Correlation of changes in cardiac calcium channels with hemodynamics in Syrian hamster cardiomyopathy and heart failure

Life Sciences, Vol. 41, pp. 153-159 Printed in the U.S.A.

Pergamon Journals

CORRELATION OF CHANGES IN CARDIAC CALCIUM CHANNELSWITH HEMODYNAMICS IN SYRIAN HAMSTER CARDIOMYOPATHYAND HEART FAILURE Mitchell S. Finkel, Eric S. Marks, Randolph E. Patterson, Edith H. Speir, Kenneth Ao Steadman and Harry R. Keiser NHLBI, NIH, Bethesda, Maryland (Received in final form April 28, 1987)

Summary We compared hemodynamics with [3H]nitrendipine (calcium channel) binding to cardiac membranes from Bio 14.6 cardiomyopathic Syrian hamsters at 4 and I0 months with t h e i r F~B controls. A 50% increase in the number (B ) of n i t r e n d i p i n e bin~ing sites (calcium channels) was seen only inmaXthe 4 month old myopathic vs controls (Bm~v=468±ll vs 309±10 fmol/mg prot with no change in a f f i n i t y (Kn) ( K n = . B ~ , I 2 vs .75±.14nM), while no differences in B or K~ were seen at I0 months (Bm:x=375±9 vs 362±7 fmol/mg p r o t ~ = . 8 2 ± ~ 1 8 vs .89±.17nM) myopathic Vs control respectively. Hemody~amic studies revealed no s i g n i f i c a n t differences in cardiac output, cardiac index, stroke volume, heart rate, mean a r t e r i a l pressure, peripheral resistance, body weight, heart weight at 4 months, but a s i g n i f i c a n t decrease in peripheral resistance (1120±360 vs 2080±240) increase in body weight (I18±2 vs 94±2 grams) and heart weight (97±5 vs 78±2gms/lOOgms body weight) in lO month myopathic vs control animals. We conclude that the onset of cardiomyopathy at 4 months is associated with a selective increase in calcium channel binding sites and heart f a i l u r e at lO months is associated with a r e l a t i v e decrease in these sites. Syrian hamsters have been extensively studied as animal models for i d i o pathic cardiomyopathy and congestive heart f a i l u r e (1,2,3). The Bio 14.6 Syrian hamster is one such genetic strain that spontaneously develops h i s t o l o g i c a l l y i d e n t i f i a b l e lesions by 4 months of age and fatal congestive heart f a i l u r e by 12 months. Although an autosomal recessive mode of inheritance has been established, a s p e c i f i c protein defect has not been found which could explain these pathophysiologic findings. Previous studies suggest that calcium overload may be involved in the path~ genesis of Syrian hamster cardiomyopathy (2,3,4). Possible mechanisms that could lead to calcium overload include increased c e l l u l a r uptake, decreased c e l l u l a r extrusion or inadequate i n t r a c e l l u l a r sequestration of calcium. Increased c e l l u l a r uptake of calcium could be caused by an increase in physiologic stimulus to uptake or increased responsiveness. Increased responsiveness, in turn, may result from abnormalities in alpha or beta adrenergic receptors, calcium channels or coupling mechanisms. Several lines of evidence, however, suggest that this animal model possesses a selective abnormality in the cardiac calcium channel (2,3,4,5). Intra-peritoneal injection of ~Ca++, for example, results in an increased myocardial uptake of labelled calcium in the myopathic compared to control 0024-3205/87 $3.00 + .00

