© 1998 American Heart Association, Inc.
Original Contributions |
Reduced Reperfusion–Induced Ins(1,4,5)P3 Generation and Arrhythmias in Hearts Expressing Constitutively Active 
1B-Adrenergic Receptors
From the Cellular Biochemistry (S.N.H., B.H.W., E.A.W.), Molecular Physiology (D.J.A.), and Experimental Cardiology Laboratories (X.-J.D.), Baker Medical Research Institute, Melbourne, Victoria, Australia, and Howard Hughes Medical Institute, Duke University Medical Center (C.M.), Durham, NC.
Correspondence to Dr E.A. Woodcock, Baker Medical Research Institute, Commercial Rd, Prahran 3181, Victoria, Australia. E-mail liz.woodcock{at}baker.edu.au
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Abstract |
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Abstract—Reperfusion of globally ischemic rat hearts causes the generation of inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and the initiation of arrhythmias. These responses are mediated by
1-adrenergic receptors
(ARs), but the subtype of receptor involved has not been identified.
Under normoxic conditions, hearts from transgenic animals expressing
constitutively active 1B-ARs in heart
(1B-constitutively active mutant [CAM]) showed higher
[3H] inositol phosphate responses to
norepinephrine (2.3-fold) than hearts from nontransgenic
animals (1B-WT) (1.6-fold). 1B-WT hearts
responded to 2 minutes of reperfusion after 20 minutes of global
ischemia by generation of Ins(1,4,5)P3 (5301±1310
to 11 413±1597 CPM/g tissue; mean±SEM; n=6; P<0.01
in [3H] labeling studies and 3.8±0.2 to 6.3±0.6 nmol/g
by mass analysis, n=6; P<0.05). In contrast to
findings in normoxia, hearts from 1B-CAM animals showed
no Ins(1,4,5)P3 response in early reperfusion. In parallel
studies, 1B-WT hearts developed ventricular
tachycardia and ventricular premature beats
(VPB) during 5 minutes of reperfusion after 20 minutes of
ischemia. The incidence of these arrhythmias was
reduced in the 1B-CAM hearts (95% to 62% for VPB and
47% to 12% for ventricular tachycardia; both
P<0.05). The resistance of the 1B-CAM
hearts was not due to 1B-AR–mediated preconditioning,
as the Ins(1,4,5)P3 response to thrombin receptor
activation during reperfusion was not different between the 2 groups.
To investigate the possibility of reduced 1A-receptor
activity in the 1B-CAM hearts, expression of the mRNA
for 1A- and 1B-receptors was measured.
1B-WT hearts contained mRNA for both receptor subtypes,
but the levels of 1B-receptor mRNA were 5-fold higher
than 1A-receptor mRNA. 1B-CAM hearts
contained very high levels of 1B-receptor mRNA (26-fold
increase), but the expression of mRNA for the
1A-receptors (0.141±0.035 amol/µg RNA; mean±SEM;
n=6) was reduced by 50% relative to 1B-WT controls
(0.276±0.046 amol/µg RNA; n=6; P<0.01). The
reduction in arrhythmogenic and Ins(1,4,5)P3 responses in
1B-CAM hearts provides evidence that these response are
not mediated by 1B-receptors.
Key Words: 1B-adrenergic receptor • 1A-adrenergic receptor • Ins(1,4,5)P3 • reperfusion • arrhythmia
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Introduction |
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Sympathetic control of cardiac function involves most importantly ß-ARs that mediate their effects primarily by activation of adenylyl cyclase and consequent stimulation of the protein kinase A cascade. The heart also contains
1-ARs that do
not play a major functional role under normal
physiological conditions but increase in relative
importance under pathological conditions, including ischemia
and postischemic reperfusion,1 as
well as some forms of heart failure.2 3
1-Receptors have been implicated in the
genesis of arrhythmias under these pathological
conditions.2 4
1-Receptors activate the
phosphatidylinositol (PtdIns)-specific phospholipase C with the
generation of inositol phosphates (InsPs) and
sn-1,2-diacylglycerol. Unlike most other cell types,
however, large increases in inositol(1,4,5)-
trisphosphate [Ins(1,4,5)P3] are not
observed when intact heart tissue is stimulated with
1-adrenergic agonists.5
The heart contains a relatively low concentration of
Ins(1,4,5)P3 receptors,6 7
and these do not appear to be localized primarily on
Ca2+ stores. Responses to
Ins(1,4,5)P3 are slow and
weak,8 and the Ca2+ so
generated does not contribute to calcium-induced calcium
release.9 Thus Ins(1,4,5)P3
is unlikely to be important in regulating beat-to-beat changes in
Ca2+ under physiological
conditions.
