Circulation. 1995;92:452-457
(Circulation. 1995;92:452-457.)
© 1995 American Heart Association, Inc.
University of Wisconsin Solution Preserves Myocardial Calcium Current Response to Isoproterenol in Isolated Canine Ventricular Myocytes
Katsushige Ono, MD;
Naoki Gondo, MD;
Makoto Arita, MD;
Harry A. Fozzard, MD;
Tetsuo Hadama, MD;
Yuzo Uchida, MD
From the Departments of Surgery (K.O., T.H., Y.U.) and Physiology (K.O.,
N.G., M.A.), Oita Medical University, Hasama, Oita, Japan, and Cardiac
Electrophysiology Laboratories and the Department of Medicine (H.A.F.), the
University of Chicago, Chicago, Ill.
Correspondence to Dr K. Ono, Second Department of Surgery, Oita Medical
University, 1-1 Idaigaoka, Hasama, Oita 879-55, Japan.
 |
Abstract
|
|---|
Background University of Wisconsin (UW) solution has been
shown
to be an effective solution for cold storage of various organs.
This
study was designed to evaluate the subcellular protective
mechanism
of UW solution during cardiac myocyte storage using
patch-clamp
techniques for the first time as a tool for the
detection of
myocyte viability.
Methods and Results The protective effects of UW solution
on the preservation of
dihydropyridine-sensitive
Ca2+ channel current response to
catecholamine were evaluated in canine cardiac
ventricular cells by measurement of single channel open
probability. Single ventricular myocytes were isolated and
stored in UW solution, in Stanford (SF) solution, or in St Thomas'
(ST) solution at 4°C for 2, 6, 12, and 24 hours, and after each
storage period, recordings were made of cell-attached
single Ca2+ channel currents. When 0.1 µmol/L
isoproterenol was applied, percent mean open probability of the
Ca2+ channel tested in freshly isolated cells was
167±4% (n=24) of controls (100%). The response was decrescent
with
increased duration of the hypothermic storage and was only 130±12%
(n=4) after 24 hours of storage in SF solution and 135±9%
(n=7) in ST
solution. However, it was significantly highly preserved as much as
165±9% (n=6) in UW solution. Ca2+ channel
kinetics
and channel conductance were not changed after up to 24 hours of
hypothermic storage.
Conclusions Hypothermic storage of canine cardiac
myocytes in UW solution preserved ß-adrenergic response, which
suggests that UW solution during cold storage preserved high-energy
phosphates in myocytes that are responsible for Ca2+
channel phosphorylations.
Key Words: calcium channels • transplantation • cardioplegia • catecholamines • electrophysiology
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Introduction
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A widely used clinical
procedure for preserving donor hearts
during transport between donor
and recipient is commonly achieved
by hypothermia and certain
preservation solutions. Although
the optimal composition of the
preservation solution remains
unknown, the use of UW solution has been
widely investigated,
and in
experimental
1 2 3 4 5
and
clinical
6 7 studies its superiority
for the
preservation
of heart at 4°C has been demonstrated.
However, the beneficial effect
of UW solution during hypothermic
heart storage has not been defined
adequately. Therefore, we
decided to evaluate UW solution by comparing
it with ST solution
and with SF solution, which are currently being
used for myocardial
preservation during routine cardiac operations and
transplantation.
Problems of myocardial protection may contribute to the significant
number of early posttransplant deaths that are attributed to heart
failure but not related to rejection or infection. Efforts to improve
the protection of donor hearts led us to study the mechanism of the
impaired hemodynamics after hypothermic heart storage
and transplantation. A decrease in cardiac performance
associated with acidosis has been attributed to a depressed
concentration of high-energy phosphates, which may be caused by a
decrease in glycolysis,8 resulting in limited delivery of
energy substrates to the mitochondria.9 10
ß-Adrenergic
stimulation by endogenous and/or exogenous
catecholamines, the major factors contributing to the
rescue of postoperative cardiac hypodynamic syndrome, leads to
increased activity of various ionic channels, upregulation of the
electrogenic Na-K pump, and consequently, hydrolysis of ATP for
mechanical work.11 However, little is known of the manner
in which Ca2+ channels of cardiac myocytes resume
normal function and respond to ß-adrenergic stimulation, after
episodes of hypothermic standstill.
