15.1. 1975

Specialia

67

E v i d e n c e f o r I n w a r d C a l c i u m C u r r e n t i n t h e A b s e n c e of E x t e r n a l S o d i u m i n R a t M y o c a r d i u m Since t h e o b s e r v a t i o n s of GII~BISCH a n d ~VEIDMANN 1 o n v o l t a g e c l a m p e d s h e e p v e n t r i c l e fibres, t h e r e h a s b e e n a g r o w i n g w e i g h t of e v i d e n c e t h a t a d e l a y e d i n w a r d c u r r e n t c o n t r i b u t e s to t h e p l a t e a u p h a s e of t h e c a r d i a c a c t i o n p o t e n t i a l . T h i s c u r r e n t is p r o b a b l y c a r r i e d b y Ca++ ions 2, 3. T h e r e is also e v i d e n c e t h a t c a l c i u m ions m o v i n g in d u r i n g t h e p l a t e a u c o n t r i b u t e directly, or i n d i r e c t l y , t o t h e rise in free i n t r a c e l l u l a r c a l c i u m t h r o u g h i n c r e a s i n g a n i n t e r n a l r e l e a s a b l e store. T h i s is t h o u g h t t o b e r e s p o n s i b l e for t e n s i o n d e v e l o p m e n t ill t h e m y o c a r d i u m 4, 5. T h e e v i d e n c e for i n w a r d c a l c i u m c u r r e n t h a s c o m e l a r g e l y f r o m s t u d i e s o n t r a b e c u l a r t i s s u e f r o m s h e e p ~ or frog 6 m y o c a r d i u m . E x p e r i m e n t s o n o t h e r species s u c h as e a t 7 a n d r a t s h a v e also b e e n r e p o r t e d . Our interest has been centred on the rat myocardium, p a r t i c u l a r l y r a t p a p i l l a r y muscle, w h i c h h a s b e e n used e x t e n s i v e l y as a m o d e l s y s t e m in i n t e r p r e t i n g m y o c a r d i a l m e c h a n i c s 9-n. T h i s is p e r h a p s u n f o r t u n a t e since i t is clear t h a t t h e r a t h e a r t b e h a v e s d i f f e r e n t l y t o m o s t o t h e r m a m m a l i a n h e a r t s i n s e v e r a l respects. I n c r e a s i n g t h e f r e q u e n c y of s t i m u l a t i o n gives a n e g a t i v e i n o t r o p i c r e s p o n s e u n d e r m o s t c o n d i t i o n s 12. I t is r e l a t i v e l y ins e n s i t i v e t o c a r d i a c glycosides ~3 a n d h a s a s h o r t a c t i o n p o t e n t i a l w i t h a l m o s t n o p l a t e a u p h a s e 1~. A d i r e c t c o m p a r a t i v e s t u d y ,of t h e r a t a n d c a t m y o c a r d i u m led to t h e s u g g e s t i o n t h a t t h e i n t r i n s i c c o n t r o l m e c h a n i c s for c o n t r a c t i l i t y in t h e r a t m a y b e q u i t e d i f f e r e n t 14. Most of t h e s e differences p o i n t to t h e p o s s i b i l i t y t h a t t h e r a t m y o c a r d i u m b e h a v e s as a 'closed s y s t e m ' w i t h r e s p e c t t o calcium. O n t h e o t h e r h a n d t h e w o r k of LEOTY e t al. ~ clearly shows a m a n g a n e s e - s e n s i t i v e slow i n w a r d c u r r e n t in s t r i p s of r a t m y o c a r d i u m . W e h a v e a t t e m p t e d t o f i n d fureher d i r e c t e v i d e n c e for a p o t e n t i a l d e p e n d e n t Ca++ c h a n n e l in t h e m y o c a r d i a l m e m b r a n e of r a t ventricle. M a t e r i a l and methods. P a p i l l a r y muscles t a k e n f r o m t h e left v e n t r i c l e of r a t s were e q u i l i b r a t e d in T y r o d e s o l u t i o n of t h e following c o m p o s i t i o n Na, 149.2; K, 5.4; Mg, 0.5; H 2 P O 4, 0.4; H C O 3 11.9; C1 146.9 meq/1 a n d glucose 5 g/l, e q u i l i b r a t e d w i t h 5 % CO2, 9 5 % O 2. W h e n tris s o l u t i o n s were used, all N a was r e p l a c e d b y tris or tris + Ca a n d t h e p H a d j u s t e d w i t h HC1, o t h e r b u f f e r s (HCO~ and H2PO~)were eliminated and the solution gassed w i t h o x y g e n T h e sucrose s o l u t i o n was m a d e b y a d d i n g 100 g sucrose a n d 5 g glucose t o 1 1 distilled w a t e r . T h e c h a m b e r c o n t a i n e d a single, c e n t r a l sucrose g a p 1.5 m m wide w h i c h was s e p a r a t e d f r o m t h e 2 e n d compartments by thin rubber membranes. The muscle was sucked into a thin plastic cannula which was passed t h r o u g h t h e holes in t h e t w o r u b b e r m e m b r a n e s a n d g e n t l y w i t h d r a w n to l e a v e t h e t i p of t h e p a p i l l a r y muscle projecting into the voltage measuring compartm e n t . C u r r e n t pulses p a s s e d t h r o u g h t h e fibres in t h e sucrose g a p f r o m t h e b a s e of t h e m u s c l e c o u l d b e u s e d t o c h a n g e t h e p o t e n t i a l of fibres in t h e t i p of t h e p a p i l l a r y muscle. A l t e r n a t i v e l y , c u r r e n t f r o m a f e e d b a c k a m p l i f i e r c o u l d b e used t o c o n t r o l t r a n s m e m b r a n e p o t e n t i a l in t h e t i p of t h e p a p i l l a r y m u s c l e m e a s u r e d b y a m i c r o e l e c t r o d e . T h e a r r a n g e m e n t was s i m i l a r to t h a t d e s c r i b e d b y ~IEBISCH and WEIDMANN 16. After perfusing the 3 chambers with Tyrode solution for i h, one end of the muscle was stimulated and potentials between the end compartments were measured. Action potentials of about 30 mV were reached at this time. The centre compartment was then perfused with sucrose. The trans gap potential increased during the next 20 min until the ratio of the trans gap action potential to the transmembrane potential recorded by microelectrode reached about 0.95. This corresponds with the average value of

