(Circulation. 1999;99:823-828.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Inhibition of Inositol(1,4,5)Trisphosphate Generation by Endothelin-1 During Postischemic Reperfusion
A Novel Antiarrhythmic Mechanism
From the Cellular Biochemistry Laboratory and the Experimental Cardiology Laboratory (X.-J.D.), Baker Medical Research Institute, Prahran, Victoria, Australia.
Correspondence to Dr E.A. Woodcock, Baker Medical Research Institute, PO Box 6492, Melbourne 8008, Victoria, Australia. E-mail liz.woodcock{at}baker.edu.au
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Abstract |
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Background—Reperfusion of ischemic rat hearts in the presence of thrombin or norepinephrine but not endothelin-1 causes the generation of inositol 1,4,5-trisphosphate (Ins 1,4,5P3) and arrhythmias. The present study investigates the effect of endothelin-1 on these responses.
Methods and Results—Ins 1,4,5P3 generation was quantified by use of [3H] labeling and high-performance liquid chromatography as well as by mass analysis. Twenty minutes of global ischemia followed by 2 minutes of reperfusion increased [3H]Ins 1,4,5P3 from 2828±265 to 5033±650 cpm/g tissue in the presence of thrombin 2.5 IU/mL and to 4561±286 cpm/g tissue in response to release of norepinephrine (n=4, P<0.01) in both cases. Reperfusion in the presence of endothelin-1 alone caused no change in Ins 1,4,5P3 (2762±240 cpm/g tissue), but when added together with thrombin or norepinephrine, endothelin-1 reduced the Ins 1,4,5P3 responses to 2313±197 and 1764±168 cpm/g tissue, respectively (n=4, P<0.01 in both cases). Similar inhibitory interactions between endothelin-1 10 nmol/L and thrombin 2.5 IU/mL were observed under normoxic conditions in nonperfused ventricle, eliminating the possibility that excessive vasoconstriction was responsible. In parallel studies, endothelin-1 suppressed the development of reperfusion arrhythmias initiated by either thrombin (ventricular fibrillation, 75% to 39%, n=16 to 18) or norepinephrine (83% to 8%, n=12 to 22) (P<0.01 in both cases).
Conclusions—Inhibition of Ins 1,4,5P3 generation during myocardial reperfusion by endothelin-1 represents a novel antiarrhythmic mechanism.
Key Words: inositol trisphosphate • arrhythmias • endothelin • thrombin • receptors, adrenergic, alpha
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Under conditions of myocardial ischemia and reperfusion, both norepinephrine and thrombin exert direct proarrhythmic effects.1 2 3 The arrhythmogenic responses to norepinephrine are mediated by
1-adrenergic receptors and can be initiated by
norepinephrine released from the sympathetic nerves under
these conditions.4 5 Both
1-receptors and thrombin receptors
activate phosphatidylinositol (PtdIns) turnover in the
myocardium with generation of inositol phosphates (InsPs)
and sn-1,2-diacylglycerol.6 7 8 Our studies
have shown that the proarrhythmic actions of both of these effectors
are related to the generation of IP3 within the
ventricle.9 10 Studies from other laboratories have also
pointed to a proarrhythmic activity of IP3. In
isolated cardiomyocytes, direct intracellular application
of IP3 but not inositol 1,3,4,-trisphosphate
[Ins(1,3,4)P3] caused
electrophysiological perturbations,
especially action potential prolongation, which could lead to
arrhythmogenesis.11 In addition, IP3
has been shown to cause Ca2+
oscillations in myocardial tissue12 and to
activate Na+/Ca2+
exchange,13 both of these actions being potentially
arrhythmogenic.
