| Part 1 CACNA1 H expression was increased in DOX induced cardiactoxicity Objective:DOX is an effective broad-spectrum anthracycline antitumor drug,but it can cause dose-dependent irreversible damage to cardiac myocytes,lead to its limited application.Cardiac myocytes,as terminal differentiated cells,do not have the ability to divide and proliferate.When the myocardium is damaged due to stimulation,compensatory hypertrophy will occur,eventually leading to decreased cardiac function and HF.During this process,the gene expression in the nucleus of the myocardium will change,resulting in the reexpression of fetal genes.T-type calcium channel is only expressed in ventricular myocytes at the development stage,but not in mature cardiomyocytes.Its core structure is α1 subunit,and there are three different α1 subunit genes in mammals:CACNA1G,CACNA1 H and CACAN1 I.The purpose of this part of the study was to construct a DOX-induced cardiac injury model in mice and to detect changes in the expression of CACNA1 H,which is one of the core subunits of the T-type calcium channel.Methods:Twelve male C57BL/6 mice,aged 7 weeks,were randomly divided into experimental group and control group,with 6 mice in each group.The experimental group received a single intraperitoneal injection of 15 mg/kg DOX,while the control group received the same amount of normal saline.The weight of all mice was measured daily,and cardiac function was evaluated by ultrasound imaging system 5 days later.Subsequently,the mice were sacrificed,and cardiac tissue was retained and weighed,then Sirius red staining and HE staining was performed on mouse heart tissues to evaluate the damage of DOX to mouse cardiomyocytes,and the difference of CACNA1 H between two groups was evaluated by RT-PCR and immunohistochemical staining.Results:Compared with the control group,the body weight and heart weight of the DOX treatment group was reduced,the values of EF and FS were significantly decreased(P< 0.05),and the cardiac function was impaired.Sirius red staining showed disordered arrangement of myocardial fibers and collagen deposition in DOX treated mice.HE staining results showed that the arrangement of myocardial cells was disordered in the DOX treatment group,and the proportion of vacuolization in myocardial cells was significantly increased compared with that in the control group(P < 0.05).Both RTPCR and immunohistochemical results showed that the expression level of CACNA1 H in the heart tissues of mice treated with DOX was significantly higher than that of the control group(P < 0.05).Conclusion:DOX can induce impaired cardiac function in mice,resulting in disordered arrangement of mouse cardiac cells.CACNA1 H is involved in DOX-induced cardiotoxicity.DOX can damage the cardiac function and the arrangement structure of cardiomyocytes in mice.The expression of CACNA1 H was increased in DOX-induced cardiac injury,which may be involved in DOX-induced cardiotoxicity.Part 2 CACNA1 H was a key molecule in DOX induced cardiotoxicity Objective:DOX can induce apoptosis of cardiomyocytes in various ways,and endoplasmic reticulum stress is closely related to apoptosis.Some studies suggest that m RNA levels of endoplasmic reticulum stress-related proteins are increased in patients with heart failure,and inhibition of endoplasmic reticulum stress can alleviate the occurrence of cardiac hypertrophy.However,the role of endoplasmic reticulum stress in DOX cardiotoxicity has been less studied.Endoplasmic reticulum,as the largest intracellular calcium ion reservoir,can not only regulate intracellular calcium ion concentration,but also its structure and functional state are affected by intracellular calcium ion concentration.In the first part of this study,it was found that CACNA1 H,one of the Ttype calcium channel subtypes,was increased in the expression of DOX-induced cardiac injury.In this part,the role of CACNA1 H in DOX-induced cardiac toxicity was explored by inhibiting CACNA1 H,and the relationship between CACNA1 H and endoplasmic reticulum stress was studied.Methods:24 mice were purchased for the preliminary experiment,and the mice were randomly divided into control group and DOX group,with 12 mice in each,and the model was constructed according to the first part of the study.Then,mice in each group were randomly divided into four groups and given 0,2,10 and 50 mg/kg ABT-639(CACNA1H inhibitor,HY-19721)by gavage for 5 consecutive days.Five days later,the mice were sacrificed and sampled to test the inhibitory effect of inhibitor ABT-639 on CACNA1H.The results showed that 10 mg/kg ABT-639 could effectively inhibit the increase of CACNA1 H expression caused by DOX.Another 24 7-week-old male C57BL/6 mice were purchased for animal experiments and randomly divided into 4groups: control group,DOX group,ABT group and DOX+ABT group,with 6 mice in each group.DOX group and DOX+ABT group were given 15 mg/kg of DOX intraperitoneal injection,control group and ABT group were given the same amount of normal saline intraperitoneal injection.From the day on,ABT group and DOX+ABT group were given a gavage of 10 mg/kg of ABT-639(HY-19721)every day,control group and DOX group were given the same amount of normal saline.The weight of each group was measured every day,5 days later the cardiac function in mice was assessed by ultrasonic imaging system.After sampling,heart tissue was weighed,then Sirius red staining and HE staining was performed on mouse heart tissues to evaluate the damage of DOX to mouse cardiomyocytes,and the difference of CACNA1 H between each group was evaluated by RT-PCR and immunohistochemical staining.Apoptosis of mouse cardiomyocytes was evaluated by TUNEL staining.Endoplasmic reticulum stress in mouse heart tissue was evaluated by CHOP immunofluorescence staining and Western Blot.Results:The results of RT-PCR and immunohistochemical