(Circulation. 1997;95:2318-2324.)
© 1997 American Heart Association, Inc.
Articles |
Negative Inotropic Effect of Adrenomedullin in Isolated Adult Rabbit Cardiac Ventricular Myocytes
From the Second Department of Internal Medicine, University of Tokyo (H.I., Y.H.) and the Research Institute of the National Cardiovascular Center, Osaka (K.K., H.M.), Japan.
Correspondence to Hiroshi Ikenouchi, MD, The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113 Tokyo, Japan. E-mail ikenouchi-2im{at}h.u-tokyo.ac.jp
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Abstract |
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Background Adrenomedullin (AM) is a potent vasodilator peptide. AM-induced vasodilatation is mediated by an increase of NO as well as cAMP. Both AM and binding sites for this peptide have been found in cardiac tissue, indicating the possible existence of an autocrine or paracrine system of AM in the heart.
Methods and Results Myocytes were isolated by use of retrograde coronary perfusion with physiological solution containing collagenase and hyaluronidase from adult rabbit ventricles. Contraction of cardiac myocytes was traced with a video motion detector, and [Ca2+]i was measured with indo 1 at 37°C. The ICa was measured with a whole-cell patch clamp at 23°C. AM and calcitonin gene–related peptide (CGRP), another member of the same peptide family, showed a concentration-dependent negative inotropic effect (10-7 mol/L AM: contraction amplitude, 64±7% of control; [Ca2+]i, 52±5% of control; n=10; 10-6 mol/L CGRP: contraction amplitude, 64±25%; [Ca2+]i, 70±3%; n=5; mean±SD). ICa was decreased to 60±39% by superfusion with AM after the cessation of NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor. Pretreatment with L-NMMA (10 µmol/L) abolished the negative inotropic effect of AM, whereas switching from AM+L-NMMA to AM+L-arginine (1 mmol/L) restored it. Superfusion with 8-bromo-cGMP also showed a negative inotropic effect. AM significantly increased the intracellular content of cGMP, a second messenger of NO, but not that of cAMP. AM (10 nmol/L) blunted the effect of 1 µmol/L forskolin.
Conclusions AM has a negative inotropic effect and decreased both [Ca2+]i and ICa, with these effects being at least partly mediated via the L-arginine–NO pathway in adult rabbit ventricular myocytes.
Key Words: adrenomedullin • myocytes • contractility • calcium • nitric oxide
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Introduction |
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Adrenomedullin is a potent vasodilator peptide that was originally discovered in pheochromocytoma tissue.1 Subsequent studies have revealed that AM immunoreactivity can be detected in the plasma2 and is widely distributed in various normal tissues, including the heart.3 Expression of mRNA for AM has also been demonstrated in these tissues.4 5 The highest levels of immunoreactive AM have been found in the adrenal medulla, atrium, and lung.6 Furthermore, abundant binding sites also exist in the ventricular tissue.7 Jougasaki et al8 reported that the circulating and ventricular levels of AM were increased in patients with congestive heart failure compared with control subjects, indicating a potential pathophysiological role of this peptide. These findings suggest that AM could be a locally acting hormone rather than a systemic agent.
Thus, AM may exert its effects on the heart via autocrine or paracrine mechanisms. Although the cardiac actions of AM have not been extensively examined, Perret et al9 showed that it had a negative inotropic effect in the isolated perfused heart. The signal transduction system for AM in myocytes remains unclear. cAMP has been suggested as a second messenger for AM, because this peptide was isolated on the basis of its activity of an increase in platelet cAMP and because it also increases cAMP in vascular smooth muscle cells, endothelial cells, and mesangial cells.1 However, an increase in intracellular cAMP in cardiac myocytes usually has a positive inotropic effect. We and others recently reported that AM increases NO release from the kidney10 and from bovine aortic endothelial cells,11 but there have been no studies concerning the effect of this peptide on NO production by cardiac myocytes. NO exerts its vasodilatory effect via activation of guanylate cyclase and the accumulation of cGMP. The effects of NO on cardiac performance are still controversial. It has been suggested that NO and cGMP could regulate the ICa in cardiac myocytes.12 13 It has also been reported that NO decreases the calcium current in myocytes, possibly via activation of the guanylate cyclase/PK-G system.12 14 Such reports suggest a potential influence of NO on cardiac performance, but the relationship between these effects on the calcium current and changes of contractility needs to be investigated. To influence cardiac performance in an autocrine or paracrine fashion via NO, AM needs to activate NO production in the heart. NO is produced by NOS, which is widely distributed in endothelial cells. A recent study revealed that cNOS is also present in cardiac myocytes.15 Taken together, these findings suggest that AM could be a hormone with a local effect on cardiac performance.
