(Circulation. 1997;96:2376-2384.)
© 1997 American Heart Association, Inc.
Articles |
Preservation of Myocyte Contractile Function After Hypothermic Cardioplegic Arrest by Activation of ATP-Sensitive Potassium Channels
From the Department of Anesthesia and Perioperative Medicine (B.H.D., L.H., R.B.H., R.C.R.) and the Department of Surgery (F.G.S.), Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston.
Correspondence to B. Hugh Dorman, MD, PhD, Department of Anesthesia and Perioperative Medicine, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425.
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Abstract |
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Background Left ventricular (LV) dysfunction can occur after hyperkalemic cardioplegic arrest and subsequent reperfusion and rewarming. Activation of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels within the myocyte sarcolemma has been shown to be cardioprotective for myocardial reperfusion injury and ischemia and may play a contributory role in preconditioning for cardioplegic arrest. Accordingly, the present study tested the hypothesis that cardioplegic arrest and activation of KATP channels by a potassium channel opener (PCO) would attenuate alterations in ionic homeostasis and improve myocyte contractile function.
Methods and Results Porcine LV myocytes were isolated and randomly assigned to the following treatment groups: normothermic control, incubation in cell culture media for 2 hours at 37°C (n=60); hyperkalemic cardioplegia, incubation for 2 hours in hypothermic hyperkalemic cardioplegic solution (n=60); or PCO/cardioplegia, incubation in cardioplegic solution containing 100 µmol/L of the PCO aprikalim (n=60). Hyperkalemic cardioplegia and rewarming caused a significant reduction in myocyte velocity of shortening compared with normothermic control values (33±2 versus 66±2 µm/s, P<.05). Cardioplegic arrest with PCO supplementation significantly improved indices of myocyte contractile function when compared with hyperkalemic cardioplegia (58±4 µm/s, P<.05). Myocyte intracellular calcium increased during hyperkalemic cardioplegic arrest compared with baseline values (147±2 versus 85±2 nmol/L, P<.05). The increase in intracellular calcium was significantly reduced in myocytes exposed to the PCO-supplemented cardioplegic solution (109±4 nmol/L, P<.05).
Conclusions Cardioplegic arrest with simultaneous activation of KATP channels preserves myocyte contractile processes and attenuates the accumulation of intracellular calcium. These findings suggest that changes in intracellular calcium play a role in myocyte contractile dysfunction associated with cardioplegic arrest. Moreover, alternative strategies may exist for preservation of myocyte contractile function during cardioplegic arrest.
Key Words: cardioplegia • potassium • contractility • myocytes
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Introduction |
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Cardioplegic arrest during cardiac surgery has traditionally been accomplished through administration of a hyperkalemic cardioplegic solution.1 However, left ventricular (LV) dysfunction has been shown to occur after hyperkalemic cardioplegic arrest and rewarming,2 which may involve alterations in ionic homeostasis.3 4 This laboratory has demonstrated that at the level of the myocyte, hypothermic hyperkalemic cardioplegic arrest causes a reduction in myocyte contractile function that may play a role in the LV dysfunction observed after cardioplegic arrest.5 6 Thus this isolated myocyte model of simulated cardioplegic arrest may be useful to determine mechanisms responsible for contractile dysfunction with reperfusion as well as potential strategies to prevent these effects. Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels exist within the myocyte that open in response to reductions in intracellular ATP or ischemia.7 8 9 10 11 The protective effects of ischemic preconditioning in regional or global ischemia have been shown to be mediated in part by the KATP channel.12 13 Recent studies have also demonstrated that a brief period of KATP channel activation by a potassium channel opener (PCO) before ischemia improves myocardial function and reduces infarct size with reperfusion.14 15 16 17 Moreover, KATP channel activation before hypothermic, hyperkalemic cardioplegic arrest or used for hyperpolarized cardioplegic arrest improves functional recovery of myocardium.18 19 20 21 22 Thus activation of KATP channels by a PCO during hyperkalemic cardioplegic arrest may improve preservation of myocyte contractile function and help restore ionic homeostasis. The objectives of the present study therefore were twofold: first, to compare the effects of cardioplegic arrest through the use of PCO-supplemented cardioplegia and traditional hyperkalemic cardioplegia on myocyte contractile function; and second, to measure intracellular calcium in the same myocyte throughout both methods of cardioplegic arrest and reperfusion to better define the mechanism of myocyte contractile dysfunction.
