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1 Division of Physiology, Hadassah Schools of Dental Medicine and Medicine, The Hebrew University, and 4 Department of Cardiology, Hadassah University Hospital, Jerusalem 91120; and 2 Department of Physical Chemistry, Tel Aviv University, Tel Aviv 69978, Israel; and 3 Division of Cardiology, Johns Hopkins Hospital, Baltimore, Maryland 21218
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Based on our observations of
energy sparing in heat-acclimated (AC) rat hearts, we investigated
whether changes in preischemic glycogen level, glycolytic rate,
and plasma thyroxine level mediate cardioprotection induced in these
hearts during ischemia-reperfusion insults. Control (C)
(24°C), AC (34°C, 30 days), acclimated-euthyroid (34°C + 3 ng/ml L-thyroxine), and control hypothyroid (24°C + 0.02% 6-n-propyl-2-thiouracil) groups were studied.
Preischemic glycogen was higher in AC than in C hearts
[39.0 ± 8.5 vs. 19.2 ± 4.2 (SE) µmol glucose/g wet wt;
P < 0.0006], and the lactate produced vs. glycogen
level during total ischemia (13C-NMR spectroscopy)
was markedly slower (AC: 
0.82x, r = 0.98 vs. C: 4.7x, r = 0.9). Time to onset of
ischemic contracture was lengthened, and the fraction of
hearts experiencing ischemic contracture was lowered. Pulse
pressure recovery was improved in AC compared with C animals
before, but not after, absolute sodium iodoacetate-induced glycolysis
inhibition. Acclimated-euthyroid hearts exhibited decreased
ischemic tolerance, whereas induced hypothyroidism in C
improved cardiotolerance. Thus higher preischemic glycogen and
slowed glycolysis are associated with hypothyroidism and are likely
important mediators of the improved ischemic tolerance exhibited by AC hearts.
ischemia; hypothyroidism; [13C]glucose nuclear magnetic resonance spectroscopy; glycolytic flux; rat
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CHRONIC EXPOSURE TO MODERATE heat [heat acclimation (AC)] results in adaptations in the mechanical and metabolic performance of the rat heart, including greater pressure generation and lower oxygen consumption (18, 20, 26, 27). These observations suggest that the acclimated heart (AC) is more energetically efficient. A sustained drop in plasma thyroid hormones during AC (12, 19) was found to mediate many of these adaptations via an effect on gene expression. The genes encoding cardiac contractile elements have been extensively studied (6, 12, 13, 19, 23, 24). An important advantageous effect of AC is the development of long-standing (2 wk) (7) cardioprotection against an ischemia-reperfusion insult (AC-ischemic cross-tolerance). During global ischemia, a longer time to onset of ischemic contracture (IC), lower ventricular pressure during peak contracture, improved systolic and diastolic recovery during reperfusion, and reduced infarct size attest to reduced injury in AC hearts (26, 28, 31). The available data indicate several cardioprotective adaptations, including changes in metabolic state, ionic handling during ischemia, and the molecular stress response (26, 27, 30). Of these, the metabolic changes (e.g., Refs. 26, 27) have been more extensively studied. Experiments using phosphorus NMR spectroscopy demonstrated that the drop in ATP and intracellular pH (pHi) during total ischemia (TI) is attenuated in hearts from AC animals and suggest that AC leads to energy sparing. Concomitantly, lactic acid production in the acclimated hearts lagged behind that of the control group, perhaps implying a slower glycolytic flux (26, 27).
During global ischemia, the primary energy source is ATP produced by glycolysis (11, 33). Because endogenous glycogen stores are a key source of glucose during ischemia, larger glycogen stores may be beneficial in this setting. Previous studies suggest conflicting effects. Although some investigations indicate that large preischemic glycogen stores provide cardioprotection, others report that glycogen depletion before the ischemic episode, probably by attenuating H+ production, has a beneficial effect (e.g., Ref. 9). There are also reports demonstrating that thyroid hormone participates in the control of glycogen production and degradation in addition to glucose mobilization and glycolytic flux. For example, a hypothyroid state leads to a lower level of 6-phosphofructo-2-kinase-2 (PFK-2) (36), which has an effect on phosphofructokinase-1 (PFK-1), the rate-limiting enzyme in glycolysis via catalyzing fructose 2,6,-bisphosphate (PFK-1 substrate) synthesis. The lower plasma concentration of thyroid hormones occurring during AC may mediate the developing AC-ischemic cross-tolerance via augmentation of endogenous glycogen levels and the downregulation of glycolytic enzymes, thereby slowing glycolytic flux and decreasing proton production.
