BIOCHEMICAL MEDICINE 18, 131-138 (1977)

Inhibition of the Cardiac Mitochondrial Calcium Pump by Adriamycin in Vitro

LEON MOORE,’ ERWIN J. LANDON, AND DAVID A. COONEY

Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, and Laboratory of Toxicology, National Cancer Institute,

Bethesda, Maryland 20014

Received October 12. 1976

Adriamycin and daunomycin are anthracycline antibiotics with excep- tional antitumor activity (l-5). One limitation to the clinical utility of these compounds is their ability to produce a total dose-dependent car- diotoxicity (3,5-8). A number of laboratory species, including the rat, exhibit pathological changes in the myocardium after treatment with adriamycin or daunomycin. Only the rabbit and monkey provide a laboratory animal model exhibiting both cardiomyopathy and congestive heart failure associated with chronic administration (9-12). In treated rabbits, electrolytes are altered (Na+ and Caz+ increase) in the ventricular myocardium before there is clinicopathologic evidence of car- diomyopathy (12). Elevations of tissue calcium are implicated in the genesis of a number of chemically induced necrotic processes (12-15). Both the mitochondria (16,17) and sarcoplasmic reticulum (isolated as part of the microsomal pellet) (l&19) of cardiac tissue have been suggested as important elements in the control of cytoplasmic calcium levels in the heart. In this report we describe in vitro inhibition by adriamycin of the energy-dependent calcium transport activity of cardiac mitochondria.

METHODS

Rat cardiac tissue was homogenized in 0.25 M sucrose with a polytron PT20; microsomes were isolated by a modification of the method of Harigaya and Schwartz (18). Cardiac mitochondria were generally pre- pared by the method of Landon (20) without EDTA and by the method of

1 Send reprint requests to Dr. Leon Moore, Laboratory of Molecular Biology, National Cancer Institute, Bldg. 37 4B19, Bethesda, Maryland 20014.

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132 MOORE. LANDON. AND COONli\l

Sordahl and Schwartz (21). The animals employed in this study were male rats of the Sprague-Dawley strain weighing approximately 250 g.

Mitochondrial calcium uptake was determined in the following medium: 100 mM potassium chloride, 30 mtvt imidazole-histidine buffer, pH 6.8, 5 mM ammonium oxalate, 5 mM Mg Cl,, 5 mM ATP (pH adjusted to 6.8 with imidazole), 20 PM 4sCaCl, (0. I ~Ciiml) in a total volume of 3 ml at 37”. At zero time the membrane fraction was added (0.025-0.05 mg of protein/ml. final concentration) to the prewarmed incubation medium. At each time point a 5OOq.d sample was removed, filtered, and 45calcium was deter- mined as previously described (22.24). In some experiments total calcium was increased to 100 PM and 5 mM sodium phosphate was added to the incubation. In one experimental series in which mitochondrial calcium uptake was determined at 100 FM calcium, oxalate was omitted from the assay mixture. In one series of experiments ATP was replaced by ADP, inorganic phosphate, and a-ketoglutarate. Microsomal (sarcoplasmic re- ticulum) calcium uptake was determined in a similar medium with 5 mM sodium azide added. The protein concentration of the microsomal incuba- tions was 0.025 to 0.05 mgiml. Nucleotides employed were obtained from the Sigma Chemical Co.. St. Louis. Missouri. 45CaCl, (8 mCi/mg of calcium) was obtained from New England Nuclear Corp., Boston, Mas- sachusetts.

Electron micrographs of the mitochondrial (20) preparations were pre- pared as follows. Samples of the preparation were fixed in 1.5% glutaral- dehyde in phosphate buffer, postfixed in osmium tetroxide, dehydrated in graded ethanols, and embedded in Epon-Araldite. Thin sections. cut at 250 to 500 8, with a Reichert Om U-2 ultramicrotome, were mounted on Formvar-coated 200-mesh copper grids. These sections were stained with lead citrate and uranyl acetate and examined with a Jeolco 1OOB electron microscope at 80 kV.

RESULTS

Electron micrographs of myocardial mitochondria prepared by the method of Landon (20) demonstrate that the preparation consists primar- ily of intact mitochondria in the orthodox configuration. The preparation also contains disrupted mitochondria, figures that resemble lysosomes, and an occasional nonvescicular structure that may have derived from disrupted mitochondria, connective tissue, or polymerized actin. None of the fields examined contain material that resemble the organized contrac- tile apparatus.

