Mechanisms and Use of Calcium-Sensitizing Agents in the Failing Heart (2006)

HomeCirculationVol. 113, No. 2Mechanisms and Use of Calcium-Sensitizing Agents in the Failing Heart Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessReview ArticlePDF/EPUBMechanisms and Use of Calcium-Sensitizing Agents in the Failing Heart David A. Kass, MD and R. John Solaro, PhD David A. KassDavid A. Kass From the Division of Cardiology, Department of Medicine, The Johns Hopkins University Medical Institutions, Baltimore, Md (D.A.K.), and the Department of Physiology and Biophysics and Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago (R.J.S.). and R. John SolaroR. John Solaro From the Division of Cardiology, Department of Medicine, The Johns Hopkins University Medical Institutions, Baltimore, Md (D.A.K.), and the Department of Physiology and Biophysics and Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago (R.J.S.). Originally published17 Jan 2006https://doi.org/10.1161/CIRCULATIONAHA.105.542407Circulation. 2006;113:305–315Depressed cardiac contractility is central to many forms of cardiac disease and reflects the heart's inability to generate adequate force despite being provided physiological activator calcium and chamber load. Yet, successful methods to enhance cardiac contractility have remained elusive. Agents such as dobutamine or milrinone that work through the β-receptor-cAMP-protein kinase A pathway are used to manage acute hemodynamic decompensation, but short-term and, particularly, long-term use can increase risks of arrhythmia and worsen outcome. These and other data have led to the conclusion that successful heart failure management should probably avoid the targeting of contractility improvement.Leading hypotheses for the failure of existing inotropic therapies is that they increase activator calcium, worsen arrhythmia, activate maladaptive Ca2+-dependent signaling cascades, and increase myocardial oxygen consumption, making hearts less efficient. An alternative approach to avoid such complications would be to directly influence the manner by which intracellular calcium is transduced into muscle force. The class of molecules that achieve this are often termed "calcium sensitizers" and have attracted growing clinical interest for more than 20 years.1 The mechanisms by which such effects are achieved vary widely and include direct activators of motor proteins such as myosin, enhancers of force generated by a cross-bridge, and agents that augment Ca2+–troponin C (TnC) binding and its consequences. To date, many such drugs have had additional effects, such as inhibiting cAMP phosphodiesterase (PDE3a), that likely contributed to their vasodilation/venodilation and Ca2+-dependent increases in heart rate and contractility. Other agents, such as levosimendan, also inhibit ATP-sensitive potassium channels, which can induce further effects.This review discusses basic molecular mechanisms for drugs that alter the myofilament response to calcium, how such agents affect muscle and whole-organ physiology, and what their clinical testing has revealed. In doing so, we attempted to bridge the gap between existing reductionist reviews to those that have focused largely on clinical outcome trials.Excitation/Contraction CouplingPhysiological and pharmacological modifications in myofilament response to Ca2+ occur as an integral mechanism of excitation/contraction coupling. In diastole, Ca2+ concentrations surrounding the sarcomere of working heart muscle cells fall below the myofilament activation threshold. With electrical stimulation, Ca2+ moves into the cytoplasmic space and binds to the myofilaments, inducing force and contraction that are &20% to 25% of maximum.2 One way to increase contractile force is to increase the Ca2+ that enters the myofilament space, and this is physiologically achieved by an elaborate set of membrane-related mechanisms that control Ca2+ fluxes. Alternatively, one can regulate how the sarcomere responds to Ca2+. Normally, changes in sarcomere length and the phosphorylation of regulatory proteins associated with thin and thick filaments modify Ca2+ sensitivity and rates of filament sliding, thereby modifying contractile reserve in concert with Ca2+ flux to and from the sarcomeres.3,4 Pharmacological approaches that further enhance the sarcomeric Ca2+ response modify this normal control mechanism.The Myofilament Force-Ca2+ RelationshipA common approach to assessing myofilament Ca2+ activation is to apply varying Ca2+ concentrations (&10−9 to 10−4 mol/L) to single cells or muscle fibers in which the membranes have been removed by detergent. Muscle or sarcomere length is held constant, and developed tension is then measured at each Ca2+ level. Alternatively, intact trabeculae can be studied by chemically inactivating intracellular Ca2+ fluxes, opening sarcolemmal Ca2+ channels, and tetanically stimulating the muscle while varying extracellular Ca2+ concentration.5 The resulting steady-state relation between Ca2+ and tension is sigmoidal in shape (Figure 1) and, once fit to the Hill equation, provides several