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ANNUAL REPORT
OF
PROGRAM ACTIVITIES
U : NATIONAL HEART AND LUNG INSTITUTE
Fiscal Year 1973
Part II
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health
INTRAMURAL RESEARCH
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 - June 30, 1973
INTRAMURAL RESEARCH
Project Reports Cardiology Branch
Summary 1
1. Phosphorylation of human platelet myosin and contractile
proteins from other sources 11
2(c) Characterization of cardiac contractile proteins from
patients with idiopathic hypertrophic subaortic stenosis 12
3(c) Echocardiographic findings in patients with idiopathic
hypereosinophilic syndromes 13
4. Effect of lidocaine and of elevating arterial pressure on the incidence of spontaneous ventricular fibrillation in
dogs during coronary occlusion 14
5. Effect of nitroglycerin on the incidence of spontaneous ventricular fibrillation during coronary occlusion in dogs- 16
6(c) Echocardiographic assessment of secondary cardiomyopathies- 17 7(c) ASH masquerading as coronary artery disease and idiopathic
myocardial hypertrophy 18
8(c) Electrocardiographic findings in 100 patients with
asjonmetric septal hypertrophy 19
9(c) Familial incidence and genetic transmission of IHSS 20
10(c) Coronary collateral function in patients without coronary
artery disease 21
11. Visualization of acute myocardial infarction by the Radionuclide Gallium-67 22
12. Lack of deleterious effects of vagally mediated brady- cardia during acute myocardial ischemia in the dog 23
13(c) Effects of nitroglycerin on coronary collateral function
in patients with coronary occlusive disease 24
14. Failure of nitroglycerin to alleviate acute myocardial
ischemia or improve collateral flow when M^02 is held con- stant in the heart-lung preparation 26
15(c) Quantification of ventricular regularization in atrial
f ibrillation — — 27
16(c) Quantitative assessment of left ventricular function in
man utilizing roentgen videodensitometry 29
17. Adrenergic influences on ventricular fibrillation threshold
and post-occlusion arrhythmias 30
18(c) Factors affecting the operative mortality in aortic
valATular disease 3 2
19(c) Mitral valve position in patients with asymmetric septal
hypertrophy 33
20. A video scanner-analog computer system for the analysis of
routine echocardiograms 35
21(c) Long-term effects of operation on obstruction and LV
hypertrophy in IHSS 36
22(c) Distribution of the cardiomyopathy in IHSS 37
23(c) Non- invasive determination of the pressure gradient in
patients with idiopathic hypertrophic subaortic stenosis 39
24(c) Echocardiographic diagnosis of IHSS by detection of
asymmetric septal hypertrophy 41
25(c) The clinical characteristics of obstructive and non- obstructive asymmetric septal hypertrophy 42
26(c) A real time system for two-dimensional echocardiography 43
27(c) Effects of operation on the cardiac response to exercise
in patients with IHSS 45
28. Reduction in extent of myocardial infarction when nitro- glycerin and methoxamine are administered during coronary occlusion 46
29(c) Preoperative predictors of the long-term results of wave
replacement in patients with aortic regurgitation 47
30(c) Characterization of the cardiac response to exercise and effects of propranolol in patients with asymmetric septal hy pe r t r ophy 4 9
31. Cholinergic innervation of the cardiac conduction system: autonomic and functional correlations 51
32. Electrical stability of acutely ischemic myocardium: Inde- pendent influences of heart rate and vagal tone 53
33. The electrical stability of acutely ischemic myocardial effects of nitroglycerin 55
34. The role of prostaglandins in the control of coronary vascular tone 56
35. Physiological and biochemical characteristics of a new
group of inotropic agents: Analogues of angiotensin II 58
36(c) Differences in distribution of morphologic abnormalities
in patients with obstructive and non-obstructive asymmetric
septal hypertrophy (ASH) : Light and electron microscopic
ob s e rva t io ns 5 9
37(c) Significance of multiple intercalated discs in hyper-
trophied human myocardium 61
38(c) Postoperative response to intense upright exercise in patients with ventricular septal defect and pulmonary hypertension: relation of impairment to age at operation 62
39(c) The occurrence of a-glycogen in human cardiac muscle cells- 63
40(c) Unusual evolution of acquired infundibular pulmonary stenosis in patients with ventricular sepal defect: Clinical and morphologic observations 64
41(c) Ultrastructural features of myocardial cell degeneration in
patients with cardiac hypertrophy 65
42. Effects of vagal stimulation on survival during experi- mental acute myocardial infarction 66
43(c) Evaluation of the ability of echocardiography to measure
alterations in left ventricular volume 67
44(c) Use of biventricular cineangiography in the evaluation of
patients with asymmetric septal hypertrophy 68
