Cardiovascular Research 1992;26:933-938
933
Regulation of tissue noradrenaline in the rat myocardial infarction model of chronic heart failure Robert Zelis, Barry Clemson, Robert Baily, and Dwight Davis
A
number of lines of evidence have suggested that sympathetic nervous system activity is increased in congestive heart failure and that it contributes significantly to the constriction of blood vesse1s.l4 Increased peroneal sympathetic efferent nerve activity has been recorded in patients with congestive heart failure and has been correlated with increases in plasma noradrenaline concentration.' It seems likely that increased nerve activity is responsible for the increased spillover of noradrenaline into the circulation which has been documented recently in patients with congestive heart failure using 'H-noradrenaline (3H-NA) kinetic methodology."' However, only half of the raised plasma noradrenaline is related to increased spillover; the remainder is caused by a decreased clearance of noradrenaline from the circulation, which in turn may be related to a neurogenically mediated reduction in regional blood flow.' In the heart, where metabolic regulation of blood flow is more dominant, the increased neuronal activity results in depletion of noradrenaline when catecholamine synthetic activity cannot keep pace with neuronal release.' ' The rate of tissue noradrenaline depletion after inhjbition of noradrenaline synthesis and the rate of decrease of H-NA specific activity after administration of 'H-NA have been
used as indices of noradrenaline turnover in congestive heart failure. Studies of noradrenaline turnover in the cardiomyopathic hamster suggested that it was increased in the whole heart.' However, turnover normalised for heart weight was not increased, and splenic noradrenaline turnover was normal. In pressure overload hypertrophr, one study reported no increase in noradrenaline turnover ; in another, the increase was small." In the failing heart of the streptozotocin diabetic rat, one group has reported high cardiac noradrenaline turnover and hi h noradrenaline % 12 Other groups content of heart, brain, spleen, and kidney. reported normal tissue content and turnover except for the pancreas where turnover tended to be reduced.13 l 4 Few tissues other than the heart have been evaluated in any congestive heart failure model. In clinical studies of regional noradrenaline kinetics, renal as well as cardiac noradrenaline spillover is increa~ed.~ The present study was designed to evaluate mechanisms that may be regulating tissue noradrenaline concentration in a number of rgans by imposing a variety of conditions known to alte noradrenaline dynamics in the rat myocardial infarction model of congestive heart failure studied 6-8 weeks following coronary ligation." This model represents the most common mechanical cause of heart failure seen
a
Division of Cardiology, Departments of Medicine and Cellular and Molecular Physiology, The Milton S Hershey Medical Center, Pennsylvania State University, PO Box 850, Hershey, Pennsylvania 17033, USA: R Zelis, B Clemson, R Baily, D Davis. Correspondence to Dr Zelis.
Downloaded from by guest on June 5, 2016
Objective: The aim was to evaluate mechanisms regulating tissue noradrenaline in congestive heart failure. Methods: Tissue noradrenaline was measured in the conscious post myocardial infarction rat model of congestive heart failure and in sham operated rats (1) under control conditions, (2) 6 h after inhibition of tyrosine hydroxylase by the intraperitoneal administration of a-methyl-paratyrosine (AMPT) (1 00 mg.kg-l every 2 h), (3) 6 h after AMPT with desipramine pretreatment (0.3 mg.kg-'), and (4) following exhaustive exercise after AMPT. Tissue noradrenaline was extracted with perchloric acid and measured by high performance liquid chromatography with electrochemical detection. Results: In control animals without drug, tissue noradrenaline concentration was lower in the following tissues in the rats with myocardial infarction compared with the sham operated group: left and right ventricles, spleen, soleus and white gastrocnemius muscles, kidney cortex, and tail artery. After AMPT, tissue noradrenaline concentration in the sham operated group was significantly lower than control; in the myocardial infarction group the fall in noradrenaline was only significant in the kidney, and group differences were no longer present. In the sham operated animals, coadministration of desipramine with AMPT attenuated the fall in tissue noradrenaline caused by AMPT in the heart and spleen. With exercise to exhaustion, cardiac noradrenaline was lower in rats with myocardial infarction than in sham operated