Free Radical Biology and Medicine 88 (2015) 39

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Iron and oxidative stress in cardiomyopathy in thalassemia

Vasilios Berdoukas a,n, Thomas D. Coates a, Zvi Ioav Cabantchik b
a
Section of Hematology, Division of Hematology, Oncology, and Blood & Marrow Transplantation, Childrens Center for Cancer and Blood Diseases, Childrens
Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA 90027, USA
b
Alexander Silberman Institute of Life Science, Hebrew University of Jerusalem, E Safra Campus at Givat Ram, Jerusalem 91904, Israel

art ic l e i nf o

a b s t r a c t

Article history:
Received 10 March 2015
Received in revised form
13 July 2015
Accepted 22 July 2015
Available online 26 July 2015

With repeated blood transfusions, patients with thalassemia major rapidly become loaded with iron,
often surpassing hepatic metal accumulation capacity within ferritin shells and inltrating heart and
endocrine organs. That pathological scenario contrasts with the physiological one, which is characterized
by an efcient maintenance of all plasma iron bound to circulating transferrin, due to a tight control of
iron ingress into plasma by the hormone hepcidin. Within cells, most of the acquired iron becomes
protein-associated, as once released from endocytosed transferrin, it is used within mitochondria for the
synthesis of protein prosthetic groups or it is incorporated into enzyme active centers or alternatively
sequestered within ferritin shells. A few cell types also express the iron extrusion transporter ferroportin,
which is under the negative control of circulating hepcidin. However, that system only backs up the
major cell regulated iron uptake/storage machinery that is poised to maintain a basal level of labile
cellular iron for metabolic purposes without incurring potentially toxic scenarios. In thalassemia and
other transfusion iron-loading conditions, once transferrin saturation exceeds about 70%, labile forms of
iron enter the circulation and can gain access to various types of cells via resident transporters or
channels. Within cells, they can attain levels that exceed their ability to chemically cope with labile iron,
which has a propensity for generating reactive oxygen species (ROS), thereby inducing oxidative damage.
This scenario occurs in the heart of hypertransfused thalassemia major patients who do not receive
adequate iron-chelation therapy. Iron that accumulates in cardiomyocytes forms agglomerates that are
detected by T2* MRI. The labile forms of iron inltrate the mitochondria and damage cells by inducing
noxious ROS formation, resulting in heart failure. The very rapid relief of cardiac dysfunction seen after
intensive iron-chelation therapy in some patients with thalassemia major is thought to be due to the
relief of the cardiac mitochondrial dysfunction caused by oxidative stress or to the removal of labile iron
interference with calcium uxes through cardiac calcium channels. In fact, improvement occurs well
before there is any signicant improvement in the total level of cardiac iron loading. The oral iron
chelator deferiprone, because of its small size and neutral charge, demonstrably enters cells and chelates
labile iron, thereby rapidly reducing ROS formation, allowing better mitochondrial activity and improved
cardiac function. Deferiprone may also rapidly improve arrhythmias in patients who do not have excessive cardiac iron. It maintains the ux of iron in the direction hemosiderin to ferritin to free iron, and
it allows clearance of cardiac iron in the presence of other iron chelators or when used alone. To date, the
most commonly used chelator combination therapy is deferoxamine plus deferiprone, whereas other
combinations are in the process of assessment. In summary, it is imperative that patients with thalassemia major have iron chelators continuously present in their circulation to prevent exposure of the
heart to labile iron, reduce cardiac toxicity, and improve cardiac function.
& 2015 Elsevier Inc. All rights reserved.

Keywords:
Thalassemia major
Iron overload
Transfusion
Reactive oxygen species
Labile plasma iron
Labile cellular iron
Iron cardiomyopathy
Iron chelation therapy
Free radicals

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Pathological iron accumulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Corresponding author.
E-mail address: vberdoukas@yahoo.com.au (V. Berdoukas).

http://dx.doi.org/10.1016/j.freeradbiomed.2015.07.019
0891-5849/& 2015 Elsevier Inc. All rights reserved.

V. Berdoukas et al. / Free Radical Biology and Medicine 88 (2015) 39

3. Pathophysiology of iron overload . . . .

4. Cardiomyopathy in thalassemia . . . . .
5. Management of cardiac iron overload
6. Summary . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
The heart is endowed with the requisite mechanisms for iron
homeostasis. Those mechanisms comprise the regulated uptake of
iron for adequate utilization, coordinately backed by a salvage
pathway for sequestration of excess iron to avoid the potential
involvement of labile iron in harmful reactions [14]. The mechanisms have evolved in cells that reside in a physiological environment (i.e., plasma or interstitial uid) that has essentially
only one source of iron, transferrin-bound iron (TBI). That iron is
maintained within a physiological range (normally at 2030%) of
transferrin saturation (TSAT) or of the total iron binding capacity,
which amounts to about
40 M [5, 6]. Cells acquire iron by receptor-mediated endocytosis (RME) of circulating transferrin (TfFe), a closely controlled mechanism. The transferrin receptors
(TfRs) on the cell surface effectively bind circulating Tf-Fe and
transfer iron to the cytosol after endocytosis, acidication and
reduction of Fe(III) to Fe(II), and its translocation by divalent metal
transporter 1, whereas apotransferrin is recycled back to the circulation chaperoned by endosomal-bound TfRs.
Cellular acquisition of iron is basically dictated by cellular
metabolic needs as reected by the level of cytosolic labile cellular
iron (LCI) that is sensed by iron-regulatory proteins (IRPs) I and II,
which subsequently alter the expression of TfR and various other
iron-regulatory proteins [13]. The transduction is based on the
interactions of IRPs with iron-responsive elements (IREs) that reside in the 3 untranslated repeated domain in TfR mRNA, but also

