(Circulation. 1995;92:452-457.)
© 1995 American Heart Association, Inc.
Articles |
University of Wisconsin Solution Preserves Myocardial Calcium Current Response to Isoproterenol in Isolated Canine Ventricular Myocytes
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.
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Abstract |
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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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TopAbstractIntroductionMethods Results Discussion References |
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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 experimental1 2 3 4 5 and clinical6 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 Ca2+-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 O2 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).
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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.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Examples of individual sweeps for Ca2+ 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
Ca2+ channel to open.14 There is
overwhelming evidence that the mechanism of ß-adrenergic
stimulation by ISO of Ca2+ channel in heart cells is
due to phosphorylation of the Ca2+
channel through enhanced protein kinase A activity.15 In
freshly isolated cells, intracellular high-energy phosphates and
cyclic nucleotides remained intact, and
Ca2+ 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 Ca2+ channel activity
on myocytes stored in the three different storage solutions for 24
hours. The cold storage periods inevitably showed a diminishing
response of Ca2+ channel to ß-adrenoceptor
stimulation. Fig 3 shows changes of percent mean NPo of
the Ca2+ channel to 0.1 µmol/L ISO as a function
of storage periods. When the storage periods were longer, response of
the Ca2+ channel to ISO was less; the percent mean
NPo of the Ca2+ 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
Ca2+ 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.
|
. 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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) 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.
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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).
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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.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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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 [Ca2+]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.
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Selected Abbreviations and Acronyms |
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References |
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