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Effects of isoproterenol infusion on myocardial structure and composition
Effects of isoproterenol infusion structure and composition *
on myocardial
Hendrick B. Burner, M.D.** Max Jellinek, Ph.D. George C. Kaiser, M.D. St. Louis, MO.
T
he clinical use of isoproterenol as a beta-adrenergic stimulator is increasing. However, there are numerous experimental studies demonstrating myocardial necrosis and compositional alteration associated with large doses of isoproterenol. This is a report of the effects of chronic isoproterenol infusion on myocardial structure and composition. Methods Six mongrel dogs, weighing 8 to 17 kilograms, were anesthetized with sodium thiopental (Pentothal), 40 mg. per kilogram, and mechanically ventilated with room air. A Silastic catheter was introduced into the jugular vein and tunneled subcutaneously to emerge at the back of the neck. The catheter joined the batteryoperated electric infusion pump (Sigmamotor, ML-S) containing a 655 days’ supply of medication. This unit was carried in a harness so that the animals could be allowed normal activity and alimentation. Isoproterenol was infused at a constant rate of 1.5 pg per kilogram per hour for six days. The animals were put to death with intravenous sodium secobarbi-
tal (Eusol), 100 mg. per kilogram, and the hearts promptly excised and placed in cracked ice. Tissue was obtained for electron microscopy. The hearts were subdivided into five segments corresponding to the chambers and the inter-ventricular septum and weighed. Myocardial samples were prepared for light microscopy and samples of the remainder were homogenized for chemical and enzymatic analysis. Catecholamines were assayed according to the method of Crout and associates.’ Total carbohydrates were determined by the direct anthrone method and glycogen by the anthrone reaction2 after isolation of glycogen from the KOH digest. Unesterified fatty acid extractions and titrations were done by the method of Dole and Meinertz,d and saponification procedures for total fatty acid determinations were those of Albrink. The phosphate content of the lipid fraction was analyzed according to the method of Youngberg and Youngbergs using the phosphate procedure of Fiske and Subbarow.’ Values were expressed as milligram of lecithin equivalents per gram of tissue. Sodium and potassium were read in a Coleman flamephotometer after the sam-
From
the Departments of Surgery, St. Louis University, and the John Cochran Veterans Administration Hospital, St. Louis. MO. 63106. Supported by the United States Public Health Service Grant HE-06312 and the John A. Hartford Foundation, Inc. Received for publication April 17. 1969. *Presented at the Western Section, American Federation for Clinical Research, Cannel, Calif.. Jan. 31. 1969. *Reprint requests to: Dr. Bamer. 1325 South Grand Blvd., St. Louis. MO. 63104.
Vol. 79, No. 2, pp. 237-243
February, 1970
American Heart Journal
237
Barrier,
238
Jellinek,
Amer. Heart 1. 1970 February,
and Kaiser
Isoproterenol
0
Fig. 1. Myocardial
RV
Infusion
Activity of phosphoenolpyruvate
t
Septum
kinase
0 control m treated
RA p= .02
RV
LV
Septum
glycogen content is shown on the left and total carbohydrate
lsoproterenol 100
LV
lnfuslon
RV
LA
-
.05
S .OOl
.Ol
Fig. 2. Isoproterenol infusion is associated with an increase. in activity of phosphoenolpyruvate kinase in all chambers.
ple was dissolved in acetic acid. Enzymatic assays were carried out by standard methods.7 Results The ratio of heart weight to body weight, grams per kilogram was 7.87 f 0.98 for the control animals and 7.96 f 0.72 for the treated animals. The results of myocardial analysis for sodium, potassium, glycogen, total carbohydrate, total lipid, phospholipid, unesteritied fatty acids, and catecholamines are summarized in Table I. These values were
content on the right.