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6
6
7
8
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8

in 5 untranslated IRE of ferritin mRNA, so that at low LCI, the

expression of TfR is favored, whereas that of ferritin is inhibited,
and vice versa when LCI is high.
Much of the iron acquired by cells physiologically is utilized for
the formation of metalloproteins either directly or via prosthetic
groups (heme or ironsulfur complexes (ISCs)), synthesized within
mitochondria to serve as enzyme cofactors. Surplus iron is generally sequestered within ferritin molecules (in the heart, primarily H-ferritin) [15,7,8], but in some cells, this protective mechanism might be also backed up by the iron exporter ferroportin
(Fpn) [5,810]. Cell iron homeostasis is largely due to a coordinated regulation of iron uptake and storage to ensure a requisite supply of metabolically essential LCI, but avoid risking cell
functions by the promiscuous generation of toxic reactive oxygen
species (ROS) from reactive oxygen intermediates (ROIs; such as
O2  and H2O2) catalyzed by labile iron [11]. LCI is just a minor
component of cells, representing a fraction (
12%) of the total
cell iron content under normal circumstances [1214]. LCI
homeostatic levels found in metabolically active cells (
0.1
1.0 M) reect not merely a balance among various factors, such as
iron uptake and utilization versus sequestration by cellular ferritin
or export by Fpn, but also the chemical composition of the compartment in question, especially its iron ligand ability, pH, and
redox potential. As up to 1% of O2 consumed by the respiratory
chain might end up as ROIs under normal conditions, mammalian
cells have adopted two complementary strategies for coping with
ROS formation: (a) control of LCI levels by a coordinated balance of

Fig. 1. Iron management in hemosiderosis. Iron normally enters the circulation (plasma) by absorption from the gut or export from reticuloendothelial (RES) cells involved in
erythrophagocytosis. The incoming iron is swiftly incorporated into transferrin, thereby raising the level of transferrin-bound iron (TBI) that serves as the circulating source
of iron for virtually all cells. TBI is transferred to cells in a highly regulated manner, raising initially the level of labile iron (LCI in the cytosol (LCIc) and mitochondria (LCImt)),
which is processed metabolically, whereas surplus iron is stored in cytosolic ferritin shells. Some cells are endowed with the iron exporter ferroportin (Fpn). As indicated,
virtually all plasma iron is TBI and nonlabile under normal conditions, whereas in hemosiderosis, iron outpours into plasma, raising the TBI beyond transferrin capacity to
absorb the metal, generating non-TBI with a labile component designated as labile plasma iron (LPI). LPI inltrates cells via different routes raising their labile iron pools and
ensuing production of toxic oxidants that can be of lethal character. Cell death evoked by iron overload can manifest as necrosis or by death paths such as oxytosis (if
inhibited by antioxidants) or ferroptosis (if inhibited by iron chelators). Original gure from Dr. Cabantchik.

V. Berdoukas et al. / Free Radical Biology and Medicine 88 (2015) 39

Fig. 2. Susceptibility of cells in systemic iron overload. In systemic iron overload, plasma gets saturated with iron that outpours from the gut and/or the RES, overwhelming
the capacity to form TBI, thus generating NTBI, which includes LPI, its labile component that can permeate cells and raise their labile iron pools in cytosol (LCIc) and
mitochondria (LCImt). Cells have a limited ability to cope with a major rise in NTBI/LPI. Labile iron catalyzes the formation of the highly reactive oxygen species (ROS), such as
hydroxyl radical (OH), by interacting with the reactive oxygen intermediates superoxide anion (O2  ) and hydrogen peroxide (H2O2). Original gure from Dr. Cabantchik.

iron uptake versus utilization and/or storage and (b) enzymatic

neutralization of ROIs by superoxide dismutases and various peroxidases as well as by antioxidant molecules. Some cells, particularly of the reticuloendothelial (RE) system and duodenocytes,
express some level of activity of the iron exporter Fpn [4,5,810].
Although classically implicated in systemic iron homeostasis, the
expression of Fpn in cells such as cardiomyocytes might provide
an important salvage route for excess cell iron extrusion that
might be essential for cell iron homeostasis [9,10]. Other cells such
as hepatocytes have an inherently high capacity to synthesize
ferritin and thereby protect cells and systems from pathological
iron accumulation (Fig. 1).

2. Pathological iron accumulation

Thalassemia is a genetic disorder characterized by a hyperactive bone marrow with ineffective erythropoiesis due to the
inability to make hemoglobin and the intramedullary apoptosis of
latter stage erythroid precursors. Patients with thalassemia major
(TM) require transfusion every 2 to 4 weeks to suppress marrow
activity. As there are no known physiological means of extruding
iron from an iron-overloaded organism [4,6], TM patients can accumulate iron very rapidly, as there is as much as 200 mg iron per
each transfused unit of blood [6]. Poorly transfused TM patients
may absorb up to 10 times more dietary iron than normal individuals because their increased marrow activity causes a reduction in the secretion of hepcidin, an erythropoietic hormone,
despite high plasma TSAT [1518]. In TM, the fast degradation of
transfused red cells (
60 days lifetime) by macrophages in conjunction with a suppressed marrow from transfusion leads to an
outpouring of iron into the plasma. When TSAT is 470%, signicant amounts of non-transferrin-bound iron (NTBI) [1923]
appear in plasma. NTBI comprises forms that are redox-active and
chelatable and are referred to as labile plasma iron (LPI) [12,24]. As
some chemical forms of NTBI can inltrate various cell types by
resident transporters or channels, or by adsorptive endocytosis,
they can be considered as indicators of impending tissue iron
overload, but also as direct therapeutic targets of chelators.
However, it must be stressed that the chemical detection of any

