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Cardiovascular Research 1998 37(1):160-170; doi:10.1016/S0008-6363(97)00220-4
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Metabolic alterations in the chronically denervated dog heart

Ger J van der Vussea,*, Marie-Louise Dubelaara, Will A Coumansa, Anne-Marie L Seymourb, Sinead B Clarkec, Arend Bonend, Angela J Drake-Hollandc and Mark I.M Noblec

aDepartment of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, 6200 MD Maastricht, Netherlands
bDepartment of Thoracic Surgery, National Heart Lung Institute at Harefield Hospital, Harefield, Middlesex, UK
cAcademic Unit of Cardiopulmonary Medicine, Charing Cross and Westminster Medical School, University of London, London, UK
dDepartment of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada

* Corresponding author. Tel. +31-43-3881086; Fax +31-43-3671028; E-mail: vandervusse@fys.unimaas.nl

Received 19 March 1997; accepted 15 August 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objectives: Previous studies have shown that chronic cardiac denervation impairs myocardial glucose oxidation. To investigate this further we tested whether the tissue content of glucose transporters, activity of glycolytic enzymes or metabolic capacity of pyruvate dehydrogenase were altered. Moreover, we investigated whether the decline in glucose utilization was associated with an upregulation of proteins and enzymes involved in fatty acid handling. Chronic cardiac denervation results also in decreased left ventricular efficiency. We explored whether alterations in mitochondrial properties could be held responsible for this phenomenon. Methods: Twelve adult dogs were included in the study. In 6 of them chronic cardiac denervation was accomplished by surgical ablation of the extrinsic nerve fibers. The other 6 dogs were sham-operated. Biopsies were obtained from the left ventricle after 4–5 weeks of denervation. The content or enzymatic activity of proteins involved in fatty acid and glucose handling was assessed. Features of glutamate oxidation were measured in freshly isolated mitochondria. Results: The content or activity of a set of fatty acid handling proteins did not change during chronic cardiac denervation. In contrast GLUT1 content significantly increased in the chronically denervated left ventricle, while the active form of pyruvate dehydrogenase declined (p<0.05). Glutamate oxidation characteristics in freshly isolated mitochondria were not affected by chronic denervation. Conclusion: The impairment of glucose oxidation in the chronically denervated myocardium is most likely caused by a decline of pyruvate dehydrogenase in its active form. It is unlikely that the decrease in work efficiency is caused by alterations in mitochondrial properties.

KEYWORDS Chronic denervation; Dog; Pyruvate dehydrogenase; Mitochondria; GLUT1; GLUT4; Carnitine acyl transferase


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Myocardial energy conversion relies heavily on the oxidation of glucose and fatty acids [1, 2]. The oxidation rate of both types of fuels and the extent to which glucose and fatty acids contribute to overall oxidative energy production depend on a variety of intracellular and extracellular factors [1, 3]. Among others, the extrinsic nervous system, consisting of parasympathetic and sympathetic fibers, seems to play an important role in controlling the selection of fuel by the heart, since chronic cardiac denervation was found to dramatically decrease the rate of myocardial glucose oxidation [4, 5]. The mechanisms underlying impaired oxidative conversion of glucose in the chronically denervated heart are not completely understood. Theoretically, the defect in glucose handling may be caused by an impaired capacity to transport the carbohydrate from the interstitial compartment into the intracellular space, a decline in flux of glucose through the glycolytic pathway, and/or a decrease in pyruvate dehydrogenase activity, resulting in a suppressed delivery of pyruvate-derived acetyl-CoA to the citric acid cycle.

Another striking effect of chronic cardiac denervation in dogs is a significant loss of mechanical efficiency, i.e., more molecular oxygen is consumed for the same amount of external work performed [5]. Although the precise mechanism underlying the decline in cardiac efficiency is unknown, suggestions have been made that this abnormality in the chronically denervated heart is caused by impaired mitochondrial coupling [6]. Solid evidence for this notion is, however, lacking. Circumstantial evidence has been provided indicating a shift from glucose to fatty acid oxidation in the chronically denervated hearts in fulfilling the energy requirements [5]. Theoretically, a shift from glucose to fatty acid oxidation in myocardial tissue may result in increased consumption of oxygen [2].

In all, a set of 9 hypotheses relevant to the two main observations on chronically denervated myocardium were formulated, dealing with i) cardiac carbohydrate metabolism, ii) fatty acid handling, and iii) mitochondrial function and energy stores. These are listed in Table 1.


