Differentiationof

Heart Purkinje Fibresimmuno- and enzyme histochemical and ultrastructural study

AKADEMISK AVHANDLINGsom med vederbörligt tillstånd

av rektorsämbetet vid Umeå universitet för avläggande av medicine doktorsexamen

kommer att offentligen försvaras i Anatomiska institutionens stora föreläsningssal

Umeå universitet torsdagen den 15 april 1982 kl 09.00

av

STURE FORSGRENleg läk

ABSTRACT

DIFFERENTIATION OF HEART PURKINJE FIBRES An immuno- and enzyme histochemical and ultrastructural study

Sture Forsgren, Institute of Anatomy, University of Umeå, Umeå, Sweden

The development of the Purkinje fibres, the cells comprising the periph­eral part 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 devel­opment are conflicting. In the present investigation bovine and human Purkinje fibres during development were studied histologically and ultra- structurally and by means of enzyme and immuno-histochemistry.

In both bovine and human fetal hearts the Purkinje fibres were distin­guished from the main myocardial mass due to a strong immunofluor­escence reaction, in sections incubated with antibodies against the inter­mediate filament subunit skeletin. Thus, a high content of skeletin, as demonstrated by immunohistochemistry, seems to be a general criterion to identify fetal Purkinje fibres. Furthermore, the Purkinje fibres were identified by their gross morphological features from an early fetal stage of the bovine hearts. In the human fetal hearts the Purkinje fibres were also identified by a high activity of cholinesterase.

By comparison with immunohistochemical observations human fetal Purkinje fibres at midgestation could be identified at the ultrastructural level: The Purkinje fibres but not the ordinary ventricular myocytes were stained in sections incubated with 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 with detectability of MM-creatine kinase. In the Purkinje fibres in the bovine fetal hearts, electron dense M-bands were observed and MM-creatine kinase was detected at an earlier stage than in ordinary ventricular myocytes.

During fetal development in the bovine hearts differences appeared be­tween Purkinje fibres and ordinary ventricular myocytes with respect to cell volumes, intercalated disks, myofibrils, mitochondria, amount of glyc­ogen and T-tubules. In all fetal stages of bovine hearts and in the human fetal hearts at midgestation the Purkinje fibres contained a greater amount of intermediate filaments.

The histochemical pattern of Purkinje fibres vs ordinary ventricular myocytes, with respect to enzymes reflecting oxidative and glycolytic activities, changed during fetal development of the bovine hearts. This could be related to the changing enzyme activities in the ordinary myo­cytes. The staining reactions of these enzymes in the Purkinje fibres seemed to remain unchanged during fetal development. Noticeably the Purkinje fibres showed higher enzyme activities than the ordinary myo­cytes at early fetal stages, indicating a higher energy demand in the form er cells.

The observations show that Purkinje fibres clearly can be identified in fetal hearts and that the use of enzyme and immuno-histochemistry has facilitated this identification. The Purkinje fibres differentiate along a different pathway from ordinary ventricular myocytes. Furthermore, 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 histochemistry, immunohisto­chemistry, electron microscopy.

UMEÂ UNIVERSITY MEDICAL DISSERTATIONSNew Series No 83 - ISSN 0346-6612

From the Institute of Anatomy, University of Umeå, Umeå, Sweden

Differentiationof

Heart Purkinje FibresAn immuno- and enzyme histochemical and ultrastructural study

by

STURE FORSGREN

Umeå 1982

3

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 sp ec 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 deve lopmen 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

embryoni c r emn an 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 and Methods .............................................................................................. 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 in t h e d e v e lo 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

Compar i son w i th o r d i n a r y v e n t r i c u l a r myocyt es . . . . 17

Compar i son w i th a t r i a l myocyt es .......................................................... 18

Comment on t h e p ropo sed "embryoni c o r g a n i z a t i o n " . . . 19

Gene ra l summary and c o n c l u s i o n s ......................................................................... 20

Acknowledgement s ............................................................................................................. 22

L i t e r a t u r e c i t e d ............................................................................................................. 23

Appendix ....................................................................................................................... 29

P ap e r I .................................. 29

P a p e r I I ......................................................................................................................41

P a pe r I I I ......................................................................................................................51

P ap e r IV . ’ .............................................................................................................. 67

Pa pe r V ............................................................................................................................ 81

