of
Heart Purkinje Fibres
immuno- and enzyme histochemical and ultrastructural study
AKADEMISK AVHANDLING
som med vederbörligt tillstånd av rektorsämbetet vid Um eå universitet för avläggande av medicine doktorsexamen
kommer att offen tligen försvaras i Anatom iska institutionens stora föreläsningssal
U m eå universitet torsdagen den 15 april 1982 kl 09.00
av
STURE FORSGREN
leg läk
D IFFER EN TIA TIO N OF H E A R T PU R K IN JE FIBRES An im m uno- and enzyme histochemical and ultrastructural study Sture Forsgren, Institute of A natom y, U niversity of Umeå, Umeå, Sweden
The developm ent of the Purkinje fibres, the cells comprising the periph
eral p art of the cardiac conduction system, has almost exclusively been studied by means of conventional histologic techniques. The descriptions of the identification and mode of differentiation of the cells during devel
opm ent are conflicting. In the present investigation bovine and hum an Purkinje fibres during developm ent were studied histologically and ultra- structurally and by means of enzyme and im m uno-histochem istry.
In both bovine and hum an fetal hearts the Purkinje fibres were distin
guished from the main myocardial mass due to a strong im m unofluor
escence reaction, in sections incubated w ith antibodies against the in ter
mediate filam ent subunit skeletin. Thus, a high content of skeletin, as dem onstrated by im m unohistochem istry, seems to be a general criterion to identify fetal Purkinje fibres. Furtherm ore, the Purkinje fibres were identified by their gross m orphological features from an early fetal stage of the bovine hearts. In the hum an fetal hearts the Purkinje fibres were also identified by a high activity of cholinesterase.
By comparison w ith imm unohistochem ical observations hum an fetal Purkinje fibres at m idgestation could be identified at the ultrastructural level: The Purkinje fibres but n o t the ordinary ventricular myocytes were stained in sections incubated w ith antibodies against MM -creatine kinase.
U ltrastructurally subendocardial cells in the ventricles showed dense M-bands. These cells represent Purkinje fibres, as presence of an electron dense M -band correlates w ith detectability of M M -creatine kinase. In the Purkinje fibres in the bovine fetal hearts, electron dense M-bands were observed and M M -creatine kinase was detected at an earlier stage than in ordinary ventricular myocytes.
D uring fetal developm ent in the bovine hearts differences appeared be
tween Purkinje fibres and ordinary ventricular myocytes w ith respect to cell volumes, intercalated disks, myofibrils, m itochondria, am ount of glyc
ogen and T-tubules. In all fetal stages of bovine hearts and in the hum an fetal hearts at midgestation the Purkinje fibres contained a greater am ount of interm ediate filaments.
The histochemical p attern of Purkinje fibres vs ordinary ventricular myocytes, w ith respect to enzymes reflecting oxidative and glycolytic activities, changed during fetal developm ent of the bovine hearts. This could be related to the changing enzyme activities in the ordinary m yo
cytes. The staining reactions of these enzymes in the Purkinje fibres seemed to rem ain unchanged during fetal development. N oticeably the Purkinje fibres showed higher enzyme activities than the ordinary m yo
cytes at early fetal stages, indicating a higher energy demand in the form er cells.
The observations show th a t Purkinje fibres clearly can be identified in fetal hearts and th a t the use of enzyme and im m uno-histochem istry has facilitated this identification. The Purkinje fibres differentiate along a different pathw ay from ordinary ventricular myocytes. Furtherm ore, it appears as if the Purkinje fibres have a specialized function already at early fetal stages.
Key words: H eart conduction system, Purkinje fibres, ordinary myocytes, differentiation, skeletin, M-band, enzyme histochem istry, im m unohisto
chemistry, electron microscopy.
Ne w Series N o 83 - ISSN 0346-6612
From the Institute of Anatomy, University of Umeå, Umeå, Sweden
Differentiation of
Heart Purkinje Fibres
An immuno- and enzyme histochemical and ultrastructural study
by
STURE FORSGREN
Umeå 1982
CONTENTS
O r i g i n a l p a p e r s ... 4
I n t r o d u c t i o n ... 5
A s p e c t s on t h e s t r u c t u r e and f u n c t i o n o f a d u l t P u r k i n j e f i b r e s ... 5
The d e v e l o p m e n t o f t h e P u r k i n j e f i b r e s ...6
The P u r k i n j e f i b r e s - h i g h l y d i f f e r e n t i a t e d c e l l s o r e m b r y o n i c r e m n a n t s ? ... . . . 7
Aims o f t h e p r e s e n t i n v e s t i g a t i o n ... 9
M a t e r i a l s a nd Me t h o d s ... 10
Main o b s e r v a t i o n s ...12
C o w ... 12
M a n ...13
D i s c u s s i o n ...15
The P u r k i n j e f i b r e s i n t h e d e v e l o p i n g h e a r t ... 15
I d e n t i f i c a t i o n ... 15
T e r m i n o l o g y ...16
F u n c t i o n a l a s p e c t s ... 16
The d i f f e r e n t i a t i o n p a t t e r n o f t h e P u r k i n j e f i b r e s . . . 17
C o m p a r i s o n w i t h o r d i n a r y v e n t r i c u l a r m y o c y t e s . . . . 17
C o m p a r i s o n w i t h a t r i a l m y o c y t e s ... 18
Comment on t h e p r o p o s e d " e m b r y o n i c o r g a n i z a t i o n " . . . 19
G e n e r a l summary and c o n c l u s i o n s ... 20
Ac k n o wl e d g e me n t s ... 22
L i t e r a t u r e c i t e d ... 23
A p p e n d i x ... 29
P a p e r I ... 29
P a p e r I I ...41
P a p e r I I I ...51
P a p e r IV . ’ ... 67
P a p e r V ... 81
ORIGINAL PAPERS
T h i s t h e s i s i s based on t h e f o l l o w i n g p u b l i c a t i o n s and manu
s c r i p t s , which w i l l be r e f e r r e d t o by t h e i r Roman numer al s:
I . F o r s g r e n , S . , T h o r n e l l , L-E. and E r i k s s o n , A.
The development o f t h e P u r k i n j e f i b r e sys t em i n t h e bo
v i n e f e t a l h e a r t .
Anatomy and Embryology 1 59, 125-135 (1980)
I I . F o r s g r e n , S. and T h o r n e l l , L-E.
The devel opment o f P u r k i n j e f i b r e s and o r d i n a r y myo
c y t e s in t h e b ovi ne f e t a l h e a r t . An u l t r a s t r u c t u r a l s t u d y .
Anatomy and Embryology 1 62, 127-136 (1981)
I I I . F o r s g r e n , S . , S t r e h l e r , E. and T h o r n e l l , L-E.
D i f f e r e n t i a t i o n o f P u r k i n j e f i b r e s and o r d i n a r y ven
t r i c u l a r and a t r i a l myocyt es i n t h e bovi ne h e a r t - An immuno- and enzyme h i s t o c h e m i c a l s t u d y .
Hi s t o c h e mi c a l J o u r n a l ( i n p r e s s )
IV. F o r s g r e n , S . , E r i k s s o n , A. , K j ö r e l l , U. and T h o r n e l l , L-E.
The c o n d u c t i o n syst em in t h e human h e a r t a t mi' dgesta- t i o n - Immunohi stochemical d e m o n s t r a t i o n o f s k e l e t i n . S u b mi t t e d f o r p u b l i c a t i o n
V. F o r s g r e n , S . , C a r l s s o n , E . , S t r e h l e r , E. and T h o r n e l l , L-E.
U l t r a s t r u c t u r a l i d e n t i f i c a t i o n o f human f e t a l P u r k i n j e f i b r e s - A c o mp a r a t i v e immunocytochemical and e l e c t r o n m i c r o s c o p i c s t udy o f c o mp o s i t i o n and s t r u c t u r e of m y o f i b r i l l a r M- r e g i o n s .
J o u r n a l of Mo l e c u l a r and C e l l u l a r Ca r d i o l o g y ( i n p r e s s )
INTRODUCTION
A s pe c t s on t h e s t r u c t u r e and f u n c t i o n o f a d u l t P u r k i n j e f i b r e s
In t h e h e a r t t h e r e i s a s ys t em, t h e c o n d u c t i o n s ys t em, which g e n e r a t e s t he s t i m u l u s f o r t h e h e a r t b e a t and c o n d u c t s t h e i mpul s e t o t h e d i f f e r e n t p a r t s o f t h e myocardium. Thereby t h e p r o p e r s u c c e s s i o n o f c o n t r a c t i o n s o f t h e a t r i a and v e n t r i c l e s i s e n s u r e d . The system c o n s i s t s o f t h e s i n o a t r i a l node (SA n o d e ) , t h e a t r i o v e n t r i c u l a r node (AY n o d e ) , t h e a t r i o v e n t r i c u l a r bundl e (AV b u n d l e , bundl e o f H i s ) , and i t s b r a n c h e s ( t h e r i g h t and l e f t bundl e b r a n c h e s ) . The p e r i p h e r a l p a r t o f t h e bundl e b r a n c h e s i s c a l l e d t h e P u r k i n j e f i b r e sys t em. The c e l l s c o mp r i s i n g t h i s s ys t e m, t h e P u r k i n j e f i b r e s , were f i r s t d i s c o v e r e d in t h e sheep h e a r t in 1845 by J E P u r k i n j e a f t e r whom t h e c e l l s were t er med.
