(Circulation. 1996;93:356-364.)
© 1996 American Heart Association, Inc.
Articles |
Increased Release of NO During Ischemia Reduces Myocardial Contractility and Improves Metabolic Dysfunction
Presented in part at the 44th Annual Scientific Session of the American College of Cardiology, New Orleans, La, March 19-22, 1995.
From the First Department of Medicine, the First Department of Physiology (H.K.), and the Department of Pathophysiology (M.T.), Department of Information Science, Osaka University School of Medicine, Osaka, Japan.
Correspondence to Masafumi Kitakaze, MD, PhD, First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565, Osaka, Japan.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background We have reported that myocardial ischemia increases nitric oxide (NO) production. Several lines of evidence suggest that NO reduces myocardial contraction. Therefore, we tested whether endogenous NO decreases the inotropic response of the ischemic myocardium and whether endogenous NO is beneficial in the metabolic function of ischemic myocardium.
Methods and Results The left anterior descending coronary artery was perfused with blood from the left carotid artery in 72 dogs. An infusion of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, did not affect fractional shortening (FS) under nonischemic conditions. After reduction of perfusion pressure so that coronary blood flow decreased to 60% of the control value, FS of the perfused area decreased, and intravenous infusion of isoproterenol increased FS. Before and during intravenous infusion of isoproterenol under conditions of coronary hypoperfusion, FS was significantly increased in the L-NAME group compared with the untreated group. Both lactate extraction ratio and the pH in coronary venous blood were significantly lower in the L-NAME–treated group than in the untreated group during coronary hypoperfusion. Infusion of L-arginine prevented the effects of L-NAME in the ischemic myocardium.
Conclusions These results indicate that endogenous NO reduces myocardial contractile function and improves myocardial metabolic function in the ischemic heart. The myocardial energy–sparing effect as well as coronary vasodilation due to NO may be beneficial to the ischemic myocardium.
Key Words: myocardial contraction • ischemia • L-NAME • nitric oxide • receptors, adrenergic
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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NO is produced in the endothelium1 2 and other tissues of the heart, where it acts as both a paracrine and autocrine autacoid. Thus, NO activates guanylate cyclase in ventricular myocytes as well as in coronary smooth muscle cells. Recent studies with cardiomyocytes, isolated papillary muscles, and isolated hearts suggest that NO and other interventions that increase cGMP concentration may influence myocardial contractility.3 4 5 6 7 8 9 Negative inotropic effects of cytokines on the heart were reduced by an inhibitor of NOS, and production of NO in septic shock decreases myocardial contractility,10 11 12 13 14 suggesting that an enhanced release of NO may reduce myocardial contractility. Myocardial ischemia increases NO production in the heart.15 16 17 18 19 20 However, it is not clear whether NO released from the ischemic myocardium contributes to the reduction of myocardial contractility. If NO decreases myocardial contractility in the ischemic heart, it also remains to be determined whether this effect is mediated directly, by activation of guanylate cyclase, or indirectly, by inhibition of norepinephrine release from sympathetic nerve endings. Finally, it is not known whether the NO-induced decreases in contractility are beneficial or deleterious for the ischemic myocardium.