In contrast to findings under physiological
conditions, reperfusion after global ischemia causes a rapid,
transient generation of
Ins(1,4,5)P3.10 This
response is dependent on norepinephrine, either exogenously
added or released from sympathetic nerves, and is mediated via
1-ARs. More importantly, generation of
Ins(1,4,5)P3 was shown to be necessary for the
initiation of reperfusion arrhythmias under these
conditions.11 12 Inhibition of the
1-receptor–mediated
Ins(1,4,5)P3 response thus provides a suitable
target for the development of antiarrhythmic agents.
Heart contains both 1A- and
1B-ARs expressed at the protein level. In rat,
80% of the expressed receptor proteins are of the
1B-subtype, whereas in human tissue, the
receptors are mostly 1A-subtype.
1D-Receptors do not appear to be expressed in
heart at the protein level.13 14 15 16 Both the
1A- and 1B-receptor
classes can generate InsPs in isolated cell
systems.17 In intact rat and rabbit heart, under
normoxic conditions, InsP generation appears to be mediated primarily
by 1B-receptors,18 but
the contribution from the 1A-subtype increases
with cardiomyocyte isolation and
culture.15 19 Furthermore,
1A-receptor activity has been associated with
pathological changes leading to hypertrophy in isolated
cells and in hearts in vivo.19 20 The current
studies were undertaken to investigate the subtype of
1-receptor involved in mediating inositol
phosphate responses in heart under conditions of
postischemic reperfusion. Studies were performed using
transgenic mice with cardiac targeted expression of constitutively
active 1B-ARs. These mutant receptors have
high activity in the absence of agonist and, in addition, show
increased agonist affinity and increased maximal activation by added
agonist.21 It was reasoned that if the
reperfusion response is mediated via 1B-ARs,
then a heightened activity would be expected in
1B-constitutively active mutant (CAM) hearts.
Thus these experiments investigated the activity of the constitutively
active 1B-ARs under normoxic conditions and
under conditions of postischemic reperfusion.
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Materials and Methods |
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Animals
Parent
1B-CAM mice were generated at
the Howard Hughes Medical Institute, Duke University. The mutant
receptor is expressed under an -myosin heavy chain promoter, as
described previously.22 Female mice derived from
F1 crosses made from SJL and C57 strains were
used to breed with heterozygotic male 1B-CAM
(transgenic/positive) mice. Animals were screened for the transgene
using Southern blot hybridization of DNA extracted from tail biopsies.
Approximately 50% of the offspring were
1B-CAM. Negative
(1B-WT) littermates were used as controls.
Both male and female mice were used in equal numbers, ranging in age
from 12 to 30 weeks. No differences in responses were observed between
male and female animals. Where indicated, mice were treated with
reserpine (3 mg/kg IP) 24 hours before experimentation to deplete
endogenous catecholamines. The procedure
resulted in a reduction of cardiac norepinephrine, measured
as described previously,23 from 559±52 to
9.5±4.7 ng/g wet weight (mean±SEM; n=4; P<0.01).
[3H]InsPs in Isolated Perfused Mouse Hearts
Adult mice were injected with heparin (5 IU/g) for 30 minutes
before killing by cervical dislocation. Hearts were removed and chilled
immediately in ice-cold saline. Cannulation of hearts was via the aorta
and carried out in ice-cold HEPES-buffered Krebs medium according to
the Langendorff method. 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. Hearts
were perfused with medium at 2 mL/min in 5 mL organ baths, kept at
37°C, and gassed constantly with 95% O2/5%
CO2. After 5 minutes of equilibration, inositol
phospholipids were labeled with [3H]inositol
(40 µCi/mL) for 2 hours. Labeled medium was removed, and hearts were
perfused with medium containing lithium chloride (LiCl, 10 mmol/L)
and propanolol (1 µmol/L) for 10 minutes.
For experiments under normoxic conditions, hearts were perfused with norepinephrine (100 µmol/L) for 20 minutes and then were frozen in liquid N2 and subsequently weighed, and InsPs were extracted.
For experiments investigating ischemia and reperfusion, hearts were subjected to global ischemia by turning off the perfusion pump and the oxygen flow. Reperfusion was achieved by restarting flow of medium and oxygen. After 2 minutes of reperfusion, hearts were frozen in liquid N2 and weighed subsequently and InsPs extracted.
