© 1995 American Heart Association, Inc.
Articles |
Arrhythmia and Delayed Recovery of Cardiac Action Potential During Reperfusion After Ischemia
Role Of Oxygen Radical–Induced No-Reflow Phenomenon
From the Department of Pharmacology and Therapeutics, University of Calgary (Alberta, Canada).
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Abstract The role of reactive metabolites of oxygen, oxygen radicals (O-Rs), as mediators of potentially arrhythmogenic alterations in cellular electrical properties and contractile dysfunction of cardiac muscle during reperfusion after ischemia was investigated. Electrical and mechanical activities of arterially perfused guinea pig right ventricular walls were recorded simultaneously with intracellular microelectrodes and a force transducer. Preparations were maintained in Krebs-Henseleit solution (perfusion rate, 1.5 mL/min) and subjected to 30 minutes of no-flow ischemia followed by 60 minutes of reperfusion or pretreated with O-R scavengers (superoxide dismutase, 50 U/mL; catalase, 600 U/mL; and mannitol, 2 mmol/L) for 10 to 20 minutes, followed by 30 minutes of ischemia and 60 minutes of reperfusion. Reperfusion in untreated preparations caused (1) depolarization of resting membrane potential by 8 to 10 mV and slow recovery of action potential duration requiring 60 minutes to attain the preischemic duration, (2) tachyarrhythmias and premature action potentials, (3) postischemic contractile dysfunction, and (4) increased coronary perfusion pressure in untreated preparations. Pretreatment with scavenger cocktail affected neither electrical nor contractile activity before or during no-flow ischemia, but it (1) accelerated recovery of resting membrane potential and action potential duration, (2) reduced the incidence of tachyarrhythmia, (3) improved contractile function, and (4) inhibited the rise in perfusion pressure on reflow. Reperfusion with an exogenous O-R–generating system containing xanthine/xanthine oxidase (X/XO, 2 mmol/L:10 mU/mL) inhibited recovery of action potential duration and contractility. Treatment of normoxic arterially perfused right ventricular walls with X/XO caused a decline in action potential duration by
20% within 30
minutes. In contrast, X/XO caused a 30% increase in the duration of
action potentials in superfused papillary muscles or small strips of
right ventricular walls over the same time period. Pretreatment with
sodium nitroprusside (10 µmol/L) inhibited the decline in duration
induced by X/XO in normoxic right ventricular walls but was without
effect on prolongation due to X/XO in papillary muscles. Reperfusion
with nitroprusside after no-flow ischemia caused (1)
accelerated recovery of preischemic action potential
configuration, (2) a significant decline in the incidence of
reperfusion arrhythmias, (3) improved postischemic contractile
performance, and (4) inhibition of the increase in perfusion pressure
associated with reflow. The data indicate that slow recovery of the
action potential duration caused by O-Rs in reperfusion cannot be
explained by the direct effects of O-Rs on cardiac myocytes. Rather,
coronary vascular injury and the no-reflow phenomenon due to O-R stress
is suggested to contribute to abnormal cardiac action potential
configuration, arrhythmogenesis, and contractile dysfunction during
reperfusion after ischemia.
Key Words: ischemia/reperfusion • cardiac action potential • nitrovasodilator • no-reflow phenomenon • oxygen radicals
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Disturbances of cardiac rhythm, including lethal ventricular arrhythmias, and postischemic contractile dysfunction are a consequence of reperfusion following pathological and/or clinical instances of myocardial ischemia.1 2 This includes arrhythmias arising from reestablishing flow after coronary spasm,3 cardiopulmonary bypass with ischemic cardiac arrest,4 and angioplastic/thrombolytic procedures.5 The clinical occurrence and possible lethal consequences of reperfusion arrhythmias and depressed contractile function have prompted considerable interest in determining the mechanisms responsible and in developing therapeutic approaches for their control.