We have tested the hypothesis that endogenous NO released from the ischemic myocardium directly reduces the inotropic response to ß-adrenergic receptor stimulation and exposure to Ca2+. Regional myocardial contractile and metabolic functions during infusions of isoproterenol and CaCl2 were assessed in ischemic canine hearts when the amount of endogenous NO was decreased by L-NAME, an inhibitor of NOS. We also examined whether the concomitant administration of L-arginine reversed any observed effect of L-NAME. To clarify whether the reduction in myocardial contractility is due to direct myocardial effects of NO or to inhibition of norepinephrine release, we performed identical procedures in chemically denervated hearts. Finally, we examined the effect of L-NAME administration on anaerobic myocardial metabolism during coronary hypoperfusion.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Instrumentation
Seventy-two mongrel dogs (body weight, 15 to 23 kg) were anesthetized with intravenous pentobarbital sodium (30 mg/kg body wt), intubated with a cuffed endotracheal tube, and ventilated with room air mixed with oxygen (1 to 2 L/min) with a respirator. A left thoracotomy was performed through the fifth intercostal space, and the heart was suspended in a pericardial cradle. After the intravenous administration of heparin 500 U/kg, a proximal portion of the LAD was cannulated and perfused with blood through an extracorporeal tube from the left carotid artery. An electromagnetic flow probe (FF-050T, Nihon Kohden) was attached to the bypass tube for measurement of CBF. CPP was measured at the proximal portion of the cannula. The femoral artery was cannulated to sample the blood for the reference to obtain the absolute value of the regional myocardial blood flow. For blood sampling, a small, short tube (diameter, 1 mm; length, 7 cm) was inserted into the epicardial vein at the center of the perfused area, and the drained coronary venous blood was returned to the jugular vein. A miniature pressure transducer (model P-5, Konigsberg) was inserted into the left ventricular cavity through a stab incision at the apex of the left ventricle. A pair of ultrasonic crystals (5 MHz, 2 mm in diameter; Schuessler) was implanted in the left ventricular anterior wall in the endomyocardial segment in the center of the perfused area to measure segment length. Hemodynamic parameters were recorded on a multichannel recorder (Rm-6000, Nihon Kohden). We calculated FS from the equation FS=[(EDL-ESL)/EDL]x100%, where EDL and ESL are end-diastolic and end-systolic segment lengths, respectively. Heart rate was 141±2 beats per minute in the intact heart and 104±2 beats per minute in the denervated heart.
The preparation of the experiment setup took 30 to 40 minutes. The hemodynamic and metabolic parameters were stable for 3 hours: There was no change in CPP (101±3 versus 100±2 mm Hg), CBF (88±3 versus 86±2 mL·100 g-1·min-1), LER (22.4±3.9% versus 21.7±2.5%), pH in the coronary venous blood (7.38±0.02 versus 7.36±0.03), or FS (25.2±1.9% versus 23.8±2.4%) between 1 and 3 hours after the experimental setup in the preliminary study (n=5).
Experimental Protocols
Protocol 1: Effect of L-NAME on
the Inotropic Response to
ß-Adrenergic Stimulation in the Ischemic
Myocardium
Twenty-seven dogs were subjected to this protocol. After
hemodynamic stabilization, left ventricular
pressure, segment length in the perfused area, CPP, and CBF were
measured. Coronary arterial and venous blood was
sampled by syringes for blood gas analysis and determination of
lactate and nitrate plus nitrite concentrations. With an occluder
attached at the extracorporeal bypass tube, CPP was reduced so that CBF
decreased to 60% of the control value. After the low CPP was set, the
occluder was manually adjusted to maintain CPP constant at the set
level. In 9 dogs (untreated group), these procedures were performed
without treatment with L-NAME. In a second group of 9 dogs (L-NAME
group), the effect of endogenous NO on the ischemic
myocardium was prevented by continuous
intracoronary administration of L-NAME. L-NAME 10
µg·kg-1·min-1
was administered into the extracorporeal bypass tube 10 minutes before
coronary hypoperfusion. In the third group of 9 dogs
(L-NAME+L-arginine group), L-arginine 1
mg·kg-1·min-1
was infused into the bypass tube in addition to L-NAME 10 µg·kg
body
wt-1·min-1
to determine whether exogenous L-arginine can restore the
effects of L-NAME. The infusions of L-arginine and L-NAME
began 5 and 10 minutes, respectively, before the onset of
hypoperfusion. Coronary hemodynamic and
metabolic parameters were measured 1 minute
before the onset of hypoperfusion. Five minutes after the onset of
coronary hypoperfusion, two doses of isoproterenol (75 and 150
ng·kg-1 ·min-1)
were infused intravenously. Measurements of the
hemodynamic and metabolic
parameters were performed before and 5 minutes after the
infusion of each dose of isoproterenol. In a preliminary study, we
confirmed that the hemodynamic and
metabolic parameters are stabilized within 4
minutes after the onset of infusion of isoproterenol. For the
assessment of the Endo/Epi flow ratio, microspheres were
injected into the left atrium before and during coronary
hypoperfusion with and without an infusion of isoproterenol 150
ng·kg-1·min-1
in each group.