0.94 r e p o r t e d b y McGuIGAN I7 u s i n g s h e e p a n d calf h e a r t t r a b e c u l a e . T h e r a t i o decreases slowly w i t h t i m e t o r e a c h a level of a b o u t 0.8 a f t e r 2 h. U n d o u b t e d l y t h i s is due t o t h e p r o g r e s s i v e rise in i n t e r n a l l o n g i t u d i n a l r e s i s t a n c e as r e p o r t e d b y KLEBER ls. T h i s f u r t h e r c o m p l i c a t e s t h e i n t e r p r e t a t i o n of c u r r e n t v o l t a g e r e l a t i o n s h i p s in p r o l o n g e d e x p e r i m e n t s . T h e t r a n s m e m b r a n e v o l t a g e change, i n r e s p o n s e t o s q u a r e c u r r e n t pulses was m e a s u r e d a t d i f f e r e n t i n t e r v a l s a l o n g t h e p r o j e c t i n g t i p w i t h a microelectrode. 2 was e s t i m a t e d b y f i t t i n g c u r v e s for a sealed e n d cable t9 t o t h e o b s e r v e d d a t a . E s t i m a t e s of 0.9 m m for d e p o l a r i z i n g pulses a n d 0.8 m m tor h y p e r p o l a r i z i n g pulses were o b t a i n e d . T h e p r o j e c t i n g t i p of t h e p a p i l l a r y muscle m u s t t h e r e f o r e b e n o m o r e t h a n 0.5 m m in o r d e r to o b t a i n s p a t i a l v o l t a g e c o n t r o l of 4- 10% w h e n t h e m i e r o e l e c t r o d e i m p a l e s t h e c e n t r a l region of t h e p r o j e c t i n g fibres a n d w h e n t r a n s m e m b r a n e r e s i s t a n c e (Rm) is u n i f o r m a n d close t o t h a t seen in t h e r e s t i n g state. W h e n p o t e n t i a l d e p e n d e n t c h a n g e s in Rm occur, c u r r e n t flow d i s t r i b u t i o n will b e c o m e n o n - u n i f o r m . T h e r a n g e of t r a n s m e m b r a n e v o l t a g e o v e r w h i c h ionic c h a n n e l s o p e n will t h u s b e s u b j e c t to cons i d e r a b l e errors in m e a s u r e m e n t . T h e m e t h o d is still h o w e v e r v a l u a b l e in i d e n t i f y i n g c u r r e n t c h a n n e l s w h e n d i f f e r e n t ions are s u b s t i t u t e d in t h e e x t e r n a l solution. Results and discussion. U s i n g s h o r t muscle l e n g t h s (0.4-0.5 m m ) c u r r e n t v o l t a g e r e l a t i o n s were m e a s u r e d in d i f f e r e n t e x t e r n a l solutions. I n T y r o d e , p e a k i n w a r d c u r r e n t s h o w e d a n a b r u p t t r a n s i e n t increase to a m a x i m u m v a l u e w i t h step d e p o l a r i z a t i o n f r o m t h e r e s t i n g p o t e n t i a l ( - - 7 0 t o --80 m V ) t o a b o u t --50 m V (Figure 1). T h e s h a r p i n c r e a s e in c u r r e n t is a l m o s t c e r t a i n l y a r e f l e c t i o n of p o o r s p a t i a l c o n t r o l of p o t e n t i a l a t t h e p o i n t w h e r e s o d i u m c o n d u c t a n c e s t a r t s to increase 2~ A n e t i n i t i a l i n w a r d c u r r e n t p e r s i s t s u p t o s t e p d e p o l a r i z a t i o n s of a b o u t + 20 m V . A t t h i s p o i n t , o u t w a r d c u r r e n t exceeds i n w a r d c u r r e n t a n d e s t i m a t e s of i n w a r d c u r r e n t b e c o m e d e p e n d e n t u p o n t h e e s t i m a t e d t i m e course of o u t w a r d c u r r e n t . T h e c u r v e s h o w n in F i g u r e 1B is b a s e d on t h e a s s u m p t i o n t h a t t h e r e is a s t e a d y n e t o u t w a r d c u r r e n t , i n d e p e n d e n t of