Reperfusion arrhythmogenesis is postulated to result from alterations
in membrane ionic currents or cell-to-cell coupling due to (1)
depletion of intracellular levels of high energy phosphates, (2)
alterations in ionic gradients, including nonhomogeneous changes in
extracellular K+ content, increased intracellular levels of
H+, Ca2+, and/or
Na+, (3) production of lysophosphoglycerides, and
(4) sympathetic catecholamine release and elevated intracellular
cAMP.1 6 7 Additionally, there is a growing body of
evidence that highly reactive metabolites of oxygen, oxygen radicals
(O-Rs), and oxidative damage may play an important role in
arrhythmogenesis and contractile dysfunction in
reperfusion.2 8 9 10 11 12 13 14 The sudden return of oxygen to
ischemic tissue is postulated to cause a "burst" of
superoxide (·O2-), singlet oxygen
(1O2), hydrogen peroxide
(H2O2), and hydroxyl radical (·OH)
generation.8 10 11 12 Consistent with this view, direct
measurements of O-Rs using electron spin resonance13 14 or
chemiluminescence15 techniques indicate elevated levels of
reactive metabolites of oxygen in the heart during reperfusion after
ischemia. Moreover, levels of malondialdehyde, a byproduct of
O-R–mediated lipid peroxidation, also apparently increase during
reperfusion.16 17 Levels of O-Rs, malondialdehyde, and
reperfusion injury may be reduced by treatment with allopurinol or a
variety of O-R scavengers, including superoxide dismutase (SOD),
catalase (CAT), ascorbate, 
-tocopherol, mannitol (MAN),
glutathione, and
N-(2-mercaptoproprionyl)-glycine.2 4 11 12 14 18 19
In addition to causing altered cardiac muscle function, evidence is accumulating that O-R stress during reperfusion provokes a marked reduction in coronary flow, the so-called no-reflow phenomenon.20 21 22 Elevated coronary resistance may result from vasoconstriction due to (1) endothelial injury and depressed basal nitric oxide release,23 24 (2) scavenging of nitric oxide in the extracellular compartment due to superoxide radical,25 26 and/or (3) coronary myocyte contraction as a consequence of abnormal Ca2+ release from the sarcoplasmic reticulum during oxidative stress.27 The importance of no-reflow to reperfusion arrhythmogenesis and recovery of cardiac electrical and contractile function after ischemia is not well defined.
Reflow arrhythmias and the role of O-Rs in their genesis have been studied extensively by using in vivo and in vitro models of ischemia/reperfusion.2 8 They are known to develop within a few seconds after the reinitiation of flow and to include premature action potentials (extrasystoles), ventricular tachycardia, and fibrillation.1 2 7 8 9 Both abnormal impulse generation and reentry are implicated in their genesis.8 28 29 A role for O-Rs in arrhythmogenesis and abnormal cellular electrical activity was identified by using intact ventricular tissue and single cardiomyocytes exposed to exogenous O-R–generating systems.9 30 31 32 33 However, the changes in action potential configuration, resting membrane potential, and/or membrane ionic currents due to O-R stress during reperfusion after ischemia per se remain to be defined. In the absence of this information, it is difficult to evaluate the pathophysiological significance of data regarding action potential configuration obtained with healthy myocardium or cardiomyocytes exposed to exogenous O-Rs.
In the present study, we sought to identify (1) the changes in resting membrane potential and action potentials during reperfusion after no-flow ischemia and (2) whether the changes were the result of O-R stress. We used an arterially perfused guinea pig right ventricular wall preparation34 ; ischemia/reperfusion was induced in the absence or presence of a cocktail of O-R scavengers (SOD, CAT, and MAN) or the endothelium-independent nitrovasodilator sodium nitroprusside. The data indicate that the changes in action potential configuration during reperfusion cannot be explained on the basis of direct oxidative injury to cardiac myocytes. It is concluded that alterations in electrical and contractile activity secondary to coronary vascular injury due to O-R stress contribute to reperfusion-induced cardiac dysfunction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparations
Guinea pigs (300 to 400 g) were anesthetized with CO2 and killed by cervical dislocation. Their hearts were quickly removed and placed in a dish containing cold Krebs-Henseleit solution composed of (mmol/L) NaCl 120, NaHCO3 25, KCl 4.8, NaH2PO4 1.2, MgSO4 1.2, CaCl2 1.8, and glucose 11.0 and a pH of 7.4 when gassed with 95% O2/5% CO2. The right ventricular walls were prepared and maintained in vitro according to a technique previously described.34 Briefly, the atria were removed, and the right ventricle was dissected from the heart, leaving the base of the aorta attached to the wall and the right coronary vasculature intact. A fine cannula was placed in the aortic opening of the right coronary artery and sutured in place, and the ventricular wall was perfused at a constant flow rate of 1.5 mL/min with 37°C Krebs-Henseleit solution. The base of the walls was pinned to the bottom of a Perspex bathing chamber with the epicardial surface up and the apex of each tissue attached to a force transducer (Gould Statham) for recording mechanical activity. Coronary perfusion pressure was monitored by a Harvard blood pressure transducer placed upstream from the cannula. Pressure values were corrected by subtraction of the cannula pressure in the absence of the preparation at the end of each experiment. The muscles were stimulated by 2-millisecond square pulses delivered from a point source at a rate of 2 Hz. Resting tension was adjusted to a level (generally 250 to 400 mg) that optimized developed tension. Action potentials recorded from arterially perfused right ventricular walls under control, ischemic, and reflow conditions had a waveform similar to that reported for other preparations,35 36 including Langendorff-perfused guinea pig hearts.37 The tissues did not exhibit abnormal rhythm or decay in mechanical function for >8 hours and were stable over the period required for the present experiments. There was no evidence of ischemic dysfunction even at the extreme edges of the tissues, implying that the dissection procedure did not damage the arterial vasculature of the right wall and that the entire preparation was well perfused.