Protocol 2: Effect of L-NAME on the
CaCl2-Induced
Inotropic Response of the Ischemic
Myocardium
Twenty-seven dogs were divided into three groups of 9 each
and subjected to the same procedures as outlined in protocol 1 with the
exception that, instead of the intravenous administration
of isoproterenol, two doses (1.5 and 3.0
µmol·kg-1 ·min-1)
of CaCl2 (Wako) dissolved in saline were infused into the
LAD via the bypass tube. The Endo/Epi flow ratio was measured before
and during coronary hypoperfusion with and without infusion of
CaCl2 (3.0
µmol·kg-1·min-1)
in each group.
Protocol 3: Effect of L-NAME on the
ß-Adrenergic
Receptor–Mediated Inotropic Response of the Ischemic
Myocardium in Chemically Denervated Hearts
Eighteen dogs were divided
into three groups of 6 and, after
chemical denervation of the heart, were subjected to the procedures
described in protocol 1. Systemic chemical
sympathectomy was performed with an
intravenous injection of 6-hydroxydopamine
50 mg/kg 5 days before the experiment. Deleterious side effects of
6-hydroxydopamine were prevented by previous injections
of propranolol 1 mg/kg and phentolamine 1 mg/kg;
three fractional doses of 6-hydroxydopamine (10, 20,
and 20 mg/kg) were administered over a 24-hour period.21
Dogs were killed immediately after the experiment, and myocardial
tissue from the perfused area was sampled for the measurement of
norepinephrine content. Norepinephrine contents
of the myocardium of systemically denervated and
innervated dogs were 11±3 and 366±28 pg/mg tissue
(P<.05), respectively.
Measurement of Regional Coronary Blood Flow
Regional
myocardial blood flow was determined by the
microsphere technique as previously
described.18 19 Nonradioactive microspheres
(Sekisui) made of inert plastic and labeled with bromine, niobium, or
zirconium were used in the present study. Specific gravities were
1.34 for bromine, 1.32 for niobium, and 1.36 for zirconium. The
microspheres were suspended in isotonic saline with 0.01%
Tween 80 to prevent aggregation, ultrasonicated for 5 minutes, and
mixed vigorously for 5 minutes immediately before injection.
Approximately 1 mL of the microsphere suspension
(2x106 to 4x106
microspheres) was injected into the left atrium, followed by
several warm (37°C) saline flushes. Immediately after the
microsphere administration, a reference blood sample was taken
from the femoral artery at a constant rate of 8 mL/min for 2 minutes.
The x-ray fluorescence activities of the stable heavy
elements were measured by a wavelength dispersive spectrometer (PW
1480, Phillips Co, Ltd), as described
previously.22 23 Because the
fluorescence is characteristic for each element when the
microspheres are irradiated by the primary x-rays, it is
therefore possible to quantify the x-ray fluorescence of
several differently labeled microspheres in a mixture.
Myocardial blood flow was calculated according to the formula times
reference flow divided by reference counts and was expressed in
milliliters per minute per gram of tissue wet mass.
Chemical Analysis
The partial pressure of oxygen, hemoglobin
content, and pH of
blood were measured with a Radiometer ABL-30. The coronary
AVO2 (mL/dL) was determined as the difference between
coronary arterial and venous oxygen contents.
M
O2 (mL·100
g-1·min-1)
was calculated from CBF (mL·100
g-1·min-1)
multiplied by AVO2. For measurement of lactate
concentration, blood (1 mL) was rapidly sampled and
centrifuged. Lactate concentration in 0.2 mL of the supernatant
was measured by enzyme assay, and LER was obtained by dividing the
coronary arteriovenous difference in lactate concentration by
the arterial lactate concentration and multiplying by
100%.
