© 1995 American Heart Association, Inc.
Articles |
Effects of NO Modulation on Cardiac Arrhythmias in the Rat Isolated Heart
From the Cardiovascular Research Laboratories, Vascular Biology Research Centre, Department of Pharmacology, Division of Biomedical Sciences, King’s College, University of London (UK).
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Abstract |
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Abstract It has been proposed that NO may function as an endogenous cardioprotectant. We have investigated whether modulation of NO levels (detected in coronary effluent by chemiluminescence) by a blocker of its synthesis, by supplementation of its precursor, and by administration of an NO donor can influence reperfusion arrhythmias in the isolated rat heart. Rat hearts were perfused with modified Krebs’ solution and subjected to 5, 35, or 60 minutes of left regional ischemia followed by 10 minutes of reperfusion. NG-Nitro-L-arginine methyl ester (L-NAME), which blocks NO synthase, increased the incidence of reperfusion-induced ventricular fibrillation (VF) from 5% in the control condition to 35% after 60 minutes of ischemia (n=20, P<.05). The profibrillatory effect of L-NAME was prevented in hearts coperfused with 1 or 10 mmol/L L-arginine (an NO precursor) but persisted in hearts coperfused with D-arginine (1 mmol/L). L-NAME did not increase VF susceptibility in hearts reperfused after 5 or 35 minutes of ischemia. L-NAME caused sinus bradycardia (264±10 versus 309±5 bpm in control groups, P<.05) and reduced coronary flow before ischemia (6.2±0.6 versus 9.2±0.6 mL · min-1 · g-1 tissue in controls, P<.05). L-NAME reduced coronary effluent NO levels after 60 minutes of ischemia; during the first minute of reperfusion, values were reduced from 1457±422 to 812±228 pmol · min-1 · g-1 (P<.05). This effect was prevented by coperfusion with L-arginine (10 344±1730 pmol · min-1 · g-1, P<.05). Qualitatively similar changes occurred with other durations of ischemia, but the effects of L-NAME were not significant. L-NAME had no effect on QT interval; eg, values after 5 minutes of ischemia were 76±7 and 85±5 milliseconds in control and L-NAME–treated groups, respectively (n=20, P=NS). The NO donor sodium nitroprusside (10 µmol/L) significantly increased coronary flow 1 minute before ischemia (15.4±1.1 versus 9.2±0.6 mL · min-1 · g-1 tissue and coronary effluent NO levels (from 1122±122 to 4093±1466 pmol · min-1 · g-1, P<.05). Sodium nitroprusside prevented the proarrhythmic effect of L-NAME and maintained coronary effluent NO levels during reperfusion. NO appears to function as an endogenous cardioprotectant antifibrillatory factor in rat heart during reperfusion following sustained ischemia.
Key Words: nitric oxide • reperfusion • ventricular fibrillation
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Introduction |
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Reperfusion of the ischemic myocardium results in ventricular arrhythmias, some of which, such as VF, are lethal.1 Susceptibility to reperfusion-induced VF is determined by the interplay between several factors. At one level, VF susceptibility is determined by the presence of a variety of biochemical substances present in the reperfused tissue.2 3 These include cAMP,4 catecholamines,5 and oxygen-derived free radicals,6 all of which have been proposed to constitute endogenous biochemical arrhythmogenic factors, and others such as bradykinin,7 adenosine,8 and acetylcholine,9 which constitute endogenous protective substances. At another level, susceptibility to VF is modulated by factors such as the duration of preceding ischemia,10 11 12 the rapidity and extent of reestablishment of flow,13 14 15 16 and the heart rate during the preceding period of ischemia.17 The complexity of the interplay between these factors is exemplified by the way that the duration of preceding ischemia apparently influences the relative role of various chemical mediators of VF. For example, the relative importance of calcium and oxygen-derived free radicals appears to vary in isolated rat hearts reperfused after brief versus more sustained periods of ischemia.18 It follows that the relative importance of endogenous protectant substances may also vary according to the duration of preceding ischemia.
