Cellular Electrophysiology of Iron-Overloaded Cardiomyocytes

Iron, the most abundant transition metal element in the human body, plays an essential role in many physiological processes. However, without a physiologically active excretory pathway, iron is subject to strict homeostatic processes acting upon its absorption, storage, mobilization, and utilization. These intricate controls are perturbed in primary and secondary hemochromatoses, leading to a deposition of excess iron in multiple vital organs including the heart. Iron overload cardiomyopathy is the leading cause of mortality in patients with iron overload conditions. Apart from mechanical deterioration of the siderotic myocardium, arrhythmias reportedly contribute to a substantial portion of cardiac death associated with iron overload. Despite this significant impact, the cellular mechanisms of electrical disturbances in an iron-overloaded heart are still incompletely characterized. This review article focuses on cellular electrophysiological studies that directly investigate the effects of iron overload on the function of cardiac ion channels, including trans-sarcolemmal and sarcoplasmic reticulum Ca²⁺ fluxes, as well as cardiac action potential morphology. Our ultimate aim is to provide a comprehensive summary of the currently available information that will encourage and facilitate further mechanistic elucidation of iron-induced pathoelectrophysiological changes in the heart.

Introduction

Iron is an essential metal element that serves many physiological functions. These range from oxygen transport by hemoglobin to catalytic activities by various iron-containing enzymes. Requirement of iron for life can also be illustrated by the ability of the immune system to restrict microbial invasion by iron-sequestrating strategies (Ganz and Nemeth, 2015). Since there is no physiologically specialized excretory pathway for iron, the total body iron content and its distribution is tightly regulated by elaborate homeostatic mechanisms acting upon iron acquisition via the gastrointestinal tract and iron recycling by the reticuloendothelial system (Crielaard et al., 2017). Primary and secondary hemochromatoses are diseases of systemic iron mishandling, which culminate in iron overload and dysfunction of iron-laden organs (Fleming and Ponka, 2012). Worldwide impact of this condition can be exemplified by complications of thalassemia, one of the most common monogenic disorders with the reported incidence exceeding 68,000 cases/year (Weatherall, 2010). The increased iron burden in thalassemic patients is the result of uncontrolled dietary iron uptake and, in severe cases, repeated blood transfusion (Cao and Galanello, 2010). Excess iron subsequently accumulates in several vital organs, with the heart being one of the major targets (Siri-Angkul et al., 2018a). Unsurprisingly, a substantial fraction (> 60%) of thalassemia-related mortality is caused by cardiac complications (Borgna-Pignatti et al., 2005).

Cardiac iron deposition is markedly heterogenous and preferentially occurs in the basal segments of the anterior and inferior ventricular walls (Buja and Roberts, 1971; Walker, 2002). Patchy myocardial fibrosis has also been reported (Buja and Roberts, 1971), but details regarding iron overload-induced structural changes are lacking. Moreover, factors other than iron toxicity also contribute to cardiac remodeling (e.g., high-output hemodynamic status in iron-overload patients with inadequate hemoglobin). Thus, the exact role of iron in cardiac structural remodeling remains to be clarified. Both iron-overload condition and heart failure can contribute to the arrhythmia risk (Kremastinos and Farmakis, 2011). The dilated and remodeled cardiac chambers become arrhythmogenic substrate. However, arrhythmia incidence in patients with transfusion-dependent thalassemia was reportedly higher than the transfusion-independent form, even though the latter group had more severe volume overload and larger ventricular volumes (Aessopos et al., 2004). This clinical finding defies simple causal relationship between heart failure and arrhythmias and suggests the independent arrhythmogenic effect of iron toxicity.

Iron-overloaded hearts exhibits gradual deterioration in both mechanical function and electrical activity. Early detectable arrhythmias include occasional premature atrial/ventricular contractions and first-degree atrioventricular (AV) block, whereas sinoatrial node dysfunction, atrial flutter/fibrillation, second- and third-degree AV block, and ventricular tachycardia/fibrillation tend to occur in advanced stages of the disease (Engle et al., 1964; Kaye and Owen, 1978; Rosenqvist and Hultcrantz, 1989; Wang et al., 1994). Repolarization abnormality including nonspecific ST-T wave changes and QTc dispersion have also been reported (Ulger et al., 2006; Russo et al., 2011; Detterich et al., 2012). Despite these well-documented abnormal electrocardiographic findings, the fundamental aspect of iron-induced arrhythmogenesis is still incompletely understood. To encourage and facilitate further research into the elusive cellular mechanisms of arrhythmias in the siderotic heart, information from electrophysiological studies is summarized and discussed in this article. For detailed clinical information, please see a clinical review by Krematinos and Farmakis (Kremastinos and Farmakis, 2011).

