Cardiovascular Research

Cardiovascular Research 1997 36(1):10-20; doi:10.1016/S0008-6363(97)00174-0
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Calcium sensitisers: mechanisms of action and potential usefulness as inotropes

J.A Lee^a,* and D.G Allen^b

^aUniversity Department of Pathology, The Medical School, Beech Hill Road, Sheffield, S10 2RX, UK
^bDepartment of Physiology, University of Sydney, Sydney NSW 2006, Australia

^* Corresponding author.

Received 22 April 1997; accepted 26 June 1997

KEYWORDS Cardiotonic agents; Calcium sensitisers; Heart failure; Inotropic agents; Cardiac muscle; Intracellular calcium

	1 Introduction

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
Heart failure remains a common and lethal condition, with treatment options which are less than satisfactory. Epidemiological studies [1–5]indicate an overall prevalence in the population of 1–3%, while mortality for all grades of heart failure is approximately 25% at 2 years and 50% at 4 years. Severe heart failure has a 2 year mortality as high as 75%. Although some cases are caused by correctable problems such as hypertension or valve lesions, the majority result either from loss of functioning myocardium due to ischaemic damage or from ineffective contraction of partially ischaemic or otherwise functionally disabled myocardium.

Therapy for heart failure thus has two main objectives [6–12]. Firstly, treatment of the failing pump and its main physiological consequences, fluid retention and vasoconstriction. Secondly treatment of the common associated problems which may be either primarily involved in causing heart failure (e.g. ischaemia) or which may be secondary complications (e.g. arrhythmias). Therapies which are currently available can reduce morbidity and have also achieved some reductions in mortality [13–18]. However, most of these treatments, including diuretics, angiotensin converting enzyme (ACE) inhibitors, vasodilators and beta-blockers, are aimed at alleviating the fluid retention and vasoconstriction associated with heart failure, rather than the primary cause.

In principle therapy aimed at supporting the pump function of the heart might also be expected to have a major role in the treatment of heart failure. In practice the benefits of positive inotropic agents have been hard to demonstrate. This therapeutic approach has a long history, beginning with Withering's introduction of digitalis in 1785 [19]. But cardiac glycosides have multiple actions and there has been protracted controversy over whether these drugs are beneficial to patients with heart failure in sinus rhythm. Recent results do point to a benefit [20], even in the presence of treatment with ACE inhibitors [21], but glycosides remain drugs with a relatively narrow therapeutic window and significant side-effects. Other inotropes have fared worse: phosphodiesterase (PDE) inhibitors have been virtually withdrawn after being found to increase mortality [22, 23]. The recent disappointments with positive inotropic drugs have been so great that many now question whether they are likely to have a significant role in the treatment of chronic heart failure [24].

In this review we suggest that the deficiencies of currently available inotropes may be, at least in part, a consequence of their cellular mode of action and that new agents utilising fundamentally different cellular mechanisms to increase force may find a useful role in heart failure therapy in the future. The drugs known as "calcium sensitisers" are a diverse group of compounds which are beginning to access such novel cellular pathways. We provide an overview of the theoretical background and experimental data relating to the effects of calcium sensitisers on heart muscle and we comment on the potential therapeutic relevance of this information. Since most calcium sensitisers are still at the experimental stage of development, it is clearly not yet possible to give a firm answer to the question of whether this inotropic approach will actually turn out to be an improvement on those we already have. Nevertheless, there seem to be grounds for optimism and we suggest that this area of investigation deserves more attention.

	2 Heart muscle activation

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
Appreciation of the advantages and disadvantages of various inotropic agents requires an understanding of heart muscle activation. This section briefly summarises some important points. Heart muscle contracts when an action potential activates an inward Ca²⁺ current across the surface membrane. The Ca²⁺ entry associated with this current is small, but it is sufficient to trigger a much larger Ca²⁺ release from the sarcoplasmic reticulum. The resulting short-lived increase in myoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) — known as the Ca²⁺ transient — causes Ca²⁺ binding to troponin C. This in turn causes conformational changes in various myofilament proteins, with the result that crossbridge attachment is stimulated and contraction occurs.

A conventional view of the latter stages of this process is that Ca²⁺ binding alters the interactions of troponin C with troponin I and troponin T in such a way that tropomyosin moves from its position in the actin filament groove, allowing myosin heads ("crossbridges") to interact with binding sites on the actin filament. In this steric hindrance model, developed force increases when [Ca²⁺]_i rises because the exposed actin binding sites recruit more crossbridges.

