane initially induced a transient increase in the Ca2ⴙ transient and twitch force before both
the Ca2ⴙ signals and contraction decreased. Reproduced with permission from Prof. M. R. Boyett and the Journal of Physiology.
.
cles that had been microinjected with aequorin. These
authors also reported that halothane did not affect resting Ca2⫹ concentrations; however, the luminescence of
aequorin is a quadratic function of [Ca2⫹] and, therefore,
is not a suitable indicator for measuring resting Ca2⫹
concentrations.124 Another potential limitation of aequorin is that its light-emitting properties may be altered
by direct interaction of the anesthetic with the photoprotein,125 although, in favor of aequorin, Housmans and
Wanek126 recently reported that neither halothane nor
isoflurane affected aequorin luminescence in the pCa
range 2– 8. In 1985, the ratiometric fluorescent Ca2⫹
indicators fura-2 and indo-1 were introduced,127 followed by fluo-3;128 these are technically less difficult to
use than aequorin.
A large number of studies using fluorescent Ca2⫹
indicators have corroborated the observations of
Bosnjak and Kampine123 that volatile anesthetics dosedependently decrease the amplitude of the intracellular Ca2⫹ transient in intact cardiac muscle.129 –136
Hence, it is now well established that volatile anesthetics indeed decrease the amount of Ca2⫹ released
into the cytosol after electrical stimulation. Figure 7
shows an example of the inhibitory effect of halothane
on peak twitch intracellular [Ca2⫹], indexed as fura-2
fluorescence ratio. Note that the anesthetic had no
effect on diastolic [Ca2⫹]. Moreover, in multicellular
cardiac muscle preparations, it has also been shown
that, after correcting fluorescent indicator signals for
autofluorescence changes, volatile anesthetics have
no effect on diastolic (resting) Ca2⫹ concentrations.129 –131 We now turn our attention to the anesthetic actions that may be responsible for the decrease
in the cytosolic Ca2⫹ transient.
NEGATIVE INOTROPIC EFFECTS OF VOLATILE ANESTHETICS
1007
Fig. 8. Effect of halothane (A) and isoflurane (B) on whole-cell Ca2ⴙ current (ICa),
measured in guinea pig ventricular mycoytes. Both anesthetics (recordings indicated by filled circles) decreased peak ICa
and enhanced the apparent rate of channel inactivation. Reproduced with permission from Pancrazio.141
Anesthetic-induced Inhibition of L-type Ca2⫹
Current and Shortening of the Action Potential
Studies using the single microelectrode voltage-clamp
technique137–139 suggested that volatile anesthetics inhibit L-type Ca2⫹ channels. Indeed, using the patchclamp technique, Bosnjak et al.140 showed that halothane, enflurane, and isoflurane reversibly decreased
whole-cell Ca2⫹ current (which is predominantly L-type
Ca2⫹ current) in canine ventricular myocytes. In wholecell and cell-attached recordings using a similar preparation (rat myocytes), Pancrazio141 confirmed and extended this work. Halothane (0.9 mM) and isoflurane (0.8
mM) decreased the peak whole-cell Ca2⫹ current by
ⵑ40% and ⵑ20%, respectively (fig. 8). In cell-attached
recordings, the anesthetics decreased both mean open
time and open probability without affecting single-channel conductance. The anesthetics also enhanced the
slow component of inactivation without affecting the
fast component. Inhibition of L-type Ca2⫹ current would
lead to a decrease in SR Ca2⫹ content (an important
determinant of contractility) over subsequent beats.
In addition to exerting a negative inotropic effect,
inhibition of L-type Ca2⫹ current should also manifest
as shortening of the action potential. Indeed, Lynch et
al.137 found that halothane (⬎2%) shortened the action potential, measured in guinea pig papillary muscle. Subsequent studies, using enflurane and isoflurane
in addition to halothane, have confirmed the observations of Lynch et al.137 that anesthetics shorten the
action potential.138,139,142,143 Rithalia et al.144 showed
that action potential duration was decreased to a
greater extent in myocytes isolated from the endocardium than those isolated from the epicardium of rat
heart. These authors speculated that this difference
might account for the greater negative inotropic effect
of halothane on the subendocardium. At lower halothane concentrations, which did not shorten action
potential duration, negative inotropy was still observed by Lynch et al.,137 suggesting that additional
actions underlie the negative inotropic actions of volatile anesthetics. In accord with Lynch et al.,137
Harrison et al.132 showed that the negative inotropic
effect of halothane on rat ventricular myocytes was
similar even when action potential duration was maintained constant by applying voltage clamp.
