© 1995 American Heart Association, Inc.
Articles |
MCI-154 Increases Ca2+ Sensitivity of Reconstituted Thin Filament
A Study Using a Novel In Vitro Motility Assay Technique
From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo (Japan).
Correspondence to Masataka Sata, MD, Department of Physiology & Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-4970.
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Abstract MCI-154 (6-[4-(4'-pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochloride trihydrate) is a potent novel cardiotonic agent whose positive inotropism is shown to be mainly based on an increase in Ca2+ sensitivity of the contractile apparatus. To elucidate the exact mechanism through which this drug acts, we investigated the movement of the reconstituted thin filament on a myosin layer in vitro. Cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the filament showed that tropomyosin-troponin complex was integrated along actin filament homogeneously. Thin filaments thus prepared were fluorescently labeled and made to slide on rat cardiac myosin fixed on a glass coverslip while varying the [Ca2+] of the medium (control, pH 7.2 at 25°C). When [Ca2+] was low, the filaments showed only brownian motion. However, above a certain level of [Ca2+] (the threshold [Ca2+]), the filaments started to slide, and the velocity increased, reaching the maximum velocity within a very narrow range of [Ca2+]. The regulation was completely abolished by using simple actin filaments without tropomyosin-troponin complex, demonstrating that the regulatory proteins are responsible for this Ca2+ regulation of the movement of the reconstituted thin filament. Under the control condition, addition of MCI-154 shifted the threshold [Ca2+] to a lower level (sensitization) in a concentration-related manner. And 10-4 mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low temperature (15°C), or the addition of inorganic phosphate (10 mmol/L). However, the maximum sliding velocity was not affected by the drug under any condition. In conclusion, MCI-154 directly sensitized the contractile apparatus under not only physiological but also pathophysiological conditions. This in vitro motility assay technique using reconstituted thin filaments is a useful tool for studying the mechanism of action of Ca2+ sensitizers.
Key Words: Ca2+ sensitizer • myosin • regulatory proteins • in vitro motility assay
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracellular free Ca2+ concentration ([Ca2+]i) plays a key role in the regulation of myocardial contraction. An increase in [Ca2+]i releases the inhibition exerted by regulatory proteins on actomyosin interaction, whereas a decrease in [Ca2+]i reverses this effect. Because of both basic and clinical interests, the relation between [Ca2+]i and the generated force has been studied extensively by using skinned cardiac muscle preparations.1 2 3 Furthermore, the recent development of various Ca2+ indicators has made it possible to study even the dynamic relation between the [Ca2+]i transient and the force transient during the cardiac cycle.4 5 6 7 One of the most important findings elucidated by these studies is that the relation between [Ca2+]i and the generated force is not always uniform, depending on the assay condition. This phenomenon, often described as the alteration in Ca2+ sensitivity of the contractile apparatus, is observed under various conditions, such as acidosis, low temperature, accumulation of intracellular inorganic phosphate, and cAMP-dependent phosphorylation of cardiac troponin I triggered by ß-adrenergic stimulation.1 2 4 8
Ca2+ sensitivity of the contractile apparatus is also known to be modulated pharmacologically by a new class of cardiotonic agents termed Ca2+ sensitizers.9 10 11 12 These drugs attract the interest of physicians because they have the potential to maintain force generation while saving energy for Ca2+ handling and thus could be promising drugs for the treatment of congestive heart failure. Although clinical trials for these drugs have been initiated,13 the precise mechanism by which these drugs exert their action is not well understood.9 Indeed, on the basis of experimental studies using whole heart or muscle fiber preparations, it has been suggested that multiple steps in excitation-contraction coupling are the sites of action of these drugs.9 10 11 12 Also, there is a possibility that the actions of these drugs are mediated by receptor or messenger systems. However, the direct identification of the mechanism of action may be difficult in these conventional experimental preparations in which complex cellular structures are preserved.