In recent years this mechanism has been challenged by an alternative idea which suggests that crossbridges are either weakly or strongly bound to actin. In this model, the main effect of increased [Ca²⁺]_i is to change the rate at which myosin heads convert from a weak attachment (WXB in Fig. 1) to a strongly bound state which produces force (SXB). This is known as the turnover kinetics model of muscle activation [25]. The evidence for this model comes from biochemical [26], mechanical [27]and X-ray diffraction studies [28]showing that crossbridges are weakly bound to the actin site in resting muscle, a finding which is incompatible with the simple steric hindrance model. Brenner [29]devised a method for measuring conversion rate of weakly bound to strongly bound crossbridges (designated f_app — the apparent rate of attachment of strongly bound crossbridges) and showed that it was sensitive to [Ca²⁺]_i. Thus in the turnover kinetics model the effect of Ca²⁺ binding to troponin-C is to accelerate the conversion of crossbridges from the weak to the strongly bound form (see Fig. 1).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Schematic diagram of processes involved in activation of heart muscle. Key: Ca — intracellular calcium; Tr — troponin; WXB — weakly bound cross-bridges (non-force-producing); SXB — strongly bound cross-bridges (force-producing). fapp is the apparent rate constant for conversion of WXB to SXB; gapp is the corresponding reverse rate constant. (i)–(v) represent possible sites at which a calcium sensitiser might act (see text).

 
The consequence of these reactions is that more force is produced as [Ca²⁺]_i rises. The shape of the resulting relation between [Ca²⁺]_i and force, as well as its sensitivity to various interventions, gives important clues to the underlying biochemical interactions. These properties are usually studied in "skinned" preparations in which the surface membrane has been removed either mechanically or chemically. When [Ca²⁺]_i is set to a series of known values, the force achieved at each value defines the steady state [Ca²⁺]-force relation. It has been known for many years that [Ca²⁺]-force curves are quite steep and show features of co-operativity. The latter property can be measured by the Hill coefficient n. For example, if Ca²⁺ bound to only one site on troponin C and force production was proportional to this binding, the Hill coefficient would be 1. Experimental data points can be fitted to the equation:

where Ca₅₀ is the [Ca²⁺]_i which causes half-maximal force production, n is the Hill coefficient and P/P_max is the force normalised to the maximum value obtained at saturating [Ca²⁺]. Thus Ca₅₀ is a measure of the Ca²⁺ sensitivity and n is a measure of the steepness or co-operativity of the relationship. The value of Ca₅₀ in skinned cardiac fibres depends on a variety of experimental factors and there is evidence that the value in intact muscle is higher than can be explained by known factors which modulate Ca₅₀ [30, 31]. The observed value of n in intact muscle is typically between 2 and 4. Since cardiac troponin has only one binding site for Ca²⁺ [32], the simplest mechanism would give an n value of 1. The source of this co-operativity remains uncertain. One possible explanation is that the attachment of crossbridges in the strongly bound form increases the affinity of troponin C for Ca²⁺ (indicated by (v) in Fig. 1) [33, 34]. A number of other theoretical possibilities — based on the various interactions which may occur between neighbouring troponin, tropomyosin and actin molecules along the thin filament — have also been considered [35–37].

In intact heart muscle the Ca²⁺ transient rises in 10–20 ms and declines over 200–400 ms [38]so that tension never reaches a steady state with [Ca²⁺]_i. Nevertheless it would be useful to be able to determine n and Ca₅₀ in intact preparations and there have been various experimental attempts to determine these values [39]. A simple method is to modify developed force by changing extracellular Ca²⁺ concentration ([Ca²⁺]_o) and to determine the peak [Ca²⁺]_i and force at each level of [Ca²⁺]_o, (Fig. 2A). Peak [Ca²⁺]_i is then plotted against peak force (Fig. 2B). Although this method does not give the correct absolute values of n and Ca₅₀, it has allowed some important observations to be made. As [Ca²⁺]_i is elevated, force initially rises steeply and then rises more slowly, with the general form shown in Fig. 2B. However, large increases in [Ca²⁺]_i cause a fall of force, which would not be predicted from skinned fibre data. This is the phenomenon of "Ca²⁺ overload" (Fig. 2A and B) [40, 41]. Along with reduced force, several other phenomena are also seen in Ca²⁺ overload — spontaneous Ca²⁺ release [i.e. Ca²⁺ release not triggered by an action potential — indicated by the arrow in Fig. 2A (iv)], aftercontractions and triggered arrhythmias. All these phenomena are now known to be caused by a tendency for the sarcoplasmic reticulum to release Ca²⁺ spontaneously when its Ca²⁺ content becomes excessive [42, 43]. From the present perspective, the important point is that any intervention which has the capacity to sufficiently elevate [Ca²⁺]_i — including all conventional positive inotropic agents — is likely to reduce force at high concentrations and also to cause arrhythmias. Furthermore since sympathetic activity itself raises [Ca²⁺]_i, doses of inotropic agents which by themselves are within the therapeutic range, may contribute to Ca²⁺ overload when endogenous sympathetic activity occurs in the patient.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Relation between [Ca2+]i and force in heart muscle during inotropic interventions. Panel A. [Ca2+]i transients and force in intact heart muscle at 4 levels of [Ca2+]o (modified from Ref. [40]). The arrow in (iv) identifies a spontaneous elevation of [Ca2+]i. These are characteristic of Ca2+ overloaded preparations. Panel B shows a typical relation between peak [Ca2+]i and peak force; the marked points correspond to the activation levels in A. Note that at the highest levels of [Ca2+]i, developed force starts to decline; this phenomenon is not observed when the steady state force-Ca2+ relation is determined in skinned fibres (e.g. Figs. 3 and 4).

