(Circulation. 1997;96:975-983.)
© 1997 American Heart Association, Inc.
Articles |
Role of Preischemic Glycogen Depletion in the Improvement of Postischemic Metabolic and Contractile Recovery of Ischemia-Preconditioned Rat Hearts
From the Peter Belfer Laboratory of the Cardiology Division, Department of Medicine (P.R.S., C.P.d.A., G.G., R.G.W.), and the Division of NMR Research, Department of Radiology (V.P.C.), The Johns Hopkins Hospital, Baltimore, Md.
Correspondence to Robert G. Weiss, MD, Carnegie 584, The Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287-6568. E-mail rgweiss{at}rad.jhu.edu
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Abstract |
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Background Ischemic preconditioning (IPC) attenuates acidosis during prolonged ischemia and improves contractile and metabolic parameters during subsequent reperfusion. Glycogen depletion induced by IPC is proposed as a potential mechanism.
Methods and Results We studied the influence of manipulations of preischemic glycogen levels (Pre-G, µmol glucose/g wet wt) on contractile and metabolic (via 31P–nuclear magnetic resonance) parameters during 30 minutes of ischemia and recovery in four groups of isovolumic rat hearts: First, control (Con, n=18, mean Pre-G, 21.5±0.8); second, after two 5-minute IPC periods (IPC, n=12, Pre-G, 11.3±0.7); third, a control group in which Pre-G was depleted by glucose-free, acetate perfusion (Con-LowG, n=9, Pre-G, 7.9±1.2); and fourth, an IPC group in which Pre-G was raised by glucose and lactate perfusion such that Pre-G was similar to Con (IPC-HiG, n=11, Pre-G, 20±1.4). Manipulation of Pre-G significantly altered the pH fall during 30 minutes of ischemia (Con, 5.76±.03, Con-LowG, 6.26±.07; IPC-HiG, 5.91±.02, IPC, 6.05±.09). IPC-HiG hearts had significantly worse metabolic recovery (PCr, 70±7 versus 91±3% initial; IPC-HiG versus IPC, P<.05) and contractile recovery (end-diastolic pressure, 52±5 versus 29±5 mm Hg, P<.05) than IPC hearts but better recovery than Con (%PCr, 56±6% and end-diastolic pressure, 72±6 mm Hg). An ischemic rise in intracellular magnesium occurred and was atttenuated in preconditioned hearts.
Conclusions Pre-G levels before ischemia influence but are not the sole determinants of the extent of acidosis during prolonged ischemia and of metabolic and contractile recovery during reperfusion in control and preconditioned hearts.
Key Words: ischemia • preconditioning • glycogen • acidosis • spectroscopy • magnesium
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Introduction |
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One or several brief ischemic episodes before a more sustained ischemic insult decreases myocyte ischemic injury and necrosis; this phenomenon has been termed "ischemic preconditioning."1 2 Since the original descriptions, ischemic preconditioning has also been shown to reduce myocardial dysfunction during reperfusion in several models.3 4 Despite intense investigation, the mechanisms by which ischemic preconditioning provides benefit remain incompletely defined. Several mechanisms are suggested, including adenosine receptor stimulation,3 5 activation of KATP channels,6
-adrenergic stimulation
with activation and translocation of protein kinase C
(PKC),7 8 9 and glycogen depletion10 11 as well
as others.
The glycogen hypothesis states that glycogen depletion occurs during
the preconditioning episodes and that the resultant lower glycogen
levels during prolonged ischemia contribute to the attenuation
of ischemic acidosis. This in turn results in less
ischemic injury and contributes to improved
postischemic metabolic and contractile
recovery. Attenuation of ischemic acidosis is observed in
preconditioned hearts10 11 12 13 14 15 16 and contributes to the
protection afforded by preconditioning.15 Wolfe and
colleagues10 observed significant glycogen depletion after
preconditioning episodes that was associated with attenuation of
subsequent ischemic acidosis and a marked reduction in infarct
size. When the time between the preconditioning interventions and the
sustained ischemic insult was increased in preconditioned rat
hearts, glycogen depletion occurred and correlated with the loss of
attenuation of ischemic acidosis and increased infarct size.
Although all of these observations are consistent with the
glycogen hypothesis, extending the time between ischemia
preconditioning episodes and prolonged ischemia may also have
permitted inactivation of other proposed transient triggers of
preconditioning such as adenosine receptor stimulation,
activation of ATP-dependent potassium channels, -adrenergic receptor
activation, and translocation of PKC. In a more recent study, Schaefer
and collaborators11 reduced preischemic
glycogen levels in control hearts to those of preconditioned hearts.