Effects of Iron Overload on Cardiac Action Potential Morphology

The first reported recording of action potential (AP) in iron-overloaded rat LV cardiomyocytes (Supplementary Table 1) demonstrated that 24-h incubation in ferric ammonium citrate (40–80 μg Fe/ml) caused reduced AP amplitude (APA), but the AP duration measured at 40% and 80% repolarization (APD₄₀ and APD₈₀) remained unchanged (Link et al., 1989). However, it has been shown later, also in rat LV cardiomyocytes, that incubation in the same concentrations of ferric ammonium citrate but with longer incubation time (72 h) was sufficient to induce shortened APD₅₀ in addition to the reduced APA (Kuryshev et al., 1999). Consistently, LV cardiomyocytes isolated from Mongolian gerbils which had been subjected to chronic subcutaneous iron injection also had decreased APA and shortened APD₅₀ (Kuryshev et al., 1999). In mouse SAN cardiomyocytes, chronic iron overload caused reduced APA, shortened APD₅₀ and APD₉₀, and a decreased spontaneous AP firing rate due to a decreased slope of diastolic depolarization (phase 4); however, the maximum diastolic potential and the slope of rapid depolarization (phase 0) were unaffected (Rose et al., 2011). Investigations into the effect of iron overload on the resting membrane potential (RMP) of ventricular cardiomyocytes in different models yielded different results. Slightly depolarized RMP (≈3–4 mV) has been observed in the LV cardiomyocytes from rats subjected to acute iron loading (80 μg Fe/ml, 24 h) (Link et al., 1989) and from Mongolian gerbils with chronic iron overload (Kuryshev et al., 1999). However, in the study by Kuryshev et al., this subtle RMP depolarization did not occur in iron-overloaded rat LV cardiomyocytes (40–80 μg Fe/ml, 72 h) (Kuryshev et al., 1999). Interspecies variation and different durations of iron exposure may account for these discordant findings.

Since the generation of reactive oxygen species (ROS) is likely to be a core pathophysiological process of iron-related cellular/organellar injury (Papanikolaou and Pantopoulos, 2005), research focusing on alterations of AP in the presence of oxidative stress should also be considered. Sequential three-phase changes of AP due to severe oxidative stress have been described in isolated rat and guinea-pig ventricular cardiomyocytes, specifically: (1) APD prolongation with early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs), (2) shortened APD and depolarized RMP, and (3) overt and persistent depolarization leading to loss of excitability (Beresewicz and Horackova, 1991). Although the oxidative stress examined in this study was induced by direct treatment with hydrogen peroxide (H₂O₂), the results may be considered to represent the downstream effects of iron-induced ROS-mediated cellular damage on the electrical properties of the cardiomyocyte. Interestingly, ROS-induced afterdepolarizations have been mechanistically linked to overactivation of Ca²⁺/calmodulin kinase (CaMK) II in rabbit ventricular cardiomyocytes (Xie et al., 2009). As expected, treatment with deferoxamine could attenuate the H₂O₂-inducedAP changes (Beresewicz and Horackova, 1991), most likely by chelating the pre-existing intracellular iron, which could catalyze the conversion of H₂O₂ and superoxide anion (^•O₂^-) to an even more potent ROS, hydroxyl radical (^•OH) (Papanikolaou and Pantopoulos, 2005).

The precise mechanism for QTc prolongation/dispersion in patients with iron overload remains obscure. Since iron loading was shown to induce APD shortening in various models (Supplementary Table 1), current cellular experimental results seem to contradict the clinical findings. However, as mentioned earlier, ROS, which is the downstream product of iron-catalyzed redox reactions, could prolong the APD and induce afterdepolarizations/triggered activities (Beresewicz and Horackova, 1991; Xie et al., 2009). In addition, given the heterogeneity of cardiac iron deposition and fibrosis (Buja and Roberts, 1971; Walker, 2002), various degrees of repolarization abnormality can occur in different myocardial regions. These could contribute to QTc prolongation/dispersion.

Effects of Iron Overload on Cardiac Calcium Channel Currents and Transporters

Altered AP parameters are the manifestation of the underlying changes in multiple trans-sarcolemmal ionic currents including those from Ca²⁺ channels. Under iron-overload conditions, voltage gated L-type and T-type Ca²⁺ channels (LTCC and TTCC) have been proposed as candidate molecular entrances into the cardiomyocyte for the ferrous form (Fe²⁺) of non-transferrin bound iron (NTBI) (Tsushima et al., 1999; Oudit et al., 2003; Kumfu et al., 2011; Lopin et al., 2012). This postulation has been supported by the fact that cardiac Fe²⁺ loading relies on electrical excitability of the heart and is sensitive to modulators of LTCC and TTCC (Tsushima et al., 1999; Oudit et al., 2003; Kumfu et al., 2011). However, the relative contribution of Ca²⁺ channels in iron uptake into the cardiomyocyte remains to be evaluated, given only a limited iron current via LTCC was observed even under a very high concentration of iron (15 mM) (Tsushima et al.,1999). Although the proof-of-concept experiment showing permeation and blockade of the TTCC by Fe²⁺in Ca_V3.1-transfected HEK293 cells has been reported (Lopin et al., 2012; Supplementary Table 2), knowledge regarding the direct electrophysiological effects of iron overload on the natively expressed cardiac TTCC is still lacking. There are only a limited number of studies concerning altered LTCC function in iron-overloaded cardiomyocytes (Supplementary Table 2).