45(c) Echocardiography during upright exercise 69
46(c) Sustained effects of nitroglycerin ointment in patients
with angina pectoris 70
47(c) Deterioration of myocardial function following aorto-
coronary bypass operation 71
48. Increased myocardial ischemia caused by reflexly induced
hypotension during coronary occlusion in the conscious dog- 72
ii
Clinic of Surgery
Summary 73
49(c) NU-5 atomic powered pacemaker — experimental and clinical
evaluation 79
50(c) Long-term follow-up of patients with congenital aortic
stenosis treated with aortic commissurotomy 80
51. The pathophysiology of intimal hyperlasia of the venous
graft in the arterial system 81
52(c) Silastic ball variance detection 82
53(c) Light and electron microscopic evaluation of hypertrophied right ventricular outflow tract muscle from patients with
; I ■ congenital heart disease 83
'"-']'.', 54(c) Assessment of aortic regurgitation in patients with the
use of the Doppler Bidirectional Velocitometer 84
55, Experimental production of progressive mitral stenosis 85
, . , 56. The effects of naloxone hydrochloride on ventricular
irritability ■ 86
57(c) Clinical effects of abrupt decrease in left atrial pressure
on renal sodium excretory patterns 87
58. Systemic and renal responses to a graded reduction in
cardiac output — ' 88
59. Blood volume expansion without altering hematocrit. Renal effects in a cross-transfusion model 89
60. Systemic and renal effects of methyl prednisolone following
a graded reduction in cardiac output 90
61. Apical-aortic valvular anastomosis for diffuse left ventricular outflow tract obstruction 91
62. Experimental evaluation of the membrane oxygenator 92
63. Transcutaneous measurement of aortic regurgitation with
a Doppler Velocitometer 93
64. A new canine model for echocardiographic evaluation of myocardial function 94
65. A comparison of left ventricular pressure, aortic flow and echocardiography in determining the response of left ventricular function or propranolol 95
66. Experimental creation of infundibular pulmonic stenosis ventricular septal defect and right ventricular hyper- tension in puppies 96
" 67(c) A review of eighteen years experience with total correction
of tetralogy of Fallot at the NIH 97
68. The experimental creation of infundibular pulmonic stenosis
and demonstration of its hemodynamic relationships 99
69. Hemodynamic and anatomic evaluation of experimental
valvular aortic stenosis 101
70. Effects of aortic stenosis on (A) coronary blood flow and
(B) regional distribution of blood flow 102
71. Hypothermic asanguineous circulatory arrest (HACA) 103
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Summa ry — -^ — ■ 105
72. A new radiochemical esterolytic assay for human urinary urokinase 117
73. Clinical biochemistry of the kallikrein-kinin system 119
74. Peptide biochemistry 122
75. Studies on the enzymes involved in the activation of human plasma kallikrein: PF/dil and Hageman Factor 124
76(c) Antigen-antibody-induced release of mediators from
passively sensitized human lung: Studies on histamine and arginine esterases 128
77. Biochemistry of the kallikrein-kininogen-kinin system 130
78. The role of prostaglandins in the vascular system 133
79. Studies on the isolation and characterization of clostridial electron transfer proteins and other iron- sulfur proteins 135
80(c) Effect of renal infarction on aromatic amino acid decar- boxylase levels in serum and kidney tissue 138
81. Characterization of bovine adrenal dopamine 3-hydroxylase- 140
82. The regulation of the hydroxyindole pathway of tryptophan
me tabolism 142
83. In vivo protein synthesis in heart, aorta, and mesenteric artery of normotensive and spontaneously hypertensive rats
84. Studies on 5,6- and 5, 7-dihydroxytryptamine — Agents that
cause degeneration of serotonergic neurons 147
85. Studies on serotonin N-acetyl-transf erase in mammalian
pineal glands 149
86. Studies on tryptophan hydroxylase — 153
87. Monoamine metabolism and blood pressure of spontaneously hypertensive rats 155
88. Strain differences of catecholamine synthesizing enzyme activity in the rat 158
89. cAMP dependent protein kinase in vascular smooth muscle
and its relationship to calcium uptake 160
90(c) The effects of varying salt intake and of acute saline
infusion on norepinephrine and dopamine excretion in man — 162
91(c) Clinical investigation of cardiovascular drugs 164
92. The role of endogenous prostaglandin synthesis in the
control of the coronary circulation 167
93(c) The role of renal prostaglandins in sodium homeostasis
and blood pressure regulation in man 170
94(c) Studies on the biological role of histamine: Alteration of
histamine metabolism in man and animals by salicylates and
other inhibitors 172
95(c) The biological role of histamine and monoamines: Studies
of histaminase and other enzyme activities in medullary
thyroid carcinoma and hyperlipoproteinemia 175