NTBI in biological uids per se should not be strictly interpreted as

an indication of potential iron toxicity or even of iron overload
[25,26].
A possibly less ambiguous term for denoting the potentially
toxic features of iron in biological uids is LPI, as it denes the
redox-active components of plasma NTBI that are also chelatable
[12]; thus, measurement of LPI suggests impending iron overload
and/or provides a direct indication of chelation efcacy of prophylactic value [24,27]. Studies with neonatal rat cardiomyocytes
exposed to medium containing LPI obtained from iron-overloaded
patients demonstrated their functional susceptibility to iron accumulation and the amenability to restoring affected contraction
by chelators, which demonstrably access cells and reduce mitochondrial iron deposits [2831]. In general, in well-transfused
TM patients, hepcidin is elevated because of the iron load and
inhibits the release of iron from cells by inhibiting Fpn activity. LCI
levels are also high in TM patients and induce the synthesis of
ferritin subunits that get organized as protein shells that can absorb large numbers of Fe(II) after their oxidation to Fe(III) via the
ferroxidase-H ferritin subunits and formation of nonlabile crystallites together with phosphate and hydroxide ions [32]. When
the levels of intracellular ferritin increase signicantly, ferritin
molecules are further processed by dehydration, followed by
proteolytic degradation leading to the formation of aggregates
known as hemosiderin [33,34]. Although the iron in ferritin and
hemosiderin is mostly nonlabile and normally in steady state with
LCI, unidirectional trafc of iron toward increased LCI can be
generated chemically by oxidation and chelation or biochemically
by proteolytic activity with lysosomal and/or proteasomal reactions. In extreme cases of iron overload, the absorptive capacity of
cellular ferritin iron can be overridden, resulting in increased levels of LCI, often toxic. In systemic iron-overload disorders, such as
in TM, the level of LCI rises, owing to inltration of iron from
plasma rich in LPI [12,14]. However, in disorders associated with
defective heme or ISC biosynthesis (sideroblastic anemia and
Friedreich ataxia, respectively), iron accumulates excessively in
mitochondria, often at the expense of the cytosolic iron. By acting
on ROIs catalytically, labile mitochondrial iron promotes increased
ROS formation, resulting in organelle damage (Fig. 2).

V. Berdoukas et al. / Free Radical Biology and Medicine 88 (2015) 39

3. Pathophysiology of iron overload

Pathological consequences of iron accumulation become apparent whenever cell and/or system pathways fail to provide the
requisite protection, resulting in cell oxidative damage and ultimately in cell death [24,6]. Siderotic pathology with footprints of
oxidative cell damage is mainly detected in systemic siderosis
(inherited or iatrogenic, as in thalassemia), whereby cells are
overwhelmed by excessive ingress of nonphysiological forms of
iron that appear in the circulation or, in regional siderosis, in
which oxidative damage accompanies mitochondrial iron accumulation [31,35]. Notoriously, both physiological TBI uptake via
RME and nonphysiological inltration of pathological NTBI in ironoverload disorders divert most of the incoming iron to mitochondria by default (Fig. 1) [4,36]. However, these organelles
have an apparently limited ability to utilize iron in biosynthetic
pathways and an even lower ability to secure the excess metal in
mitochondrial ferritin (except in testis) or extrude excess iron into
the cytosol (Fig. 2). Thus, mitochondria are among the rst cell
organelles to succumb to cellular iron overload that results in iron
agglomeration and/or precipitation within the organelle. A rise in
biochemical markers of oxidation concomitant with ROS formation is a characteristic feature of excessive iron accumulation
[14,36]. However, the causative association between ironROS and
oxidative damage in both experimental and clinical settings depends primarily on the ability of chelators to either reduce or
prevent cardiosiderosis and oxidative damage. As components of
the labile iron pool are responsible for metal-driven oxidation and
the direct targets of chelation, their neutralization has become a
primary goal in iron-chelation therapy
It should be emphasized, however, that although the direct
chemical target of chelators is labile iron, functional improvements
might result not only from direct metal detoxication, but also
from the possible deactivation of death mechanisms triggered by
siderosis as proposed for ferroptosis [35]. Irrespective of the mode
of cell rescue by chelation, the therapeutic approach to siderosis
depends on a rationale based on systemic or regional detoxication that avoids depletion of essential iron.

4. Cardiomyopathy in thalassemia
The etiology of siderotic cardiomyopathy in TM is currently
attributed to the frequent and cumulative exposure of the heart to
circulating LPI, coupled with a restricted ability of cardiomyocytes
to contain the inltration of LPI, either by restricting ingress or
increasing egress or by sequestering excess iron into ferritin (cytosolic and/or mitochondria) and/or raising antioxidant defenses
(Fig. 2 ).
Although there is pharmacological evidence that Fe(II) enters
cardiomyocytes through L-type calcium channels (LTCC) [37,38],
studies with experimental models have provided conicting results as to the membrane structures used by LPI to inltrate cardiac cells and other cells in systemic iron overload in TM [3943].
In liver and pancreas the Zn transporter ZIP14 has been proposed
to mediate transport of model iron complexes under nonpathophysiological conditions [43], as those are difcult to simulate in vitro. The natural history of TM cardiosiderosis indicates
that the heart loads iron relatively slowly compared to others (e.g.,
liver or endocrine glands), although in experimental cell models,
exposure to millimolar (suprapharmacological) concentrations of
iron salts (often supplemented with ascorbic acid as a reductant of
Fe(II)) can lead to up to 10-fold higher iron ingress rates in cardiomyocytes from neonatal rats than in broblasts, which have
few if any LTCCs [37]. However, the main difculty in dening the
precise mechanism of LPI access to cardiac cells is to ascertain the

