The Preliminary Study On The Potential Pathological Roles Of CTnI Autoantibodies In Cardiomyocyte Injury

The preliminary study on the potential pathological roles of cTnI autoantibodies in Cardiomyocyte InjuryBackground:Many cardiac diseases including acute myocardial infarction, hypertension, viral myocarditis, idiopathic cardiomyopathy, et al, developed to heart failure in the end which is due to the myocardium damage. Gene mutation, virus infection, acute and chronic myocardial ischemia can result in myocardial damage and myocardium dysfunction. Immune system can be activated during the the myocardial damage process by following factors: cardiac protein gene mutation; virus molecules have the similar epitopes as the cardiac proteins have, "new antigens" exposed after cardiac injury, et al. Elevated autoantibodies is the major character of autoimmune and many cardiac autoantibodies can be found elevated in the serum of heart disease patients, such as viral myocarditis, dilated cardiomyopathy and myocardial infarction, et al. As we know now, they are anti-adrenoceptor autoantibodies, myosin autoantibodies, anti-M₂ acetylcholine receptor autoantibodies, et al. It has been suggested that clinical improvements in patients with high cardiac autoantibodies titers are related to the disappearance of antibodies by immunoadsorption therapy. It is presumed now that these autoantibodies can damage the cardiomyocytes by some mechanisms and take part in the pathophysiological process of myocardial damage and myocardium dysfunction.Cardiac troponin I (cTnI), a regulatory protein unique to heart muscle, is released to the bloodstream after myocardial necrosis. The exposed cTnI can activate the immune system and result in the production of cTnI autoantibodies. As we know now that cTnI autoantibodies can be detected in the serum of myocardial infarction patients, but the possible pathogenic role of the cTnI autoantibodies to the cardiomyocytes is still unknown. Most researches based on animal experiments showed similar results. These findings suggested that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca²⁺ influx in cardiomyocytes, and induce severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice, et al. However the pathological roles of human cTnI autoantibodies need further investigation.Aims:To establish a method for detecting the serum level of cardiac troponin I autoantibodies with human cTnI, evaluate the serum cTnI autoantibodies level in the myocardial infarction patients, discuss the relationship between the cTnI autoantibodies level and prognosis. And make a preliminary study on the mechanisms of cardiomyocyte injury which was induced by cTnI autoantibodies by interfering the cultured cardiomyocytes of SD rat with purified mouse anti-human cTnI antibodies.Methods:1. Enrolled the testees of 38 MI patients and 38 healthy participants from the ward and outpatient clinic. Myocardial infarction was diagnosed with CAG, 18 leads ECG, cardiac biomarkers according to the MI guideline of Chinese Medical Association.2. A sandwich ELISA method detecting cTnI autoantibodies was established with human cTnI and mouse anti-human IgG. Test the effect of this method by detecting mouse anti-human antibodies of different concentrations. All the serum cTnI autoantibodies of the testees (myocardial infarction group and healthy control group) were measured by ELISA. Set the mean±3SD obtained from the healthy control to define positive for each group.3. Follow-up for discharged MI patients was 3 months. To compare the different recurring cardiac events of the two groups in 3 months.4. Cultured neonatal rat cardiomyocytes were exposed to anti-human cTnI (10μg/ml, 50μg/ml), mouse IgG(50μg/ml) and PBS buffer for 48 hours. Observed cTnI antibodies' affection to the appearance, beating ratio and cell culture supematant of cultured neonatal rat ventricular myocytes. Studied the harmful affection of cTnI antibodies by testing the concentration of CK-MB in the cell culture fluid.5. Confirmed the binding of anti-human cTnI antibodies and the cultured neonatal rat ventricular myocytes by immunohistochemistry and immunofluorescence.Results:1. 38 MI patients were diagnosed by CAG, 18 leads ECG and biochemical markers testing. The MI group 28 males and 10 females ranging in age from 50-79 (68.3±7.9), the control group had 28 males and 10 females ranging in age from 55-78 (67.9±6.3).2. The established method for measuring cTnI autoantibodies can detect the concentration beyond 0.54ng/ml. The mean value of the serum cardiac troponin I autoantibodies of the myocardial infarction group was 0.732±0.258, while the healthy control group was 0.531±0.135. 8 patients were identified as positive in the MI group and the positive rate was 21%. 3. During 3 months of follow-up, 1 positive patient died and 1 positive recurred myocardial infarction, the others had no cardiac events.4. In interference group when 50μg/ml anti-cTnI contained, the cell density decreased significantly and there were cell debris in eyesight. The cell beating rate decreased with the increase of anti-cTnI in 48 hours. The lowest beating rate was observed in 50μg/ml anti-cTnI group, which decreased by 60%, and the 10μg/ml anti-cTnI group decreased by 39%. Other groups didn't decrease in 48 hours. The color of cell culture fluid turned more and more yellow with the longer of interference time and the higher of anti-cTnI concentration. The CK-MB concentration in 50μg/ml anti-cTnI group was the highest of all the groups. There was no significant difference between subgroup with mouse IgG (50μg/ml) and subgroups without anti-cTnI.5. The result of immunohistochemistry and immunofluorescence showed that anti-cTnI antibodies could bind the neonatal rat ventricular myocytes. Negative results showed in the control group.Conclusion:1. We established an ELISA method which can detect the cTnI autoantibodies of the myocardial infarction patients. The elevated serum cTnI autoantibodies of some myocardial infarction patients may be correlated with unfavorable prognosis.2. Anti-human cTnI antibodies can damage the cultured neonatal rat cardiomyocytes.