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Myocardial function characteristics, response to isoproterenol, and calcium uptake activity in rats pretreated… Marriott, Margaret Lynne 1982

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c, f MYOCARDIAL FUNCTION CHARACTERISTICS, RESPONSE TO ISOPROTERENOL, AND CALCIUM UPTAKE ACTIVITY IN RATS PRETREATED WITH THYROID HORMONES by MARGARET LYNNE MARRIOTT B.Sc, The University of Alberta, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS .FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1982 (Q) Margaret Lynne Marriott, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pharmaceutical Sciences The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 18,1982 i i ABSTRACT Thyroid hormone pretreatment altered rat myocardial function curve c h a r a c t e r i s t i c s , showed no effect on the s e n s i t i v i t y of the heart to isoproterenol, and did not change the calcium uptake a c t i v i t y into sarcoplasmic reticulum v e s i c l e s . Hearts from rats treated either for 3 days with L-triiodothyronine (T3 ) or for 7 days with L-thyroxine (T4) were subjected to changes in a t r i a l f i l l i n g pressure from 5 cm of water to 22.5 cm of water on the modified Neely working heart apparatus. Thyroid hormone pretreated rat hearts showed increased pressure and rate parameter measurements over their vehicle pretreated counterparts at a l l a t r i a l f i l l i n g pressures. Time to peak pressure was not s i g n i f i c a n t l y lowered, but relaxation time for the T3 treated rat hearts was s i g n i f i c a n t l y lower than that of control rat hearts at a l l a t r i a l f i l l i n g pressures. T4 hearts had shorter relaxation times than did control hearts at the lower f i l l i n g pressures, but at f i l l i n g pressures over 15cm of water the TM relaxation times approached those of control. At 17.5cm of water f i l l i n g pressure T3 hearts had s i g n i f i c a n t l y shorter relaxation times than did T4 hearts. Measurement of the t o t a l pulse period or t o t a l contraction time showed similar trends, however in t h i s case T3 hearts had shorter t o t a l pulse periods than T4 hearts at a l l a t r i a l f i l l i n g pressures over. 15 cm of water. Areas i i i were measured under the l e f t ventricular pressure curve. None of the area measurements for thyroid hormone pretreated hearts were s i g n i f i c a n t l y d i f f e r e n t from control. Isoproterenol dose response curves were also obtained from the working hearts of a l l experimental groups. T3 hearts consistently showed higher values in l e f t ventricular developed pressure, rate of pressure development and rate of relaxation than did control and T4 hearts even before isoproterenol was administered. T4 hearts had values not d i f f e r e n t from control values at any dose of isoproterenol in the measurement of any parameter. Time to peak pressure and relaxation time did not vary between groups. At a sub-maximal dose of isoproterenol T3 hearts had a s i g n i f i c a n t l y decreased t o t a l time of contraction. Area under the curve from peak pressure back to baseline was s i g n i f i c a n t l y increased over control levels for T3 hearts at two low doses of isoproterenol. Total area under the curve was not changed in thyroid hormone treated hearts. In order to account for the wide difference in baseline values for a l l the parameters, the data were expressed in terms of percent maximum response, and the pD2 values (negative log ED50) were calculated. The pD2 values so obtained were not different between the groups. Calcium uptake a c t i v i t y into cardiac sarcoplasmic reticulum was measured. No differences were seen between the experimental groups. Thus, while performance of the rat heart in terms of l e f t ventricular pressure, and rate of pressure r i s e and f a l l was i v increased by thyroid hormone pretreatment, no change in s e n s i t i v i t y of the heart to isoproterenol could be shown. There was also no difference in calcium uptake a c t i v i t y . We speculate that changes in the calcium s e n s i t i v i t y of the con t r a c t i l e proteins in the rat heart may be responsible for the increased myocardial c o n t r a c t i l i t y seen after thyroid hormone pretreatment. The lack of change in s e n s i t i v i t y to isoproterenol of the thyroid pretreated hearts could then be explained by the s t e r i c hindrance of this enhanced calcium s e n s i t i v i t y by catecholamine-induced phosphorylation of the troponin I subunit by c y c l i c AMP dependent protein kinase. Calcium uptake a c t i v i t y was unchanged by thyroid hormone pretreatment. If the augmented c o n t r a c t i l i t y observed in thyroid hormone pretreated hearts over the function curve was due to increased s e n s i t i v i t y of these hearts to an unchanged concentration of calcium within the c e l l , this s i m i l a r i t y of calcium uptake a c t i v i t y would be expected. John H. McNeill Thesis Supervisor V TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES i x INTRODUCTION 1 A. Cardiac Hypertrophy 2 B. Hemodynamic Effects 5 1. Resting heart rate 5 2. Other hemodynamic effects 8 C. Electrophysiological Changes 9 D. C o n t r a c t i l i t y Effects 10 1. Isometric Studies 11 2. Isotonic Studies 16 E. Interactions with Catecholamines 17 1. Chronotropic 17 2. Inotropic 19 3. Tissue concentrations 21 4. Beta-adrenoreceptor studies 22 F. Effect on Myocardial Enzymes 26 1. Adenylate cyclase 26 2. Phosphorylase 27 3. Myocardial ATPases 30 a. Myosin ATPase 30 b. Na+K+-ATPase . 33 c. Ca + +-activated ATPase 33 v i PAGE PURPOSE OF THE INVESTIGATION 36 METHODS 39 A. Treatments 39 B. C o n t r a c t i l i t y Studies 40 1. Preparation protocol 40 2. Function curve 41 3. Isoproterenol dose response curve 43 4. pD2 value determination 43 C. Studies of Calcium Uptake A c t i v i t y 44 1. Preparation of sarcoplasmic reticulum 44 2. Measurement of calcium uptake a c t i v i t y 45 45 3. Calcium buffer preparation 47 D. S t a t i s t i c a l Analysis 47 E. Materials 48 RESULTS 49 A. Pre-experimental effects 49 1. Body and heart weights 49 2. Heart weight/body weight r a t i o 49 3. Spontaneous heart rate 50 B. C o n t r a c t i l i t y Studies 56 1. Function curve 56 2. Isoproterenol dose response curve 80 3. Determination of pD2 value 104 v i i PAGE C. Studies of Calcium Uptake A c t i v i t y 104 1. Lowry assay standard curve 107 2. Calcium uptake a c t i v i t y 107 DISCUSSION 112 A. Pre-experimental effects 112 B. C o n t r a c t i l i t y studies 116 1. Function curve 116 2. Isoproterenol dose response curve 123 C. Calcium Uptake A c t i v i t y 126 D. Integration 12 7 SUMMARY 135 BIBLIOGRAPHY 136 APPENDIX 1 145 APPENDIX 2 150 v i i i LIST OF TABLES TABLE PAGE I The effect of thyroid hormone pretreatment on r a t body and heart weight 51 I I Upper significance l i m i t s of the F-dist r i b u t i o n (P=0.05) 154 I I I Upper 5 percentage points, Q, i n the studentized range 155 • LIST OF FIGURES  FIGURE PAGE 1. Sample l e f t ventricular pressure tracing from r a t working heart,as stored to microcomputer 42 2. Flow diagram f o r the preparation of r a t cardiac microsomes enriched i n sarcoplasmic reticulum 46 3. The effect of thyroid hormone pretreatment on r a t heart weight/body weight r a t i o 53 4. The effect of thyroid hormone pretreatment on spontaneous heart rate i n r a t 55 5. The effect of thyroid hormone pretreatment on l e f t v entricular developed pressure i n the r a t working heart 61 6. The effect of thyroid hormone pretreatment on the rate of l e f t v entricular pressure development i n the r a t working heart 63 7. The eff e c t of thyroid hormone pretreatment on the rate of relaxation from peak l e f t v e n t r i c u l a r pressure i n the r a t working heart 65 8. The effect of thyroid hormone pretreatment on the time to peak l e f t v entricular pressure . i n the r a t working heart 67 9. The effect of thyroid hormone pretreatment on.relaxation time from peak pressure i n the r a t working heart 69 X F I G U E £ PAGE 10. The effe c t of thyroid hormone pretreatment on t o t a l pulse period i n the r a t working heart 71 11. The effe c t of thyroid hormone pretreatment on the time period from +dP/dt to -dP/dt i n the r a t working heart 73 12. The effect of thyroid hormone pretreatment on area under the contraction curve from baseline to l e f t v entricular peak pressure i n the r a t working heart 75 13. The effe c t of thyroid hormone pretreatment on area under the curve from peak l e f t v e n t r i c u l a r pressure back to baseline i n the rat working heart 77 1M-. The effe c t of thyroid hormone pretreatment on the t o t a l area under the entire l e f t v e ntricular contraction"curve i n r a t working heart 79 15. The effect;of isoproterenol on l e f t v e n t r i c u l a r developed pressure i n rats pretreated with thyroid hormones 85 16. The effect of isoproterenol on rate of l e f t v e ntricular pressure development i n rats pretreated with thyroid hormones 87 XI FIGURE PAGE 17. The effect of isoproterenol on rate of relaxation from peak l e f t v entricular pressure i n rats pretreated with thyroid hormones 89 18. The effect of isoproterenol on time to peak l e f t v entricular pressure i n rats pretreated with thyroid hormones 91 19. The effect of isoproterenol on relaxation time from peak l e f t ventricular pressure i n rats pretreated with thyroid hormones 93 20. The effect of isoproterenol on t o t a l l e f t v e n tricular pulse period i n rats pretreated with thyroid hormones 95 21. The eff e c t of isoproterenol on the time period from +dP/dt to -dP/dt i n working hearts from rats pretreated with thyroid hormones 97 22. The effect of isoproterenol on the area under the l e f t v entricular pressure curve from baseline to peak pressure i n rats pretreated with thyroid hormones 99 23. The effect of isoproterenol on the area under the l e f t v e n t r i c u l a r pressure curve from peak pressure back to baseline i n rats pretreated with thyroid hormones 101 FIGURE PAGE 24. The effect of isoproterenol on t o t a l area under the entire l e f t v e ntricular contraction curve i n rats pretreated with thyroid hormones 103 .25. The effect of isoproterenol as percent maximum response of rate of l e f t v entricular pressure development i n working hearts from rats pretreated with thyroid hormones 106 26. The standard curve of the Lowry protein assay 109 27. Effect of thyroid hormone pretreatment on calcium uptake a c t i v i t y i n r a t cardiac microsomes enriched i n sarcoplasmic reticulum 111 X i i i ACM0W1ITJGEMENTS I would l i k e to thank Dr. John H. McNeill f o r h i s unstinting support and guidance throughout my years i n . his laboratory. I am especially grateful to David P. H a r r i s , without whose microcomputer expertise and generosity t h i s project could not have been completed. I would also l i k e to acknowledge the help of Judy Wyne i n preparing the graphs included i n t h i s t h e s i s . 1 INTRODUCTION Hyperthyroidism produces a variety of hemodynamic and physical changes in the mammalian cardiovascular system. C l i n i c a l investigations (Graettinger et a l . 1959; Amidi et a l . 1968) have shown elevations in cardiac index, increased coronary blood flow, decreased peripheral resistance, increased myocardial oxygen consumption and increased right and l e f t ventricular work, associated with hyperthyroidism. These changes in the function of the heart and blood vessels can not only compromise individuals with pre-existing heart disease, but may also cause cardiac complications in the patient with an otherwise healthy heart (Sandler and Wilson, 1959). Although cardiovascular performance in hyperthyroidism and i t s accompanying acute state, thyrotoxicosis, have been studied in quite exhaustive d e t a i l in both c l i n i c and laboratory, many questions remain unanswered regarding the dynamic mechanisms of thyroid hormone actions upon the heart and vasculature, and indeed, also regarding the precise nature of these actions. The l e v e l of thyroid hormones within the body is regulated through the central nervous system. L-thyroxine (T4) i s deiodinated after release from the thyroid gland to the active form of the hormone L-triiodothyronine (T3). The action of T3 at the c e l l u l a r l e v e l in the myocardium i s not understood. A theory put forward by St e r l i n g (1979) proposes 2 that T3 may bind to the inner mitochondrial membrane within the myocardial c e l l . Binding i s promptly followed by an increase in oxidative metabolism. This type of binding has been shown, however the concentrations of thyroid hormone necessary to produce the effect are well out of the physiological range. Thus the importance of mitochondrial binding of T3 in physiological situations is of questionable importance. Whatever the method of interaction of T3 at the c e l l u l a r l e v e l , i t has certain undeniable actions in the myocardium once i t has arrived in s i t u . A. Cardiac Hypertrophy and Protein Synthesis Cardiac hypertrophy has been defined as an increase in the heart weight to t o t a l body weight r a t i o . The c h a r a c t e r i s t i c loss of body weight of the hyperthyroid patient, combined with the enlarged heart often seen in this condition, make cardiac hypertrophy so common a symptom in hyperthroidism as to constitute one of the markers for the disease (McEachern and Rake, 1931). Cardiac hypertrophy after thyroid hormone treatment has been demonstrated experimentally in rats (Gemmill, 1958; C a i r o l i and Crout, 1967; Frazer et a l . 1968; Sandford et a l , 1978), in guinea pigs (Muryama and Goodkind, 1968), in cats (Strauer and Scherpe, 1975), in mice (Gemmill, 1958), and in rabbits (Banerjee et a l . 1976). This, of course, is only a p a r t i a l l i s t of the demonstrations of this phenomenon. Cardiac hypertrophy in thyrotoxic patients had been suggested by aberrations in electrocardiograph tracings, such 3 as increased duration of the QRS complex and increased amplitude of the R peak (Sandler, 1959). Recent echocardiographic work by Nixon et a l . (1979), has shown a 27% reduction in l e f t ventricular mass in hyperthyroid patients after successful therapy had returned them to the euthyroid state. Thyroid hormone-induced cardiac hypertrophy has been characterized by Gemmill (1958) as a "true" cardiac hypertrophy, i . e . i t does not result from an increase in cardiac water content. Zaimis et a l . (1969), in their electron microscopic work in guinea pig heart, saw myocardial hypertrophy, enlargement of the myocardial c e l l s , and a dramatic increase in the number, size, and complexity of mitochondria inside the myocardial c e l l . A larger number of mitochondria and an increase in the amount of pleomorphism of these mitochondria, was noted by Nayler and co-workers (1971) in dog hearts subjected to electron microscopy. Callas and Hayes (1974) saw no differences from control in sarcoplasmic reticulum or sarcomere ultrastructure in hyperthyroid rat hearts. They did, however, notice marked hypertrophy of mitochondria without an increase in mitochondrial number, as well as disorientation in the c r i s t a e . These disarrangements appeared to be reversible, as the experimenters saw no u l t r a s t r u c t u r a l changes in hearts of animals who were allowed to return to the euthyroid state. Bartosova et a l . (1969) observed a proportional increases in the size of both right and l e f t ventricles in rat hearts. They also found no 4 increase in the amount of collagenous material in the heart after thyroid hormone treatment, the increase in heart weight being wholly attributed to muscle c e l l augmentation. The l a t t e r observation has been reinforced by the work of Limas and Chan-Stier ( 1 9 7 8 ) , who saw no increase in either collagen or in i n t e r s t i t i a l tissue in their work in rat heart. An increase in protein synthesis has been correlated with thyroid hormone-induced cardiac hypertrophy (Gemmill, 1 9 5 8 ; Sandford et a l . 1 9 7 8 ) . Limas and Chan-Stier ( 1 9 7 8 ) noted an increase in RNA content of hearts from T3-treated rats, and decided to examine the mechanism responsible for the increase in RNA synthesis. Increased myocardial chromatin template a c t i v i t y was observed, together with an augmentation of the number of transcription i n i t i a t i o n s i t e s . These increased functions were further l o c a l i z e d to the non-histone nuclear fraction of the myocardial preparation, while a s i g n i f i c a n t r i s e in nuclear protein kinase a c t i v i t y was also noticed. In v i t r o , stimulation of RNA synthesis by non-histone proteins was enhanced by the addition of nuclear protein kinases and c y c l i c adenosine monophosphate (cAMP). These results led the authors to postulate that, in hyperthyroidism, increased protein (RNA) synthesis i s mediated by the non-histone proteins of the nucleus, and i s dependent on phosphorylation of these proteins by nuclear protein kinases. Studies by Sandford et a l . ( 1 9 7 8 ) indicate that the increased growth of the myocardium in hyperthyroidism i s due to increased protein synthesis, rather than decreased protein 5 degradation. T4 treatment of rats caused increased protein synthesis, with s l i g h t retardation of protein degradation. When T4 treatment ceased, protein synthesis decreased while no change was noted in the l e v e l of protein degradation. Experiments by Gemmill (1958) and Sandford et a l . (1978), as well as the c l i n i c a l data of Nixon et a l . (1979) suggest that hyperthyroid cardiac hypertrophy i s a reversible phenomenon. To summarize, cardiac hypertrophy in thyroid hormone excess involves the cardiac myocytes almost exclusively, is reversible, and i s associated with increased protein (probably RNA) synthesis, increased RNA content, and with an augmentation of the transcription of DNA. B. Hemodynamic Effects of Thyroid Hormones 1. Resting heart rate Resting heart rate greater than that seen in euthyroid individuals i s a continuing observation in experimental and c l i n i c a l hyperthyroidism. Experimental evidence has been well established for this increase in heart rate at rest, in dog (Van der Schoot and Moran, 1965; Margolius and Gaffney, 1965; Brewster et a l , 1956; Taylor et a l , 1969), in cat (Strauer and Scherpe, 1975), in mouse (Gemmill, 1958), in rabbit (Lee et a l , 1965; Anton and Gravenstein, 1970), in guinea pig (Zaimis et a l , 1969; MacLeod amd McNeill, 1981), and in rat (Van der Schoot and Moran, 1965; C a i r o l i and Crout, 1967; Frazer et a l , 6 1969; Gemmill, 1958). C l i n i c a l l y , thyrotoxic tachycardia has also been exhaustively documented (McEachern and Rake, 1931; Graettinger et a l , 1959; Sandler, 1959; Wilson et a l , 1966; De Groot and Leonard, 1970). The mechanism by which thyroid hormone excess produces an increased resting heart rate i s not yet understood. C a i r o l i and Crout, in 1967, used propranolol in an attempt to block the enhanced i n i t i a l heart rate in unanaesthetized hyperthyroid rats. The hyperthyroid group exhibited less cardiac slowing in reponse to this maneuver than did the euthyroid animals. If thyrotoxic tachycardia was due to increased adrenergic nerve a c t i v i t y or to enhanced responsiveness of the s i n o - a t r i a l (SA) node to noradrenaline, i t would be expected that propranolol would have shown more effect in the thyroxine treated animals. When atropine as well was administered to the rats to check for deficiency of cholinergic input, a sharp increase in heart rate was observed. The hyperthyroid state thus appeared to have l i t t l e e ffect on vagal input to the SA node. Pretreatment with reserpine resulted in reduction of the hyperthyroid heart rate to control l e v e l s , an effect which could be reversed by the subsequent injection of atropine. Thyroid hormone induced tachycardia does not seem to be the result of an enhancement of adrenergic neural a c t i v i t y , a deficiency in cholinergic a c t i v i t y , nor an increase in s e n s i t i v i t y of cardiac beta receptors to endogenous noradrenaline. There i s , however, no direct evidence in support of these authors' conclusion that 7 thyroid hormones exert a direct effect on cardiac pacemaker c e l l s . Extensive use of propranolol in c l i n i c a l t r i a l s and recently in general practice has shown that administration of this drug w i l l improve certain of the symptoms of hyperthyroidism in man. Tachycardia can be reduced, while tremor, restlessness, anxiety, myopathy, sweating and heat intolerance a l l are ameliorated. Propranolol treatment does not, however, improve goitre, exopthalmos, and thyroid function tests, nor does i t reduce the augmented oxygen consumption of hyperthyroidism. It should be noted that while heart rate i s reduced in human hyperthyroid subjects treated with propranolol, i t i s not necessarily reduced to normal l e v e l s . (McDevitt, 1976). In T3 treated human volunteers, Wilson et al (1966) were able to abolish isoproterenol-induced tachycardia through the use of propanolol. The beta-adrenergic blocker had no e f f e c t , however, on T3-induced augmented resting heart rate. Data obtained from experimental animals in thyroid hormone excess, treated with propanolol also show only a p a r t i a l reduction of the hyperthyroid heart rate towards control, in rats ( C a i r o l i and Crout, 1967) and in guinea pigs (Goodkind, 1968). Thus, both c l i n i c a l and experimental results suggest that part of the increased resting heart rate commonly seen in hyperthyroidism i s a product of increased beta-adrenergic a c t i v i t y . An explanation for the remaining increase i s not yet a v a i l i b l e . 