Iron Metabolism in Cardiovascular Disease: Physiology, Mechanisms, and Therapeutic Targets

Abstract

The cardiovascular system requires iron to maintain its high energy demands and metabolic activity. Iron plays a critical role in oxygen transport and storage, mitochondrial function, and enzyme activity. However, excess iron is also cardiotoxic due to its ability to catalyze the formation of reactive oxygen species and promote oxidative damage. While mammalian cells have several redundant iron import mechanisms, they are equipped with a single iron exporting protein which makes the cardiovascular system particularly sensitive to iron overload. As a result, iron levels are tightly regulated at many levels to maintain homeostasis. Iron dysregulation ranges from iron deficiency (ID) to iron overload and is seen in many types of cardiovascular disease (CVD), including heart failure, myocardial infarction, anthracycline-induced cardiotoxicity, and Friedreich’s ataxia. Recently, the use of intravenous iron therapy has been advocated in patients with heart failure and certain criteria for ID. Here, we provide an overview of systemic and cellular iron homeostasis in the context of cardiovascular physiology, ID and iron overload in CVD, current therapeutic strategies, and future perspectives.

Introduction

Iron is the most abundant element by mass in the Earth, and the fourth most abundant element on planet’s crust.¹ Due to its unique oxidation-reduction properties, iron readily donates and accepts electrons and exists mainly in the ferric (Fe³⁺) and ferrous (Fe²⁺) oxidation states.²

Iron is an essential cofactor for nearly all known life forms. Mammalian cells predominantly use the less abundant ferrous iron, which has led to evolutionary regulatory mechanisms to conserve and recycle iron.³ Iron is important for cell types with high energy demand, particularly cardiomyocytes. Because iron is an ideal transporter of electrons, it facilitates many essential biochemical reactions. Important cardiovascular functions of iron include oxygen transport and storage, energy production, heme and iron-sulfur (Fe/S) cluster formation, and enzyme catalysis (Table 1).⁴ However, free unbound iron is a reactive element that generates reactive oxygen species (ROS) and oxidative stress via the Fenton reaction, which can be damaging to human cells and the cardiovascular system.⁵ Therefore, iron metabolism is tightly regulated at both the systemic and cellular levels.

Table 1.

Iron-containing proteins and their role in cardiovascular biology.

Iron-containing protein	Cardiovascular function
Heme-containing proteins
Catalase	Antioxidant defense, hydrogen peroxide detoxification
Cytochrome proteins	Electron transport chain
Cytochrome oxidase P450
Cytochrome oxidase C
Ubiquinolcytochrome c reductase
Cytochrome c1
Cytochrome b5
Cytochrome f
Succinate-ubiquinone reductase
Glutathione peroxidase 2	Cellular defense against oxidative stress
Hemoglobin	Oxygen transport
Idoleamine-pyrrole 2,3-dioxygenase	Tryptophan catabolism
Myeloperoxidase	Production of hypochlorous acid
Myoglobin	Cellular oxygen storage and transport
Nitric oxide synthase	Production of nitric oxide
Peroxidasin	Extracellular matrix formation
Prostaglandin G/H synthase	Peroxidase, cyclooxygenase
Soluble uanylate cyclase	Production of cyclic GMP
Fe/S cluster-containing proteins
Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF)	Electron transport chain
Ferrochelatase	Heme and porphyrin biosynthesis
IRP1/cytosolic aconitase (cAcn)	Iron regulation
Mitochondrial aconitase (mAcn)	Tricarboxylic acid cycle
NADH:ubiquinone oxidoreductase (complex I)	Electron transport chain
Rieske protein	Electron transport chain
Xanthine oxidase	Purine metabolism, DNA synthesis

ID manifests in two distinct forms: absolute and functional ID. In absolute ID, there is a deficit of total-body iron stores due to impaired nutrition, reduced gastrointestinal absorption, use of certain medications, and blood loss.⁶ In functional ID, there is reduced availability of iron despite sufficient iron stores as a result of the regulation of iron transport.⁷ Functional ID is often associated with inflammatory conditions, leading to reduced gastrointestinal iron absorption and iron trapping in macrophages. This mechanism helps protect the body against microorganisms which depend on iron for survival.⁸

The last three decades have seen a revolution in our understanding of the physiology and pathophysiology of iron metabolism in the cardiovascular system. In this review, we highlight the key role of iron in cardiovascular biology; we summarize the mechanisms involved in the regulation of iron homeostasis in the cardiovascular system; and we provide novel perspectives on the potential of new therapies that target iron metabolism for the treatment of CVD.

Systemic iron homeostasis

Systemic iron is regulated at the levels of iron absorption, storage, and transport to maintain homeostasis for all body systems, including the cardiovascular system (Figure 1).

Figure 1. Systemic iron regulation.

(1) Heme iron is absorbed on the luminal side of duodenal enterocytes. Within the enterocyte, heme is broken down by HO-1 and iron can either be stored as ferritin or enter the systemic circulation through ferroportin. (2) Non-heme ferric iron is reduced to ferrous iron by DCYTB, which can then enter enterocytes through DMT1. Ferrous iron is either stored or transported into the systemic circulation via FPN1. (3) In the circulation, ferrous iron is oxidized by CP to ferric iron, after which it binds tranferrin and is shuttled through circulation to the liver and other tissues. (4) In the hepatocyte, iron enters the cell and is stored in ferritin. When systemic iron levels are high, hepatocytes upregulate expression of hepcidin, which inhibits ferroportin on macrophages and enterocytes. Abbreviations: CP, ceruloplasmin; DCYTB, duodenal cytochrome b reductase 1; DMT1, divalent metal transporter 1; FPN1, ferroportin-1; FTN, ferritin; HCP1, heme carrier protein 1; HO-1, heme oxygenase-1; Tf, transferrin. (Figure credit: Sceyence Studios)

Intestinal absorption of iron

Iron is absorbed predominantly via enterocytes in the form of heme or non-heme iron.^{9, 10} Physiologically, most of the iron that leaves the body is lost through menstruation in women or sloughing of epithelial-lined surfaces such as the skin, intestinal mucosa, urinary and biliary tracts.^11–13 There are no known regulated pathways for active iron excretion, therefore, maintaining systemic and intracellular iron homeostasis is largely dependent on absorption.¹⁴

Heme-iron absorption

Heme iron is absorbed in the small intestine, however the mechanism of absorption and the responsible heme transporters are not well understood. Heme carrier protein 1 (HCP1) is a transporter expressed on luminal surface of enterocytes that transports not only heme but also other substrates, such as folate.^15–17 Once heme enters the cell, heme oxygenase-1 (HO-1) and -2 (HO-2) mediate the release of ferrous iron from heme into the cytosol.^{18, 19} The unbound ferrous iron can enter the intracellular labile iron pools, and either be stored or transported through the basolateral surface of enterocytes into the bloodstream. Intracellular heme can also be exported directly into circulation, possibly via the feline leukemia virus subgroup C cellular receptor 1 (FLVCR1) or ATP-binding cassette sub-family G member 2 protein (ABCG2) proteins.^{20, 21}