Evidence exists for basal as well as stimulated release of NO in the coronary circulation of the isolated guinea pig heart19 and rabbit heart,20 and Rees et al21 have shown basal release of NO from coronary vessels. In a recent study, Vegh et al22 proposed the novel concept that NO functions as an endogenous cardioprotectant. Blockers of the enzyme NOS resulted in a reduction in the protective effect elicited by two "preconditioning" occlusions of the left anterior descending coronary artery, implicating NO as an endogenous mediator of preconditioning against VF. However, in the study of Vegh et al, NO levels were not measured. Two important questions arise from their study: (1) Does NO function as an endogenous cardioprotectant against VF in the absence of preconditioning? (2) Does VF susceptibility correlate inversely with cardiac NO levels after treatment with drugs that purportedly affect NO synthesis?
The objective of the present study was to answer these questions and determine whether endogenous NO production protects the heart against VF induced by reperfusion after brief versus sustained ischemia. Our approach was to relate susceptibility to VF with levels of NO in coronary effluent (direct detection by chemiluminescence) and with other variables that can be affected by NO (ie, coronary flow). Interventions were used to block NO synthesis, reverse this block by substrate supplementation, and elevate NO levels independently of endogenous synthesis. The effects of these interventions were examined on VF susceptibility, NO levels, heart rate, and coronary flow.
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Materials and Methods |
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The perfusion technique, methods for induction of ischemia and reperfusion, methods for verification and quantification of OZ size, and the techniques for recording, quantifying, and analyzing data have all been described previously.15 23 All experiments were performed in accordance with the American Physiological Society guidelines and the United Kingdom Home Office "Guide on the Operation of the Animals (Scientific Procedures) Act 1986."
Animals: General Experimental Methods
Male Wistar rats (240 to 260 g; Tucks, UK) were
anesthetized with pentobarbital (60 mg/kg IP) and heparinized
with 250 U IP sodium heparin. Hearts were excised and placed in
ice-cold control perfusion solution containing (mmol/L) NaCl 118.5,
NaHCO3 25.0, MgSO4 1.2,
NaH2PO4 1.2, CaCl2 1.4, KCl 4.0,
and glucose 11.1. All solutions were filtered (5-µm pore size) before
use to remove particulate matter. Hearts were perfused according to
Langendörff,24 with perfusion solution delivered at
37°C and pH 7.4. Perfusion pressure was constant and equivalent to
1000 mm water. Mean coronary flow was therefore determined by
mean coronary resistance. Changes in flow were measured by
weighing coronary effluent collected over timed intervals (1 mL
of effluent weighs 1 g) by use of an Ohaus balance (Ohaus Corp)
accurate to ±1 mg (
0.05% of the minimum volume collected). A
unipolar ECG was recorded by implanting one stainless steel wire
electrode into the center of the region to become ischemic,
with a second electrode connected to the aorta. A traction-type
coronary occluder consisting of a silk suture (4/0, Mersilk)
threaded through a polythene guide was used for coronary
occlusion. The suture was positioned loosely around the left main
coronary artery beneath the left atrial appendage. Regional
ischemia was induced by tightening the occluder, and
reperfusion was induced by releasing it.
Experimental Protocols
Ischemia- and Reperfusion-Induced
Arrhythmias
Hearts were perfused for an initial 5 minutes with control
solution, and then the solution was switched in a randomized, blinded
fashion to one of five solutions: identical control solution, 100
µmol/L L-NAME, 100 µmol/L L-NAME plus 1 mmol/L
L-arginine, 100 µmol/L L-NAME plus 10 mmol/L
L-arginine, 10 mmol/L L-arginine, 100 µmol/L
L-NAME plus 10 µmol/L SNP, or 10 µmol/L SNP alone (Fig 1
). After a further 10-minute perfusion, the left
coronary artery was occluded to induce regional
ischemia. After 60 minutes of ischemia, the occluder
was released to allow reperfusion for 30 minutes. After this time,
hearts were perfused with L-NAME for an additional 10 minutes in order
to explore effects on the hyperemic index (a measure of
recovery of flow) and thus give an estimation of
endothelial integrity under the assumption that failure
of L-NAME to lower recovery of flow indicates a probable absence of
basal NO release secondary to endothelial injury. The
60-minute duration of ischemia was chosen because it was
expected to be associated with a low control incidence of
reperfusion-induced VF by virtue of the waning of VF susceptibility
as ischemic duration is extended.11 A low control
incidence of VF is necessary if a proarrhythmic effect on this
variable is to be demonstrated statistically. To maximize scope for
detection of possible small effects of L-NAME, we chose to use large
group sizes (n=20 per group) for this study.
