| BACKGROUND:Methamphetamine (MA) is also called methylamphetamine. Its hydrochloride is colorless transparent crystal, commonly known as "ice", was abused widely in the world. MA is a highly active stimulant in the central nervous system that can lead to addictive behavior, including euphoria, elevating mood, decreasing appetite and Sympathetic effect. The most frightening thing is use of MA carries with it a very strong possibility of addiction. Large doses produce intoxication and even sudden death. A number of clinical case reports and animal experiment data show that MA can cause severe injury to multiple organs, liver and kidney failure and heart rate imbalances in large dosage (more than20mg) used once. Long term administration of MA in low dosage can cause chronic toxicity, result in significant mental and behavior changes, such as euphoria, irritability, spiritual movement dysfunction, etc. MA has been increasingly used by drugs abusers, and numerous deleterious effects in humans including cardiovascular disease, such as cardiac arrhythmias, ischaemia, aortic dissecting aneurysm, hypertension, hypertrophic cardiomyopathy, acute coronary syndrome, congestive heart failure, and sudden death have been reported. MA induced cardiovascular system diseases and their complications, may be the most possible cause of death in MA abusers. Therefore, the cardiovascular toxicity effect of MA gradually becomes the focus and hotspot in the research of the related areas. To date, MA-induced acute myocardial infarction has been overwhelmingly reported. Cardiomyocyte apoptosis plays a critical role in the progression of heart failure. In patients with end-stage cardiomyopathy, loss of cardiomyocytes due to apoptosis can be observed that leads to the progression of cardiac dysfunction, ultimately heart failure.Some clinical reports show that the use of digoxin and calcium channel blockers can relieve symptoms and improve cardiovascular function in patients who are suffering from the cardiovascular abnormalities after MA abuse.The data indicated that MA may cause the changes of function and expression of LTCC, and plays a critical role in the progression of arrhythmia.In the present study, our aim was to analyze whether IGFBP5involved in MA-induced apoptosis as a novel target and activated the cell apoptosis pathway. In addition, the study also focused on the expression of LTCC which were correlated to the electrical remodeling caused by MA. In order to investigate the cardiotoxicity effect of MA, especially focus on the mechanism of arrhythmogenesis.OBJECTIVE:Establishment of MA treated neonatal rat ventricular myocytes toxicity model. The direct effects of MA on cardiomyocytes are still not clear. We analyzed the effect of MA on cardiomyocyte apoptosis and studied the expression of LTCC of the heart. The present study indicated that MA-induced cardiomyocyte apoptosis and remodeling of LTCC expression may be two possible factor inducing arrhythmogenesis of MA-induced cardiac lesions.In order to verify the cellular toxic effects of MA administration, acute animal model has been developed. The reversible effect of changes after MA withdrawal was evaluated by MA withdrawal animal model in different period.METHODS:This study was divided into two sections, in vitro and in vivo, to investigate cardiotoxicity induced by MA.一、MA-induced cardiotoxicity in neonatal rat ventricular myocytes (NRVMs). 1、MA-induced apoptosis in NRVMs.(1) NRVMs were prepared from0to1-day-old neonatal Sprague-Dawley rats (Experimental Animal Center of SMU, China). Ventricles sacrificed by decapitation were quickly removed, rinsed four times with modified Hank’s solution, and minced into small pieces on ice. Cultured cells were incubated with DMEM supplemented and exposure to0.5mM,1.0mM and1.5mM concentration of MA saline solution respectively. Cultured cells were incubated with DMEM supplemented without exposure to MA as the control group. Cells were grown in a humidified atmosphere of5%CO2and37℃through the experiments.(2) Observation of morphological changes after MA administration in NRVMs.(3) MA induced apoptosis in NRVMs were observed by terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assay.2%The mechanisms of MA-induced apoptosis in NRVMs.(1) Real-time PCR and Western Blot analysis:IGFBP5mRNA expression was identified in NRVMs using real-time PCR, IGFBP5protein expression was identified in NRVMs using western blot.(2) RNA interference (RNAi):siIGFBP5knockdown IGFBP5protein level in NRVMs.(3) Terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assay:Transfection with siIGFBP5reduces the apoptosis rate in NRVMs induced by MA.(4) Western Blot analysis:To investigate whether transfection with siIGFBP5reduces the apoptosis rate through caspase-3, the expression of caspase-3was identified in NRVMs using western blot.3、The effect of MA on the expression of the calcium cycling pathway proteins in NRVMs.(1) Real time PCR analysis:Real time PCR analysis was performed to assess the gene expression of LTCC alc, β2， SERCA2a, NCX and RyR2in NRVMs.(2) Western Blot analysis:Western Blot analysis was performed to assess the protein expression of LTCC alc, β2, SERCA2a, NCX and RyR2in NRVMs.二、Toxicity effect on rat ventricular myocardium induced by MA.1、Acute MA administration animal model and MA withdrawal animal model.