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1-AR-induced positive inotropic
response in heart is dependent on myosin light chain
phosphorylation
1 Department of Pharmacology, 2 Merck Sharp & Dohme Cardiovascular Research Center, and 3 Department of Cardiology, Rikshospitalet University Hospital, 4 Department of Internal Medicine, Ullerål University Hospital, University of Oslo, N-0316 Oslo, Norway
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The possible
involvement of different kinases in the 
1-adrenoreceptor
(AR)-mediated positive inotropic effect (PIE) was investigated in rat
papillary muscle and compared with 
-AR-, endothelin receptor- and
phorbol ester-induced changes in contractility. The
1-AR-induced PIE was not reduced by the inhibitors of
protein kinase C (PKC), MAPK (ERK and p38), phosphatidyl inositol
3-kinase, or calmodulin kinase II. However, PKC inhibition attenuated
the effect of phorbol 12-myristate 13-acetate (PMA) on contractility.
1-AR-induced PIE was reduced by ~90% during
inhibition of myosin light chain kinase (MLCK) by
1-(5-chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine (ML-9). Endothelin-induced PIE was also reduced by ML-9, but ML-9 had no effect on -AR-induced PIE. The Rho kinase inhibitor Y-27632 also reduced the 1-AR-induced PIE. The
1-AR-induced PIE in muscle strips from explanted failing
human hearts was also sensitive to MLCK inhibition. 1-AR
induced a modest increase in 32P incorporation into myosin
light chain in isolated rat cardiomyocytes. This effect was eliminated
by ML-9. The PIE of 1-AR stimulation seems to be
dependent on MLCK phosphorylation.
endothelin; inhibitors; 1-(5-chloronaphthalene-1-sulfonyl)1H- hexahydro-1,4-diazepine; phenylephrine; Rho kinase; -adrenoreceptor
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CATECHOLAMINES INDUCE
their effects in the heart by activation both of 1- and
-adrenoreceptors (AR) (7, 19). -ARs are responsible
for the main part of the catecholamine-induced positive inotropic
effect (PIE), but stimulation of 1-AR elicits a PIE in
most species, including failing human hearts (30). In the
rat heart this response is triphasic, consisting of a small initial
positive component, followed by a transient negative inotropic effect,
and finally a sustained PIE. Unlike the -AR-mediated response, the
mechanisms involved in the 1-AR-mediated response are
independent on cAMP (19). The 1-ARs are
Gq-coupled receptors and activation of the receptors leads to
activation of phospholipase C
and hydrolysis of
phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate
and diacylglycerol (33). Increased diacylglycerol leads to
activation of protein kinase C (PKC), which mediates a variety of
effects in the heart (24).
The mechanisms underlying the sustained PIE of 1-AR
stimulation have not been clearly elucidated so far. Activation of the 1-ARs leads to only a modest increase in systolic
intracellular [Ca2+] (13), whereas increased
myofilament Ca2+ sensitivity after 1-AR
stimulation has repeatedly been shown (4, 22, 33). This
increase in sensitivity to Ca2+ has been explained by an
intracellular alkalinization (15) and/or by increased
phosphorylation of the myofilaments (23, 33). PKC has been
proposed to be involved in the PIE of 1-AR stimulation,
but the data have been inconclusive so far (11, 12, 34).
Studies (17) with more selective PKC inhibitors are warranted.
The effect of catecholamines on smooth muscle contraction mediated by
1-AR stimulation is increased contractility induced by
activating the Ca2+-calmodulin-dependent myosin light chain
kinase (MLCK), which phosphorylates myosin light chain-2 (MLC-2,
regulatory MLC), resulting in contraction (32). MLC-2 is
dephosphorylated by myosin phosphatase, which is inactivated by
Rho-associated kinase (36). Thus, in smooth muscle, MLCK
and Rho-kinase act in concert to increase phosphorylation of MLC-2.
MLC-2 is phosphorylated by MLCK also in the heart (18),
but the role of MLC-2 phosphorylation is unclear in the heart muscle,
which, unlike smooth muscle, mainly depends on the troponin complex for
regulation of contraction.