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ORIGINAL PAPERS

This t h e s i s i s based on t he fo l l owing pu b l i c a t i o n s and manu­s c r i p t s , which wi l l be r e f e r r e d t o by t h e i r Roman numerals:

I . For sgren , S . , Th o rn e l l , L-E. and E r ik s son , A.The development of the Pu rk i n j e f i b r e system in the bo­v ine f e t a l h e a r t .Anatomy and Embryology 159, 125-135 (1980)

I I . Fo rsg ren , S. and Th o rn e l l , L-E.The development of Pu rk in j e f i b r e s and o rd inary myo­cy t e s in the bovine 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 udy.Anatomy and Embryology 162, 127-136 (1981)

I I I . Fo rsgren , S . , S t r e h l e r , E. and Th o rn e l l , L-E.D i f f e r e n t i a t i o n of Pu rk in j e f i b r e s and o rd ina ry ven­t r i c u l a r and a t r i a l myocytes in the bovine h ea r t - An immuno- and enzyme h is tochemical s t udy.Histochemical Journa l ( i n p res s)

IV. Fo rsg ren , S . , Er iks son , A. , K j ö r e l l , U. and Tho rn e l l , L-E.The conduct ion system in the human h e a r t a t mi 'dgesta- t i o n - Immunohistochemical demons tra t ion of s k e l e t i n . Submi t ted fo r pu b l i c a t i o n

V. Forsgren , S . , Ca r l s son , E . , S t r e h l e r , E. and Th o rn 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 of human f e t a l Pu rk in j e f i b r e s - A comparat i ve immunocytochemical and e l ec t ro n microscopi c s tudy of composi t ion and s t r u c t u r e of m y o f i b r i l l a r M-regions .Jou rna l of Molecular and C e l l u l a r Cardiology ( i n pr es s )

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INTRODUCTION

Aspects on t he s t r u c t u r e and func t i on of a d u l t Pu rk in j e f i b r e s

In the h e a r t t he r e i s a system, the conduct ion system, which genera t e s the s t imulus fo r the h ea r t bea t and conducts the impulse to the d i f f e r e n t pa r t s o f the myocardium. Thereby the proper succes s ion of c on t r a c t i o n s o f the a t r i a and v e n t r i c l e s i s ensured. The system c o n s i s t s of the s i n o a t r i a l node (SA node) , the a t r i o v e n t r i c u l a r node (AY node) , the a t r i o v e n t r i c u l a r bundle (AV bundle , bundle of H i s ) , and i t s branches ( t he r i g h t and l e f t bundle b r a nches ) . The pe r i phe ra l p a r t of the bundle branches i s c a l l e d the Pu rk in j e f i b r e system. The c e l l s compr is ing t h i s system, the Pu rk in j e f i b r e s , were f i r s t d i s covered in the sheep h ea r t in 1845 by J E Pu rk in j e a f t e r whom the c e l l s were termed.

H i s t o l o g i c a l l y the Pu rk in 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, in l a r ge mammals, e . g . ung u l a t e s , the c e l l s are morphologi­c a l l y d i s t i n c t i v e : They have about 3-4 t imes l a r g e r d iame ter s than o rd ina ry v e n t r i c u l a r myocytes, a vacuo la t ed appearance, spar s e m y o f ib r i l s , and m u l t i ­p l e nuc le i (1 , 17, 50, 75 ) . These c e l l s a re o f t en r e f e r r e d to as " t y p i c a l " Pu rk in j e f i b r e s . I nd iv idual c e l l s or bundles of c e l l s a re surrounded by a connec t i ve t i s s u e sheath which d e l i m i t s them from the o rd ina ry myocytes. In man and in small mammals, e . g . r a t and c a t , the c e l l s in the Pu rk i n j e f i b r e system a re sma l l e r and do not always have a cont i nuous connec t ive t i s s u e shea th . In t he se spec i e s i t i s in f a c t d i f f i c u l t to d i s t i n g u i s h t he se c e l l s from the sur rounding o rd ina ry myocytes by means of convent i onal l i g h t micro­scopy (17, 50, 76) . The spec i es v a r i a t i o n s , with r e s p e c t to the morphology o f the Pu rk i n j e f i b r e s , have l ead to confus ion in the l i t e r a t u r e rega rd ing t he terminology of the c e l l s . Some au tho rs have r e s t r i c t e d the term Pu rk i n j e f i b r e to " t y p i c a l " Pu rk in j e f i b r e s , whi le o the r s have i nc luded the c e l l s in t he pe r i phe ra l r a m i f i c a t i o n o f the bundle branches in a l l h ighe r v e r t e ­b r a t e s . Some au tho r s have p r e f e r r e d to c l a s s i f y the Pu rk i n j e f i b r e s i n to t h r e e main t ypes , mainly based on the diameter s of the c e l l s and the e x t e n t t o which the c e l l s d i f f e r from the o rd ina ry myocytes (50, 76 ) . According to Virägh and C ha l l i c e (83) the morphology of the Pu rk in j e f i b r e s changes a t spec i a l regions along the d i s t r i b u t i o n of the bundle branches and t hese a u t ­hors have c l a s s i f i e d the c e l l s i n to Pu rk i n j e I , I I , and I I I c e l l s , r e spec ­t i v e l y .

