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The effect of hyperthyroidism on rat cardiac sarcoplasmic reticulum Black, Shawn Clive 1986

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THE EFFECT OF HYPERTHYROIDISM ON RAT CARDIAC SARCOPLASMIC RETICULUM by SHAWN CLIVE BLACK B.Sc.(Pharm.), The U n i v e r s i t y of B r i t i s h Columbia. 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES FACULTY OF PHARMACEUTICAL SCIENCES DIVISION OF PHARMACOLOGY AND TOXICOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard « THE UNIVERSITY!OF BRITISH COLUMBIA October 1986 © Shawn C l i v e Black, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department O f Pharmacology and Toxicology The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 14, 1986 ABSTRACT Hyperthyroidism i s an endocrine disease which can a f f e c t the cardiovascular system. Cardiac function i s t y p i c a l l y augmented by the disease r e s u l t i n g in increased c o n t r a c t i l e force and a decrease in the relaxation time of the ventricular muscle. Since the cardiac sarcoplasmic reticulum (SR) has been shown to be intimately involved in both contraction and relaxation of the heart, i t was investigated whether i t was altered in the hyperthyroid rat heart. Hyperthyroidism was induced by subcutaneous i n j e c t i o n s of triiodothyronine (Tg) (dissolved in 0.01 N NaOH) at a dose of 500 ug/Kg/day for three days. The approach taken to investigate possible T^ mediated a l t e r a t i o n s in cardiac sarcoplasmic reticulum was to study the progression of the disease from the euthyroid state up to a point which has previously demonstrated augmented cardiac function. The e f f e c t of the treatment protocol was studied 12, 24, 48 and 72 hours a f t e r i t was i n i t i a t e d . Ventricular weight was augmented at 48 and 72 hours (p< 0.05 and p< 0.01, r e s p e c t i v e l y ) , and the SR y i e l d was s i g n i f i c a n t l y increased 24 (p< 0.05), 48 (p< 0.05), and 72 (p< 0.01) hours aft e r i n i t i a t i o n of the treatment. The r a t i o of SR y i e l d to v e n t r i c u l a r weight was greater in the treated animals indicating that the SR y i e l d was increased to a greater extent than the ventricular weight. The ATP-dependent o x a l a t e - f a c i l i t a t e d calcium transport a c t i v i t y of the SR preparation was determined at each of these times. There was no s i g n i f i c a n t difference in the rate of calcium i i uptake at 12 hours. At 24 hours, the treated rat SR calcium uptake a c t i v i t y was s i g n i f i c a n t l y (p< 0.05) higher at a l l free calcium concentrations assayed (range 0.1-5.3 pM) . At 48 and 72 hours, the SR V^ a was also s i g n i f i c a n t l y increased (p< 0.01 in each case). The K C a was not affected by the T^ treatment at any of the time points studied. Phosphorylation of the SR at 24, 48 and 72 hours indicated that the increased calcium uptake a c t i v i t y was associated with a s l i g h t , but not s i g n i f i c a n t , increase in the number of calcium pump s i t e s at 24 hours, but s i g n i f i c a n t l y more calcium pump s i t e s were l a b e l l e d at 48 and 72 hours (p< 0.01 and p< 0.05, res p e c t i v e l y ) . Therefore, the results of t h i s study suggest that hyperthyroid rat cardiac SR may contribute to the cardiac manifestations of the disease. Since long chain a c y l c a r n i t i n e s (LCAC) are known to affe c t membrane transport proteins, and l i p i d metabolism and tissue car n i t i n e content are affected by hyperthyroidism, i t was investigated whether the ca r n i t i n e derivatives l o c a l i z e d in the SR were affected by the T 3 treatment. The t o t a l carnitine content (including free, acid soluble and long chain carnitine) was s i g n i f i c a n t l y decreased 24 (p< 0.05), 48 (p< 0.05) and 72 (p <0.01) hours a f t e r i n i t i a t i o n of the treatment. Acid soluble c a r n i t i n e l e v e l s were not affected. LCAC l e v e l s were s l i g h t l y (but not s i g n i f i c a n t l y ) decreased at 24 hours, and s i g n i f i c a n t l y decreased at 48 and 72 hours (p< 0.05 and p< 0.01, res p e c t i v e l y ) . There was a strong negative c o r r e l a t i o n (r= -.93) between the increased and the decreased LCAC content of the SR. These re s u l t s suggest a possible r e l a t i o n s h i p between T^ mediated a l t e r a t i o n s in l i p i d metabolism and the increased calcium transport a c t i v i t y of the SR. However other factors may also be involved which contribute to both the augmented calcium transport and the decreased LCAC content of the hyperthyroid cardiac SR. John H. McNeill, Ph.D. Professor and Dean Faculty of Pharmaceutical Sciences Sidney KatzJ, Ph.D. Professor Faculty of Pharmaceutical Sciences TABLE OF CONTENTS page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ACKNOWLEDGEMENTS x i i i DEDICATION xiv INTRODUCTION 1 Cardiovascular E f f e c t s of Hyperthyroidism 5 Involvement of the Autonomic Nervous System 8 E f f e c t s of Hyperthyroidism on C o n t r a c t i l e Proteins 10 E f f e c t s of Hyperthyroidism on Calcium Metabolism 13 Metabolic Aspects of Hyperthyroidism 18 MATERIALS AND METHODS 23 I Materials A. Animals 23 B. Chemicals 23 II Methods 26 1. Hormone Treatment Protocol 26 2. Preparation of Cardiac Sarcoplasmic Reticulum 26 3. Measurement of Calcium Uptake A c t i v i t y 27 4. Measurement of SR Carnitines 28 A. Is o l a t i o n of Total, Acid Soluble and Long Chain Acyl Carnitines 29 B. Determination of Carnitine Content 29 v 5. Determination of SR Phosphoprotein Levels 30 6. Hydroxylamine Treatment 31 7. SDS-Polyacrylamide Gel Electrophoresis 32 8. Protein Assay 32 9. Determination of Free Calcium Concentrations 33 10. Determination of Serum Free Triiodothyronine Concentration 33 11. S t a t i s t i c a l Analysis 33 RESULTS 34 1. Characterization of the SR Preparation Employed in the Study 34 2. Studies on the Ef f e c t of T^ Treatment on Serum Free T 3, Body Weight, Ventricular Weight and SR Protein Y i e l d 39 3. Studies on the E f f e c t of T^ Treatment on Calcium Transport 48 4. Studies on the E f f e c t of T 3 Treatment on SR Carnitine Content 68 5. The Effect of T 3 Treatment on the Level of Calcium ATPase Phosphoprotein Intermediate 75 v i DISCUSSION 86 The Effe c t of T^ Treatment on Serum Free T^, Body Weight, Ventricular Weight and Sarcoplasmic Reticulum Y i e l d 86 The Ef f e c t of Hyperthyroidism on SR Calcium Transport and Phosphoprotein Levels 89 The Effe c t of Hyperthyroidism on Total, Short Chain and Long Chain Carnitine in Cardiac SR 94 CONCLUSIONS 105 BIBLIOGRAPHY 108 v i i LIST OF TABLES Table page 1 . The Eff e c t of T^ Treatment on Serum Free T^ Concentration in Control and 12, 24, 48 and 72 hour Hyperthyroid Rats 40 2. The Eff e c t of T^ Treatment on Mean Body Weight (g) 41 3. The Eff e c t of T 3 Treatment on Ventricular Weight (mg) . 42 4. The Eff e c t of T^ Treatment on Cardiac Ven t r i c l e Weight to Body Weight Ratio (mg:g) 44 5. The Eff e c t of T^ Treatment on the Amount of SR Protein Isolated (jjg) 45 6. The E f f e c t of T 3 Treatment on the SR Protein to Ventricular Weight Ratio (ug:mg) 47 7. The E f f e c t of Sodium Azide (5.0 mM) on SR Calcium Transport at 2.0 uM Free Calcium 67 v i i i LIST OF FIGURES Figure page 1. Comparison of the Sumida et a l , (1978) and McConnaughey e_t a l , (1979) SR Preparations with respect to Calcium Uptake A c t i v i t y . 35 2. Calcium Activation Curve in the Presence and Absence of 5.0 mM Sodium Azide 37 3. The E f f e c t of T 3 Treatment on SR Calcium Uptake at 12 Hours, Control vs Treated 49 4. The E f f e c t of T 3 Treatment on SR Calcium Uptake at 24 Hours, Control vs Treated 51 5. The Ef f e c t of T 3 Treatment on SR Calcium Uptake at 48 Hours, Control vs Treated 53 6. The E f f e c t of T 3 Treatment on SR Calcium Uptake at 72 Hours, Control vs Treated 55 7. The E f f e c t of T_ Treatment on V_ 3 C a vs Time (12, 24, 48 and 72 Hours) 57 8. Eadie-Hofstee Plots of 12 Hour Calcium Uptake Curve, Control vs Treated 59 9. Eadie-Hofstee Plots of 24 Hour Calcium Uptake Curve, Control vs Treated 61 10. Eadie-Hofstee Plots of 48 Hour Calcium Uptake Curve, Control vs Treated 63 11. Eadie-Hofstee Plots of 72 Hour Calcium Uptake Curve, Control vs Treated 65 ix 12. Cardiac SR Levels of Total Carnitine at 12, 24, 48 and 72 Hours. Control vs T 3 Treated at Each Time Point ....69 13. Cardiac SR Levels of Acid Soluble Carnitine at 12, 24, 48 and 72 Hours. Control vs T 3 Treated at Each Time Point 71 14. Cardiac SR Levels of Long Chain Acylcarnitine at 12, 24, 48 and 72 Hours. Control vs T 3 Treated at Each Time Point 73 15. Maximal Rate of Calcium Transport at 5.3 JJM Free Calcium vs the Level of Long Chain Acylcarnitine in the SR 76 16. SR Phosphoprotein Formation as a Function of Free Calcium Concentration (0.1 jjM-lOmM). 78 17. Calcium-Dependent Phosphoprotein Formation in Control and T 3 Treated SR at 10 mM Free Calcium. Determined at 24, 48 and 72 Hours 81 18. SDS-Polyacrylamide Gel Electrophoresis of SR From Control and 12, 24, 48,and 72 Hour T 3 Treated SR 83 x LIST OF ABBREVIATIONS AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate ATPase adenosine triphosphase C centigrade 1 4 C carbon-14 45 Ca calcium-45 i cAMP c y c l i c adenosine 5'-monophosphate CoA Coenzyme A CPT I c a r n i t i n e palmitoyltransferase I Da dalton DOC deoxycholate +dP/dT rate of development of l e f t v e n t r icular pressure -dP/dT rate of decline of l e f t v e n t r i c u l a r pressure et a l and others FFA free f a t t y acid g gram K k i l o K^a assocation constant of the enzyme for calcium L l i t e r LCAC long chain a c y l c a r n i t i n e LVDP l e f t v e n t r i c u l a r developed pressure m m i l l i micro M molar xi mg milligram min minute mL m i l l i l i t r e nmole nanomole 32 P phosphorus-32 pmole picomoles SDS sodium dodecylsulphate S.E.M. standard error of the mean SR sarcoplasmic reticulum triiodothyronine T 4 thyroxine TCA t r i c h l o r a c e t i c acid TRIS t r i s (hydroxymethyl aminomethane) w/v weight per unit volume V r maximal v e l o c i t y of calcium transport x i i ACKNOWLEDGEMENTS I am deeply grateful for the guidance and support provided to me throughout t h i s study by my supervisors, Dr.J.H.McNeill and Dr.S.Katz. I thank my committee members, Dr.R.W.Brownsey and Dr.K.M.MacLeod for their constructive comments and c r i t i c i s m s . I am f i n a n c i a l l y indebted to both the B r i t i s h Columbia and Canadian Heart Foundations for the support they have provided me throughout t h i s study. I g r a t e f u l l y acknowledge the invaluble assistance of Dr.D.Jeffery in determining the "free" calcium concentrations and Dr.M.Bridges for his assistance with the gel electrophoresis, and I thank the rest of my laboratory colleagues for their helpful comments and moral support throughout the study. I g r a t e f u l l y acknowledge the excellent typing and e d i t o r i a l assistance of L e s l i e Black. F i n a l l y , I thank the Faculty, Staff and Graduate students of the Faculty of Pharmaceutical Sciences, U.B.C. for making th i s Masters program enjoyable. xi i i DEDICATION One father i s more than a hundred schoolmasters. George Herbert, Jacula Prudentum That best academy, a mother's knee. James Russell Lowell, The Cathedral xiv INTRODUCTION Hyperthyroidism i s an endocrine disease which can af f e c t the cardiovascular system. The work of Parry in 1785 f i r s t described the association of thyroid enlargement with enlargement or palpations of the heart (Parry, 1825). These i n i t i a l observations remain v a l i d almost 200 years l a t e r as the cardiac manifestations of hyperthyroidism are amongst the more profound a l t e r a t i o n s and important c l i n i c a l features of the disease (Williams and Braunwald, 1984). The heart i s so t y p i c a l l y a f f e c t e d that the absence of a bounding precordium i s a point against the diagnosis of hyperthyroidism (DeGroot et a l , 1984). Other signs and symptoms of t h i s disease include: weight loss, weakness, dyspnea, increased t h i r s t or appetite, i r r i t a b i l i t y , profuse sweating, s e n s i t i v i t y to heat or increased tolerance to cold. Exopthalmopathy and goiter are also occasionally observed. The evidence does not support a geographical variation to the incidence of hyperthyroidism (DeGroot et a l , 1984). Epidemiological studies from England and Wales have shown the incidence of hyperthyroidism varies from 9.7 to 49.2 cases per 100,000 people (Barker and P h i l l i p s , 1984). In Iceland the incidence i s 23.6 cases per 100,000 population (Haraldsson et a l , 1985). External factors influence the incidence of the disease. The introduction of potassium iodate into commercial baking procedures caused an epidemic two months later in the exposed population (Connolly et a l , 1970). The sex d i s t r i b u t i o n 1 of the disease shows women to be from 4.2 (Harraldsson et a l , 1985) to 12 (Tunbridge et a l , 1977) times more frequently affected than males. The causes of hyperthyroidism are most often Graves' disease, toxic multinodular goiter and toxic adenoma (Werner and Ingbar, 1978). In Toronto, Canada, the d i s t r i b u t i o n of the various causes of hyperthyroidism are as follows: Graves' disease, 70%; toxic multinodular goiter and toxic adenoma, 8%; subacute t h y r o i d i t i s , 15%. (Williams et a_l, 1983). In Wales, the percent d i s t r i b u t i o n of Graves' disease i s the same but toxic multinodular goiter and toxic adenoma account for 25% of a l l cases (Williams e_t a l , 1983). Graves' disease presents as a dif f u s e enlargement of the thyroid gland, and toxic multinodular goiter as an unevenly enlarged gland. The cause of toxic multinodular goiter may be a single hyperfunctioning nodule, or a number of hyperfunctioning nodules (Werner and Ingbar, 1978). The exact etiology, of Graves' disease i s unknown, but a genetic predisposition, and abnormalities of the immune system have been implicated. Treatment of hyperthyroidism usually involves one of three forms of therapy: (1) destruction of the thyroid by i r r a d i a t i o n , (2) blocking thyroid hormone synthesis with a n t i -thyroid drugs (e.g. p r o p y l t h i o u r a c i l ) , or (3) p a r t i a l surgical ablation of the gland (DeGroot et a l , 1984). The disease results from the excess production of the thyroid hormones, triiodothyronine (T^) or thyroxine (T^), or both. The thyroid gland i s a butterfly-shaped organ located 2 a n t e r i o r l y and l a t e r a l l y to the trachea and pharnyx, the isthmus l i e s across the trachea a n t e r i o r l y just below the c r i c o i d c a r t i l a g e (DeGroot et a l , 1984). The average weight of the euthyroid gland is 30 grams, increasing to between 30 and 120 grams in hyperthyroidism (Werner and Ingbar, 1971). The functional unit of the gland i s the f o l l i c l e , a spherical arrangement of c e l l s responsible for the uptake, biosynthesis, storage and release of the thyroid hormones (DeGroot et a l , 1984). Thyroid hormone secretion i s under the influence of a negative feedback system, involving the hypothalamus, anterior p i t u i t a r y and thyroid gland. The hypothalamus secretes TRH, thyrotropin-releasing hormone, which t o n i c a l l y stimulates the anterior p i t u i t a r y . TRH receptor stimulation causes the release of TSH, thyroid-stimulating hormone. TSH binds to receptors on the surface of the f o l l i c u l a r c e l l s , stimulating the release of thyroid hormones. The primary i n h i b i t o r of TSH release i s Tg (Obregon et a l , 1980), with c i r c u l a t i n g T^ playing a role upon i n t r a p i t u i t a r y deiodination to Tg (DeGroot et at, 1984). In Graves' disease TSH lev e l s are usually low or normal, but c i r c u l a t i n g thyroid stimulators, i . e . long acting thyroid stimulator (LATS), mimic the e f f e c t of TSH at the gland and are not subject to negative feedback i n h i b i t i o n . Normal d a i l y secretion of the thyroid hormones i s 94-110 jdq T^ and 10-22 jjg Tg (DeGroot et a l , 1984). Approximately 25% of T 4 i s converted to T^ v i a peripheral 5'-monodeiodination, accounting for 80% of c i r c u l a t i n g Tg. The mean steady state concentration range of T^ is 60-100 ug/L, and for Tg the range i s 1-2.2 ug/L (DiStefano 3 