Special Article

Changes in Intracellular Ca2+ Mobilization and Ca2+ Sensitization as Mechanisms of Action of Physiological Interventions and Inotropic Agents in Intact Myocardial Cells

Masao ENDOH, MD, PhD

SUMMARY Physiological and pharmacological interventions are used to regulate cardiac contractile functions via modulation of Ca2+ signaling. The relevant regulatory mechanisms have recently been assessed in detail by use of novel experimental procedures, which include simultaneous measurements of intracellular levels of Ca2+ ions and contractile force in intact myocardial preparations loaded with the intracellular Ca2+ indicator aequorin and fluorescent dyes, namely, fura-2, indo-1 and fluo-3. Association with or dissociation from intracellular Ca2+ transients of contractile activity is taken as evidence that reflects the primary mechanism of action of individual inotropic interventions. In addition, motility assays of actin-myosin interactions in vitro have made it possible to define the site of action of Ca2+ sensitizers as troponin C and the interaction of the troponin-tropomyosin complex with actin or the actin-myosin interface at crossbridges. Frank-Starling mechanism operates at the level of the binding of Ca2+ ions to troponin C and subsequent regulatory processes, while the force- frequency relationship is mainly ascribed to an alteration in the intracellular mobilization of Ca2+ ions. Cardiotonic agents can be classified as follows: 1) agents that act via a cyclic AMP-dependent or a cyclic AMP-independent mechanism; and 2) agents that facilitate the intracellular mobilization of Ca2+ ions or increase in myofibrillar sensitivity to Ca2+ ions. Regulatory mechanisms mediated via the phosphorylation of functional proteins induced by cyclic AMP, which is responsible for the actions of novel cardiotonic agents, ƒÀ1- adrenoceptor partial agonist and selective inhibitors of phosphodiesterase

(PDE) III, have currently been clarified in more detail. Ca2+ sensitizers are of extreme therapeutic interest because of their ability to increase myocardial contractility without an increase in activation energy; they are devoid of risks of arrhythmogenicity and myocardial cell death from intracellular Ca2+ overload; and they effectively reverse contractile dysfunction under pathophysiological situations, such as acidosis or myocardial stunning. (Jpn Heart J 1998; 39: 1- 44)

From the Department of Pharmacology, Yamagata University School of Medicine, Yamagata, Japan. Supported in part by a Grant-in-Aid for Developmental Scientific Research (no. 0755193) from the Ministry of Education, Science, Sports and Culture, Japan. Address for correspondence: Masao Endoh, MD, PhD, Department of Pharmacology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-2331, Japan. Received for publication August 13, 1997. Accepted August 20, 1997. 1 Jpn Heart J 2 ENDOH January 1998

Key words: Frank-Starling mechanism, Force-frequency relationship, Cy-

clic AMP, Myofibrillar Ca2+ desensitization, Myofibrillar Ca2+ sensitization, ƒÀ1- adrenoceptor partial agonists, Selective PDE III inhibitors, Phosphoinositide hydrolysis, Ca2+ promoters, Ca2+ sensitizers

HE mechanical performance of the heart is regulated through an interlock- T ing network of regulatory systems that adjust the function of the cardiac pump so that it can respond to the continually changing needs of the body.1) These regulatory systems involve two components, namely, the intrinsic properties of myocardial cells, which play an extremely important role in the physiological adjustment of cardiac mechanical functions to the requirements of the body in vivo, and the extrinsic regulatory mechanisms, whereby the neurohumoral transmitters, autacoids, cytokines and hormones that trigger signal transduction processes allow the adjustment of cardiac contractile functions to widely changing circumstances.1,2) The intrinsic regulation involves Frank-Starling mechanism in which the strength of the cardiac contractile force changes depending on the extent of stretching of myofibrils over the physiological range of fiber lengths.3-5)

Another intrinsic mechanism involves the force-frequency relationship. In most mammalian ventricular myocardium, the amplitude of contraction is increased by an increase in the frequency of contraction (positive staircase phenomenon).6-8)

Extrinsic regulation, by contrast, is exerted via activation of membrane receptors, which is triggered by the binding of transmitters that are specific agonists for the respective receptors, such as norepinephrine, epinephrine, vasoconstrictor peptides (e.g., angiotensin II, endothelin isopeptides),2) autacoids (e.g., adenosine, prostaglandins) and cytokines [e.g., tumor necrosis factor ƒ¿(TNF-ƒ¿), interleukins], which are generated and released in response to physiological and/ or pathological interventions.

The rapid and transient increase in the intracellular concentration of free

Ca2+ ions ([Ca2+]i), which occurs prior to the generation of the force of contraction or the shortening of a myocardial cell, plays a key role in cardiac excitation- contraction (E-C) coupling.9,10) This transient increase in [Ca2+]i, subsequent to excitation of the myocardial cell membrane is known as the calcium transient

(Ca2+ transient) and it is an essential determinant of the amplitude and time course of the contraction-relaxation cycle of cardiac muscle.5,11) The amplitude

and duration of Ca2+ transients in intact cardiac myocytes are determined by

several transmembrane and intracellular factors that are related to mobilization of Ca2+ ions and/or a decrease in [Ca2+]i, including voltage-dependent L-type

Ca2+ channels (dihydropyridine receptors), Na+-Ca2+ exchange and the Ca2+ pump of sarcolemma, the Ca2+ pump and Ca2+-release channels (ryanodine receptors) of sarcoplasmic reticulum (SR), Ca2+ binding proteins, such as troponin Vol 39 No 1 Ca2+SIGNALING IN CARDIACMUSCLE CELLS 3

Figure 1. Schematic representation of the ion channels, ion-transport systems, Ca2+- binding proteins and contractile proteins that are involved in the regulation of Ca2+ signaling, as well as sites of action of some positive inotropic agents in mammalian myocardial cells. SL=sarlemmal; Tm=tropomyosin; SR=sarcoplasmic reticulum.

C, calmodulin and calsequestrin, and mitochondria (Figure 1). The binding of Ca2+ions to and their release from troponin C play a crucial role in determining the shape of Ca2+transients.3,12-14) The issues on which we shall focus in this review are the current under- standing of Ca2+signaling in myocardial cells, the relationship of such signaling to contractile regulation, the classification and mechanisms of action of cardiotonic agents, the different modes of sensitization to Ca2+ ions induced by novel cardiotonic agents, and current problems and perspectives related to the clinical application of the various novel cardiotonic agents.

CLASSIFICATIONOF INOTROPIC MECHANISMS Regulation of myocardial contractility can be classified by reference to the central role of troponin C in triggering a series of events that activate contractile proteins. Troponin C binds Ca2+ ions to release the inhibitory regulation of the sliding of thick and thin filaments in cardiac E-C coupling.5,15)According to the present classification, there are three general types of mechanism that should allow alterations in the contractile performance of cardiac muscle: 1) alteration of the amplitude or time course of Ca2+ transients ("upstream" mechanisms); 2) alteration of the affinity of troponin C for Ca2+ions ("central" mechanisms); and 4 ENDOH Jpn Heart J January 1998

3) alteration of the response of myofilaments to a given level of occupancy of Ca2+-bindingsites on troponin C, namely, alteration at the level of actin regulation that is induced by the troponin-tropomyosin complex and/or modulation of the rate of cycling of crossbridges at the actin-myosin interface ("downstream" mechanisms).5,11)While the majority of inotropic interventions alters the amplitude and duration of Ca2+ transients, it has been shown that the affinity for Ca2+ ions of troponin C and the so-called downstream mechanisms are subject to regulation by novel inotropic agents,2,16-18)which are expected to be beneficial in the treatment of myocardial failure because their use is not linked to risks of arrhythmogenicity and injury to myocardial cells due to intracellular Ca2+overload.19,20) Moreover, they have an energy-related benefit in that they do not require an increase in the activation energy that is necessary for the transport of intracellular Ca2+ions, and they are able to reverse myocardial depression in pathophysiological situations, such as ischemia with accumulation of inorganic phosphate in myocardial cells,21)as well as acidosis and myocardial stunning.2,22-26) Some inotropic interventions act by more than one mechanism. The overall regulation by individual inotropic interventions in intact myocardial cells can be analyzed by simultaneous assessment of the strength (or shortening) and time course of single (twitch) contractions and the Ca2+transient, which is now possible by use of the bioluminescent protein aequorin9) or of fluorescent dyes, such as fura-2, indo-1 and fluo-3, in preparations of myocardial tissues and single myocardial cells.27)Biochemical and electrophysiological findings, as well as findings in skinned cardiac fibers and in motility assays in vitroprovide information about the individual mechanisms that contribute to the integrated regulation of contractility in intact myocardial cells.2,16)

PHYSIOLOGICALINTERVENTIONS Frank-Starling mechanism Since myocardial contractility corresponds, by definition, to the ability of the cardiac muscle to perform stroke work (or to generate cardiac output) at constant end-diastolic fiber length (volume or pressure), the curve expressing Frank-Starling mechanism in the working heart (the ventricular function curve) defines the baseline contractility, which corresponds to the length-tension relationship in an isolated preparation of cardiac muscle.3,5,28)A change in contractility leads to a shift of the curve, so that any intervention that alters the basic property of the heart generates a new curve, which is evidence of an altered ventricular function or a new length tension relationship.4)In patients and experimental animals with heart failure, the curve is shifted downwards, and this shift Vol 39 Ca2+SIGNALING IN CARDIACMUSCLE CELLS 5 No I represents the decreased contractility that results in the reduced work of the heart at any end-diastolic fiber length.29,30) An increase or decrease in the amplitude of the contractile force induced by Frank-Starling mechanism is associated with little change in the amplitude and/or duration of intracellular Ca2+ transients, an indication that the change in contractile force is due to a process beyond the mobilization of intracellular Ca2+ ions.5) Frank-Starling mechanism is mainly due to the extent of overlapping of thin and thick filaments and Lmax (length of muscle fibers or sarcomeres at which the contractile force is maximal) is achieved at a sarcomere length of approximately 2.2-2.3ƒÊm, at which the optimum overlapping is achieved. The mechanism is also due to an increase in myofibrillar sensitivity to Ca2+ ions.3,5,28,31,32) The alteration of myofibrillar sensitivity to Ca2+ ions induced by changes in fiber length is crucial for determining the time course of

Ca2+ transients in intact cardiac muscle. The force developed at different muscle- fiber lengths influences the affinity of troponin C for Ca2+ ions, which subse-

quently affects the time course of Ca2+ transients.3,33) The regulation of Ca2+ transients induced by changes in fiber length has been well documented in

aequorin-loaded multicellular preparations of cardiac muscle.34) The time course

of the decline of Ca2+ transients at Lmax is slightly accelerated, as compared with

that at slack lengths, and this slight acceleration might be due to a force-depen-

dent increase in affinity for Ca2+ ions of troponin C at Lmax.3,5,13,14) These findings

are supported by observations in skinned cardiac muscle fibers.35)

Frank-Starling curve is shifted downwards and, actually, the curve becomes

flat in the case of ventricular muscle isolated from patients with severe myocar-

dial failure,30) an indication that loss by the heart of an intrinsic regulatory

mechanism is an essential factor in heart failure. Myofibrillar sensitivity to Ca2+

ions is unchanged or is increased to some extent in skinned cardiac fibers pre-

pared from humans with dilated cardiomyopathy,36) whereas the maximal ATPase activity of myofibrils is decreased in patients with heart failure.37) It has

been reported that Ca2+ transients show an abnormal biphasic pattern38) and that

Ca2+-transport mechanisms are impaired in such cases.22) It is assumed, therefore,

that the modulation of myocardial contractility by long-lasting and integrated

regulatory factors, which include metabolic alterations under extensive neurohumoral control, plays a crucial role in the altered Frank-Starling mechanism, primarily as a consequence of abnormal handling of Ca2+ ions during myocardial failure in situ.

