Basic Res. Cardiol. 73, 604-610 (1979) 9 1979 Dr. Dietrich Steinkopff Verlag, Darmstadt ISSN 0300-8428

Cardiothoracic Institute and National Heart Hospital, London, England

I n f l u e n c e of c o n t r a c t i l e s t a t e on the size of the e x t r a c e l l u l a r s p a c e in i s o l a t e d v e n t r i c u l a r myocardium Einflu/3 des kontraktilen Status auf die Gr6Be des Extrazellularraums b e i m isolierten V e n t r i k e l m y o k a r d P. A . P o o l e - W i l s o n ,

P. D. B o u r d i l l o n ,

a n d D. P. H a r d i n g

With 3 figures (Received J u l y 8, 1979)

Summary A m e t h o d is described with which the extraeellular space of isolated ventricular muscle can be measured accurately and continuously in the same muscle. The size of the extracellular space is shown to vary with the contractile state of the myocardium. Interventions such as quiescence, manganese and acidosis reduce myocardial contractility and increase the size of the extracellular space. Barium, ouabain and h y p o x i a cause contracture and reduce the size of the extracellular space. These changes should be taken into account when measuring intracellular electrolytes in isolated ventricular preparations.

I s o l a t e d h e a r t p r e p a r a t i o n s a r e w i d e l y u s e d to s t u d y c h a n g e s of i o n i c fluxes and intracellular electrolytes during hypoxia and ischaemia, or in t h e p r e s e n c e of d r u g s . T h e a m o u n t of c o n c e n t r a t i o n of a n e l e c t r o l y t e i n t h e i n t r a c e l l u l a r f l u i d is c a l c u l a t e d b y s u b s t r a c t i n g t h e c o n t e n t of t h e e x t r a c e l l u l a r s p a c e ( E C S ) f r o m t h a t of t h e t o t a l t i s s u e . L a r g e e r r o r s c a n a r i s e f r o m s m a l l i n a c c u r a c i e s i n t h e e s t i m a t i o n of t h e s i z e of t h e E C S , e s p e c i a l l y w i t h ions such as sodium or calcium which are present in higher concentrations i n t h e E C S t h a n i n t h e i n t r a c e l l u l a r f l u i d . T h i s p r o b l e m is of p a r t i c u l a r i m p o r t a n c e i n i s o l a t e d m y o c a r d i u m w h e r e t h e r e is a n i n c r e a s e i n t h e E C S a n d t o t a l t i s s u e w a t e r r e l a t i v e to m y o c a r d i u m in vlvo. T h e s u b j e c t h a s r e c e n t l y b e e n r e v i e w e d b y R e i c h e l (1) i n t h i s j o u r n a l . S e v e r a l i n t e r v e n t i o n s , s u c h a s c h a n g e s i n h e a r t r a t e (2) a n d d r u g s (2, 3), h a v e b e e n r e p o r t e d a s n o t a f f e c t i n g t h e s i z e of t h e E C S . I n t h e s e e x p e r i m e n t s t h e E C S w a s e s t i m a t e d f r o m t h e d i s t r i b u t i o n v o l u m e of m a r k e r substances assumed to be confined to the ECS, and a comparison was m a d e b e t w e e n m e a n v a l u e s o b t a i n e d f r o m g r o u p s of m u s c l e s . T h e a c c u r a c y of t h i s t e c h n i q u e is l i m i t e d , a n d c h a n g e s i n t h e E C S of l e s s t h a n 10% might have been undetected. 825

P o o l e . W i l s o n et al., I n f l u e n c e o f contractile state

605

This paper describes a method with which the ECS in isolated myocard i u m c a n b e m e a s u r e d c o n t i n u o u s l y w i t h a n a c c u r a c y of l e s s t h a n 1%. I n o t r o p i c i n t e r v e n t i o n s are s h o w n to c a u s e small c h a n g e s in t h e size of t h e ECS.

