Cardiac sarcoplasmic reticulum calcium ATPase, an autoimmune antigen in experimental cardiomyopathy.
Authors: Sharaf A R , Narula J , Nicol P D , Southern J F , Khaw B A 
Published: June 2012

BACKGROUND
Various myocardial cell surface and intracellular antigens have been associated with autoimmune myocarditis. Since sarcoplasmic calcium overload is a recognized pathobiochemical finding in cardiomyopathy, we reasoned that there might be a causal relation between inhibition 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 the regulation of the intracellular calcium content and the myocardial contractility, should lead to the development of cardiomyopathy and possibly myocarditis.

METHODS AND RESULTS
Monoclonal antibody 4C11-20.21 (IgM class) specific for canine cardiac SR-ATPase (M(r) approximately 110 kD) was generated by immunization of CAF1/J mice with dog heart sarcoplasmic reticulum. Antibody 4C11-20.21 inhibits 75% of the enzymatic activity of the cardiac SR-ATPase. This antibody also cross-reacts with the higher M(r) subunit of canine skeletal SR-ATPase, but the skeletal muscle SR-ATPase activity is unaffected. This antibody does not cross-react with sarcolemmal calcium ATPase (134 kD). Antibody 4C11-20.21 was used for affinity purification of cardiac muscle SR-ATPase, which did not contain sarcolemmal calcium ATPase antigen. Nine of 11 CAF1/J mice injected with purified canine cardiac SR-ATPase protein demonstrated myocardial lesions: 3 of 4 mice had occasional perivascular and/or interstitial mononuclear cell infiltrates after 3 weeks, 3 of 4 had borderline myocarditis after 6 weeks, and 3 of 3 had focal myocarditis after 12 weeks. No mononuclear infiltrates were seen in any other organ. To identify the independent effect of 4C11-20.21 antibody on cardiac muscle, 2 x 10(6) hybridoma cells producing the antibody were injected intraperitoneally into 12 severe combined immunodeficiency (SCID) mice. Eleven of 12 SCID mice showed variable cardiac myocyte degeneration without cellular infiltration between 8 and 19 days. Three control SCID mice, which received equivalent injections of hybridoma cells producing IgM anti-myosin light chain antibody, did not show any pathological lesions. Immunoperoxidase staining and/or immunoperoxidase transmission electron microscopy for detection of in vivo localization of 4C11-20.21 demonstrated staining of the subsarcolemmal myotubular system and focal staining of immediately adjacent sarcolemma in animals that received either 4C11-20.21 hybridoma cells or purified canine cardiac SR-ATPase antigen but not in controls. Immunofluorescence staining with goat anti-mouse C3 antibody revealed focal deposition of complement in the cardiac myocytes.

CONCLUSIONS
The time-dependent association between immunization with SR-ATPase antigen and the development of myocarditis in mice suggests that cardiac SR-ATPase constitutes one of several autoimmunogens capable of inducing autoimmune myocarditis. Besides antigenic specificity, since antibody to cardiac SR-ATPase also inhibits energy-dependent processes in the myocardium, it is reasonable to associate the pathological evidence of myonecrosis with the interference of calcium regulation, which controls myocardial contractility.