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Methods |
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The present study used an isolated myocyte system to examine the effects of hypothermic, hyperkalemic cardioplegic arrest and ATP-sensitive, PCO-supplemented cardioplegic arrest on myocyte contractile function.5 6 Intracellular calcium was measured in isolated myocytes throughout cardioplegic arrest and reperfusion to better understand the relation between alterations in ionic homeostasis and contractile function.
Myocyte Isolation and Contraction Analysis
Myocyte isolation and determination of myocyte contractile
function were performed with previously described
methods.5 6 23 Yorkshire swine (n=13) were the source of
myocytes for the study. A 2-mL aliquot of the isolated myocyte
suspension was plated onto coverslips previously coated with a basement
membrane substrate (Matrigel, Collaborative Research Inc) stabilized at
37°C in oxygenated media for 60 minutes, then randomly
assigned to treatment protocols after measurement of baseline
contractile function. The yield of viable myocytes was >80% and was
not affected by cardioplegic arrest and rewarming. Viable myocytes
included those that retained a rod shape, excluded trypan blue, and
remained quiescent in culture.
Isolated myocyte contractile measurements were performed as described previously.5 6 23 Contraction data for each myocyte were recorded for a minimum of 20 consecutive contractions. Parameters computed from the digitized contraction profiles included percent shortening (%), velocity of shortening (µm/s), velocity of relengthening (µm/s), total contraction duration (ms), time to 50% relaxation (ms), and velocity of shortening and relengthening normalized to resting myocyte length (µm · s-1 · µm-1). Myocyte percent shortening was determined as the percent difference between maximum and minimum cell length for each contraction. Myocyte velocity computations were obtained by differentiating the digitized contraction profiles. Myocyte velocity of shortening and relengthening were normalized to myocyte length by dividing the velocity of shortening and relengthening by the resting myocyte length measured just before contraction. All parameters were calculated for each contraction and the results averaged for the 20 contractions observed.
Experimental Design and Rationale
The objectives of this portion of the study were to define the
specific and potential interactive effects of hyperkalemic cardioplegic
arrest and cardioplegic arrest with PCO supplementation on myocyte
contractile function. Accordingly, after measurement of baseline
contractile performance, myocytes were randomly assigned to one
of six treatment protocols: (1) normothermic control group:
incubation in 37°C Ringer's solution (Na+ 130
mmol/L, Cl- 109 mmol/L, K+
4 mmol/L, Ca2+ 1.8 mmol/L)
containing 30 mEq/L HCO3-, then stored for 2
hours at 37°C in a 95% oxygen environment; (2) 24K-cardioplegia
group: incubation in Ringer's solution at 4°C containing 24 mEq/L
potassium and 30 mEq/L HCO3-, then stored at
4°C for 2 hours; (3) 12K-cardioplegia group: incubation in Ringer's
solution at 4°C containing 12 mEq/L potassium and 30 mEq/L
HCO3-, then stored at 4°C for 2 hours; (4)
PCO/24K-cardioplegia group: incubation in Ringer's solution at 4°C
containing 24 mEq/L potassium, 30 mEq/L HCO3-,
and 100 µmol/L of the PCO aprikalim, then stored at 4°C
for 2 hours; (5) PCO/12K-cardioplegia group: incubation in Ringer's
solution at 4°C containing 12 mEq/L potassium, 30 mEq/L
HCO3-, and 100 µmol/L of the PCO
aprikalim, then stored at 4°C for 2 hours; and (6) PCO group:
incubation in Ringer's solution at 4°C containing 4 mEq/L potassium,
30 mEq/L HCO3-, and 100 µmol/L
of the PCO aprikalim, then stored at 4°C for 2 hours. After each of
these incubation protocols, myocytes were resuspended in
normothermic cell culture media and myocyte contractile
function determined. After measurement of contractile function,
myocytes were exposed to 25 nmol/L isoproterenol and contractile
function measurements were repeated. This concentration of
isoproterenol (6.25 ng/mL) has been previously shown to produce
a maximal response in control, normothermic porcine
myocytes and has been shown to be within the range used
clinically.5 23
The concentration of aprikalim (100 µmol/L) that was chosen for these experiments was based on previous studies documenting sustained electromechanical arrest and improved cardioprotection after global ischemia in isolated heart preparations.10 20 21 However, in preliminary studies we observed that this concentration of aprikalim with normokalemia (4 mEq/L K+) failed to cause immediate excitation-contraction uncoupling in the isolated myocyte preparations in the presence of electrical stimulation. Accordingly, potassium at a concentration of 12 mEq/L was included with aprikalim to cause an immediate cessation of contractile activity. Myocytes were also incubated in 12 mEq/L potassium without PCO for comparison purposes. To achieve immediate electromechanical uncoupling in the cardiac surgical setting, 24 mEq/L of potassium is delivered to the myocardium. Accordingly, myocytes were exposed to 24 mEq/L of potassium during simulated cardioplegic arrest. To address potential interactive effects and a means for comparison, PCO supplementation was also used in conjunction with this increased potassium concentration.
