Cardiac repolarization: Current knowledge, critical gaps, and new approaches to drug development and patient management

American Heart Journal

Volumn 144, Issue 5, 2002, Pages 769-781

  Anderson, Mark E ^a,b   Al Khatib, Sana M ^a,b   Roden, Dan M ^a,b   Califf, Robert M ^a,b  

^a VANDERBILT UNIVERSITY MEDICAL CENTER   (United States)

^b DUKE CLINICAL RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AMIODARONE; ANTIARRHYTHMIC AGENT; CISAPRIDE; DILTIAZEM; ION CHANNEL; TERFENADINE; VERAPAMIL;

ANIMAL CELL; ARRHYTHMOGENESIS; CONFERENCE PAPER; DRUG INDUCED DISEASE; DRUG SCREENING; ELECTROCARDIOGRAM; GENE MUTATION; GENETIC DISORDER; GENOTYPE; HEART ARRHYTHMIA; HEART MUSCLE CONDUCTION DISTURBANCE; HEART REPOLARIZATION; HUMAN; LONG QT SYNDROME; NONHUMAN; PHENOTYPE; PRIORITY JOURNAL; RISK BENEFIT ANALYSIS; SINGLE NUCLEOTIDE POLYMORPHISM;

EID: 0036847942     PISSN: 00028703     EISSN: None     Source Type: Journal    
DOI: 10.1067/mhj.2002.125804     Document Type: Conference Paper

References (118)

1. Liu XK, Wang W, Ebert SN, et al. Female gender is a risk factor for torsades de pointes in an in vitro animal model. J Cardiovasc Pharm 1999;34:287-94.
2. Curran ME, Splawski I, Timothy KW, et al. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 1995;80:795-803.
3. Neyroud N, Tesson F, Denjoy I, et al. A novel mutation in the potassium channel gene KVLQT1 causes the Jervell and Lange-Nielsen cardioauditory syndrome. Nat Genet 1997;15:186-9.
4. Volders PG, Sipido KR, Vos MA, et al. Cellular basis of biventricular hypertrophy and arrhythmogenesis in dogs with chronic complete atrioventricular block and acquired torsade de pointes. Circulation 1998;98:1136-47.
5. Kaab S, Nuss HB, Chiamvimonvat N, et al. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ Res 1996;78:262-73.
6. + currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res 1993;73:379-85.
7. Walker AM, Szneke P, Weatherby LB, et al. The risk of serious cardiac arrhythmias among cisapride users in the United Kingdom and Canada. Am J Med 1999;107:356-62.
8. Griffin JP. Prepulsid withdrawn from UK and US markets. Adv Drug React Toxicol Rev 2000;19:177.
9. Morganroth J, Brown AM, Critz S, et al. Variability of the QTc interval: Impact on defining drug effect and low-frequency cardiac event. Am J Cardiol 1993;72(Suppl):26-31B.
10. Hammond TG, Carlsson L, Davis AS, et al. Methods of collecting and evaluating non-clinical cardiac electrophysiology data in the pharmaceutical industry: Results of an international survey. Cardiovasc Res 2001 ;49:741-50.
11. January CT, Riddle JM, Salata JJ. A model for early afterdepolarizations: Induction with the Ca2+ channel agonist Bay K 8644. Circ Res 1988;62:563-71.
12. January CT, Riddle JM. Early afterdepolarizations: Mechanism of induction and block. A role for L-type Ca2+ current. Circ Res 1989;64:977-90.
13. Wu Y, MacMillan LB, McNeill RB, et al. CaM kinase augments cardiac L-type Ca2+ current: A cellular mechanism for long Q-T arrhythmias. Am J Physiol 1999;276:H2168-78.
14. Wu Y, Roden DM, Anderson ME. Calmodulin kinase inhibition prevents development of the arrhythmogenic transient inward current. Circ Res 1999;84:906-12.
15. 2+ release causes myocyte depolarization - Underlying mechanism and threshold for triggered action potentials. Circ Res 2000;87:774-80.