Extraction and Quantitation of [3H]InsPs
InsPs were extracted from frozen ventricles in 2 mL of 5%
trichloroacetic acid (TCA) containing 2.5 mmol/L EDTA and 5
mmol/L phytic acid using a "Polytron" homogenizer
followed by sonication as described previously.10
After centrifugation at 5000g for 10 minutes
(4°C), supernatants were removed, and TCA
pellets were re-extracted with 1 mL TCA. The combined aqueous phases
were pooled and extracted with a 1:1 mixture of freon and
tri-N-octamylamine (0.75 mL/mL of supernatant). The final aqueous phase
was collected and treated with proteinase K (50 µg/mL; 2 hours;
50°C), and the samples then were passed through a 1-mL Dowex-50
column (4% cross-linked 4-400 mesh size) and eluted with 1 mL of
water. Urea (0.05 mol/L final) was added and samples lyophilized before
high-performance liquid chromatography (HPLC)
analysis.24
[3H]-Labeled InsPs were separated using
anion-exchange HPLC and quantitated using an on-line ß-counter
(Radiomatic Instruments Model CR) as described
previously.5 24 Recoveries of
[3H]Ins(4)P1,
[3H]Ins(1,4)P2, and
[3H]Ins(1,4,5)P3 were
determined by adding authentic standards to unlabeled tissue and
extracting as described. In all cases, recovery of
[3H]label was >90%.
Extraction and Quantitation of [3H]Inositol
Phospholipids
TCA pellets remaining after InsP extraction were extracted with
3 mL of chloroform:methanol:HCl (200:100:1) using sonication and
vigorous vortexing. EDTA (1 mL of 1 mmol/L) was added and the
phases separated by centrifugation. The interface was
re-extracted, and the final organic phase 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 re-extracted.
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] lipids remained
unhydrolyzed. The combined aqueous phases were pooled and applied to
1-mL columns of Dowex-1 (formate form). Columns were washed with 20 mL
water. Glycerophosphoinositol (deacylated PtdIns) was
eluted with 20 mL of 180 mmol/L ammonium formate and 5 mmol/L
sodium tetraborate. After an additional 20 mL of this solution,
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
glycerophosphoinositol(4,5)bisphosphate
[deacylated PtdIns(4,5)P2] was eluted with 7 mL
of 1 mol/L ammonium formate and 0.1 mol/L formic acid. Samples were
counted using a ß-counter. The [3H]-labeled
lipids were identified as PtdIns, PtdIns(4)P, and
PtdIns(4,5)P2 by removing the mobile phase with
repeated lyophilization and then performing anion-exchange HPLC as
described above.
Measurement of Ins(1,4,5)P3 and
PtdIns(4,5)P2 Mass
For measurement of Ins(1,4,5)P3 mass,
unlabeled tissue was extracted in 2 mL of 5% TCA, 2.5 mmol/L
EDTA, and 5 mol/L ATP as described above. The procedure was similar to
that described for [3H]InsPs, except that the
proteinase K step was omitted and urea was not added before
lyophilization.24 Lyophilized samples were
dissolved in water and neutralized with NaHCO3
plus NaOH, as required. Ins(1,4,5)P3 content of
the neutralized samples was quantitated by using a commercial
competitive binding assay involving an
Ins(1,4,5)P3 receptor preparation and
[3H]Ins
(1,4,5) P3, according to
the manufacturer's instructions (Amersham).
For measurement of PtdIns(4,5)P2 mass, lipids were extracted from the TCA pellets remaining after Ins(1,4,5)P3 extraction from unlabeled tissue and deacylated after the method described above. The deacylated lipids were deglycerated by adding sodium periodate (10 mmol/L) for 30 minutes, followed by 15 minutes of incubation with ethylene glycol (10% vol/vol). The reaction was completed using dimethylhydrazine (0.4%) treatment for 3 hours. Samples were neutralized and lyophilized.25 Mass assay of the Ins(1,4,5)P3 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 Ins(1,4,5)P3 [and thus the progenitor lipid as PtdIns(4,5)P2] was validated by treating aliquots of representative samples with pure Ins(1,4,5)P3 5'-phosphatase (10 ng) for 30 minutes at 37°C followed by boiling to inactivate the enzyme. This treatment removed material that displaced [3H]Ins(1,4,5)P3 in the binding assay, identifying the measured substance as Ins(1,4,5)P3 [or PtdIns(4,5)P2].
