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Cardiovascular Research 1997 35(1):90-98; doi:10.1016/S0008-6363(97)00087-4
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Glycogen utilization and ischemic injury in the isolated rat heart

Saul Schaefer* and Ravichandran Ramasamy

Division of Cardiovascular Medicine, TB 172, University of California, Davis, CA 95616, USA

* Corresponding author. Tel.: +1 (916) 752-0717; fax: +1 (916) 752-3264.

Received 20 August 1996; accepted 18 March 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Introduction: Fasting increases myocardial glycogen content and has been shown to limit injury and improve recovery following no-flow ischemia in the isolated heart. However, the protective role of glycogen loading per se in fed animals has been questioned by data in preconditioned animals showing that reduced glycogenolysis may be protective prior to no-flow ischemia. Therefore, we hypothesized that fasting protects the globally ischemic heart by mechanisms separate from glycogen loading. Methods: Isolated hearts from rats fasted for 24 h were retrogradely perfused using glucose substrate and subjected to 20 min of global no-flow ischemia. Fed rats were identically perfused either under control conditions (glucose substrate) or with an intervention chosen to increase tissue glycogen (glucose plus insulin, [insulin]) prior to ischemia. Functional recovery and creatine kinase (CK) release were measured during reperfusion. Nuclear magnetic resonance spectroscopy was used to measure intracellular pH, phosphorylated glycolytic intermediates and high-energy phosphates, while the lactate and pyruvate contents of the hearts were measured prior to and at the end of ischemia. Results: Hearts from fasted animals had significantly increased glycogen content prior to ischemia (76.6±2 vs. 40.9±3 µmol glu/gdw in control hearts, P<0.05) as did hearts exposed to insulin (88.6±10 µmol glu/gdw), but only hearts from fasted animals had greater glycogen utilization during ischemia. Hearts from fasted animals also had lower levels of lactate relative to pyruvate (L/P) under baseline conditions and, on reperfusion, reduced CK release (fasted: 183±48 versus control: 756±56 IU/gdw, P<0.05). Conversely, insulin hearts had increased CK release (1831±190 IU/gdw, P<0.001 vs control) and worse functional and metabolic recovery on reperfusion. Compared to the insulin hearts, hearts from fasted animals had both less acidosis and less rapid depletion of ATP during ischemia, as well as lower accumulation of glycolytic intermediates. Conclusion: Fasting protects the heart from ischemic injury and is associated with a lower L/P ratio and increased glycogen utilization during ischemia. In contrast, increasing glycogen content in hearts from fed animals using insulin limits glycogen utilization, increases ischemic injury, and impairs both functional and metabolic recovery under conditions of 20 min of global no-flow ischemia.

KEYWORDS Metabolism; NMR; Glycolysis; Insulin; Fasting; Rat, heart


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Myocardial ischemia, and its accompanying loss of oxygen, makes the heart dependent on anaerobic energy production in order to supply energy (ATP) for metabolic processes. Anaerobic production of ATP is largely dependent on the availability of adequate substrate for glycolysis, with the ischemic heart accelerating glycogenolysis at the onset of ischemia in order to supply glucose-1-phosphate for glycolysis [1]. Increasing glycogen stores may thus provide additional glycolytic substrate both during ischemia as well as upon reperfusion, increase the amount of ATP produced, and, potentially, improve calcium homeostasis and reduce injury [2]. This principle has been utilized with generally positive results in animal and human studies of interventions, such as glucose–insulin–potassium [GIK], that increased glycolytic flux and/or glycogen prior to or during ischemia [3, 4].

Fasting is known to increase myocardial glycogen [5, 6]and a beneficial effect of fasting prior to global ischemia has been shown in the isolated working heart [7–9]with improved time to functional recovery and lower enzyme release. Also, these studies showed that functional recovery after ischemia was correlated with greater glycogen content at the end of reperfusion in hearts from fed, but not fasted, animals. Together, these observations have suggested that interventions that increase glycogen prior to no-flow ischemia may be helpful in ameliorating the consequences of severe ischemia by providing additional substrate for ATP production. However, other than one preliminary report [10], there are no published studies in fed animals in which endogenous myocardial glycogen was increased and a protective effect demonstrated during no-flow ischemia. In contrast, studies of low-flow ischemia or hypoxia [4, 11–13]have shown protective effects using interventions that are known to increase glycogen prior to ischemia. Notably, all these studies used conditions in which flow was maintained in the myocardium, thus enabling some washout of catabolites (such as protons and lactate) that accumulate during no-flow ischemia.

