Department of Pharmacology (R.T., M.E.) and The Second Department
of Surgery (Y.S.), Yamagata University School of Medicine, Yamagata,
Japan
We studied the influence of acidosis on the positive inotropic effect
of UD-CG 212 Cl
{4,5-dihydro-6-[2-(4-hydroxyphenyl)-1H-benzimidazole-5-yl]-5-methyl-3(2H)-pyridazinone}, an active metabolite of pimobendan, in canine ventricular trabeculae loaded with aequorin. The positive inotropic effect of UD-CG 212 Cl was
markedly suppressed under acidotic conditions. The maximal contractile
response to UD-CG 212 Cl was attained at 10
5 M in the
control condition at pH 7.4, but was not achieved even at
104 M during acidosis. The maximal inotropic effect of
UD-CG 212 Cl was 18% of the maximal response to isoproterenol
(ISOmax) in association with an increase in
Ca2+ transients of 7% of ISOmax in the
control, while they are 8 and 6% of ISOmax under acidosis,
respectively. Acidosis abolished the increase in myofilament
Ca2+ sensitivity induced by UD-CG 212 Cl, whereas the
increase in Ca2+ transients induced by the compound was not
affected by acidosis. In conclusion, UD-CG 212 Cl elicited a positive
inotropic effect even under acidosis, however, UD-CG 212 Cl was much
less effective as a cardiotonic agent under acidosis mainly due to a
decrease in the Ca2+-sensitizing effect under acidotic condition.
 |
Introduction |
Cardiotonic
agents are indispensable for improvement of contractile dysfunction in
heart failure. Pimobendan {UD-CG 115 BS; 4,5-dihydro-6-[2-(p-methoxyphenyl)-5-benzimidazolyl]-5-methyl-3(2H)-pyridazinone} is a unique cardiotonic agent that has already been launched for treatment of patients with heart failure (Hagemeijer, 1993
). It has an
inhibitory action on phosphodiesterase III (Scholz and Meyer, 1986) and
prolongs the action potential duration (Honerjäger et al., 1984).
A part of the increase in force of contraction has been shown to be due
to the myofilament Ca2+ sensitization (Fujino et
al., 1988; Scheld et al., 1989; Böhm et al., 1991). In addition,
pimobendan is converted to the active metabolite UD-CG 212 Cl
{4,5-dihydro-6-[2-(4-hydroxyphenyl)-1H-benzimidazole-5-yl]-5-methyl-3(2H)-pyridazinone} by hepatic demethylation (Hagemeijer et al., 1989), which may contribute to the favorable hemodynamic effects of the mother compound
(Verdouw et al., 1987). UD-CG 212 Cl is 7.7 times more potent than
pimobendan as a phosphodiesterase III inhibitor (Böhm et al.,
1991). We have recently shown that the positive inotropic effect of
UD-CG 212 Cl is partially due to myofilament Ca2+
sensitization in aequorin-loaded canine ventricular myocardium (Takahashi and Endoh, 2001).
Acidosis affects various processes of cardiac E-C coupling (Bountra and
Vaughan-Jones, 1989; Orchard and Kentish, 1990). Acidosis decreases
myofilament Ca2+ sensitivity (Allen and Orchard,
1983; Orchard and Kentish, 1990; Palmer and Kentish, 1994), which is
partly due to a decrease in the affinity of troponin C for
Ca2+ (Palmer and Kentish, 1994), and a direct
depressant action on the crossbridge cycling (Hulme and Orchard, 1998).
In canine ventricular myocardium, acidosis markedly suppressed the
positive inotropic effect elicited by an increase in
Ca2+ mobilization, whereas the
Ca2+ sensitizer Org-30029 reversed effectively
the acidosis-induced myofilament Ca2+
desensitization (Watanabe et al., 1996).
The present study was undertaken to examine the influence of acidosis
on the positive inotropic effect of UD-CG 212 Cl. For this purpose, we
carried out experiments in isolated canine right ventricular trabeculae
loaded with aequorin. Our results indicate that the
Ca2+-sensitizing effect of UD-CG 212 Cl is
abolished under acidotic condition in the canine ventricular myocardium.
| |
Materials and Methods |
The study involving treatment of experimental animals
conforms to the institutional standards. This study was conducted in accordance with the Guidance for the Care and Use of Laboratory Animals
published by the U.S. National Institutes of Health. The approval for
the animal experiments was obtained from the Committee of Animal
Experimentation, Yamagata University School of Medicine, Yamagata,
Japan, prior to the experiments and the study was carried out also in
accordance with the Declaration of Helsinki.