rats, but higher in the soleus muscle. Conclusions: These data suggest that tissue noradrenaline depletion in congestive heart failure is not isolated to the heart, and it occurs despite activation of mechanisms that might be operating to conserve neuronal noradrenaline. One mechanism may be reduced organ blood flow to retard diffusion of noradrenaline into the circulation. If this increases interstitial noradrenaline concentration, it would facilitate prejunctional a' receptor restraint on noradrenaline release. Metabolic coronary vasdilatation during exercise reverses this process, and makes the heart most susceptible to noradrenaline depletion in congestive heart failure. Cardiovascular Research 1992;26:933-938
934
Zelis, Clemson, Bai1.y. Davis
clinically, and the time course for development of heart failure mimics the clinical situation.I6The interventions we used to alter tissue noradrenaline were parenteral a-methylparatyrosine (AMPT) to inhibit tyrosine hydroxylase acti~ity,'"'~ AMFT plus desipramine to inhibit neuronal noradrenaline reuptake and increase neuroeffector junctional noradrenaline concentration, and exercise after AMPT to increase sympathetic tone and alter regional blood flow. Methods Opercitive procedure for coronrrn ligation Studies were performed on male Sprague Dawley rats (200-225 g) (Charles River Laboratories, Wilmington, MA, USA). The investigation conforms with the Guide ,fiw the care and use of laboratory animals published by the US National Institutes of Health (NIH publication No 85-23, revised 1985). Anaesthesia was induced by placing the rat in an acrylic chamber which contained halothane (4%) in oxygen. After induction, the animals were intubated with a plastic 14 gauge cannula and maintained on a lower halothane concentration ( I .5-2.0%). Under positive pressure ventilation the chest was opened anteriorly in the fourth left intercostal space. and the left main coronary artery was identified and ligated with a 6-0 silk suture 2 mm below the atrioventricular sulcus. In the sham operated animals, the suture was left unligated. The thoracotomy was repaired and the animal allowed to recover. The perioperative mortality was 20%. The goal of the procedure was to produce a group of animals for study 6-8 weeks following coronary ligation that had a myocardial infarction of at least moderate size (>20%) and a group of sham operated controls. Our laboratory has determined that animals with an infarct size greater than 20% show m t y ~of the features of moderate compensated congestive heart failure.
Experimental protocol On the day of study, the animals were brought to the laboratory and maintained in individual cages. Sham operated animals and animals with myocardial infarction were randomly allocated to one of four groups. After prolonged acclimatisation to the laboratory. the first group of sham and myocardial infarction rats was killed by an intraperitoneal injection of pentobarbitone (200 mg) (myocardial infarction, n= I I ; sham, n= 15). These animals were used for determination of baseline tissue noradrenaline concentrations. The second erouo of animals was given DL-a-methyl-paratyrosine methyl ester hyvdrorhloride (Aldrich Chemical Company) (100 mgkg? in 1.0 ml of sterile saline) by intraperitoneal injection. Similar injections were given 2 h and 4 h later to minimise renal damage from a single injection of a high dose of the drug. The animals werekilled 6 h aft; t h e b s t injection. These animals
Determination of tissue noradrenaline concentrcltiorr For the determination of noradrenaline concentration. a portion of the frozen tissue (50-100 mg) was weighed and placed in a stainless steel pulverisation apparatus precooled in liquid nitrogen. To this was added precoolfd 0.4 M perchloric acid to achieve a tissue concentration of 25 m g m - of perchloric acid for all tissues except skeletal muscle (50 m g m - ' ) and tail artery (see below). The pulverised tissue was transferred to glass tubes and allowed to thaw in an ice bath, following which the perchloric acid concentration was reduced to 0.1 M by dilution with water. Because the smooth muscle content of the tail artery did not lend itself to easy pulverisation, noradrenaline was twice extracted on ice (30 min each) by addition of'0.4 M perchloric acid to achieve a tissue concentration of 1.25 m g m - . After pulverisation, the tissues were centrifuged for 10 min at 1090 g and the supernatants subjected to an additional centrifugation-filtration step to remove completely any particulate material. After appropriate dilution with 0. I M perchloric acid, noradrenaline concentration was determined by reverse phase high performance liquid chromatography using an 8 cm ESA HR-80 column packed with 3 p y spherical octadecylsilane and a mobile phase delivered at I .5 ml.minby an ESA 5700 solvent delivery module." The mobile phase contained the following in one litre: methanol 20 ml, I-heptane sulphonic acid 0.25 g, Na' EDTA 0.09 g, monobasic sodium phosphate 6.9 g, adjusted to pH 3.2. Noradrenaline was measured coulometrically using a series of three ESA conditioning/detector cells (models 5021 and 5011) set at the following potentials: +0.35, +0.10, and 4 . 