Original Contributions |
From the Rappaport Family Institute for Research in the Medical Sciences (B.F., M.S., H.L., G.M., R.C., O.B.), Bruce Rappaport Faculty of Medicine, The Bernard Katz Center for Cell Biophysics, Technion-Israel Institute of Technology, Haifa, Israel; the Department of Heart Surgery (I.S.), Carmel Medical Center, Haifa, Israel; the Department of Pharmacology (R.B.R.), College of Physicians & Surgeons of Columbia University, New York, NY; and the Department of Immunology (G.B.), Weizmann Institute of Science, Rehovot, Israel.
Correspondence to Ofer Binah, DSc, Rappaport Institute, POB 9697, Haifa 31096, Israel. E-mail binah{at}tx.technion.ac.il
| Abstract |
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Key Words: cytotoxic T lymphocyte Fas inositol trisphosphate ventricular myocyte perforin geneknockout mouse
| Introduction |
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Cytocidal lymphocytes (ie, CTLs and natural killer cells) possess at least two molecularly distinct fast-acting lytic mechanisms.10 In the first (secretory) pathway, granzymes cosecreted with perforin are believed to penetrate the target cell through polyperforin-induced transmembrane pores, thus bringing about the demise of the cell by activating the interleukin-1ßconverting enzyme death pathway. In the second (nonsecretory) pathway, FasL expressed on the surface of the killer lymphocytes binds to the Fas receptor (Fas/CD95/Apo-1) expressed by the target cell. This encounter triggers a cascade of intracellular protein-protein interactions and proteolytic activities culminating in apoptosis. Importantly, myocardial cells constitutively express Fas11 12 and are therefore likely to be affected by CTLs promptly and adversely.
Apoptosis, a major hallmark of Fas-mediated damage, appears to participate in the genesis and pathophysiology of paroxysmal arrhythmias, conduction disturbances, and arrhythmogenic right ventricular dysplasia.13 Myocyte destruction due to apoptosis was reported in myocarditis, in end-stage cardiomyopathy,14 and in arrhythmogenic right ventricular dysplasia.15 To investigate the mechanism(s) of Fas-induced myocyte dysfunction, we investigated the interaction of CTLs from perforin geneknockout (P-/-) mice and ventricular myocytes, thus excluding the lytic action of the pore-forming protein perforin. Action potentials, [Ca2+]i transients, and myocyte contraction were recorded from BALB/c (H-2d) ventricular myocytes conjugated to and interacting with H-2b antiH-2d PELs obtained from P-/- mice (P-/- PELs).
Because our previous studies on the interaction of murine CTLs with guinea pig myocytes demonstrated that cytocidal damage was prevented by inhibiting the IP3 pathway,16 we have now tested the hypothesis that IP3 is the intracellular agent mediating Fas-based damage to ventricular myocytes. The important finding that this damage was prevented by blocking IP3 production or IP3-operated SR channels suggests that the IP3 pathway may be targeted to attenuate the injury inflicted on the heart by killer lymphocytes, thus restoring heart function.
| Materials and Methods |
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when filled
with the pipette solution containing (mmol/L) potassium aspartate 120,
KCl 20, MgCl2 3.5,
KH2PO4 20,
Na2ATP 3, glucose 10, and EGTA 1 (pH 7.4).
Cytotoxic T Lymphocytes and Conjugate Formation
In vivoprimed H-2b
antiH-2d PELs were obtained from P-/- or
C57BL/6 (P+/+) mice 4 to 5 days after the secondary
intraperitoneal immunization with
25x106 leukemia L1210
cells.19 20 The P-/- mice used to establish the
colony at the Weizmann Institute were a gift from W.R. Clark
(University of California at Los Angeles). Perforin deficiency of
P-/- PELs was confirmed by polymerase chain reaction
analysis. On the day of the experiment, mice were killed by
cervical dislocation, and PELs were obtained by rinsing the peritoneal
cavities with PBS containing 10% heat-inactivated FCS
(PBS-FCS), as previously described.21 22 All
procedures for handling animals were in accordance with institutional
guidelines. Allogeneic CTLtarget cell conjugates were formed between
H-2b antiH-2d PELs (from
P-/- or P+/+ mice) and BALB/c (H-2d)
ventricular myocytes. Since centrifugation,
commonly used to encourage conjugation between killer and target cells,
was deleterious to myocytes, the myocytes were pretreated with the
plant lectin Con A before exposure to PEL, enabling conjugate formation
in the recording bath. After obtaining control measurements
from a nonconjugated myocyte, flow to the bath was stopped, and Con A
was added to a final concentration of 10 µg/mL. Ten minutes later, 10
to 20 µL of PEL suspension was added to the bath, resulting in
myocytes to which one to several PELs became attached. In control
experiments (not shown), we found that in Con Atreated nonconjugated
myocytes, action potential characteristics were unchanged during 60
minutes of superfusion with Tyrode's solution.