Under normoxic conditions, activation of cardiac
1-adrenergic receptors does not cause
detectable increases in IP3 content when
experiments are performed using preparations of intact heart tissue,
and InsPs accumulated appear to derive primarily from
Ins(1,4)P2.8 Under conditions of
early reperfusion, the InsP response to norepinephrine
changes so that rapid generation of IP3 is
observed.14 The thrombin-induced InsP response is
quantitatively small under normoxic conditions, but unlike the
norepinephrine response, increases in
IP3 are observed.7 Under reperfusion
conditions, the IP3 response to thrombin is
markedly enhanced, so that it is similar to the
norepinephrine response.10 In marked contrast
to findings with norepinephrine or thrombin, reperfusion of
catecholamine-depleted hearts in the presence of
endothelin-1 (ET-1) does not cause any increase in
IP3 and does not cause
arrhythmias.15 In the course of studies to
delineate the pathways mediating the responses to the different agents,
it was found that ET-1 was able to suppress the
IP3 responses both to thrombin and to
norepinephrine under reperfusion conditions. Our previous
studies would predict that inhibition of IP3
responses during myocardial reperfusion should be antiarrhythmic. The
present studies were undertaken to investigate this possibility
further.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Adult male Sprague-Dawley rats (weight, 160 to 200 g) were used. All experiments were approved by the Alfred Hospital and Baker Institute Animal Ethics Committee.
[3H]InsP Responses in Right Ventricle Strips
Right ventricles were dissected from rat hearts under ice-cold
saline, and thin strips (10 to 20 mg) were mounted in siliconized 3-mL
organ baths in HEPES-buffered Krebs medium equilibrated with 5%
CO2 /95% O2 at 37°C. The
medium contained the following, in mmol/L: HEPES buffer (pH 7.4)
20, glucose 11, Na+ 138, K+
4.5, Mg2+ 1.2,
HCO3- 25,
PO4 1.2, and Ca2+ 2. After
equilibration, the strips were labeled by incubation in medium
containing [3H]inositol 20 µCi/mL for 4
hours. After this time, labeled medium was removed, and the tissue was
washed with fresh medium and subsequently incubated for 10 minutes with
medium containing LiCl 10 mmol/L and propranolol
1 µmol/L before addition of agonists as indicated.
[3H]InsP generation was terminated after 20
minutes by placing the strips into liquid N2.
Tissue was weighed and maintained at -70°C before extraction.
[3H]InsP Responses in Perfused Rat Hearts
Where indicated, rats were treated with reserpine 5 mg/kg IP 18
hours before the experiment to deplete endogenous
norepinephrine. Catecholamine depletion was
confirmed by a reduction in heart norepinephrine content
from 582±310 to 25±18 mg/g wet weight (n=6,
P<0.01).16 All rats were given heparin (1
IU/g IP) 30 minutes before decapitation. Hearts were removed
immediately, chilled in ice-cold saline, and cannulated via the
ascending aorta to initiate Langendorff perfusion with HEPES-buffered
Krebs medium, pH 7.4, at 37°C, at 7 mL/min. After a 15-minute
equilibration period, hearts were labeled with
myo-[3H]inositol (2 µCi/mL) for 2
hours. Labeled medium was then removed and replaced with medium
containing propranolol and LiCl. For experiments under
normoxic conditions, ET-1 or thrombin was added to the
perfusate for 20 minutes, and the generation of
[3H]InsPs was terminated by freezing the
ventricles in liquid N2 after excision at the
atrioventricular junction.
For experiments under reperfusion conditions, 3H-labeled hearts were subjected to 10 minutes of perfusion with medium containing LiCl and propranolol, and then ischemia was produced by turning off the perfusion pump for 20 minutes. Reperfusion was commenced by restarting flow at 7 mL/min. Where indicated, ET-1 or thrombin was added to the perfusate immediately before reperfusion. [3H]InsP accumulation was terminated by freezing the hearts in liquid nitrogen, after excision at the atrioventricular junction. The frozen ventricles were weighed, and InsPs were extracted and quantified as described below.