Preparation of Aequorin-Loaded Canine Right Ventricular
Trabeculae.
Mongrel dogs of either sex (8-12 kg) were
anesthetized by intravenous administration of pentobarbital sodium (30 mg/kg). Hearts were rapidly excised and free-running trabeculae (<1 mm
in diameter) were dissected from the free wall of the right ventricle.
The muscle preparations had an average dimension of 14.8 ± 0.66 mm (range 13-17 mm) in length and 1.09 ± 0.11 mm2 (range 0.85-1.38 mm2)
in cross-sectional area (n = 12).
For simultaneous detection of contractile force and intracellular
Ca2+ transients, the
Ca2+-sensitive bioluminescent protein aequorin
was loaded by the modified macroinjection technique, as described
elsewhere in detail (Sawada and Endoh, 1999; Takahashi and Endoh,
2001). The muscle was electrically stimulated by square wave pulses of
5-ms duration at a voltage about 20% above the threshold at 0.5 Hz in
modified Krebs-Henseleit solution at 37°C. The composition of the
solution was as follows: 118 mM NaCl, 4.7 mM KCl, 2.5 mM
CaCl2, 1.2 mM MgSO4, 24.9 mM NaHCO3, 1.2 mM
KH2PO4, and 11.1 mM glucose
(with 0.057 mM ascorbic acid and 0.027 mM EDTA). The solution was
bubbled with 95% O2, 5%
CO2 and maintained at pH 7.4 in the control
condition. Acidosis (pH 6.6) was induced by replacing about 80% of
HCO
with Cl in modified
Krebs-Henseleit solution according to Watanabe et al. (1996). After
changing to acidotic solution, the contractile force decreased and
bioluminescence increased rapidly within 30 min and both parameters
reached a quasi-steady state after 120 min when the experiments were
started. The composition of the acidotic solution was as follows: 138 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM
MgSO4, 4.9 mM NaHCO3, 1.2 mM KH2PO4, and 11.1 mM
glucose (with 0.057 mM ascorbic acid and 0.027 mM EDTA).
Aequorin light signals were detected with a photomultiplier
(9789A; Thorn EMI Electron Tubes, Ruislip, UK) and light signals were
smoothed by a low-pass filter (cut-off frequency of 100 Hz; multichannel sarcoplasmic reticulum filter 3315; NF Electron
Instruments, Yokohama, Japan). Signals were recorded on digital
audiotape (PC-108 M; Sony Magnescale, Tokyo, Japan) for subsequent
analysis. The muscle preparation was equilibrated for about 120 min
after aequorin-loading procedure, meanwhile the bioluminescence
declined to a steady low level. During the equilibration period, the
muscles were stretched initially at a resting tension of 5 mN and the
length was later adjusted to give the developed tension of 90% of the
maximal contractile force. All experiments were carried out in
the presence of 3 × 107 M
(±)-bupranolol.
Fifty to 150 signals of Ca2+ transients and
isometric contractions were averaged to improve the signal-to-noise
ratio by means of data analysis software (Visual Designer; Intelligent
Instrumentation, Tucson, AZ) in IBM PC/AT personal computer
(FMV-Deskpower S13; Fujitsu, Tokyo, Japan). The 2.5th root of the peak
amplitude of aequorin signals was calculated as an indicator of the
peak [Ca2+]i (Blinks et
al., 1982). It was confirmed that the light emission from aequorin was
not influenced by UD-CG 212 Cl itself (Takahashi and Endoh, 2001).
In each preparation, ISOmax was determined at the
end of experiments after washout of the drugs for 2 h, and the
increase in contractile force and the amplitude of
Ca2+ transients induced by inotropic
interventions were expressed as a percentage of
ISOmax. Time courses of aequorin light transients and isometric contractions (total duration, time to peak, and time for
decline or relaxation) were calculated from the duration between the
peak and the crossings obtained by extrapolation of the steepest
portion of rising or declining phase of individual signals to the
baseline (diastolic) level (Watanabe et al., 1996).
Chemicals.
