(Circulation. 2001;103:2395.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Increased Release of Arachidonic Acid and Eicosanoids in Iron-Overloaded Cardiomyocytes
From Rammelkamp Center for Education and Research, MetroHealth Medical Center (R.M., G.P.S., N.B., Y.A.K.) and Department of Medicine (R.M.), Case Western Reserve University School of Medicine, Cleveland, Ohio.
Correspondence to Rafael Mattera, PhD, Cell Biology and Metabolism Branch, NICHD, Building 18T, Room 101, Bethesda, MD 20892. E-mail matterar{at}helix.nih.gov
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Abstract |
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Background—Patients with transfusional iron overload may develop a life-limiting cardiomyopathy. The sensitivity of lipid-metabolizing enzymes to peroxidative injury, as well as the reported effects of arachidonic acid (AA) and metabolites on cardiac rhythm, led us to hypothesize that iron-overloaded cardiomyocytes display alterations in the release of AA and prostaglandins.
Methods and
Results—Neonatal rat ventricular
myocytes (NRVMs) cultured for 72 hours in the presence of 80 µg/mL
ferric ammonium citrate displayed an increased rate of AA release, both
under resting conditions and after stimulation with agonists such as
[Sar1]Ang II. Although iron treatment did
not affect overall incorporation of [3H]AA
into NRVM phospholipids, it caused a 2-fold increase in the
distribution of precursor in phosphatidylcholine species, with a
proportional decrease in phosphatidylinositol,
phosphatidylserine, and phosphatidylethanolamine.
Increased release of AA in iron-overloaded NRVMs was reduced by the
diacylglycerol lipase inhibitor RHC80267 but was largely
insensitive to inhibitors of phospholipases
A2 and C. Iron-overloaded
cardiomyocytes also displayed increased production
of eicosanoids and induction of cyclooxygenase-2
after stimulation with interleukin-1
.
Conclusions—Iron overload enhances AA release and incorporation of AA into phosphatidylcholine, as well as cyclooxygenase-2 induction and eicosanoid production, in NRVMs. The effects of AA and metabolites on cardiomyocyte rhythmicity suggest a causal connection between these signals and electromechanical alterations in iron-overload–induced cardiomyopathy.
Key Words: cardiomyopathy • prostaglandins • signal transduction • myocytes • iron
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Introduction |
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Iron-induced myocardial disease is a life-limiting complication in patients with thalassemia major and other transfusion-dependent refractory anemias, as well as in hereditary hemochromatosis.1 Neonatal rat ventricular myocytes (NRVMs) cultured in the presence of iron and Mongolian gerbils subjected to chronic iron overload have been used as models of clinical cardiomyopathy.2 3 Acute iron loading in cultured rat myocardial cells results in peroxidative injury, decreased activity of mitochondrial respiratory enzymes, impaired contractility, and altered rhythmicity; these specific in vitro alterations can be reversed by metal chelation.2 4 5 6 Although production of hydroxyl radical (·OH) and lipid peroxidation1 7 are important in initiation of iron-overload–dependent cardiotoxicity, limited information is available regarding downstream signaling alterations. Precedents on enhanced activity of lipases and arachidonic acid (AA) release during oxidative stress8 9 10 and the role of lipid peroxides in cyclooxygenase activation,11 as well as the reported effects of eicosanoids on cardiac rhythm,12 led us to hypothesize that iron-overloaded cardiomyocytes display changes in AA release and/or production of oxidation metabolites that may contribute to the clinical cardiomyopathy.
