Deferoxamine prevents cardiac hypertrophy and failure in the gerbil model of iron-induced cardiomyopathy

Deferoxamine prevents cardiac hypertrophy and failure in the gerbil model of iron-induced cardiomyopathy TIANEN YANG, GARY M. BRITTENHAM, WEI-QIANG DONG, MATTHEW N. LEVY, CARLOS A. OBEJERO-PAZ, YURI A. KURYSHEV, and ARTHUR M. BROWN CLEVELAND, OHIO, and NEW YORK, NEW YORK

To evaluate the effects of the iron chelator deferoxamine on the functional and structural manifestations of iron-induced cardiac dysfunction, we measured cardiac power, left ventricular systolic, and diastolic function as (dP/dt)max and (dP/ dt)min, respectively, and left ventricular and septal wall thickness in isolated heart preparations derived from the Mongolian gerbil model of iron overload. We induced iron overload with weekly subcutaneous injections of iron dextran (800 mg/kg/wk); deferoxamine (DFO; 100 mg/kg) was administered twice daily by subcutaneous injection, 5 of 7 days each week; and control animals received weekly subcutaneous injections of dextran alone. Animals administered iron alone initially exhibited, at 5 weeks, increased cardiac power but by 12 to 20 weeks, cardiac power was severely diminished, with impairment of both systolic and diastolic function of the left ventricle and marked cardiac hypertrophy (P < .001 for all vs control animals). Administration of DFO with iron did not interfere with the initial augmentation of cardiac power at 5 weeks but prevented the subsequent deterioration in cardiac performance. After 12 to 20 weeks, gerbils given DFO with iron had mean values of cardiac power indistinguishable from those of control animals; both systolic and diastolic function were significantly enhanced not only in comparison with those of animals treated with iron alone but also with respect to controls. In addition, DFO prevented cardiac hypertrophy; mean ventricular and septal wall thickness in gerbils given DFO and iron were not significantly different from those in controls. In the gerbil model of iron overload, concurrent administration of DFO with iron prevents both the development of cardiac hypertrophy and the progressive deterioration in cardiac performance that are produced by chronic iron accumulation. (J Lab Clin Med 2003;142:332-40) Abbreviations: DFO ⫽ deferoxamine; dP/dtmax ⫽ the maximum value of the rate of rise of intraventricular pressure; dP/dtmin ⫽ the maximum value of the rate of fall of intraventricular pressure; EDTA ⫽ ethylenediaminetetraacetate

From the Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University; the Rammelkamp Center for Education and Research, Case Western Reserve University, MetroHealth Campus; and the Department of Pediatrics, Columbia University. Supported by US Public Health Service grants HL61642, HL62882, and DK49108. Submitted for publication January 30, 2003; revision submitted June 10, 2003; accepted June 12, 2003. Reprint requests: Arthur Brown, MD, PhD, Rammelkamp Center for Education and Research, 2500 MetroHealth Drive, R356, Cleveland, OH 44109-1998; e-mail: [email protected]. Copyright © 2003 by Mosby, Inc. All rights reserved. 0022-2143/2003/$30.00 ⫹ 0 doi:10.1016/S0022-2143(03)00135-5

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espite dramatic improvements in care, ironinduced cardiac disease remains the most frequent cause of death in thalassemia major1-3 and a major life-limiting complication of other trans-fusion-dependent refractory anemias, hereditary hemochromatosis, and other forms of iron overload.4-13 Without adequate control of iron concentrations in the body, arrhythmias and congestive heart failure eventually develop with progressive iron overload and, untreated, are often lethal within months of their initial appearance.7,12,14 Almost uniquely among causes of cardiac failure, patients who present with iron-induced heart disease sometimes can be rescued and their hearts restored to normal rhythm and function with intensive iron-chelating therapy with DFO.7,15-23 Progress in un-