Papillary muscles from the right ventricle with cross-sectional
diameters of 0.5 mm or thin strips of the right ventricular wall (1
to 2 mmx3 mmx0.5 mm) were used in some experiments. Both ends of the
papillary muscles or ventricular strips were pinned to the base of the
chamber with fine dissecting pins, and the tissues were superfused with
Krebs-Henseleit solution at 37°C (3 mL/min) and stimulated from a
point source at 0.5 Hz.
Experimental Protocols
After obtaining control recordings from right ventricular walls,
the tissues were either (1) subjected to 30 minutes of no-flow
ischemia and 60 minutes of reperfusion in the absence of
treatment (untreated preparations), (2) treated with a cocktail of O-R
scavengers including SOD (50 U/mL), CAT (600 U/mL), and MAN (2 mmol/L)
based on the method of Rosenthal and Brown38 for 10 to 20
minutes before 30 minutes of ischemia and 60 minutes of
reperfusion, (3) subjected to 30 minutes of ischemia before
reperfusion with xanthine (X, 2 mmol/L) and xanthine oxidase (XO, 10
mU/mL), or (4) subjected to 30 minutes of ischemia before
reperfusion with sodium nitroprusside (10 µmol/L). During no-flow
ischemia, the recording chamber was gassed with 95%
N2/5% CO2 to minimize O2
reaching the muscle surface.
Several experiments not involving ischemia/reperfusion were also conducted on arterially perfused right ventricular walls and superfused papillary muscles or strips of right ventricular wall. In these cases, we compared the effects of X/XO (2 mmol/L:10 mU/mL) in the presence and absence of pretreatment with sodium nitroprusside (10 µmol/L) in the arterially perfused versus superfused preparations.
Electrical Recording
Transmembrane potentials were recorded from ventricular walls,
papillary muscles, or ventricular strips with conventional
intracellular glass microelectrodes. Impalements were made from the
epicardial surface of the right ventricular walls, and action
potentials were recorded under control conditions, after 30 minutes of
ischemia, and at several times during reperfusion. Mechanical
movement of the right ventricular wall preparations precluded
continuous electrical recording from a single cell for the entire
duration of the experiments; however, it was frequently possible to
maintain an impalement for more than one or two sampling intervals
before the micropipette was dislodged. The micropipettes (20 to 30
M
) were pulled from filamented capillary tubes (outer diameter, 1.2
mm; World Precision Instruments) on a P-87 pipette puller (Sutter
Instruments), filled with 3 mol/L KCl, and connected to a WPI Duo 773
electrometer (World Precision Instruments). Electrical and contractile
activities were recorded on videotape by using a Vetter 420 analog
recorder (A.R. Vetter Co) and subsequently digitized and stored on hard
disk at a sampling frequency of 2 kHz by using a TL-1-125 Labmaster A/D
board (Axon Instruments), AXOTAPE data acquisition
software (Axon Instruments), and an IBM AT clone computer. The
following parameters of the recorded action potentials and contractions
were determined from the digitized recordings: (1) resting membrane
potential, (2) action potential duration (at 30% and 90%
repolarization [APD30 and APD90,
respectively]), (3) resting tension, and (4) developed tension. Action
potentials were selected at random from periods when the preparations
were not arrhythmic. Only those action potentials at which the
diastolic (resting membrane) potential was stable for at least three
previous cycle lengths were used.
Quantification of arrhythmias during reperfusion in the present study was accomplished by determining the percentage of time during 10-minute intervals when the tissues exhibited premature action potentials or tachyarrhythmia. Arrhythmias were considered to be premature action potentials when there were one or two nontriggered action potentials within a cycle interval. Tachyarrhythmias consisted of three or more nontriggered action potentials per cycle but were not differentiated into those with uniform or nonuniform frequency. The length of time for each arrhythmic period was measured, and the sum for all periods in each 10-minute interval during reperfusion was determined. This sum was then expressed as a percentage of the 10-minute interval.
Drugs
Chemicals for Krebs-Henseleit solution, SOD, CAT, MAN, sodium
nitroprusside, and X/XO were purchased from Sigma Chemical Co. The O-R
scavenger and generating system solutions were freshly prepared each
day.