parameters: The half-maximal activating Ca2+ concentration (Ca2+ sensitivity), steepness of the relation, and maximum tension. Each parameter varies physiologically and pathophysiologically and can be altered pharmacologically. Download figureDownload PowerPointFigure 1. A, Altered myofilament response to Ca2+ assessed by force-pCa2+ relations in skinned muscle fibers or myocytes. Solid line depicts the resting force-Ca2+ relation; line (a), enhanced Ca2+ sensitivity with pure left shift, increasing force at low and higher Ca2+ concentrations but no change in maximum (Max) or minimum tension; line (b), change in sensitivity and increase in peak tension; line (c), Ca2+-dependent sensitivity increase that is greater at systolic than diastolic levels; and line (d), increase in maximal force but no change in sensitivity once this effect is normalized relative to control. See text for further discussion. B, Differences in left ventricular pressure time course from Ca2+ sensitizer (EMD-57033, from Senzaki et al28) versus dobutamine. EMD-57033 prolongs systole with little effect on initial left ventricular pressure kinetics or relaxation rate. In contrast, dobutamine accelerates both kinetics, and systolic duration is shortened.Figure 1A demonstrates the types of changes that can occur in the force-Ca2+ relationship that reflect myofilament responsiveness to Ca2+. One is a pure leftward shift of the relation without alterations in maximal (or minimal) Ca2+-activated force (type A). Such shifts occur from a decline in protein kinase A (PKA) phosphorylation of troponin I (TnI), or myocyte alkalization. This type of change would primarily manifest in normal operating ranges of Ca2+ activation. A potential problem is that force generation is also greater at diastolic calcium levels, which could increase passive stiffness. A second type of change includes a rise in the maximal activated force (type B). Changes of this nature are observed if the cross-bridge force generation is itself altered, as has been observed with changes in redox state, with proteolytic cleavage of TnI in postischemic stunned myocardium, and with sensitizers such as EMD-50733.6 The extent to which such upward shifts occur at low calcium levels might again exacerbate diastolic effects. Lastly, a sensitizer may display Ca2+-dependent changes, so that effects at lower calcium concentrations are minimized, whereas shifts in the force-Ca2+ relation at systolic levels of calcium are manifest (type C). Levosimendan is an example of an agent thought to have this property.7 The force-Ca2+ relation can also reflect simply a rise in maximal activated force but with no actual change in calcium sensitization (type D). In this case, normalization of each relation to maximal force results in superimposable curves.In intact muscle, an increase in force by means of Ca2+ sensitization appears functionally quite different from that due to activation of cAMP-PKA-dependent mechanisms (Figure 1B). In the case of the latter, inotropy is coupled to shortening of the twitch duration and enhanced relaxation rate, due to faster E-C coupling kinetics from PKA phosphorylation of calcium handling and sarcomeric proteins (eg, voltage-gated Ca2+ channel, TnI, phospholamban, and myosin-binding protein C [MyBP-C]). In contrast, a Ca2+ sensitizer usually results in a similar rate of tension rise and net prolongation of systole. Relaxation can be slower or achieved at rates similar to those under basal conditions. At faster heart rates, these differences can become important, where diastolic time period and thus filling could be compromised. Most Ca2+-sensitizer agents have combined primary with secondary pharmacological effects, the latter often offsetting some changes that might arise from a pure sensitizer response. An example is levosimendan, which combines myofilament modifications with activation of the ATP-sensitive potassium channel and inhibition of phosphodiesterase 3A (PDE3a).Molecular Mechanisms of Actin-Myosin Cross-Bridge ActivationA rational starting point for how one might use drugs to modify the mechanical activity of cardiac sarcomeres is to consider what regulates the reaction between myosin molecular motors (cross-bridges) of thick filaments with thin-filament actin. Figure 2 illustrates this understanding in a cartoon of the working (A band) region. During diastole, active tension is essentially zero and the myosin ATPase rate low. Depending on the sarcomere length, there is a passive tension dependent largely on stress-strain relations of the giant protein, titin.8 Cross-bridges are blocked from reacting with actin by the position of tropomyosin (Tm) on the thin filament, which covers the myosin-binding sites on the thin filament and alters the reactivity of actin with myosin. The diastolic state requires the binding of the troponin complex (cTn) to the actin-Tm thin-filament backbone (Figure 2). Troponin is a complex of the following 3 proteins: cTnC, a protein that senses and binds Ca2+; cTnI, an actin-binding protein named for its ability to inhibit the actin-myosin