Methods The experimental preparation was the isolated but arterially perfused interventricular s e p t u m of the rabbit (4, 5). Male rabbits (2-3 kg) were heparinized and anaesthetized with pentobarbitone. The heart w a s r e m o v e d and a cannula inserted into the septal artery within 4 rain. All u n d e r p e r f u sed or non-contracting tissue was removed. Muscles weighed between 0.8 and 1.5 g. Th e rate of perfusion was bet ween 1.5 and 2.0 ml m i n - l g -1 wet tissue and was constant in each muscle. Temperature was held constant in each e x p e r i m e n t and was b et w een 28 and 29 ~ Muscles were electrically stimulated at 48 beats/rain. The e x p e r i m e n t s on hypoxia were performed at 32 ~ and the muscles stimulated at b e t w e e n 64 and 76 beats/rain. Muscles were perfused with a solution containing in mmol/l: lXla+, 142; K +, 5.0; Ca 2+, 1.8; Mg 2+, 1.0; H2PO4-, 0.4; HCO3-, 28; CI-, 117; and d-glucose 5.6. The solution was equilibrated with a gas m ix tu r e containing 95% O2, 5% CO2 and had a pH of 7.4. After 90 rain of equilibration muscles were labelled with 51Cr-EDTA (ethylenediamine-tetraacetic acid) (Radiochemical Centre, A m er sh am , England). Th e uptake of the isotope was followed with an 8 x 5 cm Nal crystal and g a m m a counter (J & P Engineering, Reading, England). T h e muscle was placed 2 m m in front of the crystal and effluent was returned to the vicinity of the perfusate 1.5 m away and behind 10 cm of lead shielding (5). With these precautions the b a c k g r o u n d counts were constant and were measured at the end of the e x p e r i m e n t by r em o v al of the muscle. After labelling for 40 m in the counts/min (cpm) e x c e e d e d 10,000 and were recorded each minute. Interventions were introduced after labelling for 40 rain. At the end of the e x p e r i m e n t the muscle was r e m o v e d from the apparatus, blotted, weighed and the radioactivity m e a s u r e d (ICN, Belgium). A comparison was m a d e with that of a k n o w n weight of effluent in order to calculate the exact ECS at the m o m e n t that the e x p e r i m e n t ended. The effect of a reduction of developed tension at constant resting tension was studied during a period of quiescence, respiratory acidosis (70% O2, 30% CO 2, pH -- 6.6) and in the presence of m a n g a n e s e (2.5 mmol/D. Th e effect of contracture (an increase of resting tension at constant muscle lenght) was studied by the addition of barium (8 mmol/1), ouabain (2 • 10 -6 mol/1) or h y p o x i a in the absence of substrate (PO 2 < 25 m m Hg, perfusate equilibrated with 95% N2, 5% CO2). Results are expressed as m e a n _ standard error and the statistical significance of changes analysed by S t u d e n t ' s t test.

Results U n d e r c o n t r o l c o n d i t i o n s d e v e l o p e d t e n s i o n w a s 15.6 ___ 1.0 g (n = 31) a n d r e s t i n g t e n s i o n 5.7 _-+ 0.5 g (n = 31). G e o m e t r i c a l p r o b l e m s a s s o c i a t e d w i t h c o u n t i n g of r a d i o a c t i v i t y i n s e p t a l a b e l l e d w i t h 5 1 C r - E D T A w e r e e x a m i n e d b y s t r e t c h i n g t h e a p e x a n d t w o c o r n e r s of s e p t a b y a p p r o x i m a t e l y 3 m m . T h e r e w a s n o c h a n g e i n t h e r e c o r d e d c p m o v e r a p e r i o d of 5 r ai n . T h e u p t a k e of 5 1 C r - E D T A is s h o w n i n F i g u r e 1. A r a p i d i n c r e a s e of c p m i n t h e i n i t i a l 2 m i n is a t t r i b u t a b l e t o l a b e l l i n g of t h e v a s c u l a r a n d p a r t of t h e e x t r a c e l l u l a r s p a c e s . B e t w e e n 5 a n d 20 m i n t h e r e w a s a f u r t h e r b u t s l o w e r u p t a k e of isotope. T h e s a m e result was o b t a i n e d in five contol septa. B e t w e e n 30 a n d 60, 60 a n d 90, a n d 90 a n d 120 r a i n t h e c p m i n c r e a s e d b y

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Basic Research in Cardiology, Vol. 74, No. 6 (1979) 30-

le x

E

20-

I9

I

tension

"'i 10-

g 10

0

0.J i

!