Statistics
Mean±SEM values of (1) action potential parameters, (2) resting
and developed tension, (3) percentage of time in arrhythmic activity,
and (4) perfusion pressure were determined for several tissues.
Statistical comparisons were made by using SIGMA
STAT software (Jandel Scientific Software). Paired or
unpaired Student's t tests were used for single
comparisons, ANOVA followed by Dunnett's test was used for multiple
comparisons between groups, and repeated-measures ANOVA followed by
Dunnett's test was used to compare data from different time points
within a single treatment group. The specific test used is indicated
where relevant in the text or figure legend. A value of
P<.05 was considered to be significant.
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Results |
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We first confirmed that reperfusion after 30 minutes of no-flow ischemia in guinea pig right ventricular walls led to arrhythmias (consisting of premature action potentials and tachyarrhythmias) that could be reduced with a cocktail of O-R scavengers (SOD, 50 U/mL; CAT, 600 U/mL; and MAN, 2 mmol/L). Fig 1
shows representative recordings of electrical
and mechanical activity during the first 80 seconds of reperfusion in
an untreated tissue and in a preparation treated with scavenger
cocktail for 10 minutes before the onset of the no-flow period.
Arrhythmias were initiated soon after the start of reflow in untreated
tissues but were inhibited by scavenger cocktail, as previously
reported.39 Average values for the percentage of time in
arrhythmic activity that was due to tachyarrhythmia or premature action
potentials were compared in untreated tissues and in tissues pretreated
with cocktail (Fig 2). Arrhythmic activity was observed
throughout the reflow period but was greatest over the first 30 minutes
of reperfusion, chiefly because the incidence of tachyarrhythmia
declined with time and they were not observed after 30 minutes of
reflow. Treatment with O-R scavengers reduced the incidence of
tachyarrhythmia but had little effect on premature action
potentials.
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We next sought to determine the changes in action potential
configuration during reperfusion that were affected by O-R scavenger
cocktail treatment. Representative data are shown in Fig 3, and average values for action potential and
contractility parameters in untreated and O-R scavenger
cocktail–treated tissues are given in Fig 4. When flow
to untreated right ventricular walls was stopped, several
time-dependent changes in electrical and contractile activity that were
indicative of ischemic dysfunction were observed. Specifically,
action potential duration, resting membrane potential, and developed
force declined, whereas resting tension rose over the 30 minutes of no
flow, as previously reported for this preparation.34
Pretreatment with O-R scavengers did not alter the response to no-flow
conditions (Figs 3 and 4); however, the scavenger cocktail did alter
the response of action potential duration, resting membrane potential,
and contractile function to reflow.
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) and tissues pretreated with
scavenger cocktail (
) before 30 minutes of no-flow
ischemia and 60 minutes of reperfusion. Each value is mean±SEM
for eight untreated and seven scavenger-treated tissues. Time points
are as follows: control normoxic measurement (C), after scavenger
cocktail treatment (S), after 30 minutes of no-flow ischemia
(I), and after 0.5, 1, 2, 5, 10, 15, 30, and 60 minutes of reperfusion.
*Significant difference (P<.05) from untreated preparations
by ANOVA followed by Dunnett's test.
Reperfusion of untreated preparations provoked a fall in resting
membrane potential by 8 to 10 mV; maximal depolarization was observed
between 1 and 5 minutes of reperfusion, followed by a slow
repolarization until 60 minutes of reflow, when resting potential was
not significantly different from the control level (Figs 3
and 4;
P>.05, repeated measures ANOVA). Similarly,
APD30 and APD90 also recovered slowly and
required 60 minutes of reperfusion to achieve values comparable to
those obtained before ischemia (Fig 4). Contractile performance
did not recover completely during reperfusion in untreated tissues, and
sustained postischemic dysfunction was observed at 60 minutes
of reflow. Reflow caused a rapid and significant rise in resting
tension in untreated preparations, which began within the first 0.5
minutes and reached a peak that was 200% greater than
preischemic levels between 1 and 5 minutes of reflow (Figs 3 and 4). Resting tension declined slowly back toward preischemic
levels during the remainder of the reflow period but was still
significantly elevated by >150% at 60 minutes of reperfusion
(P<.05, repeated measures ANOVA followed by Dunnett's
test). Extending the reperfusion period for an additional 2 hours in
three preparations produced no further change in resting tension (data
not shown). Similarly, developed tension recovered only slowly during
the reperfusion period, remaining depressed by >50% compared with
preischemic levels at 60 minutes of reflow. Reperfusion for an
additional 2 hours did not improve the recovery of developed tension in
three tissues.