LF+ and LF- Leukemia Cells
LF+ cells are L1210 leukemia cells of
DBA/2 mice stably transfected with the Fas expression vector, kindly
provided by P. Golstein (Marseilles-Luminy, France).
LF- cells are L1210 leukemia cells stably
transfected with the Fas antisense expression vector, kindly provided
by W.R. Clark (University of California at Los Angeles). To determine
Fas expression in in vitrocultured L1210 Fas+
(LF+) and L1210 Fas-
(LF-) leukemia, cells
(0.25x106) were washed in staining medium (0.5%
to 1% BSA in PBS+0.02% azide) and pelleted. Thirty microliters (0.25
µg) anti-Fas Armenian hamster mAb (Jo2, PharMingen) was added,
and cells were incubated on ice (30 minutes with occasional shaking).
After washing and pelleting the cells, 30 µL (1:100 dilution) of FITC
goat antiHam F(ab)2 (Jackson Immune Research)
was added, and cells were incubated as described above. After they were
washed, the cells were resuspended in PBS+0.02% azide and
analyzed by FACS.
Incubation of Myocytes With Anti-Fas mAb Jo2
To activate the Fas receptor directly, myocytes were
incubated for various periods, up to 180 minutes, at 37°C in the
presence of 10 µg/mL of the anti-Fas mAb Jo2. The incubation medium
consisted of a 1:1 mixture of the dissociation solution containing
200 µmol/L Ca2+ and 2% albumin,
and Tyrode's solution was used for the
electrophysiological experiments (see
composition above). Because of the long incubation time required for
Jo2 action, in these experiments we studied three groups of myocytes:
nonincubated myocytes (control) and myocytes incubated for various
intervals in the absence or presence of Jo2 (or another mAb).
Measurement of [Ca2+]i Transients and
Myocyte Contraction
Ventricular myocytes were loaded for 25 minutes at
room temperature (24°C to 25°C) with fura 2-AM (Molecular Probes),
at a final concentration of 5 µmol/L, in a 1:1 mixture of
Tyrode's solution and dissociation solution containing 2% bovine
albumin. Excess fura 2 was removed by rinsing twice with
Tyrode's solution. Myocytes were then transferred to a
nonfluorescent chamber mounted on the stage of an inverted
microscope (Diaphot 300, Nikon) and visualized with a x40 oil
immersion Neofluor objective.23 The chamber was
perfused (unless otherwise indicated) with Tyrode's solution at a rate
of 1 mL/min. Experiments were performed at 31°C to 32°C. Fura 2
fluorescence was measured using a dual-wavelength system
(Delta- Scan, Photon Technology Intl). Briefly, light emitted from a
xenon arc lamp was fed in parallel into two independent monochromators
to obtain quasimonochromatic light beams of two different wavelengths
exciting the cell at 340 and 380 nm. Either a 340- or 380-nm wavelength
was switched by a rotating chopper disk at a frequency enabling ratio
measurements at a rate of 150 per second. The two separate
monochromator outputs were collected by the ends of a bifurcated quartz
fiberoptic bundle. The emitted fluorescence (510 nm) was
collected by the microscope optics, passed through an interference
filter, and detected by a photomultiplier tube (710 PMT Photon Counting
Detection System, Photon Technology Intl). Raw data were stored for
off-line analysis by Felix software (Photon Technology Intl) as
340- and 380-nm counts and as the following ratio:
R=F340/F380,
where F340 and F380
indicate fluorescence at 340 and 380 nm, respectively. For
scaling the fluorescence ratio, cell-derived
autofluorescence and noncell fluorescence were
subtracted from the measured fluorescence. In these
experiments, myocytes were stimulated at 0.5 Hz using platinum wires
embedded in the walls of the perfusion
chamber.23
To monitor myocyte contraction (represented by cell shortening) while measuring [Ca2+]i transients, myocytes were simultaneously illuminated with red light, and a dichroic mirror (630-nm cutoff) placed in the emission path deflected the cell image to a video optical system (Crescent Electronic). The cursors of the optical system tracked motion of the cell edge along a raster line segment of the image during electrically stimulated contractions. The analog voltage output from the motion detector was calibrated into micrometers of motion. The motion signal obtained at 60 Hz was digitized and stored along with the fluorescence data.