Measurement of mRNA
Total RNA was prepared using the acid guanidinium thiocyanate
procedure.26 RNA concentration was determined by
absorbance at 260 nm. Levels of 1A- and
1B-mRNA transcripts were measured by RNase
protection analysis. A 746-nucleotide fragment of
the rat 1A-receptor cDNA corresponding to
nucleotides 1 to 746 of the published
sequence,27 and a 471-nucleotide
fragment of the hamster 1B- cDNA corresponding
to nucleotides 1 to 47128 were
subcloned into pGEM-4Z vectors for generation of cRNA probes as
previously described.29 Sense RNA was synthesized
in vitro from each template and served to generate a standard curve for
each RNA species so that the absolute level of these RNA transcripts
could be determined in heart RNA extracts. RNA standards and samples (5
µL), 20 µL of hybridization buffer (80% formamide; 40 mmol/L
PIPES, pH 6.7; 0.4 mol/L NaCl; 1 mmol/L EDTA) and 5 µL of
antisense probe (reconstituted in hybridization buffer) were hybridized
overnight at 45°C. After hybridization, samples were digested at
37°C for 60 minutes by addition of 300 µL of RNase buffer (300
mmol/L NaCl; 10 mmol/L Tris, pH 7.5; 5 mmol/L EDTA with 400 U
RNase T1) and the protected hybrids purified by incubation with 5 µL
proteinase K (10 mg/mL) and 20 µL sodium dodecyl sulfate
(10%) for 15 minutes at 37°C, followed by isopropanol precipitation.
RNA hybrids were then resolved on nondenaturing 4%
acrylamide gels. Absolute quantitation of unknown
1A- and 1B-mRNA
transcripts in molar terms was achieved by comparison with a standard
curve after analysis on a Fuji BAS-1000 phosphorimaging system.
Atrial natriuretic peptide (ANP) mRNA was measured by RNase
protection exactly as described previously.30
Arrhythmogenic Responses
Mice were anesthetized with pentobarbital (60 mg/kg IP)
and given heparin (20 IU IV). Hearts were cannulated in situ via the
ascending aorta, using a dissecting microscope and perfused at 2 mL/min
with Krebs-Henseleit medium constantly gassed with 5%
CO2/95% O2 (pH 7.4) at
37°C.31 The perfusate included
propranolol (1 µmol/L) to block ß-ARs and LiCl
(10 mmol/L) to replicate the conditions used in studies of
inositol phosphate generation. 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.31 The signal
electrode was superficially connected to the left
ventricular wall and the reference electrode to the aorta.
ECG was recorded throughout the experiment. A 10-minute period was
allowed to stabilize the preparation before the experiment, and then
the left main coronary artery was ligated to produce regional
ischemia. Reperfusion was achieved by removing the ligature.
The effectiveness of the ligation was assessed by 2 methods. First, a
rise in perfusion pressure (30% to 50%) was observed. The perfusion
flow rate was adjusted coincidentally with coronary occlusion
and reperfusion to maintain a constant coronary perfusion
pressure throughout the experiment. Second, at the end of the
experiment, the coronary artery was reoccluded, and the hearts
were perfused with Evans Blue (10% 0.05 mL). A nonstained area of left
ventricle (LV), indicative of lack of perfusion, was visualized. 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 coronary perfusion pressure were
monitored. Unlike our previous findings in rat
heart,11 and in agreement with the work of other
laboratories,32 ventricular
fibrillation was not observed in the mouse hearts either during the
ischemic or reperfusion periods. Ventricular
arrhythmias expressed as ventricular premature
beats (VPB) and ventricular tachycardia ([VT]
defined as at least 5 consecutive ectopic beats) were quantified
according to the Lambeth Convention guidelines and as depicted in
Figure 1
.33
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Coronary Artery Occlusion and Reperfusion In Vivo
Mice were anesthetized with a mixture of
ketamine (100 mg/kg) and xylazine (20 mg/kg IP). After tracheal
intubation, artificial ventilation with a Harvard ventilator was
started (0.5 mL tide volume; 80 strokes/min). A left thoractomy was
performed to expose the heart, and after 10 minutes of stabilization,
the left coronary artery was ligated using 7-0 silk suture
enclosing 2 releasing rings. The chest was then closed, leaving the
suture and the releasing rings accessible from outside the chest. After
40 minutes of occlusion, the ligature was released from outside the
chest to allow 60 minutes of reperfusion.
