(Circulation. 1997;96:3148-3156.)
© 1997 American Heart Association, Inc.
Articles |
Comparison of Polarized and Depolarized Arrest in the Isolated Rat Heart for Long-term Preservation
From Cardiac Surgical Research and Cardiovascular Research (M.J.S.), The Rayne Institute, St Thomas' Hospital, London, UK.
Correspondence to Dr D.J. Chambers, Cardiac Surgical Research, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK. E-mail d.chambers{at}umds.ac.uk
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Abstract |
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Background Hypothermic hyperkalemic cardioplegic solutions are currently used for donor heart preservation. Hyperkalemia-induced depolarization of the resting membrane potential (Em) may predispose the heart to Na+ and Ca2+ loading via voltage-dependent "window currents," thereby exacerbating injury and limiting the safe storage duration. Alternatively, maintaining the resting Em with a polarizing solution may reduce ionic movements and improve postischemic recovery; we investigated this concept with the reversible sodium channel blocker tetrodotoxin (TTX) to determine (1) whether polarized arrest was more efficacious than depolarized arrest during hypothermic long-term myocardial preservation and (2) whether TTX induces and maintains polarized arrest.
Methods and Results The isolated crystalloid-perfused
working rat heart preparation was used in this study. Preliminary
studies determined an optimal TTX concentration of 22 µmol/L and
an optimal storage temperature of 7.5°C. To compare depolarized and
polarized arrest, hearts were arrested with either Krebs-Henseleit (KH)
buffer (control), KH buffer containing 16 mmol/L K+,
or KH buffer containing 22 µmol/L TTX and then stored at 7.5°C
for 5 hours. Postischemic recovery of aortic flow was
13±4%, 38±2%, and 48±3%* (*P<.05 versus control and
16 mmol/L K+), respectively. When conventional 3 mol/L
KCl-filled intracellular microelectrodes were used, Em
gradually depolarized during control unprotected ischemia to
-55 mV before reperfusion, whereas arrest with 16 mmol/L
K+ caused rapid depolarization to -50 mV, where it
remained throughout the 5-hour storage period. In contrast, in 22
µmol/L TTX-arrested hearts, Em remained more polarized,
at -70 mV, for the entire ischemic period.
Conclusions Blockade of cardiac sodium channels by TTX during ischemia maintained polarized arrest, which was more protective than depolarized arrest, possibly because of reduced ionic imbalance.
Key Words: cardioplegia • electrophysiology • depolarizing
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Introduction |
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Currently, long-term preservation of the human heart for transplantation is achieved by combining profound hypothermia with a hyperkalemic cardioprotective solution. Two main types of protective solutions are used in heart transplant centers: these are either cardioplegic solutions, which have an "extracellular-type" composition, or organ preservation solutions, which have an "intracellular-type" composition. These solutions, however, were not designed or formulated for long-term preservation of the heart and thus do not address many of the unique problems associated with heart preservation. Thus, cardioplegic solutions were developed primarily to protect the myocardium over short periods of ischemia during routine cardiac surgery,1 2 3 4 whereas preservation solutions were originally developed for long-term preservation of abdominal organs such as the liver, kidney, and pancreas.5 6 Many studies have compared the effectiveness of these solutions for long-term preservation of the heart, but evidence to suggest that one type of solution offers superior preservation compared with another is inconclusive.7
Most cardioplegic and organ preservation solutions are formulated to be either moderately (16 to 25 mmol/L) or profoundly (125 mmol/L) hyperkalemic. The heart is arrested by depolarization of the myocardial Em and induction of flaccid arrest. However, Em depolarization predisposes the myocardium to accumulation of intracellular sodium8 and calcium9 via activation of voltage-dependent "window currents" and does not prevent metabolic processes that deplete cellular energy stores. Alternatively, maintenance of myocardial Em close to the resting level of -80 mV, in a polarized or hyperpolarized state, may have advantages because, at this level of Em, the voltage-dependent window currents should remain inactive. In addition, metabolic demand is reduced because of the balanced Em.
Induction of "polarized" arrest with compounds such as TTX, a specific sodium channel blocker, results in significant myocardial protection during normothermic ischemia10 11 and reduces myocardial oxygen consumption compared with hyperkalemic arrest12 ; however, Em during ischemia was not determined in any of these studies.