Taken together, we hypothesized that magnitude or rate changes in the glucose-ATP pathway, possibly mediated by the persistent drop in plasma thyroxine occurring on AC, provide metabolic strategies of cardioprotection in the acclimated hearts. Specifically, the purpose of the present investigation was twofold: 1) to study whether higher glycogen levels and slowed glycolysis in hearts from AC animals are at least in part responsible for cardiac ischemia-reperfusion tolerance; and 2) to determine whether AC-induced hypothyroidism contributes to the enhanced ischemia-reperfusion tolerance. Collectively, our data show that a larger endogenous glycogen pool resulting in enhanced glycolysis, combined with a slowed glycolytic rate, is associated with improved ischemic tolerance in the AC heart model. The results also indicate that decreased plasma thyroxine partially mediates the observed AC ischemia-reperfusion cross-tolerance.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Study Groups
Three-week-old male rats (Rattus norvegicus, Zabar strain, albino variation), initially weighing 80-100 g, fed on Ambar laboratory chow and provided with water ad libitum, were divided into four groups: 1) normothermic animals that received no treatment and served as controls (C); 2) animals that underwent AC; 3) euthyroid-acclimated rats (AT); and 4) nonacclimated hypothyroid rats (CP). The normothermic groups were maintained at an ambient temperature of 24 ± 1°C, ~50% relative humidity, whereas AC was induced by continuous exposure to 34 ± 1°C and a relative humidity of 30-40% for 1 mo (17). In the AT group, the euthyroid state was maintained in AC animals by administration of 3 ng/ml L-thyroxine (T4; Sigma Chemical) in the drinking water, as previously described. This treatment yielded plasma thyroxine concentrations (presented as means ± SD) of T4 = 3.8 ± 0.4 µg/dl and 3,3',5-triiodo-L-thyronine (T3) = 48 ± 7 ng/dl compared with 3.97 ± 0.25 µg/dl and 55 ± 4 ng/dl for T4 and T3, respectively, in the C rats (13). In the CP group, nonacclimated rats were made hypothyroid by administration of 0.02% 6-n-propyl-2-thiouracil in the drinking water for 1 mo. T4 and T3 dropped by 74 and 56%, respectively. One month of acclimation to heat resulted in a drop of 34 and 23% in plasma T4 and T3, respectively (13).Study Protocols
Animals from the C and AC groups were assigned to one of the following experimental series: 1) cardiac endurance on subjection to progressively graded ischemia; 2) cardiac endurance on TI and reperfusion after inhibition of glycolysis; 3) biochemical measurements of cardiac glycogen level on normoperfusion; and 4) on-line measurements of [1-13C]glucose metabolites. To test our hypothesis that low plasma thyroxine concentration is involved in 1) conferring AC-induced cardioprotection 2) via metabolic pathways, experimental series 1-3 were also conducted on the AT and CP rats. The entire experimental plan is presented in Fig. 1 and detailed below.
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All experimental protocols were approved by the Hebrew University Ethics Committee for Animal Experimentation.