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hamsters (2). Moreover, e a r l y treatment of these animals with various calcium channel antagonists prevents t h i s myocardial calcium overload (3). Studies have also shown a prolongation in the calcium-channel-dependent cardiac action p o t e n t i a l duration in myopathic compared to control hamsters (4). These p h y s i o l o g i c observations suggest the presence of a f u n c t i o n a l defect in a component of the v o l t a g e - s e n s i t i v e calcium channel in the hearts of c a r d i o myopathic Syrian hamster. Such a physiologic defect may be c o r r e l a t e d with a biochemical change in the numbers of calcium channels or t h e i r a f f i n i t y f o r calcium channel blockers (5). Radioligand binding techniques provide the o p p o r t u n i t y to assess the number (Bm x) and a f f i n i t y (KD) of r a d i o l a b e l l e d compounds ( l i g a n d s ) to s p e c i f i c membrane-bound proteins ( r e c e p t o r s ) ( 6 , 7 , 8 ) . The a p p l i c a t i o n of these techniques has revealed t h a t [ 3 H ] n i t r e n d i p i n e , a r a d i o l a b e l l e d calcium antagonist , binds to a protein c l o s e l y associated with the cardiac calcium channel ( 6 , 7 , 8 ) . Suc~binding has been c o r r e l a t e d with i n h i b i t i o n of potassium-stimulated ~Ca++ uptake and physiologic responses in various t i s s u e s , supporting i t s physiologic relevance ( 6 , 7 , 8 ) . More r e c e n t l y , p u r i f i c a t i o n of t h i s binding s i t e from s k e l e t a l muscle has resulted in a f u n c t i o n a l l y i n t a c t calcium channel in r e c o n s t i t u t i o n experiments (9). Thus, changes in calcium channel a c t i v i t y may be r e f l e c t e d in changes in t h i s binding s i t e . In t h i s study, we compared calcium channel binding c h a r a c t e r i s t i c s in myopathic and control hamsters at two w e l l - d e f i n e d p h y s i o l o g i c states. This was done in an e f f o r t to explore possible r e l a t i o n s h i p s between number or a f f i n i t y of [ 3 H ] n i t r e n d i p i n e bindings s i t e s and the hemodynamic status of these hamsters. Thus, p a r a l l e l changes in binding and hemodynamics would support an, as yet, undefined role f o r t h i s binding s i t e in the pathophysi~ogy of t h i s animal model. Methods Physiologic studies in i n t a c t animals were performed as f o l l o w s : Hamsters (Bioresearch Consultants, Inc. Cambridge, MA) were anesthetized with sodium p e n t a b a r b i t a l (5mg/lOOg body weight I . P . ) and both the r i g h t external j u g u l a r vein to the l e v e l of the r i g h t atrium and the l e f t femoral a r t e r y were cannulated with PE50 tubing. A t h e r m o d i l u t i o n microscope (0.33mm O.D., Columbus Instrument Corp.) was passed v i a the l e f t c a r o t i d a r t e r y to the o r i f i c e of the a o r t i c valve. Cardiac output and stroke volume were measured by a t h e r m o d i l u t i o n technique u t i l i z i n g 200ul of room temperature i n j e c t a t e . A r t e r i a l blood pressure and heart rate were continuously recorded. Core body temperature was monitored and maintained constant. Binding studies were performed as p r e v i o u s l y described (5,6) with modifications. B r i e f l y , f o l l o w i n g d e c a p i t a t i o n , hearts were removed, homogenized in 50mM T r i s pH 7.4 on ice with a Brinkman Polytron ( d i a l s e t t i n g 6) f o r 15 seconds x 3, centrifuged at 800 x g f o r I0 minutes and supernatant r e c e n t r i f u g e d at 34,000 x g f o r I0 minutes. P e l l e t s were washed x 3 with i c e cold b u f f e r and rehomogenized with the same b u f f e r (50mM T r i s pH 7.4 at 0 °) using a hand-held t e f l o n - g l a s s homogenizer. [ 3 H ] N i t r e n d i p i n e (79.5Ci/mM; New England Nuclear) binding was assayed in 50mM T r i s pH 7.4 at 25 ° x 60 minutes in the dark with approximately I00 mcg membrane protein in a f i n a l volume of O.5ml/tub~. Non-specific binding was determined by incubating in the presence of I0 ~M n i f e d i p i n e . Incubations were terminated by r a p i d l y f i l t e r i n g over GF/B glass f i b e r f i l t e r s (Whatman). The f i l t e r s were washed with 6ml of i c e - c o l d b u f f e r (50mM T r i s pH 7.4) x 3, and the r a d i o a c t i v i t y retained was determined by l i q u i d s c i n t i l l a t i o n spectrometry in Aquassure scintillation cocktail.