Consequently, we carried out a study to (1) investigate the hypothesis that polarized arrest during prolonged hypothermic ischemic storage was a more beneficial alternative to depolarized arrest for long-term preservation and (2) measure the resting Em throughout a hypothermic ischemic storage period and determine whether polarized arrest was achieved and could be maintained in comparison with depolarized arrest. Thus, we have (1) characterized the use of the reversible sodium channel blocker TTX as a cardioplegic tool for long-term hypothermic preservation of the mammalian heart, (2) investigated whether TTX induces polarized arrest throughout a prolonged period of hypothermic ischemia, and (3) compared TTX with a conventional depolarizing-type (hyperkalemic) cardioplegic solution.
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Methods |
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Animals
Hearts were obtained from male Wistar rats (Bantin and Kingman, Hull, UK) weighing 200 to 250 g. All animals received humane care in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office, London, England.
Experimental Preparation
The isolated perfused working rat heart preparation was used for
this study. Briefly, it is a left-side heart preparation in which
oxygenated KH perfusion buffer (at 37°C) enters the
cannulated left atrium at a pressure equivalent to 20 cm
H2O. The perfusate passes to the left ventricle,
where it is spontaneously ejected through an aortic cannula against a
hydrostatic pressure equivalent to 100 cm H2O. Exclusion
criteria imposed in this study rejected hearts that produced an aortic
flow <50 mL/min or a coronary flow in excess of 26 mL/min
after 20 minutes of the control (preischemic) working
period. Coronary flow from the right heart can be sampled for
enzyme analysis or pooled and recirculated with the aortic
outflow. The KH bicarbonate perfusion buffer contained (in
mmol/L) NaCl 119, NaHCO3 25, KCl 4.75,
MgSO4 1.2, KH2PO4 1.18,
CaCl2 1.4, and glucose 11.1 at a pH of 7.4 when gassed with
95% O2/5% CO2 at 37°C. This solution was
filtered through a 5-µm-porosity cellulose nitrate filter before use
and was continually passed through an in-line 5-µm-porosity filter
during the working period of the study. The filter was changed before
the reperfusion working period. During hypothermic storage, each heart
was maintained in a sealed temperature-controlled heart chamber, either
at varying temperatures or at the optimal temperature defined in a
pilot study.
Perfusion Protocol
Rats were anesthetized by diethyl ether vapor inhalation
(in 95% O2/5% CO2) and received heparin (200
IU) via the femoral vein. Hearts were rapidly excised after a median
sternotomy and placed in ice-cooled KH buffer. The aorta was cannulated
and the heart perfused in Langendorff mode for 5 minutes of
stabilization. During the stabilization period, the pulmonary
artery was cut, and the pulmonary veins to the left atrium were
cannulated. Langendorff perfusion was then stopped, and the heart was
converted to working-mode perfusion. During a 20-minute aerobic working
period, control preischemic assessment of cardiac function
(aortic flow, coronary flow, aortic pressure, heart rate) was
measured every 5 minutes. The atrial and aortic lines were then
clamped, and hearts were immediately infused with 2 mL of cardioplegia
at 20°C for 30 seconds at an infusion pressure of 30 cm
H2O via a self-sealing multi-injection port. This volume
and rate of cardioplegia delivery had previously been determined in our
laboratory as the optimum in terms of functional recovery in the rat
heart.13 The Langendorff and working mode perfusion lines
were further clamped at a distal and proximal point from the heart, and
each line was separated into two with a connector. Hearts were then
completely detached from the perfusion apparatus and stored
in 2 mL of the same hypothermic preservation solution as used for
arrest at the optimal temperature for 5 hours. After this storage
period, hearts were removed from the storage chambers and reattached to
the perfusion apparatus, with great care taken that no air
was introduced into the perfusion circuit. Hearts were reperfused in
Langendorff mode for 15 minutes, and coronary effluent was
collected on ice for determination of CK activity.14
Hearts were then converted to the working mode for 20 minutes, and
postischemic cardiac function was assessed; recovery was
expressed as a percentage of the preischemic control
values. At the end of the protocol, hearts were freeze-clamped in
Wollenberger clamps precooled in liquid nitrogen and stored in liquid
nitrogen for high-energy phosphate (ATP and CP) determination. In all
studies, hearts were treated as described above; individual study
differences or additions to this protocol are described below.
Preliminary Characterization Studies
The optimal TTX concentration and the optimal temperature for
long-term preservation were assessed in two brief preliminary
studies.
Determination of optimal TTX concentration. Hearts were arrested by infusion (at 20°C) of KH containing TTX (Sigma Chemicals) at a concentration of 1, 10, 22, 40, or 100 µmol/L and compared with hearts infused with KH alone. The 22 µmol/L concentration of TTX was obtained from previous studies10 11 15 in which TTX at this concentration was shown to enhance protection at 37°C. After arrest, hearts were stored in 2 mL of the same solution as used for arrest at a temperature of 7.5°C16 for 5 hours and then reperfused according to the reperfusion protocol described above.