16. January CT, Fozzard HA. Delayed afterdepolarizations in heart muscle: Mechanisms and relevance. Pharm Rev 1988;40:219-27.
17. Roden DM, Anderson ME. The pause that refreshes, or does it? Mechanisms in torsades de pointes. Heart 2000;84:235-7.
18. Pak PH, Nuss HB, Tunin RS, et al. Repolarization abnormalities, arrhythmia and sudden death in canine tachycardia-induced cardiomyopathy. J Am Coll Cardiol 1997;30:576-84.
19. Pogwizd SM, McKenzie JP, Cain ME. Mechanisms underlying spontaneous and induced ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. Circulation 1998;98:2404-14.
20. Pogwizd SM, Qi M, Yuan W, et al. Upregulation of Na(+)/Ca(2+) exchanger expression and function in an arrhythmogenic rabbit model of heart failure. Circ Res 1999;85:1009-19.
21. Verduyn SC, Vos MA, van der Zande J, et al. Further observations to elucidate the role of interventricular dispersion of repolarization and early afterdepolarizations in the genesis of acquired torsade de pointes arrhythmias: A comparison between almokalant and d-sotalol using the dog as its own control. J Am Coll Cardiol 1997;30:1575-84.
22. Chinushi M, Restivo M, Caref EB, et al. Electrophysiological basis of arrhythmogenicity of QT/T alternans in the long-QT syndrome: Tridimensional analysis of the kinetics of cardiac repolarization. Circ Res 1998;83:614-28.
23. El Sherif N, Caref EB, Chinushi M, et al. Mechanism of arrhythmogenicity of the short-long cardiac sequence that precedes ventricular tachyarrhythmias in the long QT syndrome. J Am Coll Cardiol 1999;33:1415-23.
24. Sicouri S, Antzelevitch C. A subpopulation of cells with unique electrophysiological properties in the deep subepicardium of the canine ventricle: The M cell. Circ Res 1991;68:1729-41.
25. Litovsky SH, Antzelevitch C. Differences in the electrophysiological response of canine ventricular subendocardium and subepicardium to acetylcholine and isoproterenol: A direct effect of acetylcholine in ventricular myocardium. Circ Res 1990;67:615-27.
26. Baker LC, London B, Choi BR, et al. Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia. Circ Res 2000;86:396-407.
27. Liu DW, Gintant GA, Antzelevitch C. Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circ Res 1993;72:671-87.
28. Ks) in canine ventricular epicardial, midmyocardial, and endocardial myocytes: A weaker lKs contributes to the longer action potential of the M cell. Circ Res 1995;76:351-65.
29. Nabauer M, Beuckelmann DJ, Uberfuhr P, et al. Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation 1996;93:168-77.
30. Antzelevitch C, Sun ZQ, Zhang ZQ, et al. Cellular and ionic mechanisms underlying erythromycin-induced long QT intervals and torsade de pointes. J Am Coll Cardiol 1996;28:1836-48.
31. Sicouri S, Antzelevitch C. Afterdepolarizations and triggered activity develop in a select population of cells (M cells) in canine ventricular myocardium: The effects of acetylstrophanthidin and Bay K 8644. Pacing Clin Electrophysiol 1991;14:1714-20.
32. Sicouri S, Antzelevitch C. Drug-induced afterdepolarizations and triggered activity occur in a discrete subpopulation of ventricular muscle cells (M cells) in the canine heart: Quinidine and digitalis. J Cardiovasc Electrophysiol 1993;4:48-58.
33. Lin Q, Srivastava D, Olson EN. A transcriptional pathway for cardiac development. Cold Spring Harb Symp Quant Biol 1997; 62:405-11.
34. Carlsson L, Drews L, Duker G. Rhythm anomalies related to delayed repolarization in vivo: Influence of sarcolemmal Ca++ entry and intracellular Ca++ overload. J Pharm Exp Ther 1996; 279:231-9.