Force-frequency relationships At a constant diastolic fiber length, an increase in the frequency of contraction above the physiological range of frequencies results in an increase in the amplitude of contractile force in intact ventricular preparations from most mam- Jpn Heart J 6 ENDOH January 1998 malian species.6,39,40)In contrast to Frank-Starling mechanism, the amplitude of contraction during operation of the force-frequency relationship is associated with a pronounced elevation of peak Ca2+transients.5,7,8,41) At least two different mechanisms are considered to be responsible for the increase in the amplitude of Ca2+ transients upon elevation of the frequency of stimulation: 1) an increase in the influx of Ca2+ ions through voltage-dependent L-type Ca2+ channels, due to an increased proportion of plateau potentials per unit time as a result of an increase in numbers of action potentials (depolarization); and 2) an increase in intracellular levels of Na+ ions due to activation of increased numbers of fast Na+ channels by depolarization, which in turn increases intracellular levels of Ca2+ ions through the Na+-Ca2+exchange system.42,43)The positive staircase phenomenon disappears or is inverted as a negative staircase with increasing severity of myocardial failure in humans,44-47)as well as in experimental animals.48,49)This phenomenon might be mainly ascribable to decreases in the amplitude of Ca2+ transients,50)due to dysfunction of Ca2+ transport in the SR, which can be reversed by forskolin.51)

MECHANISMS OF ACTION OF INOTROPIC AGENTS

Mechanisms involving cyclic AMP

The sympatho-adrenal system plays a central role in adjusting the function of the cardiac pump to the immediate requirements of the body; it provides an extrinsic facilitatory mechanism for regulation of cardiac contractility in situ.

Membrane receptors involved in this facilitatory regulation belong predominantly to the ƒÀ1-adrenoceptor subtype and to the ƒÀ2-adrenoceptor subtype to a lesser extent.52) The intracellular processes through which the activation of these receptors leads to characteristic changes in myocardial contractility have been studied extensively. Stimulation of ƒÀ1-adrenoceptors, located on the outer surface of the myocardial membrane, by sympathomimetic amines leads to activation of the catalytic subunit of adenylate cyclase via stimulatory GTP-binding protein,

Gs, and to the accumulation of cyclic adenosine 3', 5'-monophosphate (cyclic

AMP) in myocardial tissues.53,54) Accumulation of cyclic AMP is similarly promoted by positive inotropic agents that are capable either of activating adenylate cyclase [e.g., forskolin, glucagon, prostaglandin E, cholera toxin, histamine (H2- receptor agonist)] or of inhibiting the cyclic nucleotide phosphodiesterase (PDE) that catalyzes the hydrolysis of cyclic AMP to yield the inactive metabolite 5'-

AMP [e.g., isobutylmethylxanthine, theophylline, amrinone, milrinone, enoximone and other newer positive inotropic agents, such as vesnarinone, pimobendan, olprinone, and toborinone].16) All of these agents, as well as li- pophilic derivatives of cyclic AMP (e.g., dibutyryl-cAMP, 8-thio-benzyl-cAMP, 8- Vol 39 No I Ca2+SIGNALING IN CARDIACMUSCLE CELLS 7 bromo-cAMP) increase cardiac contractile function through a common mechanism, namely, the activation of cyclic AMP-dependent protein kinase A (PKA), thereby promoting the phosphorylation of functional proteins of the sarcolemma, the sarcoplasmic reticulum (SR) and myofibrils.55-57)

Functional cardiac proteins, whose phosphorylation is catalyzed by PKA, include the proteins that form L-type Ca2+ channels, phospholamban, troponin I,

C protein, ryanodine receptors, and myosin light chain (MLC). PKA activates

Ca2+ channels in association with the phosphorylation of the ƒ¿1-and ƒÀ-subunits of L-type Ca2+ channels,58-60) which are involved in a number of processes, such as channel activation, voltage-dependent inactivation, permeation and Ca2+-dependent inactivation.61-63) Phosphorylation by PKA of phospholamban and ryanodine receptors, the functional proteins involved in the uptake of Ca2+ ions into and their release from the SR,64) plays a crucial role in the cyclic AMP- mediated regulation of cardiac contractility.65) Among myofibrillar proteins, troponin I, C protein (thick filament protein), and 19-kDa (or 20-kDa) myosin light chain have been shown to be phosphorylated by PKA.55) The most important consequences of such phosphorylation are: 1) an increase in the influx of Ca2+ ions across the sarcolemma during the action potential; 2) an increase in the rate of uptake of Ca2+ ions into the SR; 3) an increase in the release of Ca2+ ions from the SR; and 4) a decrease in myofibrillar sensitivity to Ca2+ ions. In addition, it has been reported that the rate of cycling of crossbridges is increased directly by stimulation of ƒÀ-adrenoceptors.66) The net effect of these changes is a positive inotropic effect (an increase in the strength of myocardial contraction), which is associated with a prominent positive lusitropic effect (an accelerated relaxation) and an abbreviation of contraction.67-69) The major effects of ƒÀ-adrenoceptor agonists on Ca2+ transients and cardiac contraction are an increase in peak values and change in the time course. However, the extent of such effects differs among species of animals and among individual preparations.2)

In preparations of cardiac muscle isolated from the frog, cat, guinea pig, rat, rabbit, dog, and ferret, ƒÀ-adrenoceptor agonists consistently increase the peak

Ca2+ transient and the contractile force, and they reduce the duration of both the

Ca2+ transient and the force in a concentration-dependent manner.2,67-69) The interpretation of ƒÀ-adrenoceptor-mediated changes in aequorin signals, recorded from multicellular preparations of myocardial cells, is complicated by a number of properties of the Ca2+ indicator (for a detailed discussion, see refs. 9 and 10).

Aequorin luminescence increases in proportion to the concentration of Ca2+ ions, raised to the power of 2.5 and, therefore, if differences in [Ca2+]i exist within a cell, the aequorin signal will be dominated by contributions from those regions where [Ca2+]i is highest. Intracellular spatial gradients of Ca2+ ions can exaggerate these complications upon ƒÀ-adrenoceptor-mediated facilitation of the entry and Jpn Heart J 8 ENDOH January 1998 release of the Ca2+ ions are responsible for the E-C coupling.9) Detailed analysis of the relationship between Ca2+ transients and cell shortening is restricted by the experimental setup, in which single cardiomyocytes are not optimally stretched and diastolic length cannot be maintained constant when the inotropic state is greatly altered by strong stimulation of ƒÀ-adrenoceptors or elevation of [Ca2+]o. Therefore, the mechanism of regulation of myocardial contractility induced by stimulation of ƒÀ-adrenoceptors and cyclic AMP has not yet been fully elucidated in intact myocardial cells. We do not know the extent of the contributions of the decrease in the sensitivity to Ca2+ ions that is associated with phosphorylation of troponin I and of an acceleration of the Ca2+ pump of the SR, due to phosphorylation of phospholamban, to the positive lusitropic effects of ƒÀ-adrenoceptor agonists in intact myocardial cells.

Phosphorylation of functional proteins

The amplitude of peak Ca2+ transients in contracting mammalian ventricular cardiomyocytes is determined mainly by the amount of Ca2+ ions released via ryanodine receptors in the SR during the plateau of action potentials.70) There- fore, the potentiation of peak Ca2+ transients, as well as the positive inotropic and positive lusitropic responses induced by cyclic AMP, are ascribed primarily to the enhanced release of Ca2+ ions from the SR, which results from phosphorylation of functional proteins of the sarcolemma and SR.71-75) Phosphorylation and dephosphorylation of phospholamban might play a crucial role in contractile regulation in physiological situations since okadaic acid (30ƒÊM), a phosphatase inhibitor,76) has been shown to increase the contractile force by 75% and to stimulate phosphorylation of phospholamban by 325% of the control values.77) The molecular mechanisms involved in the cyclic AMP-induced enhancement of the release of Ca2+ ions have recently been elucidated in great detail. PKA phospho- rylates ryanodine receptors.78,79) Moreover, the catalytic subunit of PKA increases the probability that ryanodine receptors incorporated in a planar lipid bilayer open and it also accelerates adaptation (a rapid decline to a low steady-state level) of the ryanodine receptors to continuous elevation of Ca2+ ions.80)

The amount of influx of Ca2+ ions via voltage-dependent Ca2+ channels is increased by cyclic AMP through an increase in the probability of the voltage- sensitive opening of Ca2+ channels, which is probably related to phosphorylation of the ƒ¿1-subunit of L-type Ca2+ channels.63,81) The increase in Ca2+ ions that enter from outside of myocardial cells influences the cyclic AMP-dependent increase in peak Ca2+ transients through at least two mutually related, intracellular pathways. One pathway involves the role of influx of Ca2+ ions in the Ca2+-induced Ca2+ release (CICR) and its facilitation by cyclic AMP. The other pathway involves an increase in the amount of Ca2+ ions stored in and released from the SR by CICR Vol 39 No 1 Ca2+SIGNALING IN CARDIACMUSCLE CELLS 9

in myocardial cells. Calcium signals (an influx of Ca2+ ions via L-type Ca2+ chan-

nels) are amplified via CICR. Adachi-Akahane and coworkers determined that

the amplification factor is 24.0.82) The CICR mechanism has been assessed in

terms of local elementary Ca2+ transients by confocal microscopy in fluo-3-loaded

single myocardial cells.83-87) Application of this method should present clarifica-

tion of the regulation of ryanodine receptors by cyclic AMP in greater detail. It

has been proposed that the effectiveness of the influx of Ca2+ ions via L-type Ca2+

channels differs from that by Na+-Ca2+ exchange, the former being more effec-

tively coupled to the CICR mechanism,83) while the influx of Ca2+ ions via the

latter system is also capable of inducing the release of Ca2+ ions from ryanodine

receptors,88,89) but with less efficacy (see page 17). Furthermore, it appears that the

regulation of cardiac EC-coupling induced by activation of ƒÀ-adrenoceptors plays

a crucial role in the transition of cardiac hypertrophy to heart failure because the

same defect in EC-coupling that develops during hypertrophy might contribute

to heart failure when compensatory mechanisms induced via ƒÀ-adrenoceptors

fail.87)

The decline of the aequorin signal reflects the combined influence of the

diffusion of Ca2+ ions away from sites of their entry and release, the binding of

Ca2+ ions to troponin C and to a variety of other saturable intracellular binding

sites, and the sequestration of Ca2+ ions by the SR.46) Uptake of Ca2+ ions by the

SR is considered to be most important and it is probably the most labile, in terms

of its response to various interventions, of these different processes. Thus, the

duration of Ca2+ transients is regulated by a number of sarcolemmal, as well as

intracellular, organelles that are dynamically involved in mobilization in and

removal from the cytoplasm of Ca2+ ions during twitch contractions, and some of

these organelles operate in a voltage-dependent manner.