Determination of optimal temperature. Hearts were arrested by infusion (at 20°C) of KH containing the optimum concentration of TTX derived from the above study. They were then stored in an identical solution at either 4°C, 7.5°C, 11°C, or 15°C for 5 hours. After storage, hearts were reperfused in accordance with the reperfusion protocol described above.
Comparison of a Depolarizing Arrest Solution (High K+)
With a Polarizing Arrest Solution (TTX)
To compare the effects of depolarizing arrest with those of
polarizing arrest, the basic KH solution was used (see "Experimental
Preparation" for composition), to which either additional KCl to
increase the K+ concentration to 16 mmol/L or
TTX at a concentration of 22 µmol/L was added. Hearts
were arrested by a 30-second infusion of 2 mL of one or other of these
solutions (at 20°C) and then stored for 5 hours at 7.5°C in 2 mL of
the same solution as used for arrest. Control hearts were infused with
2 mL of KH solution over a period of 30 seconds (at 20°C) and again
stored in this solution for 5 hours. Reperfusion was carried out as
described above.
Measurement of Resting Em During Ischemic
Storage
Resting Em was measured during ischemic
storage to determine whether polarized arrest was achieved. To measure
Em during ischemia, it was necessary to design a
new heart storage chamber that would maintain a constant hypothermic
temperature and allow microelectrode measurement of Em; the
heart storage chamber design is shown in Fig 1
. After control preischemic
perfusion and cardioplegic infusion as detailed above, the heart and
perfusion cannula attachments were detached from the perfusion
apparatus and placed into the storage chamber. A platform
constructed from a stainless steel rim supporting a circle of nylon
mesh could be adjusted inside the heart chamber via a support passed
through a Teflon collar fixed in the side of the heart storage chamber.
The platform was maneuvered so that it touched the hanging heart, and
the heart and storage chamber were then rotated into a horizontal
position so that the heart was supported in the same position by the
platform while lying on its side. Preservation solution (12 mL) was
added to the chamber and subsequently perfused over the surface of the
heart via a specially designed loop. The loop was constructed of
polyvinyl tubing (3.5-mm ODx2.0-mm ID) together with a suitable
T-connector to form the shape of the letter P. On the inside of the
loop, three equidistant holes allowed the preservation solution to flow
over the surface of the heart when in use. The loop was attached to the
end of an additional stainless steel cannula passed through the
silicone rubber stopper, allowing delivery of the preservation solution
to the heart via the loop. The loop was positioned above and in contact
with the heart to allow the solution to accumulate and form a pool of
liquid inside the loop on top of the heart. The myocardial temperature
was maintained at 7.5°C by solution circulating via a cooling chamber
with a Gilson peristaltic pump (Minipuls 3). The myocardial temperature
was monitored by a thermocouple fixed in the nylon mesh of the support
platform. To obtain Em recordings, borosilicate
glass capillaries were pulled to tip resistances of 6 to 16 M
on a
DMZ Universal electrode puller. The microelectrodes were filled with 3
mol/L KCl and, with a micromanipulator (Prior), were maneuvered
into the pool of solution inside the loop to complete a circuit with
the reference electrode (Fig 1). The reference electrode was a
Ag/AgCl2 wire positioned in the stream of solution
flowing to the loop. The microelectrode tip resistances were determined
before Em measurement. The Em signal was
recorded with a purpose-built electrode amplifier and displayed on
a 20-MHz digital storage oscilloscope (Gould 1425). Em
measurements were recorded every 15 minutes throughout the storage
period on a chart recorder (Gould RS 3200).
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After storage and Em measurement, hearts were removed from the chamber and subsequently reperfused as stated in the perfusion protocol (above).
Biochemical Analysis
CK determination. The assay reagent, when stored at
-20°C, has a safe storage lifetime of 3 weeks. Samples of
reperfusate therefore were frozen at the end of each
experiment, and all samples were assayed at the end of the study with
freshly prepared assay reagent. The assay, based on that of Urdal and
Strømme,14 determined the CK content of the
reperfusate sample by following the conversion of
NADP+ to NADPH spectrophotometrically at 340 nm.
