Mimicry and Antibody-Mediated Cell Signaling in Autoimmune Myocarditis (1)

Anti-cardiac myosin (CM) antibodies cross-react to beta-adrenergic receptors (b-AR). Passive transfer of purified IgG from CM-immunized rats develops IgG deposition in the heart, resulting in a cardiomyopathic heart disease phenotype.

Autoantibodies against CM highly observed in patients with cardiomyopathy, a role of anti-CM autoantibodies in pathogenesis of the cardiomyopathy is unknown. Many studies have shown that cellular immunity is an important pathogenic determinant in autoimmune myocarditis. In animal myocarditis models, CM is used as the dominant autoantigen in genetically susceptible animals. Transfer of anti-CM mAbs induces myocarditis in several strains of mice. Antibodies against peptides of the beta-AR induce cardiac dysfunction and cardiomyopathy.

The function and binding assay using antibodies can be studied using rat primary heart cell line H9c2 from the American Type Culture Collection.

For the passive transfer, the titers of anti-CM and S2-16 (amino acid sequences 842 to 1295 of CM) IgG were measured by ELISA and range from 1/6400 to 1/12800. 6- to 8-wk-old naive Lewis rats received 0.5 mg of purified by IV injection once a week for 18 weeks.

NOTE; the induction protocol is too long with 18 weeks (longer than 4 months).

A pooled anti-S2-16 and anti-CM sera but not sera from PBS/CFA-immunized control rats increased protein kinase A activity to 50–70% above the basal level in vitro assay using H9c2 cells. The activation was inhibited by a presence of CM or S2-16 or an antagonist of beta-AR (propranolol) in the medium. These events may trigger cardiac dysfunction.

Thirteen monoclonal antibodies were established from CM immunized rats. Four antibodies cross-react to CM and beta-AR.

The CM-reactive mAbs (9B6-3E6) reacted with both the H chain of CM (200 kDa) and recombinant beta2-AR (67 kDa), and a protein in H9c2 cell lysates that had the same molecular weight as the bata2-AR in western blot analysis. The results indicated that anti-CM autoantibody in myocarditis may have pathophysiological effects on heart cells due to the cross-reactivity. Amino acid sequences between CM and beta1-AR showed a 30% identity and a 56% homology in a 57 aa overlap (CM, 98-154:beta1-AR, 300–348), and between CM and beta2-AR showed a 25% identity and a 57% homology in an 85 aa overlap (CM, 543-624: beta2-AR, 131– 213).

The defined S2-16 epitope of CM that induced Ab cross-reactive with beta-AR demonstrated a 44% identity and a 78% homology with beta1-AR in a 9 aa overlap (S2-16, 5–13: beta1-AR, 48–56) and a 34% identity and a 67% homology with beta2-AR in a 12 aa overlap (S2-16, 12–23: beta2-AR, 396 – 407).

The passive transfer of anti-CM IgG fraction resulted in increased the left ventricular cavity diameter significantly and a decrease in thickness of the septum and posterior wall. The deposition of antibodies in the heart tissues, IgG and IgG2a were observed but not IgG1. It indicated that Th1 antibodies, which can activate complements, play roles in the inflammatory and pathogenic responses.

1. Y. Li, J. S. Heuser, L. C. Cunningham, S. D. Kosanke, M. W. Cunningham, Mimicry and antibody-mediated cell signaling in autoimmune myocarditis. J. Immunol. 177, 8234–8240 (2006).