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.