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 transplant rejection as well as in viral myocarditis is well established, but the precise mechanisms whereby infiltrating CTL damage the myocardium are unknown. The aim of the study was to investigate how CTL derived perforin, the serine protease granzyme A, and the combination of both, damage guinea pig ventricular myocytes.


METHODS
Action potentials and membrane currents were recorded by means of the whole cell configuration from guinea pig ventricular myocytes.


RESULTS
Resembling the effects 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 shortening of action potential duration and reduction of resting potential and action potential amplitude, followed by complete inexcitability. Granzyme A alone was ineffective, but accelerated the deleterious effects of perforin on the morphological and electrophysiological properties of myocytes. The effects of perforin were further evaluated by measuring membrane currents by means of the whole cell voltage clamp. Perforin induced discrete changes in membrane current, reminiscent of single ion channels, with large conductance and open time of up to several seconds. Linear regression analysis of the channel I-V relations resulted in a conductance of 890 pS and a reversal potential of -7.6 mV. These results suggest that perforin induces large non-selective channels, which can account for most of the observed adverse effects.


CONCLUSIONS
As CTL participate in the immunological rejection of the transplanted heart, it is conceivable, but remains to be shown, that part of this damage is inflicted by perforin containing lytic granules.
Felzen
B
B
Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine-Technion, Haifa, Israel.
Berke
G
G
Rosen
D
D
Coleman
R
R
Tschopp
J
J
Young
J D
JD
Binah
O
O
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D053804:Granzymes; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects*; D007473:Ion Channels / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012697:Serine Endopeptidases / Q000494:pharmacology*; D013602:T-Lymphocytes, Cytotoxic; D016276:Ventricular Function
D007473:Ion Channels; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D053804:Granzymes; D012697:Serine Endopeptidases; D002118:Calcium