High-energy phosphate determination. Hearts were freeze-dried for 24 hours after freeze-clamping at the end of the protocol. Ventricular tissue was homogenized by a Polytron homogenizer in 6% perchloric acid on ice and then centrifuged at 2000 rpm for 10 minutes at 4°C. The supernatant was then removed, neutralized with 330 µL of 5 mol/L K2CO3, and centrifuged again at 2000 rpm for 10 minutes at 4°C. Aliquots of the supernatant were removed and frozen at -80°C for subsequent ATP and CP fluorometric analysis (within 1 week). Ventricular ATP was determined by hexokinase (0.14 U/mL; Boehringer Mannheim Biochemica) and ventricular CP by CK (8.8 µg/mL; Boehringer Mannheim Biochemica). Excitation and emission wavelengths were set at 365 and 460 nm, respectively, to monitor the conversion of NADP+ to NADPH. Values for ATP and CP were expressed as µmol/g dry wt tissue.
Statistical Analysis
There were 6 to 8 hearts per group in all studies, and data are
presented as mean±SEM. The preischemic control
data were compared before and after electrode insertion by an unpaired
one-tailed Student's t test. Functional recovery was first
analyzed by a one-way ANOVA, and then, if statistical
significance (P<.05) was achieved, a Dunnett's and
Tukey's modified t tests for multiple comparisons were used
to test for statistical difference between groups. Differences were
considered significant at the 95% level confidence limit.
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Results |
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Preliminary Characterization Studies
Determination of optimal TTX concentration. Fig 2A
shows the postischemic
recovery of aortic flow after 5 hours of hypothermic (7.5°C) storage.
Arrest and storage with 1 and 10 µmol/L TTX did not
improve recovery above that of control. In contrast, the higher
concentrations of TTX significantly (P<.05) improved
recovery above control; thus, recovery with 22, 40, and 100
µmol/L was 50.4±2.1%, 51.3±3.4%, and 49.3±3.5% compared
with the control group (35.4±3.9%). Interestingly, there were no
differences in postischemic recovery of function between
the three highest TTX concentrations tested, and the higher
concentrations did not have any detrimental effects.
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The time to mechanical arrest at the various TTX concentrations was concentration dependent; hearts infused with TTX at 22, 40, or 100 µmol/L arrested during the 30-second infusion period at 24.1±0.9, 10.6±0.9, and 6.3±0.9 seconds, respectively, whereas hearts treated with KH alone (control) or 1.0 or 10 µmol/L TTX did not arrest during this 30-second period.
Thus, the optimal concentration of TTX was determined to be 22 µmol/L, the lowest concentration that produced maximum recovery.
Determination of optimal storage temperature. Fig 2B
describes the postischemic recovery of aortic flow in
hearts after 5 hours of storage at different temperatures. After
storage at 4°C, 7.5°C, 11°C, and 15°C, recovery of aortic flow
was 38.4±3.8%, 54.0±9.2%, 28.8±8.1%, and 1.9±1.6%,
respectively. Recovery of aortic flow in hearts stored at 7.5°C was
significantly (P<.05) higher than at 11°C or 15°C but
was not significantly different from those stored at 4°C, despite a
higher recovery.
Myocardial ATP content (in µmol/g dry wt) in hearts stored at 4°C (17.3±0.3) was significantly (P<.05) higher than at 7.5°C (15.3±0.3), 11°C (13.9±0.4), and 15°C (9.7±1.0). In contrast, myocardial CP content (in µmol/g dry wt) in hearts stored at 4°C (36.6±2.1), 7.5°C (42.3±2.6), and 11°C (36.7±0.4) were not significantly different, whereas CP in hearts stored at 15°C (28.4±2.9) was significantly (P<.05) lower than in the 7.5°C group.
The optimum temperature for long-term storage of rat hearts arrested with TTX was determined to be 7.5°C. The conditions defined in these two preliminary studies were used in subsequent studies.
Functional and Metabolic Comparison of a Depolarizing
Arrest Solution (High K+) With a Polarizing Arrest
Solution (TTX)
There were no differences in the preischemic data
between the groups (Table 1). Fig 3 shows the postischemic
recovery of aortic flow after 5 hours of hypothermic (7.5°C) storage.
Recovery of aortic flow in both the high-K+–treated
(37.6±2.2%) and TTX-treated (48.3±2.5%) hearts was significantly
(P<.05) higher than in the control (13.0±4.1%) group of
hearts, but in addition, TTX-treated hearts recovered to a
significantly (P<.05) higher degree than the
high-K+ group. Recoveries of other functional
parameters (aortic pressure, heart rate, coronary
flow, cardiac output, stroke volume, and stroke work) for all groups
are shown in Table 1. CK leakage in the TTX-treated group of hearts
(3.7±0.3 IU/15 min) was significantly lower than in the control group
(5.2±0.5 IU/15 min) (Table 1). Myocardial ATP and CP contents at the
end of 35 minutes of reperfusion were similar in all three groups
(Table 1).
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P<.05 vs KH+K+ (16
mmol/L) value; n=8 hearts per group.