 

	3 Mechanisms of inotropy

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
As is now well-known, all major classes of inotropes currently in use act by elevating systolic [Ca²⁺]_i, i.e. by increasing the amplitude of the calcium transient [44]. However, although they produce a similar end result, a variety of cellular mechanisms are accessed by different drugs. Cardiac glycosides block the Na⁺ pump, the elevated [Ca²⁺]_i being secondary to a rise in [Na⁺]_i and involving the Na⁺/Ca²⁺ exchanger. β-adrenergic agents and PDE inhibitors both elevate cAMP and cause phosphorylation of intracellular sites on proteins. In the case of β-agonists occupancy of the β-receptor causes increased cAMP through the action of a coupled G protein and adenyl cyclase. PDE inhibitors reduce the activity of phosphodiesterases, hence reducing the rate of cAMP breakdown. Proteins which are phosphorylated as a consequence of the increased cAMP include the surface membrane Ca²⁺ channel (which increases Ca²⁺ entry and therefore sarcoplasmic reticulum Ca²⁺ loading), and troponin-I and phospholamban — which accelerate the relaxation rate [45]. Once [Ca²⁺]_i is elevated the sarcoplasmic reticulum becomes more loaded with Ca²⁺ and consequently releases more Ca²⁺ when triggered by an action potential (Fig. 2) [46].

Although conventional inotropes increase force by increasing [Ca²⁺]_i, there are in theory alternative ways of achieving the same end (see Fig. 3A). One possibility is to increase the maximum force of which the muscle is capable [curve (ii)]. This could happen if either the number of crossbridges contributing to force were increased (e.g. hypertrophy), if the force per crossbridge were to increase (the probable mechanism in alkalosis), or if crossbridges remained attached for a greater fraction of the crossbridge cycle (discussed below).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 The effects of various inotropic mechanisms on the steady state relation between force and Ca2+ (note Ca2+ is plotted on a logarithmic scale). The equations used to derive these curves are based on the schematic shown in Fig. 1. The fraction of troponin in the Ca2+-bound form [f(CaTr)] is given by: where n is the Hill coefficient and KD is the dissociation constant for the binding of Ca to troponin. Note that in this formulation co-operativity is represented by a Hill coefficient rather than an explicit interaction between troponin and cross-bridge binding (for further discussion see Refs. [36, 67]). Force (F) was calculated by assuming that fapp was a linear function of f(CaTr): where p is a factor representing the force produced by a cross-bridge and/or the number of available cross-bridges. The control curve (C) in Panel A and B has n = 2, KD=5 mM, p = 1, fapp=40 s–1, gapp=5 s–1. These values of fapp and gapp when used in a dynamic model of force production give an appropriate timecourse for the rise and fall of force in a contraction [69]. In Panel A line (i) shows the effect of increasing the affinity of troponin for calcium (KD=2.5 mM) while line (ii) shows the effect of increasing either the number of cross-bridges or the force per crossbridge (p = 1.2). In Panel B, (i) shows the effect of increasing fapp (fapp=80 s–1) or decreasing gapp (gapp=2.5 s–1) (the two curves are identical).

 
Another mechanism which could increase force would be for the curve to become more co-operative without altering Ca₅₀ or maximum force [25]. We will not discuss this possibility further since there is little direct evidence for its existence at present. In general an increase in n might be advantageous because contractile activity could then be switched on and off with less energetically costly Ca²⁺ movement. This mechanism could be an important target for drug action in the future as molecular understanding of muscle activation increases.

Finally, force could also be raised by increasing the sensitivity of the contractile proteins to [Ca²⁺]_i – [curve (i)]. In this case several different mechanisms could be involved. These are best discussed with reference to a scheme such as that shown in Fig. 1. A decrease in Ca₅₀ — that is an increase in Ca²⁺ sensitivity — could be produced in at least five different ways:

(i) An increase in the affinity of troponin C for Ca²⁺.

(ii) An increase in the effectiveness with which the troponin C–Ca²⁺ complex increases the rate of crossbridge attachment.

(iii) and (iv) A change in crossbridge kinetics. Increased force could be produced by either an increased rate of attachment (increased f_app) or a decreased rate of detachment (decreased g_app). These possibilities are illustrated in Fig. 3B which shows a control curve and two superimposed curves demonstrating the identical effect of doubling f_app or halving g_app. Both cause increased Ca²⁺ sensitivity (by a factor of 1.5) and also increased maximum force (by a factor of 1.12), with no effect on n.