Left Ventricular Mechanics
The animals were killed by cervical dislocation. Hearts were rapidly removed and placed in a Krebs-Henseleit bicarbonate buffer (KHB) at 4°C. The hearts were then mounted on a Langendorff perfusion apparatus and retrogradely perfused via the aorta at a perfusion pressure of 100 cmH2O with the KHB (containing in mM: 118 NaCl, 24 NaHCO3, 1.2 KH2PO4, 1.2 MgCl2, 2.5 CaCl2, 4.2 KCl, and 5.5 glucose) at pH 7.4, maintained at 37°C, and bubbled with 95% O2-5% CO2 (20, 26, 27). Once perfusion was started, an atrioventricular block was induced by electrical coagulation of the membranous interventricular septum with a fine-tipped soldering iron. A deflated latex balloon (Hugo Sacks Electronics, no. 3 or 4) attached to a Statham P23db pressure transducer (with PE-190 polyethylene tubing) was inserted into the left ventricle via a left atrial incision. The balloon was inflated gradually with saline until maximal systolic pressure at 0-mmHg diastolic pressure was recorded. Hence the left ventricular preload was similar for all hearts (26, 27). The hearts were paced at 300 beats/min via stainless steel electrodes by using a Grass S-88 stimulator. Left ventricular pressure was recorded by using a computerized data-acquisition system (CODAS, DATAQ). All experiments were started when a steady state was reached, usually 10-16 min after the onset of perfusion.Ischemic Tolerance
To study cardiac endurance during ischemia, two different protocols were conducted. In the first (progressive graded ischemia-reperfusion series), time to onset and peak IC pressure (maximal pressure attained on IC), diastolic and systolic pressure recovery on reperfusion, and the number of cases of IC (%) in each experimental group were measured and used for ischemic tolerance evaluation. In this experimental protocol (Fig. 1, protocol 1), perfusion pressure was decreased stepwise, to 50 and 25% of the initial perfusion pressure, with 20-min intervals between each step. The perfusion pressure was then immediately raised to its preischemic level for 20 min of recovery. Coronary flow (CF) under the conditions applied was measured with a graduated cylinder (for further details, see Refs. 26, 27). In the second series (TI-reperfusion, Fig. 1, protocol 2), time to onset, peak IC, and diastolic and systolic pressure recovery were measured during TI (30 min) and reperfusion (30 min) in the presence and absence of glycolytic inhibition. Hearts were mounted on the Langendorff apparatus as described above. For glycolysis inhibition, the hearts were perfused with KHB containing sodium-iodoacetate (IAA; 100 µM), a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor, and 5 mM pyruvic acid. The hearts underwent TI-reperfusion after 15 or 30 min of glycolysis inhibition (Fig. 1).Glycogen Measurements
Glycogen (Fig. 1, protocol 3) was extracted and hydrolyzed enzymatically by using amyloglucosidase (Sigma Chemical) according to Lo et al. (28). Glycogen levels were then measured and expressed as total hydrolizable glucose by using a no. 410 DA glucose colorimetric enzymatic detection kit (Sigma Chemical).[1-13C]Glucose Metabolism
13C-NMR study of [1-13C]glucose metabolism on normoperfusion followed by TI enables the determination of the influence of AC on glycogen synthesis and its subsequent degradation and lactate production during TI. A Langendorff perfusion apparatus adapted for a pair of hearts to increase signal-to-noise ratio was used. The pair of hearts was mounted on a bifurcated cannula, and a polyvinyl balloon was inserted into each heart, allowing the follow up of normal mechanical performance. The hearts were paced at 300 beats/min by agar-KCl polyethylene electrodes attached to a Grass S-88 stimulator. After perfusion was started (Fig. 1, protocol 4), the hearts were placed in a stoppered NMR glass tube (20-mm diameter) with lines for perfusate inflow and for continuous evacuation of the effluent. A modified glucose-free KHB containing (in mM) 121 NaCl, 5.9 KCl, 1.2 MgSO4, 1.75 CaCl2, and 23 NaHCO3, pH 7.4, was used to deplete cardiac glycogen (38). Preliminary experiments in which glycogen was assayed biochemically as above after perfusion with the glucose-free buffer indicated that 20 min of perfusion were sufficient to deplete all measurable cardiac glycogen. Immediately after glycogen depletion, the inflow line was switched, and KHB containing 5.5 mM [1-13C]glucose and 0.1 IU/ml insulin was perfused to allow the heart to synthesize [1-13C]glycogen. Perfusion was continued until the level of [1-13C]glycogen reached a plateau (monitored by on-line 13C spectrum recording; see below). Immediately thereafter, the hearts were subjected to 30 min of TI by clamping the inflow line. 13C metabolites were recorded on-line at a sampling interval of 5 min. The dry weight of the hearts was determined on termination of the experiments. To verify glycogen visibility, a series of hearts undergoing the glycogen depletion-repletion procedure was assayed biochemically for their glycogen content as above.13C-NMR Measurements
13C-NMR measurements were made by using a 20-mm dual 13C/1H probe in a Bruker AM 360-WB NMR spectrometer, operating at a frequency of 90.55 MHz for 13C. Proton-decoupled 13C-NMR spectra were measured with a 60° pulse and a relaxation delay of 1.6 s. Each spectrum was accumulated for 5 min. An aqueous methanol solution (~3 M) was used as an external reference. After each experiment, the 13C-NMR spectrum was measured with a 10-s relaxation delay for correction of partial saturation and a nuclear Overhauser effect (2). Measurements were made at 37°C. Special care was taken to prevent overheating of the hearts inside the magnet during the period of ischemia.To quantify the absolute concentrations of the metabolites by 13C-NMR, the glucose peak in Krebs-Henseleit solution placed in two glass bulbs simulating two perfused hearts was calibrated.