In isolated rat LV cardiomyocytes superfused with 2 mM Ca²⁺ plus various concentrations of iron, Fe²⁺, but not Fe³⁺, reversibly altered kinetic properties of the LTCC. Fe²⁺ (0.5–4 mM) delayed L-type Ca²⁺ current (I_Ca,L) inactivation in a dose-dependent manner (Tsushima et al., 1999), possibly due to competitive binding of Fe²⁺ at the C-terminal Ca²⁺ binding site of the LTCC, thus attenuating Ca²⁺-mediated inactivation (Imredy and Yue, 1994; de Leon et al., 1995). Lower concentration of extracellular Fe²⁺ (0.5 mM) accentuated peak I_Ca,L and increased net Ca²⁺ influx via the LTCC, but these parameters were suppressed at higher Fe²⁺ concentrations (1–4 mM) (Tsushima et al., 1999). Even though the reduced peak I_Ca,L at high Fe²⁺ concentrations could potentially be explained by competitive channel permeation between Fe²⁺ and Ca²⁺, the mechanism by which the lower concentration of Fe²⁺ increased peak I_Ca,L remains obscure. It is also not known whether or not, and how, these iron-induced perturbations of the I_Ca,L affect the T-type Ca²⁺ current (I_Ca,T). Hypothetically, in the face of impaired I_Ca,L, cellular electrophysiological remodeling that leads to the compensatory upregulation of I_Ca,T channel may occur. Proving this hypothesis will clarify the relative roles of LTCC and TTCC in cardiac iron-overload conditions, as well as the role of pharmacological modulations of TTCC.

In contrast, LV cardiomyocytes from chronically iron-overloaded mice exhibited no changes in the peak, inactivation, and current-voltage relationship of I_Ca,L (Oudit et al., 2003). Different means of iron loading and the absence of Fe²⁺ in the superfusing extracellular solution during the patch-clamp recording in this study could be responsible for this discrepancy. Single-channel recording, which can provide additional information of single-channel conductance and gating property, has not been reported in both studies.

Effects of iron overload on I_Ca,L in other locations in the heart (besides the ventricles) have also been evaluated (Supplementary Table 2). In mice, a significant difference between the heart rates of control and chronically iron-treated groups could not be eliminated by complete sympathovagal blockade, suggesting that marked bradycardia in iron-overloaded animals was due to abnormal impulse generation by the SAN instead of autonomic dysfunction (Rose et al., 2011). Further experiments in isolated mouse SAN and right atrial appendage (RAA) cardiomyocytes revealed that peak I_Ca,L was reduced and a depolarizing shift of the current-voltage relationship was observed (Rose et al., 2011). It has been proposed that the preferential reduction of I_Ca,L at negative membrane potentials was the result of differential effects of chronic iron overload on two components of I_Ca,L expressed in the atria and the conducting system, Ca_V1.2- and Ca_V1.3-mediated I_Ca,L (Mangoni et al., 2003). Ca_V1.3-mediated I_Ca,L is normally activated at a more negative membrane potential (≈-50 mV) compared to Ca_V1.2-mediated I_Ca,L (≈-30 mV), and the depolarizing shift of I_Ca,L current-voltage relationship in iron-overloaded SAN and RAA cardiomyocytes resembled the results described in Ca_V1.3-deficient mice (Zhang et al., 2002, 2005). Furthermore, I_Ca,L in LV cardiomyocytes, which does not express the Ca_V1.3-dependent component, has been shown to be unaffected by chronic iron overload (Oudit et al., 2003). Thus, selective suppression of Ca_V1.3-mediated I_Ca,L by chronic iron overload was suggested. This assumption has been supported by the finding that mRNA expression of Ca_V1.3, but not Ca_V1.2, was markedly reduced (> 40%) in RAA cardiomyocytes from chronically iron-overloaded mice. The attempt to measure the protein levels failed due to a technical error (Rose et al., 2011).

Abnormal trans-sarcolemmal and subcellular Ca²⁺ fluxes mediated by various transport mechanisms, other than the above-discussed Ca²⁺ currents, may contribute to electrical instability of the cardiomyocyte (Ter Keurs and Boyden, 2007; Zhao et al., 2012). These include the potential involvement of the Ca²⁺ handling processes in iron-induced arrhythmogenesis. Growing evidence indicates that oxidative stress accompanying iron-overload conditions can interfere with the function of multiple Ca²⁺cycling machineries (Khamseekaew et al., 2016; Gordan et al., 2018). Excess ROS could impair cytosolic Ca²⁺ removal by the Na⁺-Ca²⁺ exchanger (NCX) (Xu et al., 1997), sarcolemmal Ca²⁺ ATPase (Kaneko et al., 1989a,b), and sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) (Zeitz et al., 2002). Moreover, Fe²⁺exerts its ROS-independent inhibitory effect on ryanodine receptors (RyR) via competitive binding, thus compromising Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum (Kim et al., 1995). The causal relationship between this complex spatiotemporal Ca²⁺ dysregulation and membrane potential fluctuation (EADs, DADs, and triggered activities) is yet to be systematically proven in iron-overloaded cardiomyocytes.