normal except for myocardial glycogen depletion which accounts for the decrease in total carbohydrate (Fig. 1). The results of myocardial enzyme analysis are summarized in Table II. The activities of these enzymes were in the normal range with the exception of phosophoenolpyruvate kinase, which was statistically significantly increased in all segments but the right ventricle (Fig. 2), and increased activity of a few enzymes in one or two myocardial segments. The location of these enzymes in various metabolic pathways is shown schematically in Fig. 3. Myocardial structure and ultrastructure were normal except for glycogen depletion which can be appreciated by comparing control myocardium (Fig. 4) and treated myocardium (Fig. 5). Discussion The dosage of isoproterenol was in the low range when compared to previous experimental and clinical studies of the physiologic effects of this amine (Table I I I) in which the infusion was continued for a few minutes to a few hours.*-l4 Cardiac hypertrophy did not develop in contrast to hearts exposed to large doses of isoproterenol in which initial cardiac enlargement was related to edema and later to increases in cardiac protein and nucleic acids.i5 Hearts from animals receiving large doses of isoproterenol have shown increased Na++, Ca++, and Cl- and de-
Volume Number
79 2
Effects of isoproterenol
Table I. Myocardial
Sodium Control Potassium Control Total carbohydrate Control Glycogen Control Total lipid Control Phospholipid Control Unesterified fatty Control Catecholamines Control
on myocardium
239
analysis
6
GqlGm.
22 6 22 6 22 6 23 6 22 6 22 6 22 6
&q/Gm. mg./Gm. mg./Gm. d%/Gm. mg./Gm. acids
d%lGm. &Gm.
2.08*
0.14
121.8 130.5 25.98 24.03 19.6 15.76 0..53+ 0.54
* 23.2 * 22.9 + 0.82 * 0.58 * 2.0 + 1.27 0.1 * 0.08
41.0 44.17 79.8 78.13 1.72 4.53 0.24 3.33 66.0 69.2 26.30 25.97 16.8 15.61 0.48 0.55
* * * * * * * * * * * * * d * *
2.71 1.77 1.57 1.15 0.12 0.51 0.05 0.56 4.8 4.5 0.26 0.52 1.75 0.93 0.1 0.07
39.6 43.38 77.3 76.89* 1.35 4.13 .08 3.26 58.2 60.7 27.20 26.73 17.0 16.29 0.51 0.62
f * f * * * * * * + * * * * *
2.68 1.46 1.53 1.37 0.14 0.57 0.05 0.59 5.2 3.5 0.82 0.63 1.62 0.96 0.1 0.07
Table II. Myocardial enzyme activity (in pnoles per minute per gram of protein)
Glucose-6-phosphate dehydrogenase Control Phosphofructohinase Control Glycerophosphate dehydrogenase Control Triosephonphate isomerase* Control Aldolase* control Phosphoenolpyruvic hinase
ControI Lactic acid dehydrogeuaset Control Cytoplasmi malic enzyme Control Isocitric dehydrogenase Control Fumarase Control Mitochondriaf malio enzyme Control Malic dehydrogenaee Control
*Aldolase and tLDH activity
triosephosphate is expressed
6 21 5 11 6 21 5 11 5 11 6 16 6 21 6 11 6 15 6 16 6 15 6 14
6.9 4.65k 82.6 100.0 179.0 114.4 86.4 100.0 98.0 100.0 61.7 41.7 4.5 2.24f 51.7 37.7 35.6 24.0 31.7 35.6 51.5 41.4 1086.0 59.5
f
1.12 0.39 + 16.3 + 17.7 f 5.8 zk 9.1 f 6.5 f 6.7 f 16.2 + 11.1 2~ 6.7 I 3.9 f 0.35 0.29 _+ 11.8 3~ 6.0 + 3.7 31 1.6 5 6.7 rt 4.5 f 9.1 f 3.9 f 301.0 rt 61.0
2.6 f 0.4 2.03+ 0.2 96.8 f 24.3 100.0 k 17.3 177.3 rt 20.2 152.4 + 10.9 97.8 _+ 13.1 100.0 f 5.6 89.9 f 21.5 100.0 k 9.4 75.0 f 7.8 62.3 i 4.0 3.4 f 0.16 2.32f 0.17 24.7 f 4.1 19.3 If: 3.1 39.3 f 2.7 31.8 f 2.3 40.2 f 6.5 41.5 f 5.7 21.8 f 1.9 28.1 f 2.2 633.0 f 206.0 56.4 2~ 37.0
isomerase activities arc expressed as per cent in moles per minute per milligram of protein.