Theoretically, under conditions of no-flow ischemia, increased glycogen could be detrimental, resulting in an increase in glycolytic end-products which have been shown to diminish functional recovery of the ischemic heart after reperfusion [14]. The postulate that increased glycogen is protective during no-flow ischemia is further questioned by studies showing that glycogen depletion limits acidosis during ischemia, [15, 16]and is correlated with reduced infarct size in models of ischemic preconditioning in which glycogenolysis is reduced [16, 17]. The potential benefit of glycogen depletion would be to limit proton production and the resultant secondary increases in intracellular sodium and calcium, hence limiting ischemic injury [18]. Together, these theoretical considerations and findings with glycogen depletion suggest that increasing glycogen content could have deleterious, rather than protective, effects on myocardial tolerance to no-flow ischemia in fed animals.

A potential difference between fasted and fed animals is a lower cytosolic redox state (NADH/NAD+) in fasted animals. The redox state can profoundly influence the rate of glycolysis by inhibiting key regulatory enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [19, 20]. A lower redox state in hearts from fasted animals would result in less inhibition of GAPDH, increased glycolysis (but not necessarily glucose oxidation), increased glycogen utilization, less accumulation of glycolytic intermediates, and greater anaerobic ATP production. The cytosolic redox state is reflected in the ratio of lactate to pyruvate (L/P) assuming equilibrium of the enzyme, lactate dehydrogenase (LDH).

Therefore, the purpose of this study was to determine the functional and metabolic response to no-flow ischemia in fasted rat hearts and compare this response to hearts from fed animals under control conditions [control] and using an intervention (glucose plus insulin [insulin]) to increase glycogen content equivalent to fasted animals prior to ischemia. These experiments were designed to definitively test the hypotheses that: (a) glycogen loading provided protection during global no-flow ischemia in hearts from fasted, but not fed, animals, and (b) this protection was secondary to increased glycogen utilization in fasted animals with a lower redox state. An isolated rat heart preparation was employed, with nuclear magnetic resonance spectroscopy used to non-invasively determine intracellular phosphate compounds and pH. These measurements were coupled with measurement of creatine kinase release to determine infarct size as well as measurement of glycogen content immediately prior to, and at the end of, the ischemic period and the L/P ratio prior to ischemia.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
All experiments were performed with the approval of the University of California Davis Animal Research Committee.

2.1 Isolated heart preparation
Experiments were performed using an isovolumic isolated rat heart preparation. Non-fasted male Sprague-Dawley rats (approx 350 g) were fed with Purina Rodent Chow and water ad libitum. Fasted rats were fed identically, but the chow was removed from their cages 24 h before the experimental protocol; water ad libitum was available to the rats until the experiments. For the experimental protocol, animals were pretreated with heparin (1000 U, i.p.), followed by sodium pentobarbital (65 mg/kg i.p.). After deep anesthesia was achieved and determined by the absence of a foot reflex, the heart was rapidly excised and placed into iced saline. The arrested heart was retrogradely perfused through the aorta within 2 min. Left ventricular pressure was determined using a latex balloon placed in the left ventricle with high pressure tubing connected to a pressure transducer. Perfusion pressure was monitored using high pressure tubing off the perfusion line. Hemodynamic measurements (perfusion pressure, left ventricular end-diastolic and systolic pressure, heart rate) were recorded on a 4-channel Gold Windowgraf recorder (Gold, Inc., Valley View, OH). The perfusate was delivered using a Rainin Rabbit/Plus roller pump (Rainin Instrument Co., Emeryville, CA). The perfusate consisted of (in mM) NaCl 118, KCl 4.7, CaCl2 1.25, MgCl2 1.2, NaHCO3 25, with the standard substrate being 11 mM glucose. The perfusion apparatus was temperature-controlled, with heated baths used for the perfusate and for the water jacketing around the perfusion tubing to maintain heart temperature at 37±0.5°C under all conditions. Oxygen was directly bubbled in the perfusate container and also in the heating chambers immediately proximal to the heart using gas-permeable tubing.

2.2 Phosphorus-31 spectroscopy
All spectroscopy was performed on a GE Omega 300 vertical bore spectrometer using a dual tuned probe. After standard tuning, matching, and shimming on the water signal, phosphorus-31 spectroscopy was performed using 224 acquisitions of a 60° pulse and 1.2 s interpulse delay, with spectra processed using an exponential multiplication of 10 Hz and manual phasing. Each time point was therefore acquired over approximately 5 min. Peak intensities (areas) were determined using spectrometer software. Intracellular pH was determined from the chemical shift of the inorganic phosphate resonance using a titration curve established in this laboratory. Phosphocreatine (PCr) and adenosine triphosphate (ATP) were referenced to their control value determined in duplicate at the start of the experiment and are expressed as a fraction of baseline.