96. The biological role of histamine and other amines: Participation of histamine and serotonin in inflammation — 179
97. Studies on the biological role of histamine and poly- amines: Ornithine decarboxylase and histaminase activities
in rat thymus and other organs 182
98(c) Metabolism of hydroxyproline and collagen 185
99(c) Studies on the interrelationships between the renin- angiotensin system, urinary and plasma kallikrein and prostaglandins in normal volunteers and hypertensive
patients 188
100. Effects of tolbutamide on plasma renin activity (P.R.A.) — 190 101(c) Urinary and plasma kallikrein 192
102. Studies on renal catecholamine synthesis 195
103. Acute effects of alphamethyldopa on plasma renin activity- 197 104(c) Effects of steroid, thyroid and protein hormones, electro- lytes and psychodelic agents on neural and sensory
f unc tion 199
105(c) Trace metal metabolism 206
106(c) Taste and olfaction 217
Molecular Hematology Branch
Summary 23 7
107. Metabolism of globin mRNA transcription in bone marrow
cells 239
108. The mechanism of hemoglobin biosynthesis in rabbit reticulocyte cell-free systems 240
109(c) Regulation of hemoglobin chain synthesis in beta-
thalassemia 242
110. Evolutionary homology of components of the protein synthesizing machinery 244
111. The mechanism of hemoglobin switching in sheep and goats — 246
112. Mechanism of action of the enzjnne RNA-directed DNA
p o lyme r as e 248
Laboratory of Technical Development
Summary 251
113. Discrete cell temperature measurement study 259
114. Development of stopped-flow micro-calorimetry for the study of biochemical and cellular reactions; applications
to clinical biochemistry 262
115. The development of methods for investigating the mechanism of biochemical reactions important in cardiology, pul- monary and respiratory function, and in the circulation 265
116. An automated method for rapid bacterial and mammalian cell growth and assay 269
117. Fluorescent complexes of proteins 273
118. Methodology in fluorescence measurements 276
119. Applications of fluorescence in biochemistry 279
120. Countercurrent chromatography: Liquid-liquid partition chromatography without solid support (Part II) 282
121. Countercurrent chromatography: Liquid-liquid partition chromatography without solid support (Part I) 286
122(c) Development of the spiral coil membrane blood oxygenator and systems for long-term temporary support of pulmonary and cardiovascular systems 290
123. Blood flow measurement using nuclear magnetic resonance
te chniques 295
124. Peltier-seebeck equilibrator 298
125. Pseudo differentiator 300
126. Measurement of picomole amounts of carbon dioxide 301
127. Calorimetric measurement of carbon dioxide 303
Laboratory of Kidney and Electrolyte Metabolism
Summary 307
128. The effect of chlorpropamide on the water permeability response of the toad bladder to vasopressin 313
129. A study of the leakage of cyclic AMP from the toad
urinary bladder 315
130. A study of the concentration of cyclic AMP in the urinary bladder of the toad 317
131. Function of the early distal tubule of frog 320
132. Function of the thick ascending limb of Henle's loop 322
133. The study of ion transport in renal cortical collecting tubules 3 25
134. Mechanism of salt and water transport by proximal renal
t ubu 1 e s 328
135. Volume regulation in duck erythrocytes. Hormonal control
of cation transport in duck erythrocytes 330
136. Effect of cardiac glycosides on the excitation-contraction system in mammalian skeletal muscle 334
Pulmonary Branch
Summary - Section on Pulmonary Biochemistry 339
137(c) Sites of action and physiologic importance to some
mediators of the type I allergic reaction in man 341
138. Models of lung growth 343
139. Mechanism of collagen and non-collagen protein synthesis
in rabbit lung cell-free systems 345
140(c) The composition and synthesis of collagen in human lung 347
Laboratory of Chemistry
Summary 3 49
141. A study of organic and bio-organic systems by magnetic resonance 355
142. Mass spectrometry and structure of natural products 358
143. Structural characterization of natural products 361
144. X-ray crystallographic studies of molecules of interest for theoretical reasons and for understanding of modes
of physiological actions 364
145. Theoretical and computational investigations of the limitations of small-angle X-ray scattering 367
146. The characterization of natural materials 368
Laboratory of Biochemistiry
Summary 371
147. Studies on the anaerobic metabolism of the branched-
148. Biochemical genetics of NH^-assimilatory enzymes in
149. Kinetics and mechanisms of biochemical reactions 392
150. 1. Anaerobic metabolism of certain amino acids and other
nitrogen compounds with especial reference to the role of B]^2 coenzyme and to electron transfer and phosphory- lation reactions involved. 2. Methane biosynthesis and one-carbon compound