To address these questions, we applied a new technique by introducing Ca2+ regulation into the in vitro motility assay we have used for the study of cardiac actomyosin interaction.14 15 We reconstituted native thin filament by adding cardiac tropomyosin-troponin (Tm-Tn) complex to the actin filament. With this assay system, we could easily control the immediate environment around the crossbridges, thereby studying directly the effect of various factors, including cardiotonic agents, on the movement of the thin filament on the myosin layer. We could narrow down the action site of the Ca2+-sensitizing agents by comparing the mechanical response to Ca2+ between the reconstituted thin filament (with Tm-Tn complex) and the simple actin filament (without Tm-Tn complex) either in the presence or absence of a drug. Using this technique, we studied the effect of a potent cardiotonic agent, MCI-154 (6-[4-(4'-pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochloride trihydrate), whose inotropic action is mainly based on an increase in Ca2+ sensitivity of the myofilament.12 16
The present study, using a new in vitro motility assay technique, clearly demonstrated that MCI-154 directly increases the Ca2+sensitivity of the reconstituted thin filament sliding on cardiac myosin, under not only physiological but also pathophysiological conditions, without affecting the maximum sliding velocity of the actomyosin interaction.
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Materials and Methods |
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Preparation of Cardiac Myosin
Cardiac myosin was obtained from 4-week-old male Wistar rats, whose ventricular myocytes are known to contain only the V1 type of isomyosin.17 Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 to 50 mg/kg body wt), and the hearts were excised rapidly. All procedures described below were performed at 4°C. The hearts were washed in buffered saline (10 mmol/L sodium phosphate buffer and 0.9% NaCl, pH 7.2). Then the atria and right ventricle were removed, and only the left ventricles were used for the extraction of myosin. Because the rat hearts were small, 10 were pooled for myosin extraction. The heart muscle was homogenized in Tris/maleic acid buffer (20 mmol/L Tris/maleic acid and 1 mmol/L EDTA, pH 7.0) and collected by centrifugation (SCR 20B, Hitachi) at 1000g for 15 minutes. After the supernatant was discarded, the pellet was extracted with 3 vol Guba-Straub solution (0.3 mol/L KCl, 100 mmol/L KH2PO4, 50 mmol/L K2HPO4, 1 mmol/L ATP, 5 µg/mL leupeptin, 5 mmol/L dithiothreitol, and 1 mmol/L EDTA, pH 6.5) for 10 minutes. After the extract was centrifuged at 11 000g for 15 minutes, the supernatant was collected, and 14 vol ice-cold distilled water was added to precipitate the myosin. After 2 hours had passed, the myosin was collected by centrifugation at 11 000g for 15 minutes. The myosin was again dissolved in a high-ionic-strength solution (0.6 mol/L KCl, 10 mmol/L Tris-HCl, and 5 mmol/L dithiothreitol, pH 7.5), and the trace amount of actin was removed by centrifugation (L8M, Beckman) at 120 000g for 2.5 hours.
Preparation of Actin and Tm-Tn Complex
Monomeric actin was obtained from rat cardiac acetone powder by
the method of Spudich and Watt18 and converted to
filamentous actin by adding KCl to 50 mmol/L, MgCl2 to
1 mmol/L, and ATP to 1 mmol/L in final concentrations. Tm-Tn
complex was obtained by the method of Ebashi et al19 with
some modifications. Tm-Tn complex was extracted from rat cardiac
acetone powder in a high-ionic-strength solution (0.6 mol/L KCl, 20
mmol/L Tris-HCl, and 0.2 mmol/L ATP, pH 8.0) overnight and isolated by
acid precipitation (pH 4.6). The precipitate was dissolved in 1 mmol/L
sodium bicarbonate, and Tm-Tn complex was obtained by fraction at pH
7.4 with (NH4)2SO4 of 25 to 35
g/dL.