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raveling the mechanisms responsible for either the abrupt deterioration or the striking salvage by chelation of patients with iron overload has been halting, hindered in part by the lack of a suitable experimental model. A decade ago, Carthew and his colleagues introduced the Mongolian gerbil model of iron overload. It duplicated, for the first time, the structural cardiomyopathy and hepatic dysfunction found clinically in patients with chronic iron excess.24 In a series of studies by others25-29 and in our own laboratory,30-33 the gerbil has been found to provide an animal model that reproduces many of the essential functional and structural features of iron-induced liver and heart disease in patients. Under the original Carthew protocol for iron loading (200 mg iron/kg/wk), the development of fatal cardiac complications may take more than 1 year; in our studies, the median survival time was 68 weeks.31 Consequently, we have developed an accelerated regimen of loading, employing a dose of 800 mg iron/kg/wk, that hastens the induction of the iron-induced cardiomyopathy. Median survival time is shortened to 14 weeks but the critical features of the model are retained.31 In particular, the histopathologic pattern of cardiac iron deposition is maintained,31 left ventricular iron concentrations still overlap the range reported for patients who die of iron overload in thalassemia major (6.0-13.2 mg/g dry wt),13,25,31,32,34,35 and the bimodal pattern of cardiac electrical and contractile dysfunction is preserved.31,32 With accelerated induction, the initial phase of high cardiac output occurs at about 6 weeks rather than at 20, and the subsequent state of low-output failure appears by about 20 weeks rather than 60.31,32 Recently we used the accelerated model of iron overload in the gerbil to examine the effects of concurrent administration of DFO and found that the chelating regimen used (100 mg/kg subcutaneously twice daily, 5 of 7 days per week) reduced the iron concentration of the left ventricular apex only modestly, by about 20%.32 Nonetheless, chelator treatment substantially prolonged survival, with no deaths during the full 20 weeks of iron administration, and prevented most electrocardiographic abnormalities.32 The study reported in this article was undertaken with the accelerated model of iron overload in the gerbil to determine whether, in addition to largely preventing the electrical complications of cardiac iron overload, DFO treatment could also prevent functional and structural manifestations of iron-induced cardiac dysfunction. METHODS Experiments were performed on 2-month-old female Mongolian gerbils. The investigation conforms with the Guide for

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the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996). Three groups of animals were studied. Each group was divided into 2 subgroups: a “short-term” subgroup that was given injections for 5 weeks and a “long-term” subgroup that was given injections for 12 to 20 weeks. The first group of animals (iron group) was injected subcutaneously with iron-dextran (800 mg iron/kg [14 mmol/kg]) once a week (ferric hydroxide– dextran complex, 100 mg/mL; Sigma-Aldrich, St. Louis, Mo). The second group (control group) was injected in the same manner, but with equivalent volumes of dextran solution alone. The third group (iron/DFO group) received subcutaneous iron dextran (800 mg iron/kg [14 mmol/kg]) once a week and Desferal (DFO mesylate; Novartis, East Hanover, NJ; 100 mg DFO/kg [0.15 mmol/kg]) twice daily, 5 days a week (or a total of 1000 mg DFO/kg/ week [1.5 mmol DFO/kg/week]). DFO doses of this magnitude have been used clinically,36-39 although lower doses (ⱕ50 mg DFO/kg) are generally recommended for standard therapy.40 In studies of isolated perfused heart, after the completion of a treatment regimen, a gerbil was given heparin (500 U/kg) and then anesthetized with intrapertoneal ketamine (75 mg/ kg). The heart was exposed, and the ascending aorta was cannulated with a polyethylene catheter (PE-90). The heart was removed and immediately perfused in “nonworking” mode at 37.4°C with the use of a Langendorff apparatus.41,42 The concentrations, expressed in millimoles per liter, of the various constituents of the perfusate, were as follows: NaCl, 118; KCl, 4.7; CaCl2,2.5; MgSO4, 1.2; KH2PO4, 1.2; sodium EDTA, 0.5; NaHCO3, 25; and glucose, 5.5. The perfusate reservoir was located 70 cm above the heart, and we equilibrated the O2 and CO2 concentrations in the perfusate by continuously bubbling a mixture of 95% O2 and 5% CO2 through the solution in the reservoir. The isolated heart was immersed in a water-jacketed chamber. When the heart was perfused in nonworking mode, the perfusate passed from the elevated reservoir through polyethylene tubing and entered the coronary arterial circulation by way of a cannula in the ascending aorta.41 We sharpened the proximal end of a PE-50 catheter by cutting the catheter obliquely. This end of the catheter was then inserted into the left atrium through a pulmonary vein to permit the measurement of left ventricular pressure. The proximal end of this catheter was advanced through the mitral valve and into the left ventricle. We had flared the distal tip of this catheter by heating it with a small flame. The catheter was then advanced and forced through the ventricular apex until the flared distal end of the catheter was in contact with the internal surface of the left ventricle. To measure intraventricular pressure and its first derivative (dP/dt), the proximal end of this catheter was connected to a transducer (SPR-407, model TCB-500; Millar Instruments, Inc, Houston, Texas). The maximum and minimum values of the first derivative of left ventricular pressure, (dP/dt)max and (dP/dt)min, determined with the technique described by Grupp,41 were used to assess the maximum rates of left ventricular contraction and relaxation, respectively.41,42 We also carried out a series of experiments on the effects