Energy-dependent calcium uptake by both our mitochondrial and mic- rosomal fractions of the rat heart was similar to that observed by others (18). Calcium uptake activity of six preparation of the mitochondrial fraction prepared by the method of Landon (20) was inhibited 70 2 7.5%

ADRIAMYCIN AND CALCIUM TRANSPORT 133

by 5 mM sodium azide, the classical inhibitor of mitochondrial calcium uptake. Calcium uptake activity of mitochondria prepared by the method of Sordahl and Schwartz (21) was somewhat less inhibited by sodium azide (three preparations averaged 62%). The fraction of calcium uptake activity of these mitochondrial preparations not inhibited by azide may represent contamination with fragments of the sarcoplasmic reticulum. Both mitochondrial preparations translocate calcium at similar rates. In our hands, calcium uptake of the microsomal fraction was not inhibited by 5 mM sodium azide (six preparations, data not shown).

As demonstrated in Fig. 1, calcium uptake activity of the myocardial mitochondria was inhibited at the lowest adriamycin concentration tested (10 PM). Inhibition increased as the concentration of adriamycin in the assay medium increased. At 1 mM adriamycin, calcium uptake was 49% that of control in medium containing 20 PM calcium and only 24% that of control in medium containing 100 PM calcium. In two mitochondrial

MINUTES

FIG. I. The effect of in vitro adriamycin on rat cardiac mitochondrial calcium uptake. (A) Total calcium uptake was determined as described in Methods for mitochondria at a calcium concentration of 20 MM. (B) The conditions are similar, with 5 mM sodium phos- phate, pH 6.8, and the total calcium concentration increased to 100 pM. Each point represents the mean ? SE for the determination in six membrane preparations.

134 MOORE. LANDON. AND C'OONEY

preparations with energy-dependent calcium uptake supported by (Y- ketoglutarate, ADP, and inorganic phosphate, adriamycin (1 mM) inhib- ited calcium uptake to 47 and 34% of control In one series of experi- ments ATP-dependent mitochondrial calcium uptake was determined without oxalate in the assay mixture. At 100 PM calcium and 5 mM phosphate, mitochondrial calcium uptake was inhibited to 40% that of control in the presence of 0.5 mM adriamycin and to 30% that of control in the presence of I mM adriamycin. As expected oxalate had no effect on mitochondrial calcium uptake at the concentrations tested.

We have examined the possibility that adriamycin would produce the observed effect by promoting the passive efflux of calcium from mitochondria. ATP-dependent mitochondrial calcium uptake in the pres- ence of 100 PM calcium and 5 mM phosphate was determined in the usual manner for 20 min. After 27 min of uptake, sodium azide was added to a final concentration of 5 mM to inhibit further uptake. One minute later. adriamycin in deionized water was added to a final concentration of 1 mM. Control incubation received azide and then deionized water in place of the adriamycin solution. Samples were removed at timed intervals to deter- mine the rat of calcium efflux. Efflux was followed until greater than 95% of accumulated 45Ca2+ had been lost. There was no difference between control and adriamycin-containing incubations at all five efflux time points examined.

The lack of significant inhibition of cardiac microsomal (sarcoplasmic reticulum) calcium uptake is demonstrated in Fig. 2. At the 30-min time point, 1 mM adriamycin reduced microsomal calcium uptake to a negligi- ble degree (~20%). In four microsomal preparations challenged with adriamycin in the absence of a calcium-trapping agent (oxalate) and at 20 PM calcium, only 1 mM adriamycin had any effect on calcium uptake. Again, uptake was reduced to about 80% that of control at the 30-min time point (data not shown).

We have tested daunomycin in both cardiac subcellular fractions at a single concentration (1 mM). Daunomycin reduced calcium uptake by the microsomal fraction by less than 5%. At the same level it reduced mitochondrial calcium uptake to 47% that of control in a 20 PM calcium medium and to 26% that of control in 100 PM calcium. [In the rat, cardiac levels of daunomycin are approximately 40 PM 10 min after a single dose of 30 mg/m2 (23); the drug accumulates with repeated doses.]