H i s t o l o g i c a l l y t h e P u r k i n j e f i b r e s show wide i n t e r - and i n t r a - s p e c i e s v a r i a t i o n s . However, i n l a r g e mammals, e . g . u n g u l a t e s , t h e c e l l s a r e mor p h o l o g i c a l l y d i s t i n c t i v e : They have a b o u t 3 - 4 t i me s l a r g e r d i a m e t e r s t han o r d i n a r y v e n t r i c u l a r myocyt es, a v a c u o l a t e d a p p e a r a n c e , s p a r s e m y o f i b r i l s , and m u l t i p l e n u c l e i ( 1 , 17, 50, 7 5 ) . These c e l l s a r e o f t e n r e f e r r e d t o as " t y p i c a l "
P u r k i n j e f i b r e s . I n d i v i d u a l c e l l s o r bundl es o f c e l l s a r e s ur r o u n d e d by a c o n n e c t i v e t i s s u e s h e a t h which d e l i m i t s them from t h e o r d i n a r y myocyt es. In man and i n small mammals, e . g . r a t and c a t , t h e c e l l s i n t h e P u r k i n j e f i b r e s ys t em a r e s m a l l e r and do n o t al ways have a c o n t i n u o u s c o n n e c t i v e t i s s u e s h e a t h . In t h e s e s p e c i e s i t i s in f a c t d i f f i c u l t t o d i s t i n g u i s h t h e s e c e l l s from t h e s u r r o u n d i n g o r d i n a r y myocyt es by means o f c o n v e n t i o n a l l i g h t mi cr o
scopy ( 1 7 , 50, 7 6 ) . The s p e c i e s v a r i a t i o n s , wi t h r e s p e c t t o t h e morphology o f t h e P u r k i n j e f i b r e s , have l e a d t o c o n f u s i o n i n t h e l i t e r a t u r e r e g a r d i n g t h e t e r mi n o l o g y o f t h e c e l l s . Some a u t h o r s have r e s t r i c t e d t h e t er m P u r k i n j e f i b r e t o " t y p i c a l " P u r k i n j e f i b r e s , w h i l e o t h e r s have i n c l u d e d t h e c e l l s in t h e p e r i p h e r a l r a m i f i c a t i o n o f t h e bundl e b r a n c h e s i n a l l h i g h e r v e r t e b r a t e s . Some a u t h o r s have p r e f e r r e d t o c l a s s i f y t h e P u r k i n j e f i b r e s i n t o t h r e e main t y p e s , ma i n l y based on t h e d i a m e t e r s o f t h e c e l l s and t h e e x t e n t t o which t h e c e l l s d i f f e r from t h e o r d i n a r y myocytes ( 50, 7 6 ) . Accor di ng t o Vi r ägh and C h a l l i c e (83) t h e morphology o f t h e P u r k i n j e f i b r e s changes a t s p e c i a l r e g i o n s al ong t h e d i s t r i b u t i o n o f t h e bundl e b r a n c h e s and t h e s e a u t h o r s have c l a s s i f i e d t h e c e l l s i n t o P u r k i n j e I , I I , and I I I c e l l s , r e s p e c t i v e l y .
By use o f h i s t o l o g i c a l s t a i n s f o r g l y c o g e n , and enzyme h i s t o c h e m i c a l and im- munohi s t ochemi cal t e c h n i q u e s , t h e P u r k i n j e f i b r e s can be d i s t i n g u i s h e d from t h e o r d i n a r y myocyt es. However a l s o wi t h t h e s e t e c h n i q u e s some i n t e r s p e c i e s v a r i a t i o n s a r e o b s e r v a b l e . The P u r k i n j e f i b r e s a r e d i s t i n g u i s h e d from t h e o r d i n a r y myocyt es by a h i g h e r amount of gl y c o g e n , a h i g h e r a c t i v i t y o f g l y c o l y t i c and g l u c o n e o g e n e t i c enzymes and a l ower a c t i v i t y o f r e s p i r a t o r y en
zymes ( 46, 6 0 ) . These o b s e r v a t i o n s , as well as t h e l ower up t a k e o f oxygen and t h e l ower d e n s i t y o f c a p i l l a r i e s in t h e P u r k i n j e f i b r e syst em t han in t h e o r d i n a r y myocardium ( 18, 53) f a v o u r t h a t t h e P u r k i n j e f i b r e s have a h i g h e r r a t e o f a n a e r o b i c and a l ower r a t e o f a e r o b i c met abol i s m t han t h e o r d i n a r y myocyt es. F u r t h e r mo r e , t h e P u r k i n j e f i b r e syst em i s d i s t i n g u i s h e d from o r d i n a r y myocardium by a hi gh a c t i v i t y o f C h o l i n e s t e r a s e ( 20, 3 7 ) . I t i s , however, n ot c e r t a i n t o what e x t e n t t h i s r e f l e c t s t he r i c h ner vous com
po n e n t of t h e c o n d u c t i o n syst em or a high a c t i v i t y of t h e enzyme w i t h i n t h e P u r k i n j e f i b r e s ( 17, 7 8 ) . R e c e n t l y , t h e P u r k i n j e f i b r e s have a l s o been shown t o d i f f e r from o r d i n a r y myocyt es by a h i g h e r c o n t e n t of t h e i n t e r m e d i a t e f i l a me nt p r o t e i n s k e l e t i n , as seen wi t h i mmunof luor es cence t e c h n i q u e s ( 2 3 ) .
By e l e c t r o n mi cr os copy c l e a r d i f f e r e n c e s have been o bs e r ve d between P u r k i n j e f i b r e s and o r d i n a r y myocyt es: The P u r k i n j e f i b r e s have both e n d - t o - e n d and s i d e - t o - s i d e c o n n e c t i o n s ( t h e o r d i n a r y myocyt es a r e p r i n c i p a l l y l i n k e d end- t o - e n d ) i n c l u d i n g more f r e q u e n t n e x u s e s , abs ence of T - t u b u l e s , fewer and s m a l l e r m i t o c h o n d r i a , p r e s e n c e o f m i t o c h o n d r i a wi t h a doughnut appe a r a nc e and a l a r g e r amount o f gl ycogen g r a n u l e s as compared wi t h t h e o r d i n a r y myo
c y t e s ( 6 , 9 , 38, 4 3 , 53, 61, 6 9 ) . In p a r t i c u l a r , t h e d i f f e r e n c e wit h r e s p e c t t o T - t u b u l e s has been s t r e s s e d as a c l e a r d i s t i n g u i s h i n g f e a t u r e (61, 6 3 ) . F u r t h e r m o r e , i n t e r m e d i a t e f i l a m e n t s a r e more abundant and t h e m y o f i b r i l s ar e l e s s wel l o r g a n i z e d in t h e P u r k i n j e f i b r e s ( 6 8 , 7 1 ) . A b n o r m a l i t i e s in t he s a r c o me r e s and my o f i l a me n t - p o l y r i b o s o m e compl exes a r e t y p i c a l f o r t h e P u r k i n j e f i b r e s ( 51, 67, 6 8 ) . The d i f f e r e n c e s wi t h r e s p e c t t o m i t o c h o n d r i a and gl yc oge n may r e f l e c t t h e pr opos ed d i f f e r e n c e in c e l l met abol i s m ( s e e a bove ) . The d i s s i m i l a r i t y i n amount o f i n t e r m e d i a t e f i l a m e n t s and c o n t e n t o f s k e l e - t i n , as seen i mmu n o h i s t o c h e mi c a l l y , s u g g e s t s t h a t P u r k i n j e f i b r e s may need a d i f f e r e n t t y p e o f c y t o s k e l e t o n from o r d i n a r y myocyt es. The a b n o r m a l i t i e s in t h e s a r c o me r e s o f P u r k i n j e f i b r e s a r e pr o b a b l y due t o an imbal ance in t he s y n t h e s i s and d e g r a d a t i o n o f m y o f i b r i l l a r p r o t e i n s (6 8 ) .