The scavenger cocktail solution had no effect before or during
ischemia, as is evident from the similar values of action
potential duration, resting membrane potential, resting tension, and
developed tension during the control period and at 30 minutes of
ischemia in treated and untreated tissues (Figs 3 and 4).
However, when compared with untreated preparations, O-R scavengers
minimized the reflow-induced changes in membrane potential and led to a
rapid recovery of action potential configuration and contractile
function. For example, APD90 and resting membrane potential
recovered completely by 5 minutes of reflow compared with 60 minutes in
untreated preparations (Fig 4). Moreover, in contrast to the continued
postischemic contractile dysfunction observed in untreated
tissues, resting tension and developed tension recovered to
preischemic levels within 10 minutes of reflow in tissues
pretreated with scavenger cocktail (Figs 3 and 4). Resting tension
rapidly decreased from its ischemic level and was not
significantly different from that observed before ischemia
after 1 to 2 minutes of reperfusion (P>.05,
repeated-measures ANOVA). Developed tension recovered at the same rate
as in untreated tissues until 2 minutes, after which it continued to
improve, and by 10 minutes of reperfusion it completely recovered to
the preischemic level.
In three experiments, we limited the exposure to scavenger cocktail to the reperfusion period only (data not shown). Results similar to those described above were obtained, but complete recovery of action potential waveform to preischemic shape was delayed until 20 minutes, and the incidence of arrhythmia was reduced only after this time.
Given that O-R scavengers inhibited the decline in action potential
duration and enhanced recovery during reflow, it would be expected that
reperfusion with solution containing an exogenous O-R–generating
system should exacerbate the changes and/or prevent recovery. Fig 5 shows that reperfusion with Krebs-Henseleit solution
containing the O-R–generating system (X/XO, 2 mmol/L:10 mU/mL)
enhanced the effects of reperfusion on the action potential and
inhibited the recovery of electrical and contractile activity.
APD90 failed to recover by 60 minutes of reperfusion,
remaining significantly shorter than the preischemic duration
(P<.05, repeated-measures ANOVA followed by Dunnett's
test). Recovery of contractile activity was also reduced in reperfused
compared with untreated tissues (Fig 5).
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Previous experiments on normoxic intact myocardium and isolated
myocytes generally reported that action potential duration increases
rather than decreases in response to exogenous reactive oxygen
metabolites.9 30 40 41 42 43 44 45 However, these previous studies
generally used superfused tissues rather than arterially perfused
preparations, such as used in the present study. For this reason,
we determined the effects of X/XO (2 mmol/L:10 mU/mL) on action
potential configuration in superfused normoxic papillary muscles and
thin strips of right ventricular walls and compared the data with that
obtained for arterially perfused right ventricular walls. The results
of these experiments are shown in Figs 6 and 7. It is evident from representative action
potentials (compare Fig 6A with left panel of Fig 7A) and average data
for APD90 that O-R stress caused divergent effects on
action potential duration in superfused papillary muscles and
arterially perfused right ventricular walls. The former showed a 30%
increase and the latter showed a 20% decrease in action potential
duration over 30 minutes of X/XO treatment (Figs 6 and 7). Action
potential duration in the papillary muscles remained at a stable level
of prolongation over an additional 30 minutes of exposure to X/XO (data
not shown), but APD90 continued to decline in the right
walls, reaching a value of 40% of the control level at 60 minutes
(Fig 7). It is possible that the divergent effect of O-R stress on
action potential duration in the papillary muscle could be related to
the endocardial origin of the preparation. For this reason, we applied
X/XO to thin strips of epicardial tissue from right ventricular wall.
In three preparations, O-R stress caused a 30±5.5% increase in
APD90 similar to that observed in the papillary muscles at
30 minutes of exposure to X/XO (data not shown). The data obtained with
the right ventricular walls, papillary muscles, and ventricular strips
indicate that the same O-R–generating system had a different effect on
action potential configuration in arterially perfused compared with
superfused preparations.
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O-R stress was previously reported to contract coronary arterial
preparations in vitro and was implicated in the so-called no-reflow
phenomenon during reperfusion of intact hearts.20 21 22 We
felt it possible that O-R stress during reperfusion or X/XO treatment
could have affected the vasculature of the ventricular walls and
indirectly caused changes in cardiac action potential configuration,
arrhythmogenesis, and contractile dysfunction. To test this hypothesis,
we exposed right ventricular walls to the O-R–generating system after
pretreatment with sodium nitroprusside (10 µmol/L). This vasodilator
was previously reported to be without effect on atrial action potential
configuration at concentrations between 10 and 100 µmol/L and to
elicit action potential prolongation only in the millimolar
range.44 We also found 10 µmol/L nitroprusside to be
without effect on ventricular APD90 during 20 minutes of
normoxic perfusion of the right walls (Fig 7). Additionally, it had no
effect on APD90 in papillary muscles (n=3; data not shown).
