(Circulation. 1997;95:2303-2311.)
© 1997 American Heart Association, Inc.
Articles |
Effects of the Nitric Oxide Donor Sodium Nitroprusside on Intracellular pH and Contraction in Hypertrophied Myocytes
From the Charles A. Dana Research Institute, the Harvard-Thorndike Laboratory, and the Department of Medicine (Cardiovascular Division), Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Mass, and the Department of Medicine (K.W.S.), University of Utah School of Medicine, Salt Lake City.
Correspondence to Beverly H. Lorell, MD, Cardiovascular Division, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215.
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Abstract |
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Background We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls.
Methods and Results pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37°C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH=7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10-6 mol/L SNP caused a decrease in the amplitude of cell contraction (72±7% of baseline, P<.05) that was associated with a decrease in pHi (-0.10±0.03 U, P<.05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01±0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na+-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells.
Conclusions SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na+-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.
Key Words: calcium • cells • contractility • hypertrophy • nitric oxide
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Introduction |
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Nitric oxide donors and stimulation of the release of NO from endothelial cells depress LV pressure development in association with an earlier onset of relaxation in human studies1 2 and isolated papillary muscle preparations.3 4 Both the exogenous NO donor nitroprusside5 and the induction of NOS in vitro in response to exposure to inflammatory cytokines depress the contractility of paced adult myocytes.6
The effects of NO on the myocardial contractile response are postulated to be predominantly mediated by the intracellular messenger cGMP via a depression of myofilament sensitivity to intracellular Ca2+.2 3 4 5 6 7 8 However, the underlying mechanism is unknown, and the contribution of alterations in cytosolic Ca2+ versus pHi is controversial.
The objective of this study was to test the hypothesis that the effects of the NO donor SNP on cell contraction are mediated by changes in pHi. Because intracellular signaling and recovery from intracellular acidosis may differ in adult normal and hypertrophied cells, we also wished to determine if the response to nitroprusside differed in adult hypertrophied versus normal myocytes. Therefore, we compared the effects of SNP as well as the cGMP analogue 8-bromo-cGMP on myocyte contraction, pHi, and intracellular Ca2+ in isolated normothermic adult ventricular myocytes from aortic-banded rats and normal age-matched controls. We also examined the effects of SNP on recovery from intracellular acidification, which depends on the stimulation of Na+-H+ exchange. Our findings indicate that SNP causes a negative inotropic effect in normal adult myocytes that is predominantly mediated by intracellular acidification, which decreases myofilament sensitivity to Ca2+; in contrast, this effect does not occur in adult hypertrophied cells.
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Methods |
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Preparation of Aortic-Banded Rats
Weanling male Wistar rats (Charles River Breeding Laboratories) were banded at the age of 3 to 4 weeks (body weight, 75 to 100 g) by placing a 0.6-mm-ID stainless steel clip on the ascending aorta via a thoracic incision. Age-matched control rats underwent a sham operation. The rats were fed normal rat chow and water ad libitum and were used 12 weeks after banding. We have shown9 10 11 12 that at this stage after aortic banding, this model of pressure-overload hypertrophy is characterized by an increase in LV weight relative to sham-operated controls, concentric hypertrophy, and the absence of chamber dilatation.
Dissociation of LV Myocytes
LV myocyte isolation was performed13 by using a
modification of the methods of Capogrossi et al14 and
Haddad et al.15 Rats were anesthetized with an
injection of pentobarbital sodium (65 mg/kg body wt IP), and the heart
was rapidly excised and attached to an aortic cannula. Continuous
retrograde coronary perfusion was initiated at a perfusion
pressure of 70 cm H2O for control hearts and 100 cm
H2O for hypertrophied hearts. The heart was first perfused
with nominally Ca2+-free modified Krebs-Henseleit buffer of
the following composition (in mmol/L): NaCl 123, KCl 5.4,
MgSO4 1.2, NaH2PO4 1.2,
NaHCO3 20, and glucose 11. This medium was not recirculated
and was continuously gassed with 95% O2 and 5%
CO2 (pH 7.4) at 37°C. After 3 minutes of the initial
perfusion, the heart was perfused with recirculating Krebs-Henseleit
buffer supplemented with 0.6 mg/mL collagenase (class II,
Worthington Biochemical Corp), 0.04 mg/mL protease (type XIV, Sigma),
and 1 mg/1 mL BSA for 20 to 40 minutes. The heart was then detached
from the cannula, the LV was cut into small pieces, and dispersion of
the myocytes was performed by gentle agitation of
ventricular tissue through a serologic pipette in
Krebs-Henseleit buffer containing 100 µmol/L CaCl2
and 1 mg/mL BSA. The resulting suspension was then gently forced
through a 450-µm nylon screen filtration cloth into a 50-mL plastic
tube and rinsed twice. After this the myocytes were resuspended in
HEPES-buffered solution of the following composition (in mmol/L):
NaCl 137, KCl 5.4, MgSO4 1.2,
NaH2PO4 1.2, HEPES (free acid) 20,
CaCl2 1.2, glucose 15, and 5% FBS. The myocytes were
stored at 37°C for 1 hour.
