1 Department of Physiology and Biophysics, and 2 Section of Cardiology, Department of Medicine, College of Medicine, University of Illinois at Chicago, Chicago 60612; and 3 Department of Physiology, Midwestern University, Downers Grove, Illinois 60515
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ABSTRACT |
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We studied how the nitric oxide
(NO · ) donor 3-morpholinosydnonimine (SIN-1)
alters the response to -adrenergic stimulation in cardiac rat
myocytes. We found that SIN-1 decreases the positive inotropic effect
of isoproterenol (Iso) and decreases the extent of both cell shortening
and Ca2+ transient. These effects of SIN-1 were associated
with an increased intracellular concentration of cGMP, a decreased
intracellular concentration of cAMP, and a reduction in the levels of
phosphorylation of phospholamban (PLB) and troponin I (TnI). The
guanylyl cyclase inhibitor
1H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of
phosphorylation levels of PLB and TnI. However, the effects of SIN-1
were abolished in the presence of superoxide dismutase (SOD) or SOD and
catalase. These data suggest that, in the presence of Iso, NO-related
congeners, rather than NO · , are responsible for
SIN-1 effects. Our results provide new insights into the mechanism by
which SIN-1 alters the positive inotropic effects of
-adrenergic stimulation.
3-morpholino-sydnonimine; phospholamban; troponin I; -adrenergic
stimulation
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INTRODUCTION |
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ALTHOUGH THERE
HAVE BEEN many studies on the physiological and pathological
roles of nitric oxide (NO · ) in the regulation of
heart function, the intracellular signaling pathways are not fully
understood. There are controversial reports showing positive, negative,
or biphasic inotropic effects of NO · on the heart
(5, 13, 16, 22, 23). Some of these discrepancies may be
due to variations among species, differences in the concentrations of
NO · donors used, or to differences in the
conditions under which the experiments were performed, such as the
presence or absence of -adrenergic agonists (1, 4, 30).
Interpretation of the data on the effect of NO ·
may also be related to complex effects depending on reactions of
NO · and superoxides
(
In experiments reported here, we treated rat heart myocytes with
3-morpholinosydnonimine (SIN-1) to determine mechanisms by which
NO · and peroxynitrites affect the response to
-adrenergic stimulation. SIN-1, which is the active metabolite of
the vasodilatory drug molsidomine, alters the positive inotropic effect
of
-agonist stimulation and also generates superoxide in addition to
NO · (7, 26). The superoxide reacts
with NO · , generating a potent oxidizing product,
peroxynitrite (ONOO
)
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Peroxynitrite reacts with many biological substances and has been shown to cause several pathological conditions (2, 3, 31). Moreover, in the presence of superoxide dismutase (SOD), formation of hydrogen peroxide (H2O2) is favored, which has been shown to stimulate the cytosolic or soluble form of guanylate cyclase (9).
Our results suggest that, in single rat cardiac myocytes, SIN-1 alters
the positive inotropic effect of the -agonist isoproterenol (Iso) by
altering phosphorylation levels of the sarcoplasmic reticulum protein
phospholamban (PLB) and the myofilament protein troponin I (TnI). These
effects of SIN-1 were associated with an increased intracellular
concentration of cGMP and a decreased intracellular concentration of cAMP.
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METHODS |
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Myocyte isolation. Left ventricular myocytes were isolated from rat hearts by means of a technique previously described by Wolska et al. (32). After isolation, cells were resuspended in fresh control solution (in mmol/l: 0.2 CaCl2, 133.5 NaCl, 4 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 10 HEPES, and 11 glucose) if the cells were used for contraction experiments or in the Na-HEPES phosphate-free buffer (in mmol/l: 4.8 KCl, 1.2 MgSO4, 132 NaCl, 10 HEPES, 2.5 Na-pyruvate, 10 glucose, and 0.2 CaCl2) if the cells were used for phosphorylation experiments (33). The cells were stored in control solution or in the Na+-HEPES phosphate-free buffer with 500 µmol/l Ca2+ at room temperature (22-23°C) until used.