1 G. GIEBISCH and S. WEIDMANN, Helv. physiol, pharmac. Acta 25, CR 189 (1967). 2 D. MASCHERand K. PEPER, Pfltigers Arch. 307, 190 (1969). 8 G. W. B~ELER and H. REUTER, J. Physiol., Lond. 207, 165 (1970). 4 R. NIEDERGERKE, Experientia 75, 128 (1959). 5 E. H. WOOD, m. L. HEPPNER and S. WEIDMANN, Circulation Res. 24, 409 (1969). 6 C. LEOTY and G. RAYMOND, Pflfigers Arch. 334, 114 (1972). 7 M. KOLHARDT, B. BANER, H. KRAUSE and A. FLECKENSTEIN, Pfliigers Arch. 335, 309 (1972). s y. M. GARGOUIL,Cardiology 56, 263 (1971/72). 9 W. C. ULLRICK, Am. J. Physiol. 206, 1285 (1964). 10 B. •ORECKY, G. C. TAICHMAN, R. ~V[INELLI and M. BEZNAK, Myocardiology 1, 71 (1972). 11 R. MINELLI, V. PANAGIAand C. REGGIANI, Pfliigers Arch. 339, 79 (1973). 12 j. j. KELLY and B. F. HOFFMAN, Am. J. Physiol. 799, 157 (1960). 13 j. t{OCH-WESER and J. R. BLINKS, J. Pharmac. exp. Ther. 736, 305 (1962). 14 A. M. HENDERSON, D. L. BRUTSAERT~ W. W. PARMERLEY and E. M. SONNENBLICK, Am. J. Physiol. 217, 1273 (1969). 15 C. LEOTY, O. RAYMONDand Y. M. GARGOUIL, J. Physiol., Lond. 216, 85 P (1971). 16 G. GIEBISCn and S. WEIDMANN, J. gem Physiol. 57, 290 (1971). 1~ j. A. S. McGuIGAN, J. Physiol., Lond. 240, 775 (1974). 18 A. G. KLEBER, Pflfigers Arch. 345, 195 (1973). z9 S. WEIDMANN, J. Physiol. Lond., 118, 348 (1952). 20 j. M. KOOTSEY and E. A. JohNsoN, Biophys. J. 12, 1496 (1972).