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If NO functions as an endogenous protectant, we would predict that its role may vary according to the duration of preceding ischemia, since VF susceptibility is low with certain durations of ischemia (explicable by possible endogenous protection) and high with others (consistent with possible loss of effective protection). Thus, the protocol was repeated by using 5 minutes of ischemia in order to explore VF elicited during the waxing phase of the time-response relation, during which a low incidence of VF equivalent to that occurring with 60 minutes of ischemia is expected.23 In addition, the protocol was repeated with a 35-minute occlusion period to explore VF in hearts with a greater susceptibility to this arrhythmia.
Stereoselectivity of Actions: Experimental Protocol for
L-NAME/D-Arginine Study
To explore the selectivity of any findings with
L-arginine, experiments were performed with
D-arginine. The protocol was the same as that for the
60-minute ischemia except that the groups studied were as
follows: control, 100 µmol/L L-NAME plus 1 mmol/L
D-arginine, and 1 mmol/L D-arginine alone (n=15
per group).
Detection of NO Levels by Chemiluminescence
NO levels were measured by taking aliquots of coronary
effluent (obtained by timed collection) into 1.5-mL plastic Eppendorff
tubes (BDH Laboratory Supplies) and immediately freezing them in liquid
nitrogen. At a later date (within 1 week), NO analysis was
carried out by using a Sievers NO analyzer (model 270B, Dyson
Instruments). The technique involves an inert gas (helium) stripping NO
from the aqueous sample solution. NO is then detected by an
ozone-induced chemiluminescence reaction.25 The amount
of NO in the coronary effluent samples is determined from a
standard graph constructed by using known concentrations of NO. The NO
analyzer was connected directly to a computer (MacLab/2e,
ADInstruments) so that all analysis was automated. NO content
was expressed as picomoles per minute per gram wet weight of
perfused tissue. Values were corrected for total heart weight before
ischemia and during reperfusion and for the UZ weight during
ischemia. The rationale for this correction is that
ischemic zone tissue in isolated rat heart receives <5% of
flow per gram versus uninvolved tissue because of collateral
deficiency.26
Measurement of OZ Size and Regional Coronary
Flow
Two independent methods were used to verify occlusion and
delineate the OZ from uninvolved tissue. First, occlusion was verified
by comparing flow at 1 minute before occlusion with flow at 1 minute
after occlusion and was quantified in terms of the percentage reduction
in flow. Second, at the end of the reperfusion period, readmission of
flow was verified, and the size of the formerly occluded zone was
quantified by the disulfine blue dye exclusion method.23
OZ size was quantified as percentage of total ventricular
weight. Values of coronary flow in the UZ and the RZ were
calculated from the total coronary flow and the weight of the
OZ and the UZ, as described previously15 by using these
formulas:
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Control for Actions of L-NAME on Coronary
Flow
L-NAME was expected to lower coronary flow. If L-NAME
were found also to increase susceptibility to reperfusion-induced
VF, an argument could be made that the proarrhythmic effect was a
consequence of impairment of recovery of flow rather than a consequence
of inhibition of NO synthesis per se. To test this, we examined the
effect of deliberate restriction of flow recovery during reperfusion on
susceptibility to reperfusion-induced VF in the absence of any
administered drug. The protocol involved partially occluding the
plastic feed line connected to the aortic cannula with a screw clamp
such that coronary flow was reduced to a level equivalent to
that seen in hearts perfused with 100 µmol/L L-NAME, beginning 30
seconds before the start of reperfusion and continuing during the
entirety of the reperfusion period. This experiment was performed after
completion of the main body of the study; thus, on the basis of our
findings that L-NAME exacerbated VF only in hearts subjected to 60
minutes of ischemia (see "Results"), a 60-minute period
of ischemia was used for this protocol. Ten hearts were
used.
Exclusion Criteria
Any heart with a sinus rate of <250 bpm or a coronary
flow of >18 or <8 mL/min 5 minutes before the onset of
ischemia was excluded. This exclusion criteria did not apply to
studies involving L-NAME, since this agent was expected to reduce
coronary flow.27 Any heart not in sinus rhythm
during the 2 seconds before reperfusion was excluded from the study.