The objective of this study was to perform
electrophysiological evaluation of UW
solution compared with ST and SF solutions for the preservation of the
catecholamine response to the Ca2+
channel of the isolated canine heart myocyte.
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Methods
|
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Cell Isolation and Storage Procedure
Ventricular myocytes
were isolated from adult
mongrel dogs (weight,
14 to 33 kg). Animals were anesthetized
with sodium pentobarbital
(40 mg/kg IV), and hearts were quickly
removed and rinsed in
cold normal Tyrode's solution. A portion of
myocardium was enzymatically
dissociated using a procedure
modified from that of Salata and
Wasserstrom.
12 In brief,
a muscle column (1.6-mm diameter)
of free wall from the left
ventricular or the intraventricular
wall
was obtained by biopsy needles (Travenol Lab) and placed in
a
Ca
2+-free cardioplegic solution at 25°C with
gentle shaking
for 10 minutes. Approximately 30 muscle columns then
were incubated
in 8 mL of an enzyme solution (2.5 mg/mL, Worthington
type 2)
in a plastic test tube at 37.5°C and continuously stirred
with
O
2 bubbling. After 15 minutes, the supernatant was
discarded,
and the pellets of isolated cells were washed three times
with
8 mL of amino acid–rich medium. After each wash, the tube
was
centrifuged and the supernatant was discarded. Cells then
were
stored in each storage solution (Table
) at 4°C for
0
to 24 hours. Small aliquots of cells were added to the bath
solution in
a perfusion chamber of 200-µL volume constructed
on a glass coverslip
and mounted on the stage of an inverted
microscope. Through the
experiments, all the animals received
humane care in compliance with
the
Guide for the Care and Use of Laboratory Animals
published by the National Institutes of
Health (NIH publication 85-23,
revised 1985).
Electrical Recording
Immediately after isolation (0-hour
storage) or after 2 to 24
hours of storage in UW, ST, or SF solutions, single
Ca2+ channel recordings were made usually 15
to 20 minutes after rewarming periods with the use of the
cell-attached patch-clamp method13 at 20° to
22°C. Glass pipettes fabricated from hematocrit capillary tubes were
pulled with a multistage micropipette puller and heat-polished to a
final tip diameter of 1.5 to 3.0 µm with the use of a microforge.
When filled with pipette solutions (see below), the pipettes had
resistances of 1.0 to 1.5 M
. Patches contained typically one to
three channels, judged from the maximal number of overlaps of openings
in the entire record. This estimated number of channels was used
for the calculation of NPo. The bath solution was connected to ground
via a 150-mmol/L KCl-agar bridge and a silver–silver chloride
half-cell electrode. Electrical contact with the pipette solution
was via a chlorided silver wire. The electrode potential was adjusted
to give a zero current between the pipette solution and the bath
solution immediately before attempting to make a patch. Gigaohm (G)
seals between the pipette and the single ventricular cells
were obtained by applying gentle suction to the pipette after
contacting the cell membrane. Seal resistances for these experiments
ranged between 20 and 120 G. Single channel currents were
recorded in the cell-attached patch configuration with the use
of a patch-clamp amplifier and low-pass filtering at 2 kHz.
Data Analysis
The capacitive transient was partially
compensated by analog
circuitry, and the residual transient was removed by subtracting the
average current from equipotential steps with no channel openings. The
baseline was adjusted for each sweep so that the current averaged 0 pA
when channels were closed. An amplitude histogram was constructed from
the corrected traces. The open channel amplitude was estimated by the
peak current or by the maximal value of a gaussian function fit. An
opening or closing transition was identified by the presence of two
successive data points above or below the 50% amplitude level. Double
openings were rare, for which the subsequent closures were assigned
randomly to the openings. A diary of NPo was made throughout the
current recording, and the average NPo for more than 3 minutes
before and after application of ISO or forskolin with a 3-minute
interval was calculated and compared using percent mean NPo change. To
avoid a possible interference of the channel number over the NPo value,
actual NPo values were not used for the comparison.
Solutions
The compositions of the storage fluids are
presented in
the Table
.