The purity of each protein sample was confirmed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). Protein concentrations were determined according to Lowry et al.20
Reconstitution of Native Thin Filament
To reconstitute native thin filament, filamentous actin was
mixed with Tm-Tn complex to final concentrations of 1 and 2 mg/mL,
respectively, in a high-ionic-strength solution (0.1 mol/L KCl, 30
mmol/L Tris-HCl, 1 mmol/L ATP, 2 mmol/L MgCl2, and
0.5 mmol/L EGTA, pH 8.0), incubated at 40°C for 10 minutes, and
cooled slowly (
0.2°C per minute) to 10°C. Actin filaments to
which Tm-Tn complex was integrated, ie, "reconstituted thin
filament," were collected by centrifugation at 130 000g
for 2 hours. Pellets were suspended with a solution containing 25
mmol/L KCl, 6 mmol/L MgCl2, 25 mmol/L HEPES, and 1
mmol/L EGTA and incubated at 4°C overnight with rhodamine/phalloidin
(Molecular Probes Inc), arranging the molar ratio of actin to
rhodamine/phalloidin as 1:1.
In Vitro Motility Assay
We used the method described by Kron and Spudich21
and Harada et al22 with some
modifications.14 15 Two drops of 1% nitrocellulose in
high-purity amyl acetate were dropped on cool distilled water in a
clean 15-cm-diameter round flat jar. By removing water from the bottom
of the jar, the nitrocellulose film was fixed on the coverslip (30
mmx30 mm, Matsunami Co) placed in the jar. After cutting away excess
film with dissection forceps, the coverslip was placed on paper
toweling to dry. Cardiac myosin was treated before application
according to Warrick et al.23 Myosin (1.0 to 1.5 mg/mL)
was mixed with filamentous actin (final concentration, 0.5 mg/mL) and
with MgATP (final concentration, 2 mmol/L) in 0.6 mol/L KCl and 10
mmol/L Tris-HCl (pH 7.5). After 10 minutes on ice, the mixture was
centrifuged at 200 000g in a Beckman TL-100 centrifuge for
10 minutes to sediment the actin filament and the subset of myosin that
was irreversibly bound to the actin filament in the presence of MgATP.
The supernatant was diluted to 0.8 mg/mL. This treatment apparently
reduced the number of myosin heads that bound actin in a rigorlike
fashion. Myosin solution (60 µL) thus prepared was applied on the
nitrocellulose-coated coverslip by a micropipette and was covered by
another smaller coverslip (18 mmx18 mm). On each edge of the smaller
coverslip, 0.1 g of silicon grease (Dow Corning) was applied to create
a fluid-filled flow cell. After a 15-minute incubation on ice, 180 µL
of BSA solution (0.5 mg/mL bovine serum albumin, 30 mmol/L KCl, 20
mmol/L HEPES, and 1 mmol/L EGTA, pH 7.5) was applied to the flow cell
to wash out unbound myosin and to coat the exposed nitrocellulose.
Next, the reconstituted thin filaments suspended in the motility buffer
containing MgATP (25 mmol/L KCl, 6 mmol/L MgCl2, 25
mmol/L HEPES, 1 mmol/L EGTA, 1% 2-mercaptoethanol, 4.5 mg/mL glucose,
216 µg/mL glucose oxidase, 36 µg/mL catalase, and 2 mmol/L ATP, pH
7.2) with various concentrations of Ca2+ were
introduced onto the myosin-coated coverslip. Then 120 µL of motility
buffer was perfused to wash out unbound thin filaments. Movements of
fluorescently labeled thin filaments were observed with an inverted
fluorescence microscope (TMD-EF2, Nikon) equipped with a x100 oil
immersion objective lens (numerical aperture, 1.3; Zeiss Neofluor), a
100-W super high-pressure mercury lamp, and a rhodamine filter set. The
fluorescent image of the filament was displayed on a TV monitor
(C1846-03, Hamamatsu-Photonics) via a high-sensitive silicon
intensifier target camera (C2400-08, Hamamatsu-Photonics) and was
recorded on videotape (video recorder BR-S601M, JVC).