It has been shown that in skinned fibers from rat hearts, the addition of MLCK induces phosphorylation of MLC-2 and increases myofilament sensitivity to Ca2+ (8, 18). The addition of PKC in combination with MLCK increases Ca2+ sensitivity in skinned fibers, but it is controversial whether or not this effect of PKC is mediated by MLC-2 phosphorylation (8, 38).
The direct involvement of MLCK in the inotropic response to
1-AR stimulation has not, however, been elucidated so
far in an intact phasic contracting muscle. The studies (8,
18) on myofilament sensitivity to Ca2+ were done in
tonically contracting fibers without a plasma membrane and with the
addition of exogenous MLCK.
The main purpose of this study was to elucidate a possible involvement
of PKC, MLCK, and Rho-kinase in 1-AR-mediated PIE in rat
papillary muscle. Selective inhibitors were used to study the
involvement of the different kinases in the PIE. We also included experiments with inhibitors of other kinases that are activated by
1-AR stimulation.
To compare the effects of kinase inhibition on inotropic
responses induced by stimulation of receptors with a similar
[endothelin (ET) receptors] and a totally different (-ARs)
coupling to intracellular signal transduction pathways, experiments
using ET-1 and isoproterenol were included. A new 1A-AR
subtype-selective agonist was also used to study the involvement of
this receptor subtype in the response. Additional experiments were
performed on trabeculae from human explanted hearts. The results show
that the 1-AR-mediated PIE in the heart is dependent on
phosphorylation of MLC-2.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
The animals were cared for according to the Norwegian Animal Welfare Act, which conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). The animals were housed in a temperature-regulated room with a 12:12-h day/night cycle.Preparation of Rat Papillary Muscle
Rat heart papillary muscles were isolated as described previously with minor modifications (6). During coronary perfusion with the physiological salt solution mentioned below, left ventricular papillary muscles were excised and mounted in an organ bath with 18 ml of a slightly different physiological salt solution. During coronary perfusion, the solution contained the following (in mmol/l): 118.3 NaCl, 3.0 KCl, 0.5 CaCl2, 4.0 MgSO4, 2.4 KH2PO4, 24.9 NaHCO3, 2.2 mannitol, and 10.0 glucose. The muscle strips were mounted in four organ baths containing (in mmol/l) 119.3 NaCl, 3.0 KCl, 2.4 KH2PO4, 1.2 MgSO4, 2.0 CaCl2, 24.9 NaHCO3, 2.2 mannitol, and 10.0 glucose. Both solutions were equilibrated with 95% O2-5% CO2 at 31°C (pH 7.4). The muscles were driven electrically (field stimulation) at a frequency of 1 Hz with impulses of 5 ms duration and current ~20% above individual threshold (10-15 mA, determined in each experiment) and stretched to the maximum of the length-force relationship. Some control experiments were also performed at 0.3 Hz. Force was recorded as described previously (31). The direct current signals were analog-to-digital converted, logged, and stored in LabVIEW software data files. Areas representative for the control (basal) period and the periods with agonist stimulation could be selected to calculate averaged contraction-relaxation cycles that were representative for these periods. These cycles were then used to determine descriptive parameters such as maximal developed force, maximal development of force (dF/dtmax), time to peak force (TPF) and time to relaxation at the 20% level (TR20). Relaxation time (RT) was calculated as TR20
TPF, and
dF/dtmax was used as an index of contractility.
Changes in contractility caused by kinase inhibitors or receptor
agonists were expressed as changes in dF/dtmax.
Changes in the relaxation phase were expressed as changes in RT.