During ischemia this was associated with some attenuation of
acidosis but not to levels of preconditioned hearts, and during
reperfusion with improved recovery of creatine phosphate and
end-diastolic pressure but not developed pressure. Although
these studies also provide important insights, glycogen levels in
preconditioned hearts were not increased to those of controls to
determine the effects of preischemic glycogen levels in
preconditioned hearts, and substrate-free perfusion was used to deplete
glycogen in nonpreconditioned hearts, which itself
could potentially trigger preconditioning benefit.11 The
aim of the current study was to determine the consequences of
manipulating preischemic glycogen levels in control and
preconditioned rat hearts by means other than extending the time
between preconditioning interventions and prolonged ischemia on
ischemic acidosis and postischemic contractile and
metabolic recovery and thereby reevaluate the importance of
the glycogen depletion hypothesis as a contributing mechanism for
preconditioning benefit.
In addition, evidence suggests that ionic alterations induced by the preconditioning episodes and manifested during prolonged ischemia, such as attenuation of acidosis and cellular calcium loading, may mediate some of the benefits of ischemic preconditioning.14 15 Recent observations also demonstrate that at least some of the preconditioning metabolic and contractile benefits in rat hearts can be mimicked by administration of high extracellular magnesium.14 Magnesium is an important cofactor in reactions involving transfer of phosphate groups, is required in all processes of cellular ATP metabolism, and is also linked to altered H+ handling.17 18 During myocardial ischemia, Mgi increases with degradation of ATP, which binds most Mgi.19 20 21 22 Increased Mgi levels could be harmful to cardiac cells if they are compensated for by Mg2+ efflux through Na+-Mg2+ countertransport, which in turn would promote excess cellular Ca2+ gain through Na+-Ca2+ exchange.23 Since altered Mgi handling may play a role in mediating such ionic alterations in preconditioning, we also measured Mgi during ischemia with 31P-NMR in these control and preconditioned hearts with different preischemic glycogen levels.
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Methods |
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Isolated Rat Heart Model
Nonfasting, ex-breeder, male Wistar rats (body weight, 0.4 to 0.7 kg) were anesthetized with 250 to 325 mg/kg IP pentobarbital. The hearts were rapidly removed and perfused at a constant perfusion pressure of 70 mm Hg with a modified Krebs-Henseleit perfusate delivered at 37°C and containing (in mmol/L): sodium 144, calcium 1.5, potassium 5, bicarbonate 17.5, magnesium 1.2, chloride 134, and lidocaine 5 µg/mL. The perfusate was not recirculated, and pH was adjusted to 7.4 by bubbling with a gas mixture containing 95% O2 and 5% CO2. The hearts were paced at 4 Hz via KCl wick electrodes. To minimize temperature changes during ischemia and reperfusion, the hearts were superfused throughout the experiment with a solution identical to that of the perfusate except for the absence of substrate and insulin and in equilibrium with a 5% CO2 and 95% N2 gas mixture.15 The temperature of the air flowing around the tube that contained the heart was also controlled by a thermocouple accessory present within the nuclear magnetic resonance (NMR) probe. A latex balloon containing a solution of 100 mmol/L of phenylphosphonic acid was inserted into the left ventricle and was filled in increments to obtain the maximum developed pressure. The balloon line was connected to a Gould P23Db transducer for measurements of left ventricular pressure. Mean coronary flow was calculated by timed collections of the returning fluid.
Experimental Protocol
Hearts were rapidly excised, mounted on a Langendorff
apparatus, and retrogradely perfused with
oxygenated buffer that contained, depending on group
assignment: (a) 5 mmol/L acetate, (b) 5 mmol/L glucose and
0.05 units/mL of insulin, or (c) the combination of 5 mmol/L
lactate, 5 mmol/L glucose, and 0.05 units/mL of insulin (see
below). After 25 minutes of stabilization, baseline measurements were
obtained and hearts were submitted either to preconditioning
ischemia (see below) or to an equivalent 20-minute period of
constant perfusion pressure. All hearts were then subjected to 30
minutes of continuous normothermic ischemia caused
by clamping the perfusion lines, followed by 30 minutes of reperfusion
at the baseline perfusion pressure with glucose as the sole substrate.
The stimulator was turned off during the first 15 minutes of reflow to
reduce the incidence of arrhythmias. The effects of different
glycogen levels before ischemia were studied in four groups of
hearts (Fig 1
).
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Group 1: Control (Con). These eight hearts received only glucose with insulin as substrate. After baseline measures were obtained, perfusion pressure was maintained at 70 mm Hg for 20 minutes. Total coronary flow was then interrupted for 30 minutes followed by reperfusion at the baseline perfusion pressure.