Anesthesiology, V 101, No 4, Oct 2004
Inhibitory and Stimulatory Effects of Anesthetics on
K⫹ Channels
Halothane and isoflurane,145 as well as sevoflurane,146
have been shown to inhibit inwardly rectifying K⫹ (Kir)
channels in guinea pig ventricular myocytes; that is, at
potentials positive to the equilibrium potential for K⫹,
the anesthetics increased outward current whereas at
more negative potentials inward current was decreased.
Transient outward K⫹ current (Ito), important in the
early phase of action potential repolarization, has also
been shown to be inhibited by halothane, as well as by
isoflurane.147 Moreover, these anesthetics have been reported to inhibit delayed rectifier K⫹ current (IK)148 and
Ca2⫹-activated K⫹ (KCa) channels.149 Unlike KATP channels, which are activated primarily under pathologic
conditions, Kir channels, Ito, IK and KCa channels play
important roles in stabilizing the resting potential or
modifying the shape of the action potential under normal conditions.150
Volatile anesthetics have been reported to exert complex effects on sarcolemmal KATP channels. Using insideout patches excised from rabbit ventricular myocytes,
Han et al.151 found that isoflurane decreased the duration of KATP channel burst activity and increased the
interburst interval without affecting channel kinetics
within a burst. It should be noted that KATP channels
characteristically exhibit rundown (decreasing Po as a
function of time) and show intermittent burst activity
after patch excision.152 These characteristics have been
shown to be fully prevented by endogenous molecules
such as PIP2 (phosphatidylinositol 4,5-bisphosphate) and
long-chain acyl-CoA esters,152 suggesting that burst activity may in fact be restricted to excised patches and,
possibly, pathologic conditions.152 Han et al.151 also
showed that isoflurane shifted the relation between
[ATP] and Po to the left; that is, it decreased the sensitivity of the channel to inhibitory ATP. More recently,
Stadnicka and Bosnjak153 reported that isoflurane facilitates KATP channel opening at reduced pH. Hence, under
physiologic (and pathologic) conditions isoflurane may
facilitate KATP channel activation, which could explain,
at least in part, its ability to shorten the action potential
duration.
Volatile anesthetics have also been shown to confer
ischemic-like preconditioning via putative mitochondrial KATP channels, with downstream signaling medi-
1008
ated by reactive oxygen species.154,155 Cope et al.,156 for
example, demonstrated that halothane, enflurane, and
isoflurane decreased infarction size by about 50% (compared to control conditions) in rabbit hearts subjected to
regional ischemia either in vitro or in situ. In another
study, the cardioprotective effect of isoflurane was
shown to be blocked by 5-hydroxydecanoate, thereby
implicating mitochondrial KATP channels as mediators of
volatile anesthetic-induced preconditioning.155 In support of this conclusion, Nakae et al.157 recently reported
that isoflurane increases the open probability of mitochondrial KATP channels reconstituted in lipid bilayers.
However, there is controversy as to whether mitochondrial KATP channels play a role in preconditioning because the evidence for their involvement rests mainly on
pharmacological foundations.158 –161
Recently, volatile anesthetics have been shown to activate the two-pore domain K⫹ channels TASK and
TREK,162,163 which may be important targets contributing to cerebral depression. Meuth et al.,164 for example,
recently suggested that TASK1 and TASK3 contribute to
the activity of thalamocortical relay neurons involved in
sleep-wake cycles. TASK and TREK channels, which are
weakly expressed in heart compared with the brain, may
play a role in modulating resting membrane potential
and repolarization, such that these channels could contribute to anesthetic-induced shortening of the action
potential. Although the effects of volatile anesthetics on
various K⫹ channels in the heart are unlikely to contribute significantly to the negative inotropy, their effects on
the the action potential may contribute to anestheticinduced arrhythmias.
