Effects Of Amitriptyline On Calmodulin And Calcium Homeostasis In Rat Cardiomyocytes

[Objectives]As one of the most common antidepressants,amitriptyline is still widely used in some regions.It has complex pharmacological mechanisms and a variety of adverse reactions.Among them,the adverse reaction of cardiovascular system is the most common.Cardiotoxicity is the main cause of death.However,the mechanism of cardiotoxicity of amitriptyline is still unclear.Calcium ion has a very important status and role in the body,especially in cardiomyocytes.It is closely related to cardiovascular disease and adverse cardiovascular events.The regulation mechanism of calcium ions is also very complicated.Many calmodulins regulate calcium ions in different ways,and there are interactions between these calmodulins.At present,some studies have confirmed the effects of amitriptyline on potassium channels and sodium ion channels,but its effects on calcium channels and calmodulin have not yet been determined.And some research also just focused on the clinical changes.Therefore,further research on the cardiotoxicity mechanism of amitriptyline is essential.Differential proteins that cause myocardial damage,including some calmodulin,have been screened in vivo experiments with amitriptyline poisoning by iTRAQ technology,indicating that myocardial toxicity is related to calcium ions,and amitriptyline may affect the expression of calmodulin.Based on the existing experimental results,this study built a model of AMI infection in primary cardiomyocytes of neonatal rats,verified the expression of differential calmodulin related to the cardiac toxicity of AMI that screened by iTRAQ,and explored the cardiotoxicity mechanism of AMI poisoning.(1)To build a model of AMI exposure in primary cardiomyocytes of neonatal rats,and to observe the morphological changes of cardiomyocytes;(2)To verify the expression of differential calmodulin related to the cardiac toxicity of AMI that screened by iTRAQ,and to determine the effect of AMI on calmodulin and calcium homeostasis in primary cardiomyocytes of neonatal rats;(3)Exploring the mechanism of cardiotoxicity caused by AMI poisoning.[Methods](1)Building a model of AMI exposure in primary cardiomyocytes of neonatal rats:Primary cardiomyocytes of neonatal rats are cultured in vitro,inoculate into petri dishes,and culture until the 4th day for experimental operations.Set groups with different concentrations of 0.11/10/100μM and different intoxicate time of 2/4/6/8/12/24 h,observe the morphological changes of cardiomyocytes with a microscope,use the CCK-8 method to detect the toxic effects of AMI infection,and build a model of AMI infection of cardiomyocytes.(2)Detection of changes in calmodulin expression in cardiomyocytes by Western blotting:Primary cardiomyocytes of neonatal rats are cultured in vitro and cultured until the 4th day.The cardiomyocytes are exposed to AMI,and Western blot is used to detect the total protein of calmodulin RyR2,NCX1,Casq2 and Camk2 d in different groups.(3)Observing the steady-state changes of calcium ions in primary cardiomyocytes of neonatal rats:Primary cardiomyocytes of neonatal rats are cultured in vitro and the cardiomyocytes were exposed to cardiomyocytes on the 4th day according to the AMI model.Fluo-3 is used to label calcium ions,and changes of intracellular calcium concentration and calcium oscillation pattern in primary cardiomyocytes of neonatal rats of the AMI-exposed group and the blank group are observed with a laser confocal microscope.[Results](1)The morphological changes of cardiomyocytes in primary cardiomyocytes of neonatal rats caused by AMI exposure are characterized by cell shrinkage,pseudopodia thinning,cytoplasmic vacuoles formation,and spontaneous pulsation changes of cardiomyocytes;(2)According to the detection results of CCK-8,100μM AMI exposure cause the myocardial cell survival rate to be less than 50%.After 8 hours,the myocardial cell survival rate was inversely proportional to time,thereby a model of AMI exposure in primary cardiomyocytes of neonatal rats;(3)AMI exposure increase the expression of RyR2 in the 0.1 μM concentration group,down-regulate the expression in 1μM and 10μM groups;NCX1 down-regulate the expression in 0.1μM group;Casq2 down-regulate the expression in 0.1μM,increase the expression in 1μM and 10μM group;(4)AMI exposure significantly increases the oscillation amplitude of calcium ions and the peak value,accelerates the oscillation frequency.The oscillation mode is relatively unstable,and the average fluorescence intensity increased.[Conclusions](1)The myocardial cytotoxicity of AMI has a time-dependent relationship after 8h.There is no statistical difference in the survival rate of cells between part of intoxicate groups and blank groups before 8h.The exposure model is in 0.1/1/10μM concentration,and the exposure time is 8 hours;(2)AMI exposure affects the expression of calmodulin in the same way in in-vivo animal experiments and in-vitro cell experiments.There is a certain relationship with the concentration of AMI exposure;(3)AMI exposure can increase the concentration of calcium ions in cardiomyocytes,which may be the result of the interaction between the calmodulin.Conversely,changes in the concentration of calcium ion may also affect the function of the calmodulin,so it increase the possibility of arrhythmias,which may be one of the mechanisms of AMI poisoning and cardiotoxicity.