interaction; and cTnT, named for its ability to bind Tm. cTnC is a dumbbell-shaped protein with a single regulatory Ca2+ binding site at its N-terminal lobe and 2 structural Ca2+/Mg binding sites at its C-terminal lobe (Figure 3). The lobes are connected by a linker helix. The N-lobe of cTnC contains a ring of amino acids that coordinate binding of a single Ca2+ with high affinity and fast kinetics. Without Ca2+ bound in the ring, a sticky patch made of hydrophobic side arms of amino acids remains concealed in the structure and is unavailable to interact with its neighbor, cTnI. In diastole, cTnI is kept away from the regulatory site of cTnC by multiple protein-protein interactions among troponin components and actin-Tm that serve to anchor the troponin complex to actin and restrict Tm movement. As shown (Figure 3), cTnC is held in place by a network of protein-protein interactions of its C-lobe with both cTnI and cTnT. The interactions consist of hydrophobic interactions, charge-charge interactions, and intertwining of helixes. The versatility in the interactions allows for versatility in control and modification. In diastole, troponin is tethered to actin-Tm by the binding of 2 regions of cTnI with actin (inhibitory peptide and a second actin-binding region) and by an interaction of the N-terminal tail of cTnT to actin-Tm. The 2 actin-binding regions of cTnI flank a small hydrophobic helix (the switch peptide) that binds to the hydrophobic sticky patch formed in the cTnC N-lobe on Ca2+ binding to the regulatory site. These protein-protein interactions affect the structure of actin and restrict the range of movements of Tn-Tm on the thin filament, impeding the actin-cross-bridge reaction. Download figureDownload PowerPointFigure 2. A structural unit of the sarcomere with thick-filament cross-bridges (myosin heavy chain, MHC) shown in diastole (left) and reacting with thin-filament actin in systole (right). In diastole, Tm (tropomyosin) blocks the cross-bridge formation. In the absence of Ca2+ bound to the N-lobe of TnC, Tm is held in this position by protein-protein interactions that involve TnI, which tethers troponin to actin, and by TnT, which interacts with actin-Tm. In diastole, the N-lobe of TnC does not interact with the C-terminal region of TnI, whereas with Ca2+-TnC interaction, these regions bind triggering movement of Tm through the action of TnT. The cross-bridge is further modulated by interactions with myosin light chains (MLC) and MyBP-C, which also interacts with titin, a molecular spring protein in the sarcomere.Download figureDownload PowerPointFigure 3. Molecular mechanism of thin-filament activation. In diastole, Tm is held in blocking position by its interactions with TnT and by interactions of a TnI inhibitory peptide (green stripes) and switch peptide (red stripes) with actin. Ca2+ binding to the N-lobe of cTnI induces the opening of a hydrophobic "sticky" patch on TnC that reacts with the switch peptide of cTnI, releasing Tm and the thin filament from the inhibited state. Cardiac TnI contains a unique N-terminal peptide that also reacts with the N-lobe of TnC and regulates Ca2+ binding affinity. Term indicates terminal.Ca2+ binding to cTnC triggers systole by inducing movements of the thin-filament proteins so that blocked cross-bridges may react with actin in a cycle of interactions that impel the thin filaments on each side of the sarcomere toward the center (Figure 2). A key aspect of the molecular mechanism that triggers contraction is a Ca2+-induced hydrophobic cleft or sticky patch on the N-lobe of cTnC.9,10 The hydrophobic switch peptide of cTnI is also sticky and binds to the cTnC hydrophobic cleft and sets into motion a reversal of the diastolic state. Key steps include the release of the actin-binding regions of cTnI from actin, and most likely an altered interaction of the cTnT N-terminal tail with Tm. One of the striking features of cardiac myofilaments is that the relation between Ca2+ and tension is steep (Figure 1A), which indicates an underlying cooperative process.11 What this means is the interaction of a cross-bridge with the thin filament makes it easier for a neighboring cross-bridge to bind. This is accomplished by an influence of cross-bridge binding on the position of Tm on the thin filament. The Tm are connected end-to-end to form a continuous strand; when a cross-bridge binds the Tm strand, movement affects the reactivity of a neighboring cross-bridge for actin. With this basic review as a foundation, we turn next to how this interaction can be modified by targeted pharmacological interventions.Thin Filament Proteins as Potential Drug TargetsTargeting the cTnC-cTnI InteractionThere are 4 main interaction sites between cTnC and cTnI that are potential targets for inotropic drugs. One is the interaction of the sticky patch in the N-lobe of cTnC with the switch peptide of cTnI. An attractive aspect of this site is that the opening of the sticky patch in heart muscle by Ca2+ is fundamentally different from that of fast skeletal TnC. An example of a drug that binds to this region is