Immunohistochemical Determination of Fas Expression in
Ventricular Myocytes
Ventricular myocytes were spread on a precleaned
polylysine-coated slides. The slides were dried at room temperature,
fixed for 45 minutes with 5% paraformaldehyde, and
washed three times with PBS. Endogenous peroxidase activity
was neutralized by 20 minutes of incubation with 3%
H2O2 in methanol, followed
by a wash with PBS. Slides were then incubated at room temperature for
90 minutes with Jo2 (diluted 1:25), followed by incubations with
biotinylated antiArmenian hamster IgG secondary antibody (Jackson
Immune Research, diluted 1:750) with streptavidin-peroxidase conjugate,
and 3-amino-g-ethylcarbazol in N,N-dimethylformamide dissolved in
acetate buffer, pH 5.2 as a substrate (Histostain-SP kit, Zymed
Laboratory Inc). Incubation with nonimmune hamster serum served as a
control. Counterstaining was performed with hematoxylin.
Cytotoxicity Assay
Effector cells were incubated together with
51 Cr-labeled target cells at various ratios.
Cells were centrifuged (1000 rpm, 5 minutes, room temperature)
to facilitate conjugation and incubated at 37°C in 5%
CO2 atmosphere for different times. After
incubation, the plates were centrifuged (2000 rpm, 10 minutes),
and 100 µL of supernatant from each well was harvested and counted in
a gamma counter. The percentage of lysis was calculated as follows:
([experimental cpm-spontaneous cpm)/(total releasable
cpm-spontaneous cpm])x100.
Statistical Analysis
Results were expressed as mean±SEM. To compare means of two
populations, Student's t test for paired or unpaired
observations was used. To compare the effects of PEL or Jo2 versus
control, two-way ANOVA was performed. A value of P<.05 was
considered significant. Figures were plotted with ORIGIN software
(Microcal).
| Results |
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To explore the contribution of the FasL/Fas pathway to myocardial
immune pathology (associated with heart-infiltrating diseases), we
developed an allogeneic model of murine ventricular
myocytes interacting with P-/- PELs. After the addition of P-/-
PELs to the recording bath, a variable number of
lymphocytes promptly adhere to myocytes, forming stable conjugates (Fig 2
). It appears that soon after conjugate
formation, "reshaping" of lymphocytes occurs, a process possibly
associated with lymphocyte activation and delivery of the lethal hit.
We then investigated electrophysiological
properties of affected myocytes by recording action potentials
initially from an unbound myocyte and then during conjugate formation
with P-/- (or P+/+) PELs and throughout the cytotoxic interaction.
The main electrophysiological alterations
(Fig 3
) were reduction of
Vm and action potential amplitude and lengthening
of APD. In addition to these alterations, in 5 of 10 P-/-
PELconjugated myocytes, delayed or early afterdepolarizations
developed, as depicted by a representative experiment
(Fig 3C
). The effects of P-/- PELs on action potential
characteristics in 10 conjugated myocytes (Fig 4
) illustrate progressive changes with
the advancement of the cytotoxic interaction. In control experiments
(Fig 4
) in which action potentials from nonconjugated myocytes were
recorded, Vm and action potential amplitude
were stable during the 60-minute experiment, whereas
APD80 gradually shortened, possibly because of
the rundown of ion currents contributing to repolarization. Because
similar APD shortening occurred in all three groups during the first 40
minutes, it is unlikely that this commonly seen phenomenon affected the
outcome of the interaction with the lymphocytes. Next, we compared the
effect of perforin-deficient CTLs (P-/- PELs) with that of
perforin-containing CTLs (P+/+ PELs) by recording action
potentials from myocytes conjugated with P+/+ PELs (Figs 3
and 4
). We
found that the electrophysiological effects
of P-/- and P+/+ PELs were comparable (Fig 4
). Additionally, the
interaction of P-/- and P+/+ PELs with myocytes was associated with
typical shortening (
10%) of myocyte diastolic length,
initially observed 50 to 60 minutes after conjugation.