metabolism 398
vi
151. 1. Studies on pyridoxal-P and B-.^ coenz3rrae-dependent
amino mutases. 2. Studies on 3,5-diaralnohexanoate dehydrogenase 402
152. Menadlone-dependent p-nltrophenylphosphatase of
Clostridium stlcklandll 405
153. Ciliary structure and function.
1. Motility: Energy transducing proteins.
2. Mlcrotuble structure: a. Mutants resistant to drugs which Inhibit normal microtubule assembly, b. Tubulin affinity labeling with podophyllotoxln derivatives 407
154. Enzyme structure and mechanisms of action and control 411
155. Regulatory proteins of glutamlne synthetase from E^. coll separation, purification, and regulatory mechanisms 418
156. 1. Components Involved In the adenylylation and
deadenylylatlon of glutamlne synthetase from Pseudomonas putlda 2. Preparation and characterization of E-adenylylated
glutamlne synthetase 470
157. 1. Kinetic reaction mechanism of the y-glutamyl trans-
ferase activity of E^. coll glutamlne synthetase. 2. Purification and properties of the Pjj regulatory protein associated with the E^. coll ATP: Glutamlne synthetase adenylyl transferase 422
158. Regulation of nitrogen metabolism In E^ coll W. The application of continuous culture techniques 425
159. 1. Stereochemical studies of enzymatic reactions
2. Synthetic studies of organic compounds of biological
Interest 429
160. DNA synthesis In E^ coll 433
f'161. Studies on the binding site of mouse myeloma protein,
specific for the 2,4-dlnltrophenyl (DNP) group 436
162. Adenosine triphosphate - actln Interactions. Application of a contact-labeling technique to elucidation of the
protein binding site 438
163. Studies on the self-radlolysls of chymotrypsln, labeled with a trltlated dllsopropylphosphoryl (DIP) group. Re- lationship of free-radical distribution to binding site composition 440
164. Proteolytic fragmentation of fibrinogen 442
165. Structure of enzymatic fragments of fibrinogen 444
166. The Interaction of actln and myosin 446
167. Control of the Interaction of actln and myosin by native tropomyosin ' 449
168. Resolution and characterization of the electron transport chain of E. coll 451
169. The enzymatic propertels of SH]^ blocked myosin and its interaction with actln and ATP 454
170. Membrane biochemistry: Chemical composition and molecular organization of the amoeba plasma membrane 457
vii
171. Membrane biochemistry: Mechanism of membrane fusion in
amoeba in relation to endocytosis 460
172. Cytology of Acanthamoeba 463
Laboratory of Chemical Pharmacology
Summary 465
173. Acetaminophen-induced liver necrosis. I. Relationship between hepatic necrosis and the covalent binding of an acetaminophen metabolite to liver macromolecules 473
174. Acetaminophen-induced liver necrosis. II. Protective role
of glutathione 474
175. Acetaminophen-induced liver necrosis. III. Relationship between severity of liver necrosis and urinary metabolites
of acetaminophen 479
176(c) Acetaicinophen-induced liver necrosis. IV. Clinical
177. Acetaminophen-induced necrosis. V. N-oxidation of acetanilide analogues 485
178. The role of cytochrone P-450 in N-hydroxylation of N- acetylarylamines. Studies with 2-acetylaminofluorene
179. Role of drug-metabolism in the hemolysis caused by aniline
in rats 489
180. Role of the methemoglobinemia in hemolysis caused by
aniline and acetanilide in rats 493
181. Furosemide-induced hepatic and renal tubular necrosis. I. Occurrence of tissue lesions and modulation of the toxicity by treatments which alter drug-metabolizing
act ivi ty 496
182. Furosemide-induced hepatic necrosis. II, Comparison of necrosis with covalent binding of furosemide 498
183. Drug-induced necrosis in the kidney: Comparison of
mercuric chloride, p-aminophenol and furosemide 500
184. Possible role of cytochrome c^ reductase and cytochrome P-450 in the covalent binding of halome thanes to micro- somal protein - studies using specific antibody against cytochrome c^ reductase and by specific destruction of cytochrome P-450 502
185. Mechanism of renal toxicity-induced by chloroform 507
186. Relationship between covalent binding of aromatic hydro- carbons to lungs and production of bronchial necrosis 510
187. Biochemical changes after acetaminophen and furosemide- induced liver injury 516
188(c) Isoniazid-related hepatitis in man: Significance of ab- normal liver function tests during prophylactic therapy and correlation of susceptibility with acetylator pheno- ty pe 5 18
189. Isoniazid-induced hepatic damage: Investigations of the possible formation of an alkylating metabolite 521
190. The influence of pretreatment of rats on isoniazid metabolism 524
191. Spironolactone and testicular cytochrome P-450: Decrease of cytochrome P-450 and progesterone 17a-hydroxylase after spironolactone administration 527
viii
192. Toxic effects of l,l'-dlinethyl-4,4'-dipyrldilium di-
chloride (paraquat) in the rat and rabbit 530
193. Mechanism of dimethylnitrosamine-lnduced liver toxicity 533
194. Covalent binding as a basis of toxicity of chloramphenicol: Possible modification of structure to diminish toxicity of chloramphenicol without loss of antibacterial activity 535
195. Drug metabolism in fetal tissue 537
196. Interaction between nitrofurazone and various tissues
197. Effect of chronic treatment with phenobarbital or 3- methylcholanthrene on cytochrome P-450 and metabolic
activity in testes, adrenals, lungs and liver 541
198. Studies on the role of NADH in the metabolism of drugs
by cytochrome P-450 enzymes in liver microsomes 547
199. Development of a histochemical method for the assay of glutathione in tissues 550
200. The role of electrolytes in binding, mobilization and re- lease of norepinephrine in adrenergic neurons 552
201. Does sodium inhibit calcium-dependent mobilization of neurotransmitter at an intracellular or extracellular
site of action 555
202. The effects of exogenous metabolites of glycolysis on the retention of norepinephrine by rat heart slices 557