NO Measurement
Blood was collected into heparinized test
tubes and
centrifuged within 30 seconds for 5 minutes at
2000g. The plasma fraction was diluted 1:1 with nitrite- and
nitrate-free distilled water, and 400 µL of the diluted sample
was centrifuged at 2000g in an Ultrafree MC
microcentrifuge device (Millipore) to remove substances of
molecular weight >10 kD. The filtrate was passed through a
copper-plated cadmium column to reduce nitrate to nitrite and
reacted with the Griess reagent, and absorbance was measured at 540
nm.24 25 26 27 This value
represented the total
amount of plasma NO end products, ie, nitrate plus nitrite.
Coronary arteriovenous differences in nitrate plus nitrite
concentration [AV(NO)] reflect the amount of NO released from
the
myocardium.
Norepinephrine Measurement
The method of norepinephrine
measurement has been
described previously.28 Myocardial tissue from the area of
the LAD was sampled within 5 seconds and frozen in liquid nitrogen. The
frozen tissue was stored at -80°C for <1 week, and then
homogenized in a solution containing EDTA (0.1 mol/L),
NaHSO3 (1 mol/L), and HClO3 (0.05 mol/L). The
homogenate was centrifuged at 2000g for
10 minutes, and norepinephrine in the supernatant was
adsorbed onto alumina and separated by high-performance
liquid chromatography (LC-3A pump and Zpax-SCX column,
Shimazu Seisakusho). Norepinephrine content was determined
spectrofluorometrically by the trihydroxyindole method (Shimazu
spectrofluorophotometer RF-500LCA). The sensitivity of the assay is 10
pg/mL sample, and the intra-assay coefficient of variation is
6.8%.28
Statistical Analysis
Data are presented as mean±SEM.
Differences among
groups were tested with the modified Bonferroni test.29
ANOVA with repeated measures was also used to assess the differences in
the responses of hemodynamic and metabolic
variables to the doses of isoproterenol and CaCl2. A
value of P<.05 was considered statistically
significant.30
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Protocol 1: Effect of L-NAME on the Inotropic Response to ß-Adrenergic Stimulation in the Ischemic Myocardium
An infusion of L-NAME did not affect the coronary hemodynamic parameters under basal conditions (Table
), indicating that the effects of
L-NAME on coronary vascular tone are minimal in the
nonischemic myocardium. FS,
MO2, LER, and pH of
coronary venous blood were not affected by L-NAME
administration in the nonischemic condition (Figs 1 to 3).
Heart rate increased during infusion of isoproterenol under
ischemic conditions, but no significant differences were
apparent among the three groups (Table). CPP was not altered,
as was
designed, and CBF was increased after isoproterenol infusion, but no
differences were detected among the three groups (Table).
AV(NO)
increased in the untreated and L-NAME+L-arginine groups
during hypoperfusion and again during administration of isoproterenol
but remained unchanged in the L-NAME group (Fig 1).
Consistent with the decrease in CPP so that CBF was reduced to
60% of the baseline flow, FS,
MO2, LER,
and pH of coronary venous blood also decreased; the extent of
the decrease in LER was significantly greater in the L-NAME group than
in the untreated group, and FS in the L-NAME group was smaller than
that in the untreated group (Figs 2 and 3). In
all of the groups, FS
and MO2 increased
significantly during
intravenous infusion of isoproterenol in a
dose-dependent manner; however, FS and
MO2 achieved significantly
higher values
in the L-NAME group than in the other two groups (Fig 2). The
pH of coronary venous blood and the LER
in the L-NAME group were lower than those in the untreated group and
the L-NAME+L-arginine group both before and during infusion
of isoproterenol (Fig 3). Before the reduction in CPP,
the Endo/Epi flow ratio was 1.13±0.09 in the untreated group;
the ratio decreased to 0.84±0.09 after the reduction in CPP and
was not further affected (0.79±0.07) by the administration of
isoproterenol 150
ng ·kg-1·min-1.