Anesthetic Effects on SR Ca2⫹ Content and Ca2⫹
Release
In 1990, Herland et al.165 showed that halothane stimulated Ca2⫹ efflux from the sarcoplasmic reticulum of
chemically-skinned cardiac trabeculae. Connelly and
Coronado166 confirmed this observation using an alternative approach. After incorporating SR-rich vesicles
into artificial lipid bilayers, these authors found that
halothane and enflurane, but not isoflurane, increased
open probability of the Ca2⫹-release channel (RyR). In
accord with these findings, Lynch and Frazer167 showed
that halothane, but not isoflurane, enhanced the binding
of [3H]-ryanodine to RYRs (note that ryanodine binds to
RYRs with high affinity when channels are in the open
state). Consistent with activation of the SR Ca2⫹-release
channel, halothane, but not isoflurane, has been shown
to evoke a transient increase in [Ca2⫹] in resting cardiac
trabeculae.129 This transient effect has also been seen in
electrically stimulated myocytes. Harrison et al.132
showed that application of halothane, but not isoflurane,
transiently potentiated the Ca2⫹ transients and accompanying contractions of electrically paced myocytes
(fig. 7).
Anesthesiology, V 101, No 4, Oct 2004
HANLEY ET AL.
Halothane and, to a lesser extent, isoflurane have been
reported to decrease the Ca2⫹ content of the SR of
isolated intact myocytes132,133 (but see Hannon and
Cody136) and intact cardiac trabeculae,130,131 whereas
sevoflurane appears to have no effect.133,136 Moreover,
isoflurane and sevoflurane, but not halothane, were
found to decrease fractional release, the fraction of the
content that is released after stimulation.133,136
Anesthetic Effects on Sarcolemmal NCX
The sarcolemmal NCX is a potentially important target
of volatile anesthetics because stimulation of its Ca2⫹efflux mode (forward mode) would diminish the SR
Ca2⫹ load and thereby decrease contractility. In a radioisotope study using rat myocyte suspensions, Haworth
and Goknur168 found that halothane, isoflurane, and enflurane dose-dependently inhibited the reverse mode,
Na⫹-dependent 45Ca2⫹ influx, of the NCX. This action
could result in less trigger Ca2⫹ for CICR (see Part I).
Anesthetic sensitivity of the predominant forward mode,
Na⫹-induced Ca2⫹-efflux, was not tested. In a more recent study, halothane (1–2 MAC) and sevoflurane (1–2
MAC) were also found to inhibit both the forward and
reverse modes of the NCX in fluo-3 loaded rat cardiac
myocytes.169 On one hand, inhibition of the dominant
forward (Ca2⫹-efflux) mode would be expected to produce positive inotropy whereas, on the other hand,
inhibition of the reverse mode (Ca2⫹-influx) would produce the opposite effect. On balance, the above studies
suggest that inhibition of the NCX would have either no
net inotropic effect or, if anything, increase inotropy. It
is worth noting that volatile anesthetics could exert a
positive inotropic effect indirectly by inhibiting the
Na⫹,K⫹-ATPase,170 which would cause intracellular
[Na⫹] to increase (favoring reverse mode NCX activity).
Anesthetic Inhibition of the Sarcolemmal and SR
Ca2⫹ Pumps (Ca2⫹-ATPases)
After inhibiting the NCX with Na⫹- and Ca2⫹-free solution, Hannon and Cody136 showed that isoflurane and
sevoflurane, but not halothane, decreased the rate of
relaxation of caffeine-induced Ca2⫹ transients in ferret
ventricular myocytes. The authors concluded that isoflurane and sevoflurane inhibited the sarcolemmal Ca2⫹ATPase. However, inhibitory effects of the anesthetic on
the sarco-endoplasmic reticular Ca2⫹-ATPase isoform in
cardiac SR (sarco-endoplasmic reticular Ca2⫹-ATPase
2a)171 could have accounted, at least in part, for the
observed reduction in the rate of Ca2⫹ extrusion. In line
with the interpretation of Hannon and Cody,136 halothane and isoflurane have been reported to decrease
surface membrane Ca2⫹-ATPase activity in erythrocytes172 and neurons.173 In any case, inhibition of the
sarcolemmal Ca2⫹-pump (Ca2⫹-ATPase) would not be
expected to produce a negative inotropic action but
rather to produce positive inotropy secondary to facili-
NEGATIVE INOTROPIC EFFECTS OF VOLATILE ANESTHETICS
1009
tated SR Ca2⫹ loading. On the contrary, inhibitory effects
of volatile anesthetics on sarco-endoplasmic reticular
Ca2⫹-ATPase171,174 could contribute to the depletion of
SR Ca2⫹ stores by volatile anesthetics.