Measurement of intracellular free Ca2+ transients and cell shortening. Fura 2 fluorescence and shortening of cells were monitored simultaneously, as described in detail elsewhere (32, 33). SIN-1, 3-morpholinoiminoacetonitrile (SIN-1C), and Iso were dissolved in H2O. All stock solutions were prepared freshly and added to the superfusion solution just before use.
Labeling of mouse myocytes with 32P.
Experiments aimed at determining the level of protein phosphorylation
in myocyte preparations were done as described by Wolska et al.
(33) by use of a protocol modified from that described by
Gupta et al. (10). Myocytes were incubated in 1.0 mmol/l Ca2+-Na-HEPES phosphate-free buffer with 0.5 mCi
[32P]orthophosphate for 30 min at room temperature.
Thereafter, cells were washed twice with the Na-HEPES phosphate-free
solution with 1 mmol/l Ca2+. The myocyte suspensions (150 µl) were mixed with 150 µl of the Na-HEPES phosphate-free buffer
with 1 mmol/l Ca2+ with or without added drugs, as
described in the figure legends. After 2 min, the reaction was stopped
by adding 150 µl of SDS stop solution (in mmol/l: 1 dithiothreitol,
30 Tris · HCl, and 3 EDTA; 6% SDS, 15% glycerol, and a trace
of bromphenol blue). The samples were mixed well and stored at
20°C. Before analysis by SDS-PAGE, the samples were boiled for 10 min to convert the high molecular weight of PLB into its
low-molecular-weight form. The
1H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) stock solution was prepared in DMSO. An equivalent amount of DMSO
was added to all control samples. In experiments in which ODQ was
used cells were preincubated with these drugs for 10 min.
Determination of protein phosphorylation. Gel electrophoresis was performed using a linear 5-20% polyacrylamide gradient gel as previously described (33). An aliquot of cells containing 50 µg of protein, as determined using the Lowry method (17), was applied to each lane. The SDS-PAGE gels were scanned using Personal Densitometer SI (Molecular Dynamics), vacuum dried, and placed in Storage Phosphor Screen Cassettes (Kodak) for overnight exposure. The cassettes were scanned in a Molecular Dynamics Storm 840 PhosphorImager. The ImageQuant software from Molecular Dynamics was used for data processing. Phosphorimage and densitometric data were adjusted for background and used for calculations. Data were normalized to the maximum values. Myofilament proteins were identified by comigration with known standards. PLB was identified using a monoclonal antibody.
Determination of cGMP and cAMP levels.
Cyclic nucleotide measurements were performed on isolated myocytes by
use of a modification of the method of George et al. (8).
In this procedure, sample aliquots from myocyte preparations were
resuspended in 1.5 ml of bath solution (control or drug). After 10 min,
the myocyte samples were centrifuged at 14,000 g for 45 s, the supernatant was aspirated, and the cells were resuspended in 600 µl of cold 65% ethanol. The ethanol-myocyte solution was sonicated
and then centrifuged at 14,000 g for 20 min at 4°C. The
supernatants were aspirated and placed into separate microcentrifuge tubes, the ethanol was evaporated, and the remaining residue was stored
at 20°C. The pellets were resuspended in 1% SDS for measurement of
total protein by use of the bichinchoninic acid protein assay (Pierce,
Rockford, IL). For measurement of cGMP levels, the frozen residue was first reconstituted with 50 mmol/l acetate buffer, and the
cGMP content was determined by radioimmunoassay (RIA) with a
commercially available kit (Biomedical Technologies, Stoughton, MA).
The cAMP content was also determined by RIA with a commercially available kit (Amersham Pharmacia Biotech, Piscataway, NJ). We have
previously used these methods to measure cAMP and cGMP levels in
cardiac myocytes (35).
Statistical analysis. Data are presented as means ± SE. The significance of differences between the means was evaluated using appropriate analysis of variance (ANOVA) and the Student-Newman-Keul test for multiple comparisons. Values with P < 0.05 were considered statistically significant.
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RESULTS |
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Effects of SIN-1 and Iso on cell shortening and
Ca2+ transients.