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Effect of Anti-Fas mAb on Ventricular Myocytes
In the previous section, we demonstrated how P-/- PELs, acting
via the FasL/Fas pathway, affected conjugated myocytes. To directly
test the involvement of Fas activation in the deleterious action of
P-/- PELs, we explored the effect of the apoptosis-inducing
anti-Fas mAb Jo2 on ventricular myocytes (Fig 5
). In these experiments, action
potentials were recorded from myocytes incubated for as much as 180
minutes with 10 µg/mL Jo2 at 37°C. Whereas incubation per se (180
minutes at 37°C) did not appear to affect action potential
configuration (Fig 5A
, two different myocytes), incubation with Jo2
induced prominent action potential alterations (Fig 5B
), similar to the
alterations caused by P-/- PELs: reduction in
Vm and action potential amplitude and increase in
APD (the results are summarized in Fig 6
). Frequently, in myocytes treated with
Jo2, APD prolongation was associated with large early
afterdepolarizations (Fig 5B
). In control experiments (n=5 myocytes,
not shown), we found that 10 µg/mL hamster IgG (Jackson Immune
Research Laboratory) did not alter action potential
characteristics.
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Effect of P-/- PELs and Jo2 on [Ca2+]i
Transients and Myocyte Contraction
To further investigate Fas-mediated myocyte dysfunction, we
monitored [Ca2+]i
transients and contraction (represented by cell shortening)
in myocytes conjugated with P-/- PELs. In these experiments, fura 2
signals (fluorescence counts at 340 and 380 nm, yielding the
ratio R=F340/F380) and cell
shortening were recorded simultaneously, immediately
after addition of PELs to the bath (t=0, providing control values), and
then during 60 minutes of interaction. During the course of the
experiment, fura 2 signals were obtained for a period of 30 to 40
seconds at various time intervals. Before testing the effect of P-/-
PELs, we performed control experiments to assess the stability of
[Ca2+]i transients and
myocyte contraction during a 60-minute experiment in nonconjugated
myocytes. As shown in Fig 7A
, [Ca2+]i transients and
cell shortening were reasonably stable throughout the experiment (60
minutes). In seven nonconjugated myocytes (Table 1
), the diastolic
[Ca2+]i level
(represented by
R=F340/F380) did not change
significantly during the 60-minute experiment. In contrast, marked
changes in [Ca2+]i
transients and cell shortening were observed in a conjugated myocyte
(Fig 7B
). The cytocidal interaction with P-/- PELs (at 60 minutes;
compare with 0 minutes immediately after conjugation) resulted in two
important (and likely related) occurrences: (1) an elevation in
diastolic
[Ca2+]i, from
0.8 to
1.25, and (2) arrhythmogenic activity (indicated by the thin arrows)
represented by "aftercontractions" and nonstimulated
contractions, probably resulting from early or delayed
afterdepolarizations. A summary of PEL-induced changes in
[Ca2+]i in 13 conjugated
myocytes is shown in Table 1
, illustrating that after 60 minutes of
interaction with P-/- PELs, the fluorescence ratio was
doubled. A smaller rise in
[Ca2+]i (R=
1.25) seen
in Fig 7B
is due to the presence of a burst of arrhythmic activity,
which tends to transiently lower
[Ca2+]i, probably as a
result of enhanced extrusion mechanisms.
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Next, we examined the effects of Jo2 on
[Ca2+]i transients and
myocyte contraction (Fig 8
). The
prominent effects of Jo2, compared with control, were elevation of
diastolic
[Ca2+]i (data from 12
Jo2-treated myocytes is summarized in Table 1
), induction of
"aftercontractions" (Fig 8
), and arrhythmogenic activity
(nonstimulated contractions) (Fig 8
), closely resembling the effects of
P-/- PELs. The Fas-induced elevation in diastolic
[Ca2+]i resulted in
myocyte shortening, expressed as percent decrease of the
diastolic length. In control (n=18) and Fas-treated (n=21,
180 minutes) myocytes, diastolic length was, respectively,
114.8±3.2 and 97.7±5.3 µm (P<.05).
|
Involvement of IP3 in Fas-Mediated Myocyte
Dysfunction
Previous sections demonstrated that P-/- PELs and Jo2 caused a
marked elevation in diastolic
[Ca2+]i in affected
myocytes. An intracellular signaling molecule likely to be involved in
the Fas-mediated rise in
[Ca2+]i is
IP3. By binding to
IP3-operated Ca2+-release
channels in the SR, IP3 elevates
[Ca2+]i, which may then
trigger transsarcolemmal Ca2+
influx,24 contributing to myocyte dysfunction.