2 6 volts. Peak heights were determined by a Spectrophysics (model SP 4270) integrator. Noradrenaline tissue concentration was expressed as nmo1.g-l tissue (wet weight). Statistical anolysis Group data are expressed as mean(SEM). Differences between the myocardial infarction and sham operated group tissue noradrenaline concentration at baseline, 6 h after AMFT, and 6 h after AMFT with desipramine were determined by two way analysis of variance for independent groups with two between subject variables (myocardial infarction v sham operated) (control v AMPT AMFT + desipramine). Subsequent comparisons between the six groups were performed using the Student Newman-Keuls test. Differences were considered significant at pMI + EX
Eli1 ririery
3 I .00(2.3 I )* 22..57( 2.30)$ 23.30( 2.24)t 30.51(2.62)
Control
lhiodc,nuin S.SS(0.4S)
AM 1' AMI'T I>MI A M I T 4- EX
3.73(0.22)i 3.44(0.28)
3. I8(0.22)1
+
Mvoi~rrrrliulinfitrc!iotr
24.11(1.69) 24.49( I . I I ) 22.3" 1.39) 27.72(2.71)
5.06(0.38) 3.80(0.4I )
3.61(0.27) 3.96(0.45)
AMIT=a-methyl-paratyrosinc; u~i=desipramine:i;x=exercise
*p
933
Regulation of tissue noradrenaline in the rat myocardial infarction model of chronic heart failure Robert Zelis, Barry Clemson, Robert Baily, and Dwight Davis
A
number of lines of evidence have suggested that sympathetic nervous system activity is increased in congestive heart failure and that it contributes significantly to the constriction of blood vesse1s.l4 Increased peroneal sympathetic efferent nerve activity has been recorded in patients with congestive heart failure and has been correlated with increases in plasma noradrenaline concentration.' It seems likely that increased nerve activity is responsible for the increased spillover of noradrenaline into the circulation which has been documented recently in patients with congestive heart failure using 'H-noradrenaline (3H-NA) kinetic methodology."' However, only half of the raised plasma noradrenaline is related to increased spillover; the remainder is caused by a decreased clearance of noradrenaline from the circulation, which in turn may be related to a neurogenically mediated reduction in regional blood flow.' In the heart, where metabolic regulation of blood flow is more dominant, the increased neuronal activity results in depletion of noradrenaline when catecholamine synthetic activity cannot keep pace with neuronal release.' ' The rate of tissue noradrenaline depletion after inhjbition of noradrenaline synthesis and the rate of decrease of H-NA specific activity after administration of 'H-NA have been
used as indices of noradrenaline turnover in congestive heart failure. Studies of noradrenaline turnover in the cardiomyopathic hamster suggested that it was increased in the whole heart.' However, turnover normalised for heart weight was not increased, and splenic noradrenaline turnover was normal. In pressure overload hypertrophr, one study reported no increase in noradrenaline turnover ; in another, the increase was small." In the failing heart of the streptozotocin diabetic rat, one group has reported high cardiac noradrenaline turnover and hi h noradrenaline % 12 Other groups content of heart, brain, spleen, and kidney. reported normal tissue content and turnover except for the pancreas where turnover tended to be reduced.13 l 4 Few tissues other than the heart have been evaluated in any congestive heart failure model. In clinical studies of regional noradrenaline kinetics, renal as well as cardiac noradrenaline spillover is increa~ed.~ The present study was designed to evaluate mechanisms that may be regulating tissue noradrenaline concentration in a number of rgans by imposing a variety of conditions known to alte noradrenaline dynamics in the rat myocardial infarction model of congestive heart failure studied 6-8 weeks following coronary ligation." This model represents the most common mechanical cause of heart failure seen
a
Division of Cardiology, Departments of Medicine and Cellular and Molecular Physiology, The Milton S Hershey Medical Center, Pennsylvania State University, PO Box 850, Hershey, Pennsylvania 17033, USA: R Zelis, B Clemson, R Baily, D Davis. Correspondence to Dr Zelis.