Non-heme iron absorption

Absorption of non-heme iron begins with the reduction of ferric to ferrous iron at the apical side of the enterocyte. This is facilitated by the action of acidic gastric chyme and the action of ferrireductase enzymes, such as duodenal cytochrome b reductase 1 (DCYTB).^{22, 23} Ferrous iron can be transported into the cell via divalent metal transporter 1 (DMT1).^{24, 25} Inside the cell, iron can be stored by binding to ferritin (FTN) or released into the systemic circulation through the basolateral surface transporter, ferrorportin.^{26, 27} In the bloodstream, exported free iron is bound to transferrin, which interacts with transferrin receptor 1 or 2 (TFR1/2) on the cell surface to undergo endocytosis and be stored or utilized for various intracellular processes.²⁸

Hepcidin regulation

Hepcidin is a hormone primarily produced by the liver that controls plasma iron levels through several mechanisms including regulation of dietary iron from the intestine, release of iron from macrophages, and mobilization of iron stored into hepatocytes.²⁹ In states of iron abundance, hepcidin is released into the circulation to limit further iron absorption and the release of cellular iron stores.³⁰ Hepcidin acts by inducing endocytosis and subsequent degradation of ferroportin. Recently, the E3 ubiquitin ligase RNF217 was found to mediate ferroportin degradation.³¹ Hepcidin has also been shown to occlude the open-outward conformation of ferroportin and physically block iron export into the circulation.³²

Cellular iron homeostasis

In order to maintain cellular iron homeostasis, several proteins are involved in the cellular uptake, storage, regulation, and export of iron in the cardiovascular system (Figure 2).

Figure 2. Cardiomyocyte iron homeostasis.

Transferrin-bound ferric iron enters cardiomyocytes through TFR1 and TFR2β, while non-transferrin bound ferrous iron enters through DMT1, LTCC, TTCC, and zinc transporters. The majority of cellular iron is bound by ferritin, with the remainder stored in mitochondria or existing as labile iron. Cellular iron deficiency activates the IRP-IRE system, which upregulates TFR1 and DMT1 by binding to the 3’-UTR, while downregulating FPN1, FTH, and FTL by binding to the 5’-UTR. In extreme iron deficiency, TTP binds to AREs in the 3’-UTR of certain non-essential iron-requiring mRNA transcripts to facilitate degradation and conserve iron. The only mechanism for cellular iron export is ferroportin, which is inhibited by hepcidin. Abbreviations: ACO1, aconitase 1; ARE, AU-rich element; DMT1, divalent metal transporter 1; IRE, iron responsive element; IRP, iron regulatory protein; FPN1, ferroportin-1; FTH, ferritin heavy chain; FTL, ferritin light chain; LTCC, L-type calcium channel; NDUSF1, NADH:ubiquinone oxidoreductase core subunit S1; TF, transferrin; TFR1, transferrin receptor 1; TFR2β, transferrin receptor 2β; TTCC, T-type calcium channel; TTP, tristetraprolin; UQCRFS1, ubiquinol-cytochrome c reductase, rieske iron-sulfur polypeptide 1; UTR, untranslated region; ZIP8, zinc transporter 8; ZIP14, zinc transporter 14. (Figure credit: Sceyence Studios)

Cellular iron import

Transferrin-bound iron is imported into the cell via TFR1/2. Mice lacking cardiomyocyte-specific Tfr1 develop a severe cardiomyopathy due to dysregulated Fe/S cluster biogenesis and mitochondrial dysfunction from cardiac ID and die in the second week of life, demonstrating the crucial role for Tfr1 in maintaining cardiomyocyte homeostasis.³³ In addition to TFR1, the TFR2β isoform is also expressed in the heart and controls ferroportin transcriptional activity. Knockdown of Tfr2β protects the rodent heart from ischemia/reperfusion injury, thought to be due to an increase in cardiomyocyte antioxidative protection.³⁴

Non-transferrin bound iron can enter cardiomyocytes through DMT1, L-type and T-type calcium channels, and metal transporters. Global Dmt1 knockout mice die within 7 days of birth, demonstrating the importance of this cellular import pathway.³⁵ L-type voltage-gated calcium channels are expressed in the sinoatrial node and atrial cardiomyocytes. Under iron overload conditions, Fe²⁺ competes with Ca²⁺ for entry into cardiomyocytes via L-type calcium channels.³⁶ While T-type calcium channel expression is absent in adult human cardiomyocytes under physiological conditions, it is expressed in the embryonic heart and induced in adult cardiomyocytes in models of myocardial infarction, hypertrophy, and iron overload.³⁷ The solute carrier family 39 member 13 (SLC39A14, also known as ZIP14) has also been identified as a major non-transferrin bound iron transporter.³⁸

Cellular iron storage

Normally, cellular levels of labile iron are extremely low to minimize potential ROS formation. All cell types in the heart, particularly cardiomyocytes, are susceptible to ROS-induced damage. Excess iron is stored within the cytoplasm by complexing with ferritin, facilitated by poly-(rC)-binding proteins acting as chaperons (PCBP).³⁹ Ferritin levels are highly correlated with total cellular iron content. Ferritin-bound iron can be stored or degraded to become available for enzymatic reactions. Additionally, iron-bound ferritin is degraded by nuclear receptor coactivator 4 (NCOA4)-mediated autophagy in a process known as ferritinophagy, leading to degradation of lysosomal ferritin and release of its iron content.⁴⁰

Cardiac-specific deletion of ferritin heavy chain (Fth1) leads to iron dysregulation, increased cardiac oxidative stress, and increased susceptibility to iron overload-induced cardiac injury.⁴¹ Meanwhile, cardiac deletion of Ncoa4 in mice subjected to pressure overload improves cardiac function and attenuates ferritinophagy-mediated ferritin degradation.⁴⁰

Cellular iron regulation – iron regulatory proteins

The major regulators of cellular iron homeostasis are iron regulatory proteins (IRPs). IRP1/2 are mRNA-binding proteins that post-transcriptionally regulate cellular iron metabolism by binding to iron-responsive elements (IREs) in target mRNAs, leading to changes in gene expression. IREs are highly conserved hairpin structures of mRNAs located in the 5’- and 3’- untranslated regions (UTR) and serve as binding sites for IRPs.⁴²

IRP1 is a bifunctional protein depending on its binding to Fe/S clusters. When IRP1 is attached to Fe/S clusters, it functions as a cytosolic aconitase and cannot bind to IREs on target mRNA transcripts. However, in the setting of low cellular iron levels, Fe/S clusters dissociate from IRP1 and the apo-IRP1 can bind to IREs.⁴³ Meanwhile, IRP2 is constitutively active. However, in the setting of normal cellular iron levels, IRP2 is ubiquinated and proteolytically degraded by the FBXL5 protein.⁴⁴ Thus, in iron-replete cellular conditions, IRP1 maintains its Fe/S cluster and IRP2 is degraded, resulting in IRP system inhibition.