To ensure that any protective effects with aprikalim on myocyte contractile function were specifically caused by KATP channel activation, additional studies were performed with the KATP channel antagonist glibenclamide. Glibenclamide (1 µmol/L) was added to myocytes in the PCO/24K-cardioplegia group, and contractile function was compared with myocytes in the normothermic control group, the 24K-cardioplegia group, and the PCO/24K-cardioplegia group without glibenclamide. Sixty myocytes were examined from each treatment group. This concentration of glibenclamide has been demonstrated to block the protective effects of preconditioning in human atrial trabecular tissue.12
In the clinical setting, LV pump dysfunction after cardioplegic arrest is a time-dependent phenomenon. To more carefully examine the potential time-dependent changes in contractile function that may occur after simulated cardioplegic arrest, indices of myocyte contractile function were measured at 10, 20, and 30 minutes after cardioplegic arrest and rewarming for myocytes in the normothermic control, 24K-cardioplegia, and PCO/12K-cardioplegia groups. Specifically, immediately after rewarming, separate aliquots of isolated myocytes from each cardioplegia treatment group were incubated for either the 10,- 20-, or 30-minute period and myocyte velocity of shortening was measured. Isoproterenol was then added to myocytes in each of the designated incubation groups, and myocyte velocity of shortening measurements were repeated.
Measurement of Intracellular Calcium
Since a fundamental determinant of myocyte ionic homeostasis is
maintenance of intracellular calcium, a series of experiments
was performed in which intracellular calcium was measured throughout
cardioplegic arrest and reperfusion. Intracellular calcium measurements
were performed as described previously with Fura-2 loading and digital
fluorescence image analysis.24 Serial
determinations of myocyte intracellular calcium concentration were
performed after isolation and stabilization and then at 5 to 40 minutes
after incubation under either normothermic control
conditions, during hyperkalemic cardioplegic arrest with 24 mEq/L
potassium cardioplegia (24K-cardioplegia group), during cardioplegic
arrest with aprikalim supplementation of 12 mEq/L potassium
cardioplegia (PCO/12K-cardioplegia group), or during cardioplegic
arrest with aprikalim supplementation of 24 mEq/L potassium
cardioplegia (PCO/24K-cardioplegia group). Additional intracellular
calcium measurements were performed in each of these treatment groups
during reperfusion and then at 5, 10, and 15 minutes after reperfusion.
In this system, measurements of intracellular calcium were sequentially
recorded in the same myocyte under normothermic
conditions, throughout the period of hypothermic cardioplegic arrest,
and during rewarming and reperfusion. With the use of a
microprocessor-controlled miniature pump system and thermal temperature
controller, the media could be exchanged within the myocyte chamber to
the hyperkalemic solution with the temperature reduced and stabilized
at 4°C.6 The perfusion and temperature systems also
provided a means to return normothermic media to the
chamber and remove the cardioplegic solution, thus simulating
reperfusion and rewarming. Because the myocytes are attached to a
basement membrane substrate,5 6 23 the myocyte environment
could be altered without disturbing myocyte position or adhesion.
Data Analysis
Changes in indices of myocyte function between the control and
cardioplegia groups were examined with the use of multiway ANOVA. If
the ANOVA revealed significant differences, pairwise tests of
individual group means were compared with the use of Bonferroni's
probabilities. All statistical analysis was performed with
standard statistical software programs (BMDP Statistical Software Inc,
University of California Press). Results are presented as
mean±SEM. Values of P<.05 were considered to be
statistically significant.
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Results |
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Steady-state myocyte contractile function and representative myocyte contraction profiles for normothermic control, hyperkalemic cardioplegic, and PCO/hyperkalemic cardioplegic myocytes are summarized in Table 1
and
Fig 1.