4.2 f 0.27 3.03f 0.25 114.5 f 20.9 100.0 f 9.7 198.0 f 13.3 137.9 + 7.6 106.0 f 4.4 100.0 f 6.5 99.4 f 17.0 100.0 -+ 11.3 63.8 2~ 12.1 35.4 * 4.8 2.7 -+ 0.38 1.83? 0.15 43.0 f 8.4 27.7 + 4.2 24.0 zk 3.0 24.1 f 2.1 24.0 f 5.6 28.6 f 3.9 44.6 f 9.8 47.4 f 3.9 667.0 f 86.0 450.0 f 46.0
of normal.
2.1 * 0.71 1.615 0.1 74.5 + 10.3 100.0 f 19.4 177.1 f 29.1 150.4 f 8.3 109.2 + 9.6 100.0 f 4.7 105.4 + 9.7 100.0 f 10.8 84.9 + 1.6 64.8 zfz 5.0 2.6 * 0.42 2.41 f 0.13 21.2 f 4.4 18.4 + 3.0 42.4 f 10.3 31.0 f 2.6 45.8 f 10.1 47.6 + 5.2 24.0 f 7.7 25.7 f 2.2 513.0 f 85.0 527.0 + 35.0
2.0 zt 1.75* 74.1 i 100.0 f 129.6 If; 132.4 f 124.8 f 100.0 f 118.9 f 100.0 f 85.3 f 65.9 zk 2.6 t 2.23& 21.2 f 19.4 f 44.6 + 34.0 * 43.3 f 47.3 + 25.9 rt: 25.9 f 564.0 f 523.0 f
7.8 0.15 12.7 18.9 12.1 6.3 11.1 4.9 8.4 10.0 5.3 4.5 0.21 0.14 4.2 3.2 5.1 2.9 10.3 6.4 6.6 1.7 57.Q 33.9
240
Barrier,
Jellinek, and Kaiser
\ \ \ \ \ \ \ \ \ \ \ \ -----__
Fig. 3. The enzymes that were assayed are shown in this abbreviated metabolic scheme. GdPD, Glucose-6phosphate dehydrogenase; PFK, phosphofructokinase; FA, fumarase; AGD, d-glycerophosphate dehydrogenase; TPI, triosephosphate isomerase; PEPK, phosphoenolpyruvate kinase; LDH, lactic acid dehydrogenase; ME, malic enzyme; MD, malic dehydrogenase; ICD, isocitric dehydrogenase; F, fumarase.
Table I I I. Isoproterenol
infusion* Dosage
(pg/Kg./hr.)
Author Canine Baues Benchimolg BrownrQ Krasnowrr Mueller’* Silberschmidn Weisslerr4 Present study
6-180
2.0-24
Clinical 1.7-3.4 1.7 7.0 0 7-4.8 1.7-4.3
2.0-10 0.9 1.5
*Except in the present study these infusions for a few minutes to aeverzd hours while servations were made.
were continued physiologic ob-
creased Mg++; K+ was slightly increased by smaller doses of isoproterenol (1 to 5 mg. per kilogram) and decreased by a larger dose (80 mg. per kilogram).15 In those hearts catecholamine content was unchanged, but catecholamine concentration was decreased.15 Myocardial carbohydrate depletion was limited to glycogen and did not involve other carbohydrate fractions (Fig. 2). A large injection of isoproterenol results in initial glycogen depletion, while four days after treatment there is an increased amount of myocardial glycogen.16 Presumably, glycogen depletion is due to glycogenolysis from phosphorylase activation. The significance of the increased ac-
Fig. 4. Electron strut :ture.
photomicrograph
Fig. 5. Electron photomicrograph structure.