30

610

120

90

Minutes

Fig. 1. The uptake of SZCr-EDTA b y the interventricular septum of the rabbit. Each point represents the counts recorded each minute. A rapid uptake is followed by a slower increase of counts until approximately 20 rain. Thereafter the counts increase only slightly.

8 -- 0.9%, 6 _+ 0.8% a n d 3.5 __ 0.4% r e s p e c t i v e l y (n = 5). B y c o m p a r i s o n of t h e r a d i o a c t i v i t y of s e p t a a n d p e r f u s a t e a f t e r a t l e a s t 120 m i n of l a b e l l i n g w i t h 5~Cr-EDTA t h e e s t i m a t e d E C S w a s 480 +_ 20 m l / k g w e t t i s s u e ( n = 5). T h e t i s s u e w a t e r w a s 842 _ 35 m l / k g w e t t i s s u e ( n = 5) or 5.32 1/kg d r y t i s s u e ( n = 5). T h e e f f e c t of i n o t r o p i c i n t e r v e n t i o n s o n t h e size of t h e E C S is s h o w n i n F i g s . 2 a n d 3. R e s p i r a t o r y a c i d o s i s (70 % 02, 30% CO2, p H = 6.6) c a u s e d a fall of d e v e l o p e d t e n s i o n f r o m 17.0 _-+ 0.9 to 4.5 __ 0.5 g (n = 6). A s p r e v i ously reported the decline in tension occurred within 2 min and resting t e n s i o n w a s u n c h a n g e d (4). T h e e f f e c t w a s r a p i d l y r e v e r s i b l e (4). T h e m e a n i n c r e a s e i n c p m of 51Cr-EDTA w a s 6.7 + 0.8% ( n = 6). T h e i n c r e a s e occurred within 4 min in association with the decline in developed tension. T h e effect w a s r e v e r s i b l e (Fig. 2). I

E

25-1

3o%C02

I

quiescence I I .~.

a2+ 8 mmol/l

251 2O

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I

ouabain 5x10-6 molfi

I

4O 25-]

Mn2+ 2.5mmol/I I ~N,= I

I

hypoxia - no substrate

I

20..J 10 min t

Fig. 2. The effect of six interventions on the uptake of 5ZCr-EDTA. Each point represents the counts recorded each minute. Interventions which reduce contractility increase the cpm. Interventions which cause contracture reduce the cpm. See text for further description.

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8-

4-

Ouabai-6n 2or5x10 Ba2+ /I ~ nohypoxi a /NECS0 mol/I 8mmol substrate % Mn2+ /I 00230% ~/~'4'!quiescence~ 2.5mrnol -4-8 -12 Fig. 3. S u m m a r y of t h e e f f e c t of six i n t e r v e n t i o n s o n t h e size of t h e e x t r a c e l l u l a r s p a c e e s t i m a t e d f r o m t h e d i s t r i b u t i o n v o l u m e of 51Cr-EDTA.

