© 1997 American Heart Association, Inc.
Articles |
Mechanical Transduction of Nitric Oxide Synthesis in the Beating Heart
From the Department of Medicine (D.J.P.), Columbia University College of Physicians and Surgeons, New York, NY, and the Department of Chemistry and Institute of Biotechnology (D.J.P., S.P., S.M., V.B., E.K., S.G., T.M.), Oakland University, Rochester, Michigan.
Correspondence to Dr Tadeusz Malinski/Dr David J. Pinsky, Oakland University, Department of Chemistry and Institute of Biotechnology, Rochester, MI 48309-4401.
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Abstract |
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Abstract NO alters contractile and relaxant properties of the heart. However, it is not known whether changes in ventricular loading conditions affect cardiac NO synthesis. To understand this potential contractile-relaxant autoregulatory mechanism, production of cardiac NO in response to mechanical stimuli was measured in vivo using a porphyrinic sensor placed in the left ventricular myocardium. The beating rabbit heart exhibited cyclic changes in [NO], peaking at 2.7±0.1 µmol/L near the endocardium and 0.93±0.20 µmol/L in the midventricular myocardium (concentrations were 15±4% lower in the rat heart). In the present study, we demonstrate for the first time that increasing or decreasing ventricular preload in vivo is followed by parallel changes in [NO], which may represent a novel autoregulatory mechanism to adjust cardiac performance or perfusion on a beat-to-beat basis. To quantify the relationship between applied force and NO synthesis, intermittent compressive or distending forces applied to ex vivo nonbeating hearts were shown to cause bursts of NO synthesis, with peak [NO] linearly related to ventricular transmural pressure. Experiments in which denuding cardiac endothelial and endocardial cells abrogated the NO signal indicate that these cells transduce mechanical stimulation into NO production in the heart. Taken together, these studies may help explain load-dependent relaxation, cardiac memory for mechanical events of preceding beats, diseases associated with myocardial distension, autoregulation of myocardial perfusion, and protection from thrombosis in the turbulent flow environment within the beating heart.
Key Words: left ventricular myocardium • mechanical stimulus • porphyrinic sensor • rabbit • rat
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The beating heart responds rapidly to local changes in loading conditions, with increased cardiac output observed after simple volume infusion.1 2 Even when denervated after cardiac transplantation, the heart maintains its ability to autoregulate cardiac performance in response to increased venous return. Although several mechanisms underlying increased cardiac contractility in response to passive stretch have been identified,3 investigators have posited the existence of rapidly acting autoregulatory mechanisms to explain certain aspects of the beat-to-beat regulation of cardiac performance.4 In particular, the mechanism(s) underlying cardiac memory for mechanical events of preceding beats and load-dependent relaxation is incompletely understood.5
NO may be produced within the heart by either constitutive or inducible NO synthase.6 7 8 9 Recent studies have shown that endogenous NO agonists, exogenous NO donors, or increasing intracellular cGMP will hasten myocardial relaxation (positive lusitropy).10 11 In addition to their relaxation-hastening effects, NO and cGMP appear to directly depress ventricular contractility (negative inotropy).12 13
Conversely, the effects of changes in physiological load on instantaneous cardiac NO synthesis remain unknown. To understand this potentially important cardiac autoregulatory mechanism that may modulate both myocardial lusitropy, inotropy, and flow on a beat-to-beat basis, we considered whether altering loading conditions could modulate endogenous cardiac NO synthesis. In the present study, we demonstrate for the first time that altering ventricular filling in beating hearts in vivo or altering mechanical force on ex vivo hearts is followed by a parallel increase or decrease in cardiac NO synthesis. This appears to represent a novel autoregulatory mechanism that may be relevant to cardiac physiology as well as to clinical conditions associated with pathological myocardial distension.