of control myocardium
of treated myocardium
showing abundant
glycogen granules and normal
revealing depletion of giycogen granules and normal
242
Amer. Heart J. February, 1970
garner, Jellinek, and Kaiser
Summary
TRI -C
CYCLE
Fig. 6. This metabolic scheme indicates the pathways for breakdown of pho;phoenolpyruvate and its synthesis via the dicarboxylic acid shuttle. PEPCK, Phosphoenolpyruviccarboxylase kinase.
tivity of phosphoenolpyruvate kinase is uncertain. It may be secondary to glycogenolysis. On the other hand, it may be contributing to glycogen depletion by competition with substrate for glycogen synthesis. The direct phosphorylation of pyruvic acid to phosphoenolpyruvate may occur under ideal conditions,” but the dicarboxylic acid shuttle is considered to be the preferential pathway for lactate and amino acids to contribute to glycogen synthesis (Fig. 6). Formation of phosphoenolpyruvate through the shuttle may be followed by its conversion to pyruvic acid rather than by glycogen synthesis. Considerable additional information is necessary to determine the dynamics of these reactions. Chronic infusion of a small dose of isoproterenol was not associated with the severe alterations in myocardial structure and composition occurring after one or two injections of a large dose of isoproterenol. The major alteration, glycogen depletion, diminishes the reserve of the myocardium for anaerobic metabolism, but loss of this capacity is probably not of great pragmatic significance.
Six ambulatory dogs had continuous infusion of isoproterenol, 1.5 pg per kilogram per hour for six days. The animals were put to death and the hearts excised and subdivided into five segments corresponding to the chambers and the interventricular septum. The myocardium was studied by light and electron microscopy and analyzed for one enzyme of the hexosemonophosphate shunt, seven enzymes of the glycolytic pathway, three enzymes of the Krebs cycle, total lipid, phospholipid, unesterified fatty acids, glycogen, total carbohydrate, catecholamines, sodium, and potassium. Twelve normal dogs served as controls. Myocardial structure and ultrastructure were unaltered except for depletion of glycogen granules. Compositional alterations were limited to glycogen depletion (control, 3.5, and treated, 0.2 mg. per gram) and increased activity of phosphoenolpyruvate kinase (control, 54.0, and treated, 73.9 pmoles per minute per gram of protein). Prolonged infusion of a small dose of isoproterenol is not associated with the myocardial necrosis and profound compositional alterations occurring after large doses of this amine.
REFERENCES 1. Crout, J. R., Creveling, C. R., and Undenfriend, S.: Norepinephrine metabolism in rat brain and heart, J. Pharmacol. & Exper. Therap. 132:269, 1961. 2. Seifter, S., Dayton, S., Novic, B., and Muntweyler, E. : Estimation of glycogen with anthrone reagent, Arch. Biochem. 25:191, 1950. 3. Colowick, S. P., and Kaplan, N. 0.: Methods in enzymology, New York, 1955, Academic Press, Inc. 4. Dole, V. P., and Meinertz, H.: Microdetermination of long-chain fatty acids in plasma and tissues, J. Bid. Chem. 2$5:2595, 1960. M. I.: The microtitration of total 5. Albrink. fatty acids of serum, with notes on the estimation-of triglycerides, J. Lipid Res. 1:53, 1959. G. E., and Youngberg. M. V.: 6. Younnberg. Phosihoris metabolism: System -of blood phosphorus analysis, J. Lab. & Clin. Med. 16:158, 1930. I. Fiske, C. H., and Subbarow, Y.: The colorimetric determination of phosphorus, J. Biol. Chem. 66:375, 1925. 8. Baue, A. E., Jones, E. F., and Parkins, W. M.: The effects of beta-adrenergic receptor stimulation on blood flow, oxidative metabolism and
Y&me Number
9.
10.
11.
12.
