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(Circulation. 1999;100:675-683.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Decreased Sodium and Increased Transient Outward Potassium Currents in Iron-Loaded Cardiac Myocytes

Implications for the Arrhythmogenesis of Human Siderotic Heart Disease

Yuri A. Kuryshev, PhD; Gary M. Brittenham, MD; Hisashi Fujioka, PhD; Perry Kannan, PhD; Char-Chang Shieh, PhD; Sidney A. Cohen, MD, PhD; Arthur M. Brown, MD, PhD

From the Rammelkamp Center for Education and Research (Y.A.K., G.M.B., P.K., C.-C.S., A.M.B.), MetroHealth Campus, and the Departments of Physiology and Biophysics (Y.A.K., A.M.B.), Medicine (G.M.G.), and Pathology (H.F.), Case Western Reserve University, Cleveland, Ohio, and Centocor Inc., Molvern, Pa. (S.A.C.).

Correspondence to Arthur Brown, MD, PhD, Rammelkamp Center R301, 2500 MetroHealth Drive, Cleveland, OH 44109-1998. E-mail abrown{at}research.mhmc.org

	Abstract

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Background—Patients with chronic iron overload may develop a cardiomyopathy manifested by ventricular arrhythmias and heart failure. We hypothesized that iron-loaded cardiomyocytes may have abnormal excitability.

Methods and Results—We examined a new model of human iron overload, the Mongolian gerbil given repeated injections of iron dextran. In ventricular myocytes, we measured iron concentration and distribution, action potential, sodium and potassium currents, and sodium channel protein. We showed for the first time that (1) the iron content of gerbil ventricular cardiomyocytes was increased to amounts similar to those of patients with iron-induced cardiomyopathy; (2) the overshoot and duration of the cardiac action potential decreased; (3) sodium current was reduced, steady-state inactivation was enhanced, and single-channel currents were unchanged; and (4) transient outward potassium current was increased, but inwardly rectifying potassium current was unchanged. Neonatal rat cardiomyocytes incubated with iron for 1 to 3 days showed similar changes, and levels of cardiac sodium channel proteins were unchanged.

Conclusions—Abnormal excitability and heterogeneous cardiac iron deposition may cause the arrhythmogenesis of human siderotic heart disease.

Key Words: iron overload • cardiomyopathy • gerbil • sodium channels • potassium channels

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Iron-overload cardiomyopathy produces arrhythmias^{1 2} and heart failure.^{1 2 3 4} It is the most frequent cause of death in thalassemia major,^{1 3 4} and a life-limiting complication of transfusion-dependent refractory anemias, hereditary hemochromatosis, and other forms of iron overload.² Because iron excretion in humans is deficient, iron contained within transfused red cells or absorbed from the diet in excess of requirements progressively accumulates within the body.⁵ Eventually, a characteristic pattern of heterogeneous iron deposition develops within the heart, with the greatest amounts in the left side of the ventricular septum and free wall (especially in the epicardium), smaller amounts in the right ventricle, and still less in the atria.^{6 7} Within the myocardium, the iron is found almost entirely within myocardial cells and not within the interstitium. Past efforts to examine the effects of iron on the heart have been hampered by the lack of a suitable experimental model of human iron overload; most animal species have a far greater capacity to excrete iron than humans. Recently, Carthew et al^{8 9} reported that weekly subcutaneous injections of iron dextran to Mongolian gerbils reproduced critical features of the cardiomyopathy found in human iron overload, in part because the gerbil seems unable to excrete iron as effectively as other rodents. We hypothesized that abnormal excitability of iron-loaded heart cells might contribute to arrhythmias, and we examined membrane currents of single cardiomyocytes isolated from the hearts of iron-loaded gerbils. For comparison with a related, more extensively studied species, we used an in vitro model of cultured neonatal rat cardiomyocytes that were incubated with iron for several days.^{10 11}

	Methods

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Nonheme Iron Concentrations
Nonheme iron concentrations of heart tissue and cardiomyocytes were determined as previously described.¹²

Electron Microscopy
Cardiomyocytes were prepared according to the method of Iancu et al¹³ and examined in a CEM 902 electron microscope (Carl Zeiss Inc).

Cultures of Neonatal Rat Cardiomyocytes
One-day-old rats (Sprague-Dawley; Zivic-Miller Laboratories, Portersville, PA) were anesthetized with Nembutal (50 mg/kg body weight), and hearts were removed surgically. Ventricular cardiomyocytes were isolated using the Neonatal Cardiomyocyte Isolation System.¹⁴ Cells were plated at 2.5x10⁴ cells/cm² onto 35- or 100-mm culture dishes (Falcon) in Dulbecco's modified Eagle medium/nutrient mixture F-12 (1:1) (GIBCO) supplemented with 10% calf serum and 50 µg/mL gentamicin, and then they were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂. After incubation for 16 to 18 hours, cells were washed twice with serum-free Dulbecco's modified Eagle F-12 medium, and ferric ammonium citrate was added at a final concentration of 40 or 80 µg of elemental iron/mL. Both test and control cells were then incubated for 24 to 72 hours, and the medium was changed every 24 hours. All procedures conformed to institutional guidelines for the care and use of animals in research.