M a n g a n e s e (2.5 mmol/1) a l s o c a u s e d a r a p i d fall of d e v e l o p e d t e n s i o n a n d c o n t r a c t i o n c e a s e d w i t h i n 3 roan. R e s t i n g t e n s i o n w a s u n c h a n g e d a n d t h e effect w a s o n l y s l o w l y r e v e r s i b l e . T h e c p m of 51Cr-EDTA i n c r e a s e d b y 3.8 ___ 0.8% (n = 6). I n a f u r t h e r g r o u p of s e p t a t h e s t i m u l a t o r w a s t u r n e d off. D u r i n g q u i e s c e n c e t h e c p m i n c r e a s e d b y 5.6 ___ 1.3% ( n = 5) w i t h i n 4 m i n a n d t h e effect w a s r e v e r s i b l e (Fig. 2). Three different interventions were studied in which a contracture was i n d u c e d (Figs. 2 a n d 3). B a r i u m (8 mmol/1) c a u s e d a n i r r e v e r s i b l e c o n t r a c t u r e w h i c h w a s m a x i m a l w i t h i n 5 r a i n . R e s t i n g t e n s i o n r o s e f r o m 5.3 _ 1.3 to 26 + 1.9 g (n -- 4). A c t i v e c o n t r a c t i o n c e a s e d w h e n t h e b a r i u m c o n t r a c t u r e d e v e l o p e d . T h e c p m of 51Cr-EDTA d e c r e a s e d b y 11.6 ___ 1.7% ( n = 4). O u a b a i n w a s a d d e d t o t h e p e r f u s a t e to g i v e a c o n c e n t r a t i o n of 2 x 10 -6 mol/1 i n t w o m u s c l e s a n d 5 x 10 -6 mol/1 i n f o u r m u s c l e s . T h e s e h i g h d o s e s w e r e c h o s e n so as to c a u s e a r a p i d d e v e l o p m e n t of c o n t r a c t u r e . R e s t i n g t e n s i o n i n c r e a s e d f r o m 6.7 __ 1.2 to 26 -+_ 2.7 g i n 30 m i n . A t t h e t i m e of m a x i m a l c o n t r a c t u r e t h e c p m of 51Cr-EDTA h a d d e c r e a s e d b y 7.2 +_ 1.5% (n = 6). C o n t r a e t u r e also o c c u r r e d d u r i n g p e r f u s i o n w i t h h y p o x i c s u b s t r a t e free s o l u t i o n . U n d e r t h e s e c o n d i t i o n s d e v e l o p e d t e n s i o n d e c l i n e d a n d w i t h i n 30 r a i n t h e r e w a s a s e v e r e c o n t r a c t u r e . R e s t i n g t e n s i o n r o s e f r o m 5.3 +_ 1.1 t o 27 _ 2.2 g ( n = 5). W h e n r e s t i n g t e n s i o n w a s m a x i m a l t h e c p m h a d f a l l e n b e l o w c o n t r o l b y 6.6 _ 1.3% ( n = 5). D u r i n g t h e i n i t i a l f e w m i n u t e s of h y p o x i a w h e n d e v e l o p e d t e n s i o n fell w i t h o u t a r i s e of r e s t i n g t e n s i o n , t h e r e w a s a s m a l l i n c r e a s e i n t h e c p m (Fig. 2).

Discussion T h e e x t r a c e l l u l a r s p a c e (ECS) has, i n t h i s s t u d y , b e e n e s t i m a t e d f r o m t h e d i s t r i b u t i o n v o l u m e of S t C r - E D T A (M.W. = 343).