2.3 Measurement of creatine kinase
Measurement of creatine kinase (CK) was measured from timed 5 min collections of the effluent for 60 min of reperfusion following the ischemic period. This extended period of collection was employed because prior experiments showed that, in control hearts, CK release was maximal after approximately 30 min of reperfusion and was stabilized by 60 min [16]. Each 5 min collection was analyzed in duplicate using established spectrophotometric methods. [21]Total integrated CK activity over the reperfusion period was calculated for each heart and corrected for dry weight of the heart. CK release was expressed in IU/g dry weight.

2.4 Measurement of glycogen
Glycogen content prior to ischemia was measured from parallel experiments in separate hearts. A total of 32 hearts were perfused using the same apparatus as for the NMR experiments. Following each intervention, hearts in each group were rapidly freeze-clamped using custom aluminum tongs at the time when ischemia would be instituted (n = 4–6) and at the end of the 20 min ischemic period (n = 4–6). Tissue was assayed for glycogen content using a spectrophotometric method [22]. Glycogen content is expressed as µmol glucose equivalents per gram dry weight (µmol-glu/gdw).

2.5 Measurement of lactate and pyruvate
Tissue lactate and pyruvate were measured from parallel experiments in separate hearts. Five to 9 hearts in each group were perfused using the same apparatus as for the NMR experiments and rapidly freeze-clamped using custom aluminum tongs at the time when ischemia would be instituted and at the end of the 20 min ischemic period. Tissue was assayed for lactate and pyruvate content using a spectrophotometric method [23]. In addition, the effluent was collected in duplicate prior to ischemia and was immediately treated with perchloric acid and measured for lactate content [24]. Hearts were dried and weighed after the experiments and lactate release was expressed as µmol/ml/g dry weight (µmol/ml/gdw).

2.6 Statistical analysis
Statistical analysis was performed using INSTAT software (GraphPad, San Diego, CA) operating on a personal computer. Differences between the same measurements at different time points or between variables in all 3 groups were assessed using ANOVA, with subsequent Student-Newman-Keuls post-tests. Differences between 2 groups or measurements in one group at 2 times was assessed using Student's t-tests. A P-value of less than 0.05 was used to reject the null hypothesis. All data are expressed as mean±s.e.m.

2.7 Protocols
Three groups of hearts were studied with different preparation periods after stabilization in the magnet. All hearts were perfused at a flow rate of 12.5 ml/min prior to and following ischemia. Fasted rat hearts [fasted] and control fed rats [control] were perfused for 40 min with glucose (11 mM) as the only substrate. The fed hearts in which glycogen was increased were perfused for 40 min with glucose (11 mM) plus insulin (40 U/l) [insulin]. In each group, the preparation period was followed by 20 min of total global ischemia followed by 60 min of reperfusion. In order to eliminate the effects of different reperfusion substrates, all groups were reperfused under identical conditions using 11 mM glucose without insulin. For the spectroscopy experiments, 8 hearts were studied under control conditions, and 6 hearts under the other conditions.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Glycogen content
Compared to control hearts, hearts from fasted animals had significantly increased glycogen content prior to ischemia, as did insulin hearts (Fig. 1). Despite the similarities in pre-ischemic glycogen content, hearts from fasted animals had significantly lower glycogen content at the end of 20 min of ischemia compared to the insulin hearts. These changes in pre-ischemic and end-ischemic glycogen resulted in marked differences in glycogen utilization during ischemia, with hearts from fasted animals using almost 4 times as much glycogen (61.3 µmol-glu/gdw, P<0.0001, end-ischemia vs baseline) as control hearts (16.2 µmol-glu/gdw, P = 0.02). Insulin hearts did not have significant utilization of glycogen during ischemia (7.7 µmol-glu/gdw, P = n.s.).


Figure 1
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  		Fig. 1 Myocardial glycogen content immediately before ischemia (Baseline) and following 20 min of ischemia in the absence of reperfusion (End-ischemia) in the 3 experimental groups. Baseline glycogen content was higher in the fasted and insulin hearts than the control hearts (*P<0.05), while end-ischemic glycogen content was lower in the control and fasted hearts (+P<0.05). The differences resulted in maximal glycogen utilization in the hearts from fasted animals.

 
3.2 Tissue lactate and pyruvate
As shown in Table 1, hearts from fasted animals had a lower L/P ratio under baseline conditions than either the control or insulin hearts. Compared to hearts from control animals with equivalent lactate content, tissue pyruvate was higher in hearts from fasted animals. The insulin-treated hearts had higher levels of both lactate and pyruvate than the control hearts, resulting in an L/P ratio that was not different from control.


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  		Table 1 Tissue lactate and pyruvate measurements (µmol/gdw) in the 3 groups under baseline conditions (immediately before ischemia) and at the end of 20 min of ischemia (end-ischemia)

 
The values of tissue lactate were similar in all groups at the end of ischemia, although when compared only to either control or fasted hearts, lactate content was significantly higher in the insulin hearts. Although the tissue level of pyruvate was not significantly lower in the insulin hearts than the other groups (P = 0.06), the insulin hearts had a significantly greater L/P ratio. Despite a lower L/P ratio in the fasted hearts under baseline conditions, the L/P ratio at the end of ischemia was similar in the fasted and control hearts.