203. Studies on the potentiation of CCl/-induced hepatotoxicity in rats by pretreatment with ethyl alcohol, isopropanol,
or acetone 559
204. Mechanisms involved in thyprotective action of Dibenamine pretreatment against the hepatotoxicity of CCl, 562
205. Diene conjugation and lipid peroxidation as a mechanism
of hepatotoxicity 564
206. Temperature-dependent toxicity of adrenergic agonists in
mice 566
207. Studies on the in vivo covalent binding of CCl, and CCl^Br- 568
208. Studies on covalent binding as related to hepatotoxicity: comparison of CCL, and CBrCln 574
209. Covalent binding as a biochemical mechanism for toxicities: Covalent binding of stllbene to liver microsomes 579
210. The mechanisms of hydrazine toxicity 581
211. The role and mechanism of action of hormones in the control of cellular processes: I. The study of the effects of carbamylcholine on cyclic 3' ,5'-guanosine monophosphate
levels in the lung 584
212. The role and mechanism of action of hormones in the control of cellular processes. II. The role of GTP in the prostaglandin E-, activiation of the human blood platelet adenyl cyclase 587
213. Purification and characterization of the enzymatic ion transport system 589
214. Comparative pharmacology of receptor systems in smooth
ix
215. Studies on the chemical nature of the pharmacological receptor systems in vascular smooth muscle. I. Labeling studies of proteins associated with the a-adrenergic
receptor in rabbit aorta 595
216. Biochemical studies of the interaction of oxidant pollutants with the lung. I. The effect of N0„ on phospho- lipid bilayers 597
217. Physicochemical studies of lung surfactant lipoprotein 599
218. Physicochemical studies of complexes between drugs and biomolecules. XI. Fluorescence polarization as a tech- nique for measuring drug binding to plasma albumin 602
219. Physicochemical studies of complexes between drugs and biomolecules. XHI. Spin label studies of horse erythro- cyte carbonic anhydrases C and D 604
220. Physicochemical studies of complexes between drugs and biomolecules. XIV. A spin label study of avidin 606
221. Physicochemical studies of complexes between drugs and biomolecules. XV. A spin label study of azoester (methyl phenyldiazene carboxylate) induced damage to
human erythrocyte ghost membranes 608
Office of the Director, Intramural Research
222. Mathematical theory of renal function 611
223. Computer simulation of renal function 614
ODIR - Section of Pathology
Summary 61 7
224(c) Alcoholism — an important but unemphasized factor predis- posing to infective endocarditis with reemphasis on the syndrome of pneumococcal endocarditis, meningitis and pneumonitis in alcoholics 625
225(c) The quadricuspid semilunar valve 626
226(c) A new cause of a diastolic murmur after replacement of
the aortic valve with a caged ball prosthesis 627
227(c) Calcification of prosthetic valve anuli: A late compli- cation of cardiac valve replacement 628
228(c) Mitral valvular disease. A clinicopathologic survey
of the conditions causing the mitral valve to function
ab no rma 1 ly 629
229(c) Pathologic anatony of the cardiomyopathies (idiopathic dilated and hypertrophic types, infiltrative types and endomyocardial disease with and without eosinophilia) 630
230 Lesions observed in arterial autogenous vein grafts
light and electron microscopic evaluation 631
231(c) Cardiovascular pathology in hyperlipoproteinemia
anatomic observations in 41 necropsy patients with
normal or abnormal serum lipoprotein patterns 632
232(c) Structural features of cardiac myxomas 633
233(c) Operative treatment of hypertrophic obstructive cardio- myopathy. The case against mitral valve replacement 634
234(c) The pathologic anatomy of cardiac valve replacement 635
235(c) Structural alterations in tissue cardiac valves
implanted in hiamans and calves 636
236(c) Non-rheumatic valvular cardiac disease. A clinico- pathologic survey of 27 different conditions causing valvular dysfunction 637
237(c) Ankylosing spondylitis and aortic regurgitation
description of the characteristic cardiovascular lesion
from study of 8 necropsy patients 638
238(c) Cardiac ultrastructural changes induced by daunorubicin
therapy 639
239(c) Early human arteriosclerosis 641
240(c) The coronary arteries and myocardium in acute myocardial
infarction and shock 643
241(c) Pathology of Sipple's syndrome 645
242(c) Chylomicrons and the formation of foam cells in type I
hyperlipoproteinemia 647
243(c) Ultrastructural studies of Tangier disease 648
244(c) Ultrastructure and cytochemistry of glycogen in cardiac
d is eas es 649
245(c) Intermyo fibrillar and nuclear-myo fibrillar connections in human and canine myocardium. An ultrastructural study 650
246(c) Plasma membrane extensions in intercalated discs of human myocardium and their relationship to partial dissociation of the discs 651
247(c) Fatal tachyarrhythmias associated with myofibrillar loss, mitochondrial hyperplasia and fatty change in myocardium. A new clinico-pathologic syndrome in children 652
ODIR - Section on Experimental Atherosclerosis
Summary 653
248. Blood velocity profiles and hemodynamic stresses in the aorta and its major branches, including the large coronary arteries 657
249. Vascular mechanics: arterial wall properties 658
250. The topography of experimental atherosclerotic lesions
in the dog and pig 661
251. Development of canine and miniature swine experimental atherosclerotic animal colonies 663
252. Endothelial nuclear orientation and morphology and its relation to hemodynamic factors- 665
253. An arterial life support system for study of plasma
protein transvascular transport mechanics 667
254. Re-endothelialization of eroded vascular interface 669
255. Growth and metabolism of the aortic smooth muscle cell and its response to various factors implicated in the pathogenesis of atherosclerosis 671
256. The rate and mechanisms of regression of various crystalline lipids placed in the arterial wall 672
257. Quantitation of 4 separate isotopes following oxygen combustion of plasma lipoproteins or aorta 674