The modulation in response to ƒÀ-adrenoceptor agonists of each component

of these various organelles has to be taken into consideration as we attempt to

interpret the mode of action of ƒÀ-adrenoceptor agonists on cardiac contraction

and Ca2+ transients.67,68) Important effects of cyclic AMP in the regulation of

myocardial contractility include a positive lusitropic action, due to faster uptake

of Ca2+ ions by the SR and decreased sensitivity of myofibrils to Ca2+ ions.2) When

cyclic AMP accumulates intracellularly in response to ƒÀ-adrenoceptor agonists,

the decrease of binding of Ca2+ ions to troponin C and/or the faster dissociation

of Ca2+ ions from troponin C are considered to occur in association with the

PKA-dependent phosphorylation of troponin I, with subsequent prolongation of

the duration of and retardation of the falling phase of Ca2+ transients, which

might counteract the abbreviation of Ca2+ transients induced by the increased

rate of uptake of Ca2+ ions by the SR.57,64,68,90) There is evidence that the changes

in Ca2+ sensitivity modify the falling phase of Ca2+ transients. During the shorten- Jpn Heart J 10 ENDOH January 1998 ing deactivation of cardiac contractility, the decreased binding of Ca2+ ions to troponin C has been shown to prolong the light signal from aequorin.1,3,12,33)

In voltage-clamped and fura-2-loaded single myocytes from the rat, epinephrine (1ƒÊM) increased the rate of relaxation of Ca2+ transients at potentials at which it enhanced the Ca2+ transient.48) These results are consistent with the general belief that the faster decay of the light signal that is induced by cyclic AMP is largely due to facilitation of uptake of Ca2+ ions by the SR. The important role of the phosphorylation of phospholamban in the induction of ƒÀ- adrenoceptor-mediated positive inotropic and lusitropic effects has been demonstrated by application of molecular biological techniques to a study of this cardiac protein. In phospholamban knock-out mice, cardiac contractile function is facilitated maximally under baseline conditions and stimulation of ƒÀ-adrenoceptors fails to accelerate the contractile function.75,91) These findings imply that phospholamban, in its dephosphorylated form, acts as an inhibitory regulator of the Ca2+-ATPase of the SR and, moreover, that the PKA-induced phosphorylation promotes decreased inhibition and, thereby, accelerates the function of the

Ca2+ pump of the SR.

The C protein, a protein that is associated with thick filaments of myocardial cells, forms 7-8 transverse stripes, 10nm wide, at 43-nm intervals along thick filaments in two 300-nm zones on either side of the M-line; these zones are separated by a distance of 400nm. The stoichiometry of C protein in the thick filaments indicates that three C protein molecules are wrapped around one thick filament.57) It is postulated that C protein is involved in functional regulation by modifying the range of movement of crossbridges in such a manner that its removal from thick filaments increases the probability of the binding of myosin heads with actin at submaximally activating levels of free Ca2+ ions.73) C protein is rapidly phosphorylated by ƒÀ-adrenoceptor agonists and PKA, and it is dephosphorylated by muscarinic receptor agonists in both mammalian and amphibian hearts. The presence of phosphatase activity in partially purified preparations of

C protein indicates that the phosphorylation and dephosphorylation of this protein might be rapid.57) The physiological relevance of the phosphorylation of C protein, as well as the regulatory role of C protein itself, has not yet been clarified.57) Hartzell92) proposed a hypothetical mechanism by which C protein might regulate the relaxation rate in cardiac muscle. Phosphorylated C protein has the potential to bind to thin filaments and to inhibit actin-activated myosin

ATPase. It turns off the thin filament prematurely, thereby terminating contraction. Dephosphorylated C protein binds to thin filaments in the presence of Ca2+ ions and prevents or slows the return of tropomyosin to its blocking position as

[Ca2+]i declines and, thus, it prolongs the active state. The cardiac myosin light chain (MLC) is phosphorylated by a Ca2+- Vol 39 No 1 Ca2+SIGNALING IN CARDIACMUSCLE CELLS 11 calmodulin-dependent myosin light chain kinase (MLCK) and dephosphorylated by an LC-phosphatase. These reactions result, respectively, in an increase and a decrease in myofibrillar sensitivity to Ca2+ions in skinned cardiac muscle fibers.93) However, in contrast to the case of smooth muscle, phosphorylation of cardiac MLCK by PKA has no apparent effect.94)The functional relevance of the phosphorylation of cardiac MLC by PKA remains to be clearly defined.55)

Ca2+ desensitization Cyclic AMP causes a decrease in the sensitivity to Ca2+ ions of myofibrils in cardiac muscle. This conclusion, based on findings in intact myocardial cells,7,67-69)is supported by observations in vitrothat cyclic AMP-dependent phosphorylation of troponin I causes a rightward shift in the relationship between the level of Ca2+ ions and activation of actomyosin ATPase.95-97)In cardiac muscle that has been mechanically skinned or chemically skinned with EGTA or with detergents, the pCa (-log [Ca2+])-tensionrelationship is shifted to the right by cyclic AMP.55)The rate of dissociation (off-rate)of Ca2+ions from troponin C is, moreover, accelerated by cyclic AMP. These findings in broken cells and preparations of enzymes can be integrated to provide putative mechanisms that contribute, respectively, to the cyclic AMP-mediated positive lusitropic effect in intact cardiac muscle. It appears, however, that there exists quite a wide range of variations in the extent of the decrease in sensitivity to Ca2+ ions in intact mammalian cardiac muscle.67,68)The finding that the duration of aequorin signals is abbreviated to a lesser extent than that of isometric contraction68)can be interpreted to mean that the faster decay of Ca2+transients, induced by the facilitated uptake of Ca2+ions in the SR, might be counteracted by the faster dissociation of Ca2+ions from troponin C, while both subcellular mechanisms contribute to the cyclic AMP-mediated abbreviation of isometric contraction.2) The increased release of Ca2+ ions from the SR in respect to cyclic AMP might overcome the cyclic AMP-induced decrease in Ca2+ sensitivity to lead to the positive inotropic effect as a whole. Isoproterenol did not affect the half time of the relaxation of the Ca2+ transient, but it shortened the time from half relaxation to three-quarters relaxation of the Ca2+ transient in papillary muscle preparations of the rabbit microinjected with aequorin.68)These observations can be interpreted to mean that, during the early phase of relaxation, the influence of Ca2+ions released from troponin C is more significant in antagonizing the effect of the uptake of Ca2+ ions by the SR. Therelationship of [Ca2+]iand tensionduring twitch contraction: It is possible that the abbreviation of Ca2+transients, caused by facilitated uptake of Ca2+ ions by the SR contributes to the shift in the peak of the [Ca2+]i-tensionrelationship to the right in intact cardiac muscle since the relationship between [Ca2+]iand Jpn Heart J 12 ENDOH January 1998 tension does not reach a steady state during twitch contraction.98)It is unknown how significantly the abbreviation of Ca2+ transients via cyclic AMP-mediated signal transduction contributes to the shift of the [Ca2+]i-tensionrelationship. If Ca2+ ions are available to myofilaments for an abbreviated time because of a shorter Ca2+transient, generation of force, as measured by peak tension, might be decreased.98)If a shortened Ca2+ transient results in a decreased peak tension, then it would seem clear that a given peak Ca2+ transient should produce a decreased peak tension in the presence of an elevated level of cyclic AMP, and the relationship between the peak Ca2+ transient and peak tension would be distorted.98)In this context, McIvor and co-workers99)postulated that the binding of Ca2+ ions to troponin C is rapid enough to be largely completed by the late phase of Ca2+transients, which is abbreviated most prominently by isoproterenol. Therefore, the late phase of the Ca2+transient might not be a major determinant of the apparent shift in the [Ca2+]i-tensionrelationship. In the presence of both isoproterenol and acetylcholine (ACh) together, although the late phase of the Ca2+ transient is shortened as it is with isoproterenol alone, the [Ca2+]i-tension relationship is unchanged as compared with the control, in contrast to the case with isoproterenol alone. Yue98)showed that there is an instantaneous and approximately linear relationship between dT/dt (T: tension; t: time) and [Ca2+]i during the increase in tension in twitch contraction of the ferret papillary muscle, but the peak tension cannot be uniquely predetermined by the peak Ca2+ transient for twitches with time course equal to or faster than those slowed by ryanodine or cyclopyazonic acid.100)The faster the time course of the Ca2+ transientis, the more the relationship between peak tension and peak [Ca2+]iwill shift to the right of the steady-state [Ca2+]i-tensionrelationship. Thus, a change in the relationship between peak tension and peak Ca2+transient does not necessar- ily imply a change in myofilament sensitivity to [Ca2+]i.Yue98) suggested that as a more heuristically sensible analysis, one should look for a change in the instantaneous relationship between dT/dt and [Ca2+]ior in the dT/dtmax to peak [Ca2+]i. The so-called Yue plot apparently overcomes the problem of assessing myofibrillar sensitivity to Ca2+ ions in the face of changes in the time course of the Ca2+ transient. However, the interpretation of results is considerably less straightfor- ward; a comparison of [Ca2+]iand tension at the peak of the isometric twitch might also be promising but there exists at present no standard method for assessing the relationship.11) The steady-statepCa-tension relationship: In order to overcome the problems associated with changes in duration of Ca2+ transients, the influence of cyclic AMP on the steady-state pCa-tension relationship has been analyzed in the case of steady-state activation of contraction.11) Tetanic contraction can be induced by rapid electrical stimulation or by Na+ deficiency101)in intact heart muscle that has Vol 39 No 1 Ca2+SIGNALING IN CARDIAC MUSCLECELLS 13 been treated with ryanodine or cyclopiazonic acid, which selectively inhibits the functions of cardiac SR.67,100) The influence of cyclic AMP on Na+-deficient contracture was analyzed in aequorin-injected papillary muscles from the rat67) and the ferret.98) In the rat, when the extracellular Na+ concentration, [Na+]o, was decreased from 135mM to 17mM, light from aequorin and tension increased