8 2. Other hemodynamic changes Increases in oxygen consumption in hyperthyroid animals have been demonstrated in rats (Van der Schoot and Moran, 1965), in cats (Buccino et a l . 1967; Skelton et a l . 1970), in dogs (Brewster et a l . 1956; Van der Schoot and Moran, 1965), and in man (Graettinger et a l . 1959; Aoki et a l . 1967; Amidi et a l . 1968). Another common effect of thyroid hormone excess in man is decreased peripheral resistance, as shown by Graettinger et a l . 1959 and by Amidi et a l . 1968. Experimental demonstration of decreased peripheral resistance has also been accomplished in rats (Beznak, 1962). Cardiac index i s commonly increased in hyperthyroidism; this increase has been shown in rats (Beznak, 1962), and in cats (Buccino et a l . 1967; Strauer and Scherpe, 1975), as well as in man (Graettinger et a l . 1959; Amidi et a l . 1968). Amidi et a l . (1968) also showed increases in mean a r t e r i a l pressure in hyperthyroid patients, while similar increases have been shown in thyroid hormone treated rats (Frazer et a l , 1969) and cats (Strauer and Scherpe, 1975). In mild to moderate hyperthyroidism cardiac output is increased mainly through the mechanism of augmented heart rate. Only in severe hyperthyroidism i s an increased stroke volume added to the chronotropic al t e r a t i o n (DeGroot and Leonard, 1970). The increase in cardiac output, however far outstrips the increase in oxygen consumption in the hyperthyroid heart (Graettinger et a l . 1959). 9 Symptoms of hyperthyroidism, then, commonly involve increased resting heart rate, decreased peripheral resistance, increased oxygen consumption, increased mean a r t e r i a l pressure, and increased cardiac index, a l l indicative of a hyperkinetic, hyperdynamic circulatory state. C. Electrophysiological Changes Cardiac rhythym disturbances, p a r t i c u l a r l y an increased propensity for arrhythmias, have been appreciated as c l i n i c a l complications of the hyperthyroid condition for many years. In the experimental laboratory, Zaimis et a l . (1969) noted an increase in heart e x c i t a b i l i t y in T4 treated cat and guinea pig hearts. Arnsdorf and Childers (1970) found decreases in eff e c t i v e refractory period in isolated perfused hearts of TM treated rabbits, as well as lowered d i a s t o l i c threshold for a t r i a l responses, especially those evoked by multiple pulses. These workers also showed that no enhancement of the effect of T4 on conduction velocity or refractory period could be demonstrated with infusions of adrenaline, noradrenaline or atropine. Johnson et a l . (1973) saw an increased rate of d i a s t o l i c depolarization and decreased duration of the action potential in s i n o a t r i a l node c e l l s , in both the presence and absence of propranolol, in hyperthyroid rabbits. Hyperthyroidism shortens the refractory period and increases conduction velocity in the heart. The mechanism 10 behind this action of thyroid hormones is unknown. An explanation for this action might conceivably arise from the u l t r a s t r u c t u r a l changes in the myocardial c e l l in hyperthyroidism described by Callas and Hayes (1974) and Zaimis et a l . (1969), and their possible effect on membrane ion transport. D. C o n t r a c t i l i t y Effects Considerable controversy has developed in the study of the effects of thyroid hormone excess on the i n t r i n s i c c o n t r a c t i l e properties of the mammalian myocardium. The bulk of evidence concerning the effects of thyroid hormones on myocardial c o n t r a c t i l i t y has been obtained in in v i t r o studies of isolated cardiac tissue preparations. A b r i e f statement of some of the data obtained in the resting state of hyperthyroid myocardial preparations, concerning basal tension and pressure l e v e l s , w i l l serve to i l l u s t r a t e some of the ongoing problems involved in the experimental elucidation of the co n t r a c t i l e properties of the heart in hyperthyroidism. Levey et a l . (1969) using papillary muscle preparations from cats treated with T4 observed basal developed tensions equal to those developed by euthyroid papillary muscles, but also observed increased rate of tension development and decreased time to peak tension in hyperthyroid as compared to control. MacLeod and McNeill (1981) observed equivalent basal tensions in guinea pig l e f t a t r i a and pa p i l l a r y muscle from 11 hyperthyroid (T4 treated)and euthyroid animals. Longhurst and McNeill (1978) working with rat l e f t a t r i a and right ventricle pap i l l a r y muscle did not observe any differences in basal developed tension. However, in the same study basal developed tension in Langendorff hearts was s i g n i f i c a n t l y lower in hyperthyroid (T3 treated) animals. Nayler et a l . (1971) in contr a c t i l e studies with dog trabecular muscles and with open chest preparations, observed increased developed tension and rate of tension development in tissues from T4 treated animals as compared to controls. Thus, there are reports of decreased, increased and equivalent basal tension development in the results of these experimenters. A clear view of the cont r a c t i l e state of the hyperthyroid heart i s d i f f i c u l t to achieve. Part of this d i f f i c u l t y arises from d i f f e r i n g contributions to that c o n t r a c t i l e state from d i f f e r i n g species, treatment differences and differences in the type of tissue preparation used. This i s not to imply that species, treatment and preparation differences do not affect studies other than those into the mysteries of hyperthyroidism, but merely to make note of the wide variation possibly due to these inherent causes. 1. Isometric studies Several studies have d i r e c t l y addressed the characterization of the cont r a c t i l e properties of the hyperthyroid myocardium in an isometric isolated tissue preparation, in v i t r o . Buccino et a l . in 1967 assessed the isometric tension of papi l l a r y muscles from T4 treated cats 12 and their vehicle treated control counterparts. These experimenters saw no difference in maximum developed isometric tension. S i g n i f i c a n t increases in rate of tension development and s i g n i f i c a n t decreases in time to peak tension were c l e a r l y shown. These experiments were repeated for a range of temperatures from 21 degrees to 37 degrees Centigrade, showing d i r e c t i o n a l l y similar changes in time to peak tension and rate of tension development for hyperthyroid and euthyroid tissues i . e . as temperature rose, tension f e l l , time to peak tension shortened and rate of tension development was augmented. Parmley et al (1968) using v i r t u a l l y the same preparation of cat pap i l l a r y muscle treated with the same dose of T4, demonstrated decreased time to peak tension in hyperthyroid muscles as compared to control, but also observed a si g n i f i c a n t increase in maximum isometric tension. In 1970, Taylor examined cat papi l l a r y muscle preparations at a frequency of 12 contractions per minute, which was the frequency used by the previous two experimenters. His experiments were extended to determine papi l l a r y muscle behavior at frequencies of 30 and 60 contractions per minute as well. In hyperthyroid preparations, in agreement with the results of Parmley et a l . and Buccino et a l . , Taylor observed increased developed tension, and increased rate of tension development, with decreased time to peak tension with frequency at 12 contractions per minute. At 30 contractions per minute these increases were s t i l l noted. When frequency of contraction was 13 increased to 60 per minute however, the developed tension and rate of tension development in tissues from hyperthyroid animals were similar to those seen in muscles from euthyroid animals. This result was accompanied by the observation that the inotropic effect of increasing frequency was less in the hyperthyroid than in the euthyroid tissues, and by the proposal that the increased oxygen consumption required by high contraction frequency may induce an hypoxic depressant effect upon the hyperthyroid myocardium. In 1968, Muryama and Goodkind carried out a study in guinea pig l e f t a t r i a which examined the effects of d i f f e r i n g frequencies of contraction and d i f f e r i n g calcium concentrations on co n t r a c t i l e properties in hyperthyroid and euthyroid animals. They found developed tension in the preparations from hyperthyroid animals to be s i g n i f i c a n t l y increased over control at a l l frequencies measured (from 0.5 to 200 - contractions per minute), when the external calcium concentration was 0.625 millimolar. In 2.5 millimolar calcium at frequencies under 100 per minute, developed tension was greater in hyperthroid than in euthyroid tissues; at frequencies above 100 per minute there was no s i g n i f i c a n t difference between the two groups. When the external concentration of calcium was 5 millimolar, there a s i g n i f i c a n t increase in developed tension in the hyperthyroid over the euthyroid preparations only at frequencies below 50 per minute. Skelton et a l . in 1970 showed decreased time to peak 14 tension, increased rate of tension development, and increased t o t a l tension in pa p i l l a r y muscles from hyperthyroid as compared with euthyroid cats, associated with a considerable increase in myocardial oxygen consumption. One year l a t e r , the same group of workers repeated these experiments with the results In the second case being similar to those of the f i r s t , except for the fact that t o t a l tension was no longer s i g n i f i c a n t l y different in pa p i l l a r y muscles from T4 treated animals. The augmented c o n t r a c t i l i t y indices associated with hyperthyroidism were shown to be reduced to euthyroid levels in hypoxia (Palacios, 1979). In 1971 a similar result had been demonstrated by Skelton et a l . who suggested that hyperthyroid cardiac muscle had an altered conversion of chemical energy to mechanical work, rendering the use of energy in the myocardium an i n e f f i c i e n t process. There has been one study of the effects of T3 on the rat myocardium employing the working heart preparation f i r s t described by Neely et a l . in 1967. Brooks et a l . (1981) measured cardiac output, coronary flow,and rate of l e f t ventricular pressure development (dP/dt), at 10 centimeters of water a t r i a l f i l l i n g pressure. An increase in dP/dt was observed, but lower and higher a t r i a l f i l l i n g pressures were not examined. Hearts from thyroid hormone treated animals have also been subjected to the scrutiny of in vivo experiments. Taylor et a l . (1969) analysed the tension-velocity relationship in 15 closed chest dogs, measuring isovolumic contractions produced by sudden balloon occlusion of the aorta. Velocity of contraction was s i g n i f i c a n t l y increased in hyperthyroid animals, while t o t a l tension was s l i g h t l y increased, leading to a displacement of the tension-velocity relationship of the l e f t v entricle upwards and to the r i g h t . Time to peak tension was decreased in hyperthyroid animals, as were relaxation time and t o t a l time of contraction. An increased rate of r i s e of s y s t o l i c pressure and an increase in maximum l e f t ventricular s y s t o l i c pressure were shown in thyroxine guinea pigs which were subjected to aortic occlusion over the corresponding value in control animals (Goodkind, 1968). Strauer and Scherpe (1975) in hyperthyroid cats also saw an increase in the rate of ventricular pressure development. In man, indire c t measurements of c o n t r a c t i l i t y have been used. Left ventricular ejection time (LVET) and pre-ejection period (PEP) are the most common s y s t o l i c time intervals measured. Isovolumic contraction period (ICP), the time required in generation of pressure inside the ventricle to open the aortic valve i s also commonly reported. PEP and ICP are thought to r e f l e c t velocity of shortening and time to peak pressure.- In a t y p i c a l study of this type (Amidi et a l . 1968) decreased PEP, LVET, and ICP were reported in hyperthyroid humans. Lewis et a l . (1979) also observed decreases in PEP and ICP. Furthermore, they demonstrated increased mean velocity of circumferential fib r e shortening, using 16 echocardiographic methods. When hyperthyroid patients were returned to the euthyroid state, cardiac output and mean velocity of circumferential fibre shortening were returned to the euthyroid levels (Nixon et a l . 1979). 2. Isotonic studies Isotonic, in vi t r o studies have also been carried out, mainly in cat papi l l a r y muscle preparations. The maximum velocity of muscle fibr e shortening (Vmax) has been measured by many workers (Buccino et a l . 1967; Parmley et a l . 1968; Taylor, 1970; Skelton et a l . 1970; Strauer and Scherpe, 1975), a l l of whom found Vmax increased in the hyperthyroid state. Parmley et a l . (1968) also conducted a study assessing the compliance of the series element involved in the contraction of p a p i l l a r y muscle from hyperthyroid and euthyroid cats. They obtained i d e n t i c a l results for series element compliance for control and hyperthyroid animals, therefore concluding that the alteration in Vmax so readily demonstrable in papi l l a r y muscle from hyperthyroid cats must be due to some change in the cont r a c t i l e element of the cardiac muscle. Thus, there i s general agreement among experimenters that excess thyroid hormone does cause differences in at least some indices of heart performance. In animal studies, enhancement of maximum velocity of shortening, increased rate of tension development and decreased time to peak tension, have most often been demonstrated. C l i n i c a l studies appear to r e f l e c t similar changes in performance. 17 E. Catecholamine Interactions with Thyroid Hormones There i s s t r i k i n g s i m i l a r i t y of the cardiovascular manifestations of hyperthyroidism to the symptoms of catecholamine excess. Many experimental attempts to improve the understanding of the i n t e r r e l a t i o n of catecholamine and thyroid hormones have been undertaken. Investigation of the chronotropic and inotropic effects of catecholamines in thyroid hormone treated animals has been profuse. Catecholamine receptor studies and quantification of myocardial catecholamine content have also been explored with considerable diligence. 1. Chronotropic Interactions Work by Brewster et al in 1956 found that increases in resting heart rate observed in hyperthyroid open chest dogs were abolished by epidural pre-ganglionic blockade. An increased change in heart rate in response to single dose infusions of adrenaline (ADR) and noradrenaline (NA) was also demonstrated in the thyrotoxic animals prior to the spinal procaine epidural blockade, while induction of the blockade lowered this response to euthyroid l e v e l s . This study provided an impetus toward productive investigation of the sympatholytic agents such as guanethidine, reserpine and propranolol in the treatment of the tachycardia of hyperthyroidism. However certain of the methods of the o r i g i n a l work have been called into question. For instance, the method of blockade used i s not s p e c i f i c for sympathetic impulses, leaving open the p o s s i b i l i t y of confusion of the 18 results by influences from blockade of parasympathetic or spinal reflexes. In addition, the data were not subjected to s t a t i s t i c a l analysis. Despite some support from the work of Lee, Lee and Yoo (1965), who showed that a t r i a from thyroxine treated rabbits had a greater increase in rate and amplitude in response to NA than did a t r i a from control animals, most recent work has f a i l e d to show a potentiation of catecholamine induced chronotropic response due to thyroid hormone pretreatment. Benfey and Varma (1963) saw equivalent chronotropic responses to intravenous ADR in spinal cats pretreated either with T3 or with vehicle. In open chest dogs, Van der Schoot and Moran (1965) showed that there was no difference in heart rate response to graded doses of NA and ADR between euthyroid and hyperthyroid groups. Corroboration of this result was provided by the work of Margolius and Gaffney (1965) in intact hyperthyroid and control dogs, who saw no difference between the two groups in measuring chronotropic response to NA. Van der Schoot and Moran (1965) showed no difference between T4 treated and control rats, in absolute increase in a t r i a l rate as a response to NA. When expressed as a percent increase, this reponse was less in hyperthyroid rat a t r i a than in control. In hyperthyroid rabbits no increased chronotropic response over control was observed to NA administered either with or without cocaine (Anton and Gravenstein, 1970). Goodkind in 1969 obtained no differences in chronotropic response to isoproterenol (ISO) and NA in open chest guinea 19 pigs treated with either T4 or vehicle. On the other hand, C o l v i l l e and Telford (1970) saw enhanced heart rate responses to NA, ADR, and ISO in Langendorf isolated heart preparations from T4 treated rats, compared to control. MacLeod and McNeill (1981) saw increased pD2 values for ISO-induced chronotropy in right a t r i a from T4 treated guinea pigs, over those from euthyroid animals. Evaluation of the thyrotoxic cardiac chronotropic response to catecholamines i s further complicated by the report of Wildenthal (1972), who produced augmentation of NA-stimulated heart rate in spontaneously-beating f e t a l mouse a t r i a l cultures exposed to T3 for 2 days. It must be noted, however, that the dose of NA used to obtain this effect was very large (5 x 1 0 ~ 1 0 ) . Chronotropic responses to NA and A were measured in human volunteers treated with T3 by Aoki et a l . (1967). They found no difference in response to these catecholamines between euthyroid and hyperthyroid groups. While the majority of reports of catecholamine-thyroid hormone interaction with regard to chronotropic response appear to favor the lack of potentiation of response to catecholamines in thyroid hormone pretreated animals, the fact that several workers did see augmentation of response with this combination cannot be discounted. 