(v) A change in the feedback between attached crossbridges and troponin C affinity for Ca²⁺. An increase in feedback causes increased Ca²⁺ sensitivity (reduced Ca₅₀) and also increases cooperativity (higher n).

	4 Calcium sensitisers

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
Investigations in recent years have revealed a large number of interventions which are able to shift the Ca²⁺-force relation. Examples include changes in pH, [Mg²⁺]_i, ATP, intracellular phosphate concentration, ionic strength, muscle length, troponin I phosphorylation, myosin light chain phosphorylation, C protein phosphorylation, and many drugs of which the best known is perhaps caffeine [44, 47]. Since these interventions affect the force produced in response to a given [Ca²⁺]_i they can be described as altering the myofibrillar Ca²⁺ responsiveness. As outlined in the preceding section, it is customary to distinguish between interventions which increase Ca²⁺ sensitivity [Fig. 3A(i)] and those which increase maximum force [Fig. 3A(ii)]. In practice many interventions produce both effects, although one or the other often predominates.

With the conventional steric hindrance model of muscle activation a justification for separating these effects is that it makes explicit two potential routes by which the force of cardiac contraction could be altered independently of [Ca²⁺]_i, and in some situations separate effects are indeed observed [48]. However, as noted above, in the turnover kinetics model of activation both effects may arise from a single intervention [e.g. Fig. 3B(i)]. It is worth emphasising that the term "Ca²⁺ sensitiser" is often used in the literature to indicate compounds which increase Ca²⁺ sensitivity, maximum force or both in isolated, skinned myofibrils, i.e. as a synonym for myofibrillar Ca²⁺ responsiveness.

Inotropic agents which raise force by increasing Ca²⁺ sensitivity might avoid toxicity problems associated with Ca²⁺ overload. Thus there has been interest in developing inotropic drugs based on this mechanism of action. The finding that it was possible to alter Ca²⁺ sensitivity with synthetic compounds was made using sulmazole, a known PDE inhibitor [49, 50]. The effect was detected in a skinned fibre preparation where the drug was applied directly to the contractile apparatus. Since that time several other compounds with similar dual activity have been discovered, including isomazole, APP 201-533, DPI 201-206, adibendan, levosimendan, pimobendan, MCI 154, ORG 30029 and EMD 53998 [51–62]. Analysis of the actions of these compounds in intact preparations and in vivo is complicated, because the relative strength of the effects on Ca²⁺ sensitivity and PDE inhibition in intact heart muscle varies between different molecules and species [57, 63–67]and also because PDE inhibition has several effects in intact muscle [57]. These include reduction of calcium sensitivity in heart muscle [68], as well as vasodilation in vivo.

For study of cellular mechanisms it is clearly desirable to have compounds which increase force in intact muscle only by affecting the Ca²⁺ responsiveness of the myofibrils. Of compounds currently available, EMD 57033 — which does cause some PDE inhibition in vitro [69]— produces the most clearcut positive inotropic effect due to increased myofibrillar Ca²⁺ responsiveness yet observed in intact cardiac muscle [65, 70, 71]. It has also been studied in a wide variety of preparations. CGP 48506 is a compound which has marked calcium sensitising effects without causing PDE inhibition [72–74]. Its effects on calcium sensitivity in intact muscle are not yet known.

In the following sections we examine the evidence for the various potential mechanisms of action of agents which alter Ca²⁺ sensitivity. We also discuss the issues of energetic efficiency, slowing of relaxation and other factors relevant to the potential usefulness of these agents in chronic heart failure. Because of the extent of the information available, we concentrate on EMD 57033, mentioning other compounds where appropriate.

	5 Mechanisms of action of calcium sensitisers

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
As noted above, the definition of an intervention which increases Ca²⁺ sensitivity is that it causes a shift to the left in the relation between force and [Ca²⁺]_i, i.e. there is a reduction in the [Ca²⁺]_i which produces 50% maximum force [(i) in Fig. 3A]. Fig. 4 shows a representative example of a Ca²⁺-force relation from skinned guinea pig ventricular muscle in the presence and absence of EMD 57033 [75]. Note that EMD 57033 decreases Ca₅₀, increases maximum force and reduces the slope of the curve (n falls from 2.7 to 1.6). The lines have been calculated using a model of the type shown in Fig. 1 fitted to the data (see figure legends for details). It was assumed that the affinity of troponin for Ca²⁺ was constant in the presence of EMD 57033 and that the drug affected either f_app or g_app. The line was calculated by increasing f_app 3.6 fold or by decreasing g_app by the same ratio. In this section we consider the possible sites of action which could produce these effects and discuss which of the mechanisms identified in Fig. 1 could be responsible.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of EMD 57033 on the steady-state relation between force and Ca2+. Data points taken from Gross et al. [75]— skinned guinea pig ventricular muscle, 37°C, 10 mM EMD 57033. The continuous lines are calculated from the equations given in Fig. 3 fitted to the data points. For the control curve gapp was set to 5 s–1, and the fitted curve then has KD=11 mM, N = 2.7, fapp=23 s–1. For the EMD 57033 curve gapp and KD were maintained at the same values and the fit shown obtained with N = 1.6 and fapp=82 s–1. An identical curve was obtained if fapp was kept constant and gapp allowed to decrease.