Measurement of Area at Risk and Infarct Zone
The zone at risk (RZ) and the infarcted zone (IZ) were
determined as described elsewhere.34 In brief, at
the end of the reperfusion period, the ascending aorta was cannulated
and the heart isolated. After washing the coronary vasculature
with cold saline, the coronary artery was reoccluded, followed
by infusion of 0.1 mL 10% Evans Blue to stain the nonischemic
area. The excess dye was washed away, and large vessels, atria, and the
right ventricle were removed. The LV was then frozen, cut transversely
into 1-mm thick slices (6 to 7 slices per heart), and incubated with
1.5% triphenyltetrazolium in 100
mmol/L PBS (pH 7.4) for 15 minutes at 37°C. The viable
myocardium were stained brick red, and the infarcted
myocardium became pale white. The stained slices were
mounted on a glass plate, photographed, and enlarged. The 3 areas,
nonischemic area, RZ, and IZ, were outlined and quantified
using a software program (BioScan Optimas).
Statistics
Values presented are mean±SEM. Statistical
analysis of inositol phosphate data involved the use of 1-way
ANOVA followed by Student unpaired t test. Significance was
determined at P<0.05. Chi-square or Fisher exact test was
used for percentages of VPB and VT. The Mann-Whitney rank sum test was
used for VPB number and VT duration.
Materials
[2-3H]myo-Inositol (18
Ci/mmol),
[3H]Ins(1,4,5)P3,[14C]Ins(1)P1,
and mass assay kits for Ins(1,4,5)P3 were
obtained from the Radiochemical Center (Amersham).
[3H]-labeled Ins(1,4)P2
and Ins(4)P1 were obtained from New England
Nuclear and were supplied by Auspep. Norepinephrine,
reserpine, phytic acid, and nucleotides were obtained from
the Sigma Chemical Co. Thrombin receptor–activating peptide (TRAP) was
supplied by Auspep. Triphenyltetrazoliun was obtained from ICN
Pharmaceuticals Inc. All other chemicals were analytical reagent grade,
and reagents were dissolved in Milli Q water.
Ins(1,4,5)P3 5'phosphatase was provided by Dr
Christina Mitchell (Box Hill Hospital, Box Hill, Victoria,
Australia).
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Results |
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Effects of the
1B-CAM Receptors on Heart Weight and
ANP Gene TranscriptionHearts from the
1B-CAM animals bred in
our animal facility did not show signs of hypertrophy in
terms of tissue weight (Table 1), even
though a mild 10% to 20% increase in heart weight has been reported
previously in this strain.22 Increased ANP gene
transcription was observed, as reported previously, suggesting a
sensitivity to the development of hypertrophy. Differences
in diet or housing conditions most likely explain these
differences.
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InsP Responses in 1B-WT and 1B-CAM
Hearts Under Physiological Conditions
The effect of the CAM 1B receptors on the
contents of InsPs and inositol phospholipids was investigated in
perfused mouse hearts under normoxic conditions. Isolated mouse hearts
were labeled with [3H]inositol, pretreated with
propranolol and LiCl, and then perfused with
norepinephrine (100 µmol/L) or vehicle for 20
minutes in the continued presence of propranolol and LiCl.
[3H]InsPs and inositol phospholipids were
extracted and quantitated. In the absence of added
norepinephrine, hearts from 1B-CAM
animals contained higher levels of
[3H]inositol-labeled InsPs and phospholipids
than 1B-WT ventricles (Table 2; Figure 2). In parallel experiments, mass
contents of PtdIns(4,5)P2 and
Ins(1,4,5)P3 were measured in unlabeled hearts
from each group of animals. The mass contents of
PtdIns(4,5)P2 and
Ins(1,4,5)P3 were not different between the 2
groups (Table 2; Figure 2). Hearts from 1B-CAM
mice also showed higher total [3H]InsP
responses to norepinephrine than
1B-WT hearts. Thus the
1B-CAM receptor expressed in intact heart
shows constitutive activity and an enhanced response to
norepinephrine as described previously for this
mutant expressed in COS cells.21 Although both
1B-WT and 1B-CAM
hearts responded to norepinephrine with an increase in
[3H]InsPs, there was no detectable increase in
[3H]Ins(1,4,5)P3 or in
Ins(1,4,5)P3 mass in either preparation (Figure 2). Also, although increases in [3H]InsPs were
observed with 20 minutes of stimulation with
norepinephrine, no significant increase was observed in
either 1B-WT or
1B-CAM hearts when norepinephrine
(100 µmol/L) was perfused for 2 minutes under these normoxic
conditions (data not shown).