The solutions for the channel current
recordings were as
follows. The bath solution contained (mmol/L) potassium aspartate 140
and HEPES 10 (pH adjusted to 7.4 with CsOH). This solution depolarized
the ventricular cells to approximately 0 mV. The pipette
solution contained (mmol/L) BaCl2 110,
tetraethylammonium-Cl (TEA-Cl) 30, HEPES
10, and tetrodotoxin (TTX) 0.05 (pH adjusted to 7.4 with TEA-OH). TEA
and TTX were added to block K+ and Na+
channels, respectively. ISO 0.1 µmol/L and 1.0 µmol/L was applied
to the bath solution from a 1 mmol/L stock solution dissolved in 1 N
NaOH. Forskolin was dissolved in ethanol and added to the bath solution
at 10 µmol/L (final concentration of vehicle, 0.01%).
Statistics
Data are summarized as mean±SD in text and
figures. Responses
of Ca2+ channels to ISO or forskolin (%NPo) between
the three cardioplegic groups at each storage duration were compared by
ANOVA. Whenever significance was indicated, Scheffé's test for
multiple comparisons was used to determine significance between
different cardioplegic groups and between response of control (0 hour
storage) and of different cardioplegic groups. Channel open durations
and the single channel conductance were compared with unpaired
Student's t tests. A probability value of less than .05 was
considered significant.
|
Results
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Examples of individual sweeps for Ca
2+ channel
activity at 0
mV are shown under control conditions (Fig 1A
)
and of modulation
by ISO 0.1
µmol/L (Fig 1B
). ISO reduced the frequency
of blank
traces and consequently increased the overall probability
of a
Ca
2+ channel to open.
14 There is
overwhelming evidence
that the mechanism of ß-adrenergic
stimulation by ISO
of Ca
2+ channel in heart cells is
due to phosphorylation of
the Ca
2+
channel through enhanced protein kinase A activity.
15 In
freshly isolated cells, intracellular high-energy phosphates
and
cyclic nucleotides remained intact, and
Ca
2+ channel activity
response to ISO was prompt
(Fig 1C
). Mean NPo for the duration
sampled more than 3
minutes was increased by 0.1 µmol/L
ISO to 171% of the control (Fig
1C
) and to 167±4% in
24 cells tested. After the cells
were stored at 4°C for
24 hours in SF solution, ST solution, or UW
solution, the same
experimental protocol was applied, and the percent
mean NPo
was measured in the same fashion after 0.1 µmol/L ISO
application.
Fig 2
represents typical NPo
changes by 0.1 µmol/L ISO
of Ca
2+ channel activity
on myocytes stored in the three different
storage solutions for 24
hours. The cold storage periods inevitably
showed a diminishing
response of Ca
2+ channel to ß-adrenoceptor
stimulation.
Fig 3
shows changes of percent mean NPo of
the Ca
2+ channel
to 0.1 µmol/L ISO as a function
of storage periods. When
the storage periods were longer, response of
the Ca
2+ channel
to ISO was less; the percent mean
NPo of the Ca
2+ channel in
cells stored for 12 hours
was only 123±12% in SF solution
and 131±18% in ST solution,
whereas
it was 167±4%
immediately after isolation. Decrescent response to
ISO, however,
was highly preserved in myocytes stored in UW solution.
In cases
of 12-hour and 24-hour storage, mean NPo change in
Ca
2+ channel
activity caused by 0.1 µmol/L ISO in
myocytes stored in
UW solution was significantly higher
(
P<.05) than in cases
of SF or ST solution. Furthermore,
the increases of percent
mean NPo to ISO after 12- and 24-hour storage
in UW solution
were statistically identical to those of myocytes
obtained immediately
after isolation. The same protocol was applied
with higher concentration
of ISO (1.0 µmol/L) in Fig 4
, and
similar results were
obtained.
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Figure 1. Effects of ISO on Ca2+ channel
activity in a cell-attached patch in a freshly isolated myocyte
(0-hour storage). Examples of six consecutive sweeps recorded under
control conditions (A) and 6 minutes after application of 0.1 µmol/L
ISO (B) are shown. Single channel currents were activated by
stepping to the test potential of 0 mV for 195 ms from a holding
potential of -80 mV at 1 Hz. Onset of the voltage step is
indicated. C, NPo as the ratio of total open time to depolarization
duration was plotted against the sweep number before and after ISO.