Fig 7 shows representative data and average values for
APD90 in right ventricular walls exposed to X/XO with and
without nitroprusside pretreatment. Nitroprusside prevented the decline
in APD90 over the first 30 minutes and significantly
reduced the decline recorded at 60 minutes of O-R stress in untreated
tissues (Fig 7). However, nitroprusside at the same concentration did
not prevent the increase in APD90 in papillary muscles
after 15 minutes of X/XO superfusion (APD90 increased by
39±6.3% compared with 30±7.1% in the absence of nitroprusside).
These data suggest that (1) nitroprusside was not acting as a scavenger
since it did not block the effects of X/XO on the papillary muscle
action potential and (2) coronary vasodilation due to nitroprusside
inhibited the changes in electrical activity of normoxic cardiac muscle
observed during exposure to an exogenous O-R–generating system.
Given that nitroprusside inhibited changes in APD90 due to
an exogenous O-R–generating system in normoxic ventricular walls, we
tested whether it would similarly inhibit the changes during
reperfusion after ischemia. Fig 8 shows
representative recordings and average data for
APD90 during reperfusion with Krebs-Henseleit solution in
the absence and presence of nitroprusside. Nitroprusside treatment led
to a complete recovery of APD90 by 10 minutes of
reperfusion compared with 60 minutes of reperfusion in untreated
preparations. Additionally, contractile activity was also preserved:
(1) Resting tension recovered completely compared with sustained
contracture in untreated tissues (value at 60 minutes of reflow in
sodium nitroprusside was -13±9% of the preischemic values
and significantly lower than the 212±13% increase observed in
untreated tissues by ANOVA followed by Dunnett's test). (2) Developed
tension showed significantly improved recovery (values at 60 minutes of
reflow were 71±6% and 38±15% of preischemic values in
nitroprusside and untreated tissues, respectively; P<.05,
ANOVA followed by Dunnett's test). We also determined the incidence of
arrhythmia in tissues reperfused with nitroprusside. The rapid recovery
of action potential duration was accompanied by a complete inhibition
of tachyarrhythmia. Indeed, no preparations (n=5) reperfused with
nitroprusside showed evidence of tachyarrhythmia over the entire
reperfusion period. The incidence of premature action potentials after
the first 10 minutes of reflow was also reduced but did not achieve a
level of significant difference (Fig 2).
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The data presented above indicated the possibility that
vasoconstriction and the no-reflow phenomenon contributed to the
electrical and contractile dysfunction during reperfusion after
ischemia. Experiments were therefore performed in which
coronary perfusion pressure was monitored. Fig 9
illustrates the effects of reperfusion after ischemia and X/XO
on coronary perfusion pressure. Also indicated is the ability of the
scavenger cocktail and sodium nitroprusside to prevent the
vasoconstriction caused by reperfusion. Fig 9A shows a
representative continuous recording of perfusion pressure in an
untreated preparation during ischemia and 30 minutes of
reperfusion. A marked increase in perfusion pressure over the first 10
minutes of reflow was observed. On average, reflow caused a 136% rise
in perfusion pressure from a normoxic control value of 41.1±4.8 to a
new stable level of 97.2±20.8 mm Hg by 30 minutes of reflow (n=4).
Perfusion pressure was significantly elevated above the control
normoxic level within 0.5 minute of the initiation of reflow
(P<.05, repeated-measures ANOVA followed by Dunnett's
test). Panels B and C of Fig 9 show representative recordings
of pressure in tissues treated with scavenger cocktail and sodium
nitroprusside, respectively. Both treatments inhibited the marked rise
in perfusion pressure during reflow, with a significant reduction in
the percentage change of pressure compared with that in untreated
tissues achieved at 2 minutes for scavenger cocktail and at 5 minutes
for sodium nitroprusside (Fig 9E). No effect on perfusion pressure was
noted during treatment with O-R scavengers before ischemia
(40.4±1.1 and 40.2±1.6 mm Hg in control and cocktail-treated
tissues, respectively; n=3 each). The inset of Fig 9C shows a
representative example of the vasodilation due to sodium
nitroprusside under normoxic conditions; on average, pressure showed a
significant decline by 22.7±4.3% during sodium nitroprusside
treatment (59.2±5.6 to 45.6±4.2 mm Hg; P<.05, paired
Student's t test; n=4). Fig 9D shows a
representative recording of the rise in perfusion pressure
caused by treatment with X/XO under normoxic conditions. On average,
perfusion pressure increased to a stable value that was 121.6±38.6%
greater than under control conditions at 30 minutes (50.9±4.0 to
114.9±28.1 mm Hg; n=3; P<.05, paired Student's
t test). These data indicate that reflow and X/XO induce
vasoconstriction in this preparation and that the rise in perfusion
pressure during reperfusion can be inhibited by scavenger cocktail or
treatment with the vasodilator sodium nitroprusside.