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Methods |
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Materials
Trypsin (type I, 9000 U/mg), AA (free acid), ferric ammonium citrate, cytosine ß-D-arabinofuranoside (ara-c), Sar-Arg-Val-Tyr-Ile-His-Pro-Phe ([Sar1]Ang II), BSA, and the lactate dehydrogenase (LDH) kit were purchased from Sigma Chemical Co. Collagenase (type 2, 317 U/mg) was from Worthington. Myo-[2-3H]inositol and [5,6,8,9,11,12,14,15-3H]AA (60 to 100 Ci/mmol) were from New England Nuclear. Enzyme immunoassay kits for prostaglandin (PG) E2, PGF2
, and
6-keto-PGF1, polyclonal
anti–cyclooxygenase-2 antiserum, and
N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide
(NS-398) were purchased from Cayman, and DMEM/F12, inositol-free DMEM
(high glucose), and gentamicin were from GIBCO-BRL. Sprague-Dawley
pregnant-timed rats were purchased from Zivic-Miller (Portersville,
Pa). 1,6-Bis-(cyclohexyloximino carbonylamino)-hexane (RHC80267) was
from Biomol, and arachidonyl-trifluoromethyl-ketone
(AACOCF3),
1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)
hexyl]-1H-pyrrole-2,5-dione (U73122), and
E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one
(HELSS) were purchased from Calbiochem. Candesartan and PD123319
were obtained from J.G. Douglas (Case Western Reserve University).
Human recombinant interleukin-1 (IL-1) was a gift from R.
Chizzonite (Hoffmann-La Roche, Nutley, NJ) and Martha
Konieczkowski (Case Western Reserve University, Cleveland,
Ohio).
NRVM Preparation and Iron Treatment
Cell isolation was based on a previously reported
procedure.13 The yield of
the preparations was 
3 to 4x106
cells/heart. After isolation, NRVMs were resuspended at
4x106 cells/mL in DMEM/F12 medium
containing 50 µg/mL gentamicin (DMEM/F12/GENT) and 10% calf serum.
Cells were plated at a density of 0.33x106
cells/well of a 12-well plate or 4x106
cells per 100-mm dish and cultured at 37°C under humidified
atmosphere (95:5, air:CO2). After overnight
incubation, the serum concentration was reduced to 0.4% by 2
consecutive 5-fold dilutions with serum-free DMEM/F12/GENT. Cells were
subsequently cultured for 72 hours with or without 80 µg/mL elemental
iron (supplied as 470 µg/mL ferric ammonium citrate, based on 17%
iron content in this salt). Medium was partially renewed every 24 hours
(removal of half volume, followed by addition of an equal volume of
fresh DMEM/F12/GENT with or without iron). This resulted in serum
concentrations ranging from 0.4% to 0.1% throughout the 72-hour
culture. Cultured NRVMs displayed spontaneous
contractility after 2 days in culture. Iron treatment
decreased cardiomyocyte size, thinned its projections,
reduced the number of spontaneously beating cells, and resulted in
siderosome accumulation.13
LDH activity in extracellular medium of control and iron-treated cells
(72-hour treatment) was 14±3% and 42±2%, respectively, of total
activity in Triton X-100 cell lysates (means±SD, n=4). Control and
iron-treated NRVM cultures exhibited 13±3% and 13±1% Trypan
blue–positive cells, respectively (mean±SD, n=4); the number of cells
in the iron-treated cultures relative to the controls was 0.8±0.2
(mean±SD, n=4).
AA Release
Assays were adapted from a previous
study.14 Briefly, NRVMs
cultured in 12-well plates were labeled for 15 to 18 hours in 1.2
mL/well of DMEM/F12/GENT containing 0.7 µCi of
[3H]AA and 0.1% BSA (
90% of precursor
was incorporated). Labeled cells were washed at 37°C with
DMEM/F12/GENT plus 0.1% BSA. Assays were started by removal of wash
media and addition of 1 mL of DMEM/F12/GENT supplemented with 0.1%
BSA, 100 µmol/L unlabeled AA (unless otherwise indicated), and
agonists and/or enzyme inhibitors. After incubation at
37°C for the indicated times, samples of media were collected,
centrifuged at 14 000g
for 2 minutes, and counted. [3H]AA release
was normalized by measuring 3H dpm in
cell lysates (1 mol/L HONa) and expressed as a percentage of
incorporated 3H dpm.