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InsP Responses During Early Reperfusion in 1B-WT and
1B-CAM
Isolated perfused mouse hearts were labeled with
[3H]inositol, pretreated with
propranolol and LiCl, and then subjected to 20 minutes of
global, zero flow ischemia followed by 2 minutes of
reperfusion. Ischemia (20 minutes) did not cause any alteration
in the total [3H]InsP content (66 973±9565
CPM/g wet wt versus 60 332±9479 CPM/g; mean±SEM; P=NS)
nor in the content of
[3H]Ins(1,4,5)P3
(7407±1073 CPM/g wet wt versus 5376±1392 CPM/g; P=NS).
There was a decrease in
[3H]Ins(4)P2, as
described previously in rat heart.10 35 In
1B-WT hearts, 2 minutes of reperfusion after
20 minutes of ischemia caused generation of
[3H]InsPs (Figures 3 and 4).
As described previously in rat heart,10 increases
in [3H]Ins(1,4,5)P3 were
observed, and in parallel studies, an increase in
Ins(1,4,5)P3 mass was demonstrated (Figure 4).
This shows that the changes observed in the labeling studies reflected
mass changes rather than solely an increase in specific activity. As
described previously for rat hearts,12 the
reperfusion-induced [3H]InsP response was
quantitatively greater than observed with 2 minutes of stimulation with
maximally effective concentrations of norepinephrine. Thus
under reperfusion conditions, the [3H]InsP
response to norepinephrine is enhanced in both rat and
mouse hearts.
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Similar experiments were performed using hearts from
1B-WT animals previously treated with
reserpine to deplete endogenous norepinephrine.
Catecholamine-depleted hearts did not undergo a
reperfusion-induced increase in either
[3H]Ins(1,4,5)P3 or total
[3H]InsPs. Total
[3H]InsPs before and after 2 minutes of
reperfusion were 61±5x103 CPM/g tissue (n=6)
and 56±6x103 CPM/g tissue, respectively, and
the corresponding values for
[3H]Ins(1,4,5)P3 were
5.1±1.2x103 CPM/g tissue and
5.3±1.6x103 CPM/g tissue. Addition of
norepinephrine (100 µmol/L) to the medium used for
reperfusion caused a return of the reperfusion response (total
[3H]InsPs 102±9.7x103
and [3H]Ins(1,4,5)P3
12±2x103; n=6; P<0.01 in both
cases). Similar findings have been reported previously in rat
heart.10 This shows that the reperfusion-induced
InsP response is dependent on release of norepinephrine
from the sympathetic nerves under these conditions.
In contrast to findings in 1B-WT hearts,
reperfusion of ischemic hearts from
1B-CAM mice did not cause an increase in any
of the InsPs (total [3H]InsPs,
128±17x103 CPM/g; n=8; compared with
131±16x103 CPM/g). There was no increase in
[3H]Ins(1,4,5)P3 and no
change in Ins(1,4,5)P3 mass (Figure 4
).
Furthermore, no increase in total [3H]InsPs
(128±17 to 119±4x103 CPM/g tissue) or in
[3H]Ins(1,4,5)P3
(10.9±1.6 to 9.1±2x103 CPM/g tissue;
mean±SEM; n=4; P>0.05 in both cases) was detected when
hearts were reperfused in the presence of added excess
norepinephrine (100 µmol/L). This eliminates the
possibility of differences related to norepinephrine
availability.
Reperfusion Arrhythmias in 1B-WT and
1B-CAM Hearts
Reperfusion arrhythmias were measured over a 5-minute
period after 20 minutes of regional ischemia in perfused mouse
hearts, as described in Materials and Methods. Ventricular
fibrillation was not detected in the mouse hearts under these
conditions,32 but VPB and VT were observed in
1B-WT hearts. The incidence of
reperfusion-induced VPB and VT were significantly lower in the
1B-CAM hearts, in agreement with the finding
of lower generation of Ins(1,4,5)P3 (Figure 1;
Table 3).
