Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca²⁺-ATPase in cardiomyocytes of Adriamycin-treated rats

Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca 2+ -ATPase in cardiomyocytes of Adriamycin-treated rats first-author †E-mail:weic236@sohu.com †E-mail:weic236@sohu.com weic236@sohu.com Department of Internal Medicine, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Internal Medicine, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Objective: 2 To observe the effects of fructose-1,6-diphosphate (FDP) on serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca 2+ ]) and activity of sarcoplosnic Ca 2+ -ATPase (SRCa 2+ -ATPase) in Adriamycin (ADR)-treated rats. Methods: 2 Rats were intraperitoneally injected with ADR (2.5 mg/kg every other day for 6 times) and then with different dosages of FDP (every other day for twenty-one times). Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was performed to detect serum level of cTnI. CK-MB was detected by monoclonal antibody, Myo[Ca 2+ ] was detected by fluorescent spectrophotometry and the activity of SRCa 2+ -ATPase was detected by inorganic phosphate method. Results: 2 FDP (300, 600, 1200 mg/kg) significantly reduced the serum levels of cTnI and CK-MB, while at the same time decreased calcium concentration and increased SRCa 2+ -ATPase activity in cardiomyocytes of ADR-treated rats ( P < 0.01). Conclusions: 2 FDP might alleviate the cardiotoxic effects induced by ADR through decreasing calcium level as well as increasing SRCa 2+ -ATPase activity in cardiomyocytes. Fructose-1,6-diphosphate Adriamycin Cardiomyocyte Calcium Sarcoplosnic reticulum Ca 2+ -ATPase INTRODUCTION 1 Although Adriamycin (ADR) has been widely used in tumor treatments since the 1960s, and is now considered as one of most potent antineoplastic agents, its clinical applications remain limited owing to a series of side-effects, of which the cardiotoxicity is thought to be a major and most destructive one. Maeda et al.( 1998 1998 ) found that the cardiomyocytes of rats were Ca 2+ -overloaded and that the diastolic function could be remarkably damaged treatment with ADR, even if there were little changes in cardiac histology. Studies have confirmed in recent years that fructose-1,6-diphosphate (FDP) might reduce the myocardial damages induced by ADR via regulating the activities of oxygen free radicals in cardiomyocytes (Maeda et al., 1998 1998 ; Kang, 2003 2003 ). Yet up to now there is no information on whether FDP could relieve the Ca 2+ -overloaded state in ADR-treated cardiomyocytes. This study aims to determine the effects of FDP on serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca 2+ ]) and activity of sarcoplosnic Ca 2+ -ATPase (SRCa 2+ -ATPase) in Adriamycin (ADR)-treated rats, so that knowledge on the mechanisms of FDP’s effects against cardiotoxicity induced by ADR could be gained and the clinical applications of FDP can be further evaluated. METHODS 1 Experimental animals 2 Wistar rats ( n =40, 210~280 g, 20 males, 20 females) were supplied by Zhejiang Animal Experimental Center of China. Drugs and agents 2 ADR was purchased from HAIHUA Pharmaceuticals of Zhejiang (catalog 020305) and FDP was supplied by HUAQI Pharmaceuticals of Beijing (catalog 020116). Fura-2/AM was purchased from Sigma Company Ltd. of American. Groups and treatments 2 Rats were randomly divided into 5 groups, with 8 rats in each group. Rats in ADR group were intraperitoneally injected with ADR at dosage of 2.5 mg/kg every other day for 6 times, while in the ADR+FDP groups (I), (II), (III) they were intraperitoneally injected with ADR and then with FDP at dosage of 300, 600, 1200 mg/kg every other day for twenty-one times respectively. As a control group, rats were intraperitoneally injected with ADR and then with physiological saline following the same protocol. Twenty-four hours after treatments, rats were anesthetized