Changes of the calcium tolerance in anthracycline treated mice

Exp Toxic Pathol 1999; 51: 235-238

URBAN & FISCHER

http://www.urbanfischer.de/joumals/exptoxpath

Institute of Physiology II, Friedrich-Schiller University Jena, Germany

Changes of the calcium tolerance in anthracycline treated mice ANDREA NACHBAR and G.-A. BIEWALD With 6 figures Received: February 2, 1998; Revised: March 15, 1998; Accepted: March 22, 1998 Address for correspondence: Prof. Dr. G.-A. BIEWALD, Institute of Physiology II, Teichgraben 8, D-07740 Jena, Germany. Key words: Calcium tolerance; Anthracyclines; Cardiomyopathy; Heart, anthracycline.

Summary Anthracyclines are effective antineoplastic agents inducing cardiomyopathy as a side effect. The mechanism of the anthracycline cardiotoxicity is not completely understood, but a disturbance of the Ca2+-homeostasis seems to be involved. In our study we investigated the Ca2+-tolerance in mice after a single administration of2 mg/kg violamycin bl (vbl). Isolated hearts were perfused with increasing concentrations of Ca2+until cardiac arrest. In the hearts of control animals the highest tolerable concentration was about 19 mM. 4 and 8 days after the vbl application 8 and 12 mM Ca2+, respectively, induced cardiac arrest. On the 12th day the Ca2+tolerance was similar to control organs. 16 and 20 days after the vbI injection the highest tolerable Ca2+-concentration sank again. On the 24th day the Ca2+-tolerance was nearly the same as in controls.

Introduction The clinical use of anthracyclines in the treatment of various neoplastic diseases is limited by the risk of cardiotoxicity (BRISTOW et al. 1978). Experimental studies of cardiotoxicity induced by doxorubicin, the most commonly used anthracycline, have provided drug related alterations in the structural and functional integrity of several cellular components including sarcoplasmic reticulum (BOUCEK et al. 1993), mitochondria (SOLEM et al. 1996), and the plasma membrane (ARANCIA et al. 1995). One consequence seems to be the impairment of the calcium homeostasis inducing a calcium overload and damage of the cell. Anthracycline treated cancer patients often underwent an additional treatment, e.g. with calcium-antagonists (SAMPI 1987) or angiotensin-converting-enzyme (ACE) inhibitors (JENSEN et al. 1996) which influence the calcium homeostasis. Thus, the purpose of our study was to investigate the time course of the calcium tolerance in

the hearts of mice from the 4th to the 24th day after a single anthracycline administration. To get unequivocal results, we used violamycin BI (vbI), a highly cardiotoxic anthracycline of the rhodomycinone type (KIRCHNER et al. 1980).

Material and methods As much as 42 mice of both sexes were used in these experiments. They were 20-30 weeks old and weighed 24 ± 1 g. 30 animals were treated with 2 mg/kg vbI dissolved in sterile saline. The substance was injected into the lateral tail vein. 12 control animals only received equivalent doses of sterile saline. After 4, 8, 12, 16,20 or 24 days the mice (n = 5 vbI-treated, n = 2 controls each day) were killed by cervical dislocation. The hearts were quickly excised, via the aorta cannulated and perfused by Langendorff's technique using a buffer solution containing 150 mM NaCI, 5.4 mM KCI, 2.5 mM CaCI 2 , 0.5 mM MgS04 , 11.1 mM glucose,S mM HEPES (pH 7.4). The solution was continuously bubbled with O2 and maintained at 37°C. The perfusion pressure was about 40 em H2 0. The hearts were electrically paced at a rate of300 beats/min (5 Hz) by a stimulator (TSE Systems, 910) activated platine electrode placed on the atrial-ventricular border. The left ventricular pressure was measured using a pressure transducer (PEU, Anton Paar KG) recorded and analysed by a computer system (TIDA). The lvp at a Ca2+concentration of 2.5 mM was regarded as baseline. After a stabilisation of 20 minutes the Ca2+-concentration in the perfusion solution was reduced to 2.0, 1.5, 1.0 and 0.5 mM and then starting with 3.0 mM increased in steps of 1.0 mM until the hearts stopped beating for more than 2 minutes. The perfusion with each Ca2+-concentration lasted for 10 minutes, during the last 2 minutes the lvp was registrated. The influence of the Ca2+-concentration was expressed as per cent change from the baseline lvp at 2.5 mM Ca2+ «(lvp 2.5 mM [mmHg] - lvp Ca2+ [mmHg]/lvp 2.5 mM [mmHg] x 100». 0940-2993/99/51/03-235 $ 12.00/0