Distribution of
[3H]AA Into Individual Phospholipid
Species
Control and iron-treated NRVMs were labeled with
[3H]AA for 1 to 24 hours. Labeled cells
were washed and extracted with chloroform-methanol-water (final ratio
2:1:0.8 by volume). Lower phases were dried under nitrogen, dissolved
in chloroform:methanol:water (75:25:3 by volume), and applied onto
silica gel plates previously dipped (up to 5 cm from origin) in 0.4
mol/L boric acid and air-dried. Samples were supplemented with
phospholipid standards and subjected to ascending
chromatography with chloroform/methanol/ammonium
hydroxide/water 65:35:2:3 by volume. Plates were developed with iodine,
followed by liquid scintillation counting. Mobility of phospholipids in
this system is as follows: origin; phosphatidylinositol (PI);
phosphatidylserine (PS); phosphatidylcholine (PC);
phosphatidylethanolamine (PE); front.
Phospholipase C Activity
Cells in 12-well plates were labeled during the last
15 to 18 hours of culture with or without iron by incubation at 37°C
with 1 mL/well of inositol-free DMEM, 10 mmol/L HEPES pH 7.4, 50
µg/mL gentamicin, and 2 µCi/mL of
myo-[3H]inositol (±80 µg/mL iron).
Inositol phosphate production in
myo-[3H]inositol–labeled cells was
measured as
described.14
Eicosanoid Production
NRVMs cultured for 72 hours in the presence or
absence of 80 µg/mL iron were treated with or without 10 ng/mL
IL-1 during the last 12 to 18 hours of culture. After treatment,
extracellular medium was collected and centrifuged for 15
minutes at 1400g and room
temperature. Concentrations of PGE2,
PGF2, and
6-keto-PGF1 (stable metabolite of
PGI2) in supernatants were determined by enzyme
immunoassay.
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Results |
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Alterations in AA Release
We studied NRVMs treated with ferric ammonium citrate and low serum concentrations (
0.4% to 0.1% throughout the 72-hour
culture). This presentation of iron to
cardiomyocytes mimics the situation in patients with
transfusional iron burden, who not only display transferrin saturation
but also high levels of chelatable, low-molecular-weight,
non-transferrin-bound plasmatic
iron.7
Transferrin-independent iron uptake involves participation of
ferrireductases and low-affinity/high-capacity
mechanisms.15 Unlike
transferrin-dependent uptake, which desensitizes at high concentrations
of iron, non-transferrin-bound iron uptake becomes 300-fold higher than
the former in cultured
cells.2 7 Treatment
of NRVMs with ferric ammonium citrate results in intracellular
accumulation comparable to levels in patients with significant iron
burden.16 NRVMs treated with
this ferric complex constitute a model of the iron-overloaded heart,
given the observed metabolic alterations, ultrastructural
pathology, and changes in the overshoot of the action potential and
rhythm.2 4 5 6 13
We studied the effect of iron overload on AA release in
resting NRVMs or after activation of angiotensin II (Ang
II) receptors, a pathway relevant to cardiac pathophysiology. NRVMs
cultured for 72 hours in the presence of 80 µg/mL ferric ammonium
citrate and subjected to overnight labeling with
[3H]AA displayed a significant increase in
AA release, both under resting conditions and after stimulation with
[Sar1]Ang II (a nonselective Ang II type 1
[AT1]/Ang II type 2
[AT2] receptor agonist with reduced
susceptibility to aminopeptidases;
Table
).
Similar increases in AA release in iron-overloaded NRVMs were observed
after stimulation with thrombin or lysophosphatidic acid (not shown).
We routinely add 100 µmol/L unlabeled AA to the assay medium, to
minimize reuptake of released [3H]AA.
However, increased [3H]AA release in
iron-overloaded NRVMs was not linked to this assay condition, because
significant effects were also measured with medium containing 0 to 10
µmol/L unlabeled AA
(Table).
The effect of iron on [3H]AA release was
concentration dependent: whereas no changes were detected in NRVMs
cultured for 72 hours with 5 µg/mL iron, significant increases were
observed after treatment with concentrations 
20 µg/mL (not shown).