bepridil, which was not developed as a Ca2+ sensitizer yet was found to bind to cTnC.12 Structural studies showed that bepridil binds to the N-lobe of cTnC and induces an open configuration similar to that induced by Ca2+ in the cTnC-cTnI complex.13,14 The docking site of bepridil is different from the docking site of the switch peptide of cTnI, and thus both the drug and the switch peptide could bind to the cTnC N-lobe. Li et al13 hypothesized that bepridil may sensitize the myofilament to Ca2+ by slowing down both the closure of the hydrophobic cleft and the dissociation of cTnI on release of Ca2+ from site II of cTnC.Another point of interaction of cTnC with cTnI is at a near N-terminal peptide of cTnI that binds tightly to a hydrophobic region of the cTnC C-lobe. Activation of the myofilaments may be altered by phosphorylation of the cTnI peptide, which indicates that this region is important in transduction of the Ca2+ binding signal at the N-lobe.15 A Ca2+-sensitizing drug demonstrated to bind at this interface is the thiadiazinone EMD-53998, which is a racemic mixture of the enantiomers (+)EMD-57033 and (−)EMD-57439. The racemic EMD-53998 increases Ca2+ sensitivity of myofilament force and ATPase rate but also is a phosphodiesterase inhibitor. Ca2+ sensitization is a property of EMD-57033, and phosphodiesterase E (PD) inhibition is a property of EMD-57439.16 This evidence supported the idea of a receptor domain, which was subsequently identified as the C-lobe of cTnC.17 Determination of the nuclear magnetic resonance structure of cTnC demonstrated that EMD-57033 docks with its chiral group situated deep in the hydrophobic cleft in the cTnC C-lobe.18 This special localization suggests a structural basis for the lack of effects of the (−)enantiomer on myofilament response to Ca2+. Drug binding competes with binding of cTnI in this region in that EMD-57033 was completely displaced by cTnI(34-71) but not by a region that contained the cTnI switch peptide.A third interface important in the action of Ca2+ sensitizers is between the actin-binding inhibitory peptide of cTnI and the helix that connects the N- and C-lobes of cTnC. The Ca2+-sensitizing drug levosimendan (Simdax), a pyridazinone-dinitrile derivative, which was screened explicitly for binding to cTnC, docks at or near this region and is likely to alter the lifetime of the active state of the thin filament.19A fourth site of interaction of cTnI with cTnC is between the unique N-terminal peptide of cTnI and the N-lobe of cTnC (Figure 3). With the peptide bound, the affinity of TnC for Ca2+ is rather high; when the peptide is not bound, the affinity is lower.3 Release of the peptide from the N-lobe of cTnC occurs with phosphorylation of the cTnI N-terminal peptide associated with adrenergic stimulation. This interaction is modified physiologically and is important in regulation of the cycle time of the heart beat and in muscle shortening against an afterload. Drug-binding sites at the N-terminus have not been discovered or studied, but interactions of the N-terminal peptide of cTnI with cTnC have been reported to alter the Ca2+-sensitizing effects of levosimendan.20Targeting cTnI-Actin InteractionsIn general, there have been no attempts to alter the 2 regions of interaction of cTnI with actin by drug binding, although these regions seem especially important to consider. One region is the inhibitory peptide of cTnI, which binds to actin in diastole and is released in systole. In cTnI, this region contains a phosphorylatable threonine (Thr), which is a proline in skeletal TnI. Phosphorylation at this site alters myofilament response to Ca2+ and filament sliding velocity.15 A second binding site has been identified as critical through studies of myofilament and cardiac function in transgenic mice (ssTnI-TG) in which slow skeletal TnI (ssTnI) completely replaced cTnI. Compared with controls, ssTnI-TG myofilaments are more sensitive to Ca2+ and thus provide an animal model with myofilaments that are constitutively sensitized to Ca2+. Hearts of these mice demonstrate a remarkable resistance to acidosis,21 sepsis,22 and ischemia-reperfusion injury.23 There is evidence that a difference in a single amino acid (His/Ala) in ssTnI versus cTnI at position 162 is responsible for these functional differences.24Troponin T as a TargetA particularly sensitive region of cTnT is in a domain in the C-terminus that surrounds amino acid 206.15 Modification of this region either by phosphorylation or by mutations linked to induces changes in tension and shortening of This region appears to function in the action of cTnT as a in its of Tm away from the actin sites that react with The N-terminal tail of cTnT is also important in the from the diastolic to the systolic state, and of this interaction could a drug The position of the region away from the of as in Figure also indicates that multiple protein-protein interactions might not be altered, as is the case with a drug that binds to the Proteins as Potential Drug actin-myosin interface is also a potential site of action for Ca2+-sensitizing