2. Inotropic Interactions Catecholamine-thyroid hormone interaction in augmentation 20 of inotropic responses is also controversial. Langendorf hearts from T3 treated rats in the hands of Young and McNeill (1974) showed no differences in intropic response to NA from that of their control litter-mates. Muryama and Goodkind showed a decreased developed tension in l e f t a t r i a from guinea pigs treated with T4 in response to NA compared with response from control a t r i a . When both groups were pretreated with reserpine in order to deplete catecholamine reserves the NA responses were i d e n t i c a l to those obtained prior to reserpine treatment. Goodkind in 1969 tested the inotropic response to NA and ISO in open chest T4 and vehicle treated guinea pigs. He found greater maximum developed l e f t ventricular s y s t o l i c pressure in reponse to both agonists in euthyroid over hyperthyroid animals. Opposite trends to those obtained by Goodkind were seen by Hashimoto and Nakashima (1978) in their experiments on l e f t a t r i a from guinea pigs and papi l l a r y muscle from rabbits. They obtained a leftward s h i f t of the ISO dose response curve for hyperthyroid tissues, indicating an increase in s e n s i t i v i t y to ISO in these tissues. MacLeod and McNeill (1981) saw increases in pD2 values for isoproterenol in l e f t a t r i a and papi l l a r y muscles from T4 treated guinea pigs, also indicating supersensitivity to catecholamines in the hyperthyroid tissues. In rat, Van der Schoot and Moran (1965) saw less increase in hyperthyroid tissues in isometric tension developed in response to graded NA administration i f the results were 21 expressed in absolute values. If percent change was the method of da'ta presentation, no difference was seen. Buccino et a l . (1967) observed less inotropic response to NA in cat pap i l l a r y muscles from hyperthyroid animals than from euthyroid. Similar results were obtained by Levey et a l . (1969) In contrast, Tse et a l . (1980) showed an increased response to 1 x 10" M ISO, with a decreased maximum tension development in hyperthyroid rat ventricle s t r i p s , as compared to control. Obviously then, inotropic interrelated thyroid-catecholamine responses are not c l e a r l y defined. There i s a wealth of c o n f l i c t i n g evidence, the interpretation of which i s extremely d i f f i c u l t . 3. Catecholamine Tissue Concentrations The p o s s i b i l i t y that thyroid hormones might exert their effects upon the heart by alt e r i n g tissue levels of free catecholamines has been considered. Release of NA from sympathetic nerve endings has been reported as s l i g h t l y decreased in hyperthyroidism; decreased levels of dopamine beta-hydroxylase were also noted (Spaulding and Noth, 1975). Unaltered NA plasma levels in hyperthyroid animals have been documented (Nagel-Hiemke et a l . 1981). Wurtman et a l . 1963 have reported the a c t i v i t y of hepatic monoamine oxidase and cardiac catechol 0-methyl transferase unchanged from control 22 in hyperthyroid rats. Normal ventricular NA content has been reported in cats (Buccino et a l . 1967; Zaimis et a l . 1969), in dogs (Nayler et a l . 1971), in rats ( C a i r o l i and Crout, 1967) and in guinea pigs (Zaimis et a l . 1969). After reserpine administration, NA tissue levels were shown to be s i m i l a r l y reduced in control and hyperthyroid animals. There are reports of increased myocardial NA content, including Goodkind et al (1961) in T3 treated guinea pigs, and Lee et al (1965) in T4 treated rabbits, and of decreased concentrations of the neurotransmitter in rabbits (Anton and Gravenstein, 1970). Although no particular conclusions can be drawn from such varied data, c e r t a i n l y there i s no clearcut correlation between changes in NA tissue levels and myocardial functional performance alterations in hyperthyroid animals. 4. Beta-Adrenergic Receptor Studies If there i s potentiation of catecholamine-induced responses in tissues from thyroid hormone treated animals, an increase in number or a f f i n i t y of beta-adrenergic receptors would provide at least a p a r t i a l explanation for this phenomenon. With the development of t r i t i a t e d beta-adrenergic antagonist receptor l a b e l l i n g techniques in the late 1970's, i t became possible for experimenters to d i r e c t l y assess the quantity and s e n s i t i v i t y of beta-adrenergic receptors present in myocardial tissues. The effects of T3 and T4 pretreatment on beta-adrenergic 23 receptor c h a r a c t e r i s t i c s in rats were f i r s t chronicled in the 1977 paper of Williams and Lefkowitz. A f f i n i t y of the binding sites for both the antagonist, dihydroaloprenolol, and for the agonist, isoproterenol, were unchanged from control in both treatment groups. Up to 1 x 10~^M thyroxine incubated with the control heart membranes did not increase binding of the labelled antagonist to the receptor, nor did thyroxine i t s e l f bind at the receptor s i t e . There was, however a dramatic increase in the number of binding s i t e s found in tissues from animals pretreated with thyroid hormones. Ci a r a l d i and Marinetti (1977) provided almost immediate confirmation of this innovative finding with their work in thyroxine treated ra t s . They observed up to s i x - f o l d increases in beta-receptor number, with no change in binding a f f i n i t y . A less d e f i n i t e result was obtained by Banerjee and Kung (1977). They could show changes in beta-receptor number in thyroidectomized rats subsequently treated with thyroid hormone, but not in rats which were euthyroid at the i n i t i a t i o n of thyroid hormone treatment. Dissociation constants were v i r t u a l l y i d e n t i c a l in a l l groups. The lack of positive data concerning receptor number may have resulted from the treatment schedule used, which involved T3 injection only every other day for 3 doses. Acute incubation of T3 and T4 with rat ventricle s l i c e s was shown by Kempson et a l . (1978) to increase stereospecific dihydroalprenolol binding to beta-adrenergic receptors. This 24 action was not inhibited by cycloheximide, suggesting that i t was a post-translational event. The increased binding produced by f i f t e e n hours incubation with T3 could be inhibited by cycloheximide, however, indicating that tra n s l a t i o n a l or t r a n s c r i p t i o n a l synthesis may be involved. In cultured heart c e l l s from newborn rats, after 24 hours incubation with T3, Tsai and Chen (1978), noted an increase in beta-receptor number. It appears de novo protein synthesis i s not required for beta-receptor number increase. Further effects are noted when synthesis is allowed to occur. Ho et al (1980) confirmed the thyroid hormone-induced increase in beta-receptor number in dog heart. They also compared results of thyroxine treatment with those obtained from animals with l e f t ventricular hypertrophy of other etiology. They concluded that beta-receptor increase was not a secondary effect of hypertrophy nor a result of hemodynamic changes. Further corroborative evidence of increased beta-receptor number in hyperthyroid rats has been published by several workers (McConnaughey et a l . 1979; Tse et a l . 1980; S t i l e s and Lefkowitz, 1981). A time course study for thyroid hormone induced effects on beta-receptor number has been provided by McConnaughey et a l . (1979). Peak increases in receptor number occurred at 3 to 4 days of thyroxine treatment, then remained constant for the next 7 days. Recently, binding study methods have been modified to 25 allow accurate measurement of binding and a f f i n i t y of agonists as well as antagonists. The work of S t i l e s and Lefkowitz has shown that in hyperthyroid rat heart there are two a f f i n i t y states for the binding of ISO to the receptor, having high and low dissociation constants. The ratio of the two constants i s reported to indicate the effic a c y of hormone stimulation of adenylate cyclase. In hyperthyroidism this r a t i o i s increased, thus a more s t a b i l i z e d high a f f i n i t y state i s expected, leading to more e f f i c i e n t stimulation of adenylate cyclase. Studies of beta-adrenoreceptors in hyperthyroidism have provided much information on possible mechanisms for the myocardial alterations of thyroid hormone excess. There is general agreement on thyroid hormone induced increase in beta -adrenergic receptor number, although there are wide differences in the interpretation of significance of this finding. The recent work of S t i l e s and Lefkowitz (1981) suggests another possible mechanism of thyroid hormone effect, that of increased e f f i c a c y of transmembrane coupling. Absolute proof of such a mechanism w i l l await p u r i f i c a t i o n and characterization of the adenylate cyclase system. 26 F. Thyroid Hormone Effect on Myocardial Enzymes 1. Adenylate Cylase - Cyclic AMP' The s i m i l a r i t y in appearance of the effects of thyroid hormones and catecholamines naturally focused attention on the myocardial adenylate cyclase system. After early work in hyperthyroid animals had indicated potentiation of cardiac catecholamine actions by thyroid hormone pretreatments, this interest was increased manyfold. Levey and Epstein (1968) demonstrated a r i s e in cardiac adenylate cyclase a c t i v i t y when thyroxine was added to a cat l e f t ventricular particulate preparation. They proposed that T4 might exert i t s inotropic and chronotropic effects through adenylate cyclase. In subsequent experiments i t was found that propranolol could block catecholamine induced activation of adenylate cyclase, but had no effect on the thyroid hormone induced activation of the system. The existence of two adenylate cyclase systems was suggested as a possible explanation for this r e s u l t . Concern about the physiological significance of these in v i t r o experiments was fostered because of the high concentration of T4 used to produce the e f f e c t . It was also considered suspicious that the dose of T4 used did not augment c o n t r a c t i l i t y , and that the D analogue of thyroxine was just as effe c t i v e in producing the activation of adenylate cyclase, despite i t s much reduced physiological potency compared to the L form. Continuing studies in cats (Levey et a l . 1969; Sobel et a l . 1969), gave further question to the e a r l i e r work. Basal 27 levels of adenylate cyclase were found to be unchanged in thyroid hormone pretreated animals, while NA stimulated c y c l i c AMP accumulation in euthyroid and hyperthyroid groups to the same degree. Brus and Hess (1973) provided confirmation of the unchanged basal and NA stimulated adenylate cyclase levels in their work with T3 treated rats. McNeill et a l . (1969) also demonstrated equal basal and NA stimulated cAMP levels and adenylate cyclase a c t i v i t y in T3 treated ra t s . In 1978, McNeill showed no difference throughout the contr a c t i l e response to calcium chloride in T3 and control Langendorf hearts, either in accumulation of cAMP or in adenylate cyclase a c t i v i t y . An increased hyperthyroid response to isoproterenol induced maximum a c t i v i t y and s e n s i t i v i t y of adenylate cyclase has recently been reported by Tse et a l . (1980). Basal and sodium fluoride stimulated adenylate cyclase a c t i v i t y , and cAMP tissue levels were not diff e r e n t from control. The bulk of the evidence suggests that activation of the c y c l i c AMP system i s not an important factor in mediating thyroid hormone cardiotonic e f f e c t . 2. Phosphorylase Hyperthyroidism i s a state of augmented energy consumption. The a v a i l i b i l i t y of energy from glycogenolysis is dependent on the conversion of the inactive phosphorylase b 28 enzyme to i t s active phosphorylase a form, which is capable of catalysing the breakdown of glycogen to glucose 1-phosphate. The energy provided by this route is not used in e l i c i t i n g the i n i t i a l response to a stimulus, as the peak of the inotropic response precedes increase in phosphorylase a levels, but glycogenolysis may provide the energy needed for sustained response. Elevated tissue l e v e l s of phosphorylase a in hyperthyroidism have been well documented (Hornbrook and Brody, 1963; McNeill and Brody, 1968; Frazer et a l . 1969; McNeill et a l . 1969; McNeill, 1969; Longhurst and McNeill, 1979). Catalysis of phosphorylase kinase activation which in turn activates phosphorylase b to the a form, can occur through a cAMP dependent protein kinase (Walsh et a l . 1971). Pretreatment with propranolol has been shown to reduce basal and catecholamine stimulated phosphorylase a levels in hyperthyroid rats (McNeill and Brody, 1968; Frazer et a l . 1969). When T3 treated rats are pretreated with reserpine as well, the increased phosphorylase a levels of hyperthyroidism are reduced to euthyroid l e v e l s , while phosphorylase a levels from euthyroid animals show no change with reserpine pretreatment. Thyroid hormone induced r i s e in phosphorylase a can be demonstrated, however, without increases in cAMP levels (McNeill et a l . 1969) and without a r i s e in phosphorylase b kinase levels (Frazer et a l . 1969). Thyroid hormone induced phosphorylase activation is not related to increased 29 s e n s i t i v i t y of cardiac adenylate cyclase (McNeill et a l . 1969; Sobel et a l . 1969). Phosphorylase activation has also been shown to be independent of the increased coronary blood flow of hyperthyroidism (Longhurst and McNeill, 1979). The phosphorylase activation i s also not due to a reserpine-like supersensitivity (McNeill, 1969), nor to direct effects of thyroid hormones on cardiac phosphodiesterase in vivo (McNeill et a l , 1971 ). Dibutyryl cAMP was reported to have an increased effect on activation of phosphorylase in hyperthyroid animals as compared to control (McNeill, 1978). The p o s s i b i l i t y that this finding might be related to an increased s e n s i t i v i t y to cAMP at the protein kinase l e v e l i s s t i l l under question as cAMP dependent protein kinase levels in hyperthyroid animals have been demonstrated as normal (Katz et a l . 1977), elevated (Newcomb et a l . 1978) or decreased (Tse et a l . 1980) The activation conversion reaction of phosphorylase is also markedly dependent on calcium (Friesen et a l . 1969), but s e n s i t i v i t y to the phosphorylase activating effects of calcium was found to be not diff e r e n t throughout the calcium chloride dose response curve, in control and T3 treated guinea pigs (McNeill, 1978). There i s general acceptance of a thyroid hormone-induced increase in activation of phosphorylase. The pathway by which this increase occurs i s s t i l l much in doubt. Most of the major influences on the phosphorylase activation system have been eliminated as prime causes in the increases seen in 30 thyroid hormone treated animals. Investigation of more subtle influences remains to be done. 3. Myocardial ATPases a. Myosin ATPase Energy for myocardial contraction i s provided at the level of the myofibril by the s p l i t t i n g of ATP by myosin ATPase. Changes in myosin ATPase a c t i v i t y may be related to maximum velocity of shortening of the cardiac muscle. There i s considerable species variation in myosin ATPase a c i t i v i t y . The decreasing order of a c t i v i t y i s rat, cat, guinea pig, dog, rabbit (Yazaki and Raben,1975; Banerjee et a l . 1976). Thyrum et a l . (1970) measured myosin ATPase a c t i v i t y in T4 treated and control guinea pigs. They saw a 30% increase in calcium stimulated ATPase a c t i v i t y in guinea pigs treated with T4 over control. Increased h e l i c a l content and an altered amino acid composition of the ATPase were also shown. It was thought that de novo synthesis of a new type of myosin, or synthesis of a new protein which became associated with the myosin, might then induce greater actin activated ATPase a c t i v i t y . In 1974, Goodkind et a l . did time course studies of c o n t r a c t i l i t y and myosin ATPase a c t i v i t y in guinea pig papil l a r y muscles following T4 injec t i o n s . They saw increases in certain c o n t r a c t i l i t y indices, namely maximum developed 31 tension and rate of tension development (dT/dt), within 24 hours of the i n j e c t i o n , with no change in the myosin ATPase a c t i v i t y . After 8 days of T4 treatment the myosin ATPase a c i t v i t y was increased, coinciding with interesting changes in the measurements of c o n t r a c t i l i t y . Increased dT/dt was s t i l l noted after 8 days treatment, but maximum developed tension had dropped to euthyroid l e v e l s , with a concomitant decrease in time to peak tension. In these experiments correlation of myosin ATPase a c t i v i t y could be shown only with the late effects of T4 on c o n t r a c t i l i t y , i . e . with shortening of time to peak tension. No acute effect of T4 on myosin ATPase was observed when the hormone was incubated with myocardial ti s s u e . Yazaki and Raben (1975) could not see an effect of T4 incubated in v i t r o with rabbit cardiac tissue. They did see increases in myosin ATPase a c t i v i t y after 3 days in rabbits treated with T4, an effect which continued through 14 days of treatment. Rats pretreated with thyroid hormone in the same manner did not show increased myosin ATPase levels in response to thyroid hormone. Basal levels of a c t i v i t y in rats were, however, four times higher than the corresponding values in rabbit. This study also demonstrated changes in the amino acid composition and enzymatic properties of the myosin, providing some reinforcing evidence for the theory of Thyrum et a l . (1970) that T4 stimulates synthesis of a new myosin with altered enzymatic properties. Sulfhydryl modification i s known to augment myosin ATPase 32 a c t i v i t y . By blocking one t h i o l per myosin subunit, a c t i v i t y of the ATPase i s elevated. If a second sulfhydryl group i s blocked, however, there i s a complete cessation of a c t i v i t y . Katagiri et al in 1975 explored this phenomenon in hyperthyroid rabbits. When basal levels of myosin ATPase a c t i v i t y were measured, hyperthyroid hearts had twice the a c t i v i t y of those from euthyroid animals. When N-ethylmaleimide (NEM) was added to the incubation mixture, euthyroid myosin ATPase a c t i v i t y increased by 177%. On the other hand, addition of NEM to hyperthyroid tissues had no effect on a c t i v i t y . This study suggested that stimulation of myosin ATPase by thyroxine might occur through a conformational modification of the myosin subunit around the t h i o l group. However T4 did not act by d i r e c t l y blocking the sulfhydryl group, as these groups were accessible to modification by NEM. Rovetto et al (1972) could find no augmentation of myosin ATPase a c t i v i t y in hyperthyroid rat hearts, nor could