A significant reduction in contractile function was observed for
myocytes after hypothermic, hyperkalemic, cardioplegic arrest and
rewarming at either potassium concentration (12K-cardioplegia and
24K-cardioplegia groups). Specifically, isolated myocyte percent and
velocity of shortening were reduced by >45% in myocytes in the
12K-cardioplegia and 24K-cardioplegia groups relative to the
normothermic control group. Myocyte velocity of
relengthening was also significantly decreased by >50% in myocytes
after hyperkalemic cardioplegic arrest. The time to 50% relaxation,
which is an index of active relaxation, was significantly increased by
43% and 73% after cardioplegic arrest with either 12 or 24 mEq/L
potassium, respectively (Fig 2). Thus, consistent
with past studies, hypothermic, hyperkalemic cardioplegic arrest with
reperfusion and rewarming caused a reduction in isolated myocyte
contractile processes.5 6 23
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To determine the effect of PCO supplementation on baseline myocyte
contractile function after cardioplegic arrest, 100
µmol/L aprikalim was included in the 12- and 24-mEq/L
hyperkalemic cardioplegic solutions for the 2-hour incubation period
(PCO/12K-cardioplegia and PCO/24K-cardioplegia groups, respectively).
PCO supplementation preserved myocyte contractile function after
cardioplegic arrest. A significant increase in percent shortening
(41%) and velocity of shortening (81%) was observed in myocytes in
the PCO/12K-cardioplegia group relative to myocytes in the
12K-cardioplegia group. Significant increases in percent shortening
(63%) and velocity of shortening (73%) were also observed in myocytes
with PCO supplementation of 24 mEq/L potassium cardioplegia relative to
myocytes that underwent hyperkalemic cardioplegia arrest (24 mEq/L)
without PCO supplementation. Moreover, there were significant
reductions in the time to 50% relaxation and significant increases in
the velocity of relengthening in myocytes in the PCO/cardioplegia
groups relative to myocytes in the respective hyperkalemic cardioplegia
groups without PCO supplementation (Table 1
and Fig 2). Furthermore, there were no
differences in the velocity of shortening, velocity of relengthening,
or time to 50% relaxation between PCO/cardioplegia myocytes and
normothermic control myocytes. To determine whether PCO
alone, in the absence of elevated potassium concentrations, would
provide protective effects on myocyte contractile function, a series of
experiments was performed after exposure to PCO in a normokalemic
solution (PCO group) for 2 hours at 4°C. PCO alone did not preserve
myocyte contractile function as well as PCO-supplemented hyperkalemic
solutions. Velocity of shortening was 43±2 µm/s in the PCO
group and 58±4 µm/s in the PCO/12K-cardioplegia group
(P<.05). However, myocyte velocity of shortening was 35%
higher (P<.05) after cardioplegic arrest with PCO alone
relative to hyperkalemic cardioplegic solutions without PCO
supplementation.
To ensure that changes in myocyte length after cardioplegic arrest did
not influence myocyte contractile function, velocity of shortening and
velocity of relengthening were normalized to resting myocyte length.
Hyperkalemic cardioplegic arrest resulted in a significant reduction in
the normalized velocity of shortening by >46% in myocytes in the
12K-cardioplegia and 24K-cardioplegia groups, relative to the
normothermic control group (Table 2). Myocyte velocity of
relengthening, normalized to resting myocyte length, was also
significantly decreased by >47% after hyperkalemic cardioplegic
arrest. PCO supplementation of the cardioplegic solutions resulted in
preservation of normalized velocity of shortening. A significant
increase in normalized velocity of shortening was observed in both the
PCO/12K-cardioplegia group (109%) and the PCO/24K-cardioplegia group
(48%) relative to the respective hyperkalemic cardioplegic groups
without PCO supplementation (Table 2). Normalized velocity of
relengthening was also significantly increased by PCO supplementation
of 12 mEq/L potassium cardioplegia. Moreover, in the presence of
isoproterenol, PCO supplementation increased normalized indices of
contractile function compared with values observed with hyperkalemic
cardioplegia without PCO supplementation (Table 2).
