key: cord-0005991-f5ewl2vc
authors: Oudit, Gavin Y.; Trivieri, Maria G.; Khaper, Neelam; Liu, Peter P.; Backx, Peter H.
title: Role of L-type Ca2+ channels in iron transport and iron-overload cardiomyopathy
date: 2006-04-08
journal: J Mol Med (Berl)
DOI: 10.1007/s00109-005-0029-x
sha: 6c5a34f395f84182f69fbf12b2533994da674185
doc_id: 5991
cord_uid: f5ewl2vc

Excessive body iron or iron overload occurs under conditions such as primary (hereditary) hemochromatosis and secondary iron overload (hemosiderosis), which are reaching epidemic levels worldwide. Primary hemochromatosis is the most common genetic disorder with an allele frequency greater than 10% in individuals of European ancestry, while hemosiderosis is less common but associated with a much higher morbidity and mortality. Iron overload leads to iron deposition in many tissues especially the liver, brain, heart and endocrine tissues. Elevated cardiac iron leads to diastolic dysfunction, arrhythmias and dilated cardiomyopathy, and is the primary determinant of survival in patients with secondary iron overload as well as a leading cause of morbidity and mortality in primary hemochromatosis patients. In addition, iron-induced cardiac injury plays a role in acute iron toxicosis (iron poisoning), myocardial ischemia–reperfusion injury, Friedreich ataxia and neurodegenerative diseases. Patients with iron overload also routinely suffer from a range of endocrinopathies, including diabetes mellitus and anterior pituitary dysfunction. Despite clear connections between elevated iron and clinical disease, iron transport remains poorly understood. While low-capacity divalent metal and transferrin-bound transporters are critical under normal physiological conditions, L-type Ca(2+) channels (LTCC) are high-capacity pathways of ferrous iron (Fe(2+)) uptake into cardiomyocytes especially under iron overload conditions. Fe(2+) uptake through L-type Ca(2+) channels may also be crucial in other excitable cells such as pancreatic beta cells, anterior pituitary cells and neurons. Consequently, LTCC blockers represent a potential new therapy to reduce the toxic effects of excess iron.

Iron is a transition metal that is essential for many biochemical, metabolic and biological processes. Iron is a component of hemoglobin, myoglobin, mitochondrial electron transport chain enzymes, cytochrome p450 system and many other proteins [1] [2] [3] . The biological importance of iron, as well as its toxicity, results from rapid oxidationreduction cycling between ferric (Fe 3+ ) and ferrous (Fe 2+ ) states at physiological temperatures. Consequently, iron levels are precisely regulated under normal physiological conditions [1, 2] via several intricate feedback mechanisms involving transporters, iron-binding proteins and receptors ( Fig. 1 ) [2] . Under several clinical conditions including primary hemochromatosis and secondary iron overload, iron metabolism is perturbed, which, combined with modifying environmental factors, leads to increased morbidity and mortality [4] [5] [6] [7] . As a result of reductions in childhood mortality and increased use of blood transfusions, disease caused by iron overload is rapidly increasing in worldwide prevalence [4] [5] [6] [7] .

Under iron-overload conditions, iron in the circulation typically exceeds the capacity of iron binding by serum transferrin, leading to the appearance of highly reactive, non-transferrin-bound iron (NTBI) [8] [9] [10] . Uptake of NTBI into cells bypasses the normal negative-feedback mechanisms regulating cellular iron uptake and metabolism [2, [9] [10] [11] [12] [13] [14] . Excess uptake of NTBI combined with the lack of an effective iron excretory pathway leads to the expansion of the labile intracellular iron pool (LIP) as well as the formation of highly reactive oxygen free radicals, causing peroxidation of membrane lipids and oxidative damage to cellular proteins [1, 2, 15] . Iron-mediated cellular damage plays a key pathophysiological role in multiple disorders including acute iron toxicosis [16, 17] , iron-overload cardiomyopathy [18, 19] , Friedreich-ataxiaassociated cardiomyopathy [20] , iron-overload endocrinopathies [21] [22] [23] [24] and bone disease [25] [26] [27] , myocardial ischemia-reperfusion injury [28, 29] , atherosclerosis [30, 31] and neurodegenerative diseases [32, 33] . In this review, we will discuss the clinical disorders associated with iron overload, as well as the role of L-type Ca 2+ channels (LTCC) in iron transport and pathophysiology of ironoverload cardiomyopathy and endocrinopathy.

Primary hemochromatosis Primary hemochromatosis (hereditary or idiopathic) is a common inherited disorder that presents as four distinct subtypes (see Table 1 ) [1, 7] . In this condition, excessive iron accumulation results primarily from increased gastrointestinal absorption of iron that coupled with modifying environmental factors leads to iron overload [1, 2, 7] . Type 1 primary hemochromatosis (classic hereditary hemochromatosis) is an autosomal recessive disorder linked to mutations of the HFE gene that is involved in controlling gastrointestinal absorption of iron. Mutations of HFE genes involve either tyrosine for cysteine substitutions at position 282 (C282Y) [1, 7] or substitution of aspartate for histidine at position 63 (H63D) [1, 7] . The C282Y mutation is primarily limited to individuals of northern European ancestry and has an allele frequency of about 10%, while the H63D mutation occurs at allele frequencies greater than 5% in Mediterranean/ Middle East regions and the Indian subcontinent [34] . There is a low and variable penetrance of the C282Y mutation with 0.5-1% of homozygotes developing frank clinical hemochromatosis [1, 7, 35] . Clinical effects of H63D mutations are generally limited to 1-2% of persons with compound heterozygosity for C282Y and H63D [7, 34] .

Type 2 primary hemochromatosis is also called juvenile hemochromatosis and shows an autosomal recessive inheritance pattern. Patients with this disorder generally present at younger ages with larger iron burdens and severe organ damage before age 30 years (Table 1) [7, 36] . Most patients with juvenile hemochromatosis have mutations in the HJV gene (formerly called HFE2) that encodes for hemojuvelin, a protein with a central role in iron metabolism (see below) [7, 36] . A rarer form of juvenile hemochromatosis is caused by inactivation of hepcidin, another key regulator of iron homeostasis whose expression is modulated by hemojuvelin (Table 1) [7] . Type 3 primary hemochromatosis is another autosomal recessive condition that is linked to mutations in the transferrin receptor, TfR2 [7, 37] . In contrast to the other types of hemochromatosis, type 4 primary hemochromatosis is inherited in an autosomal-dominant manner and is linked to altered function of the iron exporter ferroportin, as discussed below (Table 1 and Fig. 1) [7, 38, 39] . Next to mutations in HFE, mutations in ferroportin are the most common cause of hemochromatosis [7, 38, 39] .

In addition to inherited hemochromatosis, high dietary iron intake is also implicated in iron overload of sub-Saharan Africans, although a genetic component for this disease has also been implicated [40] . The presence of high serum ferritin (<200 μg/l) and normal transferrin saturation [41] is associated with NTBI and possible excess myocardial iron accumulation in some men without known genetic defects in iron metabolism.