The drugs used were as follows: UD-CG 212 Cl
(Nippon Boehringer Ingelheim, Kawanishi, Hyogo, Japan);
(
)-isoproterenol hydrochloride (Sigma Chemical Co., St. Louis, MO);
(±)-bupranolol hydrochloride (Kaken Pharmaceutical Co. Ltd, Tokyo);
and pentobarbital sodium (Tokyo Kasei Kogyo Co. Ltd, Tokyo). Aequorin
was purchased from Friday Harbor Photoproteins (Friday Harbor, WA).
UD-CG 212 Cl was dissolved in dimethyl sulfoxide (Takahashi and Endoh,
2001).
Statistical Analysis.
Data are expressed as means ± S.E.M. For analysis of multiple measurements obtained from a single
preparation, we used one-way analysis of variance for repeated measures
with Bonferroni's test. A P value smaller than 0.05 was
considered to indicate statistically significant difference.
| |
Results |
Influence of Acidosis on Effects of Elevation of
[Ca2+]o.
Figure
1 shows the representative actual
tracings (A, pH 7.4; B, pH 6.6) and summarized data (C, pH 7.4; D, pH
6.6) on the influence of acidosis on the increase in aequorin light
transients and isometric contractions induced by elevation of
[Ca2+]o. At 2.5 mM
[Ca2+]o, acidosis
produced a pronounced depression of contractile force (by 64.9 ± 7.50% of the baseline level in the control; P < 0.01) and a significant increase in the amplitude of aequorin light transients (by 9.65 ± 5.11% of the baseline level in the
control; P < 0.01) in association with a prolongation
of aequorin light transients (Figs. 1, A and B, and 3A). Elevation of
[Ca2+]o increased the
force of contraction even under acidosis but to a lesser extent
compared with the control at pH 7.4 (Fig. 1, C and D; P < 0.01 versus the increase in contractile force induced by elevation
of [Ca2+]o at the
corresponding concentrations at pH 7.4). For example, at pH 7.4 the
increase in contractile force at 4.0 mM
[Ca2+]o was 26.2 ± 3.32% of ISOmax and it was associated with an
increase in Ca2+ transients by 28.8 ± 1.98% of ISOmax (n = 5). During
acidosis the increase in force at 4.0 mM
[Ca2+]o was 14.8 ± 3.27% of ISOmax, which was approximately half of the control (P < 0.01) and it was associated with an
increase in aequorin light transients by 24.0 ± 6.00% of
ISOmax (n = 5), which was not
significantly different from the control at pH 7.4 (P > 0.05).
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Fig. 1.
Influence of acidosis on the effects of elevation of
[Ca2+]o on aequorin light transients
(tracings with faster rate of rise) and of isometric contractions
(tracings with slower rate of rise). A, representative tracings at pH
7.4. Actual values for force of contraction and peak light transients:
10.6 mN/mm2 and 0.40 nA at 2.5 mM
[Ca2+]o, and 16.5 mN/mm2 and 0.65 nA at 4.0 mM [Ca2+]o. B, representative
tracings at pH 6.6. Actual values for force of contraction and peak
light transients: 3.40 mN/mm2 and 0.42 nA at 2.5 mM
[Ca2+]o, and 6.34 mN/mm2 and 0.71 nA at 4.0 mM [Ca2+]o. Each tracing in A and B
represents signal-averaged recordings of 100 successive signals
recorded from the same muscle preparation. C, summarized data at pH
7.4. Actual values for basal force of contraction and peak light
transients: 10.2 ± 5.12 mN/mm2 and 0.39 ± 0.22 nA (n = 5). D, summarized data at pH 6.6. Actual
values of basal force of contraction and peak light transients:
3.49 ± 3.35 mN/mm2 and 0.43 ± 0.16 nA
(n = 5); and ISOmax, 48.3 ± 16.7 mN/mm2 and 2.18 ± 2.52 nA (n = 5). Symbols with vertical bars represent mean ± S.E.M. Asterisks
indicate threshold concentrations for increases in each parameter
(P < 0.05); crossings indicate significant
difference compared with the increase in force of contraction induced
by the corresponding [Ca2+]o at pH 7.4.
|
|
Influence of Acidosis on Effects of UD-CG 212 Cl.