slowly (Na+-deficient contracture), and decayed slowly after reaching a peak at

about 90s. Isoproterenol (1ƒÊM), applied at the peak of Na+-deficient

contracture, suppressed tension with a slight and transient increase in the light

level. When [Na+]o was decreased from 135mM to various concentrations from

42-17mM in the absence and presence of 1ƒÊM isoproterenol, it was clear that

isoproterenol shifted the relationship to the right and downwards, a result that

suggests that it decreased the myofibrillar sensitivity to Ca2+ ions.67) The relation-

ship of tetanic tension to [Ca2+]i is shifted to the right and the Hill coefficient

(cooperativity) is decreased by isoproterenol,69,102) an indication that ƒÀ- adrenoceptor stimulation attenuates the apparent sensitivity to Ca2+ ions of the

contractile proteins and cooperativity. It has been postulated that the decrease in

sensitivity to Ca2+ ions is due to phosphorylation of troponin I, which leads to a

decrease in the binding of Ca2+ ions to troponin C.97,103)

Muscarinic inhibition One of characteristic features of the cyclic AMP-mediated regulation of contractile force is that it is antagonized both readily and selectively by activation of muscarinic and adenosine receptors.104) The inhibition in the ventricular myocardium of most mammals is observed only in the presence of cyclic AMP- mediated facilitation of cardiac contractility, and baseline contraction is scarcely affected by the inhibitory regulation. While reduction in the rate of generation of cyclic AMP through the inhibitory receptor that is coupled to an inhibitory GTP- binding protein, Gi, appears to be mainly responsible for the functional antagonistic effect of stimulation of muscarinic and adenosine receptors in most mammalian species, including the dog, rabbit and rat, in certain mammalian species, namely, in the guinea pig, levels of cyclic AMP are not lowered or are lowered only to a limited extent, and, therefore, other subcellular events, such as inhibition of PKA and activation of phosphatase, have been proposed as main mechanisms.105) In atrial muscle from mammals and in ventricular muscle from ferrets, functional antagonism by activation of K+ channels also contributes to the inhibitory regulation.2,104,106) The increase in amplitude and the abbreviation of the duration of Ca2+ transients and of isometric contractions by the accumulation of cyclic AMP that is induced by catecholamines (isoproterenol, epinephrine and norepinephrine), forskolin and selective inhibitors of PDE III (amrinone, milrinone, and 14 ENDOH Jpn Heart J January 1998 enoximone) in aequorin-injected right ventricular trabeculae from the dog were completely reversed upon stimulation of muscarinic receptors by carbachol.107)In other mammals, such as the ferret, it has been reported that certain effects of 0- adrenoceptors in ventricular muscle, namely, the abbreviation of relaxation time induced by stimulation of /3-adrenoceptors, are not reversed by stimulation of muscarinic or adenosine receptors.99,102,108,109)While the contractile force of the ventricular myocardium of most mammalian species is scarcely affected by stimulation of muscarinic or adenosine receptors under baseline condition, the ventricular myocardium of the ferret is unusual in that the baseline force of contraction is markedly depressed by activation of inhibitory receptors, even in the absence of stimulation of /~-adrenoceptors,10)probably because of development of muscarinic and adenosine receptor-activated K+ channels in this Mecies.106) It appears most likely that these differences in the basic regulatory system between the canine and ferret ventricular muscle play a crucial role in the discrepancies in the muscarinic or adenosine-induced regulation of Ca" transients and contraction after stimulation via accumulation of cyclic AMP. It has been proposed that nitric oxide (NO) and the subsequent accumulation of cyclic GMP might mediate the muscarinic inhibition of the 13- adrenoceptor-mediated acceleration of cardiac function.' 11,112)In the papillary muscle of the ferret, however, nitroprusside, which prominently increases tissue levels of cyclic GMP, and dibutyryl cyclic GMP, which permeates cell membranes, both failed to mimic the inhibitory action of ACh on the stimulation of /3- adrenoceptors.108)These results are consistent with the findings in early studies. 113,114)

Selective/31-adrenoceptor agonists Partial agonists of J3,-adrenoceptors, such as prenalterol, xamoterol and denopamine, are less effective in activation of adenylate cyclase than full agonists, such as isoproterenol and epinephrine. Therefore, for a given increase in the force of contraction, the former induces much more limited accumulation of cyclic AMP than the latter."') In other words, the functional responses to these partial agonists are much greater than those expected from accumulation of cyclic AMP if that is compared with the accumulation induced by isoproterenol.2) The partial agonist prenalterol increased levels of free and bound cyclic AMP proportionately, whereas isoproterenol caused a greater increase in levels of free than bound cyclic AMP in the rat perfused heart. "I) Thus, at functionally equally effective concentrations, prenalterol and isoproterenol increased levels of bound cyclic AMP to the same degree, while the increase in the level of free cyclic AMP was much greater with isoproterenol. These findings support the putative compartmentation of cyclic AMP in myocardial cells, and they indicate that Vol 39 No I Ca2' SIGNALINGIN CARDIACMUSCLE CELLS 15 partial agonistsof P1-adrenoceptorselevate levels of cyclicAMP in the functionally relevant compartment more effectively than isoproterenol.116)These differences in the metabolism of cyclic AMP might be responsible, in part, for the fact that partial agonists of 131-adrenoceptorshave less positive chronotropic activity than isoproterenol for a positive inotropic response of a given magnitude. We examined the relationship between the positive inotropic effect and the increase in Cal' transients induced by a partial agonist of X31-adrenoceptorsdenopamine and the full agonist isoproterenol in aequorin-injected ventricular trabeculae from doL7s.117)The relationshipswere not significantlydifferent with denopamine and isoproterenol, an indication that the partial agonist might induce the mobilization of Ca` ions and elicit Cal' desensitization in an identical manner to the full agonist isoproterenol in the functionally relevant compartment.

Selectiveinhibitors of phosphodiesteraseIII Cyclic AMP generated by the stimulation of 0-adrenoceptors is hydrolyzed rapidly by the cyclic nucleotide phosphodiesterase (PDE) in cardiac muscle, as it is in other tissues.118) The effects of 3-adrenoceptor agonists on intracellular Cal-' transients and contractile force are partially mimicked by inhibitors of PDE. Electrical effects on membranes of P-adrenoceptor agonists are also mimicked by inhibitors of PDE and derivatives of cyclic AMP. A number of novel positive inotropic agents, such as amrinone, milrinone, enoximone, piroximone, imazodan, LY 195115, vesnarinone, pimobendan, olprinone and toborinone, selectively inhibit the PDE isoenzyme, PDE III (which has a low Km for cyclic AMP and is inhibited by cyclic GMP), which is bound to the SR.2"119"120)This observation raises the possibility that the accumulation of cyclic AMP induced by selective inhibitors of PDE III might occur in a compartment different from the compartment in which stimulation of j3-adrenoceptors increases levels of cyclic AMP. The potency of the selective inhibition of PDE III is relevant to both the positive inotropic and vasodilator effects of various PDE inhibitors with different chemical structures. 121)The positive inotropic effects of inhibitors of PDE III, such as milnnone, amnnone, enoximone, vesnannone and tobonnone occur with an increase in the peak Cal' transient that is due primarily to increases in the rate of release and uptake of Ca 21 ions by the SR.2) Accumulation of cyclic AMP and increases in the peak Ca 2+ transient and the force of contraction in canine ventricular muscle are abolished by carbachol, an observation that indicates that the effects of the inhibitors of PDE III are mediated by a cyclic AMP-dependent mechanism. 112)Milrinone at elevated concentrations has a direct inhibitory effect on PKA that might contribute to the negative inotropic effect observed with high concentrations of this drug.123) Since the selective inhibition of PDE III might lead to localized increases in levels of cyclic AMP and to the localized activation Jpn Heart J 16 ENDOH January 1998 of PKA, the relationship between the Ca2+ transient and the contractile force during induction of the positive inotropic effect of inhibitors of PDE III might be different from that during induction by stimulation of ƒÀ-adrenoceptors. We ex-

amined this possibility and found that the relationships induced by toborinone

and milrinone differ from that induced by isoproterenol but they are the same as

that induced by elevation of [Ca2+]o,122) an indication that the inhibitor of PDE III

might not elicit desensitization to Ca2+ ions, as observed upon stimulation of ƒÀ-

adrenoceptors. Selective inhibitors of PDE III increase heart rate to a lesser

extent than stimulation of ƒÀ-adrenoceptors for a given increase in contractile

force. This result might likewise be due to an increase in the level of cyclic AMP

in different intracellular compartments in response to selective inhibitors of PDE

III and ƒÀ-adrenoceptor agonists.2) The type and localization of the PDE isoen-

zyme that is responsible for a functional response and the metabolism of cyclic

AMP show a wide range of variations among species of animals. Inhibition of

PDE III leads to a pronounced positive inotropic effect in the cardiac muscle of

mammals that include human, monkey, dog, cat and rabbit. It has a moderate

effect in guinea pig and hamster, and a minimal effect in rat ventricular myocar-

dium. In the rat, in contrast to other species, the inhibition of PDE IV, which has

a low Km for cyclic AMP, and is inhibited by Ro 20-1724 and rolipram, has a

much greater effect than inhibition of PDE III in terms of enhancement of the

effect of ƒÀ-adrenoceptor stimulation.124-126) Thus, it appears that, in the rat, by

contrast to other mammalian species, the PDE IV isoenzyme plays a crucial role

in the hydrolysis of cyclic AMP, which contributes to the regulation of myocar-

dial contractility in this species.