There were no significant differences in the Endo/Epi flow ratio before
hypoperfusion and during hypoperfusion in the absence or presence of
isoproterenol 150
ng·kg-1·min-1
among the three groups (L-NAME group: baseline, 1.08±0.07;
hypoperfusion, 0.81±0.10; isoproterenol, 0.72±0.12.
L-NAME+L-arginine group: baseline, 1.16±0.06;
hypoperfusion, 0.86±0.04; isoproterenol, 0.83±0.08). There were
no
significant differences in norepinephrine release
(coronary arteriovenous difference in
norepinephrinexCBF) among the three groups (untreated
group: baseline, 2.65±1.23; hypoperfusion, 1.56±0.98;
isoproterenol 150
nmol·kg-1·min-1,
1.71±1.11. L-NAME group: baseline, 2.87±0.89; hypoperfusion,
1.45±0.76; isoproterenol, 1.54±0.98.
L-NAME+L-arginine group: baseline, 2.98±1.12;
hypoperfusion, 1.39±0.43; isoproterenol, 1.55±0.67 ng·100
g-1·min-1).
The augmentation of the isoproterenol-induced inotropic
response by L-NAME was not attributable to the attenuation of
ischemia, because the pH of coronary venous blood and
the LER in the L-NAME group were lower than those in the untreated
group (Fig 3). These results indicate that endogenous NO
released from the ischemic myocardium attenuates
the inotropic response to isoproterenol.
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), L-NAME
(
), and L-NAME+L-arginine (
) groups. Values are
mean±SEM (n=9). *P<.05,
**P<.01 vs untreated
group.
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Protocol 2: Effect of L-NAME on the CaCl2-Induced
Inotropic Response of the Ischemic
Myocardium
Infusion of CaCl2 during ischemia increased
heart rate, but there were no significant differences among the three
groups (Table
). CPP was not altered, as was designed, and CBF
was
increased by CaCl2 infusion, but no differences were
detected among the groups (Table). AV(NO) increased in the
untreated
and L-NAME+L-arginine–treated groups during
hypoperfusion and again during infusion of CaCl2 but
remained unchanged in the L-NAME group (Fig 4). The
extent of the decreases in LER and the pH of coronary venous
blood in the L-NAME group was greater than those in the untreated
group, whereas FS was higher in the L-NAME group than in the untreated
group (Figs 5 and 6). The effects of
L-NAME on Ca2+-induced inotropic and metabolic
responses were blunted by the additional intracoronary
infusion of L-arginine. Before the reduction in CPP, the
Endo/Epi flow ratio was 1.14±0.08 in the untreated group; the ratio
decreased to 0.85±0.09 after the reduction in CPP and was not affected
(0.83±0.09) by the administration of CaCl2 3.0
µmol·kg-1·min-1.
There were no significant differences in Endo/Epi flow ratio among the
three groups (L-NAME group: baseline, 1.11±0.07; CaCl2 1.5
µmol, 0.80±0.08; CaCl2 3.0 µmol, 0.74±0.09.
L-NAME+L-arginine group: baseline, 1.23±0.07;
CaCl2 1.5 µmol, 0.91±0.05; CaCl2 3.0
µmol,
0.84±0.06). There were no significant differences in
norepinephrine release (in ng·100
g-1·min-1)
among the three groups (untreated group: baseline, 2.38±1.03;
ischemia, 1.80±0.68; CaCl2 3.0 µmol,
1.97±0.76. L-NAME group: baseline, 2.65±0.76; ischemia,
1.64±0.66; CaCl2 3.0 µmol, 1.76±0.87.
L-NAME+L-arginine group: baseline, 2.72±1.02;
ischemia, 1.45±0.53; CaCl2 3.0 µmol,
1.65±0.87). The augmentation of the Ca2+-induced
inotropic
response by L-NAME was not attributable to attenuation of
ischemia, because the pH of coronary venous blood and
LER in the L-NAME group were lower than those in the untreated group
(Fig 6). These results indicate that endogenous NO released
from the ischemic myocardium also attenuates the
inotropic response to CaCl2.
