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Cardiovascular Research 1999 42(3):644-650; doi:10.1016/S0008-6363(98)00334-4
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Copyright © 1999, European Society of Cardiology

Regulation of glycogen utilization in ischemic hearts after 24 hours of fasting

Li Feng Wanga, Ravichandran Ramasamya and Saul Schaefera,b,*

aDivision of Cardiovascular Medicine, University of California, One Shields Place, Davis, CA 95616, USA
bDepartment of Cardiology, VA Northern California Health Care, 10535 Hospital Way, System Mather, CA 95655, USA

* Corresponding author. Tel.: +1-916-734-5191; fax: +1-916-734-8394. E-mail address: sschaefer@ucdavis.edu (S. Schaefer)

Received 4 August 1998; accepted 16 November 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Limitations
 6 Conclusions
 References
 
Introduction: Fasting protects the ischemic heart from injury and infarction. Previous studies have shown that hearts from fasted animals have greater glycogen utilization and a lower cytosolic redox state (NADH/NAD+) during global ischemia. While the mechanisms of increased glycogen utilization in fasted animals have not been elucidated, animals that hibernate or are tolerant of anoxia are known to increase the tissue content of the active form of glycogen phosphorylase, phosphorylase a. Therefore, this study was designed to (a) determine whether hearts from fasted animals have increased activity of glycogen phosphorylase during ischemia and (b) define those mechanisms responsible for this increase. Methods: Hearts isolated from either fed or fasted (24 h) rats were perfused and freeze-clamped at baseline, and after 1 and 10 min of ischemia, for measurement of phosphorylase activity, phosphorylase kinase activity, and glucose-6-phosphate concentrations. Results: Fasting increased the phosphorylase a/b ratio under both baseline and ischemic conditions. This increase was not accompanied by an increase in the activity of phosphorylase kinase, either with maximal [Ca2+] or under physiologic [Ca2+]. Glucose 6-phosphate concentrations were lower in hearts from fasted animals under baseline, but not ischemic, conditions. Conclusions: Fasting enhances glycogen utilization during ischemia by increasing the active form of glycogen phosphorylase. This increase is not due to a change in phosphorylation by phosphorylase kinase nor end-product inhibition by G-6P. While the precise mechanism of increased glycogen phosphorylase activity in fasted animals is not clear, one likely explanation may be the lower cytosolic redox state demonstrated in the myocardium of fasted animals.

KEYWORDS Glucose-6-phosphate; Ischemic heart; Fasting; Phosphorylase; NADH


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Limitations
 6 Conclusions
 References
 
Fasting has been shown to protect against myocardial ischemia. In previous studies [1–4] hearts from fasted animals had greater functional recovery and reduced enzyme markers of cellular injury on reperfusion following global ischemia. Fasting results in numerous changes in systemic and cardiac metabolic and cellular functions, including a shift of substrate availability and utilization from glucose and fatty acids to ketone bodies [5] as well as an increase in cardiac glycogen stores and a 90% reduction in myocardial glucose utilization [6]. These changes, coupled with catecholamine stimulation in response to starvation, result in glycogen synthesis under normoxic conditions and, as recently demonstrated in this laboratory, greater glycogen utilization during ischemia [4]. These changes may provide an endogenous mechanism of protection similar to that of animals that hibernate [7] or are tolerant of ischemia (such as turtles) [8].

Since the breakdown of glycogen to glucose-1-phosphate (G-1P) is primarily mediated by the active form of glycogen phosphorylase (phosphorylase a), mechanisms that increase phosphorylase a can increase glycogen utilization during ischemia and protect the heart. For example, Mehrani and Storey [8] found that freshwater turtles, which can endure complete anoxia while diving underwater, increased phosphorylase a content in the heart under anoxic conditions without an increase in either phosphorylase kinase activity or cAMP-dependent protein kinase (PKA).