Selective inhibitors of PDE III that have been developed as therapeutic

agents for myocardial failure have common characteristics, as follows: 1) the dose

required to enhance the sympathetic stimulation mediated by ƒÀ-adrenoceptors is

much lower than that required for induction of a direct cardio-stimulatory ef-

fect;125) 2) the positive inotropic effect is associated with a vasodilator effect;121) and

3) an excessive dose results in arrhythmia. Properties that are specific to the

individual agents are responsible for ancillary effects associated with each agent.

Vesnarinone has an inhibitory effect on K+ channels, which antagonizes its cyclic

AMP-mediated positive chronotropic and vasodilator action;127) in addition, it

lowers blood levels of cytokines that might, in part, contribute to the therapeutic

effects of this drug in cases of heart failure.128) Pimobendan and MCI-145 have

Ca2+ sensitizing actions,129,130) but the therapeutic relevance of such an effect in

the clinical setting is unknown.

It should be noted that the positive inotropic effects of agents that increase

levels of cyclic AMP, including ƒÀ-adrenoceptor agonists and inhibitors of PDE

III, are greatly diminished in cardiac muscles isolated from the failing hearts of Vol 39 No 1 Ca2+SIGNALING IN CARDIACMUSCLE CELLS 17

human and experimental animals.131-135)

Intracellular accumulation of Na+ ions Na+-Ca2+exchange plays an important role in the extrusion of Ca2+ ions that have entered in myocardial cells via L-type Ca2+ channels during cardiac E- C coupling. In addition, there has been a long-standing debate as to whether or not Ca2+ions that enter cells through reverse-mode Na+-Ca2+exchange contribute to the CICR mechanism as do Ca2+ ions that enter through L-type Ca2+ channels. In cardiac muscle, the Ca2+ions that enter through L-type Ca2+ channels are primarily responsible for the CICR mechanism,84,136-139)but there is evidence that an influx of Ca2+ ions via the Na+-Ca2+ exchanger also triggers CICR.89,140)One reason for the failure to reach general agreement is the species- dependent variation in the extent of expression of Na+-Ca2+exchanger in the cardiac muscle from different experimental animals.141)Since the first cloning of the canine cardiac Na+-Ca2+exchanger, the molecular biological approach has been used to knock out the Na+-Ca2+exchanger with antisense oligonucleoti- des.142-144)Adachi-Akahane and coworkers analyzed the role of the Na+-Ca2+exchanger in the regulation of the CICR in mouse hearts that overexpressed the canine Na+-Ca2+exchanger.145) Even in such a transgenic mouse, an influx of Ca 2+ions via reverse-mode Na+-Ca2+exchange did not trigger CICR, a result that is consistent with the conclusion obtained with fluo-3-loaded cardiomyocytes in an analysis by confocal microscopy.139)Ca2+ ions that enter myocardial cells via the reverse-mode Na+-Ca2+exchanger might be taken up mainly by the SR to contribute to the elevation of the peak of the Ca2+transient and the positive inotropic effect. Thus, the regulatory relevance of an increase in [Ca2+]imight differ depending on the pathway through which Ca2+ ions enter the myocardial cell. Present findings imply that an influx of Ca2+ ions through L-type Ca2+ channels contributes to facilitation of both the CICR and the PIE, whereas the influx through reverse-mode Na+-Ca2+exchange contributes only to the PIE. Cardiac glycosides and some newly synthesized agents, such as ARL-57 and ARL-100, inhibit Na+, K+-ATPase, with the resultant cellular accumulation of Na+ ions and they might thereby elevate [Ca2+]ithrough Na+-Ca2+exchange. Other inotropic agents, such as toxins from sea anemone [e.g., anthopleurin-A and ATX II] and batrachotoxin, also appear to increase contractile function through the cellular accumulation of Na+ ions. DPI 206-106, a cardiotonic agent, is an agent that causes the cellular accumulation of Na+ions (for reviews, see refs. 2 and 16).

Hydrolysis of phosphoinositide The positive inotropic effects of sympathomimetic amines are also mediated 18 ENDOH Jpn Heart J January 1998 by a,-adrenoceptors.146~ Stimulation of myocardial a,-adrenoceptors does not result in generation of cyclic AMP but it does stimulate the hydrolysis of phosphoinositide.147> Other positive inotropic agents, such as angiotensin II, endothelin isopeptides, ATP (as an agonist of P2 purinergic receptors) and histamine (as an H2-receptor agonist) also stimulate the hydrolysis of phosphoinositide in myocardial tissues.2,148,,49)The intracellular role of phosphoinositide hydrolysis in the regulation of cardiac contractility through the activation of receptors that belong to this class has not yet been clarified. Stimulation of a,-adrenoceptors in rabbit ventricular muscle causes a positive inotropic effect in association with a relatively small increase in the peak Ca 21 transient, prolongation of the duration of contraction and abbreviation of the early phase of the Ca2+ transient, probably as the result of a decrease in the rate of dissociation of Ca 21 ions from binding sites on myofilaments (an increase in myofibrillar sensitivity to Ca 2+ions).", 151)It is of interest that the changes in Ca 21transients and in contractility induced, via a,-adrenoceptors, by phenylephrine (in the presence of 13-blockade),via angiotensin II receptors (AT, subtype) and via receptors for endothelin (ETA and ETB subtypes) in indo-l-loaded ventricular myocytes from the rabbit are very similar to each other. In each case there is an increase in contractile force in association with a relatively small increase in Ca 21 transients, an indication that these agents increase both the amplitude of Ca 2+ transients and the sensitivity of myofibrils to Ca 2+ ions. Thus, a common signaling pathway, namely, acceleration of the hydrolysis of phosphoinositide triggered by activation of this class of receptors, might have regulatory relevance."') There appear to be three pathways for an increase in the amplitude of Ca 21 transients. These pathways might be promoted by phosphorylation of regulatory proteins, catalyzed by the PKC that is activated as a result of the production of diacylglycerol via activation of phospholipase C activation. 152,153) They include direct activation of L-type Ca2+ channels, prolongation of duration of action potential that causes an increase in the voltage-dependent influx of Ca2+ ions or the intracellular accumulation of Ca2+ ions via L-type Ca 2+ channels and/or the Na+-Ca 2+exchanger, and activation of Na+-H+ exchanger with resultant intracellular accumulation of Na+ ions that increases [Ca 2+],through the Na+-Ca2+ exchanger. Experimental evidence indicates that the first mechanism does not play an important role, if it plays any role at all, in the al-adrenoceptor-mediated regulation of [Ca 2+],.114,1s5)The second '56,,s7)and, in particular, the third mechanisms appear to contribute significantly to an increase in the amplitude of Ca 2+ transients (for reviews, see refs. 158-160). Two potential subcellular mechanisms have been proposed to explain increases in Ca 2+ sensitivity of myofilaments: activation of the Na+-H+ exchanger and subsequent intracellular alkalinization; and phosphorylation of contractile Vol 39 Ca" SIGNALINGIN CARDIACMUSCLE CELLS 19 No I proteins by PKC. Evidence to support the former mechanism has been accumu- lating (e.g., refs. 161-167). However, the receptor-mediated increase in myofibrillar sensitivity to Ca" ions cannot be fully explained by alkalinization.167)It was reported recently that the development of the positive inotropic effect of angiotensin II preceded alkalinization, a result that does not support a cause and effect relationship between an increase in force and alkalinization.'68 It has been shown that PKC also catalyzes the phosphorylation of both troponin I and troponin T in vitro, l69,170>and such phosphorylation could be a mechanism for myofibrillar sensitization to Ca2+ ions. By contrast, it has also been demonstrated that the phosphorylation of troponin isoforms is associated with a decrease in the activity of actomyosin ATPase, which might lead to a negative inotropic effect .171,172) Therefore, the regulatory role of the PKC-catalyzed phosphorylation of cardiac contractile proteins in intact myocardial cells remains still unknown. The difficulties associated with the pharmacological analysis of this signaling process are mainly due to inability of activators of and inhibitors of PKC to mimic or to selectively inhibit the receptor-mediated inotropic response. Tumor- promoting phorbol esters, which have been widely used as activators of PKC, elicit a negative inotropic effect in a number of cardiac preparations, and they also inhibit the receptor-mediated positive inotropic effect (for reviews, see refs. 173 and 174). When phorbol esters or diacylglycerol are applied to intact myocardial cells, differences in intracellular localization and/or distribution, depending on the method of application, appear to be critical. There is a recent report that the photorelease using a light-sensitive caged compound, of an analog of diacylglycerol, namely, dioctanoylglycerol, within adult rat ventricular myocytes had a dose-dependent positive inotropic effect, which was blocked by the inhibitor of PKC, chelerythrine. By contrast, the external administration of the analog had a negative inotropic effect."') While inhibitors of PKC selectively inhibit the positive inotropic effect of agonists that stimulate the hydrolysis of phosphoinositide, the extent of inhibition is, at most, 20-30% of the maximal response. 116)Although the receptor agonists that stimulate the hydrolysis of phosphoinositide are of interest as positive iso- tropic drugs, in view of unique mechanism of action which involves a relatively small increase in Ca 21 transients and Ca 21 sensitization that are probably causally related to acceleration of the hydrolysis of phosphoinositide, these agonists are not clinically applicable as cardiotonic agents because of their pronounced vasoconstrictor effects on the peripheral and/or coronary vasculature. They might rather, play an important role as physiological and pathophysiological mediators of cardiovascular functional, as well as genetic, regulation. In pathological situations, inhibition of their signal transduction processes by receptor antagonists and/or pertinent enzyme inhibitors, rather than stimulation of these processes, Jpn Heart J 20 ENDOH January 1998 actually has beneficial effects on patients with heart failure.177)

Ca2+ promoters

Calcium promoters (Ca2+ agonists, for example, BAY k 8644, YC-170,

H160/51, 201-791 and CGP 28-392) bind directly to a specific binding site on the ƒ¿1-subunit of L-type Ca2+ channels and increase cardiac contractile function in association with an increase in the amplitude of Ca2+ transients through enhancement of the probability that Ca2+ channels are open.11,118) The positive inotropic effect of most of these Ca2+ agonists is, however, associated with an increase in peripheral vascular resistance that is due to peripheral vasoconstriction. In addition, these compounds cause coronary vasoconstriction by similar facilitation of the action of L-type Ca2+ channels in vascular smooth muscle cells