Preparation of Human Ventricular Trabeculae
The experimental protocol was approved by the local ethics committee, and the experiments were performed according to the institutional rules.Ventricular myocardium was obtained from heart transplant recipients immediately after explantation of the failing heart as described in detail previously (30). Ventricular strips from one unused normal donor heart and two failing explanted hearts were studied. One of the explanted hearts was failing due to ischemic heart disease, and the other one was failing due to nonischemic dilated cardiomyopathy. Both patients had severe heart failure symptoms in accordance with New York Heart Association functional class III-IV. When the tissue was being prepared, the surface was kept wet with physiological saline. Thin trabeculae were localized in the ventricular cavities, cut free, and placed in a relaxing oxygenated (95% O2-5% CO2) physiological salt solution at room temperature. To prevent contracture during transportation and further preparation, we used a Ca2+/Mg2+ concentration ratio of 1:8 comparable to that of St. Thomas' Hospital cardioplegic solution. The solution contained (in mmol/l) 118.3 NaCl, 3.0 KCl, 0.5 CaCl2, 4.0 MgSO4, 2.4 KH2PO4, 24.9 NaHCO3, 10.0 glucose, and 2.2 mannitol. The tissue was transported to the laboratory and while being immersed in this solution, muscle strips (~1 mm in diameter, 8-10 mm long, endocardium as intact as possible) were prepared. The muscle strips were mounted in organ baths containing the same oxygenated solution at 37°C, except the Ca2+ was 2.5 mmol/l and Mg2+ was 1.2 mmol/l. The muscles were driven electrically, and force was recorded in the same way as described above for the papillary muscle preparation from the rat hearts.
Experimental Design
The papillary muscles were allowed to equilibrate for 60 min before the experiments started. Basal force was 525 ± 198 mg (means ± SD, representative sample with n = 19). Kinase inhibitors, when used, were added to the muscles 20-45 min before the agonist to allow the study of the effect of the inhibitors on the basal contractility and to obtain a sufficient equilibration time. The muscles in the concentration-response study groups were exposed to cumulatively increasing concentrations of the1-AR agonist phenylephrine in the presence or absence of
inhibitors until the maximal response was obtained to evaluate the
effect of the inhibitors on the concentration-response relationship to
1-AR stimulation. The effect of the kinase inhibitors on
the inotropic response to phenylephrine was also evaluated by comparing
the time course of the inotropic response in the presence or absence of
inhibitors and, in separate experiments by adding the kinase inhibitors
after the maximal response to phenylephrine was achieved (reversal
experiments). When ET, isoproterenol, and A-61603 were used as
agonists, only experiments comparing the time course of the inotropic
response in the presence or absence of inhibitors and reversal
experiments were performed.
The ET experiments were performed in the presence of 1 µmol/l of timolol and prazosin, A-61603, and phenylephrine experiments in the presence of 1 µmol/l timolol and isoprenaline experiments in the presence of 1 µmol/l prazosin. Atropine (1 µmol/l) and ascorbate (100 µmol/l) were present in all experiments. Separate control groups with phenylephrine alone were used for each of the intervention groups with kinase inhibitors.
Isolation and Labeling of Cardiomyocytes
Ventricular cardiomyocytes were isolated from adult rat hearts by the collagenase perfusion method described previously (39). The cardiomyocytes were purified by repeated centrifugation (45 g) and then sedimented by gravity 3-4 cm through a solution containing 1% bovine serum albumin. Cardiomyocytes were plated on laminin-coated Corning dishes (100 mm × 20 mm) and incubated overnight. The final cell population contained >95% elongated cardiomyocytes.MLC-2 Phosphorylation Experiments
Phosphorylation of MLC-2 was measured in isolated cardiomyocytes to avoid the contribution from other cell types in the heart to the response. The cells were incubated for 3 h in phosphate-free minimal essential medium containing [32P]P (20 µCi/ml). After washout of extracellular [32P]P, the cells were incubated for 15 min with the appropriate inhibitors and then for 10 min with phenylephrine. At the end of the incubation period, the dishes were washed with physiological saline and the cells were scraped off the dishes in a SDS sample buffer containing phosphatase and protease inhibitors (Complete, Roche Diagnostics; Mannheim, Germany). Protein concentration was determined with bicinchoninic acid assay, and equal amounts of protein were loaded onto SDS-PAGE (15%). Incorporation of the 32P label into protein bands was quantified by using an Instant Imager (Hewlett-Packard; Meriden, CT), which measures radioactivity directly in the gel. Parallel gels were subjected to immunoblotting with a monoclonal antibody against MLC-2 to verify the correct band on the gels.Calculations and Statistics