The IP3 hypothesis was tested by investigating
whether the changes in myocyte functional properties induced by P-/-
PELs or Jo2 are modified by the PLC antagonist U-73122
(Research Biochemicals Intl)25 26 or by heparin,
a blocker of IP3-operated SR
Ca2+-release channels.27 28
In support of the IP3 hypothesis, heparin or
U-73122 included in the recording pipette solution (to exclude
a possible effect of these drugs on the killer lymphocytes) prevented
PEL-induced changes in action potential characteristics (Fig 9
). Both drugs also prevented the
occurrence of arrhythmogenic activity as well as myocyte shortening.
Next, we tested whether U-73343 (Research Biochemicals Intl), an
inactive isomer of U-73122, can interfere with P-/- PEL action.
Unlike U-73122, treating myocytes with U-73343 (2 µmol/L) before
conjugate formation did not provide protection against killer
lymphocytes. Before (t=0) and 60 minutes after conjugate formation in
the presence of U-73343 (n=3, t=60 minutes), action potential
characteristics were, respectively, as follows:
Vm, -74.4±0.5 and -60.7±0.9 mV; action
potential amplitude, 110.7±4.5 and 54.6±9.2 mV; and
APD80, 27±7 and 22±2 milliseconds (all
P<.05). For an unknown reason, APD was not increased in the
presence of U-73343. Additionally, U-73343 did not prevent P-/-
PELinduced arrhythmogenic activity and myocyte shortening. The
IP3 hypothesis was further supported by the
finding that U-73122 prevented Jo2-induced alterations in action
potential characteristics. In these experiments, U-73122 was added 30
minutes before Jo2. In Jo2-treated myocytes (10 µmol/L,
180-minute incubation) in the absence (n=7) or presence of 2
µmol/L U-73122 (n=6), action potential characteristics were,
respectively, as follows: Vm, -66.5±1.3 and
-77.5±0.7 mV; action potential amplitude, 94.1±4.8 and 137.3±7.3
mV; and APD80, 165±35 and 26±5 milliseconds
(all P<.01). Action potential characteristics of myocytes
exposed to Jo2 in the presence of U-73122 were similar to control
action potential characteristics.
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Because [Ca2+]i
elevation was a prominent effect of direct Fas activation, we tested
the ability of U-73122 (the active PLC inhibitor) and
U-73343 (the inactive form) to modify the Jo2-induced rise in
[Ca2+]i. In further
support of the IP3 hypothesis, U-73122 completely
prevented the Jo2-induced rise in diastolic
[Ca2+]i levels, whereas
U-73343 did not (Table 1
). Accordingly, aftercontractions and
arrhythmogenic activity occurred in the presence of U-73343 but not in
the presence of U-73122.
To determine whether the rise in [Ca2+]i is a necessary component of Fas-mediated cell injury, myocytes were pretreated with 10 µmol/L ryanodine (Alomone Labs) for 30 minutes before and during the entire exposure (180 minutes) to Jo2. Importantly, ryanodine prevented Jo2-induced myocyte electrophysiological and arrhythmogenic effects (ie, generation of early afterdepolarizations) as well as myocyte shortening. In the presence of ryanodine, Jo2 did not alter action potential characteristics, which were (n=6) as follows: Vm, -74.5±1.6 mV; action potential amplitude, 118.3±4.8 mV; and APD80, 22.2±3.5 milliseconds.
Direct Intracellular Effect of IP3 on the Action
Potential
To test directly the involvement of IP3 in
Fas-induced myocyte dysfunction, 1,4,5-IP3
(2 µmol/L), the Ca2+-releasing compound,
was applied intracellularly by including it in the patch pipette
solution (Fig 10
). As seen by the
representative action potential traces, intracellular
application of 1,4,5-IP3 (traces b and c) induced
oscillations in the membrane potential and arrhythmogenic
activity, which was observed during the first 10 minutes after
establishing of the whole-cell configuration. In contrast,
intracellular application of the nonfunctional derivative
1,3,4-IP3 was ineffective (Fig 10d
). The effects
of 1,4,5-IP3 and 1,3,4-IP3
on action potential characteristics are summarized in Fig 11
. Whereas
1,3,4-IP3 was ineffective,
1,4,5-IP3 significantly decreased
Vm and action potential amplitude and prolonged
APD, resembling the effects of P-/- PELs and Jo2.
