A Study On The Role Of Sphingomyelinases In Coxsackievirus B3 Infection Of Cardiomyocytes

Viral myocarditis (VMC) is a common cardiovascular disease. Although most patients have good prognosis, about 25%-30% of them will evolve into dilated cardiomyopathy (DCM) threatening health and life. Therefore, it is far-reaching to explore the pathogenesis, understanding of which can provide support for clinical treatments.The basic pathogenesis of myocarditis is immune inflammatory response which is induced by persistent virus infection and will lead to apoptosis or necrosis of myocardiocytes. The main morphological characteristics of the cells going through apoptosis include cell swelling, membrane rupture or dissolution, inflammatory factors release and so on. The membrane is the primary barrier against viruses. Sphingomyelin (SM), one of the most important components of membrane skeleton, can be synthesized with ceramide (Cer) and phosphorylcholine provided by phosphatidylcholine. Acyl CoA and Serine generate Cer in the endoplasmic reticulum catalyzed by the enzyme of reducing cofactor 2 (nicotinamide adenine dinucleotide phosphate, NADPH) and fatty acyl transferase. At the same time SM can be degraded by sphingomyelinases (SMases) into Cer and phosphorylcholine. SMases can be divided into three categories:Acid Sphingomyelinase (ASMase), Neutral Sphingomyelinases (NSMases) and Alkaline Sphingomyelinase. It has been reported that ASMase and NSMases (NSMase-1 and NSMase-2) have a high level expression in the cardiovascular system and play an important role in myocardial ischemia and reperfusion. It shows that synthesis and degradation balance of SM is important to maintain the integrity of myocardiocytes via the regulation of the SMases.In the pathogenesis of viral myocarditis, whether or not viral invasion and cell membrane damage are related to abnormal SMases expression? Whether or not the related pathways facilitate viruses infection of the cells? Whether or not process of apoptosis is triggered by the above factors? There has been no report. Coxsackievirus B3 (CVB3) is the most common virus in VMC. Therefore, we observe mRNA expression and activity changes of SMases (ASMase and NSMases) in myocardiocytes after CVB3 infection to investigate the role of SMases in VMC.Part I:SMases expression in CVB3 infected myocardiocytesObjective:To detect the changes of mRNA expression and activity of ASMase and NSMases in myocardiocytes after CVB3 infection at different time points.Methods:Myocardiocytes were separated and purified from neonate SD rats, When the cells'conditions of attachment, pulse and viability were good enough 24 hours later, they were infected with CVB3 for Oh,21,4h,8h,12h,24h,36h and 48h respectively. Control groups without CVB3 infection were set at the same time. SMases mRNA expressions in myocardiocytes were detected with Real-time quantitative polymerase chain reaction (Real-time PCR) at each time point and thin layer chromatography (TLC) was used to detect the activity of NSMases and ASMase at time points of 4h and 12h.Results:Both ASMase and NSMase-2 mRNA expression levels increased compared with the control group within 48hrs. The fluctuant trend might result from competition of raw materials. ASMase activity increased at 4hrs,12hrs after CVB3 infection; NSMase-2 activity increased significantly at 4h and decreased at 12h but still higher than normal level. Activity of NSMase-1 and mRNA expression had no significant changes at different time points after CVB3 infection.Conclusion:ASMase and NSMase-2 might play an important role in CVB3 infection and injury of myocardiocytes. NSMase-1 might have no significant effect in this process.Part II:The relationships of SMases expression, CVB3 replication and myocardial apoptosisObjective:To observe whether or not the increased expression and activity of SMases are related with virus replication and myocardial apoptosis after CVB3 infection of myocardiocytes at different time points. Method:Myocardiocytes were cultured and infected in the same way narrated in the first part. CVB3 RNA expression in myocardiocytes was detected with Real-time PCR. Virus titer was measured with TCID50 and apoptosis of myocardiocytes was detected with annexin V-FITC by flow cytometry. Relationships of virus replication and myocardiocytes apoptosis with SMases expression were studied.Results:CVB3 invasion to myocardiocytes was reflected by CVB3 proliferation (CVB3 RNA expression) and toxicity (virus titer). The overall effect was consistent with the expression of SMases. The number of CVB3 began to increase after 4h post infection and continued increasing within 48h. Virus toxicity rapidly increasd almost simultaneously after infection. Desptite volatility, it was still higher than normal levels.Conclusion:Myocadiocytes resisted to viruses through apoptosis etc. in the early stage of CVB3 infection (within 8h p.i.), so the number of CVB3 (CVB3 RNA expression) and toxicity (virus titer) were inhibited; but in a later stage (beyond 8h p.i.), the cells could not protect themselves, massive offspring viruses were generated and the enhanced toxicity caused myocardial necrosis. The gene expression of SMases reflected the viral injury degree to myocardiocytes:with the negative injury factors (including virus titer and virulence) increasing, the gene expression of SMases was upregulated, and vice versa. CVB3 infection might lead to the expression of SMases causing damage of cell membrane and facilitating the virus entry which eventually triggers apoptosis.Partâ…¢:Study of the mechanism involved in CVB3 infection induced SMases expression in myocadiocytesObjective:To observe the ERK changes at protein level and the relationship with SMases when myocardiocytes were infected by CVB3 at different times points. This preliminary study was designed to ascertain whether the expression of SMases mRNA is associated with the ERK pathway activation. Methods:Myocardiocytes were cultured and infected in the same way narrated in the first part. Gene expression of SMases at different times points of Oh, 2h,4h,8h,12h,24h,36h, and 48h post infection were detected respectively as well as ERK expression changes with western blot, the results would compare with the SMases expression.Results:The expression of ERK1 and ERK2 protein did not change after CVB3 infection. However, activated ERK proteins (p-ERK1, p-ERK2) increased in the setted time course, and the trend curve was consistent with the ASMase mRNA expression basically in the early two hours, which suggested that ERK pathway might induce the mRNA expression of ASMase. Comparatively, p-ERK1, p-ERK2 expression changes were seemingly not associated with NSMases mRNA expression.Conclusion:ERK pathway involved in the regulation of ASMase gene expression, contributing to the injury of myocardiocyte membrane, facilitating the virus entry and enhancing the virus infectivity.ConclusionFrom this research we found that gene expression and activation of SMases increased through ERK signal transduction pathway in CVB3 infected myocardiocytes, resulting from which the essential component of membrane skeleton, SM, was eventually degraded. The degeneration, dissolution or necrosis of myocardiocytes in this process resulted in the non-specific inflammation of viral myocarditis.