Effects of Iron Overload on Cardiac Sodium Channel Currents

Consistent with the decreased AP amplitude, patch clamp recording demonstrated dose- and time-dependent reduction of peak Na⁺ current (I_Na) in short-term iron-incubated (80 μg Fe/ml, 72 h) cultured rat LV cardiomyocytes and also in freshly isolated LV cardiomyocytes from chronically iron-injected gerbils (Kuryshev et al., 1999). Iron overload shifted the inactivation curve of I_Na toward negative voltages and delayed the recovery from inactivation in both species (Kuryshev et al., 1999). Single-channel recording revealed that Na⁺ channels from the iron-incubated rat LV cardiomyocytes had reduced opening probability and a decreased average single-channel current (Kuryshev et al., 1999). The amount of Na⁺ channel protein was unaffected (Kuryshev et al., 1999). These findings are summarized in Supplementary Table 2.

Effects of Iron Overload on Cardiac Potassium Channel Currents

Multiple K⁺ currents play important roles in the repolarization phase of AP and are the key determinant of the resting membrane potential. Several members of this heterogeneous group were shown to be affected by iron overload conditions (Supplementary Table 2). In rats and gerbils LV cardiomyocytes subjected to acute and chronic iron loading protocols, the peak of transient outward K⁺ current (I_to) was increased, and a small depolarizing shift (≈5 mV) of its inactivation curve was observed (Kuryshev et al., 1999). However, the inward rectifier K⁺ current (I_K1) was unaffected (Kuryshev et al., 1999).

In guinea-pig LV cardiomyocytes, the adenosine triphosphate (ATP)-sensitive K⁺(K_ATP) channel current (I_KATP) could be activated by iron-induced oxidative stress (Tokube et al., 1998). Interestingly, activating the cardiac K_ATP channel could increase ^•OH levels, whereas blocking it led to decreased ^•OH levels in rat myocardium (Obata and Yamanaka, 2000; Han et al., 2002). These findings indicate the presence of reciprocal positive-feedback interactions between ROS production and the altered function of the K_ATP channel. Moreover, the inhibitory effect of ROS on the human ether-a-go-go-related gene (hERG) K⁺ channels, which mediate the rapid delayed rectifier K⁺ current (I_Kr) in the heart, has already been studied in various cell types (Vandenberg, 2010). Nevertheless, the specific effects of iron overload on cardiac I_Kr as well as the link to cardiac repolarization abnormality have not been clearly established. Changes in I_Kr and/or I_Ks may eventually lead to altered maximum diastolic potential. In this way, indirect chronotropic effect may ensue. Thus, the relationships between various K⁺ currents and iron-derived ROS highlight another interesting domain to be explored.

Effects of Iron Overload on Hyperpolarization-Activated Pacemaker Currents

Sinus bradycardia and SAN dysfunction have been observed in iron overload patients (Detterich et al., 2012), and in chronically iron-overloaded mice (Rose et al., 2011). SAN cardiomyocytes from mice subjected to chronic iron overload exhibited decreased slope diastolic depolarization (Rose et al., 2011). Although the hyperpolarization-activated pacemaker current (I_h or I_f) is a major contributor of spontaneous depolarization in SAN cells, Rose et al. reported no significant changes in I_f density and current-voltage relationship in mice with chronic iron overload (Rose et al., 2011; Supplementary Table 2). Therefore, decreased Ca_V1.3-mediated I_Ca,L has been proposed as the underlying mechanism of the suppressed SAN automaticity (Rose et al., 2011).

Forging the Field: Additional Issues to Be Explored in Iron-Associated Cardiac Pathoelectrophysiology

In iron-overload patients, cardiac ion channels operate in the presence of both high intracellular labile iron pool and high extracellular NTBI. Most cellular electrophysiological studies exploited chronic iron loading in various species, while in some studies the cardiomyocytes were briefly incubated in iron or acutely exposed to the iron-containing solution at the time of patch-clamp recording (Supplementary Tables 1, 2). Thus, different iron-loading protocols, especially in terms of the duration of iron exposure, may lead to discrepant findings, as seen in the case of I_Ca-suppressing effects in acute and chronic iron overload models (Tsushima et al., 1999; Oudit et al., 2003). Moreover, other pathways for iron uptake into the cardiomyocyte exist, including receptor-mediated endocytosis of the well-recognized transferrin-Fe³⁺ complex and a recently described lipocalin 2-siderophore-Fe³⁺ complex (Yang et al., 2002; Xu et al., 2012; Siri-Angkul et al., 2018b). It remains to be examined whether these forms of cardiac iron-loading, bypassing the ion channel-mediated transport system, indirectly exert any effects on the electrophysiological properties of cardiomyocytes.

Additionally, investigating deeper into the genomic and molecular mechanisms (i.e., abnormal gene expression, aberrations in posttranslational modifications or trafficking of ion channels and their regulatory proteins, and interactions between the electrophysiological events and other biochemical processes) is of no less importance. This point can be exemplified by the complexity and multifaceted nature of intracellular Ca²⁺ regulation. Understanding of how deranged Ca²⁺ cycling influences the membrane excitability, in the specific context of iron-overloaded cardiomyocyte, is still incomplete.