Yazaki and Raben, in a later study (1975). Rat heart had the greatest basal myosin ATPase a c t i v i t y of the species studied. The extent of activation of myosin ATPase by thyroid hormone appears to vary inversely with the basal euthyroid myosin ATPase a c t i v i t y , which varies with the species. That i s , guinea pig and rabbit hearts, with the lowest basal a c t i v i t y , w i l l show the greatest increase in a c t i v i t y when treated with thyroid hormone. Rat heart myosin ATPase a c t i v i t y does not appear to be stimulated by thyroid hormones, possibly because 33 of i t s high a c t i v i t y in the euthyroid state. b. Na + K + -ATPase Yazaki and Raben ( 1 9 7 5 ) and Katagiri et al ( 1 9 7 5 ) reported no differences in Na + K + -ATPase a c t i v i t y from control values for rabbits treated with T 4 . These findings were confirmed in a recent study by McConnaughey et a l . ( 1 9 7 9 ) , who saw no effect on Na + K + -ATPase a c t i v i t y in the T4 treated r a t . c. C a + + activated ATPase of Sarcoplasmic Reticulum During the excitation-contraction cycle of cardiac muscle the i n t r a c e l l u l a r organelle known as the sarcoplasmic reticulum (SR) accumulates and releases calcium. It may serve as the major i n t r a c e l l u l a r calcium store. In the presence of magnesium ion and adenosine triphosphate (ATP), proteins embedded in the SR membrane and exposed to i t s external surface provide the agency for transmembrane calcium transport. Energy for calcium transport i s obtained by hydrolysis of ATP. In the absence of ATP the SR membrane i s quite impermeable to calcium. One of the major l i m i t i n g factors of cardiac relaxation i s thought to be the rate of calcium uptake into the SR from the sarcoplasm. Measurement of this uptake a c t i v i t y has attracted much interest as a possible indication of an explanation for the increased performance of the hyperthyroid heart. Nayler et a l . in 1971 were the f i r s t group to examine the 34 calcium accumulating a b i l i t y of the hyperthyroid SR. Using dogs treated with T4 for 10 days, these workers showed increases in tension development, and rate of tension development, as well as increased calcium accumulation and exchange by SR enriched microsomes. Similar results for calcium uptake a c t i v i t y were produced by Suko in 1971 in hyperthyroid rabbits. Substantial increases in t o t a l calcium uptake a c t i v i t y and in calcium activated ATPase a c t i v i t y were observed in animals treated for three to four weeks with T4. Goodkind et al (1974) were unable to see changes in parameters of calcium uptake a c t i v i t y in guinea pigs treated with thyroxine for 3 days. Limas (1978a; 1978b), in rats treated for two weeks with T4 saw increases in calcium uptake a c t i v i t y and calcium activated ATPase a c t i v i t y . These increases could be prevented through use of a protein synthesis blocker such as cycloheximide. Suko (1971) had postulated the formation of a phosphoprotein intermediate on the external surface of the SR membrane is a necessary prelude to transmembrane calcium transport. Limas (1978a) showed that increases in ATPase a c t i v i t y were preceded temporally by formation of this phosphoprotein intermediate, and that T4 induced higher steady state levels of this intermediate than were achieved in euthyroid animals. McConnaughey et a l . (1979) in rats treated for 5 days with T4, were unable to show any increase in calcium activated 35 ATPase a c t i v i t y over that seen in control animals. Increases in calcium uptake a c t i v i t y are well established in several species after at least 10 days treatment with thyroid hormone. However, increased calcium uptake a c t i v i t y has not been shown in studies of animals treated with T4 for 3 and 5 days. 36 PURPOSE OF THE INVESTIGATION The puzzle of the heart in hyperthyroidism prompted this investigation. The l i t e r a t u r e of the hyperthyroid heart i s a conglomerate of c o n f l i c t i n g r e s u l t s , interspersed with the occasional generally accepted piece of evidence. Universally endorsed changes seen in hearts from animals treated with thyroid hormones include the increase in number of beta-adrenergic receptors, the increase in phosphorylase acti v a t i o n , the increased resting heart rate, and the increased protein synthesis associated with heart enlargement. Effects known to occur in certain species, or after a certain minimum treatment length are increased myosin ATPase a c t i v i t y , increased calcium uptake a c t i v i t y into sarcoplasmic reticulum, and increased rate of tension development of isolated tissue preparations. Areas of outright disagreement encompass the entire r e l a t i o n between thyroid hormone treatment and catecholamine e l i c i t e d responses, as well as the existence of an i n t r i n s i c a l l y changed c o n t r a c t i l i t y in the hyperthyroid myocardium. The r e l a t i v e dearth of information on hyperthyroidism obtained from the isolated working heart provided the i n i t i a l d irection for this study. By producing function curves from e l e c t r i c a l l y paced working rat hearts, which could perfuse their own coronaries, and for which the peripheral resistance could be held constant i t was hoped certain insights into i n t r i n s i c c o n t r a c t i l e state of the hyperthyroid heart could be 37 gained. With the advent of microcomputer technology to our pharmacological laboratory, measurement of parameters previously unavailible could be undertaken. Elucidation of the changes in myocardial performance seen by e a r l i e r workers was our goal. Edifying perusal of the l i t e r a t u r e on the heart in hyperthyroidism i s complicated by the variety of treatments and treatment schedules used to induce hyperthyroidism in experimental animals. An useful exercise then, would be the employment of two of the more common treatments to render separate groups of animals hyperthyroid, and then to compare them to each other as well as to euthyroid control animals. Rats were chosen as the experimental species. This had been the species used in the only other study using the working heart preparation in examining effects of hyperthyroidism (Brooks et a l . 1981). There was also much information on the hyperthyroid rat heart a v a i l i b l e from isolated tissue experiments. It was deemed of interest as well to study the response to isoproterenol in the hyperthyroid rat working heart preparation. Many studies of this r e l a t i o n have been done; many are in sharp disagreement. While part of this divergence is undoubtedly due to d i f f e r i n g interpretations of similar results, our study hoped to c l a r i f y the effects of different thyroid hormone treatments on the catecholamine-thyroid hormone i n t e r r e l a t i o n . Although many workers (Nayler et a l . 1971; Suko, 1971; 38 Limas, 1978a) had previously shown increased calcium uptake a c t i v i t y in hyperthyroid cardiac sarcoplasmic reticulum, no one had ever shown such changes in rat hearts from the three day and seven day schedules of thyroid hormone treatment that we proposed to employ. It seemed of importance to measure calcium uptake a c t i v i t y in the animals of our study, so as to compare t h i s biochemical index with the results obtained from the c o n t r a c t i l e and catecholamine dose reponse work. In summary then, we proposed to examine two thyroid hormone treatment groups and a vehicle treated control group of rats with regard to their myocardial performance during function curve assessment of con t r a c t i l e state, and their response to graded doses of isoproterenol, on the working heart preparation. Calcium uptake a c t i v i t y into the cardiac sarcoplasmic reticulum would also be investigated. 39 METHODS A. Thyroid Hormone Treatments Hearts used in working heart studies and in studies of calcium uptake a c t i v i t y into cardiac sarcoplasmic reticulum were obtained from male Wistar rats weighing 200 to 250 grams. Two di f f e r e n t treatment schedules employing two di f f e r e n t thyroid hormones were used to render the treated animals hyperthyroid. One group of rats was given subcutaneous injections, once dail y for three days, of 3,3',5' t r i i o d o L-thyronine (T3) at a dose of 500 micrograms/kg administered in an alkaline saline vehicle. Control animals were given equal volume injections of vehicle alone. A second group of rats was given subcutaneous injections of L-thyroxine (T4) 500 micrograms/kg in alkaline saline once daily for 7 days. Control animals were given equal volumes of vehicle on the same dosage schedule. A l l animals received food and water ad libitum. 40 B. Contractile Studies 1. Preparation Protocol Twenty-four hours after the l a s t dose of thyroid hormone or vehicle, the rats were treated with heparin sodium (1000 U.S.P.units/kg) i.p. Ten minutes later the animals were stunned by a blow to the head and k i l l e d by c e r v i c a l d i s l o c a t i o n . The hearts were rapidly removed, and quickly attached to the stainless steel aortic cannula of a working heart apparatus (Neely et a l . 1967) as modified by Rodgers et a l . (1981). The hearts were maintained at 30 degrees Centigrade in Chenoweth-Koelle (CK) solution (Chenoweth and Koelle, 1946) of the following composition: 120 millimolar sodium chloride, 5.6 millimolar potassium chloride, 2.1 millimolar magnesium chloride, 1.8 millimolar calcium chloride, 19 millimolar sodium bicarbonate, and 10 millimolar glucose. The CK solution was aerated with a mixture of 95% oxygen and 5% carbon dioxide to maintain pH at 7.4. After the pulmonary vein was cannulated, a PE 90 tubing attached to a 25 gauge stainless steel needle was used to cannulate the l e f t v e n t r i c l e . Left ventricular presssure and aortic pressure were monitored by means of Statham pressure transducers model P23 Db, connected to a Grass model 79D polygraph. The f i r s t derivative of l e f t ventricular pressure (dP/dt) was also recorded on the polygraph. Heart rate was followed using a Grass tachograph. Spontaneous heart rate was 41 recorded during an equ i l i b r a t i o n period l a s t i n g f i f t e e n minutes. The hearts were then e l e c t r i c a l l y paced at twice threshold voltage at a rate of 300 beats per minute (5 milliseconds duration). A Mountain Hardware A/D and D/A converter was used to d i g i t i z e the l e f t ventricular pressure tracings from the Grass polygraph and to store them via an Apple II microcomputer on disk. See Appendix I for detailed descriptions of microcomputer methods used in data acquistion and analysis. See Figure 1 for a sample l e f t ventricular pressure tracing from the Apple II microcomputer. 2. Function Curves Left ventricular function was estimated by subjecting the hearts to a range of a t r i a l f i l l i n g pressures or pre-loads varying from 5 to 22.5 cm. of water. A series of function curves was generated by plot t i n g a t r i a l f i l l i n g pressures against l e f t ventricular developed pressure (LVDP), maximum rate of l e f t ventricular pressure development (+ dP/dt), maximum rate of relaxation (- dP/dt), time required to reach peak l e f t ventricular pressure development (TTP), l e f t ventricular relaxation time from peak pressure back to baseline (TTB), t o t a l time of contraction (TTC), time between attainment of maximum + dP/dt and attainment of maximum -dP/dt (TPN), area under the contraction curve from baseline to peak l e f t ventricular pressure (AP), area under the contraction curve from peak pressure back to baseline (AB), FIGURE 1. 42 I I T T S P I T M I I I I • N T E SAMPLE LEFT VENTRICULAR PRESSURE TRACING AS STORED TO MICROCOMPUTER SHOWING: S - START OF PULSE i ( s t a r t ) E - END OF PULSE i (end ) M - MAXIMUM PRESSURE i(max) P - MAXIMUM +dP/dt N - MAXIMUM - d P / d t 43 and t o t a l area under the l e f t ventricular contraction curve (AT) . 3. Isoproterenol Dose Response Curves After completion of the function curves, one dose response curve was obtained for each heart by the cumulative addition of dl - isoproterenol to the perfusion medium with the a t r i a l f i l l i n g pressure held constant at 15 cm. of water. The hearts were then c a r e f u l l y removed from the working heart apparatus, blotted dry and weighed. 4. pD2 Value Determination Values for pD2 (negative log ED 50) of isoproterenol in L-triiodothyronine (T3) treated, L-thyroxine (T4) treated, and control rat working heart preparations were determined from graded log dose response curves through linear regression as described by T a l l a r i d a and Murray (1981) using an Apple II microcomputer. 44 C. Studies of Calcium Uptake A c t i v i t y 1. Preparation of Rat Cardiac Microsomes Enriched in Sarcoplasmic Reticulum The method employed for preparation of rat cardiac microsomes enriched in sarcoplasmic reticulum is that described by Sumida et a l . (1978), modified in the following manner. Hearts were removed from hyperthyroid and control animals 24 hours after the l a s t injection of thyroid hormone or vehicle and placed in 10 millimolar T r i s maleate buffer. A l l buffer media used in this protocol were kept ice cold. A t r i a l tissue was trimmed from the hearts and discarded. After brief scissor chopping the ventricles were homogenized in 10 millimolar T r i s maleate buffer (pH 6.8), by passing six times through a Dynamix Teflon homogenizer. The homogenized tissue was then centrifuged at 5000 rpm for 10 minutes. The resultant supernatant was then strained through four layers of cheesecloth. Centrifugation was again employed, this time at 11,000 rpm for 20 minutes. After further straining through four layers of cheesecloth, the supernatant was centrifuged at 20,000 rpm for 90 minutes. The p e l l e t obtained from th i s l a s t centrifugation was then rinsed with 10 millimolar T r i s maleate buffer (pH 6.8) which contained 0.6 molar potassium chloride. The p e l l e t was resuspended using a Pasteur pipette. The resulting mixture of fine p a r t i c l e s and buffer was then subjected to three gentle 45 passes in a glass homogenize!", and centrifuged at 20,000 rpm for 90 minutes. (See Figure 2 for centrifugation flow diagram.) The supernatant obtained was discarded. The remaining p e l l e t was rinsed with 10 millimolar T r i s maleate buffer (pH 6.8) containing 40 % by weight of sucrose. After the p e l l e t had been resuspended, i t was passed gently five times through a Teflon homogenizer. Aliquots of the resulting suspension were then frozen rapidly by immersion in methybutane, cooled in dry i c e , and stored at in an ultralow temperature freezer at - 70 degrees Centigrade. 2. Measurement of Calcium Uptake A c t i v i t y ATP - dependent calcium uptake a c t i v i t y was measured according to the method of Tada et a l . (1974), with the modifications outlined below. Frozen aliquots of rat cardiac sarcoplasmic reticulum were rapidly thawed after a short period of storage, and preincubated at 30 degrees Centigrade for 7 minutes in 0.3 ml of a medium of the following composition: 0.11 molar h i s t i d i n e chloride, 2.0 molar potassium chloride, 1.0 molar magnesium chloride, 100 millimolar T r i s oxalate, 50 millimolar sodium azide, 50 millimolar Tr i s adenosine triphosphate. Oxalate f a c i l i t a t e d calcium uptake was then i n i t i a t e d with the addition of calcium 45 ++ buffer labelled with Ca . Following a 5 minute incubation period, uptake a c t i v i t y was terminated by 46 HOMOGENIZED RAT VENTRICLE 1 — CENTRIFUGE 5000 RPM x 10 MIN SUPERNATANT STRAIN CENTRIFUGE 11000 RPM x 20 MIN SUPERNATANT STRAIN CENTRIFUGE 20,000 RPM x 90 MIN SUPERNATANT (DISCARD) SUPERNATANT (DISCARD) PELLET (DISCARD) PELLET (DISCARD) PELLET RESUSPEND TRIS KC1 BUFFER GLASS HOMOGENIZER (SOLUBILIZES ACTIN + MYOSIN) CENTRIFUGE 20,000 RPM x 90 MIN PELLET RESUSPEND TRIS SUCROSE BUFFER TEFLON HOMOGENIZER ALIQUOT FREEZE STORE AT -70°C RAT CARDIAC MICROSOMES ENRICHED IN SR CENTRIFUGATION FLOW DIAGRAM PREPARATION OF RAT CARDIAC MICROSOMES ENRICHED IN SARCOPLASMIC RETICULUM FIGURE 2 47 f i l t r a t i o n of a 0.41 ml aliquot through a 0.45 micron Mil l i p o r e f i l t e r . A 0.04 molar T r i s chloride solution (pH 7.4) was used to perform two 10 ml washes of the f i l t e r . After a 3 minute drying period in an oven at 40 degrees Centigrade, the f i l t e r s were placed in v i a l s containing 10 mis of Biofluor s c i n t i l l a t i o n f l u i d , and analyzed for a c t i v i t y in a l i q u i d s c i n t i l l a t i o n counter (Isocap) over a 10 minute counting i n t e r v a l . Proteins were determined by the method of Lowry et a l . (1951 ). 3. Calcium Buffer Solution Preparation Ethyleneglycol - bis (b - amino - ethyl ether) n, n -tetra - acetic acid (EGTA) - calcium solutions were prepared in which the amount of EGTA was varied in order to provide a range of free C a + + concentrations, using a modified equation according to Katz et a l . (1970). Each solution contained a t o t a l concentration of 1.25 millimolar calcium chloride. A 45 volume of calcium chloride solution s u f f i c i e n t to y i e l d approximately 500,000 cpm / 50 ul was added to the solution for purposes of measuring extent of calcium uptake. D. S t a t i s t i c a l Analysis Analysis of variance employing the Newman-Keuls multiple comparisons test was used to analyse the experimental data. 48 The c r i t e r i o n of significance was chosen to be a probability of P < 0.05. See Appendix II for detailed methods (Snedecor and Cochran, 1967). E. Materials 45 The calcium chloride (40 mCi/mg) was supplied by Amersham Corporation. A l l other drugs and chemicals were obtained from Sigma Chemical Company. 49 RESULTS A. Pre-experimental Treatment Effects 1. Body and Heart Weights Table I shows rat t o t a l body weight preceding the f i r s t i n j e c t i o n , weight at time of s a c r i f i c e , the change in body we,ight during the treatment period, and heart weight for T3 treated, T4 treated, and vehicle treated rats. Both T3 treated and T4 treated rats exhibited a s i g n i f i c a n t decrease in body weight as compared to rats treated with alkaline saline vehicle. There was no s i g n i f i c a n t difference in weight loss between T3 treated and T4 treated rats. Final body weight of the T4 treated rats was s i g n i f i c a n t l y d i f f e r e n t from the f i n a l body weight of control rats. Hearts from both T4 treated and T3 treated rats were s i g n i f i c a n t l y d i f f e r e n t in weight from those from vehicle treated rats. 2. Heart Weight/Body Weight Ratio Heart weight was divided by body weight and the resulting number multiplied by 100. Figure 3 represents a comparison of the values thus obtained for the experimental groups in question. The vehicle treated group had a heart weight/body weight ratio of .33 ± .01, the T4 treated group .43 ± .01, 50 and the T3 treated group a value of .42 ± .03. Both thyroid hormone treated groups of animals showed a s i g n i f i c a n t difference from control heart weight/body weight r a t i o . 3. Spontaneous Heart Rate Heart rate was recorded on the polygraph/tachograph prior to i n i t i a t i o n of e l e c t r i c a l pacing of the a t r i a . Hearts from vehicle treated rats showed a spontaneous heart rate of 226 beats per minute ± 13. Heart rates for hearts from T4 treated and T3 treated rats were 294 beats per minute ± 23, and 336 beats per minute ± 27, respectively. These values were both s i g n i f i c a n t l y different from control values. There was no s i g n i f i c a n t difference in spontaneous heart rates between the T4 and T3 groups. (Figure 4) 51 TABLE I The Effect of Thyroid Hormone Pretreatment on Rat Body and Heart Weight Control T4 Treated T3 Treated (n=12) (n=7) (n=5) Rat Body Weight I n i t i a l 255 +4 (#) 250 ±7 266'+8 F i n a l 265 ±6 235 ±5" 248 ±8 A i n Body Weight +10 ±2 -13 ±3" -17 ±3" Rat Heart Weight 0.87 ±.02 1.02 ±.04* 1.03 +.06*" (#) Numbers indicate mean weight i n grams, +S.E.M. * Indicates s i g n i f i c a n t difference at P <0.05, compared to the control value 52 FIGURE 3. Effect of triiodothyronine and thyroxine pretreatment on heart weight/body weight ratio in rat. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on a ratio which consists of rat heart wet weight, obtained immediately after c o n t r a c t i l e studies were completed, divided by rat t o t a l body weight obtained immediately prior to s a c r i f i c e , this number being multiplied by 100. Each bar represents the mean heart weight/body weight ratio ± S.E.M of 5-12 observations. * indicates s i g n i f i c a n t difference from control at P<0.05. 53 HYPERTHYROID KRT HEART •CI6HT RATIO ANALYSIS .4S-S .4-T • T * .3H .2S-CONTROL T3 T4 54 FIGURE 4. Effect of triiodothyronine and thyroxine pretreatment on spontaneous heart rate in rat. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control) on spontaneous heart rate measured on the polygraph/tachograph during equi l i b r a t i o n prior to i n i t i a t i o n of e l e c t r i c a l pacing of the a t r i a . Each bar represents the mean spontaneous heart rate (beats/minute) ± S.E.M of 5-12 observations. * indicates s i g n i f i c a n t difference from control at P<0.05. 55 HYPERTHYROID RAT HERAT SPONTANEOUS HEART RATE «80n 350 H in a ^303-£250-a. 200 H ISO-CONTROL T3 T4 56 B. Contractile Studies 1. Function Curves a. Effect of T3 and T4 treatment on Left Ventricular Developed Pressure (LVDP): . Hearts from animals subjected to T3 and T4 pretreatment showed increases over control in LVDP at a l l a t r i a l f i l l i n g pressures measured. These values were s i g n i f i c a n t l y different from control for T3 hearts at a t r i a l f i l l i n g pressures of 5cm, and 7.5cm of water. For T4 hearts only at 7.5 cm of water a t r i a l f i l l i n g pressure were the values obtained s i g n i f i c a n t l y d ifferent from control. (Figure 5) b. Effect of T3 and T4 pretreatment on maximum rate of l e f t ventricular pressure development (+dP/dt): Hearts from T3 and T4 treated rats showed a s i g n i f i c a n t l y increased +dP/dt as compared to hearts from control animals at v i r t u a l l y a l l a t r i a l f i l l i n g pressures. A l l points on the graph for the thyroid hormone treated groups are s i g n i f i c a n t l y d ifferent from control except for the T4 treated group at 5 cm of water f i l l i n g pressure. (Figure 6) c. Effect of T3 and T4 pretreatment on maximum rate of relaxation (-dP/dt): An increase in -dP/dt was evident at a l l a t r i a l f i l l i n g pressures in hearts from hyperthyroid animals as compared to 57 those from control animals. This difference was s t a t i s t i c a l l y s i g n i f i c a n t for T3 hearts at 5cm, 15cm, 17.5cm, 20cm, and 22.5cm f i l l i n g pressures. For T4 hearts the values obtained for this parameter at 5cm, 7.5cm, 15cm and 17.5cm of water f i l l i n g pressures were s i g n i f i c a n t l y different from control values obtained at these settings. (Figure 7) d. Effect of T3 and T4 pretreatment on the time required to reach peak l e f t ventricular pressure development (TTP): Although in general TTP for hyperthyroid rat hearts was numerically decreased from control heart values, only two points on the function curve were s i g n i f i c a n t l y different from control. At 7.5cm and at 10cm of water a t r i a l f i l l i n g pressure the value for TTP obtained for T3 hearts was s i g n i f i c a n t l y different from control. (Figure 8) e. Effect of T3 and T4 pretreatment on l e f t ventricular relaxation time from peak pressure back to baseline (TTB): Relaxation time was decreased as a result of T3 and T4 pretreatment. S i g n i f i c a n t l y different values from control were obtained for T3 treated hearts at a l l a t r i a l f i l l i n g pressures measured. T4 relaxation time values were s i g n i f i c a n t l y different from control at 5cm, 7.5cm, 10cm, 12.5cm and 15cm of water f i l l i n g pressures. In addition, at 17.5cm of water f i l l i n g pressure, values for relaxation time for T3 hearts were s i g n i f i c a n t l y lower than those for T4 hearts. (Figure 9) 58 f. Effect of T3 and T4 pretreatment on t o t a l time per contraction (TTC): There was a decreased t o t a l time per contraction for hyperthyroid hearts as compared to control hearts. For T3 treated hearts a l l values obtained over the function curve were s i g n i f i c a n t l y d i f f e r e n t from control. In T4 treated hearts at 5cm, 7.5cm, 10cm, 12.5cm, 15cm, and 17.5cm, TTC was s i g n i f i c a n t l y different from control. Values for T3 hearts were s i g n i f i c a n t l y d i f f e r e n t from those for T4 hearts at 17.5cm, 20 cm, and 22.5cm. (Figure 10) g. Effect of T3 and T4 pretreatment on time required from maximum positive dP/dt to maximum negative dP/dt (TPN): Hearts from hyperthyroid animals showed a decreased time from maximum +dP/dt to maximum -dP/dt, as compared to hearts from control animals. Si g n i f i c a n t differences from control values occurred at 12.5cm, 15 cm, 17.5 cm, 20 cm, and 22.5 cm of water f i l l i n g pressure for hearts from T3 treated animals. At 5cm, 12.5cm and 15cm, T4 hearts had TPN values which were s i g n i f i c a n t l y decreased from control times. (Figure 11) h. Effect of T3 and T4 pretreatment on area under the contraction curve from baseline to the point of maximum LVDP (AP): While area under the l e f t ventricular pressure curve from i n i t i a t i o n of contraction to peak pressure development was 59 numerically s l i g h t l y increased in hearts from hyperthyroid animals, at no point on the function curve was this increased area s i g n i f i c a n t l y different from that seen in hearts from control animals. (Figure 12) i . Effect of T3 and T4 pretreatment on area under the contraction curve from the point of maximum l e f t ventricular pressure development to baseline (AB): There were no s t a t i s t i c a l differences seen between hearts from control and from hyperthyroid animals as regards the area under the contraction curve from peak pressure back down to baseline. (Figure 13) j . Effect of T3 and T4 pretreatment on t o t a l area under the pressure curve (AT): When values for t o t a l pulse area obtained from hearts from hyperthyroid animals were compared to those obtained from control animals, no s i g n i f i c a n t differences could be seen. (Figure 14) 60 FIGURE 5. Effect of triiodothyronine and thyroxine pretreatment on l e f t ventricular developed pressure in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on l e f t ventricular developed pressure (LVDP) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced°at 300 beats per minute. Each point represents the mean LVDP(mm Hg) ± S.E.M of 5-12 observations. * indicates s i g n i f i c a n t difference from control at P<0.05. 61 150n 125H 100H I E S 75H 0-o > 50H 25H (<) T3 treated (n=5) — (x) T4 treated (n = 7) — ( o ) vehicle treated (n=l2) T 5 10 15 20 filling pressure (cm H20) 25 62 FIGURE 6. Effect of triiodothyronine and thyroxine pretreatment on rate of l e f t ventricular pressure development in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on rate of l e f t ventricular pressure development (+dP/dt) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean +dP/dt (mm Hg/sec) ± S.E.M of 5-12 observations. * indicates T3 group s i g n i f i c a n t l y different from control at P<0.05 ** indicates both hyperthyroid groups s i g n i f i c a n t l y d ifferent from control at P<0.05. 63 500-1 1 1 1 1 1 0 5 10 15 20 25 filling pressure (cm H20) 64 FIGURE 7. Effect of triiodothyronine and thyroxine pretreatment on rate of relaxation from peak l e f t ventricular pressure in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on rate of relaxation from l e f t ventricular peak pressure (-dP/dt) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean -dP/dt (mm Hg/sec) ± S.E.M of 5-12 observations. * indicates T3 group s i g n i f i c a n t l y different from control at P<0.05. ** indicates both hyperthyroid groups s i g n i f i c a n t l y different from control at P<0.05. t * indicates T4 group s i g n i f i c a n t l y different from control at P<0.05. filling pressure (cm H20) 66 FIGURE 8. Effect of triiodothyronine and thyroxine pretreatment on time to peak l e f t ventricular developed pressure in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of -rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on the time required to achieve peak l e f t ventricular developed pressure (TTP) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean TTP (ms) ± S.E.M of 5-12 observations. * indicates s i g n i f i c a n t difference from control at P<0.05. 67 75-1 70H CO 5 > 65 CO 0) Q. E 60H 5 5 H 50 (<) T3 treated (n=5) (x) T4 treated (n=7) — ( o ) vehicle treated (n=12) 5 10 — i — 15 — i — 20 filling pressure (cm H20) 25 68 • FIGURE 9. Effect of triiodothyronine and thyroxine pretreatment on relaxation time of the l e f t ventricle in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on the time required for the l e f t ventricle to relax from peak developed pressure back to baseline (RT) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean RT (ms) ± S.E.M of 5-12 observations. * indicates s i g n i f i c a n t difference from control at P<0.05. ** indicates s i g n i f i c a n t difference from control and from T4 treated at P<0.05 69 125n 115H CO C .2 +-00 X jg 105 95H 85H 75 (<) T3 treated (n=5) (x) T4 treated (n=7) — ( o ) vehicle treated (n=12) 5 10 15 20 25 filling pressure (cm H20) 70 FIGURE 10. Effect of triiodothyronine and thyroxine pretreatment on to t a l pulse period in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on the time required for one complete l e f t ventricular contraction (TTC) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean TTC (ms) ± S.E.M of 5-12 observations. * indicates s i g n i f i c a n t difference from control at P<0.05. ** indicates s i g n i f i c a n t difference from control and from T4 treated at P<0.05. 71 190i 18CH 170 •o o *c a 160 © CO 3 a £ 150 o 140H (<)T3 treated (n=5) (x) T4 treated (n = 7)-~ -( o ) vehicle treated (n=12) 130 T 5 10 15 20 25 filling pressure (cm H20) 72 FIGURE 11. Effect of triiodothyronine and thyroxine pretreatment on time period from +dP/dt to - dP/dt in the rat working heart paced-at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on the time period in the l e f t ventricular contraction cycle from maximum +dP/dt to maximum -dP/dt (TPN) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean TPN (ms) ± S.E.M of 5-12 observations. * indicates s i g n i f i c a n t difference from control at P<0.05. 73 110-. CO Q. T3 HI > i O "v. 0. •a m > + E o 4= "O o 0) a 100-^  90H 80 70 (<) T3 treated (n = 5) — (x) T4 treated (n = 7) 1 — ( o ) vehicle treated (n=l2) 5 10 15 20 25 filling pressure (cm H20) 74 FIGURE 12. Effect of triiodothyronine and thyroxine pretreatment on area under the curve from baseline to l e f t ventricular peak developed pressure in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on area under the l e f t ventricular contraction curve from baseline to peak developed pressure (AP) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean AP (mm Hg)sec ± S.E.M of 5-12 observations. There were no s i g n i f i c a n t differences between the groups at any a t r i a l f i l l i n g pressure. 75 6000 CO 5000-O) X E E w 4000-O k. 3 CO CO CD cc CD Q. 3000J $ 2000-co 1000-(<) T3 treated (n=5) (^)T4 treated (n=7) — ( o ) vehicle treated (n= 12) — i — 15 5 10 15 20 filling pressure (cm H20) 76 FIGURE 13-Effect of triiodothyronine and thyroxine pretreatment on area under the curve from l e f t ventricular peak developed pressure back to baseline in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on area under the l e f t ventricular contraction curve from peak developed pressure back to baseline (AB) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean AB (mm Hg)sec ± S.E.M of 5-12 observations. There were no s i g n i f i c a n t differences between the groups at any a t r i a l f i l l i n g pressure. 77 (<) T3 treated (n=5) — 8000-, W T 4 treated (n=7) co 7000H O) X £ E ( o ) vehicle treated (n=12) 6000 CD § 5000 CO S 4000 a E o g 3000 CO 2000 I / V ~ i — r— 15 20 5 10 filling pressure (cm H20) 78 FIGURE 14. Effect of triiodothyronine and thyroxine pretreatment on t o t a l area under the curve for the entire l e f t ventricular contraction curve in the rat working heart paced at 300 beats per minute. The plot depicts the effects of the pretreatment of rats with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on t o t a l area under the l e f t ventricular contraction curve for the entire pulse (AT) in rat working hearts at various a t r i a l f i l l i n g pressures, while being paced at 300 beats per minute. Each point represents the mean AT (mm Hg)sec ± S.E.M of 5-12 observations. There were no s i g n i f i c a n t differences between the groups at any a t r i a l f i l l i n g pressure. 79 14000n 12000H CO E oJ 10000H E E 8000H w a *S 6000 A CO k_ co 75 o 4000 2000 (<) T3 treated (n = 5) (cx) T4 treated (n=7) — ( o ) vehicle treated (n =12)— T - r ~ 5 — i — 10 — i — 15 20 filling pressure (cm H20) 80 B. Contractile Studies 2. Isoproterenol Dose Response Curves a. Effect of isoproterenol on l e f t ventricular developed pressure in the rat working heart preparation: Throughout the isoproterenol dose response curve the LVDP response of hearts from animals treated with T3 was increased as compared to that of vehicle treated hearts. This increase was s i g n i f i c a n t for a l l doses of isoproterenol given, from 1 x -10 -7 10 Molar (M) to 1 x 10 M. Prior to the administration of isoproterenol there was also a s i g n i f i c a n t difference in LVDP achieved by hearts from T3 treated as compared to vehicle treated animals. T4 heart response was at no point different from that of the vehicle treated hearts; i t was, however s i g n i f i c a n t l y d i f f e r e n t from the response of the T3 treated hearts at a l l points on the dose response curve. (Figure 15) b. Effect of isoproterenol on the maximum rate of l e f t ventricular pressure development: Both thyroid hormone treatment groups had higher + dP/dt values than did the control group over the range of the dose response curve. Hearts from T4 treated animals did not show a s i g n i f i c a n t l y d ifferent response to isoproterenol at any dose of the drug as compared to hearts from vehicle treated animals. There was a s i g n i f i c a n t difference in the +dP/dt exhibited by hearts obtained from T3 treated animals as 81 compared to vehicle treated animals at control levels as well -10 -10 as at doses of isoproterenol of 1 x 10 M, 3 x 10 M, and -Q _ i n at 1 x 10 M. At a dose of 1 x 10 M, on the cumulative isoproterenol dose response curve, the T3 hearts also had a s i g n i f i c a n t l y greater rate of l e f t ventricular pressure development than did T4 hearts. (Figure 16) c. Effect of isoproterenol on maximum rate of relaxation: While in general, values of -dP/dt were greater in hearts from hyperthyroid animals than those from hearts of euthyroid _ Q animals, only at doses of isoproterenol of 1 x 10 M, 3 x _q _« 10 M, and 1 x 10 M were there s i g n i f i c a n t differences when comparing T3 treated hearts to vehicle treated ones. At the la s t dose mentioned the response of T3 hearts was also s t a t i s t i c a l l y d ifferent from T4 hearts in response to isoproterenol as measured by the maximum rate of relaxation. (Figure 17) d. Effect of isoproterenol on time to peak LVDP: No s t a t i s t i c a l l y s i g n i f i c a n t differences from euthyroid values of TTP were seen in hyperthyroid animals at any dose of isoproterenol. (Figure 18) e. Effect of isoproterenol on relaxation time: Although relaxation time in hearts from hyperthyroid animals was shorter than that of hearts from vehicle treated animals over the range of the isoproterenol dose-response 82 relationship, no s i g n i f i c a n t differences between groups were seen at any dose. (Figure 19) f. Effect of isoproterenol on t o t a l pulse period: The dose response curve for isoproterenol in hearts from T3 treated animals showed a decrease from values for hearts from vehicle treated rats at a l l doses given. There i s only _q one point of s i g n i f i c a n t difference, however, at 1 x 10 M isoproterenol. No differences between T4 treatment and vehicle treatment in their effects on this parameter were seen to be s t a t i s t i c a l l y s i g n i f i c a n t . (Figure 20) g. Effect of isoproterenol on time required from maximum positive dP/dt to maximum negative dP/dt: No s i g n i f i c a n t differences between the treatment groups were observed in the measurement of this variable under these experimental conditions. (Figure 21) h. Effect of isoproterenol on area to peak pressure: Values obtained for this variable were not s t a t i s t i c a l l y d i fferent between groups. (Figure 22) i . Effect of isoproterenol on area from peak pressure: Over the entire range of the dose response curve, AB in hearts from T3 animals was greater than in hearts from T4 and vehicle treated animals. Signif i c a n t differences were seen _ i o from vehicle treatment at control l e v e l s , at 1 x10 M and at 83 1 x 10-^M isoproterenol. At exactly the same doses hearts from T3 animals showed s i g n i f i c a n t differences from those from T4 treated animals. (Figure 23) j . Effect of isoproterenol on t o t a l area of pulse: No s i g n i f i c a n t differences between the treatment groups were seen in the measurement of this parameter. (Figure 24) 84 FIGURE 15. Effect of isoproterenol on l e f t ventricular developed pressure in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on l e f t ventricular developed pressure (LVDP) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean LVDP(mm Hg) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. ** indicates T3 treated group s i g n i f i c a n t l y different from vehicle treated and from T4 treated group at P<0.05. 85 negative log dose 86 FIGURE 16. Effect of isoproterenol on rate of l e f t ventricular pressure development in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on maximum rate of l e f t ventricular pressure development (+dP/dt) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean +dP/dt (mm Hg/sec) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. * indicates T3 group s i g n i f i c a n t l y d ifferent from control at P<0.05 ** indicates T3 treated group s i g n i f i c a n t l y different from vehicle treated and from T4 treated group at P<0.05. 87 6000T ( < ) T3 treated (n=5) (<x>)T4 treated (n =5)— (o) vehicle treated (n = 8 ) — 5000 CO O) X E E a. "D *C0 & 4000H 3000 2000? 9 8 negative log dose 88 FIGURE 17. Effect of isoproterenol on rate of relaxation from l e f t ventricular peak pressure in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on maximum rate of relaxation from l e f t ventricular maximum pressure (-dP/dt) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean -dP/dt (mm Hg/sec) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. * indicates T3 group s i g n i f i c a n t l y d ifferent from control at P<0.05 ** indicates T3 treated group s i g n i f i c a n t l y d ifferent from vehicle treated and from T4 treated group at P<0.05. 