© 1995 American Heart Association, Inc.
Articles |
L-Methionine Augments Mammalian Myocardial Contraction by Sensitizing the Myofilament to Ca2+
From the Second Department of Internal Medicine, Toyama (Japan) Medical and Pharmaceutical University School of Medicine (Y.K., M.I.), and the Third Division, Department of Internal Medicine, Kyoto (Japan) University Faculty of Medicine (S.S.).
Correspondence to Yasuki Kihara, MD, PhD, Third Division, Department of Internal Medicine, Kyoto University Faculty of Medicine, 54 Shogoin Kawaharacho, Sakyo, Kyoto 606, Japan.
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Abstract |
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Abstract L-Methionine is an essential amino acid that has been reported to have a potent positive inotropic effect on the mammalian myocardium. We studied the mechanisms of the inotropic effect in ventricular myocardium from the rabbit. In the isolated coronary-perfused whole heart, L-methionine in a millimolar range exerted concentration-dependent positive inotropic effects on the isovolumic left ventricle, which were associated with negative lusitropic effects (prolonged time course of relaxation). The chronotropic state and the coronary perfusion pressure were not affected. These complex effects on the isolated whole heart were not blocked by pretreatment with (µmol/L) propranolol 1, prazosin 1, carbachol 3, staurosporine 1, or [Ser1,Ile8]angiotensin II 0.1. To further study the subcellular mechanisms, isolated ventricular papillary muscles from the same species were loaded with a bioluminescent indicator, aequorin, to monitor [Ca2+]i. In the presence of 3 mmol/L L-methionine, the isometric tension showed a similar combination of the positive inotropic and negative lusitropic effects as observed in the whole heart. In contrast, the simultaneously recorded intracellular Ca2+ signals did not increase in amplitude but instead decreased. The [Ca2+]i-tension relation shifted to the left compared with that obtained in response to [Ca2+]o. In saponin (250 µg/mL)–treated skinned preparations, 3 mmol/L L-methionine also shifted the force-pCa curve to the left by 0.16 pCa units. This is the first demonstration that an essential amino acid directly acts on the myofilaments and modulates their responsiveness to Ca2+, thereby producing a positive inotropic effect.
Key Words: essential amino acids • inotropic effects • Ca2+ • aequorin • rabbits
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-Methionine is an essential amino acid and has widespread use in parenteral alimentation and foods. This amino acid exerts a positive inotropic effect on isolated coronary perfused hearts from various mammalian species.1 2 These effects appear to be associated with increases in phospholipid N-methylation of cellular membranes1 2 ; hence, it has been assumed that the composition of the membrane lipid bilayer is a modulator of intracellular Ca2+ handling. We tested whether the positive inotropic effect of this amino acid is due to an increase in the amount of Ca2+ available for activation of the contractile apparatus secondary to a net increase in Ca2+ influx through the methylated cell membranes by measuring intracellular Ca2+ transients with the bioluminescent protein aequorin. In contrast to this assumption, we found that the inotropic effects were produced without increases in the amount of Ca2+ available for activation of the contractile apparatus. The time course of the Ca2+ transient tended to shorten, while the simultaneously measured isometric tension was significantly prolonged. These observations suggest that L-methionine modulates the Ca2+ sensitivity of the myofilaments and does not exert its effects on intracellular Ca2+ handling via membrane methylation. The direct action of L-methionine on the myofilaments was further confirmed by using skinned fibers from the same species. This is the first demonstration of an essential amino acid–induced modulation of the contractile apparatus and its responsiveness to Ca2+. The results suggest interactions of the myofilaments in intact myocardial cells with intracellular amino acids or peptides that are lost in skinned fiber preparations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heart and Muscle Preparations
New Zealand White rabbits (male, 2.0 to 2.5 kg) were anesthetized by intravenous injection of 3 mg/kg sodium pentobarbital. Hearts were quickly excised and mounted on a cannula inserted into the ascending aorta. Retrograde perfusion of the coronary arteries was established via a constant-flow pump (at a rate of 24 to 30 mL/min) with the coronary perfusion pressure initially set at 80 mm Hg as monitored with a Statham P23Db transducer.3 4 Hearts were perfused with an oxygenated Tyrode's solution of the following composition (mmol/L): NaCl 135, KCl 4, Na2HPO4 0.33, MgCl2 1.0, CaCl2 1.0, dextrose 10, and HEPES 10 (pH was adjusted to 7.4). The temperature of the solution and that in an organ bath surrounding the heart was maintained at 30°C. Isovolumic left ventricular (LV) pressure was recorded via an intraventricular balloon catheter inserted into the LV through the mitral valve and connected to another Statham transducer. The volume of the balloon was kept constant at an LV end-diastolic pressure of 10 mm Hg. The right atrium was not ligated, and the preparation was allowed to contract spontaneously. As described previously,3 4 the preparation retained a stable hemodynamic condition for over 2 hours. This implied that the ongoing tissue ischemia might not supervene in this experimental setting. The isovolumic LV pressure, coronary perfusion pressure, and the surface ECG were simultaneously recorded on a chart-strip recorder (model RJG, Nihon Koden Ltd) and a pulse-code modulation (PCM) videocassette recorder (DP16, Shoshin EM Co).