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Materials and Methods |
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[NO] was measured using a catheter-protected porphyrinic microsensor prepared in a previously described manner.14 15 16 Two techniques for measuring [NO], DPV and CA, were performed with a PAR model 273 voltammetric analyzer interfaced with an IBM 80486 computer with Lab-View software, which sampled at 4 kHz and amplified and recorded the analytical signal. DPV was used to measure the basal [NO]. Briefly, in the DPV method, current versus potential curves are generated as current is plotted against a potential (range between 0.40 and 0.70 V versus SSCE). The DPV peak current at the peak potential characteristic for NO oxidation (0.63 V) was found to be directly proportional to the [NO] in the immediate vicinity of the sensor. CA, fixed at the peak potential for the oxidation of NO versus SSCE, was used for fast (resolution time, 0.1 to 1 ms) and continuous measurement of the changes of [NO] from its basal level with time. Both techniques were performed using the three-electrode mode: a catheter-protected porphyrinic microsensor working electrode, a platinum wire counter electrode, and a reference SSCE.
The volume sampled is approximately equal to the volume of the sensor (10-10 to 10-12 L). Therefore, the [NO] measured was a local or surface concentration (not a bulk or global concentration). The porphyrinic microsensor had a response time of 0.1 ms at micromolar [NO] and 10 ms at the detection limit of 1 nmol/L. Linear calibration curves were constructed for each sensor from 5x10-9 to 2x10-5 mol/L NO, before and after in vivo or in vitro measurements, with aliquots of saturated NO prepared as described previously.17
Measurement of NO amidst the dynamic in vivo conditions of cyclic breathing and heart beating is a challenging task. In order to overcome these potential interferences and to record a reliable NO signal, we had to modify the porphyrinic sensor from our previously published16 in vivo design in two ways. First, the active sensor tip was shortened to 50 to 60 µm from the previously described 3 to 5 mm.16 Also, the truncated needle from which the active sensor tip emerges was cut 50 to 60 µm shorter than its protective catheter so that the tip of the sensor was completely recessed within the ventilated catheter tip, rather than protruding from an unventilated catheter tip as previously described.16 The completed prototype appeared very similar to a previously published illustration15 ; it was tested under different experimental conditions to ensure that the sensor did not generate an analytic signal when subjected to conditions of fluid pressure, mechanical deformation of the sensor tip, or intrinsic electrical activity within the heart.
New Zealand White rabbits (4 kg) or Wistar-Kyoto rats (300 g) were anesthetized (50 mg/kg ketamine and 5 mg/kg xylazine for rabbits; 100 mg/kg ketamine and 10 mg/kg xylazine for rats), intubated, and ventilated with room air using a Harvard small animal ventilator (tidal volume of 25 mL and rate of 70 breaths/min for rabbits; tidal volume of 2.5 mL and rate of 100 breaths/min for rats). After a median sternotomy was performed, cardiac [NO] was measured as follows: To implant the porphyrinic NO sensor, ventricular tissue was pierced with a standard 0.8-mm-diameter angiocatheter needle (clad with its catheter with two 100-µm ventilation holes near the tip). The catheter/needle unit was advanced to a desired place in the heart. The position of the catheter was secured, and the placement needle was removed and quickly replaced with a porphyrinic NO sensor mounted in a truncated needle. Confirmation of myocardial localization of the active tip of the sensor was by postmortem sectioning of the heart.
Cardiac index was calculated from cardiac output measurements obtained using a Doppler flow probe (Transonics) positioned at the aortic root. A 5F micromanometer-tipped catheter (Millar Instruments) was inserted into the left ventricle via the right carotid artery under constant pressure monitoring. This was calibrated using a transducer control unit (TC-510, Millar Instruments) and zeroed to pressure at the level of the left ventricle. The left ventricular pressure signal was sampled at 4 kHz and amplified and recorded with Lab-View software (IBM computer). In certain experiments, to increase preload, saline was given as a rapid intravenous bolus (10 mL, 37°C), and myocardial NO levels were recorded in the beating heart. After several minutes, when the heart had returned to baseline conditions, preload was reduced by snaring the venae cavae for 5 to 8 seconds. For certain experiments, L-NMMA (Sigma Chemical Co) was administered intravenously or into the proximal left anterior descending artery at the indicated doses.
Measurement of NO Release and Ca2+ Flux From
Isolated Cells
Endocardial cells were mechanically removed from the surface of
the left ventricle. Coronary vascular
endothelial cells were flushed from the
coronary vasculature with lactated Ringer's solution (Baxter)
after being loosened with collagenase (Sigma) applied in a
brief pulse down the coronary arteries.18 These
two primary isolates were plated on round coverslips maintained in
HEPES-buffered saline (Sigma) at 37°C for later experiments. To
measure [NO], an L-shaped thermally sharpened14
porphyrinic microsensor (diameter, 2 to 3 µm; length, 5 to
7 µm) was positioned parallel to the cell surface within a
distance of 2 to 3 µm from the membrane surface, using a
stereotactic micromanipulator under direct visual guidance.