Extraction and Quantification of [3H]InsPs
InsPs were extracted from whole frozen ventricle (
1 g) in 3.5
mL of 5% trichloroacetic acid (TCA) containing 2.5 mmol/L EDTA
and 5 mmol/L phytic acid with a Polytron
homogenizer, followed by sonication as described
previously.14 After centrifugation at
5000g for 10 minutes at 4°C, supernatants were removed and
TCA pellets were reextracted with 1.5 mL TCA/EDTA/phytic acid. The
combined aqueous phases were pooled and extracted with a 1:1 mixture of
Freon and tri-N-octylamine 0.75 mL/mL supernatant. The final
aqueous phase was collected and treated with proteinase K 50 µg/mL (2
hours, 50°C), and the samples were then passed through a 1-mL
Dowex-50 column (4% cross-linked, 4 to 400 mesh size) and eluted with
1 mL water. Urea 0.05 mol/L final was added, and samples were
lyophilized before high-performance liquid
chromatographic (HPLC)
analysis.14 3H-labeled
InsPs were separated by anion-exchange HPLC and quantified with an
on-line ß-counter (Radiomatic Instruments, model CR) as described
previously.14
Extraction and Quantification of [3H]Inositol
Phospholipids
TCA pellets obtained as described above were extracted with 3 mL
of chloroform:methanol:HCl 200:100:1 by sonication and vigorous
vortexing. One milliliter of 1 mmol/L EDTA was added, and the
phases were separated by centrifugation. The interface
was reextracted, and the final organic phase was evaporated under
N2. The dried lipids were deacylated by treatment
with methylamine:methanol:butanol 42:47:9 for 45 minutes at 50°C,
followed by evaporation under vacuum. The residue was dissolved in
water (1 mL) and extracted with butanol:petroleum ether:ethyl formate
20:40:1. Phases were separated, and the organic phase was reextracted.
The combined aqueous phases were pooled and applied to 1-mL columns of
Dowex-1 (formate form). Columns were washed with 20 mL water.
[3H]Glycerophosphoinositol
(deacylated PtdIns) was eluted with 20 mL of 200 mmol/L ammonium
formate, 0.1 mol/L formic acid. After a further 20 mL of this solution,
[3H]glycerophosphoinositol
4-monophosphate [deacylated PtdIns(4)P] was eluted with 20 mL of
400 mmol/L ammonium formate/0.1 mol/L formic acid, and
[3H]glycerophosphoinositol
4,5-bisphosphate [deacylated PtdIns(4,5)P2] was
eluted with 7 mL of 1 mol/L ammonium formate/0.1 mol/L formic acid.
Samples were counted with a ß-counter.
The efficiency of the deacylation procedure was checked by counting the organic phase together with any remaining insoluble residue. On this basis, <5% of the 3H-labeled lipids remained unhydrolyzed. The 3H-labeled compounds were identified as deacylated PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 by removing the mobile phase by repeated lyophilization and then performing anion-exchange HPLC as described above.
Measurement of IP3 and PtdIns(4,5)P2
Mass
Tissue was extracted as described above, except that ATP 5
mmol/L replaced phytic acid in the extraction medium, the proteinase K
step was omitted, and urea was not added before lyophilization.
Lyophilized samples were resuspended in water and
neutralized with NaHCO3 plus NaOH as required.
The IP3 content of the neutralized samples was
measured with a commercial kit (Amersham).
Lipids were extracted from the TCA pellets remaining after IP3 extraction from unlabeled tissue (see above) by the method described below. The deacylated lipids were subsequently deglycerated with sodium periodate and dimethylhydrazine as described elsewhere.17 Mass assay of the IP3 resulting from deacylation and deglyceration of PtdIns(4,5)P2 was carried out as above.
The identity of the compound measured in the assay as IP3 [and thus the progenitor lipid as PtdIns(4,5)P2] was validated by treating aliquots of representative samples with pure IP3 5'-phosphatase (10 ng) for 30 minutes at 37°C, followed by boiling to inactivate the enzyme. This treatment removed material that displaced [3H]IP3 in the binding assay, identifying the measured substance as IP3 [or PtdIns(4,5)P2].