3.3 Lactate release
Compared to hearts from control animals, lactate release prior to ischemia was increased in the insulin hearts (insulin 4.37±0.45 vs control 2.61±0.27 µmol/min/gdw, P<0.05) but unchanged in the fasted hearts (3.79±0.76 µmol/min/gdw). These findings, coupled with the tissue lactate measurements, are consistent with the known effects of insulin increasing glycolysis under aerobic conditions [25]. In addition, the similar tissue lactate content and lactate release in the hearts from fasted compared to control animals indicates that, under baseline conditions, glycolysis was not altered by fasting.

3.4 Ischemic injury
Creatine kinase (CK) release was reduced in hearts from fasted animals when compared to control hearts (fasted 183±48 versus control 756±56 IU/gdw, P<0.05); conversely, insulin hearts had greater CK release on reperfusion (1831±190 IU/gdw, P<0.001 vs control and fasted).

3.5 Function
Heart rates were similar in the 3 experimental groups immediately prior to ischemia (fasted 267±14 beats/min, control 277±11, and insulin 287±13). Left ventricular developed pressure (LVDP) and end-diastolic pressure (EDP) were equal in all groups during baseline (pre-ischemic) conditions (Table 2 and Fig. 2). LVDP fell rapidly (within 5 min) in all hearts after initiation of ischemia. While recovery of left ventricular developed pressure on reperfusion was modest in the hearts from fasted animals, with a mean LVDP of 27±9 cm H2O after 60 min of reperfusion, there was no functional recovery of LVDP in any hearts perfused with insulin. Control hearts had intermediate recovery, with LVDP of 11±6 cmH2O after 60 min of reperfusion.


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  		Table 2 Left ventricular developed pressure (LVDP) (cmH2O±s.e.) in the 3 groups under baseline conditions immediately prior to the onset of ischemia (Baseline), and at 3 times after reperfusion (Reperfusion)

 

Figure 2
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  		Fig. 2 Left ventricular end-diastolic pressure (EDP) in cmH2O as a function of time for the 3 groups. Data points are given for every 5 min starting prior to ischemia, during ischemia (t = 0–20 min) and 3 reperfusion time points (immediately after reperfusion (t = 25 and 30) and at the end of the reperfusion period (t = 80). Insulin hearts had significantly lower end-diastolic pressures than both control and fasted hearts after 10 min of ischemia (#P<0.05). However, end-diastolic pressure was significantly lower in the hearts from fasted animals than both the control and insulin hearts after 10 min of reperfusion (t = 30 min) (P<0.05).

 
Left ventricular EDP was lower in the insulin hearts during the first 10 min of ischemia (Fig. 2), although all groups had similar EDP at the end of 20 min of ischemia. Concurrent with the greatest recovery of developed pressure on reperfusion, the hearts from fasted animals had a marked reduction in EDP on reperfusion. Given constant flow conditions during reperfusion, the reduced EDP on reperfusion therefore reflected decreased coronary vascular resistance in the hearts from fasted animals.

3.6 Spectroscopy
3.6.1 Intracellular pH
Intracellular pH was similar between groups under baseline conditions (Table 3). The fall in pH during ischemia was similar between groups, as was the pH at the end of the ischemic period. However, when compared only to the insulin hearts with equivalent glycogen content, hearts from fasted animals had significantly higher pH after 15 and 20 min of ischemia. Upon reperfusion with glucose substrate, intracellular pH recovered in all hearts to pre-ischemic levels within 5 min.


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  		Table 3 Intracellular pH in the 3 groups under baseline conditions immediately prior to the onset of ischemia (Baseline), during ischemia (Ischemia) and after 5 min of reperfusion (Reperfusion)

 
3.6.2 Phosphate compounds
The ratio of PCr/ATP, not corrected for partial saturation, was similar among groups under baseline conditions (fasted 2.03±0.08, glucose 1.79±0.08, and insulin 1.86±0.14).

Phosphocreatine (PCr) fell rapidly and equally in each group, with the level of PCr becoming absent by 15 min of ischemia. Reperfusion caused a rapid but only partial return of PCr in all groups within 5 min (Fig. 3), although the fasted and control hearts had significantly higher (P<0.05) PCr at this time point than the insulin hearts (fasted 0.62±0.07, glucose 0.55±0.05, and insulin 0.13±0.03). As the reperfusion period continued, PCr in the fasted hearts remained significantly higher than the insulin hearts. In contrast, there was a progressive reduction of PCr in the control hearts, such that PCr was greater in fasted hearts than the control hearts after 40 min of reperfusion.