and Fisher, 1979). Free concentrations of T 4 and T 3 in the euthyroid adult are 1-3.5 ng/dL and 0.25-0.65 ng/dL serum, respectively (DeGroot e_t a_l, 1984). The percentages of free T^ and T 3 are 0.02-0.04% and 0.2-0.45%, respectively, with the higher percentage of free T 3 due to the r e l a t i v e l y lower a f f i n i t y of plasma proteins for the hormone. In the rat, only 3% of the t o t a l T 3 pool resides in plasma while the remainder resides in slowly e q u i l i b r a t i n g pools (e.g. muscle, skin and brain; 76%) and rapidly e q u i l i b r a t i n g pools (e.g. l i v e r and kidney; 19%) (DiStefano et a l , 1982). Triiodothyronine i s believed to be the p h y s i o l o g i c a l l y active hormone (DeGroot et a l , 1984). T 3 e f f e c t s are dependent upon transport of free hormone from plasma to cytosol to nucleus. In rat heart, transport from plasma to cytosol i s a non-energy dependent, non-stereospecific mechanism but transport to the nuclear compartment i s energy dependent and ste r e o s p e c i f i c (Oppenheimer and Schwartz, 1985). The mechanism of action of T 3 involves an in t e r a c t i o n with s p e c i f i c receptors within the nucleus (Oppenheimer, 1979). Cardiac receptors are high a f f i n i t y (K D 0.9 x 10~ 1 0 M to K D 4.2 x 10~ 1 0 M) and low capacity (0.14 pmol/mg DNA to 0.5 pmol/mg DNA) (Ladenson, 1984). The receptor i s a nonhistone nuclear protein of 50,000 (Oppenheimer, 1985). That nuclear binding s i t e s are the functional T 3 receptors i s supported by data showing 1), the close c o r r e l a t i o n between the nuclear binding a f f i n i t y and the thyromimetic e f f e c t of T., analogs and 2), increases in nuclear 4 processes such as the rate of formation of polyadenylated messenger RNA, RNA polymerase a c t i v i t y and the presence of mRNA encoding for inducible protein (o<-2u globulin and p i t u i t a r y growth hormone) (Oppenheimer, 1979). There i s a po s i t i v e c o r r e l a t i o n between nuclear binding of T^ and stimulation of glucose uptake in cultured rat heart c e l l s (Tsai and Chen, 1976). T 3 therefore exerts i t s hormonal ef f e c t through nuclear interactions in T^ responsive tissues. CARDIOVASCULAR EFFECTS OF HYPERTHYROIDISM The cardiovascular consequences of experimentally induced hyperthyroidism are consonant with the expected hyperdynamic state. The maximum rate of tension development, the isometric developed tension and the rate of development of isometric tension are increased in is o l a t e d p a p i l l a r y muscle from hyperthyroid cat (Buccino et a_l, 1967; Taylor, 1970). Similar r e s u l t s have been found in hyperthyroid guinea pig p a p i l l a r y muscle (Goodkind et a l , 1974). Hyperthyroid dog l e f t v e n t r i c l e develops less tension during isovolumetric contraction, and the time to peak tension is decreased (Taylor, 1969). As well l e f t v e n t r i c u l a r c o n t r a c t i l e element v e l o c i t y i s augmented in the hyperthyroid dog heart. The hyperthyroid iso l a t e d working rat heart has an elevated spontaneous heart rate, increases in both the rate of development (+dP/dT) and the rate of decline of l e f t v e n t r i c u l a r pressure (-dP/dT), and the l e f t v entricular 5 developed pressure (Marriott and McNeill, 1983). This study also showed that the time to peak l e f t v e n t r icular pressure (LVDP), and ventricular relaxation time, are decreased. A more recent study (Brooks et a l , 1985) confirmed the results of Marriott and McNeill (1983), and demonstrated increases in cardiac output and coronary flow in the hyperthyroid isolated working rat heart. The experimental evidence in hyperthyroidism, therefore, i s equivocable with respect to changes in various indices of cardiac function in a number of d i f f e r e n t species. In man, similar cardiac a l t e r a t i o n s are observed. Cardiac output, resting heart rate, rate of development of l e f t v e n t r i c u l a r pressure and v e l o c i t y of contraction are a l l increased in the hyperthyroid state (Klein and Levey, 1984). Other cardiovascular manifestations of hyperthyroidism include p a l p i t a t i o n s and s y s t o l i c hypertension (Braunwald, 1984). Resting l e f t v e n t r icular ejection f r a c t i o n i s also augmented in man (Shafer and Bianco, 1980; Fofar et a l , 1982). The response of the hyperthyroid heart to exercise suggests the presence of a cardiomyopathy since l e f t v e n t r i c u l a r ejection f r a c t i o n increases in response to exercise in controls, but does not increase, or declines, in hyperthyroid subjects. This i s a revers i b l e myopathy, as restoration to the euthyroid state returns the exercise response to normal; th i s return to normal lags behind the return of euthyroid hormone le v e l s by several weeks (Fofar et a l , 1984). These results are in 6 c o n t r a d i s t i n c t i o n to animal studies showing l e f t v e n t r i c u l a r function to be augmented (increased LVDP and +/- dP/dt) (Marriott and McNeill, 1983) with increasing l e f t a t r i a l f i l l i n g pressures. However, because the l e f t v e n t r icular ejection f r a c t i o n was not d i r e c t l y measured ijn v i t r o (rat heart), as i t was in vivo (human heart), i t cannot be denied that the l e f t v e n t r i c u l a r ejection f r a c t i o n in v i t r o may have mimicked the in vivo response to the hyperthyroid condition. Further investigation measuring the l e f t v e n t r icular ejection f r a c t i o n in rats w i l l determine i f the myopathy exhibits similar exercise responses in species other than man. The cardiac manifestations of hyperthyroidism observed in both experimental animals and man could be due to either a d i r e c t effect of Tg on myocardial nuclear receptors and the subsequent augmentation of protein synthesis and the physiological response or a physiological adaptation of the heart to the increased demand imposed upon i t by Tg induced changes in peripheral metabolism. There i s evidence for both p o s s i b i l i t i e s . Isolated chick ventricular myocytes treated with Tg respond with an increased rate of protein synthesis (10%—16%) and rate of c e l l growth (20%-40%) (Carter et a l , 1985). Isolated v e n t r i c u l a r myocytes also spontaneously contract at a faster v e l o c i t y than control myocytes (Kim and Smith, 1985). These changes occur independently of an increased demand in work load and result from the Tg treatment. Klein and Hong (1986), however, have shown that although the i_n s i t u heart responds to 7 treatment with increases in cardiac weight, protein, and t o t a l myosin content, the rn vivo non-working heterotopically transplanted heart does not. The effect of hyperthyroidism on the heart may be a result of either p o s s i b i l i t y or a combination of the two. A number of parameters a f f e c t i n g cardiac c o n t r a c t i l i t y have been investigated in an attempt to define the biochemical mechanisms responsible for the augmented cardiac function in the disease state. INVOLVEMENT OF THE AUTONOMIC NERVOUS SYSTEM The cardiovascular manifestations of hyperthyroidism c l o s e l y resemble conditions of catecholamine excess. The accelerated heart rate of hyperthyroid patients i s attenuated by p-adrenergic receptor blockade (Grossman et a l , 1971). These observations, and those demonstrating that, (1) c i r c u l a t i n g catecholamine lev e l s are depressed in hyperthyroidism, (2) B-adrenergic receptors are not activated by thyroid hormones and (3) thyroid hormone l e v e l s are not affected by B-adrenergic antagonists (Williams and Lefkowitz, 1983), suggest the p o s s i b i l i t y that a l t e r e d catecholamine responsiveness of the cardiac tissue may be responsible for the observed cardiac a l t e r a t i o n s of hyperthyroidism. Experimental studies show cardiac responsiveness to be either unaffected (Buccino e_t a l , 8 1967; Levey et a_l, 1969; C a r r i o l i and Crout, 1967; Goodkind, 1967; Aoki et a l , 1967, 1972; Young and McNeill, 1974) or augmented ( C o v i l l e and Telford, 1970; Hashimoto and Nakashima, 1978; MacLeod and McNeill, 1981; Fox et a l , 1985) by the hyperthyroid state. Adenylate cyclase responsiveness i s also c o n t r o v e r s i a l , as both an increase (Tsai and Chen, 1978; Tse et a l , 1980) and no change (Sobel et a l , 1969; McNeill et a l , 1969) in the magnitude or the s e n s i t i v i t y of the response to catecholamines has been reported. The a c t i v i t y of cAMP-dependent protein kinase in response to both noradrenaline and isoproterenol i s s i g n i f i c a n t l y decreased in hyperthyroid rat heart (Katz et a l , 1977; Tse et a l , 1980), suggesting that t h i s step in the B-adrenergic response is not responsible for the possible s u p e r s e n s i t i v i t y of the heart to catecholamines. Studies on the influence of hyperthyroidism on B-adrenergic receptor density in rat heart report that with very few exceptions, receptor binding increases (176 +/- 98%; mean +/-S.D.) in response to thy r o i d hormone ( B i l e z i k i a n and Loeb, 1983). These changes occurr in the absence of any s i g n i f i c a n t e f f e c t on membrane a f f i n i t y for the binding ligand. In contrast to B-adrenergic receptor density, in rat heart the binding of ot-receptor ligands decreases by 40%, and ©(-antagonist binding a f f i n i t y decreases by a factor of two in rat heart. It i s apparent, therefore, that the hyperthyroid state a l t e r s adrenergic receptor pharmacology. The evidence showing an enhanced responsiveness of cardiac tissue to catecholamines and increased B-receptor density i s consistent with an 9 adrenergic receptor mediated hypothesis of altered cardiac function. However the re s u l t s concerning both the s e n s i t i v i t y of the hyperthyroid heart to catecholamines and the s e n s i t i v i t y of adenylate cyclase are not unequivicol. U n t i l the technical, species and/or other experimental differences responsible for the discrepant r e s u l t s are resolved, the adrenergic receptor hypothesis does not present a s u f f i c i e n t l y consistent case to explain the altered cardiac function of hyperthyroidism. This suggests that other mechanisms, perhaps more d i r e c t l y involved in the c o n t r a c t i l e process, may have a role to play. EFFECTS OF HYPERTHYROIDISM ON CONTRACTILE PROTEINS The augmented cardiac c o n t r a c t i l e force of hyperthyroidism may be related to changes in myosin ATPase a c t i v i t y of the heart. Several reports have documented an increase in ventricular myosin ATPase a c t i v i t y in response to thyroid hormone treatment in a number of d i f f e r e n t species including dog, rabbit and mini-pig (Conway et a l , 1976; Takeo et a l , 1984; Wiegand et a l , 1985). The hyperthyroid rat, however does not demonstrate an enhanced myosin ATPase a c t i v i t y (Rovetto e_t a l , 1972; Yazaki et a l , 1975). Rat myosin ATPase a c t i v i t y i s depressed in hypothyroidism but can be s h i f t e d to euthyroid l e v e l s by thyroid hormone treatment (Rovetto et a l , 1972), 10 indicating that the rat myosin ATPase a c t i v i t y i s also responsive to thyroid hormones under c e r t a i n conditions. Kinetic analysis of the ATPase reaction of cardiac myosin subfragment-I of rabbit v e n t r i c l e , has shown the major kinetic difference between euthyroid and hyperthyroid to be in the rate of ATP hydrolysis (Morkin et a l , 1983). Other k i n e t i c aspects of the reaction mechanism, such as the apparent ATP binding constants, and the rate constants for the binding process, were not affected by the hyperthyroid state. More d i r e c t biochemical analysis has revealed that thyroid hormone controls the d i s t r i b u t i o n of a family of ventricular myosin isoenzymes. Flink et a l (1979) have shown that thyroxine stimulates the synthesis of a cardiac myosin isoenzyme which d i f f e r s in the composition of i t s heavy chains, from that found in the euthyroid rabbit. Rat ventricular myosin has been separated into three isoenzymes (Hoh et a l , 1977). The isoenzymes are d i s t i n c t with respect to the composition of their respective heavy chains and ATPase a c t i v i t i e s . The isoenzymes, termed V"1 , V 2 and V^, have heavy chain subunits defined as Xo^^p and pB, respectively, with having the highest, V 2 intermediate and the lowest ATPase a c t i v i t y . The three isoenzymes are also present in rabbit v e n t r i c l e (Chizzonite et a l , 1984). However, the r e l a t i v e amount of each isoenzyme under euthyroid status d i f f e r s between rat and rabbit. Rabbit has a myosin d i s t r i b u t i o n of 85% and 5-10% of V 2 and , and the rat d i s t r i b u t i o n i s 80-85% V 1 , 5-10% V 2 and 10-15% V 3 (Morkin et a l , 1983). In the rabbit, the r e l a t i v e amount of increases 1 1 to 85% of the t o t a l following thyroxine administration (Martin et a l , 1981). The three myosin isoenzymes are also present in both rat and rabbit a t r i a , but in neither species is the d i s t r i b u t i o n of the isoenzymes affected by thyroid hormone treatment (Chizzonite e_t a l , 1984; Samuel et a l , 1986). The isomyosin composition however does a f f e c t the v e l o c i t y of cardiac muscle shortening (Schwartz et a l , 1981; Pagani et a l , 1984) and, therefore, in species such as the rabbit, where hyperthyroidism increases the proportion of , the e f f e c t of an increase in myosin ATPase a c t i v i t y can contribute to the augmented force of contraction. However, because rat v e n t r i c l e i s predominantly V 1 in the euthyroid condition a possible increase in the proportion of the f r a c t i o n in hyperthyroidism may not be s u f f i c i e n t to be responsible for the augmented c o n t r a c t i l e a c t i v i t y seen in the hyperthyroid state. Altered myosin ATPase a c t i v i t y cannot account for other functional changes occurring in hyperthyroidism such as the augmented rate of relaxation, hence other biochemical events such as c e l l u l a r calcium handling during contraction and relaxation of the myocardium should be considered. 