by using mebumal sodium and then sacrificed; and the hearts were separated for detection of Myo[Ca 2+ ] level and activity of SRCa 2+ -ATPase. Blood samples were collected from the tail vein to detect the serum level of cTnI and CK-MB. The detection of serum level of cTnI and CK-MB 2 Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was applied to detect serum level of cardiac troponin I (cTnI). Creatine kinase-MB (CK-MB) was detected by immunoradiometric assay using monoclonal antibody. The detection of Myo[Ca 2+ ] 2 The hearts of rats were moved to precooled Hank’s solution (NaCl 8.006 g/L, KCl 0.4026 g/L, MgCl 2 0.1.16 g/L, Hepes 2.3850 g/L, Glucose 1.800 g/L) for 5 min. Aorta cannula was separated on langendroff perfusion device for 5 min-perfusion of Hank’s solution without calcium, then incubated in Hank’s solution (containing 0.1% collagen enzyme) for 15 min, followed by a 5 min-perfusion of Hank’s solution with low concentration of calcium. The hearts were treated at 37 °C and given continuous oxygen supply. Cardiomyocytes were then shaken in preheated and oxygen-supplied Hank’s solution for 15 min and harvested to a concentration of 2×10 6 cells/L, while the concentration of calcium was adjusted to 1.2 mmol/L. Five μl Fura-2/AM solution at concentration of 1 mmol/L was added into the cell suspension, which was water bathed under 98% concentration of oxygen at 37 °C for 40 min, then washed with Hank’s solution for three times followed by centrifugation (500 r/min, 1 min). Finally the cell suspension was adjusted to a concentration of 1.25×10 6 cells/ml for fluorometric quantitation. The fluorescence intensity (F) levels were measured by spectrophotometer (LC-240) with excitation and emission settings of 340 nm and 500 nm, respectively. The F max and F min were respectively detected after 0.1% Tritionx-100 and EDTA were added. The level of Myo[Ca 2+ ] was calculated using the formula as below: [Ca 2+ ] i =( F − F min )/( F max − F )×224. Detection of SRCa 2+ -ATPase activity 2 The SRCa 2+ -ATPase was prepared using the differential centrifugation method of Yang et al.( 2002 2002 ) Coomassie brilliant blue staining was used to quantify the concentration of SRCa 2+ -ATPase and its activity was detected by inorganic phosphate method. Statistical analysis 2 Data are expressed as the mean±standard error ( x ̄± s ) Statistical differences were determined by T test and linear correlation analysis. RESULTS 1 Data and information on well-being of the rats 2 The animals showed no distress when injected with Adriamycin and/or fructose-1,6-diphosphate, their body weight decreased a little after the experiment, but there was no statistical significance ( P > 0.05). Effect of FDP on levels of cTnI, CK-MB, Myo[Ca 2+ ] and activity of SRCa 2+ -ATPase in myocardium of ADR-treated rats 2 The levels of cTnI, CK-MB and Myo[Ca 2+ ] in the ADR group were much higher, while the activity of SRCa 2+ -ATPase decreased more significantly than those in the control group ( P < 0.01). When treated with FDP, the levels of cTnI, CK-MB and Myo[Ca 2+ ] decreased and the activity of SRCa 2+ -ATPase increased remarkably, showing statistical significance when compared with those in the ADR group ( P < 0.01, Table 1 1 ). P < 0.01 vs control group P < 0.01 vs ADR group P < 0.01 vs ADR+FDP (I) group P < 0.01 vs ADR+FDP (II) group pi: inorganic phosphate Group cTnI (ng/ml) CK-MB (μg/L) Myo[Ca 2+ ] (mmol/L) SRCa 2+ -ATPase (μmol pi/(mg protein·min)) Control 0.48±0.17 172±21.31 119±5.09 408±11.21 ADR (2.5 mg/kg) 5.03±2.26 * * 612±57.15 * * 289±12.17 * * 167±9.45 * * ADR+FDP (I) (2.5 mg/kg, 300 mg/kg) 2.28±1.43 # # 431±32.27 # # 217±9.82 # # 221±11.13 # # ADR+FDP (II) (2.5 mg/kg, 600 mg/kg) 1.31±0.94 † † 302±34.41 † † 173±10.06 † † 285±8.06 † † ADR+FDP (III) (2.5 mg/kg, 1200 mg/kg) 0.73±0.24 + + 216±26.17 † † + + 143±8.53 + + 368±12.53 + + Correlation analysis of FDP