Secondary iron overload Unlike primary hemochromatosis that generally involves mutations in proteins involved in iron transport and metabolism, secondary iron overload occurs in patients with hereditary anemias including αthalassemia [42] , β-thalassemia [5] and sickle cell anemia [43] . In these patients, excessive iron exposure and secondary iron overload ensue primarily because of repeated blood transfusions coupled with increased gastrointestinal iron absorption in the setting of ineffective erythropoiesis [1, 2, 5, 44] . A reduction in childhood mortality (due to infection and malnutrition) combined with increased use of chronic blood transfusions has led to a growing incidence of iron overload in patients with hereditary anemia [4, 6, 43, 45] . αand β-thalassemias are caused by mutations resulting in defective synthesis of the αand βglobin chains of hemoglobin, respectively, and are the most common monogenetic diseases in humans (Table 1) [5, 6, 42] . Patients with thalassemia originate primarily from the Mediterranean regions, the Indian subcontinent and Southeast Asia, where the estimated gene frequency is typically 3-10% but can be as high as 30-40% within certain subpopulations [5, 6, 42] . The clinical manifestations of thalassemia range from the silent carriers to severe iron overload. Sickle cell anemia is the most common and severe form of sickle cell disease (SCD) caused by homozygous presence of a glutamate to valine mutation in the beta-globulin gene (Hgb S) ( Table 1) . Sickle cell anemia occurs most commonly in individuals of African ancestry, and in the United States, 9% of African Americans carry the sickle cell trait and one in 600 has sickle cell anemia [46, 47] .

In addition to thalassemia and SCD, several other clinical disorders are associated with secondary iron overload including sideroblastic anemia, myelodysplastic syndrome, acute myeloid leukemia, congenital dyserythropoietic anemia and chronic renal failure (Table 1) . Sideroblastic anemia is characterized by mitochondrial iron overload in erythroblast and is associated with systemic iron overload, suggesting an important role for mitochondria in cellular iron metabolism (Table 1) [44, 48] . In patients with chronic renal failure and end-stage kidney disease, anemia is common due in part to erythropoietin deficiency that often necessitates therapy with intravenous iron [49] [50] [51] . A growing recognition of the toxicity of excess iron levels has increased concerns regarding the use of parenteral iron [49] in treating these patients [50, 51] .

Acute iron poisoning Acute iron toxicosis is a common cause of pediatric drug-overdose mortality [52] [53] [54] . Acute iron poisoning in these patients leads to gastrointestinal, liver and myocardial injury coupled with microvascular ↓ Synthesis of heme [179, 180] CDA AR (types I, II) AD (type III) Liver, heart, endocrine Ineffective erythropoiesis [181] CRF Acquired (polygenic) -Oral and intravenous iron supplementation [49, 50, 51, 182] Gene is italicized with gene product in parenthesis. ↓ indicates a reduction AR Autosomal recessive, AD autosomal dominant, HFE gene encoding for an atypical member of the class I major histocompatibility protein family that heterodimerizes with â2-microglobulin, JH juvenile hemochromatosis, SCA sickle cell disease, aCDA congenital dyserythropoesis anemia, CRF chronic renal failure 1 Variable penetrance 2 Early iron accumulation occurs in macrophages/monocytes 3 Dominantly inherited form of β-thalassemia resulting from a mutation in exon 3 of the β-globin gene 4 Hemoglobin S Antilles, because of its low oxygen affinity, causes pathologic changes in heterozygotes 5 Includes both acquired and hereditary forms 6 Iron accumulates mainly in the erythroblast mitochondria dysfunction, and shock [52] [53] [54] . Mortality observed with acute iron toxicity is linked to cardiovascular effects that include impaired myocardial contractility, bradycardia and hypotension, as discussed further below [16, 17] .

The human body uses about 20 mg of iron per day for hemoglobin synthesis and the production of approximately 200 billion erythrocytes [1, 2] . Consequently, most of the iron in the body is contained in hemoglobin. An additional 4-5 mg of iron is utilized daily for the production of other cellular proteins such as mitochondrial proteins, muscle myoglobin and others. A significant amount of iron is also stored at relatively high levels in hepatocytes and macrophages of the reticulo-endothelial system. Hepatocytes extract dietary iron delivered from the intestines, while reticulo-endothelial macrophages recycle iron from ingested senescent red blood cells. Once in the body, iron is delivered to most cells of the body via the blood as ferric iron bound to the abundant serum protein transferrin (i.e. diferric-transferrin). Despite large daily iron fluxes within the body, the total iron content in normal individuals is relatively fixed, with men (50 mg Fe/kg) having slightly higher iron levels than women (40 mg/kg). Movement of iron within the body is mediated by a combination of transporters whose activity is complexly regulated and modulated by various factors [2] . The best described "traditional" transporters include dimetal transporter 1 (DMT1), transferrin receptors (TfR1 and TfR2), ferroportin and the putative "heme receptor". Our recent studies have established that iron can also be transported in excitable cells like cardiomyocytes by voltage-dependent L-type Ca 2+ channels, which are promiscuous divalent transporters. Generally, iron transport involves redox cycling between ferrous (Fe 2+ ) and ferric (Fe 3+ ) states ( Fig. 1 and Table 2 ) that are catalyzed by several ferric reductases such as duodenal-cytochrome b (Dcytb) as well as ferroxidases such as ceruloplasmin and hephaestin ( Fig. 1) [2, 7] . Both ceruloplasmin and hephaestin are multi-copper-containing ferroxidases that are present in the serum and as a transmembrane protein in the basolateral surface of the duodenum, respectively, and are necessary for iron egress from intestinal enterocytes into the circulation and binding to transferrin [2, 55] .

Transferrin-transferrrin receptor system The best characterized and most prevalent method for iron transport involves the binding of plasma diferric-transferrin iron (i.e. transferrin-bound iron) to type I or type II transferrin receptors (i.e. TfR1 or TfR2). In individuals with normal iron levels, plasma iron is almost exclusively bound to transferrin (Tf), an abundant plasma protein with an extraordinarily high affinity for ferric iron at normal plasma pH [1, 2] . Transferrin-bound ferric iron is relatively "nonreactive" and is extracted from the plasma by most cells of the body following binding of ferritin to transferrin receptors (TfR1 and TfR2) in clathrin-coated regions of the cell membrane [1, 2] . Complexes of transferrin and TfR1/2 are internalized into acidic endosomes where the iron is reduced (to Fe 2+ ) and released into the cytosol by the divalent metal transporter 1, DMT-1 (see below) [2] . While the type 1 transferrin receptor, TfR1, is ubiquitously expressed, the TfR2 expression is restricted to hepatocytes, duodenum and erythroid precursors [2, 56] . Both receptors are expressed at relatively high levels in hepatocytes of the liver and enterocytes of the small intestine, especially the duodenum. The expression of TfR1 is profoundly down-regulated under iron-overload conditions, particularly in the liver, through the action of iron-response elements [1, 2] . By contrast, TfR2 protein expression is up-regulated when iron is elevated, making TfR2 a potential sensor or marker of iron status [56, 57] . TfR2 upregulation is probably an important contributor to excessive hepatic iron uptake in iron overload [56, 57] .