1 2 EFFECT OF HYPERTHYROIDISM ON CALCIUM METABOLISM Cardiac function i s dependent upon the temporal flux of calcium ions through membrane systems governing the concentration of ionic calcium within the myoplasm. The three major membrane systems of the cardiac c e l l are the sarcolemma, sarcoplasmic reticulum and mitochondria. Mitochondrial calcium handling does not contribute to the normal regulation of cardiac contraction because (1) the i n h i b i t i o n of mitochondrial calcium uptake in intact hearts does not a l t e r the time course of relaxation and (2) the kin e t i c s of calcium uptake by the mitochondria i s too slow for the organelle to be important in normal calcium c y c l i n g (Winegrad, 1982). In certain pathological conditions, such as myocardial ischemia, mitochondrial calcium metabolism can a f f e c t c o n t r a c t i l i t y (Bourdillon e_t a l , 1981). The sarcolemmal membrane contributes to the regulation of free calcium through a number of mechanisms including the Na/Ca exchanger and the Ca-ATPase (Winegrad, 1982) and the glycocolyx coating external to the sarcolemmal membrane (Langer, 1978). Thyroid hormone mediated al t e r a t i o n s to any of these sarcolemmal processes has not been reported. Of pi v o t a l importance is the cardiac sarcoplasmic reticulum (SR), a membrane li m i t e d r e t i c u l a r structure of continuous vesicles and tubules forming a network through the myocyte surrounding the my o f i b r i l s . The SR i s responsible for the accumulation of calcium to promote relaxation (Tada et a l , 1978) and calcium release from the SR i s responsible for inducing contraction 1 3 (Fabiato, 1983). The enzyme responsible for the energy-dependent vectoral transport of calcium from the myoplasm into the lumen of the sarcoplasmic reticulum i s the calcium-activated, magnesium-dependent adenosine triphosphatase (Ca + Mg ATPase). The enzyme accounts for up to 40% of cardiac SR protein and has a molecular weight of approximately 100,000 daltons (Tada et a l , 1978). Other protein components of the SR membrane are phospholamban, a 22,000 dalton molecular weight protein involved in the regulation of calcium transport and ATPase a c t i v i t y (Ambudkar and Shamoo, 1984), calsequestrin, a 57,000 dalton calcium binding protein (Campbell et a l , 1982), a 53,000 dalton band, accounting for 10-15% of SR protein (Chamberlain et a l , 1983), and h i g h - a f f i n i t y calcium-binding protein ( C o l l and Murphy, 1984). Depending on the preparative technique used, between 20 and 40 protein bands have been detected in is o l a t e d SR membranes (Jones and Besch, 1979; Chamberlain et a l , 1983). The a c t i v i t y of the. calcium pump enzyme in the SR i s regulated by ions and regulatory proteins within the c e l l . Calcium ions autoregulate calcium transport by the SR such that increases in cytoplasmic free calcium stimulate ATP hydrolysis and calcium transport (Hasselbach, 1964). The rate of calcium uptake by the SR follows t y p i c a l Michealis-Menton k i n e t i c s , with a half-maximal stimulation by 1 pM calcium ( K C a 1 pM) (Tada et a l , 1974). Calcium transport by cardiac SR i s stimulated by monovalent cations; potassium augments both calcium ATPase and 1 4 calcium transport f i v e - f o l d (Jones et a_l, 1977). Potassium ion enhances the rate of dephosphorylation of the enzyme, thereby increasing the rate of enzyme turnover, expressed as increases in ATPase and calcium uptake a c t i v i t y . The augmentation of cardiac c o n t r a c t i l i t y and relaxation by p-adrenergic receptor stimulation i s well documented (Tsien, 1977). p-adrenergic receptor stimulation increases adenylate cyclase a c t i v i t y , r e s u l t i n g in elevated cAMP levels (Tsien, 1977). Cyclic-AMP promotes the release of the c a t a l y t i c subunit of c y c l i c AMP dependent protein kinase from the regulatory subunit. The c a t a l y t i c subunit catalyses the phosphorylation of phospholamban (Tada et a l , 1975). The close association of phospholamban with the Ca-ATPase enzyme, and the st r u c t u r a l perturbation induced by phosphorylation, r e s u l t s in augmentation of calcium uptake, with phospholamban serving as either an activator (Tada et a l , 1 978) or derepressor (Hicks et a l , .1979) of the enzyme. SR calcium transport and calcium ATPase a c t i v i t y are also regulated by calmodulin, a 17,000 dalton a c i d i c protein (Katz and Remtulla, 1978; Lopaschuk et a l , 1980; Davis et a l , 1983). Calmodulin requires the presence of calcium and possibly a s p e c i f i c calmodulin dependent protein kinase to augment SR calcium transport. The mechanism by which calmodulin stimulates calcium transport i s sim i l a r to that of cAMP-dependent protein kinase: the receptor protein for the kinase mediated phosphorylation i s phospholamban (LePeuch et a l , 1979), and the increased pump a c t i v i t y i s due to the interaction of the 1 5 covalently modified phospholamban with the enzyme. C y c l i c AMP-dependent protein kinase and calcium-calmodulin dependent protein kinase phosphorylate phospholamban at d i s t i n c t s i t e s and th e i r e f f e c t s are additive. It has also been suggested that calmodulin acts d i r e c t l y on the Ca-ATPase enzyme i t s e l f and not through the kinase phospholamban system (Katz, 1980). Cardiac SR i s also regulated by a calcium-activated, phospholipid-dependent protein kinase (protein kinase C). This enzyme catalyses the phosphorylation of SR r e s u l t i n g in augmented calcium transport (Limas, 1980) and calcium ATPase (Movsesian e_t a l , 1984) a c t i v i t y . This e f f e c t may also be mediated by phospholamban phosphorylation (Movsesian et a l , 1984). The cardiac sarcoplasmic reticulum i s , therefore, intimately involved in contraction and relaxation processes that take place in the heart. SR calcium transport i s a dynamic process affected by calcium, potassium and the three regulatory proteins described above. Previous studies have shown augmented cardiac SR calcium uptake and calcium ATPase from hyperthyroid rat and rabbit heart (Suko, 1974; Limas, 1978a; Guarnieri et a l , 1980). The r e s u l t s describing changes in hyperthyroid cardiac SR are not unequivocal however, as a decreased calcium uptake a c t i v i t y has been shown in both rat (Takacs et a l , 1985) and canine (Conway et a l , 1976) heart. The investigations of Limas (1978a) probed for possible mechanisms of the augmented SR function, and demonstrated an increase in the steady-state lev e l s of the 1 6 calcium transport phosphoprotein intermediate formed during the translocation reaction. This response, in addition to calcium transport, was prevented by i n h i b i t o r s of protein synthesis. The enhanced cAMP-dependent protein kinase phosphophorylation of phospholamban observed in hyperthyroid rat heart SR may indicate increased l e v e l s , or a c t i v i t y of, endogenous cAMP-dependent protein kinase (Limas, 1978b). These results are at variance with a subsequent report (Guarnieri et a_l, 1980) where, although basal calcium uptake was augmented in hyperthyroid rat heart SR, the response to exogenous cAMP-dependent protein kinase was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s . This l a t t e r study had results in accord with those of Katz e_t a l (1977), as no difference in c o n t r a c t i l e response to d i b u t r y l cAMP could be discerned between control and hyperthyroid rat heart. Katz e_t a_l (1977) also showed that the a c t i v a t i o n of cAMP-dependent protein kinase in response to d i b u t y r l cAMP was not altered by the hyperthyroid state. These studies on the eff e c t of thyroid hormone on the SR calcium pump indicate that more information i s required before a l l mechanisms can be defined which explain the enhanced SR function in hyperthyroidism. METABOLIC ASPECTS OF HYPERTHYROIDISM Hyperthyroidism induces changes in cardiac protein, carbohydrate and l i p i d metabolism (Muller and S e i t z , 1984a, 1984b, 1984c) and results in increased cardiac protein content. This i s due in part to a stimulation of protein synthesis (Carter et a l , 1985) and no concomitant, or a reducing e f f e c t on, protein catabolism (Carter et a l , 1980). The e f f e c t s of hyperthyroidism on protein metabolism are organ-specific, as s k e l e t a l muscle mass and sarcoplasmic r e t i c u l a r protein l e v e l s are decreased in response to the hormone. With respect to carbohydrate metabolism, cardiac glycogen catabolism i s sen s i t i v e to the tissue l e v e l s of phosphorylase a, the active form of the enzyme responsible for the formation of glucose -1-phosphate from glycogen. The c a t a l y t i c a c t i v i t y of phosphorylase i s dependent upon i t being in the active phosphorylase a, or phosphorylated, state. Phosphorylation of the enzyme i s mediated by phosphorylase kinase, which in turn i s regulated by calcium and cAMP-dependent protein kinase (Taegtmeyer, 1985). Adrenergic agents which increase tissue cAMP l e v e l s , as well as the influx of calcium during depolarization, therefore, influence the catabolism of glycogen. In the hyperthyroid rat heart, i t has consistently been demonstrated that basal phosphorylase a l e v e l s are increased and that the elevation in phosphorylase a in response to norepinephrine i s augmented (McNeill and Brody, 1968; McNeill e_t a l , 1969; Young and McNeill, 1974). Calcium ion mediated 18 a c t i v a t i o n of phosphorylase a i s not affected by hyperthyroidism (Hartley and McNeill, 1976). The metabolic consequence of elevated phosphorylase a a c t i v i t y would be decreased tissue glycogen stores which has been reported in hyperthyroid guinea pig heart by Bressler and Wittels (1966). In spite of lowered glycogen, which may have raised the oxidizable pool of glucose, glucose metabolism i s decreased. Lactate metabolism i s also depressed in hyperthyroid rat heart owing to the i n h i b i t i o n of pyruvate dehydrogenase r e s u l t i n g from an elevation in free fatty acid metabolism ( F i n t e l and Burns, 1982).Long chain fatty acid metabolism i s elevated in hyperthyroid guinea pig heart as well (Bressler and Wittels, 1966). Hyperthyroidism may a f f e c t cardiac l i p i d metabolism because of the increased serum free f a t t y acid concentration occurring in the disease (Muller and S e i t z , 1984). This would result in greater substrate delivery to the heart, as the heart derives a l l of i t s free fatty acids from the c i r c u l a t i o n either v i a passive d i f f u s i o n or active transport across the sarcolemma (Bieber and F i o l , 1985). Hyperthyroidism also influences l i p i d metabolism by i t s e f f e c t on c a r n i t i n e palmitoyltransferase I. Carnitine palmitoyltransferase I i s an inner mitochondrial membrane bound enzyme responsible for the transacylation of acyl-CoA thioesters to a c y l c a r n i t i n e esters (Schulz, 1985). Acyl c a r n i t i n e formation i s required for transfer of the acyl groups into the mitochondrial matrix by c a r n i t i n e : a c y l c a r n i t i n e translocase for subsequent B-oxidation. Carnitine 19 palmitoyltransferase I has been suggested to be the rate l i m i t i n g enzyme for free f a t t y acid metabolism (McGarry et a l , 1978). In hyperthyroid rat l i v e r (Stakkestad and Bremer, 1982) and guinea pig heart (Bressler and Wittels, 1966) the a c t i v i t y of the enzyme i s enhanced. Malonyl-CoA, an endogenous i n h i b i t o r of the enzyme (McGarry et_ a l , 1978), retains i t s i n h i b i t o r y effect in hyperthyroidism but the magnitude of i n h i b i t i o n i s reduced (Stakkestad and Bremer, 1982). Long chain a c y l c a r n i t i n e s , metabolic intermediates of fatty acid metabolism, have been shown to be potent endogenous in h i b i t o r s of a number of membrane bound enzymes. Palmitoyl car n i t i n e i s the most abundant of the a c y l c a r n i t i n e s and most frequently studied. DL-palmitoylcarnitine has been shown to i n h i b i t calcium-independent phosphodiesterase (Katoh et a l , 1982), calcium-dependent phospholipid-sensitive protein kinase (Wise and Kuo, 1983) and Na,K-ATPase (Wood et a l , 1977) from cardiac tissue. Sarcoplasmic r e t i c u l a r calcium uptake and calcium ATPase a c t i v i t i e s are also i n h i b i t e d by palmitoylcarnitine ( P i t t s et_ a l , 1978). Accumulation of long chain a c y l c a r n i t i n e s in the ischemic heart has been documented (Idell-Wenger et al_, 1978) and i n h i b i t i o n of sarcoplasmic r e t i c u l a r function by these intermediates proposed as a mechanism for the loss of c o n t r a c t i l i t y observed following ischemia ( P i t t s et a l , 1978). Accummulation of long chain acy l c a r n i t i n e s in SR fract i o n s of diabetic rat heart and the concomitant depression both in SR calcium transport and cardiac function has been proposed as a contributing factor to the 20 depressed cardiac function observed in diabetes (Lopaschuk et a l , 1983). Long chain a c y l c a r n i t i n e l e v e l s are, therefore, dynamic. I_n v i t r o studies have demonstrated that long chain a c y l c a r n i t i n e s have the capacity to i n h i b i t calcium fluxes across the sarcoplasmic reticulum. In hyperthyroid rat heart tissue t o t a l c a r n i t i n e l e v e l s are depressed (Suzuki et a l , 1983) and free and short-chain a c y l c a r n i t i n e lev e l s are depressed in mouse cardiac tissue (Cederblad and Engstrom, 1978); long chain a c y l c a r n i t i n e l e v e l s were not reported in either paper. Levels of long chain a c y l c a r n i t i n e s are detected in SR membrane fractions from control heart indicating that the metabolic intermediates are not a consequence of disease alone. The p o s s i b i l i t y e xists that the presence of endogenous long chain a c y l c a r n i t i n e s in SR membranes could play a role in calcium transport _i_n v i t r o , and possibly in cardiac function vivo» As mentioned e a r l i e r , cardiac c o n t r a c t i l i t y i s increased and relaxation time decreased in hyperthyroid rat heart. The close association of cardiac SR in these processes indicates that i t may be involved in these changes. Since studies have shown both an increase (Limas,1978a) and a decrease (Takacs et a l , 1985) in hyperthyroid rat cardiac SR calcium transport, i t was decided to determine the e f f e c t of hyperthyroidism on rat cardiac SR using an experimental model which has previously demonstrated the above cardiac a l t e r a t i o n s . To more clos e l y monitor the possible e f f e c t of hyperthyroidism on the SR i t was decided to study the SR during the progression of the disease. It has been suggested 21 that l i p i d m e t abolic changes c o u l d a f f e c t c a r d i a c f u n c t i o n through t h e i r e f f e c t s on membranes (Katz and Messineo, 1981). Since there i s an a l t e r a t i o n i n c a r d i a c f r e e f a t t y a c i d metabolism and a decrease i n the l e v e l s of the e s s e n t i a l c o f a c t o r c a r n i t i n e i n the h y p e r t h y r o i d h e a r t , i t was de c i d e d to determine i f the l e v e l s of the c a r n i t i n e d e r i v a t i v e s a s s o c i a t e d with the SR membrane are a f f e c t e d by the h y p e r t h y r o i d s t a t e , and i f a l t e r a t i o n s can be d e t e c t e d , the r e l a t i o n s h i p to p o s s i b l e changes i n c a l c i u m t r a n s p o r t a c t i v i t y determined. 22 MATERIALS AND METHODS I . M a t e r i a l s A. Animals Male Wistar r a t s i n the weight range of 250-300g were u t i l i z e d i n t h i s study. The r a t s were o b t a i n e d from the U.B.C. Animal Care f a c i l i t i e s . B. Chemicals Chemicals, p r o t e i n s and m a t e r i a l s used were purchased from the f o l l o w i n g s o u r c e s : 1 . Sigma Chemical Co. A c e t y l Coenzyme A Bovine Serum Albumin L - C a r n i t i n e C a r n i t i n e A c e t y l T r a n s f e r a s e Copper S u l f a t e Deoxycholic A c i d Disodium ATP Dowex 1X8-400 EGTA F o l i n - C i o c a l t e u Phenol Reagent H i s t i d i n e Hydroxylamine Magnesium C h l o r i d e 23 Sodium Azide Sodium Bicarbonate Sodium Potassium Tartrate Sucrose T r i c h l o r o a c e t i c Acid L-3,5,3'-Tri iodothyronine Tris-ATP Tris-Base Tris-HCl Tris-Oxalate 2. BDH Calcium Chloride Charcoal (Norit A (alkaline)) Potassium Chloride Sodium Acetate Sodium Hydroxide 3. Amersham Amerlex T-3 RIA k i t 4 5 C a 32 J P-ATP 4. Fisher Potassium Hydroxide Scintiverse II 24 5. Chemonics S c i e n t i f i c Sodium Chloride 6. Amachem Potassium Phosphate Monobasic 7. Pierce Chemical Co. Sodium Tetrathionate 8. J.T.Baker Chemical Co. Hydrochloric Acid 9. New England Nuclear Aquasol 10. ICN 1 4 1- C-Acetyl Coenzyme A 11. A l l i e d Chemical Perchloric Acid 12. M i l l i p o r e Corporation HA 0.45 u f i l t e r s 13. Whatman Ltd. GF/A 2.4 cm f i l t e r s 25 I I . Methods 1. Hormone Treatment Protocol Animals were randomnly assigned to either control (vehicle treated) . or test (L-3,5,3'-triiodothyronine (Tg) treated) groups. To induce the hyperthyroid state, the male Wistar rats were treated with T^ at a dose of 500 jjg/Kg/day. The T^ was dissolved in 0.01 N NaOH (1.0 mg/mL) and injected subcutaneously. Following treatment (between 0800 and 0900 hours) the animals were housed in group cages (3-4 animals/cage) where food and water were avail a b l e ad libitum. Animals were s a c r i f i c e d for study 12, 24, 48 and 72 hours after hormone treatment was initiated.Animals in the 12 and 24 hour groups received a single dose of T^ and were studied 12 and 24 hours after t h i s single dose, respectively. Animals in the 48 hour group received two doses and were s a c r i f i c e d 24 hours after the second dose. Those in the 72 hour group received three doses and were s a c r i f i c e d 24- hours after the l a s t dose. 2. Preparation of Cardiac Sarcoplasmic Reticulum Cardiac sarcoplasmic reticulum (SR) was prepared as previously described (McConnaughy et a l , 1979) with s l i g h t modifications (Dr.G.D.Lopaschuk, personal communication). Following decapitation of the rat, the heart was quickly removed and rinsed of blood in ice cold 10 mM NaHCO^, pH 7.4. The heart was trimmed of p e r i c a r d i a l f a t , connective tissue, large vessels and a t r i a , blotted dry on absorbent tissue paper and weighed. 