The main purpose of this article is to provide the molecular/cellular basis of iron-induced cardiac pathoelectrophysiology. However, it should be noted that additional data from other aspects are also essential for bridging the gap of knowledge between the cellular phenomena and the clinical phenotypes. These include, but not limited to, alterations in intercellular coupling, roles of non-parenchymal cells (e.g., fibroblasts and macrophages), structural remodeling, systemic immunoinflammatory responses, and coincident extracardiac pathology (e.g., iron-induced endocrinopathies and autonomic disturbances) (Kremastinos and Farmakis, 2011). Due to lack of information, the extent and the characteristics of interactions between these phenomena and cellular electrophysiological changes are difficult to be determined at this time.

Despite incomplete characterization of iron-induced K⁺ current alterations, amiodarone has been used to treat supraventricular arrhythmias commonly found in iron-overload patients (Pennell et al., 2013). Potential role of LTCC blockers as an inhibitor of cardiac iron uptake is also being evaluated in clinical trials (Fernandes et al., 2013, 2016; Shakoor et al., 2014; Eghbali et al., 2017; Sadaf et al., 2018). It is still unknown whether the use of LTCC blockers will exert any significant electrophysiological effects. Clinically, diseases of the cardiac rhythm can be classified according to their ECG features and/or functional anatomical characteristics (e.g., atrial arrhythmias, ventricular arrhythmias, etc). However, for siderotic hearts, current data are still insufficient to formulate specific pathophysiological explanations for such clinical entities. We hope that, with more information in the future, iron overload-induced arrhythmias could be revisited with this useful functional anatomy-based approach.

Conclusion

Although some perturbations of ionic channels/currents of iron-induced electrical disturbances in the heart have already been characterized, current electrophysiological knowledge regarding the siderotic heart is still far from complete, especially the lack of data in human cardiomyocytes. Further cellular electrophysiological insights are certain to provide a firm base for our in-depth understanding and for future investigations in more complicated systems (e.g., myocardial tissue, whole-heart, and clinical cardiac electrophysiology), with identification of potential therapeutic targets and development of practical treatment strategies being the extremely worthwhile goals.

Author Contributions

NS-A, L-HX searched the literature, collected the data, analyzed the data, and wrote the manuscript. SC, NC studied the design, searched the literature, analyzed the data, and wrote the manuscript.

Funding

This work was supported by the National Institute of Health (R01s HL97979 and HL133294 to L-HX), the American Heart Association (Grant-in-Aid to L-HX), the Thailand Research Fund (RTA6080003 to SC), the National Science and Technology Development Agency Thailand (NSTDA Research Chair grant to NC), and the Chiang Mai University Center of Excellence Award (NC).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2018.01615/full#supplementary-material

References

Aessopos, A., Farmakis, D., Hatziliami, A., Fragodimitri, C., Karabatsos, F., Joussef, J., et al. (2004). Cardiac status in well-treated patients with thalassemia major. Eur. J. Haematol. 73, 359–366. doi: 10.1111/j.1600-0609.2004.00304.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Beresewicz, A., and Horackova, M. (1991). Alterations in electrical and contractile behavior of isolated cardiomyocytes by hydrogen peroxide: possible ionic mechanisms. J. Mol. Cell Cardiol. 23, 899–918. doi: 10.1016/0022-2828(91)90133-7

PubMed Abstract | CrossRef Full Text | Google Scholar

Borgna-Pignatti, C., Cappellini, M. D., De Stefano, P., Del Vecchio, G. C., Forni, G. L., Gamberini, M. R., et al. (2005). Survival and complications in thalassemia. Ann. N. Y. Acad. Sci. 1054, 40–47. doi: 10.1196/annals.1345.006

PubMed Abstract | CrossRef Full Text | Google Scholar

Buja, L. M., and Roberts, W. C. (1971). Iron in the heart. Etiology and clinical significance. Am. J. Med. 51, 209–221. doi: 10.1016/0002-9343(71)90240-3

CrossRef Full Text | Google Scholar

Cao, A., and Galanello, R. (2010). Beta-thalassemia. Genet. Med. 12, 61–76. doi: 10.1097/GIM.0b013e3181cd68ed

PubMed Abstract | CrossRef Full Text | Google Scholar

Crielaard, B. J., Lammers, T., and Rivella, S. (2017). Targeting iron metabolism in drug discovery and delivery. Nat. Rev. Drug Discov. 16, 400–423. doi: 10.1038/nrd.2016.248

PubMed Abstract | CrossRef Full Text | Google Scholar

de Leon, M., Wang, Y., Jones, L., Perez-Reyes, E., Wei, X., Soong, T. W., et al. (1995). Essential Ca²⁺-binding motif for Ca²⁺-sensitive inactivation of L-type Ca²⁺ channels. Science 270, 1502–1506. doi: 10.1126/science.270.5241.1502

CrossRef Full Text | Google Scholar

Detterich, J., Noetzli, L., Dorey, F., Bar-Cohen, Y., Harmatz, P., Coates, T., et al. (2012). Electrocardiographic consequences of cardiac iron overload in thalassemia major. Am. J. Hematol. 87, 139–144. doi: 10.1002/ajh.22205

PubMed Abstract | CrossRef Full Text | Google Scholar

Eghbali, A., Kazemi, H., Taherahmadi, H., Ghandi, Y., Rafiei, M., and Bagheri, B. (2017). A randomized, controlled study evaluating effects of amlodipine addition to chelators to reduce iron loading in patients with thalassemia major. Eur. J. Haematol. 99, 577–581. doi: 10.1111/ejh.12977