Furthermore, any heart with an OZ size of <30% or >55% was
discarded. All excluded hearts were immediately replaced.
Arrhythmia Diagnosis and ECG Analysis
A digital storage–type oscilloscope (model DSO400, Gould)
and a chart recorder (model RS3200, Gould) were used in the
identification and analysis of waveforms and diagnosis of
arrhythmias. Arrhythmias were defined according to the
Lambeth conventions (Walker et al),28 with slight
modification.29 Ventricular premature beats
were defined as discrete and identifiable premature QRS complexes; a
run of four or more ventricular premature beats was defined
as ventricular tachycardia. VF was defined as a
signal from which individual QRS deflections vary in amplitude and
coupling interval on a cycle-to-cycle basis. From the ECG, the
incidence of ventricular arrhythmias, the RR
interval, and the QT interval (measured at the point of 100%
repolarization with on-screen cursors) were obtained.
Measurement of all variables was performed blind, permitting use of sampling-based statistics (see below). Stock solutions were prepared by the experimenter and then coded by another person. Data were analyzed blind, and codes were revealed after data analysis.
Drugs and Materials
All drugs were obtained from Sigma Chemical Co and stored as
stock solutions in deionized water. SNP is light sensitive; hence,
precautions were taken to ensure its stability (the perfusion
apparatus was wrapped in aluminium foil). Water for
preparing perfusion solution was obtained by use of a
reverse-osmosis system (Milli-RO 10 and Milli-Q 50, Millipore Ltd),
which provides water of >18 M
resistivity. Water for use as part of
the NO analyzer was of "super pure" quality (Romil
Chemicals).
Statistics
Measurement of all variables was performed in a blinded
manner, permitting use of sampling-based statistics.
Gaussian-distributed variables were expressed as mean±SEM and
were subjected to ANOVA. If treatment constituted a significant source
of variance, each group was compared with the control by Dunnett’s
test. A paired t test was used for comparing the
hyperemic index before versus after the administration of
L-NAME. A value of P<.05 was taken as significant.
Mainland’s contingency tables30 were used for
nonparametric analyses (eg, analysis of VF
incidence).
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Results |
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Effects of NO Modulators on ECG Intervals and Coronary Flow
In hearts subjected to 60 minutes of regional ischemia, L-NAME increased the incidence of reperfusion-induced VF (P<.05, Fig 2
). However, it did not have any
effect on other reperfusion-induced arrhythmias, ie,
ventricular premature beats (90% versus 100% in
controls), bigeminy (70% versus 90% in controls), salvos (65% versus
80% in controls), or ventricular tachycardia
(65% versus 60% in controls). The profibrillatory effect of L-NAME
was dependent on the duration of the preceding ischemia; L-NAME
did not increase VF incidence in hearts reperfused after 35 or 5
minutes of ischemia (Fig 3). L-NAME had no
significant effect on the incidence of ischemia-induced VF
(40% versus 20% in controls).
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L-NAME had no effect on the QT interval (eg, 74±3 versus 69±3
milliseconds in controls 1 minute before reperfusion) but caused
significant bradycardia both before and during ischemia (Table 1). However, L-NAME had no effect on the PR interval at
any time during the experiment (eg, 37±1 versus 37±2 milliseconds in
controls 1 minute before reperfusion).
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L-NAME reduced coronary flow before and during ischemia
(Table 2). Reduction of flow during reperfusion by
L-NAME suggests an involvement of NO in hyperemia. This is
further supported by the observation that introduction of L-NAME to
control hearts 30 minutes after the start of reperfusion reduced the
hyperemic index within 5 minutes (Fig 4).
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L-Arginine (10 mmol/L) alone had no significant effect on
the incidence of reperfusion-induced VF (Fig 2
) (or
ischemia-induced VF, 20% versus 20% in controls) or QT
interval (eg, 75±3 versus 69±3 milliseconds in controls 1 minute
before reperfusion), PR interval, or heart rate before or during
ischemia (Table 1). L-Arginine significantly
increased coronary flow (Table 2).
The proarrhythmic effect of L-NAME in hearts reperfused after 60
minutes of ischemia was attenuated by coperfusion with 1 and 10
mmol/L L-arginine (Fig 2), which rendered VF incidence no
different from that in the control group. Since in hearts reperfused
after 35 or 5 minutes of ischemia L-NAME was without
proarrhythmic activity, there was no scope for amelioration of
proarrhythmia by L-arginine (Fig 3).