Divalent metal transporter 1 In addition to transferrinbound iron, iron also enters cells by DMT1 transporters (also called Nramp2, DCT1 or SLC11A2), which are H + / divalent metal symporters capable of transporting many other divalent metals including Mn 2+ , Cu 2+ , Zn 2+ and Cd 2+ [58] [59] [60] . DMT1 is highly expressed in the kidney and intestines while being expressed at much lower levels in brain and heart [58] [59] [60] . In the brush borders of the duodenum, DMT1 transports iron into enterocytes from the intestinal lumen following reduction in dietary non-Tfbound ferric to ferrous iron by intestinal ferric reductase Dcytb, a process that is facilitated by low pH in the duodenum where most of the dietary iron is absorbed ( Fig. 1 ) [58] [59] [60] . As already mentioned, DMT1 also mediates iron export from the intracellular endosomes following iron uptake via the transferrin and heme uptake systems.

Heme-iron transport Iron can also be imported as a hemeiron complex into selected tissues such as intestinal enterocytes and reticulo-endothelial macrophages by a putative "heme receptor", whose molecular identity remains unclear. A membrane receptor called feline leukemia virus type C receptor (FLVCR) can facilitate transport of heme into erythrocytes and may also impact on heme transport in the intestine and liver [61] . Once internalized, heme-bound iron is released by heme oxygenase.

Ferroportin Ferroportin (Fpn), a divalent iron export protein that is also known as iron-regulated protein (IREG1), metal-transporter protein (MTP1) or SLC11A3, is highly expressed on the surface of enterocytes, macrophages, hepatocytes and placental cells [7, 38, 39] . In these cells, Fpn exports reduced iron into the plasma, whereupon the iron is oxidized by ceruloplasmin or hephaestin, which are copper-containing ferroxidases found in the serum as well as the basolateral surface of enterocytes. Once oxidized, iron binds to transferrin [2, 55] . Fpn levels are regulated by the binding of hepcidin, a critical iron regulatory protein (see below), which leads to Fpn internalization and degradation, thereby decreasing export of cellular iron [38, 39] . As already mentioned, type 4 primary hemochromatosis is associated with Fpn mutations leading to impaired export of cellular iron and the subsequent accumulation of iron and iron overload (see Fig. 1 ) [7, 38, 39] . There are multiple Fpn mutations leading either to defective cell surface localization, decreased ability to be internalized and degraded in response to hepcidin, or dominant negative mutants, which may explain the phenotypic variation as well as the dominant inheritance of the disease [38, 39] . In addition to controlling the exit and release of intracellular iron, ferroportin also controls the intestinal uptake of extracellular iron at the apical membrane, possibly by modulating the activity of DMT1 [62] .

Although iron absorption is normally tightly regulated in order to maintain iron at levels that prevent tissue damage due to oxidative stress, elevated body iron does occur under several disease conditions due to excessive intestinal iron absorption or repeated blood transfusions leading to surplus iron accumulation in tissues such as liver, spleen, bone marrow, pancreas, heart, pituitary and central nervous system. As transferrin becomes iron-saturated, iron increasingly resides as NTBI, which is readily transported into selected tissues of the body, particularly heart, endocrine tissue and brain [8, 9] . In patients with secondary iron overload, total serum iron levels range from 20 to 60 μM (normal range=8-15 μM) with estimated NTBI levels ranging from 1 to 10 μM [2, 8, 9, 63] . Excessive iron levels in the cell saturate the ferritin binding capacity, leading to iron binding to low molecular weight compounds that form the LIP. This iron is highly catalytically active and participates in free-radical-generating reactions [2, 14] . Although the mechanism for the transport of NTBI into cells remains unclear, it is clear that selected tissues are particularly susceptible to excess iron uptake when NTBI is present. Several transport mechanisms have been proposed to participate in NTBI uptake. Recent evidence suggests that TfR2 is responsible for the excessive iron uptake into liver hepatocytes since TfR2 expression levels increase in iron overload, leading to liver cirrhosis and hepatic tumors [56, 57] . However, NTBI uptake into excitable cells such as cardiomyocytes, neurons, beta cells of the pancreas and pituitary cells requires reduction of ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ) by a membrane-associated ferri-reductase system, properties that clearly distinguish it from transferrin-receptor-mediated ferric iron uptake [11, 12, 64] . In these excitable tissues, it has been suggested that NTBI uptake is DMT1-dependent [59] . However, the maximum NTBI uptake rate (i.e. V max ) in cardiomyocytes increases without changes in sensitivity to iron (i.e. K D ), which contrasts with DMT1 whose protein expression decreases when iron is elevated [60] . In addition, DMT1 is expressed at extremely low levels in most excitable tissue including the heart [59, 60] . We have shown that NTBI uptake into cardiomyocytes is dependent on L-type Ca 2+ channels, which are known to be promiscuous divalent metal transporters [65] [66] [67] [68] . Importantly, L-type Ca 2+ channels satisfy all the criteria consistent with the NTBI transporter, and most importantly, this transporter readily explains the pattern of tissues at risk for damage and dysfunction under iron-overload conditions. Specifically, tissues with the greatest risk in iron overload are excitable tissues with high duty cycles for L-type Ca 2+ channel activity: cardiomyocytes, anterior pituitary cells, pancreatic beta cells and neurons (see Table 3 ). Evidence supporting a critical role for NTBI uptake by L-type Ca 2+ channels in cardiomyocytes of the heart is reviewed below.

Mechanisms for the regulation of iron Iron homeostasis necessitates tight control of iron uptake, storage and export as well as management of intracellular iron distribution [1, 2] . A major requirement for proper iron homeostasis is limiting the labile intracellular iron while simultaneously ensuring iron availability for many essential iron-containing proteins and enzymes. Since the loss of iron from the body is, under normal circumstances, largely independent of the total body iron, alterations in total body iron are primarily set by modulation of its absorption in the small intestines, especially in the duodenum. Iron uptake by the small intestine appears to be regulated by two interrelated processes: crypt programming and hepcidin expression levels [2, 7] . Crypt programming involves setting the iron uptake rates in mature villus enterocytes based on the total body iron levels sensed by developing enterocytes within the intestinal crypts via TfR1/2. Specifically, iron levels in the developing enterocytes influences the expression pattern of various iron transporters. Intestinal iron uptake also appears to be regulated in immature enterocytes by HFE, the gene involved in type I hemochromatosis [7] . With mutations in HFE in human (as well as in HFE knockout mice), transferrin-bound iron uptake is decreased in association with enhanced expression and activity of FPN1, resulting in increased iron export from enterocytes into the blood [69, 70] . Regulation of iron uptake in enterocytes may also involve beta 2-microglobulin that forms a macromolecular complex with HFE and TfR1.

Modulation of iron transport in small intestinal enterocytes and reticulo-endothelial macrophages by hepcidin represents an alternate mechanism for regulating iron.