Activation of glycogen phosphorylase may be considered as resulting from either (a) phosphorylation of phosphorylase b by phosphorylase kinase or (b) a direct effect on phosphorylase, either by glucose-6-phosphate (G-6P) [9] or the cytosolic redox state [10]. In the first mechanism (a), phosphorylase kinase may be activated by increased cAMP due to β-receptor stimulation, with full activity requiring the binding of calcium to calmodulin (the {delta} subunit of phosphorylase kinase) [11]. For example, Dobson and Mayer [12] found that phosphorylase a formation increased rapidly (~20 s) secondary to cAMP-dependent transformation of phosphorylase kinase under either ischemic conditions or following β-adrenergic stimulation with epinephrine in the isolated working rat heart. In the second mechanism (b), activation of phosphorylase can be regulated either by end-products such as G-6P, which is an allosteric inhibitor of phosphorylase b [13] and an activator of phosphorylase phosphatase, [14] or by the cytosolic redox state (NADH/NAD+), with Stura et al. [10] providing evidence that NADH and NAD+ can bind directly to glycogen phosphorylase.

Therefore, this study aimed to test the following hypotheses: (1) fasting for 24 h increases glycogen utilization during myocardial ischemia by increasing glycogen phosphorylase a activity and (2) that in parallel to the adaptive response in turtles, this increase is not due to an enhanced phosphorylation by phosphorylase kinase, but is rather a direct effect on phosphorylase by either G-6P or the cytosolic redox state NADH/NAD+. These hypotheses were tested using a previously described isolated rat heart model of global ischemia which has shown protection of fasting from ischemia injury. Biochemical measurements of phosphorylase and phosphorylase kinase activity were made under baseline and ischemic conditions, coupled with measurement of G-6P. The data showed that, under these conditions, the hearts from fasted animals resulted in a higher activity of phosphorylase a without any changes in the activity of phosphorylase kinase.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Limitations
 6 Conclusions
 References
 
All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 8523) and under a protocol approved by the University of California, Davis Animal Use and Care Administrative Advisory Committee.

2.1 Isolated rat heart preparation
As described previously [4], hearts from fed and fasted male Sprague–Dawley rats were isolated and retrograde perfused using Krebs–Henseleit buffer consisted of (in mM) NaCl 118, KCl 4.7, CaCl2 1.25, MgCl2 1.2, NaHCO3 25, with 11 mM glucose as the carbon substrate. Left ventricular developed pressure and end-diastolic pressure were measured throughout the protocols. 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±0.3°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 Fasting protocol
Rats were fed ad libitum on standard rat chow (Purina) and water. Fed control animals were allowed to eat until the experimental time, while fasted animals were allowed only water for 24 h prior to the experimental protocol. This period of time was chosen because most of the metabolic changes occur within 24 h of fasting [5, 6] and this period of fasting was significant enough to show protection during ischemia in our experiments as well as those of Taegtmeyer and co-workers [1, 2].

2.3 Experimental protocols
Hearts were perfused under constant flow (12.5 ml/min) conditions for 15 min and then subjected to 1 and 10 min of global no-flow ischemia. Hemodynamic data (heart rate, developed pressure and end-diastolic pressure) was obtained under baseline conditions using a computer-based physiologic recording system (BIOPAC Systems, Santa Barbara, CA, USA). Hearts in each experimental group were freeze-clamped at the end of the baseline perfusion period and after 1 and 10 min of ischemia using modified Wollenberg clamps cooled in liquid nitrogen. At each time point (baseline, ischemia 1 min and ischemia 10 min), tissue was analyzed for phosphorylase activity, maximal phosphorylase kinase activity, the activity of phosphorylase kinase over a physiologic range of calcium concentrations, and G-6P concentrations. Cardiac glycogen content was measured at the end of the baseline perfusion period to verify that fasting increased glycogen content [4].

2.4 Glycogen assay
Cardiac glycogen content was measured in six hearts from each group from freeze-clamped tissue using a spectrophotometric method [4, 15]. Glycogen content is expressed as µmol glucose equivalents per gram dry weight (µmol-glu/gdw).