(1) Acute MA administration group (AM group):A total of10Sprague-Dawley male rats, weighting from200to220g, were raised per cage with alternating light-dark cycle (lighting14hours per day) at26℃room temperature. Food and water were provided ad libitum. Rats were habituated to the animal facilities for7days before use. Each of rat was injected intraperitoneally with MA in1ml of saline (0.9%) at the dose of5mg per kg body weight twice daily (at12h intervals) for1week. Rats were killed24h after the last injection of MA and the hearts were quickly removed.(2) Acute MA withdrawal group (AWgroup):A total of40Sprague-Dawley male rats, weighting from200to220g, were raised per cage with alternating light-dark cycle (lighting14hours per day) at26℃room temperature. Food and water were provided ad libitum. Rats were habituated to the animal facilities for7days before use. Each of rat was injected intraperitoneally with MA in1ml of saline (0.9%) at the dose of5mg per kg body weight twice daily (at12h intervals) for1week. Rats were subdivided into4groups (AW1. AW2、AW4and AW8) and sacrificed after1,2,4and8weeks.(3) Acute control group (AC group):A total of10Sprague-Dawley male rats, weighting from200to220g, were raised per cage with alternating light-dark cycle (lighting14hours per day) at26℃room temperature. Food and water were provided ad libitum. Rats were habituated to the animal facilities for7days before use.1ml of0.9%normal saline injected into each rat twice daily in the same way as with the acute MA administration group. Rats were killed24h after the last injection of MA and the hearts were quickly removed. 2、Pathological studies of cardiac lesions after acute MA administration and MA withdrawal in rat ventricular myocardium. Ventricles were taken and preserved in10%formalin in room temperature for24h. For paraffin sections, Harris hematoxylin and eosin (H&E) staining was performed to assess the pathological analysis.3、The effect of acute MA administration and MA withdrawal on the expression of the ion channel proteins in rat ventricular myocardium(1) Real time PCR analysis:Real time PCR analysis was performed to assess the gene expression of LTCC alc, β2after acute MA administration and MA withdrawal in rat ventricular myocardium.(2) Western Blot analysis:Western Blot analysis was performed to assess the protein expression of LTCC alc, β2after acute MA administration and MA withdrawal in rat ventricular myocardium.Result:一、MA-induced cardiotoxicity in NRVMs.1、Morphological changes of NRVMsAfter24-48h of culturing, cells contact with each other to form cell clusters. Pulse synchronization. Frequency in70to90times/min. More than95%NRVMs were beat rhythmically.MA administration for0to24h, no obvious morphological changes in0.5mM group was observed, but the pulse frequency was increased. For48to72h, the cell beat became regular (70to90times/min). Very few cells appear round, flat, spindle form and refraction enhancement. No obvious morphological changes in1.OmM group were observed, but the pulse frequency was increased. For48to72h, the pulse frequency was gradually became slower (50to70times/min) and became irregular shape. According to the time of MA administration, Cell damage was aggravating, at the end, stop beating and death. And, we also found that,1.5mM group were more serious than1.OmM group.2, Using TUNEL staining demonstrated that MA induces apoptosis in NRVMs. A TUNEL results indicate that the apoptosis rate increased in NRVMs. No TUNEL-positive cells were observed in the control group. Apoptosis rate of0.5mM MA group was5.01±0.225%,1.0mM MA group was7.50±0.340%,1.5mM MA group was31.43±0.896%.0.5mM、1.0mM、1.5mM MA group relative to control group was considered statistically significant (P<0.01).3、The mechanisms of MA-induced apoptosis in NRVMs.The mRNA and protein levels of IGFBP5increase in MA dose-dependent manner in NRVM. The efficiently knockdown of IGFBP5expression also presented in NRVMs with MA treatment preceded by siIGFBP5transfection. TUNEL data demonstrated that silencing of IGFBP5expression protected NRVMs from MA-induced apoptosis to some degree. IGFBP5knockdown attenuates MA-induced cardiomyocyte apoptosis.Conclusion:The myocardial damage elicited by MA was thought to be due to a direct action of MA, though it seems likely that MA directly modulates the functioning of neonatal rat ventricular myocytes independent of neurotransmitters. MA is known to trigger myocardial damage following the higher concentration of MA administration.MA increased the expression of caspase-3. Silencing of IGFBP5with small interfering RNA significantly suppressed the expression of caspase-3in NRVMs after MA treatment. This indicates that the suppression or induction of apoptosis is mediated by a caspase-3dependent pathway.MA interferes with intracellular Ca2+homeostasis possibly through calcium cycling pathway proteins modulation. The LTCC and calcium handling proteins belong to important functional groupings that have been implicated in the pathogenesis of MA induced arrhythmias.Ica-L was another important ionic current in the repolarization of ventricular myocytes. The early after depolarizations and delayed after depolarizations caused by the enhancement of Ica-L in the repolarization were important incentives of the reentrant arrhythmia. Our findings suggest that remodeling of CACNA1C expression may be a possible factor inducing arrhythmogenesis in late convalescence period of MA-induced cardiac lesions. |
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