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Statistical analysis of the data: Statistical comparisons between untreated and anthracycline-treated groups were made by Student's t-test; all data are expressed as mean values ± SD. Statistical significance was assessed for P values below 0.05.

fusion with a solution containing 11.0 mM Ca2+ induced irregular contractions, at 12.0 mM Ca2+ the hearts stopped beating. At Ca2+-concentrations of7.0 mM and more the lvp in vbI-treated hearts was significantly lower than in control hearts. 200

Results

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Control: The reduction of the Ca 2+-concentration of the solution under 2.5 mM reduces the Ivp to 94 ± 16 % at a concentration of2.0 mM, to 67 ± 14 % at 1.5 mM and 35 ± 6 % of the starting value at 0.5 mM. After an increase of the Ca2+-concentration to 3.0 mM the hearts developed about 160 ± 10 % of the contraction force they reached at 2.5 mM. Ca 2+-concentrations as high as 14.0 mM did not change the Ivp any longer. A concentration of 15 mM reduced the lvp to 19 ± 10 %,16.0-19.0 mM induced short term cardiac arrest, at 20.0 mM the hearts stopped beating permanently. 4 days after anthracycline treatment (fig. 1): At Ca 2+_ concentrations under 2.5 mM the hearts reacted like the hearts of untreated animals. The reduction to 2.0 mM decreased the Ivp to 88 ± 8 %. At 1.5 mM Ca2+ the Ivp was 74 ± 12 %, at 1.0 mM 50 ± 7 % and at 0.5 mM 32 ± 6 % of the baseline value. The increase in the Ca2+-concentration to 3.0 mM led to a doubling of the lvp and induced a significantly higher rise of the contraction force than in control hearts. The lvp sank to 143 ± 13 % at 4.0 mM, to 124 ± 3 % at 5.0 mM and to 117 ± 8 % at 6.0 mM. At 7.0 mM the lvp reached 12 ± 2 % of the starting level and was significantly lower than the control value. The perfusion with 8.0 mM Ca2+containing solution caused cardiac arrest.

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Fig. 2. The lvp in dependance on the Ca2+-concentration 8 days after the vbI treatment, the broken line indicates irregular contractions, * lvp significantly lower than in controls.

12 days after anthracycline-treatment (fig. 3): There was no significant alteration in the lvp measured during the perfusion with 2.0 mM compared to the baseline value at 2.5 mM or the lvp at 2 mM in control organs. The further reduction of the Ca2+-concentration did not induce a significantly greater decrease (42 ± 13 % at 1.5 mM, 29 ± 25 % at 1.0 mM and cardiac arrest at 0.5 mM) than in control organs. 3.0 mM and 4.0 mM Ca2+ did not alter the lvp compared to the baseline value at 2.5 mM. At concentrations of 5.0 and 7.0 mM the lvp reached his maximum of about 140 ± 12 % of the baseline. Ca 2+-concentrations of 8.0 and higher induced a decrease of the lvp that was significantly lower than the control value only at 10-14 mM. At 20 mM Ca2+ the hearts developed 58 ± 1 % of the initial value, but they did not always follow the pacing. At 21 mM the hearts stopped beating. 200

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Fig. 1. The lvp in dependance on the Ca +-concentration 4 days after the vbI treatment, the broken line indicates irregular contractions, * lvp significantly lower than in controls.

8 days after anthracycline treatment (fig. 2): Similar to the control organs in the vbI-treated hearts the reduction of the Ca2+-concentration to 2.0 mM caused a decline in the lvp to 81 ± 20 % of the starting value. At a concentration of 1.5 mM the contraction force was 40 ± 12 %, at 1.0 mM 26 ± 7 % of the lvp at 2.5 mM and was in both cases significantly lower than in control organs at these concentrations. At 0.5 mM the hearts did not beat any longer. An increase of the Ca2+-concentration caused a rise in the lvp to 126 ± 35 % at 3.0 mM and 149 ± 30 % at 5.0 mM. Afterwards the lvp sank to 36 ± 15 % at 10.0 mM, the per236

Exp Toxic Pathol51 (1999) 3

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Fig. 3. The lvp in dependance on the Ca2+-concentration 12 days after the vbI treatment, the broken line indicates irregular contractions, * lvp significantly lower than in controls.

16 days after anthracycline treatment (fig. 4): The hearts developed at 2.0 mM Ca2+73 ± 6 %, at 1.5 mM 39 ± 9 %, at 1.0 mM 28 ± 8 % and 0.5 mM 6 ± 6 % of the contraction force they did at 2.5 mM. Ca2+-concentrations of less than 2.5 mM induced a significantly stronger decrease in the lvp than in untreated organs.