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Protocol 3: Effect of L-NAME on the ß-Adrenergic
Receptor–Mediated Inotropic Response of the Ischemic
Myocardium in Chemically Denervated Hearts
Infusion of L-NAME in the
absence or presence of
L-arginine had no effect on coronary
hemodynamic or metabolic
parameters under baseline conditions in any of the three
groups with denervated hearts (Table). Heart rate increased
during
infusion of isoproterenol under ischemic conditions, but no
significant differences were apparent among the three groups
(Table).
CPP was not altered, as was designed, and CBF was increased after
isoproterenol infusion, but no differences were detected among the
three groups (Table). AV(NO) increased in the untreated
and
L-NAME+L-arginine–treated groups during hypoperfusion
and again during isoproterenol administration but remained unchanged in
the L-NAME group (Fig 7). CPP, FS,
MO2, LER, and pH in
coronary venous blood also decreased; the extent of the
decrease in LER in the L-NAME group was significantly greater than that
in the untreated group, whereas FS was significantly higher in the
L-NAME group than in the untreated group (Figs 8 and
9). Both FS and
MO2 were
further increased during infusions of 75 and 150 ng·kg body
wt-1·min-1
of isoproterenol compared with the untreated group, in which they were
blunted by concomitant infusion of L-arginine (the
L-NAME+L-arginine group). pH in the coronary venous
blood and LER in the L-NAME group were lower than those in the
untreated group and the L-NAME+L-arginine group both before
and during infusion of isoproterenol (Fig 9). In the untreated
group,
the Endo/Epi flow ratio was 1.10±0.07 before the reduction in CPP. The
Endo/Epi flow ratio decreased to 0.85±0.11 after the reduction in CPP,
and it remained unchanged (0.76±0.06) after administration of
isoproterenol 150
ng·kg-1·min-1.
There were no significant differences in Endo/Epi flow ratio before
hypoperfusion or during CPP reduction before and during administration
of isoproterenol 150
ng·kg-1·min-1
among the three groups (L-NAME group: baseline, 0.98±0.11;
hypoperfusion, 0.72±0.14; isoproterenol, 0.70±0.12.
L-NAME+L-arginine group: baseline, 1.13±0.08;
hypoperfusion, 0.82±0.10; isoproterenol, 0.78±0.04). The change
in norepinephrine release (in ng·100
g-1·min-1)
was not observed among the three groups (untreated group: baseline,
0.87±0.53; ischemia, 0.56±0.34; isoproterenol 150
nmol·kg-1·min-1,
0.65±0.31. L-NAME group: baseline, 1.12±0.34; ischemia,
0.65±0.57; isoproterenol, 0.72±0.23.
L-NAME+L-arginine group: baseline, 1.01±0.56;
ischemia, 0.46±0.33; isoproterenol, 0.53±0.27). These results
indicate that endogenous NO released from the
ischemic myocardium attenuates myocardial
contractility directly, not by inhibiting
norepinephrine release.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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We have shown that L-NAME, which inhibits endogenous NO production, enhances the inotropic responses to isoproterenol and CaCl2 in the ischemic myocardium, which are restored by L-arginine. L-NAME increased anaerobic myocardial metabolism, which was also reversed by L-arginine. These results suggest that endogenous NO released from the ischemic myocardium attenuates positive inotropic responses and thereby inhibits the progression of anaerobic myocardial metabolism.
Cellular Mechanism by Which NO Affects Myocardial
Contractility
Several possible mechanisms may account for the effect
of L-NAME
on myocardial contractility during ischemia:
(1) L-NAME per se may have a positive inotropic effect. However, this
possibility is not likely, given that FS was not affected by this
compound under nonischemic conditions. (2) Inhibition of NO
production by L-NAME decreases the degree of ischemia.