35. Mozur A, Roden DM, Anderson ME. Systemic administration of calmodulin antagonist W-7 or protein kinase A inhibitor H-8 prevents torsade de pointes in rabbits. Circulation 1999;100:2437-42.
36. Verduyn SC, Vos MA, Gorgels AP, et al. The effect of flunarizine and ryanodine on acquired torsades de pointes arrhythmias in the intact canine heart. J Cardiovasc Electrophysiol 1995;6:189-200.
37. Bouchard RA, Clark RB, Giles WR. Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes: Action potential voltage-clamp measurements. Circ Res 1995;76:790-801.
38. Zuhlke RD, Pitt GS, Deisseroth K, et al. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 1999;399:159-62.
39. Peterson BZ, DeMaria CD, Adelman JP, et al. Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels. Neuron 1999;22:549-58.
40. Pate P, Mochca-Morales J, Wu Y, et al. Determinants for calmodulin binding on voltage-dependent Ca2+ channels. J Biol Chem 2000;275:39786-92.
41. Braun AP, Schulman H. The multifunctional calcium/calmodulindependent protein kinase: From form to function. Annu Rev Physiol 1995;57:417-45.
42. Anderson ME, Braun AP, Wu Y, et al. KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharm Exp Ther 1998;287:996-1006.
43. Kaab S, Dixon J, Duc J, et al. Molecular basis of transient outward potassium current downregulation in human heart failure: A decrease in Kv4.3 mRNA correlates with a reduction in current density. Circulation 1998;98:1383-93.
44. Beuckelmann DJ, Erdmann E. Ca(2+)-currents and intracellular [Ca2+]i-transients in single ventricular myocytes isolated from terminally failing human myocardium. Bas Res Cardiol 1992; 87(1 Suppl):235-43.
45. Kruger C, Erdmann E, Nabauer M, et al. Intracellular calcium handling in isolated ventricular myocytes from cardiomyopathic hamsters (strain BlO 14.6) with congestive heart failure. Cell Calcium 1994;16:500-8.
46. Bailey BA, Houser SR. Calcium transients in feline left ventricular myocytes with hypertrophy induced by slow progressive pressure overload. J Mol Cell Cardiol 1992;24:365-73.
47. Nuss HB, Kaab S, Kass DA, et al. Cellular basis of ventricular arrhythmias and abnormal automaticity in heart failure. Am J Physiol 1999;277:H80-91.
48. Wu Y, Temple J, Zhang R, et al. Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy. Circulation 2002;106:1288-93.
49. Kong W, Po S, Yamagishi T, et al. Isolation and characterization of the human gene encoding lto: Further diversity by alternative mRNA splicing. Am J Physiol 1998;275:H1963-70.
50. Wang Q, Curran ME, Splawski l, et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat Genet 1996;12:17-23.
51. ks potassium channel. Nature 1996;384:80-3.
52. Sanguinetti MC, Jiang C, Curran ME, et al. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the lKr potassium channel. Cell 1995;81:299-307.
53. Franqueza L, Lin M, Shen J, et al. Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with mink subunits. J Biol Chem 1999;274:21063-70.
54. Napolitano C, Schwartz PJ, Brown AM, et al. Evidence for a cardiac ion channel mutation underlying drug-induced QT prolongation and life-threatening arrhythmias. J Cardiovasc Electrophysiol 2000;11:691-6.
55. Sanguinetti MC. Long QT syndrome: Ionic basis and arrhythmia mechanism in long QT syndrome type 1. J Cardiovasc Electrophysiol 2000;11:710-2.
56. Li H, Chen Q, Moss A J, et al. New mutations in the KVLQT1 potassium channel that cause long-QT syndrome. Circulation 1998; 97:1264-9.
57. Tanaka T, Nagai R, Tomoike H, et al. Four novel KVLQT1 and four novel HERG mutations in familial long-QT syndrome. Circulation 1997;95:565-7.
58. Chen J, Zou AR, Splawski I, et al. Long QT syndrome-associated mutations in the Per-Arnt-Sim (PAS) domain of HERG potassium channels accelerate channel deactivation. J Biol Chem 1999;274:10113-8.