PubMed Abstract | CrossRef Full Text | Google Scholar

Engle, M. A., Erlandson, M., and Smith, C. H. (1964). Late cardiac complications of chronic, severe, refractory anemia with hemochromatosis. Circulation 30, 698–705. doi: 10.1161/01.CIR.30.5.698

PubMed Abstract | CrossRef Full Text | Google Scholar

Fernandes, J. L., Loggetto, S. R., Verissimo, M. P., Fertrin, K. Y., Baldanzi, G. R., Fioravante, L. A., et al. (2016). A randomized trial of amlodipine in addition to standard chelation therapy in patients with thalassemia major. Blood 128, 1555–1561. doi: 10.1182/blood-2016-06-721183

PubMed Abstract | CrossRef Full Text | Google Scholar

Fernandes, J. L., Sampaio, E. F., Fertrin, K., Coelho, O. R., Loggetto, S., Piga, A., et al. (2013). Amlodipine reduces cardiac iron overload in patients with thalassemia major: a pilot trial. Am. J. Med. 126, 834–837. doi: 10.1016/j.amjmed.2013.05.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Fleming, R. E., and Ponka, P. (2012). Iron overload in human disease. N. Engl. J. Med. 366, 348–359. doi: 10.1056/NEJMra1004967

PubMed Abstract | CrossRef Full Text | Google Scholar

Ganz, T., and Nemeth, E. (2015). Iron homeostasis in host defence and inflammation. Nat. Rev. Immunol. 15, 500–510. doi: 10.1038/nri3863

PubMed Abstract | CrossRef Full Text | Google Scholar

Gordan, R., Wongjaikam, S., Gwathmey, J. K., Chattipakorn, N., Chattipakorn, S. C., and Xie, L. H. (2018). Involvement of cytosolic and mitochondrial iron in iron overload cardiomyopathy: an update. Heart Fail Rev 23, 801–816. doi: 10.1007/s10741-018-9700-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Han, J., Kim, N., Park, J., Seog, D. H., Joo, H., and Kim, E. (2002). Opening of mitochondrial ATP-sensitive potassium channels evokes oxygen radical generation in rabbit heart slices. J. Biochem. 131, 721–727. doi: 10.1093/oxfordjournals.jbchem.a003157

PubMed Abstract | CrossRef Full Text | Google Scholar

Imredy, J. P., and Yue, D. T. (1994). Mechanism of Ca²⁺-sensitive inactivation of L-type Ca²⁺ channels. Neuron 12, 1301–1318. doi: 10.1016/0896-6273(94)90446-4

CrossRef Full Text | Google Scholar

Kaneko, M., Beamish, R. E., and Dhalla, N. S. (1989a). Depression of heart sarcolemmal Ca²⁺-pump activity by oxygen free radicals. Am. J. Physiol. 256, H368–H374.

Google Scholar

Kaneko, M., Elimban, V., and Dhalla, N. S. (1989b). Mechanism for depression of heart sarcolemmal Ca²⁺ pump by oxygen free radicals. Am. J. Physiol. 257, H804–H811.

Google Scholar

Kaye, S. B., and Owen, M. (1978). Cardiac arrhythmias in thalassaemia major: evaluation of chelation treatment using ambulatory monitoring. Br. Med. J. 1:342. doi: 10.1136/bmj.1.6109.342

CrossRef Full Text | Google Scholar

Khamseekaew, J., Kumfu, S., Chattipakorn, S. C., and Chattipakorn, N. (2016). Effects of iron overload on cardiac calcium regulation: translational insights into mechanisms and management of a global epidemic. Can. J. Cardiol. 32, 1009–1016. doi: 10.1016/j.cjca.2015.10.012

PubMed Abstract | CrossRef Full Text | Google Scholar

Kim, E., Giri, S. N., and Pessah, I. N. (1995). Iron(II) is a modulator of ryanodine-sensitive calcium channels of cardiac muscle sarcoplasmic reticulum. Toxicol. Appl. Pharmacol. 130, 57–66. doi: 10.1006/taap.1995.1008

PubMed Abstract | CrossRef Full Text | Google Scholar

Kremastinos, D. T., and Farmakis, D. (2011). Iron overload cardiomyopathy in clinical practice. Circulation 124, 2253–2263. doi: 10.1161/CIRCULATIONAHA.111.050773

PubMed Abstract | CrossRef Full Text | Google Scholar

Kumfu, S., Chattipakorn, S., Srichairatanakool, S., Settakorn, J., Fucharoen, S., and Chattipakorn, N. (2011). T-type calcium channel as a portal of iron uptake into cardiomyocytes of beta-thalassemic mice. Eur. J. Haematol. 86, 156–166. doi: 10.1111/j.1600-0609.2010.01549.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Kuryshev, Y. A., Brittenham, G. M., Fujioka, H., Kannan, P., Shieh, C. C., Cohen, S. A., et al. (1999). Decreased sodium and increased transient outward potassium currents in iron-loaded cardiac myocytes. Implications for the arrhythmogenesis of human siderotic heart disease. Circulation 100, 675–683. doi: 10.1161/01.CIR.100.6.675

PubMed Abstract | CrossRef Full Text | Google Scholar

Link, G., Athias, P., Grynberg, A., Pinson, A., and Hershko, C. (1989). Effect of iron loading on transmembrane potential, contraction, and automaticity of rat ventricular muscle cells in culture. J. Lab. Clin. Med. 113, 103–111.

