| Research Background: Adriamycin was antitumor agent employed clinically for the treatment of several types of cancer.However,according to a large number of clinical therapeutic effects and experimental research,adriamycin has the ability to phagocyte myocardium,which cause cardiotoxicity in the heart,thus its clinical application is limited.Cardiomyocardial toxicity caused by adriamycin was chronic toxicity,continuous or gradual accumulation,with dose dependence,and this damage was irreversible,severe lead to patients with heart failure and other cardiac diseases.It was clinically called adriamycin–induced cardiotoxicity.Calumenin,a member of the CREC family,is commonly found in endoplasmic reticulum/sarcoplasmic reticulum of mammalian cardiomyocytes.According to relevant literature reports,Calumenin can alleviate endoplasmic reticulum stress response of cardiomyocytes to a certain extent,it reduced the apoptosis rate of cardiomyocytes by endoplasmic reticulum stress.In recent years,the role of Calumenin in regulating calcium homeostasis in the endoplasmic reticulum has been widely recognized in studies of adriamycin–induced cardiotoxicity.With the deepening of the study on adriamycin–induced cardiotoxicity.These and other studies have suggested its occurrence and development mechanism is very complex,however,the conclusion remains to be established by in vivo and in vitro experiments to explore the role of Calumenin in the pathogenesis of adriamycin–induced cardiotoxicity,and to provide a theoretical basis for the treatment of adriamycin–induced cardiotoxicity.Objective: 1.In the present study,we have examined to investigate the role of Calumenin in the occurrence and development of adriamycin–induced cardiotoxicity and its possible mechanism.Methods: 1.Animal experiment: adult SD rats(180-200 g)were selected to establish the blank control(CON)group and adriamycin–induced cardiotoxicity(ADR)group at different time points(2week,4week and 8week).The rat model of adriamycin-induced cardiomyopathy was established by intraperitoneal injection of adriamycin solution.When the model was built,the changes of body weight and heart weight of rats in each group were observed.We have further estimated the cardiac progressive damage of rats in each group.Cell experiment: the Suckling mice of healthy adult rats were selected,SPF grade,healthy male,1-3 d;Cells were isolated and cultured to establish control(CON)group and adriamycin–induced cardiotoxicity(ADR)group,and ADR group(5 mol/L adriamycin + cardiomyocytes).2.After the successful preparation of adriamycin–induced cardiotoxicity rat model in each group,the rats were anesthetized by intraperitoneal injection.The apical part of the heart was extracted and put into the tissue fixation solution.The damage of adriamycin on rat myocardium was observed by pathological section,he staining and Masson staining.3.After the successful preparation of adriamycin–induced cardiotoxicity rat model in each group,the rats were anesthetized by intraperitoneal injection.The apical part of the heart was extracted and put into the tissue fixation solution,the relationship between Calumenin and mitochondrial morphological changes was explored by transmission electron microscopy.4.After the successful preparation of adriamycin–induced cardiotoxicity rat models in each group,the rats were anesthetized by intraperitoneal injection.The apical part of the heart was prepared the single cell suspension of cardiac cells,Ca2+ concentration was detected by Rho D-2-AM in vitro and in vivo,a mitochondrial calcium ion specific fluorescent probe,to study the relationship between adriamycin and Ca2+ concentration.5.After the successful preparation of adriamycin–induced cardiotoxicity rat models in each group,the rats were anesthetized by intraperitoneal injection.The apical part of the heart was extracted for mitochondrial purification and the single cell suspension of cardiac cells was prepared.The mitochondrial membrane potential was detected by JC-1 staining and the permeability of mitochondrial conversion pores of cardiomyocytes in each group was determined by calcein-AM green fluorescence staining in vitro and in vivo,so as to observe the effects of ADR on both.6.Cell proteins of each group in the in vitro experiment and myocardial tissue proteins of rats in the CON group,2-week ADR group,4-week ADR group and 8-week ADR group,respectively.ADR groups in the in vivo experiment were extracted,the expression levels of Calumenin protein,mitochondrial dynamics-related proteins and apoptotic related proteins were detected by Western blot.Further to observe protein expression between different time nodes.Results: 1.In animal experiment,compared with con group,the weight and heart weight of rats in ADR group were significantly decreased;With the delay of feeding time,,the ratio of heart weight to body weight was the largest in 8-week ADR group compared with CON group,2-week ADR group,4-week ADR group.2.The results of HE and Masson showed that the myocardial fibrosis in ADR group was more serious than that in CON group;compared with CON group,2-week and 4-week ADR group,myocardium fibrosis was the most serious in 8-week ADR.3.The ultrastructure of myocardium in each group was observed by transmission electron microscopy.The results showed that in the CON group,the ultrastructure of myocardium was normal,the running disc was clear,the mitochondria were orderly,the morphology was normal without swelling,and the muscle filaments were complete.However,in each group of ADR group,mitochondrial morphology was irregular,and the mitochondrial ridges were destroyed,the mitochondria of cardiomyocytes were swollen and disordered,and the myofilaments were dissolved and broken;compared with the 2-week ADR group and the 4-week ADR group,the above situation was most serious in the 8-week ADR group with the continuous accumulation of adriamycin in rats.4.According to the data measured by the luciferase plate instrument,both in the cell experiment and in the animal experiment,compared with the CON group,the Ca2+ concentration in the mitochondria of the ADR group was increased;in animal experiments,Ca2+ concentration was higher in 8-week ADR than in 2-week ADR and 4-week ADR.5.According to the data measured by the luciferase plate instrument,both in the cell experiment and in the animal experiment,compared with the CON group,the mitochondrial membrane potential in the ADR group showed a downward trend and the mitochondrial MPTP was significantly open;In animal experiments,compared with 2-week ADR and 4-week ADR,the decreasing trend of mitochondrial membrane potential and the open state of mitochondrial MPTP were more obvious in 8-week ADR.6.According to western blot results showed that the expressions of calumenin and mitochondrial fusion protein OPA-1 in ADR group were lower than those in CON group,while the expressions of mitochondrial fission protein DRP-1、Cytochrome C(Cyt C)、Chop and Bax were higher than those in CON group,both in the cell experiment and in the animal experiment.In animal experiments,in 8-week ADR,the expressions of Calumenin、mitochondrial fusion protein OPA-1 and Bcl-2 were lower than those in 2-week ADR and 4-week ADR,and the expressions of mitochondrial fusing proteins DRP-1、Cyt C、Chop and Bax were higher than those in2-week ADR and 4-week ADR.Conclusions: 1.Adriamycin could reduce the expression of Calumenin and promote Ca2+ overload in cardiomyocytes,further lead toincrease the expression of mitochondrial fission protein DRP-1,and decrease the expression of mitochondrial fusion protein OPA-1.2.Adriamycin could reduce the expression of Calumenin,increase the expression of apoptotic proteins,and then lead to the occurrence of Adriamycin–induced cardiotoxicity. |
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