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, n=3) or SNP
(
, n=4). *Significant difference (P<.05) from
untreated tissues by ANOVA followed by Dunnett's test. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study reports data indicating that reduced coronary vascular perfusion due to O-R stress contributes to electrical and contractile dysfunction during reperfusion after no-flow ischemia. O-R stress has been implicated as a mediator of cardiac dysfunction, including coronary arterial dysfunction leading to the so-called no-reflow phenomenon.2 7 8 9 10 11 12 13 14 20 21 22 28 31 32 33 39 44 However, the consequences of no-reflow to arrhythmogenesis and recovery of electrical and contractile function during reperfusion are not well defined. In the present study, reperfusion of guinea pig right ventricular walls after 30 minutes of no-flow ischemia was found to cause a slow recovery of action potential configuration, arrhythmias, postischemic contractile dysfunction, and a rise in coronary perfusion pressure. Treatment with a cocktail of O-R scavengers improved recovery, reduced coronary perfusion pressure, and inhibited arrhythmic activity, indicating that O-R stress contributes to cardiac dysfunction in this model of ischemia/reperfusion. However, improved recovery during reperfusion was also achieved with sodium nitroprusside, which caused vasodilation but lacked a direct effect on the cardiac action potential or O-R scavenger action. This indicates the possible involvement of altered coronary vascular perfusion as a contributor to electrical and contractile dysfunction associated with reperfusion.
Evidence that O-R stress may lead to alterations in cardiac function secondary to reduced coronary perfusion during reflow was obtained in the present study by considering the effects of reperfusion on action potential duration and comparing the actions of exogenous O-Rs on action potential configuration in arterially perfused versus superfused preparations of cardiac ventricular muscle. Recovery of action potential duration to a value comparable to that recorded before ischemia required 60 minutes of reflow in untreated preparations, whereas APD90 recovered fully by 5 minutes after treatment with a cocktail of O-R scavengers. These data indicate that endogenous O-R stress during reperfusion inhibited lengthening and recovery of action potential duration. Consistent with this observation, we found that (1) reperfusion with an exogenous O-R–generating system prevented recovery of APD90 and (2) perfusion with exogenous O-Rs caused a decline in action potential duration in normoxic arterially perfused ventricular preparations. In contrast, direct oxidative injury due to superfusion of papillary muscles or thin strips of right ventricular walls with exogenous O-Rs caused prolongation, not shortening, of action potential duration. These results are consistent with previous studies that generally reported increased action potential duration during exposure of normoxic preparations to exogenous O-R stress.9 30 40 41 42 43 44 For example, increased action potential duration was observed in several studies using either superfused papillary muscles, isolated strips of ventricle, or trabeculae exposed to H2O241 42 44 or photoilluminated rose bengal.45 However, no effects on action potential duration in ventricular strips47 and papillary muscles48 or action potential shortening accompanied by marked depolarization in papillary muscles31 49 were also reported. Purkinje fibers consistently show shortening of action potential duration,31 48 whereas isolated ventricular myocytes invariably show prolongation followed by shortening after longer times of exposure.9 30 40 41
The divergent observations concerning the effects of O-Rs on cardiac action potential configuration in the literature may be related to differences in the species of O-R present, the level of O-R stress, metabolic status of the myocytes, and/or regional differences in the response to O-Rs.9 However, these explanations would appear to inadequately account for the differences between the effects of X/XO on arterially perfused right ventricular walls versus superfused papillary muscles or strips of ventricular muscle in the present study. The perfused and superfused preparations were exposed to the same level of exogenous stress due to X/XO, yet opposite effects on APD90 were observed. It is unlikely that differences in metabolic status arising as a result of the perfused versus superfused nature of the two preparations were involved; APD90 was similarly depressed in perfused preparations in response to exogenous O-Rs due to X/XO under normoxic and postischemic conditions. We also do not attribute the divergent effect of exogenous O-R stress to regional differences in action potentials. Regional differences in response to O-R stress are known, eg, the consistent decline in duration in Purkinje fibers31 48 versus the early lengthening in ventricular myocytes.9 30 40 41 42 43 44 47 However, we observed a similar prolongation of action potential duration in papillary muscles and strips of right ventricular walls superfused with X/XO. Moreover, there is little difference in action potential configuration of epicardial and endocardial tissues in guinea pig, and H2O2 caused a similar increase in action potential duration in both epicardium and endocardium of nonperfused ventricular strips from guinea pig right ventricular walls, although the former displayed a greater sensitivity.42