The right ventricular papillary muscles were isolated from the same strain of rabbits as described above. Muscles <0.3 mm in diameter were selected for skinned-fiber studies, and those <0.8 mm in diameter were used in the aequorin investigations.5 6 The base of each preparation was attached to a muscle holder; the other end was tied to a force transducer (BG-10, BG-1, Kulite Semiconductor Products, Inc) with an 8-0 Tevdek thread in a bath continuously perfused with Tyrode's solution at 30°C. The preparation was stimulated with a square-wave pulse of 1-millisecond duration at threshold voltage for 2 hours and at a frequency of 0.33 Hz. During the initial half of this equilibration period, the muscle length was repeatedly stretched to attain the maximal tension generation (Lmax).5 6
[Ca2+]i Monitored With
Aequorin
Muscles were loaded with the
Ca2+-regulated bioluminescent indicator aequorin by
a modified chemical-loading procedure (macroinjection), as described
elsewhere.4 6 7 8 Briefly, muscles were first exposed to
low-Ca2+ solution containing (mmol/L) NaCl 135, KCl
4, Na2HPO4 0.33, MgCl2 5,
CaCl2 0.1, dextrose 20, EDTA 0.1, and HEPES 10 (pH was
adjusted to 7.3) for 2 to 4 minutes at 25°C. Instead of immersing the
muscle into aequorin solution, 1 to 1.5 µL aequorin solution (1
mg/mL) was then pressure-injected into muscles just beneath the
endocardium through a glass micropipette. CaCl2 was
gradually increased to 1.0 mmol/L within 40 minutes, and the
temperature of the bath was slowly returned to 30°C. Finally, the
EDTA-containing perfusate was replaced with standard Tyrode's
solution. The light emitted by the Ca2+-aequorin
interaction was detected with a photomultiplier (R585, Hamamatsu
Photonics). The incident window of the cathode was directly attached to
a polished quartz cylinder (10 mm in diameter, 30 mm in length,
Fujitoku Co. Ltd).6 8 The opposite end of the quartz
cylinder was submerged in the bath solution to a level just above that
of the horizontally mounted preparation. The surface of this light
guide covered the entire upper surface of the preparation, which
minimized the motion artifacts.6 8 The minimum current
that can be reliably detected in this system is 
0.1 nA. The
percentage of successful signal detection included in the present
study was 75% (9 of 12 preparations). In successful preparations, the
aequorin signal did not show any signs of Ca2+
overload, even when the highest concentration of extracellular
Ca2+ was supplied. The light and force signals were
simultaneously recorded on chart-strip and PCM recorders. To delineate
changes in these signals, the series of signals at each steady state
condition was played back for averaging 16 to 64 times by an off-line
computer (Signal Processor 7T18A, NEC-Sanei Co).6 8
Skinning Procedure and Solutions
The thin papillary muscle was chemically skinned by exposure to
a solution containing 250 µg/mL saponin, 5 mmol/L K2ATP,
7 mmol/L MgCl2, 5 mmol/L EGTA, 60 mmol/L KCl, 60
mmol/L imidazole, 12 mmol/L creatine phosphate, and 15 U/mL creatine
phosphokinase (pH 7.1) at room temperature.9 10 This
concentration of saponin was used to remove the sarcoplasmic reticulum
(SR) membrane as well as the sarcolemma.9 The total salt
concentrations necessary to obtain the desired pCa, pMg, pMgATP, and pH
at a constant ionic strength were calculated by using software
(SPECS) described and supplied by Dr A.