E l e c t r o p h y s i o l o g i c a l l y , i t has been shown t h a t c o n d u c t i o n i s r a p i d t h r o u g h o u t t h e bundle b r a n c h e s and t h e p e r i p h e r a l P u r k i n j e f i b r e s a t a r a t e o f 1 . 5 - 5 . 0 m/ sec ( 17, 3 2 ) . In c o n t r a s t , t h e v e l o c i t y in o r d i n a r y v e n t r i c u l a r myo
car di um i s 0 . 3 - 1 . 0 m/ s e c . Numerous a t t e m p t s have been made t o e x p l a i n t h e s e p h y s i o l o g i c a l d i f f e r e n c e s . The l a r g e d i a m e t e r s o f t h e P u r k i n j e f i b r e s and t h e i r a g g r e g a t i o n i n t o t i g h t l y packed b u n d l e s f a v o u r a g r e a t e r c o n d u c t i o n v e l o c i t y i n t h e s e c e l l s t han i n o r d i n a r y myocyt es ( 6 3 ) . The n e x u s e s , which a r e f r e q u e n t between t h e P u r k i n j e f i b r e s , pr o b a b l y r e p r e s e n t l o w- r e s i s t a n c e pat hways (8 ) . The P u r k i n j e f i b r e s a r e a l s o i n f l u e n c e d by bot h s y mp a t h e t i c and p a r a s y m p a t h e t i c br a n c h e s o f t h e autonomic ner vous sys t em. A d i r e c t c e l l u l a r b a s i s f o r c h o l i n e r g i c ant agoni sm o f t h e e l e c t r o p h y s i o l o g i c a l e f f e c t s o f c a t e c h o l a m i n e s on t h e P u r k i n j e f i b r e s i s documented ( 7 ) . Be s i de s r a p i d c o n d u c t i o n , t h e P u r k i n j e f i b r e s a l s o have t h e p r o p e r t y o f spont a ne ous de
p o l a r i z a t i o n ( 1 8 ) . C o n t r a c t i o n s a r i s i n g whol l y w i t h i n t h e v e n t r i c l e s , as in c o mp l e t e AY b l o c k , o r i g i n a t e i n P u r k i n j e f i b r e s .
I t i s t h u s c l e a r t h a t in t h e a d u l t h e a r t t h e P u r k i n j e f i b r e s d i f f e r h i s t o l o g i c a l l y , ul t r a s t r u c t u r a l l y , h i s t o c h e m i c a l l y , i mmunohi s t oc he mi c a l l y and e l e c t r o p h y s i o l o g i c a l l y from t h e o r d i n a r y myocyt es.
The d evel opment o f t h e P u r k i n j e f i b r e s
The o r i g i n and development of t h e d i f f e r e n t p a r t s of t h e c o n d u c t i o n system has f o r a l ong ti me been t h e s o u r c e o f c o n t r o v e r s y . However, i t i s c l e a r t h a t a c o n d u c t i o n system can n ot be i d e n t i f i e d m o r p h o l o g i c a l l y a t t h e s t a g e when c a r d i a c c o n t r a c t i o n s s t a r t . C o n t r a c t i o n s o c c u r soon a f t e r t he f o r ma t i o n o f t h e s i n g l e he*art t u b e (57) or even e a r l i e r , b e f o r e t h e two e n d o c a r d i a l t u b e s have begun t o f u s e ( 2 7 ) . The pacemaker r e g i o n i n t h e t u b u l a r h e a r t has been pr opos ed t o s h i f t d u r i n g devel opment , i n i t i a l l y be i n g l o c a t e d in t he v e n t r i c l e ( 5 5 ) . Ot h e r s have ar gued t h a t t h e i mpul s e from t h e b e g i n n i n g i s g e n e r a t e d in t h e p r o s p e c t i v e s i n o a t r i a l r e g i o n (45, 8 0 ) .
With r e s p e c t t o t h e o r i g i n of t h e c o n d u c t i o n system components one t h e o r y has been t h a t a new s u p r a v e n t r i c u l a r growth a r e a in t h e embryonic h e a r t de
v e l o p s , from which t h e AY b undl e and t h e bundl e b r a n c h e s devel op ( 59, 8 6 ) . Ac c or di ng t o t h i s t h e o r y t h e end o f t h e AV bundl e i s an a c t i v e l y growing t i s s u e . Ot he r a u t h o r s have con t e n d e d t h a t t h e AV bundl e (42) and t h e P u r k i n j e f i b r e s ( 3 , 34, 84) a r i s e i n s i t u in t h e AV canal and in t h e v e n t r i -
c l e s , r e s p e c t i v e l y . Wenink, Ande-rson and c o l l e a g u e s ( 4, 87) have propos ed t h a t t h e p h y l o g e n e t i c and o n t o g e n e t i c o r i g i n of t h e d i f f e r e n t p a r t s of the co n d u c t i o n syst em i s a s e r i e s o f myocar di al r i n g s between segments of t he t u b u l a r h e a r t . Accor di ng t o t h e s e t h e o r i e s t h e P u r k i n j e f i b r e s de ve l op from t h e b u l b o v e n t r i c u l a r r i n g , formed between t h e bul bus and t h e p r i m i t i v e ven
t r i c l e . In a r e c e n t s t udy t h e s e i d e a s c o u l d , however, not be conf i r me d ( 4 2 ) . The mo r phol ogi c a l d i f f e r e n c e s between c o n d u c t i v e t i s s u e e l e me n t s and f u n c t i o n i n g myocardium i s so s l i g h t in embryonic h e a r t s , t h a t d i s t i n c t i o n be
tween t h e two t y p e s i s e x t r e me l y d i f f i c u l t a t t h i s s t a g e ( 8 1 ) . Mai nly f o r t h a t r e a s on t h e r e a r e d i f f e r e n t o p i n i o n s a b o u t t h e s t a g e o f development a t which t h e d i f f e r e n t p a r t s o f t h e c o n d u c t i o n syst em can be i d e n t i f i e d as d i s c r e t e s t r u c t u r e s . Most a u t h o r s , however, a g r e e t h a t t h e P u r k i n j e f i b r e s ap
p e a r a t a l a t e r s t a g e t h a n t h e more proximal p a r t s of t h e c o n d u c t i o n sys t em.
The devel opment o f t h e " t y p i c a l " P u r k i n j e f i b r e s , as well as t h a t o f t he P u r k i n j e f i b r e s o f man, has been s t u d i e d wi t h c o n v e n t i o n a l h i s t o l o g i c t e c h n i q u e s b u t no t by means o f h i s t o c h e m i s t r y o r e l e c t r o n mi c r os c opy. The c r i t e r i o n f o r i d e n t i f i c a t i o n of t h e P u r k i n j e f i b r e s wi t h h i s t o l o g i c t e c h n i q u e s has been t h e a p p e a r a n c e o f c e l l s whi ch, as compared wi t h t h e o r d i n a r y myo
c y t e s , a r e more r ounded and l a r g e r and have m y o f i b r i l s onl y in t h e p e r i p h e r y . Using such c r i t e r i a t h e P u r k i n j e f i b r e s in human h e a r t s can n ot be d i s t i n g u i s h e d from o r d i n a r y myocytes a t ni ne weeks of g e s t a t i o n (40) and ar e f i r s t d i s t i n g u i s h e d a t a bout 14 weeks (8 6 ) . At l a t e r s t a g e s of f e t a l d e v e l opment t h e y become more c l e a r l y d i s t i n g u i s h a b l e ( 56, 8 6 ) . Anderson and T a y l o r ( 3 ) , however, d e s c r i b e two c e l l p o p u l a t i o n s - one s u b e n d o c a r d i a l and one o u t e r l a y e r o f c e l l s - which can a l r e a d y be r e c o g n i z e d in t he v e n t r i c l e s a t e a r l y embryonic s t a g e s in human h e a r t s . These a u t h o r s pr opos e t h a t t he s u b e n d o c a r d i a l c e l l p o p u l a t i o n forms t h e P u r k i n j e f i b r e s and t h e o u t e r l a y e r t h e o r d i n a r y v e n t r i c u l a r myocardium. The r e a s o n f o r t h e d i v e r g e n t d e s c r i p t i o n s i s pr o b a b l y r e l a t e d t o t h e b a s i s f o r i d e n t i f i c a t i o n of t he P u r k i n j e f i b r e s : Al t hough Anderson and T a y l o r a l s o d e s c r i b e d a d i f f e r e n c e in c e l l d i a me t e r i t i s mer el y t h e o b s e r v a t i o n o f an e x t e n s i v e l a y e r s u b e n d o c a r d i a l l y i n t h e v e n t r i c l e s which i s t h e i r b a s i s f o r i d e n t i f i c a t i o n . The devel opment o f t h e " t y p i c a l " P u r k i n j e f i b r e s has been s t u d i e d in t h e sheep: The P u r k i n j e f i b r e s a r e d e s c r i b e d as d i s t i n g u i s h a b l e from o r d i n a r y myocardium a t 70 mm (49) r e s p e c t i v e l y 100 mm (24) CR- l engt h wi t h c o n v e n t i o n a l h i s t o l o g i c t e c h n i q u e s .
For c l a r i t y i t must be p o i n t e d o u t t h a t n o t a l l a u t h o r s s t u d y i n g d e v e l o p i n g h e a r t s ter m t h e c o n d u c t i o n c e l l s i n t h e v e n t r i c l e s P u r k i n j e f i b r e s . I t must be remembered t h a t t h e c r i t e r i a f o r d i f f e r e n t i a t i n g P u r k i n j e f i b r e s from o r d i n a r y myocytes ( s e e above and D i s c u s s i o n ) a r e v a l i d f o r a d u l t h e a r t s . The P u r k i n j e f i b r e s - h i g h l y d i f f e r e n t i a t e d c e l l s o r embryonic r emnant s?
