EFFECTS OF PURIFIED PERFORIN AND GRANZYME-A FROM CYTOTOXIC T-LYMPHOCYTES ON GUINEA-PIG VENTRICULAR MYOCYTES

Objective: Involvement of cytotoxic T lymphocytes (CTL) in heart trans plant rejection as well as in viral myocarditis is well established, b ut the precise mechanisms whereby infiltrating CTL damage the myocardi um are unknown. The aim of the study was to investigate how CTL derive d perforin, the serine protease granzyme A, and the combination of bot h, damage guinea pig ventricular myocytes. Methods: Action potentials and membrane currents were recorded by means of the whole cell configu ration from guinea pig ventricular myocytes. Results: Resembling the e ffects of CTL derived lytic granules, perforin caused gradual myocyte shortening and contracture, leading to complete loss of the rod shaped morphology and to cell destruction. These changes were preceded by sh ortening of action potential duration and reduction of resting potenti al and action potential amplitude, followed by complete inexcitability . Granzyme A alone was ineffective, but accelerated the deleterious ef fects of perforin on the morphological and electrophysiological proper ties of myocytes. The effects of perforin were further evaluated by me asuring membrane currents by means of the whole cell voltage clamp. Pe rforin induced discrete changes in membrane current, reminiscent of si ngle ion channels, with large conductance and open time of up to sever al seconds. Linear regression analysis of the channel I-V relations re sulted in a conductance of 890 pS and a reversal potential of -7.6 mV. These results suggest that perforin induces large non-selective chann els, which can account for most of the observed adverse effects. Concl usions: As CTL participate in the immunological rejection of the trans planted heart, it is conceivable, but remains to be shown, that part o f this damage is inflicted by perforin containing lytic granules.