By use of h i s t o l o g i c a l s t a i n s f o r glycogen, and enzyme h is tochemical and im- munohistochemical t echn iques , the Pu rk i n j e f i b r e s can be d i s t i ng u i sh ed from the ord inary myocytes. However a l so with t he se t e chniques 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 observab l e . The Pu rk in j e f i b r e s are d i s t i ng u i sh ed from the o rd ina ry myocytes by a h igher amount of glycogen, a h igher a c t i v i t y of g ly ­c o l y t i c and g luconeogene t ic enzymes and a lower a c t i v i t y of r e s p i r a t o r y en­zymes (46, 60 ) . These ob se r va t i on s , as well as the lower uptake of oxygen and the lower dens i t y of c a p i l l a r i e s in the Pu rk in j e f i b r e system than in t he o rd ina ry myocardium (18, 53) favour t h a t the Pu rk in j e f i b r e s have a h igher r a t e of anaerobi c and a lower r a t e of ae rob i c metabolism than the o r­d ina ry myocytes. Fur thermore, the Pu rk in j e f i b r e system i s d i s t i n gu i s h ed from o rd inary myocardium by a high a c t i v i t y of Ch o l i ne s t e r a se (20, 37 ) . I t i s , however, not c e r t a i n to what e x t e n t t h i s r e f l e c t s the r i ch nervous com­ponent of the conduct ion system or a high a c t i v i t y of the enzyme wi th in the Pu rk in j e f i b r e s (17, 78) . Recent l y, the P u rk in j e f i b r e s have a l so been shown to d i f f e r from o rd ina ry myocytes by a h igher con t e n t of the i n t e rmed ia t e f i ­lament p ro t e in s k e l e t i n , as seen with immunofluorescence t echn iques (23) .

6

By e l e c t r o n microscopy c l e a r d i f f e r e n ce s have been observed between Pu rk in j e f i b r e s and o rd ina ry myocytes: The Pu rk in j e f i b r e s have both end- to-end and s i d e - t o - s i d e connec t i ons ( t he o rd ina ry myocytes are p r i n c i p a l l y l i nked end- t o- end) i nc lud ing more f r equen t nexuses , absence of T - tub u l e s , fewer and sma l l e r mi tochond r ia , presence of mi tochondr ia with a doughnut appearance and a l a r g e r amount of glycogen granu les as compared with the o rd ina ry myo­cy t e s (6 , 9 , 38, 43, 53, 61, 69) . In p a r t i c u l a r , the d i f f e r e nc e with r e s pec t t o T - tub 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, 63) . Fur thermore, i n t e rmed ia t e f i l amen t s are more abundant and the m yo f i b r i l s are l e s s well organi zed in the Pu rk i n j e f i b r e s (6 8 , 71 ) . Abnormal i t ies in the sarcomeres and myofi lament -polyr ibosome complexes are t yp i ca l f or the Pur­k i n j e f i b r e s (51, 67, 68 ) . The d i f f e r e n ce s with r e s pe c t to mi tochondr ia and glycogen may r e f l e c t the proposed d i f f e r e n c e in c e l l metabolism (see above) . The d i s s i m i l a r i t y in amount of i n t e rmed ia t e f i l amen t s and con t en t of ske l e- t i n , as seen immunohis tochemical ly , sugges ts t h a t Pu rk i n j e f i b r e s may need a d i f f e r e n t type of cy to ske l e ton from o rd ina ry myocytes. The abno rm a l i t i e s in t he sarcomeres of Pu rk in j e f i b r e s are probably due to an imbalance in the s y n t h e s i s and deg rada t i on of m y o f i b r i l l a r p ro t e i n s (68 ) .