79 2
survival in hemorrhagic shock, Ann. Surg. 167:403, 1968. Benchimol, A., Lucena, E. G., and Dimond, E. G.: Stroke volume and peripheral resistance during infusion of isoproterenol at a constant fixed heart rate, Circulation 31:417, 1965. Brown, R. S., Carey, J. S., Woodward, N. W., Mohr, P. A., and Shoemaker, W. C.: Hemodynamic effects of sympathomimetic amines in clinical shock, Surg. Gynec. & Obst. 122:303, 1966. Krasnow, N., Rolett, E. L., Yurchak, P. M., Hood, W. B., Jr., and Gorlin, R.: Isoproterenol and cardiovascular performance, Am. J. Med. 37:514, 1964. Mueller, H., Giannelli, S., Jr., Ayres, S. M., Conklin, E. F., and Gregory, J. J.: Effect of isoproterenol on ventricular work and myocardiai metabolism in the postoperative heart, Circulation 37 and 33 (Supp. II): II, 146, 1968.
Effects of isoproterenol on myocardium
243
13. Silberschmid, M., Smith, L. L., Staeheiin, H. B., and Hinshaw, D. B.: Isoproterenol and cardiac response to experimental lactic acidosis, Surgery 63:181, 1968. 14. Weissler, A. M., Leonard, J. J., and Warren, J. V.: The hemodynamic effect of isoproterenol in man with observations on the role of the central blood volume, J. Lab. & Clin. Med. 53:921, 1959. 15. Stanton, H. C., Brenner, G., and Mayfield, E. D., Jr.: Studies on isoproterenol-induced cardiomegaly in rats, AM. HEART J. 77:72, 1969. 16. Mar&o, C. A.: Fine structural study of myocardial changes induced by isoproterenol in rhesus monkeys, Am. J. Path. 50:27, 1967. 17. Krimsky, I.: Phosphorylation of pyruvate by the pyruvate kinase reaction and reversal of glycolysis in a reconstructed system, J. Biol. Chem. 234:232, 1959.
on myocardial
Hendrick B. Burner, M.D.** Max Jellinek, Ph.D. George C. Kaiser, M.D. St. Louis, MO.
T
he clinical use of isoproterenol as a beta-adrenergic stimulator is increasing. However, there are numerous experimental studies demonstrating myocardial necrosis and compositional alteration associated with large doses of isoproterenol. This is a report of the effects of chronic isoproterenol infusion on myocardial structure and composition. Methods Six mongrel dogs, weighing 8 to 17 kilograms, were anesthetized with sodium thiopental (Pentothal), 40 mg. per kilogram, and mechanically ventilated with room air. A Silastic catheter was introduced into the jugular vein and tunneled subcutaneously to emerge at the back of the neck. The catheter joined the batteryoperated electric infusion pump (Sigmamotor, ML-S) containing a 655 days’ supply of medication. This unit was carried in a harness so that the animals could be allowed normal activity and alimentation. Isoproterenol was infused at a constant rate of 1.5 pg per kilogram per hour for six days. The animals were put to death with intravenous sodium secobarbi-
tal (Eusol), 100 mg. per kilogram, and the hearts promptly excised and placed in cracked ice. Tissue was obtained for electron microscopy. The hearts were subdivided into five segments corresponding to the chambers and the inter-ventricular septum and weighed. Myocardial samples were prepared for light microscopy and samples of the remainder were homogenized for chemical and enzymatic analysis. Catecholamines were assayed according to the method of Crout and associates.’ Total carbohydrates were determined by the direct anthrone method and glycogen by the anthrone reaction2 after isolation of glycogen from the KOH digest. Unesterified fatty acid extractions and titrations were done by the method of Dole and Meinertz,d and saponification procedures for total fatty acid determinations were those of Albrink. The phosphate content of the lipid fraction was analyzed according to the method of Youngberg and Youngbergs using the phosphate procedure of Fiske and Subbarow.’ Values were expressed as milligram of lecithin equivalents per gram of tissue. Sodium and potassium were read in a Coleman flamephotometer after the sam-
From
the Departments of Surgery, St. Louis University, and the John Cochran Veterans Administration Hospital, St. Louis. MO. 63106. Supported by the United States Public Health Service Grant HE-06312 and the John A. Hartford Foundation, Inc. Received for publication April 17. 1969. *Presented at the Western Section, American Federation for Clinical Research, Cannel, Calif.. Jan. 31. 1969. *Reprint requests to: Dr. Bamer. 1325 South Grand Blvd., St. Louis. MO. 63104.