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of iron and of DFO in the relationships between preload pressure and left ventricular power. These pressure-flow experiments were similar technically to those of one of our previous studies31 and to the protocol of Grupp.41 When the heart was perfused in “working mode,” one end of a PE-90 catheter was inserted into the left atrium through a pulmonary vein, and the other end of this catheter was connected to the tubing from the elevated perfusate reservoir with the use of a stopcock. The flow generated by the left ventricle was monitored continuously with an electromagnetic flow meter (FM 501D; Carolina Medical Electronics, Inc, King, NC). A micrometer clamp was applied to the tubing just proximal to the polyethylene catheter inserted into the pulmonary vein. This clamp was used to adjust the preload pressure of the isolated perfused heart. The preload pressure was measured with a transducer (model PT 300; Grass-Teletactor Division, AstroMed, Inc, West Warick, RI). Tygon tubing (internal diameter 3/32 inches), which represents the “systemic arterial system,” was connected to a cannula in the aorta by way of a stopcock. We incorporated a bubble trap in this systemic artery analog to introduce suitable “arterial compliance.” A second pressure transducer was connected to the arterial-pressure analog to measure the “afterload pressure.” A micrometer clamp was included to adjust the “peripheral vascular resistance” and thereby to vary the “afterload pressure.”41,42 To compare the effects of changes in cardiac preload on cardiac power, we increased the preload pressure in increments of 5 mm Hg over a range of 10 to 40 mm Hg while the afterload pressure was held constant at 60 mm Hg. The overflow from the water-jacketed heart chamber was collected during the experiment; the rate of overflow constituted the coronary flow. The aortic and coronary flows were collected and measured throughout each experiment. Cardiac work per minute (ie, cardiac power) was calculated as the product of the afterload pressure and the total flow (aortic plus coronary flows).41

Fig 1. Effects of iron and iron/DFO on cardiac power. A, 5-week treatment; B, 12- to 20-week treatment. Changes in cardiac power per gram of heart weight in isolated heart preparations were derived from control (n ⫽ 10, dark circles) and iron-loaded (n ⫽ 17, open circles) and iron/DFO-treated (n ⫽ 15, triangles) gerbils as a function of preload pressure. Afterload pressure was maintained constant at 60 mm Hg. *P ⬍ .01; **P ⬍ .001.

Histologic evaluation and biochemical measurements. To evaluate liver function and determine serum iron

concentration, we took blood samples (1-2 mL) from the femoral artery before the chest was opened in animals from each of the long-term groups. After data on coronary flow, aortic flow, (dP/dt)max and (dP/dt)min data had been collected from the working-heart preparations, the heart was filled with saline solution at a pressure of 5 mm Hg. The distended heart was then fixed in 10% formalin for at least 24 hours.43 A 1-mm-thick slice of the heart was obtained halfway between the base and apex of the left ventricle. The thickness of the left ventricular wall between the papillary muscles and of the interventricular septum between anterior and posterior ventricular septa44 were measured with the Scion Image for Windows technique (Scion Corp, Frederick, Md). Aspartate aminotransferase, alanine aminotransferase, and serum iron were meaured (BM/Hitachi Analyzer, Diamond Diagnostics, Inc, Holliston, Mass) in the blood samples obtained before the animal’s chest was opened. Data analysis. Data are expressed as mean ⫾ SEM; we determined the statistical significance of the data using anal-