Simultaneous Measurement of
[Ca2+]i and Cell Contraction
[Ca2+]i was measured with the
Ca2+-sensitive fluorescence indicator indo
1-AM16 (Molecular Probes, Inc) as
described13 17 by using a modification of the method of
duBell et al18 and Ikenouchi et al.19 First,
10 mL FBS was mixed with 234 µL of 25% pluronic F-127 (BASF
Wyandotte Corp) dissolved in dimethyl sulfoxide. After this, 1 mL of
1 mmol/L indo 1-AM in dimethyl sulfoxide was added to 9 mL of an
FBS–pluronic F-127 mixture, sonicated, and divided into 400-µL
aliquots that were stored at 37°C. Myocytes were attached on
coverslips with cell adhesive (Cell-Tak, Collaborative Research, Inc)
and loaded with 5 µmol/L indo 1-AM in HEPES-buffered solution at
room temperature for 30 minutes. The coverslip was rinsed with indo
1-AM–free buffer solution and placed in a flow-through heated (37°C)
cell-superfusion chamber on the stage of an inverted microscope
(Nikon). The instrumentation for fluorescence measurement has
been described.16 17 19 20 The excitation source was a
high-pressure mercury arc lamp that provided an intense emission peak
at 360 nm. Further selection of this excitation was made with
narrow-bandwidth interference filters. The excitation beam was chopped
at 360 Hz to reduce bleaching, and the myocyte was illuminated via
epifluorescent optics by using a Fluor x40 objective lens
(Nikon). The fluorescence light was collected by the objective
lens and transmitted to a custom-modified spectrofluorometer (FM-1000,
Rincon Scientific Instruments) for simultaneous measurement
of both 400- and 500-nm wavelengths by using two separate
photomultiplier tubes. The spectrofluorometer provided analog signals
representing the fluorescence intensity at both
wavelengths and the ratio of emitted fluorescence (400/500 nm).
The subtraction of background autofluorescence was done by
offsetting the photomultiplier tube outputs during the measurement of
fluorescence from an unloaded myocyte at the beginning of each
experiment. An adjustable iris was used to restrict the optical image
to only one myocyte of interest in each experiment to minimize
background fluorescence from other myocytes. The image of the
beating myocyte was obtained by illumination via the 50-W standard
microscope light source passed through a 645-nm band-pass filter. This
wavelength was long enough not to interfere with the
fluorescence detection at 400 and 500 nm. The motion of the
myocyte was monitored by using a solid-state camera (GP-CD60,
Panasonic) and a custom-modified video detector system20
(Crescent Electronics). The analog output of the cell-motion signal was
monitored and recorded continuously with the analog signal of the
[Ca2+]i-sensitive fluorescence ratio
(F400/500 nm). Two platinum electrodes placed in the bathing fluid were
connected to a stimulator (SD9G, Grass Instruments) and were used to
stimulate the myocyte at 0.5 Hz with 3-ms pulses.
