The mechanisms of reversible and irreversible myocyte injury that occurs during cardiac transplant rejection would be studied in cultured myocytes, dissociated adult myocytes, and intact ventricular trabeculae. Lymphocytes would be obtained from spleens of mice during rejection of an abdominal cardiac allograft, and cultured in the presence of donor cell antigens in vitro. The interaction of these sensitized recipient lymphocytes with cultured ventricular myocytes obtained from donor strain animals would be investigated by measurement of myocyte contraction and relaxation (video motion detector), membrane potential, and (Ca2+)i transients (indo-1 fluorescence). Myocyte injury would be quantified by 51Cr release. A major hypothesis to be tested is that early alterations in myocyte function can occur without marked 51Cr release, and are reversible. Reversibility of myocyte injury would be assessed by recovery of contractile function, cation contents, and ATP contents, after 24 to 48 hours in tissue culture. The role of the cytotoxic T lymphocyte (CTL) in the injury process would be investigated by cloning CTLs from sensitized lymphocyte cultures and investigating their effects on myocytes. Changes in contraction and relaxation of ventricular trabeculae obtained from rejecting abdominal cardiac allografts would be studied in a muscle bath to relate alterations in cultured myocyte function to altered function of intact tissue. The effects of pore-forming proteins isolated from CTL granules on physiological function, cation contents, and membrane currents of cultured fetal and isolated adult myocytes would be investigated to determine if inward current induced by perforin alters contraction and relaxation by prolonging the duration of the action potential and/or increasing intracellular (Na+). The importance of extracellular Ca2+ on CTL-induced injury would be studied, and it would be determined if effects on Ca2+ homeostasis are reversible. In addition the effects of Ca channel blocking drugs would be examined. Finally, the effects of lymphokines on myocyte function and on the intensity of the CTL-myocyte interaction would be investigated.