Mechanical force was generated by a pulse of saline solution injected
from a computer-controlled nanoliter injector positioned at a fixed
vertical distance from the cell surface. Applied force was measured
with a piezoelectric microtransducer.
Ca2+ was measured using a fura 2-AM fluorescent method as described previously.19 The cells were loaded with 0.5 µmol/L of the indicator fura 2-AM (Sigma) in physiological salt solution for 25 minutes. Typically, [Ca2+]i images were calculated from an average of two successive background-subtracted video frames recorded after 360- and 380-nm excitation.
Ex Vivo Measurement of [NO] in the Nonbeating Heart in Response
to Applied Compression and Endothelial Denudation
A heart was rapidly excised from a 2.5-kg male New Zealand White
rabbit after pentobarbital anesthesia and placed between
two Lucite plates connected to a force transduction system, which was
immersed in Hanks' balanced salt solution without phenol red (Sigma)
at 37°C. [NO] was measured, with the intermittent application of
mechanical compression, using a catheter-protected porphyrinic
microsensor placed deeply between septal trabeculae and CA
recording (at 0.63 V) using a PAR model 264A voltammetric
analyzer. For certain experiments, cardiac
endothelial cells were denuded before [NO]
measurement by giving a brief (<20-s) pulse of the detergent Triton
X-100 (Sigma) down the coronary arteries, followed by a
lactated Ringer's flush. This treatment has been shown to denude
coronary microvascular endothelial cells,
leaving adjacent cells (including myocytes) intact.20
Histological confirmation of the effects of the Triton
X-100 pulse were obtained by fixing the heart in 10% formalin,
embedding in paraffin, and performing thin sectioning followed by
hematoxylin and eosin staining.
Measurement of NO Release in Response to Stretch
Male Sprague-Dawley rats (250 g) were euthanized and heparinized
intravenously (300 U heparin, Sigma), followed by rapid
cardiectomy. Hearts were incised, and a catheter-protected porphyrinic
microsensor was embedded deeply between septal trabeculae
in an orientation tangential to the applied force and immersed in 100
mL of lactated Ringer's solution (Baxter) at 37°C. The edges of the
opened left ventricle were secured with serrated arms attached to a
strain-gauge transducer for application and measurement of applied
stretch. The stretch was applied and released at the time points
indicated. In the last 60 s of each 120-s interval, a DPV
voltammogram (current versus potential) was recorded at a scan rate
of 5 mV/s from 0.40 to 0.70 V. Peak current was observed at 0.63 V,
which is the characteristic potential for NO oxidation on the
sensor.9 14 15
Release of [NO] in response to circumferential stretch was measured by obtaining rat hearts as described above, inserting a cannula into the cross-clamped aortic root, and flushing the coronary vasculature free of blood using an infusion of lactated Ringer's solution (1.6 mL/min controlled with a roller pump). A small incision was made in the left atrium, and a balloon-tipped catheter was introduced into the left ventricle through the mitral valve. The catheter was connected to a pressure manometer attached to a valved balloon inflation device, which enabled left ventricular end-diastolic pressures to be maintained and monitored at desired levels. The coronary arteries were perfused with lactated Ringer's solution under constant flow conditions (1.6 mL/min controlled with a roller pump); [NO] was measured in the coronary sinus effluent by DPV using a porphyrinic sensor without the protective catheter.
Ca2+ Dependence of Load-Dependent Cardiac [NO]
Release
Rat hearts were prepared for ex vivo measurement of [NO]
release in response to circumferential stretch (as described above).
Intracavitary pressure was varied by inflating the left
ventricular balloon. After baseline measurements were
obtained, a Ca2+ ionophore (A23187, 47.5 µmol,
Sigma) was introduced into the coronary perfusate. In
separate experiments, to determine the effects of Ca2+
chelation on load-dependent cardiac NO synthesis, intracellular and
extracellular Ca2+ chelation was accomplished by adding
EGTA (200 µmol/L, Sigma) and MAPTAM (200
µmol/L, Molecular Probes)21 to the
coronary perfusate, after which the load (intracavitary
pressure) was increased by inflating the left ventricular
balloon.