Arrhythmogenic Responses
Rats were anesthetized with pentobarbital 60 mg/kg IP
and given heparin 200 IU IV. Hearts were cannulated in situ via the
ascending aorta and perfused at 7 mL/min with Krebs-Henseleit medium
constantly gassed with 5% CO2/95%
O2, pH 7.4, at 37°C. The perfusate
included propranolol 1 µmol/L to block
ß-adrenergic receptors and LiCl 10 mmol/L to replicate the
conditions used in studies of InsP release. This preparation is
essentially similar to a standard Langendorff preparation, except that
the heart remains attached to the open chest of the animal and the
perfusion flow rate is controlled by a peristaltic pump.9
A 10-minute period was allowed to stabilize the preparation before the
experiment, then the left coronary artery was ligated to
produce regional ischemia, involving 30% to 40% of the heart,
judged from the reduction in coronary flow required to maintain
coronary perfusion pressure (CPP). After 20 minutes, the
ligature was released to initiate reperfusion. The perfusion flow rate
was adjusted coincidentally with coronary occlusion and
reperfusion to maintain a constant CPP. We have previously demonstrated
that the incidences of ischemic ventricular
tachycardia (VT) and ventricular fibrillation
(VF) were significantly reduced by propranolol to 40% and
30%, respectively, compared with 80% and 75% in the control group
(both P<0.05). Ischemic ventricular
arrhythmias were unaffected by LiCl.9 The
incidence of reperfusion arrhythmias was not altered by
propranolol or LiCl alone or in combination. During the 20
minutes of ischemia and 5 minutes of reperfusion, the
epicardial ECG and the CPP were monitored. Ventricular
arrhythmias were quantified according to the Lambeth Convention
guidelines.18
Materials
Reserpine, propranolol, norepinephrine,
thrombin, and LiCl were obtained from Sigma Chemical Co. ET-1 was
obtained from Peninsula Laboratories. Thrombin receptor–activating
peptide (TRAP, SFLLRN) was from Auspep.
myo-[3H]Inositol,
[3H]IP3,
[14C]Ins(1)P1, and
IP3 mass assay kits were from Amersham.
[3H]Ins(1,3,4)P3,
[3H]Ins(1,4)P2, and
[3H]Ins(4)P1 were from
New England Nuclear and were supplied by Auspep. All other reagents
were analytical reagent grade, and solutions were prepared in
Milli-Q water.
Statistics
Group differences for nonparametric data (incidence
and duration of VT and VF) were examined with a
2 test with Fisher's exact calculation or by
a Mann-Whitney rank-sum test. Parametric data (InsP responses)
were examined by ANOVA followed by Student's unpaired t
test if significant group differences were found. A value of
P<0.05 was considered significant.
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Results |
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InsP Responses in Normoxic Ventricle in the Presence of ET-1 and Thrombin
Strips of right ventricle were labeled with [3H]inositol, preincubated in medium containing LiCl 10 mmol/L and BSA 0.1% for 10 minutes, and then stimulated with ET-1 10 nmol/L or thrombin 2.5 IU/mL for 20 minutes. [3H]InsPs were extracted and quantified. ET-1 caused increases in [3H]Ins(1)P1, [3H]Ins(4)P1, and [3H]Ins(1,4)P2, but there was no increase in [3H]IP3. Thrombin caused a smaller overall increase in labeled InsPs but, unlike ET-1, caused an increase in [3H]IP3 in addition to increases in the lower InsPs. When ET-1 was added together with thrombin, the overall [3H]InsP response was higher than the thrombin response but lower than the response to ET-1. Importantly, no increase in [3H]IP3 was observed (Figure 1
, top).
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Similar experiments were performed in perfused rat ventricles.