Velocity Measurement
The measurement of the velocity was performed during a replay of
the videotape recording. Each video frame was digitized at a rate of 3
frames per second into a 480x360 pixel array by a video grabber card
(Personal Vision, Orange Micro Inc) equipped in a personal computer
(Macintosh II fx, Apple). The filaments were 0.5 to 5 µm in length.
More than 95% of the simple actin filaments or reconstituted thin
filaments in the presence of enough free Ca2+
continued to move independent of their lengths, although some of the
moving filaments suddenly stopped and resumed movement at the same
velocity as before. The investigator, using a mouse, located the
leading edge of a thin filament in successive snapshots, allowing the
computer to calculate the mean velocity of the filament from the
movement distance and the elapsed time. To reduce quantification errors
by the confounding effects of discontinuous movement of the filaments,
only continuous movements for >3 seconds were scored.
Experimental Protocol
First, the motility assay was performed while varying the
[Ca2+] of the motility buffer under the control
condition (pH 7.2 at 25°C) in the absence or presence of MCI-154
(10-6, 10-5, and
10-4 mol/L). [Ca2+] was adjusted by
using a Ca2+/EGTA buffer system and
calculated by the method of Fabiato and Fabiato.24
[Ca2+] values of the medium were expressed by pCa
(-log [Ca2+]). Next, to clarify how
[Ca2+] regulates the movement of the reconstituted
thin filament, we also performed a similar in vitro motility assay by
using simple actin filaments instead of reconstituted thin filaments,
as previously described,14 15 21 22 while varying the
[Ca2+] of the motility buffer under the control
condition (pH 7.2 at 25°C) in the presence or absence of
10-4 mol/L MCI-154. Furthermore, to investigate the
effects of experimental conditions on the movement of the reconstituted
thin filament, the same experiments were performed under acidosis (pH
6.8 at 25°C), at a low temperature (pH 7.2 at 15°C), and in the
presence of 10 mmol/L inorganic phosphate (pH 7.2 at 25°C) without
changing other factors in the absence or presence of MCI-154
(10-4 mol/L). For each condition, the experiments were
repeated three times with different proteins, which were purified
independently.
Double Staining of Reconstituted Thin Filament
The distribution of Tm-Tn complex along fibrous actin in the
reconstituted thin filament was determined by double labeling of the
same filament by fluorescent dyes. Tm-Tn complex was incubated with
fluorescein isothiocyanate (Sigma Chemical Co) at a ratio of 3:1
(wt/wt) in a Tris/maleate buffer (40 mmol/L, pH 8.5) at room
temperature for 3 hours and dialyzed against 1 mmol/L sodium
bicarbonate to remove unreacted fluorescent material.25
The physiological activities of Tm-Tn complex are known to be well
preserved after labeling.25 Tm-Tn complex thus labeled was
incubated with filamentous actin to form reconstituted thin filament,
and actin in the reconstituted thin filament was also labeled with
rhodamine/phalloidin as described above. These doubly stained thin
filaments were introduced onto the myosin-coated coverslip and observed
with the fluorescence microscope via a high-sensitive CCD camera
equipped with an image intensifier (model C2400-87,
Hamamatsu-Photonics) and a computed image processor (Argus-50,
Hamamatsu-Photonics); filter systems specific for rhodamine
fluorescence (excitation, 510 to 560 nm; emission, >580 nm) and
fluorescein fluorescence (excitation, 450 to 490 nm; emission, >510
nm) were used.
Statistical Analysis
The mean velocity for each [Ca2+] was
determined from three different preparations in which 20 to 30
different reconstituted thin filaments were scored. Under the same
condition, sliding velocities at various [Ca2+]
values were compared with one-way ANOVA. Sliding velocities of
reconstituted thin filaments and simple actin filaments at the same pH
and [Ca2+] were compared by using Student's
t test. A value of P<.01 was considered to be
significant.