chemical nature of the permeating iron forms. This is because the

composition of LPI varies with concentration, which is mostly in
the lower micromolar range and in forms complexed to citrate
and/or albumin, all in a background of 430 M TBI ( 470% TSAT).
Cardiotoxicity is attributed to a rise in LCI induced by exposure
to high LPI (or LPI simulants), by targeted suppression of Fpn expression in cardiomyocytes, or by mutations that affect the utilization of iron in the biosynthesis of heme or ironsulfur clusters in
particular cell types [44,45]. The fact that a rise in LCI per se and
ensuing damage can be prevented by neutralization of LPI with an
impermeative chelator or corrected with a permeative chelator
implicates labile iron in cardiosiderosis and places it as a pharmacological target, i.e., LPI and LCI in systemic siderosis and LCI in
regional siderosis [4549].
Until two decades ago, the only chelator approved for treating
transfusional hemosiderosis was deferoxamine (DFO) [50]. Because of its poor oral bioavailability and short half-life in the circulation, DFO was usually given parenterally by slow infusion with
portable pumps 5 days per week and for 810 hours per day. It is
understandable that a regimen of chelation that efciently reduces
hepatosiderosis needs to have success on cardiosiderosis [51,52]
as: (1) iron loading of the liver differs from that of the heart, not
only in terms of extent and rapidity (owing to a different repertoire of transporters and channels), but also etiologically, e.g.,
the liver draws iron from enhanced erythrophagocytosis in the RE
and from NTBI/LPI in the parenchyma, whereas the heart loads
iron primarily from NTBI/LPI; and (2) liver and heart cells respond
differently to iron chelators with limited permeation capacity,
such as DFO. Moreover, chelation with agents of short lifetime that
are not given continuously (day and night) leaves extrahepatic
tissue essentially exposed to inltration by LPI during periods of
no chelation [51,52]. This problem is even more acute in patients
who do not adhere to recommended regimens [53,54].
The amount of myocardial iron accumulation predicted to occur
over 20 years exposure to LPI was estimated at approximately
10 mg of iron per gram of myocardial tissue [37]. This matches
closely with the cardiac iron concentration (CIC) found in postmortem studies of cardiac iron in TM and in animal models of iron
overload [55,56]. The mean lethal level of CIC in deceased TM
patients who died of cardiac failure was 5.98 72.42 mg/g dry wt,
and the range was 3.19 to 9.5 mg/g dry wt in one recent study [55]
and 6.0 to 13.2 mg/g dry wt in an older study [56]. The 5.98 mg/g
dry wt value is equivalent to a T2* MRI value of about 4 ms [55],
which is well into the T2* range predictive of severe cardiomyopathy [57]. Cardiac iron accumulation can appear early in life (age
o10 years) in patients with transfusion-dependent conditions
that have suppressed erythropoiesis and hepatic iron overload
(Fig. 3). This is consistent with the notion that when iron is not
adequately used by the bone marrow for erythropoiesis, it might
lead to a rise in LPI and ensuing cardiac and pancreatic iron
overload [58]. Moreover, patients may experience arrhythmia even
without detectable cardiac iron overload, but given that chelation
therapy can swiftly resolve the condition, it implicates labile iron
as a key etiopathological factor in cardiosiderosis.
The increased LCI observed in model cardiomyocytes exposed
to LPI may explain the profound contractile dysfunctions observed
experimentally and clinically, in which ensuing Ca2 overload and
impaired diastolic function appear during the early stages of iron
overload. Elevations in LCI from exposure to various iron sources
or mutations that affect the mitochondrial ability to use Fe for
biosynthetic purposes also lead to regional increase in labile iron
and ROS production in mitochondria and an ensuing plethora of
biochemical changes reective of mitochondria oxidation and
dysfunction [37].
The fact that continuous chelation, which immediately reduces
the labile iron pool, results in improvement in arrhythmias at

LIC (mg/g dw)

Pancreas R2* (Hz)

Cardiac R2* (Hz)

V. Berdoukas et al. / Free Radical Biology and Medicine 88 (2015) 39

100
75
50

25

Disorders with
ineffective
erythropoiesis (black)
have greater exposure
to free iron

CDA
DBA
TM
SCD
PK

100

Total iron loading is

independent of marrow
activity

10

1
0

10

Age (yrs)
Fig. 3. MRI studies in children less than 10 years of age with transfusion-dependent anemias showing the R2* values (1000/T2*) for cardiac, pancreatic, and liver iron. The
stars are patients with DiamondBlackfan anemia, the triangles are patients with congenital dyserythropoietic anemia, the black dots are thalassemia, and the crosses are
patients with sickle cell anemia. The black markers are for patients who have erythroid suppression either because of their disease, such as DiamondBlackfan anemia, or
because of the aim of their transfusions to suppress erythropoiesis. The red markers are for those without suppressed erythropoiesis, most of whom are patients with sickle
cell anemia. Although many patients demonstrate iron overload as seen in their liver iron concentration, only those with suppressed erythropoiesis show cardiac and
pancreatic iron load. There are ve thalassemia patients who have excess cardiac iron [58, 68].