89 (<) T3 treated (n=5) 0*) T4 treated (n=5) — (o) vehicle treated (n = 8) — 4000n 1000H . . . r-C 10 9 8 7 negative log dose 90 FIGURE 18. Effect of isoproterenol on time to peak l e f t ventricular pressure development in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on time required to achieve peak l e f t ventricular pressure (TTP) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean TTP (ms) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. There were no s i g n i f i c a n t differences between groups at any dose of isoproterenol. 91 ISOPROTERENOL DRC - RflT WORKING HEART W T3 TREATED (N-S) C") T4 TREATED (N-5) Co) VEHICLE TREATED (N-8) NEGATIVE LOG DOSE 92 FIGURE -19. Effect of isoproterenol on relaxation time from peak l e f t ventricular pressure development in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on time required for the l e f t ventricle to relax frrom peak pressure (RT) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean RT (rns) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. There were no s i g n i f i c a n t differences between groups at any dose of isoproterenol. 93 ISOPROTERENOL DRC - RflT WORKING HEART H) T3 TREATED CN-5) I") T4 TREATED CN-5) Co) VEHICLE TREATED CN-8) 110-1 94 FIGURE 20. Effect of isoproterenol on t o t a l pulse period in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on time required for one complete l e f t ventricular contraction (TTC) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean TTC (ms) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. * indicates s i g n i f i c a n t difference from control at P<0.05. 95 (<)T3 treated (n=5) (x)T4 treated (n-5) — (o) vehicle treated (n=8) — 180 n 1304- • — , r C 10 9 8 negative log dose 96 FIGURE 21. Effect of isoproterenol on time period from +dP/dt to -dP/dt in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on the time period in the l e f t ventricular contraction cycle from maximum +dP/dt to maximum -dP/dt (TPN) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean TPN (ms) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. There were no s i g n i f i c a n t differences between groups at any dose of isoproterenol. 97 ISOPROTERENOL DRC - RAT WORKING HEART H) T3 TREATED (N-5) (") T4 TREATED (N-5) (o) VEHICLE TREATED (N-8) 120n l l f l - t 70H NEGATIVE LOG DOSE 98 FIGURE 22. Effect of isoproterenol on area under the curve from baseline to l e f t ventricular peak developed pressure in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on area under the l e f t ventricular contraction curve from baseline to peak developed pressure (AP) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean AP (mm Hg)sec ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working, heart apparatus. There were no s i g n i f i c a n t differences between groups at any dose of isoproterenol. 99 6000-1 ISOPROTERENOL DRC - RAT WORKING HEART («) T3 TREATED (N-5) («) T4 TREATED (N-5) (o) VEHICLE TREATED CN-8) NEGATIVE LOG DOSE 100 FIGURE 23. Effect of isoproterenol on area under the curve from l e f t ventricular peak developed pressure back to baseline in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on area under the l e f t ventricular contraction curve from peak developed pressure back to baseline (AB) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean AB (mm Hg) sec ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. ** indicates T3 treated group s i g n i f i c a n t l y different from vehicle treated group and from T4 treated group at P<0.05. 101 (<) T3 treated (n=5) ..... (x) T4 treated (n=5) — (o) vehicle treated (n=8) 3000 —r~ 10 9 8 7 negative log dose 102 FIGURE 24. Effect of isoproterenol on t o t a l area under the curve for the entire l e f t ventricular contraction curve in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on t o t a l area under the l e f t ventricular contraction curve (AT) in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean AP (mm Hg)sec ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. There were no s i g n i f i c a n t differences between groups at any dose of isoproterenol. 103 12000-, ISOPROTERENOL ORC - RflT WORKING HEART W T3 TREATED (N-5) C«) T4 TREATED (N-5) (o) VEHICLE TREATED (N-6) T 9 8 NEGATIVE LOG DOSE 104 B. Contractile Studies 3. Determination of pD 2 Values The data plotted in Part B, 2, showed s i g n i f i c a n t differences in the numerical values for the parameters measured at control levels i . e . before isoproterenol was added to the system. In order to p a r t i a l l y correct for this difference, and in order to analyse the data with respect to pD2 values (- log ED 50), the data were replotted for each parameter in terms of percent maximum response to isoproterenol. No immediate c l a r i f i c a t i o n of the situation resulted. However, the difference in dose of isoproterenol at which the groups reached maximum +dP/dt was clear enough to warrant calculation of pD2 values using maximum positive dP/dt as the index. Hearts from vehicle treated animals showed a pD2 value for isoproterenol of 8.18 ± .12, those from T4 treated hearts had a pD2 value of 8.25 ± .40, and T3 treated hearts had a pD2 value of 8.80 ± .15. There was no si g n i f i c a n t difference in pD2 values for isoproterenol between experimental groups. As the parameter chosen on which to do this calculation had exhibited the largest differences in response to isoproterenol of a l l the parameters measured, i t was assumed that no s i g n i f i c a n t differences would be found in analysis of any of the other parameters for pD2 values. (Figure 25) 105 FIGURE 25. Effect of isoproterenol as % maximum response of rate of l e f t ventricular pressure development in working hearts from triiodothyronine, thyroxine and vehicle pretreated rats paced at 300 beats per minute. The plot depicts the effect of various doses of isoproterenol on maximum rate of l e f t ventricular pressure development (+dP/dt) expressed as % of maximum response in rat working hearts from animals pretreated with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (vehicle). Each point represents the mean +dP/dt (% max. response) ± S.E.M of 5-8 observations. Dose response curves were obtained at a constant a t r i a l f i l l i n g pressure of 15 cm of water on the working heart apparatus. pD2 values were 8.8 ± .15 for T3, 8.25 ± .40 for T4, and 8.18 ± .12 for control hearts. There were no s i g n i f i c a n t differences in pD2 values for isoproterenol between the groups. 106 negative log dose isoproterenol 107 C. Studies of Calcium Uptake A c t i v i t y 1. Lowry Assay Standard Curve The correlation c o e f f i c i e n t obtained for the bovine serum albumin standard curve used in determining protein content in samples of rat cardiac sarcoplasmic reticulum was .9979. (Figure 26) 2. Calcium Uptake A c t i v i t y Sarcoplasmic reticulum (SR) prepared from hearts of T3 treated animals showed a trend towards higher calcium uptake a c t i v i t y than did SR from hearts of vehicle treated animals. Calcium uptake a c t i v i t y of hearts from T4 treated animals was v i r t u a l l y i d e n t i c a l to that of hearts from control animals. There were no points of s t a t i s t i c a l l y s i g n i f i c a n t divergence of hyperthyroid heart calcium uptake a c t i v i t y values from those of control hearts. (Figure 27) 108 FIGURE 26. The standard curve of the Lowry protein assay using freshly prepared bovine standard albumin The plot depicts the protein assay standard curve constructed from values of op t i c a l density or absorbance at 700nm (x 1000) of various concentrations of bovine serum albumin The correlation c o e f f i c i e n t obtained from the plot was •9979. 109 STANDARD CURVE OF LOWRY PROTEIN ASSAY ~~I ' ~> r 1 1 1 , 25 60 75 100 125 150 175 200 micrograms BSA 110 FIGURE 27. Effect of triiodothyronine and thyroxine pretreatment on calcium uptake a c t i v i t y in rat cardiac microsomes enriched in sarcoplasmic reticulum. The plot depicts the effect of pretreatment with triiodothyronine (T3) for 3 days, with thyroxine (T4) for 7 days, and with vehicle (control), on calcium uptake a c t i v i t y in rat cardiac microsomes enriched in sarcoplasmic reticulum at various concentrations of free calcium. Each point represents the mean calcium uptake a c t i v i t y (nmol/mg protein/minute) of 4-8 observations. There were no s i g n i f i c a n t differences between groups at any concentration of free calcium. I l l HYPERTHYROID RAT HEART (A) T3 treated (n=4) <x) T4 treated (n=4) (o) control (n=8) i i i i i i i 1 1 -1 0 log free calcium (micromolar) 112 DISCUSSION A. Pre-Experimental Treatment Effects The treatment of rats with either T3 or T4 caused a s i g n i f i c a n t loss of body weight. Vehicle treated rats gained in body weight over the course of the study. Heart weights of treated animals were greater than those of controls. The heart weight/body weight ratio was increased from control values in both T3 and T4 treated rats (Figure 3). These data are in agreement with previous reports regarding heart and body weight s t a t i s t i c s in thyroid hormone treated rats (Gemmill, 1958; C a i r o l i and Crout, 1967; Frazer et a l . 1969; McNeill et a l . 1969; Sandford et a l . 1978). The increased heart weight/body weight ratio i s indicative of cardiac hypertrophy. This increase in the r e l a t i v e size of the heart i s thought to be associated with increased protein synthesis in the hyperthyroid animal (Sandford et a l . 1978). The results obtained in our study for changes in weight parameters are e n t i r e l y in accordance with the assumption that the animals had indeed become hyperthyroid as a result of the thyroid hormone treatment schedules employed. Spontaneous heart rate, measured prior to i n i t i a t i o n of e l e c t r i c a l pacing on the working heart apparatus, was found to be increased from control in both T3 and T4 hearts (Figure 4). This effect of thyroid hormone treatment in the rat has been reported many times previously (Gemmill, 1958; Van der Schoot and Moran, 1965; C a i r o l i and Crout, 1967; Frazer et a l . 1969). 113 Increase in heart rate i s an extremely important c l i n i c a l consideration in thyrotoxicosis. In most hyperthyroid patients tachycardia i s the major physiological mechanism employed by the heart to produce the increase in cardiac output required by the condition. In our experiments with hyperthyroid and control working hearts, heart rate was held constant through e l e c t r i c a l pacing of the a t r i a , and thus could not contribute to the changes in function seen as effects of thyroid hormone treatment. Spontaneous heart rates are reported here mainly as an index of thyrotoxicosis. Given the tachycardia of our treated animals, we are provided with further j u s t i f i c a t i o n in believing them hyperthyroid after treatment. It should be noted that the working hearts of thyroid hormone treated animals exhibited signs of electrophysiological changes. Tachycardia was accompanied by an increased tendency of the hyperthyroid hearts toward the development of arrhythmias. This propensity for arrhythmia i s well noted in the c l i n i c a l and experimental l i t e r a t u r e concerning hyperthyroidism (De Groot and Leonard, 1970; Freedberg et a l . 1970; Johnson et a l . 1973), and i s thought to be associated with a lowered d i a s t o l i c threshold for evocation of a t r i a l responses especially those from multiple pulses (Arnsdorf and Childers, 1970). Major delays in our attempts to characterize the function of the hyperthyroid myocardium were created by this tendency toward arrhythmia in the thyrotoxic animals. Among the 114 interim solutions employed to deal with the problem was an attempt to destroy the s i n o - a t r i a l (SA) node of the heart with formalin after recording the spontaneous heart rate and before i n i t i a t i o n of e l e c t r i c a l pacing. This method was not successful. When enough formalin to destroy the SA node and slow the heart was given, the function of the heart was most often severely depressed as well, due to the non-specific nature of the maneuver. Removal of the entire right atrium of each heart was also t r i e d as a remedy for the tachycardia and arrhythmia of thyrotoxicosis. This method too, was unsatisfactory. The surgery was r e l a t i v e l y crude and hard to control, also we found that removal of the entire atrium often did not slow the thyrotoxic hearts in the le a s t . Many of them continued to beat rapidly due to some other, ectopic pacemaker. The control hearts, on the other hand, seemed often to be mechanically compromised by a t r i a l excision. A satisfactory conclusion to the dilemma was accomplished by reducing both the concentration of calcium in the buffer, and the temperature of the preparation. O r i g i n a l l y we had used the standard Chenoweth-Koelle buffer containing 2.2mM calcium. By reducing the buffer concentration to 1.8mM calcium, s t i l l well within the physiological range, the tendency toward arrhythmia was diminished. By decreasing the temperature to 30 degrees Centigrade, arrhythmia was reduced to a minimum. Both control and hyperthyroid hearts, under these new experimental conditions, provided stable 115 preparations which could be e l e c t r i c a l l y paced at 300 beats per minute. S t a b i l i t y of heart function was also preserved while the manipulations of the function curve were performed and while the isoproterenol dose response curve was completed. Research of the l i t e r a t u r e produced the information that this problem had been faced by e a r l i e r workers using di f f e r e n t tissue preparations. Buccino et a l . (1967) had varied temperature from 21 degrees to 37 degrees Centigrade, eventually choosing 30 degrees as their experimental setpoint. Their temperature studies showed that while measurements of functional parameters might not remain numerically the same at different temperatures, the changes seen at different temperatures were d i r e c t i o n a l l y the same. Relative differences between treatment and control groups remained s i g n i f i c a n t at lower temperatures. The effect of the temperature change was not different for hyperthyroid as compared to control hearts. In the working heart preparation, Baisch et a l . (1981), have shown that temperatures ranging from 29 through 37 degrees Centigrade provide reasonable and reproducible values for assessing myocardial function. We therefore f e l t quite confident in the v a l i d i t y of the measurements made under the experimental conditions of this study. 116 B. C o n t r a c t i l i t y Studies 1. Function Curves Hearts from both the T3 and T4 treatment groups developed more l e f t ventricular pressure at faster rates of r i s e and f a l l for shorter periods of time than did controls (Figures 5 through 10). This data i s consistent with certain of the reports of the l i t e r a t u r e (Amidi et a l , 1968; Parmley et a l , 1968; Muryama and Goodkind, 1968;Taylor, 1970; Goodkind et a l . 1974; Brooks et a l . 1981). Both hyperthyroid groups could develop a higher maxiumum ventricular pressure than could controls in reponse to the highest a t r i a l f i l l i n g pressure (22.5cm of H20) of the function curve (Figure 5). Over the range of the function curve hyperthyroid hearts consistently exhibited an increased c o n t r a c t i l e response over euthyroid hearts, measured in terms of rate of pressure development (+ dP/dt, Figure 6) or rate of relaxation (- dP/dt, Figure 7). Values for a l l parameters of the function curve experiments are expressed in terms of unit performance index per heart e.g. l e f t ventricular pressure per heart. Since the hyperthyroid hearts were invariably larger than the control hearts (Table I ) , the p o s s i b i l i t y that the data might be biased by this fact was considered. Perhaps the a b i l i t y of the hearts to contract was the same in hyperthyroid and control animals, and the differences seen due only to the increased weight of the hyperthyroid hearts. Analysis of our data in terms of grams wet weight of heart is somewhat spurious in our view, as prior to treatment a l l three groups 117 of rats had similar body and heart weights. Thus the enlarged heart of the hyperthyroid rat i s part of the effect of treatment with thyroid hormone and the proper control from which to measure such an effect i s a heart from an otherwise i d e n t i c a l animal treated with vehicle. Nevertheless, in an e f f o r t to achieve completeness, the calculations were done to express the functional parameters in terms of unit weight. As might be expected, the values of LVDP, and positive and negative dP/dt's when corrected per unit weight were not di f f e r e n t for hyperthyroid hearts compared to control. The differences in a l l time parameters, however, were magnified rather than diminished. Thus, every point on the time to peak tension curve would become s i g n i f i c a n t l y d i f f e r e n t from control for both hyperthyroid groups (Figure 8). In relaxation time and t o t a l contraction time, where s i g n i f i c a n t differences had already been present, these differences would be exaggerated (Figures 9 and 10). Time to peak tension is at least as acceptable as a measure of the con t r a c t i l e response as is +dP/dt. Therefore, the statement that at each point on the working heart function curve a difference i s seen between hyperthyroid and control heart function, does not seem unduly unwarranted, whether in terms of +dP/dt per heart or TTP per gram wet weight. While certain of the parameters measured in this study are commonly reported, others are not as familiar, while s t i l l others are reported here for the f i r s t time. Some explanation of their possible significance i s in order. 