To investigate the possible contribution of AM to cardiac performance via the NO-cGMP system or the adenylate cyclase–cAMP system, we examined the effects of this peptide on contraction, [Ca2+]i, ICa, and intracellular cGMP and cAMP in isolated adult rabbit ventricular myocytes.
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Methods |
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Isolation of Cardiac Myocytes
A modification of the method of Haddad et al17 and our previous method18 was used. Briefly, New Zealand White rabbits weighing 2.0 to 2.5 kg were given 500 U heparin IV and were anesthetized with 50 mg/kg sodium pentobarbital. The heart was isolated, and coronary perfusion was initiated via a short cannula inserted into the aortic root immediately after the chest was opened. After the rabbit heart was mounted on an aortic cannula, perfusion was initiated with MKRBB containing nominal zero Ca2+ and continued for 5 minutes. The composition of MKRBB was as follows (in mmol/L): NaCl 91.7, KCl 30, MgSO4 1.2, NaH2PO4 1.2, dextrose 15, taurine 20, adenosine 0.5, and NaHCO3 19. The buffer was gassed with 95% O2/5% CO2 (pH 7.4). Then perfusate was switched to 50 µmol/L Ca2+ plus 0.2 mg/mL collagenase (class II, Worthington Biochemical) and 0.4 mg/mL hyaluronidase (type I-S, Sigma Chemical Co). Flow was adjusted to give perfusion pressure of 50 mm Hg, and each heart was perfused with recirculated collagenase and hyaluronidase for
15 minutes. Then the hearts were removed and
rinsed in 50 µmol/L Ca2+ MKRBB, after which the
ventricles were cut into small pieces in the same buffer and incubated
in the same solution containing 0.2 mg/mL collagenase for 5
to 10 minutes. The cell suspensions were subsequently mixed with the
same amount of inhibitor solution containing 50
µmol/L Ca2+ and 12% FCS to stop digestion, followed by
centrifugation at 250 rpm for 5 minutes. The
supernatant was discarded, and the cells were resuspended in culture
medium, which consisted of 95% MEM (Gibco Laboratories), 0.1%
penicillin-streptomycin, and 5% FCS (all percentages calculated as
wt/vol). The cells were attached to coverslips with cell adhesive
(Cell-Tak, Collaborative Research, Inc) and were incubated in a 5%
CO2/95% air atmosphere for several hours at 37°C. All
studies were performed within several hours after isolation.
Measurement of Contractility and
[Ca2+]i in Isolated Myocytes
The instrumentation used to obtain fluorescence signal
from the myocytes has been described in detail
elsewhere.19 [Ca2+]i was
measured with the Ca2+ fluorescent dye indo 1. To
load the cells with the fluorescent indicator, indo 1
acetoxymethyl ester (Dojindo Chemical Inc) was used. The dye was
dissolved by sonication in DMSO–pluronic acid (25% wt/vol) in
modified Krebs-Henseleit buffer at a final concentration of 4
µmol/L in the presence of 5% (wt/vol) FCS. The cells were exposed
for 5 minutes to indo 1 at a final concentration of 4 µmol/L and
then washed for 30 minutes. Next, the cells were placed in a heated
(37°C) superfusion chamber on the stage of an inverted microscope
equipped with epifluorescence optics (Nikon Diaphot). The
excitation wavelength was 360 nm. Cell images and fluorescence
were collected by a Fluor x40 objective lens and transmitted to a
spectrophotometer system attached to the video port of the
microscope.18 Cells were quiescent without stimulation. A
bipolar field stimulation electrode was placed near the cell and
electrically paced the cell at a rate of 0.5 Hz. The image of the
beating cells was obtained with a video camera (model 540, Pulnix)
attached to the microscope by illumination via the standard microscope
light source passed through a 700-nm bandpass filter. This wavelength
was long enough not to interfere with the fluorescence
detection. Motion of myocytes was detected with a custom-made video
motion analyzer. Cell motion was tracked along a selected video
monitor raster line. The video signal was processed as described
previously,18 19 with the signals of fluorescence
intensity at 400 and 480 nm as well as the analog signal of the ratio
of intensities at the two wavelengths.