59. Splawski I, Shen J, Timothy KW, et al. Spectrum of mutations in long-QT syndrome genes: KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Circulation 2000;102:1178-85.
60. Wang Q, Shen J, Splawski I, et al. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 1995;80:805-11.
61. Bennett PB, Yazawa K, Makita N, et al. Molecular mechanism for an inherited cardiac arrhythmia. Nature 1995;376:683-5.
62. Kambouris NG, Nuss HB, Johns DC, et al. A revised view of cardiac sodium channel "blockade" in the long-QT syndrome. J Clin Invest 2000;105:1133-40.
63. Abbott GW, Sesti F, Splawski I, et al. MiRP1 forms I-Kr potassium channels with HERG and is associated with cardiac arrhythmia. Cell 1999;97:175-87.
64. Yang P, Wei J, Murray KT, et al. Frequency of ion channel mutations and polymorphisms in a large population of patients with drug-associated long QT syndrome [abstract]. Pacing Clin Electrophysiol 2001;24:579.
65. K9 potassium channels. Circulation 1999;100:1-495.
66. Zhang L, Timothy KW, Vincent GM, et al. Spectrum of ST-T-wave patterns and repolarization parameters in congenital long-QT syndrome: ECG findings identify genotypes. Circulation 2000; 102:2849-55.
67. + channel blockade and to increases in heart rate: Implications for gene-specific therapy. Circulation 1995;92:3381-6.
68. Compton SJ, Lux RL, Ramsey MR, et al. Genetically defined therapy of inherited long-QT syndrome: Correction of abnormal repolarization by potassium. Circulation 1996;94:1018-22.
69. Schwartz PJ, Priori SG, Spazzolini C, et al. Genotype-phenotype correlation in the long-QT syndrome: Gene-specific triggers for life-threatening arrhythmias. Circulation 2001;103:89-95.
70. Wilde AA, Jongbloed RJ, Doevendans PA, et al. Auditory stimuli as a trigger for arrhythmic events differentiate HERG-related (LQTS2) patients from KVLQT1-related patients (LQTS1). J Am Coll Cardiol 1999;33:327-32.
71. Moss AJ, Robinson JL, Gessman L, et al. Comparison of clinical and genetic variables of cardiac events associated with loud noise vs. swimming among subjects with the long QT syndrome. Am J Cardiol 1999;84:876-9.
72. Moss AJ, Zareba W, Benhorin J, et al. ECG T-wave patterns in genetically distinct forms of the hereditary long QT syndrome. Circulation 1995;92:2929-34.
73. Vincent GM, Timothy KW, Leppert M, et al. The spectrum of symptoms and QT intervals in carriers of the gene for the long-QT syndrome. N Engl J Med 1992;327:846-52.
74. Zareba W, Moss AJ, Schwartz PJ, et al. Influence of genotype on the clinical course of the long-QT syndrome. N Engl J Med 1998; 339:960-5.
75. Priori SG, Napolitano C, Schwartz PJ. Low penetrance in the long-QT syndrome: Clinical impact. Circulation 1999;99:529-33.
76. Locati EH, Zareba W, Moss AJ, et al. Age- and sex-related differences in clinical manifestations in patients with congenital long-QT syndrome: Findings from the International LQTS Registry. Circulation 1998;97:2237-44.
77. Lehmann MH, Timothy KW, Frankovich D, et al. Age-gender influence on the rate-corrected QT interval and the QT-heart rate relation in families with genotypically characterized long QT syndrome. J Am Coll Cardiol 1997;29:93-9.
78. Moss AJ, Schwartz PJ, Crampton RS, et al. The long QT syndrome: Prospective longitudinal study of 328 families. Circulation 1991;84:1136-44.
79. Moss AJ, Zareba W, Hall WJ, et al. Effectiveness and limitations of beta-blocker therapy in congenital long-QT syndrome. Circulation 2000;101:616-23.
80. Dorostkar PC, Eldar M, Belhassen B, et al. Long-term follow-up of patients with long-QT syndrome treated with beta-blockers and continuous pacing. Circulation 1999;100:2431-6.