(for reviews, see refs. 2 and 63). These vascular effects prevent the clinical use of these compounds as positive inotropic agents for the treatment of heart failure. A

novel Ca2+ promoter, BAY y 5959[(R)-isopropyl 2-amino-5-cyano-6-methyl-1,4-

dihydro-4-(3-phenyl-quinolinone-5-yl)-pyridine-3-carboxylate] has been reported

to possess selectivity for cardiac as distinct from vascular smooth muscle, which

raises the possibility that this new compound might have an application as a positive inotropic agent in a clinical setting. Indeed, the results of experiments in animal models suggest the potential clinical effectiveness of BAY y 5959.178,179)

Regulation of Ca2+ sensitivity and Ca2+ sensitizers It is evident that the force of contraction is dissociated from [Ca2+]iunder various pathophysiological conditions and after application of certain classes of inotropic agents. For example, hypoxia,21,180)acidosis,181) the accumulation of inorganic phosphate, 182)and the phosphorylation of troponin I that is induced by PKA183)decrease myofibrillar responsiveness to Ca2+ ions, which is defined as a "decrease in myofibrillar Ca2+ sensitivity" , a "decrease in the Ca2+ sensitivity of

Table I. Classification of Ca2+ Sensitizers Vol 39 Cap' SIGNALING IN CARDIAC MUSCLE CELLS Nn I 21 the contractile apparatus (or proteins)" or "myofibrillar Ca 2+desensitization". The following experimental procedures can be used to allocate the nature of the modulation of Ca" sensitivity to one of three classes (Table I): class I) interaction with the Ca 2'-binding site of troponin C with increases or decreases in the Ca"-binding affinity, by a so-called "central mechanism"; class II) interfer- ence with the interaction of the troponin-tropomyosin complex with actin by a tt downstream mechanism"; and class III) interaction with cycling of crossbridges, beyond the regulation by Ca 2+ ions, another "downstream mechanism".') Experimental procedures available for the differentiation of Ca 21 sensitizers whose actions belong to the different classes include the following: 1) the application of various Ca 2+indicators (aequorin, indo-1, fura-1, and fluo-3) to intact myocardial cells to examine the relationship between the force and the Ca 2+ transient;9,27)2) motility assays in vitro to investigate directly the interaction of myosin with actin, in which the effects of various interventions (that include application of cardiotonic agents) on the movement of visualized thin filaments or actin over a fixed layer of myosin when the immediate environment around the crossbridges can be controlled; 18,24,25,184)3) assessment of the pCa-tension relationship in skinned cardiac muscle fibers;"') 4) determination of amount of 45Ca2+ bound to troponin C; and 5) determination of the dependence on Ca 2+ions of activation of actomyosin ATPase activity, which is not always a good index of mechanical alterations. 184,186)Ca2+ sensitizers whose actions belong to the different classes can be distinguished on the basis of differences in the results obtained by these various experimental methods (Table I). In the following section the characteristics of individual Ca2+ sensitizers are described in detail (for reviews, see refs. 2, 187-189). Methylxanthines: Methylxanthines, including caffeine and theophylline, increase the sensitivity to Ca2+ ions of contractile proteins in skinned cardiac fibers. This effect is due to the ability of these compounds to increase the affinity of troponin C for Ca 21 ions by decreasing the rate of dissociation of Ca 21 ions from troponin C.19) However, details of the mechanism of Call sensitization are un- clear. A recent study failed to show that caffeine increases the Ca 2+ -binding affinity of troponin C in skinned cardiac fibers.'" It was proposed, therefore, that caffeine might act directly on the actin-myosin interaction. Because methylxanthines have multiple actions in cardiac E-C coupling and receptor- mediated processes, for example, myofibrillar Ca 2+ sensitization, nonselective inhibition of PDE, suppression of transport of Ca 2' at the SR and antagonism of adenosine Al receptors, the inotropic responses to methylxanthines were com-

2+ plex. Theophylline has a definite inhibitory action on the amplitude of Ca transients in intact canine cardiac muscle that has been microinjected with aequorin.192) This result is essentially consistent with the findings in skinned or Jpn Heart J 22 ENDOH January 1998

Figure 2. Chemical structures of some novel Ca' sensitizers.

Table II. Potencies of Cat} Sensitizers with Respect to Inhibition of PDE III and Cat Sensitization

hyperpermeable preparations of cardiac musc1e.190,193J Sulmazole: Sulmazole (AR-L 115 BS; 2-[(2-methoxy-4-methylsulfinyl)phenyl]-lH-imidazo[4,5b]pyridine) was the first cardiotonic agent to be shown to have a Ca" sensitizing effect (Figure 2). Although sulmazole has an inhibitory action on PDE III, it is more effective as a cardiotonic agent than other novel V of 39 Ca" SIGNALING IN CARDIAC MUSCLE CELLS No I 23 inhibitors of PDE 111.194,'9') Findings in studies in vitro imply that sulmazole has a Ca 21 sensitizing action. '96,'9') The positive inotropic effect of sulmazole was found to be mediated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms, depending on its concentration in ventricular myocardium of the dog. '98) The positive inotropic effect of sulmazole at concentrations up to 0.3 mM was mediated by the accumulation of cyclic AMP, which was associated with an increase in the amplitude of Ca' transients and was abolished by carbachol, a muscarinic receptor agonist. The effect of sulmazole at concentrations of 1 mM and higher was associated with a decrease in the peak Ca' transient and a further increase in contractile force, with prolongation of the duration of contraction, and it was not inhibited by carbachol in the dog ventricular myocardium. 107) While (+) and (-) isomers of sulmazole are equipotent in the inhibition of PDE, only the (+) enantiomer has a significant Ca2+ sensitizing action in vitro and a positive inotropic effect. By contrast, no stereoselectivity was found in the vasodilator effect by the drug.'99)In the closed-chest dog, sulmazole at 1 mg/kg body weight increased the maximum rate of rise of intraventricular pressure, dP/dtm., with no alteration in heart rate or the myocardial oxygen consumption and it reduced peripheral vascular resistance.200,201) Further development of sulmazole for clinical application was discontinued because of its unfavorable effects. A structurally related compound, pimobendan, with a similar pharmacological profile has been developed to replace sulmazole. Pimobendan: Pimobendan (UD-CG 115 BS; 4,5-dihydro-6-[2-(4- methoxyphe-nyl)-1H-benzimidazol-5-yl]-5-methyl-3(2H)-pyridazinone), a conge- ner of sulmazole, has potent cardiotonic and vasodilator activity (Figure 2).202) Pimobendan has both a selective PDE III-inhibitory action and a Ca 2+sensitizing action.203-206) Fujino and coworkers201) showed that the Ca 2+sensitizing action of pimobendan is due to a stereo-specific effect on binding of Ca2+ ions to troponin C. In partially depolarized preparations of cardiac muscle, pimobendan increased force with little or no effect on the slow action potentials that reflect, predominantly, the activity of Ca 2+ channels.208)Since the IC50 value for the inhibition of PDE III is 2.4 µM,209)whereas the EC50value for Ca 2+sensitization is 40 µM,208)the drug seems more likely to act as an inhibitor of PDE III to accumulate cyclic AMP that leads to an increase in Ca' transients (Table II). In addition, its demethylated derivative, UD-CG 212 Cl (a major metabolite of pimobendan in the liver), does not have any Ca 2+sensitizing action and is more effectiveas an inhibitor of PDE III than the mother compound .210)Therefore, the contribution of cyclic AMP-mediated effect of pimobendan may be more pronounced in vivothan that expected from isolated preparations of cardiac muscle in vitro. The positive inotropic effect of pimobendan at 1 mM was associated with Jpn Heart J 24 ENDOH January 1998 an accumulation of cyclic AMP in the ventricular muscle of the dog205) and a positive chronotropic effect was observed with pimobendan at concentrations up to 300 µM in the guinea pig.21) However, the magnitude of both effects was much lower than that induced by isoproterenol. The ranges of concentration at which pimobendan inhibits PDE III and induces Ca 21 sensitization are closely overlapping in intact myocardial cells. The positive inotropic effect of pimobendan at 25 µM was associated with a marked increase in the amplitude of Ca 2+ transients in the ferret ventricular myocardium,130) which was ascribed to its inhibitory action on PDE III. The duration of Ca 2+ transients was abbreviated by pimobendan, whereas that of isometric contraction was unchanged or was slightly prolonged in the ferret."') These results support the hypothesis that the effect of the accumulation of cyclic AMP might be masked by the Ca 2+sensitizing effect of the compound. Pimobendan at 20 µM shifted the pCa-tension curve to the left in skinned ventricular muscle fibers from the ferret.130) As the Ca 2+ sensitizer, pimobendan is considered to belong to the class of agents that increase the affinity of Ca 21 ions for troponin C. It shifts the pCa-tension curve to the left with no effect on the maximal tension. 130,208)It also requires the troponin-tropomyosin complex for its Ca2+ sensitizing action in motility assay in vitro,25)and it increases the amount of 45 Ca 2+ bound to the single low-affinity class of Ca 2+ -binding site of troponin C. 208) Nonetheless, in intact myocardial cells, the relationship between [Ca 2+];and tension developed is not shifted by pimobendan'30,212)probably because of the contribution of inhibition of PDE III and subsequent accumulation of cyclic AMP. In a clinical setting, the drug improved exercise tolerance and quality of life2 ") and it reversed the subsensitivity to catecholamines214)in patients with heart failure. Pimobendan has been approved as an oral agent for the treatment of heart failure in Japan. EMD 53998.• EMD 53998 is a racemic mixture of the stereoisomers of thiadiazinone (5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]6- methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one) with a pronounced positive iso- tropic effect, and it acts as a Ca 2+sensitizer (Figure 2).215)Experimental evidence indicates that it acts directly on contractile proteins. '11,216-221)EMD 53998 has both a Ca 21 sensitizing action and a PDE III-inhibitory action ,21S)and it has been shown that the former action is due to the (+) enantiomer (EMD 57033) and the latter to the (-) enantiomer (EMD 57439) (Table II).111,219,221)In skinned cardiac muscle fibers, the pCa-tension relationship was shifted to the left and maximal tension was increased in the presence of EMD 53998.215,217,218)In intact myocardial cells, contractile force or shortening was increased without or with only a small increase11,220,221) or with a decrease 216)in the amplitude of Ca 21 transients. The small increase in peak Ca 21transients was due mainly to inhibition of PDE Vol 39 Cat' SIGNALING IN CARDIAC MUSCLE CELLS 25 No I