Gap Junctions
Halothane and isoflurane have been shown to uncouple electrically pairs of myocytes, indicating that these
agents block gap junctions.175 In accord, Burt and
Spray176 showed that halothane (2 mM) decreased gap
junction conductance by reducing the number of open
channels without affecting unitary conductance. In further work, He and Burt177 have shown that halothane
gates gap junctions to the closed state, the extent of
which is dependent on anesthetic concentration and the
connexin composition of the channel. Heteromeric
channels were found to be more sensitive to inhibition
by halothane than homomeric channels composed of
either connexin 40 or connexin 43. The ability of halothane to induce uncoupling would not inhibit the contractility of individual myocytes, but it may perturb the
normal spread of excitation, rendering the heart more
prone to arrhythmias.
Anesthetic Effects on the Contractile Machinery and
the pCa-force Relation
In addition to reducing Ca2⫹ availability, volatile anesthetics may also decrease the responsiveness of the contractile proteins to a given amount of Ca2⫹. Bosnjak and
Kampine123 examined the effects of halothane on [Ca2⫹]
(measured with aequorin) and tension, simultaneously
measured in intact papillary muscle. Halothane decreased force proportionately more than peak Ca2⫹,
suggesting that the anesthetic was having a direct inhibitory action at the level of the contractile proteins. In
agreement with Bosnjak and Kampine,123 Housmans178
demonstrated that isoflurane depressed force, even
when the Ca2⫹ transient (measured using aequorin) was
restored to control concentrations by elevating extracellular [Ca2⫹].
Many recent studies using various muscle preparations
(single myocytes and thin ventricular trabeculae) have
established that halothane and other anesthetics do indeed inhibit the Ca2⫹-responsiveness of the contractile
proteins. For example, using thin ventricular trabeculae
(diameter ⬍250 ␮m) loaded with fluo-3, Hanley and
Loiselle129 showed that force remained depressed when
the cytosolic Ca2⫹ transient was restored to control
concentrations by increasing extracellular [Ca2⫹] in the
presence of halothane or isoflurane (fig. 9). Similar results with halothane and isoflurane were obtained by
Jiang and Julian,130,131 who also used rat trabeculae.
Furthermore, Davies et al.133 likewise found that halothane and isoflurane, but not sevoflurane, decreased
Ca2⫹-responsiveness of the contractile proteins in fura2-loaded rat ventricular myocytes. In contrast to Davies
Anesthesiology, V 101, No 4, Oct 2004
Fig. 9. Simultaneous measurement of force and fluo-3 fluorescence, an index of [Ca2ⴙ], in a rat cardiac trabecula. Application
of isoflurane decreased force and [Ca2ⴙ]. Restoration of the Ca2ⴙ
transient amplitude by elevation of external [Ca2ⴙ] did not recover force, indicating that, in addition to decreasing Ca2ⴙ availability, the anesthetic decreased Ca2ⴙ-responsiveness of the
contractile proteins. Adapted with permission from Hanley and
Loiselle.129
et al.,133 Bartunek and Housmans179 found that sevoflurane, in addition to decreasing Ca2⫹-availability, decreased myofibrillar Ca2⫹-responsiveness (see also Graham et al.180).