The control superfusate for adult rabbit ventricular myocytes was a bicarbonate-buffered normal Tyrode's solution containing (in mmol/L): NaCl 126, KCl 4.4, MgCl2 1.0, CaCl2 0.9, dextrose 11, and NaHCO3 18, gassed with 95% air/5% CO2 (pH 7.4). The buffer was superfused at a rate of 3 mL/min throughout the study. After a 30-minute equilibrium period, the control superfusate was switched to buffer containing test agents. The effects of graded doses of AM, CGRP, 8-bromo-cGMP, or forskolin on cell motion and [Ca2+]i were examined with cells on different coverslips. The effects of AM were also studied in the presence or absence of L-NMMA, an inhibitor of NOS, and methylene blue, an inhibitor of soluble guanylate cyclase.
Intracellular cGMP and cAMP
The intracellular contents of cAMP and cGMP before and after
exposure to AM (10-7 to
10-10 mol/L) or forskolin
(10-6 mol/L) were measured with an enzyme
immunoassay kit (Biotrak, Amersham Life Science), and the absorbance
was read on a spectrophotometer (ImmunoReader NJ2001, Japan Inter Med
Inc) at 450 nm. Cardiac myocytes were isolated as described above,
suspended in 50 mL of control bicarbonate-buffered normal Tyrode's
solution, and placed into polypropylene tubes in 2-mL aliquots. Under
microscopic observation, the extent of rod-shaped cells, that is,
viable myocytes, in the cell suspension was 30% to 40%; the rest were
roundup cells. AM or forskolin was added from stock solution, and the
tubes were incubated in a CO2 incubator for 10 minutes at
37°C and then centrifuged at 1500 rpm for 5 minutes at 4°C.
The supernatant was discarded, and 500 µL 6% trichloroacetic acid
was added on ice to stop the reaction. To adjust the pH, 360 µL 1N
NaOH was added to each tube. Then the cells were
homogenized by a motor-driven homogenizer,
the tubes were centrifuged at 3000 rpm for 5 minutes at 4°C,
and the supernatant was stored at -20°C until assay. Both cAMP and
cGMP were measured in the same preparation after
acetylation.
Measurement of ICa
We used established methods for recording the
ICa.20 21 Briefly, adult rabbit
ventricular myocytes were voltage-clamped at 23°C by use
of a current-to-voltage convertor (Axopatch 200, Axon Instruments). The
patch pipettes had a resistance of 1 to 1.5 M
and contained the
following (in mmol/L): CsCl 130, NaCl 10, MgCl2 0.1,
dextrose 5.5, K2ATP 5, KOH 11, and HEPES 10. The pH was
adjusted to 7.1 with KOH, and the total K+ concentration
was 21 mmol/L. The control external solution contained (in
mmol/L): NaCl 138, KCl 4.4, CaCl2 0.9, MgCl2
1.0, dextrose 11, HEPES 12, and NaOH 6.4 (pH 7.4). The L-type
Ca2+ current was measured several times at 1-minute
intervals during perfusion with the control solution to confirm the
stability of the preparation by depolarizing cells from -40 to +10 mV
for 450 ms. Between each measurement, the membrane potential was held
at -80 mV. After superfusion with a solution containing 10 nmol/L AM
and 10 µmol/L L-NMMA for 2 minutes, the solution was changed to
one containing 10 nmol/L AM and 1 mmol/L L-arginine,
and the ICa was measured at 1-minute intervals.
