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The pathogenesis of dilated cardiomyopathy in Emery- Dreifuss muscular dystrophy (EDMD) is still unknown. Autoimmune mechanisms have recently been taken into account. The aim of this investigation was to determine whether the level of circulating antibodies to heart proteins which were previously detected, correlates with disease progression. Troponin I was chosen as the target. Ten patients with EDMD and 10 age-matched normal controls were tested. An enzyme linked immunoassay (ELISA) technique was used to determine the possible relation between the level of anti-troponin I antibodies at diagnosis and at follow-up. Autoantibodies against troponin I were detected in all EDMD patients. At diagnosis the level was higher in the X-linked EDMD form (X-EDMD), as compared to the autosomal dominant form (AD-EDMD). At follow-up the elevated level of the autoantibodies persisted in all the EDMD cases. However, in the AD-EDMD form, the level was found to be significantly rising with disease progression, in the X-EDMD form, on the other hand, it was declining. No clear-cut relationship between the level of the circulating antibodies and cardiac symptomatology was present. Detection of anti-troponin I antibodies may provide a non-invasive marker of early stages of dilated cardiomyopathy in EDMD.
A deficit of emerin or lamins A/C is related to a very rare, genetically transmitted, Emery-Dreifuss muscular dystrophy (EDMD). Even if the defect is generalized to all tissues skeletal muscle, heart and joints are selectively affected. Muscle atrophy, joint contractures, and dilated cardiomyopathy are the leading symptoms. Cardiac disease, although often silent, usually precedes skeletal muscle involvement. The pathogenesis of dilated cardiomyopathy in EDMD has not been recognized, yet. Activation of mitogen protein kinase (MAPK) in the development of cardiomyopathy has already been suggested (1, 2). Activation of MAPK in a mice model of EDMD, prior to cardiomyopathy has been described (3, 4). The question is, if it is the basic abnormality, which leads to the development of cardiac disease in human EDMD. Other mechanism(s) should be also taken into account. It is already known that in a subset of patients with idiopathic dilated cardiomyopathy (DCM), autoimmune mechanism(s) are involved (5–1,1). In EDMD, autoimmune mechanism(s) may also participate in evoking DCM. In our previous report (11), we have already demonstrated that, in EDMD patients, different heart muscle proteins, including troponin I, are immunogenic.
In order to establish whether the presence of autoantibodies is related to the progression of the disease we have examined anti-troponin I level at diagnosis and at follow-up. In our opinion, testing the anti-troponin I antibodies may define the role of anti-heart autoantibodies in predicting the susceptibility at risk of dilated cardiomyopathy in EDMD.
A total of 10 patients (6 with emerin deficiency – X-EDMD, 4 with lamin A/C deficiency – AD-EDMD) and 10 healthy age-matched controls were examined. The diagnosis of EDMD was based on neurological and cardiologic examinations, DNA analysis, electromyographic, biochemical, histological, histochemical, ultrastructural, immunocytochemical and immunochemical emerin/lamins determinations.
Fasting blood for testing the antibodies level was taken at the first neurological and cardiologic diagnosis of EDMD and, later, at follow-up (one to six years after diagnosis), centrifuged and the serum preserved at -30 °C until used. The enzyme linked immunosorbent assay (ELISA) procedure for the detection of autoantibodies was based on that described by Caforio et al. (5) with small modifications. In our work, instead of α-myosin, troponin I as a representative cardiac protein was used. The multiwell plates (Sigma) were coated with 100 µl troponin I from human heart (Sigma) at a concentration of 5 µg/ml. This was found to be the optimal concentration. Serum was diluted 1:40, 1:80, 1:160, 1:320, and 1:640. Serum dilution 1:320 was chosen as appropriate for the anti-troponin I antibody screening. The plates were incubated for 1 h at 37 °C and washed once with phosphate buffered saline (PBS) solution (Sigma), containing 0.1% Tween 20 (PBS-T). The wells were blocked with 200 µl PBST, containing 2% bovine serum albumin fraction V (BSA, Sigma), incubated for 30 min at 37 °C and washed 3 times with 200 µl of PBS-T. They were then coated with 100 µl of each serum diluted 1:320 with PBS-T containing 1% BSA, incubated for 1 h at 37 °C and washed for five times with PBS-T. Afterwards the plates were coated with 100 µl of anti-human IgG γ chain biotin conjugate (Sigma), diluted 1:1000 in PBS-T, incubated for 1 h at 37 °C and washed five times with PBS-T. Avidin-peroxidase complex (10 µg/ml, Sigma) was prepared by dilution 1:20 with PBS-T before use, added to each well and incubated for 1 h in the dark, washed four times with PBS-T, coated with a developing solution of o-phenylene diamine in Na2HPO4-citric acid buffer pH 5.0-5.5 (Sigma), and incubated in the dark for 30 min. The absorbance was assessed immediately using a Sigma Diagnostics EIA Microwell Reader II at 450 nm.
Data were presented as mean ± standard deviation (SD) and range of the values. Differences in variable values were assessed with Mann-Whitney U test and Wilcoxon matched pair test. The relationships between variables were analyzed using Spearman’s correlation coefficient (ρ). A P < 0.05 value was considered statistically significant.
Clinical neurological and cardiologic features are outlined in Table Table1.1. In all patients, neurologic symptoms and cardiac parameters indicating dilated cardiomyopathy were present. Both the initial symptoms and skeletal muscle involvement, as well as cardiac parameters indicate AD-EDMD as the more severe progressing form of EDMD, as compared to the X-linked type.