The values after agonist responses were generally expressed as a percentage of the control period (100%) before the addition of kinase inhibitors. With the exception of 1-(5-chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine (ML-9), none of the inhibitors influenced basal contractility. In the presence of ML-9, the contractility stabilized at a new and lower steady-state level during the equilibration period. Responses to agonists were calculated as differences between the steady-state contractility level after and before addition of agonists and expressed as stated above. This calculation method avoided any bias in evaluating the effects of agonists due to changes in basal contractility.Responses to phenylephrine in the presence of the different kinase
inhibitors were compared with and statistically evaluated against
responses to phenylephrine in separate control groups in the absence of
inhibitors. For convenience, the data from all the control groups
(n = 9) in the absence of inhibitors were combined and
are presented in the text and Table 1 as
phenylephrine in the absence of inhibitors representing a total number
of 72 experiments. When appropriate, the maximal responses to
phenylephrine in each of the kinase inhibitor groups were adjusted
according to this representative value. The apparent affinity
(pD2) values for phenylephrine in each of the kinase
inhibitor groups were compared with and expressed as shifts relative to
the relevant control group. The concentration-response curves
obtained from the papillary muscle experiment were constructed
according to Ariëns et al. (3) by estimating
centiles (EC10 to EC100) for each single
experiment and calculating the corresponding means. This calculation
provides mean curves that express the response as a fractional response or the percentage of maximum and display correct horizontal positioning and mean slopes of the curves. Horizontal positioning of the curves was
expressed by pD2 values (pD2 = log
EC50).
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The significance levels of differences were expressed by calculating
P according to Student's one-sample test or two-sample test
as appropriate. A value of P 
0.05 was considered to
reflect significant differences.
Drugs
32P (carrier free) was purchased from Amersham Pharmacia Biotech. Bisindolylmaleimide I (BIM), LY-294002, and SB-203580 were purchased from Calbiochem-Novabiochem (San Diego, CA). ML-9 was also purchased from Sigma (St. Louis, MO), together with phenylephrine hydrochloride, timolol, ET-1, l-isoproterenol hemisulfate, atropine, L-ascorbic acid, and phorbol 12-myristate 13-acetate (PMA). N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxy-benzenesulfonamide (KN-63) was purchased from from Biomol Research Laboratories (32a). A-61603 hydrobromide was purchased from Tocris. Monoclonal antibody to MLC-2 was purchased from Biocytex (Marseille, France). Y-27632 was a kind gift from Welfide (Osaka, Japan).Stock solutions were prepared in double-distilled water, ethanol, or
dimethyl sulfoxide and kept at 20°. When dimethyl sulfoxide or
ethanol was used to solve the inhibitor or agonist, appropriate control
experiments including the solvent were included. Further dilutions of
the drugs were made fresh daily and kept cool (0-4°) and dark.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1-AR-Induced PIE
-AR-antagonist), 1-AR stimulation
by phenylephrine induced a triphasic PIE, as shown before
(20). Both concentration-response curves and time curves
were constructed from the experiments. The maximal sustained PIE of
phenylephrine was a 37 ± 1.4% increase above control (means ± SE of all control experiments, n = 72, P < 0.0001). The transient negative inotropic
component of the phenylephrine response observed in time course
experiments amounted to a 17 ± 1.7% decrease below control value
(n = 10, P < 0.001). A representative
example of signal-averaged contraction-relaxation cycles is shown in
Fig. 1A. Concentration-response experiments showed that
phenylephrine induced a PIE with a pD2 value (log EC50) of 5.2 ± 0.05 (n = 21) (Fig.
1B).
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Effects of Inhibitors of PKC on 1-AR-Induced PIE
1-AR-mediated PIE is not mediated by
PKC activation.
Effect of PKC Inhibition on PMA-Induced Changes in Contractility
The effect of the PKC activator PMA on contractility in the absence or presence of BIM was studied to verify the ability of BIM to inhibit effects of PKC activation in our experimental system. PMA (100 nmol/l) induced a 22 ± 4.6% reduction (n = 8, P < 0.01) below control contractility after 30-min exposure (Fig. 2A). PMA reduced the contractility by only 4 ± 1.1% (n = 6, P < 0.01 vs. in the absence of BIM) when BIM (2 µmol/l) was added 20 min before the addition of PMA (Fig. 2B). The sustained PIE of phenylephrine was not influenced by PMA treatment [37 ± 2.7% increase above control, n = 4; not significant (NS) vs. in the absence of PMA].