When IRPs bind to IREs in the 3’-UTR of target transcripts (i.e., TFR1), mRNA expression is stabilized leading to increased translation. However, IRPs binding to IREs in the 5’-UTR of target transcripts (i.e., ferroportin) lead to mRNA destabilization, steric blockade of ribosomal entry, and reduced translation.⁴⁵ Thus, in iron-deficient cellular conditions, IRPs stabilize the messenger RNA (mRNA) of TFR1 and DMT1 to increase iron import and inhibit mRNA translation of ferroportin to reduce iron export. Additional proteins regulated by the IRP/IRE system include DMT1, ferroportin, 5-aminolevulinic acid synthase 2 (ALAS2; heme biosynthesis), hypoxia inducible factor 2 alpha (HIF2α).⁴⁶ The IRPs are important for cardiomyocyte homeostasis, and loss of cardiac IRPs is associated with reduced systolic function, exacerbated heart failure (HF) after myocardial infarction, and impaired mitochondrial ability to generate energy for cardiomyocytes.⁴⁷

Cellular iron regulation – tristetraprolin

In the setting of severe prolonged ID, a secondary iron regulatory pathway is activated to ration and conserve iron. ID induces tristetraprolin (TTP) expression, which binds to AU-rich elements (AREs) in the TFR1 mRNA transcript and promotes transcript degradation via the CCR4-NOT transcription complex subunit 1 (CNOT1) deadenylase complex.^{48, 49} In addition to its binding to TFR1, TTP also binds and promotes the degradation of non-essential mRNA transcripts encoding Fe/S-containing proteins in the mitochondrial electron transport chain. Thus, under ID conditions, TTP conserves iron use to only essential cardiac proteins. Consistent with the essential role for TTP in regulating iron homeostasis, mice with Ttp deletion develop cardiac dysfunction in response to ID and human cultured cells with TTP deletion have increased cell death in ID conditions.⁵⁰

Cellular iron regulation – hepcidin

The heart has the second highest expression of hepcidin in the body. Systemic hepcidin produced by the liver inhibits ferroportin-mediated cellular iron export, and a similar role for cardiac hepcidin exists for cardiomyocyte iron efflux.^{51, 52} While cardiac hepcidin reduces cardiomyocyte iron export through modulation of ferroportin, the precise mechanistic regulation of cardiac hepcidin on cellular iron homeostasis remains to be elucidated.

Cardiomyocyte-specific deletion of hepcidin in mice leads to shortened lifespan, systolic dysfunction, and cardiac hypertrophy. At the cardiomyocyte level, hepcidin deletion reduces mitochondrial complex I and IV activity, leading to mitochondrial metabolic dysfunction.⁵³ Hepcidin has also been reported to have anti-apoptotic, anti-hypertrophy, and anti-fibrotic effects in models of HF.⁵⁴

Cellular iron export – ferroportin

The only known iron-exporting protein in mammals is ferroportin. This makes cardiomyocytes particularly vulnerable to iron overload. Mice lacking cardiomyocyte-specific Fpn1 develop a fatal dilated cardiomyopathy due to toxic levels of cardiac iron accumulation.⁵⁵ Interestingly, sv129 mice with cardiac and skeletal muscle Fpn1 deletion driven by the muscle creatine kinase (MCK) Cre allele show normal cardiac phenotype and iron homeostasis, suggesting there may be differences in phenotype based on mouse strain, Cre driver, and the exact Fpn1 deletion.⁵⁶

Mitochondrial iron homeostasis

Mitochondrial iron import in cardiomyocytes is regulated via mitoferrin 2 (MFRN2) and the mitochondrial calcium uniporter (MCU).⁵⁷ Mitochondria are the major site of heme and Fe/S cluster biosynthesis, both of which require iron. Because Fe/S clusters are critical components of the mitochondrial complexes I-V, mitochondrial iron is essential for normal functioning of the respiratory chain.⁵⁸

In the mitochondria, iron can also be stored in the form of mitochondrial ferritin. Mitochondria also contain mitochondrial ferritin (FTMT) to sequester mitochondrial free iron and minimize ROS production.⁵⁹ FTMT is not regulated by IRPs. Mutations in FTMT lead to mitochondrial iron overload and cytoplasmic ID, demonstrating that FTMT may shuttle iron to the cytoplasm in addition to mitochondrial iron storage.⁶⁰

While the mechanism of mitochondrial iron export is not clear, it is likely regulated by ATP-binding cassette subfamily B (ABCB) proteins. Ablation of Abcb7 in animal and cell models is associated with iron overload, mitochondrial oxidative stress, and impaired mitochondrial function in cardiac tissues and cardiomyoblasts.⁶¹ Deletion of Abcb8 in mice leads to mitochondrial iron overload, ROS production, impaired cytosolic maturation of Fe/S proteins, and cardiomyopathy.⁶² In addition to ABCB proteins, it is postulated that mitochondrial iron export may also occur in the form of Fe/S clusters or heme conjugated to glutathione.⁶³

Cellular and systemic iron deficiency

The function of the cardiovascular system, particularly the heart, is strongly linked to mitochondrial function which relies on sufficient iron levels. Clinically, the standard tests used for the diagnosis of ID include serum ferritin, serum iron, transferrin, and transferrin saturation (TSAT). However, the interpretation of serum ferritin can be challenging because its expression is significantly influenced by many factors, including inflammation, infection, and malignancy.⁶⁴ Ferritin may become falsely elevated in diseases associated with low-grade inflammation or oxidative stress, including several CVD. Similarly, functional ID may be associated with ‘normal’ iron indices.⁶⁵ Additionally, systemic ID does not necessarily correlate with ID at the cellular level in cardiovascular tissues.⁶⁶

Considerations in defining iron deficiency in cardiovascular disease

A major challenge in the diagnosis of ID in patients with CVD is the lack of an accurate and precise definition. The American Heart Association (AHA) and European Society for Cardiology (ESC) define ID in patients with HF as ferritin <100ug/L (absolute ID), or 100–300 ug/L with a TSAT <20% (functional ID) (Table 2).^67–69 This definition of ID was extrapolated from patients with chronic kidney disease (CKD) and subsequently used in early pilot studies using intravenous (IV) iron in HF.⁷⁰

Table 2.

Current guideline recommendations for intravenous iron in heart failure.

Organization	Year	Diagnosis of Iron Deficiency	Recommendation	Class/Level of Evidence
European Society of Cardiology (ESC)	2021	Ferritin <100 ng/mL (absolute), or ferritin 100–300 ng/mL if TSAT <20% (functional)	IV ferric carboxymaltose should be considered in symptomatic patients with HFrEF and ID to alleviate HF symptoms, improve exercise capacity and quality of life	IIa/A
American College of Cardiology (ACC); American Heart Association (AHA)	2022	Ferritin <100 ng/mL (absolute), or ferritin 100–300 ng/mL if TSAT <20% (functional)	In patients with HFrEF and ID with or without anemia, IV iron replacement is reasonable to improve functional status and quality of life	IIa/B

Abbreviations: HF, heart failure; HFrEF, heart failure with reduced ejection fraction; ID, iron deficiency; TSAT, transferrin saturation.