 
Current evidence suggests that available Ca²⁺ sensitisers do not increase the affinity of troponin C for Ca²⁺ [mechanism (i) in Fig. 1]. If this were the case measurements of Ca²⁺ binding to either isolated troponin C or to the myofilaments under conditions in which feedback of crossbridges was prevented should demonstrate an increased occupancy. Ca²⁺ binding measurements with isolated troponin C in the presence of sensitisers have not been reported to our knowledge, while Ca²⁺ binding to isolated myofibrils failed to show increased binding in the presence of sensitising concentrations of EMD 57033 [71], levosimendan [56]or CGP 48506 [76]. Furthermore if increased affinity of troponin C for Ca²⁺ were the sole mechanism of action it would be expected to cause a parallel shift in the Ca²⁺-force curve, whereas increased force at saturating [Ca²⁺]_i is observed.

Another possibility is that a sensitiser might increase the force produced for a given level of Ca²⁺ bound to troponin C, but in the absence of a change in the maximum value of f_app [mechanism (ii) in Fig. 1]. No direct measurements of this type are available, but an alternative approach is to test whether sensitisers can affect actin–myosin interactions in the absence of troponin and tropomyosin. Solaro et al. [71]showed that the ATPase rate of pure actomyosin (i.e. in the absence of troponin and tropomyosin) was enhanced about 2 fold in the presence of 10 µM EMD 57033. They also demonstrated that purified actin filaments increased their velocity of motion along myosin filaments in the presence of the drug in an in vitro motility assay. Both these experiments indicate that EMD 57033 can enhance actin–myosin interactions in the absence of troponin and tropomyosin and suggest that mechanism (iii) may be important, i.e. an increase in the maximum value of f_app. These findings have been confirmed in several related studies, either with [Ca²⁺]_i so low that Ca²⁺ bound to troponin is negligible [77]or after a procedure which extracts troponin I and renders the preparation insensitive to Ca²⁺ [78, 79]. All these studies indicate that the compound acts directly on actin–myosin interactions rather than on the activating proteins.

A number of studies have investigated the possible contribution of increased f_app or decreased g_app [mechanism (iii) or (iv)]. Leijendekker and Herzig [80]measured the rate of tension redevelopment after a quick stretch and also the oscillatory frequency at which muscle stiffness was at a minimum. Both processes were slower in the presence of 25 µM EMD 53998. This suggests that crossbridge cycling rates are slower in the presence of drug, indicating that (f_app+g_app) is reduced. The energetic efficiency (force/ATP consumed, see below) was also measured in this study and was increased, but only at force levels below 60% of maximum. Since energetic efficiency is proportional to 1/g_app [29], it was concluded that the most likely effect of EMD 53998 was to reduce g_app.

Simnett et al. [81]measured the rate of relaxation in a skinned cardiac preparation when Ca²⁺ was rapidly lowered with diazo-2, a caged Ca²⁺ chelator. They found that EMD 57033 had either no effect or caused a slight acceleration of relaxation rate. Under such circumstances one would expect relaxation to be dominated by g_app. This result therefore suggests that g_app is little affected by EMD 57033, indicating that if EMD has a site of action on crossbridge kinetics it is likely to increase f_app. Arner et al. [78]measured the rate of tension redevelopment in skinned fibres activated from a rigor state with caged ATP. They found that EMD 53998 (half maximal concentration 10 µM) increased the rate of force redevelopment, consistent with an increase in (f_app+g_app). On the grounds that Simnett et al. found no change in g_app, they proposed that EMD 53998 might increase f_app. Clearly some of these results are mutually inconsistent and it is not yet possible to reach a consensus on whether EMD 57033 increases f_app or decreases g_app. A note of caution relevant to the interpretation of these results with photolabile probes is sounded by the recent finding that EMD 57033 is itself photolabile [82].