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Effects of MLCK Inhibitors on 1-AR-Induced PIE and
Basal Contractility
Effect of ML-9 on 1-AR-induced PIE.
In the presence of the MLCK inhibitor ML-9 (10 and 50 µmol/l,
respectively), the sustained PIEs of phenylephrine (30 µmol/l) were
25 ± 1.8% (n = 4) and 4 ± 1.8%
(n = 6, P < 0.0001 vs. phenylephrine without ML-9) above control, respectively (Table 1). A representative example is shown in Fig. 3A,
and the data are summarized in Table 1 and Fig. 3B. The
presence of ML-9 had no effect on the transient negative inotropic
component of the response observed in time-course experiments: 15 ± 2.0% (n = 6) and 15 ± 5.1%
(n = 6) reduction below control in the absence and
presence of ML-9, respectively. Separate concentration-response
experiments were performed, and the maximal PIE of phenylephrine in
these experiments was 40 ± 3.9% (n = 5) and
8 ± 0.9% (n = 5, P < 0.0001)
above control, in the absence and presence of ML-9 (50 µmol/l),
respectively (Fig. 3C), with no effect of ML-9 on the
pD2 value (Fig. 3D and Table 1). When ML-9 (50 µmol/l) was added after phenylephrine, the PIE induced by
phenylephrine was reversed to 7 ± 3.6% (n = 6, P < 0.001) above control (Fig.
4A).
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Effect of ML-9 on basal contractility and on
1-AR-induced PIE at reduced stimulation frequency.
Addition of ML-9 (50 µmol/l) influenced also the basal contractility
at 1 Hz. The basal force development was reduced by 29 ± 4.6%
(n = 10, P < 0.0001), and the
relaxation time (RT) was increased by 41 ± 4.7%
(n = 6, P < 0.001). The prolonged RT
resulted in a cumulative increase in diastolic tension depending on the stimulation frequency. At 0.3 Hz the stimulus interval allowed the
ML-9-treated muscles to relax completely, thus giving no effect on
diastolic tension. To avoid the effect of ML-9 (50 µmol/l) on the
diastolic tension, its effect on 1-AR-induced PIE was also studied at 0.3 Hz. These experiments were done to ensure that the
inhibitory effect of ML-9 was not due to a nonspecific increase in
diastolic stiffness. At 0.3 Hz, the PIE of phenylephrine was 37 ± 6.9% (n = 4) and 4 ± 5.3% (n = 4) above control, in the absence or presence of ML-9, respectively
(P < 0.01). When ML-9 was given after phenylephrine,
the response was reversed to 8 ± 4.4% above control
(n = 4, P < 0.001).
Effects of MLCK Inhibition on 1A-AR-induced PIE
1A-AR agonist A-61603 induced a triphasic
response similar to that of phenylephrine. The sustained PIE of A-61603
(10 nmol/l) was 25 ± 2.7% above control (n = 6, P < 0.01), and this response was reduced to 5 ± 1.7% above control (n = 8) in the presence of 50 µmol/l ML-9 (P < 0.0001 vs. without ML-9). When ML-9
was added at steady-state response to A-61603, the response was
reversed to 1 ± 0.8% above control (n = 6, P < 0.001) (Fig. 4B). The transient
negative inotropic component of the response to A-61603 was not
influenced by ML-9 (5 ± 1.6%, n = 6, and 8 ± 0.9%, n = 8, below control without or with ML-9,
respectively). At 100 nmol/l of A-61603, the PIE was 57 ± 7.7%
(n = 6) and 20 ± 6.8% (n = 6)
above control without or with ML-9, respectively (P < 0.01).