Group 2: Ischemic preconditioning (IPC). These seven hearts also received only glucose with insulin as substrate, but glycogen levels were lowered by preconditioning ischemic episodes. After baseline measures, seven hearts underwent an ischemic preconditioning protocol consisting of two 5-minute periods of total global ischemia, each followed by 5 minutes of reperfusion. Subsequently, each heart underwent 30 minutes of total ischemia followed by reperfusion at the baseline perfusion pressure. This preconditioning protocol has been shown to improve contractile and metabolic recovery in the perfused rat heart.15
Group 3: Control, low glycogen (Con-LowG). To observe the effects of lower glycogen levels per se, ie, in the absence of prior preconditioning episodes, on contractile and metabolic measures during ischemia and reperfusion, nine hearts underwent the same protocol as the control group except that 5 mmol/L acetate was used as substrate, which prevented glycogen accumulation. Subsequently, the substrate was switched to 5 mmol/L glucose with insulin 5 minutes before ischemia. The following ischemia and reperfusion periods were conducted as in the control group with glucose as the sole exogenous substrate.
Group 4: Ischemic preconditioning, high glycogen (IPC-HiG). To determine the effects of increased glycogen levels in hearts that had been preconditioned before the prolonged ischemic period, nine hearts were perfused with lactate 5 mmol/L, glucose 5 mmol/L, and insulin for 65 minutes, since combined lactate/glucose infusion has been shown to increase myocardial glycogen synthesis by at least sixfold.24 This was followed by a 5-minute period with insulin and glucose as sole substrate and a subsequent preconditioning protocol identical to that described for group 2 (IPC). This was followed by 30 minutes of total ischemia and reperfusion, as described above.
Thus, all four groups were perfused with 5 mmol/L glucose and insulin for at least 5 minutes before ischemia and throughout reperfusion so that no differences in substrates existed immediately before preconditioning or during ischemia. By experimental design, two groups were expected to have high glycogen levels and the other two low glycogen levels, with one group at each glycogen level being preconditioned.
31P-NMR Spectroscopy
Hearts were positioned in a 20-mm probe of a Bruker AM 360-WB
spectrometer (8.5 T). Magnetic field homogeneity was optimized during
observation of the water proton signal using the decoupler coil.
Proton-decoupled minimally saturated 31P-NMR spectra
were obtained with a 2.1-second delay between pulses of 22-ms duration
at a flip angle of 60 degrees.15 25 26 Cycles of 64 pulses
were collected during 
2.5 minutes. Relative metabolite
quantification was obtained by the use of an automated iterative
time-domain nonlinear least-squares fitting routine as previously
described.27 Baseline spectra were acquired during glucose
perfusion before the onset of ischemia. Results are expressed
as percent of these baseline values. Intracellular pH was measured by
the chemical shift of the inorganic phosphate peak relative to the
phosphocreatine peak. The chemical shift values in parts per million
were converted to pH units as previously reported.28
The calculation of [Mgi] as described by Gupta and
Moore29 uses the chemical shift separation between the
- and ß-phosphate resonances of ATP in the 31P-NMR
spectrum (
-ß).
-ß is used to calculate the
fraction of free ATP (
) by the previously reported equation:
 | (1) |
(-ß)
MgATP represents the
(-ß)
in excess of Mg2+ and
(-ß)ATP
in the absence of magnesium, [ATP]free is the sum of
unchelated ATP species, and
[ATP]total=ATPfree+ MgATP. The values of
(-ß)ATP
and
(-ß)MGATP
used were those reported by Mosher et al,30 which also
take pH effects into account. To calculate the free intracellular
magnesium from the calculated fraction of free ATP (), we used the
equation
 | (2) |
 | (3) |
Glycogen Assays
To assess glycogen levels before ischemia, 50 additional
hearts were studied in parallel, identical experiments with the
exception that immediately before the sustained ischemic
period, the hearts were perfused with substrate-free perfusate
for 30 seconds to clear the coronary circulation of glucose and
frozen by rapid compression between Wollenberg tongs cooled to the
temperature of liquid nitrogen. Twenty-two other hearts were frozen at
the end of the 30 minutes of total ischemia. Glycogen levels
were determined in the frozen samples using the extraction method of
Walaas and Walaas.32 Glucose was measured
spectrophotometrically using a modified glucose- 6-phosphate
dehydrogenase and hexokinase assay in which glucose was omitted and
replaced by 1 mmol/L ATP.33
Statistics
Results of continuous variables are reported as mean±SEM.
Unless specified, each parameter was evaluated initially by
one-way ANOVA for differences among the groups. When the resulting
F values indicated that significant differences were
present among the groups, the analysis proceeded by the use
of Bonferroni's multiple comparisons procedure at an appropriate level
of significance. Two-tailed values of P<.05 were
considered significant.