2.5 Phosphorylase assay
Cardiac glycogen phosphorylase activity was measured by a method modified from Gilboe et al. [16]. Briefly, about 200 mg of frozen powdered tissue was homogenized in 1 ml solution containing 20 mM NaF and 1 mM EDTA in 60% glycerol, pH 6.7, at –20°C. The homogenate was diluted (1:10) with MES buffer containing 1% of acid-wash charcoal to remove any nucleotide and then centrifuged at 12 000 g for 10 min at 0–4°C. To assay for total phosphorylase activity, 20 µl of enzyme preparation was added to 30 µl of an assay mixture (pH 6.1) containing 100 mM KF, 50 mM MES, 100 mM [14C]glucose-1-phosphate (about 0.0025 µCi/µmol), 1% rabbit liver glycogen, and 3 mM 5'-AMP. For the phosphorylase a assay, the 5'-AMP was omitted from the assay mixture and 0.75 mM caffeine was added. After incubation at 30°C for 20 min in a shaking water bath, a 40-µl aliquot was removed and spotted immediately on a 2-cm2 square of Whatman 31 ET filter paper. The filter paper was held for 2 or 3 s after spotting to ensure complete absorption of the sample, then dropped into a beaker containing 50% cold ethanol and stirred with a PTFE-covered stirring bar. This was followed by several continuous washes with 50% ethanol at room temperature. After a 5 min wash with acetone, the papers were dried and counted in a liquid scintillation counter. The activity was expressed both in µmol of the 14C-glycosyl moiety from G-1P incorporated into glycogen per minute per milligram protein as well as the phosphorylase a/b ratio (i.e. phosphorylase a activity /(total phosphorylase–phosphorylase a activity).

2.6 Phosphorylase kinase assay
The activity of cardiac phosphorylase kinase was determined by a method modified from Cooper et al. [17] and expressed as the ratio of activity at pH 6.8 to that at pH 8.2 [18]. Approximately 200 mg of frozen heart tissue was homogenized in 1 ml of extraction buffer containing 30 mM Tris (pH 7.5), 30 mM KCl, 5 mM EDTA, 100 mM NaF, and 1 mM PMSF, and centrifuged at 40 000 g for 15 min at 4°C. The supernatant was diluted (1:5) with buffer (pH 6.8) containing 10 mM β-glycerophosphate, 5 mM EDTA, 125 mM NaF and 45 mM 2-mercaptoethanol. The assay solution contained 50 mMβ-glycerophosphate, 50 mM Tris, 3.6 mM [{gamma}-32P]ATP, 12 mM MgAc2, 18 mM 2-mercaptoethanol, 6 mg/ml phosphorylase b and 0.2 mM CaCl2. After preincubation at 30°C for 2 min, the reaction was initiated by addition of 10 µl of sample to 50 µl of assay solution. The reaction was allowed to proceed for 10 min and was terminated by spotting 50 µl of the mixture onto 2-cm2 Whatman ET-31 filter paper, which was immediately washed with 10% TCA for 20 min at 4°C and subsequently washed three times (20 min each) with 5% TCA and once (5 min) with 95% ethanol. The filter paper was dried and counted in a scintillation counter. To determine the sensitivity of phosphorylase kinase to physiologic [Ca2+], the calcium concentration was varied using 0.5 mM EGTA to buffer Ca2+, followed by addition of CaCl2 from 0.05 to 0.7 mM to give the calculated free Ca2+ concentrations shown [17].

2.7 Glucose-6-phosphate
Glucose-6-phosphate in frozen heart tissue was measured using a standard spectrophotometric method [19].

2.8 Protein concentration
The concentration of protein in the enzyme solutions was determined by modified Lowry assay method using the Sigma protein assay kit. Bovine serum albumin was used as a standard.

2.9 Statistics
Statistical analysis was performed using INSTAT (GraphPad, San Diego, CA, USA) operating on a personal computer. Differences between groups were assessed using Student’s unpaired two-tailed t-test. In the instance of repetitive measures (e.g. phosphorylase kinase activity), ANOVA for repeated measures was used with Student–Newman–Keuls post-test if ANOVA was significant. A level of P<0.05 was used as the threshold for significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Limitations
 6 Conclusions
 References
 
3.1 Hemodynamics
There was no difference between groups in heart rate (fed: 274±16, fasted: 261±36 bpm), left ventricular developed pressure (fed:117±11, fasted: 112±10 mmHg) or end-diastolic pressure (fed: 12±2, fasted: 12±2 mmHg) under baseline conditions (n=6 in each group). In addition, there was no difference in end-diastolic pressure between groups after 10 min of ischemia (fed: 16±1, fasted: 17±1 mmHg).