If the steal phenomenon of coronary flow from the
ischemic area to the nonischemic area or from
endocardium to epicardium in the ischemic area is
attenuated by L-NAME, then the drug may reduce the extent of
ischemia by improving myocardial perfusion, resulting in the
observed increase in myocardial contractility. However,
this possibility is also unlikely, because administration of L-NAME did
not affect the myocardial Endo/Epi flow ratio and resulted in a
decrease in both LER and pH in coronary venous blood. These
results indicate that L-NAME did not alter myocardial perfusion
and improved myocardial ischemia in the present study. (3)
Given that NO inhibits norepinephrine release from
sympathetic nerve endings,31 L-NAME may enhance the
release of norepinephrine and thereby increase contractile
function. However, in the present study, L-NAME did not affect
norepinephrine release from the ischemic
myocardium. Furthermore, the increased inotropic response
to isoproterenol in the L-NAME group was also observed in chemically
denervated hearts. Thus, the contribution of an increase in
norepinephrine release from sympathetic nerve endings may
be minimal. (4) NO inhibits platelet
aggregation32 33 34 35
and neutrophil adherence to the coronary
vasculature,36 37 which may enhance myocardial
anaerobic metabolism. On this basis, myocardial
contractility should be decreased in the L-NAME group
as a result of progressive aggregation of platelets and adherence
of neutrophils. However, increased platelet aggregation and
leukocyte adherence attributable to L-NAME appear unlikely because we
observed an increase in myocardial contractility with
worsening myocardial ischemia. (5) L-NAME has been shown to
modulate cholinergic effects in addition to the inhibition of NO
synthase,38 which may affect myocardial
contractility in ischemic
myocardium. Buxton et al38 reported that the
antagonistic effects of muscarinic receptors by L-NAME are
not blunted by L-arginine; however, the increases in the
inotropic response of ischemic myocardium due to
L-NAME was blunted by L-arginine in the present study,
suggesting that NO may be responsible for the inotropic effect of
L-NAME observed in the present study. Thus, we conclude that NO
decreases myocardial contractility in ischemic
myocardium by its direct action.
Although we observed that myocardial inotropic effects of isoproterenol and CaCl2 are enhanced by the L-NAME, Balligand et al5 reported the selective effects of NO on ß-adrenergic stimulation. This difference between the Balligand et al and the present studies may be attributable to the differences in the species and models of the experiments (rat cardiomyocyte versus canine hearts) and the differences in the conditions of the heart (normoxic versus ischemic conditions).
Increases in cellular cGMP concentration decrease
myofilament
sensitivity to Ca2+ in intact cardiac
myocytes6 and may also modify the intracellular
Ca2+ concentration, suggesting that cGMP plays a role in
modulating myocardial contraction. Biochemical and pharmacological
studies with ventricular myocytes have shown that the
negative inotropic action of NO is mediated by cGMP
accumulation.39 In the adult myocardium,
an increase in cGMP inhibits Ca2+ influx and reduces the
positive inotropic effect of an increase in cAMP induced by
ß-adrenergic receptor stimulation,5
consistent with the results of the present study.
Alternatively, the effect of NO inhibition may change myocardial
contractility through alteration of heart rate. The
inotropic effect of NO synthase inhibition has been reported to be
related to the stimulation frequency through the modulation of ion
channels in isolated papillary muscle preparations.40
However, this mechanism is not likely, because heart rate did not
change with and without L-NAME administration in the ischemic
heart (Table).
Our study provides functional and biochemical evidence for the generation of NO by an isoform of NOS that appears to be constitutively present in canine myocardium. Although NOS is induced in rat vascular smooth muscle cells by interleukin-1ß,41 it is unlikely that myocardial NOS is induced by cytokines in the present study, because the NO-mediated response to ischemia in the present study is rapid.
Pathophysiological Relevance
The question arises as to
whether the increase in myocardial
contractility induced by L-NAME is beneficial or
deleterious to the ischemic heart. Our study suggests that
L-NAME treatment is deleterious, because we observed that the LER and
the pH of coronary venous blood during infusion of
isoproterenol were lower in the L-NAME–treated group than in the
untreated group, indicating that anaerobic myocardial
metabolism in the ischemic heart is increased by
L-NAME treatment. The ischemic myocardium thus
appears to be forced to contract at the expense of a further increase
in myocardial anaerobic metabolism.