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Results |
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Baseline Contractile and Metabolic Parameters
The baseline measures of developed pressure, end-diastolic pressure, and coronary flow were similar and did not differ among the four groups of hearts. The mean values for all hearts were 151±4 mm Hg, 14±1 mm Hg, and 19.3±0.4 mL/min, respectively. A representative 31P-NMR spectrum is shown in Fig 2
and demonstrates the
spectral quality routinely obtained. The mean baseline
PCr/ATP ratios and intracellular [Mg+2] also did not
differ among the groups and averaged 1.78±.07 and 0.54±.02
mmol/L, respectively. Mean intracellular pH was somewhat higher in
Con-LowG hearts (7.17±0.01) than in those randomized to Con
(7.14±0.01), but this small difference was not considered
physiologically important. Mean intracellular
pH was 7.15±.01 for all hearts studied. All of these contractile and
metabolic parameters are consistent
with those in prior reports of isolated perfused rat
hearts,11 15 19 34 supporting the viability of the
preparations routinely obtained.
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Preconditioning Episodes
Preconditioning ischemic episodes resulted in a modest but
statistically significant depression in contractile recovery at 5
minutes of reflow in both preconditioned groups (IPC and IPC-HiG),
which was more prominent in IPC-HiG hearts. After the first
preconditioning episode (see Fig 3), developed pressure (DP) recovered
to 111±7 mm Hg in IPC but to only 87±5 in IPC-HiG hearts
(P<.01). After the second preconditioning
episode, DP recovered to 119±5 mm Hg and to 91±5 mm Hg in
IPC and IPC-HiG, respectively (P<.0025).
End-diastolic pressures (EDP) were modestly higher in
IPC-HiG hearts during the second reflow period (18±2 mm Hg) than
in IPC hearts (11±2 mm Hg, P<.035). Coronary
flow was lower in IPC-HiG hearts before prolonged ischemia than
in preconditioned hearts without enhanced glycogen levels (14.8±1
versus 19.6±1.1 mL/min, P<.005). Ischemic
preconditioning transiently decreased PCr levels (see Fig 4), and there
was a modest but statistically significant difference in PCr between
the IPC (24±2% of baseline) and IPC-HiG (16±2%, P=.027)
groups only during the first ischemic episode.
ATP levels did not change significantly during preconditioning
interventions, and there were no differences among the groups
immediately before prolonged ischemia. Intracellular pH fell
transiently during both ischemic episodes (Fig 5) in hearts
submitted to preconditioning, but there were no differences between
these two groups. Mgi increased during both
ischemic preconditioning episodes with return to baseline
levels during reflow (Fig 6). Just before prolonged
ischemia, mean Mgi was higher in IPC hearts
(0.58±0.05 mmol/L) than in the IPC-HiG hearts
(0.46±0.03 mmol/L, P=.032).
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Preischemic and End-Ischemic Glycogen
Levels
In parallel experiments, glycogen levels were measured in five
hearts immediately after they were mounted on the perfusion
apparatus and in four groups of hearts that were submitted
to the four protocols described above at a time just before that when
prolonged ischemia would start. The mean initial glycogen
concentration was 12.7±0.6 µmol glucose/g wet wt (n=5). In the
group submitted to the control protocol, it was 21.5±0.8 µmol
glucose/g wet wt (n=18) just before the time when prolonged
ischemia would start, indicating that glycogen accumulated
during 55 minutes of normal perfusion conditions. This was
significantly higher than the glycogen level in hearts submitted to the
IPC protocol (11.3±0.7 µmol glucose/g wet wt, n=12,
P<.01 versus Con). The Con-Low G protocol decreased mean
glycogen in nonpreconditioned hearts to a level of
7.9±1.2 µmol glucose/g wet wt (n=9), and the IPC-HiG protocol
raised mean glycogen in preconditioned hearts to 20±1.4 µmol
glucose/g wet wt (n=11), a level that was similar to that of the
nonpreconditioned hearts. These data indicate that
glycogen levels before ischemia in preconditioned hearts were
matched to those of control hearts without increasing the interval
between preconditioning episodes and prolonged ischemia and
without subjecting the hearts to a substrate-free period.
Glycogen was also measured at the end of 30 minutes of ischemia in other hearts to assess mean ischemic glycogen degradation. Glycogen was 10.6±0.7 µmol glucose/g wet wt (n=5) at the end of ischemia in Con hearts, for a mean loss of (21.5-10.6=) 10.9 µmol glucose per gram wet wt during ischemia in that group. At the end of ischemia in the other groups glycogen was 3.4±0.8 µmol/g wet wt in IPC (n=5), 2.4±1.2 µmol/g wet wt in Con-LowG (n=6), and 8.1±1.6 µmol/g wet wt in IPC-HiG (n=6), suggesting mean glycogen ischemic degradation of 7.9 µmol/g wet wt in IPC, 5.5 µmol/g wet wt in Con-LowG, and 11.9 µmol/g wet wt in IPC-HiG hearts.