The current definition of ID in patients with HF has been criticized due to the lack of validation against the gold standard of bone marrow staining, and may not accurately reflect true ID. Among patients with HF undergoing coronary bypass surgery, iron staining performed on bone marrow aspirated from the sternum demonstrated poor positive predictive value for ID based on the ferritin-TSAT definition, and that the most sensitive and specific cutoffs for ID were TSAT <19.8% or serum iron <13 umol/L. Additionally, several patients with HF with reduced ejection fraction (HFrEF) and isolated hypoferritinaemia (ferritin <100 μg/mL with a TSAT >20%) labeled as ID by the AHA and ESC definitions were found to be iron sufficient when compared to bone marrow staining.⁷¹ Thus, serum ferritin may be falsely elevated in inflammatory states like HF and other CVD and not correlate with iron availability. The identification of patients with true ID is critical because unnecessary treatment may dilute any potential treatment benefits and put patients at unnecessary risk. Therefore, it is important to reassess the current diagnostic criteria for ID in CVD and to continue designing more specific criteria to accurately identify patients with ID, particularly in HF.

Novel biomarkers may allow for more accurate and reproducible measures of true ID in patients with HF and other CVD. Serum soluble transferrin receptor (sTfR) is a promising novel ID-related biomarker since circulating sTfR levels quantitatively reflect iron demand and the erythroid proliferation rate.⁷² ID defined as serum sTfR of ≥1.25 mg/L was accurate in identifying ID when compared to bone marrow staining in clinically stable patients with HF. Similarly, serum sTfR was significantly correlated with myocardial and mitochondrial iron status, which was not seen with ferritin, serum iron, or TSAT.⁷³ An alternative approach to defining ID is using serum hepcidin. Low serum hepcidin levels (<14.5ng/mL) reflect iron depletion. Among patients with a recent episode of acute HF, the combination of low serum hepcidin and elevated sTfR was strongly predictive of all-cause mortality at one year while ferritin <100 ng/mL or TSAT <20% was not predictive of outcomes.⁷⁴

Iron overload

Primary iron overload

Primary iron overload occurs when iron levels outpace the body’s intracellular storage capacity.⁷⁵ The excess iron can deposit into various organs leading to multi-organ dysfunction with a particular predilection for the liver, heart and endocrine glands.⁷⁶

Hemochromatosis

Hemochromatosis is a genetic disorder resulting from dysfunctional regulation of iron absorption in the intestines leading to progressive iron overload with iron deposition in various organs. In a retrospective cohort study of hospitalized patients with hemochromatosis, nearly 30% had CVD, namely arrhythmias, HF, and pulmonary hypertension.⁷⁷ Hemochromatosis is most commonly due to mutations in the homeostatic iron regulator (HFE) protein. HFE is highly expressed on the hepatocyte cell surface and competes with transferrin for TFR1 binding, thereby negatively regulating iron uptake. This competitive inhibition is overcome as iron-bound transferrin concentrations increase and displace HFE from TFR1, allowing for endocytosis of iron.⁷⁸ The most common HFE genetic alteration is an amino acid substitution from cysteine to tyrosine at the 282 position (C282Y) which inhibits HFE function.

Friedrich’s ataxia

Friedreich’s ataxia (FA) is an autosomal recessive mutation of the mitochondrial protein frataxin that presents early in childhood with progressive spinocerebellar ataxia, cardiac hypertrophy, liver dysfunction, kyphoscoliosis, dysarthria, hearing impairment, and diabetes.^79–81 The underlying mutation in FA is most commonly a trinucleotide repeat expansion of GAA within the first intron of the frataxin gene, with severity of disease associated with GAA repeat length. Deficiency of frataxin impairs Fe/S cluster formation which negatively affects many mitochondrial enzymes, resulting in dysfunctional mitochondrial respiration, increased oxidative damage, and iron overload.^82–84 Fe/S cluster biosynthesis is essential for mitochondrial and cytosolic aconitase, subunits of mitochondrial respiratory chain complexes (complex I, II, and III), ferrochelatase (involved in heme synthesis), and various metabolic enzymes.⁸⁵ The most common cardiac complication of FA is hypertrophic cardiomyopathy, which affects up to 85% of patients by early adulthood and is also the most common cause of death.⁸⁶ Despite the introduction of novel mouse models of FA, a reliable animal model that recapitulates the diverse clinical features of the disease seen in humans remains elusive.^{87, 88}

Secondary iron overload

Causes of secondary iron overload include iatrogenic administration of iron (dietary or parenteral), ineffective erythropoiesis due to hematologic diseases (i.e., thalassemia, myelodysplastic disease, and sickle cell disease), and repeated transfusion of packed red blood cells.^89–91 Various methods for iron chelation therapy are under investigation for treating secondary iron overload, including deferiprone and the use of nanochelators.^{92, 93}

Ferroptosis

Ferroptosis is an iron-dependent form of cell death characterized by increased oxidative stress and lipid peroxidation.^{94, 95} Mechanistically, ferroptosis integrates metabolic and ROS pathways.⁹⁶ Increases in the labile iron pool through increased iron import, heme degradation, and ferritinophagy lead to the generation of ROS via the Fenton reaction which promotes lipid peroxidation.^{97, 98} The common final pathway that ultimately mediates cell death in ferroptosis is the incorporation of lipid peroxides in the cell membrane which increases membrane permeability and promotes eventual rupture (Figure 3).^{99, 100} The anti-oxidative enzyme glutathione peroxidase 4 (Gpx4) is downregulated in both in vivo and in vitro models of myocardial infarction and leads to lipid peroxidation and ferroptosis^{101, 102}. Additionally, murine ischemia/reperfusion models have shown that reduction of Gpx4 plays a central role in cell death via activation of ferroptosis.¹⁰³

Figure 3. Overview of ferroptosis in cardiovascular disease.

Increased iron import, heme degradation, and ferritinophagy increase the labile iron pool which leads to the generation of reactive oxygen species via the Fenton reaction, ultimately promoting lipid peroxidation and ferroptosis. Abbreviations: SLC39A14, solute carrier family 39 member 13. (Figure credit: Sceyence Studios)

Iron metabolism dysregulation in cardiovascular disease

Iron homeostasis plays a critical role in cardiovascular physiology, and both ID and iron overload have detrimental effects on the cardiovascular system. In this section, we discuss the iron dysfunction that occurs in several CVD.

Heart failure with reduced ejection fraction (HFrEF)

HFrEF is associated with elevated inflammatory cytokine levels including interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α.¹⁰⁴ While it was originally believed that HFrEF patients have elevated levels of circulating hepcidin similar to other chronic inflammatory states, recent studies have demonstrated that hepcidin levels are reduced in advanced HFrEF.¹⁰⁵ Because elevated levels of serum hepcidin are necessary for functional ID, it is unlikely that the low serum hepcidin levels associated with the chronic inflammation of HFrEF promote functional ID. This suggests that HFrEF has an inflammatory profile distinct from other chronic inflammatory diseases characterized by high serum hepcidin, such as CKD, and may have a different iron regulatory response to inflammation compared to other chronic inflammatory conditions.¹⁰⁶

At the cellular level, iron homeostasis is dysregulated in failing myocardial tissue. While some studies of explanted failing human hearts undergoing cardiac transplantation show reduced mitochondrial iron levels, our group found increased mitochondrial iron and total cellular heme iron levels in failing human hearts.^107–109 Additionally, our group showed increased mitochondrial iron in mice after ischemia/reperfusion injury and in human hearts with ischemic heart disease.¹¹⁰ Thus, cellular and mitochondrial iron stores are deranged in failing human hearts, and the exact derangement may be dependent on the subtype of cardiomyopathy.

