Investigation On The Role Of Calpain In The Pathogenesis Of Acute Virus Myocarditis

Viral myocarditis, especially acute viral myocarditis, is a common disease of cardiovascular system with a partial tendency to progress into dilated cardiomyopathy (DCM) which has a high mortality due to an irreversible outcome of heart failure. Lacking of full understanding of the pathogenesis mechanism, effective treatments are still out of reach worldwide.The known mechanisms causing DCM so far include the hereditary factors, the sustained virus infection, the autoimmune reaction to myocardium, mechanical stress and toxicants etc. Except the autoimmune and hereditary causes, virus (esp. coxsackievirus B3) infection is the most key factor leading to an eventual progression to DCM. Continuous injury to the heart tissue induced by sustained virus infection may be an important etiology of the disease which involved bimolecular events such as cardiocyte apoptosis and myocardium fibrosis etc.Calpain is a family of calcium-dependent intracellular cysteine proteases with a wide range of substrates including various cell skeletal proteins and enzymes involved in intracellular signal transduction. They functions in physiological processes like cell motility, cell cycle regulation, gene expression, and apoptosis etc. Via these functions in abnormal conditions, Calpain attributes to many pathological conditions including muscle dystrophy, cancers, Alzheimer's disease, ischemic-reperfusion injury, atherosclerosis and diabetes etc.Whether or not Calpain involves in the pathogenesis of viral myocarditis? Whether or not there exists a correlation between Calpain and bimolecular events like cardiocyte apoptosis, sustained virus replication and myocardium fibrosis etc. in the progression of virus myocarditis and DCM? Such information is unavailable so far. Here, serious experiments were designed and carried out to seek for evidence of Calpain's involvement in the pathogenesis of acute virus myocarditis.PartⅠ:A Study on the correlation between Calpain mRNA expression and Cardiocyte apoptosis in acute murine Coxsackievirus B3 myocarditisObjective:To detect the expression of Calpain-1 and Calpain-2 mRNA and study its relationship with cardiocyte apoptosis in acute murine coxsackievirus B3 (CVB3) myocarditis.Methods:Balb/c mice were single-infected with CVB3 to establish the experimental model of acute virus myocarditis. Meanwhile, drug (N-acetyl-Leu-Leu-Norleucinal, ALLN) intervention group (n=12) and vehicle control group (n=10) were set. Normal controls (n=8) were prepared with same volume of Dulbecco's Modified Eagle Media (DMEM) without CVB3. The cardiocyte apoptosis was detected through Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method and the apoptosis rate of each slide was calculated. The expression level of Calpain-1 and Calpain-2 mRNA were determined by RT-PCR.Results:Compared with the normal group and the vehicle control group, both the Calpain-1 (P=0.02 and P=0.04) and Calpain-2 (P<0.01 and P=0.02) mRNA expression level of the virus group were increased significantly followed by a significant apoptosis rate increase (both P values<0.01) respectively as well. No significant statistic difference was achieved between the normal control group and the vehicle control group as for both the Calpain-1 (P>0.05) and Calpain-2 (P>0.05) mRNA expression level and the cardiocyte apoptosis rate. The drug intervention group was significantly different with the normal control group (both P values<0.01) and the virus group (P=0.04 and P<0.01) respectively with regard to both the Calpain-1 and Calpain-2 mRNA expression level and the cardiocyte apoptosis rate (both P values<0.01). Calpain-1 and Calpain-2 mRNA expression level were significantly correlated with the cardiocyte apoptosis rate respectively.Conclusion:Coxsackievirus B3 participated in the onset of acute myocarditis via its promotion effect on Calpain mRNA expression, as well as a certain mechanism correlated with cardiocyte apoptosis inhibition.PartⅡ:Preliminary investigation on the effect of Calpain on the Coxsackievirus B3 replication in infected cardiocytes in vitroObjective:To investigate