118 Measurements of developed force, whether pressure or tension, and measurements of the rate of development of that force have been commonly used to indicate the state of c o n t r a c t i l i t y of the experimental myocardium. The use of these parameters in assessing c o n t r a c t i l i t y i s standard practice, but has been called into question by certain experimenters. (Abel, 1976). His contention, simply stated, is that assessment of c o n t r a c t i l i t y is often biased by the use of parameters and experimental procedures which involve dimensional change in response to Starling's law as an essential c h a r a c t e r i s t i c of their magnitude. That i s , not only i s the i n t r i n s i c c o n t r a c t i l i t y of the myocardial c e l l being measured by these parameters, but also included in the measurement i s the response of the c e l l to stretch. In this regard, the working heart preparation i s d e f i n i t e l y g u i l t y , as the very essence of the function curve is to subject the heart to varying f i l l i n g pressures causing varying amounts of stretch of the muscle f i b e r s , resulting in varying amounts of tension being generated in response. Abel (1976) advocated the use of parameters which throughout the course of such an experimental manipulation as the working heart function curve, maintain a r e l a t i v e l y constant value, i . e. have r e l a t i v e l y l i t t l e of their value due to the contribution of the response to stretch. Time to peak tension is t y p i c a l of this sort of measurement, while +dP/dt can be used as an example of a parameter whose value increases considerably over the range of the function curve in response to stretch. With this 119 controversy in mind and with new measurements made possible through the use of microcomputer software technology, we decided to augment the three standard measurements of LVDP, +dP/dt, and - dP/dt, by seven more or less novel ones. Time to peak tension was an obvious choice. It has often been reported in the l i t e r a t u r e , and exhibits the cha r a c t e r i s t i c s considered necessary to evaluate the accurate measurement of c o n t r a c t i l i t y by Abel. We also examined relaxation time and to t a l contraction time (very seldom measured in the li t e r a t u r e ) and time taken by the heart between the maximum +dP/dt and maximum -dP/dt (never before reported). Area under the l e f t ventricular pressure curve was measured, and measurments of area under the curve to peak pressure, and from peak pressure back to baseline were also carried out. Area under the curve has been previously reported by Seigel and Sonnenblick (1963) as IIT or integrated isometric time tension index. This parameter varies d i r e c t i o n a l l y as end d i a s t o l i c volume i s changed, and i s therefore not considered a c o n t r a c t i l i t y index of repute by Abel (1976). However, Seigel and Sonnenblick have reported IIT as a measure of the t o t a l impulsive force exerted by the muscle. Further, use of this index rather than either of i t s components (time or tension) provides an indication of the t o t a l mechanical energy a v a i l i b l e in each muscle contraction. If these assertions are correct, measurement of this index might prove useful in our study, so as to ascertain whether t o t a l mechanical energy per beat i s increased in hyperthyroid hearts. In monitoring a l l 120 of these pressure, time and area indices, we hoped to be able to detect any subtle differences that might surface not only in comparing function of hyperthyroid hearts to controls, but also in comparison of the function of the two treatment groups. This exercise had varied success. We found LVDP (Figure 5 ) , +dP/dt (Figure 6 ) , -dP/dt Figure 7 ) and TTP (Figure 8 ) ) to have almost i d e n t i c a l values in the T3 and T4 treated groups. It was not expected that they be quite so similar, as the animals were treated with different hormones for different lengths of time. No e f f o r t had been made to achieve exactly the same le v e l of hyperthyroidism, only to reproduce the most common treatments used. While most of the parameters measured showed equal values for T3 and T4 rats, there were some interesting differences between the two groups when relaxation time was considered (Figure 9 ) . Evaluation of this parameter showed that hyperthyroid working hearts in general relaxed much faster than their control counterparts. At high f i l l i n g pressures, however, while T3 hearts continued to relax very quickly, T4 hearts behaved in a manner more similar to the control than to the T3 hearts, with their relaxation times approaching those of control. Relaxation in T3 hearts seems to have been expedited in some manner. T4 hearts show the same sort of enhanced relaxation at low to normal f i l l i n g pressures, but at high f i l l i n g pressures, this enhancement was no longer present. As the uptake of calcium into the sarcoplasmic 121 reticulum i s thought to be a major determinant of relaxation e f f i c i e n c y in the myocardial c e l l , an obvious direction for further examination lay in measuring this uptake a c t i v i t y . This study was undertaken and w i l l be discussed l a t e r . Previous reports of relaxation time in hyperthyroidism include a study by Taylor et a l . in 1969, in which isovolumic contractions produced by sudden balloon occlusion of the ascending aorta were measured in T4 treated dogs. Decreased relaxation times in comparison to control were seen in hyperthyroid animals, in agreement with our data. Guarnieri et a l . (1980) also saw decreased relaxation times in their measurements in ventricle s t r i p s from hyperthyroid rats. Trends seen in the measurement of t o t a l pulse period, or t o t a l time of contraction, mainly r e f l e c t the differences between hyperthyroid and euthyroid hearts seen in the relaxation time values (Figure 10). Thus, a l l T3 contraction times are reduced from control, as are a l l T4 times up to the two highest a t r i a l f i l l i n g pressures (20 and 22.5cm. of H20). For these two pressures and for 17.5 cm. of water, T3 values are d i f f e r e n t from T4 times. Taylor et a l . (1969) and Guarnieri et a l . (1980) also measured duration of contraction, finding i t reduced in hyperthyroid preparations. The measurement of time from +dP/dt to -dP/dt was inspired by the work of Allen and Blinks (1978) using aequorin in the measurement of calcium transients in myocardial c e l l s . Their work appeared to indicate that cataloguing of t h i s time value might approximate the time at which calcium was being 122 actively accumulated by the cardiac sarcoplasmic reticulum. The results obtained (Figure 11), however, did not p a r t i c u l a r l y add to the information already gained from monitoring relaxation time, time to peak tension, and t o t a l pulse period. Further reinforcement of the differences between hyperthyroid and control heart function was obtained, but only at r e l a t i v e l y high f i l l i n g pressures. Differences between T3 and T4 function did not show up as cl e a r l y in this measurement as they did with relaxation time. Area under the l e f t ventricular pressure curve was measured (Figures 12,13,14). There were no differences observed between groups in the values obtained. This result suggests that the mechanical energy a v a i l i b l e during contraction was the same in euthyroid as i t was in hyperthyroid myofibrils. This equivalence might be expected, as in essence, this parameter consists of time multiplied by tension. Thus, in hyperthyroid function curves, although developed pressure was increased, time of contraction was decreased from control, so the product of the two values w i l l probably not be diff e r e n t from the product of the two control values. The most important results of the function curve studies are the marked increases in rates of r i s e and f a l l of developed pressure in hyperthyroid hearts over control hearts (Figures 6,7). Relaxation times and t o t a l contraction times were reduced from control values for both hyperthyroid groups, with the major differences in performance between the two 123 treatments being evident in the differences for these two parameters seen at high f i l l i n g pressures on the working heart (Figures 9 and 10). 2. Isoproterenol Dose Response Curves We had hoped the isoproterenol dose response curves obtained in this study might add to our understanding of myocardial function in hyperthyroidism. Variation within the groups was considerable, however, making analysis and interpretation d i f f i c u l t . The major difference between the groups seems to be a baseline difference between the T3 treatment group and the other two groups. In retrospect, although the outward performance of the two hyperthyroid goups through the function curve was very similar, perhaps the differences seen in relaxation time between T3 and T4 hearts was a presaging of the decline in function of the T4 hearts observed during the isoproterenol dose response curve. That the depression in function was a secondary change related to hypoxia i s ce r t a i n l y a p o s s i b i l t y in hyperthyroid tissue in which the oxygen consumption is markedly increased. However, in this case lack of oxygen seems unlikely as the cause of functional shortcomings. Although the two thyroid groups were treated in exactly the same fashion, the T3 hearts continued to demonstrate increased performance throughout the drug experiment, while T4 hearts exhibited declining performance. A l l groups did respond to isoproterenol, attesting to the 124 potency of the drug preparation. Maximum response to isoproterenol expressed in absolute terms was very similar for the hyperthyroid and euthyroid groups. LVDP's of T3 hearts were s i g n i f i c a n t l y d i f f e r e n t from both control and T4 values at a l l doses of isoproterenol and also prior to the administration of the drug (Figure 15). At low doses of isoproterenol +dP/dt was also d i f f e r e n t from control; at the lowest dose of isoproterenol T3 heart response was di f f e r e n t from that of T4 hearts as well (Figure 16). Guarnieri et a l . (1930) reported supersensitivity to isoproterenol for T4 treated rats at low doses of isoproterenol. No absolute values for dT/dt were given, however the authors reported no baseline differences. No differences from control were seen in measurements of time to peak tension (Figure 18) and relaxation time (Figure 19) in hyperthyroid hearts as compared to controls. At a dose of isoproterenol of 1 x 10E-9M in the measurement of t o t a l pulse period there was a s i g n i f i c a n t decrease for the T3 treated animals as compared to control (Figure 20). The results obtained with t h i s parameter are worthwhile examining in another regard. Control hearts in developing maximum response to isoproterenol showed a decrease in t o t a l pulse period of approximately 35 milliseconds, or a 20% reduction. The T3 group showed a decrease in t o t a l pulse period due to iso was 16 milliseconds or 10% reduced; T4 pulse period decreased about 14 milliseconds or an 8% decrease. A l l groups at maximum response had very similar t o t a l pulse period 125 values, despite the large spread of these values before drug treatment. No differences were seen in measurements of area (Figures 22 and 24) except at low doses of ISO in area from peak pressure back to baseline (Figure 23). These differences appeared to be baseline derived. Because of the major differences in baseline in a l l the measurements done in t h i s part of the study, a l l parameters were further graphed in terms of percent maximum response. When analysed in this manner, the clearest differences between groups were seen in the measurements of +dP/dt. Accordingly +dP/dt was used to calculate pD2 values. No s i g n i f i c a n t difference in this value was obtained in comparing controls and hyperthyroid animals. This result agrees with that shown by several experimenters (Margolius and Gaffney, 1965; Young and McNeill, 1974; Longhurst and McNeill 1979). Disagreement does occur with the results of others (Hashimoto and Nakashima, 1978; MacLeod and McNeill, 1981), who saw supersensitivity to catecholamines in guinea pigs and in rats (Tse et a l . 1980; Guarnieri et a l . 1980). Buccino et a l . 1967 and Van der Schoot and Moran, 1965 on the other hand saw catecholamine subsensitivity in cats and rats respectively. In this area of greatest controversy i t i s unfortunate that our own data are not more clear. 126 C. Studies of Calcium Uptake into Cardiac Sarcoplasmic Reticulum Rate of calcium removal from the sarcoplasm i s determined by the ATP dependent calcium transport mechanisms of the cardiac sarcoplasmic reticulum. This uptake a c t i v i t y can influence the rate of myocardial relaxation although a direct correlation between the two has not yet been shown. In various species, treatment with thyroid hormones for 10 days or more has been shown to increase the a c t i v i t y of calcium uptake into the SR (Nayler et a l . 1971; Suko, 1971; Limas, 1978a). In rats treated for 5 days with T4 however, McConnaughey et a l . (1979) were not able to show calcium uptake a c t i v i t y increased over control. In addition Goodkind et a l . (1974) saw no changes in a c t i v i t y of calcium uptake in guinea pigs treated with T4 for 3 days. Our results agree with the results of the l a t t e r two groups. In our rats treated for 7 days with T4 or treated for 3 days with T3 we could see no differences from control animals in calcium uptake a c t i v i t y into cardiac sarcoplasmic reticulum (Figure 27). 127 D. Integration Synthesis of the information gleaned from these experiments into one coherent picture is not a simple task. This study measured one biochemical parameter, calcium uptake a c t i v i t y of the sarcoplasmic reticulum. Dozens of other biochemical parameters may have had bearing on the functional changes seen in our studies of the hyperthyroid myocardium. As we have mentioned, the variety of treatments used makes extrapolation from one set of experiments to another set very ar b i t r a r y . It is also very risky to make the assumption based on observed results from one species that similar changes w i l l occur in another species. Indeed, experiments in which more than one species was studied tend to indicate the opposite, as most often there are large species dependent differences. Nevertheless some integration of our data with that collected by other laboratories i s necessary, i f only to provide direction to future studies. Our studies in rat have shown an increased rate of l e f t ventricular pressure development in T3 and T4 treated animals over controls (Figure 6 ) . Maximum velocity of shortening of muscle may be affected by changes in myosin ATPase a c t i v i t y . Although in several other species myosin ATPase a c t i v i t y i s known to increase as a res u l t of hyperthyroidism, in the rat experimenters have not been able to demonstrate such a change (Rovetto et a l . 1972; Yazaki and Raben, 1 9 7 5 ) . Even in species where changes in myosin ATPase have been shown, these biochemical changes were preceded temporally by changes in 128 c o n t r a c t i l i t y indices (Goodkind et a l . 1974). These performance differences have been seen within 24 hours of the i n i t i a t i o n of thyroid hormone treatment. Thus, later changes in c o n t r a c t i l i t y may involve myosin ATPase a c t i v i t y augmentation, but especially in the rat, some other mechanism must be responsible for the e a r l i e r function changes. Thyroid hormones do not cause increases in adenylate cyclase activation, nor in tissue levels of c y c l i c adenosine monophosphate (McNeill et a l . 1969; Brus and Hess, 1973) in rats. Tissue catecholamine levels have not been shown to be increased in T3 or T4 treated rats ( C a i r o l i and Crout, 1967). Activation of phosphorylase a has repeatedly shown to be increased in rats treated with thyroid hormones ( McNeill and Brody, 1968; Frazer et a l . 1969; McNeill et a l . 1969; Longhurst and McNeill, 1979). Phosphorylase activation can occur through the indi r e c t actions of cAMP dependent protein kinase. The suggestion has been made that in hyperthyroidism, s e n s i t i v i t y to cAMP at the protein kinase l e v e l i s increased. There i s no direct evidence for this hypothesis as yet. Consideration of the a v a i l i b l e reports make this an unlikely mechanism for the augmented performance of hyperthyroid rat hearts (Katz et a l . 1977; Tse et a l . 1980). Calcium can d i r e c t l y activate phosphorylase in the myocardial c e l l . Perhaps the mechanism of enhanced c o n t r a c t i l i t y in hyperthyroidism relates to increased influx of calcium into the c e l l by means of the slow inward calcium current. However, Skelton et al (1976) have used cat 129 papillary muscles skinned with glycerol to demonstrate the preservation of the increased c o n t r a c t i l e performance of hyperthyroid tissues even in the absence of sarcolemmal influences. Furthermore, i f the mechanism of increased contraction i s increased concentrations of calcium within the c e l l , why i s there no increase in calcium uptake a c t i v i t y to take this extra calcium back up out of the sarcoplasm? In the later stages of thyroid hormone treatment, perhaps, increases in the amount of calcium in the sarcoplasm are important to the functional changes seen, possibly due to perturbation of the sarcolemma. This would be consistent with the increased uptake of calcium into SR seen in animals treated for longer periods of time with thyroid hormones (Nayler et a l . 1971; Suko, 1971; Limas 1978a). Rate of force development depends on the shortening properties of the c o n t r a c t i l e element and the s t i f f n e s s of the series e l a s t i c component. Early studies by Parmley et al (1967) eliminated changes in the series element or s t i f f n e s s of the myocardium as a mechanism for the increased c o n t r a c t i l i t y of hyperthyroidism. Very recent work by Allen and Kurihara (in print, as reported by Jewell, 1982) has used aequorin fluorescence to characterize the calcium transients or fluctuating calcium levels within the mammalian myocardial c e l l . One of these newly reported studies shows the tension response of skinned ventricular muscle to calcium with the muscle having di f f e r e n t sarcomere lengths. The curve r e l a t i n g tension production to 130 calcium concentration can be shifted to the l e f t , that is the s e n s i t i v i t y to calcium increased i f the muscle i s stretched. Moving into the realm of speculation, perhaps the action of thyroid hormone within the myocardial c e l l i s to produce changes in the co n t r a c t i l e element so as to favor more tension development. This change need not be a stretched sarcomere, i t could as easily be some change in binding a f f i n i t y for calcium at troponin C. Enhanced contractile performance in hyperthyroidism would r e f l e c t an increase in calcium s e n s i t i v i t y at the co n t r a c t i l e protein, generating more tension for the same concentration of calcium. This mechanism would be congruent with the lack of difference shown in our studies of the sarcoplasmic reticulum calcium uptake a c t i v i t y (Figure 27). The calcium concentration at the myofibril being the same in the euthyroid as the hyperthyroid, the need for increased calcium uptake a c t i v i t y would be obviated. Several pieces of evidence are at odds with the mechanism of increased s e n s i t i v i t y to calcium at the contractile element. F i r s t l y , McNeill (1978), did co n t r a c t i l e studies in guinea pig Langendorf hearts which showed no increase in calcium s e n s i t i v i t y for hyperthyroid hearts over control hearts. Also, Goodkind (1969) showed a greater positive inotropy to calcium in euthyroid rather than hyperthyroid animals. Secondly, what of the increases in rate of relaxation seen in the hyperthyroid hearts? Increases in calcium s e n s i t i v i t y would not necessarily increase relaxation in conjunction with contraction. 131 However,in view of recent work by Ray and England (1976) and others (Solaro et a l . 1976) on phosphorylation of troponin I by cAMP dependent protein kinase activated through catecholamines, some form of calcium supersensitivity at the cont r a c t i l e protein l e v e l remains an attractive theory. Inotropic intervention by catecholamines causes increased slow calcium current and cAMP production, leading to increased tension. The amount of tension produced however, is less than might be expected for the large calcium transients which have been seen after catecholamine administration (Jewell, 1982). The phosphorylation of troponin I by cAMP dependent protein kinase has been shown to decrease calcium s e n s i t i v i t y (Mope et a l . 