It is important to note that systemic iron status does not necessarily correlate with cellular iron status at the myocardial level. Reduced myocardial iron content may occur despite normal systemic iron stores. Conversely, cardiac mitochondrial iron overload may occur in HF patients despite systemic ID. While markers of systemic ID show varying association with mortality, myocardial ID as assessed by T2 cardiac MRI is a strong predictor of major cardiovascular events in patients with HFrEF.¹¹¹

Recent studies suggest that systemic iron levels are also very dynamic in HFrEF over time. While many patients with HFrEF develop ID, the rates of spontaneous resolution are also high. Approximately half of ambulatory patients with chronic HF and ID had natural resolution of ID over the course of a year.¹¹² Interestingly, the prevalence of ID also increases with worsening New York Heart Association (NYHA) functional class and in acutely decompensated HF. Although persistent ID is associated with higher mortality, there is no mortality difference between patients with HFrEF and resolution of ID compared to patients with HFrEF who never developed ID.¹¹³ This suggests that there is some natural fluctuation of systemic iron levels in HF, potentially as a compensatory response to the severity of clinical HFrEF. Thus, it remains unclear whether ID serves as a marker of HFrEF clinical severity or a causal driver of HFrEF progression. Longitudinal, mechanistic epidemiological and intervention studies are needed to investigate the contribution of various factors implicated in the pathophysiology of ID in HFrEF.

Heart failure with preserved ejection fraction (HFpEF)

There are few studies comparing circulating iron indices with mortality in patients with HFpEF, raising questions about the clinical significance of current ID definitions in HFpEF.¹¹⁴ In a cohort of HFrEF and HFpEF patients, the current ID definition of TSAT <20% was significantly associated with all-cause mortality in all patients with HF, but the definition of ferritin <100 ug/L was not associated with all-cause mortality in patients with either HFrEF or HFpEF. Meanwhile, ID defined by sTfR >1.76 mg/L was significantly associated with all-cause mortality in patients with HFpEF or HFrEF.¹¹⁵ Thus, ID definitions using novel biomarkers such as sTfR in lieu of ferritin may have important implications in determining which patients with HFrEF and HFpEF derive the most benefit from correction of ID. Given the emerging subtypes of HFpEF, it remains unclear if a particular subgroup of patients with HFpEF is at particular risk of systemic or cellular iron dysregulation.

Anthracycline-induced cardiomyopathy

Since the late 1960s, anthracyclines such as doxorubicin have been used to treat a wide variety of malignancies, including breast cancer and leukemia. While anthracyclines reduce cancer burden for many patients, their use is limited by the risk of cardiomyopathy.¹¹⁶ Although the mechanisms of doxorubicin-induced cardiomyopathy are complex, a growing body of evidence suggests iron dysregulation as a key driver. Anthracyclines preferentially accumulate in cardiac mitochondria due to their high affinity for the inner mitochondrial membrane protein cardiolipin. The molecular structure of anthracyclines promotes redox cycling between the quinone and semiquinone moieties, leading to accumulation of superoxide anions which can ultimately be converted to highly toxic hydroxyl radicals in the presence of iron, ultimately resulting in myocardial oxidative damage.¹¹⁷

Multiple in vitro and in vivo models of doxorubicin treatment demonstrate a consistent signature of mitochondrial iron accumulation, leading to increased oxidative stress. Our group found that doxorubicin suppresses the mitochondrial iron exporter ABC protein-B8 (ABCB8), leading to increased mitochondrial iron accumulation.^{62, 118} Several other models of systemic iron accumulation through either high-iron diet or genetic manipulation in mice also significantly increase the susceptibility to anthracycline-induced cardiotoxicity.^{119, 120}

Viral myocarditis

Dysregulated iron metabolism may be involved in the development of inflammatory viral heart disease. In animal models of acute and chronic viral myocarditis, histological staining and transmission electron microscopy revealed impaired mitochondrial iron metabolism and iron deposits within necrotic myocytes.¹²¹ At the systemic level, serum indices of iron homeostasis are deranged in humans with acute myocarditis suggesting dysregulated iron metabolism, which normalize within 6 weeks.¹²²

Coronary artery disease and atherosclerosis

When circulating iron levels exceed the carrying capacity of TFR, non-transferrin bound iron circulates throughout the body and can lead to vascular endothelial cell injury. In cultured vascular cells, excess non-transferrin bound iron promotes atherosclerosis and vascular dysfunction through ROS generation, apoptosis, and inflammatory cell recruitment.^{123, 124} Epidemiological studies also demonstrate an association between iron overload and atherosclerosis burden.¹²⁵

Cardiac ischemia/reperfusion injury

Although reperfusion of acutely ischemic myocardium is necessary to prevent myocardial infarction, reperfusion can paradoxically result in additional myocardial damage known as “reperfusion injury.”^{126, 127} Mitochondrial iron is increased in murine hearts subjected to ischemia/reperfusion injury, which stimulates the iron-dependent overproduction of ROS, leading to inhibition of respiratory chain enzymes and energy production.¹²⁸ Additionally, in patients presenting with ST-elevation myocardial infarction (STEMI) who were reperfused and developed intramyocardial hemorrhage, residual myocardial iron was associated with adverse left ventricular remodeling.¹²⁹

Arrhythmias

Chronic iron overload is associated with heart block and atrial fibrillation in both mice and humans.¹³⁰ The L-type calcium channel, voltage-gated sodium channels, and ryanodine sensitive calcium channels are affected by iron in isolated cardiomyocytes in animal models.¹³¹ Cellular iron can displace calcium from its intracellular binding sites which prevents calcium-dependent channel inactivation and prolongs the calcium influx that can disrupt cardiac conduction and induce arrhythmias.¹³⁰ Additionally, sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase 2a (SERCA2a) levels and activity are reduced in iron overload disease, which promote arrhythmias and abnormal cardiac relaxation.¹³²

Valvular disease

Valvular calcification can be precipitated by oxidative stress, including iron-mediated ROS.¹³³ The aortic valve is the most commonly affected heart valve, with iron accumulation seen in human calcified aortic valves.¹³⁴ In histologic samples, heme and iron colocalized with valvular microhemorrhages and identified a macrophage-induced mineralization of valvular interstitial cells.¹³⁵ In valvular interstitial cells from patients with aortic stenosis, non-heme-bound intra-valvular iron was associated with extracellular matrix remodeling and calcification.¹³⁶

Pulmonary hypertension

Nearly 40% of patients with pulmonary artery hypertension (PAH) are ID, however a clear mechanism behind PAH and ID remains unclear.¹³⁷ Bone morphogenetic protein receptor type 2 (BMPR2) mutations affect pathways involved in iron regulation through hepcidin production.¹³⁸ In addition, mice expressing ferroportin with a hepcidin resistance mutation (C326Y) develop PAH with increased levels of endothelin-1, which is a known mediator of pulmonary vascular smooth muscle constriction.¹³⁹

Stroke

The brain is at particular risk for ischemic damage due to ID given its high metabolic activity. ID in the brain leads to neuronal dysfunction, impaired synaptogenesis, reduced neurotransmitter production and aberrations in myelination.¹⁴⁰ Moreover, ischemia/reperfusion injury in the brain can lead to an iron overload state that induces neuronal cell ferroptosis pathways.¹⁴¹

Targeting iron dysregulation in cardiovascular disease

Maintaining iron homeostasis is essential for normal cardiovascular function and correcting systemic and cellular iron dysregulation is a critical therapeutic approach to CVD. Here, we summarize the current strategies to manage iron dysregulation in CVD.