Isolation of Gerbil Cardiomyocytes
Mongolian gerbils (females, 6 to 8 weeks old; from Charles River Laboratories, Portage, Mich) were given subcutaneous injections of iron dextran at 200 mg Fe · kg^-1 · wk^-1. Hearts were prepared using Langendorff perfusion and the modified enzymatic method.^{15 16} After perfusion, the left ventricular epicardium was excised, minced, and gently shaken. Dissociated myocytes were stored for 2 to 24 hours at 4°C in a solution containing (in mmol/L): KCl 85, K₂HPO₄ 30, MgCl₂ 5, creatine 5, taurine 20, glucose 20, EGTA 0.125, ß-OH-butyric acid 5, Na₂ATP 2, pyruvic acid 5, and CaCl₂ 0.02 and 50 g/L PVP-40 (pH 7.2). Quiescent, rod-shaped cells were selected for electrophysiology.

Electrophysiology
The cardiac action potential (CAP) of gerbil myocytes was recorded using the whole-cell giga-seal technique.¹⁷ Pipette and bath solutions were, respectively, (in mmol/L): potassium aspartate 140, MgCl₂ 5, HEPES 10, EGTA 10, glucose 10, and Na₂ATP 2 (pH 7.2) and NaCl 140, KCl 5.4, MgCl₂ 1, CaCl₂ 2, HEPES 10, and glucose 10 (pH 7.4). The measured liquid junction potential (LJP) was -10 mV. CAP from cultured neonatal rat myocytes was recorded using the perforated patch method with amphotericin B.¹⁸ The pipette solution was (in mmol/L): KCl 8, potassium aspartate 25, sodium aspartate 5, K₂SO₄ 60, MgCl₂ 1, HEPES 10, EGTA 1, glucose 10, and sucrose 20 (pH 7.2). The measured LJP was -7 mV. CAPs were evoked by depolarizing current pulses (150 pA and 5 ms for gerbil cells or 10 pA and 2 ms for neonatal rat cells) at 1 Hz, with 30-s intervals between series. The first CAP in each series was used to compare parameters, and we always corrected for LJP.

For neonatal rat Na⁺ currents, the pipette and bath solutions were, respectively, (in mmol/L): CsCl 120, NaCl 10, CaCl₂ 1, MgCl₂ 2, EGTA 11, HEPES 10, and Na₂ATP 2 (pH 7.2) and NaCl 140, CsCl 10, KCl 5, MgCl₂ 5, CaCl₂ 0.02, 4-aminopyridine 5, HEPES 10, and glucose 10 (pH 7.4). External Ca²⁺ was reduced to minimize Ca²⁺ currents. Gerbil Na⁺ current was much larger, so we reduced the Na⁺ concentration. For gerbil Na⁺ currents, the pipette and bath solutions were, respectively, (in mmol/L): CsCl 120, CaCl₂ 1, MgCl₂ 2, EGTA 11, HEPES 10, and Na₂ATP 2 (pH 7.2) and NaCl 20, N-methyl-D-glucamine 75, aspartate 75, CsCl 20, KCl 5, MgCl₂ 5, CaCl₂ 0.02, 4-aminopyridine 5, HEPES 10, and glucose 10 (pH 7.4). The series resistance was 3 to 4 megohms, and 90% series resistance compensation was used. Records were low-pass filtered at 10 kHz and digitized at 20 kHz. LJP was 2 mV and was uncorrected.

Single Na⁺ channel currents were registered in cell-attached mode. Pipettes had about 5 megohms resistance, were coated with Sylgard (Dow Corning), and filled with a solution containing (in mmol/L): sodium aspartate 150, CaCl₂ 0.5, MgCl₂ 3, and HEPES 10 (pH 7.4). To zero membrane potential, the bath solution contained (in mmol/L): potassium aspartate 140, sodium aspartate 10, MgCl₂ 5, EGTA 0.5, HEPES 10, and glucose 10 (pH 7.4). Records were filtered and sampled as above. For macropatches, the cell-attached configuration was used; electrodes had a constant diameter and a resistance of 1 megohm. Currents were analyzed with Transit software.¹⁹

K⁺ currents were recorded in whole-cell configuration. Pipette and bath solutions were, respectively, (in mmol/L): potassium aspartate 140, MgCl₂ 5, HEPES 10, EGTA 10, glucose 10, and Na₂ATP 2 (pH 7.2) and NaCl 140, KCl 5.4, MgCl₂ 5, CaCl₂ 0.02, HEPES 10, and glucose 10 (pH 7.4). Series resistance was 3 to 10 megohms, and 90% series resistance compensation was used. Records were low-pass filtered at 1 kHz and sampled at 2 kHz. Data were corrected for a LJP of -10 mV.