Volatile anesthetics could depress myofibrillar Ca2⫹responsiveness by decreasing Ca2⫹-sensitivity or maximal Ca2⫹-activated force of the contractile proteins. At
least part of the depressive action of halothane and
isoflurane is attributable to a reduction in maximal Ca2⫹activated force because Hanley and Loiselle129 showed
that these agents decreased maximum force when trabeculae were tetanized in the presence of ryanodine and
high extracellular [Ca2⫹] (fig. 10). Whether volatile anesthetics shift the pCa-force relation in “intact” cardiac
muscle remains to be elucidated.
Studies using chemically or mechanically skinned cardiac muscle preparations have shed light on the direct
effects of volatile anesthetics on the contractile proteins.
Halothane, enflurane, and isoflurane have been shown to
decrease maximal Ca2⫹-activated force in detergentskinned human181 and rat cardiac fibers.182 Herland et
al.183 found that high doses of halothane, but not enflurane or isoflurane, decreased maximal Ca2⫹-activated
force in mechanically disrupted or saponin-skinned rat
cardiac muscle preparations. When membranes were
further disrupted with Triton X-100, maximal Ca2⫹-activated force was decreased by both halothane and isoflu-
Fig. 10. Halothane and isoflurane decrease maximal Ca2ⴙ-activated force. Ryanodine-induced tetani were used to obtain maximal Ca2ⴙ-activated force in a trabecula (A). In the presence of
halothane or isoflurane, maximal Ca2ⴙ-activated force was decreased (B). Reproduced with permission from Hanley and
Loiselle.129
1010
rane but not by enflurane. Using Triton X-100-skinned rat
cardiac muscle, Prakash et al.184 found that halothane
and sevoflurane, at 1 or 2 MAC, decreased maximal
Ca2⫹-activated force. Hence, the majority of data using
skinned cardiac muscle, as well as the study of Hanley
and Loiselle129 using intact trabeculae, indicate that volatile anesthetics decrease maximal Ca2⫹-activated force.
The next question is whether volatile anesthetics decrease the Ca2⫹-sensitivity (pCa for half-maximal activation) of the contractile proteins. Studies using skinned
fibers have produced conflicting results. Murat et al.182
reported that halothane, enflurane, and isoflurane each
increased the [Ca2⫹] for half-maximal activation in
skinned cardiac fibers. In accord, Tavernier et al.181
found that halothane and isoflurane, each at 1 MAC,
decreased Ca2⫹-sensitivity of the contractile proteins.
However, Herland et al.183 found that the effect of anesthetics on Ca2⫹-sensitivity depended on the method of
skinning employed. When the muscle was mechanically
skinned or treated with saponin, sensitivity was increased by the anesthetics, whereas addition of Triton
X-100 abolished the sensitizing effects of halothane and
isoflurane and reduced the effect of enflurane. Hence,
the disparate observed effects of anesthetics on Ca2⫹sensitivity of the contractile proteins may reflect differences in technique, including differences in skinning
procedure, species, muscle preparation, and anesthetic
concentrations. Further studies using intact muscle preparations and graded tetani may help resolve whether
volatile anesthetics decrease, increase, or have no effect
on Ca2⫹-sensitivity of the contractile system. It should be
noted that force will be reduced at any given [Ca2⫹]
when sensitivity is unchanged but maximal Ca2⫹-activated force is reduced.
Anesthetic Effects on Cross-bridge Cycling
Murat et al.185 examined the effects of volatile anesthetics on force and stiffness during rapid length perturbations at controlled levels of contractile activation.
Halothane, enflurane and isoflurane each decreased active stiffness, increased the stiffness/force ratio and increased the time constant of force recovery. These findings indicated that the anesthetics decreased the number
of force-generating cross-bridges, decreased the force
per cross bridge and reduced the rate of actomyosinATPase activity. Halothane and isoflurane, as well as
sevoflurane, were also found to decrease dynamic stiffness, as well as shortening amplitude, in intact ferret
papillary muscles.186 In further work, Hannon et al.,135
using intact papillary muscles and ryanodine-induced
tetani, found that isoflurane shifted the relation between
[Ca2⫹] and rate of tension redevelopment toward higher
[Ca2⫹], suggesting that the anesthetic reduced the rate
of cross-bridge turnover. Thus, direct inhibitory effects
of volatile anesthetics at the level of actin-myosin protein
Anesthesiology, V 101, No 4, Oct 2004
HANLEY ET AL.