Iron supplementation in heart failure

In the past decade, the effects of iron supplementation have been evaluated in patients with HFrEF. Here, we review the major trials of IV iron in patients with HFrEF, potential risks associated with IV iron, and strategies for alternative routes of iron administration (Table 3).

Table 3.

Major clinical trials of iron supplementation therapy in heart failure with reduced ejection fraction.

Trial	Year	Design	Patients	Treatment	Duration	Inclusion criteria	Change in primary endpoint
Toblli et al.	2007	Double-blind RCT	40	IV iron sucrose weekly for 5 weeks	24 weeks	LV EF ≤45% NYHA II-IV CrCl <90 Hgb <12.5g/dL (men) or <11.5 g/dL (women)	Decrease in NT-proBNP and CRP
FERRIC-HF	2008	Observer-blind RCT	35	IV iron sucrose weekly until ferritin >500, then monthly	16 weeks	LV EF ≤45% NYHA II-III	Improvement in pVO2 only in pre-specified group with baseline Hgb <12.5 g/dL
FAIR-HF	2009	Double-blind RCT	459	IV FCM weekly until repletion, then monthly	24 weeks	LV EV ≤45% (if NYHA III) LV EV ≤40% (if NYHA II) Hgb 9.5–13.5 g/dL	Improvements in PGA and NYHA class
IRON-5 HF	2013	Double-blind RCT	54	Oral ferrous sulfate 200mg TID	90 days	LV EF <50% NYHA II-III	Change in 6MWT – terminated early
CONFIRM-HF	2015	Double-blind RCT	304	IV FCM at baseline and week 6, then at weeks 12, 24, and 36 if ID	52 weeks	LV EF ≤45% NYHA II-III	Improvement in 6MWT at 24 weeks
IRONOUT-HF	2017	Double-blind RCT	225	Oral iron polysaccharide 150mg BID	16 weeks	LV EF ≤40% NYHA II-IV Hgb 9–14 g/dL (men) or 9–13.5 g/dL (women)	No improvement in pVO2
EFFECT-HF	2017	Open-label RCT	172	IV FCM at baseline and weeks 6 and 12	24 weeks	LV EF ≤45%	No improvement in pVO2*
AFFIRM-HF	2020	Double-blind RCT	1,132	IV FCM at baseline and week 6, then at weeks 12 and 24 if ID	52 weeks	Patients admitted for ADHF with LV EF <50%	No improvement in composite endpoint of recurrent HF admissions and CV death

Abbreviations: 6MWT, 6-minute walk test; ADHF, acute decompensated heart failure; CrCl, creatinine clearance; CRP, C-reactive protein; Hgb, CV, cardiovascular; hemoglobin; FCM, ferric carboxymaltose; HF, heart failure; ID, iron deficient; IV, intravenous; LV EF, left ventricular ejection fraction; NT-proBNP, N-terminal (NT)-pro hormone BNP; NYHA, New York Heart Association; pVO2, peak oxygen uptake; PGA, patient global assessment; RCT, randomized-controlled trial.

^*No improvement in pVO2 after removing 4 patient deaths assigned a pVO2 value of zero in the placebo group.

Major trials of IV iron in HFrEF

FAIR-HF was the first trial of 459 ambulatory symptomatic HFrEF patients (NYHA Class II-III) with ID randomized to IV ferric carboxymaltose (FCM) or placebo (normal saline). The definition of ID used in FAIR-HF was adopted from the CKD guidelines, despite the very distinct mechanisms of disease in CKD and HF, as described above.¹⁴² After 24 weeks, IV FCM therapy was associated with improvement in NYHA functional class and patient-measured disease burden, including 6-minute walk test (6MWT) and quality of life.¹⁴³

CONFIRM-HF was the second major trial that randomized 304 patients with ambulatory symptomatic HFrEF (NYHA Class II-III) and ID to IV FCM or placebo. After 52 weeks, IV FCM replicated the improvements in NYHA class, 6MWT, and quality of life seen in FAIR-HF.¹⁴⁴ The results of the FAIR-HF and CONFIRM-HF trials prompted the 2016 European Society of Cardiology and 2017 AHA and ACC guidelines to recommend IV iron supplementation in symptomatic HFrEF patients with concomitant ID.^{67, 68}

In 2017, the EFFECT-HF trial randomized 174 participants with ambulatory symptomatic HFrEF (NYHA Class II-III) and ID to either IV FCM or standard of care in an open-label fashion. After 24 weeks, the primary endpoint of change in peak oxygen uptake (VO₂) was decreased by 1.19±0.38 ml/kg/min in the standard of care group but was maintained in the FCM group. However, there were four deaths in the standard of care group which were assigned a peak VO₂ value of 0, while there were no deaths in the FCM group. In an analysis with the four deaths removed, there was no significant difference in the primary endpoint of peak VO₂ between individuals who received IV FCM or standard of care.¹⁴⁵

It is important to note that the FAIR-HF, CONFIRM-HF, and EFFECT-HF trials were all sponsored by the manufacturer of FCM, Vifor Pharma (Zurich, Switzerland). In the FAIR-HF and CONFIRM-HF trials, representatives from the pharmaceutical sponsor were involved in the trial design, implementation, and oversight. Also, FCM is the only recommended form of IV iron in patients with HFrEF by the European guidelines.⁶⁸

In a meta-analysis of IV iron supplementation in HF patients, iron administration was associated with improved quality of life and reduced hospitalization, but no change in all-cause mortality.¹⁴⁶ Similarly, the recent AFFIRM-AHF study demonstrated that IV FCM did not improve mortality among patients with acute HF and ID.¹⁴⁷ In a pre-specified analysis of AFFIRM-AHF, IV FCM modestly improved subjective health status in patients with HFrEF, but this became non-significant after 24 weeks of IV FCM initiation.^{148, 149}

Of note, alternative formulations of IV iron have less evidence for improving subjective endpoints. In the United States, the most common formulation of IV iron available in major hospitals is iron sucrose. The FERRIC-HF trial randomized 35 patients with symptomatic HFrEF (NYHA Class II-III) and ID to iron sucrose or no iron therapy. After 18 weeks of treatment, IV iron sucrose improved peak VO₂ and NYHA class, however these changes were only significant in the pre-specificized subgroup of patients with anemia (hemoglobin <12.5 g/dL).¹⁵⁰

In summary, the use of IV iron is associated with symptomatic benefits in patients with HFrEF and ID. HF is a chronic progressive disease, and improvements in symptoms and patient-reported outcomes are important. However, therapies that only improve symptoms require significant efficacy and safety evidence before widespread use, particularly if there are potential safety risks associated with those therapies.