although for agents that interact with myosin has not been a EMD-57033 is to increase the actin-myosin interaction in that lack There are also proteins in thick-filament proteins that modify the actin-cross-bridge reaction but that have not been explicitly as targets for Ca2+-sensitizing These include the and the regulatory light chains of myosin and (Figure 3). The light chains of myosin are an important of myofilament sensitivity to Ca2+ and the kinetics of the reaction of cross-bridges with actin by structural to the cross-bridge and also by the extent of movement of cross-bridges away from the thick-filament This movement is by the state of phosphorylation of in phosphorylation of the in regions of the heart important in cardiac in MyBP-C, which both structural and regulatory is of a protein network that the myosin the myosin and (Figure The cardiac is unique in sites in response to altered adrenergic with hearts that it is critical for the of and of muscle systolic both and regulate cross-bridge kinetics, and modifications of their protein-protein interactions may be a to the of and Agents and are a of that sarcomeric proteins and a growing of and that this and these are also targets for modifying the myofilament response to Ca2+. This approach has not yet been developed for inotropic and its the of this an example serve to this growing of an of protein kinase is an agent developed by with potential to and increases in heart failure and can cTnT and cTnI, to a myofilament response to inhibition of might be to enhance Ca2+ and this has been in animal The with this approach is of which requires of key steps in the signaling pathway that to activation of and of on the 4 several of the key features of calcium sensitizers in the intact EMD-57033 by way of One is the ability of the sensitizer to enhance contractility in normal or in striking to (Figure the of Ca2+ sensitizers is to they display in the failure state. such as are less by EMD-57033 than whereas that would more likely reflect enhanced cross-bridge such as are enhanced in (Figure In failure the response of both and to dobutamine are whereas increases with as in control This the targeting of mechanisms to the Download figureDownload PowerPointFigure of a calcium sensitizer on in cardiac function in normal and A, from control and heart failure at and inotropic with either EMD-57033 or dobutamine. EMD-57033 the relation leftward and in both control and failure whereas there is of the dobutamine response in B, of the differences in inotropic response to dobutamine and EMD-57033 as by and in control and The increase in both with dobutamine was in and EMD-57033 induced similar increases in by EMD-57033 greater than those in in both types of indicates heart versus control versus change from dobutamine. Relaxation rates are more with than with Ca2+ sensitizer in normal although filling rates are enhanced by indicates dobutamine. versus of response in normal hearts by Ca2+ sensitizer data from Senzaki et sensitizers have effects on diastolic function on their of action and the type of at lower EMD-57033, as a sensitizer with inhibitory effects, increases diastolic and relaxation in cardiac muscle or in the agent not diastolic are to enhance the rate of pressure yet they can enhance diastolic filling rates (Figure The latter and the lack of a net increase in diastolic in are likely due to effects from greater which hearts to to and thus have enhanced Agents with more complex mechanisms of such as levosimendan, may also have offsetting effects on diastole to inhibition and whereas with more targeted agents (eg, diastolic function with relaxation may their a sensitizer force of Ca2+ the of that augment calcium heart rate or stimulating calcium release can further augment the inotropic effect in a This is shown for EMD-57033 in response to a change in heart rate (Figure the rate greater contractility by its effects on Ca2+ and thus as as by effects TnI with EMD-57033 this effect it targets a more mechanism.The of a sensitizer to inotropy under of physiological was demonstrated by et These levosimendan to with heart During an increase in and little inotropic Levosimendan enhanced contractility at but contractility also further (Figure As shown in Figure the inotropic and effect of the drug was at peak with the observed at This of Ca2+ sensitizers a problem with that work by increasing activator calcium, that the inotropic effect can be to be less under and more when Download figureDownload PowerPointFigure of levosimendan on function in A, systolic reserve shift to and in heart failure without levosimendan, the response is altered, so there is a left shift of the with and B, and inotropic response to levosimendan at and at varying of There is a response of the drug to further enhance effects on both features of heart indicates heart from et with important of Ca2+ sensitizers is their ability to contractility without a increase in myocardial can be into 3 used for basal used for coupling and used for force generation (myosin As demonstrated by the is to cardiac work and the latter