Data acquisition and analyses were performed with an Axopatch-1D amplifier, TL-1 interface, and pClamp 5.5.1 or 6 software (Axon Instruments). All experiments were performed at room temperature (20°C to 23°C)

Western Blot Analysis
Equal amounts of total membrane protein (5 µg) isolated from control and iron-treated cardiomyocytes were separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane using an electrical field of 45 V for 16 hours at 4°C. The membrane was probed with the sodium-channel rH1 subtype–specific polyclonal antibody D-492.²⁰ Signals were detected using anti-rabbit secondary antibody followed by electrogenerated chemiluminescence (Amersham Life Sciences Inc) detection methods.

Statistics
The Student's t test was used to compare 2 population samples, and ANOVA was used for >2.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Iron Loading
In the left ventricle and liver from gerbils loaded with iron by weekly injections of iron dextran, the iron level¹² increased progressively (Figure 1), whereas control gerbils were unchanged. In whole hearts from gerbils loaded with iron for 20 weeks, the concentration increased almost 10-fold above controls (from 0.3±0.1 mg of Fe/g of heart, dry weight, to 2.9±1.6 mg of Fe/g of heart; n=5). For comparison, arrhythmias and heart failure develop in patients with left ventricular nonheme iron concentrations of 2.0 mg of Fe/g heart, dry weight.⁶ In cultured neonatal rat cardiomyocytes incubated with 80 µg/mL Fe for 3 days, the increase was also 10-fold (from 0.7±0.2 mg of Fe/g of cells, dry weight, to 8.7±0.9 mg of Fe/g of cells; n=4). In control cells and during incubation with iron, the cell density remained constant. Iron loading also produced cytological changes. Iron was present only in loaded neonatal rat cardiomyocytes (Figure 2), and it appeared as electron-dense granules scattered throughout the cytoplasm or deposited within membrane-bound cytoplasmic organelles known as siderosomes, as reported previously.¹³ In 2 control gerbils, 100 myocytes were examined, and a total of 8 siderosomes, averaging 0.7x0.6 µm, were observed. In 2 iron-loaded gerbils (8 weeks), 100 myocytes contained 39 siderosomes, averaging 3.2x2.0 µm.

View larger version (10K): [in this window] [in a new window]   Figure 1. Iron concentrations in left ventricle (A) and liver (B) of Mongolian gerbils after weekly administration of iron dextran in 3 controls () and 3 iron-loaded animals (•). Approximate threshold for development of cardiac failure in patients with transfusional iron overload, as found in autopsy studies,6 is indicated by horizontal line.

View larger version (138K): [in this window] [in a new window]   Figure 2. Representative electron micrographs of rat neonatal cardiomyocytes after 72 hours of incubation in control medium (A) or 80 µg/mL Fe (B) and gerbil cardiomyocytes taken from control left ventricular epicardium (C) or after weekly injections of iron dextran for 8 weeks (D). Cytosol of control rat cardiomyocytes (A) contains no visible electron-dense particles. In iron-loaded cardiomyocytes (B), membrane-bound bodies (siderosomes; arrows) contain many electron-dense ferritin particles. Some cardiomyocytes from control gerbil (C) contain single siderosomes (arrow). In cardiomyocytes from iron-loaded gerbil (D), number of siderosomes is increased. Note that cytosol of gerbil cardiomyocytes contains visible, electron-dense ferritin particles (C and D; arrowheads). Unstained. Bars=0.5 µm.