Data Analysis
Results are expressed as mean±SEM. In each set of experiments,
n is the number of animals studied. Statistical analysis was
performed using an unpaired Student's t test or by ANOVA
followed by Scheffé's F test. Means were considered
significantly different at P<.05.
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Results |
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By use of a porphyrinic microsensor to detect rapid fluctuations in [NO],16 in vivo measurements were performed in a beating rabbit or rat heart. Although this sensor has been used for both in vitro and in vivo measurements in prior published studies,9 14 16 several characteristics of the sensor were tested to ensure that recordings in the beating heart would reflect authentic [NO] rather than artifacts due to intramyocardial placement and consequent piezoelectric artifacts due to pressure changes or mechanical deformation of the sensor tip.
To demonstrate that the sensor does not respond with an electrical
signal to changes in pressure, recordings were made in an
electrolyte-filled sealed chamber, within which pressure was rapidly
altered. As can be seen in Fig 1a
, alterations in pressure have no measurable effect on the amperometric
current generated by the sensor, which was immersed in the confined
electrolyte solution.
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To rule out potential piezoelectric electrical interference due to
mechanical deformation of the sensor tip, a micromanipulator was used
to deform the sensor tip at a physiologically
relevant frequency (3 Hz). This generated only small electrical noises,
six times smaller than the signal recorded by the sensor at low (50
nmol/L) [NO] (Fig 1b
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To eliminate the possibility that the sensor might respond to the
electrical current generated within the intrinsic cardiac conduction
system or within the depolarizing myocardium, the
porphyrinic sensor was used as the exploring electrode connected to a
standard ECG. As is apparent from Fig 1c, when used in this manner, the
porphyrinic sensor is barely able to detect an electrical signal. In
these experiments defining the analytic response of the sensor to
potential piezoelectric or in vivo electrical current interferants, the
measured peak [NO] signals were in most cases at least 30 to 100
times larger and temporally shifted from the conservatively estimated
background noises.
An anesthetized rabbit was used to measure local fluctuations
in [NO] in the beating heart, with the sensor placed (via apical
puncture) in the apical left ventricular endocardium or
myocardium, as is shown schematically in Fig 2a. Controlling the depth of penetration
and postmortem confirmation of the location of the sensor tip were more
easily accomplished in rabbit than in rat hearts, and relatively
accurate data were produced in relation to the distribution of [NO]
in the wall of the rabbit heart. However, this method was not precise
enough for measurement of [NO] distribution in the relatively thin
wall of the rat heart. Therefore, most of the in vivo [NO]
measurements that required higher precision in relation to the
localization of the sensor were performed in the rabbit heart. Rapid
changes in cardiac [NO] related to the cardiac cycle were observed in
both rabbit and rat hearts. In the rabbit heart, each cardiac cycle
(period, 326±16 ms) began and ended with an intercycle [NO] of
0.67±0.16 µmol/L (n=20) near the endocardium (Fig 2b, upper tracing). During early systole, [NO] reached a basal level of
0.62±0.05 µmol/L (n=20), followed by a slow increase
(20±1 nmol · L-1 ·
ms-1) to a semiplateau. Early
diastolic filling was accompanied by a brisk rise of [NO]
(46±3 nmol · L-1 ·
ms-1), with a peak diastolic
[NO] of 2.7±0.1 µmol/L (n=20) that was attained at
239±17 ms into the cardiac cycle. After this peak, there was a sharp
decay (-30±3 nmol · L-1 ·
ms-1, n=20) to the intercycle [NO].