Data Analysis
The obtained 13C-NMR signals of glycogen, glutamate, and lactate were analyzed with the aid of an NMR imaging curve-fitting subroutine (New Methods Research, Syracuse, NY). The peaks were simulated by Lorentzian line shapes for correction of their partial overlap and relatively low signal-to-noise ratios. A high convergence was achieved, and the simulated peaks were integrated. The integral peak intensities were then normalized to the dry heart weight; saturation factors were also accounted for. Absolute concentrations of the metabolites are expressed as micromoles per gram dry weight.Statistics
To assess significant changes, a commercial software package was used to calculate one-way and two-way ANOVA and Student's t-test. Treatments were taken as the fixed effects, and the individual hearts were assumed to be random samples from the animal heart population. For ratio tests, the nonparametric
2
test for two samples was employed. Values of P < 0.05 were considered to be statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Heart weights, the pressure generated at normoperfusion, and the
concomitant CF are presented in Table 1.
The mean left ventricular systolic pressure developed by AC hearts was
significantly greater than that of the C group. Administration of
thyroxine during the acclimation period to maintain AT rats (12,
19) eliminated the generation of the greater pressure that
characterizes the AC state (13). The CP hearts developed a
pressure similar to that of the C group. Basal CF was similar in all of
the experimental groups, except for the CP group in which a marked
elevation was recorded (Table 1).
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Cardiac Endurance During Progressive Graded Ischemia: Effects of AC
A perfusion pressure of 50 cmH2O resulted in a drop of 47-50% in the systolic pressure of the experimental hearts (not shown). None of the hearts developed IC. A further decrease in the perfusion pressure to 25% of normoperfusion induced a marked difference between the AC and the C groups (Table 2). A total of 36% of the 14 hearts in the AC group compared with 70% in the C group (n = 14) developed IC. Similarly, the time to onset of IC was markedly longer in the AC than in the C hearts. Passive ventricular pressure was used as an additional parameter of the severity of the induced ischemia. During ischemia, the ventricular pressure in the C group was twice as high as that in the AC hearts. On reperfusion, diastolic pressure was restored by 34 and 64% in the C and the AC hearts, respectively.
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TI, Glycolysis Inhibition, and Glycogen Level: Effects of AC
Figure 2 depicts the effects of TI and reperfusion on ventricular and systolic pressures in C and AC hearts in the absence of glycolysis inhibition and subsequent to each of the two induced periods of glycolysis inhibition.
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On TI, ventricular pressures at peak IC were markedly higher than those developed in hearts subjected to the progressive graded ischemia protocol. AC hearts endured ischemia far better than C hearts did. During TI, this was reflected by the delayed onset of IC (15 ± 6.5 min compared with 10 ± 4.1 min in the C hearts; P < 0.003) and by better recovery of the pulse pressure (Fig. 2A). After 15 min of IAA administration (Fig. 2B), the onset of IC in both C and AC hearts was immediate. However, peak IC in the AC group was delayed compared with that in the C hearts (18.3 ± 6.5 min in AC heart vs. 12.2 ± 4 min in C hearts; P < 0.005). Similarly, on reperfusion, pulse pressure of the AC hearts dropped by only 15.8 ± 3.6% compared with pulse pressure recovery in the absence of IAA, whereas that of the C hearts fell by 40.0 ± 7.2% (P < 0.00001). Perfusion of IAA for 30 min before TI was necessary to abolish the differences between the AC and the C groups (Fig. 2C).