Cardiac Action Potential
In rats, iron loading significantly reduced the overshoot of CAP (from 50.7±2.8 mV [n=6] to 25.5±4.8 mV [n=10]; P<0.002) and shortened the duration ([action potential duration at 50% repolarization] from 263.3±44.3 to 108.2±45.3 ms; P<0.038) without significant change in resting membrane potential (-68.1±0.4 and -67.2±0.6 mV, respectively). The results were consistent with earlier reports of this model that used monolayers rather than single cells.¹¹ In gerbils, data were pooled from 3 control and 3 iron-loaded animals. The overshoot was reduced (from 27.5±9.0 mV [n=7] to -1.5±6.4 mV [n=10]; P<0.02). The resting membrane potential was depolarized (from -71.8±0.3 to -67.5±0.9 mV; P<0.02) but, despite depolarization, the action potential duration at 50% repolarization was shortened (from 6.3±1.4 to 5.2±0.6 ms) (Figure 3A). In iron-loaded cells, the overshoot increased with repetitive pulsing, perhaps related to the reduced availability of the transient outward K⁺ current (I_to). In both rat and gerbil cells, all electrophysiological measurements were recorded in the absence of iron in the extracellular solution and were not influenced by the addition of iron as ferric ammonium citrate at 80 µg/mL. Control values of resting potential and action potential durations for neonatal rat cells are consistent with those from reports using similar conditions.^{21 22} Likewise, the control resting potential and action potential durations of gerbil cardiomyocytes are similar to values reported for adult rats.^{21 22}

View larger version (20K): [in this window] [in a new window]   Figure 3. Action potentials and Na+ currents in cardiomyocytes isolated from control and iron-loaded gerbils. A, Control and iron-treated (8 weeks) gerbils; 10 action potentials were produced with depolarizing current injections at 1 Hz. Broadening is due to inactivation of K+ current (arrows indicate first action potential). B, Whole-cell Na+ currents in cardiomyocytes isolated from control and iron-loaded (8 weeks) gerbils; currents were elicited with test potentials from -80 mV to 30 mV in 10-mV steps; holding potential was -90 mV (with 250-ms prepulse) to -140 mV; external solution contained 20 mmol/L Na+. INa measured at -30 mV was 122.1±14.5 pA/pF (n=9) in control cells and 70.2±7.7 pA/pF (n=6) in iron-loaded cardiomyocytes (P<0.029). C, In each experiment, peak currents at each potential tested were normalized to the maximum peak current. The results were then averaged in the control and iron-loaded gerbils and expressed as I/Imax or fraction of Imax in cardiomyocytes from control () and iron-loaded (•) gerbils. D, Steady-state inactivation in cardiomyocytes from control (; V0.5, -82.5±2.1 mV; k=6.8±0.3 mV; n=7) and iron-loaded gerbils (•; V0.5, -101.2±1.1 mV; k=6.6±0.2 mV; n=4; P<0.05). Inactivation curves were done in 0.02 mmol/L Ca2+. E, Recovery from inactivation at -140 mV in cardiomyocytes from control () and iron-loaded gerbils (•).

Sodium Currents
Iron overload significantly reduced Na⁺ currents in both gerbil and rat models (Figures 3B and 4A). The measurements in control and iron-loaded myocytes were made at equally brief times <5 minutes after penetration using pipettes with similar access resistances. In this way, time-dependent, hyperpolarizing shifts in inactivation were minimized and kept similar among the different groups of cells. Under these conditions, no differences existed in the voltage-dependence of the peak currents between control and iron-loaded cells, and the current-voltage relationships were simply scaled (Figures 3C and 4B). For rat cells, it was possible to show that the effects of incubation with iron were concentration-dependent and cumulative with time. At 80 µg/mL Fe, currents were unaffected on day 1, reduced by day 2, and significantly reduced by day 3 (P<0.01, Figure 4E). At 40 µg/mL Fe, the depression on day 3 was still statistically significant, but it was about half of that produced by the 80 µg/mL Fe dose.

View larger version (21K): [in this window] [in a new window]   Figure 4. Na+ currents in iron-treated neonatal rat cardiomyocytes. A, Whole-cell currents in control and iron-treated (80 µg/mL Fe for 72 hours) cells were elicited with test potentials from -80 mV to 80 mV in 10-mV steps; holding potential was -90 mV (with 250-ms prepulse) to -140 mV; external solution contained 140 mmol/L Na+. B, Normalized (I/Imax) and averaged peak currents from control () and iron-treated (•) rat cardiomyocytes. C, Steady-state inactivation in control (; V0.5, -69.9±1.1 mV; k=7.1±0.2 mV; n=14) and iron-treated (•; V0.5, -79.0±1.9 mV; k=8.1±0.4 mV; n=15) cardiomyocytes (P<0.05). D, Recovery from inactivation in control () and iron-treated (•) cardiomyocytes. E, Density of INa (at test potential of -10 mV) in control (open bars) and iron-treated (40 µg/mL Fe, crossed bar; 80 µg/mL Fe, filled bars) rat cardiomyocytes plotted as function of cell treatment with iron; currents were recorded 24 to 38, 48 to 62, and 72 to 88 hours after addition of iron into media. *P<0.031. Numbers above bars indicate number of cells.

