Excitation-contraction Coupling in the Heart and the Negative Inotropic Action of Volatile Anesthetics

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 (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 (Na^v1.1, Na^v1.3, and Na^v1.6) and intercalated disks (Na^v1.5).2The depolarization of the cell leads to the activation of L-type Ca²⁺channels, which are primarily encoded by the α^1Cgene (Ca^v1.2),3and 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 ∼300 ms. The action potential spreads from cell to cell, a process that is facilitated by Na^v1.5 channels (in the intercalated disks) and extensive gap junctions between cells, each of which is connected to ∼15 of its neighbors.4In 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.6–9Ca²⁺-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.11,12The 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).15This 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.

The NCX, which serves as the primary route for Ca²⁺extrusion at rest,17,18can 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.19–21Although this possibility was first demonstrated under pathologic conditions of calcium overload, where the cell is highly sensitive to trigger calcium concentrations,19subsequent experiments showed that conditions which would lead to NCX reversal (such as strong depolarization or increased intracellular Na⁺concentrations) could also trigger CICR.20–22It has even been suggested that the NCX might be a major source of trigger Ca²⁺during the normal action potential,21although this has been debated.23Blockade of NCX with an inhibitory peptide suggested that only up to 27% of the trigger might be attributable to NCX.22In addition to the membrane potential, internal Na⁺concentrations will also be critical for determining the NCX contribution.24During the upstroke of the action potential, Na⁺influx might increase Na⁺concentrations locally to promote reverse mode exchange20,25as well modify SR Ca²⁺content.26Some researchers have been unable to demonstrate potent triggering of CICR at negative potentials by reverse mode exchange when the Na⁺current is activated,24,27and 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.

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,”28and such structures will increase the reliability of CICR by limiting diffusional loss of the trigger Ca²⁺signal within them.29–31Although the dihydropyridine receptors (L-type Ca²⁺channels) and the RyRs (Ca²⁺-release channels) are colocalized,32,33a combination of immunofluorescence and data deconvolution techniques have led to the suggestion34that neither voltage-gated Na⁺channels nor the NCX are located in the dyad. On the contrary, more recent immunolocalization data from Thomas et al.  35suggest that the NCX is indeed concentrated in t-tubules.

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.36In this model, it is not the global cytosolic concentrations of Ca²⁺(or any other messenger) that are important but rather the micro-environment around the RyR. The importance of local rather than global signals is based on the idea that most 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 RyRs5,37,38has 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.37,39From the site of release (near z-lines) the Ca²⁺spark diffuses to cover a region of the cell approximately 2 μm in diameter5with a slightly greater spread in the longitudinal direction40(fig. 2). When measured at room temperature, the Ca²⁺spark reaches a peak in ∼10 ms and lasts ∼40–80 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.41When 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.37,42,43It has been estimated that during excitation-contraction coupling the spontaneous spark rate is increased by a factor of ∼10,000 by a “local” 100-fold increase in Ca²⁺resulting from the trigger Ca²⁺influx.44Such a local increase in Ca²⁺is consistent with some computer simulations of changes in Ca²⁺attributable to L-type Ca²⁺channels.31Moreover, the latency for SR Ca²⁺release is <2 ms,45consistent with the rate of RyR opening expected from such high local trigger Ca²⁺concentrations.30Although strictly speaking, Ca²⁺sparks cannot be elemental if attributable to the concerted activation of a number of local RyRs in a cluster,40the fact that evoked Ca²⁺sparks appear to have a modal amplitude distribution at fixed locations within the cell46suggests 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.  48have 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.  46and Soeller and Cannell49suggest 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.

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.50,51For 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,52,53which may explain why transient inward currents, afterdepolarizations, and 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 β¹-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 β¹-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.

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.50,54,55The activity of the SR Ca²⁺-ATPase can be increased by cAMP-dependent phosphorylation of the endogenous inhibitor phospholamban,56whereas the activity of the NCX can be modulated by phosphorylation via  protein kinase C.57The 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.58,59Indeed, 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 cells60or 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 by Ca²⁺is mediated by its binding to the regulatory protein troponin C (TnC).62The contractile proteins (myofibrils), which occupy around 60% of the cell volume,63,64consist 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 ∼1 μm in length and protrude from anchoring points on the z-line.65,66Tropomyosin lies in the groove between the two actin chains of the thin filament and its movements are regulated by the Ca²⁺-sensitive troponin complex.67Interdigitating 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 tropomyosin-binding 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 cAMP-dependent protein kinase A.68When troponin I is bisphosphorylated, the Ca²⁺off-rate of TnC is increased, which enhances the relaxation rate of a contraction.68,69Cardiac TnC has three Ca²⁺-binding sites, unlike the skeletal isoform, which has four.56,70Two 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 (∼70 nm)71and the Ca²⁺-specific site of TnC (with a dissociation constant of ∼500 nm)55is unoccupied.72In 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.74This mechanism has been extensively reviewed.70,75As 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.76,77The biochemical steps that drive cross-bridge cycling (hydrolysis of ATP via  actomyosin-ATPase) have been largely characterized.78Basically, 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.  79have simultaneously measured the mechanical events and actomyosin-ATPase activity of a single-headed myosin molecule interacting with an actin filament. Under these artificial 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 

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.56The [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.  81showed 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. ,82who determined the intracellular [Ca²⁺]-force relation before and after skinning in the same muscle preparation (rat cardiac trabecula). The [Ca²⁺] required for half-maximal force was considerably higher after skinning, suggesting that the skinning procedure may damage or remove components involved in the regulation of actin-myosin interaction. Hence, data obtained from experiments employing skinned muscle preparations cannot be readily extrapolated to the intact system.