81. Priebe L, Beuckelmann DJ. Simulation study of cellular electric properties in heart failure. Circ Res 1998;82:1206-23.
82. Berger RD, Kasper EK, Baughman KL, et al. Beat-to-beat QT interval variability: Novel evidence for repolarization lability in ischemic and nonischemic dilated cardiomyopathy. Circulation 1997; 96:1557-65.
83. Mazur A, Anderson ME, Bonney S, et al. Pause-dependent polymorphic ventricular tachycardia during long-term treatment with dofetilide: A placebo-controlled, implantable cardioverter-defibrillator-based evaluation. J Am Coll Cardiol 2001;37:1100-5.
84. Soffer J, Dreifus LS, Michelson EL. Polymorphous ventricular tachycardia associated with normal and long QT intervals. Am J Cardiol 1982;49:2021-9.
85. Morganroth J, Brozovich FV, McDonald JT, et al. Variability of the QT measurement in healthy men, with implications for selection of an abnormal QT value to predict drug toxicity and proarrhythmia. Am J Cardiol 1991;67:774-6.
86. Bazett HC. An analysis of the time-relations of electrocardiograms. Heart 1920;7:353-62.
87. Brendorp B, Elming H, Jun L, et al. QTc interval as a guide to select those patients with congestive heart failure and reduced left ventricular systolic function who will benefit from antiarrhythmic treatment with dofetilide. Circulation 2001;103:1422-7.
88. Viswanathan PC, Rudy Y. Pause induced early afterdepolarizations in the long QT syndrome: A simulation study. Cardiovasc Res 1999;42:530-42.
89. The European Agency for the Evaluation of Medicinal Products, Committee for Proprietary Medicinal Products (CPMP). Points to Consider: The Assessment of the Potential for QT Interval Prolongation by Non Cardiovascular Medicinal Products Accessed: December 17, 1997. Available at: www.emea.eu.intpdfshumanswp09869en.pds.
90. Priori SG, Napolitano C, Diehl L, et al. Dispersion of the QT interval: A marker of therapeutic efficacy in the idiopathic long QT syndrome Circulation 1994;89:1681-9.
91. Day CP, McComb JM, Campbell RW. QT dispersion: An indication of arrhythmia risk in patients with long QT intervals. Br Heart J 1990;63:342-4.
92. Hii JT, Wyse DG, Gillis AM, et al. Precordial QT interval dispersion as a marker of torsade de pointes. Disparate effects of class la antiarrhythmic drugs and amiodarone. Circulation 1992;86:1376-82.
93. Kors JA, van Herpen G, van Bemmel JH. QT dispersion as an attribute of T-loop morphology. Circulation 1999;99:1458-63.
94. Zabel M, Klingenheben T, Franz MR, et al. Assessment of QT dispersion for prediction of mortality or arrhythmic events after myocardial infarction: Results of a prospective, long-term follow-up study. Circulation 1998;97:2543-50.
95. Brendorp B, Elming H, Jun L, et al. QT dispersion has no prognostic information for patients with advanced congestive heart failure and reduced left ventricular systolic function. Circulation 2001;103:831-5.
96. Brooksby P, Batin PD, Nolan J, et al. The relationship between QT intervals and mortality in ambulant patients with chronic heart failure - The United Kingdom Heart Failure Evaluation and Assessment of Risk Trial (UK-HEART). Eur Heart J 1999;20:1335-41.
97. Nowinski K, Jensen S, Lundahl G, et al. Changes in ventricular repolarization during percutaneous transluminal coronary angioplasty in humans assessed by QT interval, QT dispersion and T vector loop morphology. J Intern Med 2000;248:126-36.
98. Lehmann MH, Suzuki F, Fromm BS, et al. T wave "humps" as a potential electrocardiographic marker of the long QT syndrome. J Am Coll Cardiol 1994;24:746-54.