ysis of variance. P values of .05 or less were considered statistically significant. RESULTS Effects of iron and iron/DFO on cardiac power. Fig 1, A, shows the changes in cardiac power in response to alterations in preload pressure in the isolated perfused hearts from the short-term control, iron, and iron/DFO groups. After 5 weeks of treatment, cardiac power in the isolated hearts derived from the short-term iron group and those from the short-term iron/DFO group did not differ significantly. In contrast, hearts from both the iron and iron/DFO groups achieved greater levels of cardiac power than did the hearts from the control group (P ⬍ .001). Fig 1, B, shows the changes in cardiac power in response to alterations in preload pressure in the isolated perfused hearts from the long-term control, iron,

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more negative value signifies a greater rate of relaxation. Fig 2, B and D, shows the maximal rates of the measures of left ventricular contraction, (dP/dt)max, and of left ventricular relaxation, (dP/dt)min, in the hearts from the long-term control, iron, and iron/DFO groups. After 12 to 20 weeks of treatment, the mean value of (dP/dt)max in the hearts from the iron group (Fig 2, B) was significantly less (P⬍0.001) than that in hearts from the control group. By contrast, in the hearts from the iron/DFO group, ventricular contraction, (dP/dt)max, was substantially increased compared with that in the iron and control groups (P ⬍ .001 for both). In a similar manner, as shown in Fig 2, D, left ventricular relaxation, (dP/dt)min, was increased in the hearts from the iron group with respect to the control group but greatly decreased in the hearts from the iron/DFO group compared with hearts from the iron and control groups (P ⬍ .001 for both). Effects of iron and iron/DFO on structure and selected biochemistry. Fig 3 shows representative unstained

Fig 2. Effects of iron and iron/DFO on contractility. Changes in (dP/dt)max and (dP/dt)min in the left ventricles were derived from the same animals from which the data shown in Fig 1 were obtained. *P ⬍ .01; **P ⬍ .001.

and iron/DFO groups. After 12 to 20 weeks of treatment, cardiac power in the isolated hearts from the long-term iron group was significantly lower than that in the hearts from the long-term iron/DFO or control group (P ⬍ .001). After long-term treatment, the performance of hearts from the iron/DFO group and those from the control group did not differ significantly. Effects of iron and iron/DFO on contractility. Fig 2, A and C, shows the maximal rates of the measures of left ventricular contraction, (dP/dt)max, and of left ventricular relaxation, (dP/dt)min, in the preparations derived from the short-term control, iron, and iron/DFO groups. Fig 2, A, shows that the mean values of our measure of left ventricular contraction, (dP/dt)max, were virtually identical in the preparations derived from the control and iron/DFO groups after 5 weeks of treatment but the mean (dP/dt)max from the iron group was increased with respect to both (P ⬍ .001 for both). In similar fashion, Fig 2, C, shows that the mean values of the measure of left ventricular relaxation, (dP/dt)min, were also almost the same in the preparations derived from the control and iron/DFO groups, whereas while the mean (dP/dt)min from the iron group was decreased with respect to both (P ⬍ .001 for both). Note that the

cross-sections of the central regions of the ventricles from gerbils in the long-term control, iron, and iron/ DFO groups at 16 weeks. The ventricular cross-section from a gerbil treated with iron alone (Fig 3, B) is dark brown, indicating marked hemosiderin accumulation and severe concentric hypertrophy. The specimen from a gerbil treated with iron and DFO (Fig 3, C), is pale brown, suggesting less hemosiderin accumulation. However, Prussian blue staining revealed only a modest decrease in intensity for iron/DFO hearts (Fig 3). Furthermore, ventricular wall thickening in Fig 3, C, is less than that in Fig 3, B, the sample from a gerbil treated with iron alone. Table I shows the mean body, heart, and liver wet weights at the time animals were killed, along with the thickness of the left ventricular wall and the interventricular septum for the control, iron, and iron/DFO gerbils in both the short-term (5 weeks) and long-term (12-20 weeks) groups. In the short-term animals, there were no significant differences in body or heart weight among the groups. By contrast, liver weight and both left ventricular wall and septum thickness were increased compared with those of controls in both the iron and iron/DFO groups. In each case, the iron/DFO group showed less of an increase than the iron group. With long-term treatment, all the measurements listed in Table I were significantly increased in the iron group compared with those in the control group. Specifically, measurements of mean ventricular and septal thickness confirmed the development of significant hypertrophy in the long-term iron group. By contrast, the addition of DFO (long-term iron/DFO group) resulted in mean heart weights and left ventricular and septal thicknesses

