Articles |
From the Rappaport Family Institute for Research in the Medical Sciences (B.F., R.L., O.B.), Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel; Merck Research Laboratories (M.G.), Rahway, NJ; the Department of Cell Biology (G.B.), Weizmann Institute of Science, Rehovot, Israel; and the Department of Molecular Pharmacology (P.G.), Stanford (Calif) University School of Medicine.
Correspondence to Ofer Binah, DSc, Rappaport Institute, PO Box 9697, Haifa 31096, Israel.
| Abstract |
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Key Words: cytotoxic T lymphocytes peritoneal exudate cytotoxic C lymphocytes ventricular myocytes heart transplant rejection Ca2+ channel blockers
| Introduction |
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Lymphocyte-induced damage to the cardiac muscle is not restricted to heart transplant rejection but is evident in other heart diseases. It is commonly thought that myocarditis, initiated by a viral infection, often coxsackievirus B3, may progress in a significant number of cases to dilated cardiomyopathy. Although the precise pathological mechanisms of dilated cardiomyopathy are far from clear, there is a rather wide agreement that CTL-mediated immune response contributes to the ongoing cardiac damage, often leading to terminal heart failure.3 4 5 In addition, CTLs have been implicated in myocardial damage induced in Chagas' heart disease in humans and also in the chronic model of Chagas' heart disease in Trypanosoma cruziinfected mice.6 7
To understand the precise nature of the interaction of infiltrating lymphocytes and myocytes during heart transplant rejection as well as during the course of other heart diseases associated with infiltrating CTLs, in a recent study we established an in vitro model of interaction of CTLs and guinea pig ventricular myocytes.8 We used patch-clamp techniques to monitor action potentials and membrane currents in single ventricular myocytes undergoing cytocidal interaction with killer lymphocytes. We showed that CTLs caused general decline in action potential characteristics and in myocyte contracture; these effects can account, at least in part, for the decline in cardiac function during heart transplant rejection and in related heart diseases. On the basis of these findings, we proposed that cytotoxicity was essentially caused by [Ca2+]i overload in conjugated myocytes.
In spite of inherent limitations of an isolated CTL-myocyte conjugate as a model of CTL-mediated heart diseases, it is currently the only experimental model enabling electrophysiological recordings from an "identified" CTL-myocyte conjugate undergoing cytocidal interaction.8 Therefore, we thought that this model was a valuable tool for investigating drugs that may interfere with the cytocidal interaction either at the killer and/or at the target cell site. Because we have previously suggested that PEL-induced damage is associated with [Ca2+]i overload in the myocyte, in the present study we investigated the effects of Ca2+ channel blockers. We hypothesized that blocking ICa,L by means of common Ca2+ channel blockers, such as CoCl2 and verapamil, will eliminate the main trigger for Ca2+ release from intracellular stores, mainly the sarcoplasmic reticulum, and will therefore reduce [Ca2+]i overload and myocyte deterioration.
| Materials and Methods |
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Electrophysiological
Recordings
After 1 to 2 hours of incubation in KB medium, myocytes
were
transferred to the recording chamber (0.5 mL), which was
mounted on the stage of the inverted microscope (Zeiss IM). The bath
was superfused with Tyrode's solution at a rate of 1 to 2 mL/min, and
all experiments were carried out at room temperature (24.0°C to
25.0°C). Membrane potentials and currents were measured by means of
the whole-cell recording technique10 with an
EPC-7 (List Medical Electronics) or Axon 200A (Axon Instruments, Inc)
patch-clamp amplifier. Electrodes were prepared from glass
micropipettes (H15/10, Jencons Scientific), pulled on a vertical puller
(Narashige PP-83), and had a tip resistance of 2 to 4 M
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.
PELs
In vivo primed PELs were obtained from BALB/c mice 4 to
5 days
after the secondary intraperitoneal immunization
with 25x106 C57BL leukemia EL4
cells.11 At the day of the experiment, mice were killed by
cervical dislocation, and peritoneal exudates were obtained by rinsing
the peritoneal cavities with PBS containing 10%
heat-inactivated NCS. The peritoneal exudate cells were
centrifuged, resuspended in RPMI+NCS+HEPES (10 mmol/L), and
incubated in Petri dishes at 37.0°C to cause adherence of B cells and
macrophages to the plastic. After 60 minutes, the nonadherent
cells (PELs) were centrifuged again, then eluted by
RPMI+NSC+HEPES, and kept on ice. The cellular composition and
surface
markers (analyzed by FACS) of PELs are as follows: small to
medium-sized lymphocytes (91%), histiocytes (<2%), and
polymorphonuclear cells (<2%); Thy 1.2 (95%), Lyt-2 (82%), and
L3T4 (19%).12 13 All procedures for handling animals
were
in accordance with institutional guidelines.