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Results Studies comparing body and heart weights, cardiac outputs, cardiac indices, stroke volumes, heart rates, mean a r t e r i a l pressures and peripheral resistances revealed minor differences in these parameters between the Bio 14,6 and F B control hamsters at 4-6 months of age (n=5) (Table I ) . However, at lO-121months of age, body and heart weights were increased and peripheral resistances were decreased in the Bio 14,6 animals compared to t h e i r F B c o n t r o l s (p<.05, n=4), Moreover, m o r t a l i t y approached 50% among the I~-12 month-old myopathic hamsters, whereas none of the c o n t r o l s died (n=lO), In our p r e l i m i n a r y studies we found [ 3 H ] n i t r e n d i p i n e to bind with high a f f i n i t y to a s i n g l e class of binding s i t e s over a dosage range from 0,03 to 8nM (Figure I ) , E q u i l i b r i u m was reached w i t h i n 30 minutes (not shown) and s p e c i f i c binding was l i n e a r over a range of 25 to 200mcg of membrane protein (not shown), Ligand competition experiments revealed complete displacement of [ 3 H ] n i t r e n d i p i n e binding by n i t r e n d i p i n e = n i f e d i p i n e with verapamil only weakly and p a r t i a l l y displaced binding; while d i l t i a z e m potentiated binding at 37 ° (Figure 2). These studies, t h e r e f o r e , established t h a t we are studying the same [ ' H ] n i t r e n d i p i n e binding s i t e p r e v i o u s l y described in heart and noncardiac t i s s u e ( 6 , 7 , 8 ) . Table I FIB Controls (4-6 mos)

Bio 14.6 (4-6 mos)

FIB Controls " (10-12 m o s )

Bio 14,6 (10-12 mos)

Weights Body wgt Dry Heart

123±8 72±3

120±9 *69±5

94±2 78±2

°118±12 °97±5

Hemodynamics CO Cl SV HR MAP PR

44±19 36.2±16 ,146±,05 306±44 130±15 3.3±1.2

48±16 40,1±12 ,145±,05 337±53 98±18 2,2±,7

39.4±3,5 42,1±4,4 ,126±0.1 318±38 101.5±19 2.6±.3

54±13 45±7 ,175±.06 321±38 72.5±14 °I,4±,45

Legend ° = (10-12) Bio 14.6 s i g n i f i c a n t l y d i f f e r e n t from FIB control at 0,05 l e v e l * (4-6 mos) Bio 14.6 s i g n i f i c a n t l y d i f f e r e n t from 10-12 mos Bio 14.6 at 0,05 l e v e l (N=5; unpaired " t - t e s t " ) Body weight in grams,

l=per lO00mg dry body weight

CO = Cardiac output in ml/min CI = Cardiac index in ml/min/lOOgm SV = Stroke volume in ml

HR = Heart rate in beats/min MAP = Mean a r t e r i a l pressure(mmHg) PR = Peripheral resistance(MAP/C~

We then compared Scatchard p l o t s derived from s a t u r a t i o n experiments of 4-6 month old male Bio 14,6 cardiomyopathic hamsters with sex and age-matched FIB c o n t r o l s . Whereas the slopes of these p l o t s are s i m i l a r , t h e i r X±intercepts are markedly d i f f e r e n t , r e f l e c t i n g s i m i l a r a f f i n i t i e s (K D) but a greater number of s i t e s (B ) in myopathic hearts, Table I I summarlzes max r e s u l t s of 7 separate experlments (n:7; 6 animals/experiments x 7 : 42 animals) revealed K 65±.12 vs ,75±,14nM and B :468±II vs 309±lOfmol/mg ma prot, (±SEM) (n=7) ~o~ 4-6 month old Bio 14.6 myopa~hlc hamsters and t h e i r

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age-matched FIB c o n t r o l s , r e s p e c t i v e l y . S t a t i s t i c a l analyses using Student's t t e s t f o r unpaired data and two-way a n a l y s i s of variance demonstrated a s i g n i f i c a n t increase in the number of [ 3 H ] n i t r e n d i p i n e b i n d i n g s i t e s in the hearts of these myopathic hamsters vs t h e i r FIB c o n t r o l s ( p < . O l ) . The KD values, however, were not s i g n i f i c a n t l y d i f f e 6 e n t . S i m i l a r experiments comparing 10-12 month-old Bio 14.6 hamster hearts ( i n heart f a i l u r e ) w i t h t h e i r FIB c o n t r o l s revealed no s i g n i f i c a n t d i f f e r e n c e s in e i t h e r the Kn ( 82± IS vs .89±.17nM)or B (375±8 9 vs 362±6.7 fmol/mg p r o t . ) (±SEM)~ • " max " ( n : 4 ; 6 anlmals/experlment x 4 = 24 animals) r e s p e c t i v e l y (Table I I ) .

300

(Z

250

E

200 Z o

o5 m u u.,

~

150

lOO

ul

--

N

10o

p.