staining showed that the expression levels of m RNA and protein of CACNA1 H in DOX-treated mice were significantly higher than those in the control group(P < 0.05).The addition of ABT-639 could significantly alleviate the DOX-induced increase in CACNA1 H expression(P < 0.05).The body weight and heart weight of mice in the ABT+DOX group was increased compared with that in the DOX group,and the values of EF and FS was significantly increased compared with that in the DOX group(P < 0.05).Sirius red staining showed that the myocardial fibers of mice in the ABT+DOX group were in orderly arrangement,and HE staining results showed that the myocardial cells in the ABT+DOX group were aligned,and the vacuolization ratio of myocardial cells was significantly lower than that in the DOX treatment group(P < 0.05).Western blot showed that in the heart tissue of DOX group,the expression of endoplasmic reticulum stress marker protein p-PERK,ATF6,GRP78,the proteins participate in the endoplasmic reticulum stress related to apoptosis pathway ATF4,CHOP,and the apoptosis protein caspase12 were significantly increased than the control group(P <0.05),the expression of these proteins in the heart tissue of ABT + DOX group were significantly decreased compared with that in the DOX group(P < 0.05).TUNEL staining showed that the number of apoptotic cardiomyocytes in the DOX treatment group was significantly increased compared with the control group(P < 0.05),and the number of apoptotic cardiomyocytes in the ABT+DOX group was significantly decreased compared with the DOX treatment group(P < 0.05).Immunofluorescence staining showed that the number of CHOP positive cells in the heart tissues of mice treated with DOX increased significantly compared with the control group(P < 0.05),and the number of CHOP positive cells in the ABT+DOX group decreased significantly compared with the DOX treatment group(P < 0.05).Conclusion:DOX can induce cardiac toxicity by inducing myocardial cell apoptosis and endoplasmic reticulum stress.Inhibition of CACNA1 H can reduce the cardiotoxic effect of DOX.Part 3 CACNA1 H participated in DOX induced cardiomyocyte apoptosis through endoplasmic reticulum stress pathway Objective:DOX damages the myocardial cell membrane nonspecifically,causing calcium ions to flow through the nonspecific channel,leading to calcium overload,which leads to the occurrence of myocardial injury.Exogenous compounds can cause changes in the calcium channels on ER membrane,leading to the phenomenon of calcium depletion or calcium overload of ER,resulting in the dysfunction of ER,and finally irreversible apoptosis.This part of the study aims to explore whether calcium channel CACNA1 H can influence the apoptosis of cardiomyocytes through endoplasmic reticulum stress pathway by using endoplasmic reticulum stress inhibitors in cell experiments.Methods:H9C2 cells were cultured in vitro,after synchronous treatment,1 μM of DOX was used to interfere with the cells for 12 hours(DOX treatment group).On this basis,10μM CACNA1 H inhibitor ABT-639 were used to treat cells(ABT+DOX group),and another group was given 30 μM ER stress inhibitor UR-906 treated cells(UR+DOX group).CCK-8 assay was used to detect the cell survival of each group after different treatments,and RT-PCR was used to detect the expression of CACNA1 H in each group.The expression of CHOP protein in H9C2 cells of each group was evaluated by CHOP immunofluorescence staining,the expression of ER stress and apoptosis-related markers in H9C2 cells of each group was evaluated by western blot.The production of reactive oxygen species in H9C2 cells was evaluated by DHE,and the concentration of calcium in H9C2 cells was evaluated by Fluo 4-AM.Results:CCK-8 result showed that 1μM DOX had obvious cytotoxic effect on H9C2 cells,while 10 μM ABT-639 and 30 μM UR-906 could significantly improve the cell damage caused by DOX.RT-PCR showed that ABT-639 could significantly inhibit the increase of CACNA1 H expression caused by DOX(P < 0.05).Immunofluorescence staining showed that compared with the control group,the expression of CHOP protein was increased in the dox-treated group(P < 0.05),and it was more integrated with the nucleus.Compared with the DOX treated group,the expression of CHOP protein in the ABT+DOX group and UR+DOX group significantly decreased(P < 0.05),and the decrease was more significant in the UR+DOX group.Western Blot showed that in the cells of DOX group,the expression of endoplasmic reticulum stress marker protein pPERK,ATF6,GRP78,the proteins participate in the endoplasmic reticulum stress related to apoptosis pathway ATF4,CHOP,and the apoptosis protein caspase12 and caspase3 were significantly increased than the control group(P < 0.05),the expression of these proteins in the cells of ABT+DOX group and UR+DOX group were significantly decreased compared with that in the DOX group(P < 0.05),and UR-906 had stronger effect.DHE staining showed that the production of reactive oxygen species was increased significantly in the DOX treatment group compared with the control group(P < 0.05).Compared with the DOX treatment group,the production of reactive oxygen species decreased significantly in the ABT+DOX group and the UR+DOX group(P < 0.05),and the effect of UR-906 was stronger.Fluo 4-AM staining showed that the intracellular calcium concentration in the DOX treatment group was significantly higher than that in the control group(P < 0.05),and the calcium concentration in the ABT+DOX group and the UR+DOX group was significantly lower than that in the DOX treatment group(P < 0.05),and the reduction was more significant in the ABT+DOX group.Conclusion:Dox-induced up-regulation of CACNA1 H can induce excessive endoplasmic reticulum stress in cardiomyocytes,leading to apoptosis of cardiomyocytes... |
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