Cellular Mechanism Of Bradykinin In Alleviating The Decrement Of Ventricular Myocytes Contractility Induced By Doxorubicin In Guinea-pigs

Objective: Doxorubicin, a broad-spectrum antitumor drug, plays an important role in the treatment of acute leukemia and breast cancer. Unfortunately, clinical application indicates that although doxorubicin is effective in the treatment of tumor cells, cardiac arrhythmia and heart failure can be found in large doses or long-term application, which limits its clinical dosage increment. Bradykinin is a vasoactive peptide containing nine amino acid residues. A previous study indicates that it has protective effect on heart in ischemia-reperfusion injury model. While there is no evidence showing that bradykinin has protective effects or not against the myocardial toxicity caused by doxorubicin. To explore this question, the effects of doxorubicin on the sarcomere length and calcium transients of ventricular myocyte in guinea pig in the presence or absence of bradykinin were observed. The goal of this study is to investigate the protective effects of bradykinin against acute cardiotoxicity induced by doxorubicin in guinea-pig. The study provides a new sight on the treatment of cardiotoxicity induced by doxorubicinMethods:1 Preperation of ventricular myocytes: Male adult guinea-pigs were anesthetized by abdominal injection, then hearts were rapidly excised and left ventricular myocytes were isolated freshly via Langendorff perfusion apparatus.2 Detemination of sarcomere length and calcium transient: The left cardiac ventricular myocytes were loaded with Fluo-2AM for 25³0 min without daylight. Then the myocardial cells were put into the chamber and sarcomere length and calcium transient were detected with the video-based, motion edge detection system. The effects of doxorubicin on the sarcomere length and [Ca²⁺]i transient amplititudes were analyzed in the presence or absence of bradykinin. The parameters of the sarcomere length and [Ca²⁺]i transient amplititudes of ventricular myocytes were calculated and analyzed by ANOVA analysis.3 Detemination of action potential and I_Ca-L: Male adult guinea-pigs were anesthetized by abdominal injection, then hearts were rapidly excised and left cardiac ventricular myocytes were isolated. The ventricular myocardial cells were put into the chamber, and the action potentials were recorded by whole cell patch-clamp technique in current-clamp mode. I_Ca-L was recorded in voltage-clamp mode. The effects of doxorubicin on the action potential duration and I_Ca-L were analyzed with or without bradykinin. The parameters of action potential duration(APD₅₀ and APD₉₀) and I_Ca-L were calculated.4 Statistical design and data analysis: The baseline cell length and the fluorescence ratio before the drug administration were assigned a value of 100% to calculate the percentages of changes in cell shortening and the fluorescence ratio after drug administration. Data were presented as mean±SD. Values were compared by paired or unpaired Student's t-test. Statistical significance was considered to be P < 0.05.Results: 1 Effects of doxorubicin on cell shortening with or without bradykinin. The experiments were done by using a video-based, motion edge detection system. The cell shortening and the maximal rate of contraction?d L/dtContrac? were decreased by doxorubicin-treatment in a concentration-dependent manner?P < 0.01?. The decrements of sarcomere length and the maximal rate of contraction by doxorubicin were alleviated by pretreated myocytes with bradykinin?P < 0.01?; 2 Effects of doxorubicin on calcium transients in the presence or absence of bradykinin. Diastolic [Ca²⁺]i was increased by doxorubicin dose-dependently?P < 0.05 or P < 0.01? while systolic [Ca²⁺]i had no obvious change in all doxorubicin concentrations. The amplitude of calcium transients was decreased by doxorubicin Concentration-dependently?P < 0.05 or P < 0.01?. The elevation of diastolic [Ca²⁺]i and reduction of calcium transient amplitude caused by doxorubicin were relieved by bradykinin; 3 Effects of doxorubicin on action potential duration in the presence or absence of bradykinin. Both APD₅₀ and APD₉₀ of ventricular myocytes were reduced Concentration-dependently in the presence of doxorubicin?P < 0.01?. The decrements of APD₅₀ and APD₉₀ induced by doxorubicin were also improved by pretreatment the myocytes with bradykinin?P < 0.01?; 4 Effects of doxorubicin on I_Ca-L in the presence or absence of bradykinin. I_Ca-L amplitudes were decreased by doxorubicin-treatment in a concentration-dependent manner?P < 0.01?. The inhibitory effects of doxorubicin on I_Ca-L were caused by delaying the activation process. The decrements of I_Ca-L caused by doxorubicin were alleviated in the pretreated myocytes with bradykinin via blunting the activation process.Conclusion:1 Doxorubicin decreases sarcomere length and intracellular calcium transient, elevates diastolic [Ca²⁺]i, shortens action potential duration and reduces I_Ca-L. The results demonstrate that the toxic effects of doxorubicin on myocardial cells are caused by dysfunction of intracellular calcium hemostasis.2 Bradykinin has protective effect against cardiotoxicity caused by doxorubicin. In fact, we do find that the shortened sarcomere length, elevated diastolic [Ca²⁺]i and reduced amplitude of calcium transients caused by doxorubicin were alleviated by bradykinin. Bradykinin blunts the shortening of action potential duration and diminution of I_Ca-L induced by doxorubicin.