Myocytes from 21 hearts from sham-operated rats and 16 hearts from aortic-banded rats were studied. The yield of viable myocytes, defined as the percentage of rod-shaped myocytes with clear striations and exclusion of Trypan blue, was 60% to 70% in control myocytes and 50% to 60% in hypertrophied myocytes. To prevent bias when we selected a myocyte to be analyzed among myocytes in a microscopic field, we chose a rod-shaped myocyte with very clear striations, without any spontaneous cell-motion oscillations, and with visually moderate cell-motion amplitude of contraction (5.0 to 10.0 µm) at a pacing rate of 0.5 Hz. This selection procedure was used to counter the potential bias of selecting excessively vigorous rather than depressed myocytes. One to three experiments were performed in sequence from separate coverslips of myocytes isolated from one heart. We have recently documented the stability of both cell motion and [Ca2+]i for the 15-minute duration of the protocols used in the present study and have demonstrated minimal compartmentalization of indo 1 in intracellular organelles in chemically skinned myocytes.13 To determine the absolute values of peak-systolic and end-diastolic [Ca2+]i under baseline conditions in hypertrophied and control myocytes, we performed calibration studies by using a modification of the methods of Cheung et al21 and Borzak et al.22 These methods have been described in detail.13
Measurement of pHi
pHi was measured with the pH-sensitive indicator
SNARF-1 AM (Molecular Probes, Inc).17 23 24 25 First, 50 µg
SNARF-1 AM was added to 50 µL dimethyl sulfoxide that had been mixed
with 450 µL FBS. Loading of SNARF-1 AM was done by exposing the
myocytes on coverslips to a final concentration of 8 µmol/L
SNARF-1 AM for 15 minutes at 37°C. The coverslip was rinsed with
SNARF-1 AM–free solution and placed on a flow-through cell chamber as
described above. Excitation was performed at 540 nm, and
fluorescence emission was collected simultaneously
at 580 and 640 nm by using the same optics system as described above
except for the substitution of different dichroic mirror and
interference filters. At the end of the experiment, the emission ratio
from each cell was calibrated in situ by exposing cells to solutions of
varying pH. Each solution contained (in mmol/L) K+ 140
(adjusted to keep [K+] constant), MgCl2 1.0,
HEPES 4.0, EGTA 2.0, 2,3-butanedione monoxime 30, BAPTA-AM (Molecular
Probes, Inc) 50 µmol/L, and nigericin (Sigma) 14 µmol/L
and was titrated to varying pH values (6.7, 6.85, 7.0, 7.2, and 7.4) by
using 1.0N KOH. Since changes in pH are not linear to the ratio of
fluorescence emission,25 pHi was
calculated by the equation
pHi=(ax+c)/(1+bx), where
x is the measured emission ratio, and a,
b, and c are constant
parameters.24 25 26
Experimental Protocol
Effects of Nitroprusside on Cell Contraction and
[Ca2+]i
Hypertrophied (n=16) and control (n=26) myocytes were superfused
with oxygenated HEPES-buffered normal Tyrode's solution of
the following composition (in mmol/L): NaCl 137, KCl 3.7,
MgCl2 0.5, HEPES (free acid) 4.0, CaCl2 1.2,
glucose 11, and probenecid 0.5 (final pH, 7.40). Probenecid, a blocker
of organic anion transport, was added because it inhibits the secretion
of both indo 1 and fura 2 free acids from loaded cells.27
The myocytes were maintained at 36°C to 37°C and were paced at 0.5
Hz. After baseline data were recorded, the cells were superfused
with an oxygenated HEPES-buffered solution containing SNP
(Sigma) over a concentration range of 10-10,
10-8, and 10-6 mol/L.
Each SNP concentration was studied in a separate group of myocytes
(five to nine cells per group) in the presence of a 1.2-mmol/L
[Ca2+] perfusate for 10 minutes. The analog
cell-motion signals and the F400:500-nm analog signals were
recorded simultaneously. The F400/500 values were
converted to [Ca2+]i by using the procedure
described in the previous section.
Effects of Nitroprusside on pHi
To determine the effects of 10-6 mol/L
SNP on pHi, separate experiments were performed in
hypertrophied (n=7) and control (n=7) myocytes loaded with SNARF-1.
Myocytes were first superfused with the same HEPES-buffered solution
that was used for baseline superfusion to eliminate the effects of
Cl-/HCO3- exchange and
Na+/HCO3- exchange on
pHi. The superfusate was then switched to a
solution with 10-6 mol/L SNP for 6 minutes. In
additional experiments in hypertrophied (n=11) and control (n=14)
myocytes, the effects of an NH4Cl pulse in the presence and
absence of 10-6 mol/L SNP was examined by
abrupt exposure and washout of HEPES-buffered solution containing
10.0 mmol/L NH4Cl that had been prepared by equimolar
replacement of NaCl with NH4Cl. The abrupt exposure to
NH4Cl initially increases pHi as basic
NH3 rapidly enters the myocyte.24 25
pHi then falls as changed NH4+
enters the cell via K+ channels and dissociates. Upon
washout of extracellular NH4Cl, intracellular acidosis is
created as internal NH3 leaves the cell, causing the
intracellular retention of H+. The initial recovery from
this intracellular acidosis depends predominantly on forward
Na+-H+ exchange.24 28 To determine
the relationship between cell shortening and pHi in the
absence of SNP, transient intracellular acidification was induced by a
pulse and washout of NH4Cl in additional adult myocytes
(n=26). The same protocol was performed sequentially in the myocytes to
assess pHi and contraction since optical considerations do
not permit simultaneous measurements of SNARF-1
fluorescence and detection of cell motion.