68

Specialia

time, d u r i n g t h e 150 msec d e p o l a r i z i n g pulse. T h i s p r o b a b l y r e s u l t s i n some u n d e r e s t i m a t e of t h e i n w a r d c u r r e n t since t h e r e a p p e a r s t o b e a s m a l l i n i t i a l t r a n s i e n t outward current component after sodium has been w a s h e d o u t (Figure 1A, c o l u m n 2). W i t h i n 1-2 rain of t h e s t a r t of t h e w a s h o u t w i t h s o d i u m - f r e e tris s o l u t i o n t h e i n i t i a l i n w a r d t r a n s i e n t disappears. It may be argued that a smatl delayed i n w a r d c u r r e n t r e m a i n s e v e n a f t e r 10 m i n s o a k i n g in Nafree s o l u t i o n since o u t w a r d c u r r e n t p a s s e s t h r o u g h a m i n i m u m a t a b o u t 50 msee. F o r t h e sake of c o m p l e t e n e s s , t h i s c o m p o n e n t was c a l c u l a t e d a s s u m i n g a s t e a d y o u t w a r d c u r r e n t e q u a l t o t h e n e t c u r r e n t a t t h e e n d of t h e pulse. I n t h i s e x p e r i m e n t a s m a l l a m o u n t of c a l c i u m (0.1 m M ) w a s a d d e d to t h e tris solution. T h i s h e l p e d t o m a i n t a i n s t a b i l i t y of m e m b r a n e p o t e n t i a l d u r i n g t h e p r o l o n g e d w a s h o u t periods. U p o n r e p l a c e m e n t of t h i s tris s o l u t i o n w i t h a s o l u t i o n c o n t a i n i n g large a m o u n t s of c a l c i u m (10-50 m M ) large i n w a r d c u r r e n t s b e c a m e a p p a r e n t w i t h i n 1-2 m i n a n d p e r s i s t e d for as l o n g as t h e e x p e r i m e n t c o n t i n u e d (30 min).

EXPERIENTIA 31/1

H e r e a g a i n t h e r e is e v i d e n c e of p o o r s p a t i a l c o n t r o l a t c l a m p p o t e n t i a l s b e t w e e n --20 m V a n d zero. T h e s u d d e n increase i n i n w a r d c u r r e n t b e t w e e n --20 m V a n d --10 m V a n d t h e s m a l l h u m p i n t h e v o l t a g e r e c o r d are t y p i c a l i n d i c a t o r s of this. W h i l e t h e s e a r t e f a c t s are a n u n d e s i r a b l e f e a t u r e for precise c l a m p a n a l y s i s t h e y n e v e r t h e l e s s are a c o n s e q u e n c e of t h e r a t h e r large c a l c i u m d e p e n d e n t i n w a r d c u r r e n t . N e t i n w a r d c u r r e n t was a g a i n o n l y m a i n t a i n e d for p o t e n t i a l s u p t o a b o u t + 20 m V . A t h i g h e r p o t e n t i a l s t h e r e a p p e a r e d to b e a n i n i t i a l i n w a r d current component superimposed on a steady outward current. The inward component persisted even at voltage s t e p s of + 80 m V . T h e a r g u m e n t s a g a i n s t t h e i n w a r d c u r r e n t c o m p o n e n t in Ca-tr/s s o l u t i o n b e i n g due t o sodium, seem considerable. Finest t h e i n w a r d c u r r e n t d i s a p p e a r s w i t h i n 1-2 rain of s w i t c h i n g to Na-free tris golntions i n d i c a t i n g a r e a s o n a b l y r a p i d s o d i u m w a s h out. S e c o n d l y t h e t i m e course of t h e i n w a r d c u r r e n t in h i g h Ca is q u i t e d i f f e r e n t t o t h a t in h i g h s o d i u m a n d t h i r d l y t h e a p p a r e n t r e v e r s a l p o t e n t i a l of t h e i n w a r d c u r r e n t in t h e p r e s e n c e of e x t e r n a l Ca is so

TRIS 0 . t rnM Ca (13 rain O Na]

TYRODE

TRIS 5 0 mM Ca [50 rain O Na)

v m (my) --80

--60

--40

--20

0

+20

40

60

80

100

120

B --2O

{,u.A)

--40

/,'

T~,s 15o ~

Col

I,

..... i

9 --60

TYRooE

_Z

k___

Fig. 1. A) Typical current and voltage records from rat papillary muscle (see text). Lower trace; microelectrode recording of transmembrane potential during step depolarizations. Upper trace; current records. After washing out tyrode solution with tris, the initial inward current component disappears (column 2). After 20 rain, the 0.1 mM Ca-iris solution is replaced by 50 mM Ca-tris. Large inward currents appear within a few minutes and persist for at least 30 min after the change (column 3). B) A plot of inward current (zlI ; see inset) as a function of transmembrane potential in the 3 different solutions, sequence as in A.