Fabiato.11 The absolute stability constants used for
calculating the compositions of the solutions were as reported in the
accessory program (ABSTAPP).11 The solutions
were prepared at room temperature, with a pMg of 3.0, pMgATP of 2.5,
EGTA concentration of 10 mmol/L, ionic strength of 0.18 mol/L, and pH
of 7.1, which was adjusted by using 30 mmol/L BES. The solutions also
contained 12 mmol/L creatine phosphate and 15 U/mL creatine
phosphokinase. The skinned muscle was initially subjected to a
relaxation-activation cycle by using the method described by Moisescu
and Thieleczek.12 The relaxation solution had a pCa of
8.0, whereas the activation solution had a pCa of 4.5. During the
relaxation cycle, the muscle length was readjusted to the
Lmax at which an increase in resting tension was first
observed, as described by Maughan et al.13 The pCa of each
solution was obtained by replacing EGTA with CaEGTA.
L-Methionine was added to these buffer solutions of
different pCa values at a concentration of 3 mmol/L.
The tension-Ca2+ relations obtained in the skinned
preparations were fitted to the modified Hill relation:
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Chemicals
The following drugs and chemicals were used:
L-methionine (Wako Pure Chemicals);
DL-propranolol HCl, carbamylcholine HCl
(carbachol), prazosin HCl, and staurosporine (Sigma Chemical Co); and
[Ser1,Ile8]angiotensin II (Bachem
Inc). Aequorin was purchased from the laboratory of Dr J.R. Blinks,
Friday Harbor, Wash. Possible direct effects of
L-methionine on aequorin luminescence were tested in
vitro as described by Blinks et al.14 In brief, 300 µL
of the Ca2+-buffered solution with pCa of 6.0 and
pMg of 3.0 was placed in a cuvette of a light-tight and heat-controlled
aggregometer equipped with a photodiode array (FS-100, Kowa Co Ltd).
While the solution was continuously stirred at 250 rpm at 30°C, 20
µL of aequorin solution was added with a Hamilton microsyringe. At
steady state aequorin luminescence (this condition lasted >5 minutes
with a gradual decline by its consumption), 10 µL of 0.1 mol/L
L-methionine solution was slowly injected into the
solution with the microsyringe. L-Methionine injection,
however, did not change the level of aequorin luminescence nor modulate
the time course of aequorin consumption (data not shown). This result
indicated not only that L-methionine at this
concentration did not interact with aequorin in such a manner as to
interfere with its luminescence but also that
L-methionine did not modulate the Ca2+
buffer capacity of the solutions that we used for the skinned-muscle
study.
Data Analysis and Statistics
The results were analyzed with computer software
(STATISTICA/w, StatSoft) and presented as mean±SD.
The statistical significance of differences between data before and
after administration of 3 mmol/L L-methionine was
determined by Student's paired t test. Comparisons of data
from the cumulative L-methionine concentration effects
were analyzed by one-way repeated-measures ANOVA and multiple
comparisons with the Newman-Keuls test. Data from skinned fibers at
different pCa levels before and after L-methionine
administration were analyzed with two-way repeated-measures ANOVA. The
level of statistical significance was set at P<.05. Curve
fittings of the force-pCa relation to the Hill equation were assisted
by computer software (SIGMAPLOT 5.1/w,
Jandel Scientific Software).
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Results |
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Effects of L-Methionine on Isolated Coronary-Perfused Rabbit Hearts
We initially found that L-methionine in the concentration ranges reported previously (up to 600 µmol/L)1 2 exerted relatively modest hemodynamic effects under our experimental conditions. In contrast, L-methionine at millimolar concentrations exerted clear positive inotropic effects. The cumulative concentration effects of L-methionine on the peak LV pressure are shown in Fig 1
, top left. When 3 mmol/L of
L-methionine was added to the coronary perfusate (Fig 1,
top right), the peak LV pressure began to increase within 5 minutes.