We attribute the divergent effects of O-R stress on the perfused right ventricular walls and superfused papillary muscle or strips of right ventricular walls identified in the present study to vasculature injury in the perfused preparations and effects on cardiac action potential secondary to reduced coronary perfusion. Our data show that the endothelium-independent vasodilator sodium nitroprusside inhibited the decline in action potential duration in normoxic right ventricular walls due to exogenous O-Rs and led to rapid recovery of APD90 during reperfusion. We do not attribute these effects to a direct action of the drug on cardiac action potential or to scavenging of reactive oxygen metabolites. Nitroprusside was used in the present study because it was previously reported to be without effect on atrial action potential duration below millimolar concentrations,46 and we found it to be without effect on the right ventricular walls or papillary muscle at 10 µmol/L. The absence of a direct effect on action potential configuration suggests that the improved recovery of electrical and contractile function obtained with sodium nitroprusside during reflow derives from noncardiac myocyte effects of the drug. Additionally, since nitroprusside did not prevent action potential prolongation due to X/XO in papillary muscles, it is also unlikely that the cardioprotection provided by sodium nitroprusside was due to the drug acting as an O-R scavenger. It is possible that nitric oxide released by nitroprusside interacted with superoxide radicals produced during X/XO or reperfusion. However, this would not appear to be important in the present study because the reaction product of superoxide and nitric oxide is peroxynitrate, which is a highly reactive and toxic molecule,50 and would have provoked enhanced damage rather than the cardioprotection that we observed with sodium nitroprusside.
There is ample evidence that ischemia/reperfusion may cause vascular injury in a variety of organs, including the heart,20 21 and O-Rs are implicated as mediators of this damage.22 For example, vasodilator reserve and reactivity to several vasoactive substances are reported to be depressed by brief periods of coronary occlusion in several preparations (eg, see References 23, 24, 51, and 5223 24 51 52 ). Moreover, Bolli et al22 observed increased vascular resistance and depressed regional blood flow monitored by microspheres in postischemic compared with nonischemic endocardium, which could be reversed by O-R scavengers,21 22 and exogenous O-Rs are known to cause coronary vascular dysfunction53 and endothelial injury.54 55 56 In the present study, we focused on the potential role of O-R stress–induced vascular injury on action potential duration because the slow O-R scavenger–sensitive recovery of APD90 during reflow and the decline in duration with exogenous O-R stress could not be explained by a direct effect of reactive oxygen metabolites on cardiac myocytes. However, the data also suggest that depressed coronary flow due to O-Rs during reperfusion also contributed to arrhythmogenesis and postischemic contractile dysfunction. When the rise in coronary pressure during reflow was inhibited by scavenger cocktail or nitroprusside, action potential configuration recovered rapidly, the incidence of arrhythmias was reduced, and contractile function was significantly improved compared with untreated tissues.
The ability of nitroprusside to inhibit reperfusion arrhythmogenesis and improve recovery of contractile function in the absence of a direct action or inherent O-R–scavenging properties provides strong evidence for the involvement of coronary vasoconstriction and the no-reflow phenomenon. Although a burst of nitric oxide release at reflow may be deleterious to postischemic recovery,57 slow release of nitric oxide by nitroprusside may maintain basal coronary vasodilation, improve perfusion of the myocardium, and, as a consequence, improve the recovery of action potential configuration, reduce the incidence of arrhythmia, and reduce postischemic contractile dysfunction. Further experiments are required to determine the mechanism by which vasodilatation during reflow leads to action potential prolongation, reduces the incidence of arrhythmia, and improves contractile function.
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Acknowledgments |
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This study was supported by a grant (DG-N026) from the Medical Research Council (MRC) of Canada. Dr Cole was a Scholar of the MRC, and Dr Jabr was a trainee of the Canadian Heart Foundation. The authors express their thanks for the expert technical assistance of Caroline McPherson and Rodel Padua.
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Footnotes |
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Reprint requests to Dr W.C. Cole, Associate Professor, Department of Pharmacology and Therapeutics, HMRB, University of Calgary, 3330 Hospital Dr, NW, Calgary, Alberta, Canada, T2N 4N6.
Previously published in part as an abstract (Biophys J. 1994;66:A83).
Received March 7, 1994; accepted March 31, 1995.
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