PubMed Abstract | Google Scholar

Lopin, K. V., Gray, I. P., Obejero-Paz, C. A., Thevenod, F., and Jones, S. W. (2012). Fe²⁺ block and permeation of CaV3.1 (alpha1G) T-type calcium channels: candidate mechanism for non-transferrin-mediated Fe²⁺ influx. Mol. Pharmacol. 82, 1194–1204. doi: 10.1124/mol.112.080184

PubMed Abstract | CrossRef Full Text | Google Scholar

Mangoni, M. E., Couette, B., Bourinet, E., Platzer, J., Reimer, D., Striessnig, J., et al. (2003). Functional role of L-type Cav1.3 Ca²⁺ channels in cardiac pacemaker activity. Proc. Natl. Acad. Sci. U.S.A. 100, 5543–5548. doi: 10.1073/pnas.0935295100

PubMed Abstract | CrossRef Full Text | Google Scholar

Obata, T., and Yamanaka, Y. (2000). Block of cardiac ATP-sensitive K⁺ channels reduces hydroxyl radicals in the rat myocardium. Arch. Biochem. Biophys. 378, 195–200. doi: 10.1006/abbi.2000.1830

PubMed Abstract | CrossRef Full Text | Google Scholar

Oudit, G. Y., Sun, H., Trivieri, M. G., Koch, S. E., Dawood, F., Ackerley, C., et al. (2003). L-type Ca²⁺ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy. Nat. Med. 9, 1187–1194. doi: 10.1038/nm920

PubMed Abstract | CrossRef Full Text | Google Scholar

Papanikolaou, G., and Pantopoulos, K. (2005). Iron metabolism and toxicity. Toxicol. Appl. Pharmacol. 202, 199–211. doi: 10.1016/j.taap.2004.06.021

PubMed Abstract | CrossRef Full Text | Google Scholar

Pennell, D. J., Udelson, J. E., Arai, A. E., Bozkurt, B., Cohen, A. R., Galanello, R., et al. (2013). Cardiovascular function and treatment in beta-thalassemia major: a consensus statement from the American Heart Association. Circulation 128, 281–308. doi: 10.1161/CIR.0b013e31829b2be6

PubMed Abstract | CrossRef Full Text | Google Scholar

Rose, R. A., Sellan, M., Simpson, J. A., Izaddoustdar, F., Cifelli, C., Panama, B. K., et al. (2011). Iron overload decreases CaV1.3-dependent L-type Ca²⁺ currents leading to bradycardia, altered electrical conduction, and atrial fibrillation. Circ. Arrhythm Electrophysiol. 4, 733–742. doi: 10.1161/CIRCEP.110.960401

PubMed Abstract | CrossRef Full Text | Google Scholar

Rosenqvist, M., and Hultcrantz, R. (1989). Prevalence of a haemochromatosis among men with clinically significant bradyarrhythmias. Eur. Heart J. 10, 473–478. doi: 10.1093/oxfordjournals.eurheartj.a059512

PubMed Abstract | CrossRef Full Text | Google Scholar

Russo, V., Rago, A., Pannone, B., Papa, A. A., Di Meo, F., Mayer, M. C., et al. (2011). Dispersion of repolarization and beta-thalassemia major: the prognostic role of QT and JT dispersion for identifying the high-risk patients for sudden death. Eur. J. Haematol. 86, 324–331. doi: 10.1111/j.1600-0609.2011.01579.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Sadaf, A., Hasan, B., Das, J. K., Colan, S., and Alvi, N. (2018). Calcium channel blockers for preventing cardiomyopathy due to iron overload in people with transfusion-dependent beta thalassaemia. Cochrane Database Syst. Rev. 7:CD011626. doi: 10.1002/14651858.CD011626.pub2

PubMed Abstract | CrossRef Full Text | Google Scholar

Shakoor, A., Zahoor, M., Sadaf, A., Alvi, N., Fadoo, Z., Rizvi, A., et al. (2014). Effect of L-type calcium channel blocker (amlodipine) on myocardial iron deposition in patients with thalassaemia with moderate-to-severe myocardial iron deposition: protocol for a randomised, controlled trial. BMJ Open 4:e005360. doi: 10.1136/bmjopen-2014-005360

PubMed Abstract | CrossRef Full Text | Google Scholar

Siri-Angkul, N., Chattipakorn, S. C., and Chattipakorn, N. (2018a). Diagnosis and treatment of cardiac iron overload in transfusion-dependent thalassemia patients. Expert Rev. Hematol. 11, 471–479. doi: 10.1080/17474086.2018.1476134

PubMed Abstract | CrossRef Full Text | Google Scholar

Siri-Angkul, N., Chattipakorn, S. C., and Chattipakorn, N. (2018b). Roles of lipocalin 2 and adiponectin in iron overload cardiomyopathy. J. Cell. Physiol. 233, 5104–5111. doi: 10.1002/jcp.26318