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A similar cyclic fluctuation of [NO] was observed in the
myocardium 1 to 2 mm beneath the endocardium (Fig 2b, middle tracing). However, the intercycle [NO] at this depth within
the myocardium was smaller (0.26±0.08
µmol/L, n=20), with a lower peak diastolic [NO]
of 0.93±0.2 µmol/L (n=20) and a slower decay rate
(-5±1 nmol · L-1 ·
ms-1, n=20). A similar pattern of [NO]
fluctuation was observed in the rat heart with the sensor placed in the
midventricular myocardium (Fig 2c). The
intercycle [NO] was 0.22±0.03 µmol/L (n=20), with a
lower peak diastolic [NO] of 0.79±0.04
µmol/L (n=20). Both of these concentrations were 15±4%
(P<.05) lower than that observed in the
myocardium of the rabbit. The [NO] signal recorded by
sensors in both rabbit and rat hearts disappeared when monitored at a
potential 0.40 V (which is 230 mV below the peak potential of NO
oxidation), clearly indicating that the signal measured at 0.63 V is
due to NO, not mechanical noises, which are applied independent of
potential (Fig 2b, lower tracing). To demonstrate the relationship
between intracavitary left ventricular pressure, the ECG
signal, and instantaneous [NO], synchronous recordings of
each of these were performed as described in "Materials and
Methods." Fig 3 represents a
simultaneous display of these three measured
parameters.
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To confirm that these measurements reflected authentic [NO], L-NMMA,
an inhibitor of NO synthase, was administered
intravenously (Fig 4a). Both
basal and peak [NO] were gradually (over several beats) but
significantly reduced; administration of L-NMMA (40 mg/kg)
diminished the peak [NO] in the endocardium to 0.95±0.07
µmol/L (n=20). In the underlying myocardium, peak
[NO] was also reduced (to 0.33±0.05 µmol/L, n=20).
Intravenous infusion of L-NMMA produced a
consistent rise in mean arterial pressure (from
76±2 to 89±3 mm Hg) and duration of the cardiac cycle (from
326±16 to 363±18 ms) and a consistent fall in the cardiac
index (from 123±7 to 109±5 mL ·
min-1 · kg-1),
left ventricular end-diastolic pressure (from
11±2 to 8±1 mm Hg), and left ventricular
systolic pressure (from 92±3 to 77±3 mm Hg).
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In order to separate the vasoconstrictive effect of L-NMMA in the peripheral vasculature from its effects on the heart itself, a separate experiment was performed: L-NMMA (20 µL, 200 µmol/L) was directly injected into the proximal left anterior descending coronary artery of the rabbit. The pattern of NO release as well as the peak [NO] was identical, within experimental error, to that observed after the intravenous infusion of L-NMMA: inhibition of NO production (from 1.57±0.06 to 0.90±0.07 µmol/L), decrease of cardiac index (from 123±7 to 108±4 mL · min-1 · kg-1), decrease of left ventricular end-diastolic pressure (from 11±1 to 8±1 mm Hg), and decrease of left ventricular systolic pressure (from 92±3 to 75±3 mm Hg) were observed. However, in contrast to the intravenous infusion of L-NMMA, mean arterial pressure in the first 30 s decreased from 76±3 to 65±2 mm Hg. These experiments suggest that L-NMMA, by inhibiting the release of NO, decreased the efficiency of the heart.
When preload was increased by a rapid intravenous bolus of
physiological saline, there was a gradual but
significant increase in both peak and basal [NO] in the beating heart
to 3.9±0.3 (n=7) and 0.80±0.12 µmol/L (n=7),
respectively (Fig 4b). When ventricular filling was reduced
by temporary ligation of the venae cavae, the [NO] decreased, also
gradually, over several beats to 0.81±0.05 µmol/L for
peak [NO] (n=7) and 0.10±0.05 µmol/L for basal [NO]
(n=7) (Fig 4c).
To determine which of the cell types within the heart that express
constitutive NO synthase activity might be responsible for
load-dependent cardiac [NO], additional studies were performed.
Endothelial cells or endocardial cells produced peak
[NO] of 0.97±0.25 µmol/L (n=9) and 0.50±0.18
µmol/L (n=9), respectively, after application of an external
mechanical force of 15±3 dyne/cm2 normal to the cell
membrane (as measured by a porphyrinic microsensor placed 2 to 3
µm from the cell membrane) (Fig 5a and 5b, upper tracings). In these
experiments, the [NO] decreased exponentially with increasing
distance, becoming undetectable at a distance of 35 to 50 µm
from the studied cell. Mechanically transduced [NO] release also
decreased (50±18%) in the presence of L-NMMA (Fig 5b, lower tracing).