The time course of sodium current activation and inactivation was unchanged. At steady-state, the activation-voltage relationships were similar, but inactivation shifted significantly toward hyperpolarized potentials in both gerbil and rat cells (Figures 3D and 4C). Recovery from inactivation at -140 mV was slowed significantly in both species: in gerbils, from 3.39±0.24 ms (n=7) to 6.83±1.48 ms (n=4; P<0.013; Figure 3E) and in rats, from 2.06±0.26 ms (n=5) to 3.84±0.66 ms (n=5; P<0.044; Figure 4D).

Experiments on cell-attached macropatches from rat cardiomyocytes loaded with the higher dose of iron for 72 hours gave results consistent with whole-cell experiments. Currents were significantly reduced by iron, and steady-state inactivation was shifted toward hyperpolarized potentials (voltage of half-maximal inactivation [V_0.5], -78.6±3.2 mV [n=3] to -88.9±4.8 mV [n=4]; P<0.05; slope factors, 5.6±0.75 and 4.8±1.3 mV) (Figure 5A).

View larger version (25K): [in this window] [in a new window]   Figure 5. A, Steady-state INa inactivation in control () and iron-treated (•) rat cardiomyocytes recorded in cell-attached configuration. B, Averaged INa measured in cell-attached configuration at test potential of -10 mV. C1, Currents of single Na+ channels recorded at test potential of -60 mV. Iron was loaded at 80 µg/mL Fe for 72 hours. C2, Averaged currents from 32 single records.

Single-channel events at suprathreshold negative potentials (-60 and -70 mV) were sufficient for current and open-time histograms to be collected and analyzed. Single-channel conductance was 13.8±1.9 pS (n=4) and 14.6±1.5 pS (n=4) in control and iron-loaded cells, respectively. The mean open times were 0.34±0.03 ms (n=4) and 0.41±0.07 ms (n=4) for control and iron-loaded cells, respectively; they agreed with values reported by others at similar potentials in isolated neonatal rat cardiomyocytes.²³ The opening probability was reduced in iron-loaded cells, and this was reflected in the averaged single-channel currents (Figure 5C). At a test potential of -10 mV (Figure 5B), the average current per patch was 47±13 pA (n=5) and 15±6 pA (n=9) for control and iron-loaded cells, respectively, which is consistent with the results at -60 mV (Figure 5C).

Sodium Channel Protein
To test whether Na⁺ channel protein was changed, we analyzed Western blots on rat cardiomyocytes incubated with 80 µg/mL Fe for 3 days. We found that the amount of Na⁺ channel protein was similar between control and iron-treated cardiomyocytes (Figure 6A) and, on average, the latter showed an increase of 22±7%, which was not statistically significant (Figure 6B).

View larger version (17K): [in this window] [in a new window]   Figure 6. Sodium-channel protein in control and iron-treated rat cardiomyocytes. A, Western blot of Na+ channel protein in controls and after iron-loading. B, Average values of densitometry on blots from 4 control and 4 test Western blots.

Potassium Currents
Representative K⁺ currents from control and iron-loaded gerbil cardiomyocytes are shown in Figure 7A. Iron loading had no effects on either the non-inactivating K⁺ outward current (I_ss) or the inward K⁺ current (I_K1) (Figure 7B) measured at 400 ms. In contrast, peak current (I_peak) was significantly increased (Figure 7B). I_peak was measured at positive potentials to exclude contamination with sodium current (I_Na). Similar results were obtained in cultured rat myocytes (not illustrated). Time constants for I_to at 50 mV were 57.7±1.9 ms (n=6) and 52.8±2.6 ms (n=6) for control and iron-loaded myocytes, respectively. Steady-state inactivation shifted positively 5 mV (Figure 7C), but recovery from inactivation at -100 mV was not affected (Figure 7D). The voltage-dependence of steady-state inactivation and recovery of I_to in gerbils are different from values reported for adult rats²⁴ but similar to those in neonatal rats.²¹