Effects of MLCK Inhibition on 1-AR-induced PIE
In Human Explanted Hearts
1-AR-induced
PIE in isolated ventricular strips from human explanted hearts were
studied in four trabeculae from one normal donor heart with the use of norepinephrine (100 µmol/l) as an 1-AR agonist and in
eight trabeculae from two different explanted hearts with terminal
heart failure with the use of phenylephrine as the agonist. The
experiments were done in the presence of 6 µmol/l timolol. The PIE of
phenylephrine and norepinephrine was 60 ± 11.6%
(n = 4, P < 0.01) and 94 ± 21.0% (n = 2) above control, respectively (NS,
phenylephrine vs. norepinephrine) (Fig.
5). The presence of ML-9 reduced the
response by ~50% to 27 ± 8.5% (n = 4, P < 0.05) and 47 ± 2.0% above control
(n = 2) after phenylephrine and norepinephrine
stimulation, respectively.
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Effect of Rho-Associated Kinase Inhibition on
1-AR-induced PIE
1-AR-mediated PIE was 34 ± 4.4%
(n = 8) and 22 ± 3.0% (n = 8)
above control (P < 0.05) in the absence and presence
of Y-27632 (50 µmol/l) added 30 min before the agonist, respectively
(Table 1 and Fig. 6). When Y-27632 was
added after the agonist, the PIE of phenylephrine was reversed to
5 ± 2.3% above control (n = 6, P < 0.001) (Fig. 6).
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Effects of Inhibitors of MAPK Family on
1-AR-Induced PIE
Effects of Inhibitors of
Ca2+-Calmodulin-Dependent Kinase II and
Phosphatidyl Inositol 3-Kinase on 1-AR-Induced PIE
Effect of MLCK Inhibition on -AR-Induced PIE
-AR stimulation was studied. These experiments were also
done to decide whether inhibition by ML-9 was due to a nonspecific depression of the muscle, making it nonresponsive to agonist
stimulation in general. Isoproterenol was given in the presence of 0.1 µmol/l prazosin (1-AR antagonist). Maximal PIE of
isoproterenol (10 µmol/l) stimulation was 118 ± 6.4% above
control (n = 16, P < 0.01). ML-9 (50 µmol/l) had no effect on isoproterenol-induced PIE: 118 ± 14.0% above control (n = 6, NS vs. without ML-9) (Fig. 3B). ML-9 had no significant effect on isoproterenol-induced
PIE when added after isoproterenol in an attempt to reverse the
response (6 ± 2.7% reduction of maximal response,
n = 5, NS). A representative experiment is shown in
Fig. 4C. ML-9 had the same effect on basal contractility in
these experiments as in the phenylephrine experiments (data not shown).
Effect of MLCK Inhibition on ET Receptor-Induced PIE
ET-1 (50 nmol/l) induced PIE was 33 ± 6.1% (n = 6) and 8 ± 2.9% (n = 6, P < 0.01) above control in the absence and presence of ML-9, respectively (Fig. 3B). When ML-9 was added after the agonist, the PIE of ET-1 was reversed to 5 ± 2.5% (n = 6, P < 0.001) above control (Fig. 4D).MLC-2 Phosphorylation Levels After 1-AR
Stimulation
1-AR stimulation (data
not shown). Phosphatase inhibition by calyculin A (0.1 µmol/l) increased the phosphorylation level of MLC-2 by 90 ± 7.5% above control (n = 3, P < 0.01).
1-AR-induced increase in MLC-2 phosphorylation was
146 ± 21.2% above control (n = 3, P < 0.05) in the presence of calyculin A.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The novel findings of this study are that the mechanism for the
1-AR-mediated PIE in the adult rat heart is dependent on MLCK and to a lesser extent Rho kinase and is apparently independent on
PKC. 1-AR-induced PIE in the human heart was also
sensitive to MLCK inhibition. The present results are also to our
knowledge the first demonstration of the effect of the MLCK inhibitor
ML-9, the Rho kinase inhibitor Y-27362, and the selective
1A-AR agonist A-61603 on contractility in the rat heart.