In experiments using fura 2–loaded endothelial cells
to detect increases in [Ca2+]i, a rapid
increase in [Ca2+]i was observed to precede
the [NO] pulse (Fig 5a, lower tracing). When cardiac
endothelial cells were denuded in an ex vivo heart
using a brief pulse of Triton X-100 (a procedure that leaves myocytes
unscathed) (see Fig 6d),20
cardiac NO synthesis in response to applied force was greatly
diminished (compare Fig 6a with 6b). When a similar ex vivo heart was
examined without endothelial denudation, L-NMMA
significantly diminished the [NO] response to applied force,
confirming the measurements to be authentic [NO] (compare Fig 6a with
6c). Taken together, these experiments strongly suggest that
endothelial and endocardial cells within the heart are
the transducing cells responsible for load-dependent cardiac
[NO].
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In order to quantify the relationship between mechanically
applied forces and cardiac [NO], ex vivo studies were performed.
Initial observations were made in freshly excised nonbeating rabbit
hearts subjected to mechanical deformation, with local [NO] measured
with a sensor placed deep between septal trabeculae (Fig 6a). Each incremental application of force, normal to the exterior of
the heart (compressive force mimicking that of systole), was followed
by a CA signal after 50 ms, indicating a burst of NO. A linear
relationship between peak [NO] and applied force was observed, with
an increase of peak [NO] (from 0.14±0.02 to 1.10±0.08
µmol/L) (n=7) over the range of applied force (from
4.70x10-2 to
28.2x10-2 N/cm2). Also, cardiac
NO production was significantly inhibited by L-NMMA (Fig 6b).
In the presence of 100 µmol/L L-NMMA, [NO] decreased by
80±3% to 70±5% (n=7) over the range of applied forces from
4.70x10-2 to
28.2x10-2 N/cm2. As a control,
cardiac endothelial cells were denuded,20
and subsequent cardiac NO bursts in response to applied forces were
greatly diminished (Fig 6c). The efficiency of the denudation process
was tested with acetylcholine. Acetylcholine (1
µmol/L)–stimulated NO release decreased by 85±15%.
To characterize the cardiac NO synthesis that occurs in response to the
circumferential stretch (mimicking forces during diastole),
a balloon was inserted into the left ventricle through the left
atrium/mitral valve. Left ventricular transmural pressure
was varied by inflating the balloon, and [NO] was measured directly
in the coronary effluent (to estimate the portion of [NO]
that was released into the blood stream) with the porphyrinic
microsensor. During constant flow perfusion with lactated Ringer's
solution (1.6 mL/min), circumferential stretch was accompanied by an
increase in [NO] (Fig 7a and 7b). This
increase in [NO] was linearly related to transmural pressure over the
range of pressures studied (40±10 nmol/L of NO [n=5] at
0 mm Hg to 167±20 nmol/L [n=5] at 60 mm Hg).
Spiking the perfusate with the
[Ca2+]o chelator EGTA and the
membrane-permeable [Ca2+]i chelator
MAPTAM21 caused a decrease of [NO], both at baseline and
after application of transmural pressure (Fig 7c). In experiments
designed to facilitate [Ca2+]o entry, with
ventricular transmural pressure maintained at 0
mm Hg, addition of the Ca2+ ionophore A23187 to the
perfusate increased the [NO] more than that observed after
the application of maximal circumferential stretch (compare the
rightmost tracings of Fig 7a and 7c), suggesting maximal stimulation of
Ca2+-dependent NO synthesis. Taken together, these results
suggest a role for [Ca2+]i in the mechanical
transduction of load-dependent cardiac NO synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There have been a growing number of recent publications addressing the influence of NO on the contractile (inotropic) and relaxation (lusitropic) properties of cardiac myocytes and the heart.7 10 12 22 Endothelial shear stress has been shown to increase [NO] by stimulating a constitutive NO synthase.18 23 However, to date, there is no evidence that indicates that altering cardiac loading conditions independent of coronary blood flow affects NO synthesis in vivo. The present study demonstrates for the first time that NO is released in pulsatile fashion from the beating heart and that its synthesis is directly related to ventricular loading conditions in vivo. These results support the existence of a novel cardiac autoregulatory mechanism that may facilitate ventricular filling in times of increased demand. Furthermore, these data may help to explain certain aspects of the beat-to-beat regulation of cardiac performance and flow and provide insights into the pathophysiology of diseases associated with increased myocardial distension, such as valvular heart disease or heart failure.