26 The ventricular tissue was f i r s t minced with sc i s s o r s , followed by two 15 second homogenizations with a Kinematica tissue homogenizer (5 seconds rest between homogenizations) at speed 4. The homogenate was d i l u t e d to a volume of 25 mL with the homogenization buffer, and centrifuged at 500 xg for 5 minutes ( a l l centrifugation at 4°c). The r e s u l t i n g p e l l e t was discarded and the supernatant centrifuged at 7000 xg for 15 minutes. The p e l l e t was discarded and the supernatant centrifuged at 31,000 xg for 30 minutes. Following t h i s step the supernatant was discarded and the p e l l e t resuspended in 12 mL of media of 0.6 M KC1 and 30 mM h i s t i d i n e - C l , pH 7.0, and centrifuged at 31,000 xg for 30 minutes. The f i n a l p e l l e t was resuspended in 0.75 mL of a media consisting of 0.25 M sucrose, 0.3 M KC1 and 0.1 M T r i s - C l , pH 7.2. Aliquots of the SR were either quick frozen in l i q u i d nitrogen and stored at -80°c or, assayed immediately following i s o l a t i o n . 3. Measurement of Calcium Uptake A c t i v i t y SR calcium uptake a c t i v i t y was determined as follows: 5-10 jjg of SR protein was incubated at 30°c for 5 minutes in a reaction media containing (in mM): h i s t i d i n e - C l , pH 6.8 40; KC1 110; MgCl 2 5; NaNg 5; t r i s - o x a l a t e 2.5; tris-ATP 5. The reaction was started with the addition of EGTA buffered calcium (0.1 5.3 uM free, f i n a l concentration, 126 nmoles t o t a l added, 45 / containing CaCl 2; 100,000-200,000 dpm/tube to monitor calcium transport). The reaction proceeded for 5 minutes and was terminated with the f i l t r a t i o n of an aliquot of the reaction 27 mixture through a M i l l i p o r e HA 0.45 u f i l t e r . The f i l t e r was washed once with 15 mL 40 mM T r i s - C l , pH 7.2, dryed and counted in 5 mL l i q u i d s c i n t i l l a t i o n c o c k t a i l . The rate of calcium uptake a c t i v i t y was determined according to the following equation: Calcium Uptake (nmoles/mg SR protein/minute)= (S.C.-B.C.)/(T.C.-B.C.) x (D.F./R.T.) x (Total Ca 2 +/mg SR) Where, S.C. = Sample Counts = dpm obtained in sample T.C. = Total Counts = t o t a l dpm added to reaction B.C. = Background Counts = dpm present in 5 mL s c i n t i l l a t i o n c o c k t a i l alone D.F. = d i l u t i o n factor to correct for the volume of the reaction counted =1.22 R.T. = reaction time = 5 minutes 2 + Total Ca = t o t a l calcium present in the reaction media mg SR = mg of SR protein present in the reaction 4. Measurement of SR Carnitines The determination of the le v e l s of the t o t a l , acid soluble and long chain acyl c a r n i t i n e s were made following i s o l a t i o n of these fractions from an aliquot of SR. 28 A. Isolation of Total, Acid Soluble and Long Chain Acyl  Carnitines An aliquot of SR (100-200 jjg protein) was centrifuged at 40,000 xg for 45 minutes, the supernatant discarded, and the p e l l e t resuspended in 0.6 mL ice cold 6% perchloric a c i d . From t h i s suspension an aliquot of 0.1 mL was i s o l a t e d for determination of t o t a l c a r n i t i n e . This aliquot was neutralized with 0.075 mL 2 M T r i s base and placed on i c e . The remaining suspension was centrifuged at 12,000 xg for 10 minutes. A 0.20 mL aliquot of t h i s supernatant was i s o l a t e d for the determination of acid soluble c a r n i t i n e , neutralized with 0.15 mL 2 M T r i s base and placed on i c e . The p e l l e t , containing the long chain a c y l c a r n i t i n e f r a c t i o n , was washed with ice cold 6% perchloric acid, and resuspended in 0.1 mL d i s t i l l e d water. To both the t o t a l c a r n i t i n e and the long chain a c y l c a r n i t i n e f r a c t i o n s , 0.1 mL 1 M T r i s base and 0.05 mL 0.4 N KOH were added and the samples hydrolysed at 70°C for one hour. Following the hydrolysis the samples were neutalized with 0.2 mL 0.575 N HC1, and assayed as free c a r n i t i n e . B. Determination of Carnitine Content The method used to determine the amount of c a r n i t i n e present in the sample was that of McGarry and Foster, (1976). A volume of 0.15 mL of the c a r n i t i n e sample ( t o t a l , acid soluble or long chain) was added to 1.05 mL of reaction media containing 120 uM T r i s - C l pH 7.3, 2 uM sodium tetrathionate and 25 nM acetyl-CoA (0.025 uCi 1 - 1 4C~acetyl-CoA). The reaction was i n i t i a t e d by the 29 addition of 0.01 mL c a r n i t i n e acetyl transferase suspension (1 u n i t ) , and proceeded for 30 minutes at room temperature. The reaction was terminated by the addition of 0.3 mL of a suspension of Dowex 1X8-400 anion exchange resin (0.22 mL water in 0.3 mL suspension),vortexed and the reaction vessel placed in an ice bath. The tubes were vortexed twice more at 10 minute in t e r v a l s , each time being replaced into the ice bath. Following the vortexing the tubes were centrifuged at 1500 xg for 10 minutes. A 0.5 mL aliquot of the supernatant was added to 5 mL l i q u i d s c i n t i l l a t i o n f l u i d and counted. The amount of 1 4C-acety l - L - c a r n i t i n e formed i s st o i c h i o m e t r i c a l l y related to the amount of ca r n i t i n e present in the sample. 5. Determination of SR Phosphoprotein Levels The SR (0.15 - 0.35 mg/mL) was preincubated at 30°c for 5 minutes, then transferred to a 10°C water bath for a further 5 minute preincubation. The reaction was i n i t i a t e d by the addition of the SR membranes to the reaction media containing ( f i n a l concentration) h i s t i d i n e - C l , pH 6.8, 40 mM; MgC^, 0.01 mM; 32 Tris-EGTA, pH 7.4, 0.1 mM and Tris-ATP 2 jM (containing P-ATP at a s p e c i f i c a c t i v i t y of 2500 dpm/pmole t o t a l ATP). The phosphorylation reaction proceeded for 15 seconds and was terminated by the addition of 0.4 mL of an ice cold stop solution (5% w/v TCA, 5 mM Na2ATP and 2 mM KH2PC>4), vortexing and placing the reaction vessel in an ice bath. The reaction mixture was f i l t e r e d through a Whatman GF/A 30 f i l t e r , the f i l t e r washed with 30 mL ice cold 5% w/v TCA and dryed and counted in 5 mL l i q u i d s c i n t i l l a t i o n f l u i d . The amount of phosphoprotein formed i s calculated according to the following equation: Phosphoprotein (pmol/mg protein) = (S.C.-B.G.)/(S.A.)(R.V.)(P.T.) Where, S.C. = Sample Counts = dpm obtained in sample B.G. = Background = dpm from s c i n t i l l a t i o n c o c k t a i l alone 5. A. = Sp e c i f i c A c t i v i t y = (Media Counts - Background)/Total ATP R.V. = Reaction Volume = 0.2 mL P.C. = Protein Concentration (mg SR protein/mL) 6. Hydroxylamine Treatment The SR was phosphorylated under the reaction conditions described above and the reaction stopped by the addition of 0.4 mL of ice cold 15% w/v TCA. The SR membranes were centrifuged at 1500 xg for 10 minutes, and the supernatant discarded. The p e l l e t was resuspended in 0.5 mL of either 0.6 M hydroxylamine/ 6.8 M sodium acetate, pH 5.2, or 0.6 M sodium c h l o r i d e / 6.8 M sodium acetate, pH 5.2, and incubated at room temperature for 10 minutes, followed by the addition of 2.0 mL of ice cold 5% w/v TCA. The SR was p e l l e t e d by centrifugation at 1500 xg for 10 minutes, the supernatant discarded and the p e l l e t resuspended in 0.5 mL 5% w/v TCA. The suspension was f i l t e r e d on a Whatman GF/A f i l t e r which was dryed and counted in 5 mL of l i q u i d s c i n t i l l a t i o n c o c k t a i l . 31 7. SDS-Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed in 1.5 mm thick gels according to a modification of the method of Laemmli and Favre (1973): The sample buffer was i d e n t i c a l to the 0.0625 M T r i s - C l , pH 6.8 buffer used by these workers. The stacking gel was 5% (w/v) acrylamide, 0.1% (w/v) SDS and 0.315 M T r i s -HC1, pH 6.8; the separating gel was a 5-20% (w/v) acrylamide and a 0.13-1.0% (w/v) bis-acrylamide gradient in 0.375 M Tris-HCl, pH 8.8 and 0.1% (w/v) SDS. Following electrophoresis at 15 ma/slab for 16 hours, gels stained with AgNO^ (0.1%), according to Morrisey (1981). 8. Protein Assay SR protein was q u a n t i f i e d using a DOC/TCA p r e c i p i t a t i o n modification of the Lowry (Lowry et a_l, 1951) protein assay, as described by Peterson, (1977). A 1 0 uL aliquot of the f i n a l SR suspension was added to 1.490 mL of d i s t i l l e d water. To t h i s was added 12.5 uL of 2% DOC, the test tube vortexed and allowed to stand 10 minutes at room temperature. Following the incubation, 0.5 mL of ice cold 24% TCA was added, the tube vortexed, then centrifued at 1500 xg for one hour. The supernatant was c a r e f u l l y aspirated and discarded. To the p e l l a t was added 1.0 mL of copper reagent (1 mL 2% NaHC03 in 0.1 N NaOH, 0.01 mL 2% NaK t a r t r a t e and 0.01 mL 1% CuS0 4 per mL of reagent), the tubes vortexed and incubated at room temperature for 10 minutes. This was followed by the addition of 0.1 mL of a 50/50 F o l i n -Ciocalteu phenol reagent/water mixture. After a minimum of one 32 hour incubation at room temperature, the absorbance was read at 660 nm. 9. Determination of the Free Calcium Concentrations Free calcium concentrations were calculated using a FORTRAN program, TCATIONS.BC (modified s l i g h t l y from Goldstein, (1979)). Association constants were taken from Martell and Smith (1979-1982); except in the case of monoprotonated ligands which were calculated as described by Blinks e_t a l (1982). Prior to application of the program, constants were corrected for temperature according to Tirtoco et a_l ( 1978), using enthalphy values tabulated in Martell and Smith (1978-1982). The constants were also adjusted for ionic strength as described by Martell and Smith (1978-1982) and Blinks et a l , (1982). These l a t t e r three corrections were done using a BASIC program, APPK. 10. Determination of Serum Free Triiodothyronine Concentration The concentration of free T^ in rat serum samples c o l l e c t e d at the time of s a c r i f i c e were determined with an Amersham Amerlex R T-3 RIA k i t . 1 1 . S t a t i s t i c a l Analysis When two groups were compared, s t a t i s t i c a l analysis was performed using the unpaired Student's t - t e s t . For multiple comparison, one-way analysis of variance followed by Newman-Keuls' test was used. P r o b a b i l i t i e s of p< 0.05 and p< 0.01 were used to define the l e v e l of si g n i f i c a n c e . 33 RESULTS 1. Characterization of the SR Preparation Employed in the Study A comparison was made of the SR calcium uptake a c t i v i t y from two d i f f e r e n t SR preparative techniques; Sumida et a_l (1978) vs. McConnaughey et a_l ( 1979). The results shown in figure 1 demonstrate that at a l l free calcium concentrations tested, the SR prepared according to the modified McConnaughey et a_l (1979) method transported calcium with a greater v e l o c i t y and with a V"Ca approximately f i v e - f o l d greater than the Sumida et. a l (1978) preparation. The enhanced calcium transport a c t i v i t y of the preparation (McConnaughey et a_l, 1979) was considered important when attempting to discern possible differences in SR calcium transport a c t i v i t y between the control and T^ treated animals. The McConnaughey et a l (1979) preparation was therefore used throughout the study. To ascertain the possible contribution of mitochondrial membrane contamination in the SR preparation, calcium uptake a c t i v i t y in the presence and absence of 5 mM Na N3 was determined. Figure 2 shows that the calcium uptake a c t i v i t y determined under the experimental conditions employed i s independent of mitochondrial calcium transport processes. Since the objective of the study was to determine i f differences could be detected between control and T^ treated rat heart SR with respect to a number of parameters, i t was decided that a l l parameters would be assayed from each in d i v i d u a l SR sample prepared. Using t h i s technique interanimal v a r i a t i o n in 34 F i g u r e 1. Comparison of the Sumida et a l (1978) and the McConnaughey e t a l SR p r e p a r a t i v e t e c h n i q u e s w i t h r e s p e c t t o c a l c i u m t r a n s p o r t a c t i v i t y . R e s u l t s shown are a t y p i c a l e x p e r i m e n t . Sumida (•-•), McConnaughey ( o - o ) . 35 36 F i g u r e 2. C a l c i u m a c t i v a t i o n c u r v e of c a l c i u m uptake i n the absence (o-o) and p r e s e n c e (•-•) of 5 mM sodium a z i d e . C a l c i u m uptake a c t i v i t y d e t e r m i n e d as d e s c r i b e d i n M e t h o d s . R e s u l t shown i s a t y p i c a l e x p e r i m e n t . 37 CALCIUM UPTAKE + / ~ 5 m M SODIUM AZIDE vs FREE [Ca++] the cardiac response to hyperthyroidism was more readily apparent, and differences lost or gained were not masked by a pooling of the cardiac tissues. Therefore, for each group described below, the n value represents the results obtained from separate determinations of the various parameters from the SR y i e l d of single hearts. Preliminary results (data not shown) indicated that the SR y i e l d using the modified McConnaughey e_t al,(l979) SR preparation would be s u f f i c i e n t for these purposes. 2. Studies on the E f f e c t of T 3 Treatment on Serum Free T^ Body  Weight, Ventricular Weight and SR Protein Y e i l d Table 1 shows the serum free T^ concentrations of the control and treated groups. The data indicate that the animals were in fact c l i n i c a l l y hyperthyroid, as they had s i g n i f i c a n t l y elevated serum concentrations of T^. The e f f e c t of T^ treatment on body weight i s shown in Table 2, where the vehicle treated controls are compared to the T^ treated rats. Except for the 12 hour time point, where a s l i g h t drop in the mean body weight was observed, the control rats gained weight over the period of study (2%-4%). The mean body weight of the T 3 treated rats decreased over the study period (2%-6%). At no point was the mean body weight observed at s a c r i f i c e , s i g n i f i c a n t l y d i f f e r e n t from the i n i t i a l body weight. The wet ventricular weight of the control and T^ treated groups i s shown in Table 3. There was no s i g n i f i c a n t difference between the wet ventricular weight of the control and the T^ treated group 12 hours aft e r a single dose of T_. At 24 hours, 39 Table 1. The Eff e c t Of Tg Treatment On Serum Free T 0 Concentration TIME SERUM FREE Tg CONCENTRATION POINT (HR) (pmol/1) Control 4.9+/- 0.56 (9) 12 23.5+/- 7.5 (3) 24 16.0+/- 1.9 (3) 48 29.8+/- 3.2 (4) 72 121.7+/- 9.6 (7) Results expressed as the mean +/- S.E.M., Bracketed values (n)= the number of animals. 40 Table 2. The Effect of T ? Treatment on Mean Body Weight GROUP TIME CONTROL T 3 TREATED POINT (HR) BODY WEIGHT (g) BODY WEIGHT (g) INITIAL AT SACRIFICE INITIAL AT SACRIFICE 12 289+/-14 287+/-8 (-1) 288+/-8 275+/-4 (-5) 24 287+/-14 293+/-11 (+2) 286+/-12 280+/-8 (-2) 48 256+/-5 266+/-6 (+4) 253+/-4 246+/-4 (-3) 72 310+/-7 317+/-6 (+2) 308+/-12 289+/-9 (-6) Control refers to those animals receiving vehicle (0.01 N NaOH) alone. Results are mean +/- S.E.M.,n=6. The bracketed values represent the percentage increase (+) or decrease (-) in mean body weight at s a c r i f i c e r e l a t i v e to i n i t i a l weight. 41 Table 3. The Eff e c t of T., Treatment on Ventricular Weight (mg) TIME GROUP POINT (HR) CONTROL Tg TREATED 12 903+/-23 861+/-16 (N.S.) 24 808+/-84 860+/-30 (N.S.) 48 841+/-16 917+/-20 * 72 906+/-21 1046+/-29 ** The ve n t r i c u l a r weight was determined as described in Methods. The results shown are the mean +/- S.E.M., n=6. N.S., non-s i g n i f i c a n t ; *, s i g n i f i c a n t l y d i f f e r e n t from control p< 0.05; **, s i g n i f i c a n t l y d i f f e r e n t from control p< 0.01. 