3.2 Glycogen content
Consistent with previous investigations [4, 5, 20], fasting significantly increased tissue glycogen content from 57.6±4.7 to 83.2±2.0 µmol-glu/gdw (P=0.0006, n=6 in each group) under baseline conditions.

3.3 Phosphorylase activity
As shown in Fig. 1, fasting did not change phosphorylase b activity, but significantly increased phosphorylase a activity, expressed as both absolute activity (µmol/min/mg protein) and the phosphorylase a/b ratio under both baseline and ischemic conditions. The difference persisted into 10 min of ischemia, although the activity ratio fell as the period of ischemia lengthened.


Figure 1
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  		Fig. 1 Effects of fasting on phosphorylase activity were determined at baseline, after 1 and 10 min of ischemia in the two groups (n=6 in each group at each time point, data shown as mean±S.E.M.). Phosphorylase was assayed as described in Section 2. Bottom: total phosphorylase b activity, middle: phosphorylase a activity; top: phosphorylase a/b ratio. *P<0.05 vs. fed.

 
3.4 Phosphorylase kinase activity
In order to determine whether the increase in the phosphorylase a/b ratio was due to phosphorylation by phosphorylase kinase, the activation state of phosphorylase kinase was measured as the pH 6.8/8.2 activity ratio at these same baseline and ischemia time points. As seen in Fig. 2, there were no significant differences in phosphorylase kinase activation which might be attributable to covalent modification under either baseline or ischemic conditions.


Figure 2
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  		Fig. 2 Effects of fasting on phosphorylase kinase activity determined at baseline, and after 1 and 10 min of ischemia in the two groups (n=6 in each group at each time point, data shown as mean±S.E.M.). Phosphorylase kinase was assayed as described in Section 2. The activity was expressed as the phosphorylase kinase ratio at pH 6.8/8.2. There were no significant differences between fed and fasted hearts at all time points.

 
3.5 Phosphorylase kinase activity under varying [Ca2+]
Using methods described previously [17], the activity of phosphorylase kinase was measured under physiologic [Ca2+] (0.1 to 316 µM) at these same time points. As seen in Fig. 3, there was a minimal effect of fasting on phosphorylase kinase activity under baseline conditions, with a marginally higher activity of phosphorylase kinase only at a calculated [Ca2+] of 6.5 µM (P=0.08). Under ischemic conditions, there was no significant difference in phosphorylase kinase activity between hearts from fed or fasted animals at any [Ca2+]. Thus, in combination (Figs. 2 and 3Go), the data demonstrate that the increased activity of glycogen phosphorylase induced by fasting cannot be attributed to the activation of phosphorylase kinase by either covalent modification by phosphorylation or a change in the sensitivity to the allosteric regulator Ca2+.


Figure 3
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  		Fig. 3 Phosphorylase kinase sensitivity to Ca2+ at baseline, after 1 and 10 min of ischemia were determined in the two groups (at pH 6.8) in the presence of 0.5 mM of EGTA (n=4 in each group at each time point, data shown as mean±S.E.M.). CaCl2 concentrations from 0.05 to 0.7 mM were added to give the calculated free Ca2+ concentrations. There were no significant differences between fed and fasted hearts at all time points.

 
3.6 Glucose-6-phosphate (G-6P)
Since G-6P is known to inhibit phosphorylase b, lower levels of G-6P would result in higher phosphorylase a/b ratios. Hence, G-6P levels were measured in the two groups. As seen in Fig. 4, G-6P levels were lower in hearts from fasted animals under baseline conditions, but this difference was lost during ischemia.


Figure 4
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  		Fig. 4 Glucose-6-phosphate (G-6P) levels were measured at baseline, after 1 and 10 min of ischemia in the two groups (n=6 in each group at each time point, data shown as mean±S.E.M.). G-6P was higher in FASTED hearts at baseline (*P<0.05 vs. fed hearts), but no significant differences were observed between the two groups under ischemic conditions.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Limitations
 6 Conclusions
 References
 
The major finding of this study is that fasting for 24 h increased myocardial glycogen phosphorylase a activity during global ischemia, thus providing an explanation for the higher glycogen utilization previously demonstrated under these conditions [4].