In a separate group of hearts, myocardial content of ATP and CP were
measured at the end of the 5-hour storage period (Fig 4). Myocardial ATP contents (in
µmol/g dry wt) in control hearts (6.3±1.0) and
high-K+–treated hearts (6.9±1.0) were not significantly
different, whereas they were significantly (P<.05) higher
(9.6±1.4) in TTX-treated hearts than in controls. Myocardial CP
contents (in µmol/g dry wt) in both
high-K+–treated (8.1±1.2) and TTX-treated (10.7±0.2)
hearts were significantly (P<.05) higher than in the
control (5.3±0.1) group. CP content in the TTX-treated group of hearts
was also significantly (P<.05) higher than in the
high-K+–treated group of hearts.
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Determination of Resting Em Throughout Ischemic
Storage
The resting Em measured throughout the 5 hours of
storage is illustrated in Fig 5. The
initial recording was made as soon after the onset of
ischemia as possible (5 minutes), and the final
recording was made 5 minutes before the end of ischemia
to allow the heart and cannulas to be reconnected back to the
apparatus before reperfusion. The resting Em in
control (ischemia alone) hearts steadily depolarized throughout
the 5-hour storage period from an initial recording of
-70.4±1.7 mV to a final value of -52.9±1.2 mV. In contrast, both
the high-K+– and TTX-treated hearts rapidly reached a
relatively constant resting Em. The initial values obtained
for the high-K+–treated (-54.0±0.7 mV) and TTX-treated
(-74.0±1.0 mV) hearts depolarized to final levels before reperfusion
of -50.2±0.5 and -65.4±0.7 mV, respectively.
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), depolarized arrest (
), and polarized arrest (
).
All values represent mean±SEM; n=8 hearts per group.
Recovery of function after Em measurement.
Preischemic function data for hearts in which resting
Em was measured is shown in Table 2; there were no differences between
groups. The aortic flow in TTX-treated hearts recovered to 48.2±3.1%,
which was significantly (P<.05) higher than hearts in the
control group (28.2±8.2%) but, although higher than hearts in the
high-K+ group (37.8±4.9%), did not reach statistical
significance. Recovery of other functional parameters
(aortic pressure, heart rate, coronary flow, cardiac output,
stroke volume, and stroke work) were also calculated for all groups
(Table 2).
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Discussion |
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This study has demonstrated that arrest with an optimal concentration (22 µmol/L) of TTX significantly improved postischemic recovery of rat hearts stored for 5 hours at an optimal temperature (7.5°C) compared with hearts arrested with 16 mmol/L K+ or ischemic arrest (control). In addition, we have demonstrated, to the best of our knowledge for the first time, that resting Em during extended hypothermic storage in TTX-arrested hearts was maintained at a more polarized potential of
-70 mV compared with the depolarized
arrest induced by 16 mmol/L K+ (Em
maintained at -50 mV) or the gradual change in resting
Em of ischemic arrest hearts.
Optimal TTX Concentration and Storage Temperature
Tyers and colleagues,10 11 in the mid 1970s, were the
first to demonstrate the cardioprotective properties of TTX during
cardioplegic arrest and ischemia in the isolated,
crystalloid-perfused rat heart. They reported that 14 µg of
intracoronary TTX (7 µg/mL; 22 µmol/L) was the
minimum concentration required to induce arrest. At this concentration,
TTX-induced arrest resulted in significant preservation of high-energy
phosphates and cardiac function of hearts subjected to 60 minutes of
37°C ischemia. Tyers and colleagues suggested that TTX
afforded cardioprotection by attenuating the decline of high-energy
phosphates during ischemia and speculated, because there was no
experimental evidence, that "Continued membrane polarization is an
advantage of TTX over potassium arrest..." that prevented
activation of a calcium current.15 Sternbergh and
colleagues12 observed lower myocardial oxygen consumption
and resting tension during 60 minutes of continuous perfusion (37°C)
of hearts with a solution containing 25 µmol/L TTX than
in hearts perfused with a solution containing 20 mmol/L
K+. There was no assessment of cardiac function or
Em either before or after infusion of the cardioplegic
solutions. In the present study, we also observed a significant
improvement in protection above hypothermic ischemia alone at a
TTX concentration of 22 µmol/L. Interestingly, at higher
concentrations of TTX (up to 100 µmol/L), we were unable
to demonstrate any further improvement in protection.