The basal glycogen level in untreated hearts was significantly higher
(twofold) in the AC than in the C hearts (Fig.
3).
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[13C]Glucose Metabolism Using NMR Spectroscopy
Figure 4 shows a typical 13C-NMR spectrum obtained from two simultaneously perfused hearts from the end of [13C]glucose loading throughout the TI session. Figure 4A represents the 13C-NMR spectrum accumulated during the first 5 min of TI, starting on termination of [13C]glucose loading. The various peaks of the glucose metabolites from left to right are of labeled glycogen (peak 1),
-glucose (peak 2),
-glucose (peak 3), C2-labeled glutamate
(peak 4), C4-labeled glutamate (peak
6), C3-labeled glutamate (peak 8), lactate
(peak 9), and alanine (peak 10).
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During [13C]glucose loading under normoperfusion (Fig.
4B), the uptake of [1-13C]glucose by the AC
hearts was markedly greater than in the C hearts. As in the biochemical
analyses, the [13C]glycogen levels in the AC hearts were
higher than in the C hearts (by ~72%; 36.6 ± 3.8 vs. 21.2 ± 2.7 µM/g dry wt [13C]glucose; P < 0.005). [13C]glutamate levels were also significantly
higher (Fig. 4C). Glutamate C4 labeling appears
earlier than glutamate C2 (and glutamate C3) in
the Krebs cycle (38). Thus a difference in the ratio of
the accumulated glutamate C4 to the subsequently
accumulated glutamate C2 among experimental groups is
indicative of differences in their metabolic rates (5,
39). In this investigation, the [13C]glutamate
C4-to-glutamate C2 ratio on termination of the
[13C]glucose loading was lower in the C than in the AC
hearts (0.79 in C hearts vs. 0.98 in AC hearts), implying a lower
metabolic rate in the AC hearts. During TI, there was a marked fall in
[13C]glycogen, coincident with a pronounced increase in
[13C]lactate, the glycolysis end point. Individual
spectra recorded for pairs of C and AC hearts and the summarized data
are presented in Figs. 4B and
5, respectively. It is evident from our
data that the lactic acid produced exceeded the capacity of the
measured glycogen as a [13C]glucose source (e.g., in AC
hearts, [13C]glycogen was 34.6 ± 3.5 µM
[13C]glucose vs. 126 ± 9.8 µM
[13C]glucose for [13C]lactate). This
discrepancy raised a question regarding NMR glycogen visibility
{[13C]glycogen invisibility has been shown in other
tissues and has been extensively discussed by Kunnecke and Seelig
(25), e.g., with respect to liver glucose metabolism}.
To address this issue, a biochemical glycogen assay was performed in
nonacclimated hearts undergoing the glycogen depletion and repletion
procedure, as above. There were no significant differences between the
basal glycogen level measured in untreated C hearts and that in hearts that underwent the glycogen depletion and repletion protocol, suggesting glycogen invisibility in our experimental system. Based on
previous studies (e.g., Ref. 15), the method used in our experiments seems to be reliable for detection of temporal changes in
glycogen synthesis and degradation during ischemia.
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The ratio between the biochemically assayed and NMR-assayed glycogen
was 3.4 for both C and AC hearts, suggesting a consistent difference
between the enzymatic and the spectroscopic quantification. The dashed
lines in Fig. 5 represent calculated lines for the glycogen level
corrected according to the biochemically assayed-to-NMR-assayed glycogen ratio. The calculated peak glycogen levels at the end of the
repletion procedure matched the levels of the lactic acid produced.
Glycogen levels plotted vs. the lactic acid produced (Fig.
6) suggest that the major difference
between the two groups is quantitative and that glycolysis arrest in AC
hearts occurs when glycogen depletion is detectable, whereas in C
hearts glycolysis arrest occurred before glycogen depletion was
detected. This is reflected by the slope of the lactate accumulated vs.
the glycogen depleted (only data before glycolysis arrest were taken):
AC 0.82x, r = 0.98 vs. C
4.7x, r = 0.9.