Contribution of Na+-H+ Exchange
To further assess the contribution of
Na+-H+ exchange to SNP-induced acidification,
additional SNARF-1–loaded control (n=5) and hypertrophied (n=5)
myocytes were perfused with 10 µmol/L EIPA (Sigma), which
inhibits Na+-H+ exchange. As described above,
all experiments were performed using HEPES-buffered solutions with an
extracellular pH of 7.40. The pHi was measured at baseline
and after 6 minutes of exposure to 10 µmol/L EIPA, followed by 6
minutes of exposure to 10-6 mol/L SNP in the
presence of EIPA. To directly examine the effects of inhibition of
forward Na+-H+ exchange, control (n=7) and
hypertrophied (n=6) myocytes were abruptly exposed to a solution
containing 0 Na+–0 Ca2+ (no added
Ca2+) in which
N-methyl-D-glucamine
(Sigma)24 was substituted for NaCl. Calcium was removed to
prevent Ca2+ entry via Na+–0 Ca2+
exchange, which may modify pHi. After pHi
showed stability (
15 minutes), the cells were also perfused with
10-6 mol/L SNP for 6 minutes.
Effects of 8-Bromo-cGMP on pHi
To examine the potential role of cGMP in control and
hypertrophied myocytes, the effects of the cGMP analogue 8-bromo-cGMP
(10-5 mol/L; Sigma) on pHi were
compared in control (n=7) and hypertrophied (n=5) myocytes loaded with
SNARF-1. The same protocol was performed in an additional series of
control (n=5) and hypertrophied (n=6) myocytes to measure cell
contractions. The effect of 8-bromo-cGMP on recovery from the
intracellular acidosis induced by an NH4Cl pulse was also
examined in control (n=11) and hypertrophied (n=13) myocytes.
Statistical Analysis
Two-way ANOVA with repeated measures was used to compare the
values measured in response to exposure to SNP or 8-bromo-cGMP for
control and hypertrophied myocytes. ANOVA for repeated measures was
used to compare the responses of control and hypertrophied cells during
recovery from the acidification induced by an NH4Cl pulse.
An unpaired Student's t test was used for comparisons of
the values between the groups at baseline. A probability value of <.05
was considered significant. Results are expressed as mean±SEM.
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Results |
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Characteristics of Isolated Normal and Hypertrophied Myocytes
The extent of hypertrophy in the aortic-banded and sham-operated control rats is shown in Table 1
; Table 2 shows the baseline function of LV control myocytes
from sham-operated rats and hypertrophied myocytes from aortic-banded
rats. Myocytes were superfused with oxygenated
HEPES-buffered solution (pH 7.40) containing 1.2 mmol/L
CaCl2 and were paced at 0.5 Hz at 37°C.
Diastolic cell length was slightly increased in
hypertrophied myocytes compared with controls. The amplitude of cell
shortening and percent fractional cell shortening were not
significantly different between the groups. The time to peak shortening
and the time to 50% relengthening were slightly prolonged in the
hypertrophied cells relative to the control cells. Under the
experimental conditions of this study, systolic and
diastolic [Ca2+]i values using
the indicator indo 1 were similar between the two groups; these values
are similar to those reported by us13 and
others22 29 in adult rat myocytes. The values of
pHi using the indicator SNARF-1 were similar in control
(n=19) and hypertrophied (n=15) myocytes; these values are similar to
measurements in isolated ventricular
myocytes.19 25 26
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Effects of SNP on Cell Shortening
The effects of SNP on fractional cell shortening were examined in
the presence of 10-10,
10-8, and 10-6 mol/L
SNP in separate groups of control and hypertrophied myocytes. SNP
caused a maximal negative inotropic effect at a concentration of
10-6 mol/L in the control myocytes that was
manifested as a reduction in the amplitude of contraction to 72±7% of
baseline (P<.05). In contrast,
10-6 mol/L SNP did not depress fractional cell
shortening in the hypertrophied myocytes (87±5% of baseline, NS). Fig 1 illustrates that the depressant effect of SNP on cell
shortening in the control myocytes was associated with an abbreviation
of the duration of cell contraction and a slight increase in
diastolic cell length, whereas these effects were not
observed in the hypertrophied myocytes. SNP
(10-6 mol/L) significantly decreased the time
to 50% relengthening in the control but not the hypertrophied myocytes
(Table 3). The effects of 10-6
mol/L SNP on intracellular Ca2+ were examined by using the
indicator indo 1 in the presence of 1.2 mmol/L Ca2+.