Epochs of six consecutive sweeps in control (A) and with ISO (B) are
indicated by  . Mean NPo (NPo) values calculated in control and
with ISO during the period indicated by the thick bars for more than 3
minutes are shown. Dots above the time scale indicate blank sweeps.
Number of channels was estimated as one from the maximum overlapping
events in the particular patch.
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Figure 2. Effect of ISO (0.1 µmol/L) on NPo of the
Ca2+ channel using the same voltage-clamp
protocol as in Fig 1. Diaries of NPo changes after
application of 0.1 µmol/L ISO in a myocyte stored in SF (A), ST (B),
and UW (C) solutions for 24 hours are illustrated. Mean NPo values
calculated during the time indicated by thick bars for more than 3
minutes are shown.
|
|
We wanted to determine if adrenergic ß-receptors of myocytes
became dysfunctional after hypothermic storage, and if so, whether the
direct increase of intracellular cAMP concentration would mimic the
intact response of ISO on Ca2+ channel activity. The
effect of forskolin is known to be a direct activator of
adenylate cyclase16 ; therefore,
Ca2+ channel response to forskolin was observed and
mean NPo change was measured on freshly isolated myocytes and on
myocytes stored in SF, ST, and UW solutions for 2 to 24 hours in the
same fashion as in Figs 3 and 4. In Fig
5, the channel
responses to forskolin were attenuated after prolonged storage, and the
effectiveness of UW solution on the maintenance of the response
was similar to responses obtained with ISO (0.1 and 1.0 µmol/L).
To determine whether or not the cold storage would alter the channel
opening/closing behavior per se, channel kinetics were studied in cells
with and without storage. The mean open time durations of
Ca2+ channels at 0 mV in cells with or without
hypothermic storage for 24 hours were studied and compared: 0.36±0.06
ms in freshly isolated myocytes (n=16); 0.36±0.02 ms in SF
solution
(n=5); 0.38±0.03 ms in ST solution (n=5); and
0.38±0.04 ms in UW
solution (n=7). Among these, no statistical difference was detected
between each group (.43<P<.98). Moreover, the single
channel conductance (24.2±2.2 pS in freshly isolated myocytes;
n=8)
remained unchanged regardless of the type of storage solution after 24
hours: P=.16 versus SF, 26.3±1.2 pS (n=4);
P=.24
versus ST, 25.8±1.1 pS (n=4); P=.13 versus UW,
26.1±0.7 pS
(n=5). Since the mean open time duration and the channel conductance
values measured after 24 hours of storage were statistically identical
to those of control (without storage), it was suggested that the
transmembrane potential was not altered by hypothermic storage. Thus,
we consider that cold storage of cardiac myocytes for periods of up to
24 hours does not modify Ca2+ channel kinetics.
|
Discussion
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Although several mechanisms underlie myocardial dysfunction
after
prolonged hypothermic preservation in stock solutions
in cases of heart
transplantation, many are thought to be a
direct consequence of either
a decrease in high-energy phosphate
compounds such as ATP or an
increase in H
+ concentration, both
of which occur with the
cessation of normothermic aerobic
metabolism.
10 Decreased levels of ATP and the
loss of protein kinase activity
are expected to occur during cold
storage, and these events
may contribute to the inability to maintain
transmembrane cation
homeostasis and mechanical performance of
myocytes leading to
increases in [Na
+]
i
and
[Ca
2+]
i.
17 18
This
disarrangement of
intracellular cation homeostasis impairs the
mechanical performance
of myocytes, and on reperfusion, severe
cell injury or cell
death can ensue. The notion that myocardial
protection may be
achieved by maintaining cellular ATP levels and
protein kinase
activity has been entertained.