Figure 3
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  		Fig. 3 Changes in phosphocreatine (PCr) during the ischemic period and reperfusion as a fraction of baseline for the 3 groups. PCr fell rapidly during ischemia in all groups. PCr recovery was greatest in the hearts from fasted animals, followed by the control perfused hearts, and was lowest in the insulin hearts. There were no significant differences between control and fasted hearts by ANOVA. Abbreviations same as in Fig. 1. *P<0.05, control and fasted greater than insulin. +P<0.05, fasted greater than insulin

 
Adenosine triphosphate (ATP) content was similar between groups at the onset of ischemia, with similar rates of depletion during the first 10 min of ischemia (Fig. 4). When compared to other groups, the insulin hearts had significantly lower levels of ATP at 10 min of ischemia (P<0.05), while the hearts from fasted animals had greater levels of ATP than the insulin hearts after 15 min of ischemia (P<0.05). Although end-ischemic ATP levels were similar, reperfusion resulted in significantly greater recovery of ATP after 5 min of reperfusion in the hearts from fasted animals compared to all other groups (P<0.05). There was no significant recovery of ATP in the insulin hearts. Thus, metabolic recovery during reperfusion was significantly greater in the fasted than the hearts perfused with insulin, manifest primarily by greater recovery of PCr. Conversely, despite the marked reduction in infarct size in the hearts from fasted animals, metabolic recovery was only marginally improved compared to control animals.


Figure 4
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  		Fig. 4 Changes in adenosine triphosphate (ATP) during the ischemic period and reperfusion as a fraction of baseline for the 3 groups. ATP was higher in the fasted and control hearts than the insulin hearts after 10 min of ischemia and after 10 min of reperfusion (*P<0.05). ATP was higher in the hearts from fasted animals than the insulin hearts after 15 min of ischemia (+P<0.05). Reperfusion resulted in an increase of ATP in the hearts from fasted animals, with significantly higher levels of ATP in the hearts from fasted animals than the control or insulin hearts after 5 min of reperfusion (t = 25) (@P<0.01). Although ATP levels slowly fell throughout reperfusion in all the groups, higher levels of ATP persisted for 50 min of reperfusion in both the fasted and control hearts, resulting in significantly higher levels of ATP in the fasted and control hearts compared to the insulin hearts after 10 min of reperfusion (*P<0.05).

 
Phosphomonoesters: The phosphomonoester (PME) resonances at the end of ischemia were defined as a fraction of the Pi resonance at the end of ischemia (PME/Pi). PME resonances in the hearts from fasted animals (0.24±0.02) were significantly lower than those in the control (0.45±0.06) or insulin hearts (0.43±0.05, P<0.05). These results are consistent with prior experiments showing that interventions that reduce ischemic injury are characterized by lower PME resonances [24].


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Protective effect of fasting
This study has confirmed the previously reported protective effect of fasting on myocardial performance, metabolism and injury following global no-flow ischemia [7–9]. As in the present study, fasting in those experiments increased cardiac glycogen content in rat hearts prior to ischemia. Following 15 min of global ischemia and 30 min of reperfusion, hearts from fasted rats had a shorter time to return of function following reperfusion and reduced enzyme markers of cellular injury, as well as higher adenine nucleotide content at the end of ischemia (primarily ATP and ADP). These prior experiments also showed that recovery in the hearts from fasted animals was related to post-ischemic glycogen levels and preservation of the glucose metabolic rate, suggesting that the increased glycogen content in these hearts was protective.

4.2 The role of glycogen in ischemia
Glycogen has been shown to be a primary substrate of the heart during ischemia [26], with increased glycogen stores prior to ischemia postulated to provide increased substrate for glycolysis during ischemia. The role of increasing glycogen prior to or during low-flow ischemia or hypoxia has been examined indirectly using interventions such as infusion of glucose and insulin to increase glycolytic substrate [3, 4, 11, 27]or reserpine to increase glycogen content [11]. While these studies have generally established an important protective role for glycolytic substrate under low-flow conditions, contrasting data showing worse functional recovery on reperfusion have also been observed in glycogen-loaded compared to glucose-perfused hearts [13].

Furthermore, while fasting has been shown to protect the ischemic heart [7–9], the evidence supporting a protective role for increased glycogen content in fed animals under no-flow conditions of global ischemia is scarce, with one published study and one abstract in the literature showing a beneficial effect [10, 28]. In concordance with the present data, glycogen breakdown during ischemia in one preliminary study [10]was significantly lower in the insulin-treated hearts than the control hearts, showing that insulin limited glycogenolysis during ischemia. In addition, inhibition of the glycogen debranching enzyme, 1,6-amyloglucosidase, limited functional recovery in both the control and lactate hearts, but not in the insulin hearts, consistent with a protective effect of glycogenolysis. Despite this evidence of a protective effect of increased glycogen in hearts from fed animals, there is a theoretical basis for the postulate that increased substrate supply provided by glycogen may injure the myocardium by worsening acidosis and increasing lactate accumulation under no-flow conditions, thus countering any beneficial effects of increased ATP production from anaerobic glycolysis [19]. Combined with data that reduced glycogenolysis is associated with protection from global ischemia [17], the precise benefit of increased glycogen content in the absence of fasting is unclear.