ICa during vehicle superfusion was measured to
detect the extent of spontaneous rundown in our experiment. In another
experiment, 10 nmol/L AM together with 1 µmol/L forskolin was
superfused for 4 minutes, then switched to solution with forskolin
without AM. ICa was measured at 2-minute
intervals.
Statistical Analysis
The effects of the agents were assessed by ANOVA for repeated
measurements, followed by Dunnett's test. Statistical significance was
set at P<.05.
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Results |
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Effects of AM and CGRP on Myocyte Contractility and [Ca2+]i
A representative tracing of the effect of AM on myocyte contractility and [Ca2+]i is shown in Fig 1A
.
Superfusion with AM had a concentration-dependent negative inotropic
effect and decreased the amplitude of
[Ca2+]i. These effects appeared within 1
minute after application of AM, whereas washout of AM resulted in
recovery of the amplitude of cell motion and
[Ca2+]i to baseline values. The averaged
effects of AM are shown in Fig 1B.
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Superfusion with CGRP, another peptide in the same family as AM,1 also showed a concentration-dependent (10-9 to 10-6 mol/L) negative inotropic effect and decreased the amplitude of [Ca2+]i (CGRP 10-6 mol/L, motion amplitude 64±25%, [Ca2+]i 70±3%, n=5, P<.05, mean±SD). Both contractility and [Ca2+]i returned to baseline after washout of CGRP.
To examine the possibility that AM exerts its negative inotropic effect through stimulation of cGMP, we studied the effects of 8-bromo-cGMP on [Ca2+]i and motion of the myocytes. Superfusion with 8-bromo-cGMP (10-6 to 10-4 mol/L) had a dose-dependent negative inotropic effect (10-4 mol/L, 68±3%, P<.001, n=4) and decreased the amplitude of [Ca2+]i (67±4%, P<.004). These changes resembled the effects of AM and CGRP, suggesting that the NO-cGMP system may contribute to the actions of AM.
To confirm this hypothesis, we studied the effects of L-NMMA and
methylene blue. L-NMMA alone tended to decrease the amplitude of cell
motion, but this change was not statistically significant. In the
presence of 10 µmol/L L-NMMA, addition of
10-8 mol/L AM did not influence cell
contraction. However, addition of 1 mmol/L L-arginine
reproduced the negative inotropic effect of
10-8 mol/L AM (motion amplitude, 73±9% of
control, n=7, P<.001, mean±SD, Fig 2). Cell
motion returned to baseline after washout of these agents. The effects
of L-NMMA on the [Ca2+]i could not be
determined because of deterioration of the calcium signal, possibly by
interference with indo 1–evoked fluorescence or some other
factors in these cells. Therefore, the effects of methylene blue on
AM-induced changes of [Ca2+]i were examined.
Methylene blue alone slightly increased cell motion amplitude, then AM
was added to a methylene blue–containing solution (Fig 3). Addition of 10-8 mol/L AM
in the presence of 10-5 mol/L methylene blue
influenced neither cell motion nor [Ca2+]i.
However, AM began to reduce both amplitudes of cell motion and
[Ca2+]i after the cessation of methylene blue
infusion.
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Intracellular cAMP and cGMP
Exposure of the myocytes to AM for 10 minutes increased the
intracellular cGMP level in a concentration-dependent manner. The
maximal intracellular level was obtained with
10-7 mol/L AM and was 182% of the control
value (Fig 4, top). In contrast, exposure to forskolin
for 10 minutes did not increase the intracellular cGMP content. AM did
not increase the intracellular cAMP content at any concentration. In
contrast, forskolin caused a marked increase in intracellular cAMP
content (Fig 4, bottom).
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Effect of AM on ICa
Superfusion of AM alone caused a significant decrease in
ICa (65±30%, P<.03, n=5) after
subtraction of spontaneous rundown, associated with a significant
decrease in motion amplitude (48±31%, P<.05). To further
investigate whether AM regulates the calcium current in these cells via
the NO system, we measured the ICa during
superfusion of AM with or without L-NMMA in the same cells.