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Cardiac Endurance During Ischemia, Glycolysis Inhibition, and Glycogen Levels in AT and CP Hearts
To gain insight into the involvement of thyroid hormones in the enhanced cardioprotection observed in the AC hearts, AT and CP hearts were subjected to progressive graded ischemia or TI, as before. In the graded ischemia experimental paradigm, similar to the C and the AC hearts, a drop in the perfusion pressure by 50% decreased systolic pressure of the AT and the CP hearts by 52 and 45% without induction of IC (not shown). Further drop to 25% of the initial pressure induced IC in 55 and 40% of the AT and CP hearts, respectively (Table 2). The time to onset of IC in the AT group was similar to that observed for the nonacclimated hearts, whereas that of the CP group was markedly longer and exceeded that seen in the AC hearts (Table 2). In the AC hearts, recovery of diastolic pressure was the most pronounced among all the experimental groups studied.Figure 7 shows the systolic and
ventricular pressures for the AT and CP hearts subjected to TI after
15-min IAA perfusion. This duration of IAA perfusion was found to be
the most suitable for detecting differences between the groups
undergoing glycolysis inhibition. Euthyroidism during acclimation
completely blunted the pulse pressure recovery, as observed in the AC
hearts (AT, Fig. 7 vs. AC, Fig. 2B; P < 0.0001). In the CP hearts as in the AC hearts, peak IC was markedly
delayed, and the recovered pulse pressure on reperfusion was somewhat
greater than that in the C hearts (P < 0.02 vs. C
hearts subjected to 15-min glycolysis inhibition). The basal glycogen
level in the CP hearts was the highest among all studied groups. The
glycogen level of AT hearts was also significantly higher than that of
the C group but did not differ significantly from that of the AC hearts
(Fig. 3).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The major issue of this investigation is the metabolic aspects of cardioprotection developed with AC. We show that, in the AC heart, enhanced cardiac protection during ischemia-reperfusion insult is associated with the 1) preischemic larger endogenous glycogen stores; and 2) greater glycolytic capacity during TI but a slower glycolytic rate than in the preacclimation state. There is causal evidence suggesting that the sustained drop in the plasma thyroxine level occurring on AC is at least partly responsible for the observed metabolic changes.
AC and Ischemic Tolerance: Cardioprotection Induced by Metabolic Changes
This investigation confirmed our previous observations (26, 27) that AC confers cardioprotection during ischemia-reperfusion insult. In the graded ischemia protocol, there were fewer cases with a delayed onset of IC, as well as better recovery of diastolic and systolic function in the AC group. During TI, the onset of IC in the AC group was also delayed, and systolic pressure recovery was somewhat better in this group. Our laboratory's previous 31P-NMR studies (27, 28) showed that conspicuous AC-induced cardioprotection is achieved via an attenuated drop in ATP and pHi during the ischemic insult. In the present study, evidence obtained from two independent experimental paradigms, glycolysis inhibition (Fig. 2) and 13C-NMR glucose metabolite detection before and during TI (Figs. 5 and 6), implied that, in the totally ischemic AC hearts, enhanced glycolytic capacity, but at a slower rate, and larger endogenous glycogen stores contribute to the attenuated drop in ATP level.Although ischemia and oxygen deprivation cause the heart to shift to anaerobic energy production, during severe ischemia, glycolysis is arrested by its own end product (protons), thus leading, together with Na+ and Ca2+ accumulation, to the onset of IC and ischemic injury. Glycogen breakdown may provide the heart with the glucose necessary for glycolysis. There are also indications that cellular homeostasis in the ischemic heart is better preserved as long as glycogen is present (e.g., Refs. 8, 9, 22, 35). It has, therefore, been questioned whether a large preischemic glycogen pool is beneficial or whether it has a "toxic" effect on the ischemic heart. The large number of studies on this subject has provided controversial results (9). Schneider and Taegtmeyer (34) and Cross et al. (8), for example, maintain that there is a positive correlation between the glycogen level and enhanced ischemic tolerance. In contrast, several other investigators [e.g., King et al. (22)] pointed to the deleterious effects of a higher proton level leading to the inhibition of glycolysis and halting ATP production during the ischemic event. It is noteworthy that, in the study by King et al., enhanced concentrations of glycogen metabolites were simulated by the high-glucose level in the perfused solution. This situation differs from that in the AC heart in which endogenous glycogen is constitutively elevated. In addition to a pure quantitative difference, glycolytic degradation of glucose-6-phosphate derived from glycogen generates 3 ATP vs. 2 ATP when exogenous glucose is degraded, we suggest that the acclimation-induced augmentation of constitutive glycogen is accompanied by parallel enzymatic changes, which may affect the rate of glycogenolysis and, in turn, the rate of proton production via glycolysis. The latter argument is supported by the response of the glycolytic system (primarily GAPDH) to IAA inhibition and by our observations of altered lactic dehydrogenase and PFK-2 transcript levels in the normoxic AC heart (7). Provided that elevated PFK-2 mRNA, as found in the AC heart, is associated with altered expression of the enzyme, a change in glycolytic flux could be envisioned.