Hepcidin is a small (20-25 amino acids) cysteine-rich antimicrobial peptide that is expressed and secreted into the blood by the liver in response to elevated iron and inflammation [2, 7, 36] . Hepcidin inhibits iron excretion in macrophages and enterocytes by binding to Fpn1, leading to internalization and loss of function of Fpn1 [38] . By contrast, in iron deficiency and anemia as well as in type 1 primary hemochromatosis, hepcidin levels are reduced, which increases Fpn1 expression as well as iron release from enterocytes and macrophages [70] . Indeed, loss of HFE function in type 1 primary hemochromatosis impairs the ability of hepcidin to regulate duodenal iron absorption, which may be the mechanism responsible for excessive iron absorption under this condition [70, 71] . Thus, posttranslational regulation of Fpn1 by hepcidin forms a homeostatic loop whereby iron regulates secretion of hepcidin, which in turn controls the concentration of Fpn1 and thereby iron export at the enterocyte surface [38, 72] . This loop also operates in the regulation of iron levels under inflammatory conditions, which may explain the anemia in chronic inflammation as well as the antimicrobial actions of hepcidin [2, 7, 70, 73] . Although the precise physiological role of hemojuvelin (HJV; also known as RGMc and HFE2), a glycosylphosphatidylinositol-an- [7, 36, 74, 75] . This connection helps explain the central role of hepcidin (and HJV) in the pathogenesis of juvenile hemochromatosis as well as anemia in chronic diseases (see Table 1 ) [7, 36, 74, 75] . The regulation of iron crypt programming and hepcidin, as well as the regulation of iron uptake in many cells of the body, involves tight negative-feedback regulation [2, 7, 9, 58] via iron regulatory proteins (IRP1 and IRP2) that recognize iron-responsive elements (IRE) located on the 3′ or 5′ untranslated regions (UTR) of mRNA for many ironregulated genes. For example, the transferrin receptors and H and L subunits of the intracellular iron-storage protein, ferritin [76, 77] , contain IREs in the 3′-UTR and 5′-UTR of their mRNAs, respectively [2, 78] . Under conditions of low iron, IRP binding to IREs blocks translation of ferritin mRNA and stabilizes transferrin receptor mRNA. When labile intracellular iron increases, IRP1 binding to IREs leads to IRP2 degradation, resulting in efficient translation of ferritin and rapid degradation of transferrin receptor mRNA [2, 77, 78] . DMT1 and FPN1 also contain IREs in their mRNA UTRs that are suggested to be responsible for the decreased DMT1 expression in the heart (and intestine) observed with iron overload (Table 2) [2, 7, 59, 60] .

Clinical description Iron-overload cardiomyopathy is a common cause of death due to cardiovascular complications especially in the second and third decades of life [4-6, 79, 80] . Indeed, in European [81] , North American [79, 80] and Chinese [24] patients with thalassemia major, the severity of cardiac dysfunction is the primary determinant of survival, while in patients with primary hemochromatosis, cardiovascular disease also contributes significantly to their mortality and morbidity [82] [83] [84] [85] . In addition, cardiac iron accumulation in beta-thalassemia patients correlates directly with both heart disease and mortality [5, 79, 80] . Although iron chelation therapy is widely used for treating iron-overload patients, cardiomyopathy and mortality are still common in these patients [24, 86] . Regardless of its origin, iron overload in patients [13, 87, 88] and animal models [18, 19] leads to a restrictive cardiomyopathy with prominent early diastolic dysfunction that invariably progresses to end-stage dilated cardiomyopathy characterized by impaired systolic function and reduced mean arterial blood pressure often accompanied by arrhythmias including atrioventricular (AV) block, conduction defects, bradyarrhythmias, tachyarrhythmias and sudden cardiac death (Fig. 3) [79, 80, 84, [89] [90] [91] [92] . AV nodal block is a particularly common occurrence, which is due to an especially high accumulation of iron in the AV nodal compared to other tissues of the heart (see Fig. 3 and Table 3 ) [89] .

Pathophysiology The precise mechanism underlying cardiomyocyte dysfunction induced by iron overload is not entirely clear. Iron within cells can be divided into the "unavailable" or tightly bound pool, which includes iron in ferric hydroxide precipitates and iron complexed with ferritin, and the "available" LIP. The LIP is more readily available for Fenton-type reactions whereby the conversion of reduced iron (Fe 2+ ) into oxidized iron (Fe 3+ ) Fig. 2 Interaction between ironmediated oxidative stress and the excitation-contraction coupling in a cardiomyocyte. ROS reactive oxygen species, SER-CA2a sarcoplasmic reticulum Ca 2+ ATPase isoform 2, NCX sodium-calcium exchanger, SR sarcoplasmic reticulum generates various free radicals [15, 93] . Under normal physiological conditions, the generation of free radicals is minimized by a repertoire of enzymatic and non-enzymatic antioxidant mechanisms [93] . However, when iron levels are chronically elevated, excessive free radical generation leads to depletion of antioxidants and increased cellular damage due to oxidation of lipids, proteins and nucleic acids [19, 93] . in animal models [18, 19, 94] as well as in patients with primary hemochromatosis, betathalassemia major and end-stage kidney disease [95] [96] [97] . Oxidation of lipids (i.e. lipid peroxidation) is of particular importance in iron overload that leads to marked increases in unsaturated (malondialdehyde and hydroxynonenal) and saturated (hexanal) aldehydes in the heart and plasma [18, 19] . These compounds are associated with cellular dysfunction, cytotoxicity and cell death [18, 19, 93, 98, 99] . Consistent with a critical role of oxidative stress in iron-overload cardiomyopathy, the risk of developing ironoverload cardiomyopathy in patients with primary hemochromatosis is strongly influenced by polymorphisms of the manganese-superoxide dismutase (MnSOD) gene [100] . Additional factors that may also modulate the amount of oxidative stress observed under iron-overload conditions include inflammation [101, 102] and ApoE polymor-phisms (indicator of low intrinsic antioxidant status) [103] .

Iron-induced oxidative damage can have multiple effects on cardiac function. For example, iron overload has been linked to cardiomyocyte loss due to apoptosis [19, 29, 93] , which can be reduced by antioxidant treatment [19] . Enhanced apoptosis and/or necrosis in iron-overload cardiomyopathy could be linked to altered mitochondrial function as seen in Friedreich ataxia [93, 104, 105] . Increased myocyte loss is probably a major contributor to the prominent interstitial fibrosis seen in iron-overload cardiomyopathy, although iron-mediated effects on cardiac fibroblasts might also contribute [9] . Regardless, reduced cardiomyocyte numbers combined with increased myocardial interstitial fibrosis are predicted to contribute to impaired systolic and diastolic function in iron-overloaded hearts. Increased iron-mediated oxidative stress can also directly alter myocyte electrical and contractile properties by affecting the function of several regulators of cardiac excitation-contraction coupling [106] [107] [108] . Specifically, with elevated oxidative stress, sarcoplasmic reticulum (SR) Ca 2+ leak through ryanodine receptors (Ca 2+ release channels) [109] is increased, sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) activity is inhibited, sodiumcalcium exchanger (NCX) [110] currents are elevated, LTCCs are reduced [111, 112] and sodium as well as potassium currents can be either enhanced or depressed (see Fig. 2 ) [113] . These effects can occur by direct protein oxidation or by the generation of lipid peroxidation products [114, 115] . Collectively, elevated oxidative stress leads to reduced peak systolic Ca 2+ levels, slowed rates of Ca 2+ relaxation and elevated diastolic Ca 2+ levels, which probably contribute to the impaired systolic and diastolic function observed in iron-overload cardiomyopathy and also leads to arrhythmias, impaired electrical conduction and sudden death [116] .