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DISCUSSION

Fig 3. Effects of iron and iron/DFO on cardiac structure. Left column: representative cross-sections of hearts from (A) a control (dextran only) gerbil (n ⫽ 6); (B) an iron-overloaded (800 mg/kg/wk for 16 weeks) gerbil (n ⫽ 6); and (C) a gerbil administered iron (800 mg/kg/wk for 16 weeks) plus DFO (200 mg/kg, 5 days a week for 16 weeks) (n ⫽ 6). Arrows denote papillary muscles. Right column: Similar cross-sections from hearts stained with Prussian blue (representative of 3 animals in each group). Note that this iron/DFO specimen stains with almost the same intensity as the iron specimen.

that were substantially and significantly less that those in the long-term iron group but not significantly different from those in the control group. Table II shows the biochemical results. Aminotransferase activities and serum iron concentrations were greatly increased in the iron group compared with those in the other 2 groups. Serum iron was significantly higher in the iron/DFO group than in the control group, but aminotransferase activities did not differ significantly. Cardiac iron was significantly increased in both the iron and iron/DFO groups compared with the controls. However, we found only a 20% difference between the iron and iron/DFO groups.

Concurrent DFO administration prevented both the development of cardiac hypertrophy and the progressive deterioration in cardiac performance produced by chronic iron accumulation. In the experimental groups administered iron alone, the results in these studies of isolated heart preparations underscore our earlier findings in gerbils treated with an accelerated regimen of iron loading. An initial state of high cardiac output is produced that then evolves into a subsequent state of low-output failure,31 in a pattern similar to the course of cardiomyopathy in patients with transfusional iron overload.45-47 Using the same gerbil model, we recently established that concurrent treatment with DFO abolished most electrocardiographic abnormalities and averted iron-induced cardiac death.32 The results of the studies of isolated heart preparations reported here demonstrate that DFO therapy can also (a) prevent the structural remodeling of the ventricular wall that underlies the development of cardiac hypertrophy and (b) counteract the progressive depression of cardiac function with iron loading that culminates in frank congestive failure. It is notable that concurrent DFO treatment did not interfere with the apparent augmentation of cardiac power that develops during the initial phase of iron loading. With the accelerated regimen of iron administration, cardiac power was increased at 5 weeks compared with that in controls in both the iron and iron/ DFO groups (Fig 1, A). At the same time, DFO maintained left ventricular systolic and diastolic function in the gerbils administered iron and DFO, as assessed on the basis of mean (dP/dt)max and (dP/dt)min, respectively, at a level almost identical to that in the controls, even as these values in the group given iron alone were significantly enhanced (Fig 2, A and C). By 12 to 20 weeks, with continued iron loading, cardiac power had significantly diminished in the animals treated with iron alone, but DFO given along with the iron maintained cardiac power at levels indistinguishable from those of the control animals (Fig 1, B). In concert with the deterioration in cardiac power in the gerbils treated with iron alone, both left ventricular systolic and diastolic function were impaired compared with these parameters in control animals (Fig 2, B and D). We found it intriguing that gerbils treated with iron and concurrent DFO then displayed a surge in both systolic and diastolic function—in mean (dP/dt)max and (dP/dt)min, respectively— greater than that in either the control animals or those treated with iron without DFO (Fig 2, B and D). The magnitude and pattern are reminiscent of the changes seen at 5 weeks in animals administered iron alone (Fig 2, A and C), as if the DFO