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To ensure that the effects of aprikalim on myocyte contractile function
were specifically caused by KATP channel activation,
simulated cardioplegic arrest was performed with a cardioplegic
solution containing the KATP channel antagonist
glibenclamide. The inclusion of glibenclamide (1 µmol/L)
to the 24 mEq/L hyperkalemic cardioplegic solution containing 100
µmol/L aprikalim caused a significant reduction in myocyte
velocity of shortening (26.3±2.0 versus 58.9±1.8 µm/s)
compared with normothermic control values (Fig 3). Glibenclamide
supplementation of the PCO/24K-cardioplegia group yielded steady-state
myocyte velocity of shortening values similar to the 24K-cardioplegia
group (29±1.9 µm/s). More importantly, myocyte velocity of
shortening was significantly reduced in the glibenclamide-supplemented
PCO/24K-cardioplegia group relative to the PCO/24K-cardioplegia group
without glibenclamide (Fig 3). Thus glibenclamide abolished the
beneficial effects of PCO supplementation of hyperkalemic cardioplegic
arrest, which indicates that the protective effects on myocyte
contractile function achieved by aprikalim appear to be mediated by the
KATP channel.
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Isoproterenol (25 nmol/L) was added to normothermic
control, hyperkalemic cardioplegia, and PCO/hyperkalemic cardioplegia
myocytes to determine the effects of PCO supplementation on
ß-adrenergic responsiveness after cardioplegic arrest (Table 1).
Isoproterenol caused a significant increase in all indices of myocyte
contractile function in normothermic control, hyperkalemic
cardioplegia, and PCO/hyperkalemic cardioplegia myocytes. Percent
shortening and velocity of shortening increased by >75% in all of the
myocyte groups. However, all contractile function indices of
12K-cardioplegia and 24K-cardioplegia myocytes after isoproterenol
administration remained significantly decreased relative to
normothermic control myocytes. Supplementation of
hyperkalemic cardioplegia with the PCO aprikalim significantly improved
ß-adrenergic responsiveness after cardioplegic arrest compared with
values observed with hyperkalemic cardioplegia without PCO
supplementation. Moreover, velocity of relengthening was significantly
higher and time to 50% relaxation was significantly lower after
cardioplegic arrest and isoproterenol administration for myocytes in
the PCO/12K and PCO/24K-cardioplegia groups relative to the 12K and
24K-cardioplegia groups. ß-Adrenergic responsiveness was similar
between myocytes in the PCO/12K-cardioplegia group and
normothermic control group. However, both percent and
velocity of shortening were significantly reduced in myocytes in the
PCO/24K-cardioplegia group relative to the normothermic
control group after isoproterenol administration.
Myocyte contractile function was measured every 10 minutes for 30
minutes after cardioplegic arrest and rewarming to determine any
time-dependent changes in contractile function after cardioplegic
arrest with and without PCO supplementation (Fig 4). In the
normothermic control and PCO/12K-cardioplegia groups,
myocyte contractile function and ß-adrenergic responsiveness were
stable throughout the time course study. With hyperkalemic cardioplegic
arrest and rewarming (24K-cardioplegic group), steady-state myocyte
contractile function and ß-adrenergic responsiveness were reduced
compared with normothermic control and PCO/12K-cardioplegia
values and remained lower for the 30-minute period of observation after
rewarming. Thus, the protective effect of PCO supplementation on
myocyte contractile function extended beyond the initial period of
reperfusion and rewarming.
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In light of the significant preservation of myocyte contractile
function and ß-adrenergic responsiveness after cardioplegic arrest
with PCO-supplemented cardioplegia, repeated measurements of
intracellular calcium were performed serially in the same myocyte
throughout hyperkalemic cardioplegic arrest and reperfusion
(24K-cardioplegia group, n=30), PCO-supplemented cardioplegic arrest
and reperfusion with 12 mEq/L potassium cardioplegia
(PCO/12K-cardioplegia group, n=30), PCO-supplemented cardioplegic
arrest and reperfusion with 24 mEq/L potassium cardioplegia
(PCO/24K-cardioplegia group, n=30), and under normothermic
control conditions to better understand the mechanism underlying the
observed changes in myocyte contractile function. Intracellular calcium
concentration remained at baseline levels in normothermic
control myocytes, ranging from 78±2 to 85±2 nmol/L. A
significant increase in intracellular calcium occurred at the onset of
hyperkalemic cardioplegic arrest (24K-cardioplegia group) to 147±2
nmol/L (Fig 5).