Altered function of ion channels and electrogenic transporters in response to iron overload and elevated oxidative stress can lead to increased susceptibility and generate arrhythmias in the heart [113, 116, 117] , which are commonly observed under iron-overload conditions [79, 113, 116] . For example, spontaneous Ca 2+ release combined with slowed Ca 2+ re-uptake in the SR and increased NCX activity predisposes to increased triggered arrhythmias and delayed after-depolarization that are observed under ironoverload conditions. In iron-overloaded gerbils, ventricular action potential duration has been shown to be abbreviated in connection with increased transient outward K + current (I to ) densities coupled with reduced Na + currents [113] , although reduced L-type Ca 2+ currents might also contribute. In iron-treated mice and gerbils, common electrophysiological observations include QRS prolongation accompanied by slowed conduction velocity, bradycardia and prolongation of the PR interval in association with firstand second-degree AV block (Fig. 3) [92] . Slowed conduction in the heart may be related to the observation that elevated oxidative stress reduces cardiac voltage-gated Na + currents [118] . Action potential shortening and abnormal impulse conduction [92, 113] in the setting of increased interstitial fibrosis observed in iron overload [18, 19] Fig. 3 Telemetric recordings from conscious mice that were injected with placebo or iron (n=6) over a 4-week period as previously reported [18] ; baseline heart rate=500-600 bpm. increase the propensity for unidirectional conduction block, wavefront breakup and creation of arrhythmic ventricular re-entry circuits [92, 117] . The reduced heart rates and AV block may be related to a combination of preferential iron deposition and increased interstitial fibrosis in these regions [18, [89] [90] [91] . In addition, iron deposition in the myocardium is heterogeneous, which can further promote arrhythmias and sudden cardiac death by increasing the heterogeneity of ventricular repolarization and QT dispersion, which is linearly correlated with iron burden [89, 113, 119] . In addition to the direct effects on the heart, elevated iron can also affect the function of vascular smooth muscle cells and endothelial cells. Indeed, endothelial dysfunction and increased arterial stiffness are commonly observed in patients with primary hemochromatosis [120] , beta-thalassemia major [121] and sickle cell anemia [122] . These vascular changes can in turn affect myocardial perfusion and function.

The basis for the high sensitivity of the heart (and other excitable tissues) to excessive iron uptake, damage and dysfunction under conditions of iron overload was an enigma for many years. However, several classical characteristics of NTBI transport and of the cardiomyopathy under iron-overload conditions provide important insights into the possible mechanism of iron transport into the heart under these conditions. For example, NTBI transport in cardiomyocytes requires divalent ferrous, not trivalent ferric, iron, and its rate increases markedly with chronic iron elevations [12, 64, 68] . These two features argue strongly against TfR-mediated or DMT1-mediated mechanisms for NTBI transport in the heart. Specifically, TfR1 transports ferric iron and TfR1 mRNA expression is reduced in association with a 63% decline in IRP1 in cardiomyocytes exposed to chronic elevations in iron [123] . Similarly, while DMT1 can transport ferrous iron, myocardial DMT1 mRNA expression has been shown either to decrease with elevated iron in adult hearts [60] or to be uncorrelated with changes in iron accumulation in cultured myocytes despite the absence of mRNA changes [123] . The lack of involvement of these iron transporters in myocardial NTBI uptake is further supported by low-level TfR1-dependent iron uptake into heart as well as the extremely low expression levels of TfR1 and DMT1 in the heart [60, 123] .

Unlike DMT1 and TfR, L-type Ca 2+ channels satisfy all the known properties of NTBI transport. Indeed, while LTCCs are primarily utilized for the transport of Ca 2+ , they are able to transport many other divalent, but not trivalent, cations including Fe 2+ , Zn 2+ , Co 2+ , Sr 2+ , Ba 2+ and Mn 2+ [66] [67] [68] 124] . Consistent with a dominant role of LTCC in myocardial iron transport, iron uptake is increased by the selective LTCC agonist, Bay K 8644, as well as following beta-adrenergic stimulation in proportion to the degree of LTCC current enhancement while being inhibited by LTCC blockers [68] . Moreover, low Fe 2+ levels slow the rate of LTCC current inactivation, resulting in increased Fe 2+ entry per heartbeat [68] , and L-type Ca 2+ currents are not decreased in iron overload [18] , consistent with the absence of IREs in the mRNA of the pore-forming subunits of cardiac LTCCs (i.e. Ca V 1.2) (see Table 2 ). The Fe 2+mediated slowing of Ca 2+ current inactivation could arise from competition between Fe 2+ and Ca 2+ for the C-terminal cytoplasmic Ca 2+ binding site involved in Ca 2+ -mediated inactivation of LTCCs [108] . Thus, LTCCs are the only Fe 2+ transporter whose activity increases with elevated iron, consistent with NTBI uptake in the heart under ironoverload conditions [9, 12, 18, 68] . Remarkably, the amount of myocardial iron accumulation predicted to occur via LTCCs over 20 years, a relevant period for patients with iron overload, is estimated to be about 10 mg of iron per gram of myocardial tissue [68] , which matches closely to the 3-10 mg/gm of myocardial iron measured in patients with iron-overload cardiomyopathy [89] and in animal models [18, 19] .

A more definitive link between LTCCs and myocardial iron deposition was obtained in mouse studies where LTCC blockers, like amlodipine and verapamil, reduced intracellular myocardial iron accumulation and reduced oxidative stress while protecting diastolic and systolic cardiac function [18] . Total myocardial iron level was determined by atomic absorption spectrophotometry, while intracellular iron deposition was examined by Prussian blue histological staining coupled with analytical electron microscopy [18, 19] . Moreover, cardiac-specific LTCC overexpression caused increases in myocardial iron accumulation and oxidative damage in proportion to the elevated Ca 2+ currents that were, again, reduced with LTCC blockers [18] . These beneficial cardiac effects of LTCC blockers in ironoverload mice were not observed in the liver, as would be expected, since hepatocytes express minimal levels of functional LTCCs [125] . Involvement of LTCC in iron transport is also supported by the protective actions of taurine (i.e. 2-aminoethane-sulphonic acid) supplementation against myocardial iron accumulation, oxidative damage, and altered cardiac structure and function [19] . The link between LTCCs and iron uptake in excitable tissues is consistent with the observation that the maximal rates of iron uptake (V max ) are approximately tenfold higher in cardiomyocytes than in fibroblasts [12, 64, 123 ] that have few if any LTCCs. The link between LTCC and myocardial iron transport is further substantiated by the high susceptibility to iron-mediated injury and disease observed in excitable cells that require high LTCC activity for proper functioning such as excitation-contraction coupling in vascular smooth muscle cells [106] [107] [108] as well as in excitation-secretion coupling in osteoblasts [126] , endocrine cells and neurons [106, [127] [128] [129] .

The molecular basis for iron permeation in LTCC has not previously been examined. However, ion selectivity, permeability and blocking properties of LTCCs are dictated by electrostatic binding of ions within the pore [130, 131] . Strong electrostatic interactions between ions and the selectivity filter combined with a large pore radius (i.e. 6.2 nm) allows many divalent cations to readily permeate LTCCs, independent of their ionic radius (i.e. r Ca =2.4 nm vs r Fe =2 nm), although some permeant ions also "block" LTCC currents depending on the relative binding affinity [65, 124] . By contrast, under normal biological conditions, monovalent cations cannot easily permeate LTCC, while trivalent cations are often potent blockers of LTCCs. These biophysical properties of cardiac LTCCs originate from the pore-forming α1 subunits that are comprised of four internal repeat domains (I-IV) each made up of six membrane-spanning segments (S1-S6) [106, 108, 132] and which co-assemble with β-subunits and α 2 /δ-subunits to form functional LTCCs [106, 108] . The cardiac α1 subunits are members of a highly homologous family of highvoltage-activated Ca 2+ channel genes (i.e. the Ca v 1 family) that includes Ca v 1.1, Ca v 1.2 (α 1C ) and Ca v 1.3 (also called α 1S , α 1C and α 1D ) [106, 108, 132] Cardiomyocytes express Ca v 1.2 [128, 133, 134] , which is also expressed (as spliced variants) in vascular smooth muscle [128] , brain [128] and endocrine tissue including pancreatic beta cells [127, 128] .

