Fas (CD95/Apo-1)–Mediated Damage to Ventricular Myocytes Induced by Cytotoxic T Lymphocytes From Perforin-Deficient Mice: A Major Role for Inositol 1,4,5-Trisphosphate

Abstract —Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene–knockout (P−/−) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca 2+ ] i transients, and contractions of myocytes conjugated to P−/− PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P−/− PELs were, respectively, as follows: V m , −73.2±1.5 and −53.6±6.4 mV (mean±SEM); action potential amplitude, 117.9±3.9 and 74.3±21.2 mV; and action potential duration at 80% repolarization, 17±6 and 42±13 milliseconds (all P 2+ ] i increased during the cytocidal interaction with P−/− PELs, from a fluorescence ratio of 0.82±0.05 (n=7) to 1.98±0.09 (n=13) ( P 3 )–operated sarcoplasmic reticulum Ca 2+ channels, or U-73122 (2 μmol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP 3 analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P−/− PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP 3 . We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.