50

0

, 50

, ~ i . ~'~ * 100 150 200 250 300 350 BOUND

I

I

I

I

2

4

6

8

i3HiNIT FREE (riM)

Figure 1 S a t u r a t i o n and Scatchard p l o t ( i n s e t ) of [ 3 H ] n i t r e n d i p i n e binding to cardiac membranes prepared from 6 month-old FIB c o n t r o l hamsters. Discussion We have c a r e f u l l y c h a r a c t e r i z e d the hemodynamic s t a t u s of the Syrian Hamster model of cardiomyopathy by c a n n u l a t i n g e x t e r n a l j u g u l a r v e i n , femoral a r t e r y and c a r o t i d a r t e r y using a percutaneous method f o r the f i r s t time. We have i d e n t i f i e d two d i s t i n c t p h y s i o l o g i c states at 4-6 and 10-12 months of age. At 4-6 months o f age these hamsters demonstrate a decrease in p e r i p h e r a l r e s i s t a n c e and mean a r t e r i a l pressure as w e l l as an increase in heart rate. Because of the small sample size none of these d i f f e r e n c e s achieved s t a t i s t i c a l s i g n i f i c a n c e , however. At 10-12 months of age a statistically s i g n i f i c a n t decrease in p e r i p h e r a l r e s i s t a n c e and increases in body and heart weights are apparent• Moreover, increases in cardiac o u t p u t and stroke volume are suggested• As w e l l as . a decrease in mean a r t e r i a l

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120 110 IO0

ii" 6O

NITREN

u

20 10

0 11

10

9

8

7

6

5

- L O G [DISPLACER] M

Figure 2 Displacement curves of calcium channel antagonists competing for [3H]nitrendipine binding revealing appropriate pharmacologic s p e c i f i c i t y . Table I I *_KD(nM)

Biax(fmol/mg prot.)

#Expts.

#animals

FIB Controls (4-6 mos)

.75±.14

°309±10

7

42

Bio 14.6 (4-6 mos)

.65±.12

°468±11

7

42

F.B Controls

.89±.17

362±6.7

5

30

Bio 14.6 (10-12 mos)

.82±.18

375±8.9

5

30

(I0-12 mos)

Legend *Values represent mean ± SEM. °4-6 mos Bio 14.6 s i g n i f i c a n t l y d i f f e r e n t from 4-6 mos Control at p<.Ol level (unpaired " t - t e s t " ) . pressure. However, the small sample sizes again preclude demonstrating s t a t i s t i c a l significance. At 4-6 mos the myopathic hamster appear to be reasonably well-compensated despite the presence of h i s t o l o g i c a l l y

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i d e n t i f i a b l e lesions (1,2,3). At 10-12 months they lapse into f l o r i d congestive heart f a i l u r e despite increases in cardiac output and stroke volume and decreases in mean a r t e r i a l pressure and peripheral resistance. Our findings are completely consistent with those of Abelman et al in which hemodynamic measurements were made by a completely d i f f e r e n t technique employing transthoracic cardiac puncture ( I 0 ) . On the basis of h i s t o l o g i c and hemodynamic studies i t appears that t h i s animal model undergoes a stage of hemodynamically compensated tissue necrosis followed by a stage of hemodynamic decompensation and hypertrophy. We then compared calcium channel binding c h a r a c t e r i s t i c s between myopathic and control hearts in both the compensated and decompensated states using the radioligand, [3H]nitrendipine. We found a s i g n i f i c a n t increase in the Bmax of the hemodynamically compensated myopathic hearts compared to controls, while also finding a decrease in B to control values in the decompensated state, max I n t e r p r e t a t i o n of these findings is s i g n i f i c a n t l y aided by considering several other complementary reports. F i r s t . i t has been shown that the B for [3H]nitrendipine binding to cardiac tissue can be increased by the a ~ t i o n of d i l t i a z e m to the incubation medium ( I I ) . This suggests that a l l o s t e r i c modulation of t h i s binding s i t e in v i t r o can change the apparent B by making more s i t e s a v a i l a b l e for [3H]nitrendipine binding. This c ~ d be accomplished by changing from a lower to a higher a f f i n i t y conformation in v i t r o . Second. Bean has shown that [3H]nitrendipine binds p r e f e r e n t i a l l y to the inactivated state of the channel (12). Thus, an increase in B may r e f l e c t a greater frequency of the inactivated st~t e per

max . channel as apposeG to a greater t o t a l number of channel sites. Third, calcium channels are subject to physiologic modulation that may involve phosphorylation-dependent conformational states that regulate calcium fluxes (9,