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Table I. Body, heart and liver wet weights at the time of death, plus thickness of left ventricular wall and the interventricular septum for the control, iron, and iron/DFO gerbils in the short-term and long-term groups Group

5 wk Iron Iron/DFO Control 12-20 wk Iron Iron/DFO Control

Terminal body wt (g)

74.1 ⫾ 1.1 (n ⫽ 23)* 71.2 ⫾ 1.3 (n ⫽ 34) 68.6 ⫾ 2.2 (n ⫽ 16)

Liver wt (g)

Left ventricular wall (mm)

Septum (mm)

4.9 ⫾ 0.2 (n ⫽ 23)** 4.4 ⫾ 0.1 (n ⫽ 34)† 2.7 ⫾ 0.1 (n ⫽ 16)‡‡

2.52 ⫾ 0.16 (n ⫽ 11)** 2.01 ⫾ 0.07 (n ⫽ 12)†† 1.76 ⫾ 0.11 (n ⫽ 12)

2.23 ⫾ 0.16 (n ⫽ 11)* 1.82 ⫾ 0.05 (n ⫽ 12)† 1.79 ⫾ 0.11 (n ⫽ 12)

7.0 ⫾ 0.2 (n ⫽ 32)** 6.3 ⫾ 0.2 (n ⫽ 19)†† 2.9 ⫾ 0.2 (n ⫽ 15)‡‡

2.65 ⫾ 0.11 (n ⫽ 12)** 2.22 ⫾ 0.13 (n ⫽ 11)† 2.0 ⫾ 0.1 (n ⫽ 11)

2.54 ⫾ 0.08 (n ⫽ 12)** 2.16 ⫾ 0.11 (n ⫽ 11)† 1.96 ⫾ 0.07 (n ⫽ 11)

Heart wt (mg)

578.0 ⫾ 10.9 (n ⫽ 23) 558.7 ⫾ 13.1 (n ⫽ 34) 552.4 ⫾ 14.4 (n ⫽ 16)

80.3 ⫾ 1.5 (n ⫽ 32)** 596.3 ⫾ 18.7 (n ⫽ 32)* 77.1 ⫾ 1.7 (n ⫽ 19) 526.8 ⫾ 14.9 (n ⫽ 19)†† ‡‡ 68.2 ⫾ 1.5 (n ⫽ 15) 524.7 ⫾ 13.7 (n ⫽ 15)

Data expressed as mean ⫾ SEM. Data analysis: *iron vs control; †Iron/DFO vs iron; ‡Iron/DFO vs control; *†‡P ⬍ .05; **††‡‡P ⬍ .001.

Table II. Biochemical data of the long-term (12-20 weeks) control, iron and Iron/DFO groups Aminotransferase concentrations Group

Iron (n ⫽ 9) Iron/DFO (n ⫽ 8) Control (n ⫽ 13)

ALT (U/L)

AST (U/L)

Serum iron (␮g/dL)

Cardiac Iron (␮g/mg dry wt)

1124.6 ⫾ 463** 173.1 ⫾ 55.3† 67.2 ⫾ 10.0

2779.8 ⫾ 1220.6** 417.6 ⫾ 83.2† 181.9 ⫾ 20.7

3744.2 ⫾ 461.6** 1856.2 ⫾ 339.1‡ 311.5 ⫾ 25.3

12.9 ⫾ 1.2 (n ⫽ 12)** 10.6 ⫾ 0.6 (n ⫽ 12)‡ 0.3 ⫾ 0.1 (n ⫽ 12)

ALT ⫽ alanine aminotransferase; AST ⫽ aspartate aminotransferase. Data expressed as mean ⫾ SEM. Data analysis: *Iron vs control, †iron/DFO vs iron, ‡iron/DFO vs control; *†P ⬍ .05, **‡P ⬍ 0.001.