Fig. 11. Mitochondrial sites of action of volatile anesthetics.
Halothane, isoflurane, and sevoflurane partially inhibit complex I of the electron transport chain. Halothane is also a weak
inhibitor at complex II, whereas N2O (which is commonly coadministered with volatile anesthetics), like NO, has been reported to exert an inhibitory action at complex IV. Reproduced
with permission from Hanley et al.194
interaction may account for the ability of these agents to
decrease maximal Ca2⫹-activated force.
Inhibitory Effects of Anesthetics on Mitochondrial
Function
Volatile anesthetics have long been known to impair
nicotinamide adenine dinucleotide
(NADH) oxidation by mitochondria,187–190 suggesting
that these agents may inhibit complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain.
Using isolated liver mitochondria, Hall et al.190 showed
that halothane inhibited NADH oxidation at concentrations that did not affect succinate oxidation. Consistent
with inhibition of NADH oxidation, halothane and isoflurane,129,191,192 as well as sevoflurane,193 have been
shown to increase NADH fluorescence in intact cardiac
muscle.
Recently, Hanley et al.,194 using heart submitochondrial particles, have shown that halothane, isoflurane,
and sevoflurane indeed inhibit the activity of NADH:
ubiquinone oxidoreductase (complex I), which accounts for about 40% of the proton pumping capacity of
the respiratory chain (fig. 11). Halothane also inhibited,
albeit less potently, succinate dehydrogenase, the catalytic component of complex II (fig. 11). At concentrations equivalent to 2 MAC, halothane and isoflurane
decreased activity of complex I by ⵑ20%, whereas
sevoflurane decreased activity by ⵑ10%. Volatile anesthetics are usually administrated in combination with
nitrous oxide (N2O), which has itself been reported to
inhibit the respiratory chain, albeit at complex IV (cytochrome c oxidase).195,196
Could inhibitory effects on electron transport chain
activity account for the negative inotropic action of
volatile anesthetics? In 31P nuclear magnetic resonance
studies, 1.5% halothane (2 MAC) was shown to not
decrease creatine phosphate or ATP concentrations.197,198 Moreover, the observed ⵑ50% decrease in
rate pressure product (an index of cardiac mechanical
work) induced by halothane was not accompanied by an
increase in the concentrations of Pi (inorganic phos-
NEGATIVE INOTROPIC EFFECTS OF VOLATILE ANESTHETICS
phate) or H⫹, inhibitors of excitation-contraction coupling and the contractile apparatus,94 indicating that
impairment of oxidative metabolism is not the major
mechanism by which volatile anesthetics exert negative
inotropy. Although effects of volatile anesthetics on mitochondrial function cannot explain the negative inotropy, the inhibition of the respiratory chain (energy
supply) will decrease cardiac reserve.194 Inhibition at
complex I may explain how volatile anesthetics increase
the rate of production of reactive oxygen species, which
are thought to mediate anesthetic-induced, ischemic-like
preconditioning.154,199
Conclusion
Although volatile anesthetics have been shown to target multiple sites in heart muscle cells, the sites of action
responsible for negative inotropy are predominantly the
L-type Ca2⫹ channels, the SR, and the contractile apparatus. Volatile anesthetics reduce L-type Ca2⫹ current
(the trigger for SR Ca2⫹ release) and, depending on the
agent used, deplete SR Ca2⫹ content or decrease fractional release of Ca2⫹ from the SR. These anesthetic
actions depress the elevation in cytosolic [Ca2⫹] after
membrane depolarization. This negative inotropic effect
of reduced Ca2⫹-availability is compounded by inhibitory effects of the volatile anesthetics at the level of the
contractile apparatus (decreased Ca2⫹-responsiveness).
Volatile anesthetics impair cross-bridge cycling, decrease
maximal Ca2⫹-activated force, and, possibly, shift the
[Ca2⫹]-force relation to higher [Ca2⫹]. Anesthetic actions on mitochondrial function, K⫹ channels, the sarcolemmal Ca2⫹-ATPase, and the NCX probably contribute minimally to the negative inotropy.
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