CARDIAC SARCOPLASMIC-RETICULUM CALCIUM-ATPASE, AN AUTOIMMUNE ANTIGEN IN EXPERIMENTAL CARDIOMYOPATHY

Background Various myocardial cell surface and intracellular antigens have been associated with autoimmune myocarditis. Since sarcoplasmic c alcium overload is a recognized pathobiochemical finding in cardiomyop athy, we reasoned that there might be a causal relation between inhibi tion of sarcoplasmic calcium exclusion and pathogenesis of the disease and that immunization with sarcoplasmic reticulum calcium ATPase (SR- ATPase) or antibody specific for SR-ATPase, which can interfere with t he regulation of the intracellular calcium content and the myocardial contractility, should lead to the development of cardiomyopathy and po ssibly myocarditis. Methods and Results Monoclonal antibody 4C11-20.21 (IgM class) specific for canine cardiac SR-ATPase (M(r) approximate t o 110 kD) was generated by immunization of CAF1/J mice with dog heart sarcoplasmic reticulum. Antibody 4C11-20.21 inhibits 75% of the enzyma tic activity of the cardiac SR-ATPase. This antibody also cross-reacts with the higher M(r) subunit of canine skeletal SR-ATPase, but the sk eletal muscle SR-ATPase activity is unaffected. This antibody does not cross-react with sarcolemmal calcium ATPase (134 kD). Antibody 4C11-2 0.21 was used for affinity purification of cardiac muscle SR-ATPase, w hich did not contain sarcolemmal calcium ATPase antigen. Nine of 11 CA F1/J mice injected with purified canine cardiac SR-ATPase protein demo nstrated myocardial lesions: 3 of 4 mice had occasional perivascular a nd/or interstitial mononuclear cell infiltrates after 3 weeks, 3 of 4 had borderline myocarditis after 6 weeks, and 3 of 3 had focal myocard itis after 12 weeks. No mononuclear infiltrates were seen in any other organ. To identify the independent effect of 4C11-20.21 antibody on c ardiac muscle, 2x10(6) hybridoma cells producing the antibody were inj ected intraperitoneally into 12 severe combined immunodeficiency (SCID ) mice. Eleven of 12 SCID mice showed variable cardiac myocyte degener ation without cellular infiltration between 8 and 19 days. Three contr ol SCID mice, which received equivalent injections of hybridoma cells producing IgM anti-myosin light chain antibody, did not show any patho logical lesions. Immunoperoxidase staining and/or immunoperoxidase tra nsmission electron microscopy for detection of in vivo localization of 4C11-20.21 demonstrated staining of the subsarcolemmal myotubular sys tem and focal staining of immediately adjacent sarcolemma in animals t hat received either 4C11-20.21 hybridoma cells or purified canine card iac SR-ATPase antigen but not in controls. Immunofluorescence staining with goat anti-mouse C3 antibody revealed focal deposition of complem ent in the cardiac myocytes. Conclusions The time-dependent associatio n between immunization with SR-ATPase antigen and the development of m yocarditis in mice suggests that cardiac SR-ATPase constitutes one of several autoimmunogens capable of inducing autoimmune myocarditis. Bes ides antigenic specificity, since antibody to cardiac SR-ATPase also i nhibits energy-dependent processes in the myocardium, it is reasonable to associate the pathological evidence of myonecrosis with the interf erence of calcium regulation, which controls myocardial contractility.