Figure
2 shows the representative actual
tracings (A, pH 6.6) and summarized data (B, pH 7.4; C, pH 6.6) on the
influence of acidosis on the increase in Ca2+
transients and isometric contractions induced by UD-CG 212 Cl. Figure
2A shows actual tracings with application of UD-CG 212 Cl, which
indicates that the compound is able to induce a positive inotropic
effect in association with a moderate increase in aequorin light
transients even under acidosis. Figure 2, B and C, show the
concentration-response curve for increases in
Ca2+ transients and contractile force induced by
UD-CG 212 Cl in the control (Fig. 2B) and acidotic (Fig. 2C)
conditions. In the control condition the concentration-response curve
for UD-CG 212 Cl was bell-shaped: the maximal response to UD-CG 212 Cl
was achieved at 105 M, amounted to 17.6 ± 2.43% of ISOmax, and was associated with an
increase in Ca2+ transients by 6.85 ± 1.68% of ISOmax (n = 7 each). In
the control condition at pH 7.4 the EC50 value
for the positive inotropic effect of UD-CG 212 Cl was 3.38 × 107 M and the EC50 for
the increase in amplitude of Ca2+ transients was
2.35 × 106 M. Under acidosis the maximal
response to UD-CG 212 Cl was not achieved even at
104 M; the positive inotropic effect at
104 M was 7.53 ± 0.90% of
ISOmax (P < 0.01 versus the
increase induced by UD-CG 212 Cl at 104 M at pH
7.4) and was associated with an increase in Ca2+
transients by 6.06 ± 2.84% of ISOmax that
was not significantly different from the increase at pH 7.4 (n = 5; P > 0.05 versus the control).
The EC50 value for the positive inotropic effect of UD-CG 212 Cl was supposed to be higher than 3.18 × 106 M, and the value for
Ca2+ transients was >2.79 × 106 M. Overall, the extent of increases in
Ca2+ transients induced by UD-CG 212 Cl under
acidosis was essentially similar to that at pH 7.4 (P > 0.05), whereas the positive inotropic effect of UD-CG 212 Cl was
much smaller under acidotic condition (P < 0.01).
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Fig. 2.
Influence of acidosis on the effects of UD-CG 212 Cl
on aequorin light transients (tracings with faster rate of rise) and of
isometric contractions (tracings with slower rate of rise). A,
representative tracings at pH 6.6. Each tracing represents
signal-averaged recordings of 100 successive signals. Actual values for
contractile force and peak light transients: 3.31 mN/mm2
and 0.42 nA in baseline condition, and 5.11 mN/mm2 and 0.48 nA at 105 M UD-CG 212 Cl. B, summarized data at pH 7.4. Actual values for basal force of contraction and peak aequorin light
transients: 9.58 ± 4.71 mN/mm2 and 0.33 ± 0.17 nA; and ISOmax, 51.6 ± 13.9 mN/mm2 and
2.33 ± 2.61 nA (n = 7). C, summarized data at
pH 6.6. Actual values of basal force of contraction and peak light
transients: 3.45 ± 2.91 mN/mm2 and
0.40 ± 0.14 nA; and ISOmax, 48.3 ± 16.7 mN/mm2 and 2.18 ± 2.52 nA
(n = 5). Symbols with vertical bars represent mean ± S.E.M. Asterisks indicate threshold concentrations for increases in
each parameter (P < 0.05); crossings indicate
significant difference compared with the increase induced by the
corresponding concentrations of UD-CG 212 Cl at pH 7.4. Data in B are
taken for comparison from Takahashi and Endoh (2001) with permission.
|
|
Influence of Acidosis on Duration of Aequorin Light
Transients and Contraction.
Figure 3
shows alterations of the amplitude and time course of aequorin light
transients and isometric contractions induced by acidosis at 2.5 mM
[Ca2+]o (Fig. 3A) and by
UD-CG 212 Cl at 105 M under acidosis (Fig. 3B).