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Results |
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Protein Samples
Fig 1
shows the results of SDS-PAGE of the
contractile and regulatory proteins used in the present study. We
confirmed the purity of each protein sample.
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Double Staining of the Filaments
Fig 2 shows how the doubly stained reconstituted
thin filaments were observed under the fluorescence microscope. The top
panel shows the reconstituted thin filaments observed with a filter
system specific for rhodamine fluorescence, indicating the structure of
actin filaments. The bottom panel shows the identical filaments
observed with a filter system specific for fluorescein fluorescence,
indicating the distribution of Tm-Tn complex. Because these two panels
presented similar images, we considered the possibility that the
Tm-Tn complex was integrated along the actin filaments homogeneously to
form reconstituted thin filaments. This result was consistent with the
previous finding by Ishiwata and Kondo.26
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Movement of the Reconstituted Thin Filament
The top panel of Fig 3 shows the sliding velocity
of the reconstituted thin filament (pH 7.2 at 25°C) as a function of
pCa in the absence (control) or presence of MCI-154 (10-4
mol/L). Under either condition, when the pCa value was large (low
[Ca2+]), most filaments showed only brownian
motion and never showed smooth sliding movement on the myosin layer. At
a certain level of pCa (threshold pCa level), however, all filaments
suddenly started to move at submaximum velocity. A further decrease in
the pCa value (increments in [Ca2+]) raised the
sliding velocity slightly to reach the maximum velocity. The relation
between pCa and sliding velocity was very steep. These results were
similar to those previously reported with proteins from rabbit skeletal
muscle.22 27 Although responses of filament sliding to
Ca2+ were qualitatively similar, the addition of
10-4 mol/L of MCI-154 slightly increased the threshold pCa
value from 6.1 (control) to 6.3 (in the presence of MCI-154),
indicating that this drug directly increased the sensitivity of the
contractile proteins to Ca2+. However, MCI-154 did
not change the sliding velocity at higher [Ca2+]
values (maximum velocity).
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) or presence (
) of 10-4 mol/L
MCI-154 under the control condition (pH 7.2 at 25°C). The
concentration of free Ca2+ is expressed as pCa
(-log10 [Ca2+]). Curves were fit by
eye. *Submaximum point at which the velocity was significantly lower
than that of the maximum points (P<.01). Bottom, Graph
showing the relation between the sliding velocities of the actin
filaments without tropomyosin-troponin complex and the pCa value in the
absence (
Fig 4 shows the effect of various concentrations of
MCI-154 on the relation between pCa and the sliding velocity of the
reconstituted thin filament. MCI-154 increased the threshold pCa value
at which the reconstituted thin filament started to move in a
concentration-related manner. However, MCI-154 did not affect the
sliding velocity at higher [Ca2+] values. In three
different preparations, the threshold pCa levels were identical in the
presence of various concentrations of MCI-154 as well as in the absence
of the drug.
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), 10-5 mol/L (
), or
10-4 mol/L (
To confirm that the regulatory proteins are responsible for this on-off
regulation, we also performed similar experiments using actin filament
without Tm-Tn complex under the control condition (pH 7.2 at 25°C).
The bottom panel of Fig 3
shows the sliding velocity of the actin
filament over a wide range of pCa values. Actin filaments slid at a
constant velocity independent of the [Ca2+] value
within the whole pCa range examined. Sliding velocities were not
significantly different from those of the reconstituted thin filament
at high [Ca2+] values under the same pH and
temperature. Furthermore, the addition of 10-4 mol/L of
MCI-154 did not affect the sliding velocity of actin filament
significantly, suggesting that MCI-154 has no effect on the actomyosin
interaction step.