The peak LV pressure continued to increase in a monophasic fashion and
reached a steady state after 20 minutes of perfusion. The inotropic
effect was associated with a small but persistent prolongation of the
pressure time course (Fig 1, bottom). Despite the inotropic effect and
changes in the pressure time course, the heart rate, coronary perfusion
pressure, and LV diastolic pressure showed no significant changes. The
grouped data from 11 hearts are presented in Table 1. Thus, L-methionine exerted positive
inotropic and negative lusitropic actions without affecting the
chronotropic state or the coronary vascular tone. These effects of
L-methionine on the LV contraction, coronary perfusion,
and chronotropic state were consistently observed even when the heart
was pretreated with (µmol/L) propranolol 1, prazosin 1,
carbachol 3,15 staurosporine 1, or
[Ser1,Ile8]angiotensin II (an
angiotensin II analogue16 ) 0.1. (These interventions were
examined separately for each drug, and the test was repeated in two
isolated hearts.)
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In five isolated hearts, the effects of L-methionine were further tested at 37°C. By this increase in temperature, the heart rate increased to 234±30 beats per minute, and the peak LV pressure decreased to 67±9 mm Hg. Administration of 3 mmol/L L-methionine augmented the LV pressure to 94±12 mm Hg (increased by 40%). Quantitatively, this positive inotropic effect was more remarkable than that observed at 30°C; however, even at this temperature, the inotropic effect was accompanied by negative lusitropic effects (time to peak LV pressure, from 122±16 to 128±19 milliseconds; time to 80% regression of LV pressure, from 140±25 to 151±23 milliseconds), and the chronotropic state was not affected (230±25 beats per minute).
By again replacing the coronary perfusate with that without
L-methionine, both the LV pressure development and the
pressure time course returned to their control levels by 30 minutes
(Fig 1
, top right and bottom left). The reversibility of these effects
suggested that L-methionine by itself, and not its
intracellular metabolites, might be primarily responsible for these
changes.
Effects of L-Methionine on Intracellular
Ca2+ Transients and Tension in Papillary Muscles
The subcellular mechanisms of the inotropic effects of
L-methionine were investigated in aequorin-loaded
papillary muscles in which intracellular Ca2+
transients and the corresponding tension tracings were simultaneously
recorded. As shown in the whole-heart study, L-methionine
at concentrations of 1 and 3 mmol/L increased the peak of developed
tension by 16% to 25% (Table 2). The increase in
tension induced by L-methionine perfusion was monophasic
and reached a steady state within 30 minutes. The resting tension was
not affected. The inotropic effect was also associated with a slight
but significant prolongation of the relaxation time course. These
positive inotropic and negative lusitropic effects of
L-methionine were reversed to the control conditions by
20 to 30 minutes after the solution was replaced with that not
containing L-methionine (Fig 2, top).