10
In this context, evaluation of Ca2+ channel current
response to isoproterenol after cold storage is important. For studies
of cardiac energy metabolism, nuclear magnetic resonance
spectroscopy is useful because it makes use of nondestructive and
quantitative measures of high-energy phosphated metabolites;
however, it is incompetent for the analysis of functionally
different components of ATP, cAMP, or protein kinase, ie, substrates or
catalysts needed for various biomechanical reactions. Our measurements
of Ca2+ channel activity served as a tool to
estimate phosphorylation, which plays a decisive role
in regulating cardiac excitability and
contractility.14 15 Although it is not yet
possible to observe [Ca2+]i transients
and force development in single isolated mammalian
ventricular myocytes, the bulk of evidence favors the
phenomenon of Ca2+-induced release of
Ca2+ as an important component of
excitation-contraction coupling in mammalian heart.19 The
salient feature of that phenomenon, as observed in single skinned
cardiac cells, is that it is the rate of change of free
[Ca2+]i resulting directly from the
Ca2+ entering the cytoplasm via L-type
Ca2+ channels that "triggers" release of
Ca2+ from the sarcoplasmic reticulum.20
Based on these findings, measurements of Ca2+
channel response to catecholamine are twofold: (1)
contractility of myocardium and (2) overall
phosphorylation ability of the myocytes. Since we used
single channel recording in cell-attached mode and no
artificial intracellular solution for the current-recording
pipette, change in composition of intracellular metabolites or ions
during and after storage should straightforwardly reflect the amount of
Ca2+ channels phosphorylated via
ß-adrenergic stimulation.14 21
This study was to test whether cardioplegia and storage with UW
solution, when compared with commonly used SF cardioplegic solution and
ST solution, would result in improved myocyte preservation from the
view- point of electrophysiological study.
We first studied the effect of hypothermic storage in SF or ST
solutions on the Ca2+ channel response to ISO. This
protocol was chosen to address a possible adverse effect of prolonged
hypothermic cardiac standstill on the characteristics of the
Ca2+ channel current. We found that
Ca2+ current response to 0.1 µmol/L ISO in cells
stored in SF or ST solution progressively declined as a function of the
storage period, reaching two thirds of that measured in freshly
isolated cells (Fig 3). In contrast,
Ca2+ currents obtained from cells stored in UW
solution responded to ISO more prominently than did those in SF or ST
solution after 12- to 24-hour storage periods. Preservation of ISO
response in the Ca2+ channel activity may reflect
the net result of cAMP formation, protein kinase A–dependent
phosphorylation, and
dephosphorylation.14 15 The formation
of cAMP and the phosphorylation event are dependent on
intracellular local concentration of ATP, whereas the
dephosphorylation would not be. The effect of UW
solution could have no relation to the activity of phosphatase because
the inhibition of phosphatase would have resulted in a prolongation of
phosphorylation duration of the Ca2+
channel and would have led to an upregulation of the channel activity.
This is inconsistent with our results indicating that the
response to ISO was decreased toward the prolongation of the storage
period. On the other hand, direct effects of UW solution on myocyte
surface proteins were unlikely. The kinetics of the
Ca2+ channel such as the mean open time at 0 mV or
the single channel conductance remained unchanged even after 24 hours
of hypothermic storage in comparison with those from freshly isolated
myocytes. Since an increase of intracellular cAMP level by forskolin
without stimulating ß-adrenoceptors (Fig 5)
mimicked the effect of ISO, the function of the adrenoceptor might not
be involved in the effect of UW solution. The best and simplest
possibility that is consistent with the data is that UW
solution maintained high-energy phosphates such as ATP and creatine
phosphate in the myocyte pool that is relevant to
phosphorylation of Ca2+ channels.
This is consistent with nuclear magnetic resonance studies in
rat and pig myocardial tissue extracts showing that ATP was better
maintained in UW solution–preserved hearts than those preserved in
SF or ST solutions,4 5 which do not contain
adenosine (Table). Moreover, adenosine and phosphate in
the UW solution have been shown to stimulate ATP synthesis in the
hypothermically perfused canine tissue.22 This may account
partially for the better preservation of
phosphorylation reaction of the Ca2+
channel by ISO.
Rewarming of myocytes after cold storage initiates retrieval of
electrical excitability and mechanical contractility.
Cardiac ion transports in sarcolemma, through channels or
carrier-mediated, require energy consumption in most cases. Since
we used nonworking cells throughout the storage and current
recording periods, the intracellular ATP may be consumed only
for the maintenance of intracellular metabolic and
ionic consistency; besides, it decreased as a result of the
diffusion to outside the plasma membrane and the degradation thereof.