Representative traces of ICa and
simultaneous motion tracings are shown in Fig 5A, with the averaged data and time course being shown
in Fig 5B. Cell contraction did not change significantly in the
presence of L-NMMA, but AM exerted a significant negative inotropic
effect after addition of excess L-arginine (Fig 5B, bottom). There was a similar effect on ICa (Fig 5B, top), which was decreased significantly by AM only after the
addition of L-arginine. This change was significant after
subtraction of spontaneous rundown of ICa in
control cells (Fig 5B).
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We also examined the counteraction of AM on the effect of forskolin. Forskolin (1 µmol/L) had a significant positive inotropic effect in these cells (contraction amplitude, 191±33%; n=7, P<.05) associated with an increase of [Ca2+]i (122±8%, n=7, P<.05). Motion and [Ca2+]i changes were reversible after cessation of forskolin superfusion. In the patch-clamp experiment, forskolin by itself markedly increased ICa (232±57%, P<.05, n=4). Cosuperfusion of 10 nmol/L AM with 1 µmol/L forskolin significantly blunted the increase of ICa (114±2%, n=4, P<.01), whereas the amplitude of motion did not change significantly (100±21%, n=4, P=NS). Four minutes after the washout of AM, forskolin increased the amplitude of contraction (124±17%) and ICa (132±19%, P<.01). Contraction and ICa returned to the control levels after cessation of forskolin.
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Discussion |
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This study showed that AM had a concentration-dependent negative inotropic effect in isolated adult rabbit ventricular myocytes, as did CGRP and 8-bromo-cGMP. The amino acid sequence of AM shows partial homology with that of CGRP. CGRP is a potent vasoactive, hypotensive peptide secreted from C nerve terminals and increases cAMP in rat platelets.1 An NOS inhibitor, L-NMMA, blocked the negative inotropic effect of AM, and addition of L-arginine restored the effect. A guanylate cyclase inhibitor, methylene blue, also blocked the negative inotropic effect of AM. After exposure to AM, the intracellular cGMP content increased in a concentration-dependent manner, suggesting the possible contribution of NO and subsequent cGMP production to the negative inotropic effect of AM. In addition, AM decreased both [Ca2+]i and ICa in these cells, changes that may also have contributed to its negative inotropic effect.
A negative inotropic effect of AM was previously found in an isolated heart preparation.9 Activation of the NO-cGMP system by AM was recently reported in kidney10 and bovine aortic endothelial cells,11 whereas the present study suggested a possible contribution of the NO-cGMP system to the effect of AM in adult rabbit cardiac ventricular myocytes. The precise mechanism underlying the cGMP-evoked negative inotropic effect remains unclear. However, recent studies have provided evidence that NO or cGMP regulates the calcium current in cardiac myocytes.12 Méry et al13 reported that cGMP had no significant effect on the basal ICa in rat myocytes but had a strong dose-dependent inhibitory effect after stimulation of ICa by cAMP. We found that AM had a small but significant effect on basal ICa, and this reduction of ICa could also contribute to the negative inotropic effect of AM. It is difficult to explain the differences between the present and previous studies regarding the effects of cGMP on basal ICa, but differences in the experimental preparation or species used may be involved, because we previously reported that the species or the maturity of cardiac myocytes greatly affected the inotropic response to endothelin-1 or angiotensin II.22 L-NMMA blocked the effect of AM on ICa in the present study, suggesting a significant contribution of NO. A recent study showed that cGMP has an inhibitory effect on basal ICa.23 It was suggested that PK-G activity may vary from cell to cell and that basal ICa is regulated by the balance between PK-A and PK-G activity. If the endogenous PK-G activity is low, addition of exogenous 8-bromo-cGMP to the bath should inhibit ICa. Conversely, if the endogenous PK-G activity is already high, there would be little or no effect. Thus, basal endogenous PK-G activity might be relatively low in isolated rabbit ventricular myocytes.