There was no effect of 10-6 mol/L SNP on peak
systolic [Ca2+]i in either control
(99.4±1.0% of baseline, NS) or hypertrophied (100.2±0.1% of
baseline, NS) myocytes, nor was there any effect on the time course of
the [Ca2+]i transient (Fig 1).
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Effects of SNP on pHi
The effects of 10-6 mol/L SNP on
pHi in control and hypertrophied myocytes are shown in Fig 2. At baseline, pHi was similar in control
and hypertrophied myocytes (7.10±0.01 versus 7.07±0.02 U, NS). SNP
caused a significant fall in pHi (-0.10±0.03 U,
P<.05) in the control myocytes. In contrast, the
hypertrophied myocytes showed no change in pHi
(-0.01±0.01 U, NS) in response to SNP.
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Additional experiments were done to determine if a similar degree of
intracellular acidification depresses contractility in
adult myocytes when it is caused by an intervention other than SNP. Fig 3 shows the relationship between pHi and
fractional shortening expressed as a percent of baseline in normal and
hypertrophied myocytes obtained during maximal acidification and after
an initial 1 minute of recovery after an NH4Cl pulse in the
absence of SNP. The mean values of pHi and fractional
shortening are redisplayed for the normal myocytes described above,
which were studied at baseline and in response to
10-6 mol/L SNP. These data show that there is
a linear relationship between the decrease in pHi and the
depression of contractility in both normal and
hypertrophied myocytes. Thus, the absence of a depressant effect of SNP
on contractility in the hypertrophied myocytes is not
due to a difference in the relationship between intracellular
acidification and contractility in normal and
hypertrophied cells. Second, these data show that a similar magnitude
of intracellular acidification caused by the intervention of an
NH4Cl pulse compared with the intracellular acidification
caused by SNP depresses contractility to a similar
extent in normal myocytes.
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To assess the potential contribution of Na+-H+
exchange on the effects of SNP, additional cells were exposed to
10 µmol/L EIPA for 6 minutes (to inhibit
Na+-H+ exchange) and then exposed to
10-6 mol/L SNP for 6 minutes during continued
perfusion with EIPA. EIPA alone did not modify baseline pHi
in either the control (-0.01±0.01 U, NS) or hypertrophied
(-0.01±0.02 U, NS) myocytes. In the presence of EIPA,
10-6 mol/L SNP failed to cause intracellular
acidification in either the control or hypertrophied myocytes. To
directly compare the contributions of forward
Na+-H+ exchange on pHi in the
control and hypertrophied myocytes, forward
Na+-H+ exchange was abruptly inhibited by
exposure to HEPES-buffered solution containing 0 Na+–0
Ca2+. Abrupt inhibition of forward
Na+-H+ exchange caused a more rapid and larger
intracellular acidification in control than hypertrophied myocytes (Fig 4, left). Steady-state levels of pHi were
reached after 15 minutes of inhibition of
Na+-H+ exchange, after which exposure to
10-6 mol/L SNP caused no further acidification
in control or hypertrophied myocytes (Fig 4, right).
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To further assess the potential contribution of an NO donor on recovery
from intracellular acidosis, additional control and hypertrophied
myocytes were exposed to an abrupt pulse and washout of
NH4Cl in the presence and absence of
10-6 mol/L SNP. Abrupt exposure to
NH4Cl initially increases pHi as basic
NH3 rapidly enters the cell.25 26 Charged
NH4+ then enters the cell via K+
channels and dissociates. Upon rapid washout of extracellular
NH4Cl, intracellular acidosis is created as NH3
leaves the cell, causing the intracellular retention of H+.