All subsequent studies were performed with NRVMs cultured for 72 hours
with or without 80 µg/mL iron. Time courses of
[3H]AA release showed significantly higher
rates in iron-treated cells either in presence or absence of agonist
(Figure 1). As shown in
Figure 2, increased response to
[Sar1]Ang II in iron-overloaded NRVMs was
characterized by a higher efficacy and unchanged potency compared with
control cells (EC50 0.2±0.1 nmol/L and
0.16±0.03 nmol/L for control and iron-treated cells, respectively;
average and range, n=2). The AA release driven by
[Sar1]Ang II in NRVMs exhibited a
pharmacology consistent with participation of
AT1 receptors (antagonized by candesartan, but
insensitive to PD123319;
Figure 3). The iron-overload–dependent increase in AA
release was observed either in NRVMs cultured under standard conditions
(low serum, maintaining nonmyocyte cardiac cells <15% to
20%) or with the antimitotic agent ara-c, to further minimize their
contribution
(Figure 4).
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Predominant routes for production of AA in mammalian
cells include hydrolysis of the
sn-2 ester bond of
glycerophospholipids by phospholipase A2
(PLA2) and the activity of diacylglycerol (DAG)
lipase on DAG generated by phospholipase C (PLC) or phospholipase
D/phosphatidic acid phosphohydrolase. Selective inhibitors
were used to study the contribution of these pathways to increased AA
release in iron-overloaded NRVMs. Whereas incubation with DAG lipase
inhibitor RHC80267 inhibited
[3H]AA release in control cells, it also
resulted in 65% inhibition of the enhancement in release triggered
by iron treatment
(Figure 5A). On the other hand, increased
[3H]AA release in iron-overloaded NRVMs
was not blocked by various selective and nonselective
PLA2 inhibitors, including
aristolochic acid, AACOCF3, HELSS, and
quinacrine (not shown). In contrast to the effects on AA release,
iron-overloaded NRVMs showed no differences in PLC activity compared
with control cells
(Figure 5C, left panel). Consistent with this, the
PLC inhibitor U73122 reduced production of inositol
phosphates
(Figure 5C) but did not normalize increased release of
[3H]AA in iron-loaded NRVMs
(Figure 5B).
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Phospholipid Labeling
Although iron treatment of NRVMs did not significantly
affect overall incorporation of [3H]AA
into phospholipids (not shown), the distribution showed a 2-fold
increase in the incorporation of [3H]AA in
PC species, together with a proportional decrease in labeling of PI,
PE, and PS
(Figure 6). Similar increases in PC labeling (2- to 2.2-fold)
were detected in cells incubated with
[3H]AA for either 6.5 or 24 hours
(Figure 6) or after shorter incubations (1 to 6 hours
interval, not shown). We also observed a time-dependent shift in the
PE/PC ratio in both experimental groups consistent with
time-dependent remodeling of AA from PC to
PE.17 However, increased
labeling of PC in iron-overloaded NRVMs results in this being the
predominantly labeled phospholipid class, as opposed to higher
labeling of PE in controls
(Figure 6).
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Prostanoid Production
We also studied whether iron overload triggered changes
in production of PGE2,
PGF2, and PGI2, the
major eicosanoids secreted by cultured
cardiomyocytes.18 19
Cells were stimulated with IL-1, which markedly increases prostanoid
production in NRVMs compared with agonists such as Ang II or
endothelins (not shown). Iron-overloaded cells stimulated with IL-1
displayed increased production of PGE2,
PGF2, and
6-keto-PGF1 (stable metabolite of
PGI2;
Figure 7) compared with controls. The increase in
IL-1–induced PGF2 production
driven by iron overload was sensitive to 30 µmol/L
indomethacin, consistent with this effect
representing cyclooxygenase (COX)
activity; however, a fraction of the PGF2
production in cytokine-stimulated control cells was
insensitive to this treatment and may reflect isoprostanes (not shown).
The higher resting levels of prostaglandin
production in iron-overloaded cells seemed to originate from
increased COX activity, as measured after 15-minute incubation with 50
µmol/L unlabeled AA (production of
PGF2 was 1.1±0.1 and 2.1±0.1 ng/mg in
control and iron-treated NRVMs, respectively; means±SEM; n=4). Iron
treatment also enhanced IL-1–dependent COX-2 induction in NRVMs
(bottom panel of
Figure 7), whereas increased production of
PGE2 in cytokine-stimulated cells
(either control or iron-overloaded) was blocked by the COX-2–selective
inhibitor NS-398 (not
shown).