Ca v 1.3 shows a far more restricted expression pattern, being found in sinoatrial (SA) nodal cardiomyocytes [133] as well as pancreatic beta cells where it plays a relatively minor role in insulin release [128] . Ca v 1.1 subunits are expressed in skeletal muscle [106] . Given the high degree of amino acid identity and thereby structural similarity between the Ca v 1 family members, it seems likely that LTCCs also participate in NTBI uptake in excitable tissues other than the heart. Indeed, as already mentioned, the cells most susceptible to damage and dysfunction in iron overload are precisely those having high densities of LTCC. One potential glaring exception to this pattern is skeletal muscle cells, which have high densities of LTCC. However, skeletal muscle cells utilize LTCCs for excitationcontraction coupling without requiring ion permeation [106] , consistent with the relative resistance of skeletal muscle to the effects of iron overload.

Involvement of LTCC in iron transport helps explain several key clinical features of iron-overload cardiomyopathy. For example, second-and third-degree heart blocks are commonly observed in iron overload, and these patients often require pacemaker device implantation [89] [90] [91] . Prominent AV node pathology is consistent with the essential requirement of LTCC in AV nodal conduction [135] that is reflected in the very high baseline LTCC current densities recorded in AV nodal myocytes (i.e. 14-18 pA/pF) [136, 137] . Iron transport by LTCC could explain the observation that the L-type Ca 2+ channel blocker diazepam protects the heart against acute iron-induced toxicity and mortality without affecting iron absorption and excretion [138, 139] . Similarly, Ca 2+ channel blockers protect the myocardium from ischemia-reperfusion injury [140, 141] , which has been linked to iron-mediated injury [29, 142] .

NTBI entering cardiomyocytes is effectively trapped in the cytosol following rapid redox cycling to ferric iron and binding to ferritin [76, 143] , conversion to the insoluble ferric iron (hemosiderin) deposits [113] or expansion of the reactive LIP leading to iron-mediated oxidative damage. Permeation of reduced, reactive iron (Fe 2+ ) through LTCCs in iron overload may help explain the profound contractile dysfunction, Ca 2+ overload and impaired diastolic function commonly observed during the early stages of iron overload [13, 42, 84, 88] . Specifically, the reduced iron entering through LTCCs has preferential access to the major regulators of excitation-contraction coupling (Fig. 2) . Moreover, Fe 2+ -induced slowing of LTCC current inactivation by Fe 2+ leads to increased Ca 2+ entry and Ca 2+ overload [68] . Since elevated Ca 2+ , particularly through LTCCs, is involved in the initiation and progression of heart disease in a calcineurin-dependent manner [144] , it is conceivable that the slowing of LTCC inactivation by NTBI may contribute to the cardiomyopathy seen in iron overload. Additionally, slowed inactivation of LTCCs positively feedback to further increase NTBI Fe 2+ entry into cardiomyocytes, thereby amplifying alterations in cellular function. Elevations in cytosolic iron also lead to iron uptake into mitochondria where it normally becomes incorporated into Fe-S clusters and is utilized for heme biosynthesis [3] . Iron homeostasis in the mitochondria requires frataxin, a ferroxidase that detoxifies redox-active iron, thereby providing a critical anti-oxidative defense against iron-derived radicals in the mitochondria [145, 146] . Frataxin deficiency causes a clinical syndrome called Friedreich ataxia that involves neurodegeneration, dilated cardiomyopathy and diabetes mellitus [147] .

Although our previous results provide unequivocal evidence for NTBI transport by LTCC in heart, the application of LTCC blockers failed to alter iron uptake rates in cultured rat neonatal myoctes [64] . However, these studies should be interpreted with caution for several reasons. First, neonatal myocytes have much smaller L-type Ca 2+ current compared to adult myocardium [148] . Second, when culturing neonatal cardiomyocytes at low densities, the fraction of beating myocytes is very low and beating rates are slow compared to normal heart rates. Therefore, it is imperative that electrical pacing be used in conjunction with high-density cultures when examining the role of LTCC in iron transport. Third, neonatal cultures suffer from high rates of myocytes death, particularly in the presence of iron, making it essential to consider the number of viable cells in the calculation of iron uptake rates. Fourth, our results predict that LTCCs also contribute to ferrous iron export. Consequently, at non-physiologically high iron loads (as used in the cultured myocyte studies), LTCC blockers are predicted to have a limited impact on the net iron uptake. Collectively, the validity of the neonatal cultured myocyte system as a tool to test the role of LTCCs as iron transporters is questionable [64] .

Endocrine dysfunction in patients with iron-overload: a potential role for LTCC L-type Ca 2+ channels are present in large numbers in pancreatic beta cells [127, 128] and in various cell types of the anterior pituitary gland including gonadotrophs [149] [150] [151] , thyrotrophs [152] , corticoptrophs [153] and in the parathyroid-hormone-producing cells of the parathyroid gland (Table 3 ) [154] . In these endocrine cells, LTCCs are critically involved in excitation-secretion coupling whereby membrane depolarization activate (open) LTCCs, allowing extracellular Ca 2+ entry and stimulation of exocytosis [127, 149, 151] . Given the important role of LTCC in mediating cellular NTBI uptake, it seems plausible to suggest a link between LTCCs and iron-overload-induced dysfunction in these endocrine tissues. Similar to cardiomyocytes, excessive Fe 2+ entry through LTCCs is expected to impair secretion and increase cell loss (see Table 3 ). Consistent with this mechanism, histological and immunohistochemical analyses have confirmed deposition of iron in human beta cells [155] , while imaging studies have shown iron accumulation in the anterior pituitary gland in patients with iron overload [156] .

These observations help explain the observation that endocrinopathies are common co-morbidities present in patients with iron overload and include diabetes mellitus [21, 22, 24, 155] , hypoparathyroidism [22, 157] and anterior pituitary dysfunction associated with hypopituitary hypogonadism [22] [23] [24] , impaired adrenocorticotrophin reserve [21, 23] and hypothyroidism [22] . Diabetes mellitus in patients with iron overload is associated with reduced insulin reserve and impaired insulin release. Consequently, most patients often progress to be insulin-dependent relatively early during the course of illness [21, 22, 158] . In addition, up to 50% of patients with beta-thalassemia major have a short stature and failure of puberty associated with primary and secondary amenorrhea in women [22, 24] .

Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases in both pediatric [159] and adult [160, 161] populations. The recognition that LTCCs are critical transporters of iron under conditions of iron overload opens up the possibility of using CCBs in the treatment and/ or prevention of iron-overload cardiomyopathy in combination with standard chelation therapy. Unlike iron chelators that are effective in removing excess iron accumulation in cells, CCBs are expected to be particularly effective early in iron overload by reducing tissue iron uptake and preventing disease progression. In advanced iron overload, the potential effectiveness of CCBs may be limited, but they should block further iron uptake, thereby enhancing the effectiveness of iron chelation therapies. However, the applicability of CCBs in the setting of advanced dilated cardiomyopathy or conduction block may be limited due to their negative inotropic and chronotropic effects.