Intracellular calcium concentrations remained significantly elevated
throughout the period of hyperkalemic cardioplegic arrest and continued
at increased levels during reperfusion; intracellular calcium
normalized 10 minutes after reperfusion. In contrast, incubation with
PCO supplementation of 12 mEq/L potassium cardioplegia
(PCO/12K-cardioplegia group) did not result in any significant change
in intracellular calcium concentration from baseline values during the
entire period of cardioplegic arrest and with reperfusion (Fig 5). In
the presence of PCO and 24 mEq/L of potassium (PCO/24K-cardioplegia
group), intracellular calcium significantly increased from
normothermic baseline values (109±4 nmol/L) 5
minutes after initiation of cardioplegic arrest but returned to
baseline values in 10 minutes and remained significantly lower than
intracellular calcium levels that were obtained in the presence of 24
mEq/L potassium without PCO supplementation throughout the period of
cardioplegic arrest and reperfusion. Thus PCO supplementation prevented
the rise in intracellular calcium levels induced by cardioplegic arrest
when included with 12 mEq/L potassium cardioplegia and attenuated the
increase in intracellular calcium concentration when included with a
potassium level (24 mEq/L) commonly used to initiate cardioplegic
arrest.
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Discussion |
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Since the first description of survival after cardiopulmonary bypass, improved myocardial preservation during periods of absent coronary perfusion has been the goal of many research efforts. The vast majority of basic and clinical studies in myocardial preservation techniques for cardiac surgery have focused on different mechanisms for cardioplegia delivery, the temperature of the solution, and substrate additives to the hyperkalemic cardioplegic solution.1 However, all of these studies were based on hyperkalemic cardioplegic arrest, which may be a mechanism for LV dysfunction after rewarming. The present study examined whether activation of the KATP channel through the use of a PCO during cardioplegic arrest would preserve intracellular calcium homeostasis and improve myocyte contractile function after reperfusion. Myocyte contractile function and intracellular calcium concentration were measured after hyperkalemic cardioplegic arrest, after cardioplegic arrest with PCO supplementation, and under control, normothermic conditions at baseline. The most significant findings of the present study were (1) hyperkalemic cardioplegic arrest resulted in a significant reduction in myocyte contractile function, ß-adrenergic responsiveness, and active relaxation processes and caused a significant increase in intracellular calcium and (2) cardioplegic arrest with PCO supplementation preserved myocyte contractile function, ß-adrenergic responsiveness, and active relaxation activity and attenuated increases in intracellular calcium. This study demonstrated for the first time, therefore, that the addition of PCO to cardioplegic solutions can prevent the deleterious effects of cardioplegic arrest on both myocyte contractile function and ionic homeostasis.
The PCO aprikalim has the capacity to produce sustained electromechanical arrest in isolated heart preparations that is reversible with reperfusion.10 20 21 The onset of asystole after administration of aprikalim in whole hearts, however, is significantly delayed to >220 seconds. Initiation of mechanical arrest with aprikalim alone in the isolated myocyte model in the presence of electrical stimulation was also delayed when compared with hyperkalemic cardioplegic arrest. Moreover, during cardioplegic arrest with aprikalim, there is prolonged electrical activity in the form of spikelike action potentials that persist after action potential shortening has occurred and mechanical function has ceased.20 The delay in the onset of electromechanical arrest and persistent electrical activity observed with aprikalim may detract from the clinical utility of cardioplegic arrest with PCO compounds. In the present study, PCO (aprikalim) alone in a normokalemic cardioplegic solution did not preserve contractile function to the same extent as PCO supplemented hyperkalemic cardioplegic solutions. This is probably due to the fact that aprikalim alone failed to extinguish isolated myocyte contractile activity for up to 5 minutes and thereby prolonged myocyte metabolic processes. Therefore, a reduced concentration of potassium (12 mEq/L) was added to the cardioplegia solution containing aprikalim to cause a more rapid electromechanical arrest without persistent electrical or contractile activity. A combination of 24 mEq/L potassium cardioplegia with aprikalim was also examined to determine any beneficial effects of PCO supplementation on myocyte contractile function in the presence of the potassium concentration (24 mEq/L) that is commonly used clinically to initiate cardioplegic arrest. PCO supplementation of both hyperkalemic cardioplegia solutions significantly preserved myocyte contractile function relative to hyperkalemic cardioplegia alone, which agrees with a recent report that showed that PCO provided additional protection to that afforded by hyperkalemic cardioplegia in isolated rat hearts.19