The degr e e o f d i f f e r e n t i a t i o n o f t h e P u r k i n j e f i b r e s i s a c o n t r o v e r s i a l t o p i c : The c e l l s a r e d e s c r i b e d t o be " embr yoni c" , or to be h i g h l y " d i f f e r e n t i a t e d ^ . I t i s a l s o d e b a t e d w h e t h e r t h e P u r k i n j e f i b r e s and o r d i n a r y myo
c y t e s d i f f e r e n t i a t e al ong t h e same r o u t e b u t a t d i f f e r e n t r a t e s or d i f f e r e n t i a t e al o n g d i f f e r e n t r o u t e s ( 1 8 ) . F u r t h e r t h e d e s c r i p t i o n o f t h e t h r e e main t y p e s o f P u r k i n j e f i b r e s , as i d e n t i f i e d h i s t o l o g i c a l l y ( s e e a b o v e ) , in t er ms o f d i f f e r e n t i a t i o n i s c o n f u s i n g : The c e l l t y p e s a r e r e f e r r e d to as f u l l y d i f f e r e n t i a t e d , l e s s d i f f e r e n t i a t e d or l e a s t d i f f e r e n t i a t e d ( 50, 7 6 ) .
In o l d l i t e r a t u r e t h e P u r k i n j e f i b r e s ar e s a i d t o be c e l l s which remain un
d e ve l ope d and which r es embl e embryonic c a r d i a c muscle c e l l s ( e . g . 3 3 , 4 8 ) .
F i e l d (24) con t e n d e d t h a t t h e P u r k i n j e f i b r e s were h e l d up in t h e i r d e v e l o p ment . Also in more r e c e n t s t u d i e s t h e P u r k i n j e f i b r e s have been d e s c r i b e d as
"embryoni c" and " r e t a r d e d " in devel opment . What a r e t h e b a s e s f o r t h e s e sug
g e s t i o n s ? I t i s a f a c t t h a t many o f t h e b i oc he mi c a l and m e t a b o l i c c h a r a c t e r i s t i c s d i s t i n g u i s h i n g c o n d u c t i o n t i s s u e from o r d i n a r y myocardium in t h e a d u l t h e a r t (amount o f g l y c o g e n , r e s i s t a n c e t o a n o x i a , a c t i v i t i e s o f o x i d a t i v e and g l y c o l y t i c enzymes) a l s o d i f f e r e n t i a t e embryonic from a d u l t myocar
dium ( 1 8 ) . In a d d i t i o n , t h e r e a p p e a r t o be s i m i l a r i t i e s between t h e u l t r a s t r u c t u r e o f t h e a d u l t P u r k i n j e f i b r e s and t h a t o f u n d i f f e r e n t i a t e d embryon
i c myocyt es ( 6 , 4 7 , 5 2 ) . Thi s has l e a d t o t h e c o n c l u s i o n t h a t t h e P u r k i n j e f i b r e s have an " emb ryo n i e o r g a n i za t i o n" . Vi r âgh and C h a l l i c e (82) have f u r t h e r propos ed t h a t t h e m y o f i b r i l l a r devel opment i n t h e P u r k i n j e f i b r e s i s
" i n c o m p l e t e " , as an e x p l a n a t i o n o f t h e f i l a m e n t o u s components in t h e c e l l s . The " d e f e c t s " in m y o f i b r i l l o g e n e s i s in t h e P u r k i n j e f i b r e s ( e x e m p l i f i e d by e . g . t h e my o f i l a me n t - p o l y r i b o s o m e compl exes) mi ght r e p r e s e n t c e s s a t i o n of m y o f i b r i l l o g e n e s i s a t an embryonic s t a g e ( 5 1 ) . I t has a l s o been pr opos ed t h a t t h e c o n d u c t i n g t i s s u e r e p r e s e n t s remnant s o f embryonic t i s s u e on t he b a s i s o f i t s s p e c i a l p h y s i o l o g i c a l p r o p e r t i e s ( 4 1 ) .
A l t e r n a t i v e l y , t h e P u r k i n j e f i b r e s and t h e o r d i n a r y myocyt es have been de
s c r i b e d as r e p r e s e n t i n g d i f f e r e n t pathways o f d i f f e r e n t i a t i o n , by which t h e P u r k i n j e f i b r e s r e t a i n t h e hi gh c o n d u c t i v e p r o p e r t i e s and t h e spont a ne ous c o n t r a c t i l i t y ( 2 9 ) . The a p p e a r a n c e o f t h e m y o f i b r i l l a r m a t e r i a l in t h e P u r k i n j e f i b r e s has been a t t r i b u t e d t o an i mbal ance in s y n t h e s i s and d e g r a d a t i o n o f m y o f i b r i l l a r p r o t e i n s . Th i s imbal ance may r e f l e c t a p r o c e s s of d i f f e r e n t i a t i o n in which t h e devel opment o f " n e r v e - l i k e " p r o p e r t i e s has t aken p r e c e d e n c e over t h e devel opment o f c o n t r a c t i l i t y ( 7 0 ) . F u r t h e r m o r e , t h e f a c t t h a t t h e P u r k i n j e f i b r e s change by age, e . g . i n c r e a s e mar kedl y in s i z e , would f a v o u r t h a t t h e c e l l s a r e no t embryonic r emnant s ( 1 6 ) . Robb e t al (56) even propos ed t h a t t h e c o n d u c t i o n c e l l s undergo a more e x t e n s i v e change t han do t h e o r d i n a r y myocyt es , as t h e c o n d u c t i o n c e l l s were h i s t o l o g i c a l l y more d i f f e r e n t from o r d i n a r y myocyt es in human h e a r t s o f l a t e f e t a l s t a g e s t han o f e a r l y f e t a l s t a g e s . Bogusch (11) c onc l ude d i n an u l t r a s t r u c t u r a l s t udy on c h i c k e n P u r k i n j e f i b r e s d u r i n g devel opment t h a t t h e c e l l s go t hr o u g h a d i s t i n c t d i f f e r e n t i a t i o n p r o c e s s . He e . g . o b s e r v e d t h a t t y p i c a l a d u l t f e a t u r e s such as l e p t o m e r i c f i b r i l s and l e p t o m e r i c complexes d i d n o t ap p e a r u n t i l a f t e r h a t c h i n g and t h a t t h e amount o f gl ycogen in t h e P u r k i n j e f i b r e s de
c r e a s e d d u r i n g devel opment . However, u l t r a s t r u c t u r a l i n f o r m a t i o n on d e v e l o p i n g P u r k i n j e f i b r e s in t h e mammalian h e a r t i s needed, as t h e r e a r e d i f f e r e n c e s between a d u l t c h i c k e n and mammalian P u r k i n j e f i b r e s , e . g . wi t h r e s p e c t t o amount o f gl y c o g e n .
AIMS OF THE PRESENT INVESTIGATION
The p r i n c i p a l aims were t o answer t h e f o l l o w i n g q u e s t i o n s :
Can enzyme and i mmun o - h i s t o c h e mi s t r y be employed in o r d e r t o f a c i l i t a t e t h e i d e n t i f i c a t i o n o f f e t a l P u r k i n j e f i b r e s a t t h e l i g h t m i c r o s c o p i c l e v e l ? I s i t p o s s i b l e t o u l t r a s t r u c t u r a l l y d e f i n e t h e human f e t a l P u r k i n j e f i b r e s ?
What a r e t h e mor p h o l o g i c a l c h a r a c t e r i s t i c s o f f e t a l Pur k i n j e f i b r e s compared wi t h a d u l t ones?
How do t h e P u r k i n j e f i b r e s d i f f e r e n t i a t e as compared wit h t h e o r d i n a r y v e n t r i c u l a r and a t r i a l myocyt es? Do P u r k i n j e f i b r e s show an "embryoni c o r g a n i z a t i o n " ?
MATERIALS AND METHODS
The o r i g i n a l p a p e r s a r e r e f e r r e d t o by t h e i r Roman n ume r a l s . In t he o r i g i n a l p a p e r s f u r t h e r d e s c r i p t i o n s can be found.
MATERIALS
F e t a l b ovi ne h e a r t s ( I - I I I ) : T we n t y - e i g h t f e t u s e s were o b t a i n e d d u r i ng r o u t i n e s l a u g h t e r o f cows. The cr own- r ump( CR) - l e ngt hs o f t h e f e t u s e s v a r i e d be
tween 5 cm ( g e s t a t i o n a l age a b o u t 50 days) and 100 cm ( f u l l t e r m, 260-270 d a y s ) .
The h e a r t s o f t h e f e t u s e s were d i s s e c t e d o u t i mmedi at el y a f t e r or w i t h i n 1-2 ti o f s a c r i f i c e . From t h e for mer h e a r t s , speci mens were f i x e d in a s l i g h t l y s t r e t c h e d s t a t e i n 2 . 5 % g l u t a r a l d e h y d e in T y r o d e ' s b u f f e r and f u r t h e r p r o c e s s e d f o r l i g h t mi cr oscopy ( I ) and t r a n s m i s s i o n e l e c t r o n mi cr oscopy ( I I ) . These h e a r t s were from f e t u s e s wi t h CR- l e ngt hs 7-100 cm. The speci mens from h e a r t s d i s s e c t e d o u t wi t h some de l a y a f t e r s a c r i f i c e were p r o c e s s e d f o r en
zyme h i s t o c h e m i s t r y and i mmunof l uor es cence mi cr oscopy ( I , I I I ) . These h e a r t s were from f e t u s e s wi t h CR- l e ngt hs 16-100 cm ( I ) and 5-72 cm ( I I I ) . The s m a l l e s t h e a r t s p r o c e s s e d f o r c r y o - s e c t i o n i n g ( CR- l e ngt hs 5 and 8 . 5 cm) were f r o z e n as whole p i e c e s t o al l o w s e c t i o n i n g i n t h e f r o n t a l p l a n e o f t he h e a r t s ( I I I ) .