258. Aortic metabolism of plasma lipoproteins 675
xl
259 Characterization of canine plasma lipoproteins in the
normal and hyperlipemic animal 677
260 Triglyceride lipase associated with the aortic endo-
thel ium 679
Molecular Disease Branch
S ummary 681
261(c) NHLI Type II coronary intervention study 693
262(c) The study of the lipid storage disease metachromatic
leulcodys trophy 696
263(c) Enzymatic studies in tissue lipid storage diseases 700
26A(c) Glycolipids and other lipid constituents of normal
human liver 705
265(c) Tissue lipidoses: Abnormal biochemistry in tissue lipid
storage diseases 708
266(c) The biochemistry and metabolism of plasma lipoproteins:
The metabolism of plasma lipoproteins in the rat 711
267 The biochemistry of plasma lipoproteins: The structure
of plasma lipoproteins in the rat 713
268(c) The biochemistry of plasma lipoproteins: The structure
of very low density lipoproteins 715
269(c) The biochemistry and metabolism of plasma lipoproteins:
Post-heparin lipolytic enzymes and their role in normal
and abnormal lipid transport and clearance 718
270 Structure-function relationships of the plasma apolipo-
pr o t eins 722
271 1. Chemistry and physical properties of human parathyroid
hormone (HPTH) . 2. Mass spectroscopic identification of the methyl and phenyl thiazolinone and thiohydantoin amino acids ob- tained from the automated edman degradation of poly- peptides and proteins. 3. Chemistry of the high density lipoprotein, apo-Lp-G-I
(A-I ) 7 24
272(c) The biochemistry and metabolism of plasma lipoproteins: The turnover and function of very low density, low
density and high density lipoproteins 731
273(c) Tissue lipidoses: Microscopic studies in tissue lipid
storage diseases 735
274(c) The biochemistry and metabolism of plasma lipoproteins:
Studies of familial hyperlipoproteinemia 738
275 The biochemistry and metabolism of plasma lipoproteins: A
study of a differentiated mouse liver cell line 747
276 The effect of insulin and dexamethasone on the metabolism
of mammalian cells in culture 751
277 Release of histamine from mast cells 754
278 Adipose tissue metabolism: Lipoprotein lipase 756
279 Role of cyclic AMP and cyclic CMP in modulation of growth, differentiation and aging of cells in culture 758
280 Effects of hormones on metabolism of adipose tissue
studied in vitro 760
xll
281 Investigation of the control of cyclic A^G' phospho- diesterase (PDL) activity in fat cells 762
282 Metabolism of Guanosine 3 *, 5 '-monophosphate in lung 76A
283 Adipose tissue hormone-sensitive lipase: Mechanisms for regulation of activity 767
Laboratory of Biochemical Genetics
284 Gene expression in neuroblastoma x glioma hybrid cells 777
285 Biochemical assays for action potential or receptor ionophores 778
286 Selection for cells of neuronal origin synthesizing
specific transmitters 779
287 Development of cell lines with neuronal phenotypes 780
288 Study of acetylcholine receptors on muscle cells grown
in vitro 781
289 Probing neuronal acetylcholine receptors with a—
bungaro toxin 782
290 The acetylcholine receptors of the nervous system in
Aplysia 783
291 Development of cell lines with neuronal properties 785
292 Development of cell lines expressing neuronal phenotypes by treating embryonic neuronal tissue with ethylnitro-
sourea 786
293 The storage and release of molecules required for synaptic communication 787
294 Ultrastructural investigation of the neuronal properties of somatic cell hybrids between neuroblastoma and other
cell types 788
295 Cell recognition and adhesion as early events in synapse formation 790
296 Glycolipids of neuroblastoma and neuroblastoma x glioma
hybrid cells 791
297 Dissociated cell culture assay of nerve growth factor 792
298 Neural models for a two dimensional nerve net 793
299 Chemistry and function of microtubules 795
300 A general method for mapping mammalian genes using somatic
cell hybrids 797
301 Development of an in vitro model of the Lesch-Nyhan
Syndrome 798
302 Genetic analysis of A particles using somatic cell hybrids- 799
303 Genetic analysis of adenosine 3',5'-cyclic monophosphate
(cAMP) in mammalian cells 800
304 Disseminated intravascular coagulation 801
305 Genetic analysis of differentiated functions using somatic
cell hybrids 803
306 The biology of cyclic AMP in E. coli 804
307 Mechanisms in protein synthesis 806
308 Cultured cell systems for neurobiology 808
309 A histof luorescence study of cultured chromaffin cells 810
ANNUAL REPORT OF THE CARDIOLOGY BRANCH NATIONAL HEART AND LUNG INSTITUTE July 1, 1972 through June 30, 1973
The experimental interests of the Cardiology Branch developed over the past few years have continued. These relate to the pathogenesis, pathophysiology, and treatment of coronary artery disease; the ultras true tural and molecular mechanisms responsible for impaired contractile function of the heart; the de- velopment of the potential capabilities of echocardiography; and the applica- tion of echocardiographic and electron microscopic techniques to define the basic developmental pathophysiology, genetics, and epidemiology of asymmetric septal hypertrophy, or ASH (idiopathic hypertrophic subaortic stenosis).
CORONARY ARTERY DISEASE
Mechanisms of Sudden Death and Pharmacologic Treatment of Acute Myocardial Infarction
I) Atropine in Acute Myocardial Infarction. Approximately 60% of deaths caused by acute myocardial infarction (AMI) occur within the first two hours of onset of symptoms and before medical aid arrives. Since bradycardia 1) oc- curs in a high percentage of patients seen soon after the onset of AMI, and 2) is believed to predispose to the development of ventricular ectopic rhythms and ventricular fibrillation (VF) , it has been suggested that a substantial decrease in the number of prehospital arrhythmic deaths would result if atro- pine were administered shortly after onset of ischemic pain in patients with bradycardia. We have performed several studies to test this hypothesis.