Vol. 79, No. 2, pp. 237-243
February, 1970
American Heart Journal
237
Barrier,
238
Jellinek,
Amer. Heart 1. 1970 February,
and Kaiser
Isoproterenol
0
Fig. 1. Myocardial
RV
Infusion
Activity of phosphoenolpyruvate
t
Septum
kinase
0 control m treated
RA p= .02
RV
LV
Septum
glycogen content is shown on the left and total carbohydrate
lsoproterenol 100
LV
lnfuslon
RV
LA
-
.05
S .OOl
.Ol
Fig. 2. Isoproterenol infusion is associated with an increase. in activity of phosphoenolpyruvate kinase in all chambers.
ple was dissolved in acetic acid. Enzymatic assays were carried out by standard methods.7 Results The ratio of heart weight to body weight, grams per kilogram was 7.87 f 0.98 for the control animals and 7.96 f 0.72 for the treated animals. The results of myocardial analysis for sodium, potassium, glycogen, total carbohydrate, total lipid, phospholipid, unesteritied fatty acids, and catecholamines are summarized in Table I. These values were
content on the right.
normal except for myocardial glycogen depletion which accounts for the decrease in total carbohydrate (Fig. 1). The results of myocardial enzyme analysis are summarized in Table II. The activities of these enzymes were in the normal range with the exception of phosophoenolpyruvate kinase, which was statistically significantly increased in all segments but the right ventricle (Fig. 2), and increased activity of a few enzymes in one or two myocardial segments. The location of these enzymes in various metabolic pathways is shown schematically in Fig. 3. Myocardial structure and ultrastructure were normal except for glycogen depletion which can be appreciated by comparing control myocardium (Fig. 4) and treated myocardium (Fig. 5). Discussion The dosage of isoproterenol was in the low range when compared to previous experimental and clinical studies of the physiologic effects of this amine (Table I I I) in which the infusion was continued for a few minutes to a few hours.*-l4 Cardiac hypertrophy did not develop in contrast to hearts exposed to large doses of isoproterenol in which initial cardiac enlargement was related to edema and later to increases in cardiac protein and nucleic acids.i5 Hearts from animals receiving large doses of isoproterenol have shown increased Na++, Ca++, and Cl- and de-
Volume Number
79 2
Effects of isoproterenol
Table I. Myocardial
Sodium Control Potassium Control Total carbohydrate Control Glycogen Control Total lipid Control Phospholipid Control Unesterified fatty Control Catecholamines Control
on myocardium
239
analysis
6
GqlGm.
22 6 22 6 22 6 23 6 22 6 22 6 22 6
&q/Gm. mg./Gm. mg./Gm. d%/Gm. mg./Gm. acids
d%lGm. &Gm.