A d u l t bovi ne h e a r t s ( I I I ) : The h e a r t s o f t h r e e a d u l t cows were o b t a i n e d d u r i n g r o u t i n e s l a u g h t e r o f cows and were d i s s e c t e d w i t h i n 1-2 h o f s l a u g h t e r . The speci mens were p r o c e s s e d f o r enzyme h i s t o c h e m i s t r y and immunohist ochemi- s t r y . Some speci mens were f r o z e n t o g e t h e r wi t h f e t a l specimens from c o r r e s po n d i n g r e g i o n s .
F e t a l human h e a r t s (I V, V ) : The h e a r t s o f t e n human f e t u s e s ( g e s t a t i o n a l age 17-21 w e e k s ) , o b t a i n e d a t l e g a l i n t e r r u p t i o n s o f pr egnancy per f or med by a p r o s t a g l a n d i n i n s t i l l a t i o n p r o c e d u r e ( f i v e h e a r t s ) or by hys t er ot omy ( f i v e h e a r t s ) , were i n v e s t i g a t e d . The for mer h e a r t s were p r o c e s s e d f o r enzyme and i m m u n o - h i s t o c h e m i s t r y . These h e a r t s were c u t i n t o 3 - 4 b l o c k s t o a l l ow s e c t i o n i n g in t h e t r a n s v e r s e and f r o n t a l p l a n e s o f d i f f e r e n t r e g i o n s of t he h e a r t s . One h e a r t was n o t d i v i d e d t o a l l ow s e r i a l s e c t i o n i n g in t he f r o n t a l p l a n e o f t h e e n t i r e h e a r t .
The h e a r t s from t h e f e t u s e s o b t a i n e d by hys t er ot omy were i mmedi at el y removed from t h e f e t u s e s and w i t h i n a few mi n u t e s p e r f u s i o n o f t h e h e a r t s wi t h an o x y ge na t e d R i n g e r ' s s o l u t i o n ad modum L a n g e n d o r f f was s t a r t e d a t room tem
p e r a t u r e . These h e a r t s were p r o c e s s e d f o r t r a n s m i s s i o n e l e c t r o n mi c r os c opy.
A d u l t human h e a r t s ( V ) : H e a r t s from two human a u t o p s i e s were o b t a i n e d . The s peci mens were p r o c e s s e d f o r enzyme and i m mu n o - h i s t o c h e mi s t r y .
T i s s u e s s t u d i e d ( I - V ) : The t i s s u e s s t u d i e d in t h e bovi ne h e a r t s were t he P u r k i n j e f i b r e sys t em ( I , I I , I I I ) , t h e o r d i n a r y v e n t r i c u l a r myocardium ( I , I I , I I I ) and t h e o r d i n a r y a t r i a l myocardium ( I I I ) . In t h e human h e a r t s t he SA- and AV-nodes, t h e AV-bundle and bundl e b r a nc he s ( I V) , t h e P u r k i n j e f i b r e sys t em (IV, V), t h e o r d i n a r y v e n t r i c u l a r myocardium (IV, V) and t he o r d i n a r y a t r i a l myocardium (IV, V) were s t u d i e d . D e t a i l e d d e s c r i p t i o n s o f how t he speci mens were c u t can be found in t h e o r i g i n a l p a p e r s .
ENZYME AND IMMUNO-HISTOCHEMISTRY
G e n e r a l : The speci mens were f r o z e n i n f r e on o r propane c h i l l e d wi t h l i q u i d n i t r o g e n . S e r i a l s e c t i o n s were c u t in a c r y o s t a t a t -20°C and s t a i n e d f o r t h e d e m o n s t r a t i o n o f enzyme a c t i v i t i e s , t h e i n t e r m e d i a t e f i l a m e n t s u b u n i t s k e l e t i n o r t h e M- l i n e p r o t e i n s myomesin or MM-creatine k i n a s e ( s e e D i s c u s s i o n ) . Ot h e r s e c t i o n s were t r e a t e d wi t h c o n t r o l s e r a o r s t a i n e d wi t h r o u t i n e h i s t o l o g i c a l s t a i n s . From s e v e r a l o f t he speci mens a l a r g e s e r i e s o f s e c t i o n s was c u t . By t h e s e r i a l s e c t i o n i n g p r o c e d u r e i t was p o s s i b l e t o pr o p e r l y i d e n t i f y t h e d i f f e r e n t r e g i o n s o f t h e c o n d u c t i o n system (I V) .
Enzyme h i s t o c h e m i s t r y : S e c t i o n s were s t a i n e d t o d e mo n s t r a t e t h e a c t i v i t y of m y o f i b r i l l a r ATPase ( I , IV), NADH-t etrazolium r e d u c t a s e (NADH-TR) ( I , IV), s u c c i n i c dehydr ogenas e (SDH) ( I , I I I , IV), m e n a d i o n e - l i n k e d a - g l y c e r o p h o s - p h a t e dehydr ogenas e ( I , I I I , IV) or p h o s p h o r y l a s e ( I ) . The h i s t o c h e m i c a l methods were a c c o r d i n g t o Dubowitz and Brooke ( 1 9 ) . S e c t i o n s were a l s o s t a i n e d t o d e mo n s t r a t e t h e a c t i v i t y of C h o l i n e s t e r a s e (IV, V): The p r o c e du r e s were mai nl y a c c o r d i n g t o Anderson and T a y l o r ( 3 ) , u s i n g Gomor i ' s medi
um ( 2 6 ) . Ot he r s e c t i o n s were s t a i n e d wi t h PAS ( I ) or van Gi eson ( I , IV). The s e c t i o n s were viewed in a L e i t z Di a l u x 20 P h o t omi c r os c ope .
Immunohi st ochemi s t r y : R a b b i t a n t i s e r a a g a i n s t bovi ne P u r k i n j e f i b r e s k e l e t i n ( s e e 2 2 , 23) and a g a i n s t t h e ch i c k e n b r e a s t muscl e Mr 165, 000 M- p r o t e i n myomesin ( s e e 21, 64, 65) were p r e p a r e d . Sheep a n t i serum a g a i n s t human MM- c r e a t i n e k i n a s e was p u r c ha s e d from B o e h r i n g e r (FRG). Sera from normal r a b b i t s and sheep s e r v e d as c o n t r o l s . S e c t i o n s were i n c u b a t e d wi t h a n t i s k e l e t i n a n t i b o d i e s ( I , I I I , IV, V) o r wi t h a n t i b o d i e s a g a i n s t MM-creatine k i n a s e or myomesin ( I I I , V). The s e c t i o n s were examined under a L e i t z Or t h o p l a n e Pho
t o m i c r o s c o p e equi pped wi t h e p i f l u o r e s c e n c e o p t i c s .
CONVENTIONAL LIGHT MICROSCOPY AND TRANSMISSION ELECTRON MICROSCOPY
F e t a l b ovi ne h e a r t s ( I I ) : The g l u t a r a l d e h y d e f i x e d speci mens were c u t i n t o small b l o c k s , p o s t f i x e d in 1% OSO4 , r i n s e d i n b u f f e r and d e h y d r a t e d . Em
beddi ng was c a r r i e d o u t in Ves t opal W. Survey semi t h i n and f i n e s e c t i o n s were c u t in a LKB Ul t r ot ome I o r I I I . The s ur vey s e c t i o n s were s t a i n e d wi t h t o i u i d i ne b l u e and t h e f i n e s e c t i o n s wi t h ur anyl a c e t a t e and l e a d c i t r a t e . Some o f t h e f i n e s e c t i o n s were c o l l e c t e d on gol d g r i d s and s t a i n e d wi t h pe
r i o d i c a c i d - t h i o s e m i c a r b a z i d e - s i l v e r p r o t e i n a t e (PA-TSC-SP) ( s e e 6 6 , 6 9 ) . L i g h t mi c r o s c o p i c e x a mi na t i on was c a r r i e d o u t in a L e i t z Di a l u x 20 Photomic
r o s c o p e and e l e c t r o n mi c r o s c o p i c e x a mi n a t i o n i n a P h i l i p s EM 300 o r in a Siemens Elmiskop 1A.
F e t a l human h e a r t s ( V ) : The h e a r t s were i n i t i a l l y f i x e d by a d d i t i o n of 2.5%
g l u t a r a l d e h y d e t o t h e p e r f u s i o n f l u i d . A f t e r t h e p e r f u s i o n , t he h e a r t s were p l a c e d i n g l u t a r a l d e h y d e f o r a f u r t h e r 2 h. The h e a r t s were t hen r i n s e d in T y r o d e ' s b u f f e r and s e l e c t e d a r e a s were c u t o u t . P o s t f i x a t i o n and f u r t h e r p r o c e d u r e s were as d e s c r i b e d f o r t h e bovi ne h e a r t s .