The present results indicate that the sustained PIE induced by
1-AR stimulation is sensitive to an inhibitor of MLCK,
whereas the transient negative inotropic response is not. The results indicate an important role of MLC-2 phosphorylation in
1-AR-induced PIE. The results were mainly obtained by
the use of ML-9, which has been shown to be a selective inhibitor of
MLCK (25). For interpretation of these results it is
essential to consider the selectivety of ML-9. ML-9 inhibits MLCK at
10-15 times lower concentration than PKA and PKC. Although a
potential inhibition by ML-9 of PKC and PKA is possible at the
concentrations used in the study, it is unlikely that such effects
contributed to the influence of the inhibitor because of the following:
1) all effects of 1-AR stimulation reported
so far have been shown to be cAMP/PKA independent (34);
2) ML-9 exerted no effect on the inotropic effect of
isoproterenol, which is mediated by cAMP/PKA; and 3) a more
selective inhibitor of PKC used at high concentrations in the present
study had no significant effect on the 1-AR-induced PIE.
Thus the inhibitory effect of ML-9 is most likely due to a direct
effect on MLCK.
The PKC inhibitor BIM did not reduce the 1-AR-induced
PIE in the papillary muscle preparation. To show that BIM was able to
inhibit PKC-mediated effects in this preparation, we studied the effect
of the PKC activator PMA in the absence or presence of BIM. Short-term
exposure to PMA induced a sustained negative inotropic response that
was inhibited by BIM. The results show that BIM is able to inhibit
PKC-mediated effects and that PKC activation by PMA and
1-AR stimulation by phenylephrine have opposite
sustained effects on contractility, as previously shown by others
(9). The results also showed that the
phenylephrine-induced PIE was not inhibited by the presence of PMA
further indicating that PKC is not involved in the
1-AR-mediated sustained PIE in the adult rat heart.
The MLCK-dependent PIE induced by 1-AR stimulation was
reproduced by selective stimulation of the 1A-AR subtype
as revealed by the sensitivity to ML-9. A-61603 has been shown to be a
selective 1A-AR agonist (16) and was
reported to induce a PIE in ventricular strips from neonatal rats
(10). The present results demonstrate that selective
activation of the 1A-AR subtype mediates a PIE that is
dependent on MLCK activation, revealing a possible link between this
subtype and activation of MLCK. The results further indicate that the
1A-AR subtype is involved in both the negative and the
positive component of the inotropic response to 1-AR stimulation.
Inhibition of MLCK influenced the basal contractility significantly
with a reduced maximal development of force and an increased relaxation
time at 1 Hz. Only a low frequency of stimulation allowed the muscles
to fully relax and keep the diastolic tension unchanged. Dephosphorylated MLC-2 downregulates the attachment rate of cross bridges and phosphorylation of MLC-2 increases cross-bridge cycling kinetics (18). Decreased MLC-2 phosphorylation levels have
been associated with a negative inotropic state, and the present
results are in agreement with an important influence of MLC-2
phosphorylation on the dynamics of the contraction-relaxation cycle.
The data indicate especially an important role of the phosphorylation
level of MLC-2 with consequences for the diastolic tension. ML-9
inhibited the PIE of 1-AR stimulation also during
experimental conditions where no effect of ML-9 on diastolic tension
was seen (i.e., at 0.3 Hz).
To investigate the possibility that the inhibitory effect of ML-9 was
due to a general depressant effect on contractile function, we studied
the effect of MLCK inhibition on -AR-induced PIE. The results
indicate that the ML-9 inhibition was selective because it did not
interfere with -AR-mediated PIE. Thus the results show that the
inhibitory effect of ML-9 on agonist-induced PIE was not due to a
nonspecific effect of the inhibitor on basal force development. The
muscles were still fully responsive to -AR-induced PIE, which is
dependent on increased intracellular Ca2+ transients and is
not related to increased myofilament sensitivity (22). ET
receptors on the other hand couple to the same signal transduction
pathways as 1-ARs and have been shown to induce a PIE
probably mediated by increased Ca2+ sensitivity of the
myofilaments (14, 27). We found in the present study that
ML-9 inhibited ET-1-stimulated PIE to a similar degree as the
1-AR-induced response suggesting a dependence on MLCK of
positive inotropic responses mediated by Gq coupled
receptors in general.