Furthermore, there is a possibility that endogenous NO directly affects the anaerobic metabolism of ischemic myocardium. Ljusegren et al42 suggested that cGMP reduces lactate accumulation in the hypoxic, nonbeating ventricular muscle: Not only increased blood supply but also diminished lactate production due to NO and cGMP may be responsible for the attenuation of myocardial ischemia. Furthermore, Beitner et al43 reported that cGMP inhibits phosphofructokinase of cardiac and skeletal muscles and suggested that the inhibition of activity of phosphofructokinase may decrease the whole glycogenolysis and glycolysis. These actions of cGMP in ischemic myocardium may contribute to the NO-dependent inhibition of anaerobic metabolism.
The administration of NOS inhibitors to anesthetized or conscious animals has been shown to induce cardiac depression as well as systemic hypertension and marked systemic vasoconstriction,44 45 which appears to contradict the present observations. Various mechanisms may underlie this apparent discrepancy. Hypertension induced by NOS inhibitor may stimulate a baroreceptor reflex and a consequent reduction in cardiac output. Increased afterload may also result in a decrease in ventricular stroke volume. These systemic hemodynamic effects of NOS inhibitors may reduce cardiac output. Furthermore, NOS inhibitors induce coronary vasoconstriction and thereby reduce myocardial oxygen supply.46 47 In our experimental model, coronary perfusion pressure was maintained constant during hypoperfusion, and because L-NAME was selectively infused into the LAD, it did not affect systemic blood pressure or afterload. Thus, our results might have differed if we had administered L-NAME systemically.
Recently, Weyrich
et al48 showed that
physiologically relevant concentrations of NO
did not induce physiologically significant
negative inotropic effects acutely; NO exerted a statistically
significant negative inotropic effect only in the presence of high
concentrations of norepinephrine. In our study, FS,
MO2, LER, and pH of
coronary venous blood were not affected by L-NAME or
L-arginine administration under nonischemic
conditions, which may correspond to the control conditions of Weyrich
et al.48 An increase in vagal nerve activity reduces the
contractile response to sympathetic stimulation49 and
infused ß-adrenergic
agonists.50 51 52 Studies have
also shown that NO53 and NO donors54 reduce
myocardial contraction in an in vivo model. We have also shown that NO
modulates myocardial contractility, suggesting that
increased release of NO during ischemia reduces myocardial
contractility or that the ischemic
myocardium is more sensitive to NO.
Clinical Relevance
In the clinical setting, myocardial
ischemia is often
followed by reperfusion with percutaneous transluminal
coronary revascularization and
percutaneous transluminal coronary angioplasty.
Hasebe et al55 showed that endogenous NO
attenuates myocardial stunning in dogs. Furthermore, exogenous NO
precursors56 and angiotensin-converting
enzyme inhibitors57 58 reduce the infarct
size, with attenuation of myocardial ischemia and
reperfusion.59 The present findings suggest that the
beneficial effects of NO donors or
angiotensin-converting enzyme inhibitors
are partially attributable to a reduction in the extent of myocardial
ischemia because of a myocardial energy-sparing effect as
well as coronary vasodilation. Beckman and
colleagues60 proposed that NO combined with superoxide can
yield the peroxide anion (ONOO-) and that this anion
decomposes into the highly reactive hydroxyl radical. These free
radicals may be involved in myocardial cellular injury. During
reperfusion after sustained ischemia, NO may have bidirectional
effects on myocardium because of the coexistence of NO and
O2-. Because we used the hypoperfusion
model, ONOO- may not have contributed to our results.
Further investigation is required to determine whether
endogenous NO participates in a negative feedback system to
enhance the contractility induced by ß-adrenergic
receptor stimulation and exerts a protective effect in
ischemia-reperfusion models before the present
observations can be applied to the clinical setting.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
We thank Noriko Tamai and Shinya Suzuki for their technical assistance.
Received May 23, 1995; revision received July 26, 1995; accepted August 29, 1995.
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References |
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