MAIN OBSERVATIONS
The o b s e r v a t i o n s on t h e P u r k i n j e f i b r e s a r e d e s c r i b e d in r e l a t i o n t o t he s p e c i e s s t u d i e d and t h e t e c h n i q u e s used. The t i s s u e s s t u d i e d in p a r a l l e l a r e a l s o commented on. I l l u s t r a t i o n s and f u r t h e r d e s c r i p t i o n s of t he r e s u l t s a r e found in t h e o r i g i n a l p a p e r s .
COW
Semi t h i n p l a s t i c s e c t i o n s ( I ) : Even a t t h e e a r l i e s t s t a g e s t u d i e d (CR- l engt h 7 cm) t h e P u r k i n j e f i b r e s were d i s t i n g u i s h a b l e from o r d i n a r y v e n t r i c u l a r myoc yt e s . The d i s t i n g u i s h i n g f e a t u r e s f o r t h e P u r k i n j e f i b r e s were t h e i r ag
g r e g a t i o n i n t o b u n d l e s , t h e c o n n e c t i v e t i s s u e s h e a t h and t h e c l e a r c yt opl a s m o f t h e c e l l s . However, in t h e l a t e f e t a l s t a g e t h e P u r k i n j e f i b r e s were more e a s i l y d i s t i n g u i s h e d , due t o a d i f f e r e n c e in c e l l d i a m e t e r : They had l a r g e r d i a m e t e r s t h a n t h e o r d i n a r y v e n t r i c u l a r myocyt es a t t h i s s t a g e , wh i l e in e a r l y f e t a l s t a g e s t h e two c e l l t y p e s had a p p r o x i ma t e l y t h e same d i a m e t e r s . F u r t h e r m o r e , i n f e t u s e s wi t h CR- l engt hs 20 cm and more t h e P u r i n j e f i b r e s were d i s t i n g u i s h e d from o r d i n a r y v e n t r i c u l a r myocyt es by a s t r o n g e r PAS-re- a c t i o n , w h i l e i n s m a l l e r f e t u s e s t h e t i s s u e s showed a compar able PAS-reac- t i o n . The P u r k i n j e f i b r e s seemed t o become b i n u c l e a t e more f r e q u e n t l y as age i n c r e a s e d . Occas i onal m i t o t i c f i g u r e s were o bs e r ve d in t h e P u r k i n j e f i b r e s , w h i l e t h e y were more f r e q u e n t in t h e o r d i n a r y v e n t r i c u l a r myocyt es.
I mmunohi s t ochemi s t r y ( I , I I I ) : In a l l t h e f e t a l s t a g e s s t u d i e d t he P u r k i n j e f i b r e s were d i s t i n g u i s h e d from t h e o r d i n a r y v e n t r i c u l a r myocytes by an i n t e n s e f l u o r e s c e n c e in s e c t i o n s i n c u b a t e d wi t h a n t i b o d i e s a g a i n s t t he i n t e r m e d i a t e f i l a m e n t s u b u n i t s k e l e t i n . The s t r o n g f l u o r e s c e n c e in t he P u r k i n j e f i b r e s emanated from t h e c e n t r a l p a r t o f t h e cyt o p l a s m o f the c e l l s . ’ The n u c l e i were devoi d o f f l u o r e s c e n c e and t h e f l u o r e s c e n c e was weak a t c e l l b o r d e r s f a c i n g o t h e r c e l l s in t h e b u n d l e s .
The M- l i n e p r o t e i n MM-creat ine k i n a s e was d e t e c t e d a t an e a r l i e r s t a g e in P u r k i n j e f i b r e s and a t r i a l myocytes than in o r d i n a r y v e n t r i c u l a r myocyt es.
The o t h e r M- l i ne p r o t e i n , myomesin, c o u l d be d e t e c t e d in a l l t h r e e c e l l t y p e s even a t t h e e a r l i e s t s t a g e s t u d i e d (CR- l engt h 5 cm). At t h i s s t a g e , however, d e t e c t a b i l i t y o f myomesin was more obvi ous in t h e P u r k i n j e f i b r e s t h a n i n t h e o r d i n a r y v e n t r i c u l a r myocyt es.
Enzyme h i s t o c h e m i s t r y ( I , I I I ) : The P u r k i n j e f i b r e s showed a h i g h e r a c t i v i t y o f SDH t h a n t h e o r d i n a r y v e n t r i c u l a r myocytes in f e t u s e s wi t h CR- l engt h 32 cm or l e s s . In l a r g e r f e t u s e s t h e a c t i v i t y o f SDH seemed t o be equal in t he two c e l l p o p u l a t i o n s , wh i l e in a d u l t h e a r t s t h e P u r k i n j e f i b r e s showed a l owe r a c t i v i t y . The P u r k i n j e f i b r e s showed a h i g h e r a c t i v i t y o f « - g l y c e r o p h o s p h a t e de hydr ogenas e (a high a c t i v i t y c o r r e l a t e s wi t h g l y c o l y t i c a c t i v i t i e s ) t h a n t h e o r d i n a r y v e n t r i c u l a r myocyt es, wi t h t h e e x c e p t i o n t h a t , in h e a r t s o f s t a g e s 11-32 cm, t h e o r d i n a r y v e n t r i c u l a r myocytes e v e n t u a l l y ex
h i b i t e d an equal a c t i v i t y . The a c t i v i t y o f SDH and a - g l y c e r o p h o s p h a t e de
hydr oge na s e of P u r k i n j e f i b r e s seemed t o remain unchanged from e a r l y t o l a t e f e t a l s t a g e s . The a c t i v i t y o f SDH g r a d u a l l y i n c r e a s e d f o r both o r d i n a r y ven
t r i c u l a r and a t r i a l myocyt es. The a c t i v i t y o f a - g l y c e r o p h o s p h a t e dehydro
genase was h i g h e r in t h e a t r i a t han in t h e v e n t r i c l e s o f t h e s m a l l e s t f e t u s e s ( CR- l engt h 5 and 8 . 5 cm), lower in t h e a t r i a o f l a r g e r f e t u s e s and low i n both t i s s u e s o f a d u l t h e a r t s .
E l e c t r o n mi cr os copy ( I I ) : In t h e e a r l i e s t s t a g e s ( CR- l engt h 7 and 10 cm) t h e a p p e a r a n c e of t h e i n t e r c a l a t e d d i s k s and t h e m y o f i b r i l s was q u i t e s i m i l a r in P u r k i n j e f i b r e s and o r d i n a r y v e n t r i c u l a r myocyt es. With i n c r e a s i n g g e s t a t i o n a l age t h e l a t t e r c e l l s became more packed wi t h m y o f i b r i l s and a t t a i n e d more s t e p - w i s e formed i n t e r c a l a t e d d i s k s . The m y o f i b r i l l a r M-band became more e v i d e n t wi t h age, t h e P u r k i n j e f i b r e s c l e a r l y p r e c e e d i n g t h e o r d i n a r y v e n t r i c u l a r myocytes in t h i s r e s p e c t . L e p t o f i b r i l s were obs e r ve d in bot h c e l l t y p e s onl y in o l d f e t u s e s . I n t e r m e d i a t e f i l a m e n s were more a bundant in t h e P u r k i n j e f i b r e s . My o f i l a me nt - pol yr i bos ome compl exes t y p i c a l of a d u l t cow P u r k i n j e f i b r e s were no t ob s e r v e d . The m i t o c h o n d r i a were few and s c a t t e r e d i n t h e cyt o p l a s m in bot h c e l l t y p e s in e a r l y s t a g e s . In t h e o l d e s t f e t u s e s t h e m i t o c h o n d r i a formed l o n g i t u d i n a l rows between t h e m y o f i b r i l s and p e r i n u c l e a r masses ar ound t h e n u c l e i in t h e o r d i n a r y v e n t r i c u l a r myocyt es, wh i l e t h e y were s t i l l s c a t t e r e d in t h e c yt opl a s m in t h e P u r k i n j e f i b r e s . In t h e e a r l i e s t s t a g e s bot h c e l l t y p e s had abundant gl ycogen g r a n u l e s , w h i l e in l a t e r s t a g e s t h e amount was h i g h e r in t h e P u r k i n j e f i b r e s . T - t u b u l e s were seen onl y in t h e o r d i n a r y v e n t r i c u l a r myocyt es o f t h e l a r g e s t f e t u s .