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  		Table 1
 

   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 General outline of the experiments
Experiments were performed on twelve adult Beagle dogs (10 males, 2 females, ranging in weight from 12 to 15 kg). The hearts of six dogs were denervated and the remaining six dogs underwent a sham-operation (see below). Four to five weeks after denervation or sham-operation, the thorax of the animals was reopened under standard anesthesia (see below). At least 6 biopsies were taken from the left ventricular wall of the heart. Biopsies were used for the determination of i) high-energy phosphates; ii) glucose transporters; iii) enzymes of the glycolytic pathway; iv) pyruvate dehydrogenase; v) proteins/enzymes involved in cardiac fatty acid handling, and vi) cardiac lipid content, respectively. Care was taken that the respective biopsies were collected from corresponding sites of the twelve left ventricles included in this study. Biopsies, taken with an electrically driven biopsy bore, ranged in weight from 100 to 500 mg wet weight of tissue. The tissue specimens were rapidly frozen in liquid nitrogen and stored at –80°C for further biochemical analysis.

After sampling of biopsies, the heart was removed from the thorax and a piece of tissue (0.5 to 1 g) was taken for mitochondrial isolation. Tissue fractionation and measurement of mitochondrial properties (see below) were performed immediately, while the whole measurement was finished within two hours after taking the tissue specimen to avoid ageing of the mitochondria.

Arterial blood samples were taken via a catheter inserted into the femoral artery. Blood samples were collected two minutes before commencement of the procedure to obtain biopsies from the left ventricle. The samples were immediately centrifugated to remove blood cells. The blood plasma was transferred to glass test tubes and frozen in liquid nitrogen.

The protein content of the myocardial tissue specimen and mitochondrial fractions was analyzed according to Lowry and colleagues [7]with bovine serum albumin as the standard.

2.2 Cardiac denervation procedure and sham-operation
After overnight fasting, dogs were anesthetized with halothane which was maintained throughout the operative procedure. The lungs were ventilated with a mixture of O2 and N2O (40/60, by volume). The chest was opened between the fourth and fifth rib, the pericardium was removed and the denervation procedure was performed according to Donald and colleagues [8], as described by Drake and co-workers [9]. After denervation by surgical ablation, the chest was closed and the animals were allowed to recover. The whole operation procedure lasted 3 to 4 hours and was performed under strictly sterile conditions. Sham-operations were identical to the denervation procedure, except for surgical ablation of the cardiac sympathetic and parasympathetic nerves. Post-operative pain was suppressed according to standard procedures. To avoid microbiological infections, the animals received ampicillin peri- and post-operatively. The surgical procedures have all been approved by the Institutional Animal Care and Use Committee of the Maastricht University.

2.3 Biochemical analyses
2.3.1 Glucose transporters in left ventricular tissue (Hypothesis 1, Table 1)
The presence of the glucose transporters 1 and 4 (GLUT 1 and GLUT4, respectively) in denervated hearts and hearts of sham-operated dogs was assessed according to Ploug and co-workers [10]. Using this method, frozen biopsies (50 mg) were homogenized in buffer (30 mmol/l HEPES, pH 7.4, 210 mmol/l sucrose, 2 mmol/l EGTA, 40 mmol/l NaCl and 2 mmol/l PMSF), and mixed with 3 ml of KCl (1.16 mol/l) and pyrophosphate (58.3 mmol/l) and left on ice for 15 min. Total membranes were recovered by centrifugation (256,000xg for 75 min at 4°C). GLUT1 and GLUT4 were determined using Western blotting procedures as described previously [11, 12]. Antibodies against both glucose transporters were obtained from East Acres Biologicals (Southbridge, Ma, USA). GLUT4 and GLUT1 proteins were visualized using an enhanced chemiluminescence detection system (Amersham Canada, Oakville, Ontario) according to the instructions of the manufacturer. Western blots were quantified using a Macintosh LC with an Abaton scanner and appropriate software (Scan Analysis, Biosoft, Cambridge, UK). Samples from sham-operated and denervated dog hearts were always run within the same gel. With this technique the tissue content of GLUT1 and GLUT4, respectively, is quantified in arbitrary chemiluminescence units.

2.3.2 Enzymes involved in the glycolytic pathway (Hypothesis 2, Table 1)
Enzyme activities were measured in homogenates prepared according to the following standard technique unless otherwise indicated. Aliquots of frozen tissue were thawed in a buffer containing 0.25 mol/l sucrose, 2 mmol/l EDTA, and 10 mmol/l TRIS (pH 7.4) and subsequently homogenized with the use of an Ultra Turrax T25 (IKA, Staufen, Germany). Thereafter, the 10% homogenate (w/v) was sonicated for 1 min (4 times 15 s, with 15 s rest) at 0°C with the use of an Ultrasonic disintegrator (Sanyo Gallenkamp PLC, Leicester, UK). The sonicated homogenate was centrifuged at 1200xg and 4°C for 10 min, and the supernatants were stored at –80°C for further biochemical analysis.