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P-/- PELs Damage Ventricular Myocytes by Activating
the Fas Receptor
To ascertain initiation of Fas-based cytotoxicity by P-/- PELs,
we have tested their cytocidal activity against Fas-expressing and
-nonexpressing target cells (LF+ and
LF-, respectively). The
LF+ and LF- cell lines
were derived from leukemia L1210 of DBA/2 mice, transfected with Fas
overexpression and anti-sense constructs, respectively. By means of
routine FACS analysis, we have found that 77% of
LF+ cells were positively stained for Fas, versus
only 2.2% of the LF- cells. To test whether
retarded Fas expression abrogated the susceptibility of
LF- to Fas-based apoptosis, we examined
the effect of Jo2 on LF+ and
LF- cells. Indeed, the results (Table 2
) reveal refractoriness to Fas-induced
apoptosis of the low-level Fasexpressing
LF- cells compared with the
LF+ cells. Although these results clearly show
refractoriness to Jo2, we further established comparable differential
refractoriness to Fas-based CTL-mediated apoptosis. To
ascertain clear-cut Fas-based CTL action, P-/- PELs were used. The
results (Table 3
) clearly show that
whereas LF+ cells were effectively lysed by
P-/- PELs, LF- cells were not. Hence,
LF- cells are refractory to Fas-based
apoptosis induced either by Jo2 or by CTLs.
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Finally, we tested the effect of prior occupation of Fas by Jo2 (10 µg/mL, 10 minutes, 24.0°C to 25.0°C) on P-/- PELmyocyte interaction. In P-/- PELconjugated myocytes (60 minutes after conjugation) untreated or treated with Jo2 (before conjugation), action potential characteristics were, respectively, as follows: Vm, -52.6±5.5 and 74.4±1.2 mV; action potential amplitude, 84.6±12.2 and 116.8±3.9 mV; and APD80, 42.9±17.2 and 10.8±1.2 milliseconds (all P<.05). Thus, prior occupation of the Fas receptor completely prevented PEL-induced myocyte dysfunction, providing a clear-cut proof that P-/- PELs damage myocytes by activating the Fas receptor.
| Discussion |
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The novel observation made was that myocyte dysfunction caused by
P-/- PELs or by the apoptosis-inducing anti-Fas mAb Jo2 was
prevented by blocking the IP3 pathway.
Importantly, it was demonstrated that freshly dissociated
ventricular myocytes express Fas (Fig 1
). We have found
that in P-/- PELconjugated or Jo2-treated myocytes,
Vm and action potential amplitude were reduced,
APD was markedly prolonged, and delayed or early afterdepolarizations
(frequently culminating into triggered arrhythmias) were
generated. Comparable electrophysiological
effects were induced by PELs from perforin-containing (P+/+) mice
interacting with guinea pig ventricular
myocytes,16 suggesting that at least in this in
vitro setting, the absence of the pore-forming protein perforin did not
diminish the capacity of P-/- PELs. Additionally,
diastolic
[Ca2+]i levels were
elevated in myocytes interacting with P-/- PELs or treated with Jo2.
We have previously reported16 that in conjugated
myocytes P+/+ PELs induced higher (R=
4.0)
[Ca2+]i elevation than in
the present study (R=
2.0). This difference can result from the
following: (1) a contribution of Ca2+ influx
through perforin channels (in P+/+ PELs) to the
[Ca2+]i accumulation, (2)
an IP3-induced Ca2+ release
stronger in guinea pig than in murine ventricular myocytes,
and (3) the use of 1.8 mmol/L
[Ca2+]o in the previous
study compared with 1.0 mmol/L
[Ca2+]o in the
present study.
In support of the IP3 hypothesis, heparin, an antagonist of IP3-operated Ca2+ SR channels, and U-73122, a specific PLC inhibitor, prevented P-/- PELinduced and Jo2-induced action potential changes and arrhythmogenic activity, as well as myocyte shortening. Accordingly, U-73122 prevented Jo2-induced elevation in diastolic [Ca2+]i levels. Clear-cut support for the involvement of IP3 in Fas-based myocyte dysfunction is found in the novel observation that intracellular application of the active IP3 analogue, 1,4,5-IP3, but not the inactive analogue, 1,3,4-IP3, caused electrophysiological changes resembling those brought about by P-/- PELs and Jo2. These results are in accord with recent work16 on the cytocidal interaction of mouse P+/+ PELs with guinea pig ventricular myocytes. The major electrophysiological difference was that the APD of conjugated guinea pig ventricular myocytes was decreased16 compared with the marked increase seen in the present study. This difference may reflect the experimental models used.