Our preliminary study showed that the optimal temperature for long-term preservation with TTX was 7.5°C, supporting the findings of Takahashi and colleagues,16 who used STH No. 2 (containing 16 mmol/L K+) for arrest and long-term preservation in Langendorff-perfused rat hearts. Keon and colleagues17 reported that profound hypothermia (<4°C) was associated with elevated resting tension (contracture) in human right atrial trabeculae muscles, and this was also seen in the study by Takahashi et al16 in hearts stored at 5°C. It is possible that hypothermia-induced release of calcium from the sarcoplasmic reticulum may be a factor in the increase in contracture upon cooling to temperatures of <4°C.18 After reperfusion, myocardial ATP content of TTX-protected hearts showed a progressive decline with increasing temperatures, again confirming the results obtained by Takahashi and colleagues16 using STH for long-term preservation. In contrast, myocardial CP content exhibited a bell-shaped profile over the temperature range of 5°C to 20°C, with a peak at 7.5°C, also supporting observations by Takahashi et al.16 This is particularly relevant because inhibition of cellular creatine transport reduces contractile function, suggesting that cellular levels of creatine and CP are important determinants of cardiac function.19 Decreased levels of myocardial total creatine and CP contents have been suggested to be markers of failing myocardium.20 More recently, slower recovery of CP measured by 31P NMR after cardioplegic arrest was found to be associated with poor diastolic function.21 The close correlation between the profiles of cardiac function and myocardial CP after an ischemic insult in our studies supports the theory that CP may be an important determinant of cardiac function during reperfusion.
The optimal temperature and TTX concentration for long-term cardioplegic preservation were determined to be 7.5°C and 22 µmol/L, respectively. These conditions were then used to compare long-term preservation of hearts arrested with a KH solution alone (control), a KH solution containing 16 mmol/L K+, or a KH solution containing 22 µmol/L TTX.
Cardiac Metabolism During Storage
Earlier studies demonstrated that TTX-arrested hearts exhibited
higher postischemic levels of ATP and CP after
ischemia and reperfusion compared with hearts subjected to
unprotected ischemia,11 and this was supported by
the ATP and CP values obtained in the present study. However, we
also determined levels of myocardial ATP and CP at the end of
ischemia, and TTX-treated hearts had significantly higher
values than either control or high-K+–treated hearts. It
is generally believed that during periods of metabolic
demand, CP acts as a reservoir of high-energy phosphate that maintains
ATP levels via the CK reaction and that during ischemia, CP
levels fall much more rapidly than ATP levels.22 23 In
this study, CP was lower than ATP after 5 hours of ischemia in
control hearts; in contrast, however, CP remained higher than ATP in
the high-K+– and TTX-treated hearts. It is possible that
this reflects compartmentalization of the CK enzyme system. Within the
myocyte, isoforms of CK are associated with sites of energy
production and utilization, such as the contractile
apparatus, nuclei, sarcoplasmic reticulum, ion
translocating systems, and the mitochondria.24 The CK
isoform coupled to mitochondria (CK-Mi) is different from the cytosolic
isoform (CK-MM) in that the CK-Mi reaction is driven unidirectionally
to form CP from ATP.25 Thus, maintained activity of the
CK-Mi system in the high-K+– and TTX-protected hearts may
have utilized ATP to form CP, resulting in myocardial CP levels being
elevated above that of ATP in these groups.
Potassium-induced depolarization elevates intracellular calcium ([Ca2+]i) through voltage-dependent calcium channels26 and augments cellular energy consumption.12 It has been proposed that these two observations are inextricably linked, with the elevated [Ca2+]i stimulating the energy-dependent calcium ATPase of the sarcoplasmic reticulum and thereby increasing the utilization of cellular ATP.27 We have shown that myocardial levels of ATP and CP are higher in TTX-treated hearts at the end of ischemia and therefore speculate that TTX-treated hearts, in which the Em was maintained at a more polarized level, were able to conserve cellular energy stores better than control and high-K+–treated hearts because of a reduced calcium influx.
Resting Em and Cardioprotection
Rapid depolarization of Em (to -40 mV) together
with [Na+]i accumulation has been observed
when the Na pump has been inhibited by ouabain28 ; in
addition, profound hypothermia is known to inhibit the Na
pump.29 In our study, rapid depolarization was observed in
the control group of hearts, and it is likely that this occurred as a
result of the hypothermic storage temperature mediating Na pump
inhibition. In contrast, the rapid depolarization occurring in the
K+-treated hearts can be accounted for almost exclusively
by the infusion of 16 mmol/L K+. By the Nernst
equation, assuming that the sarcolemma acts as a semipermeable membrane
to K+ alone, the measured Em achieved the
predicted level for this extracellular K+ concentration.