Potential risks associated with IV iron

IV iron supplementation rapidly corrects systemic iron levels by bypassing gastrointestinal absorption. By circumventing physiological pathways of iron absorption and homeostasis, IV iron delivers large amounts of iron to all tissues, including those of the cardiovascular system. The excess amount of systemic iron overwhelms the iron-carrying capacity of transferrin, leading to accumulation of non-transferrin bound iron and highly reactive labile iron pools within tissues and cells.¹⁵¹

At the level of the cardiomyocyte, labile iron can react with hydrogen peroxide and generate toxic hydroxyl radicals, ultimately leading to lipid peroxidation and ferroptosis which can directly contribute to cardiomyopathy. In apolipoprotein E-deficient mice, excess iron induced a pro-inflammatory state in endothelial cells, exacerbating the progression of atherosclerosis.¹⁵² Among healthy human volunteers, IV iron administration led to significant increases in circulating non-transferrin bound iron and was associated with transient endothelial dysfunction and biomarkers of oxidative stress.¹⁵³

The use of IV iron is also associated with elevated levels of the biologically active form of fibroblast growth factor (FGF)-23. This hormone is important for phosphate homeostasis, and elevated FGF-23 levels reduce renal phosphate absorption and lead to hypophosphatemia, bone resorption, and in severe cases, osteomalacia. In humans, the use of IV FCM, but not other IV iron formulations, has been associated with hypophosphatemia and osteomalacia.¹⁵⁴ Additionally, elevated FGF-23 levels have been associated with left ventricular hypertrophy and all-cause mortality in patients with CKD.¹⁵⁵

There is also emerging evidence for an association between excessive iron levels and neoplastic initiation and proliferation. In animal models, the administration of excessive amounts of IV iron increases the development of primary neoplasms at sites of iron deposition, particularly adenocarcinomas, colorectal tumors, hepatomas, mesotheliomas, renal tubular cell carcinomas, and sarcomas.¹⁵⁶ In patients with hemochromatosis, elevations in hepatic iron are associated with a 200-fold increase in the risk of hepatic carcinoma. Blood transfusions can also result in high circulating iron levels.¹⁵⁷ In multiple studies, blood transfusion recipients had an increased future risk of developing non-Hodgkin’s lymphoma compared to frequency-matched controls.^{158, 159}

Strategies for alternative routes of iron supplementation

Oral iron supplementation is most common therapy for ID. Oral iron is inexpensive, widely available, and safe. The most common formulations of oral iron include ferrous fumarate, ferrous gluconate, and ferrous sulfate. The use of oral iron supplementation is limited by gastrointestinal side effects, including metallic taste, nausea, bloating, and constipation. However, oral iron preparations differ in their tolerability and efficacy in correcting ID, and newer oral iron formulations, such as ferric maltol, are better tolerated.¹⁴²

There have been no completed trials evaluating oral and IV iron in a head-to-head fashion in HF. The IRON-HF study attempted to compare oral ferrous sulphate versus IV iron sucrose supplementation in patients with HFrEF and ID, but the study was terminated early due to prolonged recruitment and lack of funding.¹⁶⁰

In the IRONOUT-HF trial, 16 weeks of treatment with oral iron polysaccharide (150mg twice daily) improved ferritin and TSAT levels compared with placebo in HFrEF patients with ID, but had no significant effect on the primary endpoint of change in VO₂ max or 6MWT.¹⁶¹ However, there are several caveats in interpreting the results of the IRONOUT-HF trial. First, the duration of treatment may have been insufficient to observe a change in the primary outcome of change in VO₂ max. Second, the cumulative amount of oral iron administered during the study period exceeded the recommended IV dosage to correct ID by more than 15-fold, and the dosing strategy may have important implications for iron absorption. Finally, patients with reduced hepcidin at baseline (<6.6 ng/ml) derived the most benefit from oral iron supplementation, suggesting that HF patients with more specific biomarkers of true ID may benefit from oral iron supplementation

Recently, a small non-randomized open-label study showed that low-dose oral sucrosomial iron improved iron indices, exercise capacity, and quality of life in patients with HFrEF and ID.¹⁶² These promising preliminary results prompted two ongoing randomized controlled trials comparing oral sucrosomial iron with IV FCM in patients with HFpEF (PREFER-HF, NCT03833336) and HFrEF (IVOFER-HF, EudraCT 2017-005053-37). These trials will provide insight regarding the utility of oral sucrosomial iron as stand-alone or adjuvant therapy to IV iron supplementation in patients with HF.

The dosing amount and schedule is critically important for optimal absorption of oral iron. Traditional multi-daily high-dose oral iron supplementation rapidly increases hepcidin levels and reduces iron bioavailability. Recent evidence suggests that lower daily doses of 15–20mg elemental iron is efficacious in repleting systemic iron stores in elderly patients and pregnant women with ID.¹⁶³ Every-other-day oral iron supplementation in single doses was found to have fewer gastrointestinal side effects and reduced hepcidin levels compared to daily dosing.¹⁶⁴

Given the benefits of oral iron, patients with CVD and uncomplicated ID should be considered for oral iron formulations. Meanwhile, IV iron should be reserved for select symptomatic HFrEF patients with known GI malabsorption, IBD, heavy blood loss, moderate-to-severe CKD and end-stage renal disease (ESRD), or those who have previously not tolerated oral iron. In fact, the current FDA label for IV FCM indicates that this therapy for the treatment of ID in “adult patients who have intolerance to oral iron or have had an unsatisfactory response to oral iron [or who have] non-dialysis dependent CKD.”

Reducing iron through chelation therapy or dietary approaches

The human body has no mechanism for the removal of excess systemic or myocardial iron. Numerous studies have examined the role of iron chelators and dietary iron restriction in CVD with and without iron overload.^{165, 166}

Anthracycline-induced cardiomyopathy

In anthracycline-induced cardiomyopathy, the iron chelator dexrazoxane is approved for cardioprotection in patients with a cumulative doxorubicin dose >350 mg/m² or >300 mg/m² in the United States and Europe, respectively.¹¹⁷ The findings of increased cardiomyocyte iron specifically in the mitochondria suggest that cell-permeable iron chelators which preferentially reduce cardiac mitochondrial iron and mitochondria-directed antioxidants may be novel therapeutic targets to reverse anthracycline cardiotoxicity.¹¹⁸ Several groups have reported that iron chelation and ferroptosis inhibitors reduce anthracycline cardiotoxicity by improving mitochondrial function.^{167, 168}

Ischemia/reperfusion injury

Genetic or pharmacological reduction of mitochondrial iron prevents both ischemia/reperfusion-mediated free radical overproduction and cardiac injury. Mitochondria-specific overexpression of the anti-ferroptosis Gpx4 was also found to be cardioprotective in response to ischemia/reperfusion injury.¹⁶⁹ We have previously shown that reduction in baseline cardiac mitochondrial iron levels, either by genetic overexpression of the mitochondrial iron export protein ATP-binding cassette transporter protein B8 (Abcb8) or pharmacologically with the cell-permeable iron chelator 2,2’-bipyridyl, protected against ischemia/reperfusion-induced cardiomyopathy in a mouse model.^{62, 110} Thus, targeted therapies aimed at mitochondria-specific iron chelation, ferroptosis, or the reduction oxidative stress may improve cardiac function after ischemia/reperfusion injury.