The present data show that, in contrast to hearts from fasted animals with increased glycogen content, increasing glycogen content in hearts from fed animals worsened the metabolic and functional response to no-flow ischemia. This impaired response to ischemia in the fed animals was associated with accumulation of phosphomonoesters (as indicated by increased PME/Pi ratios) and, compared to hearts from fasted animals, greater acidosis and more rapid ATP depletion during ischemia. Since earlier studies showed a protective effect of glycogen loading with shorter [10]or less severe [28]ischemic stress, the finding of worse recovery in the glycogen-loaded fed animals may be due to a deleterious effect only under more severe or prolonged ischemic conditions.

One explanation for the differences between hearts from fed and fasted animals is the greater glycogen utilization in the hearts from fasted animals than either of the fed animal groups, suggesting an important protective role of glycogen utilization during ischemia. The precise mechanism for the protection of increased glycogen utilization is unknown. Although glycogen may have particular benefit in providing substrate for ATPases such as the SR Ca2+ ATPase and maintaining calcium homeostasis during ischemia [29, 30], the benefit of increased glycogenolysis may simply be due to the greater amount of ATP produced from glycogen than from glucose [31]

4.3 Regulation of glycogenolysis
Potential regulatory mechanisms of glycogenolysis include the cascade from receptor stimulation to activation of cAMP and cyclic-AMP-dependent phosphorylase kinase, calcium stimulation of phosphorylase kinase b, as well as inhibition of protein phosphatase-1 [31, 32]. In addition, the lower L/P ratio (and hence NADH/NAD+) in the hearts from fasted animals may have contributed to increased glycogen utilization, as well as the lower PME/Pi ratios, observed in these hearts. Since key glycolytic regulatory enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) require NAD+ [19, 20], a lower L/P ratio would increase flux through glycolysis. Coupled with less glucose utilization [9], this effect on GAPDH could increase glycogenolysis via a reduction in glucose-6-phosphate (G6-P) [31]. This postulate is supported by the lower levels of PME's in the hearts from fasted animals, an observation linked to both lower levels of G6-P and reduced ischemic injury [16, 24]. Conversely, the higher L/P ratio, as seen in the insulin hearts, would limit flux through glycolysis.

4.4 Effect of fasting on glycolysis
Despite different levels of glycogen, the similar tissue lactate content and lactate efflux under baseline conditions in the glucose-perfused hearts from fasted and fed animals indicate that glycolysis (but not necessarily glucose oxidation) was not altered by fasting in the absence of ischemia. The similar levels of tissue lactate at the end of ischemia (Table 1) indicate that the differences in glycogen utilization between hearts from fed and fasted animals were not reflected in differences in lactate accumulation.

In the fed hearts using insulin, lactate release under baseline conditions was greater than the control hearts, consistent with the known effects of insulin stimulating glucose uptake and glycolytic flux [33]. As in the control hearts, lower glycogen utilization during ischemia in the insulin hearts did not result in a decrease in tissue lactate accumulation during ischemia. Together, these data suggest that differences in glycogen utilization are not necessarily reflected in conventional measures of glycolysis such as lactate accumulation. While there is no apparent explanation for this discordance, other studies in both fed and fasted animals have shown similar disparities between glycogen utilization and lactate accumulation during ischemia [9, 34].

4.5 Limitations
While this study has focused on the role of glycogen loading and utilization, there are clearly other mechanisms operative during ischemia and reperfusion in these hearts. For example, the use of insulin to increase glycogen stores in the heart has numerous metabolic effects independent of the level of tissue glycogen [33]. Conversely, the effects of fasting are numerous, and the role of glycogen utilization is likely only one factor in the protective mechanism. For example, recent studies have shown that ATP consumption by the mitochondrial F1F0 ATPase can be affected by active regulation during ischemia [35, 36]. It is possible, therefore, that the relative ATP preservation in the hearts from fasted animals could be secondary to lower F1F0-ATPase activity.

These experiments used an isovolumic rat heart preparation and, hence, measures of functional recovery may not be comparable to previous studies [7–10]using an isolated working heart preparation. However, the findings of greater or lesser functional recovery in the different groups of hearts were supported by concordant measurement of creatine kinase release as an indicator of myocardial injury. Thus, the internal consistency of these data strongly supports the protective or deleterious effects of the interventions.