In vitro inhibition of mitochondrial calcium uptake is not limited to cardiac mitochondria. In liver mitochondrial preparations adriamycin (1 mM) inhibited calcium uptake by about 50% when the assay medium contained 20 WM calcium. The liver microsomal fraction has also been shown to sequester calcium by an energy-dependent process (24). In vitro adriamycin has little effect on calcium uptake by the liver microsomal

ADRIAMYCIN AND CALCIUM TRANSPORT 135

2500 04 CONTROL M A IO-% &Cl A l6%4 &ii A lCi3W

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FIG. 2. Adriamycin in vitro and calcium uptake by rat cardiac sarcoplasmic reticulum. Calcium uptake was determined as described in Methods for sarcoplasmic reticulum (micro- somes). The assay contained 100 pM calcium and each point represents the mean 2 SE for the determination in six membrane preparations.

preparation at 10 or 100 pM; adriamycin reduced calcium uptake less than 5%. At 1 mM, adriamycin reduced liver microsomal calcium uptake to about 75% that of control.

DISCUSSION

Organ-specific necrosis produced by chemical agents has prompted study of the toxic reaction to a number of compounds. In the liver, compounds such as carbon tetrachloride (13,25) and thioacetamide (25,26) evoke a necrotic lesion. In the heart, isoproterenol (14,27), adriamycin, and daunomycin (lO- 12) produce organ-specific necrosis. After adminis- tration of these agents, an elevation of tissue calcium has been noted. Carbon tetrachloride (28,29) and isoproterenol (14,27) produce an in- crease of tissue calcium within an hour. Several investigators have suggested that this increase of tissue calcium may be important in the genesis of the necrotic process (12-14,25-27). Our recent demonstration of energy-dependent calcium uptake by liver microsomes (24) and the prompt, marked, and prolonged inhibition of this activity after administra-

136 MOORE. LANDON. AND COONEY

tion of carbon tetrachloride in vivo (30) suggested an investigation of the in vitro effect of adriamycin on the calcium uptake activity of mitochon- drial and microsomal fractions of the rat heart.

In muscle, calcium serves as the message coupling excitation and contraction. In skeletal muscle the sarcoplasmic reticulum serves as the sink for calcium necessary to the coupling of excitation and contraction. In addition, the sarcoplasmic reticulum. utilizing an energy-dependent transport process, sequesters cytoplasmic calcium to allow relaxation of the muscle (3 1,32). In cardiac muscle it is less clear which cellular or- ganelle or organelles are responsible for the control of cytoplasmic cal- cium levels. Some investigators suggest that the mitochondria play an important role in calcium sequestration (17.33). while others stress the importance of the sarcoplasmic reticulum (18.19). An alteration of cal- cium transport of either the mitochondria or sarcoplasmic reticulum of cardiac tissue by adriamycin might contribute to the alteration of electro- lyte metabolism apparent after chronic administration of the anthracy- cline antibiotics ( 12).

Daunomycin and adriamycin are inhibitors of mitochondrial function. These compounds are quinones; they inhibit coenzyme Q10 (34) and uncouple oxidative phosphorylation by cardiac mitochondria at concen- trations in excess of 100 PM (35-37). On the basis of this information one would predict that these compounds could alter mitochondrial calcium transport. We have observed an inhibition of mitochondrial calcium ac- cumulation by adriamycin and daunomycin. Decreased net calcium ac- cumulation could occur if calcium uptake were inhibited or if the passive efflux of calcium were stimulated by these compounds. Our results suggest that adriamycin and daunomycin inhibit mitochondrial calcium uptake, but have little if any effect on the passive efflux of accumulated calcium. Nevertheless, any relation between this biochemical phenome- non and the development of drug-induced cardiomyopathy remains to be established.

SUMMARY

Adriamycin and daunomycin are clinically useful antineoplastic agents, known for their ability to produce a delayed cardiomyopathy upon chronic administration. Recently it has been suggested that an alteration of myocardial calcium metabolism precedes the cardiomyopathy. This study demonstrates that these cardiotoxic antibiotics inhibit one of the subcellular systems thought to be important in the regulation of calcium metabolism in the heart. In the present communication we report that calcium translocation by cardiac mitochondria is inhibited by these an- thracyclines, but that calcium translocation by the cardiac sarcoplasmic reticulum is not inhibited.

ADRIAMYCIN AND CALCIUM TRANSPORT 137

ACKNOWLEDGMENTS

This work was supported in part by USPHS Grant HL 14681, Grant GMOOO58, and a grant from the Middle Tennessee Heart Association.

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