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 ∼1.9 μm and can be extended to SLs of up to ∼2.3–2.4 μm. 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 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.62,72An increase in overlap of thick and thin myofilaments, a reduction in interfilament lattice spacing,62,85and an increase in intracellular Ca²⁺release86,87are 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 Smith72argue 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.88Fitzsimons and Moss85previously 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. ,89suggests 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,90is 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,84,93,94as 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 μm,95is presumed to underlie this end-stop effect (figs. 4C and 4D).

At SLs < 2.1 μm, where passive force is less than 5% of maximal twitch force, titin overshadows collagen as the main contributor (>90%) to passive force.95This 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.96,97The elasticity of titin resides in its I-band portion, which spans from the z-line to the tip of the myosin filament.98This 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 

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.100–102Mitochondria occupy ∼30% of the myocyte volume4,63,64and 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 ∼200 mV, as electrons are transferred along the various complexes of the electron transport chain. The energy stored in this electrochemical 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,103it 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.  104deduced 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 Stienen105measured 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⁺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.108Ca²⁺influx into the mitochondria is thought to regulate metabolism because an increase in mitochondrial [Ca²⁺] stimulates ATP synthase109and the activity of pyruvate dehydrogenase and α-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.  111were able to demonstrate mitochondrial Ca²⁺transients using confocal microscopy. In accord, in work with patch-clamped ferret or cat ventricular myocytes, Zhou et al.  112were 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,113using 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.56Hence, small changes in mitochondrial Ca²⁺permeability could greatly influence Ca²⁺distribution in the cell.

Although neurohumoral regulation is outside the scope of this review (see, for example, Morris and Malbon114or 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 α-helices.116,117The G protein-coupled receptors regulate the activity of various membrane-bound enzymes including adenylate cyclase, guanylate cyclase, and phospholipase C, which produce, respectively, the secondary messengers cAMP, cGMP, and DAG.118In 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 α¹-receptors, three α²-receptors, and three β-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 β¹-receptors, the Gαs 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),119troponin 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 Gαi, which inhibits adenylate cyclase, producing the opposite effect to adrenergic receptor stimulation.

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 explain 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.121Anesthetic 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).122When 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.

In 1986, Bosnjak and Kampine123showed that halothane decreased the Ca²⁺transient in cat papillary muscles 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.124Another potential limitation of aequorin is that its light-emitting properties may be altered by direct interaction of the anesthetic with the photoprotein,125although, in favor of aequorin, Housmans and Wanek126recently 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,127followed by fluo-3;128these are technically less difficult to use than aequorin.

A large number of studies using fluorescent Ca²⁺indicators have corroborated the observations of Bosnjak and Kampine123that volatile anesthetics dose-dependently decrease the amplitude of the intracellular Ca²⁺transient in intact cardiac muscle.129–136Hence, it is now well established that volatile anesthetics indeed decrease the amount of Ca²⁺released into the cytosol after electrical stimulation. Figure 7shows 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.129–131We now turn our attention to the anesthetic actions that may be responsible for the decrease in the cytosolic Ca²⁺transient.

Studies using the single microelectrode voltage-clamp technique137–139suggested that volatile anesthetics inhibit L-type Ca²⁺channels. Indeed, using the patch-clamp technique, Bosnjak et al.  140showed that halothane, enflurane, and isoflurane reversibly decreased whole-cell Ca²⁺current (which is predominantly L-type Ca²⁺current) in canine ventricular myocytes. In whole-cell and cell-attached recordings using a similar preparation (rat myocytes), Pancrazio141confirmed and extended this work. Halothane (0.9 mm) and isoflurane (0.8 mm) decreased the peak whole-cell Ca²⁺current by ∼40% and ∼20%, 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.  137found 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.  137that anesthetics shorten the action potential.138,139,142,143Rithalia et al.  144showed 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. ,137suggesting that additional actions underlie the negative inotropic actions of volatile anesthetics. In accord with Lynch et al. ,137Harrison et al.  132showed 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.