Downloaded from by guest on June 5, 2016
Objective: The aim was to evaluate mechanisms regulating tissue noradrenaline in congestive heart failure. Methods: Tissue noradrenaline was measured in the conscious post myocardial infarction rat model of congestive heart failure and in sham operated rats (1) under control conditions, (2) 6 h after inhibition of tyrosine hydroxylase by the intraperitoneal administration of a-methyl-paratyrosine (AMPT) (1 00 mg.kg-l every 2 h), (3) 6 h after AMPT with desipramine pretreatment (0.3 mg.kg-'), and (4) following exhaustive exercise after AMPT. Tissue noradrenaline was extracted with perchloric acid and measured by high performance liquid chromatography with electrochemical detection. Results: In control animals without drug, tissue noradrenaline concentration was lower in the following tissues in the rats with myocardial infarction compared with the sham operated group: left and right ventricles, spleen, soleus and white gastrocnemius muscles, kidney cortex, and tail artery. After AMPT, tissue noradrenaline concentration in the sham operated group was significantly lower than control; in the myocardial infarction group the fall in noradrenaline was only significant in the kidney, and group differences were no longer present. In the sham operated animals, coadministration of desipramine with AMPT attenuated the fall in tissue noradrenaline caused by AMPT in the heart and spleen. With exercise to exhaustion, cardiac noradrenaline was lower in rats with myocardial infarction than in sham operated rats, but higher in the soleus muscle. Conclusions: These data suggest that tissue noradrenaline depletion in congestive heart failure is not isolated to the heart, and it occurs despite activation of mechanisms that might be operating to conserve neuronal noradrenaline. One mechanism may be reduced organ blood flow to retard diffusion of noradrenaline into the circulation. If this increases interstitial noradrenaline concentration, it would facilitate prejunctional a' receptor restraint on noradrenaline release. Metabolic coronary vasdilatation during exercise reverses this process, and makes the heart most susceptible to noradrenaline depletion in congestive heart failure. Cardiovascular Research 1992;26:933-938
934
Zelis, Clemson, Bai1.y. Davis
clinically, and the time course for development of heart failure mimics the clinical situation.I6The interventions we used to alter tissue noradrenaline were parenteral a-methylparatyrosine (AMPT) to inhibit tyrosine hydroxylase acti~ity,'"'~ AMFT plus desipramine to inhibit neuronal noradrenaline reuptake and increase neuroeffector junctional noradrenaline concentration, and exercise after AMPT to increase sympathetic tone and alter regional blood flow. Methods Opercitive procedure for coronrrn ligation Studies were performed on male Sprague Dawley rats (200-225 g) (Charles River Laboratories, Wilmington, MA, USA). The investigation conforms with the Guide ,fiw the care and use of laboratory animals published by the US National Institutes of Health (NIH publication No 85-23, revised 1985). Anaesthesia was induced by placing the rat in an acrylic chamber which contained halothane (4%) in oxygen. After induction, the animals were intubated with a plastic 14 gauge cannula and maintained on a lower halothane concentration ( I .5-2.0%). Under positive pressure ventilation the chest was opened anteriorly in the fourth left intercostal space. and the left main coronary artery was identified and ligated with a 6-0 silk suture 2 mm below the atrioventricular sulcus. In the sham operated animals, the suture was left unligated. The thoracotomy was repaired and the animal allowed to recover. The perioperative mortality was 20%. The goal of the procedure was to produce a group of animals for study 6-8 weeks following coronary ligation that had a myocardial infarction of at least moderate size (>20%) and a group of sham operated controls. Our laboratory has determined that animals with an infarct size greater than 20% show m t y ~of the features of moderate compensated congestive heart failure.