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Effect of Thrombin Receptor Activation During Reperfusion
Failure to detect a reperfusion response in
1B-CAM hearts could mean either that
1B-receptors are not involved in mediating the
InsP response to norepinephrine under reperfusion
conditions or that the presence of the active
1B-receptors has had a preconditioning
effect.36 In previous studies, we have shown that
under reperfusion conditions, thrombin receptor activation causes
similar Ins(1,4,5)P3 and arrhythmogenic
responses.12 Any preconditioning effect of
1B-CAM receptors would be expected to affect
responses to 1-receptor or thrombin receptor
activation. Thus responses to thrombin receptor stimulation were
investigated in 1B-WT and
1B-CAM hearts.
Catecholamine-depleted hearts from
1B-WT or 1B-CAM mice
were subjected to 20 minutes of ischemia followed by
reperfusion in the presence of thrombin receptor activating peptide
(TRAP, SFLLRN; 50 µmol/L). Ins(1,4,5)P3
content was measured after 2 minutes. As shown in Figure 5, addition of TRAP to
catecholamine-depleted 1B-WT
hearts caused generation of Ins(1,4,5)P3. The
TRAP response was quantitatively similar to the response to
norepinephrine. Similar experiments were performed using
1B-CAM hearts. Addition of TRAP (50
µmol/L) to 1B-CAM hearts for the 2-minute
reperfusion period caused an increase in
Ins(1,4,5)P3 similar to that observed in
1B-WT hearts. Thus the
1B-CAM receptor reduces the
reperfusion-induced Ins(1,4,5)P3 response only
when this is activated by 1-adrenergic
agonists. This argues against preconditioning as an explanation.
|
Infarct Size Measurements in 1B-WT and
1B-CAM Hearts
Preconditioning would be expected to reduce infarct size after
coronary artery ligation as well as reducing
arrhythmias.36 Experiments were performed
to measure infarct size in hearts from 1B-WT
and 1B-CAM animals after
ischemia/reperfusion in vivo. There was no difference between
control and 1B-CAM groups (n=7 per group) in
the RZ (45±4% versus 50±4% of LV; P=NS) or IZ (32±4%
versus 31±3% of LV; P=NS). The infarct size, calculated
from the ratio of IZ/RZ (%) was also similar between the 2 groups
(67±4% versus 62±6%; P=NS).
Expression of 1A-Receptors in
1B-CAM Hearts
1B-CAM hearts did not generate
Ins(1,4,5)P3 in early reperfusion, providing
evidence that the response is not
1B-receptor–mediated. As hearts express only
1B- and 1A-receptors
with minimal expression of 1D-receptors at the
protein level,15 this implicates
1A-receptor involvement in mediating the
reperfusion response and implies that
1A-receptor activity is depressed in
1B-CAM hearts. Thus the effect of the
1B-CAM receptors on the expression of
1A-receptors was investigated. RNA was
extracted from 1B-CAM and
1B-WT hearts, and the content of mRNA for
1A- and 1B-receptors
was quantitated by RNase protection. The content of
1B-receptor mRNA in the
1B-CAM hearts, expressed under the myosin
heavy chain promoter, was markedly increased (26-fold) relative to
1B-WT hearts. The level of expression of
1B-receptor mRNA under the -MHC promoter in
the 1B-CAM hearts was very constant between
animals, but there was greater variation between animals in expressions
of 1A- and
1B-receptor mRNAs expressed under their own
promoters. Hearts from 1B-WT animals contained
mRNA for both receptor classes, but the
1B-receptors were expressed more strongly,
accounting for 5 times more mRNA than the
1A-receptors. More importantly, although there
was considerable variation between animals, the expression of the mRNA
for 1A-receptors on average was depressed in
the 1B-CAM hearts (Figure 6), raising the possibility of lower
levels of expression of 1A-receptors in the
1B-CAM hearts. The expression of mRNA for
GAPDH was similar in 1B-WT and
1B-CAM hearts (Figure 6).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Reperfusion of rat hearts after global ischemia causes release of norepinephrine from the sympathetic nerves, the transient generation of Ins(1,4,5)P3 within the myocardium, and the initiation of arrhythmias.10 11 37 As increases in Ins(1,4,5)P3 are not detectable in intact heart under physiological conditions in response to norepinephrine, this marks a major change in the operation of the inositol phosphate pathway. Delineating the relative roles of the
1-receptor subtypes in mediating InsP
responses under normoxic and reperfusion is thus of central
importance.