Heart failure and recovery with intensive chelation

Thalassaemia Centre
University of Torino

Fig. 4. Rapid clinical improvement in cardiac function with chelation of therapy. Response to chelation treatment in an adult Italian TM patient. In 2004, the cardiac MRI
showed a cardiac T2* of about 5 ms, consistent with severe iron loading. Soon after, his LVEF fell to 17% and he was in overt cardiac failure. Chelation therapy with
deferoxamine and deferiprone resulted in rapid improvement in LVEF to over 52%. A cardiac T2* MRI about 9 months later was about 6 ms (severe iron load), indicating that
the relief of cardiac dysfunction occurred before a signicant improvement in the cardiac iron concentration. (Reproduced with kind permission from Professor Antonio Piga,
from Torino, Italy.).

times within hours and in heart failure symptoms within weeks,

long before total cardiac iron load decrease, attests to the importance of these labile iron species in the genesis of iron-induced
cardiac disease.

5. Management of cardiac iron overload

Many studies have shown a reduction in the level of cardiac iron
with the use of the currently available chelators, either as

V. Berdoukas et al. / Free Radical Biology and Medicine 88 (2015) 39

monotherapy or in combination. Importantly, iron cardiomyopathy

is almost always reversible if aggressive treatment is started before
the patient is in extremis [5966]. The implementation of effective
iron chelation in combination with the ability to directly measure
tissue iron by MRI has resulted in a change in the median survival of
patients with TM and other transfusional iron overload from less
than 20 years in the 1970s to probably over 60 years currently. Our
understanding of iron homeostasis has dramatically increased over
the past 2 decades and has greatly informed our approach to clinical
management, and the clinical responses to treatment have conrmed the basic iron hemostasis facts (reviewed in [58]). Based on
our current understanding of the entry of labile iron by nonregulated routes, we now recommend that some chelator be present in the circulation at all times. The authors have seen a patient
with mild heart failure and cardiac iron normalize both parameters
by keeping the same total weekly dose of deferoxamine, but simply
changing administration from 5 days a week to 7 days a week. This
response is totally consistent with the cardiac iron loading physiology described here. Fig. 4 further shows the critical points made
here regarding the role of labile iron in a TM patient who had a
sudden drop in left-ventricular ejection fraction (LVEF; green line)
while relatively compliant with DFO therapy. The cardiac iron was
very high (low T2* o10 ms; pink line) when continuous DFO and
the mitochondrial-permeative iron chelator deferiprone was started. The ejection fraction (green line) rapidly recovered within a
month or so, but the cardiac iron by T2* MRI did not increase up to a
safe level of 410 ms for a year and did not normalize to 420 ms
for over 2 years. This theme of the relationship of reactive labile iron
to organ damage is repeated many times in medical practice and
the medical literature, even though the mechanism is often not
appreciated. In fact, whereas elevated transferrin saturation has
long been used as a marker in the hemochromatosis literature as
well as in the epidemiology literature as a marker of poor outcome,
the realization that this is a surrogate for labile plasma iron is underappreciated (reviewed in [67]). Transferrin saturation and labile
plasma iron measurements can change within hours of ingestion of
iron or of starting chelation, making neither of them suitable for
routine therapeutic monitoring of iron treatment. However, pancreatic and cardiac iron loads assessed by MRI seem to be excellent
surrogates for area under the curve time integral of exposure to
labile iron, as these organs essentially load iron only when exposed
to labile iron species.
Overall, the general consensus is that chelators should be circulating continuously in patients with iron-overload disorders to
avoid the presence of LPI and LCI and therefore prevent cardiac
iron load, alleviate cardiac dysfunction, and prevent loading and
damage to endocrine organs.

6. Summary
Labile iron is the toxic iron species that interacts with ROIs in cells
to form powerful oxidants, which directly damage proteins, DNA,
RNA, and associated functions. Clinically, a TSAT over 70% indicates
the presence of plasma NTBI, of which its labile component (LPI)
comprises various chemical species of variable composition, depending on the degree of iron overload. Those labile iron species
inltrate cells via different pathways that are not related to the
physiological TfRME transferrin pathway. In the pancreas and liver,
Zn transporters (ZIP14) have been proposed to be involved in iron
transport, whereas other resident transporters or channels are at play
in the heart. A rise in LCI levels leads to increased ROS production
and cardiac complications such as arrhythmia and reduced LVEF,
even without signicantly excessive cardiac iron load.
It seems essential to minimize labile iron in the plasma by
using chelation therapy that reduces exposure to LPI to a

minimum for most of the day while leaving some basal LCI levels
for cellular iron metabolism. This should allow for better mitochondrial function and reduce the risk of cardiac failure and
rhythm disturbances. The chelator deferiprone, either as monotherapy or in combination with deferoxamine, has thus far provided the best results in terms of cardiac iron detoxication and
functional recovery from cardiosiderosis, and its combination with
DFO exhibited the best results in hepatosiderosis.
In summary, MRI and access to adequate continuous ironchelation therapy aided by periodic LPI measurement that could
guide chelation therapy should further reduce the risk of cardiac
dysfunction and cardiac-related deaths in transfusion-related iron
overload such as TM.

Acknowledgments
The authors thank Dr. Martine Torres for her suggestions and
careful review of the manuscript. T.D. Coates has consultant
agreements with Novartis, Apo-Pharma, Shire Pharmaceuticals,
and Sideris Pharma. Vasili Berdoukas is a consultant for ApoPharma. Z.I. Cabantchik is a consultant for Afferix.