If the heart were to function as a purely systolic (extrusion)
pump, without the ability to transduce changes in preload into parallel
changes in [NO], then within certain
physiological limits, abrupt changes in preload
should produce abrupt changes in cardiac output. In the present
study, we offer an explanation for why this is not so: we demonstrate
that abruptly increasing or decreasing ventricular preload
in vivo is gradually (not abruptly) followed by parallel changes in
[NO] over several cardiac cycles (Fig 4). This model may also provide
an explanation for several deviations observed from the Frank-Starling
model (a relatively static systolic pump model, which does not
reflect the dynamic interplay between systole and
diastole).1 2 24
There is also likely to be an additional beneficial reason for
increased cardiac [NO] under conditions of increased mechanical
stimulation. The turbulent blood flow within the beating heart provides
a potent stimulus for platelet adhesion and
aggregation.25 Since both platelet adhesion to
endocardial cells and platelet aggregation are inhibited by
NO,26 the unusually high load-dependent local [NO] (Fig 2) observed near the endocardium is likely to be of considerable
importance in inhibiting local platelet adhesion and aggregation in
the highly turbulent blood flow in the heart. It is possible that a
lack of this effect in poorly contracting hearts and artificial hearts
and on the surface of prosthetic cardiac valves may contribute
to the prothrombotic diathesis observed under these conditions.
Although different cell types within the heart express constitutive NO synthase activity, cardiac microvascular endothelial cells appear to be the predominant source of load-dependent cardiac NO synthesis. They are abundant within the heart and are located close to all cardiac myocytes. The endocardium itself also contributes significantly to load-dependent cardiac NO synthesis. However, rapid effects due to endocardial NO in the heart wall are only likely to occur in a narrow zone of subjacent myocardium because of diffusion constraints.14 27 Therefore, most of the endocardium-derived NO is rapidly dissipated into the intracavitary flow of ventricular blood, where it inhibits local platelet aggregation; this situation may be relevant to the possibility that hemoglobin may serve as a systemic carrier for NO.28
Myocytes also possess a constitutive Ca2+-dependent NO synthase,7 although in the present experiments, they do not represent a significant source of NO synthesis, at least in response to an applied load. These data are in good agreement with previous data reported for spontaneously beating neonatal ventricular myocytes, which do not release detectable [NO] in culture in the absence of adrenergic stimulation.29 These data are not surprising, given that the large [Ca2+]i transients during excitation and contraction are likely to dwarf increases in [Ca2+]o entry via mechanically gated cation channels.30 Furthermore, stimulation of the constitutive NO synthase in intact hearts causes lusitropic effects that are not observed in similarly stimulated ventricular papillary muscle preparations,10 suggesting a role for cardiac endothelial cells in NO-dependent effects on cardiac muscle.
There are likely to be several physiological
consequences of mechanical transduction of cardiac NO synthesis. Cells
within the heart are subjected to tremendous mechanical deformation
during filling and beating. In addition, there is substantial evidence
that NO directly affects mechanical properties of cardiac myocytes. NO
acts directly on myocytes via increases in cGMP to facilitate
relaxation and to mediate an acetylcholine-stimulated decrease in
contractility (negative inotropy).13
Coincidentally, previously published data clearly show that cGMP
concentration in myocytes fluctuates during each cardiac cycle,
reaching a sustained maximum just after the T wave.31 This
is in excellent agreement with the present study, which observes
the peak [NO] just after the T wave (Fig 3).
The present study is the first to identify load-dependent mechanical transduction of NO synthesis in the beating heart in vivo and to identify phasic changes in [NO] that occur during the cardiac cycle. Because NO and cGMP have negative inotropic and positive lusitropic actions on the heart, these studies suggest that mechanical transduction of cardiac NO synthesis and its short-term accumulation may provide an important autoregulatory mechanism for the modulation of myocardial contractility and relaxation in response to abrupt changes in preload.
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Selected Abbreviations and Acronyms |
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-monomethyl-L-arginine
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Acknowledgments |
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This study was supported in part by grants from the National Aeronautics and Space Administration, the American Heart Association, and the Public Health Service (HL-55397). We thank Y. Naka and S.T. Pinsky for expert technical assistance with these studies. E. Kubaszewski is on sabbatical leave from Poznan University of Technology.
Received November 18, 1996; accepted June 11, 1997.
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References |
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