2.08*
0.14
121.8 130.5 25.98 24.03 19.6 15.76 0..53+ 0.54
* 23.2 * 22.9 + 0.82 * 0.58 * 2.0 + 1.27 0.1 * 0.08
41.0 44.17 79.8 78.13 1.72 4.53 0.24 3.33 66.0 69.2 26.30 25.97 16.8 15.61 0.48 0.55
* * * * * * * * * * * * * d * *
2.71 1.77 1.57 1.15 0.12 0.51 0.05 0.56 4.8 4.5 0.26 0.52 1.75 0.93 0.1 0.07
39.6 43.38 77.3 76.89* 1.35 4.13 .08 3.26 58.2 60.7 27.20 26.73 17.0 16.29 0.51 0.62
f * f * * * * * * + * * * * *
2.68 1.46 1.53 1.37 0.14 0.57 0.05 0.59 5.2 3.5 0.82 0.63 1.62 0.96 0.1 0.07
Table II. Myocardial enzyme activity (in pnoles per minute per gram of protein)
Glucose-6-phosphate dehydrogenase Control Phosphofructohinase Control Glycerophosphate dehydrogenase Control Triosephonphate isomerase* Control Aldolase* control Phosphoenolpyruvic hinase
ControI Lactic acid dehydrogeuaset Control Cytoplasmi malic enzyme Control Isocitric dehydrogenase Control Fumarase Control Mitochondriaf malio enzyme Control Malic dehydrogenaee Control
*Aldolase and tLDH activity
triosephosphate is expressed
6 21 5 11 6 21 5 11 5 11 6 16 6 21 6 11 6 15 6 16 6 15 6 14
6.9 4.65k 82.6 100.0 179.0 114.4 86.4 100.0 98.0 100.0 61.7 41.7 4.5 2.24f 51.7 37.7 35.6 24.0 31.7 35.6 51.5 41.4 1086.0 59.5
f
1.12 0.39 + 16.3 + 17.7 f 5.8 zk 9.1 f 6.5 f 6.7 f 16.2 + 11.1 2~ 6.7 I 3.9 f 0.35 0.29 _+ 11.8 3~ 6.0 + 3.7 31 1.6 5 6.7 rt 4.5 f 9.1 f 3.9 f 301.0 rt 61.0
2.6 f 0.4 2.03+ 0.2 96.8 f 24.3 100.0 k 17.3 177.3 rt 20.2 152.4 + 10.9 97.8 _+ 13.1 100.0 f 5.6 89.9 f 21.5 100.0 k 9.4 75.0 f 7.8 62.3 i 4.0 3.4 f 0.16 2.32f 0.17 24.7 f 4.1 19.3 If: 3.1 39.3 f 2.7 31.8 f 2.3 40.2 f 6.5 41.5 f 5.7 21.8 f 1.9 28.1 f 2.2 633.0 f 206.0 56.4 2~ 37.0
isomerase activities arc expressed as per cent in moles per minute per milligram of protein.
4.2 f 0.27 3.03f 0.25 114.5 f 20.9 100.0 f 9.7 198.0 f 13.3 137.9 + 7.6 106.0 f 4.4 100.0 f 6.5 99.4 f 17.0 100.0 -+ 11.3 63.8 2~ 12.1 35.4 * 4.8 2.7 -+ 0.38 1.83? 0.15 43.0 f 8.4 27.7 + 4.2 24.0 zk 3.0 24.1 f 2.1 24.0 f 5.6 28.6 f 3.9 44.6 f 9.8 47.4 f 3.9 667.0 f 86.0 450.0 f 46.0
of normal.
2.1 * 0.71 1.615 0.1 74.5 + 10.3 100.0 f 19.4 177.1 f 29.1 150.4 f 8.3 109.2 + 9.6 100.0 f 4.7 105.4 + 9.7 100.0 f 10.8 84.9 + 1.6 64.8 zfz 5.0 2.6 * 0.42 2.41 f 0.13 21.2 f 4.4 18.4 + 3.0 42.4 f 10.3 31.0 f 2.6 45.8 f 10.1 47.6 + 5.2 24.0 f 7.7 25.7 f 2.2 513.0 f 85.0 527.0 + 35.0
2.0 zt 1.75* 74.1 i 100.0 f 129.6 If; 132.4 f 124.8 f 100.0 f 118.9 f 100.0 f 85.3 f 65.9 zk 2.6 t 2.23& 21.2 f 19.4 f 44.6 + 34.0 * 43.3 f 47.3 + 25.9 rt: 25.9 f 564.0 f 523.0 f
7.8 0.15 12.7 18.9 12.1 6.3 11.1 4.9 8.4 10.0 5.3 4.5 0.21 0.14 4.2 3.2 5.1 2.9 10.3 6.4 6.6 1.7 57.Q 33.9
240
Barrier,
Jellinek, and Kaiser
\ \ \ \ \ \ \ \ \ \ \ \ -----__
Fig. 3. The enzymes that were assayed are shown in this abbreviated metabolic scheme. GdPD, Glucose-6phosphate dehydrogenase; PFK, phosphofructokinase; FA, fumarase; AGD, d-glycerophosphate dehydrogenase; TPI, triosephosphate isomerase; PEPK, phosphoenolpyruvate kinase; LDH, lactic acid dehydrogenase; ME, malic enzyme; MD, malic dehydrogenase; ICD, isocitric dehydrogenase; F, fumarase.