Experimental protocol On the day of study, the animals were brought to the laboratory and maintained in individual cages. Sham operated animals and animals with myocardial infarction were randomly allocated to one of four groups. After prolonged acclimatisation to the laboratory. the first group of sham and myocardial infarction rats was killed by an intraperitoneal injection of pentobarbitone (200 mg) (myocardial infarction, n= I I ; sham, n= 15). These animals were used for determination of baseline tissue noradrenaline concentrations. The second erouo of animals was given DL-a-methyl-paratyrosine methyl ester hyvdrorhloride (Aldrich Chemical Company) (100 mgkg? in 1.0 ml of sterile saline) by intraperitoneal injection. Similar injections were given 2 h and 4 h later to minimise renal damage from a single injection of a high dose of the drug. The animals werekilled 6 h aft; t h e b s t injection. These animals
Determination of tissue noradrenaline concentrcltiorr For the determination of noradrenaline concentration. a portion of the frozen tissue (50-100 mg) was weighed and placed in a stainless steel pulverisation apparatus precooled in liquid nitrogen. To this was added precoolfd 0.4 M perchloric acid to achieve a tissue concentration of 25 m g m - of perchloric acid for all tissues except skeletal muscle (50 m g m - ' ) and tail artery (see below). The pulverised tissue was transferred to glass tubes and allowed to thaw in an ice bath, following which the perchloric acid concentration was reduced to 0.1 M by dilution with water. Because the smooth muscle content of the tail artery did not lend itself to easy pulverisation, noradrenaline was twice extracted on ice (30 min each) by addition of'0.4 M perchloric acid to achieve a tissue concentration of 1.25 m g m - . After pulverisation, the tissues were centrifuged for 10 min at 1090 g and the supernatants subjected to an additional centrifugation-filtration step to remove completely any particulate material. After appropriate dilution with 0. I M perchloric acid, noradrenaline concentration was determined by reverse phase high performance liquid chromatography using an 8 cm ESA HR-80 column packed with 3 p y spherical octadecylsilane and a mobile phase delivered at I .5 ml.minby an ESA 5700 solvent delivery module." The mobile phase contained the following in one litre: methanol 20 ml, I-heptane sulphonic acid 0.25 g, Na' EDTA 0.09 g, monobasic sodium phosphate 6.9 g, adjusted to pH 3.2. Noradrenaline was measured coulometrically using a series of three ESA conditioning/detector cells (models 5021 and 5011) set at the following potentials: +0.35, +0.10, and 4 . 2 6 volts. Peak heights were determined by a Spectrophysics (model SP 4270) integrator. Noradrenaline tissue concentration was expressed as nmo1.g-l tissue (wet weight). Statistical anolysis Group data are expressed as mean(SEM). Differences between the myocardial infarction and sham operated group tissue noradrenaline concentration at baseline, 6 h after AMFT, and 6 h after AMFT with desipramine were determined by two way analysis of variance for independent groups with two between subject variables (myocardial infarction v sham operated) (control v AMPT AMFT + desipramine). Subsequent comparisons between the six groups were performed using the Student Newman-Keuls test. Differences were considered significant at pMI + EX
Eli1 ririery
3 I .00(2.3 I )* 22..57( 2.30)$ 23.30( 2.24)t 30.51(2.62)
Control
lhiodc,nuin S.SS(0.4S)
AM 1' AMI'T I>MI A M I T 4- EX
3.73(0.22)i 3.44(0.28)
3. I8(0.22)1
+
Mvoi~rrrrliulinfitrc!iotr
24.11(1.69) 24.49( I . I I ) 22.3" 1.39) 27.72(2.71)
5.06(0.38) 3.80(0.4I )
3.61(0.27) 3.96(0.45)
AMIT=a-methyl-paratyrosinc; u~i=desipramine:i;x=exercise
*p