1) When AMI is produced in conscious closed-chest dogs by inflation of a balloon cuff previously implanted around the left anterior coronary artery (LAD), arrhythmias, including VF, tend to develop more frequently in dogs treat- ed with atropine than in randomly selected control dogs. We also found that the type of ventricular ectopic beats occurring during the early phase of AMI in dogs can be characterized as malignant arrhythmias (those that when present often lead to VF and death) and benign arrhythmias (those that never are asso- ciated with the eventual development of VF) . Using this information, we demon- strated that while benign arrhythmias are successfully suppressed by increasing heart rate with either atropine or atrial pacing, these interventions rarely abolish malignant arrhythmias. We next determined the influence of pronounced and persistent bradycardia, produced by vagal stimulation, on the incidence of benign and malignant arrhythmias during AMI. In the same closed-chest dog prep- aration heart rate was held between 80 and 100 beats /min in the control group and between 40 and 60 in the bradycardia group. Our findings demonstrated that the overall incidence of arrhythmias as well as the incidence of malignant ar- rhythmias was not increased by bradycardia during AMI. Moreover, when arrhyth- mias occurred in the bradycardia group, they were more short-lived, with sig- nificantly fewer arrhythmias present 45 minutes after AMI in the bradycardia as compared to the normal heart rate dogs. Thus a) bradycardia appears to ex- ert a favorable influence on the incidence of ventricular arrhythmias during experimental AMI, b) increasing heart rate with atropine tends to have a dele- terious effect on arrhythmias, c) not all ventricular arrhythmias are potenti-
ally lethal, and d) interventions may have very different effects on benign and malignant arrhythmias, a finding indicating that the clinical efficacy of antiarrhythmic drugs cannot be based on the assumption that a decrease in over- all arrhythmia incidence is equivalent to a reduction in mortality.
2) Additional studies were designed to determine the mechanisms responsible for the apparent protective effects of bradycardia and deleterious effects of increasing heart rate on serious ventricular arrhythmias during AMI.
a) First, we found a highly significant direct correlation between per cent increase in heart rate produced by atropine and per cent increase in is- chemic injury (as determined by ST segment elevation recorded from intramyo- cardial electrodes) over a heart ratf range of 30 to 180 beats/min. This find- ing indicates that the increase in MVO2 produced by a faster rate is sufficient to unfavorably alter the critical balance between myocardial oxygen demand and supply, thereby producing increased ischemia.
b) Previous studies from other laboratories showed that bradycardia and that acute myocardial ischemia lower VF threshold and increase disparity of myocardial refractory periods, findings indicating diminished electrial stabil- ity and leading to the now commonly accepted concept that increasing heart rate during AMI protects against serious ventricular arrhythmias. However, the elec- trophysiologic effects of a faster heart rate during AMI were not explored, an important omission since elevating heart rate during AMI increases the degree of ischemic injury. We thus measured VF threshold and disparity of refractory periods during AMI in open-chest dogs. As we suspected, fibrillation threshold was lower at faster heart rates and disparity of refractory periods was great- er. Thus, lower heart rates during AMI lead to electrophysiologic changes that protect against the development of serious ventricular arrhythmias. In addi- tion, we found that vagal stimulation per se, independent of changes in heart rate, enhanced electrical stability of the ventricle. This was an unexpected result, since it commonly is believed that vagal innervation of the ventricles is not physiologically important. To localize the cholinergic pathways respon- sible for this stabilizing electrophysiologic effect, vinblastine, a neurotoxic agent, was injected into the para-aortic area adjacent to the AV node. This area contains cholinergic ganglia that supply postganglionic fibers to the ven- tricle. Five to 7 days later, vagal stimulation caused atrial slowing but did not change VF threshold, signifying that vagal efferents to the atria were in- tact but those to the ventricle (responsible for increasing electrical stabil- ity) were functionally inoperative. The anatomic correlate of this electro- physiologic finding was obtained by studying the morphologic relationship of the ventricular conducting system and cholinergic nerves within the ventricu- lar septum. In control dogs Purkinje fibers were encapsulated by numerous cholinergic nerves. However, few cholinergic fibers were found in vinblastine treated dogs. We conclude that 1) stimulation of the vagus nerve decreases vulnerability of the ventricle to fibrillation, and 2) this action is mediated by a rich network of cholinergic nerves intimately related to Purkinje fibers
in the ventricular septum.
3) To determine whether the electrophysiologic effects of vagal stimulation are of functional significance, the incidence of VF is being assessed in dogs with and without vagal stimulation during 30 min of LAD occlusion. In one
series of experiments heart rate is allowed to slow in the vagally stimulated dogs. While 87% of the control dogs have fibrillated after 30 minutes of AMI, only 407o of the vagally treated dogs have. Moreover, when heart rate is held constant (to assess the effects of vagal stimulation per se ) , 88% of control dogs have developed VF during coronary occlusion, vihile only 45% of the vagally stimulated dogs have.