We studied the effects of the NO · donor SIN-1 on
twitch contractions and on fura 2 fluorescence ratios as a measure of
intracellular free Ca2+ in single cardiac myocytes,
isolated from rat hearts. Measurements were made before and after
stimulation with the -adrenergic agonist Iso. A representative
example of the effects of 3-min perfusion with 1 µM Iso alone on the
Ca2+ transient is shown in Fig.
1A and the effects on cell
shortening during twitch contraction in Fig. 1B.
Three-minute perfusion with Iso resulted in a significant steady-state
increase in cell shortening (to 237 ± 9% of control;
n = 6) and an increase in the amplitude of the
Ca2+ transient (to 157 ± 15% of control;
n = 6). A representative example of the effects of
3-min perfusion with a combination of 1 µM Iso and 200 µM SIN-1 on
the Ca2+ transient is shown in Fig. 1C and on
those on cell shortening in Fig. 1D. When the cells were
perfused for 3 min with 1 µM Iso and 200 µM SIN-1, cell shortening
increased only to 167 ± 17% of control (n = 6),
and the amplitude of the Ca2+ transient was increased to
124 ± 15% of control (n = 6). The reduced
response to Iso was also observed when the cells were perfused for 5 or
7 min with addition of 200 µM SIN-1. Figure 2 summarizes the effects of 1 µM Iso in
the presence and absence of 200 µM SIN-1 on the peak amplitude of the
Ca2+ transient and amplitude of twitch contraction at three
different time points (Fig. 2A) and on changes in time to 50 and 75% of relaxation of cell shortening after 3 min perfusion with
Iso or a combination of Iso and SIN-1 (Fig. 2B).
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Effects of SIN-1 and Iso on protein phosphorylation.
There are a number of mechanisms that could account for the altered
Ca2+ transient and shortening of cells in the presence of
SIN-1. One possibility is that SIN-1 may alter phosphorylation levels
of proteins involved in excitation-contraction coupling. To determine this possibility directly, we measured phosphorylation of myocyte proteins under control conditions and after treatment with
1) 0.1 µM Iso; 2) 0.1 µM Iso + 200 µM
SIN-1; 3) 0.1 µM Iso + 200 µM SIN-1C; 4)
0.5 µM Iso; 5) 0.5 µM Iso + 200 µM SIN-1; and
6) 0.5 µM Iso + 200 µM SIN-1C (Fig.
3A). SIN-1C, the final product of SIN-1 oxidation and which does not release
NO · , was used as a negative control. Treatment
with Iso resulted in a significant increase in 32P
incorporation into PLB, cTnI, and C protein. Maximal phosphorylation of
PLB and TnI (taken as 100%) was observed at 0.5 µM Iso. The level of
phosphorylation of light chain 2 (LC2) was not altered by
0.5 µM Iso. SIN-1 (200 µM) or SIN-1C (200 µM) did not alter the
basal levels of phosphorylation of PLB, cTnI, or LC2.
However, 200 µM SIN-1 resulted in a significant decrease in the
levels of phosphorylation of PLB and cTnI caused by Iso. There was no significant change in the levels of phosphorylation of LC2
or C protein. Addition of SIN-1C (200 µM) did not significantly
reduce the phosphorylation of cTnI and PLB induced by Iso. Figure
3B summarizes the effects of buffer with vehicle alone
(control), 0.5 µM Iso, 0.5 µM Iso plus 200 µM SIN-1, and 0.5 µM
Iso plus 200 µM SIN-1C on the PLB, cTnI, and LC2
phosphorylation.
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Effects of SIN-1 on protein phosphorylation in the presence of ODQ.
To test whether soluble guanylyl cyclase is involved in the effect of
SIN-1 on the protein phosphorylation induced by Iso, we performed a
series of experiments in the presence of 10 µM ODQ, a selective
inhibitor of soluble guanylyl cyclase. Figure 4 shows a SDS-PAGE analysis (Fig.