42 there was a s l i g h t but not s i g n i f i c a n t increase (9%) in the wet ventricular weight of the treated animals. At the 48 hour (P< 0.05) and 72 hour (P< 0.01) time points the wet v e n t r i c u l a r weight was s i g n i f i c a n t l y greater in the treated groups r e l a t i v e to the respective controls. The heart weight to body weight r a t i o i s an i n d i c a t i o n of the r e l a t i v e size of the heart. Administration of thyroid hormones has been associated with cardiac hypertrophy and increases in the heart weight to body weight r a t i o (Marriott and McNeill, 1983; Morkin et a l , 1983). Table 4 shows that the ventricular weight to body weight r a t i o (mg:g) increased with T^ administration after 24 hours of treatment. The difference increased with the duration of the T^ treatment. However because the control animals gained weight and the T^. treated animals l o s t weight, the r a t i o s in Table 4 may not be a r e l i a b l e index of hypertrophy . To correct for t h i s , a comparison was made based on i n i t i a l body weight where there was no difference between the treated and control groups. The r a t i o of the T^ treated animals remained greater than the respective c o n t r o l s . The magnitude of the difference was, however, decreased. A further indication that the T^ treatment affected the rat heart i s given in Table 5, where the amount of SR protein isolated i s shown. There was no s i g n i f i c a n t increase in the amount of SR protein i s o l a t e d 12 hours aft e r a single dose of T^. However, 24 hours aft e r a single dose, the amount of SR protein i s o l a t e d from the T^ treated hearts was s i g n i f i c a n t l y greater (p< 0.05) than that of the control group. At 48 and 72 43 Table 4. The Eff e c t of Tg Treatment on Cardiac V e n t r i c l e Weight to Body Weight Ratio (mg:g) TIME POINT (HR) CONTROL GROUP Tg TREATED DIFFERENCE 12 24 48 72 3.15 2.76 3.16 2.86 3.13 3.07 3.73 3.62 0.1 0.3 0.5 0.7 44 Table 5. The Ef f e c t of T^ Treatment on the Amount of SR Protein Isolated TIME GROUP POINT (HR) CONTROL Tg TREATED n 12 1046+/-76 24 792+/-38 48 960+/-60 72 1108+/-42 1005+/-59 (N.S.) 6 902+/-36 * 6 1146+/-75 * 13 1494+/-107 ** 6 The re s u l t s shown are in jug and are the mean +/- S.E.M., n= sample s i z e . N.S., non-significant difference between control and treated; *, s i g n i f i c a n t l y d i f f e r e n t from control p< 0.05; **, s i g n i f i c a n t l y d i f f e r e n t from control p< 0.01. 45 hours there was a s i g n i f i c a n t (p< 0.05 and p< 0.01, r e s p e c t i v e l y ) e l e v a t i o n i n the amount of SR p r o t e i n i s o l a t e d from the t r e a t e d group r e l a t i v e to the c o n t r o l s . To address the q u e s t i o n of whether the changes i n the amount of SR p r o t e i n i s o l a t e d are due to the o v e r a l l i n c r e a s e d c a r d i a c mass a s s o c i a t e d w i t h the T^ t r e a t e d animals or to a T^ mediated a l t e r a t i o n i n the SR p r o t e i n , the r a t i o of SR p r o t e i n i s o l a t e d to v e n t r i c u l a r weight (ug:mg) was c a l c u l a t e d (Table 6 ) . The r a t i o of SR p r o t e i n to v e n t r i c u l a r weight i n c r e a s e d i n the T^ t r e a t e d groups w i t h i n c r e a s i n g d u r a t i o n of the treatment, i n d i c a t i n g t h a t the SR was a l t e r e d by the treatment. 46 Table 6. The E f f e c t of T^ Treatment on SR Protein to  Ventricular Weight Ratio (uqtmq) TIME GROUP POINT(HR) CONTROL T 3 TREATED DIFFERENCE 12 1.13 1.17 0.04 24 0.98 1.05 0.07 48 1.14 1.25 0.11 72 1.22 1.43 0.21 47 3. S t u d i e s on the E f f e c t of Treatment on SR C a l c i u m T r a n s p o r t The r a t e of c a l c i u m t r a n s p o r t was d e t e r m i n e d i n SR a s s a y e d i m m e d i a t e l y a f t e r i s o l a t i o n over a f r e e c a l c i u m c o n c e n t r a t i o n range of 0.1 p M - 5.3 u M . At t w e l v e hours f o l l o w i n g a s i n g l e i n j e c t i o n of T^, t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the r a t e of c a l c i u m t r a n s p o r t a t any of t h e f r e e c a l c i u m c o n c e n t r a t i o n s a s s a y e d ( F i g u r e 3 ) . At 24 hours f o l l o w i n g the i n i t i a l dose of T^, the r a t e of c a l c i u m t r a n s p o r t was s i g n i f i c a n t l y (p< 0.05) g r e a t e r a t a l l f r e e c a l c i u m c o n c e n t r a t i o n s a s s a y e d ( F i g u r e 4 ) . At 48 h o u r s , ( F i g u r e 5) t h e r e was a f u r t h e r i n c r e a s e i n c a l c i u m t r a n s p o r t i n the T^ t r e a t e d group (p<0.0l a t a l l f r e e c a l c i u m c o n c e n t r a t i o n s , e xcept 0.5 J JM, where the s i g n i f i c a n c e remained a t p< 0.05). At 72 h o u r s , t h e c a l c i u m t r a n s p o r t r a t e was f u r t h e r augmented and was s i g n i f i c a n t l y (p< 0.01) g r e a t e r i n the T^ t r e a t e d SR a t a l l f r e e c a l c i u m c o n c e n t r a t i o n s a s s a y e d ( F i g u r e 6 ) . I t was d e t e r m i n e d t h a t the d i f f e r e n c e s o b s e r v e d i n the r a t e s of c a l c i u m t r a n s p o r t between c o n t r o l and T^ t r e a t e d groups were not due t o d i f f e r e n c e s i n non-ATP dependent c a l c i u m uptake ( d a t a not shown). F i g u r e 7 shows t h a t when the maximal c a l c i u m t r a n s p o r t r a t e (a t 5.3 jM f r e e c a l c i u m ) i s p l o t t e d as a f u n c t i o n of t i m e , the a ugmentation of c a l c i u m t r a n s p o r t by T^ t r e a t m e n t c o r r e l a t e s p o s i t i v e l y (r=0.997) w i t h the p r o g r e s s i o n of the d i s e a s e . 48 Figure 3 . The e f f e c t of T^ treatment on the rate of calcium transport 12 hours aft e r the f i r s t dose. Calcium transport a c t i v i t y of control (o-o) and T^ treated (•-•) SR was determined as described in Methods. The results are the mean +/- S.E.M. with an n=6 in each group. 49 CALCIUM UPTAKE at 12 HOURS 100-1 — \ 804 o QL 70-O-f 1 - I 1 1 p 0 1 2 3 4 5 FREE Ca++ CONCENTRATION (yixM) 50 F i g u r e 4 . T h e e f f e c t o f T ^ t r e a t m e n t o n t h e r a t e o f c a l c i u m t r a n s p o r t 2 4 h o u r s a f t e r i n i t i a t i o n o f t h e t r e a t m e n t . C a l c i u m t r a n s p o r t a c t i v i t y o f c o n t r o l ( o - o ) a n d T ^ t r e a t e d ( • - • ) S R w a s d e t e r m i n e d a s d e s c r i b e d i n M e t h o d s . R e s u l t s a r e t h e m e a n + / - S . E . M . w i t h a n n = 6 i n e a c h g r o u p . * , s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l p < 0 . 0 5 ) . 51 CALCIUM UPTAKE at 24 HOURS 100-1 "I 1 1 ! 1 1 P 0 1 2 3 4 5 FREE Ca++ CONCENTRATION (yuM) 52 F i g u r e 5 . The e f f e c t o f T^ t r e a t m e n t on t h e r a t e o f c a l c i u m t r a n s p o r t 48 h o u r s a f t e r i n i t a t i o n o f t r e a t m e n t . C a l c i u m t r a n s p o r t a c t i v i t y o f c o n t r o l ( o - o ) a n d T^ t r e a t e d ( • - • ) SR was d e t e r m i n e d a s d e s c r i b e d i n m e t h o d s . R e s u l t s a r e t h e mean + / - S . E . M . , w i t h a n n=6 i n e a c h g r o u p . * , s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l p< 0 . 0 5 ; * * , s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l p< 0 . 0 1 . 53 CALCIUM UPTAKE at 48 HOURS 100-1 0-| ; , , 1 1 r 0 1 2 3 4 5 FREE Ca++ CONCENTRATION (/xM) 54 F i g u r e 6. The e f f e c t o f T^ t r e a t m e n t on t h e r a t e o f c a l c i u m t r a n s p o r t 72 h o u r s a f t e r i n i t i a t i o n o f t h e t r e a t m e n t . C a l c i u m t r a n s p o r t a c t i v i t y o f c o n t r o l ( o - o ) a n d T^ t r e a t e d (•-•) SR was d e t e r m i n e d a s d e s c r i b e d i n M e t h o d s . R e s u l t s a r e t h e mean +/- S.E.M., w i t h an n=6 i n e a c h g r o u p . **, s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l p< 0.01. 55 CALCIUM UPTAKE at 72 HOURS O-f i 1 1 1 1 p 0 1 2 3 4 5 FREE Ca++ CONCENTRATION (/xM) 56 Figure 7. Graph showing the c o r r e l a t i o n of disease progression and augmented calcium transport a c t i v i t y . Control (o-o) and T 3 treated (•-•) values were obtained from the 5.3 JJM free calcium point on figures 3 -6 . Results are expressed as mean +/- S.E.M.. MAXIMAL CALCIUM TRANSPORT RATE vs TIME 110 - i 20-10-0 _ | , 1 1 1 1 12 24 36 48 60 72 T I M E . h o u r s 57 The K^a was calculated from the slope of Eadie-Hoffstee plots of the calcium a c t i v a t i o n curves at 12, 24, 48 and 72 hours (Figures 8-11, respectively. The K^a of the SR calcium transport process was not altered by the T^ treatment at any of the time points studied, and remained within the range of previously reported values (Tada et a l , 1978). The V"Ca was augmented at 24, 48 and 72 hours (y intercept). Table 7 shows that for both control and T^ treated groups there i s no s i g n i f i c a n t difference in calcium uptake a c t i v i t y either in the presence or absence of 5.0 mM sodium azide. 58 F i g u r e 8. E a d i e - H o f s t e e p l o t s of the 12 hour c a l c i u m uptake d a t a r e p r e s e n t e d i n F i g u r e 3, of c o n t r o l (o-o) and t r e a t e d (•-•) g r o u p s . The K C a d e t e r m i n e d from these p l o t s f o r b o t h the c o n t r o l and T^ t r e a t e d groups was 0.6 uM. 59 EADIE-HOFSTEE PLOT OF 12 HOUR CALCIUM UPTAKE CURVE 100-"5 c V/S (nmoles/mg prot./min)/(/LtM)) 60 Figure 9. Eadie-Hofstee plots of the 24 hour calcium uptake data represented in Figure 4 of control (o-o) and treated (•-•) groups. The K C a determined from these plots for control and T^ treated groups were 1.2 pM and 1.0 uM, respectively. 61 EADIE-HOFSTEE PLOT OF 24 HOUR CALCIUM UPTAKE CURVE V/S (nmoles/mg prot./min)/(/zM)) Figure 10. Eadie-Hofstee plots of the 48 hour calcium uptake data represented in Figure 5, of control (o~o) and treated (•-•) groups. The K C a determined from these plots for the control and T^ treated groups were 0.9 jiM and 0.8 uM, respectively. 63 EADE HOFSTEE PLOT OF 48 HOUR CALCIUM UPTAKE CURVE 120-1 100 H N / S ( n m o l e s / m g p r o t . / m i n ) / ( / 2 , M ) ) 64 F i g u r e 11. E a d i e - H o f s t e e p l o t s o f t h e 72 h o u r c a l c i u m u p t a k e d a t a r e p r e s e n t e d i n F i g u r e 6, o f c o n t r o l ( o - o ) a n d Tg t r e a t e d (•-•) g r o u p s . The K C a d e t e r m i n e d f r o m t h e s e p l o t s f o r t h e c o n t r o l a n d Tg t r e a t e d g r o u p s were 0.9 jM a n d 0.8 pM, r e s p e c t i v e l y . 65 EADIE-HOFSTEE PLOT OF 72 HOUR CALCIUM UPTAKE CURVE 120-1 W/S (nmoles/mg prot./min)/(/zM)) 66 Table 7. The E f f e c t of Sodium Azide (5 mM) on SR Calcium Transport At 2 jiM Free Calcium CALCIUM TRANSPORT (nmoles/mg protein/minute) TIME GROUP (+) NaN3 (-) NaN^ POINT (HR) 12 Control 41.2+/-2.5 37.9+/-3.8 T 3 Treated 39.7+/-2.3 38.1+/-4.3 24 Control 40.2+/-6.5 43.7+/-4.7 T 3 Treated 60.1+/-11.4 60.4+/-11.3 48 Control 43.0+/-3.6 40.3+/-4.5 T 3 Treated 57.9+/-2.7 54.3+/-4.4 72 Control 40.7+/-3.5 39.3+/-4.3 T 3 Treated 71.5+/-2.8 74.2+/-3.4 Results shown are the mean +/- S.E.M.. 67 4. S t u d i e s on t h e E f f e c t o f T r e a t m e n t on SR C a r n i t i n e L e v e l P r e v i o u s r e p o r t s h a v e i n d i c a t e d t h a t p a l m i t o y l c a r n i t i n e i n h i b i t s c a r d i a c SR c a l c i u m u p t a k e ( P i t t s e t a l , 1978) a n d t h a t d i a b e t i c r a t h e a r t SR d e m o n s t r a t e s d e p r e s s e d c a l c i u m u p t a k e a c t i v i t y c o n c o m i t a n t w i t h i n c r e a s e d l o n g c h a i n a c y l c a r n i t i n e l e v e l s ( L o p a s c h u k e_t a l , 1 9 8 3 ) . I t was t h e r e f o r e i n v e s t i g a t e d w h e t h e r t h e SR l e v e l s o f c a r n i t i n e e s t e r s c o u l d p l a y a r o l e i n t h e c a l c i u m t r a n s p o r t a l t e r a t i o n s a s s o c i a t e d w i t h t h e h y p e r t h y r o i d s t a t e . The SR l e v e l s o f t o t a l c a r n i t i n e ( w h i c h i n c l u d e s f r e e , s h o r t c h a i n a c y l a n d l o n g c h a i n a c y l c a r n i t i n e s ) , a c i d s o l u b l e ( s h o r t c h a i n ) a n d l o n g c h a i n a c y l c a r n i t i n e s were d e t e r m i n e d f r o m a f r a c t i o n o f t h e SR i s o l a t e d f o r c a l c i u m t r a n s p o r t s t u d i e s . T h e r e was no s i g n i f i c a n t d i f f e r e n c e i n t o t a l c a r n i t i n e l e v e l s 12 h o u r s a f t e r t h e i n i t i a l d o s e ( F i g u r e 1 2 ) . The T 3 t r e a t m e n t t h o u g h , r e s u l t e d i n a s i g n i f i c a n t d e p r e s s i o n i n t h e l e v e l o f t o t a l c a r n i t i n e ( F i g u r e 12) p r e s e n t i n t h e SR a t 24 h o u r s (p< 0 . 0 5 ) , 48 h o u r s (p< 0.05) a n d 72 h o u r s (p< 0.01) f o l l o w i n g i n i t i a t i o n o f t h e t r e a t m e n t . The l e v e l o f a c i d s o l u b l e c a r n i t i n e ( F i g u r e 13) was n o t a f f e c t e d by t h e T^ t r e a t m e n t , a s no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e c o n t r o l a n d T^ t r e a t e d g r o u p s c o u l d be d i s c e r n e d a t a l l t i m e p o i n t s s t u d i e d . The l e v e l s o f l o n g c h a i n a c y l c a r n i t i n e s ( F i g u r e 14) were n o t a l t e r e d a t 12 h o u r s a n d o n l y s l i g h t l y d e p r e s s e d ( n o t s i g n i f i c a n t ) a t 24 h o u r s . A t 48 a n d 72 h o u r s , t h e r e was a s i g n i f i c a n t d e p r e s s i o n i n t h e l e v e l s o f l o n g c h a i n a c y l c a r n i t i n e s (p< 0.05 a n d p< 0.01, r e s p e c t i v e l y ) . The a b s o l u t e v a l u e o f t h e t o t a l , a c i d s o l u b l e 68 Figure 12. The e f f e c t of T^ treatment on the l e v e l of t o t a l carnitine measured in the SR. Total c a r n i t i n e l e v e l s were determined as described in Methods. Results shown are the mean +/- S.E.M.. The sample sizes, for both control and T^ treated groups, at the various time points are as follows: 12 hours, 5; 24 hours, 5; 48 hours,4; 72 hours, 6. 69 TOTAL SR CARNITINE 220-200-UJ 180-O 12HR 24HR 48HR 72HR TIME POINT OF DETERMINATION 70 Figure 13. The e f f e c t of T^ treatment on the l e v e l of acid soluble c a r n i t i n e measured in the SR. Acid soluble carnitine l e v e l s were determined as described in Methods. Results shown are the mean +/- S.E.M.. The sample sizes for control (C) and T^ treated (T) groups at the various time points are as follows: 12 hours, (C)=(T)=5; 24 hours, (C)=5, (T)=6; 48 hours, (C)=(T)=4; 72 hours, (C)=(T)=6. 71 ACID SOLUBLE SR CARNITINE 60 54-z U J 1— 48-o cn Q_ 42-cr tn o> 36-30-z f— z 24-cr < (J 18-a> o 12-E c 6-12HR 24HR 48HR 72HR TIME POINT OF DETERMINATION 72 Figure 14. The e f f e c t of Tg treatment on the l e v e l of long chain a c y l c a r n i t i n e s determined in the SR. Long chain a c y l c a r n i t i n e l e v e l s were determined as described in Methods. Results shown are the mean +/- S.E.M.. The sample sizes for the control (C) and Tg treated (T) groups are as follows: 12 hours, (C)=(T)=5; 24 hours, (C)=5, (T)=6; 48 hours, (C)=(T)=4; 72 hour, (C)=(T)=6. 73 LONG CHAIN SR CARNITINE and long c h a i n a c y l c a r n i t i n e s v a r i e d q u i t e s i g n i f i c a n t l y , as shown by the s c a l e of the y - a x i s of F i g u r e s 12, 13 and 14 . The reason f o r t h i s v a r i a t i o n i s not known at t h i s time. There i s a str o n g n e g a t i v e c o r r e l a t i o n (r= - 0 . 9 3 ) between the r a t e of c a l c i u m t r a n s p o r t at maximal f r e e c a l c i u m ( 5 . 3 uM) and the l e v e l of long c h a i n a c y l c a r n i t i n e s present i n the SR (F i g u r e 1 5 ) . Thus, i t appears that the augmented r a t e of SR c a l c i u m t r a n s p o r t observed i n the T^ t r e a t e d animals may i n some way be r e l a t e d to the d e p r e s s i o n i n the SR l e v e l of long c h a i n a c y l c a r n i t i n e s . 5. The E f f e c t of T^ Treatment on The L e v e l of Calcium ATPase  Phosphoprotein Intermediate. F i g u r e 16 shows SR phosphoprotein formation as a f u n c t i o n of f r e e c a l c i u m c o n c e n t r a t i o n (range 0 .1 iuM-10 mM) i n SR from c o n t r o l heart t i s s u e . The r e a c t i o n time was 15 seconds to maximize steady s t a t e c a l c i u m ATPase phosphoprotein formation and to minimize kinase-mediated p h o s p h o r y l a t i o n of SR p r o t e i n . Under these c o n d i t i o n s , the phosphoprotein formed was p r i m a r i l y the phosphoprotein i n t e r m e d i a t e a s s o c i a t e d with the c a l c i u m pump as hydroxylamine treatment r e s u l t e d i n a marked r e d u c t i o n i n the c a l c i u m dependent a c y l phosphate phosphoprotein l e v e l s (68%) yet had l i t t l e e f f e c t on c a l c i u m independent phosphoprotein formation (15%). Calcium-dependent phosphoprotein formation i n c r e a s e d with i n c r e a s i n g f r e e c a l c i u m . Subsequent p h o s p h o r y l a t i o n experiments 75 Figure 15. Correlation of maximal calcium transport a c t i v i t y (determined at 5.3 uM free calcium as described in Methods) with the SR l e v e l of long chain a c y l c a r n i t i n e s . Results are expressed as the mean +/- S.E.M.., control (o-o) and T_ treated (•-•) groups. 