References
[1] J. Wang, K. Pantopoulos, Regulation of cellular iron metabolism, Biochem. J.
434 (2011) 365381.
[2] T.A. Rouault, The role of iron regulatory proteins in mammalian iron homeostasis and disease, Nat. Chem. Biol. 2 (2006) 406414.
[3] M.W. Hentze, M.U. Muckenthaler, N.C. Andrews, Balancing acts: molecular
control of mammalian iron metabolism, Cell 117 (2004) 285297.
[4] D.J. Lane, A.M. Merlot, M.L. Huang, D.H. Bae, P.J. Jansson, S. Sahni, D.
S. Kalinowski, D.R. Richardson, Cellular iron uptake, trafcking and metabolism: key molecules and mechanisms and their roles in disease, Biochim.
Biophys. Acta 1853 (2015) 11301144.
[5] T. Ganz, Systemic iron homeostasis, Physiol. Rev. 93 (2013) 17211741.
[6] R.E. Fleming, P. Ponka, Iron overload in human disease, N. Engl. J. Med. 366
(2012) 348359.
[7] N.C. Andrews, Disorders of iron metabolism, N. Engl. J. Med. 341 (1999)
19861995.
[8] E. Gammella, P. Buratti, G. Cairo, S. Recalcati, Macrophages: central regulators
of iron balance, Metallomics 6 (2014) 13361345.
[9] S. Lakhal-Littleton, M. Wolna, C.A. Carr, J.J. Miller, H.C. Christian, V. Ball,
A. Santos, R. Diaz, D. Biggs, R. Stillion, P. Holdship, F. Larner, D.J. Tyler, K. Clarke,
B. Davies, P.A. Robbins, Cardiac ferroportin regulates cellular iron homeostasis
and is important for cardiac function, Proc. Natl. Acad. Sci. USA 112 (2015)
31643169.
[10] X.H. Ge, Q. Wang, Z.M. Qian, L. Zhu, F. Du, W.H. Yung, L. Yang, Y. Ke, The iron
regulatory hormone hepcidin reduces ferroportin 1 content and iron release in
H9C2 cardiomyocytes, J. Nutr. Biochem. 20 (2009) 860865.
[11] B. Halliwell, J. Gutteridge, Free Radicals in Biology and Medicine, Oxford Univ.
Press, London, 2007.
[12] Z.I. Cabantchik, Labile iron in cells and body uids: physiology, pathology, and
pharmacology, Front. Pharmacol 5 (2014) 45.
[13] A. Jacobs, Low molecular weight intracellular iron transport compounds, Blood
50 (1977) 433439.
[14] M. Kruszewski, Labile iron pool: the main determinant of cellular response to
oxidative stress, Mutat. Res. 531 (2003) 8192.
[15] G.J. Anderson, D. Darshan, S.J. Wilkins, D.M. Frazer, Regulation of systemic iron
homeostasis: how the body responds to changes in iron demand, Biometals 20
(2007) 665674.
[16] G.J. Anderson, Mechanisms of iron loading and toxicity, Am. J. Hematol. 82
(2007) 11281131.
[17] C. Hershko, Iron loading and its clinical implications, Am. J. Hematol. 82 (2007)
11471148.
[18] Cabantchik, Z.I., S.Y., Breuer, W., Esposito, B. The Molecular and Cellular Basis of
Iron Toxicity in Iron Overload Disorders: Diagnostic and Therapeutic
Approaches.2013.
[19] C. Hershko, G. Graham, G.W. Bates, E.A. Rachmilewitz, Non-specic serum iron
in thalassaemia: an abnormal serum iron fraction of potential toxicity, Br. J.
Haematol. 40 (1978) 255263.
[20] W. Breuer, C. Hershko, Z.I. Cabantchik, The importance of non-transferrin
bound iron in disorders of iron metabolism, Transfus. Sci. 23 (2000) 185192.
[21] P. Brissot, M. Ropert, C. Le Lan, O. Loreal, Non-transferrin bound iron: a key
role in iron overload and iron toxicity, Biochim. Biophys. Acta 1820 (2012)
403410.