The activity of hexokinase was measured according to Sols and co-workers [13]. The activity of phosphofructokinase was monitored according to Ling and co-workers [14]. The activity of fructose-biphosphate aldolase was measured according to Spolter et al. [15]. The activity of glyceraldehyde-3-phosphate dehydrogenase was assessed according to Furfine and Velick [16]. 3-Phosphoglycerate kinase activity was measured according to Bucher [17]. Pyruvate kinase activity was assessed as described by Gutmann and Bernt [18]. The activity of lactate dehydrogenase was determined according to Bergmeyer and Bernt [19]. The activities of the glycolytic enzymes were assessed at 25°C.

2.3.3 Pyruvate dehydrogenase (Hypothesis 3, Table 1)
To monitor pyruvate dehydrogenase (Pdh) activity in cardiac samples the following homogenization procedure was followed. Aliquots of frozen heart tissue were ground to a fine powder under liquid nitrogen. Each sample ({approx}200 mg of powdered tissue) was homogenized in an appropriate buffer (see below) using a polytron tissuemiser. The sample was then frozen in liquid nitrogen, allowed to thaw and rehomogenized. This freeze-thaw/homogenization cycle was repeated once more to release Pdh from mitochondrial membranes. The sample was subsequently centrifuged in a cooled microcentrifuge for 8 min. The supernatant was used in the Pdh assay. To measure total Pdh activity, tissue was homogenized in a buffer containing (mmol/l): HEPES (75), dichloroacetate (5), MgCl2 (5), ADP (1), dithiothreitol (1), leupeptin (0.05) and Triton-X (1%, v/v), pH 7.0. The buffer used to monitor the active form of Pdh consisted of (mmol/l): HEPES (25), KH2PO4 (25), KF (25), dichloroacetate (1), EDTA (3), ADP (1), dithiothreitol (1), leupeptin (0.05) and Triton-X (1%, v/v), pH 7.0.

The activity of Pdh was assayed in the presence of a pyruvate generating system, consisting of lactate, NAD+ and lactate dehydrogenase. To this end, aliquots of the homogenates were incubated at 30°C in an incubation buffer, containing (mmol/l): HEPES (50), MgCl2 (1), EGTA (0.08), dithiothreitol (1), rotenone (0.004), NAD+ (1.67), thiamine pyrophosphate (0.2), lactate (16.7) and lactate dehydrogenase (2 U/l), pH 7.2. The production of NADH was continuously followed at 340 nm for 5 min. Pdh activity was calculated from the linear portion of the curve, i.e., 0–30 s after start of the reaction by addition of an aliquot of the tissue supernatant sample to the incubation buffer. The above-mentioned assay system was developed by Seymour and Chatham (unpublished; see acknowledgements) and was partly based on the techniques described by Paxton and Sievert [20]and Hansford et al. [21].

2.3.4 Glycogen handling (Hypothesis 4, Table 1)
Glycogen synthase. After thawing, tissue aliquots were homogenized in 50 mmol/l TRIS, 20 mmol/l NaF and 2 mmol/l EDTA (pH 7.8) with the use of an Ultra Turrax and subsequently sonicated. Thereafter the sonicated homogenate was centrifuged at 1200xg and 4°C for 10 min and the supernatant was stored at –80°C. The activity of glycogen synthase was estimated at 37°C according to Danforth [22]. When the D-form of the enzyme, i.e., the inactive form, was measured, 10 mmol/l glucose 6-phosphate was added to the incubation medium. In case of the active or I-form, the latter substance was omitted.

Glycogen phosphorylase. Aliquots of thawed tissue were homogenized in 20 mmol/l TRIS, 1 mmol/l EDTA and 0.5 mmol/l dithiothreitol. The total activity of glycogen phosphorylase was estimated at 25°C according to Schreiber and Bowling [23]. The inactive form of the enzyme (phosphorylase b) was measured in the presence of 5 mmol/l AMP.

2.3.5 Enzymes and proteins involved in fatty acid metabolism (Hypothesis 5, Table 1)
Enzyme activities were measured in homogenates prepared according to the following standard technique. Aliquots of frozen tissue were thawed in a SET buffer, containing (mmol/l), sucrose (250), EDTA (2), and TRIS (10) (pH 7.4), and subsequently homogenized with the use of an Ultra Turrax. Thereafter, the {approx}10% homogenate (w/v) was sonicated for 1 min (4 x 15 s, with 15 s rest) at 0°C with the use of an Ultrasonic disintegrator. The sonicated homogenates were centrifuged at 1200xg and 4°C for 10 min, and the supernatants were stored at –80°C for further biochemical analysis.

The activities of carnitine acyltransferase I and II were assessed employing a radiochemical method as previously described by Scholte and colleagues [24]. 3-Hydroxyacyl-CoA dehydrogenase activity was determined according to the method of Bradshaw and Noyes [25]. Acetyl-CoA carboxylase was assayed using acetyl-CoA as substrate in the presence of 10 mmol/l Mg citrate according to Saddik et al. [26]. The activities of enzymes involved in fatty acid metabolism were assayed at 37°C.