We showed in the present article that a relatively low concentration of AM blunted the effect of forskolin, that is, AM suppressed the increase of contraction amplitude and delayed the increase of ICa by forskolin. This suggested that AM could modify the effects of the activated PK-A system in these cells.
Shah et al24 reported that 8-bromo-cGMP exerts a negative inotropic effect in isolated rat myocytes, but they detected no change of the intracellular calcium concentration or pH and concluded that there was a reduction of the myofilament response to Ca2+. Although 8-bromo-cGMP showed a similar negative inotropic effect in our preparation, the [Ca2+]i decreased significantly, and the decrease in the amplitude of motion was proportionate. This decrease in intracellular calcium may explain the negative inotropic effect of AM in these cells, although there is still a possibility that myofilament sensitivity to Ca2+ could change simultaneously. A change in intracellular pH is one possibility that could also affect the Ca2+ sensitivity of contractile elements in ventricular myocytes, as we reported previously.25
AM increases intracellular cAMP in various types of cells, such as kidney cells, endothelial cells, and platelets. The AM receptor and the signal transduction pathway are still under investigation. There are reports that AM receptors of rat vascular smooth muscle cells are functionally coupled to adenylate cyclase, which interacts with CGRP,26 and the vasoactive effects of AM are inhibited by CGRP or a CGRP receptor antagonist.27 Owji et al7 suggested that there are at least two subtypes of AM receptors and that the heart has a specific receptor. They found the highest level of AM binding in the heart. They also found that AM could bind to CGRP receptors and suggested that the increase in cAMP may be due to cross-activation of CGRP receptors, whereas signal transduction via specific AM receptors may not involve the cAMP pathway.
A recent study showed that the iNOS induction in cardiac microvascular endothelial cells affects the contractility of cocultured myocytes.16 Production of NO by iNOS is supposed to be much greater than by cNOS, and the resulting high levels of NO could be harmful to cells. Conversely, the small amount of NO produced by cNOS could act as a superoxide scavenger.28 In our study, the majority of the cells were myocytes, which were not adjacent to other types of cells, including endothelial cells, and the effect of AM appeared immediately after superfusion with rapidly flowing solution. Thus, AM may activate cNOS in cardiac myocytes. The mechanism of activation of cNOS in the present study is unclear. In endothelial cells, a transient increase of intracellular calcium may activate cNOS.10 However, in beating cardiac myocytes, the peak systolic calcium concentration is already elevated to around 600 to 800 nmol/L.29 In the present study, [Ca2+]i decreased after superfusion with AM. It seems unlikely that a transient increase in [Ca2+]i triggers the activation of cNOS, so an alternative pathway for cNOS activation may exist in cardiac myocytes.
Previous investigations have detected mRNA for AM in heart tissue,4 and there is also evidence that cNOS activity is present in the heart.15 These findings provide a molecular biological basis for a possible AM-NO-cGMP system in cardiac tissue. The present study indicated that such an AM-NO-cGMP system may be responsible for the effects of this peptide on cardiac myocytes, but the clinical significance of these findings remains unclear. It has been reported that the plasma AM level is increased in patients with hypertension, especially hypertension associated with organ failure, or renal dysfunction and that the increase is proportional to the plasma norepinephrine level,30 suggesting that AM may act to counterbalance sympathetic activity. A recent report indicated that AM immunoreactivity is markedly increased in the ventricles of patients with congestive heart failure and that plasma AM levels are also increased,8 suggesting a potential role for this peptide in the neurohumoral activation that characterizes human congestive heart failure. It is also uncertain whether or not the negative inotropic effect of AM in such a setting is beneficial. A recent study using an ischemic heart model showed that intravenous infusion of SPM-5185, an NO donor, significantly reduced the area of myocardial necrosis after coronary occlusion, suggesting the possibility that supply of NO could be beneficial.31 AM may act as an in situ NO donor in the heart, but further investigations are necessary to determine the clinical importance of this peptide.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants 07670757 and 07557055 from the Japanese Ministry of Education, Culture, and Science.
Received September 23, 1996; revision received November 25, 1996; accepted December 21, 1996.
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