V. Berdoukas et al. / Free Radical Biology and Medicine 88 (2015) 39

[22] A.M. Kolb, N.P. Smit, R. Lentz-Ljuboje, S. Osanto, J. van Pelt, Non-transferrin
bound iron measurement is inuenced by chelator concentration, Anal. Biochem. 385 (2009) 1319.
[23] D.H. Lee, D.Y. Liu, D.R. Jacobs Jr, H.R. Shin, K. Song, I.K. Lee, B. Kim, R.C. Hider,
Common presence of non-transferrin-bound iron among patients with type
2 diabetes, Diabetes Care 29 (2006) 10901095.
[24] G. Zanninelli, W. Breuer, Z.I. Cabantchik, Daily labile plasma iron as an indicator of chelator activity in Thalassaemia major patients, Br. J. Haematol. 147
(2009) 744751.
[25] Z.I. Cabantchik, E. Rachmilewitz, Labile iron: potential toxicity in iron overload
disorders, Hematologist 12 (2015) 6.
[26] I. Slotki, Z.I. Cabantchik, The labile side of iron supplementation in CKD, J. Am.
Soc. Nephrol (2015).
[27] F. Danjou, Z.I. Cabantchik, R. Origa, P. Moi, M. Marcias, S. Barella, E. Defraia,
C. Dessi, M.L. Foschini, N. Giagu, G.B. Leoni, M. Morittu, R. Galanello, A decisional algorithm to start iron chelation in patients with beta thalassemia,
Haematologica 99 (2014) e38e40.
[28] G. Link, C. Hershko, Rat heart cells in culture: a model of iron toxicity and
chelation, J. Lab. Clin. Med. 122 (1993) 1415.
[29] J. Moreb, C. Hershko, Y. Hasin, Effects of acute iron loading on contractility and
spontaneous beating rate of cultured rat myocardial cells, Basic Res. Cardiol.
83 (1988) 360368.
[30] H. Glickstein, R.B. El, M. Shvartsman, Z.I. Cabantchik, Intracellular labile iron
pools as direct targets of iron chelators: a uorescence study of chelator action
in living cells, Blood 106 (2005) 32423250.
[31] D.M. Templeton, Y. Liu, Genetic regulation of cell function in response to iron
overload or chelation, Biochim. Biophys. Acta 1619 (2003) 113124.
[32] R. Crichton,. Inorganic Biochemistry of Iron Metabolism from Molecular Mechanisms to Clinical Consequences 2014.
[33] E. Miyazaki, J. Kato, M. Kobune, K. Okumura, K. Sasaki, N. Shintani, P. Arosio,
Y. Niitsu, Denatured H-ferritin subunit is a major constituent of haemosiderin
in the liver of patients with iron overload, Gut 50 (2002) 413419.
[34] M.P. Weir, J.F. Gibson, T.J. Peters, Haemosiderin and tissue damage, Cell. Biochem. Funct. 2 (1984) 186194.
[35] M. Kruszewski, The role of labile iron pool in cardiovascular diseases, Acta
Biochim. Polon 51 (2004) 471480.
[36] J.P. Munoz, M. Chiong, L. Garcia, R. Troncoso, B. Toro, Z. Pedrozo, J. Diaz-Elizondo, D. Salas, V. Parra, M.T. Nunez, C. Hidalgo, S. Lavandero, Iron induces
protection and necrosis in cultured cardiomyocytes: role of reactive oxygen
species and nitric oxide, Free Radic. Biol. Med. 48 (2010) 526534.
[37] G.Y. Oudit, M.G. Trivieri, N. Khaper, P.P. Liu, P.H. Backx, Role of L-type Ca2
channels in iron transport and iron-overload cardiomyopathy, J. Mol. Med. 84
(2006) 349364.
[38] E. Gammella, S. Recalcati, I. Rybinska, P. Buratti, G. Cairo, Iron-Induced Damage
in Cardiomyopathy:Oxidative-Dependent and Independent Mechanisms,
Oxidative Medicine and Cellular Longevity Volume (2015), Article ID 30182,
http://dx.doi.org/10.1155/2015/230182.
[39] M.P. Chen, Z.I. Cabantchik, S. Chan, G.C. Chan, Y.F. Cheung, Iron overload and
apoptosis of HL-1 cardiomyocytes: effects of calcium channel blockade, PLoS
One 9 (2014) e112915.
[40] N. Chattipakorn, K.S. Fucharoen, S. Chattipakorn, S. Calcium channels and iron
uptake into the heart, World J. Cardiol. 3 (2011) 215218.
[41] S. Kumfu, S. Chattipakorn, K. Chinda, S. Fucharoen, N. Chattipakorn, T-type
calcium channel blockade improves survival and cardiovascular function in
thalassemic mice, Eur. J. Haematol. 88 (2012) 535548.
[42] J.P. Liuzzi, F. Aydemir, H. Nam, M.D. Knutson, R.J. Cousins, Zip14 (Slc39a14)
mediates non-transferrin-bound iron uptake into cells., Proc. Natl. Acad. Sci.
USA 103 (2006) 1361213617.
[43] S. Kumfu, S. Chattipakorn, S. Srichairatanakool, J. Settakorn, S. Fucharoen,
N. Chattipakorn, T-type calcium channel as a portal of iron uptake into cardiomyocytes of beta-thalassemic mice, Eur. J. Haematol. 86 (2011) 156166.
[44] T.A. Rouault, Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease, Dis. Models Mech 5 (2012) 155164.
[45] O. Kakhlon, W. Breuer, A. Munnich, Z.I. Cabantchik, Iron redistribution as a
therapeutic strategy for treating diseases of localized iron accumulation, Can.
J. Physiol. Pharmacol. 88 (2010) 187196.
[46] Y. Liu, J.G. Parkes, D.M. Templeton, Differential accumulation of non-transferrin-bound iron by cardiac myocytes and broblasts, J. Mol. Cell. Cardiol. 35
(2003) 505514.
[47] X. Gao, M. Qian, J.L. Campian, J. Marshall, Z. Zhou, A.M. Roberts, Y.J. Kang, S.
D. Prabhu, X.F. Sun, J.W. Eaton, Mitochondrial dysfunction may explain the
cardiomyopathy of chronic iron overload, Free Radic. Biol. Med. 49 (2010)
401407.
[48] C.J. Murphy, G.Y. Oudit, Iron-overload cardiomyopathy: pathophysiology, diagnosis, and treatment, J. Card. Failure 16 (2010) 888900.
[49] Z.I. Cabantchik, A. Munnich, M.B. Youdim, D. Devos, Regional siderosis: a new

challenge for iron chelation therapy, Front. Pharmacol 4 (2013) 167.