III and the subsequent accumulation of cyclic AMP indeed by the (-) enantiomer BM 57439.18,220,22')Neither the (+) enantiomer EMD 57033 nor the (-) enantiomer EMD 57439 affected binding of Ca 2+ions to troponin C in skinned fibers of canine heart.") With respect to the subcellular mechanism of Ca 2+sensitization, Zhao and Kawai showed that EMD 53998 suppressed the binding of nucleotides to crossbridges and increased the resistance of crossbridges to the accumulation of phosphate in the myocardium. They proposed that EMD 53998 thereby decreases the number of detached crossbridges and increases the number of attached crossbridges.222)EMD 53998 restored contractile dysfunction that had been induced by ischemia at concentrations up to 1 pM but at 10 µM it markedly elevated left ventricular end-diastolic pressure in the isolated perfused 223) guinea-pig heart. EMD 57033: EMD 57033 is the (+) enantiomer of EMD 53998. It has a predominant Ca 2+sensitizing action with lower PDE III-inhibitory activity, in contrast to the (-) enantiomer EMD 57439 which has dominant PDE III-inhibitory action and no Ca' sensitizing effect (Table 11).111,210,221,224,225) Neither enantiomer enhances the binding of Ca 2+ions to troponin C. EMD 57033 induced a leftward shift in the pCa-Mg-ATPase activity relationship (EMD 57439 was ineffective in this respect).") Thus, it seems that EMD 57033 might act on a process subsequent to binding of Ca 2+ ions to troponin C a "downstream" mechanism 5) (Table I). The stimulatory effect of EMD 57033 on Mg-ATPase activity was observed even in preparations from which the troponin-tropomyosin complex had been extracted,18)indicating that EMD 57033 is effective even in the absence of a Ca2+ regulatory site. Thus, this compound might act directly at the actin- myosin interface. Indeed, EMD 57033 is the first Ca 2+sensitizer that has been shown to act directly to facilitate the actin-myosin interaction in the absence of Ca2+ ions ("class III") in motility assays in vitro.18,57)EMD 57033 stimulated the velocity of sliding of actin filaments on myosin heads that had been attached to nitrocellulose-coated coverslips, whereas EMD 57439 was ineffective. From these findings, Solaro and coworkers proposed that the mechanism of action of EMD 57033 on cardiac contractility involves the direct promotion of the actin-myosin interaction and, further, that the Ca2+ sensitization induced by EMD 57033 is due to the crossbridge-dependent activation of thin filaments.18J EMD 57033 and EMD 53998 prolong the duration of contraction and elicit a negative lusitropic effect that is due to their Ca" sensitizing action in mammalian cardiac muscle,' 16,221,226)which could impair diastolic relaxation. In other word, in myopathic hearts in which [Ca 21],is elevated during diastole,227) Ca 21 sensitizers might increase diastolic pressure. Indeed, it has actually been demonstrated that some Ca 2+ sensitizers, in particular at higher concentrations can shorten diastolic cell length in single cardiomyocytes;18'218,221) they can also Jpn Heart J 26 ENDOH January 1998 elevate resting tension in preparations of papillary muscle216,22) and increase end- diastolic pressure in perfused guinea-pig and rat hearts.218,223,227)These observations imply that Ca 21sensitizers might elevate end-diastolic pressure as a result of increased diastolic tone, namely, in cases of heart failure .223,22')This phenomenon would lead to venous congestion and aggravation of the symptoms of heart failure. Tobias and colleagues showed, in the isovolumically beating isolated rabbit heart, that EMD 57033 at an inotropic concentration (2 µM) caused a modest prolongation of the duration of contraction that was due to slowed relaxation but had no effect on diastolic tone .22')However, two Ca 2+sensitizers, EMD 57033 and Org 30029 (described below), impaired relaxation in isolated failing human myocardium to a greater extent than in nonfailing human myocardium.229) Therefore, further detailed study is required to clarify the effects of Ca 2+ sensitizers on relaxation and diastolic properties of cardiac muscle during heart failure.210) Ca 2+ sensitizers should have an energetic advantage over Ca 2+-mobilizing cardiotonic agents because the former do not require the increased expenditure of activation energy.231)For a given increase in contractile force, EMD 57033 has been shown to require less myocardial oxygen consumption than any of the following: dihydroouabain in the guinea-pig heart '232)and dobutamine,233)elevation of [Ca 2+]oor isoprotereno1234)in the isolated heart or cardiomyocytes from rats. In addition, EMD 57033 was able to fully reverse the myocardial depression induced by acidosis without increasing the amplitude of Ca' transients. Thus such Ca 21 sensitizers appear promising as drugs for treatment of heart failure. 23) Levosimendan:Levosimendan (LS; R-[[(1,4,5,6-tetrahydro-4-methyl-6-oxo- 3-pyridazinyl)-phenyl]hydrazono]propanedinitrile) binds to troponin C with high affinity (Figure 2).235)LS was identified by screening with an affinity column of immobilized troponin C. Racemic simendan was discovered by this procedure and the active enantiomer was purified as LS. It has been postulated that LS elicits a positive inotropic effect by an increase in the affinity for binding of Ca 2+ ions to troponin 0.236)LS binds to the amino-terminal domain of recombinant human cardiac troponin C, stabilizes the change in the conformation of troponin after binding of Ca' ions'21') and amplifies the trigger for contraction that is induced by troponin C in a Ca2+-dependent manner. This mode of action might result in a positive inotropic effect without an increase in [Ca 21],.218)It is notewor- thy that LS does not enhance the binding of Ca 2+ ions to troponin 0239)but stabilizes the conformational change in troponin after binding of Ca 2+ ions. 237) Thus, it appears that LS belongs to class II of Ca 2+ sensitizers, which modulate the interaction of the troponin-tropomyosin complex with actin. Since the binding of LS to troponin C is Ca 2+-dependent,235) Ca 2+ sensitization induced by LS should be maximal during systole and minimal during diastole. This specificity V of 39 Ca" SIGNALING IN CARDIAC MUSCLE CELLS 27 No I circumvents a potential major disadvantage of Ca 2+sensitizers, namely, impairment of relaxation and elevation of end-diastolic pressure, and it might explain the finding that LS does not impair relaxation .2') LS had a positive inotropic effect in the Langendorff perfused heart '239)as well as in isolated cardiac muscle.241) LS is also a potent inhibitor of PDE with an IC5o of 25 nM242)which might contribute in part to the positive inotropic effect of the compound (Table I) 209,243,244)The following observations in the guinea-pig cardiac muscle indicate a significant contribution of cyclic AMP-mediated signaling to the inotropic effect of LS: 1) LS at 1.0.tM increased significantly the level of cyclic AMP; 2) LS increased the extent of phosphorylation of phospholamban, troponin I and C protein; 3) LS at 0.1 µM enhanced the positive inotropic effect of isoproterenol; 4) carbachol, a muscarinic receptor agonist, at 10 µM diminished the positive inotropic effect of LS; 5) LS elicited a positive chronotropic effect over a range of concentrations at which it had a positive inotropic effect (> 0.1 AM); 6) LS at 10 µM increased the amplitude of the L-type Ca 2+current four-fold;239,24') and 7) in studies in humans and dogs, LS had a vasodilator action that reduced both the preload and the afterload to the heart. 240,246,247)A positive lusitropic effect, as expected from an increase in the extent of phosphorylation of phospholamban and troponin I, was not, however, induced by LS. This finding might have been due to a balance of the inhibition of PDE and the Ca 21 sensitizing action of LS 245) The finding that the energetic cost of inotropism induced by LS was similar to that obtained with isoproterenol also implies a contribution of cyclic AMP to the effect of LS in vivo.248)The attractive hypothesis was proposed, based on findings in the perfused guinea-pig heart, that LS at low concentrations (< 0.1 µM) acts predominantly as a Ca 21 sensitizer, whereas at higher concentrations its action as an inhibitor of PDE III contributes to the positive inotropic effect. 188,239)However, the original findings were not supported by later results, namely, that LS at 10 nM increased the extent of phosphorylation of phospholamban, while at 0.1 µM it markedly enhanced the positive inotropic effect of isoproterenol and by itself elicited a positive chronotropic effect in the guinea-pig cardiac muscle.245)These results can be ascribed to inhibition of PDE III and the subsequent accumulation of cyclic AMP in response to LS at relatively low concentrations. MCI-154: MCI-154 (6-[4-(4'-pyridyl) aminophenyl]-4,5-dihydro-3(2H)- pyridazinone hydrochloride) is a cardiotonic agent with Ca 21 sensitizing action and selective PDE III-inhibitory action (Figure 2).'29,249)The IC50 value of MCI- 154 for inhibition of PDE III has been reported as 4 µM250) and 10 µM251) in vitro while the Ca 2+sensitizing action in cardiac skinned muscle fibers was observed at 100.tM (Table II).244,252)A much smaller Ca 21sensitizing effect has been observed at lower concentrations of MCI-154 (1-10µM) in guinea-pig skinned cardiac muscle fibers. 129)In preparations of isolated cardiac muscle from various mamma- Jpn Heart J 28 ENDOH January 1998 lian species MCI-154 has a positive inotropic action and in anesthetized and conscious dogs it also has a vasodilator action. 129,249,253,254)These results suggest the contribution of both actions to the cardiovascular pharmacological profile of the compound. MCI-154 increases the amplitude of Ca" transients prominently in aequorin-injected intact cardiac muscle'211,116) and this increase is markedly at- tenuated by carbachol, a muscarinic receptor agonist.256)Therefore, the accumulation of cyclic AMP due to inhibition of PDE III might contribute, in part, to the positive inotropic effect of MCI-154.256) By contrast, a clear-cut Ca 21 sensitizing action of MCI-154 has been observed in saponin-treated skinned fibers of guinea-pig ventricles 121) and of canine ventricles '251) and of human failing hearts.252) MCI-154 is 100 times more potent than sulmazole as a Ca 21 sensitizer in skinned cardiac muscle fibers from the guinea pig."') MCI-154 increased the affinity of troponin C for Ca 21 ions in canine heart myofilaments, but it did not affect the activity of actin-activated myosin and myosin ATPase in preparations from dog hearts."') In addition, MCI-154 enhanced directly the binding of Ca 2+ ions to troponin C.257) These observations indicate that MCI-154 might mainly act as a Ca2+ sensitizer at Ca2+ binding sites on troponin C to enhance the affinity of the latter for Ca 21 ions (class I in Table I). In the motility assay in vitro, in which fluorescently labeled thin filaments, reconstituted from actin and the tropomyosin-troponin complex, slide over myosin filaments fixed on nitrocellulose-coated coverslips, MCI-154 at 1 to 100 µM increased the threshold value of -log [Ca21], (pCa) at which the thin filaments start to move on the myosin layer, in a concentration-dependent manner.24)Thus, MCI-154 seemed to act directly on the reconstituted thin filaments. MCI-154 had a similar effect on the movements of thin filaments even under simulated pathophysiological conditions, such as acidosis, low temperature and an increased level of inorganic phosphate. However, MCI-154 did not restore the maximal sliding velocity after its depression by these pathophysiological interventions. Furthermore, MCI-154 did not have any effect on the movement of actin filaments alone.24)These findings indicate that MCI-154 does not act directly on the cycling of crossbridges. The effectiveness of MCI- 154 in reversing myocardial dysfunction induced by pathophysiological interventions has been demonstrated by other experimental procedures, including procedures that involved skinned cardiac muscle fibers. 252,258,259) ORG 30029: Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2- carboximidamide hydrochloride) has a positive inotropic activity with an extremely high efficacy in cardiac muscle from various mammalian species, such as rabbit, rat, guinea pig and dog. 260.261)For example, in the right ventricular trabeculae isolated from the dog261)and in rat papillary muscle'160) the maximal contraction achieved by Org 30029 was 160% of the maximal response to Vol 39 Ca' SIGNALING IN CARDIAC MUSCLE CELLS No I 29 isoproterenol. Org 30029 has both Ca2+ sensitizing action and PDE-inhibitory action (Table II). Org 30029 inhibits both PDE III and PDE IV, and the IC 50 values of Org 30029 for inhibition of PDE III and IV are 37 tM and 20 AM, respectively. In detergent (Triton X 100) skinned trabeculae isolated from the right ventricle of rats, Org 30029 shifted the pCa-tension relationship to the left at 100 pM and it increased the maximal tension activated by Ca2+ first at 10 µM. 262) In detergent-skinned right ventricular trabeculae from rats and human atria, Org 30029 at 1 mM increased the maximal tension activated by Ca 2+by 90% and 60%, respectively.260)In aequorin-injected right ventricular trabeculae of the dog, Org 30029 increased the amplitude of Ca2+transients and levels of cyclic AMP in cells. Since carbachol abolished the response of Ca' transients and the accumulation of cyclic AMP induced by Org 30029, they must have been due to the PDE-inhibitory action of Org 30029.261)Org 30029 increased the contractile force up to a level 20% higher than the maximal response to isoproterenol, even in the presence of carbachol, and this result might have been due to the Ca 2+ sensitizing action of the compound.261)The positive inotropic effect of Org 30029 in the presence of carbachol was associated with a decrease in the amplitude of Ca 2+transients; Org 30029 prolonged the duration of contraction without alterations in the time course of Ca 2+transients, an indication that the compound acts as a Ca 21sensitizer.261) While the site of Org 30029-induced Ca 21sensitization has not yet been identified, evidence from recent studies implies that it acts on the cycling of crossbridges.260,261)Org 30029 can reverse the myocardial depression induced by acidosis and 2,3-butanedione monoxime (BDM) in dog ventricular trabeculae, with little alteration of the amplitude of Ca 2+transients.26) Org 30029, resembling EMD 57033, impaired relaxation in a concentration-dependent manner in failing human myocardium to a greater extent than in nonfailing human myocardium. 229) CGP 48506: CGP 48506 [(+)5-methyl-6-phenyl-1,3,5,6-tetrahydro-3,6- methano-1,5-benzodiazocine-2,4-dione] is a Ca 21sensitizer that has no inhibitory effect on the activity of PDE from the guinea-pig myocardium and failing human hearts (Figure 2).16,263)Its stereoisomer, CGP 48508, did not affect the force of contraction in failing human myocardium..213) CGP 48506 (1 to 100 .LM) caused a parallel shift of the pCa-tension relationship to the left in a concentration- dependent manner with a decrease in maximal tension at the highest concentration (100 µM) in mammalian skinned cardiac muscle fibers. 16,261-261)The EC 50 values of CGP 48506 for Ca2+sensitization in skinned trabeculae from the guinea pig and the failing human myocardium were 22 pM and 10µM, respectively, whereas CGP 48506 at up to 300 µM did not affect the activity of PDE isoen- zymes I to IV from both species (Table II).'6,263)Furthermore, CGP 48506 (up to 100 LM)did not increase the extent of phosphorylation of the cardiac regulatory Jpn Heart .1 30 ENDOH January 1998 proteins that are phosphorylated by PKA nor did it increase levels of cyclic AMP in guinea-pig ventricular cardiomyocytes.76) When CGP 48506 was administered to isolated rat cardiomyocytes that had been loaded with fura-2, it significantly increased the amplitude of cell shortening with little or no change in the Ca 2+ transient, indicating that CGP 48506 exerts a Ca 21 sensitizing effect in intact myocardial cells.261) CGP 48506 did not affect the binding of Ca 21ions to troponin C and it was able to reverse the inhibition of contraction induced by BDM both in intact cells and in skinned fiber bundles isolated from the rat.264)These findings indicate that CGP 48506 acts as a Ca 2+sensitizer by a "downstream" mechanism.') Since CGP 48506 potentiated the Ca 2+-independent rigor tension in skinned ventricular trabeculae from pigs, it has been proposed that the compound acts partly through a Ca2+-independent mechanism, namely, on cycling of crossbridges265)(class III in Table I). CGP 48506 at 100.tM increased resting tension by slowing relaxation in isolated rat papillary muscle.26') Nonetheless, for a given positive inotropic effect, CGP 48506 had a much smaller effect on diastolic length than the thiadiazinone EMD 57033.264)Studies in skinned cardiac muscle fiber bundles from rats to determine the influence of Ca 21 sensitizers on the intrinsic relaxation rate by photolysis of diazo-2, a caged chelator of Ca2+ions indicated that CGP 48506 slightly accelerated relaxation, in contrast to caffeine, which decreased the initial high rate of relaxation after photolysis.266)