The Experimental Model: Conjugate Formation Between Myocytes
and CTLs
CTL-myocyte conjugates were formed between mouse BALB/c
anti-EL4
CTLs (PELs) and guinea pig ventricular myocytes pretreated
with the plant lectin Con A before exposure to PELs. The
sugar-binding lectin "bridges" over the specific recognition
step between the killer and the xenogeneic (guinea pig)
myocytes.14 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, a drop
(10 to 20 µL) of PEL suspension was added to the bath, resulting in
myocytes to which several PELs were attached (see Fig 1A
). That
PELs
and myocytes were firmly bound (ie, conjugated) was verified by gently
moving the recording pipette in the bath; in the presence of
Con A, PELs strongly adhered to the "cruising" myocyte; in its
absence, the lymphocyte detached.
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Statistical Analysis
Results are expressed as
mean±SEM. To compare means of two
populations, we used Student's t test for paired or
unpaired observations.
| Results |
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To test the hypothesis that
blocking ICa,L, which
triggers Ca2+ release from the sarcoplasmic reticulum, will
reduce [Ca2+]i overload and the
subsequent
cytocidal damage, we determined whether the potent Ca2+
channel blocker CoCl2, recently found to attenuate
the lytic effects of CTL lytic granules on ventricular
myocytes,15 can modify PEL-myocyte cytocidal interaction.
In control experiments, we determined the effect of CoCl2
(3 mmol/L) on action potential characteristics of nonconjugated
myocytes (Fig 2A
, upper trace) and found, as expected,
that CoCl2 attenuated the plateau and shortened APD. As
shown in the Table
, whereas Vm and APA were
unchanged
during 60 minutes of superfusion with CoCl2,
APD50 was reduced. To test the ability of CoCl2
to modulate PEL-myocyte cytocidal interaction, the drug was introduced
to the superfusate immediately after the addition of PELs
and conjugate formation and remained in the bath for the entire
duration of the experiment. This procedure was used to prevent a
possible effect of CoCl2 on conjugate formation. As seen by
the representative recordings in Fig 2A
(lower
trace), the action potential of the CoCl2-treated
conjugated myocyte is indistinguishable from that of
CoCl2-treated nonconjugated myocyte, indicating that
CoCl2 provided considerable protection to the conjugated
myocyte. Aside from the expected APD shortening by CoCl2 in
the conjugated myocyte (as in the nonconjugated myocyte),
Vm and APA were not reduced, the plateau was not abolished
(compare with Fig 1B
) (summarized in the Table
),
and most important,
delayed afterdepolarizations were not induced. In agreement with these
findings, PEL-induced myocyte contracture and destruction did not occur
in CoCl2-treated conjugated myocytes.
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To determine whether
the protective potency of CoCl2 was
due to a block of ICa,L, we tested whether
verapamil (2 µmol/L), another Ca2+ channel
blocker, had a comparable protective effect. Unlike
CoCl2, verapamil failed to protect
myocytes against killer lymphocytes (Fig 2B
and
Table
), and action
potential deterioration occurred as under drug-free conditions.
Accordingly, verapamil did not prevent the induction of
delayed afterdepolarizations (Fig 2B
, lower trace) or myocyte
contracture and destruction, indicating that ICa,L was not
directly involved in the damage induced in myocytes by killer
lymphocytes.
Changes in IK During PEL-Myocyte Interaction: Effect of
CoCl2
Marked APD shortening and attenuation of the plateau
are the major
electrophysiological alterations in
conjugated myocytes. Hence, we addressed two related questions: (1)
Does an increase in IK (affecting the plateau and
repolarization of the action potential) in conjugated myocytes
contribute to the above changes in action potential? (2) Did
CoCl2 prevent the changes in action potentials of
conjugated myocytes because of its effect on IK per se? In
other words, can a direct inhibitory effect of
CoCl2 on IK potentially oppose a PEL-induced
([Ca2+]i-dependent) increase in
IK? Theoretically, it can be argued that experiments
testing the effect of CoCl2 on IK could have
been more conclusive if performed with conjugated myocytes rather than
with nonconjugated myocytes; however, we chose the second option, since
the possibility of an "indirect" effect of CoCl2 on
IK (in myocytes), mediated through the drug's effect on
the lymphocyte, could not be excluded. Fig 3A
depicts
representative membrane currents in a nonconjugated
myocyte at 0 and 60 minutes (after the beginning of the experiment);
these experiments served as the control for testing the changes in
IK in conjugated myocytes. To prevent a possible effect on
the cytocidal process (in the myocyte and/or the lymphocyte), we did
not use ion channel blockers (eg, tetrodotoxin) or ion-substitution
protocols in order to obtain "pure" IK. The decline
in IK with time seen in this experiment probably resulted
from the natural current "run-down," commonly occurring with
other currents (eg, ICa,L). A summary of the decline in
IK over time (0 to 60 minutes) in nonconjugated myocytes is
depicted by IK I-V relations (Fig 3B
). In contrast
to the
moderate decline seen in control experiments, in conjugated myocytes
(Fig 3B
), the interaction with PELs resulted in a marked
increase in
IK, which can account, at least in part, for APD
shortening and for the plateau attenuation. The mechanism responsible
for the increase in IK in conjugated myocytes was not
explored directly in the present study. We next addressed the
second question, testing whether CoCl2 affects
IK in nonconjugated myocytes. As seen by the
representative membrane currents (Fig 4A
) and by the I-V
relations (Fig 4B
),
CoCl2 only minimally affected IK.