View larger version (19K): [in this window] [in a new window]   Figure 7. K+ currents in gerbil cardiomyocytes. A, Whole-cell K+ currents in cardiomyocytes isolated from control and iron-loaded (8 weeks) gerbils. Currents were elicited with test potentials from -130 mV to 50 mV in 10-mV steps; holding potential was -100 mV. B, Average steady-state (filled symbols) and peak currents (open symbols) in control (squares; n=6) and iron-loaded (circles; n=6) cardiomyocytes. *P<0.05. C, Steady-state inactivation of Ito in control (squares; V0.5, -57.7±0.5 mV; k=5.1±0.4 mV; n=5) and iron-loaded (circles; V0.5, -52.3±1.8 mV; k=5.2±0.4 mV; n=5) cardiomyocytes (P<0.05). Ito was measured as difference between Ipeak and ISS. D, Recovery of Ito from inactivation at -100 mV in control (squares; =1396±53 ms; n=5) and iron-loaded (circles; =1428±73 ms; n=6) cardiomyocytes.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These studies are the first to examine the membrane currents of single cardiomyocytes loaded with iron, either in vivo or in vitro. Iron overload, both in vivo and in vitro, had similar effects on cellular iron accumulation and on Na⁺ and K⁺ currents of single cardiomyocytes from 2 closely related rodent species. The gerbils treated with repeated injections of iron dextran over a period of months developed a cardiomyopathy that mimics the cardiomyopathy that develops over a period of years in patients with chronic iron overload.^{8 9} Because knowledge of cardiac electrophysiology in the gerbil is limited, we also studied the rat, a species in which knowledge of cardiac electrophysiology is extensive.^{25 26} Because the rat can excrete excess iron, cardiac iron deposition could not be produced in vivo in this species. Instead, isolated rat cardiomyocytes were exposed to iron in vitro. Given the 2 different methods of iron loading, the correlation of the changes in Na⁺ and K⁺ currents between the 2 models suggests that acutely loaded rat cardiomyocytes may be useful surrogates for determining the specific ion channel targets of iron cardiotoxicity. This is especially important for K⁺ currents because a number of molecular candidates for I_to have been described in rats²⁵ and humans.²⁶

The mechanism whereby iron reduces the number of functional cardiac Na⁺ channels and enhances the inactivated state of those channels that remain functional is unknown. Iron may produce peroxidative damage to DNA²⁷ and to membrane lipids and proteins.^{28 29} Our antibody experiments suggest that channel production is unaffected. The enhancement of the inactivated state is also consistent with direct modification of Na⁺ channels, although involvement of the membrane lipid in which the channels are embedded cannot be excluded.³⁰ In cultured rat cardiomyocytes, in vitro treatment with iron altered membrane fatty acids³¹ and suppressed mitochondrial respiratory enzymes, with a concomitant reduction in cellular ATP.³² The decrease in ATP might alter phosphorylation of Na⁺ channel protein, which affects steady-state inactivation and recovery from inactivation.^{33 34} Whatever the underlying mechanism, the loss of functional Na⁺ channels and enhanced Na⁺ channel inactivation cause the reduction in the overshoot of CAP in the iron-loaded cardiomyocytes.

The relatively slow recovery from inactivation of I_to in gerbil cardiomyocytes resembles neonatal rat cardiomyocytes and differs from human epicardial myocytes. However, I_to recovery in human subendocardial myocytes is also slow³⁵ and, in humans, heart rate is 5 times slower than in gerbils.

We do not know the molecular species responsible for K⁺ currents in gerbil cardiomyocytes and their involvement in the increase of I_to. In neonatal rats, as well as in humans, possible candidates for I_to are Kv4.2/4.3, Kv1.4,^{36 37} and a calcium-activated Cl^- current. Involvement of the latter in our experiments is unlikely because the cells were dialyzed with Ca²⁺-free, EGTA-buffered solution, and the extracellular solution contained only 20 mmol/L Ca²⁺. We do not know the mechanism for the increase, although altered K⁺ channel phosphorylation might contribute.³⁸ The contrast with the reduction in Na⁺ current makes it clear that iron does not simply produce nonspecific damage to ion-channel proteins in general. Moreover, the effect of iron on K⁺ currents was specific; I_ss and I_K1 were unaffected. The partial depolarization of iron-loaded gerbil cardiomyocytes is not due to a change in I_K1 and may result from loss of Na/K ATPase activity.³⁹

The reduction in action potential overshoot will compromise propagation of the cardiac impulse, and increase in I_to may foreshorten CAP. These effects, together with a heterogeneous pattern of iron deposition, may enhance QT dispersion; clinical evidence of this exists in thalassemia major patients with iron overload. In a preliminary study, QT dispersion was measured as the difference in QT interval among 12 leads of the surface ECG and calculated as QT_max-QT_min; 14 of 24 patients had increased (>60 ms) QT dispersion (G.M. Brittenham, unpublished data). Reduced propagation and increased QT dispersion may provide a substrate for the cardiac arrhythmias of iron cardiomyopathies. The specific K⁺ channel affected by iron may, therefore, be a target for treatment of the arrhythmias^{1 2} caused by iron-overload cardiomyopathy.

	Acknowledgments

 
Supported by grants from the National Institutes of Health (HL36930, HL55404, and HL61642 to A.M.B.; HL07147 and HL52447 to G.M.B.); the American Heart Association, National (AHA EI 9640126N to S.A.C.) and Northeast Ohio affiliates (NEOHA 96-244 to C.-C.S.; NEOHA 97-231 to Y.A.K.); and Cooley's Anemia Foundation (to Y.A.K.).