CLINICAL APPLICATIONS OF POSITIVE INOTROPIc AGENTS

The role of positive inotropic agents in the treatment of patients with heart failure has not yet been settled. It has been shown repeatedly and clearly estab- lished that agents with a positive inotropic action are effective for the treatment of patients with acute heart failure. Classical as well as novel cardiotonic agents are able to improve the quality of life (QOL) of patients by correcting the hemodynamic disorders and enhancing the capacity for exercise. However, to date, no positive inotropic drugs have succeeded in improving the survival rates of the patients with chronic congestive heart failure by long-term administration. While digitalis has been employed as a therapeutic agent for the treatment of chronic heart failure and has been recognized as a cardiotonic agent for more than 200 years, it is only very recently that a large-scale clinical trial, with 6,800 patients who had heart failure with left ventricular ejection fractions of 0.45 or less, and with sinus rhythm, was conducted to examine the clinical effectiveness of digoxin.267)The patients received administration of digoxin (at median dose of 0.25 mg per day; average follow up, 37 months) in addition to diuretics and angiotensin-converting-enzyme inhibitors. Digoxin had no effect on overall mortality, but the risk of hospitalization, especially for worsening heart failure, was Vol 39 Ca" SIGNALINGIN CARDIACMUSCLE CELLS 31 No I reduced with digoxin therapy. When the combined outcome was analyzed, the incidence of death from worsening heart failure or hospitalization for that diag- nosis was markedlv reduced 267) The beneficial effects of digoxin might be due, in part, to its sympathoinhibitory action that can mitigate adverse effects of long- term excessive sympathetic adrenergic stimulation in heart failure .2") Further detailed evaluation of clinical data may be required before a final conclusion is reached. The clinical effectiveness of long-term administration of positive inotropic agents that act via the accumulation of cyclic AMP has also not been demonstrated. The most serious problem associated with this class of agents is that they increase the rate of oxygen consumption by the heart and elicit arrhythmias at higher doses. Although the agents described in this review affect a common signal transduction process. their nharmacoloeical profiles are different. Selective inhibitors of PDE III and partial agonists of /3l-adrenoceptors increase the heart rate much less than full agonists of 0-adrenoceptors in animal models and in patients with heart failure. Thus, the former agents might have an energetic advantage over the latter because tachycardia is an important factor in increased consumption of oxygen. Unfortunately, most of the clinical trials with novel inhibitors of PDE III have failed to demonstrate prolongation of the life span of patients with severe congestive heart failure. Rather, these drugs tended to shorten it. During the course of development of novel positive inotropic agents it has become clear that the long-term administration of these agents at the doses that produce acute hemodynamic effects upon single application is harmful for the long-term prog- nosis of patients, leading, possibly, to decreased survival rates of patients even compared with a placebo. The evaluation of the effects of long-term administration of low doses of novel cardiotonic agents with mixed or novel pharmacological actions, such as vesnarinone (with PDE III-inhibitory action, inhibition of K+ channels, inhibition of inactivation of Na+ channels, suppression of plasma levels of cytokines), pimobendan (with PDE III-inhibitory and Ca" sensitizing actions) and BAY v 5959 (Ca2+ promoter) awaits further large-scale clinical trials. Calcium sensitizers are expected to be suitable for the clinical application because they do not increase the activation energy required for increased mobilization and removal of intracellular Ca' ions, and they avoid the Ca2+overload that can lead to arrhythmias and ultimate injury to myocardial cell, in contrast to catecholamines and digitalis that increase [Ca2+];.Furthermore, Ca2+ sensitizers are able to reverse the myocardial dysfunction encountered in pathophysiological conditions, such as myocardial ischemia, acidosis and stunning, when Ca2+-mobilizing agents fail to increase the contractile force. A potential clinical disadvantage could be an impairment of diastolic relaxation of the heart due to an in- Jpn Heart J 32 ENDOH January 1998

crease in Ca2+ sensitivity during diastole, namely, in the failing heart in which diastolic levels of Ca2+ ions could be elevated compared with those in the normal heart. There is insufficient evidence, as yet, to allow us to come to any conclu- sions about the clinical effectiveness of Ca2+ sensitizers. Further basic and clinical studies are required to elucidate the significance of the various potential problems. 230,269) It has been shown recently that endogenous compounds such as the amino acid L-methionine,270) growth hormone and insulin-like growth factor-1,271,272) elicit a positive inotropic effect mainly via an increase in myofibrillar sensitivity to Ca2+ ions in mammalian cardiac muscle. These findings are of interest in at least two respects; they suggest the presence of endogenous systems that contribute to the protection of myocardial cells against the development and progression of myocardial failure; and may suggest the potential therapeutic relevance of the signal transduction processes that involve these endogenous compounds.

ACKNOWLEDGMENT Preparation of this review was supported in part by a Grant-in-Aid for Develop- mental Scientific Research (no.07557193) from the Ministry of Education, Science, Sports and Culture, Japan.

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