76 MAXIMAL CALCIUM TRANSPORT RATE vs SR LCAC CONCENTRATION F i g u r e 16. SR p h o s p h o p r o t e i n f o r m a t i o n as a f u n c t i o n of f r e e c a l c i u m c o n c e n t r a t i o n . P h o s p h o r y l a t i o n performed a c c o r d i n g t o p r o t o c o l d e s c r i b e d i n Methods. R e s u l t s shown a r e t h e mean +/- S.E.M. of two s e p a r a t e e x p e r i m e n t s p erformed i n d u p l i c a t e . 78 CALCIUM DEPENDENT PHOSPHOPROTEIN vs LOG FREE [Ca++] 250-n OH 1 1 1 1 1 1 -7 -6 -5 -4 -3 -2 -1 LOG FREE [Ca++] 79 were ca r r i e d out at 10 mM free calcium to label a maximal number of s i t e s under these conditions. Figure 17 shows the calcium dependent phosphoprotein formation in control and Tg treated SR membranes at 10 mM free calcium. There was no s i g n i f i c a n t difference between the phosphoprotein l e v e l s determined in any of the three control groups. Twenty-four hours following the f i r s t dose there was a s l i g h t but not s i g n i f i c a n t increase in the phosphoprotein levels detected in the Tg treated group. By 48 hours, phosphoprotein levels in the Tg treated group were s i g n i f i c a n t l y (p< 0.01) greater than control l e v e l s and at 72 hours the Tg treated phosphoprotein l e v e l s remained s i g n i f i c a n t l y (p< 0.05) greater than the control l e v e l . The hydroxylamine s e n s i t i v i t y and the calcium-dependence of the phoshoprotein formed indicated that i t was primarily the calcium pump protein that was labeled. The number of active pumping s i t e s was, therefore, increased after 24 hours and s i g n i f i c a n t l y increased after 48 and 72 hours of the Tg treatment regimen. Figure 18 shows the results of the SDS polyacrylamide gel electrophoretic separation of the SR proteins v i s u a l i z e d by R s i l v e r s t a ining. Track A shows the Biorad molecular weight marker proteins; myosin (200 kDa), B-galactosidase (116.3 kDa), phosphorylase B (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin i n h i b i t o r (21.5 kDa) and lysozyme (14.4 kDa). Tracks B, C, D, E, and F represent control, 12, 24, 48 and 72 hour SR respectively. The gel indicates that there i s no q u a l i t a t i v e 80 Figure 17. Phosphoprotein l e v e l s in control and treated SR fractions determined at 10 mM free calcium. Phosphoprotein determinations were performed as described in methods, and the res u l t s shown are the mean +/- S.E.M., N.S.= non-significant, *= s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , p< 0.05, **= s i g n i f i c a n t l y d i f f e r e n t from control, p<0.0l. n values: 24 hour control and treated= 10, 48 hour control and treated= 9, 72 hour control and treated= 10. 81 CALCIUM-DEPENDENT PHOSPHOPROTEIN vs TIME 240-1 2 4 H R 4 8 H R 7 2 H R T I M E P O I N T O F D E T E R M I N A T I O N Legend CZ3 C o n t r o l EZ3 T3 T reated 82 Figure 18. SDS gel electrophoresis and s i l v e r stain of co n t r o l , 12, 24, 48 and 72 hour SR membranes. Gel electrophoresis and staining were done as described in methods. Track A represents the protein standards: Myosin, 200 kDa; B-galactosidase, 116.3 kDa; phosphorylase B, 92.5 kDa; bovine serum albumine, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; soybean trypsin i n h i b i t o r , 21.5 kDa; lysozyme, 14.4 kDa. Tracks B, C, D, E and F represent control, 12, 24, 48 and 72 hour SR samples, respectively. 83 Figure IB A B C D • F 84 d i f f e r e n c e s i n the p r o t e i n s i s o l a t e d from e i t h e r c o n t r o l or any of t h e T 3 t r e a t e d groups. P r o t e i n bands i n d i c a t i v e of SR can be d e t e c t e d a t a p p r o x i m a t e l y 100 kDa ( c a l c i u m ATPase), 57 kDa ( c a l s e q u e s t r i n ) and 22kDa (phospholamban). 85 DISCUSSION The E f f e c t of Treatment on Serum Free , Body Weight, Ventricular Weight and Sarcoplasmic Reticulum Y i e l d The aim of t h i s project was to investigate biochemical a l t e r a t i o n s in the heart occurring during the progression of the hyperthyroid state. Therefore, an appropriate experimental model was required in which hyperthyroidism could be reproducibly induced. The animal chosen was the male Wistar rat and hyperthyroidism was induced by the d a i l y subcutaneous i n j e c t i o n of 500 Jjg/Kg T^. This p a r t i c u l a r treatment protocol had been u t i l i z e d previously to study cardiac a l t e r a t i o n s due to hyperthyroidism (McNeill and Brody, 1968; McNeill et a l , 1969; Marriott and McNeill, 1983) and shown to produce cardiac a l t e r a t i o n s c l o s e l y resembling aspects of the human condition. The T^ treatment protocol used in t h i s study did in fact r e s u l t in elevated serum free T^ values (Table 1) at the 12, 24, 48 and 72 hour time points studied. The serum h a l f - l i f e of T^ i s 24 hours (DeGroot e_t a^L, 1984), which may account for the observed decrease in serum free T^ at 24 hours compared to the 12 hour concentrat ion. As in the human disease, the rats in t h i s study responded to the T^ treatment with a loss of body weight (Table 2); the maximum loss was after the f u l l treatment, at 72 hours, where the treated rats lost an average of 19 g. This is approximately the same weight loss experienced by the same sex and s t r a i n of 86 rat subjected to the i d e n t i c a l treatment in a previous study (Marriott and McNeill, 1983). The T 3 treated rats also demonstrated increased " i r r i t a b i l i t y " , in that they were more sensit i v e to external s t i m u l i ! than controls. This parameter was not measured, but only observed, and since i t i s subjective i s intended only as an indication of hyperthyroidism in the rat p a r a l l e l i n g a sign of the human disease. The wet ventricular weight was increased by the T^ treatment 48 and 72 hours aft e r i n i t i a t i o n of the treatment (Table 3). Others have also reported s i g n i f i c a n t increases in heart weight following the treatment protocol used in t h i s study (Marriott and McNeill, 1983) and with other thyroid hormone treatment protocols (Korecky and Beznak, 1971; Morkin et a l , 1983; Fox et a l , 1985). Although the finding of cardiac hypertrophy in experimental hyperthyroidism i s common, t h i s anatomical change may represent a difference between the animal model and the human disease. Ventricular hypertrophy i s generally absent in patients dying from thyrotoxicosis (Morkin et a l , 1983), and the transverse diameter of the heart i s usually normal in l i v i n g patients (DeGroot et a_l, 1984). However, cardiac hypertrophy can occur in humans with long standing hyperthyroidism (Klein and Levey, 1984). The rapidly induced hypertrophy seen experimentally i s l i k e l y to be a result of the pharmacological doses of thyroid hormones t y p i c a l l y administered and the short onset of thyrotoxicosis associated with these doses, r e l a t i v e to the longer onset of the human disease. The ventricular weight to 87 body w e i g h t r a t i o s r e p r e s e n t e d i n T a b l e 4 a r e a f u r t h e r i n d i c a t i o n t h a t t h e r e was c a r d i a c h y p e r t r o p h y i n t h e T^ t r e a t e d g r o u p , a n d t h a t t h e d e g r e e o f h y p e r t r o p h y i n c r e a s e d w i t h i n c r e a s i n g d u r a t i o n o f t h e d i s e a s e . T h e r e f o r e , t h e v a r i o u s i n d i c e s o f h y p e r t h y r o i d i s m e x p r e s s e d by t h e r a t s i n d i c a t e t h a t t h e y were h y p e r t h y r o i d , a n d t h a t t h e T^ t r e a t m e n t p r o t o c o l was a p p r o p r i a t e f o r t h e p u r p o s e s o f t h e s t u d y . T h e r e was a d i f f e r e n t i a l e f f e c t o f t h e T^ t r e a t m e n t on t h e v e n t r i c u l a r w e i g h t a n d t h e y i e l d o f SR p r o t e i n . T h e r e was no s i g n i f i c a n t i n c r e a s e i n t h e v e n t r i c u l a r w e i g h t 24 h o u r s a f t e r t h e f i r s t d o s e , y e t t h e y i e l d o f SR was s i g n i f i c a n t l y g r e a t e r a t t h i s t i m e i n t h e T^ t r e a t e d g r o u p . A t t h e l a t e r t i m e p o i n t s o f 48 a n d 72 h o u r s , t h e y i e l d o f SR was a l s o s i g n i f i c a n t l y g r e a t e r t h a n i n t h e c o n t r o l s . T h y r o i d hormone t r e a t m e n t h a s been shown t o i n c r e a s e t h e a b s o l u t e a r e a o f s a r c o t u b u l a r membrane i n r a t v e n t r i c l e ( M c C a l l i s t e r a n d P a g e , 1 9 7 3 ) . T h i s s t u d y a l s o showed t h a t t h e membrane a r e a p e r u n i t c e l l v o l u m e a n d p e r u n i t m y o f i b r i l l a r v o l u m e r e m a i n e d c o n s t a n t , i n d i c a t i n g t h a t m y o f i b r i l l a r v o l u m e a n d s a r c o t u b u l a r membrane b o t h r e s p o n d t o t h y r o i d hormone. The r e s u l t s o f t h i s p r o j e c t i n d i c a t e t h a t t h e r a t i o o f SR p r o t e i n y i e l d t o v e n t r i c u l a r w e i g h t i n c r e a s e s w i t h i n c r e a s i n g d u r a t i o n o f T^ t r e a t m e n t ( T a b l e 6 ) . T h i s r e s u l t may i m p l y t h a t t h e T^ t r e a t m e n t i s i n c r e a s i n g SR p r o t e i n t o a g r e a t e r e x t e n t t h a n o v e r a l l c a r d i a c mass. The SDS- g e l e l e c t r o p h o r e t i c s e p a r a t i o n o f SR f r o m c o n t r o l a n d h y p e r t h y r o i d s a m p l e s ( F i g u r e 18) t h o u g h , shows t h a t t h e r e a r e no q u a l i t a t i v e d i f f e r e n c e s i n d i s t r i b u t i o n o f t h e SR p r o t e i n s i s o l a t e d f r o m any 88 of the time points studied. In contrast to the r e s u l t s shown in Table 6, Limas (1978a) did not observe any substantial difference in the y i e l d of SR protein between control and hyperthyroid groups. The reason for t h i s difference i s not known. The Effect of Hyperthyroidism on SR Calcium Transport and  Phosphoprotein Levels The finding in t h i s study that cardiac SR calcium transport i s increased following thyroid hormone treatment i s in agreement wiht previously published reports (Suko, 1974; Limas, I978a,b; Guarnieri et a_l, 1980). Enhanced SR calcium transport a c t i v i t y i s consistent with the thesis that t h i s organelle may in part be responsible for the augmented cardiac function t y p i c a l l y observed in both experimental and human hyperthyroidism. Currently, there are two possible mechanisms by which functional and biochemical changes in the heart could be induced in the hyperthyroid state: The f i r s t i s that cardiac function i s altered due to an i n t r e r a c t i o n between cardiac nuclear T^ receptors and the subsequently augmented protein synthesis. The second i s that cardiac manifestations of hyperthyroidism are the result of the increased work load imposed upon the heart by increased peripheral metabolism. With respect to the f i r s t p o s s i b i l i t y , the well defined control over isomyosin d i s t r i b u t i o n by thyroid hormones (Morkin et a l , 1983), and the 89 a u g m e n t e d c o n t r a c t i l e p r o p e r t i e s o f e a c h o f t h e s e i s o m y o s i n s ( S c h w a r t z e t a l , 1981; P a g a n i e t a l , 1 9 8 3 ) , s u g g e s t t h a t t h i s may be t h e c a s e i n s p e c i e s o t h e r t h a n r a t . I n r a t , m y o s i n V 1 a c c o u n t s f o r a b o u t 8 5 % o f t o t a l m y o s i n i n t h e e u t h y r o i d s t a t e , t h e r e f o r e , t h e a u g m e n t e d c o n t r a c t i l i t y c a n n o t r e s u l t f r o m t h e s h i f t i n i s o m y o s i n s a s s e e n , f o r e x a m p l e , i n r a b b i t . I n t h i s s t u d y , t h e l a t e n c y p e r i o d f o r m e d i a t e d a u g m e n t a t i o n o f SR c a l c i u m t r a n s p o r t was b e t w e e n 12 a n d 24 h o u r s a f t e r a d m i n i s t r a t i o n ( s i g n i f i c a n t i n c r e a s e a t 24 h o u r s , F i g u r e 4 ) . T h i s p e r i o d o f t i m e i s i n a g r e e m e n t w i t h T^ m e d i a t e d i n c r e a s e s i n r a t c a r d i a c f u n c t i o n ( B r o o k s e t a l , 1 9 8 5 ) , a n d t h e i n d u c t i o n o f t h e s y n t h e s i s o f T 3 r e s p o n s i v e enzymes ( O p p e n h e i m e r , 1 9 8 3 ) . B r o o k s e t a l ( 1 9 8 5 ) s t a t e d t h e T^ m e d i a t e d i n c r e a s e s i n c a r d i a c c o n t r a c t i l i t y o c c u r r e d p r i o r t o c h a n g e s i n w h o l e body f u n c t i o n . I n t h i s s t u d y t h e i n c r e a s e d c a l c i u m t r a n s p o r t a c t i v i t y was s e e n t o o c c u r p r i o r t o i n c r e a s e s i n v e n t r i c u l a r w e i g h t . T h e r e f o r e , t h e r e s u l t s o f t h i s s t u d y s u g g e s t a d i r e c t e f f e c t o f T^ on t h e h e a r t , a n d s p e c i f i c a l l y on SR c a l c i u m t r a n s p o r t . SR c a l c i u m t r a n s p o r t was i n c r e a s i n g l y a u g m e n t e d w i t h t h e d u r a t i o n o f t h e h y p e r t h y r o i d t r e a t m e n t ( F i g u r e 7 ) . The d i f f e r e n c e s i n t h e o b s e r v e d r a t e s o f c a l c i u m t r a n s p o r t b e t w e e n t h e c o n t r o l a n d T^ t r e a t e d g r o u p s were n o t due t o a l t e r a t i o n s i n m i t o c h o n d r i a l membrane c o n t a m i n a t i o n , a s t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n t h e r a t e o f c a l c i u m t r a n s p o r t i n e i t h e r t h e a b s e n c e o r t h e p r e s e n c e o f 5mM s o d i u m a z i d e ( T a b l e 7 ) . The maximum i n c r e a s e i n c a l c i u m u p t a k e a c t i v i t y o c c u r r e d a t 90 72 hours, which i s the same time that cardiac c o n t r a c t i l i t y has been shown to be augmented following the same regimen (Marriott and McNeill, 1983). Therefore, augmented SR calcium transport probably contributes to both the increased c o n t r a c t i l e a c t i v i t y and the decreased relaxation time observed in the hyperthyroid rat heart. To further characterize the eff e c t of T 3 treatment on the SR, the le v e l s of the calcium-ATPase phosphoprotein intermediate were determined. A high calcium concentration which competitively i n h i b i t s the magnesium-dependent dephosphorylation of the ATPase (Tada et a l , 1978) was used in order that a l l the calcium ATPase s i t e s present in the SR could be l a b e l l e d with substrate. In t h i s way the number of s i t e s in the SR of control and T^ treated rats could be compared. Under these conditions there was maximal i n h i b i t i o n of dephosphorylation; 68% of the phosphoprotein formed was hydroxylamine-sensitive, indicating that the calcium pump was accounting for the majority of the phosphoprotein detected. At 48 and 72 hours, the phosphoprotein levels detected in hyperthyroid SR were s i g n i f i c a n t l y greater than control l e v e l s (p< 0.01 and p<0.05, r e s p e c t i v e l y ) . This indicates that there are more phosphorylated calcium pumps in the hyperthyroid SR. This may resu l t from, (1) an increased number of active pumping s i t e s (2) an increase in the t o t a l number of pumping s i t e s or, (3) an increased turnover of the pump. Calcium ATPase a c t i v i t y was not determined, therefore the d i s t i n c i t o n between these p o s s i b i l i t i e s cannot be made. However, because of the increased y i e l d of SR protein at 24 91 hours, i t i s probable that there i s an increased number of calcium ATPase s i t e s . Other, in d i r e c t evidence supporting t h i s p o s s i b i l i t y comes from work showing that thyroid hormone induced increases in calcium transport are prevented by the administration of i n h i b i t o r s of RNA and protein synthesis (Limas, 1978a). Thyroid hormone has also been shown to influence the a c t i v i t y and expression of the Na/K-ATPase transport enzyme (Curfman et a_l, 1977; Guernsey and Edelman, 1983). Calcium transport in cardiac SR i s regulated by a number of d i f f e r e n t mechanisms. Conceivably, d i f f e r e n t degrees of regulator a c t i v i t y could at least p a r t l y account for the differences in calcium uptake a c t i v i t y observed between control and hyperthyroid cardiac SR. C y c l i c AMP-dependent protein kinase, calcium-calmodulin dependent protein kinase and calcium-sensitive phospholipid-dependent protein kinase a l l augment calcium transport through phospholamban phosphorylation (LePeuch et a l , 1979; Movsesian et a l , 1984). Since only 68% of the phosphoprotein detected was hydroxylamine sen s i t i v e , d i f f e r e n t i a l phospholamban phosphorylation could have contributed to the remaining 32% since the phosphate bond associated with phospholamban i s hydroxylamine i n s e n s i t i v e . As mentioned e a r l i e r the effect of hyperthyroidism on the responsiveness of SR calcium uptake a c t i v i t y to cAMP-dependent protein kinase i s c o n t r o v e r s i a l . Limas (1978b) has shown that hyperthyroid rat cardiac SR calcium transport responds to cAMP-92 dependent protein kinase and the magnitude of the augmentation is approximately two-fold greater than the control response. The cAMP-dependent protein kinase-mediated incorporation of phosphate into a 22 KD SR protein (phospholamban) p a r a l l e l e d the calcium uptake response. An increased l e v e l or a c t i v i t y of endogeneous cAMP-dependent protein kinase was suggested as the explanation for these r e s u l t s . The work of Guarnieri et a l (1980), showed that the c o n t r a c t i l i t y of hyperthyroid rat i n t e r v e n t r i c u l a r s t r i p s in response to d i b u t y r l cAMP did not d i f f e r from that of controls, and that hyperthyroid tissue could not respond to the same extent as control in the presence of isoproterenol. They also showed that the SR calcium uptake response of hyperthyroid rats to exogeneous cAMP and protein kinase was s i g n i f i c a n t l y less than c o n t r o l s . Basal calcium uptake a c t i v i t y in the hyperthyroid rats was greater than control by two-fold. The lack of response to cAMP-dependent protein kinase was suggested to be due to t h i s high basal a c t i v i t y . Calmodulin may exert some ef f e c t over the observed calcium transport a c t i v i t y , as the amount of t h i s protein in heart has been shown to be increased in hyperthyroidism (Segal et a l , 1985). Studies in our laboratory, though, indicate that calmodulin does not stimulate SR calcium transport in rats (R.Mahey, personal communication). 