In view of the fact that effects of L-NAME on VF incidence were
observed to be significant only in hearts reperfused after a sustained
(60-minute) period of ischemia, studies with SNP were
restricted to hearts subjected to 60 minutes of ischemia. The
effects of SNP were qualitatively similar to those of
L-arginine, with no significant effect on ischemia-
or reperfusion-induced arrhythmias (Fig 2). However,
perfusion with SNP resulted in a significant increase in heart rate
both before the onset of ischemia and throughout the
ischemic period (Table 1) and a significant increase in
coronary flow (Table 2). Coperfusion of SNP with L-NAME led to
a diminution of the profibrillatory effect of L-NAME during reperfusion
such that VF incidence was no longer significantly different from
control incidence (Fig 2).
Effects of D-Arginine
The effects of D-arginine were investigated to explore
the stereoselectivity of arginine in hearts reperfused after 60 minutes
of ischemia. D-Arginine alone had no significant
effect on the incidence of reperfusion-induced VF (20% versus 0%
in controls). In addition, D-arginine was without any
effect on the QT interval (eg, 72±2 versus 70±3 milliseconds in
controls 1 minute before reperfusion), heart rate (Table 3), or coronary flow (Table 4).
D-Arginine also failed to attenuate the profibrillatory
effects of L-NAME on reperfusion-induced VF (47% VF in hearts
perfused with L-NAME plus D-arginine versus 0% in
controls) and failed to influence any effects of L-NAME on heart rate
(Table 3) or coronary flow (Table 4). These data contrast with
those for L-arginine and show that the effects of the
arginine enantiomers on responses to L-NAME were stereoselective.
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Relation Between VF Incidence and NO Levels
Stock Krebs’ solution (not used to perfuse hearts) contained no
detectable NO when processed according to the same procedure as for
coronary effluent. In rat hearts that were not perfused with
drug or subjected to regional ischemia but simply perfused with
Krebs’ solution to time match the ischemia/reperfusion
protocol, basal levels of NO in the coronary effluent were
steady for at least the first 35 minutes of perfusion (Fig 5A). In control hearts, regional ischemia reduced
NO to a minimum level reached 15 minutes after the onset of
ischemia. This was then followed by a steady rise in NO to
levels exceeding basal (Fig 5B). L-NAME caused a time-dependent
reduction of NO levels, significant from 45 minutes after the onset of
ischemia and reaching a maximum effect of >95% inhibition
(Fig 5C). NO levels were significantly reduced by L-NAME during
reperfusion. The effects of L-NAME on NO levels were completely
surmounted by coperfusion with either L-arginine or SNP
(Fig 5D, 5E, and 5H), and during reperfusion, there was a significant
rise in coronary effluent NO levels in these groups that
paralleled the hyperemic response.
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Effect of Deliberately Restricting Recovery of Flow in
Untreated Hearts
In 10 additional hearts, coronary flow was reduced by
partially occluding the cannula delivering the perfusion solution to
the heart in an attempt to match the impairment of flow recovery caused
by L-NAME. L-NAME had reduced the recovery of flow at 1 minute after
the start of reperfusion by 27%, from 9.4±0.5 to 5.9±0.6
mL · min-1 · g-1 in the initial
experiment (see Table 2). Thus, we obtained an equivalent mean flow
reduction of 31±2% at 1 minute before the start of reperfusion in
these additional hearts. Impairment of flow recovery at 1 minute after
reperfusion was also successfully achieved in these hearts; the value
was 6.3±0.4 mL · min-1 · g-1,
which is not significantly different from the value in L-NAME–perfused
hearts. In contrast to our findings with L-NAME, deliberate impairment
of recovery of flow did not elevate VF incidence to levels seen in
L-NAME–perfused hearts (35%). In fact, the incidence of
reperfusion-induced VF was 10%, very similar to the value in
control hearts reperfused without restriction in flow recovery
(5%).
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Discussion |
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Various biochemical substances including NO have been proposed as "endogenous cardioprotective" mediators.3 Evidence for NO functioning in this role is indirect. NO may play a role in preconditioning against reperfusion-induced VF,22 although this has not been supported by measurement of NO production. In the present study, we examined whether NO plays a role as a cardioprotective mediator by using pharmacological tools and direct detection of cardiac NO production.