PubMed Abstract | CrossRef Full Text | Google Scholar

Ter Keurs, H. E., and Boyden, P. A. (2007). Calcium and arrhythmogenesis. Physiol. Rev. 87, 457–506. doi: 10.1152/physrev.00011.2006

PubMed Abstract | CrossRef Full Text | Google Scholar

Tokube, K., Kiyosue, T., and Arita, M. (1998). Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes. Pflugers Arch. 437, 155–157. doi: 10.1007/s004240050760

PubMed Abstract | CrossRef Full Text | Google Scholar

Tsushima, R. G., Wickenden, A. D., Bouchard, R. A., Oudit, G. Y., Liu, P. P., and Backx, P. H. (1999). Modulation of iron uptake in heart by L-type Ca²⁺ channel modifiers: possible implications in iron overload. Circ. Res. 84, 1302–1309. doi: 10.1161/01.RES.84.11.1302

CrossRef Full Text | Google Scholar

Ulger, Z., Aydinok, Y., Levent, E., Gurses, D., and Ozyurek, A. R. (2006). Evaluation of QT dispersion in beta thalassaemia major patients. Am. J. Hematol. 81, 901–906. doi: 10.1002/ajh.20679

PubMed Abstract | CrossRef Full Text | Google Scholar

Vandenberg, J. I. (2010). Oxidative stress fine-tunes the dance of hERG K⁺ channels. J. Physiol. 588, 2975. doi: 10.1113/jphysiol.2010.195651

CrossRef Full Text | Google Scholar

Walker, J. M. (2002). The heart in thalassaemia. Eur. Heart J. 23, 102–105. doi: 10.1053/euhj.2001.2850

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, T. L., Chen, W. J., Liau, C. S., and Lee, Y. T. (1994). Sick sinus syndrome as the early manifestation of cardiac hemochromatosis. J. Electrocardiol. 27, 91–96. doi: 10.1016/S0022-0736(05)80114-1

PubMed Abstract | CrossRef Full Text | Google Scholar

Weatherall, D. J. (2010). The inherited diseases of hemoglobin are an emerging global health burden. Blood 115, 4331–4336. doi: 10.1182/blood-2010-01-251348

PubMed Abstract | CrossRef Full Text | Google Scholar

Xie, L. H., Chen, F., Karagueuzian, H. S., and Weiss, J. N. (2009). Oxidative-stress-induced after depolarizations and calmodulin kinase II signaling. Circ. Res. 104, 79–86. doi: 10.1161/CIRCRESAHA.108.183475

PubMed Abstract | CrossRef Full Text | Google Scholar

Xu, G., Ahn, J., Chang, S., Eguchi, M., Ogier, A., Han, S., et al. (2012). Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation. J. Biol. Chem. 287, 4808–4817. doi: 10.1074/jbc.M111.275719

PubMed Abstract | CrossRef Full Text | Google Scholar

Xu, K. Y., Zweier, J. L., and Becker, L. C. (1997). Hydroxyl radical inhibits sarcoplasmic reticulum Ca²⁺-ATPase function by direct attack on the ATP binding site. Circ. Res. 80, 76–81. doi: 10.1161/01.RES.80.1.76

CrossRef Full Text | Google Scholar

Yang, J., Goetz, D., Li, J. Y., Wang, W., Mori, K., Setlik, D., et al. (2002). An iron delivery pathway mediated by a lipocalin. Mol. Cell. 10, 1045–1056. doi: 10.1016/S1097-2765(02)00710-4

CrossRef Full Text | Google Scholar

Zeitz, O., Maass, A. E., Van Nguyen, P., Hensmann, G., Kogler, H., Moller, K., et al. (2002). Hydroxyl radical-induced acute diastolic dysfunction is due to calcium overload via reverse-mode Na⁺-Ca²⁺ exchange. Circ. Res. 90, 988–995. doi: 10.1161/01.RES.0000018625.25212.1E

CrossRef Full Text | Google Scholar

Zhang, Z., He, Y., Tuteja, D., Xu, D., Timofeyev, V., Zhang, Q., et al. (2005). Functional roles of Cav1.3(alpha1D) calcium channels in atria: insights gained from gene-targeted null mutant mice. Circulation 112, 1936–1944. doi: 10.1161/CIRCULATIONAHA.105.540070

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhang, Z., Xu, Y., Song, H., Rodriguez, J., Tuteja, D., Namkung, Y., et al. (2002). Functional Roles of Ca(v)1.3 (alpha(1D)) calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ. Res. 90, 981–987. doi: 10.1161/01.RES.0000018003.14304.E2

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhao, Z., Wen, H., Fefelova, N., Allen, C., Baba, A., Matsuda, T., et al. (2012). Revisiting the ionic mechanisms of early afterdepolarizations in cardiomyocytes: predominant by Ca waves or Ca currents? Am. J. Physiol. Heart Circ. Physiol. 302, H1636–H1644. doi: 10.1152/ajpheart.00742.2011

PubMed Abstract | CrossRef Full Text | Google Scholar