4.1 Phosphorylase a activity
The measurements of phosphorylase a activity in the fed hearts are consistent with those published by Cornblath et al. [21] in the isolated perfused heart and Dobson and Mayer [12] in the in vivo non-working heart. Hearts from fasted animals had higher phosphorylase a activity than fed animals under both baseline and ischemic conditions (Fig. 1). While the higher phosphorylase a activity during ischemia is consistent with the greater glycogen utilization seen in this model [4], the higher baseline activity is unexplained since glycogen is synthesized in hearts during fasting. It should be recognized that protein phosphatases (PP1G and PP-2A) also regulate glycogen synthase and that net glycogen synthesis is a complex interplay of relative rates of synthase and phosphorylase activity. Therefore, one possible explanation for this finding is that baseline glycogen synthase activity, as well as phosphorylase activity, was higher in the hearts from fasted animals.

4.2 Regulation of phosphorylase — phosphorylase kinase
As noted earlier, activation of phosphorylase may be due to either (1) phosphorylation by phosphorylase kinase or (2) a direct effect on the phosphorylase enzyme by either G-6P or NADH and NAD+ [10]. Significant differences and increases in phosphorylase a activity and phosphorylase a/b ratio were observed in the hearts from fasted animals despite the absence of any differences between groups in phosphorylase kinase activity status or calcium dependency. Thus, there is no evidence that phosphorylation of the enzyme was a mechanism of the increase in phosphorylase a activity in fasted animals. The absence of any increase in phosphorylase kinase activity parallels observations in the turtle which is tolerant of long periods of anoxia [8].

These findings may be compared to those of Dobson and Mayer [12] who measured phosphorylase and phosphorylase kinase activity during ischemia and anoxia in both working and non-working rat hearts. In their studies, phosphorylase a activity increased rapidly (~20 s) with the onset of ischemia, but was associated with a significant increase in phosphorylase kinase activity only in working hearts. Since the increase in phosphorylase kinase activity was replicated by administration of epinephrine and abolished by β-adrenergic receptor blockade, these investigators concluded that, at least in the working rat heart, the ischemia induced increase in phosphorylase a activity was due to a cAMP-dependent transformation of phosphorylase kinase. However, in the non-working heart, as in the current experiments, a statistically significant increase in phosphorylase kinase activity was not observed with ischemia, implicating other mechanisms in the increase in phosphorylase a activity.

Consistent with these prior data, the present data showed that both baseline and ischemic activities of phosphorylase kinase were similar in both groups, suggest that preceding components of the cascade leading to phosphorylase kinase activation (e.g. increases in adenylate cyclase, cAMP, or cAMP-dependent protein kinase) were not altered either by fasting or ischemia. These data may be compared to data showing that intracellular levels of cAMP were increased in non-cardiac tissues (liver, kidney, muscle, fat, and islets of Langerhans) of animals fasted for 48 h [22].

In addition to phosphorylation by cAMP-dependent protein kinase, Newsholme et al. [11] have shown that phosphorylase kinase activity may be increased 1.5–3 times by exogenous calmodulin, and that inhibition of calmodulin-dependent enzymes (using trifluoperazine) can attenuate phosphorylase activation with either calcium or isoproterenol. This activation may occur in the absence of any changes in cAMP [23] and is a result of a change in Vmax without any changes in Km [11]. In the current study, determination of phosphorylase kinase activity under conditions of varying [Ca2+] did not demonstrate significant differences between hearts from fed and fasted animals during ischemia, indicating that it is unlikely that the interaction of calcium and calmodulin (the {delta} subunit of phosphorylase kinase) was altered by fasting.

4.3 Regulation of phosphorylase b to a conversion — direct effects of G-6P
In the absence of significant changes in phosphorylase kinase, the increase in the phosphorylase a/b ratio must be due to a direct effect on the enzyme. In this study, we examined the regulation of G-6P on phosphorylase.