L-NAME blocks NOS-mediated formation of NO from L-arginine.31 It is 4 to 100 times more potent than the range of its analogues.31 32 33 34 Thus, we have used L-NAME as a specific potent tool to explore the cardioprotective role of NO. The concentration of L-NAME used in the present study has been previously shown to cause >80% block of NOS and abolish pharmacological responses mediated by NO.34
L-NAME caused a fourfold increase in the incidence of reperfusion-induced VF following 60 minutes of ischemia. This proarrhythmic effect was found to be absent in hearts coperfused with L-arginine. This effect could be attributed specifically to the ability of L-arginine to compete with and overcome the inhibitory action of L-NAME on NOS, since D-arginine failed to mimic L-arginine in any respect. The endothelium-independent NO donor SNP35 had actions similar to those of L-arginine, confirming that the proarrhythmic effects of L-NAME could be surmounted by agents that elevate NO levels by different mechanisms. The proarrhythmic effect of L-NAME was associated with a significant reduction in NO levels after 1 minute of reperfusion. This effect was also surmounted by L- but not D-arginine. In hearts reperfused after 5 or 35 minutes of ischemia, L-NAME had no significant effect on reperfusion-induced VF incidence or NO levels. These data provide the first direct evidence that endogenous NO protects against reperfusion-induced VF in the absence of preconditioning.
Hyperemia (in the guinea pig heart) is mediated in part by release of NO,19 and the tissue source is most likely the coronary endothelium. If the source of cardioprotective NO is also the coronary endothelium, a functional coronary endothelium and sensitivity to the vasoconstrictor effects of L-NAME would need to be demonstrable in control hearts during reperfusion. We tested this by introducing L-NAME 10 minutes after the start of reperfusion to hearts subjected earlier to 60 minutes of ischemia in the absence of L-NAME. The hyperemic index was significantly reduced by L-NAME. This is consistent with the presence of coronary endothelium capable of basal release of NO in control hearts subjected to 60 minutes of ischemia and reperfusion.
Inhibitors of NO synthesis have previously been shown to cause coronary vasoconstriction in rabbit isolated hearts and in conscious dogs and rabbits.36 37 38 We found L-NAME to cause vasoconstriction, which was prevented by L-arginine but not D-arginine. The vasoconstriction is not likely to be responsible for the profibrillatory effects observed, since impairment of recovery of coronary flow during reperfusion would reduce, not increase, susceptibility to reperfusion-induced VF.16 L-NAME also caused sinus bradycardia. This effect was perhaps surprising, given that (1) in ventricular myocytes, slowing of spontaneous beating rate by cholinergic agonism appears to be NO-mediated39 and related to the inhibition of L-type calcium current40 and (2) in sinoatrial node cells, L-NAME (albeit at a tenfold higher concentration than that we used) inhibits cholinoreceptor-mediated inhibition of the L-type calcium current.41 Nevertheless, none of the cited studies actually shows that L-NAME increases sinus rate, particularly under conditions relevant to the present study (ie, in the absence of endogenous cholinomimetic substance). Other data actually report bradycardic effects of NOS blockers in the in vivo setting,42 43 44 data more consistent with our own. Since the bradycardia we observed with L-NAME was prevented by coperfusion with L-arginine but not D-arginine, it does appear to involve inhibition of NO synthesis. We can rule out as a mechanism for the bradycardia the possibility of a drop in sinoatrial nodal temperature as a consequence of the reduction in coronary flow caused by L-NAME, since the hearts were superfused throughout at 37°C. The important point to emphasize is that regardless of the mechanism and whether the observations were surprising or not, the bradycardia cannot itself account for the proarrhythmic effect of L-NAME, since bradycardia is not proarrhythmic in reperfused hearts.17
It may be argued that despite the evident relation between NO and the proarrhythmic effects of L-NAME, perhaps L-NAME exacerbates reperfusion-induced VF by virtue of its ability to impair recovery of coronary flow during reperfusion. It has been shown previously that after a brief period (10 minutes) of ischemia, reperfusion-induced VF incidence is barely related to the magnitude of flow recovery when tested by deliberate restriction of flow recovery, and impairment of recovery of flow tends to reduce, rather than increase, susceptibility to VF.16 Moreover, in hearts reperfused after a sustained (240-minute) period of ischemia, an inherent defect in flow recovery exists, leading to a 40% reduction in coronary flow in the RZ compared with the adjacent UZ, yet the incidence of reperfusion-induced VF in such hearts is zero.45 However, since the relation between flow recovery and susceptibility to reperfusion-induced VF has not been examined in hearts reperfused after an intermediate duration of ischemia (ie, 60 minutes, as used in the present study), it is conceivable, albeit unlikely, that L-NAME increases VF incidence by impairing recovery of flow and that the impairment in NO content has no direct influence on VF susceptibility. To test this speculative hypothesis, we deliberately restricted flow recovery in a set of untreated hearts subjected to 60 minutes of ischemia followed by reperfusion. The incidence of reperfusion-induced VF was no different from that in the original control group. Therefore, we not surprisingly conclude that L-NAME does not increase susceptibility to reperfusion-induced VF by impairing recovery of flow during reperfusion.