Higher concentrations than 2.5 mM Ca2+ did not cause any further increase in the lvp. Between 3.0 and 5.0 mM Ca2+ the hearts reached 85 ± 7 % of the initial value at 2.5 mM, at 6.0 mM there were short term cardiac arrests, at 7.0 mM the hearts stopped beating. Concentrations of more than 2.5 mM Ca2+induced significantly lower lvps than in untreated hearts.

compared to the initial value. At 6.0 mM an increase of the contraction force started and the Ivp reached about 167 ± 20 % at 7.0-10.0 mM Ca2+. Afterwards the Ivp decreased to 112± 14 % at 15.0mM. Under perfusion with 16.0-18.0mM Ca2+the contraction became irregularly and there were transient cardiac arrests, at 20 mM the hearts stopped beating.

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Fig. 4. The lvp in dependance on the Ca2+-concentration 16 days after the vbI treatment, the broken line indicates irregular contractions, * lvp significantly lower than in controls.

Fig. 6. The Ivp in dependance on the Ca2+-concentration 24 days after the vbI treatment, the broken line indicates irregular contractions, * Ivp significantly lower than in controls.

20 days after anthracycline treatment (fig. 5): At 2.0 mM Ca2+ the lvp was 60 ± 8 %, at 1.5 mM 37 ± 7 %, at 1.0 mM ± 1 % of the initial value at 2.5 mM, at 0.5 mM they did not beat any longer. The perfusion with concentrations under 2.5 mM induced a significantly stronger decrease in the Ivp than in control organs. Higher concentrations than 2.5 mM did not cause increase of the Ivp compared to the initial value at 2.5 mM. Between 3.0 and 5.0 mM the lvp reached about 70 ± 12 % ofthe baseline value, at 6.0 mM the Ivp sank to 50 ± 21 %. At 7.0 mM the hearts did not beat any longer. Higher Ca2+-concentrations than 2.5 mM were asso~iated with significantly lower Ivps than in control organs.

Discussion

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Fig. 5. The lvp in dependance on the Ca2+-concentration 20 days after the vbI treatment, the broken line indicates irregular contractions, * Ivp significantly lower than in controls.

24 days after anthracycline treatment (fig. 6): The hearts developed at 2.0 mM 67 ± 12 %, at 1.5 mM 43 ± 7 % and at 1.0 mM 8 ± 1 % of the initial value at 2.5 mM. Similar to the 20th day after the vbI treatment the reduction of the Ca2+concentration induced a significantly stronger decrease of the Ivp than in control hearts. The perfusion with solution containing between 3.0 and 5.0 mM Ca2+ did not alter the Ivp

The alteration of the calcium tolerance in the hearts of anthracycline-treated mice shown in our experiments may be connected with the ability of the intracellular calcium regulation. The latter could be influenced by the damages of the mitochondria by the decrease of the cellular ATPamount and the following inactivation of calcium transport processes as well as by the impairment of the mitochondria in their function as calcium store. The disturbance of the cellular energy state could be in parts caused by the high affinity of anthracyclines for the binding of cardiolipin, a phospholipid of the inner mitochondrial membrane. This inactivated the cytochrome-c-oxidase, the NADH-dehydrogenase and the cytochrome-creductase that require a specific cardiolipin environment (GOORMAGHTIGH et al. 1980; FRY & GREEN 1980; FRY & GREEN 1981; GOORMAGHTIGH et al. 1982; PRAET et al. 1984). The cellular ATP and GTP levels sank without clear signs of lipid peroxidation (CINI-NERI et al. 1993). But the formation of free superoxide radicals should also contribute to the cellular energy deficit by alterations of the electron pump systems, the mitochondrial membrane and the oxidative phosphorylation (GOORMAGHTIGH & RUYSSCHAERT 1984). Decreasing cellular ATP amount should impair the function of the Ca2+-ATPase and, via the N a+-K+ -ATPase, the N a+-Ca2+-exchanger. Additionally the Ca2+-dependent ATPase of the sarcoplasmic reticulum and the sarcolemmal N a+-K+ -ATPase are directly damaged by anthracyclines (OLSEN et al. 1988). If and how far a calcium overload of the mitochondria is a primary cardiotoxic anthracycline effect, is discussed. OLSEN et al. (1974) suggest, that change in the mitochondrial calcium handling is involved in the pathogenesis, in an other study (ECKENHOFF & SOMLYO 1989) the mitoExp Toxic Pathol51 (1999) 3