	Footnotes

 
Dr Shieh's current address is Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Ill 60064; Dr Brittenham's current address is College of Physicians and Surgeons, Columbia University, Harkness Pavilion, Room HP550, 630 W 168th St, New York, NY 10032.

Received December 31, 1998; revision received March 23, 1999; accepted April 9, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Wolfe L, Olivieri N, Sallan D, Colan S, Rose V, Propper R, Freedman MH, Nathan DG. Prevention of cardiac disease by subcutaneous deferoxamine in patients with thalassemia major. N Engl J Med. 1985;312:1600–1603.[Abstract]
   
2. Witte DL, Crosby WH, Edwards CQ, Fairbanks VF, Mitros FA. Practice guideline development task force of the College of American Pathologists: hereditary hemochromatosis. Clin Chim Acta. 1996;245:139–200.[Medline] [Order article via Infotrieve]
   
3. Brittenham GM, Griffith PM, Nienhuis AW, McLaren CE, Young NS, Tucker EE, Allen CJ, Farrell DE, Harris JW. Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major. N Engl J Med. 1994;331:567–573.[Abstract/Free Full Text]
   
4. Olivieri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. Blood. 1997;89:739–761.[Free Full Text]
   
5. Brittenham GM. Disorders of iron metabolism: deficiency and overload. In: Hoffman R, Benz EJ, Shattil SJ, Furie B, Cohen HJ, Silberstein LE, eds. Hematology: Basic Principles and Practice. New York, NY: Churchill Livingstone; 1995:492–523.
   
6. Buja LM, Roberts WC. Iron in the heart: etiology and clinical significance. Am J Med. 1971;51:209–221.[Medline] [Order article via Infotrieve]
   
7. Fitchett DH, Coltart DJ, Littler WA, Leyland MJ, Trueman T, Gozzard DI, Peters TJ. Cardiac involvement in secondary haemochromatosis: a catheter biopsy study and analysis of myocardium. Cardiovasc Res. 1980;14:719–724.[Medline] [Order article via Infotrieve]
   
8. Carthew P, Dorman BM, Edwards R, Francis JE, Smith AG. A unique rodent model for both the cardiotoxic and hepatotoxic effects of prolonged iron overload. Lab Invest. 1993;69:217–222.[Medline] [Order article via Infotrieve]
   
9. Carthew P, Smith AG, Hider RC, Dorman B, Edwards RE, Francis JE. Potentiation of iron accumulation in cardiac myocytes during the treatment of iron overload in gerbils with the hydroxypyridinone iron chelator CP94. Biometals. 1994;7:267–271.[Medline] [Order article via Infotrieve]
   
10. Link G, Pinson A, Hershko C. Heart cells in culture: a model of myocardial iron overload and chelation. J Lab Clin Med. 1985;106:147–153.[Medline] [Order article via Infotrieve]
    
11. Link G, Athias P, Grynberg A, Pinson A, Hershko C. Effect of iron loading on transmembrane potential, contraction, and automaticity of rat ventricular muscle cells in culture. J Lab Clin Med. 1989;113:103–111.[Medline] [Order article via Infotrieve]
    
12. Overmoyer BA, McLaren CE, Brittenham GM. Uniformity of liver density and nonheme (storage) iron distribution. Arch Pathol Lab Med. 1987;111:549–554.[Medline] [Order article via Infotrieve]
    
13. Iancu TC, Shiloh H, Link G, Bauminger ER, Pinson A, Hershko C. Ultrastructural pathology of iron-loaded rat myocardial cells in culture. Br J Exp Pathol. 1987;68:53–65.[Medline] [Order article via Infotrieve]
    
14. Toraason M, Luken ME, Breitenstein M, Krueger JA, Biagini RE. Comparative toxicity of allylamone and acrolein in cultured myocytes and fibroblasts from neonatal rat hearts. Toxicology. 1989;56:107–117.[Medline] [Order article via Infotrieve]
    
15. Powell T, Twist VW. A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium. Biochem Biophys Res Commun. 1976;72:327–333.[Medline] [Order article via Infotrieve]
    
16. Mitra R, Morad M. A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. Am J Physiol. 1985;249:H1056–H1060.
    
17. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high resolution current recording from cell and free membrane patches. Pflugers Arch. 1981;391:85–100.[Medline] [Order article via Infotrieve]
    
18. Kuryshev YA, Childs GV, Ritchie AK. Corticotropin releasing hormone stimulation of Ca²⁺ entry in corticotropes is partially dependent of protein kinase A. Endocrinology. 1995;136:3925–3935.[Abstract]
    
19. VanDongen AJM. A new algorithm for idealizing single ion-channel data containing multiple unknown conductance levels. Biophys J. 1996;70:1303–1316.[Abstract/Free Full Text]
    