Effect of Different Experimental Conditions
Fig 5 shows the relations between pCa and the
sliding velocity of the reconstituted thin filament under different
experimental conditions in the absence or presence of 10-4
mol/L MCI-154. The relation was similar to that under the control
condition (top panel of Fig 3), and the threshold pCa levels were
identical in the different preparations under the same condition. Under
acidic conditions (pH 6.8 at 25°C), the threshold pCa value decreased
greatly to 5.1, with a concomitant decrease in maximal sliding velocity
to less than half of that under the control condition (pH 7.2 at
25°C). The addition of 10-4 mol/L MCI-154 increased the
threshold pCa value to 5.5 without changing the maximal sliding
velocity (Fig 5A). When the temperature was decreased to 15°C at pH
7.2, the threshold pCa value decreased mildly to 5.8, with a
concomitant decrease in maximal sliding velocity to less than one third
of that under the control condition (pH 7.2 at 25°C). The addition of
10-4 mol/L MCI-154 increased the threshold pCa value to
6.1 without changing the sliding velocity (Fig 5B).
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The addition of 10 mmol/L inorganic phosphate in the assay buffer
decreased the threshold pCa value moderately to 5.5, but the sliding
velocity did not change significantly from that in the absence of
inorganic phosphate (pH 7.2 at 25°C). The addition of
10-4 mol/L MCI-154 increased the threshold pCa to 6.1
without changing the sliding velocity (Fig 5C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we succeeded in introducing Ca2+ regulation into the in vitro motility assay system and directly evaluated the Ca2+ sensitivity of the contractile system. The movement of the actin filament combined with Tm-Tn complex, ie, reconstituted thin filament, is regulated in an on-off fashion by the [Ca2+] of the motility buffer. Using this assay system, we found that a novel Ca2+sensitizing agent, MCI-154, increased the Ca2+ sensitivity of the contractile system via direct action on regulatory proteins under both physiological and pathophysiological conditions.
Importance of Ca2+ Sensitivity
Studies using skinned cardiac muscle preparations or intact heart
muscle loaded with Ca2+-sensitive bioluminescent
dyes have demonstrated that the relation between
[Ca2+]i and the force generated by
cardiac myofilament can be modified by various changes in the
intracellular condition.1 2 3 4 5 6 7 The most well-known clinical
setting in which the modulation of Ca2+ sensitivity
plays an important role is the hypoxic or ischemic
condition.3 5 7 Using a papillary muscle preparation,
Allen and Orchard7 showed that during the early phase of
hypoxia, tension decreased quickly, whereas
[Ca2+]i remained unchanged. It was
also reported that abnormal inhibitory function of regulatory proteins
is partly responsible for the impaired cardiac function in
cardiomyopathic hamsters.28 29
The concept of Ca2+ sensitivity is also important in the treatment of congestive heart failure, because a new class of cardiotonic agents, known as Ca2+ sensitizers, has been introduced in this field recently. These drugs have not only basic but also clinical importance for the following reasons: (1) Long-term drug trials for the treatment of congestive heart failure have proved that conventional cardiotonic agents, which achieve their effect by increasing [Ca2+]i, actually shorten the prognosis of the patients.30 31 32 (2) The only drugs that improve patient prognosis are vasodilating agents, which, at least in part, function by decreasing the energy expenditure of the heart.33 34 35 If Ca2+-sensitizing agents can help cardiac muscle generate more force with minimal increase in [Ca2+]i, they may save the energy consumed for Ca2+ handling and are hoped to improve the prognosis of the patients with congestive heart failure.