MAN
Immunohi s t ochemi s t r y ( IV, V) : In s e c t i o n s i n c u b a t e d wi t h a n t i b o d i e s a g a i n s t t h e i n t e r m e d i a t e f i l a m e n t s u b u n i t s k e l e t i n a s u b e n d o c a r d i a l c e l l p o p u l a t i o n was d i s t i n g u i s h e d from t h e o r d i n a r y v e n t r i c u l a r myocardium in t h e human f e t a l h e a r t s a t m i d g e s t a t i o n by an i n t e n s e f l u o r e s c e n c e . By s e r i a l s e c t i o n i n g i t was o b s e r v e d t h a t t h e bundl e br a n c h e s took c o n t a c t wi t h t h i s c e l l popu
l a t i o n ( s e e bel ow) . In f a c t , p a r t s o f t he bundle br a n c h e s were a l s o composed o f i n t e n s e l y f l u o r e s c i n g c e l l s . A s h i f t from moder at e f l u o r e s c e n c e ( o f com
p a r a b l e magni t ude t o t h a t of t h e o r d i n a r y myocardium) t o i n t e n s e f l u o r e s cence o c c u r r e d in t h e i n i t i a l p a r t o f t he l e f t bundle br anch and in t h e d i s t a l i n t r a s e p t a l p a r t of t he r i g h t bundl e b r a n c h . The i n t e n s e l y f l u o r e s c i n g c e l l s a r e i n t e r p r e t e d as t he P u r k i n j e f i b r e s .
Al so t h e o r d i n a r y v e n t r i c u l a r and a t r i a l myocytes showed f l u o r e s c e n c e , which emanated from t h e Z d i s k s and t h e i n t e r c a l a t e d di s k r e g i o n s , in s e c t i o n s i n c u b a t e d wi t h a n t i s k e l e t i n a n t i b o d i e s . The f l u o r e s c e n c e in t h e more i n t e n s e l y f l u o r e s c i n g P u r k i n j e f i b r e s was more un i f o r ml y s p r e a d in t h e cyt o p l a s m.
The P u r k i n j e f i b r e s and t h e o r d i n a r y v e n t r i c u l a r and a t r i a l myocyt es o f t h e f e t a l h e a r t s a t m i d g e s t a t i o n showed f l u o r e s c e n t c r o s s - s t r i a t i o n s in s e c t i o n s i n c u b a t e d wi t h a n t i b o d i e s a g a i n s t t h e M- l i ne p r o t e i n myomesin. The P u r k i n j e f i b r e s were i d e n t i f i e d in s e r i a l s e c t i o n s due t o t h e i r high s k e l e t i n r e a c t i v i t y and high C h o l i n e s t e r a s e a c t i v i t y ( s e e be l o w) . In s e c t i o n s i n c u b a t e d wi t h a n t i b o d i e s a g a i n s t t h e M- l i n e p r o t e i n MM-creatine k i n a s e t h e P u r k i n j e f i b r e s and some a t r i a l myocyt es showed a c r o s s - s t r i a t e d p a t t e r n , wh i l e t h e o r d i n a r y v e n t r i c u l a r myocyt es d i d n o t . In a d u l t h e a r t s s t r i a t i o n s were e v i d e n t in t h e myocytes o f a l l t h r e e t i s s u e s in s e c t i o n s t r e a t e d t o d e mo n s t r a t e p r e s e n c e of MM-creat ine k i n a s e .
Enzyme h i s t o c h e m i s t r y (I V, V ) : S e c t i o n s s e r i a l t o t h o s e i n c u b a t e d wi t h a n t i - b o d i e s were p r e p a r e d f o r enzyme h i s t o c h e m i s t r y . Thi s e n a b l e d t h e i d e n t i f i c a t i o n o f t h e d i f f e r e n t p a r t s o f t h e c o n d u c t i o n sys tem ( I V) . All p a r t s of t he c o n d u c t i o n syst em showed a hi gh a c t i v i t y o f C h o l i n e s t e r a s e .
E l e c t r o n mi cr os copy ( V) : Ult r a s t r u c t u r a l l y two c e l l p o p u l a t i o n s were d i s t i n - g u i s h e d i n t h e v e n t r i c l e s in t h e human f e t a l h e a r t s : A s u b e n d o c a r d i a l c e l l p o p u l a t i o n in which t h e m a j o r i t y o f t h e m y o f i b r i l s showed dense M-bands and t h e r e s t of t h e myocardium in which t h e m y o f i b r i l s showed no dense M-bands.
The s u b e n d o c a r d i a l c e l l p o p u l a t i o n i s i n t e r p r e t e d t o r e p r e s e n t t h e P u r k i n j e f i b r e s . Also in some r e g i o n s in t h e a t r i a t h e m y o f i b r i l s showed dense M-bands.
P u r k i n j e f i b r e s formed b u n d l e s or l o o s e l y a r r a n g e d c e l l f o r ma t i o n s s u r rounded by abunda nt c o n n e c t i v e t i s s u e and few c a p i l l a r i e s . Some o f them were l y i n g in c o n t a c t w i t h o r d i n a r y myocar di al t i s s u e . In g e ne r a l t h e P u r k i n j e f i b r e s had a more rounded c e l l u l a r and n u c l e a r shape and l a r g e r d i a m e t e r s t h a n t h e o r d i n a r y v e n t r i c u l a r and a t r i a l myocyt es. The number of s p e c i a l i z e d c e l l c o n t a c t s seemed t o be h i g h e s t between t h e P u r k i n j e f i b r e s . I n t e r m e d i a t e f i l a m e n t s were most f r e q u e n t in t h e P u r k i n j e f i b r e s . The m y o f i b r i l s occupi e d onl y p a r t s of t h e cyt o p l a s m and had a v a r y i n g ap p e a r a n c e in a l l t h r e e c e l l t y p e s s t u d i e d ; howewer t he y v a r i e d t o t h e g r e a t e s t e x t e n t in t h e P u r k i n j e f i b r e s . Sever al f e a t u r e s were common t o t h e t h r e e c e l l t y p e s : eg. a s i n g l e n u c l e u s in a l m o s t a l l c e l l s , no w e l l - f o r m e d s t e p w i s e i n t e r c a l a t e d d i s k s , p r e s e n c e o f e l e c t r o n dense masses (mai nl y composed o f f i l a m e n t s and r i b o s omes ) , few and s c a t t e r e d m i t o c h o n d r i a , a bundant gl ycogen g r a n u l e s and no T- t u b u l e s . In t h e a t r i a l myocyt es t h e r e were numerous so c a l l e d a t r i a l s p e c i f i c g r a n u l e s .
DISCUSSION
THE PURKINJE FIBRES IN THE DEVELOPING HEART
The devel opment o f " h i g h l y d i f f e r e n t i a t e d " ( " t y p i c a l " ) ( bovi ne ) and " l e s s d i f f e r e n t i a t e d " (human) P u r k i n j e f i b r e s ( c f . 5 0 , 76) has been s t u d i e d in t h e p r e s e n t i n v e s t i g a t i o n . A s u b e n d o c a r d i a l c e l l p o p u l a t i o n in t h e v e n t r i c l e s , i n t e r p r e t e d t o r e p r e s e n t t h e P u r k i n j e f i b r e s , c o u l d be d i s t i n g u i s h e d i n both t h e bovi ne and human f e t a l h e a r t s . The a c c u r a c y o f t h i s i n t e r p r e t a t i o n can be d i s c u s s e d i n r e l a t i o n t o t h e i d e n t i f i c a t i o n , t h e t e r mi n o l o g y and t h e f u n c t i o n o f t h e c e l l s .
I d e n t i f i c a t i o n
One way t o i d e n t i f y t h e P u r k i n j e f i b r e s in t h e v e n t r i c l e s i s t o t r a c e t h e d i s t r i b u t i o n of t h e bundl e b r a n c h e s p e r i p h e r a l l y . Th i s was e n a b l e d by s e r i a l s e c t i o n i n g and by t h e f a c t t h a t t h e bundl e b r a n c h e s were c l e a r l y d i s t i n g u i s h e d from o r d i n a r y myocardium by enzyme h i s t o c h e m i s t r y . For both t h e bo
v i n e and t h e human f e t a l h e a r t s i t was c l e a r l y d e mo n s t r a t e d t h a t t h e bundl e br a n c h e s became d i s t r i b u t e d i n t o t h e s u b e n d o c a r d i a l c e l l p o p u l a t i o n s . Thus t h e s e c e l l p o p u l a t i o n s r e p r e s e n t t h e c o n d u c t i o n c e l l s in t h e v e n t r i c l e s . Anot her way t o " i d e n t i f y " P u r k i n j e f i b r ë s in t h e v e n t r i c l e s in t i s s u e s e c t i o n s would be t o s e a r c h f o r c e l l s w i t h " P u r k i n j e f i b r e - l i k e " mo r phol ogi c a l c h a r a c t e r i s t i c s . That was per f or med in speci mens o f s u b e n d o c a r d i a l r e g i o n s o f t h e v e n t r i c l e s and in t r a n s v e r s e s e c t i o n s t hr ough t h e v e n t r i c l e s . The c h a r a c t e r i s t i c s l ooked f o r were t h e ones d e s c r i b e d t o be t y p i c a l o f a d u l t P u r k i n j e f i b r e s , such as t h e i r g r o s s mor phol ogi c a l f e a t u r e s , a high c o n t e n t o f s k e l e t i n , a high c o n t e n t o f gl ycogen and t y p i c a l enzyme h i s t o c h e m i c a l c h a r a c t e r i s t i c s wi t h r e s p e c t t o o x i d a t i v e and g l y c o l y t i c a c t i v i t i e s ( c f . 1 , 17, 23, 4 6 , 50, 60, 7 5 ) .