4A) and phosphorimage (Fig. 4B) of proteins from
isolated cardiac myocytes in the presence of 10 µM ODQ with and
without 0.5 µM Iso and 200 µM SIN-1. Even in the presence of ODQ,
SIN-1 significantly decreased the level of phosphorylation of cTnI and
PLB caused by Iso. The summary of the phosphorylation levels of cTnI
and PLB relative to the level of phosphorylation in the presence of 10 µM ODQ and 0.5 µM Iso is presented in Fig. 4C.
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Effects of SIN-1 on shortening and
Ca2+ transient in the presence of SOD.
As shown in Fig. 5, the presence of SOD alone prevented SIN-1 from
decreasing PLB and cTnI phosphorylation. We also wanted to know whether
preventing changes in phosphorylation of PLB and cTnI would be
sufficient to reverse functional changes in myocyte shortening and
Ca2+ transients. We compared cell shortening and
Ca2+ transients in myocytes exposed to the following
conditions: 1) 35 U/ml SOD; 2) 35 U/ml SOD and 1 µM Iso; or 3) 35 U/ml SOD, 1 µM Iso, and 200 µM SIN-1.
In the presence of SOD, SIN-1 did not significantly reduce the extent
of shortening or the Ca2+ transient that were increased by
Iso (Fig. 6). These data suggest that
alteration in PLB and cTnI phosphorylation may be major mechanisms responsible for SIN-1 effects on shortening and the Ca2+
transient.
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Effects of SIN-1 on intracellular levels of cAMP and cGMP.
To test the extent to which alterations in Ca2+ transient,
cell shortening, and levels of protein phosphorylation are due to altered activities of cAMP- and cGMP-dependent kinases, we measured the
levels of cAMP and cGMP in isolated rat ventricular myocytes with
different treatments. Treatment of myocytes with Iso resulted in
significant increases in the intracellular level of cAMP and no change
in the level of cGMP. In the presence of Iso, addition of SIN-1
resulted in a significant increase in cGMP and a decrease in cAMP. Data
from different treatments are summarized in Table 1.
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DISCUSSION |
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To the best of our knowledge, our results are the first to show
directly that, in the presence of -adrenergic stimulation, SIN-1
alters phosphorylation of proteins involved in excitation-contraction coupling, specifically PLB and TnI. Important questions are
1) what the mechanism is by which NO is able to alter
phosphorylation of regulatory proteins PLB and TnI; and 2)
how alterations in phosphorylation levels of PLB and TnI can explain
the results of functional studies with isolated cells.
The effect of NO on cardiac contractility may be biphasic, depending on
the concentration of the NO · donor used
(22). In the present study, we focused on the mechanisms responsible for altered cardiomyocyte contractility by high
concentrations of SIN-1 in the presence of the -agonist Iso.
Incubation of rat cardiac myocytes with SIN-1 resulted in an increased
cGMP concentration in both the presence and absence of Iso. This first
set of data suggests that the guanylyl cyclase and/or cGMP-stimulated
or -inhibited phosphodiesterases (PDEs) may be involved in the
observed effects of SIN-1. In addition to an increase in cGMP levels,
SIN-1 caused a decrease in the concentration of cAMP, which
suggests stimulation of PDEII and increased degradation of cAMP.
However, after blocking soluble guanylyl cyclase by ODQ we found that,
although cGMP level was now unchanged by SIN-1, SIN-1 still reduced the
level of phosphorylation of PLB and TnI. These data indicate a
cGMP-independent pathway for alterations in phosphorylation and are
consistent with recently published results of Sandirasegarane and
Diamond (25). They have shown that, in the presence of
Iso, high concentrations of the NO donors
S-nitroso-N-penicillamine and DEA
resulted in marked attenuation of Iso-mediated increase in cell
shortening, in both the presence and absence of ODQ.
Moreover, Weiss et al. (29) found that, in isolated
Langendorff-perfused rat hearts, effects of intracoronary perfusion of
nitroprusside were unrelated to an increase in cGMP- or cGMP-dependent
changes in cAMP.
Another important finding on the mechanism of SIN-1 action comes from our experiments with catalase and SOD. Our data show that the effects of SIN-1 on phosphorylation of PLB and TnI, as well as on blunting the Ca2+ transient and myocyte shortening, are related to NOx, and H2O2 is not involved in its action.