In the present study, a significant reduction in myocyte contractile processes and increased intracellular calcium occurred after hyperkalemic cardioplegic arrest. The prolonged extracellular hyperkalemia associated with traditional cardioplegia causes membrane depolarization and subsequent increases in intracellular calcium by several mechanisms including a potentiation of calcium influx through the opening of voltage-dependent calcium channels, which subsequently induces additional release of calcium from the sarcoplasmic reticulum.25 26 27 28 An additional contributory mechanism for increased intracellular calcium during hyperkalemic cardioplegic arrest and reperfusion may involve sodium-calcium exchange activity.28 29 30 31 32 33 During ischemia and reperfusion, an inward sodium flux occurs, which has been shown to linearly correlate with subsequent intracellular calcium uptake and increases in intracellular calcium concentration caused by sodium-calcium exchange. Increased intracellular calcium appears to play an important role in myocardial stunning and reperfusion injury with reduced mechanical function and may contribute to the reductions in myocyte contractile function observed in the present study.30 34 35 36 37 38 Moreover, ongoing metabolic processes such as energy-requiring ionic pumps continue to operate during a depolarized membrane state that can deplete energy supplies.39 40 Prolonged metabolic inhibition and ischemia during cardioplegic arrest further increases cytosolic levels of free calcium and thereby may contribute to the deleterious effects of increased intracellular calcium observed during cardioplegic arrest.37 38 39 41 42
PCO supplementation of cardioplegia reduced intracellular calcium accumulation during cardioplegic arrest and preserved myocyte contractile function after reperfusion and rewarming. The mechanism by which PCO supplementation of hyperkalemic cardioplegia attenuated calcium levels may include modulation of membrane potentials and energy-dependent processes. The membrane depolarizing effects of extracellular hyperkalemia are ameliorated by PCO, which may reduce the voltage-dependent accumulation of intracellular calcium by maintaining membrane potential above the gating level and thereby decreasing calcium influx. For example, a PCO can shift the resting membrane potential in muscle cells 15 mmol/L to the negative in the presence of 16 mmol/L extracellular potassium.43 The reduction in intracellular calcium accumulation by a PCO during hyperkalemic cardioplegic arrest may also involve an inhibitory effect on sodium channels with a reduction in sodium flux. In a recent study, PCOs were shown to inhibit inward sodium current in atrial cardiomyocytes, which was independent of the effects on potassium conductance.44 Such reductions in sodium channel activity by a PCO may reduce intracellular sodium accumulation and subsequent sodium-calcium exchange activity, resulting in a decrease in intracellular calcium levels during arrest and reperfusion. The reduction in intracellular calcium by a PCO during hyperkalemic cardioplegic arrest agrees with recent reports that demonstrate that the intracellular calcium elevation and intracellular calcium wave propagation in ventricular myocytes induced by moderate extracellular hyperkalemia (16 mmol/L K+) could be prevented by a PCO through a glyburide-sensitive mechanism.45 46 The prevention of intracellular calcium accumulation during cardioplegic arrest observed in the present study may play a central role in the protective effect on myocyte contractile function. Support for this hypothesis is provided in part by the proposed participation of increased intracellular calcium in myocardial stunning and dysfunction with reperfusion.30 34 35 36
It has been demonstrated that PCOs protect the myocardium from the deleterious effects of ischemia and reperfusion injury.10 15 16 17 20 21 The protection provided by ischemic preconditioning appears to involve activation of the KATP channel.12 13 Furthermore, KATP channel activation before ischemia or hypothermic, hyperkalemic cardioplegic arrest improves functional recovery of papillary muscle or isolated heart preparations.14 18 19 Thus one possible mechanism for the beneficial effects of PCO involves KATP channel activation during the transition from normothermia to hypothermic arrest as well as during the rewarming period. Finally, the protective effects of PCO compounds such as aprikalim on myocyte contractile processes during cardioplegic arrest may include preservation of myocardial energy reserves. At hyperpolarized membrane potentials, transmembrane ion gradients are minimized, which allows for reduced myocyte metabolic demand.40 47 This maintenance of energy substrate by PCOs was recently confirmed in ischemic myocardium in which ATP was preserved by pretreatment with cromakalim.48 In the present study, the velocity of relengthening and time to 50% relaxation, which reflects active relaxation processes, were significantly impaired after hypothermic, hyperkalemic cardioplegic arrest. An increase in intracellular calcium concentration in diastole after reperfusion has been reported to be associated with impaired active relaxation processes and decreased ATP levels.49 50 The preservation of ATP levels during arrest by PCO may play a role in improved diastolic function, since active relaxation processes are energy dependent.48 Future studies examining PCO pretreatment and specific PCO-induced ionic fluctuations and associated changes in ATP levels would be valuable in further understanding the precise biochemical and cellular mechanisms involved in the improved myocardial protection observed with PCO compounds during cardioplegic arrest. In the present study, PCO supplementation of cardioplegia normalized myocyte active relaxation processes to normothermic control values, which may be related to improved calcium homeostasis. This agrees with studies in intact heart preparations in which improved LV diastolic function was reported with PCO-induced cardioplegic arrest.10 20