Besides these changes in isometric tension, the simultaneously measured
aequorin signals showed alterations in opposite directions (Fig 2,
top). The peak level of the intracellular Ca2+
transient decreased by 10.2% (P<.05) in the presence of 3
mmol/L L-methionine. This decrease in the aequorin signal
was not due to consumption of loaded aequorin, since the decay of peak
signals during the 30 minutes preceding L-methionine
perfusion was 4% at the most (n=9). Besides, the decrease by
L-methionine perfusion was reversed toward the control
level during the subsequent washout period (Fig 2, top). Thus, the
increases in peak tension mediated by L-methionine were
not accompanied by a corresponding increase in peak levels of
intracellular Ca2+ transients. This observation was
in contrast to the inotropic effects produced by increasing
[Ca2+]o of the solution to 2 mmol/L,
which was tested in the same muscle preparation (Fig 2, middle). The
time course of the aequorin signal was not affected by this increase in
[Ca2+]o. On the other hand, the
descending phase of the intracellular Ca2+ transient
during L-methionine perfusion tended to be abbreviated,
and the mean value of the time to 80% regression was slightly reduced
from 209 to 196 milliseconds (P=.15). The time courses of
the tension tracings and aequorin signals are presented in Fig 2,
bottom, by electronically adjusting their peak levels to an equal
magnitude. A comparison of the effects on the peak
[Ca2+]i–peak tension relation at 3
mmol/L L-methionine and those at 2 mmol/L
[Ca2+]o from grouped data is shown in
Fig 3, right. The effects of
[Ca2+]o on this relation and their
modulations in response to 3 mmol/L L-methionine from a
representative experiment are illustrated in Fig 3, left. At a
given [Ca2+]o,
L-methionine produced an increase in tension without an
increase in the amount of intracellular Ca2+
available for the contractile apparatus, which resulted in a leftward
shift of the relation. This shift of the
[Ca2+]i-tension relation together with
changes in the time courses suggested that with
L-methionine, myofilaments increased their affinity for
Ca2+ and produced more force without the aid of an
increase in the amount of Ca2+ supplied from
internal Ca2+ stores.5 17 18
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Effects of L-Methionine on the
Ca2+-Force Relation of Skinned-Muscle
Preparations
The aequorin study suggested an interaction of
L-methionine with the myofilaments. However, the
Ca2+-sensitization phenomenon observed in the
working preparation may be due to indirect effects, which include
intracellular alkalization by activated H+
extrusion.19 20 Moreover, it is not clear whether the
effects were caused by L-methionine itself or by its
intracellular metabolites, such as
S-adenosyl-L-methionine.1 2 21
Thus, the effects of L-methionine were further tested in
skinned-muscle preparations from the same animal. Fig 4,
left panel (left and middle tracings), shows activation-relaxation
cycles of a skinned-muscle preparation with or without 3 mmol/L
L-methionine in the bathing solution. In the right
tracing, the effects of L-methionine were tested when the
muscle was partially activated at a pCa of 6.0. With
L-methionine, the force generated by muscle significantly
increased over the range of pCa between 5.5 and 7.0 in a reversible
manner. In contrast, the resting tone (pCa 8.0) and the maximal
activated force (pCa 4.5) were not affected by the presence of
L-methionine. Data from eight muscles were fitted to the
Hill equation, and the curves are shown in Fig 4, right panel. With
3 mmol/L L-methionine, the
[Ca2+]1/2 was shifted to the
left by a pCa value of 0.16 (5.89 versus 6.05, P=.0189),
whereas the cooperativity factor (n) was not affected (1.31 versus
1.34, P=.7630). The results indicate that the positive
inotropic and negative lusitropic effects of L-methionine
are due at least in part to a direct interaction of the amino acid with
the contractile proteins, which results in an alteration of
responsiveness to Ca2+.
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indicates control;
, 3 mmol/L L-Me), there is a significant difference
(P=.0037, two-way ANOVA followed by Newman-Keuls test). A
vertical bar attached to each circle indicates the SD. With 3 mmol/L
L-Me, the [Ca2+] value yielding 50% tension
activation [Ca2+]1/2 was shifted to
the left by 0.16 pCa (5.89 vs 6.05, P=.0189 by paired
t test), whereas the cooperativity factor (n) was not
affected (1.31 vs 1.34, P=.7630). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we found that L-methionine alters the responsiveness of myofilaments so that they require less Ca2+ for equivalent force generation. The results from aequorin-loaded intact myocardium and from skinned preparations were consistent. This sensitization effect may also explain the tension prolongation that occurred along with the inotropic effects in the whole heart and in papillary muscle preparations.5 15 16 In contrast, L-methionine did not increase the magnitude of intracellular Ca2+ transients during muscle contraction. Thus, as for the inotropic mechanisms, it is unlikely that membrane methylation by metabolites of this amino acid modulates the Ca2+ transport systems of the cell membrane to increase the amount of Ca2+ for activation of the contractile proteins.1 2 We believe that this is the first demonstration of essential amino acid–mediated modulation of myofilaments and of its effects on the contractile state of the ventricular myocardium.