[3H]Inositol-labeled hearts were perfused with
medium containing LiCl and BSA for 10 minutes, and then ET-1 10 nmol/L,
thrombin 2.5 IU/mL, or both agonists were added and perfusion was
continued for 20 minutes. Responses to the 2 agonists were similar to
those described for right ventricle strips. ET-1 caused increases in
3H-labeled InsPs without any increase in
IP3. Thrombin caused a smaller stimulation but
did produce an increase in
[3H]IP3. Addition of ET-1
together with thrombin prevented the thrombin-induced
IP3 response (Figure 1
, bottom).
InsP Responses During Postischemic Reperfusion: Effects
of Endothelin and Thrombin
We have previously reported that, under conditions of early
reperfusion, the IP3 response to thrombin is
markedly enhanced.10 Experiments were performed to
determine whether ET-1 inhibited the IP3 response
to thrombin under reperfusion conditions. Hearts from
catecholamine-depleted rats were labeled with
[3H]inositol and subsequently subjected to 20
minutes of global zero-flow ischemia followed by 2 minutes of
reperfusion with oxygenated medium at 7 mL/min. In the
absence of added agonists, the [3H]InsP level
after 2 minutes of reperfusion was not different from that after 20
minutes of ischemia, as described previously.14
When thrombin 2.5 IU/mL or TRAP 50 µmol/L was added to the
perfusate immediately before perfusion was restarted,
generation of [3H]IP3 was
observed. When ET-1 10 nmol/L was added, there was no increase in
[3H]IP3, as described
previously.15 Experiments were performed in which both
thrombin 2.5 IU/mL and ET-1 10 nmol/L were added to the
perfusate immediately before reperfusion. In the presence of
both agonists, the rise in
[3H]IP3 seen in the
presence of thrombin alone was eliminated (Figure 2, top left). A similar reduction in the
IP3 response to thrombin was observed when ET-1
was added at 3 nmol/L (data not shown).
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Similar experiments were performed using unlabeled hearts and
quantifying IP3 mass. Two minutes of reperfusion
of catecholamine-depleted ischemic hearts in the
presence of thrombin 2.5 IU/mL or TRAP 50 µmol/L caused an
increase in IP3 content. No such increase was
observed in hearts perfused with ET-1 10 nmol/L (Figure 2, top
right).
Phospholipid Changes During Reperfusion in the Presence of ET-1
and Thrombin
Competition for phospholipid precursors might provide an
explanation for the inhibitory effect of adding ET-1 and
thrombin in combination. To address this possibility, the levels of
3H-labeled PtdIns, PtdIns(4)P, and
PtdIns(4,5)P2 were measured in the hearts
subjected to the procedures described above. In
catecholamine-depleted ventricles, levels of
PtdIns(4,5)P2, both
3H-labeled and mass, were high, and these levels
decreased when thrombin or TRAP was added during reperfusion. ET-1
caused no decrease in PtdIns(4,5)P2 and prevented
the decrease observed in the presence of thrombin (Figure 2, bottom left). Thus, competition for substrate is an unlikely
explanation for the observed mutually inhibitory
interactions between thrombin and ET-1.
Interaction Between ET-1 and Norepinephrine During
Reperfusion
Under reperfusion conditions, InsP responses to
norepinephrine and thrombin appear similar, and both agents
initiate arrhythmias.10 Experiments similar to
those involving ET-1 and thrombin were performed in which the effects
of ET-1 on the IP3 response to
norepinephrine were investigated. Intact
3H-labeled hearts (without
catecholamine depletion) were subjected to the
ischemia/reperfusion procedure in the presence or absence of
ET-1 10 nmol/L. In the absence of ET-1, reperfusion caused the
generation of [3H]IP3 as
described previously,9 and ET-1 prevented this response.
Similar results were obtained when catecholamine-depleted
hearts were perfused with norepinephrine 100 µmol/L
(Figure 3). In contrast to the
inhibitory action of ET-1, addition of thrombin 2.5 IU/mL
during reperfusion did not reduce the IP3 caused
by endogenously released norepinephrine (Figure 3).