Metabolic and Ionic Consequences for Prolonged
Ischemia
During the 30 minutes of no-flow ischemia, developed
pressure was quickly abolished and end-diastolic pressure
eventually increased in all groups. PCr levels fell rapidly and were
nearly undetectable after 5 minutes of prolonged ischemia in
all groups. ATP levels fell more slowly than PCr during
ischemia in all groups and trended lower in the IPC and IPC-HiG
groups (Fig 4
).
During prolonged ischemia, intracellular pH (Fig 5) decreased
but differed significantly among the four groups (P<.001).
Intracellular pH decreased significantly more in control hearts than in
the other groups (P<.05). There was a significant
attenuation of the intracellular acidosis in IPC and Con-LowG hearts as
compared with all other hearts (P<.05), but the slightly
higher mean pH in Con-LowG than in IPC was not significant. IPC-HiG
hearts had an intermediate attenuation of ischemic acidosis
with a pH of 5.91±0.02 at 30 minutes of ischemia. Mean
intracellular pH during ischemia in IPC-HiG was
significantly higher than in Con hearts but lower than that of the
IPC and Con-LowG groups (P<.05). Thus,
preischemic glycogen levels affect but are not the sole
determinants of the extent of ischemic acidosis in this
model.
Mgi increased in all groups during the first 20 minutes of
prolonged ischemia (Fig 6) and differed significantly among the
groups (P<.005). After 20 minutes of ischemia, ATP
depletion did not allow reliable measurements of Mgi.
During the first 20 minutes of ischemia, Mgi
increased significantly more in Con hearts compared with the other
three groups (P<.05). IPC and IPC-HiG had the lowest
Mgi during ischemia. Con-LowG evidenced higher
Mgi than preconditioned hearts, although ATP depletion only
permitted Mgi determinations in five of nine Con-LowG
hearts at the last time point in ischemia. Therefore, the
Mgi increase during prolonged ischemia in the
hearts that underwent preconditioning (IPC and IPC-HiG) was
significantly attenuated compared with control hearts.
Metabolic and Contractile Consequences for
Postischemic Recovery
During the subsequent reperfusion period, recovery of DP differed
significantly among the four groups of hearts (P<.001). DP
recovery was similar in the hearts submitted to preconditioning
protocols (DP at 30 minutes for IPC-HiG, 63±12 mm Hg; for IPC,
86±10 mm Hg, P=NS), which was significantly higher
than that of both control groups (Con, 15±3 mm Hg and Con-LowG,
39±9 mm Hg; IPC versus Con, IPC versus Con-LowG, IPC-HiG versus
Con, IPC-HiG versus Con-LowG, all P<.05, whereas Con versus
Con-LowG, P=NS). Although there was a trend for reduced DP
recovery in IPC-HiG compared with IPC and for improved recovery in
Con-LowG (39±9 mm Hg) compared with Con, these differences were
not significant at the .05 level by multiple comparison testing over
the entire reperfusion period with the Bonferroni correction. During
reperfusion, end-diastolic pressure differed significantly
among the four groups (P<.001). EDP was highest in Con and
lowest in IPC hearts (P<.05 by repeated-measures ANOVA).
Glycogen loading significantly increased EDP in preconditioned hearts
(IPC-HiG versus IPC, P<.05) and glycogen depletion
significantly lowered EDP in control hearts (Con-LowG versus Con,
P<.05; Con-LowG versus IPC-HiG, P=NS).
Coronary flow during reperfusion was significantly higher in
IPC hearts (14.1±0.7 mL/min at 30 minutes of reflow,
P<.05) than in all other groups (Con, 10.2±0.9 mL/min;
Con-LowG, 11.5±0.7 mL/min; IPC-HiG, 9.0±0.8 mL/min).
Although there was some postischemic recovery of PCr in all hearts, the extent of PCr recovery differed significantly among the four groups (P<.001). The best recovery was observed in ischemia-preconditioned hearts (mean, 91±3% at 30 minutes of reperfusion, P<.05 versus other groups). Glycogen loading in preconditioned hearts significantly depressed PCr recovery (to 70±7% in IPC-HiG compared with IPC, P<.05) but not to the level of control hearts (56±7%, P<.05 for IPC-HiG versus Con). Low preischemic glycogen levels did not increase PCr recovery in control hearts (Con-LowG, 59±3% versus Con, P<.05). There were no significant differences in ATP recovery during reperfusion among the four groups. During reflow, intracellular pH increased in all groups and Mgi returned to baseline levels by 30 minutes, with no differences among all groups.