A second approach to the question of whether NO protects the reperfused rat heart against VF was addressed by examining the effects of SNP, which functions as a precursor of exogenous NO release.35 SNP elevated coronary flow and prevented the profibrillatory action of L-NAME. SNP relaxes smooth muscle via NO-dependent cGMP elevation.46 47 48 This indirect evidence of pharmacological activity of SNP attributable to NO release was substantiated by direct evidence from measured levels of NO in coronary effluent. These data further support the hypothesis that NO can function as a cardioprotective agent.
Although our data clearly show that endogenous NO protects against reperfusion-induced VF, the cellular mechanism involved has not been examined. One possibility relates to effects of NO on oxygen-derived free radicals. Although superoxide radicals can inactivate NO49 50 such that during reperfusion this effect contributes to increased neutrophil aggregation and adherence,51 NO may itself inactivate superoxide radicals,52 thereby reducing injury associated with superoxide and its reactive metabolites. Oxygen-derived free radicals including superoxide may play a role in the initiation of reperfusion-induced VF.11 Therefore, it is possible that the cardioprotective effects of endogenous NO may be associated with vitiation of superoxide radicals. To test this hypothesis, direct detection of free radical production would be required. Another possible explanation of action of NO is elevation of cardiac cGMP, since stimulation of endothelium-derived relaxing factor/NO production results in increased guanylate cyclase activity. However, the ability of cGMP to reduce susceptibility to reperfusion-induced VF is not established.3 The present study was not designed to examine the electrophysiological mechanism for the antifibrillatory actions of NO. To address this question, patch-clamp and activation-mapping studies would be required.
In summary, NO satisfies several of the criteria proposed recently for establishing that a substance functions as an endogenous mediator of cardioprotection.3 (1) NO was found to be present in the heart. (2) Modulation of NO levels by enhancement of synthesis/release and by inhibition of production led to corresponding modulation of susceptibility to VF. (3) Exogenous administration of (a precursor of) NO mimicked the effects of increasing production of endogenous NO. Therefore, NO appears to function as an endogenous cardioprotectant in the isolated rat heart. Although this role appears to be restricted to reperfusion following sustained (60-minute) ischemia, it is substantial, since block of NO synthesis by L-NAME quadrupled susceptibility to VF.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was funded by the Wellcome Trust. R. Pabla is the recipient of a Prize Studentship from the Wellcome Trust. We thank Dr Phil Moore (King’s College, London, UK) for his helpful comments during the preparation of the manuscript. We also gratefully acknowledge Dr Jack Botting (RDS, London, UK), who in 1989 originally suggested to us that NO may function as an endogenous antiarrhythmic substance.
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Footnotes |
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Reprint requests to Michael J. Curtis, PhD, Cardiovascular Research Laboratories, Vascular Biology Research Centre, Department of Pharmacology, Division of Biomedical Sciences, King’s College, University of London, Manresa Rd, London SW3 6LX, UK.
Previously reported in preliminary form to the British Pharmacological Society (Br J Pharmacol. 1993;110:115P; Br J Pharmacol. 1994;112:381P) and The Physiological Society (J Physiol. 1994;475P:63P; J Physiol. 1995;483P:10P-11P).
Received June 20, 1994; accepted June 23, 1995.
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