15.1. 1975

69

Speeialia

Fig. 2. Recordings of membrane potential responses to depolarizing pulses in rat papillary muscle. At zero time the tyrode solution was replaced by 10 mM Ca-tris soiution. Subsequent changes as indicated.

.s I

50mv

t

I

50msec

I 20

40

TIME

iN

60

100

80

ONa(min)

high t h a t it would exceed the sodium equilibrium p o t e n t i a l even if no sodium were washed out of the extracellular space in 50 rain. I t follows t h a t the large inward currents seen in t h e high calcim-n-tris solution should lead to regenerative spikes if large enough depolarization pulses are passed t h r o u g h the preparation. The typical action p o t e n t i a l in response to small depolarization pulses (15-20 mV, 2 msec) disappears w i t h t h e same t i m e course as t h a t required for the disappearance of the t r a n s i e n t inward current w h e n T y r o d e is replaced w i t h tris solutions. If t h e d u r a t i o n of the depolarizing pulse is increased .to a b o u t 30 msec and the a m p l i t u d e of t h e pulse progressively increased, a p o t e n t i a l is reached at which regenera t i v e spikes appear. A t y p i c a l e x p e r i m e n t is shown in F i g u r e 2. A t t i m e zero, T y r o d e is e x c h a n g e d for tris + 10 m M Ca. The spikes are generated by an initial depolarizat i o n of a b o u t 50 m V and can still be generated after a b o u t 30 rain in this solution, w i t h a slight decrease in p e a k a m p l i t u d e . After washing w i t h tris containing no calcium, t h e r e g e n e r a t i v e spikes disappear in a b o u t 5 rain and c a n n o t be o b t a i n e d w i t h larger depolarizing pulses. M e m b r a n e p o t e n t i a l begins to decline slowly after a b o u t 10 rain. U p o n readmission of Ca-iris, r e g e n e r a t i v e spikes can again be elicited and h a v e been observed for at least a n o t h e r 40 rain. The Calcium d e p e n d e n c y of b o t h the inward current in t h e clamp c o n d i t i o n and t h e r e g e n e r a t i v e spikes in n o n - c l a m p e d tissue seem to preclude the possibility t h a t the inward current is carried by o t h e r ions. The possibility t h a t the inward current is carried b y tris ions seems unlikely as such a channel would h a v e to be d e p e n d e n t upon e x t e r n a l calcium levels. Two questions r e m a i n unanswered, a) Is there still a considerable inward Ca++ current ill the presence of the n o r m a l constituents of T y r o d e solution ? b) W h y does t h e r a t m y o c a r d i a l action p o t e n t i a l h a v e such a brief p l a t e a u in t h e presence of an inward calcium current ? Our observations on s t e a d y state current suggest t h a t t h e range of a n o m a l o u s rectification is considerably less in rat m y o c a r d i u m t h a n in o t h e r species. The larger

o u t w a r d p o t a s s i u m current m a y account for the brief action p o t e n t i a l even in the presence of inward calcium current. The e x p e r i m e n t s reported here p r o v i d e further support for t h e existence of a channel in the r a t m y o c a r d i a l m e m b r a n e which carries an inward calcium current. I n Ca-Iris solutions an inward current is generated at depolarization steps a b o v e a b o u t --20 inV. I t remains to be seen how i m p o r t a n t this current is, as a c o m p o n e n t of the n o r m a l rat m y o c a r d i a l action potential.

Zusammen[assung: An P a p i l l a r m u s k e l n y o n R a t t e n herzen ist bet Depolarisation mittels S p a n n u n g s k l e m m e ein betr~ichtlicher Einw~irtsstrom messbar, dessen St~irke in Na+-freier L6sung yon der extrazellul~iren Ca~+-Kon z e n t r a t i o n abh~ingt. Die kurze D a u e r des A k t i o n s p o t e n rials kleiner N a g e t i e r e wird d a r a u f zuriickgefiihrt, dass die K + - K o n d u k t i v i t ~ i t bet Depolarisation der Zellmemb r a n nicht so stark absinkt wie bet den meisten andern Herzen. G. W.

MMNWOOD

21 and

J. A. S. McGuIGAN

Physiologisches Institut der Unive~sitgit, Bi~hlplatz 5, CH-3012 Bern (Switzerland), 5 August 1974.

21 Present address of G. W. MAINWOOD, Dept. of Physiology, University of Ottawa, Ottawa, Canada KIN 6N5, to whom reprint requests have to be sent. Supported in part by the iKedieal Research Council of Canada. Our thanks to Professor Weidmann for his help and advice.