Attenuation of Ischemic Mgi2+
Increase
Since cardiac Mgi levels during ischemia have
not been reported before in this preconditioning setting, previously
published 31P-NMR data15 obtained in a similar
model were analyzed for Mgi to determine whether
evidence existed to confirm these observations. The results are shown
in Fig 7 and also show significant Mgi differences among
the three groups during the ischemic interval
(P<.001); all three groups were different from each other
(P<.05). These observations demonstrate that
the largest ischemic increase in Mgi occurs in
control hearts and that an attenuation of the rise in Mgi
is seen in hearts preconditioned with ischemia or with
metabolic inhibition with cyanide. Since acidosis can
affect Mgi and since hearts exhibiting the largest fall in
pH during ischemia have the greatest Mgi increase
during ischemia (Fig 7), the effects of acidosis per se on
Mgi were retrospectively evaluated in hearts exposed to
respiratory acidosis.35 Respiratory acidosis induced by
perfusion with perfusate bubbled with a 30% CO2
and 70% O2 gas mixture resulted in a modest fall in
intracellular pH (mean change in pH 0.4 units35 ) and a
modest increase in Mgi in control and preconditioned hearts
(Fig 8) of 0.1 to 0.15 mmol/L. Intracellular pH and
Mgi changes were less than those observed during total
ischemia, were each similar in control and preconditioned
groups, and suggest that acidosis per se may contribute to increases in
Mgi in this model.
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Discussion |
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In this study, the metabolic and contractile consequences of altering preischemic glycogen levels in control and ischemia-preconditioned rat hearts without increasing the time interval between preconditioning episodes and prolonged ischemia were determined. Glycogen levels before prolonged ischemia influenced the extent of ischemic acidosis in both control and preconditioned hearts but were not the sole determinants of acidosis or of postischemic functional and metabolic recovery. Glycogen-loaded preconditioned hearts exhibited less attenuation of ischemic acidosis and poorer metabolic and functional recovery compared with conventional preconditioned hearts but higher ischemic pH and better functional and metabolic recovery than did control hearts. It is concluded that preischemic glycogen levels can explain some but not all of the beneficial effects of ischemic preconditioning in this model.
Although the glycogen hypothesis of preconditioning protection has been
investigated before,10 11 the current study reexplores the
hypothesis in a novel fashion. Prior experiments in which the time
interval between the last preconditioning episode and the prolonged
ischemic interval were increased provided strong evidence in
support of the glycogen hypothesis because that intervention resulted
in preischemic glycogen repletion that was associated with
loss of the attenuation of ischemic acidosis and decreased
postischemic recovery.10 However, glycogen
repletion may not have been the only factor contributing to reduced
protection; several recently reported mediators of ischemic
preconditioning, such as adenosine receptor
stimulation,3 5 activation of KATP
channels,6 -adrenergic stimulated activation, and
translocation of protein kinase C7 8 9 are receptor mediated
and also transient events. The progressive loss of such factors, in
addition to glycogen repletion, may have contributed to the loss of
protection when the interval between the final preconditioning episode
and sustained ischemia was significantly prolonged. In the
current studies, control levels of glycogen were achieved after
preconditioning by prior glycogen loading without prolonging the
interval between preconditioning and sustained ischemia. This
independent manipulation of glycogen levels would not be expected to be
confounded by the other above-mentioned potential mediators of
preconditioning. Increased glycogen levels in preconditioned hearts
resulted in an intermediate degree of ischemic acidosis and of
functional and metabolic postischemic recovery.
The low glycogen levels in control hearts (Con-LowG) matched those
observed after ischemic preconditioning and were associated
with improved contractile recovery. This too is consistent with
the conclusion that the preischemic glycogen level plays an
important but not necessarily a unique role in ischemic
preconditioning.
Although glycogen is the ultimate source of glycolytic catabolites
(lactate, and indirectly protons from hydrolysis of glycogen-derived
ATP), which contribute to ischemic injury, it is not obvious
that glycogen always plays a detrimental role during total myocardial
ischemia because glycogen also provides the predominant source
for ATP generation. The findings that higher preischemic
glycogen stores are associated with a decrease in recovery after
ischemia (higher EDP in Con versus Con-LowG and higher EDP and
lower %PCr in IPC versus IPC-HiG) are consistent with some
prior studies of glycogen depletion in rat hearts.11 36
The current findings may superficially appear to contradict the
findings of other studies in which fasting-induced glycogen
accumulation was associated with improved postischemic
recovery and lessened loss of adenine
nucleotides.37 However, the differences
between the studies may be explained in part by the different methods
of glycogen loading. In the present study total glycogen stores
were increased >200% by perfusion with glucose and insulin in control
hearts, and this was associated with significantly more catabolite
accumulation during the subsequent ischemic episode (see Fig 5), in agreement with prior observations.38 In contrast,
fasting increased myocardial glycogen content in rats by only
25%,37 and this was not associated with relatively
more catabolite accumulation. The mechanism of the important beneficial
effects of fasting remains unknown but could not be attributed to
increased ischemic glycogenolysis.37 In addition
to differences in the magnitude of preischemic glycogen
accumulation between fasting and prolonged glucose and insulin infusion
protocols, there may be differences in the type of glycogen formed by
each protocol, which may also affect ischemic glycogenolysis
and hence catabolite accumulation. Bailey and
collaborators38 suggested but did not prove that insulin
treatment increases the accessibility of glycogen to
phosphorylase during ischemia and that this may be
explained by insulin increasing the rate of synthesis of
1-4 linkages while leaving unaffected the activity of
the branching enzyme (which forms 1-6 linkages).