As reported previously in studies using isolated coronary-perfused
whole-heart preparations,1 2 we observed a positive
inotropic effect in the presence of L-methionine. At the
same time, we found that the inotropic effect was accompanied by a
prolonged time course of isovolumic LV contraction, although neither
the chronotropic state nor coronary vascular tone was altered. This set
of hemodynamic changes with L-methionine is inconsistent
with the known effects of positive inotropic drugs, which include those
mediated by 
- and ß-adrenergic receptors, cAMP, and
Ca2+. ß-Adrenergic stimulation and cAMP would be
expected to shorten the tension time course and induce a positive
chronotropic action.17 22 23 Agents that simply increase
[Ca2+]i, including elevated
[Ca2+]o and the cardiotonic steroids,
would not be expected to affect the time course.24 25
-Adrenergic stimulation prolongs the time course of
contraction.17 18 However, -agonists would also be
expected to exert a vasoconstrictive effect on the coronary
vasculature. Pretreatment with prazosin did not block the effects of
the amino acid. Moreover, neither an angiotensin II antagonist
([Ser1,Ile8]angiotensin II) nor an
inhibitor of protein kinase C (staurosporine) affected the inotropic
response. These observations suggested that the actions of
L-methionine were mediated by mechanisms different from
those governed through these representative membrane receptors
and their intracellular second messengers.
It has been reported that
S-adenosyl-L-methionine, an intracellular
metabolite of L-methionine, induces successive
methylation of phosphatidylethanolamine into
phosphatidylcholine.1 2 21 Phospholipid methylation
modulates the functional state of several membrane proteins, which
include ß-adrenergic receptors,26
Ca2+ pumps of the sarcolemma2 27 and of
the SR,2 28 and the
Na+-Ca2+ exchanger.2
Enhancement of sarcolemmal Ca2+ channel activity has
also been suggested in smooth muscles29 and in the
diaphragm,30 although a similar response has not been
confirmed in cardiac muscle preparations. Since such diverse actions of
phospholipid methylation might modulate both the transmembrane
Ca2+ influx and efflux simultaneously but at
different magnitudes, global changes in the level of membrane
methylation may not show a simple correlation with the subsequent
changes in the level of [Ca2+]i.
Measurements of transmembrane Ca2+ efflux in vivo
during L-methionine perfusion2 may provide
some information. However, the results should be carefully
interpreted, because each subcellular function in such a system might
also be affected by activities of other subcellular factors, which
include [Ca2+]i. A more direct means
of addressing this issue is to document intracellular
Ca2+ transients in a contracting ventricular muscle
during L-methionine perfusion. Our aequorin study showed
that with L-methionine perfusion, the amplitude of the
intracellular Ca2+ transient decreases and the time
course tends to be shortened. This decrease in intracellular
Ca2+ amplitude could be primarily caused by
activation of the sarcolemmal and SR Ca2+ pumps,
namely, a relative increase in Ca2+ efflux as a net
effect of membrane methylation. However, this change in
[Ca2+]i by itself could not explain
the positive inotropic effects. Alternatively, Ca2+
sensitization of myofilaments as shown in the peak
[Ca2+]i–peak tension relation (Fig 3)
might produce similar changes in the intracellular
Ca2+ transient. With an increase in myofilament
sensitivity to Ca2+, the apparent peak of
intracellular Ca2+ transient might decrease without
an actual decrease in the amount of Ca2+ released
from intracellular storage sites, because of the more rapid and
effective trapping of Ca2+ from the cytosolic
space.18 The intracellular Ca2+
transient tended to be shortened, which was also expected to occur in
the presence of more powerful Ca2+ buffers. Thus,
these observations regarding intracellular Ca2+
transients in the working myocardium are consistent with other
observations in the present study. The apparent decrease in peak
[Ca2+]i might be due at least in part
to such effects. In the presence of these sensitization effects, our
aequorin study could not conclusively determine the role of membrane
methylation by L-methionine on
[Ca2+]i. However, it is unlikely that
an increase in the transsarcolemmal Ca2+ influx was
the primary cause of the inotropic effect during
L-methionine perfusion. Methylation of the membrane
Ca2+ transporters may favor extrusion of
Ca2+ from the cytosolic space or may cancel each
other to maintain homeostasis over
[Ca2+]i. We also recorded the surface
action potentials of some aequorin-loaded papillary muscles (n=5) by
using the standard glass microelectrode technique (3 mol/L KCl in the
pipette, 15- to 20-M
tip resistance in the control solution).