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Effect of ET-1 on Reperfusion Arrhythmias Initiated by
Norepinephrine or Thrombin
Intact perfused hearts (without reserpine pretreatment) were
subjected to 20 minutes of regional ischemia, followed by
reperfusion. An ECG was recorded during the first 5 minutes of
reperfusion, and all reperfusion VT and VF occurred within the first 2
minutes after reperfusion was commenced. ET-1 at 10 nmol/L was added at
reperfusion and maintained for a period of 4 minutes. The addition of
ET-1 largely prevented the onset of VT and VF
(Table), a finding in keeping with
the inhibition by ET-1 of reperfusion-mediated
IP3 release (Figure 3). In hearts treated
with ET-1 at 10 nmol/L, a progressive rise in CPP was observed (from
33±1 mm Hg at 0 minutes to 44±2, 54±3, 63±4, and 67±4
mm Hg at 1, 2, 3, and 4 minutes of reperfusion, respectively, n=8,
P<0.01 by ANOVA). Because hearts were perfused at a
constant flow rate, the increase in CPP indicated vasoconstriction.
Although the flow was delivered by a pump, an uneven distribution of
myocardial flow might ensue in the presence of ET-1. To address this
question, ischemic hearts were perfused with ET-1 at 3 nmol/L,
a concentration that has been shown to effectively activate the
release of InsPs during reperfusion.10 Although ET-1
at this dose had minimal vasoconstrictor effects (CPP increased from
34±1 mm Hg to 38±1, 41±2, 44±2, and 45±3 mm Hg at 1,
2, 3, and 4 minutes of reperfusion, respectively, n=8,
P<0.01), reperfusion arrhythmias were essentially
eliminated (Table). Thus, this finding argues against
coronary vasoconstriction with resultant slower reperfusion as
an explanation for the antiarrhythmic action of ET-1 under reperfusion
conditions.
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Similar experiments were performed in
catecholamine-depleted hearts perfused with thrombin during
reperfusion. Depletion of catecholamines caused a reduction
in reperfusion arrhythmias from 100% (VT incidence) and 83%
(VF incidence) in intact hearts to 17% (VT) and 0% (VF) in the
reserpine-treated group. The addition of thrombin 2.5 IU/mL increased
the incidence and duration of reperfusion arrhythmias relative
to the catecholamine-depleted controls (Table).
Addition of ET-1 10 nmol/L alone did not increase either the incidence
or duration of arrhythmias over the 5-minute reperfusion
period.10 However, when added together with thrombin 2.5
IU/mL, ET-1 10 nmol/L reduced the arrhythmogenic action of thrombin
(Table).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Under conditions of ischemia and reperfusion, both norepinephrine and thrombin are arrhythmogenic, and ET-1 is not.1 3 15 Our previous studies have provided evidence that the onset of arrhythmias is related to the generation of IP3 under reperfusion conditions in the presence of thrombin or norepinephrine but not in the presence of ET-1. Under reperfusion conditions, ET-1 induces a robust InsP response that does not involve an increase in IP3; furthermore, our data indicated that the InsPs generated after ET-1 stimulation did not derive from IP3.15 The marked difference between the InsP responses initiated by norepinephrine or thrombin on the one hand and those by ET-1 on the other indicates that the changes caused under reperfusion conditions are specific to certain receptor classes. Furthermore, the finding that ET-1 is not arrhythmogenic even though it causes InsP generation points to IP3 specifically and as being related to arrhythmogenic responses.
In the present study, ET-1 was shown to inhibit the
IP3 responses to both thrombin and
norepinephrine during early reperfusion. This
inhibitory action of ET-1 did not appear to be due to
intense vasoconstriction preventing effective reperfusion, for 2
reasons. First, similar inhibition of IP3
responses was observed under normoxic conditions and in nonperfused
ventricle strips (Figure 1). Second, ET-1 inhibited the
reperfusion-induced IP3 response at
concentrations below those that caused observable vasoconstriction.