Alterations in the composition of glycogen by glucose and insulin
perfusion, with more 1-4 linkages, could increase
glycogen accessibility and the degree of ischemic
acidosis.38 It may be that fasting, which is associated
with low insulin levels, results in a higher proportion of branched
1-6 linkages, which may be less accessible during
ischemia and may therefore generate fewer potentially
deleterious products such as lactate and protons from ATP
hydrolysis. Thus the amount of glycogen, the conditions under which it
was synthesized,37 the relative proportion of
1-4 and 1-6 glucosyl linkages, and the
time of day of the experiments39 all may contribute to the
extent of glycogen degradation during ischemia and to
postischemic mechanical recovery. On the basis of very
recent studies of glycogen-loaded and glycogen-depleted hearts
reperfused after short and long periods of low-flow ischemia in
the absence and presence of glucose, it has been persuasively argued
that the contradictory findings of past studies of the effects of high
glycogen in ischemic myocardium may be due to
differences in the extent of glycogen depletion during
ischemia.40 The factors and mechanisms controlling
ischemic glycogen degradation under such varied conditions,
including ischemic preconditioning and fasting, remain
incompletely understood.
Ischemic glycogen degradation was recently studied in the setting of ischemic preconditioning. Finegan et al41 observed that an ischemic preconditioning protocol lowered preischemic glycogen levels, glycolysis, and calculated proton production under aerobic conditions in working rat hearts. Ischemic preconditioning was also associated with reduced glycogen degradation during 30 minutes of ischemia and less glycolysis and proton production during subsequent reperfusion.41 The observations were considered consistent with the hypothesis that the protective effects of ischemic preconditioning are associated with depletion of glycogen and an inhibition of glycolysis and proton production during both ischemia and early reperfusion. In a more recent study using serial measures of glycolysis and glycogenolysis in the same hearts during ischemia, it was demonstrated that the reduction in glycolysis and proton accumulation in preconditioned hearts occurs throughout ischemia and not just after depletion of all glycogen stores and is due to a primary reduction in glycogenolysis.42 An attenuation of the conversion of the enzyme primarily responsible for myocardial glycogen degradation, glycogen phosphorylase, to the a or "active" form is also observed during early ischemia in preconditioned hearts and likely contributes to the primary reduction in glycogenolysis.42 Modest reductions in preischemic 13C-glycogen levels in control hearts in that study did not reduce glycogenolysis, glycolysis, or proton accumulation, unlike the more severe preischemic total glycogen depletion in control hearts (Con-LowG) in this study, which attenuated ischemic proton accumulation. Taken together, all of these studies demonstrate that ischemic preconditioning reduces preischemic glycogen levels and slows glycogenolysis and glycolysis during ischemia. Prior ischemic preconditioning per se and glycogen depletion can each affect glycogenolysis and proton accumulation during ischemia, possibly by different mechanisms, and both contribute to attenuated proton accumulation in conventional preconditioning.