The content of fatty acid-binding protein (FABP) in dog left ventricular tissue was estimated with an enzyme-linked immuno-sorbent assay (ELISA), specifically developed to monitor the content of muscle-type FABP (H-FABP) in canine tissues. To this end, H-FABP was isolated and purified from dog myocardium largely according to Van Nieuwenhoven and colleagues [27]. The purified H-FABP was used to raise polyclonal antibodies against dog H-FABP in rabbits [28]. After purification an aliquot of the antibodies was used as catcher, another part was labelled with horse radish peroxidase (HRP) and subsequently used as detector in the ELISA system [28]. The amount of dog H-FABP used for calibration of the assay ranged from 0 to 17.5 ng/ml, corresponding with 0 to 0.9 ng per well of the PVC plate. Ortho-phenylene diamine (OPD) and H2O2 were used for monitoring the activity of HRP present in the detector attached to H-FABP. The optical density of the reaction product of OPD was measured with a Titertek Multiskan MK II (Labsystems, Finland) at 492 nm.

2.3.6 Tissue and blood lipid content (Hypothesis 6, Table 1)
The content of fatty acyl moieties in fatty acids, triacylglycerols and phospholipids in myocardial tissue and blood plasma was assayed with the use of a gas chromatographic system as described earlier [29].

2.3.7 Mitochondrial properties (Hypothesis 7, Table 1)
Left ventricular samples of approximately 0.5 to 1.0 g wet weight were homogenized in 10 to 20 ml of homogenization buffer, consisting (mmol/l) of sucrose (250), TRIS (10) and EDTA (2) (SET buffer, pH 7.4) at 4°C. The crude homogenate was centrifuged at 600xg for 5 min. The pellet was discarded and the supernatant was centrifuged at 7000xg for 10 min. The latter step was repeated twice after resuspension of the mitochondrial pellet in the SET buffer. Thereafter, the mitochondrial pellet was resuspended in {approx}0.75 ml of a medium consisting (mmol/l) of KCl (100), MgCl2 (1), EDTA (0.2) and TRIS (50), pH 7.4. The rate of glutamate oxidation in freshly isolated mitochondria ({approx}1 mg protein per incubation flask) was assessed with a polarographic method using a Gilson oxygraph (Gilson Medical Electronics FNC, Middleton, WI, USA). The incubation mixture consisted (mmol/l) of KCl (15), KH2PO4 (30), TRIS (25), sucrose (45), mannitol (10), MgCl2 (5), EDTA (7), glucose (20), NAD+ (0.5), cytochrome C (0.015) and 0.2% bovine serum albumin (fatty acid and citrate free, Sigma A7030 St. Louis, MO, USA), pH 7.4. L-glutamate (11 mmol/l; Sigma G-1501) was used as substrate. To measure the maximal glutamate oxidation rate (state 3 respiration) the reaction was started with the addition of 400 nmol ADP (Boehringer 102164, Mannheim, Germany). P/O ratio was calculated from the amount of ADP utilized (to form ATP) and the amount of molecular oxygen consumed during state 3 respiration [30]. Respiratory control index (RCI) was calculated by dividing the glutamate oxidation rate in excess of ADP (state 3 respiration) by the rate measured after all added ADP was converted to ATP (state 4 respiration).

2.3.8 Mitochondrial density (Hypothesis 8, Table 1)
The mitochondrial enzyme citrate synthase was estimated at 37°C using oxaloacetic acid as substrate according to Shephard and Garland [31].

2.3.9 Left ventricular content of glycogen (Hypothesis 4), phosphocreatine and adenine nucleotides (Hypothesis 9, Table 1)
Biopsies, ranging in wet weight from 80 to 100 mg, were freeze-dried overnight with the use of a GT2 freeze-dryer (Lebold Heraeus, Köln, Germany). After freeze-drying, adherent blood and connective tissue were removed. A part of the freeze-dried material ({approx}5 mg dry weight) was used for glycogen analysis as described before [32]. Another part ({approx}10 mg dry weight) was used for determination of adenine nucleotides and related compounds, and creatine and phosphocreatine by HPLC with the use of a modified method after Wynants and Van Belle [33], as described by Van der Vusse et al. [34].