93 The E f f e c t of Hyperthyroidism on Total, Short Chain and Long Chain Carnitine in Cardiac SR The i n h i b i t o r y effect of long chain a c y l c a r n i t i n e s (LCAC), p a r t i c u l a r l y palmitoylcarnitine, on membrane bound enzymes was discussed in the introduction. To r e i t e r a t e b r i e f l y , isolated cardiac SR incubated in the presence of exogenous palm i t o y l c a r n i t i n e , exhibited depressed calcium uptake and ATPase a c t i v i t y ( P i t t s et a_l, 1978). Cardiac sarcolemmal Na/K-ATPase a c t i v i t y was also found to be depressed in the presence of exogenous palmitoylcarnitine (Adams et a l , 1979). Since the amount of LCAC in the c e l l i s increased both following an ischemic episode (Idell-Wenger e_t a l , 1978) and in the diabetic myocardium (Feuvray et a l , 1979), i t has been suggested that these l i p i d metabolic intermediates play a role in the cardiomyopathies associated with these conditions. Lopaschuk e_t a l , (1983) has also shown increased SR LCAC le v e l s concomittant with depressed calcium transport in SR i s o l a t e d from diabetic rat heart. The r e s u l t s presented in t h i s thesis indicate a possible r e l a t i o n s h i p between reduced SR LCAC and an increase in SR calcium transport a c t i v i t y in hyperthyroid rat heart. The metabolic a l t e r a t i o n s associated with hyperthyroidism and possible mechanisms of augmentation of SR calcium transport under conditions of reduced sarcoplasmic r e t i c u l a r LCAC, warrant further discussion. To support t h i s hypothesis there i s 94 e v i d e n c e t h a t m e t a b o l i c a l t e r a t i o n s a s s o c i a t e d w i t h h y p e r t h y r o i d i s m f a v o u r a r e d u c t i o n i n c a r d i a c LCAC. As p r e v i o u s l y m e n t i o n e d , p r i o r t o m i t o c h o n d r i a l o x i d a t i o n , t h e a c t i v a t e d f a t t y a c y l - C o A d e r i v a t i v e s o f F F A ' s a r e t r a n s a c y l a t e d by c a r n i t i n e a c y l t r a n s f e r a s e I (CAT I ) . Ah e s s e n t i a l c o f a c t o r i n t h e t r a n s a c y l a t i o n r e a c t i o n i s c a r n i t i n e ( 4 - t r i m e t h y l a m i n o - 3 - h y d r o x y b u t y r a t e ) ( B r e m e r , 1 9 8 3 ) . C a r n i t i n e m e t a b o l i s m i s i n f l u e n c e d by t h y r o i d h o r m o n e s i n a way w h i c h , i n c o n j u n c t i o n w i t h o t h e r m e t a b o l i c a l t e r a t i o n s , may s u p p o r t t h e d e c r e a s e o f SR LCAC c o n t e n t : T h y r o i d hormone a d m i n i s t r a t i o n t o r a t s a n d m i c e r e s u l t s i n - d e c r e a s e d c a r n i t i n e s y n t h e s i s ( S u z u k i e t a l , 1 9 8 3 ) , w i t h h y p e r t h y r o i d r a t c a r d i a c t i s s u e h a v i n g r e d u c e d t o t a l c a r n i t i n e c o n t e n t . T h e s e r e s u l t s a r e i n a g r e e m e n t w i t h t h o s e o f C e d e r b l a d a n d E n g s t r o m ( 1978) w h i c h show d e c r e a s e d s h o r t c h a i n and f r e e c a r n i t i n e c o n t e n t i n h e a r t t i s s u e f r o m h y p e r t h y r o i d m i c e . The r e s u l t s c o n c e r n i n g c a r d i a c c a r n i t i n e l e v e l s a r e n o t c o n s i s t e n t : B r e s s l e r a n d W i t t e l s ( 1 9 6 6 ) h a v e shown f r e e a n d l o n g c h a i n a c y l c a r n i t i n e t o be i n c r e a s e d i n h y p e r t h y r o i d g u i n e a p i g h e a r t . The d i f f e r e n c e s may r e p r e s e n t a s p e c i e s v a r i a t i o n i n c a r d i a c c a r n i t i n e m e t a b o l i s m . C a r n i t i n e h a s b een shown t o be m e t a b o l i z e d t o p - m e t h y l c h o l i n e by an i n t r a m i t o c h o n d r i a l enzyme, c a r n i t i n e d e c a r b o x y l a s e , t h e a c t i v i t y o f w h i c h i s c o n t r o l l e d by t h e r a t e o f f r e e f a t t y a c i d o x i d a t i o n ( K h a i r a l l a h a n d W o l f , 1 9 6 7 ) . I n man h y p e r t h y r o i d i s m h a s b e e n a s s o c i a t e d w i t h i n c r e a s e s i n u r i n a r y c a r n i t i n e e x c r e t i o n ( M a e b a s h i e_t a _ l , 1 9 7 7 ) . T h e r e f o r e i t h a s b een d o c u m e n t e d t h a t c a r n i t i n e l e v e l s i n t h e h y p e r t h y r o i d h e a r t a r e 95 e i t h e r d e c r e a s e d ( S u z u k i e t a l , 1983; C e d e r b l a d a n d E n g s t r o m , 1978) o r i n c r e a s e d ( B r e s s l e r a n d W i t t l e s , 1 9 6 6 ) , t h a t u n d e r a m e t a b o l i c s t a t e o c c u r r i n g i n h y p e r t h y r o i d i s m c a r n i t i n e m e t a b o l i s m i s i n c r e a s e d ( K h a i r a l l a h a n d W o l f , 1 9 6 7 ) , a n d t h a t i n man h y p e r t h y r o i d i s m r e s u l t s i n i n c r e a s e d e x c r e t i o n o f c a r n i t i n e . H o wever, b e c a u s e K h a i r a l l a h a n d W o l f ( 1 9 6 7 ) d i d n o t d e t e r m i n e m i t o c h o n d r i a l d e g r a d a t i o n o f c a r n i t i n e i n h y p e r t h y r o i d a n i m a l s , t h e e x t r a p o l a t i o n o f t h e i r o b s e r v a t i o n s t o t h e h y p e r t h y r o i d s t a t e may n o t be a c c u r a t e ; t h e r e may be c h a n g e s a s s o c i a t e d w i t h h y p e r t h y r o i d i s m w h i c h r e d u c e t h e c a t a b o l i s m o f c a r n i t i n e u n d e r c o n d i t i o n s o f i n c r e a s e d f r e e f a t t y a c i d m e t a b o l i s m . More r e c e n t r e s u l t s s u g g e s t t h a t t h e a c t u a l m e t a b o l i t e d e t e r m i n e d was t r i m e t h y l a m i n o a c e t o n e , a n d t h a t t h e c o n c l u s i o n r e g a r d i n g c a r n i t i n e d e g r a d a t i o n may n o t be a c c u r a t e ( B r e m e r , 1 9 8 3 ) . T h e r e f o r e , d a t a c o n c e r n i n g t h e c a r d i a c c o n t e n t o f c a r n i t i n e i n h y p e r t h y r o i d i s m a r e c o n t r o v e r s i a l . I n none o f t h e t h r e e r e p o r t s d e s c r i b i n g t h e c a r d i a c c a r n i t i n e l e v e l s were a t t e m p t s made t o a s c r i b e t h e l e v e l s d e t e c t e d t o e i t h e r c y t o s o l i c o r s u b c e l l u l a r c o m p a r t m e n t s . The d i s t r i b u t i o n o f t h e v a r i o u s c a r n i t i n e d e r i v a t i v e s may be i n f l u e n c e d by t h e h y p e r t h y r o i d s t a t e . F u r t h e r r e s e a r c h i s r e q u i r e d t o a n s w e r t h e q u e s t i o n o f w h e t h e r o r n o t c e l l u l a r d i s t r i b u t i o n a n d t h e r e l a t i v e a m o u nts o f t h e v a r i o u s c a r n i t i n e d e r i v a t i v e s a r e a f f e c t e d by h y p e r t h y r o i d i s m , a n d i f s o , what t h e s i g n i f i c a n c e o f s u c h c h a n g e s t o e i t h e r o v e r a l l c a r d i a c m e t a b o l i s m o r c a r d i a c f u n c t i o n may b e . 96 In addition to a l t e r a t i o n s in c a r n i t i n e metabolism, the hyperthyroid heart also exhibits an increase in free fatty acid metabolism (Bressler and Wittels, 1966; F i n t e l and Burns, 1982). This increase may in part be accounted for by the increased serum free fatty acid concentrations associated with hyperthyroidism (Muller and S e i t z , 1984c) and the thyroid hormone stimulated a c t i v i t y of c a r n i t i n e palmitoyltransferase I (Bressler and Wittels, 1966; Stakkestad and Bremer, 1983). Since the heart derives i t s FFA supply from the c i r c u l a t i o n , increased c i r c u l a t i n g FFA r e s u l t s in increased substrate delivery to the heart. Although the heart i s an omnivorous organ capable of adapting to a number of d i f f e r e n t substrates for energy production, the preferred substrate i s generally that most avail a b l e in the a r t e r i a l c i r c u l a t i o n (Berne and Levey, 1981). Under euthyroid conditions, approximately 60% of the metabolic energy i s supplied by various free fatty acids (Opie, 1968). Increased supply of free f a t t y acids, in addition to the lack of other substrates, r e s u l t s in an increase in FFA u t i l i z a t i o n . The rate l i m i t i n g step of FFA oxidation i s the transacylation reaction leading to the synthesis of LCAC- from long chain acyl-CoA. By increasing CPT I a c t i v i t y , thyroid hormones are accelerating the flux of activated FFA into the mitochondria. Thyroxine treatment also increases mitochondrial content in rat heart (McCallister and Page, 1973), so that there is an increased capacity for B-oxidation of acyl-CoA. The decrease in available c a r n i t i n e and increase in substrate delivery and f r e e . f a t t y acid metabolism are conditions which may 97 p r o v i d e a r a t i o n a l e f o r t h e d e c r e a s e d LCAC d e t e c t e d i n t h e SR o f h y p e r t h y r o i d r a t h e a r t . S i n c e c a r n i t i n e s y n t h e s i s may be d e c r e a s e d , a n d / o r m e t a b o l i s m a n d e x c r e t i o n i n c r e a s e d , c a r d i a c l e v e l s o f t h i s c o f a c t o r a r e d e c r e a s e d . I n c o n j u n c t i o n w i t h t h i s , CPT I a c t i v i t y a n d f r e e f a t t y a c i d m e t a b o l i s m a r e i n c r e a s e d a n d l a c t a t e m e t a b o l i s m c o n c o m i t t a n t l y i s d e c r e a s e d . The f o l l o w i n g h y p o t h e s i s i s t h e r e f o r e s u g g e s t e d : s i n c e ( 1 ) t h e r a t e o f s y n t h e s i s o f LCAC i s a u g m e n t e d a n d , (2) t h e m e t a b o l i c s t a t e o f t h e h e a r t i s s u c h t h a t t h e demand f o r i n t r a m i t o c h o n d r i a l t r a n s p o r t o f LCAC i s h i g h , a n d (3) c a r n i t i n e l e v e l s a r e d e c r e a s e d , t h e p r o b a b i l i t y t h a t LCAC w i l l d i f f u s e away f r o m t h e i n n e r m i t o c h o n d r i a l membrane l o c a l e , where t h e c a r n i t i n e p o o l i s t h e most m e t a b o l i c a l l y a c t i v e , i s d e c r e a s e d , h e n c e , LCAC d e p o s i t i o n i n t o o t h e r s u b c e l l u l a r o r g a n e l l e s ' i s d e c r e a s e d . T h i s h y p o t h e s i s , a n d t h e m e t a b o l i c a l t e r a t i o n s o b s e r v e d i n h y p e r t h y r o i d i s m upon w h i c h i t i s b a s e d , a p p e a r t o be u n i q u e t o t h e h y p e r t h y r o i d s t a t e . I n o t h e r d i s e a s e s t a t e s , c a r n i t i n e d e f i c i e n c y i s a s s o c i a t e d w i t h c a r d i o m y o p a t h i e s . D i p t h e r i t i c m y o c a r d i t i s i s a s s o i c a t e d w i t h t r i g l y c e r i d e a c c u m m u l a i t o n , a d e p r e s s e d r a t e o f FFA o x i d a t i o n a n d d e c r e a s e d c a r n i t i n e c o n t e n t ( O p i e , 1 9 6 8 ) . F a m i l i a l c a r d i a c c a r n i t i n e d e f i c i e n c y h a s b e e n a s s o c i a t e d w i t h a c a r d i o m y o p a t h y ( T r i p p ejt a _ l , 1 9 8 1 ) . T r e a t m e n t o f t h e p a t i e n t s s u r v i v i n g t h i s c a r d i o m y o p a t h y w i t h L - c a r n i t i n e r e s u l t e d i n r e s t o r a t i o n o f c a r d i a c f u n c t i o n , a l t h o u g h , c a r d i a c l e v e l s o f t o t a l , f r e e a n d e s t e r f i e d c a r n i t i n e were n o t c h a n g e d 98 f r o m p r e t r e a t m e n t v a l u e s . T h e r e a r e o t h e r e x a m p l e s o f h e a r t f a i l u r e a n d s u c c e s s f u l t r e a t m e n t w i t h o r a l c a r n i t i n e ( R e b o u c h e , 1 9 8 6 ) . The i n f l u e n c e o f h y p e r t h y r o i d i s m on c a r n i t i n e m e t a b o l i s m i n one r e s p e c t f o l l o w s t h e a b o v e o b s e r v a t i o n s . Of t h e t o t a l b ody c a r n i t i n e , a t l e a s t 9 0 % r e s i d e s i n s k e l e t a l m u s c l e ( R e b o u c h e , 1 9 8 6 ) . T h e r e may be a r e l a t i o n s h i p b e t w e e n t h e a u g m e n t e d e x c r e t i o n o f c a r n i t i n e i n h y p e r t h y r o i d p a t i e n t s ( M a e b a s h i e_t a l , 1977) a n d t h e s k e l e t a l m u s c l e w e a k n e s s o b s e r v e d i n h y p e r t h y r o i d i s m ( D e G r o o t e t a l , 1 9 8 4 ) . I t i s p a r a d o x i c a l t h a t c a r d i a c c a r n i t i n e i s d e c r e a s e d i n h y p e r t h y r o i d i s m ( C e d e r b l a d a n d E n g s t r o m , 1978; S u z u k i e t a l , 1983) a n d t h a t c a r d i a c f u n c t i o n i s a u g m e n t e d i n t h e d i s e a s e . H o w e v e r , t h e c a r d i a c d y s f u n c t i o n a m e l i o r a t e d by c a r n i t i n e t r e a t m e n t was n o t a s s o c i a t e d w i t h an i n c r e a s e i n c e l l u l a r c a r n i t i n e , a n d t h e b e n e f i c i a l e f f e c t h a s been a t t r i b u t e d t o r e p l a c e m e n t o f i n t r a c e l l u l a r LCAC w i t h f r e e c a r n i t i n e ( R e b o u c h e a n d E n g e l , 1 9 8 3 ) . T h e r e f o r e a c o n s i d e r a t i o n t h a t may be more i m p o r t a n t t h a n t h e t o t a l c a r n i t i n e c o n t e n t o f t h e h e a r t , i s t h e r e l a t i v e amount o f t h e v a r i o u s c a r n i t i n e d e r i v a t i v e s ( f r e e , s h o r t a n d l o n g c h a i n ) . A l e s s s u b j e c t i v e h y p o t h e s i s c o n c e r n i n g t h e o b s e r v e d d e c r e a s e i n SR LCAC f r o m h y p e r t h y r o i d h e a r t r e l a t e s t o m e t a b o l i c p r o c e s s e s o n g o i n g d u r i n g t h e i s o l a t i o n o f t h e SR. O x i d a t i o n o f l o n g c h a i n a c y l d e r i v a t i v e s may be t a k i n g p l a c e b e t w e e n t h e t i m e o f i s o l a t i o n o f t h e v e n t r i c u l a r t i s s u e a n d t h e t i m e when t h e SR i s s e p a r a t e d f r o m m i t o c h o n d r i a l c o m p o n e n t s o f t h e c e l l . The a d d i t i o n o f c y a n i d e , t o p r e v e n t t h e o x i d a t i o n o f a c y l 99 c a r n i t i n e s , r e s u l t s i n an a p p r o x i m a t e t h r e e - f o l d i n c r e a s e i n t h e l e v e l o f l o n g c h a i n a c y l c a r n i t i n e d e t e r m i n e d i n a m i t o c h o n d r i a l f r a c t i o n o f r a t h e a r t ( I d e l l - W e n g e r e t a l , 1 9 7 8 ) . A d d i t i o n o f c y a n i d e d i d n o t a f f e c t t h e l e v e l o f t o t a l c a r n i t i n e i n t h e m i t