All these events might account for the loss of high-energy
phosphates during and early after storage. SF solution and ST solution
are so-called extracellular cardioplegic solutions, in that their
K+ concentration (27 and 16 mmol/L, respectively) are
approximately one seventh of that in the intracellular milieu. In
ventricular myocytes, the membrane potential is close to a
K+ diffusion potential or the Nernst potential for
K+.23 With hypothermia, the transmembrane
Na+,K+-ATPase becomes inactive, allowing
a flux of K+ out of the cell and Na+ into the
cell.24 Unexpectedly, single unitary current conductance
and resulting estimated cytosolic K+ concentrations
remained unchanged even after 24-hour cold storage in all storage
groups. It is conceivable that the ATP-dependent Na/K pump
(Na+-K+ ATPase) was fully activated
during the rewarming period in order to maintain sufficient
concentrations of intracellular K+ as well as
high-energy phosphates responsible for Ca2+
channel phosphorylation that was once deficient during
storage periods.
On the other hand, UW solution is an intracellular type of solution
with a high concentration of K+ (125 mmol/L) and a
relatively low concentration of Na+ (20 mmol/L). A high
concentration of K+ and a low concentration of
Na+ could reduce the movement of Na+ and
K+ across the cell membrane, hence conserving the ATP used
in maintaining Na+-K+ ATPase. Besides this
effect, UW solution contains components that theoretically should
improve preservation by decreasing (1) the edema associated with
hypothermic storage and (2) cytotoxic oxidants generated on
reperfusion. Although intracellular edema formation resulting from
hypothermia and oxygen-derived free radical formation are thought
to play important roles in causing myocardial dysfunction during
storage and reperfusion, relations of cellular edema or free radicals
and Ca2+ channel activity have never been clear.
Finally, caution is warranted in generalizing these data to humans.
These experiments were performed in isolated canine myocytes, which is
not an equivalent physiological model for the whole
human heart. Although numerous studies report that isolated myocytes
are similar to intact cardiac muscle in terms of their ATP
content,25 oxygen demand,26 substrate
utilization,26 and so on, functional changes in myocytes
during isolation or maintenance in storage solutions may make
the cell nonrepresentative of cells in intact
muscle.27 An example of this is the localization and
coupling of receptors for hormones and neurotransmitters such as
catecholamines. Receptors for hormones and
neurotransmitters are regulated by a variety of factors. Receptor
density and affinity for agonists are known to be affected by the
presence of agonists. Myocytes exposed to catecholamines,
for example, reduce the apparent number of adrenergic receptors in the
sarcolemma.28 This process of downregulation may occur
quickly and is significant within minutes of cell exposure to an
agonist. The phenomenon of receptor regulation is
physiological and is not unique to isolated cells.
Other processes, which have not been identified, may occur in the
isolated myocytes because they are removed from the tissue or they are
autonomically innervated.27
Summary
We made use of the probability of
Ca2+ channel
openings to evaluate the cellular function maintained after different
periods of hypothermic storage in isolated canine myocytes. Despite its
limitations, a quantitative analysis of the
phosphorylation ability of the Ca2+
channel can be made with the use of various storage solutions and
application of different durations of cold storage.
Ca2+ channel activity obtained from myocytes stored
in UW solution (4°C) was as responsive to ISO as those from freshly
isolated myocytes and was significantly higher than those of myocytes
stored in SF and ST solutions after prolonged storage (12 to 24 hours).
Such beneficial effects of UW solution may be associated with
preservation of intracellular high-energy phosphate pool during and
after cold storage periods.
|
Selected Abbreviations and Acronyms
|
|---|
| ISO |
= |
isoproterenol |
| NPo |
= |
open probability |
| SF |
= |
Stanford |
| ST |
= |
St Thomas' |
| UW |
= |
University of Wisconsin |
|
|
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|
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Top
Abstract
Introduction
) and in myocytes stored for various
durations (2, 6, 12, and 24 hours) in SF (
), ST (
), and in
UW (
) solutions, where each mean NPo before ISO was assigned as
100%. Numbers of cells tested are given in parentheses.
*P<.05, **P<.01.