Coronary artery disease

In the TACT trial, patients with stable coronary artery disease at least 6 months after MI were randomized to the divalent anion chelator ethylenediamine tetraacetic acid (EDTA; in addition to ascorbate, B vitamins, electrolytes, procaine, and heparin solution) or placebo. Iron chelation with EDTA was associated with a significant reduction in the composite outcome of mortality, myocardial infarction, stroke, coronary artery revascularization, and hospitalization due to angina. Patients with diabetes and anterior myocardial infarction history derived the most benefit from chelation therapy.¹⁷⁰ The promising results of the TACT trial has prompted the larger ongoing TACT-II trial to confirm these results in diabetic patients with prior myocardial infarction. Because of its affinity for calcium, EDTA may promote hypocalcemia and renal injury. However, there was no significant difference in serious adverse events in the TACT trial.

In patients with coronary artery disease, the iron chelator deferoxamine (DFO) improved endothelial-mediated vasodilation.¹⁶⁵ Additionally, in patients undergoing coronary artery bypass graft surgery, infusion with DFO during cardioplegia was associated with reduction in oxidative stress and improvement in cardiac function for up to 12 months.¹⁷¹ Adverse effects of DFO therapy include deafness, visual changes, and infusion site reactions, however newer iron chelators such as deferasirox have improved safety profiles.

Future directions

In addition to iron chelation and supplementation, several other strategies have been employed to target iron homeostasis, especially in the field of cancer which may also be relevant to CVD. These potential therapies can be divided into three molecular targets: 1) PDH-HIF axis, 2) hepcidin-ferroportin axis, and 3) ferroptosis inhibitors.

Modulation of the prolyl-4-hydroxylase/hypoxia inducible factor (PDH/HIF) axis

Sequestered iron can be mobilized by downregulating the synthesis or function of hepcidin, or by stabilizing hypoxia inducible factors (HIFs) through prolyl-4-hydroxylase (PHD) inhibition. HIF2α leads to an increase in cellular iron content, and HIFs have been shown to play a key role in cancer growth and CVD.¹⁷²

Many HIF-stabilizing compounds (vadadustat, daprodustat, roxadustat) have completed or are in late-stage clinical trials for CKD patients with anemia.¹⁷³ A recent meta-analysis demonstrated that PHD inhibitors increase circulating hemoglobin, serum transferrin, and intestinal iron absorption while reducing circulating hepcidin levels independent of inflammation.¹⁷⁴ Additional benefits of PHD/HIF pathway modulators include oral administration, reduction in cholesterol levels, and improvements in blood pressure.¹⁷⁵

Given the role of HIFs in the regulation of proteins involved in energy metabolism, angiogenesis, and cellular growth, HIF stabilization may promote cancer growth. Thus, long-term clinical trials are needed to better understand the effects of systemic HIF pathway activation, particularly in the setting of CVD. However, activation of HIF signaling may allow for a physiological approach to re-establish iron homeostasis in patients with functional ID and CVD.

Modulation of the hepcidin-ferroportin axis

In preclinical and early clinical trials, targeting hepcidin directly (LY2787106, lexaptepid pegol, anticalins) or indirectly via the targeting of inflammation (ziltivekimab) or bone morphogenic protein (BMP)-6 (LY3113593) increases intestinal iron absorption and mobilization.¹⁷⁶ Additionally, ferroportin antibodies (LY2928057) have been utilized to block the interaction between hepcidin and ferroportin and increase the mobilization of sequestered iron.¹⁷⁷ Antibodies targeted against hepcidin or hemojuvelin (to inhibit hepcidin synthesis) may also be effective in reducing cellular iron.¹⁷⁸

Because the hepcidin-ferroportin axis regulates intestinal iron absorption and mobilization of sequestered iron, therapeutic agents targeting this axis may be particularly effective in functional ID but less so for absolute ID.

Ferroptosis

Excessive ferroptosis has been linked to several CVD, including hemochromatosis, doxorubicin-induced cardiotoxicity, ischemia/reperfusion injury, atherosclerosis, and arrhythmias.¹⁷⁹ Ferrostatin-1 (fer-1) is a ferroptosis inhibitor that acts as an antioxidant within the lipid bilayer to prevent lipid oxidation, and has been shown to attenuate hydrogen peroxide-induced injury in primary cardiomyocytes and reduce ischemic injury in a mouse heart transplantation model of ischemia/reperfusion injury. While ferrostatin-1 strongly inhibits ferroptosis in vitro, its low stability in plasma leads to reduced in vivo function which limits clinical applicability.^{180, 181} Future research is also needed to develop reliable biomarkers specific to ferroptosis in CVD to determine therapeutic efficacy in clinical trials. Recently, the sodium-glucose cotransporter 2 (SGLT2) inhibitor canagliflozin was found to decrease ferroptosis-associated gene expression in a rat model of HFpEF.^{182, 183}

Conclusions

Iron is essential for cardiovascular processes, including bioenergetics, electrical activity, and programmed cell death. Thus, the human cardiovascular system has evolved to conserve iron with multiple redundant iron import proteins and regulatory mechanisms to maintain iron homeostasis. Meanwhile, humans have only one cellular iron export mechanism which makes the cardiovascular system, particularly the heart, highly susceptible to iron overload.

Despite more than three decades of research, the biological mechanisms underlying ID and iron overload-mediated cardiotoxicity at the cellular level are not completely understood. Mechanistic studies show that even baseline cardiac mitochondrial iron levels participate in cardiovascular dysfunction, and precise modulation of cellular iron levels is essential for physiological cardiovascular functions. It also remains to be seen whether the dysregulated systemic iron homeostasis in CVD is a driver of disease, a compensatory mechanism to maintain cellular iron levels, or both.

The emergence of various iron-modulating therapeutics demonstrates the importance of maintaining iron homeostasis in the cardiovascular system. However, many iron therapeutics, such as IV iron supplementation, can have adverse effects and future research is needed in identifying groups of individuals with true ID who would most benefit from treatment. Additional research is also needed in understanding the underlying mechanisms of ID-mediated damage at the cellular level in cardiovascular tissues in order to develop novel therapeutic approaches.

Future studies of iron dysregulation in CVD will focus on understanding the role of ID and iron overload on cardiovascular health, revealing the underlying causes of ID and iron overload for the onset or progression of CVD, and identifying the pathways downstream of ID or iron overload that contribute to cardiovascular dysfunction which may ultimately serve as potential therapeutic targets.