However, we found no detectable changes in the action potential
duration, the resting membrane potential, or the peak depolarization
level during 3 mmol/L L-methionine perfusion (data not
shown). This observation adds supplemental evidence against the
sarcolemma as the primary source of the inotropic effects.
The skinned-muscle study demonstrated a direct interaction of L-methionine with the contractile proteins. The force-pCa curve showed a parallel leftward shift without changes in the relaxation (pCa 8.0) or maximal activation (pCa 4.5) levels of force. Although Ca2+ binding sites are exclusively located in troponin C in the range of pCa >5,31 32 such a shift in force-pCa relation may occur in several ways. The possible sites of L-methionine interaction include the Ca2+-binding sites on troponin C,32 33 the phosphorylation site of troponin I, binding sites of troponin I which affect the interaction between troponin I and troponin C, or the troponin T–tropomyosin complex, as suggested by the DPI 201-106–mediated leftward shift of the force-pCa curve in the failing human myocardium.10 Despite these possibilities, the interaction site of L-methionine with the contractile protein was not elucidated in the present study. This determination requires further molecular-based studies. However, L-methionine may be useful as a tool with which to uncover additional regulatory mechanisms of contractile proteins.
Little is known about the membrane transport of essential amino acids
in the mammalian myocardium. In several cell lines from the liver,
brain, and kidney, it has been demonstrated that active amino acid
transport systems, part of which are dependent on the
[Na+]i (Na+ cotransport system),
are working.34 35 In cardiac myocytes, taurine, an amino
acid that is not incorporated into proteins, was reported to be taken
up through this transport mechanism and to reach concentrations as high
as 10 to 30 mmol/L in the cytosol, whereas the plasma concentration was
60 µmol/L. The intracellular level was subjected to change by
interventions such as transient ischemia, low-Na+
perfusion, or
low-Ca2+/low-Mg2+
perfusion.36 Thus, it is reasonable to assume the presence
of such membrane transport systems for essential amino acids, enabling
accumulation of these amino acids into the cell against gradients
across the membrane. Although we did not examine this systematically in
the present study, we felt that during the isolated whole-heart
experiments, when more time had been consumed for stabilizing the
preparation before the initiation of protocols, more remarkable
positive inotropic effects by L-methionine subsequently
emerged. This may suggest the preexistence of an
L-methionine pool in the cytosol, which might be affected
by the time-dependent protein metabolism and/or the reverse
cotransportation of the amino acids out of the cell. Since the
L-methionine concentrations in the perfusate that we
examined in this study were >20 times higher than the plasma level in
humans (30 to 60 µmol/L),37 the physiological as well as
the clinical roles of L-methionine as a modulator of the
inotropic state remain to be clarified. However, if such a cytosolic
pool of this amino acid was present in the intact myocardium, the
level of extracellular concentration per se may not directly regulate
the inotropic effects.
The force-Ca2+ relations estimated in the intact myocardium are known to lie to the left of those obtained in skinned myocardial preparations.4 22 38 This difference does not appear to be artifactual.4 38 It has been assumed that there could be modulatory substances in the cytosol that were removed during the skinning process.39 Results of our skinned-muscle study imply that with a supply of an essential amino acid that might be abundantly present in the cytosol, the force-pCa relation was partially shifted leftward, toward that in intact cardiac cells. Determination of the actual role of L-methionine in the regulation of force-pCa relations in physiologically intact cells awaits further studies. However, our results demonstrated the presence of a putative cytosolic substance that modulates force generation of the contractile apparatus at a given [Ca2+]i.
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Acknowledgments |
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This study was supported in part by the Japan Heart Foundation, Tamura Foundation, Yokoyama Foundation, and grants-in-aid from the Ministry of Education, Culture, and Science of Japan (Nos. 02770482 and 03770485) to Dr Kihara. We thank Isao Kamae, MD, DPH, for assistance in statistical analysis.
Received September 6, 1994; accepted March 6, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wexler LF, Weinberg EO, Ingwall JS, Apstein CS.
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