G-6P may increase the conversion of phosphorylase b to a by two mechanisms. First, G-6P may directly bind to phosphorylase b, modify its conformation and inhibit its phosphorylation by phosphorylase kinase [13]. Second, G-6P may activate phosphorylase phosphatase in muscle [14], thus increasing dephosphorylation of phosphorylase a. The measurements in this study showed that, consistent with these observations, G-6P levels were lower in hearts from fasted animals under baseline conditions when the phosphorylase a/b ratio was higher. However, a regulatory role for G-6P is unlikely since the higher phosphorylase a/b ratio was observed during ischemia despite similar G-6P levels in hearts from fed and fasted animals. Alternatively, it may be that the effects of lower baseline levels of G-6P on phosphorylase persisted into ischemia despite the increase in G-6P.

4.4 Regulation of phosphorylase b to a conversion — the cytosolic redox state
The cytosolic redox state (NADH/ NAD+) may be a potential regulator of in situ phosphorylase activity. In previous experiments, the ratio of NADH/ NAD+, as measured by the lactate/pyruvate ratio, was significantly lower in hearts from fasted animals than fed animals [4]. Since the lower NADH/NAD+ ratio was largely due to an increase in pyruvate concentrations, these data suggested that the fasted animals had higher NAD+ concentrations than fed animals [4]. Kinetic studies have shown that both NADH and NAD+ can inhibit AMP activation of phosphorylase b by binding directly to phosphorylase b [10]. The calculated [NAD+] in fasted hearts, derived from the lactate/pyruvate ratio [4] and published values of [NADH] in the heart [24], is about 1.5 mM. At this concentration, NAD+ may activate phosphorylase b more than NADH over a range of physiologic concentrations of AMP (1–100 µM) [25]. Hence, the higher [NAD+] in the hearts from fasted animals could result in a higher phosphorylase a/b ratio.


   5 Limitations
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Limitations
6 Conclusions
 References
 
The primary limitation of this study is the use of the non-working isolated perfused heart to examine the mechanisms of glycogen regulation during myocardial ischemia in hearts from fasted animals. First, mechanisms regulating glycogenolysis in the intact animal may differ from those of the isolated heart since regulation of phosphorylase kinase may be different under in vivo working conditions, where β-adrenergic stimulation has been shown to result in increases in phosphorylase kinase activity [12].

Second, hearts were perfused with buffer containing only glucose as a substrate, contrasting with the in situ conditions of increased fatty acid and ketone body oxidation during fasting [26]. Since the presence of fatty acids are known to increase G-6P concentrations and limit glycogenolysis [9], the addition of fatty acids to the perfusate likely would have altered the G-6P measurements. Hence, the absence of a regulatory role for G-6P under ischemic conditions can only be concluded for this model and not extrapolated to other substrate conditions.

Finally, although the data conclusively show that fasting increased the phosphorylase a/b ratio in the absence of any change in phosphorylase kinase activity, the precise mechanisms of this increase are not proven. Specifically, the roles of protein phosphatases (PP1G and PP-2A) [14] were not examined, and it is possible that cAMP mediated increases in protein phosphatase inhibitor-1 could decrease the dephosphorylation of active phosphorylase a in hearts from fasted animals.


   6 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Limitations
 6 Conclusions
References
 
These experiments show that fasting increased the activity of glycogen phosphorylase a, thus providing a mechanism for the increased glycogen utilization during ischemia previously described in this model. This increase cannot be attributable to the activation of phosphorylase kinase by either covalent modification by phosphorylation or a change in the sensitivity to the allosteric regulator Ca2+. These results parallel findings in studies of turtles that are tolerant of long periods of anoxia [8] which also showed higher phosphorylase a activity in the absence of changes in phosphorylase kinase activity. These data are consistent with the hypothesis that changes in glycogen utilization during fasting are part of a common endogenous adaptive mechanism to protect myocardium under conditions of reduced oxygen availability.

Time for primary review 22 days.


   Acknowledgements
 
The authors wish to thank Dr. Donal Walsh for his intellectual guidance and experimental assistance in the conduct of this study. Dr. Schaefer was supported by a grant from the American Heart Association. Dr. Ramasamy was supported by grants from the American Diabetes Association, the Juvenile Diabetes Foundation, and the University of California, Davis.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Limitations
 6 Conclusions
 References
 

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