ß-Adrenergic responsiveness was significantly impaired in myocytes after hyperkalemic cardioplegic arrest. ß-Receptor uncoupling with dampened adenyl cyclase activity and downregulation of ß-receptors has been shown to occur after hyperkalemic cardioplegic arrest and may contribute to the decreased ß-adrenergic responsiveness observed.51 Since the period after hyperkalemic cardioplegic arrest and cardiopulmonary bypass is associated with decreased ventricular performance, such reduced ß-adrenergic responsiveness observed in the present study may be clinically relevant, particularly because adrenergic agonists are frequently used to facilitate a difficult wean from cardiopulmonary bypass. PCO-supplemented cardioplegia preserved ß-adrenergic responsiveness after cardioplegic arrest, which may reflect prevention of changes in ionic homeostasis and associated deleterious cellular effects downstream from the ß-receptor.30 37 52 53 Thus the preservation of myocyte contractile function and ß-adrenergic responsiveness with PCO-supplemented cardioplegia observed in the present study may have important clinical applications. A significant decrease in LV performance occurs after hyperkalemic cardioplegic arrest and cardiopulmonary bypass.2 Moreover, a greater proportion of patients are presenting for cardiac surgery with preexisting LV dysfunction who are at an even greater risk for the development of LV pump dysfunction in the immediate postoperative setting.54 However, there are limitations with respect to clinical application of PCO compounds. First, the onset of electromechanical arrest with PCO agents is significantly delayed relative to hyperkalemic arrest. The inclusion of a low concentration of potassium (12 mEq/L) in the cardioplegic solution with a PCO, as described in the present study, may accelerate mechanical arrest without altering the membrane potential and ionic balance characteristic of PCO agents. Second, some PCO compounds such as aprikalim appear to have proarrhythmic sequelae consisting primarily of an increased incidence of ventricular fibrillation on reperfusion.20 Further evaluation of PCO compounds is clearly indicated to determine the safety and efficacy of PCO agents in cardioplegic arrest.
The isolated myocyte model used in the present study allows for a direct examination of contractile function in a precisely controlled milieu so that the effects of compounds such as aprikalim can be assessed. Moreover, an examination of isolated myocyte contractile properties has additional advantages including removal of neurohormonal influences, loading conditions, and alterations in coronary perfusion encountered in vivo, which could influence ventricular performance. There are, however, limitations of this isolated myocyte model. Optimal solute diffusion between the cytosol and extracellular milieu that is present in the isolated myocyte system does not exist in vivo, in which coronary artery disease and hypertrophy alter capillary diffusion distances. Moreover, continuous exposure to the hyperkalemic environment may differ from typical clinical conditions during aortic cross-clamping. However, such drawbacks also define the strengths of the isolated myocyte model, as an assessment can be made of the direct effects of cardioplegic arrest on the basic functional unit of the heart, the cardiac myocyte.
In summary, PCO-supplemented cardioplegia provided effective electromechanical arrest and prevented the reductions in myocyte contractile function and ß-adrenergic responsiveness observed after traditional hyperkalemic cardioplegic arrest. This study also demonstrated for the first time that the accumulation of intracellular calcium during traditional hyperkalemic cardioplegic arrest and reperfusion could be prevented with a PCO-supplemented cardioplegic solution. The maintenance of reduced intracellular calcium concentrations during PCO-supplemented cardioplegic arrest suggests that a mechanism for preservation of myocyte contractile processes is improved calcium homeostasis. Although further studies are needed, PCO compounds may have clinical utility in providing improved myocardial protection during cardioplegic arrest.
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Acknowledgments |
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This study was supported by a South Carolina American Heart Association Grant (B.H.D.), a National American Heart Association Grant (B.H.D.), and National Institutes of Health grant HL-45024 (F.G.S.). Dr Spinale is an Established Investigator of the American Heart Association.
Received January 9, 1997; revision received May 1, 1997; accepted May 5, 1997.
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