99. Ghadebo DT, Trimble RW, Khoo MSC, et al. Calmodulin inhibitor W-7 unmasks a novel electrocardiographic parameter that predicts initiation of torsade de pointes. Circulation 2002;105:770-4.
100. El-Sherif N, Zeiler RH, Graelius W, et al. QTU prolongation and polymorphic ventricular tachyarrhythmias due to bradycardia-dependent early afterdepolarizations. Circ Res 1988;63:286-305.
101. Antzelevitch C, Sicouri S. Clinical relevance of cardiac arrhythmias generated by afterdepolarizations: Role of M cells in the generation of U waves, triggered activity and torsade de pointes. J Am Coll Cardiol 1994;23:259-77.
102. Carlsson L, Almgren O, Duker G. QTU-prolongation and torsades de pointes induced by putative class III antiarrhythmic agents in the rabbit: Etiology and interventions. J Cardiovasc Pharm 1990;16:276-85.
103. Kurita T, Ohe T, Shimizu W, et al. Early afterdepolarization-like activity in patients with class IA induced long QT syndrome and torsades de pointes. Pacing Clin Electrophysiol 1997;20:695-705.
104. Rosenbaum DS, Jackson LE, Smith JM, et al. Electrical alternans and vulnerability to ventricular arrhythmias. New Engl J Med 1994;330:235-41.
105. Pasture JM, Girouard SD, Laurita KR, et al. Mechanism linking T-wave alternans to the genesis of cardiac fibrillation. Circulation 1999;99:1385-94.
106. Roden DM, Lazzara R, Rosen M, et al. Multiple mechanisms in the long-QT syndrome: Current knowledge, gaps, and future directions. Circulation 1996;94:1996-2012.
107. Crumb WJ Jr. Rate-dependent blockade of a potassium current in human atrium by the antihistamine loratadine. Br J Pharm 1999; 126:575-80.
108. Mitcheson JS, Chen J, Lin M, et al. A structural basis for drug-induced long QT syndrome. Proc Nat Acad Sci 2000;97:12329-33.
109. Rampe D, Wible B, Fedida D, et al. Verapamil blocks a rapidly activating delayed rectifier K+ channel cloned from human heart. Mol Pharmacol 1993;44:642-8.
110. Wegener JW, Nawrath H. Action of tertiary phenylalkylamines on cardiac transient outward current from outside the cell membrane. Naunyn-Schmiedebergs Arch Pharmacol 1996;354:746-54.
111. MacNeil DJ, Davies RO, Deitchman D. Clinical safety profile of sotalol in the treatment of arrhythmias. Am J Cardiol 1993;72:44-50A.
112. Czekalla J, Beasley CM Jr, Dellva MA, et al. Analysis of the QTc interval during olanzapine treatment of patients with schizophrenia and related psychosis. J Clin Psychiatry 2001;62:191-8.
113. Cui G, Sen L, Sager P, et al. Effects of amiodarone, sematilide, and sotalol on QT dispersion. Am J Cardiol 1994;74:896-900.
114. Zabel M, Hohnloser SH, Behrens S, et al. Differential effects of D-sotalol, quinidine, and amiodarone on dispersion of ventricular repolarization in the isolated rabbit heart. J Cardiovasc Electrophysiol 1997;8:1239-45.
115. Makkar RR, Fromm BS, Steinman RT, et al. Female gender as a risk factor for torsades de pointes associated with cardiovascular drugs. JAMA 1993;270:2590-7.
116. Stanton MS, Prystowsky EN, Fineberg NS, et al. Arrhythmogenic effects of antiarrhythmic drugs: A study of 506 patients treated for ventricular tachycardia or fibrillation. J Am Coll Cardiol 1989;14:209-15.
117. Simons GR, Eisenstein EL, Shaw LJ, et al. Cost effectiveness of inpatient initiation of antiarrhythmic therapy for supraventricular tachycardias. Am J Cardiol 1997;80:1551-7.
118. Lehmann MH, Hardy S, Archibald D, et al. Sex difference in risk of torsade de pointes with d,l-sotalol. Circulation 1996;94:2535-41.