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Discussion |
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Ubiquitous iron-regulated RNA-binding proteins sense intracellular chelatable pools of iron and control expression of target genes by regulating either translation or stability of mRNAs containing 1 or more iron-responsive elements in their 5' or 3' regions, respectively.20 Genes regulated by this mechanism include H- and L-ferritin, erythrocyte 5-aminolevulinic synthase, mitochondrial aconitase, transferrin receptor, and Nramp2/DCT1 intestinal iron transporter.20 Although significant advances were made in understanding regulation of genes affecting iron homeostasis, limited information is available regarding signals altered by iron overload. Exceptions to this situation are the effects of iron on expression of protein kinase C-ß21 and fibrogenic genes.22 This limitation hinders the treatment of transfusional iron-dependent cardiomyopathy, a life-limiting complication in inadequately chelated patients.1
We showed that iron overload increases AA release and PC
labeling, as well as COX activity, COX-2 induction, and eicosanoid
production, in NRVMs. Release of AA is catalyzed by
PLA2 enzymes and/or DAG lipase. The
PLA2 superfamily includes at least 10 different
groups displaying differences in primary structure, cellular
localization, and Ca2+
sensitivity.23 Most of the
PLA2 activity in cardiomyocytes is
Ca2+-independent.24
The results obtained with the inhibitors
AACOCF3 (selective for both group IV
PLA2 and group
VI-Ca2+–independent
PLA2), HELSS (selective for group
VI-Ca2+–independent
PLA2), and RHC80267 (DAG lipase-selective) are
consistent with an at least partial participation of the latter
in increased AA release in iron-overloaded cells. However, 2 forms of
Ca2+-independent PLA2
(82- and 40-kDa, respectively), exhibiting differences in
intracellular localization and HELSS sensitivity, were evidenced in
cardiomyocytes.24
The possibility that iron overload may affect a HELSS-insensitive
Ca2+-independent PLA2
deserves further exploration.
Interestingly, the distribution of labeled
[3H]AA incorporated in phospholipids of
iron-overloaded NRVMs shows an increase in PC
(Figure 6
). Incorporation of
[3H]AA may predominantly reflect
phospholipid remodeling through the deacylation-acylation Lands cycle,
as opposed to de novo glycerophospholipid biosynthesis through the
Kennedy cycle,17 whereas
time-dependent remodeling of [3H]AA from
PC into PE may represent coenzyme A–independent transacylase
activity. Iron treatment of NRVMs may increase availability of
lysophospholipid acceptors for formation of PC and/or decrease AA
remodeling into PE.
The association of changes in AA release and activity of AA-releasing enzymes with myocardial alterations is not unprecedented. Levels of unsterified fatty acids, particularly AA, increase in ischemic myocardium, preceding losses in total phospholipids. Similarly, translocation of Ca2+-independent PLA2 from cytosol to membranes25 and protection by its selective inhibitor HELSS26 were reported in ischemic hearts.
The observation that iron overload sensitizes NRVMs to COX-2
induction
(Figure 7) is also significant. Altered eicosanoid
production in NRVMs may be responsible for either phenotypic
protection or damage during iron overload. Cotreatment with 50 to 500
nmol/L PGF2 prevented morphological changes
and the decreased beating in iron-overloaded NRVMs, whereas opposite
effects were observed with the PGI2 analog
beraprost (R.M. and Y.A.K., unpublished data, 2000). The observations
with PGF2 are consistent with its
hypertrophic effects in
cardiomyocytes27
and with the compensatory role of COX-dependent signals during
oxidative
stress.19
The reported effects of AA metabolites on myocardial
rhythmicity12 suggest a
causal connection between our observations and iron-overload–induced
electromechanical changes. Indeed, PGD2,
PGE2, PGF2, and
thromboxane A2–mimetic compounds
trigger reversible tachyarrhythmias, characterized by
increased beating rate and
contractures.12 Conversely,
prostacyclin (PGI2) reduces contraction rate in
cardiomyocytes.12
Interestingly, iron overload not only potentiated IL-1–induced
eicosanoid release but also modified the relative ratio of eicosanoid
production in cytokine-stimulated NRVMs. Although
PGI2 was the most abundant product in
resting NRVMs (controls or iron-overloaded), a relatively higher
increase in PGE2 was observed after IL-1
treatment of iron-overloaded cells compared with smaller changes in
either PGI2 or the less abundant
PGF2 (30-fold versus 3- to 4-fold,
respectively, in
Figure 7). Consequently, PGE2 was the
predominant eicosanoid in cytokine-stimulated iron-overloaded
NRVMs. This is significant because the specific pattern of eicosanoid
production in iron-overloaded cardiomyocytes may be
ultimately responsible for the final balance of proarrhythmogenic or
antiarrhythmogenic effects.