[50] J.B. Porter, Practical management of iron overload, Br. J. Haematol. 115 (2001)
239252.
[51] A. Aessopos, C. Fragodimitri, F. Karabatsos, A. Hatziliami, J. Yousef,
A. Giakoumis, A. Dokou, E.D. Gotsis, V. Berdoukas, M. Karagiorga, Cardiac
magnetic resonance imaging R2* assessments and analysis of historical
parameters in patients with transfusion-dependent thalassemia, Haematologica 92 (2007) 131132.
[52] M.A. Tanner, R. Galanello, C. Dessi, M.A. Westwood, G.C. Smith, S.V. Nair, L.
J. Anderson, J.M. Walker, D.J. Pennell, Myocardial iron loading in patients with
thalassemia major on deferoxamine chelation, J. Cardiovasc. Magn. Reson. 8
(2006) 543547.
[53] C.K. Lee, M.R. Stockler, A.S. Coates, V. Gebski, S.J. Lord, R.J. Simes, Australian
New Zealand Breast Cancer Trials, G. Self-reported health-related quality of
life is an independent predictor of chemotherapy treatment benet and
toxicity in women with advanced breast cancer, Br. J. Cancer 102 (2010)
13411347.
[54] K.A. Payne, M.P. Desrosiers, J.J. Caro, J.F. Baladi, N. Lordan, I. Proskorovsky,
K. Ishak, D. Rofail, Clinical and economic burden of infused iron chelation
therapy in the United States, Transfusion 47 (2007) 18201829.
[55] J.P. Carpenter, T. He, P. Kirk, M. Roughton, L.J. Anderson, S.V. de Noronha, M.
N. Sheppard, J.B. Porter, J.M. Walker, J.C. Wood, R. Galanello, G. Forni, G. Catani,
G. Matta, S. Fucharoen, A. Fleming, M.J. House, G. Black, D.N. Firmin St, T.
G. Pierre, D.J. Pennell, On T2* magnetic resonance and cardiac iron, Circulation
123 (2011) 15191528.
[56] B. Modell, V. Berdoukas, The Clinical Approach to Thalassaemia, Grune &
Stratton, New York, 1984.
[57] P. Kirk, M. Roughton, J.B. Porter, J.M. Walker, M.A. Tanner, J. Patel, D. Wu,
J. Taylor, M.A. Westwood, L.J. Anderson, D.J. Pennell, Cardiac T2* magnetic
resonance for prediction of cardiac complications in thalassemia major, Circulation 120 (2009) 19611968.
[58] T.D. Coates, Physiology and pathophysiology of iron in hemoglobin-associated
diseases, Free Radic. Biol. Med. 72 (2014) 2340.
[59] M.A. Tanner, R. Galanello, C. Dessi, G.C. Smith, M.A. Westwood, A. Agus,
M. Roughton, R. Assomull, S.V. Nair, J.M. Walker, D.J. Pennell, A randomized,
placebo-controlled, double-blind trial of the effect of combined therapy with
deferoxamine and deferiprone on myocardial iron in thalassemia major using
cardiovascular magnetic resonance, Circulation 115 (2007) 18761884.
[60] V. Berdoukas, G. Chouliaras, P. Moraitis, K. Zannikos, E. Berdoussi, V. Ladis, The
efcacy of iron chelator regimes in reducing cardiac and hepatic iron in patients with thalassaemia major: a clinical observational study, J. Cardiovasc.
Magn. Reson. 11 (2009) 20.
[61] D.J. Pennell, J.B. Porter, A. Piga, Y. Lai, A. El-Beshlawy, K.M. Belhoul, M. Elalfy,
A. Yesilipek, Y. Kilinc, T. Lawniczek, D. Habr, M. Weisskopf, Y. Zhang,
Y. Aydinok, Investigators, C. s. A 1-year randomized controlled trial of deferasirox vs deferoxamine for myocardial iron removal in beta-thalassemia major
(CORDELIA), Blood 123 (2014) 14471454.
[62] L.J. Anderson, M.A. Westwood, S. Holden, B. Davis, E. Prescott, B. Wonke, J.
B. Porter, J.M. Walker, D.J. Pennell, Myocardial iron clearance during reversal
of siderotic cardiomyopathy with intravenous desferrioxamine: a prospective
study using T2* cardiovascular magnetic resonance, Br. J. Haematol. 127
(2004) 348355.
[63] L.J. Anderson, B. Wonke, E. Prescott, S. Holden, J.M. Walker, D.J. Pennell,
Comparison of effects of oral deferiprone and subcutaneous desferrioxamine
on myocardial iron concentrations and ventricular function in beta-thalassaemia, Lancet 360 (2002) 516520.
[64] D.J. Pennell, V. Berdoukas, M. Karagiorga, V. Ladis, A. Piga, A. Aessopos, E.
D. Gotsis, M.A. Tanner, G.C. Smith, M.A. Westwood, B. Wonke, R. Galanello,
Randomized controlled trial of deferiprone or deferoxamine in beta-thalassemia major patients with asymptomatic myocardial siderosis, Blood 107
(2006) 37383744.
[65] V. Berdoukas, S. Carson, A. Nord, A. Dongelyan, S. Gavin, T.C. Hofstra, J.C. Wood,
T. Coates, Combining two orally active iron chelators for thalassemia, Ann.
Hematol. 89 (2010) 11771178.
[66] M.S. Elalfy, A.M. Adly, Y. Wali, S. Tony, A. Samir, Y. Elhenawy, Efcacy and
safety of a novel combination of two oral chelators deferasirox/deferiprone
over deferoxamine/deferiprone in severely iron-overloaded young beta thalassemia major patients, Eur. J. Haematol (2015).
[67] M. Puliyel, A.G. Mainous 3rd, V. Berdoukas, T.D. Coates, Iron toxicity and its
possible association with treatment of cancer: lessons from hemoglobinopathies and rare, transfusion-dependent anemias, Free Radic. Biol. Med. 79C
(2015) 343351.
[68] V. Berdoukas, A. Nord, S. Carson, M. Puliyel, T. Hofstra, J. Wood, T.D. Coates,
Tissue iron evaluation in chronically transfused children shows signicant
levels of iron loading at a very young age, Am. J. Hematol. 88 (2013)
E283E285.