An important factor in the control of glycolysis and its failure during ischemia is the decrease in ATP (leading to PFK-1 acceleration) and the fall in pHi (proton effects on GAPDH causing the arrest of glycolysis). In addition to our laboratory's findings of a delayed reduction of ATP and a drop in pHi (26, 27) in the totally ischemic heart, we recently conducted experiments in which pHi was measured by 31P-NMR spectroscopy during TI after IAA glycolysis inhibition. In the AC hearts, pHi was significantly higher than that in the C hearts (e.g., following TI of 20 min, pHi in AC and C hearts was 6.58 ± 0.07 vs. 6.35 ± 0.06), suggesting that, in the former, attenuated ATP hydrolysis or enhanced pHi regulation takes place, favoring the enhanced and continuing glycolysis observed in the present investigation. Weiss et al. (38) showed that slowing down the development of intracellular acidosis by attenuated glycogenolysis (because of the lower activation of glycogen phosphorylase) is one of the underlying mechanisms of ischemic preconditioning-induced cardioprotection. Both AC and ischemic preconditioning slow down the development of intracellular acidosis as part of their cardioprotective repertoire. This outcome could be achieved, however, by different mechanisms under the different physiological conditions used.
In conclusion, one important cardioprotective pathway developed on AC is mediated via the production of a larger glycogen pool and a prolonged period of attenuated glycolysis, both allowing improved ATP supplementation with attenuated development of intracellular acidosis. In turn, longer preservation of cellular integrity is maintained.
AC and Ischemic Tolerance: Does a Lower Thyroxine Level Play a Role?
Our present study provides causal evidence that the enhanced ischemic tolerance achieved on AC is associated, at least partly, with the decrease in plasma thyroxine level occurring on AC (12). This was implied by the finding that the maintenance of a euthyroid state throughout the acclimation period (AT group) diminished ischemic tolerance in the AT hearts compared with the matched AC hearts. In the progressively graded ischemia protocol, this was displayed by a higher number of IC cases with earlier onset within the AT compared with the AC group. During TI after 15 min of IAA administration, the AT hearts did not show any recovery of pulse pressure. As opposed to this, in the AC hearts, 15 min of IAA glycolysis inhibition were insufficient to completely blunt pulse pressure recovery from ischemia-reperfusion insult. Enhanced ischemic tolerance by the CP hearts, e.g., pronounced delay in the developed IC, decreased responsiveness to glycolysis inhibition, and fewer cases of IC in the progressive graded ischemia protocol compared with C hearts, provides a further link between enhanced ischemic tolerance and low thyroxine in the AC hearts.Several other investigators (1, 3, 14) also reported hypothyroidism-induced ischemic cardioprotection and demonstrated that hearts from CP rats similar to our AC hearts show ATP sparing when TI is applied (3, 14). However, in the CP state, despite the presence of some favorable effects, pathological consequences prevail. In contrast, on AC, the harmful consequences of the CP state are probably overridden by concerted beneficial adaptive traits leading to enhanced mechanical and metabolic performance, as already shown in our laboratory's previous studies (12, 13).
Interestingly, euthyroidism during AC (AT group) 1) did not
interfere with the augmentation of the endogenous glycogen pool induced
by AC and 2) did not decrease GAPDH responsiveness to IAA as
observed in the AC hearts. Whereas the resemblance in hypo- and
hyperthyroidism interventions in glycogen content is beyond the scope
of this investigation, these findings emphasize that elevated glycogen
without matched changes in the glycolytic flux is insufficient to
render metabolically induced cardioprotection. This is depicted in Fig.