20. Cohen SA, Levitt LK. Partial characterization of the rH1 sodium channel protein from rat heart using subtype-specific antibodies. Circ Res. 1993;73:735–742.[Abstract/Free Full Text]
    
21. Kilborn MJ, Fedida D. A study of the developmental changes in outward currents of rat ventricular myocytes. J Physiol. 1990;430:37–60.[Abstract/Free Full Text]
    
22. Wahler GM, Dollinger SJ, Smith JM, Fleman KL. Time course of postnatal changes in rat heart action potential and in transient outward current is different. Am J Physiol. 1994;267:H1157–H1166.[Abstract/Free Full Text]
    
23. Kunze DL, Lacerda AE, Wilson DL, Brown AM. Cardiac Na⁺ currents and the inactivating, reopening and waiting properties of single cardiac Na⁺ channels. J Gen Physiol. 1985;86:691–719.[Abstract/Free Full Text]
    
24. Apkon M, Nerbonne JM. Characterization of two distinct depolarization-activated K⁺ currents in isolated rat ventricular myocytes. J Gen Physiol. 1991;97:973–1011.[Abstract/Free Full Text]
    
25. Kass RS. Ionic basis of electrical activity in the heart. In: Sperelakis N, ed. Physiology and Pathophysiology of the Heart. Boston, Mass: Kluwer Academic Publishers; 1995:77–90.
    
26. Brown AM. Cardiac potassium channels in health and disease. Trends Cardiovasc Med. 1996;4:118–124.
    
27. Henle ES, Linn S. Formation, prevention, and repair of DNA damage by iron/hydrogen peroxide. J Biol Chem. 1997;272:19095–19098.[Free Full Text]
    
28. Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem. 1997;272:20313–20316.[Free Full Text]
    
29. Fridovich I. Superoxide anion radical (O₂-), superoxide dismutases, and related matters. J Biol Chem. 1997;272:18515–18517.[Free Full Text]
    
30. Weiland SJ, Gong Q-H, Poblete H, Fletcher JE, Chen L-Q, Kallen RG. Modulation of human muscle sodium channels by intracellular fatty acid is dependent on the channel isoform. J Biol Chem. 1996;271:19037–19041.[Abstract/Free Full Text]
    
31. Link G, Pinson A, Kahane I, Hershko C. Iron loading modifies the fatty acid composition of cultured rat myocardial cells and liposomal vesicles: effect of ascorbate and a-tocopherol on myocardial lipid peroxidation. J Lab Clin Med. 1989;114:243–249.[Medline] [Order article via Infotrieve]
    
32. Link G, Saada A, Pinson A, Konijn AM, Hershko C. Mitochondrial respiratory enzymes are a major target of iron toxicity in rat heart cells. J Lab Clin Med. 1998;131:466–476.[Medline] [Order article via Infotrieve]
    
33. Qu Y, Rogers J, Tanada T, Scheuer T, Catteral W. Modulation of cardiac Na⁺ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C. Proc Natl Acad Sci U S A. 1994;91:3289–3293.[Abstract/Free Full Text]
    
34. Watson CL, Gold M. Modulation of Na⁺ current inactivation by stimulation of protein kinase C in cardiac cells. Circ Res. 1997;81:380–386.[Abstract/Free Full Text]
    
35. Nabauer M, Beuckelmann DJ, Uberfuhr P, Steinbeck G. Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation. 1996;93:168–177.[Abstract/Free Full Text]
    
36. Xu H, Dixon JE, Barry DM, Trimmer JS, Merlie JP, McKinnon D, Nerbonne JM. Developmental analysis reveals mismatches in the expression of K⁺ channels a-subunits and voltage-gated K⁺ channel currents in rat ventricular myocytes. J Gen Physiol. 1996;108:405–419.[Abstract/Free Full Text]
    
37. Kaab S, Dixon J, Duc J, Ashen D, Nabauer M, Beuckelmann DJ, Steinbeck G, McKinnon D, Tomaselli GF. Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current density. Circulation. 1998;98:1383–1393.[Abstract/Free Full Text]
    
38. Murray KT, Fahrig SA, Deal KK, Po SS, Hu NN, Snyders DJ, Tamkun MM, Bennet PB. Modulation of an inactivating human cardiac K⁺ channel by protein kinase C. Circ Res. 1994;75:999–1005.[Abstract/Free Full Text]
    
39. Link G, Pinson A, Hershko C. Ability of the orally effective iron chelators dimethyl- and diethyl-hydroxyperid-4-one and deferoxamine to restore sarcolemmal thiolic enzyme activity in iron-loaded heart cells. Blood. 1994;83:2692–2697.[Abstract/Free Full Text]
    

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