In t h e bovine f e t a l h e a r t s , g r o s s mor phol ogi cal c r i t e r i a f o r i d e n t i f i c a t i o n o f t h e P u r k i n j e f i b r e s were a p p l i c a b l e a t t h e e a r l i e s t s t a g e i n v e s t i g a t e d ( CR- l e ngt h 7 cm). The d i s t i n c t i o n was, however, c l e a r e r i n l a t e r s t a g e s . The c e l l s were a l s o d i s t i n g u i s h e d from o r d i n a r y myocardium by a h i g h e r c o n t e n t o f s k e l e t i n , as seen i mmun o h i s t o c h e mi c a l l y . The m e t a b o l i c c r i t e r i a f o r t h e i d e n t i f i c a t i o n o f bovine P u r k i n j e f i b r e s changed d u r i n g devel opment.
Also i n t h e human f e t a l h e a r t s , s u b e n d o c a r d i a l c e l l s s ur r o u n d e d by c onnec
t i v e t i s s u e were d i s t i n g u i s h e d i n t h e v e n t r i c l e s in semi t h i n p l a s t i c s e c t i o n s ( u n p u b l i s h e d o b s e r v a t i o n s ) . . However, i t was not p o s s i b l e t o unequi vo
c a l l y i d e n t i f y t h e s e c e l l s as P u r k i n j e f i b r e s ( c f . 4 0 , 56, 8 6 ) . On t h e o t h e r hand, t h e P u r k i n j e f i b r e s were i d e n t i f i e d due t o t h e i r high c o n t e n t o f s k e l e t i n , as seen i mmu n o h i s t o c h e mi c a l l y . Also wi t h o t h e r t e c h n i q u e s a c l e a r s ep
a r a t i o n between two c e l l t y p e s in t h e v e n t r i c l e s was p o s s i b l e . The P u r k i n j e f i b r e s , in compar i son wi t h t he c e l l s in t h e r e s t of t he v e n t r i c u l a r myocar
dium, showed a h i g h e r a c t i v i t y o f C h o l i n e s t e r a s e , were l a b e l l e d by a n t i b o d i e s a g a i n s t MM-creat ine k i n a s e and showed M-bands u l t r a s t r u c t u r a l l y . The use o f immuno- and enzyme h i s t o c h e m i s t r y had t h u s enhanced t h e p o s s i b i l i t y t o i d e n t i f y P u r k i n j e f i b r e s in t h e human f e t a l h e a r t a t t h e l i g h t m i c r o s c o p i c l e v e l . In f a c t , a l r e a d y a t t e n weeks g e s t a t i o n a l age an e x t e n s i v e c e l l popu
l a t i o n i s d i s t i n g u i s h e d subendocar di a l l y in t h e v e n t r i c l e s , due t o a high c o n t e n t o f s k e l e t i n and a high a c t i v i t y o f C h o l i n e s t e r a s e ( u n p u b l i s h e d ob
s e r v a t i o n s ) . F u r t h e r mo r e , i t was p o s s i b l e t o u l t r a s t r u c t u r a l l y i d e n t i f y t he
human f e t a l P u r k i n j e f i b r e s a t m i d g e s t a t i o n as t h e s e c e l l s bu t not t he o r d i nar y myocytes in t h e v e n t r i c l e s had a t t a i n e d e l e c t r o n dense M-bands and con
t a i n e d MM-creat i ne k i n a s e . Immunohi stochemical d e t e c t i o n o f MM-creatine k i nas e c o r r e l a t e s wi t h t h e p r e s e n c e o f an e l e c t r o n dense M-band ( 64, 65, 8 5 ) . The f u n c t i o n of t h e M- l i n e bound MM-creat i ne k i n a s e i s propos ed t o be a p a r t i c i p a t i o n in t h e r e g u l a t i o n o f t h e ATP me t a bol i s m r e l a t e d t o mu s c u l a r con
t r a c t i o n ( 65, 8 5 ) . Termi nol ogy
I f we c o nc l ude t h a t t h e c e l l s i n t h e s u b e n d o c a r d i a l c e l l p o p u l a t i o n s ar e c o n d u c t i on c e l l s and a t l e a s t p a r t l y can be d i s t i n g u i s h e d from o r d i n a r y myo
car di um on t h e same b a s i s as a d u l t P u r k i n j e f i b r e s , i s i t a c c u r a t e t o c a l l t h e c e l l s P u r k i n j e f i b r e s ? C l e a r l y t he c e l l s in t h e bovi ne f e t a l h e a r t s , from an e a r l y f e t a l s t a g e , can be c a l l e d P u r k i n j e f i b r e s , mai nl y as t hey c o u l d be d i s t i n g u i s h e d by g r o s s mor phol ogi c a l f e a t u r e s and a high c o n t e n t of s k e l e t i n . These f e a t u r e s a r e two main c r i t e r i a o f a d u l t P u r k i n j e f i b r e s ( e . g. 23, 7 6 ) .
A high c o n t e n t o f s k e l e t i n , as d e mo n s t r a t e d immunohi stochemical l y ( I V) , can a l s o be used as a c r i t e r i o n t o d e f i n e human P u r k i n j e f i b r e s ( c f . 2 3 ) . Thus from a t l e a s t t e n weeks g e s t a t i o n a l age, which i s t h e e a r l i e s t s t a g e we have s t u d i e d h i t h e r t o , and onwards, c o n d u c t i o n c e l l s which m e r i t t h e name P u r k i n j e f i b r e s a r e p r e s e n t in human f e t a l h e a r t s . F u r t h e r mo r e , we obs er ved t h a t t h e s h i f t i n t o s k e l e t i n - r i c h c e l l s o c c u r r e d a t s p e c i a l l e v e l s : In t he i n i t i a l p a r t o f t h e l e f t bundl e branch and in t h e d i s t a l p a r t o f t he i n t r a mural r i g h t bundl e b r a n c h . Thus from t h e s e l e v e l s and f u r t h e r p e r i p h e r a l l y t h e c on d u c t i o n c e l l s mi ght be c a l l e d P u r k i n j e f i b r e s . Thi s i s of s i g n i f i ca nc e f o r t h e human c o n d u c t i o n system as t h e b o r d e r between bundl e branch and p e r i p h e r a l P u r k i n j e f i b r e c e l l t y p e s f o r a long ti me has been d i s c u s s e d and n o t been d e t e r mi n e d ( c f . 28, 6 2 ) .
F u n c t i o n a l a s p e c t s
What do t h e o b s e r v a t i o n s in t h e p r e s e n t s t u d i e s imply wi t h r e s p e c t t o t he f u n c t i o n o f f e t a l P u r k i n j e f i b r e s , as compared wi t h a d u l t ones? E l e c t r o p h y s - i o l o g i c a l l y t h e b e h a v i o u r of t h e human f e t a l h e a r t i s compar able t o t h a t of t h e a d u l t h e a r t ( 2 5 , 35, 7 9 ) . J a n s e e t al (35) a r gue d t h a t t h e e l e c t r o p h y s i ol o g i c a l m a t u r i t y of t h e human f e t a l h e a r t was produced d e s p i t e a "morphol o
g i c a l i mma t ur i t y" o f t h e c o n d u c t i o n sys tem. However, t h e p r e s e n t s t u d i e s i n d i c a t e t h a t t h e f e t a l P u r k i n j e f i b r e s a r e no t "i mmat ur e" . Mo r p h o l o g i c a l l y i t a p p e a r s t h a t t h e c e l l s i n s t e a d have a s p e c i a l i z e d f u n c t i o n a l r e a d y a t e a r l y f e t a l s t a g e s .
The f o r ma t i o n a f bu n d l e s a l r e a d y a t t h e e a r l y f e t a l s t a g e of bovi ne h e a r t s may f a v o u r t h a t t h e P u r k i n j e f i b r e s e a r l y have f a s t c o n d u c t i o n p r o p e r t i e s . Whether nexuses a r e more abundant between P u r k i n j e f i b r e s t han o r d i n a r y myo
c y t e s a l r e a d y in f e t a l h e a r t s , which i s our p r e l i m i n a r y o p i n i o n (V), must a w a i t f u r t h e r s t u d i e s . The high a c t i v i t y of SDH and a - g l y c e r o p h o s p h a t e de
h ydr ogenas e and t h e p r e s e n c e of dense M-bands in t h e bovi ne P u r k i n j e f i b r e s a t e a r l y f e t a l s t a g e s shows t h a t t h e c e l l s a t t h e s e s t a g e s a r e more d i f f e r e n t i a t e d t han t h e o r d i n a r y myocyt es ( s e e bel ow) . The high a c t i v i t y o f t he two enzymes f a vo u r s t h a t t h e energy demand i s h i g h e r in t h e P u r k i n j e f i b r e s a s compared wi t h t h e o r d i n a r y myocytes a t e a r l y f e t a l s t a g e s . The f a c t t h a t s k e l e t i n i s p r e s e n t in l a r g e amounts in f e t a l P u r k i n j e f i b r e s , p o s s i b l y i n s t e a d o f m y o f i b r i l l a r p r o t e i n s , s u g g e s t s t h a t t h e c e l l s e a r l y a r e no t d i r e c t e d t o c o n t r a c t i l i t y .