II, Nitroglycerin and Acute Myocardial Infarction. Nitroglycerin (TNG) gen- erally is believed contraindicated in the treatment of AMI because of the po- tential deleterious effects of a decrease in blood pressure (and thereby coro- nary perfusion pressure) and reflex increase in heart rate. If TNG dilates coronary collateral vessels, however, its net effect may be beneficial. We have now completed several studies, the results of which suggest that TNG may favorably alter the course of AMI.
1) We first demonstrated that hypotension, either reflexly induced (by stim- ulating the carotid baroreceptors) or induced by hemorrhage, increases the de- gree of ischemic injury occurring during AMI. In contrast, when identical lev- els of hypotension are produced by TNG, the degree of ST elevation (and presum- ably, the degree of ischemia) is reduced during 15 minutes of coronary occlu- sion. Moreover, v^en arterial pressure is held constant by simultaneous infu- sion of the alpha receptor agonists methoxamine or phenylephrine, the current of injury is reduced further. To confirm that the beneficial action on ST seg- ments reflects a decrease in myocardial ischemia, another series of randomly treated closed-chest dogs was studied. In these animals the effects of TNG and methoxamine on development of myocardial infarction was examined after 5 hrs of coronary occlusion. Ten minutes after onset of occlusion, ST elevation was measured in each of several previously implanted intramyocardial electrodes, and dogs were randomized into control and treated groups. Treated dogs received iv TNG during the remainder of the 5 hr occlusion, and arterial pressure was maintained at preinfusion levels with iv methoxamine. Six of 13 control dogs died; 2 of 14 treated dogs died. After 24 hours severity of infarct was as- sessed in survivors by measurement of creatine phosphokinase (CPK) content of myocardium subjacent to each electrode. In controls, a significant reduction
in CPK occurred in 48 of 57 electrode sites where ST elevation was present af- ter 10 minutes of occlusion. Only 19 of 69 such sites showed significant CPK reduction in treated dogs (p < .001). We conclude that in experimental coro- nary occlusion TNG reduces myocardial ischemia, an effect greatly potentiated by preventing the decrease in coronary perfusion pressure and reflex tachy- cardia with methoxamine.
2) It currently is believed that TNG does not significantly increase myocar- dial blood flow in patients with diseased coronary arteries. Thus, relief of angina pectoris by TNG is ascribed to a lowering of myocardial oxygen demands. The studies just presented, however, demonstrated that even when 2 of the more important determinants of myocardial oxygen consumption were held constant (arterial pressure and heart rate), TNG still reduced ischemic injury. There- fore, to determine \Aiether TNG can increase blood flow to ischemic regions, the capacity of coronary collaterals to supply blood to areas of potential ischemia was assessed before and after TNG in dogs in which MVO2 was held constant, and
in patients undergoing saphenous vein bypass surgery. In the animal experiments.
we found that whenever TNG reduced the current of injury recorded by epicardial electrodes after LAD occlusion, the reduction in ischemic injury always corre- lated with an increase in retrograde coronary flow, a measure of the capacity of the collaterals to supply blood to ischemic myocardium. In patients under- going coronary artery bypass operation, flow and pressure measurements were made from the distally (but not proximally) attached vein graft while the fi- brillating heart was sustained by cardiopulmonary bypass. Following TNG coro- nary collateral resistance fell. Thus, TNG can reduce ischemic injury during experimental AMI as a result of increased collateral flow. Moreover, TNG also is capable of diminishing resistance to collateral flow in man despite severe multivessel disease.
3) We next studied the electrophysiologic effects of TNG during AMI produced in the open-chest dog. VF threshold was measured during ischemia in the pres- ence and absence of TNG infusion, and during ischemia when phenylephrine was administered with TNG to maintain arterial pressure at control levels. We found that TNG enhanced electrical stability of the heart during AMI; moreover when arterial pressure was restored by simultaneous infusion of phenylephrine, VF threshold during AMI returned to control values.
4) Finally, a highly malignant model of AMI in dogs was developed to deter- mine whether the beneficial effects of TNG on ischemic injury, collateral re- sistance, and electrical stability could alter the incidence of VF and death during AMI. Twelve of 13 control dogs died from VF during 30 minutes of AMI; in contrast, only 7 of 14 dogs treated with TNG and methoxamine died (p < .05). As a result of these studies, we conclude that the long-standing clinical ca- veat not to administer TNG to patients with AMI may be in error; indeed, TNG may be uniquely valuable in the treatment of AMI, having the potential to di- minish ischemic injury and reduce the incidence of fatal arrhythmias.
Surgical Therapy of Coronary Artery Disease
Despite the enormous number of aorto-coronary bypass grafts being performed worldwide, little is known of the effects of operation on overall and regional myocardial function. We have studied 22 consecutive patients who underwent cardiac catheterization before and one week to 9 months after aorto-coronary bypass. We found that all patients, regardless of whether grafts were patent or occluded, improved clinically. In many angina was relieved totally. How- ever, overall LV performance deteriorated in 10 of these patients and improved in only one. In patients whose grafts were occluded, ejection fraction de- creased an average of 187o (p "^ .05). Twenty-eight LV wall segments were sup- plied by patent grafts; function improved in 6, was unchanged in 13, and dete- rioBted in 9. Twenty-two segments were supplied by occluded grafts; none im- proved, 14 were unchanged, and 8 deteriorated. We conclude that regardless of graft patency, most patients improve clinically. Despite this, deterioration of myocardial function