Glycogen, lactate and pyruvate measurements were performed in parallel experiments separate from the other measurements. Thus, it is possible that the degree of glycogen loading was different in these hearts compared to the hearts used in the metabolic and functional experiments. To limit any differences, however, the freeze-clamp hearts used for glycogen measurements were perfused in the same apparatus as used in the NMR experiments and the effect of fasting on glycogen levels was similar to that previously seen [5].

4.6 Conclusion
This study tested the hypothesis that glycogen loading in fasted, but not fed, hearts provides protection to the globally ischemic heart under no-flow conditions. In addition to 24-hour fasting, insulin was employed in hearts from fed animals to increase myocardial glycogen stores prior to global ischemia. While fasted rat hearts had less ischemic injury than control fed hearts, high glycogen fed animals treated with insulin had worse metabolic and functional recovery, and increased tissue injury. Hearts from fasted animals had a lower cytosolic L/P ratio, greater glycogen utilization during ischemia, and less accumulation of glycolytic intermediates. These data show that, under conditions of 20 min of global no-flow ischemia, interventions such as fasting that increase glycogen content with a concurrent increase in glycogen utilization are beneficial to the heart. Conversely, increasing glycogen content while limiting glycogen utilization, as in the fed animals treated with insulin, is detrimental to the ischemic heart.

Time for primary review 42 days.