Table I I I. Isoproterenol
infusion* Dosage
(pg/Kg./hr.)
Author Canine Baues Benchimolg BrownrQ Krasnowrr Mueller’* Silberschmidn Weisslerr4 Present study
6-180
2.0-24
Clinical 1.7-3.4 1.7 7.0 0 7-4.8 1.7-4.3
2.0-10 0.9 1.5
*Except in the present study these infusions for a few minutes to aeverzd hours while servations were made.
were continued physiologic ob-
creased Mg++; K+ was slightly increased by smaller doses of isoproterenol (1 to 5 mg. per kilogram) and decreased by a larger dose (80 mg. per kilogram).15 In those hearts catecholamine content was unchanged, but catecholamine concentration was decreased.15 Myocardial carbohydrate depletion was limited to glycogen and did not involve other carbohydrate fractions (Fig. 2). A large injection of isoproterenol results in initial glycogen depletion, while four days after treatment there is an increased amount of myocardial glycogen.16 Presumably, glycogen depletion is due to glycogenolysis from phosphorylase activation. The significance of the increased ac-
Fig. 4. Electron strut :ture.
photomicrograph
Fig. 5. Electron photomicrograph structure.
of control myocardium
of treated myocardium
showing abundant
glycogen granules and normal
revealing depletion of giycogen granules and normal
242
Amer. Heart J. February, 1970
garner, Jellinek, and Kaiser
Summary
TRI -C
CYCLE
Fig. 6. This metabolic scheme indicates the pathways for breakdown of pho;phoenolpyruvate and its synthesis via the dicarboxylic acid shuttle. PEPCK, Phosphoenolpyruviccarboxylase kinase.
tivity of phosphoenolpyruvate kinase is uncertain. It may be secondary to glycogenolysis. On the other hand, it may be contributing to glycogen depletion by competition with substrate for glycogen synthesis. The direct phosphorylation of pyruvic acid to phosphoenolpyruvate may occur under ideal conditions,” but the dicarboxylic acid shuttle is considered to be the preferential pathway for lactate and amino acids to contribute to glycogen synthesis (Fig. 6). Formation of phosphoenolpyruvate through the shuttle may be followed by its conversion to pyruvic acid rather than by glycogen synthesis. Considerable additional information is necessary to determine the dynamics of these reactions. Chronic infusion of a small dose of isoproterenol was not associated with the severe alterations in myocardial structure and composition occurring after one or two injections of a large dose of isoproterenol. The major alteration, glycogen depletion, diminishes the reserve of the myocardium for anaerobic metabolism, but loss of this capacity is probably not of great pragmatic significance.
Six ambulatory dogs had continuous infusion of isoproterenol, 1.5 pg per kilogram per hour for six days. The animals were put to death and the hearts excised and subdivided into five segments corresponding to the chambers and the interventricular septum. The myocardium was studied by light and electron microscopy and analyzed for one enzyme of the hexosemonophosphate shunt, seven enzymes of the glycolytic pathway, three enzymes of the Krebs cycle, total lipid, phospholipid, unesterified fatty acids, glycogen, total carbohydrate, catecholamines, sodium, and potassium. Twelve normal dogs served as controls. Myocardial structure and ultrastructure were unaltered except for depletion of glycogen granules. Compositional alterations were limited to glycogen depletion (control, 3.5, and treated, 0.2 mg. per gram) and increased activity of phosphoenolpyruvate kinase (control, 54.0, and treated, 73.9 pmoles per minute per gram of protein). Prolonged infusion of a small dose of isoproterenol is not associated with the myocardial necrosis and profound compositional alterations occurring after large doses of this amine.
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