Under normoxic conditions, the constitutively active
1B-receptors conferred on the expressing
hearts a heightened InsP response in the presence or absence of
norepinephrine (Figure 2). If
1B-receptors mediated the
norepinephrine response during reperfusion, then generation
of very large amounts of Ins(1,4,5)P3 would be
expected in 1B-CAM hearts. However, this was
not found to be the case. Hearts from 1B-WT
animals responded to reperfusion by generation of
Ins(1,4,5)P3 as demonstrated previously in rat
hearts but, in marked contrast to findings under normoxic conditions,
the 1B-CAM hearts showed no detectable
Ins(1,4,5)P3 response during early reperfusion.
Together with the reduced Ins(1,4,5)P3 response,
the 1B-CAM hearts had a reduced incidence of
reperfusion arrhythmias. This agrees with previous findings
from our laboratory of a relationship between
Ins(1,4,5)P3 generation and arrhythmias
in rat heart. Rat hearts develop more substantial arrhythmias
under these conditions of early reperfusion than mouse hearts, with
ventricular fibrillation being observed in addition to VPB
and VT.11 12 Ischemia has been shown to
cause the translocation and activation of PKC,38
and this together with any sn-1,2-diacylglycerol generated
along with Ins(1,4,5)P3 might be important in
arrhythmogenesis.39 However, our studies in rat
hearts showed no antiarrhythmic action of inhibitors of
protein kinase C during reperfusion.11 More
importantly, recent studies from other laboratories have demonstrated a
direct proarrhythmic action of Ins(1,4,5)P3 when
applied intracellularly.40 Thus it seems most
likely that Ins(1,4,5)P3 itself, presumably via
perturbations in cytosolic Ca2+, initiates
electrophysiological changes that culminate
in the development of arrhythmias.
The reduced Ins(1,4,5)P3 response during
reperfusion of 1B-CAM hearts suggests that the
norepinephrine response is not mediated by
1B-receptors under these conditions. However,
it is also possible that the 1B-CAM receptors
have caused a preconditioning effect. We have shown previously that
preconditioning can reduce the reperfusion
Ins(1,4,5)P3 response,41
and others have demonstrated a preconditioning effect of
1B-receptor activation under some
conditions.36 42 However, 2 different experiments
argue against preconditioning as an explanation of the reduced
reperfusion response in the 1B-CAM hearts.
First, 1B-CAM hearts responded to thrombin
receptor activation under reperfusion conditions in a similar manner to
1B-WT hearts. Preconditioning would be
expected to reduce responses to all effectors similarly. Second,
preconditioning would also be expected to reduce infarct size after
coronary artery ligation,36 but infarct
sizes were found to be similar in the 2 groups. Thus no evidence was
found for a preconditioning effect of the
1B-CAM receptors under the conditions of our
experiments.
The data presented indicate the involvement of
1-receptors other than
1B-subtype as mediators of the
reperfusion-induced Ins(1,4,5)P3 response to
norepinephrine. Hearts, at least human and rat, express
only the 1A- and
1B-subtypes of
1-receptors at the protein
level.15 If also true of mouse heart, this
implies an involvement of 1A-receptors. Such
an interpretation however, suggests a reduction in
1A-receptor activity in the
1B-CAM hearts. Some evidence for this was
supplied by the experiments showing reduced
1A-receptor mRNA expression in the
1B-CAM hearts (Figure 1). However, it must be
stressed that an involvement of 1D-receptors
cannot be discounted at this stage. We have reported previously that
stimulation of 1A-receptors causes a reduction
in 1B-receptor mRNA.43
Together with data reported here, this points to a reciprocal
relationship between these 2 receptor subclasses, at least in the
myocardium.
An involvement of receptors with
1A-specificity in the generation of
arrhythmias under ischemic/reperfusion conditions has
been suggested previously on the basis of effects of selective
antagonists.44 45 46 47 In addition,
increased activity of 1A-receptors has been
shown to be associated with the development of other pathological
conditions such as hypertrophy and failure, with
hypertrophic agents causing an increase in expression of
1A-receptor mRNA together with a decrease in
1B-receptor
expression.20 1A-ARs in
heart thus appear to be involved in several different pathologies, and
subtype-selective antagonists might prove useful in
protecting the heart under several different pathological conditions.
|
Acknowledgments |
|---|
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. We thank Dr Christina Mitchell (Box Hill Hospital, Box Hill, Victoria, Australia) for providing the Ins(1,4,5)P3 5'phosphatase and Professor R.M. Graham (Victor Chang Institute, Sydney, Australia) for a critical evaluation of the manuscript. We are grateful to Dr R.J. Lefkowitz (Duke University, Durham, NC) for providing the transgenic lines and for helpful discussion.
Received May 22, 1998; accepted September 14, 1998.
|
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