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Effects of Verapamil and CoCl2 on
IK in PELs
The observation that two potent
Ca2+ channel blockers,
CoCl2 and verapamil, differed markedly in their
ability to interfere with PEL-induced cytotoxicity implied that a block
of ICa,L in conjugated myocytes is unlikely to provide
protection against the cytotoxic damage. This observation, along with
the finding that IK, which is markedly increased in
conjugated myocytes, is also not a target for CoCl2,
suggested that the protection provided by CoCl2 may have
been due to the drug's effect on membrane currents of the killer
lymphocyte. Ideally, testing the effect of CoCl2 on PEL
currents should have been performed on active lymphocytes conjugated to
a myocyte; however, since in every experiment, several lymphocytes
adhered to a myocyte (Fig 1A
), it could not be determined
whether
a randomly monitored PEL is indeed the one killing the myocyte.
Furthermore, it is extremely difficult (if not impossible) to determine
how the presence of a patch electrode on the lymphocyte and dilution of
the lymphocyte intracellular content affect the cytotoxic process. To
bypass this uncertainty, experiments were performed on nonconjugated
PELs. Typical PEL whole-cell membrane currents are depicted in Fig
5A
. The expansion of the first 5 milliseconds (Fig
5B
)
demonstrates that the membrane current closely resembles common
IK, with a relatively rapid onset and
voltage-dependent kinetics. The I-V relations of current measured
at 22 milliseconds are depicted by the open circles in Fig 6
.
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Since a variety of membrane currents, and specifically
K+
currents, have been associated with lymphocyte activation and
proliferation,16 17 18 it was of
importance to determine
whether the ability of CoCl2 and the inability of
verapamil to protect myocytes coincide with their effects
on PEL membrane currents. As seen by the effect of both drugs on the
I-V relations (Fig 6
), whereas verapamil was practically
ineffective, CoCl2 almost completely blocked IK
in PELs, providing a plausible explanation for the drug's ability to
protect myocytes against PEL-induced cytotoxic damage.
Margatoxin Protects Conjugated Myocytes and Blocks IK
in PELs
Because the above experiments suggested that blocking
IK in PELs prevented damage induced by the lymphocytes, we
used margatoxin, a specific blocker of IK in T lymphocytes.
Margatoxin, a peptide isolated from the venom of the scorpion
Centruroides margaritatus, has been shown to block cloned
rat Kv1.3 channels19 20 as well as
IK in PEL hybridomas (O. Binah, P. Gardner, unpublished
data, 1995). First, we tested whether margatoxin has a protective
effect comparable to that of CoCl2. As seen in Fig 7
(and summarized in the Table
), the presence of 10
nmol/L margatoxin (a concentration previously shown to block
IK) prevented the typical PEL-induced deterioration in
action potential characteristics and the induction of delayed
afterdepolarizations. Margatoxin also prevented myocyte contracture. To
exclude an effect of margatoxin (at 10 nmol/L) on myocytes that might
be related to its protective efficacy, we determined whether margatoxin
affects the action potential or membrane current of nonconjugated
myocytes. At the same concentration that margatoxin blocked PEL-induced
damage, it did not affect action potential characteristics
(Table
) or
IK (Fig 8
). Finally, we determined whether
the protective capacity of margatoxin corresponds with its ability to
block IK in PEL (Fig 9
). As anticipated,
margatoxin blocked IK almost completely in PELs, leaving a
small current component, most likely due to a "leakage current."
Therefore, this observation strongly supports the findings with
CoCl2 (and verapamil) suggesting that blocking
IK in PELs prevents delivery of the cytotoxic message (ie,
the "lethal hit") to the myocyte.
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| Discussion |
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We have recently reported that PEL-myocyte interaction results in a reduction in Vm, APA, and APD, as well as contracture and cell destruction. We concluded that some effects may be accounted for by [Ca2+]i overload. We suggested that the marked APD shortening could result from (1) [Ca2+]i-dependent increase in the repolarizing outward current, IK,21 and (2) decreased ICa,L resulting from Ca2+-dependent inactivation.22 In the present study, we demonstrated that PEL-myocyte interaction was associated with a marked increase in IK, probably caused by increased [Ca2+]i. The possibility that APD shortening was due to Ca2+-dependent ICa,L inactivation is less likely, since shortening occurred in the presence of verapamil and in the notable absence of ICa,L.