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chondrial calcium transport and the permeability were not altered by anthracyclines. Additionally a receptor mediated lesion of the sarcoplasmic reticulum by anthracyclines was shown. The binding of anthracyclines to the ryanodine receptor of the sarcoplasmic reticulum increases the affinity of the receptor channel activator site for calcium. The channel can be activated by low calcium concentrations and the calcium release is enhanced (PESSAH et al. 1990; BOUCEK et al. 1991). The binding seems to be fully reversible. The succession of these events is not completely understood. In our experiments the calcium tolerance first decreased, was nearly normal at the 12th day after the vbItreatment and decreased again afterwards. It might be possible, that the first reduction was indirectly caused by the vbI binding to cardiolipin and the ryanodine receptor. The normalisation of the Ca2+ tolerance could therefore be a result of the elimination of vb!. The reason for the second impairment could be the vbI-induced generation of free radicals and the following oxidative damage of enzymes and mitochondria. Acknowledgement: This study was supported by the Bundesministerium fUr Bildung, Wissenschaft, Forschung und Technologie BMBF (01 ZZ 9602). The authors thank Mrs. S. BERNHARDT for excellent technical support.

References 1. ARANCIA G, BORDI F, CALCABRINI A, et al.: Ultrastructural and spectroscopic methods in the study of anthracyclinemembrane interaction. Pharmacol Res 1995; 32: 255-272. 2. BOUCEK RJ JR, BUCK SH, SCOTT F, et al.: Anthracyclineinduced tension in permeabilized cardiac fibers: evidence for the activation of the calcium release channel of sarcoplasmic reticulum. J Mol Cell Cardio11993; 25: 249-259. 3. BRISTOW DG, CLARK JW, JR.: A mathematical model of the vagally driven primary pacemaker. Am J Physio1 1983; 244: H150--161. 4. CrNI-NERI G, BANDINELLI M, NERI B: Free-radical production and nucleotide loss in anthracycline-induced cardiac damage. Med Sci Rev 1993; ???: 905-907. 5. ECKENHOFF RG, SOMLYO AP: Cardiac mitochondrial calcium content during fatal doxorubicin toxicity. Toxico1 ApplPharmacol 1989;97: 167-172.

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6. FRY M, GREEN DE: Cardiolipin requirement by cytochrome oxidase and the catalytic role of phospholipid. Biochem Biophys Res Commun 1980; 93: 1238-1246. 7. FRY M, GREEN DE: Cardiolipin requirement for electron transfer in complex I and III of the mitochondrial respiratory chain. J BioI Chern 1981; 256: 1874-1880. 8. GOORMAGHTIGH E, CHATELAIN P, CASPERS J, et al.: Evidence of a specific complex between adriamycin and nagatively-charged phospholipids. Biochim Biophys Acta 1980; 597: 1-14. 9. GOORMAGHTIGH E, BRASSEUR R, RUYSSCHAERT JM: Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment. Biochem Biophys Res Commun 1982; 104: 314-320. 10. GOORMAGHTIGH E, RUYSSCHAERT JM: Anthracycline glycoside-membrane interactions. Biochim Biophys Acta 1984;779:271-288. 11. JENSEN BV, NIELSEN SL, SKOVSGAARD T: Treatment with angiotensin converting enzyme inhibitor for epirubicin-induced dilated cardiomyopathy. Lancet 1996; 347: 297-299. 12. KIRCHNER E, HARTL A, GUTTNER J, et al.: Comparative studies on cardiotoxicity of some anthracycline antitumor antibiotics. Arch Toxicol Supp11980; 4: 399-401. 13. OLSON HM, YOUNG DM, PRIEUR DJ, et al.: Electrolyte and morphologic alterations of myocardium in adriamycin-treated rabbits. Am J Patho11974; 77: 439-454. 14. OLSON RD, MUSHLIN PS, BRENNER DE, et al.: Doxorubicin cardiotoxicity may be caused by its metabolite, doxorubicinol. Proc Nat! Acad Sci USA 1988; 85: 3585-3589. 15. PESSAH IN, DURIE EL, SCHIEDT MJ, et al.: Anthraquinone-sensitized Ca2+ release channel from rat cardiac sarcoplasmic reticulum: possible receptor-mediated mechanism of doxorubicin cardiomyopathy. Mol Pharmacol 1990;37:503-514. 16. PRAET M, POLLAKIS G, GOORMAGHTIGH E, et al.: Damages of the mitochondrial membrane in Adriamycin treated mice. Cancer Lett 1984; 25: 89-96. 17. SAMPI K: Cancer chemotherapy combined with a calcium antagonist in patients with hematologic malignancies and solid tumors resistant to standard chemotherapy. Gan To Kagaku Ryoho 1987; 14: 951-955. 18. SOLEM LE, HELLER LJ, WALLACE KB: Dose-dependent increase in sensitivity to calcium-induced mitochondrial dysfunction and cardiomyocyte cell injury by doxorubicin. J Mol Cell Cardio11996; 28: 1023-1032.