Since many Ca²⁺ sensitisers are also PDE inhibitors (see above) they may elevate cAMP and thus have effects similar to β-adrenergic stimulation, including elevation of [Ca²⁺]_i. It is therefore important to test the effects of apparent Ca²⁺ sensitisers in intact cardiac preparations while simultaneously measuring [Ca²⁺]_i. Such experiments can reveal whether part or all of the inotropic effect of a putative sensitiser is in fact attributable to increased Ca²⁺ transients. It is also important to establish that a sensitising effect is comparable in magnitude in intact as opposed to skinned preparations. For example, it is possible that a sensitising effect could be lost in an intact preparation if the drug could not cross the surface membrane or had additional desensitising effects in the intact cell. For EMD 57033 results in intact muscle convincingly demonstrate an inotropic effect which is due to increased calcium responsiveness of the myofilaments [65, 71]. The PDE effects of this drug must be relatively small in the intact muscle preparations used, since the Ca²⁺ transients were reduced in the presence of the drug, whereas significant PDE inhibition would be expected to increase the Ca²⁺ transients.

	6 Effects on energetic efficiency

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
When force is increased by raising [Ca²⁺]_i, a greater fraction of available crossbridges are activated. With this mechanism it is therefore expected that more force is associated with raised metabolic consumption, since greater ATP breakdown is entailed both by Ca²⁺ movement and by crossbridge cycling [83]. Thus the appropriate comparison for a novel inotropic agent is whether the extra force per unit of energy consumed is more or less than for a simple manoeuvre such as raising [Ca²⁺]_o. In theory the greater the increase of force per unit energy consumed, the more clinically effective a novel inotrope would be, especially since in ischaemic heart disease there is usually only a limited capacity to increase the supply of oxygen and nutrients.

Because the heart must produce work (pressure x volume change), measuring force produced per unit of energy consumed in isometric contractions may not be representative of effects on the ejecting heart. For example the ‘catch state’ of many shellfish muscles [84], allows force maintenance for long periods with little energy consumption, but the muscle cannot shorten or perform work. Ideally measurements of the "energetic efficiency" of an inotrope should thus be made on ventricular muscle which is both producing force and shortening. However, few studies to date have used this approach to assess inotropic drugs.

An important theoretical consideration is that in a two-state model of crossbridges (e.g. Fig. 1) force is proportional to f_app/(f_app+g_app), while energy consumption is proportional to f_appxg_app/(f_app+g_app). Thus energetic efficiency (force per unit energy) is proportional to 1/g_app [29]. Hence if an inotrope increases the energetic efficiency of isolated contractile proteins, the implication is that it reduces g_app, assuming that neither the number of cross-bridges nor the force/cross-bridge are affected. Note, however, that in an intact heart this conclusion cannot be drawn, since changes in energetic efficiency may also involve altered Ca²⁺ movements. Under some circumstances, it may be possible to enhance the force per cross-bridge [85]. Although this is an attractive target for the future, the concentration of EMD 57033 used to achieve this result in skinned skeletal muscle (50 µM) was much higher than appears to be physiologically relevant in cardiac muscle.

Several studies have shown that for intact papillary muscles in which energy consumption was assessed by heat output, drugs with sensitising activity (including pimobendan [86], EMD 53998 [87]and EMD 57033 [75]) increased the efficiency of ventricular muscle by comparison with either isoprenaline or digitalis. Studies on skinned fibres have reached the same conclusion, although the increase in energetic efficiency was small and seen only at low or intermediate force levels [80, 88]. Since energy consumption associated with Ca²⁺ release and uptake is eliminated in fully skinned fibres (where all the membranes have been removed), these results indicate that the most important component of increased energetic efficiency in intact preparations arises from reduced Ca²⁺ movements.

Other studies have been performed in isolated perfused hearts using O₂ consumption as an indicator of energy utilisation. In a study using blood-perfused dog hearts, no difference was found between the O₂ consumption produced by equi-effective inotropic doses of Ca²⁺ or EMD 53998 [89]. However, since EMD 53998 combines sensitising properties with PDE inhibition (which reduces energetic efficiency) this result is likely to reflect the mixed mechanisms of action. Consistent with this interpretation, a study of EMD 57033 (which has the same chemical formula as EMD 53998, but is the enantiomer with all the calcium sensitising activity and relatively little PDE inhibition) produced a 40% increase in developed force with no change in O₂ consumption [90].

A recent study [91]extended this experimental approach to intact pig hearts undergoing normal contraction, so that any effects of on energetic efficiency would necessarily be produced in muscle with complete working crossbridge cycles. Left ventricular external work was calculated (the area under a shortening/developed pressure loop) and compared to O₂ consumption. Addition of the predominantly sensitising EMD 60263 increased force but did not significantly affect energetic efficiency. Hearts were then exposed to several short periods of ischaemia, following which external work and energetic efficiency was substantially reduced ("myocardial stunning"). In this situation EMD 60263 produced a striking increase in energetic efficiency. If this result is confirmed it indicates that although this compound may not affect the energetic efficiency of normal working myocardium, it can increase energetic efficiency of myocardium affected by preceding ischaemia. Interestingly there is a precedent for this finding in that earlier studies on skinned cardiac tissue indicated that EMD 53998 could overcome the reduction in energetic efficiency produced by elevated phosphate [88], one of the main factors accounting for depression of force in hypoxic heart muscle [92].