1980). The release of the bonds holding troponin I to actin and in s t e r i c hindrance of the myosin is the la s t step before crossbridge formation. With catecholamine administration phosphorylation of the troponin I by cAMP dependent protein kinase would tend to prolong this hindrance. Thus, i f the c o n t r a c t i l i t y increases seen in hyperthyroidism were due to increased calcium s e n s i t i v i t y at the calcium binding troponin C, phosphorylation of troponin I would mask the increased calcium s e n s i t i v i t y . This would lead to no difference in the s e n s i t i v i t y of hyperthyroid rat hearts to isoproterenol over control rat hearts, just as seen in our results (Figure 25). In p r a c t i c a l l y any other receptor system the large increase in receptor number seen with beta-adrenergic receptors in hyperthyroidism would lead to greatly increased 132 s e n s i t i v i t y to the agonist. However with the large number of spare beta receptors in the heart the effects of number increase w i l l not be great. By the law of mass action a certain increase in binding i s predicted by the increase in receptor number. It may be a f a i r l y subtle change however. This may explain to a certain extent the varied experimental results seen in the l i t e r a t u r e . The pD2 values seen by MacLeod and McNeill in 1981 in ^^ treated guinea pigs are almost i d e n t i c a l numerically to those obtained in this study for T3 treated rats. Values for control animals are also s i m i l a r . Yet in our study there were no s i g n i f i c a n t differences between groups, while in the e a r l i e r study supersensitivity to isoproterenol was shown. The variation in s t a t i s t i c s used accounts for a certain part of the d i f f e r e n t conclusion. There was considerably more variation within the groups in our study as compared to the e a r l i e r one as well. Nevertheless, these results are indicative of the continuing problem. When the differences one i s trying to examine are expected to be small, and the complications of d i f f e r i n g treatments, species and experimental design are added to the picture, the chances of obtaining clearcut s t a t i s t i c a l differences are remote. The differences in functional a b i l i t y of hyperthyroid hearts and in reported s e n s i t i v i t y of these hearts to catecholamines are not found in this study to be due solely to treatment differences. If our experiments had been done recording only the parameters previously measured in our 133 laboratory ( LVDP, +dP/dt, -dP/dt) the two thyroid hormone treatment groups would have been reported to have no difference in their performance throughout the function curve. The large v a r i a b l i t y encountered in the isoproterenol studies cannot allow major decisions to be made concerning s e n s i t i v i t y of the treatment groups to ISO. If supersensitivity had been defined in this study as an increased response at a sub-maximal dose of isoproterenol, as o i t appears to have been defined by Guarnieri et a l . (1980), we too would have supersensitivity by the T3 treated hearts at 1 -9 x 10 M ISO in measurement of t o t a l pulse time. If only T3 treated hearts had been compared with control hearts in calculation of pD2 values, and therefore t-test s t a t i s t i c s could have been used, we would have shown a leftward s h i f t of the dose response curve in +dP/dt, a supersensitivity very similar to that seen by MacLeod and McNeill (1981). Rather than continuing the age old battle over s e n s i t i v i t y to catecholamines in future studies, i t might prove more edifying to examine the i n t r i n s i c c o n t r a c t i l e properties of the hyperthyroid myocardium. The obvious extension of the present studies i s to involve other species. Guinea pigs, unlike rats, have been shown to develop increased myosin ATPase a c t i v i t y when treated with thyroid hormones. Would the functional differences in hyperthyroid hearts observed in this study in rats be magnified under the influence of this added biochemical parameter? Another obvious direction for future experimentation i s the 134 determination of calcium s e n s i t i v i t y of the hyperthyroid rat working heart beginning with simple dose response curves to calcium chloride. Skinned muscle preparations could also be used to determine i f calcium s e n s i t i v i t y remained the same in the absence of sarcolemmal influences. Measurements of LVDP, +dP/dt, -dP/dt, time to peak tension, relaxation time and t o t a l contraction time, a l l provided valuable information and should be monitored in future studies of this type. The information provided by the recording of time from +dP/dt to - dP/dt appears to be redundant; continued measurement of this parameter seems unwarranted. The value of measurement of areas under the contraction curve i s not yet established. It would probably be useful to continue to record these measurements for the next studies for the purpose of comparison especially i f the measurements obtained in this manner were compared with work determinations such as stroke work. 135 SUMMARY 1. Thyroid hormone treatment increased rate of l e f t ventricular pressure development and rate of relaxation in rat working hearts. 2. Hyperthyroid rat hearts had decreased relaxation times and t o t a l contraction times. 3 . Hearts from T3 treated animals differed from those from T4 treated animals in relaxation time at high a t r i a l f i l l i n g pressures. T4 hearts at these high f i l l i n g pressures had increased relaxation times, approaching control heart values. 4. 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Data Acquisition A simple operational amplifier c i r c u i t conditioned the l e f t ventricular pressure signal from the Grass polygraph. The output from this c i r c u i t was connected to one channel of the Analog to D i g i t a l (A/D) converter. A machine language routine was used to c o l l e c t the data and to store i t to disk. The pressure signal was sampled every 1.5 milliseconds for 1024 sample points, thus ensuring the capture of at least five complete pressure pulses at each c o l l e c t i o n time. An Applesoft Basic program provided a simple user interface to the machine language routine, c a l l i n g the routine repeatedly and saving the raw data through the use of a sequential naming convention. By means of another Applesoft Basic program, the stored raw data could be retrieved at w i l l , read back, and plotted on the high-resolution graphics of the microcomputer for v e r i f i c a t i o n of the sampling process. This process allowed the user to consecutively store data throughout an experiment in which the apparatus and/or protocol demanded constant attention. Post-experimentally, the stored data could be recalled for perusal and analysis. 146 B. Data Analysis A complete pressure pulse was i d e n t i f i e d in the data by means of yet another Applesoft Basic program. Maximum and miniumum pulse pressures were found, and 10% and 90% pressure values calculated from these. For purposes of measuring the time parameters, the start and f i n i s h of a pulse were defined as the time at which the pressure was evaluated at 1% of the maximum pressure. These values were found by f i t t i n g the best f i t exponential to twenty time points around the beginning of the curve. The time point of peak pressure was located by f i t t i n g a cubic equation to four points on the top 10% of the pressure curve, and then solving for the zero of the derivative of the res u l t i n g quadratic equation. Maximum positive and negative slopes of the pressure curve were determined by calculating areas of maximum change over points separated by several samples. Area under the curve was calculated by use of Simpson's Rule, that i s by f i t t i n g quadratic equations to each set of three consecutive data points (Barrodale et a l . 1971). The above calculations were done on two more complete curves from each raw data f i l e , and the averaged results stored to disk. Other programs written in Applesoft Basic allowed similar data from the same heart to be collected from separate disk f i l e s and averaged e.g. successive function curve determinations at a particular a t r i a l f i l l i n g pressure. Data from 'different t r i a l s of the same experiment could then 147 be collected and averaged into compiled f i l e s . These compiled f i l e s were used to compare d i f f e r e n t l y treated groups, and to plot the values obtained. An ANOVA program, which included Newman-Keuls Studentized Range S t a t i s t i c s , was used for s t a t i s t i c a l comparison of the different treatments. C. Mathematical Basis of Curve Analysis The analysis of individual pulses began by i s o l a t i n g three pulses from each set of fiv e d i g i t i z e d pulses sequentially into an array f ( i ) , with 1<=i<=200. Then the following values and times were determined. 1. The maximum and minimum data values: f(max) = MAXIMUM ( f(i),1<=i<=200 ) f(min) = MINIMUM ( f(i),1<=i<=200 ) 2. The following values and their positions: f(range) = f(max) - f(min), f(10%) = 0.1 ( f(range) ) s i m i l a r l y for f(50%),f(90%) and f ( 1 % ) . i +(10%) = ( i , such that f(i)<=f(10%) and f(i+1)>f(10%)), s i m i l a r l y for i + ( 9 0 % ) , i"(10%), and i"(90%). 3. To determine the start and f i n i s h of the pulse ( i ( s t a r t ) and i(end)), we assumed that the beginning of a pulse can be dx described by an exponential of the form: y = ce . This equation can be linearized by taking the natural logarithm of both sides to give: ln(y) = dx + l n ( c ) . Linear regression was performed on ln(y) against x to obtain the slope, m, and the 148 intercept, z ( i n t ) , giving: ln(y) = mx + z ( i n t ) . Therefore we could solve for the value of x which would give any given y. In this case we were interested in y = f ( 1 % ) , so solving the equation gave the time at which the curve was at 1% of i t s maximum, or i + ( 1 % ) . 4. Maximum positive and negative rates of change of pressure were determined as: +dP/dt = MAXIMUM ( (f(i+4) - f ( i - 4 ) )/8, i + ( 1 0 % X = i< = i +(90%) ), i(+dP/dt) = ( i ' - i ' ' ) / 2 , where i ' i s the largest i,and i ' 1 i s the smallest i,such that f ( i ) = f(+dP/dt). Sim i l a r l y for -dP/dt amd i(-dP/dt). 5. The time of maximum pressure, i(max), was determined by calculating the c o e f f i c i e n t s of the cubic equation passing through four points around the top of the pulse. More e x p l i c i t l y , find the equation g'(i) = q i J + r i + s i + t ,such that g(i) = f ( i ) at four equally spaced i ' s in the inte r v a l ( i +(90%) to i~(90%)) The maximum of this cubic p occurs at the zero of i t s derivative: g'(i) = 3qi + 2 r i + s. By l e t t i n g a=3q, b=2r, c=s, y=g'(i), and x=i this becomes p the familiar quadratic: y = ax + bx + c, and substituting the c o e f f i c i e n t s into the quadratic equation gave i(max). 6. The areas under the curve (AP,AB, and AT) were found using Simpson's rul e . This method approximates the area under a curve by f i t t i n g a series of quadratic equations to succeeding sets of three consecutive points. Fortunately,this procedure reduces to the following formula: 149 A = (h / 3 ) (f(1)+4f(2)+2f (3)+4f(4)+... + 4f(n -3)+2f(n-2)+4f(n-1)+f(n))) where n i s odd and h i s the time between samples; h = 1.5 milliseconds in our case. 150 APPENDIX II S t a t i s t i c a l Methods A. Newman-Keuls Test for Multiple Comparisons (Calculations for an example are shown in Part B) 1. One way ANOVA c l a s s i f i c a t i o n was carried out on the groups being tested. 2. The following data were tabulated: Degrees of freedom between groups df(B) Degrees of freedom within groups df(W) Mean square within groups MS(W) F value F The F value obtained was checked against the F value given in the P = 0.05, F-distribution tables (Table I I ) . If the calculated F value was less than the tabulated F value, none of the treatment groups was s i g n i f i c a n t l y d i f f e r e n t from each other, and the test was terminated at this point. 3. If the calculated F value indicated that there was a difference between the groups the Newman-Keuls test was used to determine whether the differences between the group means were s i g n i f i c a n t . 4. To this end, because the experimental groups were not of equal siz e , the harmonic mean of the group sizes was calculated for use in the test, through the use of the following formula: 151 HM = a/( 1/n(2) + ...1/n(a)) where a = number of experimental groups n = number of subjects in each group 5. The harmonic mean was substituted into the following f ormula: d = SQR (MS(W)/HM) 6. The Studentized Range S t a t i s t i c s (Q1, Q2, etc) were calculated from the Studentized Range Table (Table III) for number of comparisons made (e.g. a,a-1 etc.), versus the df(W) tabulated above. These s t a t i s t i c s (Q1, Q2, etc) were multiplied by the value of d obtained in step 5, to obtain the c r i t i c a l values (C1, C2, etc), which the difference between each pair of means must exceed to d i f f e r s i g n i f i c a n t l y at P<0.05. 7. The group means were arranged in order from lowest to highest and the difference between each pair of means was calculated. 8. The largest difference between group means was compared with the largest c r i t i c a l value i . e . C1 obtained for "a" comparisons by multiplying Q1 x d. If this difference exceeded i t s corresponding c r i t i c a l value, the means were s i g n i f i c a n t l y d i f f e r e n t at P<0.05. The next largest difference between means was then compared to i t s corresponding c r i t i c a l value i . e . C2 obtained for "a-1" comparisons by multiplying Q2 x d, u n t i l a l l groups were compared. If the largest difference between group means was not larger than C1 the testing ceased at that point. An 152 example from the data follows. B. Calculation Example: The Effect of Thyroid Hormone Pretreatment on Spontaneous Heart Rate in Rat. Control T3 treated T4 treated 195 # 300 355 195 260 285 155 350 210 240 420 335 220 350 210 220 335 325 210 225 335 220 330 210 290 215 n 12 5 7 Mean 226 336 294 ± SEM 13 27 23 // numbers indicate spontaneous heart rate in beats per minute from individual rat hearts. 1. From AN0VA: df(B) = 2 df(W) = 21 MS(W) = 2792 F calc =8.825 F tab = 3.47 153 Therefore there i s a difference between the groups 2. Calculation of Harmonic Mean: HM = 3/(1/12 + 1/5 + 1/7) = 7 3. Calculation of d: d = SQR (2792/7) = 19.97 4. Determination of Studentized Range S t a t i s t i c : Q1 = 3.58 (for a = 3) Q2 = 2.95 (for a-1 = 2) 5. Calculation of c r i t i c a l value: C1 = Q1 x d C2 = Q2 x d = 3.58 x 19.97 = 2.95 x 19.97 = 71.49 =58.91 6. Group Means Control = 226 T4 = 294 T3 = 336 T3 - Control = 110 > C1 * T4 - Control = 68 > C2 * T3 - T4 = 42 < C2 7. S i g n i f i c a n t differences at P<0.05 are indicated by an asterisk. In this example spontaneous heart rate in animals treated with T3 is s i g n i f i c a n t l y different from heart rate in control animals treated with vehicle. T4 treated animals also showed a s i g n i f i c a n t l y higher spontaneous heart rate than did control animals. There was no s i g n i f i c a n t difference between heart rates of T3 and T4 treated animals. 154 Table I I . T A B L E A 15 UPPbR 5°„ PERCLNTACI: POINTS. Q. IN T H t S l U D K N T I Z t U R A N G K * Degrees ol" Freedom./ I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 ' 18 i 19 | 20 ', 24 30 40 60 120 Number of Treatments, a 18.0 6.09 4.50 3.93 3.64 3.46 3.34 3.26 3.20 3.15 3.11 3.08 3.06 3.03 3.01 3.00 2.98 2.97 2.96 2.95 2.92 2.89 2.86 2.83 2.80 2.77 27.0 32.8 37.2 8.33 9.80 10.89 5.91 6.83 7.51 5.04 5.76 6.29 4.60 4.34 4.16 4.04 3.95 3.88 3.82 3.77 3.73 3.70 3.67 3.65 3.62 3.61 3.59 3.58 3.53 3.48 3.44 3.40 3.36 3.32 5.22 4.90 4.68 4.53 4.42 4.33 4.26 4.20 4.15 4.11 4.08 4.05 4.02 4.00 3.98 3.96 3.90 3.84 3.79 3.74 3.69 3.63 5.67 5.31 5.06 4.89 4.76 4.66 4.58 4.51 4.46 4.41 4.37 4.34 4.31 4.28 4.26 4.24 4.17 4.11 4.04 3.98 3.92 3.86 40.5 11.73 8.04 6.71 6.03 5.63 5.35 5.17 5.02 4.91 4.82 4.75 4.69 4.64 4.59 4.56 4.52 4.49 4.47 4.45 4.37 4.30 4.23 4.16 4.10 4.03 43.1 12.43 8.47 7.06 6.33 5.89 5.59 5.40 5.24 5.12 5.03 4.95 4.88 4.83 4.78 4.74 4.70 4.67 4.64 4.62 4.54 4.46 4.39 4.31 4.24 4.17 45.4 13.03 8.85 7.35 6.58 6.12 5.80 5.60 5.43 5.30 5.20 5.12 5.05 4.99 4.94 4.90 4.86 4.83 4.79 4.77 4.68 4.60 4.52 4.44 4.36 4.29 47.3 13.54 9.18 7.60 6.80 6.32 5.99 5.77 5.60 5.46 5.35 5.27 5.19 5.13 5.08 5.03 4.99 4.96 4.92 4.90 4.81 4.72 4.63 4.55 4.47 4.39 10 49.1 13.99 9.46 7.83 6.99 6.49 6.15 5.92 5.74 5.60 5.49 5.40 5.32 5.25 5.20 5.15 5.11 5.07 5.04 5.01 4.92 4.83 4.74 4.65 4.56 4.47 II 50.6 14.39 9.72 8.03 7.17 6.65 6.29 6.05 5.87 5.72 5.61 5.51 5.43 5.36 5.31 5.26 5.21 5.17 5.14 5.11 5.01 4.92 4.82 4.73 4.64 4.55 12 51.9 14.75 9.95 8.21 7.32 6.79 6.42 6.18 5.98 5.83 5.71 5.61 5.53 5.46 5.40 5.35 5.31 5.27 5.23 5.20 5 10 5.00 4.90 4.81 4.71 4.62 13 53.2 15.08 10.16 8.37 7.47 6.92 6.54 6.29 6.09 5.93 5.81 5.71 5.63 5.56 5.49 5.44 5.39 5.35 5.32 5.28 5.18 5.08 4.98 4.88 4.78 4.68 14 54.3 15.38 10.35 8.52 7.60 7.04 6.65 6.39 6.19 6.03 5.90 5.80 5.71 5.64 5.57 5.52 5.47 5.43 5.39 5.36 5.25 5.15 5.05 4.94 4.84 4.74 15 55.4 15.65 10.52 8.67 7.72 7.14 6.75 6.48 6.28 6.12 5.98 5.88 5.79 5.72 5.65 5.59 5.55 5.50 5.46 5.43 5.32 5.21 5.11 5.00 4.90 4.80 16 56.3 15.91 10.69 8.80 7.83 7.24 6.84 6.57 6.36 6.20 6.06 5.95 5.86 5.79 5.72 5.66 5.61 5.57 5.53 5.50 5.38 5.27 5.17 5.06 4.95 4.84 17 57.2 16.14 10.84 8.92 7.93 7.34 6.93 6.65 6.44 6.27 6.14 6.02 5.93 5.86 5.56 5.44 5.33 5.22 18 58.0 16.36 10.98 9.03 8.03 7.43 7.01 6.73 6.51 6.34 6.20 6.09 6.00 5.92 5.79 5.85 5.73 5.79 5.61 5.50 5.38 5.27 19 20 58.8 59.6 16.57 16.77 11.12 11.24 9.14 9.24 8.12 7.51 7.08 6.80 6.58 6.41 6.27 6.15 6.06 5.98 5.91 5.84 5.68 5.74 5.79 5.63 5.69 5.59 5.65 5.74 5.70 5.66 5.55 5.43 5.32 5.11 5.15 5.20 5.00 5.04 5.09 4.89 4.93 4.97 8.21 7.59 7.16 6.87 6.65 6.47 6.33 6.21 6.11 6.03 5.96 5.90 5.84 5.79 5.75 5.71 5.59 5.48 5.36 5.24 5.13 5.01 Reproduced from Snedecor and Cochran 's S t a t i s t i c a l Methods, 6th E d i t i o n , 1967. Iowa S ta te U n i v e r s i t y P r e s s , Ames, Iowa, page 568. 155 TABLE III. "2 1 2 3 4 5 6 8 12 24 X 1 161.4 199.5 215.7 224.6 230.2 234.0 238.9 243.9 249.0 254.3 2 18.51 19.00 19.16 19.25 19.30 19.33 19.37 19.41 19.45 19.50 3 10.13 9.55 9.28 9.12 9.01 8.94 8.84 8.74 8.64 8.53 4 7.71 6.94 6.59 6.39 6.26 6.16 6.04 5.91 5.77 5.63 5 6.61 5.79 5.41 5.19 5.05 4.95 4.82 4.68 4.53 4.36 6 5.99 5.14 4.76 4.53 4.39 4.28 4.15 4.00 3.84 3.67 7 5.59 4.74 4.35 4.12 3.97 3.87 3.73 3.57 3.41 3.23 8 5.32 4.46 4.07 3.84 3.69 3.58 3.44 3.28 3.12 2.93 9 5.12 4.26 3.86 3.63 3.48 3.37 3.23 3.07 2.90 2.71 10 4.96 4.10 3.71 3.48 3.33 3.22 3.07 2.91 2.74 2.54 11 4.84 3.98 3.59 3.36 3.20 3.09 2.95 2.79 2.61 2.40 12 4.75 3.88 3.49 3.26 3.11 3.00 2.85 2.69 2.50 2.30 13 4.67 3.80 3.41 3.18 3.02 2.92 2.77 2.60 2.42 2.21 14 4.60 3.74 3.34 3.11 2.96 2.85 2.70 2.53 2.35 2.13 15 4.54 3.68 3.29 3.06 2.90 2.79 2.64 2.48 2.29 2.07 16 4.49 3.63 3.24 3.01 2.85 2.74 2.59 142 2.24 2.01 17 4.45 3.59 3.20 2.96 2.81 2.70 2.55 2.38 2.19 1.96 18 4.41 3.55 3.16 2.93 2.77 2.66 2.51 2.34 2.15 1.92 19 4.38 3.52 3.13 2.90 2.74 2.63 2.48 2.31 2.11 1.88 20 4.35 3.49 3.10 2.87 2.71 2.60 2.45 2.28 2.08 1.84 21 4.32 3.47 3.07 2.84 2.68 2.57 2.42 2.25 2.05 1.81 22 4.30 3.44 3.05 2.82 2.66 2.55 2.40 2.23 2.03 1.78 23 4.28 3.42 3.03 2.80 2.64 2.53 2.38 2.20 2.00 1.76 24 4.26 3.40 3.01 2.78 2.62 2.51 2.36 2.18 1.98 1.73 25 4.24 3.38 2.99 2.76 2.60 2.49 2.34 2.16 1.96 1.71 26 4.22 3.37 2.98 2.74 2.59 2.47 2.32 2.15 1.95 1.69 27 4.21 3.35 2.96 2.73 2.57 2.46 2.30 2.13 1.93 1.67 28 4.20 3.34 2.95 2.71 2.56 2.44 2.29 2.12 1.91 1.65 29 4.18 3.33 2.93 2.70 2.54 2.43 2.28 2.10 1.90 1.64 30 4.17 3.32 2.92 2.69 2.53 2.42 2.27 2.09 1.89 1.62 40 4.08 3.23 2.84 2.61 2.45 2.34 2.18 2.00 1.79 1.51 60 4.00 3.15 2.76 2.52 2.37 2.25 2.10 1.92 1.70 1.39 120 3.92 3.07 2.68 2.45 2.29 2.17 2.02 1.83 1.61 1.25 x 3.84 2.99 2.60 2.37 2.21 2.10 1.94 1.75 1.52 1.00 Reproduced from Manual of Pharmacologic Calculations with Computer Programs, 1981. Springer-Verlag, New York, Heidelberg, Berl in, Ronald J . Tallarida and Rodney B. Murray, Page 144. 

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