Mechanism Responsible for the Modulation of Ca2+
Sensitivity
Despite its basic and clinical importance, the mechanism of
Ca2+ sensitivity modulation in cardiac muscle still
remains obscure. Experiments using cardiac muscle or cell showed that
the change in Ca2+ sensitivity during hypoxia or
ischemia is associated with changes in the levels of intramyocardial
metabolites,36 eg, the decrease in pH,5
decline in phosphocreatine concentration,2 or accumulation
of inorganic phosphate.2 However, in these intact
preparations, it is hard to exclude the possibility that a concomitant
change in other factors may take place. Although skinned muscle
preparations allow us to change the intracellular condition as
desired,1 2 this approach may not be so powerful in
studying the site of action of Ca2+-sensitizing
agents because these drugs are supposed to act at one or more of the
steps in the excitation-contraction coupling process. The proposed
steps are (1) Ca2+ binding to troponin C, (2) steps
in the process by which the inhibitory activity of troponin I, troponin
T, and tropomyosin on the crossbridge reaction is reversed, and (3) a
direct effect on either actin filament or myosin itself. Furthermore,
the contribution of receptors and the intracellular messenger system
should also be considered.
It may resolve these problems, to some extent, to study the relation between actin-activated myosin ATPase activity and [Ca2+] while changing the condition of the reaction solution.8 37 However, the myosin ATPase activity in solution is not always a good index of the mechanical properties of contractile proteins.14 27
In Vitro Motility Assay System
To circumvent those problems described above, we used a new form
of in vitro motility assay in which movement of the reconstituted
thin-filament sliding was observed under Ca2+
regulation. This type of assay system has already been used by other
investigators with skeletal muscle proteins.22 27 Since a
simple actin filament without integration of Tm-Tn complex was
insensitive to [Ca2+], Tm-Tn complex actually gave
Ca2+ sensitivity to the actin filament. The thin
filaments reconstituted in the present study were physiological
both structurally and functionally. First, the double staining of the
reconstituted thin filament suggested that the Tm-Tn complex was
integrated homogeneously along the actin filament as in native thin
filament. Second, experiments performed while changing the assay
conditions showed that acidosis, low temperature, and the addition of
inorganic phosphate decreased the Ca2+ sensitivity
of the contractile proteins in a manner similar to that reported to
occur with muscle or cell preparations.
Finally, the way actomyosin sliding was regulated in this assay may
require comment. The sliding velocity increased to the maximum velocity
within a very narrow pCa range, showing clear contrast to the tension
versus pCa relation of cardiac muscle or to the ATPase activity versus
pCa relation of cardiac myofibril. Tension or ATPase activity increases
gradually as pCa value decreases, because an increase in
[Ca2+] promotes the crossbridge formation,
resulting in a proportional increase in force or ATPase activity. On
the other hand, according to studies about the unloaded actin filament
velocity over a sparsely coated myosin surface,38 39 actin
filament velocity (V) measured in the in vitro motility assay was a
function of the number of crossbridges capable of interacting with the
actin filament (N) and the proportion of the stroking time to the total
ATPase cycle time (f), ie, duty ratio, as indicated
below22 38 39 :
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Effect of a Ca2+-Sensitizing Agent, MCI-154
MCI-154 is a potent nonglycoside and nonsympathomimetic
cardiotonic agent with a pyridazinone
structure.12 16 40 41 42 This drug has been shown to have a
positive inotropic effect with vasodilator property, in anesthetized or
conscious dogs, and in isolated cardiac muscle of various mammalian
species.16 41 42 Experiments with chemically skinned
cardiac fibers from guinea pigs suggested that the mechanism of
positive inotropism is mainly based on an increase in
Ca2+ sensitivity of the contractile
apparatus.16 Perreault et al12 demonstrated
that MCI-154 has a similar effect also in myopathic myocardium from
patients with end-stage heart failure. Regarding the potency for
increasing the sensitivity to Ca2+, MCI-154
has been reported to be 100 times more potent than sulmazole in skinned
myocardial fibers from guinea pigs.16 Although a shift in
the [Ca2+] versus developed force relation
introduced by this drug has been confirmed by many investigators, the
mechanism of action remained to be elucidated.40 41 42 43 One of
the problems to be answered is whether the effect of this drug is
exerted directly or mediated by second-messenger systems. Kitada et
al43 demonstrated that MCI-154 enhances
Ca2+ binding to troponin C, but it has been unknown
how this drug affects the mechanical properties of the contractile
proteins.