Although these results support the suggestion that Ca²⁺ sensitisers might be capable of increasing energetic efficiency of contraction, perhaps especially in metabolically compromised hearts, it is not yet clear to what extent this improvement derives from Ca²⁺ handling as opposed to crossbridge cycling. Further studies of effects on energetic efficiency are required, since compounds which could increase force while also increasing efficiency could be particularly useful in the clinical setting.

	7 Slowing of relaxation

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
A consistent finding in intact isolated preparations is that compounds which increase force by increasing calcium sensitivity also slow the relaxation rate [37, 65, 67, 70]. Usually the time to peak force is little affected, while early relaxation shows moderate slowing. At high drug concentrations there can be substantial slowing of the late phase of relaxation and even a failure of force to return to resting levels in the diastolic period [65, 70]. Observations on relaxation rate are significant since they may help to identify mechanisms of action and also because slowing of relaxation could be therapeutically disadvantageous in certain clinical situations (see below).

In theory slowing of relaxation could be caused by several mechanisms: (i) slower decline of [Ca²⁺]_i, (ii) slower dissociation of Ca²⁺ from troponin, or (iii) slower detachment of crossbridges. White et al. [65]showed that the Ca²⁺ transient timecourse was abbreviated with EMD57033, excluding mechanism (i). Simnett et al. [81]used skinned fibres in which Ca²⁺ was lowered very rapidly using a photoactivated caged Ca²⁺ buffer. Under these circumstances EMD 57033 did not slow relaxation (in fact it slightly accelerated it, although this might have been caused by photolysis of the drug [82]). In a similar study on CGP 48506 [93], Palmer and Kentish found no change in relaxation, and therefore crossbridge detachment, in the presence of the drug. Thus the most likely cause of slowing of relaxation in the intact preparation would appear to be mechanism (ii), even though the overall binding of Ca²⁺ to troponin appears to be unaffected by sensitisers (see above). Slower dissociation of Ca²⁺ from troponin could be either a direct effect on troponin in force-producing myofilaments or the result of co-operative interactions on the myofilaments as discussed previously.

The main factor causing cardiac filling in diastole is the relatively small pressure gradient between the central veins and the ventricles. Thus it is important that the ventricle should be compliant as possible during diastole. Also, at elevated heart rates the duration of diastole becomes rate-limiting for cardiac filling, so it is important that relaxation should be as fast as possible to maximise effective filling. Both these considerations suggest that an inotrope which significantly slowed relaxation or increased diastolic stiffness would be undesirable [94], especially since in some patients heart failure is associated with deterioration of diastolic properties [95–97].

On this basis it has been argued that calcium sensitising inotropes might be deleterious rather than advantageous in heart failure [94]. However, potential effects on relaxation must be weighed against the other possible advantages of sensitisers. If they achieve an inotropic effect with a smaller increase in O₂ consumption and without inducing arrhythmias (see below) then the overall advantages could be substantial, especially since diastolic dysfunction is of primary importance in only a small minority of cases. Also it is important to note that concentrations of EMD 57033 which could exert clinically useful effects (e.g. 25–50% increase in force) have been found to have little effect on the time course of relaxation [65, 90, 98]. Even large increases in force (e.g. 100%) are associated with only a 10–20% prolongation of relaxation [65, 98]. Interestingly a study on perfused rabbit hearts showed that while there was a substantial slowing of relaxation with isovolumic contraction, slowing was no longer apparent when the hearts were allowed to eject blood [99]. In addition, in vivo studies have established that sensitisers are able to significantly increase cardiac output (see below). It should also be noted diastolic stiffness data is often interpreted solely in terms of myocyte properties. However, diseased hearts usually contain a greatly increased connective tissue component [100], which is probably the major contributor to altered diastolic distensibility in many cases.

As noted above, many currently available sensitisers also have PDE inhibitory activity. Since this effect causes acceleration of relaxation, it is possible that a judicious mixture of the two activities could be useful in certain circumstances. Alternatively it might be advantageous to combine use of sensitisers with drugs which slow the heart rate.

	8 Potential usefulness of calcium sensitisers as inotropes

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
Several additional issues are of importance if the inotropic effects demonstrated by calcium sensitisers in normal cardiac muscle are to be translated into clinically useful effects in diseased hearts.