While the data supporting the potential effectiveness of CCBs in the treatment of iron-overload cardiomypathy has been obtained in rodents, pharmacological blockade of LTCC might have an even greater therapeutic benefit in humans for two reasons: (a) humans lack iron excretory pathways that are present in rodents [1, 2] , and (b) humans have proportionately larger trans-sarcolemmal I Ca,L influx per cardiac cycle [162] . The translation of the rodent studies to humans is bolstered by the observation that the plasma concentration of CCBs required to reduce iron accumulation in murine hearts [18] is similar to levels reported in patients [163] [164] [165] . Blockade of the LTCC may also provide additional benefits beyond a direct inhibition of Fe 2+ entry into cardiomyocytes and endocrine tissues. For example, CCBs could potentially reduce Fe 2+ -induced Ca 2+ overload as a result of LTCC inactivation [68] and thereby facilitate diastolic ventricular filling [166] . Finally, calcium channel blockers may promote myocardial microvascular perfusion by vasodilating coronary arterioles while improving coronary endothelial function [132, 167] . LTCC blockers like amlodipine also possess antioxidant properties that could help offset the oxidative effects of iron overload [167] . Given the high degree of oxidative stress, diastolic dysfunction and possibility for coronary endothelial dysfunction in iron-overload cardiomyopathy, these effects may increase the potential therapeutic benefits of CCBs in patients with iron overload. In addition to protecting the heart, pharmacological blockade of LTCCs may also reduce the endocrinopathies observed in iron-overload patients. Clearly, clinical trials are warranted to establish the clinical efficacy and safety of CCB treatment in patients with iron overload.

Disorders of iron metabolism

Balancing acts; molecular control of mammalian iron metabolism

Iron trafficking in the mitochondrion: novel pathways revealed by disease

Thalassemia-a global public health problem

The beta-thalassemias

Inherited haemoglobin disorders: an increasing global health problem

Hereditary hemochromatosis-a new look at an old disease

The importance of non-transferrin bound iron in disorders of iron metabolism

Genetic regulation of cell function in response to iron overload or chelation

Labile plasma iron in iron overload: redox activity and susceptibility to chelation

Regulation of the transferrin-independent iron transport system in cultured cells

Uptake of non-transferrin-bound iron by both reductive and nonreductive processes is modulated by intracellular iron

Iron overload cardiomyopathies: new insights into an old disease

Serum markers of stored body iron are not appropriate markers of health effects of iron: a focus on serum ferritin

Low-molecular-weight iron complexes and oxygen radical reactions in idiopathic haemochromatosis

Depression of contractility in isolated rabbit myocardium following exposure to iron: role of free radicals

Hemodynamic effects of experimental iron poisoning

L-type Ca(2+) channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy

Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model

Idebenone delays the onset of cardiac functional alteration without correction of Fe-S enzymes deficit in a mouse model for Friedreich ataxia

Clinical consequences of acquired transfusional iron overload in adults

Multicentre study on prevalence of endocrine complications in thalassaemia major. Italian Working Group on Endocrine Complications in Non-endocrine Diseases

Mineralocorticoid status and endocrine dysfunction in severe hemochromatosis

Morbidity and mortality patterns of thalassaemia major patients in Hong Kong: retrospective study

Bone and joint involvement in genetic hemochromatosis: role of cirrhosis and iron overload

Iron lactateinduced osteomalacia in association with osteoblast dynamics

Bone histomorphometry in children and adolescents with beta-thalassemia disease: iron-associated focal osteomalacia

Iron-chelating agents in non-iron overload conditions

HFE mutation and dietary iron content interact to increase ischemia/reperfusion injury of the heart in mice

Iron, atherosclerosis, and ischemic heart disease

Iron and peripheral arterial disease: revisiting the iron hypothesis in a different light

Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson's disease

Redox metals in Alzheimer's disease

Multicentric origin of hemochromatosis gene (HFE) mutations

Disease-related conditions in relatives of patients with hemochromatosis

Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis

Hemochromatosis due to mutations in transferrin receptor 2

Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization

The molecular basis of ferroportinlinked hemochromatosis

African iron overload

Determination of non-transferrin-bound iron in genetic hemochromatosis using a new HPLC-based method

Genetic and clinical features of hemoglobin H disease in Chinese patients

Progression of iron overload in sickle cell disease

Iron overload in sideroblastic and other non-thalassemic anemias

Survival of children with sickle cell disease

Management of sickle cell disease

Sickle cell disease

The genetics of inherited sideroblastic anemias

Parenteral iron formulations: a comparative toxicologic analysis and mechanisms of cell injury

Serum ferritin is a marker of morbidity and mortality in hemodialysis patients

Iron overload and cardiovascular complications in dialysis patients

Acute iron intoxication: treatment controversies

Myocardial failure and shock in iron poisoning

Iron poisoning

Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse

Regulation of transferrin receptor 2 protein levels by transferrin

Diferric transferrin regulates transferrin receptor 2 protein stability

Cloning and characterization of a mammalian proton-coupled metal-ion transporter

Iron-dependent regulation of the divalent metal ion transporter

Post-transcriptional expression of DMT1 in the heart of rat

Identification of a human heme exporter that is essential for erythropoiesis

Ferroportin/IREG-1/MTP-1/ SLC40A1 modulates the uptake of iron at the apical membrane of enterocytes

Non-transferrin-bound iron in the serum of hemodialysis patients who receive ferric saccharate: no corretion to peroxide generation

Characterization of Fe2+ and Fe3+ transport by iron-loaded cardiac myocytes

Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore

Block of current through single calcium channels by Fe, Co, and Ni

Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels

Modulation of iron uptake in heart by Ltype Ca2+ channel modifiers: possible implications in iron overload

Role of HFE in iron metabolism, hereditary haemochromatosis, anaemia of chronic disease, and secondary iron overload

Effect of hepcidin on intestinal iron absorption in mice

Regulatory networks for the control of body iron homeostasis and their dysregulation in HFE mediated hemochromatosis

Inhibition of iron transport across human intestinal epithelial cells by hepcidin

Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis

Hemojuvelin: a supposed role in iron metabolism one year after its discovery

Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload

Control of cellular iron homeostasis by ironresponsive elements in vivo

Iron metabolism and the IRE/IRP regulatory system: an update

Mammalian tissue oxygen levels modulate iron-regulatory protein activities in vivo

Survival in medically treated patients with homozygous beta-thalassemia

Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major

Survival and causes of death in thalassaemia major

Long-term survival in patients with hereditary hemochromatosis

Cardiac alterations in 36 consecutive patients with idiopathic haemochromatosis: polygraphic and echocardiographic evaluation

Cardiac abnormalities in hemochromatosis

The role of Doppler left ventricular filling indexes and Doppler tissue echocardiography in the assessment of cardiac involvement in hereditary hemochromatosis

Survival in betathalassaemia major in the UK: data from the UK Thalassaemia Register