Thus, it is likely that any influence on Em of the
temperature-induced inhibition of the Na pump would be masked. In the
TTX-treated hearts, there was attenuation of the depolarization
response; this may be related to inhibition of Na current activity or
to attenuation of the rapid ischemia–induced
[K+]e accumulation preventing a significant
depolarization of the cell membrane, as we have recently
demonstrated.30
The extent to which Em finally depolarizes during
hypothermic ischemia observed in the control hearts of our
study closely agrees with previous work performed at normothermia.
Kléber31 showed, in guinea pig hearts, a gradual
depolarization of Em to -50 mV after only 15 minutes of
normothermic, unprotected global ischemia. In our
study, Em depolarized more slowly, and only after 5 hours
of hypothermic storage did we observe a comparable degree of
depolarization. This difference in the rate of Em
depolarization may be explained by the different storage temperatures
during ischemia in the two studies.
Hyperkalemia-Induced Arrest
Cardioplegic arrest and storage of hearts with KH plus
K+ (16 mmol/L) induced rapid depolarization of
the myocardium (to -50 mV), which was maintained for
the duration of the ischemic storage period. Kurihara and
Sakai18 measured the effects on resting Em of
changing the extracellular K+ concentrations between 6 and
200 mmol/L in guinea pig ventricular muscle
maintained at 4°C. At these K+ concentrations,
Em depolarization varied from -80 to -10 mV; 16
mmol/L K+ also caused the Em to
depolarize to -50 mV, which agrees with the measured Em
during depolarized arrest in this study. Previously, Krohn and
coworkers,32 using conventional 3 mol/L KCl-filled
glass microelectrodes, measured Em of sheep Purkinje fibers
during cardioplegic arrest by Bretschneider HTK solution (which
contains 10 mmol/L K+). Exposure to the HTK
solution induced a depolarization of the Em to -60
mV.
Depending on the K+ concentration, depolarization of Em with cardioplegic solutions (such as STH) may activate Na and Ca influx via steady-state voltage-dependent window currents. López and colleagues26 showed that [Ca2+]i was elevated by exposure of isolated myocytes to a hyperkalemic challenge equivalent to the potassium content of our high-K+ group (16 mmol/L). This elevated [Ca2+]i was prevented by aprikalim or nicorandil, openers of the K+ATP channel. They speculate, despite the lack of Em measurements, that aprikalim and nicorandil prevented the increase in [Ca2+]i by inhibiting a Ca current via voltage-dependent Ca channels due to Em hyperpolarization.
Sodium window currents8 have been shown to exist when Em is between -65 and -15 mV, whereas Ca window currents9 have a more positive activation window of -40 to -15 mV. However, many studies have shown that depolarization of the cardiac Em by either rubidium,33 hyperkalemic solutions,34 35 or voltage clamping36 reduces intracellular sodium ([Na+]i), which is in contrast to our hypothesis that depolarization may increase sodium influx. An important consideration is that the observations by Ellis34 and by January and Fozzard36 were made in tissue preparations that were both nonischemic and incubated at 37°C, when the sodium pump would almost certainly remain active and would tend to extrude Na+, because the driving force of the pump may be increased when extracellular potassium is increased.37 Stinner and colleagues35 also demonstrated a reduction of [Na+]i while superfusing sheep Purkinje fibers with the depolarizing hyperkalemic STH. They mimicked hypothermia-mediated sodium pump inhibition by adding 0.1 mmol/L dihydro-ouabain to STH. Under these conditions, STH superfusion was associated with Em depolarization and a rise in [Na+]i activity. Furthermore, addition of procaine, a sodium channel blocker, reduced the depolarization-induced increase in [Na+]i activity, suggesting that the increase is partly mediated via the sodium channel. However, at this Em, sodium influx via the sodium channel would be mediated only via the sodium window current, because the fast inward sodium current would be fully inactivated.38 In the context of ischemic injury and contractile dysfunction, recent evidence39 suggests that the sodium window current may be an important contributor to sodium accumulation during early ischemia.
TTX-Induced Arrest
TTX induces arrest by blocking the cardiac sodium channels
responsible for the fast inward sodium current
(INa) initiating the cardiac action potential.
Blockade of this channel should prevent Em depolarization
induced by sodium ion flux into the myocyte during the first phase of
the cardiac action potential; consequently, maintenance of
resting Em during the action potential prevents the inward
flux of calcium ions required for contraction via
voltage-activated calcium currents. We have shown that resting
Em during polarized arrest remains at -70 mV, close to
the normal resting Em of the myocyte. At this
Em, myocyte ion transport systems are relatively inactive
and the sodium and calcium window currents should be inhibited.