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This m a r k e r has p r e v i o u s l y b e e n u s e d to m e a s u r e the E C S of r a b b i t m y o c a r d i u m i n v i v o a n d the values o b t a i n e d were similar to t h o s e with sucrose, greater t h a n with inulin a n d smaller t h a n with chloride (6). I n the present s t u d y the size of the E C S of the interventricular s e p t u m i n v i t r o (48%) is large b y c o m p a r i s o n with the E C S of v e n t r i c u l a r m u s c l e i n v l v o (24.6% see ref. 6). T h e total tissue w a t e r (840 m l / k g w e t tissue) is also greater i n y l t r o t h a n /n v i v o (78.7 - 0.3 see ref. 6). Tissue o e d e m a is a feature of m o s t if n o t all isolated tissues a n d the subject has b e e n r e v i e w e d in this journal (1). The precise values for total a n d extracellular w a t e r in isolated m u s c l e s v a r y w i t h the degree to w h i c h surface w a t e r is b l o t t e d f r o m the w e t tissue before weighing. T h e u p t a k e c u r v e of ~ICr-EDTA (Fig. 1) s h o w s that the isotope is r a p i d l y d i s t r i b u t e d into w h a t is p r e s u m e d to be the v a s c u l a r a n d extracellular spaces. E q u i l i b r a t i o n of a small a m o u n t of ~ICr-EDTA (approxim a t e l y 10%) is slower a n d n o t c o m p l e t e for 20 min. S l o w diffusion of the i s o t o p e into T-tubules, the intercalated disc a n d the p e r i n u c l e a r s p a c e m a y be an e x p l a n a t i o n b u t w h a t e v e r the m e c h a n i s m , values for the E C S o b t a i n e d f r o m isotopic labelling for 5 m i n w o u l d be c o n s i d e r a b l y less t h a n t h o s e o b t a i n e d after 20 min. B e t w e e n 30 a n d 120 m i n the c p m of 51CrE D T A c o n t i n u e d to increase, albeit m o r e slowly. S u c h a p h e n o m e n o n has b e e n r e p o r t e d p r e v i o u s l y in cardiac m u s c l e (7) a n d m a y be d u e to isotope r e a c h i n g m o r e slowly e x c h a n g i n g parts of the ECS, crossing t h e cell m e m b r a n e or e n t e r i n g the intracellular space of s o m e partly d a m a g e d cells o n the edge of the preparation. It is n o t possible to d i s t i n g u i s h b e t w e e n t h e s e possibilities b u t n o n e invalidates the use of the isotope in s h o r t - t e r m e x p e r i m e n t s as in this study. T h e effect of h y p o x i a on the distribution v o l u m e of ~ C r - E D T A (Fig. 2) s u g g e s t s that cell d a m a g e m u s t be substantial before the isotope crosses the cell m e m b r a n e since, e v e n in the p r e s c e n c e of severe contracture, the isotope does n o t a p p e a r to h a v e free access to the intracellular fluid. Alterations in the size of the E C S in this s t u d y h a v e b e e n e x p r e s s e d as percentages. The control c p m were t a k e n as t h o s e before a n y i n t e r v e n t i o n after s u b t r a c t i o n of the b a c k g r o u n d o b t a i n e d w h e n the m u s c l e was r e m o v e d f r o m the e x p e r i m e n t a l apparatus. A n y c h a n g e in the E C S may, therefore, be slightly u n d e r e s t i m a t e d since the c o n t r o l c p m will i n c l u d e surface w a t e r o n the muscle, the perfusate in the v a s c u l a t u r e a n d d r o p s of effluent f o r m i n g u n d e r the muscle. F o r these reasons the p e r c e n t a g e c h a n g e in the E C S c o u l d be u n d e r e s t i m a t e d b y u p to a p p r o x i m a t e l y 2%. The use of 51Cr-EDTA h a s the a d v a n t a g e over o t h e r m a r k e r s that the isotope is a g a m m a - e m i t t e r a n d can be c o n t i n u o u s l y m o n i t o r e d as in this study. C h a n g e s in the E C S can be followed in the s a m e septum. Since the c p m e x c e e d 10,000, a c h a n g e of less t h a n 1% c a n be d e t e c t e d whereas, if c h a n g e s in the ECS h a d b e e n studied b y c o m p a r i s o n of m e a n values for g r o u p s of muscles, f u r t h e r variability is i n t r o d u c e d a n d c h a n g e s in the E C S of less t h a n 10% m i g h t be overlooked. Most p r e v i o u s studies h a v e u s e d this latter m e t h o d . Cardiac glycosides (3), c a t e c h o l a m i n e s (2), a n d hart rate (2) have b e e n r e p o r t e d n o t to alter the ECS, a l t h o u g h c a t e c h o l a m i n e s a n d increase of h e a r t rate do a p p e a r to r e d u c e tissue w a t e r (8). The difference b e t w e e n these a n d o u r results is p r o b a b l y d u e to the different sensitivity of the t e c h n i q u e s . A s e c o n d r e a s o n is t h a t earlier