   Acknowledgements
 
Supported by grants to Dr. Schaefer from the National Institutes of Health (HL 02131) and the California Affiliate of the American Heart Association (93-231) and to Dr. Ramasamy from the American Diabetes Association.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Bailey I.A., Radda G.K., Seymour A.M., Williams S.R. The effects of insulin on myocardial metabolism and acidosis in normoxia and ischaemia. A 31P-NMR study. Biochim Biophys Acta (1982) 720(1):17–27.[Medline]
  2. Mallet R.T., Hartman D.A., Bunger R. Glucose requirements for postischemic recovery of perfused working heart. Eur J Biochem (1900) 188(2):481–493.
  3. Eberli F.R., Weinberg E.O., Grice W.N., Horowitz G.L., Apstein C.S. Protective effect of increased glycolytic substrate against systolic and diastolic dysfunction and increased coronary resistance from prolonged global under perfusion and reperfusion in isolated rabbit hearts perfused with erythrocyte suspensions. Circ Res (1991) 68:466–481.[Abstract/Free Full Text]
  4. Runnman E.M., Lamp S.T., Weiss J.N. Enhanced utilization of exogenous glucose improves cardiac function in hypoxic rabbit ventricle without increasing total glycolytic flux. J Clin Invest (1990) 86:1222–1233.[ISI][Medline]
  5. Segel L.D., Chung A., Mason D.T., Amsterdam E.A. Cardiac glycogen in Long-Evans rats: diurnal pattern and response to exercise. Am J Physiol (1975) 229:398–401.[Abstract/Free Full Text]
  6. Arnall D.A., Palmer W.K., Miller W.C., Oscai L.B. Effect of fasting on myocardial substrates in male and female rats. Am J Physiol (1988) 254(23):C560–C563.[ISI][Medline]
  7. Goodwin G.W., Taegtmeyer H. Metabolic recovery of isolated working rat heart after brief global ischemia. Am J Physiol (1994) 36:H462–H470.
  8. Schneider C.A., Taegtmeyer H. Fasting in vivo delays myocardial cell damage after brief periods of ischemia in the isolated working rat heart. Circ Res (1991) 68:1045–1050.[Abstract/Free Full Text]
  9. Doenst T., Guthrie P.H., Chemnitius J., Zech R., Taegtmeyer H. Fasting, lactate, and insulin improve ischemic tolerance in rat heart: a comparison with ischemic preconditioning. Am J Physiol (1996) 270:H1607–1615.[Medline]
  10. Depre C, Hue L. Improvement of post-ischemic functional recovery by increasing glycogen content in the isolated working rat heart (Abstract). J Am Coll Cardiol 1994;special issue:263A.
  11. Scheuer J., Stezoski S.W. Protective role of increased myocardial glycogen stores in cardiac anoxia in the rat. Circ Res (1970) 28:835–849.
  12. Downing S.E., Chen V. Myocardial hibernation in the ischemic neonatal heart. Circ Res (1990) 66:763–772.[Abstract/Free Full Text]
  13. Cross H.R., Opie L.H., Radda G.K., Clarke K. Is high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved. Circ Res (1996) 78:482–491.[Abstract/Free Full Text]
  14. Neely J.R., Grotyohann L.W. Role of glycolytic products in damage to ischemic myocardium. Circ Res (1984) 55:816–824.[Abstract/Free Full Text]
  15. Kupriyanov V.V., Lakomkin V.L., Steinschneider A.Y., et al. Relationships between pre-ischemic ATP and glycogen content and post-ischemia recovery of rat heart. J Mol Cell Cardiol (1988) 20:1152–1162.
  16. Schaefer S., Carr L.J., Prussel E., Ramasamy R. Effects of glycogen depletion on ischemic injury in the isolated rat heart: insights into preconditioning. Am J Physiol (1995) 268:H935–944.[ISI][Medline]
  17. Weiss R.G., de Albuquerque C., Vandegaer K., Chacko V.P., Gerstenblith G. Reduced glycogenolysis rather than glycogen depletion contributes to decreased lactate accumulation after preconditioning. Circ Res (1996) 79:435–446.[Abstract/Free Full Text]
  18. Steenbergen C., Murphy E., Watts J.A., London R.E. Correlation between cystolic free calcium, contracture, ATP, and irreversible ischemic injury in perfused rat heart. Circ Res (1990) 66:135–146.[Abstract/Free Full Text]
  19. Rovetto M.J., Lamberton W.F., Neely J.R. Mechanisms of glycolytic inhibition in ischemic rat hearts. Circ Res (1975) 37:742–751.[Abstract/Free Full Text]
  20. Lagunas R., McLean P., Greenbaum A.L. The effect of raising the NAD+ content on the pathways of carbohydrate metabolism and lipogenesis in rat liver. Eur J Biochem (1970) 15:179–190.[ISI][Medline]
  21. Rosalski S.B. An improved procedure for serum creatine phosphokinase determination. J Lab Clin Med (1967) 69:696–701.[ISI][Medline]
  22. Roe J.H., Dailey R.E. Determination of glycogen with the Anthrone Reagent. Anal Biochem (1966) 15:245–250.[CrossRef][ISI][Medline]
  23. Noll F, Bergmeyer HJ, editors. Methods of enzymatic analysis, 6th ed. Weinheim: Verlag Chemie, 1984;582pp.
  24. Jeffrey F.M.H., Storey C.J., Malloy C.R. Predicting functional recovery from ischemia in the rat myocardium. Basic Res Cardiol (1992) 87:548–558.[CrossRef][ISI][Medline]
  25. Newsholme E.A., Randle P.J. Regulation of glucose uptake by muscle. Biochem J (1964) 93:641–651.[ISI][Medline]
  26. Stanley W.C., Hall J.L., Stone C.K., Hacker T.A. Acute myocardial ischemia causes a transmural gradient in glucose extraction but not glucose uptake. Am J Physiol (1992) 262:H91–96.[ISI][Medline]
  27. Apstein C.S., Gravino F.N., Haudenschild C.C. Determinants of a protective effect of glucose and insulin on the ischemic myocardium. Circ Res (1983) 52:515–526.[Abstract]
  28. Seymour A.L., Bailey I.A., Radda G.K. A protective effect of insulin on reperfusing the ischemic rat heart shown using 31P-NMR. Biochim Biophys Acta (1983) 762:525–530.[Medline]
  29. Entman M.L., Kaniike K., Goldstein M.A., et al. Association of glycogenolysis with cardiac sarcoplasmic reticulum. J Biol Chem (1976) 251:3140–3146.[Abstract/Free Full Text]
  30. Tani M., Neely J.R. Hearts from diabetic rats are more resistant to in vitro ischemia: Possible role of altered Ca2+ metabolism. Circ Res (1988) 62:931–940.[Abstract/Free Full Text]
  31. Salway, JG. Metabolism at a glance. Oxford: Blackwell Science Ltd. 1994;.
  32. Begum N., Graham A.L., Sussman K.E., Draznin B. Role of cAMP in mediating effects of fasting on dephosphorylation of insulin receptor. Am J Physiol (1992) 262:E142–E149.[ISI][Medline]
  33. Sato K., Kashiwaya Y., Keon C.A., et al. Insulin, ketone bodies, and mitochondrial energy transduction. FASEB J (1995) 9:651–658.[Abstract]
  34. Hoekenga D.E., Brainard J.R., Hutson J.Y. Rates of glycolysis and glycogenolysis during ischemia in glucose–insulin–potassium-treated perfused hearts: A 13C, 31P nuclear magnetic resonance study. Circ Res (1988) 62:1065–1074.[Abstract/Free Full Text]
  35. Das A.M., Harris D.A. Reversible modulation of the mitochondrial ATP synthase with energy demand in cultured myocytes. FEBS Lett (1989) 256:97–100.[CrossRef][ISI][Medline]
  36. Vuorinen K., Ylitalo K., Peuhkurinen K., Raatikainen P., Ala-Rami A., Hassinen I.E. Mechanisms of ischemic preconditioning in rat myocardium. Roles of adenosine, cellular energy state, and mitochondrial F1F0 ATPase. Circulation (1995) 91:2810–2818.[Abstract/Free Full Text]

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