When attempting to extrapolate these studies to humans, one should bear in mind that there may be considerable species differences between ion transport mechanisms. Certainly, in many respects the physiologies of the rat and human heart are very different. However, there have been many studies in other animals31 35 40 41 42 43 which have demonstrated that either [Na+]i or [Ca2+]i can increase during ischemia. There were no differences in the behavior of the sodium current in rabbit and rat ventricular tissue during exposure to the ischemic metabolite lysophosphatidylcholine.44 Shattock and Bers45 demonstrated that at diastole, the [Na+]i of rat myocardium is higher than that in rabbit and that Em was more positive with respect to ENa/Ca. This relationship is thought to favor calcium uptake and sodium extrusion and thereby load the cell with calcium. Conversely, in the rabbit, calcium extrusion is favored by Em at rest being more negative with respect to ENa/Ca. However, it is difficult to predict the effects of ischemia on this relationship in the rabbit heart; if we assume that Em becomes depolarized and that [Na+]i also increases during ischemia, then ENa/Ca may become more negative than Em and begin to resemble that of the rat heart during ischemia. Therefore, changes observed in different species may be quantitatively different, but the mechanisms responsible may be qualitatively similar.
A reduced calcium and sodium influx during ischemia may be beneficial to eventual recovery of cardiac function during reperfusion. Tani and Neely,46 using 23Na NMR, demonstrated that [Na+]i does increase during ischemia and that 45Ca2+ uptake during reperfusion is linearly correlated with the level of [Na+]i at the end of ischemia. This evidence supports the hypothesis that sodium may be exchanged for calcium, possibly by Na+/Ca+ exchange, and that this would result in a rise in intracellular calcium during reperfusion. Reduction of [Na+]i accumulation during long-term ischemia should attenuate calcium overload during reperfusion and prove beneficial to the eventual functional recovery of hearts.
LCAs have been shown47 to increase [Na+]i upon addition to rabbit ventricular myocytes, contributing to an increase in intracellular calcium via Na+/Ca+ and the generation of delayed afterdepolarizations and triggered activity during reperfusion. Release of LCAs has previously been shown to occur minutes after the onset of ischemia.48 The resulting ionic imbalance may prove detrimental to the eventual recovery of the heart upon reperfusion. Inhibition of LCA production during ischemia delays cellular uncoupling, extracellular potassium accumulation, and the onset of ischemic contracture.49 Addition of exogenous palmitoyl carnitine to ventricular myocytes has been shown to trigger a TTX-sensitive inward sodium current similar to the sodium window current.50 It is possible that TTX may have antagonized the effects of LCAs and reduced the sodium accumulation via this route during ischemia in this study. However, in depolarized hearts, the actions of LCAs would not have been antagonized, and these hearts might have been more prone to sodium accumulation during ischemia.
Potassium Channel Openers
Agents that induce hyperpolarization of the
cell membrane have recently been advocated as potentially beneficial
alternatives to hyperkalemic depolarization for myocardial protection.
Agents such as adenosine51 or a relatively new
class of drugs that activate ATP-dependent potassium channels
(potassium channel openers52 53 ) have been used to provide
improved protection compared with high-potassium cardioplegic
solutions. In these studies, however, Em has not been
measured, and the mechanism by which the improved protection occurs
remains unclear. In particular, it is unknown whether
hyperpolarization of the membrane is maintained
throughout the ischemic period.
Summary
Cardioplegic arrest and long-term preservation with TTX at a
concentration of 22 µmol/L and a temperature of 7.5°C
was shown to be optimal for the recovery of postischemic
cardiac function and metabolic status in the isolated
crystalloid perfused rat heart. At this concentration and temperature,
microelectrode recordings of resting Em have shown,
for the first time in isolated globally ischemic rat hearts,
that polarized arrest and preservation were achieved and maintained for
the duration of the preservation period (5 hours). Induction of
depolarized arrest by 16 mmol/L potassium was also shown to
be maintained throughout the ischemic period. The superior
myocardial preservation demonstrated by a polarizing preservation
solution may be attributed to the attenuation of sodium accumulation
via (1) the sodium window current, (2) the LCA-mediated sodium current,
and (3) better preservation of high-energy phosphates during
ischemia. Further studies into the pharmacological manipulation
of the cardiac resting Em and the use of compounds that
prevent accumulation of sodium during ischemia, by techniques
that allow measurement of intracellular sodium, may lead to a long-term
preservation solution that is more suited to extending viable storage
of the heart.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Andrew K. Snabaitis is supported by a British Heart Foundation PhD Studentship; Michael J Shattock is supported by a British Heart Foundation Senior Lectureship.
Received January 22, 1997; revision received May 20, 1997; accepted June 6, 1997.
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References |
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