Recovery from this intracellular acidosis depends predominantly on
forward Na+-H+ exchange.24 28 The
rate of recovery from maximum intracellular acidification is similar in
control and hypertrophied cells at baseline.26 In the
presence of SNP, the rate of recovery from the intracellular
acidification was depressed in the control compared with the
hypertrophied myocytes (P<.004) (Fig 5, left).
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Effects of 6-Bromo-cGMP on Cell Shortening and
pHi
The effects of NO on myocardial contractions are postulated to be
partly mediated by the intracellular messenger cGMP, which
activates protein kinase G and modulates cAMP levels via
inhibition of cAMP phosphodiesterases. To examine this potential
mechanism, additional control and hypertrophied myocytes were exposed
to the cGMP analogue 8-bromo-cGMP (10-5
mol/L); steady-state changes in fractional cell shortening and
[Ca2+]i were measured after 6 minutes of
perfusion. Exposure to 8-bromo-cGMP caused a significant depression of
fractional cell shortening in the control myocytes (74±9% of
baseline, P<.05) in the absence of a change in peak
systolic [Ca2+]i (101±1% of
baseline, NS). In the control myocytes, 8-bromo-cGMP caused a
significant fall in pHi (-0.04±0.01 U,
P<.05), suggesting that its negative inotropic effects were
related to intracellular acidification. In contrast, in the
hypertrophied myocytes, 8-bromo-cGMP had no effect on fractional cell
shortening (97±6% of baseline), peak systolic
[Ca2+]i (104±1% of baseline), or
pHi (-0.01±0.02 U).
We also studied the effects of 10-5 mol/L
8-bromo-cGMP on recovery from the intracellular acidification induced
by an NH4Cl pulse. Like SNP, 8-bromo-cGMP depressed the
rate of recovery from acidification in the control compared with the
hypertrophied myocytes (Fig 5
, right). Thus, the effects of the NO
donor SNP and 8-bromo-cGMP on cell shortening, pHi, and
recovery from intracellular acidification are similar in normal adult
myocytes, whereas these depressant effects on
contractility and pHi are not observed in
adult hypertrophied myocytes.
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Discussion |
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In the present study, the NO donor SNP depressed the amplitude of cell shortening and promoted an earlier onset of relaxation in isolated normal adult rat myocytes. The predominant mechanism of this depression of the contractile response was due to myofilament desensitization to calcium due to intracellular acidification rather than changes in intracellular calcium. Moreover, exposure to the cAMP analogue 8-bromo-cGMP caused similar changes in myocyte contractility and pHi in control myocytes. In contrast, the effects of SNP as well as 8-bromo-cGMP were strikingly different in adult hypertrophied myocytes from rats with chronic aortic banding. SNP and 8-bromo-cGMP had no significant effects on myocyte contractility or pHi in hypertrophied myocytes.
Effects of NO on Cardiac Contraction
In the human heart, acute exposure to the exogenous NO donor SNP
via intracoronary infusion depresses LV developed pressure in
association with an earlier onset of relaxation.1 2
Similar cardiodepressant effects have been observed in humans in
response to the intracoronary infusion of substance P, which
releases NO from the coronary
endothelium.2 In clinical studies and
studies using isolated hearts from experimental animals, interpretation
of the direct effects of NO donors on contractile function is
confounded by potential simultaneous changes in
coronary blood flow, which modify coronary vascular
turgor and load.