Anti-troponin I level in serum, at diagnosis, was elevated in all patients with EDMD (Table (Table2).2). It was higher in the X-EDMD than in the AD-EDMD patients (P < 0.05). At follow-up, in the X-EDMD group, the antibody level was tending to decline. On the contrary, in AD-EDMD, the absorbance of the anti-troponin I antibodies was rising (Table (Table2).2). The correlation coefficient between the absorbance values, at diagnosis and at follow-up, in X-EDMD, was negative and moderate (ρ = - 0.77, P = 0.072) (Fig. (Fig.1).1). In AD-EDMD, a very strong positive significant linear correlation between the progression of the disease (interval of time from diagnosis to the follow-up) and the absorbance change of the autoantibodies appeared (ρ = 0.98, P = 0.001) (Fig. (Fig.2).2). No such correlation was present in X-EDMD (ρ = - 0.23, P = 0.658).
There was no significant correlation between the level of antibodies and cardiologic symptomatology.
EDMD is a rare genetically transmitted disease, characterized by progressive muscle weakness, joint contractures and dilated cardiomyopathy. DCM progresses in a relevant proportion of both the X-linked EDMD form (12), as well as the AD-EDMD form (13, 14). Cardiac disease usually precedes skeletal muscle involvement. In the X-EDMD form, atrio-ventricular block predominates. This ranges from sinus bradycardia, prolongation of the PR intervals up to complete block, which often leads to sudden death. Atrial muscles are involved earlier than the left ventricle muscles. In the AD-EDMD form, dilatation of the left ventricle and changes in contraction-relaxation time predominate. The pathogenesis of dilated cardiomyopathy and differences in cardiologic symptoms in both EDMD-forms is as yet unknown. Recently, activation of MAKP, which is present in a mouse model of EDMD, is thought to be responsible for the development of cardiomyopathy in both forms of EDMD (3, 4). It has also been indicated that heart symptomatology is evoked by disruption of the cytoskeletal desmin network in cardiomyocytes (15). This possibility, however, in EDMD should be rejected, as desmin is not abnormally expressed and localized in the EDMD muscles (16). It should be taken into account that, the immune system may contribute to the development of dilated cardiomyopathy in the EDMD patients, due to the presence of autoantibodies against heart proteins (11).
Autoimmune mechanism(s) are known to be active in a subset of patients with idiopathic dilated cardiopmyopathies. In DCM, myocarditis and also after myocardial infarction anti-heart antibodies are present in the serum. They indicate that autoimmunologic mechanism(s) are participating in these diseases (6, 8–10, 17, 18). The type of heart proteins, which are acting as antigens, and the frequency of their appearance in DCM is a matter of controversy. High frequency of anti-myosin antibodies up to 86% (6), but also lower frequency up to 20% (9) is presented. There are also reports that in DCM the autoantibodies are directed mainly against cardiac specific α-myosin isoform and tropomyosin (8, 10, 17). Among the antibodies directed against other heart muscle proteins there are also those against troponin I (11). Recently, also autoantibodies to cardiac troponin I in patients with idiopathic and ischemic dilated cardiomyopathy have been described (19). The presence of anti-heart antibodies is usually related to clinical parameters and is associated with more severe impairment of the left ventricular systolic function and diastolic stiffness (20). The appearance of the autoantibodies may serve as early markers of the disease, when heart dysfunction is still unrecognized and also for the disease predisposition. The level of anti-α-myosin antibodies has been reported to be lower at follow-up than at diagnosis, and, in some patients, they are even undetectable with disease progression (21, 22).
The question to be answered is, whether the anti-heart antibodies are the cause, or the consequence of DCM. This problem has been disputed for several years. It has not yet been defined, whether anti-heart antibodies play a substantial role in the development of DCM. Their primary role, in the development of dilated cardiomyopathy, may be suggested by the fact that they occur early in the course of the disease and are cytotoxic to myocytes (23). The autoantibodies are directed against some cardiac structural components and promote myocardial damage either by inducing inflammation, or increasing the Ca2+ currents and activation of receptors on the surface of cardiomyocytes (19). The latter hypothesis is supported by experiments indicating that administration of monoclonal antibodies to troponin I, in wild-type mice induces staining of the surface of cardiomyocytes and increases the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. This leads to chronic stimulation of Ca2+ influx in cardiomyocytes, heart dysfunction and dilated cardiomyopathy (24, 27). It is also possible that anti-heart antibodies are produced as a result of release of heart proteins into the bloodstream and may reflect only damage to cardiac muscle cells and a non-specific immune response. The differences between both forms of EDMD in the level of antibodies at diagnosis and at follow-up is hard to explain. They are possibly related to the fact that, in AD-EDMD, damage of the left ventricle muscle cells predominates and is increasing with disease progression, in the X-EDMD, mainly disturbances in heart conductivity are present. In X-EDMD, autoimmunity gradually subsides, which is also observed in some autoimmune disorders such as insulin dependent diabetes type 1 (28). This is not the case in AD-EDMD.
Whatever the role of anti-heart antibodies, their level in the blood might be a useful non-invasive marker in predicting the susceptibility of the EDMD patients at risk of developing DCM, especially as it often causes sudden death of the patients even with no evident preceding cardiac symptoms.
The study was conducted after approval (No KB 2/2005) of the ethics committee for human research at the Medical University of Warsaw. The study was supported by a grant from the State Committee for Scientific Research (No 2PO5B 106 29) to Prof. Irena Hausmanowa-Petrusewicz).