2.4 Statistical analyses
The statistical analysis assumes that inhibition of glucose utilization and enhancement of metabolic rate in the chronically denervated heart are established facts [5]leading to a number of sub-hypotheses of these phenomena. The case of sub-hypothesis 8 (Table 1), i.e., a potential increase in mitochondrial density, was tested with only one variable: citrate synthase activity. This variable was compared between the sham and denervated groups by Students t-test. The other eight hypotheses (each of which involved more than one dependent variable) were tested using Hotelling's T2 (MANOVA); p<0.05 was considered to reflect a statistically significant difference.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 The content of glucose transporters in left ventricular tissue (Hypothesis 1)
Western blot analysis of homogenates of left ventricular biopsies revealed that the content of GLUT1 significantly increased by approximately 70% after four to five weeks of cardiac denervation (Fig. 1). In contrast, the tissue content of GLUT4 did not change significantly.


Figure 1
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  		Fig. 1 The relative content of GLUT1 and GLUT4 in left ventricular tissue. The mean of the content, measured in arbitrary chemiluminescence units, of both GLUT1 and GLUT4 in hearts of sham-operated dogs was set at 100%. The values measured in the denervated hearts were expressed relative to the corresponding mean value measured in the sham-operated group. Data refer to mean±S.D. (n = 6). Asterisk indicates significantly different from values in the sham-operated group (p<0.05).

 
3.2 Activities of enzymes in the glycolytic pathway (Hypothesis 2)
The activity of a selected number of key enzymes of the cardiac anaerobic glycolytic pathway in both sham-operated and chronically denervated animals is summarized in Table 2. Four to five weeks of cardiac denervation did not affect the maximal activity of hexokinase, 6-phosphofructo-1-kinase, fructose-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, or lactate dehydrogenase.


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  		Table 2 The activity of selected enzymes involved in the glycolytic pathway and glycogen handling in left ventricular tissue

 
3.3 The enzymatic activity of the pyruvate dehydrogenase complex (Hypothesis 3)
Chronic cardiac denervation did not influence the total activity of the pyruvate dehydrogenase complex, as measured in homogenates of left ventricular tissue (Fig. 2). However, pyruvate dehydrogenase present in its active form was significantly lower in chronically denervated cardiac tissue than in the corresponding sham-operated group. On average, the percentage pyruvate dehydrogenase in the active form declined from 48% to 12% after four to five weeks of denervation.


Figure 2
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  		Fig. 2 The total activity (t) and active form (a) of pyruvate dehydrogenase in sham-operated and chronically denervated cardiac left ventricular tissue. Asterisk indicates significantly different (p<0.05) from the corresponding value in the sham-operated group. Results are expressed as mean±S.D. (n = 6).

 
3.4 Tissue glycogen content and activity of glycogen phosphorylase and synthase (Hypothesis 4)
The left ventricular content of glycogen amounted to 204±95 and 216±61 µmol glucose residues/g dry weight in the sham-operated and denervated groups, respectively (N.S.). Chronic cardiac denervation did not affect the activity (either total or percentage in the active form) of glycogen phosphorylase and glycogen synthase (Table 2).

3.5 Fatty acid-binding protein and enzymes involved in cardiac fatty acid utilization (Hypothesis 5)
In Table 3 the tissue content of fatty acid-binding protein (FABP) and the maximal activity of a selection of enzymes involved in the fatty acid oxidative pathway are shown. Four to five weeks of denervation had no significant effect on the tissue content of the putative cytoplasmic fatty acid carrier FABP. Moreover, the maximal activities of carnitine acyltransferase I and II, 3-hydroxyacyl-CoA dehydrogenase and acetyl-CoA carboxylase did not differ between denervated hearts and hearts of sham-operated dogs.


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  		Table 3 The content of fatty acid-binding protein (FABP) and the maximal activity of fatty acid handling enzymes in left ventricular tissue

 
3.6 Lipid content and fatty acyl composition of arterial plasma and myocardial tissue (Hypothesis 6)
The lipid concentration in arterial blood plasma of sham-operated and cardiac denervated dogs is summarized in Table 4. The arterial fatty acid content was of the order of 200 µmol/l in sham-operated and cardiac denervated dogs. The differences in mean values were not statistically significant. The same holds for the arterial plasma concentrations of triacylglycerols and phospholipids. Palmitic acid, oleic acid and linoleic acid were found to be the most abundant fatty acids in the plasma fatty acid pool. Plasma triacylglycerols were also mainly composed of palmitic, oleic and linoleic acyl residues. Plasma phospholipids predominantly consisted of palmitic, stearic, linoleic and arachidonic acyl chains (data not shown). With respect to the relative fatty acyl composition of the various plasma lipid pools, no significant differences could be observed between sham-operated and cardiac denervated dogs.