Reasonable targets for a role of altered production/metabolism of AA in the electromechanical derangement in iron-overloaded hearts include Na+ and K+ currents. Precedents supporting this possibility are the reduced Na+ currents and increased transient outward K+ current (Ito) in iron-overloaded gerbil cardiomyocytes,13 together with the inhibition of cardiac Na+ currents by acute treatment with exogenous AA and other polyunsaturated fatty acids.28 In support of this hypothesis, we observed that chronic treatment of NRVMs with AA (72 hours, 10 µmol/L) decreased Na+ currents and increased Ito density (Y.A.K. and R.M., unpublished data, 2000). Interestingly, iron-overloaded gerbil hearts exhibit decreased conduction velocity (K. Laurita, PhD, unpublished data, 2000), consistent with reduced Na+ currents in isolated cardiomyocytes.13 Whereas reduced cardiac Na+ currents may underlie antiarrhythmic effects of n-3 polyunsaturated fatty acids during delayed afterdepolarizations and triggered activity,29 a slowing in conduction velocity and the possible shortening of action potentials and refractoriness (due to increased Ito), combined with increased size of iron-overloaded hearts, may allow reentry arrhythmias in this cardiomyopathy.
The possible consequences of increased release of AA in Ang
II–stimulated iron-overloaded NRVMs on cardiac phenotype
deserve consideration. This peptide regulates
contractility and growth of cardiomyocytes,
as well as hyperplasia and matrix production in cardiac
fibroblasts.30 Activation of
AT1 receptors is relevant to mechanical
strain–dependent hypertrophy (through recruitment of
mitogen-activated protein
kinases31 ) and
stretch-mediated apoptosis in
myocytes.32 Given the
effects of AA on activation of mitogen-activated protein
kinases33 and
apoptosis,34 it is
possible to speculate that increased Ang II–dependent AA release may
contribute to progression from hypertrophy to heart failure
in iron-overload–induced cardiomyopathy. The
interaction between cardiac nonmyocyte cells and
cardiomyocytes and the relative expression of Ang II
receptors in these cell types received
attention.35 The
identification of different mechanisms responsible for activation of
extracellular signal–regulated kinases by Ang II in rat neonatal
cardiac fibroblasts and myocytes supports expression of receptors in
both cell types.31 Using
NRVMs cultured with ara-c (to minimize fibroblast proliferation), we
observed similar iron overload–dependent increases in AA release and
also increased production of PGE2 and
PGF2 (albeit to a lower extent than in
standard cultures; not shown), consistent with a significant
role of cardiomyocytes in these responses. However,
interactions between cardiomyocytes and nonmyocytes
in the alterations driven by iron overload cannot be
excluded.
Additional studies are required to determine whether the present observations extend to the animal model of cardiac overload and their incidence in human cardiomyopathy. Although the connection between altered production/metabolism of AA and electromechanical alterations in this cardiomyopathy requires additional investigation, the present observations represent an advance in the identification of pathways that either cause or represent adaptive responses to cardiac iron overload.
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Acknowledgments |
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This study was supported by American Cancer Society grant RPG-97–008–01-BE to Dr Mattera, hematology training grant HL07147-22 and Cooley’s Anemia Foundation fellowship to Dr Kuryshev, and NIH grant HL61642 to Dr A.M. Brown (VP Research Metrohealth Medical Center).
Received October 5, 2000; revision received December 14, 2000; accepted January 10, 2001.
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