8, which shows time to onset of IC vs. preischemic glycogen pool. The AC and the CP hearts, which
acquire both an elevated glycogen pool and altered glycolysis, are the most protected.
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What step(s) then, in the "glucose pool-glycolysis" pathway might
be affected by the AC-sustained low-thyroxine level? Based on the
present investigation, additional data accumulated in our laboratory,
and cited studies on CP hearts, the already known common targets for AC
and hypothyroidism seem to be distributed among several steps, as shown
in Fig. 9.
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The endogenous glucose pool (glycogen) is augmented in both AC and CP hearts. AC hearts also have the ability to increase glucose uptake, as indicated in Fig. 5. This finding agrees with the observed increases in GLUT-4 level in AC hearts (O. Teper, S. Sasson, and M. Horowitz, unpublished observations) and with Nagasawa et al. (32), documenting an increased whole body sensitivity to insulin in AC rats. Adult hearts have the machinery to regulate GLUT-4-mediated glucose uptake at the translational or posttranslational level (4). James et al. (21) and Weinstein and Haber (37) reported on the occurrence of similar processes in the CP heart. Hence we can hypothesize that similar adaptations, possibly mediated by a sustained low-thyroxine level, upregulate glucose uptake and, in turn, the glycogen pool in the AC heart.
Rate-limiting enzymes. Gualberto et al. (16) reported that hypothyroidism is associated with a dramatic loss in the activity of PFK-1, the rate-limiting glycolytic enzyme, as well as PFK-2 activity and fructose 2,6-bisphosphate level. This effect was partly reversed by the addition of triiodothyronine, suggesting that the drop in thyroxine could account for the decrease in carbohydrate metabolism in the hearts of the CP rats. Cohen et al. (7) showed that PFK-2 mRNA provides a target for the adaptive influence. This is partially blunted by persistent administration of thyroxine during the acclimation. This finding may place PFK-2 as a target for the hypothyroidism-mediated acclimatory response, leading to an attenuated glycolytic rate, as observed in the present investigation.
Lower responsiveness of GAPDH. The present investigation also suggests that both AC and CP affect the responsiveness of GAPDH to IAA inhibition. This may be inferred from the same recovery of the pulse pressure in the reperfused, 15-min IAA glycolysis-inhibited AC and CP hearts compared with that of the AT hearts and the completely abolished reperfusion recovery in the AC hearts after 30-min IAA glycolysis inhibition. These results may imply that differences in the level or affinity of GAPDH limit the glycolytic flux during no-flow ischemia via their inhibition by proton accumulation (9).
We can thus summarize that a larger preischemic glycogen pool in conjunction with quantitatively increased glycolysis, but at a slower rate, are likely important mediators of the improved ischemic tolerance exhibited by AC hearts. Sustained low-thyroxine level is probably associated with some of these metabolic features. In the setup of AC, the metabolic adaptations provide one cardioprotective strategy only. Other pathways, such as enhanced 72-kDa heat shock protein or antioxidant cytoprotection, also play an important role. Our laboratory's finding that 72-kDa heat shock protein content in the hypothyroid state (which shows cardioprotection in the presence of ischemia) is very low (29) may suggest that the two pathways, the metabolic and the cytoprotective, are apparently independent.| |
ACKNOWLEDGEMENTS |
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This study was supported by United States-Israel Binational Fund Grant 9100158/1-3.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Horowitz, Dept. of Physiology, Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel (E-mail: horowitz{at}cc.huji.ac.il).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 9, 2002;10.1152/japplphysiol.00304.2002
Received 8 April 2002; accepted in final form 6 August 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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, Systolic pressure; 
,
diastolic pressure. Significant difference in the recovery of the pulse
pressure between the CP and the AT, * P < 0.01. Significant difference in the time-to-peak contracture pressure between
the CP and the AT groups, +++ P < 0.0001. No significant difference was observed between AT and C after
30-min IAA administration or between CP and AC after 15-min IAA
administration (see Fig. 
, Increase; 
, decrease; 
,
attenuated effect.