8.1 Effects on diseased muscle
Although diseased hearts often contain normal muscle, substantial areas may be affected by relative hypoxia or low flow ischaemia, so it is important that an inotrope is beneficial (or at least not harmful) to these areas as well. Most of the short-term negative inotropic effect attributable to hypoxia and ischemia is caused by accumulation of intracellular phosphate and hydrogen ions [92]. Than et al. [98]examined the effects of several inotropes in normal cardiac muscle and in muscle exposed to hypoxia or a moderate metabolic acidosis. Interestingly the results obtained were broadly similar for inotropes acting by increasing [Ca²⁺]_i and for calcium sensitisers (including EMD 57033). A sufficient inotropic stimulus supplied by either class of inotrope could fully reverse the negative inotropic effect of acidosis [70, 98]. With traditional inotropes this was achieved by increasing the Ca²⁺ transient amplitude. With EMD 57033 the negative inotropic effect of acidosis was reversed with a simultaneous reduction in Ca²⁺ transient amplitude [70, 98].

In pure hypoxia (i.e. no flow restriction) force depression is due mainly to intracellular phosphate accumulation, with only a minor contribution from acidosis [101]. Both classes of inotropes were relatively ineffective in reversing force depression in this situation, implying that hypoxia reduces force by mechanisms downstream [47]of the point of action of all the inotropes tested. Thus sensitisers can reverse at least one of the major mechanisms causing force depression in diseased muscle and they do so in a way which is both energetically favourable (reduced Ca²⁺ cycling) and less likely to induce arrhythmias.

8.2 Arrhythmias
In many organs malfunction of a small volume of tissue is of little or no consequence, but in the heart even a tiny arrhythmogenic focus can have disastrous consequences. Although arrhythmias can be caused by many mechanisms [102]those associated with raised [Ca²⁺]_i [103]are of particular importance in the current context. [Ca²⁺]_i is likely to be already raised in diseased muscle [104]and conventional inotropes raise it still further. It is thus perhaps not surprising that arrhythmias have been a problem with positive inotropic therapy in clinical practice. Calcium sensitisers would be expected not to increase [Ca²⁺]_i-associated arrhythmias and thus might avoid at least some of these problems. However, in one study which attempted to investigate this point experimentally, ventricular arrhythmias associated with acutely increased wall-stress in the isolated working rat heart were increased both by raising [Ca²⁺]_o and by EMD 57033 [105]. Further work using a variety of models to investigate the arrhythmogenic potential of sensitising inotropes in working hearts is clearly needed.

8.3 In vivo studies
Whole animal studies show that it is possible to improve cardiovascular parameters in normal, stunned and failing hearts with no overt increase in arrhythmias, at least in the short term [91, 106–108]. In particular, EMD 57033 can increase cardiac output in both normal hearts [106]and in a pacing-induced model of heart failure [107]. Since LVdP/dt_max also increased in these experiments, any deleterious effect due to slowing of relaxation was clearly outweighed by the positive inotropic effect of the drug. The sensitising compound MCI-154 was also effective in a pacing-induced model of heart failure, where it improved diastolic distensibility and relaxation as well as systolic function [108].

Available evidence thus suggests that current sensitisers can produce an inotropic effect in diseased muscle and should also have energetic advantages. They seem to be at least no worse than conventional inotropes with respect to early arrhythmogenesis and theoretical considerations suggest that they may possess advantages in this area. Also increased cardiac output has been produced in heart failure without evidence of diastolic dysfunction.

	9 Conclusions

Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 
Prevention, arrest and reversal of underlying ischemic damage would be the best way to reduce the incidence of heart failure and to slow its progress. Unfortunately clinically proven treatments producing these effects are not yet available. Also, it seems inevitable that factors such as late diagnosis and damaging acute events will mean that there will always be substantial numbers of patients with incipient or overt heart failure. In such cases it would be useful to have agents which could safely enhance myocardial contractility. Enhancement of myofibrillar calcium responsiveness represents a novel approach to this goal which has several theoretical advantages. It has not yet been directly tested in the clinical setting. The mixed performance of pimobendan [109]may reflect the fact that its sensitising activity is combined with PDE inhibition.

Our knowledge of the molecular mechanisms involved in cardiac muscle force production remains sketchy, although great strides are now being made with the application of molecular techniques. Most current inotropes increase force by increasing [Ca²⁺]_i, but some compounds, such as the sensitisers discussed in this article, are beginning to access the possibilities for increasing force inherent in the myofilament machinery. As we have outlined, there are reasons for believing that this approach to positive inotropy may have greater success than traditional methods. In the final analysis, the degree to which pharmacological supportive therapy can make an impact on heart failure will be determined by the extent to which it can be shown to make controlled beneficial changes to quality of life and patient survival. This in turn depends on drug safety and hence the disease stage at which a risk/benefit analysis mandates that supportive treatment should be started. Although there have been past disappointments, it is clear that we should not reject the possibilities offered by substances which manipulate myofibrillar Ca²⁺ responsiveness.

Time for primary review 28 days.

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Top 1 Introduction 2 Heart muscle activation 3 Mechanisms of inotropy 4 Calcium sensitisers 5 Mechanisms of action... 6 Effects on energetic... 7 Slowing of relaxation 8 Potential usefulness of... 9 Conclusions References

 

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