o c h o n d r i a l c o m p a r t m e n t ( t h e i n c r e a s e i n l o n g c h a i n c a r n i t i n e was r e f l e c t e d by a d e c r e a s e i n f r e e c a r n i t i n e ) , n o r d i d i t a f f e c t t h e l e v e l s o f t o t a l c a r n i t i n e i n t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t . P r e s u m a b l y , t h i s l a t t e r f r a c t i o n w o u l d c o n t a i n t h e SR membranes, h o w e v e r t h e d i s t r i b u t i o n o f t h e v a r i o u s c a r n i t i n e d e r i v a t i v e s was n o t d e t e r m i n e d . I t i s p o s s i b l e t h a t t h e p r o c e d u r e s e m p l o y e d i n t h i s s t u d y w o u l d a l l o w m i t o c h o n d r i a l o x i d a t i o n o f l o n g c h a i n a c y l c a r n i t i n e a f t e r r e m o v a l o f t h e h e a r t f r o m t h e r a t . S i n c e c y a n i d e was n o t i n c l u d e d i n t h e h o m o g e n i z a t i o n m e d i a , l o n g c h a i n a c y l c a r n i t i n e may h a v e b e e n m e t a b o l i z e d , a n d s i n c e t h e r e i s e v i d e n c e o f e n h a n c e d CPT I a c t i v i t y i n t h e h y p e r t h y r o i d h e a r t , t h e r a t e o f m e t a b o l i s m o f l o n g c h a i n a c y l c a r n i t i n e i n t h e h y p e r t h y r o i d homogenate may h a v e been g r e a t e r t h a n i n c o n t r o l . The n e t e f f e c t , t h e r e f o r e , b e i n g d e c r e a s e d LCAC d e t e c t e d i n t h e SR. A l t h o u g h t h i s p o s s i b i l i t y p r o v i d e s an a l t e r n a t e e x p l a n a t i o n f o r t h e d e c r e a s e d l e v e l s o f LCAC d e t e c t e d i n h y p e r t h y r o i d SR, i t d o e s n o t n e c c e s s a r i l y o b v i a t e t h e p o s s i b l e r e l a t i o n s h i p b e t w e e n i n c r e a s e d SR c a l c i u m t r a n s p o r t a n d d e c r e a s e d SR LCAC. T h e r e a r e a number o f c o n s i d e r a t i o n s t o be made i n e x t r a p o l a t i n g in v i t r o d a t a t o t h e i r i v i v o s i t u a t i o n , n o t t h e l e a s t o f w h i c h i s t h e e f f e c t o f t h e i s o l a t i o n t e c h n i q u e s on t h e 100 o r g a n e l l e , enzyme o r o t h e r b i o c h e m i c a l p a r a m e t e r u n d e r s t u d y . When t h e a b s c i s s a e o f f i g u r e s 12, 13 a n d 14 a r e c o m p a r e d , i t i s a p p a r e n t t h a t t h e r e i s a l a r g e v a r i a t i o n i n t h e v a l u e s o f t h e v a r i o u s c a r n i t i n e d e r i v a t i v e s d e t e c t e d . The i s o l a t i o n t e c h n i q u e u t i l i z e d t o s e p a r a t e t h e a c i d s o l u b l e a n d l o n g c h a i n f r a c t i o n s f r o m t o t a l c a r n i t i n e may a c c o u n t f o r t h e v a r i a t i o n . As d e s c r i b e d i n t h e m e t h o d s , t h e i s o l a t i o n o f LCAC i n v o l v e s w a s h i n g t h e a c i d i n s o l u b l e p r e c i p i t a t e p r i o r t o h y d r o l y s i s t o remove r e s i d u a l f r e e a n d a c e t y l c a r n i t i n e . O t h e r w o r k e r s h a v e d o c u m e n t e d a l o s s o f LCAC due t o t h i s w a s h i n g s t e p ( I d e l l - W e n g e r e t a l , 1 9 7 8 ) . Of t h e t o t a l LCAC c a r n i t i n e p r e s e n t i n t h e c y t o s o l , t h e m a j o r i t y i s p r i m a r i l y b o und t o membrane s t r u c t u r e s s i n c e i t s h y d r o p h o b i c c h a r a c t e r makes i t t h e r m o d y n a m i c a l l y more f a v o u r a b l e t o r e s i d e i n t h e h y d r o p h o b i c d o m a i n s t h e y a f f o r d . F i g u r e 12 shows a s i g n i f i c a n t d e p r e s s i o n i n t o t a l c a r n i t i n e 24,48 a n d 72 h o u r s a f t e r i n i t i a i o n o f t h e T^ t r e a t m e n t , a n d f i g u r e 14 shows a d e p r e s s i o n i n LCAC a t 24 ( n o n -s i g n i f i c a n t ) , 48 .and 72 h o u r s . The c o n c e n t r a t i o n o f LCAC d e t e r m i n e d i n t h e c o n t r o l c a r d i a c SR ( 6 . 3 - 8 . 3 nmoles/mg SR p r o t e i n ) were a p p r o x i m a t e l y 6 - f o l d h i g h e r t h a n t h o s e r e p o r t e d by L o p a s c h u k e t a l ( 1 9 8 3 ) , y e t a r e l o w e r t h a n t h e e s t i m a t e d c o n c e n t r a t i o n s u g g e s t e d by P i t t s e t a l ( 1 9 7 8 ) o f 30 n m o l e s LCAC/mg SR p r o t e i n . A c i d s o l u b l e c a r n i t i n e r e m a i n s u n a f f e c t e d by t h e d u r a t i o n o f T^ t r e a t m e n t ( F i g u r e 1 3 ) . The o b v i o u s t r e n d i s t h a t b o t h LCAC a n d t o t a l c a r n i t i n e l e v e l s d e c l i n e w i t h i n c r e a s i n g d u r a t i o n o f T^ t r e a t m e n t . I t may be p o s s i b l e t h a t t h e p r i m a r y d e t e r m i n a n t o f t o t a l c a r n i t i n e i n t h e SR i s t h e LCAC 101 f r a c t i o n , a n d t h a t t h e d i s c r e p e n c y b e t w e e n t o t a l a n d LCAC a r i s e s b e c a u s e o f t h e i s o l a t i o n p r o c e d u r e s e m p l o y e d . F u r t h e r s t u d i e s w h i c h a v o i d t h e w a s h i n g o f t h e a c i d i n s o l u b l e p r e c i p i t a t e may p r o v i d e e v i d e n c e s u p p o r t i n g t h i s h y p o t h e s i s . A n o t h e r c o n s i d e r a t i o n t o be made i n i n t e r p r e t i n g t h e s e d a t a i s t h a t t h e l e v e l s o f t h e c a r n i t i n e d e r i v a t i v e s a r e r e p o r t e d a s nmoles/mg SR p r o t e i n . S i n c e t h e s t u d y a l s o showed i n c r e a s e d SR y e i l d f r o m h y p e r t h y r o i d r a t h e a r t , i t may be p r u d e n t t o c o n s i d e r t h a t t h e r e s u l t s o b t a i n e d b a s e d on a p r o t e i n s t a n d a r d a r e n o t a t r u e r e f l e c t i o n o f t h e amount o f c a r n i t i n e i n t h e SR. A l t h o u g h t h e r e s u l t s r e p o r t e d a r e a c c u r a t e , when b a s i n g t h e v a l u e o f one p a r a m e t e r on t h e amount o f a n o t h e r , i t i s more i d e a l i f t h e l a t t e r d o e s n o t c h a n g e b e t w e e n c o n t r o l a n d d i s e a s e d g r o u p s . U n d e r c i r c u m s t a n c e s w here an a p p r o p r i a t e b a s e p a r a m e t e r c a n n o t r e a d i l y be a c q u i r e d , b a s i n g t h e p a r a m e t e r o f i n t e r e s t on a number o f d i f f e r e n t b a s e s may f u r t h e r i n d i c a t e t h e a c t u a l s i t u a t i o n . I f t h e r e s u l t s r e p o r t e d i n t h i s s t u d y a r e skewed b e c a u s e o f an u n r e l i a b l e s t a n d a r d , t h e n t h e s i t u a t i o n c o n c e r n i n g t h e a c i d i n s o l u b l e c a r n i t i n e f r a c t i o n w o u l d c h a n g e s u c h t h a t t h e s e l e v e l s c o u l d be i n c r e a s e d r a t h e r t h a n n o t c h a n g e d by t h e h y p e r t h y r o i d s t a t e . H y p o t h e t i c a l l y , i f t h e l o n g c h a i n a c y l c a r n i t i n e i n t h e SR i n t e r a c t e d i n some s p e c i f i c manner w i t h t h e c a l c i u m pump t o i n h i b i t i t s a c t i v i t y , t h e n a r e l a t i v e i n c r e a s e SR p r o t e i n i n t h e a b s e n c e o f a c o n c o m m i t a n t i n c r e a s e i n l o n g c h a i n a c y l c a r n i t i n e c o u l d a c c o u n t f o r t h e o b s e r v e d i n c r e a s e i n c a l c i u m u p t a k e r e p o r t e d i n t h i s s t u d y . T h e r e i s no e v i d e n c e 1 02 i n t h i s s t u d y t o s u p p o r t s u c h a h y p o t h e s i s . The i n h i b i t o r y e f f e c t o f p a l m i t o y l c a r n i t i n e on SR c a l c i u m t r a n s p o r t a n d A T P a s e a c i t v i t y i s due t o i t s d e t e r g e n t e f f e c t s on t h e SR membrane (Adams e t a l , 1 9 7 9 ) . The a c y l e s t e r i n s e r t s i n t o t h e l i p i d m a t r i x o f t h e SR e x e r t i n g i t s e f f e c t s t h r o u g h p h y s i c a l d i s r u p t i o n o f t h e l i p i d b i l a y e r . Adams e t a l (1979) h a v e p r o p o s e d t h a t a t l o w c o n c e n t r a t i o n s ( b e l o w t h e c r i t i c a l m i c e l l e c o n c e n t r a t i o n o f a p p r o x i m a t e l y 15 jM) t h e a c y l c a r n i t i n e p e r t u r b s t h e SR membrane i n s u c h a way a s t o i n c r e a s e t h e " l e a k i n e s s " o f t h e membrane t o c a l c i u m . F i g u r e 15 shows a c o r r e l a t i o n ( r = - 0 . 9 3 ) b e t w e e n t h e r a t e o f c a l c i u m t r a n s p o r t a n d t h e SR l e v e l o f LCAC. The f i g u r e s u g g e s t s a r e l a t i o n s h i p b e t w e e n a u g m e n t e d c a l c i u m t r a n s p o r t a n d r e d u c e d e n d o g e n o u s LCAC i n t h e SR membrane. U n d e r e u t h y r o i d c o n d i t i o n s , t h e amound o f LCAC i s o l a t e d w i t h t h e SR may r e s u l t i n a v e s i c l e p o p u l a t i o n w i t h a p e r m e a b i l i t y t o c a l c i u m . As t h e h y p e r t h y r o i d s t a t e p r o g r e s s e s , t h e m e t a b o l i c a l t e r a t i o n s i n d u c e d r e s u l t i n a d e c r e a s e d amount o f LCAC i s o l a t e d i n t h e SR, r e d u c i n g t h e p e r m e a b i l i t y o f t h e v e s i c l e t o c a l c i u m a n d , t h e r e f o r e , i n c r e a s i n g t h e a p p a r e n t r a t e o f c a l c i u m t r a n s p o r t . E x t r a p o l a t i n g t h e s e in v i t r o r e s u l t s t o t h e i r i v i v o s i t u a t i o n , a l o w e r e d c a l c i u m p e r m e a b i l i t y o f t h e SR w o u l d a l l o w more e f f i c e n t s e q u e s t r a t i o n o f c a l c i u m d u r i n g r e l a x a t i o n , a n d t h e r e f o r e more c a l c i u m a v a i l a b l e f o r r e l e a s e d u r i n g d e p o l a r i z a t i o n . H o w e v e r , b e c a u s e t h e c o n t r i b u t i o n o f LCAC t o t h e t o t a l SR l i p i d must be s m a l l , a s j u d g e d by t h e l i p i d c o m p o s i t i o n o f t h e SR, t h i s may n o t be t r u e . P h o s p h o l i p i d s 1 0 3 a c c o u n t f o r a p p r o x i m a t e l y 8 0 % o f SR l i p i d , a n d n e u t r a l l i p i d s t h e r e m a i n d e r , o f w h i c h 9 5 % i s c h o l e s t e r o l ( T a d a e t a l , 1 9 7 8 ) . T h i s l e a v e s a b o u t 1% o f t h e t o t a l l i p i d , o f w h i c h a component w i l l be LCAC. T h e r e f o r e when v i e w e d i n t h e p e r s p e c t i v e o f t h e t o t a l l i p i d c o n t e n t o f t h e SR, a n d s i n c e p a s s i v e p e r m e a b i l i t y t o c a l c i u m i s e x t r e m e l y l o w u n d e r n o r m a l c o n d i t i o n s , i t d o e s n o t seem p r o b a b l e t h a t t h e d e c r e a s e d LCAC c o n t e n t c o u l d a l t e r t h e p e r m e a b i l i t y o f n a t i v e SR membrane t o c a l c i u m . 1 04 CONCLUSIONS 1. The e x p e r i m e n t a l m o d e l o f h y p e r t h y r o i d i s m u s e d i n t h i s s t u d y r e s u l t e d i n a l t e r a t i o n s i n t h e c a r d i a c t i s s u e . The e f f e c t o f t h e Tg t r e a t m e n t p r o t o c o l on t h e v e n t r i c u l a r w e i g h t was a p p a r e n t a f t e r 48 h o u r s . V e n t r i c u l a r w e i g h t r e m a i n e d s i g n i f i c a n t l y a b o v e t h e c o n t r o l 72 h o u r s a f t e r t h e t r e a t m e n t was s t a r t e d . The d e g r e e o f h y p e r t r o p h y , a s r e f l e c t e d by t h e c a r d i a c v e n t r i c u l a r t o body w e i g h t r a t i o , i n c r e a s e d w i t h t h e p r o g r e s s i o n o f t h e d i s e a s e . The Tg t r e a t m e n t h a d a d i f f e r e n t e f f e c t on t h e SR c o m p a r e d t o t h e v e n t r i c l e • a s a w h o l e . S i g n i f i c a n t l y g r e a t e r y i e l d s o f SR p r o t e i n were o b t a i n e d 24, 48 a n d 72 h o u r s a f t e r i n i t i a t i o n o f t h e t r e a t m e n t . The e a r l y a l t e r a t i o n i n t h e SR, o c c u r i n g b e t w e e n 12 a n d 24 h o u r s a f t e r t h e f i r s t Tg i n j e c t i o n , h a s a t i m e c o u r s e s i m i l a r t o o t h e r t h y r o i d hormone i n d u c i b l e e f f e c t s a n d may r e p r e s e n t a d i r e c t e f f e c t on SR p r o t e i n s y n t h e s i s . S D S - p o l y a c r y l a m i d e g e l - e l e c t r o p h o r e s i s i n d i c a t e d no o b v i o u s d i f f e r e n c e b e t w e e n c o n t r o l a n d t r e a t e d SR membrane p r o t e i n s a t a n y o f t h e t i m e p o i n t s a s s a y e d . T h e r e f o r e , i f Tg d o e s h a v e a n e f f e c t on SR p r o t e i n i t i s w i t h o u t a c h a n g e i n t h e p r o t e i n p r o f i l e o f t h e o r g a n e l l e . 2. The SR c a l c i u m u p t a k e a c t i v i t y was d e t e r m i n e d d u r i n g t h e p r o g r e s s i o n o f t h e d i s e a s e . I t was f o u n d t h a t t h e V C a was s i g n i f i c a n t l y i n c r e a s e d 24, 48 a n d 72 h o u r s a f t e r t h e i n i t i a t i o n o f t h e hormone t r e a t m e n t . The i n c r e a s e i n V C a was p r o g r e s s i v e , c o r r e l a t i n g p o s i t i v e l y w i t h t h e t i m e o f t h e d i s e a s e . The T 105 t r e a t m e n t was w i t h o u t an e f f e c t on t h e K^ a o f t h e c a l c i u m pump a t a n y o f t h e t i m e p o i n t s s t u d i e d . P h o s p h o r y l a t i o n e x p e r i m e n t s i n d i c a t e d a s l i g h t i n c r e a s e i n t h e number o f c a l c i u m pump s i t e s 24 h o u r s a f t e r t h e f i r s t d o s e , a n d a f t e r 48 a n d 72 h o u r s t h e number of. c a l c i u m pump s i t e s i n t h e t r e a t e d g r o u p s was s i g n i f i c a n t l y g r e a t e r t h a n t h e r e s p e c t i v e c o n t r o l s . T h e s e r e s u l t s , i n c o n j u n c t i o n w i t h t h o s e s h o w i n g an i n c r e a s e d y i e l d o f SR p r o t e i n i n t h e T^ t r e a t e d g r o u p s , s u g g e s t t h a t h y p e r t h y r o i d i s m i n t h e r a t i s a s s o c i a t e d w i t h an a u g m e n t a t i o n i n t h e amount o f SR i n t h e h e a r t , a n d i n t h e c a l c i u m t r a n s p o r t a c t i v i t y o f t h e SR. T h e s e a l t e r a t i o n s i n t h e SR a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t a l t e r e d SR f u n c t i o n i n h y p e r t h y r o i d i s m c a n e x p l a i n t h e a u g m e n t e d c o n t r a c t i l e b e h a v i o u r o f t h e h y p e r t h y r o i d r a t h e a r t . 3. The l e v e l s o f t o t a l c a r n i t i n e a n d l o n g c h a i n a c y l c a r n i t i n e d e t e c t e d i n t h e SR were d e c r e a s e d f o l l o w i n g T^ t r e a t m e n t . T o t a l c a r n i t i n e was s i g n i f i c a n t l y d e c r e a s e d 24 h o u r s a f t e r t h e t r e a t m e n t was i n i t i a t e d , a n d r e m a i n e d s o a t 48 a n d 72 h o u r s a s w e l l . L o n g c h a i n a c y l c a r n i t i n e l e v e l s w e r e n o t s i g n i f i c a n t l y l o w e r t h a n c o n t r o l u n t i l 48 a n d 72 h o u r s . The T^ t r e a t m e n t d i d n o t a f f e c t t h e l e v e l s o f a c i d s o l u b l e c a r n i t i n e d e t e c t e d i n t h e SR a t a n y o f t h e t i m e p o i n t s s t u d i e d . T h e r e was a s t r o n g n e g a t i v e c o r r e l a t i o n b e t w e e n t h e m a x i m a l c a l c i u m u p t a k e a c t i v i t y d e t e r m i n e d i n t h e SR a t e a c h o f t h e t i m e p o i n t s s t u d i e d a n d t h e l e v e l o f l o n g c h a i n a c y l c a r n i t i n e d e t e c t e d i n t h e SR. T h e s e 1 0 6 r e s u l t s suggest the p o s s i b l e r e l a t i o n s h i p between the l e v e l of l o n g c h a i n a c y l c a r n i t i n e i n the SR and the c a l c i u m pump a c t i v i t y . These r e s u l t s a r e d i f f e r e n t from p r e v i o u s l y p u b l i s h e d r e p o r t s c o n c e r n e d w i t h the e f f e c t of l o n g c h a i n a c y l c a r n i t i n e s on SR f u n c t i o n , where the norm i s t h a t l o n g c h a i n a c y l c a r n i t i n e s a r e i n c r e a s e d i n c a r d i o m y o p a t h i e s and c o n t r i b u t e to the d e p r e s s e d c a r d i a c f u n c t i o n . I t i s r e c o g n i z e d t h a t these r e s u l t s s h o u l d be i n t e r p r e t e d w i t h c a u t i o n because of p o s s i b l e t e c h n i c a l r e a s o n s and the c o n t r i b u t i o n of l o n g c h a i n a c y l c a r n i t i n e to the t o t a l membrane l i p i d . 1 0 7 BIBLIOGRAPHY Ambudkar, I.S. and Shamoo, A.E. 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