with cTnI, CK-MB, Myo[Ca 2+ ] and SRCa 2+ -ATPase 2 The effects of FDP on cTnI, CK-MB, Myo[Ca 2+ ] and SRCa 2+ -ATPase were dose-dependent ( P < 0.01) (Table 2 2 ). P < 0.01 vs FDP cTnI CK-MB Myo[Ca 2+ ] SRCa 2+ -ATPase FDP −0.913 * * −0.874 * * −0.925 * * 0.831 * * Correlation analysis of Myo[Ca 2+ ] with cTnI, CK-MB and SRCa 2+ -ATPase 2 There was negative relationship between Myo[Ca 2+ ] and SRCa 2+ -ATPsae ( P < 0.01), but positive relationship was found between Myo[Ca 2+ ] and cTnI or CK-MB ( P < 0.01, P < 0.05, respectively) (Table 3 3 ). P < 0.01 vs cTnI and SRCa 2+ -ATPase P < 0.05 vs CK-MB cTnI CK-MB SRCa 2+ -ATPase Myo[Ca 2+ ] 0.817 * * 0.632 # # −0.819 * * DISCUSSION 1 The precise mechanisms of how ADR causes cardiotoxicity remains unclear even today, but evidences suggested free radicals play a pivotal role in the process (Yang et al., 2002 2002 ). Increasing proofs indicated that calcium overload in myocardial cells could be closely correlated to ADR-induced cumulative cardiotoxitit (Li et al., 2002 2002 ; Huang et al., 2003 2003 ): ADR depolymerizes membrane phospholipids inlaid protein of myocardial cells, enhances the membrane permeability and increases the calcium influx; it also inhibits the activity of Na + -K + ATPase, reduces the Na + -K exchange and enhances the Na + -Ca 2+ exchange, therefore induces the calcium overload (Huang et al., 2003 2003 ). At the same time, ADR down-regulates the calcium intake of sarcoplasmic reticulum, leading to an overload state of calcium in myocardial cells (Maeda et al., 1998 1998 ). Present studies showed that ADR might increase the serum levels of cTnI, CK-MB and Myo[Ca 2+ ]–a positive correlation was found between the latter and the former two–and decreased the activity of SRCa 2+ -ATPase, whereas the effects of ADR was inhibited by FDP in a dose-dependant manner, suggesting that FDP could reduce the concentration of calcium and improve the activity of SRCa 2+ -ATPase in myocardial cells, therefore alleviate the ADR-induced myocardial damages. The mechanisms are presumed to be as described below: (1) To enhance the energy supply of myocardial cells. When myocardial cells are injured because of anoxia, low-energy supply or other harmful factors, the extrinsic FDP may supply energies by producing substance such as ATP to participate in the myocardial metabolism (Zhou et al., 1999 1999 ; Hua et al., 2003 2003 ); (2) To reduce the reperfusion cardiomyopathy caused by oxygen free radicals by FDP’s antioxidant effects (Maeda et al., 1998 1998 ; Kang, 2003 2003 ); (3) To relieve the calcium overload state. Although calcium is a key factor in mediating the excitement activities of myocardial cells, an extraordinary high concentration of calcium in myocardial cells will result in Ca 2+ -overload and myocardial function failure. FDP enhances the synthesis of ATP and calcium transportation so that the concentrations of calcium in myocardial cells could be controlled (Hua et al., 2003 2003 ). A study (Galzigna et al., 1989 1989 ) revealed that besides its transmembrane activity, FDP might combined with the membrane of myocardial cells to inhibit the Ca 2+ influx under conditions of anoxia or other myocardial injuries. The result is confirmed by Bickler and Kellecher ( 1992 1992 ) who worked on cortex cells and astrocytes of rats by fluorescent probe technique. In conclusion, we provide evidence that ADR leads to a higher serum levels of cTnI and CK-MB, which may in part be related with calcium overload in myocardial cells. FDP reduces the high level of calcium in myocardial cells, increases the activity of SRCa 2+ -ATPase, and finally alleviate the myocardial injuries caused by ADR. References PE Bickler , JA Kellecher . Fructose-1,6-diphosphate stabilizes brain intracellular calcium during hypoxia in rats . Stroke , 1992 . 23 1617 - 1622 . . 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