For this reason, several studies have examined the direct effects of NO donors on contractile function in isolated cardiac muscle preparations and cardiac myocytes. Smith et al3 have shown that SNP depresses isometric tension development and causes premature tension decline in isolated papillary muscles. Similar effects on the contractile function of isolated papillary muscles have been observed in response to the paracrine action of endogenous NO released from endocardial cells by stimulation with bradykinin and cytokines.3 4 Weyrich et al30 failed to observe an effect of either NO donors or NOS inhibitors on the contractile function of unstimulated isolated adult rat papillary muscles or myocytes. However, the majority of recent studies have confirmed that NO donors depress adult cardiomyocyte shortening and promote premature myocyte relengthening. Brady et al5 report that SNP depresses the amplitude of isolated myocyte shortening. The induction of NOS in vitro in response to exposure to inflammatory cytokines depresses the contractility of paced adult rat myocytes.6 The depressant effects of NO are even more pronounced in the presence of ß-adrenergic stimulation. Both the exposure to NO donors and the induction of endogenous NOS activity by cytokines blunt the enhanced contractile response of isolated myocytes to ß-adrenergic agonists,7 31 and similar inhibitory effects of NO on ß-adrenergic stimulation have been observed in humans.32 The effects of NO on the myocardial contractile response are postulated to be predominantly mediated by the intracellular messenger cGMP, which activates protein kinase G and modulates cAMP levels via inhibition of cAMP phosphodiesterases7 8 ; however, non-cGMP effects, including the generation of reactive oxygen species, may also occur.33 34
Several downstream mechanisms could potentially contribute to the depressant effects of NO on myocardial contractile function. ß-Adrenergic stimulation is well recognized as increasing the amplitude of myocyte contraction and the intracellular calcium transient via increases in the inward L-type calcium current, and NO depresses ß-adrenergic–mediated increases in intracellular calcium.35 However, the mechanism of the depressant effect of NO donors on contractility in the absence of ß-adrenergic stimulation is controversial. Recent studies in isolated myocytes show that cGMP, acting through protein kinase G or the inhibition of cAMP phosphodiesterase, may decrease the inward L-type calcium current,36 37 38 an effect that could reduce free intracellular calcium. On the other hand, NO has also been reported to modulate calcium release from the sarcoplasmic reticulum in skeletal muscle and papillary muscles,32 39 which would be expected to promote an increase in intracellular calcium. Thus, the net effect of NO on the intracellular calcium transient is difficult to predict.
In the present study, we observed that neither the NO donor SNP nor 8-bromo-cGMP caused steady-state depression of the amplitude of the intracellular calcium transient. In contrast, both SNP and 8-bromo-cGMP decreased the contractile response to calcium in normal adult myocytes. These observations are consistent with the findings of Shah et al,8 who have observed that in intact isolated rat myocytes loaded with the fluorescence indicator indo 1, exposure to 8-bromo-cGMP causes a very transient initial positive inotropic effect and an increase in the calcium transient. These initial effects are followed by a sustained, steady, stable negative inotropic effect that is not associated with a reduction in the amplitude of the calcium transient. Using analyses of steady-state tetanic contracture and the response to KT5823, a relatively specific inhibitor of protein kinase G, they concluded that 8-bromo-cGMP depresses the myofilament response to calcium via activation of protein kinase G, but they did not elucidate the underlying mechanism.8
NO and Intracellular Acidification
Our novel observations provide support for the theory that the
predominant mechanism of the negative inotropic effect of NO in adult
myocytes is the induction of intracellular acidification. Intracellular
acidification is well established as a mechanism that depresses
myofilament sensitivity to calcium and causes reductions in tension
development and contraction amplitude and an increase in cell
length.25 40 41 42 Using washout of an NH4Cl
pulse to cause abrupt intracellular acidification, we showed that a
similar degree of intracellular acidification caused by an intervention
other than SNP depressed contractility to a similar
extent in normal adult myocytes. These data lend support to a
cause-and-effect relationship between the intracellular acidification
caused by SNP and the depression in contractility. Our
findings do not exclude the possibility that other factors, such as the
presence of inorganic phosphate or the effects of protein kinase G on
the calcium sensitivity of troponin C,43 44 may contribute
to the modulation of myofilament sensitivity by NO. We observed that
inhibition of the sodium-proton exchange with EIPA prevented
SNP-induced intracellular acidification in normal myocytes.
In addition, NO markedly impaired recovery from the acute intracellular acidification induced by an NH4Cl pulse in control compared with hypertrophied myocytes, which depends on the stimulation of the forward sodium-proton exchange. These findings suggest that NO, via cGMP, may acutely impair contractility by disabling the forward sodium-proton exchange. In the present study, the direct inhibition of forward Na+-H+ exchange by exposure to a solution containing 0 Na+–0 Ca2+ also caused a more rapid and severe intracellular acidification in control than hypertrophied cells. Since extracellular Ca2+ was excluded, this difference is not related to Ca2+ entry via the Na+-Ca2+ exchange, which can modify pHi.45 Nor is this difference due to differences in intrinsic buffering power (Bi), since we have shown that Bi is comparable in control and hypertrophied cells from this model.26 The source of intracellular proton accumulation in response to disabling the forward Na+-H+ exchange is likely to be related to metabolic acid production, although a small contribution from transient reverse Na+-H+ exchange and passive proton influx due to the membrane potential cannot be excluded.