Acidosis produced a pronounced depression of contractile force with a
small abbreviation of contraction (Fig. 3A, top), while
Ca2+ transients were markedly prolonged by
acidosis (Fig. 3A, bottom). In summarized data the total duration of
aequorin light transients was prolonged from 184.5 ± 5.67 ms
significantly to 230.1 ± 5.77 ms by 24.8 ± 3.13%
(n = 5; P < 0.01) with a significant
prolongation of decline time from 133.1 ± 8.01 to 172.1 ± 8.97 ms (n = 5; P < 0.01). The time to
peak light was not significantly altered by acidosis: 51.5 ± 3.05 ms in the control and 57.8 ± 3.71 ms in acidosis
(n = 5; P > 0.05). In contrast, the
duration of isometric contractions was rather shortened, although the
difference was statistically not significant: the total duration of
contraction was 370.6 ± 31.1 ms in the control and 322.4 ± 24.0 ms in acidosis; the time to peak force was 157.5 ± 7.33 ms
in the control and 141.6 ± 8.81 ms in acidosis; and the
relaxation time was 213.1 ± 24.2 ms in the control and 180.8 ± 15.3 ms in acidosis (n = 5 each).
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Fig. 3.
Influence of acidosis on the time course of aequorin
light transients (tracings with faster rate of rise) and isometric
contractions (tracings with slower rate of rise). A, superimposed
tracings of the influence of acidosis at 2.5 mM
[Ca2+]o. Top, actual tracings; bottom,
normalized tracings. B, superimposed tracings of the effects of UD-CG
212 Cl under acidosis (pH 6.6). Top, actual tracings; bottom,
normalized tracings. Each tracing represents signal-averaged recordings
of 100 successive signals.
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|
During acidosis UD-CG 212 Cl at 105 M induced a
moderate positive inotropic effect in association with a small but
definite increase in the amplitude of Ca2+
transients (Fig. 3B, top). UD-CG 212 Cl induced little alteration of
the time course of Ca2+ transients and isometric
contractions during acidosis (Fig. 3B, bottom).
Influence of Acidosis on Myofilament Ca2+
Sensitivity during Application of Different Inotropic
Interventions.
Figure 4 shows the
relationship between the peak Ca2+ transients and
contractile force during the elevation of
[Ca2+]o and
administration of UD-CG 212 Cl in the control and acidotic conditions.
The relationship for elevation of
[Ca2+]o under acidosis
was shifted to the right and downward compared with the control. In the
control condition UD-CG 212 Cl shifted markedly the relationship of the
amplitude of Ca2+ transients and force to the
left and upward, an indication that the compound elicits an increase in
myofilament Ca2+ sensitivity (Takahashi and
Endoh, 2001). In contrast, under acidosis the relationship during
administration of UD-CG 212 Cl was superimposable to that for the
elevation of [Ca2+]o, an
indication that the increase in myofilament Ca2+
sensitivity induced by UD-CG 212 Cl in the control condition was
abolished under acidosis in canine ventricular myocardium.
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Fig. 4.
Relationship between the peak Ca2+
transients and force of contraction during application of different
inotropic interventions, including elevation of
[Ca2+]o and UD-CG 212 Cl under the control
(pH 7.4) and acidotic (pH 6.6) conditions in isolated canine right
ventricular trabeculae loaded with aequorin. Open symbols, pH 7.4;
closed symbols, pH 6.6; circles, [Ca2+]o; and
triangles, UD-CG 212 Cl. The numbers in parentheses represent numbers
of preparations. Ordinate, the increase in force of contraction induced
by the respective inotropic interventions expressed as a percentage of
ISOmax; abscissa, the increase in Ca2+
transients. Symbols with vertical and horizontal bars represent
mean ± S.E.M. Data on UD-CG 212 Cl at pH 7.4 are taken for
comparison from Takahashi and Endoh (2001) with permission.
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| |
Discussion |
Important findings in the present study are that the increase in
myofilament Ca2+ sensitivity induced by UD-CG 212 Cl was abolished under acidosis in canine ventricular myocardium (Fig.
4). While acidosis has multiple effects on the process of cardiac E-C
coupling, including Ca2+ mobilization and
Ca2+ sensitivity, the increase in
Ca2+ transients induced by UD-CG 212 Cl at pH 6.6 was not decreased significantly compared with the increase at pH 7.4 (Fig. 2). This finding indicates that the increase in
Ca2+ sensitivity induced by UD-CG 212 Cl is
highly labile to acidosis, which is in strong contrast to previous
findings that the Ca2+ sensitization induced by
EMD 57033 (Lee et al., 1993) or Org-30029 (Watanabe et al., 1996) was
not attenuated by acidosis. Thus, the Ca2+
sensitization induced by UD-CG 212 Cl may occur through a mechanism different from that for EMD 57033 or Org-30029.
Influence of Acidosis on Myofilament Ca2+
Sensitivity.