How can alterations in phosphorylation levels of PLB and TnI explain the results of functional studies with isolated cells? PLB is an inhibitory protein of the SR Ca2+ pump in its unphosphorylated state, and phosphorylation or ablation releases this inhibition (6, 14, 18). Reduction in PLB phosphorylation, as was observed in our experiments, would result in a reduction in Ca2+ transients and decrease contractility. At the same time, we found lower levels of TnI phosphorylation, which would result in increased myofilament Ca2+ sensitivity (24, 27). Thus TnI phosphorylation has an opposite effect on contractility compared with reduced phosphorylation of PLB. In isolated single myocytes, we found decreased contractility, which suggests that reduction in PLB phosphorylation is more important in determining effects of SIN-1 on contractility than on the increase myofilament sensitivity to Ca2+. Reduced levels of phosphorylation of PLB and TnI by SIN-1 in the presence of Iso would also result in the smaller effect on the kinetics of relaxation of cell shortening, as was observed in our experiments.
Although our experiments strongly suggest that effects of SIN-1 on rat
cardiac myocyte contractility and levels of protein phosphorylation are
largely cGMP independent, the cGMP-dependent mechanism of SIN-1 that
has been previously postulated by us (28) and others
(5) cannot be completely excluded and may play a significant role in different experimental conditions. For example, in
frog, rat, and guinea pig ventricular myocytes, SIN-1 had no effect on
ICa in the basal state (20, 21,
28). However, depending on the dose, SIN-1 had a stimulatory or
inhibitory effect on ICa previously stimulated
by forskolin, cAMP, or Iso (21, 28). In guinea pig and rat
myocytes, it has previously been found that the inhibitory effect of
SIN-1 on Ca2+ current was PDE independent (21,
28). In rat Langendorff-perfused hearts, Klabunde et al.
(15) found that the effect of the NO inhibitor
N-methyl-L-arginine on myocardial contractility
was through an alteration in cGMP level, which led to an increase in
the PDE activity and to a decreased cAMP level. Decreased concentration of cAMP would be expected to result in decreased activity of protein kinase A (PKA) and reduced phosphorylation of both cTnI and PLB. However, in our experiments, the decreased concentration of cAMP was
probably not due to changes in cGMP-dependent PDE activity. Another
possible mechanism by which SIN-1 reduced cAMP levels and PKA activity
is its direct or indirect effect on phosphatase activity. It is
important to note that Yokoyama et al. (34) reported that
tumor necrosis factor-, which is known to be elevated in heart
failure and to work, at least in part, through a
NO · pathway, caused a reduction in the
phosphorylation levels of TnI and PLB with no change in phosphorylation
of LC2 by a concentration-dependent activation of
phosphatase type 2A and no effect on type 1 phosphatase activity; both
phosphorylated PLB and TnI are excellent substrates for phosphatase
type 2A (19).
In summary, the results of the present study demonstrate that, in rat
cardiac myocytes and during -adrenergic stimulation, the negative
inotropic effect of the exogenous NO · donor SIN-1
in cardiac myocytes is associated with a reduction in the peak
amplitude of the Ca2+ transient and phosphorylation levels
of TnI and PLB. These effects depend on both cAMP- and cGMP-dependent
protein kinases. The results presented in this paper add important new
insights into our understanding of pathophysiological conditions such
as human heart failure in which the NO · signaling
pathway is altered (11, 12).
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grants R29 HL-58591 (B. M. Wolska) and T32 HL-07692 (M. O. Stojanovic and M. T. Ziolo). B. M. Wolska was supported by an Established Investigator Grant of the American Heart Association. G. M. Wahler was supported by a grant from the American Heart Association of Metropolitan Chicago and from funds provided by the Research Affairs Office of Midwestern University.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. M. Wolska, Section of Cardiology, Dept. of Medicine (M/C 787), Univ. of Illinois at Chicago, 840 S. Wood St. Chicago, IL 60612 (E-mail: bwolska{at}uic.edu).
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.
Received 19 December 2000; accepted in final form 26 February 2001.
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