This study has several potential limitations. The changes in contraction amplitude and the earlier onset of relaxation are similar to changes observed in isolated papillary muscles and isolated hearts in response to "physiologically" derived NO from endothelial and endocardial cells. Nonetheless, it is important to recognize that the actual concentration of biologically active NO delivered to the myocyte target in response to exposure to SNP is difficult to estimate and to correlate with in vivo concentrations.46 Second, the effects of exogenous NO donors on contractility may be modulated or blunted by activation of the constitutive calcium-sensitive nitrate oxide synthase that is present in adult rat myocytes.35 In this regard, Kaye et al47 report that high pacing frequencies (3 Hz) increase constitutive NOS (NOS3) activity, NO production, and intracellular cGMP content in isolated adult rat myocytes, whereas this induction is not observed at lower pacing rates (0.5 Hz). For this reason, the present study was performed at a pacing frequency of 0.5 Hz to avoid the confounding variable of pacing-induced activation of constitutive NOS.
Differing Effects in Hypertrophied Versus Normal Myocytes
Our observation that the NO donor SNP did not modify
contractility or promote intracellular acidification in
hypertrophied rat myocytes is novel and unsuspected. This model of
pressure-overload hypertrophy due to ascending aortic
banding has been extensively characterized by our laboratory. At this
stage of chronic pressure overload, animals are characterized by
concentric LV hypertrophy with preserved systolic
shortening indices, the absence of chamber dilation, and a predominant
increase in myocyte width with a slight increase in myocyte
length.9 10 11 12 26 Consistent with these
characteristics, fractional cell shortening was similar in the control
and hypertrophied myocytes in the present study. Thus, the failure
to observe a negative inotropic effect of SNP in the hypertrophied
myocytes is unlikely to be due to severe depression of contractile
function at baseline. The different effects of SNP on intracellular
acidification in control and hypertrophied cells could also potentially
be related to differences in intrinsic Bi in hypertrophied
and normal myocytes. However, we have demonstrated26 that
Bi is comparable in control and hypertrophied myocytes from
this aortic-banded model. In addition, we have shown that the effects
of intracellular acidification per se on contractility
are similar in normal and hypertrophied myocytes. In the present
study, the observation that the cGMP analogue 8-bromo-cGMP simulated
the effects of SNP on pHi in the normal but not the
hypertrophied cells suggests that downstream signaling effects of cGMP
on the sodium-proton exchange may be blunted in pressure-overload
hypertrophy, which could be related to increased
constitutive NOS activity.47 In this regard, Kelm et
al48 have shown an increased basal production of
NO in hypertrophic hearts from spontaneously hypertensive rats. A
limitation of the present study is the possibility that our
observations may apply only to hypertrophied adult rat cardiac
myocytes. Thus, additional studies will be needed to confirm our
observations in other species and models of pressure-overload
hypertrophy.
Integrated Cardiac Paracrine Signaling: Role of
pHi
The potential importance of the modulation of pHi in
the regulation of the contractile response in
well-oxygenated myocardium is not unique to the
actions of NO. Several studies have shown that the positive inotropic
effects of both endothelin and angiotensin II in adult
myocytes are predominantly mediated by an increase in myofilament
calcium sensitivity due to intracellular alkalization rather than a net
increase in intracellular calcium.17 23 24 26 Relevant to
the potential role of sodium-proton exchange in the signaling of NO,
the positive inotropic effects of endothelin and
angiotensin II appear to be related to activation of the
sodium-proton exchanger. Taken together, these observations suggest the
presence of complex and opposing paracrine signaling pathways of NO and
endothelin in the heart, the acute effects of which on contractile
function, and potentially cell growth, are mediated by opposing effects
on pHi. Our observations support the hypothesis that the
coupling of NO signaling with the Na+-H+
exchange differs in adult rat normal and hypertrophied myocytes.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by National Heart, Lung, and Blood Institute grants HL-38189 (B.H.L. and N.I.) and HL-42873 (K.S.) and a US Fogarty Fellowship (J.B.). We greatly appreciate the suggestions of Dr William Barry regarding the analysis of pHi. We also appreciate the help of Tony L. Baptista in the preparation of the manuscript.
Received October 9, 1996; revision received November 22, 1996; accepted November 27, 1996.
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References |
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