Another novel observation of this study is that ischemic
preconditioning significantly attenuates the increase in intracellular
Mg2+ observed during prolonged is-chemia (Fig 6). We
also analyzed previously published 31P-NMR data
from prior preconditioning experiments in this model15 for
Mgi measurements for the first time (see Fig 7) and also
observed a significant attenuation of the ischemic rise in
Mgi in ischemia-preconditioned and
metabolic inhibition–preconditioned rat hearts compared
with controls. Those data therefore independently confirm the current
observations of an attenuation of the ischemic rise in
Mgi with preconditioning. Intracellular Mg2+,
as measured by both 31P- and 19F-NMR
spectroscopy, normally increases threefold to fourfold in rat hearts
during ischemia, from 0.5 to about 1.5 to 2.0
mmol/L,19 22 although some estimates range
higher.20 Mgi decreases with subsequent
postischemic reperfusion but remains somewhat elevated
compared with preischemic values19 20 and may
contribute to myocardial stunning by decreasing sarcoplasmic reticular
Ca2+ transport.43 The ischemic rise in
intracellular Mg2+ is typically attributed to ATP depletion
because most Mg2+ is bound to ATP and there is a temporal
correlation between the fall in ATP and the rise in free
Mg2+.19 20 21 22 44 However, since the increase in
Mgi (1 to 2 mmol/L) is much less than the decrease in
ATP (10 mmol/L), intracellular binding or sequestration of
Mg2+17 and/or extrusion of cellular
Mg2+ likely contribute to cellular Mg2+
homeostasis under ischemic conditions. The controlling
mechanisms and the significance of the attenuation of the
ischemic rise in Mg2+ with preconditioning are not
known. ATP levels are not relatively preserved in preconditioned
compared with control hearts (Fig 4), and inorganic phosphate increases
during ischemia to a similar extent in all groups in this model
(Pi as percent of baseline: Con, 1000±216; IPC, 1284±184;
Con-LowG, 1018±168; IPC-HiG, 1190±109). Therefore, the decreased
release of ATP-bound Mg or increased accumulation of inorganic
phosphate cannot account for the attenuated rise in Mg2+ in
preconditioned hearts. Altered intracellular binding of
Mg2+ to ADP or other adenine nucleotides as
well as cellular extrusion could be considered as contributing
mechanisms.
Differences in intracellular pH between preconditioned and control
hearts during ischemia may partly explain the attenuation of
the ischemic rise in Mg2+. Acidosis in isolated
myocytes modestly increases intracellular
Mg2+,17 18 and mild respiratory acidosis in
the intact rat heart (Fig 8) also increases
Mgi2+. It has been suggested that
Mg2+ and H+ share common intracellular binding
sites17 18 and therefore the primary attenuation in
ischemic acidosis in preconditioning could result in less
displacement of bound Mg2+ and a smaller increase in
Mgi. However, the data from high glycogen–preconditioned
(IPC-HiG) hearts suggest that pH is not the only factor affecting the
ischemic rise in Mg2+. In high
glycogen–preconditioned hearts, ischemic acidosis was
significantly more severe than in conventionally preconditioned hearts,
but a similar attenuation of the ischemic rise in
Mgi was observed in both groups. Another explanation for
the attenuation of the Mgi increase may be displacement of
bound Mg2+ by Ca2+.45 Measurements
of [Cai] in perfused rat hearts obtained with 5F-BAPTA
and 19F-NMR demonstrate a significant attenuation of the
rise in [Cai] during ischemia in preconditioned
compared with nonpreconditioned hearts.14
Taken together, an attenuation of the ischemic rise in
[Cai] during ischemia in preconditioned hearts
could displace less bound Mg and increase [Mgi] less.
Since both Ca2+ and Mg2+ compete for the same
binding sites, it is difficult to be certain of which ionic change is
primary. Increased Mgi levels could be harmful to cardiac
cells if they increase Mg efflux through
Na+-Mg2+ countertransport, which in turn
promotes cellular Ca2+ gain through
Na+-Ca2+ exchange.23
Unfortunately, even extreme manipulations of extracellular
[Mg2+] do not result in significant changes in cardiac
intracellular [Mg2+].18 Therefore, studies
in which Mgi is independently changed to determine the
significance of the attenuation of the ischemic rise in
Mgi in preconditioning cannot be conducted at this time.
The effect of preconditioning on Mgi was investigated
before by Bradamante et al,46 who reported no
differences in Mgi between control and
ischemia-preconditioned hearts during ischemia. Such
findings do not agree with our observations, and the reasons for this
discrepancy are not obvious although they may be due to differences in
the preconditioning protocols. The prior study did not report an
attenuation of acidosis during ischemia in preconditioned
hearts, which has been reported in other
studies10 11 12 13 14 15 35 47 and may contribute to the attenuation
of the ischemic increase in Mgi. The current
observations from two independent sets of experiments demonstrate for
the first time an attenuation of the Mgi increase during
ischemia in preconditioned hearts.
In summary, changes in glycogen levels before prolonged ischemia affected the extent of ischemic acidosis in both control and preconditioned hearts but were not the sole determinants of acidosis or postischemic functional and metabolic recovery. It is concluded that preischemic glycogen levels can explain some but not all of the beneficial effects of ischemic preconditioning in this model.
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Acknowledgments |
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This work was supported by National Institutes of Health grant HL-52315-02 and an American Heart Association Grant-in-Aid. The research was performed during the tenure of Dr Weiss as an Established Investigator of the American Heart Association and Scientist for the Samuel J. Katcef Memorial. The authors wish to thank Olive Stebbing for assistance preparing the manuscript.
Received October 28, 1996; revision received February 4, 1997; accepted February 10, 1997.
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