In the present study, we used a new form of in vitro motility assay by using only myosin and thin filament reconstituted with actin and Tm-Tn complex to evaluate the effect of MCI-154 without considering the effect on other proteins. MCI-154 increased the threshold pCa value at which the reconstituted thin filament started to move on the myosin layer in a dose-dependent manner, demonstrating that the MCI-154 directly acts on the reconstituted thin filament to increase the sensitivity to Ca2+. MCI-154 also increased the Ca2+ sensitivity of the reconstituted thin filament under pathophysiological conditions, such as acidosis, low temperature, and the addition of inorganic phosphate but without changing the maximum sliding velocity under any condition. Because there is neither adenylate cyclase nor phosphodiesterase in this simplified assay system, the effect of this drug is definitely not mediated by elevation of the cAMP level. Furthermore, the addition of 10-4 mol/L MCI-154 to the motility buffer had no effect on the maximum velocity of the simple actin filament without Tm-Tn complex and that of the reconstituted thin filament under any condition, suggesting that the unloaded actomyosin interaction step is not influenced by this drug.
Limitation of the Present Study
In the present study, we used rhodamine/phalloidin to label
reconstituted thin filaments. Phalloidin has been shown to interact
specifically with filamentous actin and stabilize the bonds between
actin monomers.44 According to Dancker et
al45 and Prochniewicz-Nakayama et al,46 the
ability of filamentous actin to activate myosin ATPase or
Ca2+ sensitivity of the contractile apparatus is not
affected by phalloidin. However, Bukatina and Fuchs47
suggested that phalloidin can change the kinetic parameters of the
crossbridge cycle and may also enhance the Ca2+
sensitivity of the myofibrils of the striated muscle, cardiac more than
skeletal. Further study will be needed to clarify whether phalloidin
actually affects the Ca2+ sensitivity of the
reconstituted filament. Accordingly, we must be careful when we apply
the results in the present study to the in vivo situation.
We suggested that Ca2+ sensitivity of the cardiac contractile apparatus is modulated at the regulatory protein level. Although double staining of the reconstituted thin filament indicated that Tm-Tn complex is integrated along actin filament homogeneously, more detailed study is necessary to determine whether the structure of the thin filament reconstituted in this study is exactly the same as that of native thin filament.
We found that MCI-154 has no effect on the sliding velocity of the actin filament or the reconstituted thin filament on the myosin layer. It is well known that the actin sliding velocity measured in the in vitro motility assay correlates with the maximum shortening velocity of the fully unloaded myofilament rather than the isometrically generated force.21 22 23 Kitada et al16 demonstrated that MCI-154 increases maximal Ca2+-activated force, suggesting that this drug also has an effect on actomyosin interaction. We consider that this discrepancy originated from the difference in experimental conditions, ie, unloaded or isometric shortening. To clarify this point, measurement of drag force generated at one crossbridge may be necessary.
Conclusion
We introduced Ca2+ regulation into the in vitro
motility assay and showed how Ca2+ sensitivity of
the contractile system was modulated under various conditions. Using
this assay system, we investigated the mechanisms of action of a novel
potent cardiotonic agent, MCI-154. The addition of MCI-154 sensitized
the contractile system via direct action on regulatory proteins. The
sensitizing effect was present under not only physiological but
also pathophysiological conditions. This in vitro motility assay
technique using reconstituted thin filament proved to be a useful tool
in studying Ca2+ regulation of cardiac
contraction.
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Acknowledgments |
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This study was supported in part by a grant-in-aid from the Vehicle Racing Commemorative Foundation and a Japan Heart Foundation Research Grant for 1993. We are grateful to Hisako Oh-hata for her technical assistance.
Received June 24, 1994; accepted December 15, 1994.
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