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  		Table 4 Lipid content of arterial plasma and left ventricular tissue

 
The content of fatty acids in the left ventricular wall was found to be very low and not significantly different between sham-operated and cardiac denervated dogs (Table 4). The absolute values (ranging from 20–40 nmol/g wet weight) were of the same order of magnitude as previously found in hearts of acutely operated control dogs [35]. The tissue fatty acid pool was mainly composed of palmitic, stearic and linoleic acid (data not shown). The triacylglycerol content of the cardiac left ventricle of sham-operated and cardiac denervated animals was of the order of 17 µmol fatty acyl moieties per g wet weight of tissue (N.S.) (Table 4). This lipid pool predominantly consisted of palmitic, oleic and linoleic acyl moieties (data not shown). Cardiac tissue phospholipids in both groups of animals amounted to {approx}19 µmol fatty acyl residues/g wet weight (N.S.) (Table 4). Palmitic, stearic, oleic, linoleic and arachidonic acyl residues were the main constituents of the latter esterified lipid pool (data not shown). With respect to the relative fatty acyl composition of the left ventricular lipid pools, no significant difference between sham-operated and cardiac denervated dogs could be observed.

3.7 Mitochondrial properties: oxidation rate of glutamate and degree of coupling (Hypothesis 7)
The glutamate oxidation rate was of the order of 500 natom O/mg mitochondrial protein per min (Table 5). Denervation had no effect on the rate of mitochondrial glutamate oxidation. Moreover, both the P/O ratio and the respiratory control index indicate that chronic denervation did not influence the degree of coupling of respiratory chain activity to ATP production in mitochondria freshly isolated from left ventricular tissue.


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  		Table 5 Mitochondrial glutamate oxidation rate, P/O ratio and respiratory control index

 
3.8 Mitochondrial density (Hypothesis 8)
The activity of the mitochondrial citric acid cycle enzyme citrate synthase in homogenates of left ventricular tissue obtained from sham-operated and cardiac denervated dogs amounted to 132±52 and 152±25 mU/mg tissue protein, respectively. The statistically non-significant difference strongly suggests that cardiac mitochondrial density did not change during four to five weeks of chronic denervation.

3.9 Tissue content of high-energy phosphates and related compounds (Hypothesis 9)
HPLC analysis of tissue specimens of denervated hearts and hearts of sham-operated dogs revealed that cardiac denervation did not affect the level of adenine nucleotides (ATP, ADP and AMP) in the left ventricle (Table 6). The same holds for the tissue levels of GTP and NAD. The contents of IMP, adenosine, inosine, hypoxanthine, xanthine and uric acid were below the detection limit. Levels of cardiac phosphocreatine and creatine did not differ significantly between the denervated and sham-operated group (Table 6). The phosphocreatine/creatine ratio in cardiac tissue was also similar in both groups.


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  		Table 6 Cardiac tissue content of high-energy phosphates and related compounds

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
In the clinical setting, cardiac transplantation results in chronic denervation of the ventricles due to ablation of the extrinsic nerve fibers. Although no gross alterations in cardiac mechanical performance have been documented after transplantation, distinct changes in cardiac metabolic properties have to be anticipated due to chronic denervation. In this respect, it is worth mentioning that animal studies indicate a decline in cardiac efficiency, i.e., more molecular oxygen is consumed for the same amount of external work [5]. This reduced efficiency was found to be associated with a decrease in the contribution of glucose oxidation to overall oxidative energy conversion [36]. The mechanisms underlying impaired glucose utilization and decline in efficiency in the denervated left ventricles are, however, not completely understood.

The present study was designed to explore in more detail cardiac metabolism in order to identify possible causes of the decline in rate of glucose oxidation in the chronically denervated heart. In addition, we investigated whether a decrease in cardiac glucose utilization is accompanied by an upregulation of the content or activities of proteins or enzymes involved in handling of cardiac fatty acids. Finally, attempts were made to seek an explanation for the decrease in cardiac efficiency. To this end, the degree of mitochondrial coupling was investigated. In the present study, a duration of 4 to 5 weeks denervation was chosen, as previous studies have shown that this time interval is sufficiently long to cause almost complete depletion of the cardiac noradrenaline stores and to evoke alterations in cardiac glucose handling and work efficiency [4, 5].

Theoretically, the denervation-induced decrease in glucose oxidation could be caused by a multiple set of changes. Uptake of glucose by cardiac myocytes is governed by sarcolemmal glucose transporting proteins, GLUT4 and GLUT1 being the most important candidates [37, 38]. The slight, but statistically non-significant decrease in total tissue content of GLUT4 makes it less likely that GLUT4 is responsible for the previously observed decline in glucose consumption in the chronically denervated myocardium. It should be emphasized that chronic cardiac denervation was found to be associated with a significant increase in the GLUT1 content. This finding parallels the results observed in rat skeletal muscles subjected to chronic denervation [39]. At present, it is unknown whether the enhanced content of GLUT1 in the denervated heart positively affects the total capacity of the cardiomyocytes to extract glucose from the extracellular environment. If so, the altered GLUT1 content does not explain the previously observed decline in overall glucose utilization in the chronically denervated heart.