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e x p e r i m e n t s (2, 3) were p e r f o r m e d on atrial a n d n o t v e n t r i c u l a r m u s c l e as in our e x p e r i m e n t s . The t h i c k n e s s of v e n t r i c u l a r m u s c l e is greater t h a n atrial; wall t e n s i o n a n d c o m p r e s s i o n of the tissue on c o n t r a c t i o n will be larger. The results of our e x p e r i m e n t s in an isolated p r e p a r a t i o n d e m o n s t r a t e that the size of the ECS is altered by the contractile state of the muscle. I n t e r v e n t i o n s w h i c h r e d u c e c o n t r a c t i o n increase t h e ECS; c o n t r a c t u r e decreases the ECS. T h e c h a n g e s o c c u r within 5 rain of t h e altered contractile state. The findings c a n n o t be a t t r i b u t e d to g e o m e t r i c a l c h a n g e s associated w i t h m e c h a n i c a l activity, since passive s t r e t c h i n g of the m u s c l e did n o t alter the crop over 5 rain. A l t h o u g h passive s t r e t c h i n g increased tension in the muscle, the increase was only in the p l a n e of the three points at w h i c h the m u s c l e was held and is n o t e q u i v a l e n t to the c o m p r e s s i o n effects of contracture. Isolated ventricular m u s c l e appears, therefore, to b e h a v e as a " s p o n g e " in w h i c h the size of the E C S is partly d e t e r m i n e d b y the contractile state a n d c o m p r e s s i o n of the muscle. A similar effect has b e e n s h o w n (9) in s m o o t h m u s c l e s u b j e c t e d to high external p r e s s u r e w h e r e the a m o u n t of fluid w h i c h c o u l d be r e m o v e d was similar to the v o l u m e of the ECS. A l t h o u g h the results are applicable to isolated preparations, t h e y do n o t necessarily a p p l y to the m y o c a r d i u m in vivo. C h a n g e s in the E C S i n v l y o m u s t be d u e to transfer of fluid t h r o u g h the l y m p h a t i c s y s t e m or capillaries a n d c a n n o t be d u e to fluid m o v e m e n t s across the tissue surface as here, or into a p e r f u s i o n b a t h as in o t h e r i n v i t r o preparations. F u r t h e r more, the ECS i n v i v o is smaller t h a n i n v i t r o ( c o m p a r e our results with ref. 6), a n d c o n s e q u e n t l y errors in the size of the E C S i n v i v o will h a v e a smaller effect on the calculation of intracellular electrolytes. T h e results h a v e implications for the a c c u r a t e m e a s u r e m e n t of intracellular electrolytes in the p r e s e n c e of inotropic interventions. F o r ' e x a m p l e , u n d e r our e x p e r i m e n t a l c o n d i t i o n s a 10 % increase in the E C S w o u l d cause an increase of 6.8 m m o l of s o d i u m a n d 0.09 m m o l of c a l c i u m per kg wet tissue. Calculated values for intracellular electrolytes w o u l d be substantially in error if c h a n g e s in the size of the E C S were not d e t e c t e d a n d t a k e n into account.

Acknowledgements

This w o r k was supported by The British Heart Foundation and Medical Research Council.

Zusammenfassung Es wird eine Methode beschrieben zur kontinuierlichen Messung des extrazellulfiren R a u m e s in isolierter Ventrikelmuskulatur. Die Gr6Be des ExtrazeUul~rraumes variiert mit d e m kontraktilen Zustand des Muskels. Eingriffe wie Stillstellung, M a n g a n und Azidose reduzieren die Kontraktilit~t und vergr6Bern den Extrazellul~rraum. Barium, Ouabain und Hypoxie verursachen Kontraktur und verkleinern den Extrazellul~krraurn.Diese Anderungen sollten ber(ieksichtigt werden, w e n n intrazellul~re Elektrolyte in isolierten Ventrikelpr~paraten bestimmt werden.

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Basic Research in Cardiology, Vol. 74, No. 6 (1979) References

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Authors' address: Dr. P. A. Poole-WHson, Cardiothoracic Institute and National Heart Hospital, 2 B e a u m o n t Street, L o n d o n W l N 2DX (England)