Halothane and isoflurane,145as well as sevoflurane,146have been shown to inhibit inwardly rectifying K⁺(K^ir) 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 (I  ^to), important in the early phase of action potential repolarization, has also been shown to be inhibited by halothane, as well as by isoflurane.147Moreover, these anesthetics have been reported to inhibit delayed rectifier K⁺current (I  ^K)148and Ca²⁺-activated K⁺(K^Ca) channels.149Unlike K^ATPchannels, which are activated primarily under pathologic conditions, K^irchannels, I  ^to, I  ^Kand K^Cachannels 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 K^ATPchannels. Using inside-out patches excised from rabbit ventricular myocytes, Han et al.  151found that isoflurane decreased the duration of K^ATPchannel burst activity and increased the interburst interval without affecting channel kinetics within a burst. It should be noted that K^ATPchannels characteristically exhibit rundown (decreasing P^oas a function of time) and show intermittent burst activity after patch excision.152These characteristics have been shown to be fully prevented by endogenous molecules such as PIP²(phosphatidylinositol 4,5-bisphosphate) and long-chain acyl-CoA esters,152suggesting that burst activity may in fact be restricted to excised patches and, possibly, pathologic conditions.152Han et al.  151also showed that isoflurane shifted the relation between [ATP] and P^oto the left; that is, it decreased the sensitivity of the channel to inhibitory ATP. More recently, Stadnicka and Bosnjak153reported that isoflurane facilitates K^ATPchannel opening at reduced pH. Hence, under physiologic (and pathologic) conditions isoflurane may facilitate K^ATPchannel 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 K^ATPchannels, with downstream signaling mediated by reactive oxygen species.154,155Cope et al. ,156for 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 K^ATPchannels as mediators of volatile anesthetic-induced preconditioning.155In support of this conclusion, Nakae et al.  157recently reported that isoflurane increases the open probability of mitochondrial K^ATPchannels reconstituted in lipid bilayers. However, there is controversy as to whether mitochondrial K^ATPchannels 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,162,163which may be important targets contributing to cerebral depression. Meuth et al. ,164for 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 anesthetic-induced arrhythmias.

In 1990, Herland et al.  165showed that halothane stimulated Ca²⁺efflux from the sarcoplasmic reticulum of chemically-skinned cardiac trabeculae. Connelly and Coronado166confirmed 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 Frazer167showed 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.129This transient effect has also been seen in electrically stimulated myocytes. Harrison et al.  132showed that application of halothane, but not isoflurane, transiently potentiated the Ca²⁺transients and accompanying contractions of electrically paced myocytes (fig. 7).

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,130,131whereas sevoflurane appears to have no effect.133,136Moreover, isoflurane and sevoflurane, but not halothane, were found to decrease fractional release, the fraction of the content that is released after stimulation.133,136 

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 Goknur168found 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.169On 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,170which would cause intracellular [Na⁺] to increase (favoring reverse mode NCX activity).

After inhibiting the NCX with Na⁺- and Ca²⁺-free solution, Hannon and Cody136showed 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)171could 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,136halothane and isoflurane have been reported to decrease surface membrane Ca²⁺-ATPase activity in erythrocytes172and neurons.173In 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 facilitated SR Ca²⁺loading. On the contrary, inhibitory effects of volatile anesthetics on sarco-endoplasmic reticular Ca²⁺-ATPase171,174could contribute to the depletion of SR Ca²⁺stores by volatile anesthetics.

Halothane and isoflurane have been shown to uncouple electrically pairs of myocytes, indicating that these agents block gap junctions.175In accord, Burt and Spray176showed that halothane (2 mm) decreased gap junction conductance by reducing the number of open channels without affecting unitary conductance. In further work, He and Burt177have 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.

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 Kampine123examined 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,123Housmans178demonstrated 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 μm) loaded with fluo-3, Hanley and Loiselle129showed 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,130,131who also used rat trabeculae. Furthermore, Davies et al.  133likewise found that halothane and isoflurane, but not sevoflurane, decreased Ca²⁺-responsiveness of the contractile proteins in fura-2-loaded rat ventricular myocytes. In contrast to Davies et al. ,133Bartunek and Housmans179found 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 Loiselle129showed 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 detergent-skinned human181and rat cardiac fibers.182Herland et al.  183found 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 isoflurane but not by enflurane. Using Triton X-100-skinned rat cardiac muscle, Prakash et al.  184found 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 Loiselle129using 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.  182reported that halothane, enflurane, and isoflurane each increased the [Ca²⁺] for half-maximal activation in skinned cardiac fibers. In accord, Tavernier et al.  181found that halothane and isoflurane, each at 1 MAC, decreased Ca²⁺-sensitivity of the contractile proteins. However, Herland et al.  183found 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.

Murat et al.  185examined 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 actomyosin-ATPase 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.186In further work, Hannon et al. ,135using 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 interaction may account for the ability of these agents to decrease maximal Ca²⁺-activated force.

Volatile anesthetics have long been known to impair nicotinamide adenine dinucleotide

(NADH) oxidation by mitochondria,187–190suggesting that these agents may inhibit complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain. Using isolated liver mitochondria, Hall et al.  190showed that halothane inhibited NADH oxidation at concentrations that did not affect succinate oxidation. Consistent with inhibition of NADH oxidation, halothane and isoflurane,129,191,192as well as sevoflurane,193have been shown to increase NADH fluorescence in intact cardiac muscle.

Recently, Hanley et al. ,194using 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 ∼20%, whereas sevoflurane decreased activity by ∼10%. 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.197,198Moreover, the observed ∼50% 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 phosphate) or H⁺, inhibitors of excitation-contraction coupling and the contractile apparatus,94indicating 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.194Inhibition 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 

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.