The decline in glucose oxidation in the chronically denervated heart may be caused by an impaired flux of carbohydrate intermediates through the glycolytic pathway. The present and previous observations [6]do not indicate changes in maximal activity of key enzymes in the glycolytic pathway. Previous findings of Drake and colleagues [36]showed a change in the tissue content of some intermediates of the glycolytic pathway, which might suggest altered activity due to allosteric regulation in vivo of one or more key enzymes involved in the conversion of glucose to pyruvate in the chronically denervated heart.

The pyruvate dehydrogenase complex is responsible for the conversion of pyruvate to acetyl-CoA, which is subsequently degraded to carbon dioxide and water by concerted action of the citric acid cycle and respiratory chain. This study clearly shows that the total activity of the enzyme complex remained unaffected in the chronically denervated heart, excluding a regulatory effect of the extrinsic nervous system on the enzyme complex at the transcriptional and translational level. However, the proportion of the enzyme in its active form was significantly depressed, which offers a feasible explanation for the decline in overall rate of glucose oxidation in the chronically denervated dog heart as previously reported by Drake and colleagues [36]. Phosphorylation of pyruvate dehydrogenase is known to reduce the catalytic activity of the enzyme complex, whereas dephosphorylation enhances the catalytic activity. Catecholamines are known to promote dephosphorylation of the enzyme complex with a concomitant increase in the capacity to convert pyruvate into acetyl-CoA [40]. In this respect it is of interest to note that in the normal rat heart elevated extracellular catecholamine levels have been shown to stimulate the activity of the pyruvate dehydrogenase complex [41]. It is tempting to speculate that, in particular, the virtually depleted endogenous noradrenaline and adrenaline stores in the denervated heart [4, 5]exert a negative action on the activity of pyruvate dehydrogenase by keeping a substantial proportion of the enzyme complex in the phosphorylated state.

The present findings strongly suggest that chronic cardiac denervation does not affect glycogen metabolism, since both the tissue content of glycogen and the activity of glycogen synthase and glycogen phosphorylase were found to be similar in the sham and denervated dog heart.

Previous studies [36]have shown that chronic cardiac denervation exerts a significant effect on substrate selection by the heart. Drake and co-workers [36]reported that the contribution of glucose oxidation to overall substrate combustion declined from {approx}40% to {approx}10%. Because glucose and fatty acids are the main sources of oxidizable fuel for the cardiomyocytes [2], it has been suggested that the decline in glucose oxidation is compensated by an enhanced contribution of fatty acid oxidation to overall oxidative energy conversion [42]. The supposed increase in fatty acid utilization might be caused by alterations in the content of proteins and activity of enzymes involved in cellular fatty acid metabolism. Evidence is accumulating that fatty acid-binding protein (FABP) plays a pivotal role in the intracellular transport of fatty acids from the sarcolemma to their site of conversion, i.e., the mitochondrial outer membrane in the cardiomyocyte [2, 43, 44]. The present observation that the cardiac content of FABP was not affected by denervation of the heart excludes a possible decisive role of this protein in the supposed increase of fatty acid utilization in the denervated heart. Moreover, the absence of an increase in the maximal activities of fatty acid handling enzymes, such as carnitine acyltransferases I and II, and 3-hydroxyacyl-CoA dehydrogenase (Table 3) does not favor the idea that a possible increase in cardiac fatty acid oxidation is caused by upregulation of the synthesis and, hence, cardiac content of enzymes involved in the fatty acid catabolizing pathway.

Recent studies [45, 26]have disclosed that the rate of fatty acid oxidation in the heart can be manipulated in a subtle way by varying the content of malonyl-CoA. Malonyl-CoA is a potent inhibitor of carnitine acyltransferase I, a key enzyme in cardiac fatty acid metabolism. It has been suggested [46, 26]that the actual cellular content of malonyl-CoA is primarily dependent on the activity of acetyl-CoA carboxylase, an enzyme catalyzing the conversion of acetyl-CoA to malonyl-CoA. The present findings indicate that chronic cardiac denervation does not affect the activity of this enzyme measured in homogenates prepared from biopsies of left ventricular tissue. This observation makes a potential role of malonyl-CoA in regulating the rate of fatty acid oxidation in denervated myocardial tissue less likely.

Gas chromatographic analysis of arterial plasma samples and tissue specimens of control and cardiac denervated animals revealed no significant alterations in the content of fatty acids in blood and tissue. As the difference in fatty acid content in blood and tissue represents a driving force for the uptake of fatty acids by cardiac cells [2], the present data indicate that the supposed increase in cardiac fatty acid consumption is not caused by alterations in the content of these oxidizable substrates in blood plasma and tissue. Moreover, the unchanged fatty acyl composition of the cellular triacylglycerol and phospholipid pool indicates that myocardial non-oxidative fatty acid metabolism is apparently not altered during chronic denervation. The unchanged fatty acid composition of membrane phospholipids also makes alterations in membrane properties of the chronically denervated myocardium due to, among others, fatty acyl dependent differences in fluidity, less likely.