was able to delay but not prevent this effect of iron loading. In the absence of an understanding of the underlying mechanisms, it is impossible to determine whether any relationship exists with the apparent ability of DFO to delay but not prevent the iron-induced prolongation of PR and QRS intervals in our earlier electrocardiographic studies in gerbils.32 In any event, the overall conclusion is clear: concurrent DFO administration prevents the progressive depression of function that is finally responsible for the development of congestive heart failure and death. In addition to these favorable functional effects, DFO treatment abolished the gross structural consequences of iron loading. Concurrent DFO treatment prevented iron-induced cardiac hypertrophy and thickening of the ventricular wall, as evidenced both by the appearance of the cardiac cross-sections in Fig 3 and the measurements of heart weight and wall thickness listed in Table I. Although we did not examine the effects of DFO on the liver, the marked decrease in transaminase concentrations is consistent with beneficial effects of DFO treatment in the gerbil model of iron overload that have been reported by other investigators.29 Finally, we should emphasize that the serum iron assay used for the measurements in Table II could not distinguish between transferrin-bound iron, ferrioxamine (in the iron/DFO

group), circulating iron dextran, and other forms of non–transferrin-bound plasma iron that likely made variable contributions to the measurements.29 These profound protective benefits of DFO were evident in these experiments despite the modest decrement in cardiac iron produced by the chelator treatment, a mean decrease of only about 20% as determined in our previous studies.32 The limited decrease in cardiac iron in these studies is a consequence, in part, of the quantitative relationships between the amounts of iron and chelator administered. In the gerbil model subjected to the accelerated regimen used here, the rate of iron loading was 800 mg/wk [14 mmol/kg] over 20 weeks for a total of 16,000 mg/kg [286 mmol/kg]. This rate of iron loading is much greater than that in patients who receive transfusions on a long-term basis. For comparison, the rate of iron loading from transfusion in thalassemia major is about 1.8 to 3.5 mg/kg/wk (0.3-0.6 mmol/kg/wk].3 DFO is a hexadentate chelator, and 1 molecule of DFO is needed to bind 1 atom of ferric iron. On a molar basis, the amount of DFO administered in these studies was substantially less than the amount of iron given. In the gerbil studies, DFO was administered in subcutaneous bolus injections of 100 mg/kg (1.5 mmol/kg) 2 times a day, 5 days a week or, over 20 weeks, a total of 20,000 mg/kg (30 mmol/kg).

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Thus the amount of iron administered was more than 9 times greater than the maximum amount that could be chelated by the DFO. We also note that the large doses of DFO used in this study would, in normal animals without iron overload, produce growth retardation and even death.48,49 The comparisons are of necessity inexact, in part because DFO is an inefficient chelator. In clinical use, only 5% to 10% of the drug administered binds iron and leads to its excretion,50 although the proportion may have been higher under the conditions of these studies. Moreover, for the maximum chelating effect to be achieved, DFO must be given in a continuous parenteral infusion.15,51 In our studies, the chelator was given by subcutaneous bolus injection twice daily. In human subjects, subcutaneous administration of DFO twice a day has been reported to result in urinary iron excretion comparable to that obtained with prolonged (8 to 12 hours/day) subcutaneous infusion52 but likely less than what could be achieved with continuous infusion. A further factor to consider is that a proportion of the iron administered during these studies probably remains metabolically inaccessible as complexes of iron dextran sequestered within macrophages of the reticuloendothelial system,53-55 with 70% to 80% of these cells located within the liver.56 We also note that the cardioprotective benefits of DFO observed in the gerbil model are consistent with earlier observations by Hershko and colleagues in iron-loaded rat heart cells in culture that indicated favorable effects of the chelator on mitochondrial enzymes.57-59 Overall, the results of these studies add to the growing evidence of the utility and relevance of the gerbil model of iron overload for the study of both the functional and structural consequences of iron-induced cardiac disease.24,25,29-32,60 Studies to date have provided evidence that concurrent administration of DFO can protect against iron-induced cardiac disease.29,32 Future investigations will examine the extent to which the cardiomyopathy of iron overload is reversible with iron chelation. The observations that modest decreases in cardiac iron with DFO treatment may greatly enhance survival, protect against potentially lethal arrhythmias, and prevent both cardiac hypertrophy and congestive failure may also have clinical relevance in the design of optimal chelating regimens.40,56 Finally, the gerbil model may be especially useful in the evaluation of iron-chelating agents being considered for clinical application in the management of transfusional iron overload.62-66 REFERENCES

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