ML-9 was also able to reduce the PIE of 1-AR stimulation
in ventricular strips from explanted failing and normal human hearts. It is not possible from the present data to address whether this mechanism of stimulating contraction in human hearts is more important in failing than in normal hearts.
It has been shown that the Rho kinase inhibitor Y-27632 inhibits
phenylephrine-induced calcium sensitization and contraction in smooth
muscle (36). Ca2+ sensitization is caused by a
decrease in myosin phosphatase activity due to phosphorylation of the
phosphatase by Rho kinase (32). Rho kinase is activated by
receptors like the 1-AR, which activates the small G
protein RhoA (29). The effect of Y-27632 on cardiac muscle
performance has been unknown so far, but the present results indicate
that the 1-AR-induced PIE is mediated also by
inactivating the myosin phosphatase, with an increased phosphorylation
level of MLC-2 as the result. The inhibitory effect of Rho kinase
inhibition was modest when added before the agonist but prominent when
added at the steady-state response to the agonist to reverse the
response, probably reflecting an increased sensitivity to phosphatase
reactivation when the substrate is already phosphorylated during
exposure to the agonist.
Our results indicate that the PIE of phenylephrine and ET-1 is
dependent on MLCK, and to a lesser extent Rho kinase, but not on PKC.
Phosphorylation of MLC-2 should then be important in
1-AR and ET-receptor-induced increase in contractility.
In the present study we found a modest but significant increase in
MLC-2 phosphorylation after 1-AR stimulation. ML-9
eliminated this increase completely. These results indicate that MLC-2
phosphorylation is essential for the PIE of 1-AR
stimulation, possibly by increasing the sensitivity to calcium.
A disagreement exists regarding whether the phosphorylation level of
MLC-2 is increased by 1-AR or -AR stimulation
(18, 26, 37). A recent study (2), however,
showed that both angiotensin II and phenylephrine induced
phosphorylation of MLC-2 in neonatal rat cardiomyocytes
(2). This activation was not dependent on PKC activation.
Increased phosphorylation of MLC-2 after ET-1 stimulation has also been
shown in rat papillary muscle (27). The effects on MLC-2
phosphorylation in the cited studies are modest. In the present study,
the effect of 1-AR stimulation on the phosphorylation
level of MLC-2 was amplified by the phosphatase inhibitor calyculin-A.
The effect of calyculin-A on basal phosphorylation demonstrates a high
phosphatase activity in the cardiomyocytes.
Our results suggest a PKC-independent activation of MLCK as the main
mechanism for 1-AR-induced PIE with an additional
activation mediated by the Rho kinase/myosin phosphatase pathway, both
actions leading to increased MLC-2 phosphorylation levels.
Phosphorylation of MLC has been known for a long time to be important
for vascular smooth muscle contraction (32), but the role
of MLC in the heart has not been fully characterized (18).
MLC-2 phosphorylation and increased Ca2+ sensitivity of the
myofilaments after activation of Gq coupled receptors could
be an important aspect of understanding the PIE of agonists, which
stimulates the heart independently of the cAMP/PKA system and with only
a modest increase in intracellular Ca2+ (34).
A possible important role of the 1-AR in supporting the
heart of patients at risk for heart failure has been proposed (21), and further understanding of the molecular
mechansims involved in such stimulation will be a challenge in future research.
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ACKNOWLEDGEMENTS |
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This work was supported by The Norwegian Research Council on Cardiovascular Diseases, the Norwegian Research Council and the EU Biomed II Concerted Action Alpha-1 Heart Project Contract BMHC-CT96-0287.
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FOOTNOTES |
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Parts of this study have appeared previously in abstract form: Andersen GØ, Schiander IG, Osnes JB, and Skomedal T. The alpha-1-adrenoceptor mediated positive inotropic response in the rat heart is dependent on myosin light chain kinase. Eur Heart J 21: 61, 2000.
Address for reprint requests and other correspondence: G. Ø. Andersen, Dept. of Pharmacology, Univ. of Oslo, PO Box 1057, Blindern, N-0316 Oslo, Norway (E-mail: g.o.andersen{at}labmed.uio.no).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 16, 2002;10.1152/ajpheart.00232.2002
Received 18 March 2002; accepted in final form 13 May 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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