Involvement of IP3 in Fas-Mediated Myocyte
Dysfunction
An important question regarding the finding that Fas-based damage
of myocytes was inhibited by blocking the IP3
pathway is how IP3 accumulation can cause myocyte
dysfunction. It is well established that the second messenger
IP3 plays an important role in
[Ca2+]i mobilization by
opening IP3-operated SR (or ER)
Ca2+ channels.29 30 31
Recently, several laboratories have suggested that
IP3 also has a role in intracellular
Ca2+ homeostasis in cardiac
preparations.32 33 34 For example, Borgatta et
al32 demonstrated the occurrence of a
low-conductance Ca2+-release channel sensitive to
IP3 in SR vesicles from the canine
ventricular septum. It appears that at least under normal
conditions, the heart differs from other tissues. Whereas in T
lymphocytes35 IP3 triggers
a rapid and transient release of Ca2+ followed by
transmembrane Ca2+ influx, in both atria and
ventricular muscle IP3 causes a slow
leakage of Ca2+ from intracellular
stores.36 37 38 39 Several studies support the
association of IP3 accumulation and functional
(mostly electrophysiological) derangements
in a number of disease states.23 40 41 For
example, IP3-enhanced spontaneous
Ca2+ oscillations (characteristics of
Ca2+-overloaded SR) occur in saponin-skinned rat
papillary muscle.39 Ca2+
oscillations, commonly seen in postischemic
reperfusion, contribute to ventricular arrhythmogenesis and
are perhaps compatible with Fas-induced early and delayed
afterdepolarizations seen in the present study. Interestingly,
adding PLC to guinea pig ventricular myocytes caused
functional alterations resembling those seen in PEL-bound myocytes,
including a reduction of Vm, induction of delayed
afterdepolarizations, increased
[Ca2+]i, and cell
destruction.42 Strong support for the
IP3 hypothesis is drawn from recent studies
investigating the association between IP3 and
arrhythmogenic activity resulting from reperfusion arrhythmias
in rat hearts.40 43 44 Jacobsen et
al44 have found that reperfusion following acute
ischemia causes a rapid transient increase in
IP3 levels, which is dependent on local release
of norepinephrine. They have also shown that the PLC
inhibitor U-73122 (but not its inactive isomer U-73343)
inhibits IP3 generation and thrombin-induced
arrhythmias, but not those initiated by epinephrine,
thus establishing the requirement for IP3
production in arrhythmogenesis under these conditions. It
should be noted that U-73343 differs chemically from U-73122 only in
one double bond and that the only functional difference lies in
their efficacy for inhibiting IP3-specific
PLC.45 Thus, the effectiveness of U-73122 in
preventing Fas-mediated myocyte dysfunction in the present study
indicates the involvement of PLC.