Our original working hypothesis was that blocking ICa,L will attenuate PEL-induced damage to conjugated myocytes as a result of decreased [Ca2+]i overload. That this assumption was incorrect was suggested by the findings that two potent Ca2+ channel blockers differed in their ability to protect myocytes; although verapamil was ineffective, CoCl2 provided significant protection. We then concluded that the conjugated myocyte was not the target for the protective action of CoCl2 and directed our experiments to the lymphocyte. Under normal experimental conditions, we recorded one type of membrane current in PEL, which is characterized by rapid-onset voltage-dependent kinetics and the absence of inactivation (even at pulses of 200-millisecond duration [not shown]). We found that in complete agreement with the drug's ability (or inability) to protect conjugated myocytes, PEL membrane current, carried mainly by IK, was blocked by CoCl2 but not by verapamil. Although we have not carried out a comparative analysis of the current characteristics, of the three known types of IK in lymphocytes, the current sensitivity to CoCl2 and margatoxin and insensitivity to verapamil suggest that it is the l-type IK.16 17 18 23
Having found that blocking IK in PELs prevented damage to
conjugated myocytes, we tested the protective efficacy of margatoxin, a
specific K+ channel blocker in
lymphocytes.24 25 26 As anticipated from
previous reports,
margatoxin blocked IK in PELs and provided complete
protection to conjugated myocytes. At the same time, margatoxin did not
affect the action potential or IK of nonconjugated
myocytes, suggesting that the myocyte was not the target for the
protective action of margatoxin. Margatoxin is a 39amino-acid
peptide toxin recently isolated from the New World scorpion
Centruroides margaritatus, and its primary structure has
been determined.26 27 Margatoxin is structurally
related
to other well-known scorpion toxins and displays 44% sequence
identity with charybdotoxin, 41% with iberiotoxin, and 79% with
noxiustoxin. Previous studies have shown that margatoxin blocks
voltage-dependent Kv1.3 current in human peripheral T
lymphocytes with high affinity (half block at
50
pmol/L)27 and has no effect on the small-conductance
Ca2+-activated K+ channels in human T
lymphocytes.24 In smooth muscle sarcolemma, margatoxin,
unlike charybdotoxin, is inactive on the high-conductance
Ca2+-activated K+ channel even at 1
µmol/L.27
Why Does Blocking IK in Killer Lymphocytes Prevent the
Cytotoxic Damage to Conjugated Myocytes?
The involvement of different
ion channels in T-lymphocyte
activation and proliferation is well
established,16 17 18 but
less is known about the role of ion channels in cytotoxic function.
Several observations suggest that K+ currents are involved
in cytotoxicity. 86Rb efflux from CTLs was increased under
conditions of specific target cell lysis.28 Pretreatment
of natural killer cells with quinidine,
4-aminopyridine, verapamil, and
CdCl2, at concentrations that block K+
channels, inhibited the killing of target cells.29
Similarly, the K+ channel blockers quinidine and
4-aminopyridine inhibited target cell lysis by
lymphokine-activated killer cells.30 Strong
support for the involvement of K+ channels in lymphocyte
activation and cytotoxic function comes from a recent study testing the
effects of margatoxin in human T lymphocytes.24 At
concentrations at which margatoxin blocked voltage-dependent
K+ channels in lymphocytes (0.5 to 5 nmol/L), the toxin
inhibited interleukin-2 production of ionomycin+phorbol
12-myristate 13-acetatestimulated T lymphocytes, as well
as the anti-CD3induced rise in
[Ca2+]i.
These observations strongly support the findings of the present
work, ie, that blocking IK prevented killer lymphocyte
activation and delivery of the lethal hit to the target cell. Finally,
it should be noted that at present the precise role of
K+ channels in lymphocyte activation and cytotoxic function
is not entirely clear. Since depolarizing the cell with high external
K+ enhanced killing whereas hyperpolarizing the cell with
valinomycin or low external K+ inhibited killing, the
primary role of K+ channels is probably not only to
maintain a negative resting potential.31 Thus, although
there is a correlation between K+ channel blockade and
inhibition of lymphocytotoxicity, the mechanism(s) whereby
K+ channels are involved in killing is yet to be
determined.18
In summary, we investigated the cytocidal interaction of PELs and ventricular myocytes and demonstrated that blocking IK in PELs by CoCl2 and margatoxin prevented PEL-induced damage to the myocytes. This pharmacological modulation of lymphocytotoxicity in vitro can be considered a potential modality for a novel immunosuppression therapy.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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Received May 5, 1995; accepted October 31, 1995.
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