13). Thus, an increase in B ayma r e f l e c t phosphorylation of the calcium channel by an exogenous r e g u l a t o r , y And fourth, Wagner, et. al. have shown an increase in [3H]nitrendipine binding (B ) in the brain, skeletal muscle max and heart of these myopathic hamsters before the onset of h i s t o l o g i c necrosis (14). This suggest that the increase in Bm x may r e f l e c t a primary defect in calcium channel structure in t h i s animal mo~el. We compared [3H]nitrendipine binding and hemodynamics in post-necrotic animals at two well-defined hemodynamic states for the f i r s t time. Viewing our present study in l i g h t of these others suggests that t h i s animal model may, in fact, s u f f e r from a genetic defect in the calcium channel i t s e l f or a regulator of the channel. As noted previously, any number of e x t r i n s i c mechanisms could lead to an apparent increase in B in [3H]nitrendipine max binding assays. To d i s t l n g u l s h between a real i n t r l n s i c defect in the calcium channel as apposed to an e x t r i n s i c regulatory e f f e c t w i l l require p u r i f i c a t i o n and r e c o n s t i t u t i o n studies. •

References I. 2. 3. 4.

F. HOMBURGER. Myopathy of Hamster Dystrophy: History and Morphologic Aspects. Annals of the New York Academy of Sciences 317: 2-17, (1979). A. FLECKENSTEIN. Calcium Antagonists Against Hamster Cardiomyopathy In: Calcium Antagonism in Heart and Smooth Muscle. Wiley-lnterscience 153-164, (1983). S.M. FACTOR, T. MINASE, R. DOMINITZ, E.H. SONNENBLICK. Microvascular Spasm in the Cardiomyopathic Hamster: A Preventable Cause of Focal Myocardial Necrosis. C i r c u l a t i o n 66 (No.2): 342-354, (1982). K.L. ROSSNER and H.G. SACHS. Electrophysiological Study of Syrdan Hamster Hereditary Cardiomyopathy. Cardiovascular Research 12: 436-443,(1978).

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5. 6. 7. 8. 9. I0.

II. 12. 13. 14.

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M.S. FINKEL, E.S. MARKS, R.E. PATTERSON, E.H. SPEIR, K.A. STEADMANand H.R. KEISER. Increased Cardiac Calcium Channels in Hamster Cardiomyopath~ American Journal of Cardiology 57: 1205-1206, (1986). A. SCHWARTZAND D.J. TRIGGLE. Cellular Action of Calcium Channel Blocking Drugs. Ann Rev Med 35: 325-339, (1984). W.G. NAYLER and J.D. HOROWITZ. Calcium Antagonists: A New Class of Drugs. Pharm Ther 20: 203-263, (1983). R.A. JANIS and D.J. TRIGGLE. New Development in Ca++ Channel Antagonists. J Medicinal Chemistry 26 (No.6): 775-785, (1983). V. FLOCKER~, H.J. OEKEN, F. HOFMAN, D. PELZER, A. CAVALIE, W. TRAUTWEIN, Purified Dihydropyridine-binding Site From Skeletal Muscle t-Tubules is a Functional Calcium Channel. Nature 323: 66-68, (1986). W.H. ABELMAN, F.E.JEFFREY and R. WAGNER. Hemodynamics of the Hereditary Cardiomyopathy of Syrian Hamsters. Recent Advances in Studies on Cardiac Structure and Metabolism. Myocardiology. E. Bajusz and G. Rona (ed.) University Park Press, Baltimore I: 225-234, (1972 a). A. SCHWARTZ, I.L. GRUPP, J.S. WILLIAMS and P.L. VAGHY. Effects of Dihydropyridine Calcium Channel Modulators in the Heart: Biochem. Biophys. Res. Comm. 125: 387-394, (1984). B.P. BEAN. Nitrendipine Block of Cardiac Calcium Channels: High A f f i n i t y Binding to the Inactivated State. Proc. Natl. Acad. Sci. U.S.A. 81: 6388-6392, (1984). H. REUTER. Calcium Channel Modulation by Neurotransmitters, Enzymes and Drugs. Nature 301: 569-574, (1983). J.A. WAGNER, l . J . REYNOLDS, H.F. WEISMAN, P.DUDECK, M.L. WEISFELDT and S.H. SNYDER. Calcium Antagonist Receptors in Cardiomyopathic Hamster: Selective Increase in Heart. Muscle, Brain. Science 232: 515-518, (1986).