A probable candidate mediating IP3-based damage is elevated [Ca2+]i, known to be cytotoxic to a variety of cell types, including cardiac myocytes.46 To the best of our knowledge, no other adverse action of IP3 has been reported. Electrophysiologically, increased [Ca2+]i has at least three important consequences: (1) stimulation of a transient inward current evoking delayed afterdepolarizations, so that triggered activity can develop in otherwise quiescent ventricular muscle, (2) generation of Ca2+-dependent slow responses in depolarized fibers, so that conditions for reentry are favored, and (3) intracellular uncoupling with slowing of conduction.47 Although [Ca2+]i elevation in myocytes cannot account for all of the Fas-induced (IP3-mediated) adverse effects, such as attenuated Vm and action potential amplitude, it can trigger the arrhythmogenic activity seen in myocytes conjugated with P-/- PELs or exposed to Jo2. Additionally, [Ca2+]i elevation can cause morphological changes in myocytes. Nevertheless, although Fas activation indeed caused prominent [Ca2+]i elevation, it is questionable whether it is sufficient to trigger cell destruction (and apoptosis), suggesting that other elements of the Fas cascade may contribute to myocyte dysfunction. In addition to direct effects, elevated [Ca2+]i can affect myocyte function indirectly, by activating a variety of intracellular components, such as Ca2+-dependent phosphatases and endonucleases.46 48
That [Ca2+]i elevation
may be associated with Fas-based action (eg, apoptosis) was
suggested by several groups. Oshimi and
Miyazaki49 have found that in the human B cell
line FMO, apoptosis induced by anti-Fas mAb is associated with
[Ca2+]i elevation, which
is proposed by the authors (on the basis of chelation of
[Ca2+]i) to be a
prerequisite for DNA and chromatin fragmentation. The SR-ER
Ca2+-ATPase blocker thapsigargin, which initiates
[Ca2+]i rise by depleting
[Ca2+]i stores and thus
generating Ca2+ influx through specific plasma
membrane Ca2+ channels
(ICRAC), commits to apoptosis cells
belonging to different lineages, such as human hepatoma
cells50 and mouse lymphoma
cells.51 Furthermore, Rovere et
al52 have suggested that in normal human
V
9/vd2+ T-cell clones, engagement of the Fas
receptor causes [Ca2+]i
mobilization, resulting from activation of
ICRAC, which is required for the subsequent
apoptosis. Accordingly, the bcl-2 gene product,
which represses apoptosis, has been suggested to interfere with
the [Ca2+]i mobilization
that is associated with apoptosis induced by growth factor
withdrawal and, in particular, to protect from apoptosis via
inhibition of Ca2+ release and subsequent
ICRAC
activation.51
An important question, yet to be answered, is how Fas activation causes
IP3 accumulation. An alternative explanation of
the IP3 hypothesis (although unlikely) is that
both heparin and U-73122 prevent Fas-mediated damage by interfering
with one or more downstream mediators of the Fas signaling pathway. At
least theoretically, none of the cardiac receptors (eg,
1-adrenergic, muscarinic, endothelin,
angiotensin II, and thrombin) that are coupled to
IP3 turnover23 53 54 55 are
likely to be directly triggered by Fas activation; therefore,
determining how the IP3 pathway is
activated by FasL requires further investigation. Finally,
support for the involvement of the IP3R in
myocyte damage has been provided by Jayaraman and Marks.56
Based on the findings that IP3R1-deficient T cells
are resistant to apoptosis induced by dexamethasone, TCR stimulation,
ionizing radiation and Fas, they suggest that intracellular calcium
release via the IP3R1 is a critical mediator of
apoptosis.
Fas Activation Is Directly Responsible for Myocyte Dysfunction
Induced by P-/- PELs or by Jo2
Of the two (granzyme and perforinbased and FasL/Fas-mediated)
mechanisms of lymphocytotoxicity, discussed in the present study,
we investigated the latter. To this end, CTLs from gene-knockout
(P-/-) mice have been used (P-/- PELs). That P-/- PELs affected
target cells by activating Fas was demonstrated by the following
findings: (1) only L1210 leukemia cells transfected with Fas
overexpression (LF+; 77% Fas-positive,
determined by FACS analysis) were lysed by P-/- PELs or Jo2,
but not L1210 cells transfected with the Fas antisense construct
(LF-; 2% Fas-positive), and (2)
ventricular myocytes in which Fas was initially occupied by
Jo2 were subsequently refractory to the damaging action of P-/-
PELs.
Although activation of the FasL/Fas pathway may eventually lead to cell death, it does not preclude the possibility that Fas-based myocardial dysfunction can also result from contribution of FasL-affected diseased myocytes to the global decline in cardiac function. That Fas-induced myocyte damage is potentially reversible is supported by the frequent recovery of cardiac function in many cases of clinical myocarditis, a T-celldependent disease, and by the observations that systolic dysfunction occurring during heart transplant rejection may be reversed by treating the rejection process.
In summary, the finding that IP3 is involved in Fas-induced damage to myocytes contributes to the understanding of mechanisms of lymphocytotoxicity as they relate to myocardial pathologies in which heart-infiltrating lymphocytes play a key role, such as heart transplant rejection and DCM. Hence, the intracellular messenger IP3 mediating myocardial damage may be a target for pharmaceuticals aimed at attenuating the injury inflicted to the affected heart by killer lymphocytes.
| Selected Abbreviations and Acronyms |
|---|
|
| Acknowledgments |
|---|
| Footnotes |
|---|
Received July 28, 1997; accepted December 16, 1997.
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