Animal studies, conducted to mimic the denervated state of the transplanted heart, have revealed that cardiac denervation results in reduced efficiency of cardiac function, i.e., more oxygen is consumed to perform the same amount of external work. In dogs, the increase of oxygen utilization was of the order of 25% after four weeks of cardiac denervation [5]. Loss of cardiac work efficiency might be caused by a variety of alterations in the chronically denervated heart [5]. From a metabolic point of view, in the chronically denervated heart increased oxygen consumption may be due to less tightly ‘coupled’ mitochondria, i.e., more molecular oxygen is consumed for the same amount of ATP produced, and by a shift from glucose to fatty acid oxidation as prime source of metabolic energy. The present findings do not favor the notion that cardiac mitochondrial properties per se change during chronic denervation, as the values of both the P/O ratio and the respiratory control index, two variables reflecting the degree of coupling of oxygen consumption to ATP production [30], were found to be similar in denervated and sham-operated animals. Earlier findings of Butcher and co-workers [6], however, suggested that the maximal rate of mitochondrial oxygen consumption is increased in the denervated dog heart. In their study, oxygen consumption of minced tissue was analyzed in the presence of an artificial uncoupler. This observation may indicate that the sensitivity of mitochondria in denervated hearts towards uncoupling agents is increased, but does not necessarily imply that the degree of coupling of mitochondria is altered. On the other hand, it cannot be excluded that mitochondria are less tightly coupled in denervated hearts in vivo, because of specific alterations in the micro-environment of the mitochondria in situ interfering with their energy converting activity. During fractionation of the tissue and isolation of the mitochondria this (these) factor(s) may be lost. The present findings also indicate that chronic cardiac denervation does not affect mitochondrial density in the left ventricle.

On the basis of studies performed on isolated rat hearts [47]it can be concluded that enhanced utilization of fatty acids as fuel is accompanied by a higher increase in oxygen consumption than can be explained by the amount of ATP produced per fatty acid molecule oxidized. Hence, a shift from glucose to fatty acid oxidation may contribute to the apparent decline in efficiency of the chronically denervated heart. Recently, Sato and colleagues [48]showed that, in the isolated rat heart, enhanced generation of acetyl-CoA via the pyruvate dehydrogenase complex significantly improves cardiac work efficiency. If extrapolation of their findings to the dog heart in situ is allowed, the decrease in pyruvate dehydrogenase activity, as shown in the present study, and, hence, reduced production in pyruvate-derived acetyl-CoA may offer an attractive explanation for the earlier reported decline in efficiency in the chronically denervated heart.

The unchanged levels of cardiac high-energy phosphates, i.e., ATP, GTP and phosphocreatine, indicate that also in the denervated heart mitochondrial energy conversion can easily keep up with the amount of energy required for electro-mechanical processes. Hence, these findings do not favor the notion that cardiac denervation might exert an adverse effect on mitochondrial functioning [5].

In summary, the present study indicates that chronic cardiac denervation exerts significant effects on some proteins and enzymes involved in myocardial glucose handling. The appreciable decrease of the active form of pyruvate dehydrogenase in the left ventricular wall offers an attractive explanation for the previously observed decline in the rate of glucose oxidation in the chronically denervated heart. The decline in work efficiency in chronically denervated heart is most likely not caused by alterations in the oxidative properties of left ventricular mitochondria. Although no indications were found for increased intracellular fatty acid transport and oxidative capacity in the denervated heart, it cannot be excluded that in vivo subtle regulating factors, giving rise to enhanced fatty acid utilization, have to be considered, the nature of which will have to be elucidated in future experiments.

Time for primary review 15 days.


   Acknowledgements
 
The help of Claire Bollen in preparing the manuscript and Theo Roemen in preparing the graphs is greatly appreciated. The authors are greatly indebted to Ruud Kruger, John Hynd and Theo van der Nagel in their expert biotechnical help. The development of the Pdh assay was made possible by a NATO Collaborative Research Grant # 940624 to Drs. A.-M.L. Seymour and D.J.C. Chatham. Dr. J.F.C. Glatz and Miss Yvonne de Jong are acknowledged for their help in analyzing tissue FABP.

This study is supported by the European Community, project # SCI *CT920806 and in part by the British Heart Foundation, PG 95174 (to A. Drake-Holland).


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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I. Vergroesen, D. Merkus, J. W.G.E van Teeffelen, J. Dankelman, J. A.E Spaan, H. B van Wezel, M. I.M Noble, and A. J Drake-Holland
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