Furthermore, our studies showed that the inhibitory action
of ET-1 was not caused by competition for phospholipid precursors,
because the content of the immediate precursor of
IP3, PtdIns(4,5)P2, was
high in the presence of both ET-1 and thrombin (Figure 2). Thus,
it seems likely that ET-1 receptor activation can inhibit the
generation of IP3 when this is stimulated by
either norepinephrine or thrombin. However, whether this
inhibitory action occurs as a direct consequence of
ET-receptor activation or whether it is mediated by some distal
component of the ET-receptor signal transduction pathway remains to be
established. Furthermore, the inhibition of IP3
generation might be directed at the level of the receptors, the G
proteins, or the phospholipase C enzymes. A recent study described the
phosphorylation and partial inactivation of
1B-adrenergic receptors after stimulation of
ETA receptors in Rat-1
fibroblasts,19 and a similar mechanism might be involved
in this instance. However, the finding that responses to
norepinephrine and thrombin were similarly affected argues
for an action at a target common to both receptor systems.
Although norepinephrine and thrombin are
proarrhythmic under reperfusion conditions
(Table),2 3 there are conflicting reports
concerning the arrhythmogenic potential of ET-1. Direct proarrhythmic
activity was reported in studies of perfused pig hearts, but these
arrhythmias may have been secondary to
vasoconstriction.20 Other studies have reported
potentially antiarrhythmic actions of ET-1.21 We have
found that prolonged infusion with ET-1 (>10 minutes) causes
arrhythmias even under normoxic conditions, and these are most
likely secondary to severe vasoconstriction. During reperfusion in the
absence of other proarrhythmic agents, ET-1 has no effect on
arrhythmogenesis,15 but the present studies show that,
in the presence of thrombin or norepinephrine, ET-1 is
effectively antiarrhythmic (Table). Thus, the apparent
discrepancies in the observed effects of ET-1, proarrhythmic,
antiarrhythmic, or nonarrhythmic, are related to the different
conditions studied. The effects of ET-1 in vivo would comprise a
complex mixture of these opposing activities. Although ET-1 is clearly
unsuitable for use as an antiarrhythmic agent, the antiarrhythmic
action of ET-1 points to a novel antiarrhythmic mechanism that might
provide scope for the development of entirely novel agents that inhibit
the reperfusion-induced generation of IP3 and
thus prevent the initiation of arrhythmias. Although our
studies have implicated IP3 in the genesis of
arrhythmias under ischemic22 and
reperfusion conditions,9 10 recent studies have extended
the pathological role of IP3 to inflammatory
heart diseases by showing that inhibitors of the
IP3 pathway prevent the development of
electrophysiological disturbances
in cardiomyocytes caused by the presence of
activated lymphocytes.11 23
Responses to combinations of thrombin and ET-1 or norepinephrine and ET-1 together are different from the responses to either effector alone, and this applies to InsP generation as well as arrhythmogenesis. In the in vivo situation, all of these factors are likely to be present in the ischemic myocardium. In addition, the presence of inflammatory lymphocytes could further complicate such interactions by causing IP3 generation within the myocardium. Thus, IP3 levels in the ischemic and reperfused myocardium in vivo are under multifactorial control, and the overall arrhythmogenic response may well depend critically on combinations of effectors. Despite the complexities, the present findings stress the importance of IP3 in initiating arrhythmias. Agents that prevent IP3 generation may prove useful in preventing the development of intractable arrhythmias.
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Acknowledgments |
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This work was supported by the Australian National Health and Medical Research Council and by a grant-in-aid from the National Heart Foundation of Australia.
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Footnotes |
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Guest Editor for this article was Hein J.J. Wellens, MD, University Hospital Maastricht, Netherlands.
Received April 23, 1998; revision received September 3, 1998; accepted October 1, 1998.
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