Blood pressure and heart rate in young thalassemia major patients

Left ventricular diastolic Doppler characteristics in beta-thalassemia major

Iron in the heart. Etiology and clinical significance

The cardiomyopathy of idiopathic hemochromatosis

Prevalence of a haemochromatosis among men with clinically significant bradyarrhythmias

Optical mapping reveals conduction slowing and impulse block in ironoverload cardiomyopathy

Molecular bases of cellular iron toxicity

Iron supplementation generates hydroxyl radical in vivo. An ESR spin-trapping investigation

Antioxidant status and lipid peroxidation in hereditary haemochromatosis

Oxidative stress and antioxidant status in beta-thalassemia major: iron overload and depletion of lipid-soluble antioxidants

Lipophilic antioxidants and iron status in ESRD patients on hemodialysis

Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes

Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins

The mitochondrial superoxide dismutase A16V polymorphism in the cardiomyopathy associated with hereditary haemochromatosis

Myocarditis in beta-thalassemia major. A cause of heart failure

Association of heart failure in homozygous beta-thalassemia with the major histocompatibility complex

Apolipoprotein E epsilon4 allele as a genetic risk factor for left ventricular failure in homozygous beta-thalassemia

Friedreich's ataxia: disease mechanisms, antioxidant and coenzyme Q10 therapy

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death

Structure and regulation of voltage-gated Ca2+ channels

L-type calcium channel abundance and function with cardiac hypertrophy and failure: a review

Excitation-contraction coupling and cardiac contractile force

NADH oxidase activity of rat cardiac sarcoplasmic reticulum regulates calcium-induced calcium release

Oxygen free radicals and excitation-contraction coupling

Effect of sulfhydryl oxidation on ionic and gating currents associated with L-type calcium channels in isolated guinea-pig ventricular myocytes

Suppression of Ca2+ influx through L-type voltage-dependent calcium channels by hydroxyl radical in mouse cerebral cortical neurons

Decreased sodium and increased transient outward potassium currents in iron-loaded cardiac myocytes. Implications for the arrhythmogenesis of human siderotic heart disease

The antioxidant effects of a novel iron chelator salicylaldehyde isonicotinoyl hydrazone in the prevention of H(2)O(2) injury in adult cardiomyocytes

Malondialdehyde inhibits cardiac contractile function in ventricular myocytes via a p38 mitogen-activated protein kinase-dependent mechanism

Earliest cardiac toxicity induced by iron overload selectively inhibits electrical conduction

What causes sudden death in heart failure?

Slowly inactivating sodium currents are reduced by exposure to oxidative stress

The effect of iron stores on corrected QT dispersion in patients undergoing peritoneal dialysis

Association between increased iron stores and impaired endothelial function in patients with hereditary hemochromatosis

Arterial stiffness and endothelial function in patients with beta-thalassemia major

Arterial stiffness and wave reflections in patients with sickle cell disease

Differential accumulation of non-transferrin-bound iron by cardiac myocytes and fibroblasts

Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells

Plasma membrane Ca2+ release-activated Ca2+ channels with a high selectivity for Ca2+ identified by patch-clamp recording in rat liver cells

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

Insulin granule dynamics in pancreatic beta cells

Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels

Differential expression of ionic channels in rat anterior pituitary cells

Calcium channels: mechanisms of selectivity, permeation, and block

Differences in apparent pore sizes of low and high voltage-activated Ca2+ channels

Calcium-antagonist drugs

L-type calcium channels: highs and new lows

Ionic remodeling of cardiac Purkinje cells by congestive heart failure

Cardiac HCN channels: structure, function, and modulation

Modulation by mexiletine of action potentials, L-type Ca current and delayed rectifier K current recorded from isolated rabbit atrioventricular nodal myocytes

Ionic remodeling of sinoatrial node cells by heart failure

The efficacy of diazepam in the treatment of acute iron overload in rats

Negative inotropic effect of diazepam in isolated guinea pig heart

Enhancement of salvage of reperfused ischemic myocardium by diltiazem

Stunned myocardium and the attenuation of stunning by calcium antagonists

Iron-mediated cardiovascular injury

Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression

Inhibition of calcineurin and sarcolemmal Ca2+ influx protects cardiac morphology and ventricular function in K(v)4.2N transgenic mice

Assembly of human frataxin is a mechanism for detoxifying redox-active iron

Friedreich's ataxia, no changes in mitochondrial labile iron in human lymphoblasts and fibroblasts: a decrease in antioxidative capacity

Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin

Developmental regulation of the L-type calcium channel alpha1C subunit expression in heart

Stimulation of Ca2+ influx in alpha T3-1 gonadotrophs via the cAMP/PKA signaling system

Characterization of the gonadotrophin-releasing hormone calcium response in single alpha T3-1 pituitary gonadotroph cells

Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone

Thyrotropin (TSH)-releasing hormone stimulates TSH beta promoter activity by two distinct mechanisms involving calcium influx through L type Ca2+ channels and protein kinase C

Spontaneous transients of [Ca2+]i depend on external calcium and the activation of Ltype voltage-gated calcium channels in a clonal pituitary cell line (AtT-20) of cultured mouse corticotropes

Parathyroid cells express dihydropyridinesensitive cation currents and L-type calcium channel subunits

The haemochromatotic human pancreas: a quantitative immunohistochemical and ultrastructural study

Pituitary gland height evaluated by MR in patients with beta-thalassemia major: a marker of pituitary gland function

Hypoparathyroidism in beta-thalassemic patients

Insulin sensitivity and beta-cell secretion in thalassaemia major with secondary haemochromatosis: assessment by oral glucose tolerance test

Calcium channel blockers: pharmacology and place in therapy of pediatric hypertension

A calcium antagonist vs a non-calcium antagonist hypertension treatment strategy for patients with coronary artery disease. The International Verapamil-Trandolapril Study (INVEST): a randomized controlled trial

Outcomes in hypertensive patients at high cardiovascular risk treated with regimens based on valsartan or amlodipine: the VALUE randomised trial

Cardiac excitation-contraction coupling

Interaction of verapamil and cimetidine: stereochemical aspects of drug metabolism, drug disposition and drug action

Effects of grapefruit juice and smoking on verapamil concentrations in steady state

Persistence of antihypertensive efficacy after missed doses: comparison of amlodipine and nifedipine gastrointestinal therapeutic system

Diastolic dysfunction

Novel vascular biology of third-generation L-type calcium channel antagonists: ancillary actions of amlodipine

A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis

The C282Y mutation causing hereditary hemochromatosis does not produce a null allele

Screening hepcidin for mutations in juvenile hemochromatosis: identification of a new mutation (C70r)

The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22

Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis

A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis

Autosomaldominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene

Lethal thalassemia after insertional disruption of the mouse major adult betaglobin gene

A Mouse Model for {beta}0-Thalassemia

Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease

Knockouttransgenic mouse model of sickle cell disease

Animal models for X-linked sideroblastic anemia

Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload

Congenital dyserythropoietic anemia type II: epidemiology, clinical appearance, and prognosis based on long-term observation

Enhanced oxidative stress in haemodialysis patients receiving intravenous iron therapy

Functional properties of Cav1.3 (alpha1D) L-type Ca2+ channel splice variants expressed by rat brain and neuroendocrine GH3 cells

Generation of action potentials in a mathematical model of corticotrophs

Electrophysiology of stimulus-secretion coupling in human beta-cells