Acidosis decreases the Ca2+
sensitivity at the process of Ca2+ binding to
troponin C (Orchard and Kentish, 1990; Palmer and Kentish, 1994) and
decreases also directly the crossbridge cycling (Hulme and Orchard,
1998). Acidosis decreases the Ca2+ binding to
troponin C through modulation of C-terminal domain of troponin I
(Westfall et al., 1997, 2000). Since EMD 57033 binds the C terminus of
troponin C in a region of interaction with troponin I (Li et al.,
2000), the acidosis-induced decrease in Ca2+
sensitivity and the reversal induced by Ca2+
sensitizers may occur through integrated mechanisms of thin filament regulation that ultimately lead to the structural alterations of
troponin C to decrease or reverse the Ca2+.
It has been controversial whether UD-CG 212 Cl increases
Ca2+ sensitivity in cardiac muscle. In skinned
cardiac cells, UD-CG 212 Cl increased Ca2+
sensitivity under the condition where inorganic phosphate level was
elevated (Westfall et al., 1993; Fraker et al., 1997) but not under
normal condition (Böhm et al., 1991; Komukai and Kurihara, 1996).
These findings suggest that the effect of the compound may be
preferentially exerted under pathophysiological conditions such as
ischemia-reperfusion where acidosis plays an important pathological
role (Vanheel et al., 1989). The present observation, however, does not
support such a beneficial effect of UD-CG 212 Cl and indicates that the
expression of Ca2+ sensitization induced by UD-CG
212 Cl may be extremely sensitive to the experimental conditions. Since
the regulation of Ca2+ sensitivity in intact
cells is different from that in skinned cardiac fibers (Gao et al.,
1994; Hulme and Orchard, 1998; Komukai et al., 1998), further study is
necessary to identify the site of
Ca2+-sensitizing action of the compound in
relation to acidosis- and/or inorganic phosphate-induced
Ca2+ desensitization mechanisms.
Myofilament Ca2+ Sensitization and Cyclic AMP.
It
is noteworthy that the Ca2+-sensitizing action of
UD-CG 212 Cl is inhibitable with the muscarinic receptor agonist
carbachol (Takahashi and Endoh, 2001). Carbachol has been used as a
pharmacological tool to differentiate the cyclic AMP-independent from
cyclic AMP-mediated process, in which the former has been defined as
Ca2+ sensitizers (Endoh, 1987, 1999), since it
has been established that cyclic AMP decreases
Ca2+ sensitivity due to phosphorylation of
phospholamban and troponin I. More recently, however, we found that the
Ca2+-sensitizing effect of certain agents is
susceptible to carbachol. Carbachol abolished the
Ca2+-sensitizing effect of levosimendan (Sato et
al., 1998), OR-1896, the active metabolite of levosimendan (Takahashi
et al., 2000a,b) and UD-CG 212 Cl (Takahashi and Endoh, 2001) in dog
and rabbit ventricular myocardium. These findings together indicate
that the subcellular mechanism for the above-mentioned agents may
involve cyclic AMP for the expression of the
Ca2+-sensitizing effect. While the role of cyclic
AMP in Ca2+ sensitization has been unknown,
myosin binding protein C that is phosphorylated by protein kinase A to
lead to an activation of actomyosin ATPase activity (Winegrad, 1999)
may be a potential candidate. In a clinical setting it is postulated
that these agents are free from a risk of serious adverse effect on
diastolic function because an acceleration of relaxation induced by
moderate accumulation of cyclic AMP resulting from phosphodiesterase
III inhibition may counteract the Ca2+
sensitization at diastole (Sugawara and Endoh, 1999).
As a potential mechanism for alteration of cyclic AMP-mediated
regulation under acidosis, it is noteworthy that
Mundina-Weilenmann et al. (1996) have shown that the
phosphorylation of phospholamban and troponin I is facilitated under
acidosis probably due to acidosis-induced inhibition of type 1 phosphatase in rat cardiac muscle. The
Ca2+-sensitizing effect of levosimendan that is
also sensitive to carbachol (Sato et al., 1998) has been shown to be
attenuated by pretreatment with isoproterenol (Haikala et al., 1997).