the effect of Calpain on the replication of Coxsackievirus B3 (CVB3) in cultured cardiocytes.Methods:Cardiocytes were separated from neonate SD rats, inoculated in the culture plates. When the cells'conditions of attachment, pulse and viability were satisfactory 24 hours later, cultured the cells with culture medium containing serially-diluted ALLN (40μg/ml,20μg/ml, 10μg/ml,5μg/ml,2.5μg/ml,1.25μg/ml and 0.625μg/ml) without serum for 0.5 hour. Then the medium was changed to one containing serially-diluted ALLN with 20% fetal calf serum and cultured in the incubator for another 48 hours. The same control groups without ALLN intervention were set at the same time. Cell viability was measured using Cell Counting Kit-8 (CCK-8) to detect drug's toxicity to the cardiocytes followed by the measurement of the change of viability of the cardiocytes infected by CVB3. Infected cardiocytes by CVB3 were inoculated with culture medium containing drugs with different concentrations of 1ug/ml; 2.5μg/ml and 5μg/ml respectively for 48 hours before the supernatant was harvested and virus 50% tissue culture infective dose (TCID50) were tested.Results:Compared with normal control group, lower concentrations (5μg/ml, 2.5μg/ml,1.25μg/ml,0.625μg/ml) of ALLN intervention made no difference in cells' absorbance (P>0.05), while higher concentrations brought about significant (P<0.05) decrease indicating drug's toxicity to the cardiocytes. Safe concentrations of ALLN significantly (P<0.01) enhanced the absorbance of CVB3-infected cardiocytes demonstrating its protective effect against virus infection. ALLN with a concentration of 5μg/ml significantly (P=0.02) decreased the titers of CVB3 in the culture medium supernatant while only a tendency of decrease was achieved with regard to the ALLN concentration of 1μg/ml and 2.5μg/ml.Conclusion:Safe concentrations of ALLN could preserve cardiocytes'viability from CVB3 infection in vitro probably through interfering with CVB3 replication. Calpain may play a certain role in the life cycle of CVB3.PartⅢ:Preliminary investigation on Calpain's effect on myocardium fibrosis in acute Coxsackievirus B3 myocarditisObjective:To observe the Calpain activity inhibitor's effect on the myocardium fibrosis degree in acute murine coxsackievirus B3 (CVB3) myocarditis and the proliferation and migration ability of myocardium fibroblast in vitro.Methods:Balb/c mice were single-infected with CVB3 to establish the experimental model of acute virus myocarditis. Meanwhile, drug (N-acetyl-Leu-Leu-Norleucinal, ALLN) intervention group (n=12) and vehicle control group (n=10) were set. Normal controls (n=8) were prepared with same volume of Dulbecco's Modified Eagle Media (DMEM) without CVB3. Mice were harvested on the seventh day and hearts were taken out and slices were prepared for HE staining to ascertain the establishment of the model, for Mallory staining to observe the degree of myocardium fibrosis and the total fibrosis area was calculated through Leica imaging system.Myocardium fibroblasts were separated from neonate SD rats and synchronized via a 24-hour's fast before inoculating with culture medium containing serially-diluted ALLN (40μg/ml, 20μg/ml, 10μg/ml,5μg/ml,2.5μg/ml,1.25μg/ml,0.625μg/ml) and 20% fetal calf serum for another 24-hour's inoculation. The same control groups without ALLN intervention were set at the same time. The effects of Calpain inhibitor on the proliferation and migration of cardiac fibroblasts were respectively detected by Cell Counting Kit-8 (CCK-8) and cell scarification experiment.Results:Total degree of myocardium fibrosis of acute virus myocarditis group is significantly (P<0.01) more severe than normal controls and was ameliorated by Calpain activity inhibition (P<0.01). Experiments in vitro indicated that Calpain activity inhibition decreased absorbance of fibroblast (P<0.001) and demonstrated an inhibition effect on the migration of fibroblast (P<0.001) as well in a concentration dependent manner.Conclusion:Calpain is involved in the pathogenesis of fibrosis in acute Coxsackievirus B3 myocarditis probably through its promotion effect on both cardiac fibroblasts'proliferation and migration.