This finding indicates that a strong activation of cyclic AMP-mediated signaling process is able to suppress the cyclic AMP-related
Ca2+ sensitization. Such a mechanism could
contribute also to the acidosis-induced attenuation of the UD-CG 212 Cl-induced Ca2+ sensitization. Actually,
accumulation of cyclic AMP induced by UD-CG 212 Cl at pH 7.4 reached
first a significant level of approximately 30% of the baseline level
at the highest concentration of 3 × 104
M, which was much less than the accumulation induced by isoproterenol (>100% at 105 M) in canine ventricular muscle
(Endoh et al., 1991).
General Considerations.
The relationship of
Ca2+ and force was examined by the use of
plotting the relation of developed force and peak
Ca2+ transients (Blinks, 1993). Analysis and
explanation of the graph such as that in Fig. 4, however, require great
care when there are changes in the time course of
Ca2+ transients because the alteration of
equilibration kinetics between Ca2+ and the
myofilaments in intact cardiac cells can elicit an apparent shift of
the relation without changing Ca2+ sensitivity
(Yue, 1987). Namely, 
-adrenergic stimulation produces apparent shift
of the relation to the direction of Ca2+
desensitization due to the abbreviation of duration of
Ca2+ transients (Endoh and Blinks, 1988). In the
present study acidosis prolonged Ca2+ transients
that could cause an apparent increase in Ca2+
sensitivity. Nonetheless acidosis shifted the relationship to the
direction of Ca2+ desensitization (Fig. 4), an
indication that the acidosis-induced Ca2+
desensitization may have overcome the apparent shift due to
equilibration kinetics in the present study. UD-CG 212 Cl did not
affect the time course of Ca2+ transients, which
may have no influence on the equilibration kinetics of
Ca2+-troponin C binding during twitch contraction
in intact cardiac cells.
The present study was carried out in canine ventricular trabeculae.
Given the size of the muscles used, the temperature, and the
stimulation rate, it is not completely excluded that the core of the
muscle preparation could possibly be hypoxic. Thus, the core of the
muscle would have a raised inorganic phosphate, which has been shown to
alter the response to UD-CG 212 Cl (Westfall et al., 1993; van Meel et
al., 1995; Fraker et al., 1997). Therefore, it is likely that the cells
in the muscle core that contribute to the force response are responding
in a manner different from the surface cells, from which aequorin
signals were recorded. To get insight into this important issue we
compared the effects of levosimendan in indo-1-loaded rabbit
ventricular myocytes and aequorin-loaded papillary muscles (Sato et
al., 1998). Levosimendan elicited an identical
Ca2+-sensitizing effect on both preparations.
Furthermore, the response of aequorin-loaded rabbit and dog ventricular
trabeculae to the Ca2+ sensitizer OR-1896 was
very similar (Takahashi et al., 2000a,b). These observations altogether
imply that hypoxia in cells in the core of the muscle preparation used
may not have crucial influence on the findings in the current study.
In this study the acidotic solution contained a different
[Cl] than the control solution. Given that
Cl is involved in pH regulation, this could
complicate the interpretation of the data. Although this is an
important issue, the present study could not determine the role of
[Cl] in this respect. Nevertheless, it was
revealed that 1) the results obtained in the current study are
qualitatively very similar to those reported previously in the acidosis
induced by alteration of CO2 levels (Allen and
Orchard, 1983; Orchard and Kentish, 1990; Komukai et al., 1998); and 2)
acidosis elicited a differential effect on the action of Org-30029
(Watanabe et al., 1996) and UD-CG 212 Cl under the same experimental condition.
In conclusion, the increase in myofilament Ca2+
sensitivity induced by UD-CG 212 Cl, the active metabolite of
pimobendan, was abolished under acidotic condition in canine
ventricular myocardium. Such a modification of the positive inotropic
effect of cardiotonic agents that act through an increase in
myofilament Ca2+ sensitivity have to be taken
into consideration when these agents are applied under
pathophysiological condition.
We are grateful to Nippon Boehringer Ingelheim (Kawanishi,
Hyogo, Japan) for a generous supply of UD-CG 212 Cl.
This research was supported in part by grants-in-aid for
Scientific Research (B) (11557203) from the Ministry of Education, Science, Sports, and Culture, Japan.
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Abstract
Introduction
- and 
a cardiotonic pyridazinone which elevates cyclic AMP and prolongs the action potential in guinea-pig papillary muscle.
Naunyn-Schmiedeberg's Arch Pharmacol
325:
259-269[Medline].