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 conduct ion i s rap id through­out the bundle branches and the per i phe ra l Pu rk in j e f i b r e s a t a r a t e of 1 .5- 5 . 0 m/sec (17, 32 ) . In c o n t r a s t , t he ve lo c i t y in o rd ina ry v e n t r i c u l a r myo­cardium i s 0 . 3 - 1 . 0 m/sec. Numerous a t t emp ts have been made to exp la in these phys i o log i ca l d i f f e r e n c e s . The l a rg e diameter s of the Pu rk in j e f i b r e s and t h e i r agg rega t ion i n to t i g h t l y packed bundles favour a g r e a t e r conduct ion v e l o c i t y in t hese c e l l s than in o rd ina ry myocytes ( 63) . The nexuses , which a r e f r equ en t between the Pu rk in j e f i b r e s , probably r e p r e se n t low-re s i s t ance pathways (8 ) . The Pu rk in j e f i b r e s a r e a l s o i n f l uenced by both sympathet ic and parasympathe t i c branches of the autonomic nervous system. A d i r e c t c e l ­l u l a r ba s i s f o r ch o l i n e rg i c antagonism of the e l e c t ro p h y s io lo g i ca l e f f e c t s o f ca techo lamines on the Pu rk i n j e f i b r e s i s documented (7 ) . Besides r ap id conduct i on , the Pu rk in j e f i b r e s a l s o have the p roper ty of spontaneous de­p o l a r i z a t i o n (18) . Con t r ac t i ons a r i s i n g wholly w i thin the v e n t r i c l e s , as in complete AY b lock , o r i g i n a t e in Pu rk in j e f i b r e s .

I t i s thus c l e a r t h a t in the a d u l t h ea r t the Pu rk in 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 ic a l l y , immunohistochemical ly and e l e c t r o p h y s i o l o g i c a l l y from the o rd ina ry myocytes.

The development o f t he Pu rk in j e f i b r e s

The o r i g i n and development of the d i f f e r e n t p a r t s of the conduct ion system has fo r a long t ime been the source of con t rove r sy . However, i t i s c l e a r t h a t a conduct i on system can not be i d e n t i f i e d morpholog ica l l y a t the s t age when ca r d i ac c o n t r a c t i o n s s t a r t . Cont r ac t i ons occur soon a f t e r the format ion of the s i ng l e he*art tube (57) or even e a r l i e r , b efor e the two endocardi al t ubes have begun to fuse (27) . The pacemaker region in the t u b u l a r he a r t has been proposed t o s h i f t dur ing development, i n i t i a l l y being l oca t ed in the v e n t r i c l e (55) . Others have argued t h a t the impulse from the beginning i s genera t ed in the p ro spec t i ve s i n o a t r i a l region (45, 80 ) .

With r e s p e c t to the o r i g i n of the conduct ion system components one theory 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 area in the embryonic he a r t de­ve lops , from which the AY bundle and the bundle branches develop (59, 8 6 ) . According t o t h i s theory the end of the AV bundle i s an a c t i v e ly growing t i s s u e . Other au tho rs have contended t h a t the AV bundle (42) and the Pur ­k in j e f i b r e s (3 , 34, 84) a r i s e in s i t u in the AV canal and in the v e n t r i -

7

c l e s , r e s p e c t i v e l y . Wenink, Ande-rson and co l l e agues (4, 87) have proposed t h a t the phy logene t i c and on togene t i c o r i g in of the d i f f e r e n t p a r t s of the conduct i on system i s a s e r i e s of myocardial r i ngs between segments of the t u b u l a r h e a r t . According to t he se t h e o r i e s the Pu rk i n j e f i b r e s develop from the bu lb o v en t r i cu l a r r i n g , formed between the bulbus and the p r i m i t i v e ven­t r i c l e . In a r e cen t study these ideas could, however, not be confi rmed (42) .

The morphological d i f f e r e n ce s between conduct i ve t i s s u e elements and func­t i o n in 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 be­tween the two types i s extremely d i f f i c u l t a t t h i s s t age (81) . Mainly for t h a t reason t he r e are d i f f e r e n t opinions about the s t age of development a t which the d i f f e r e n t pa r t s of the conduct ion system 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 au tho r s , however, agree t h a t the Pu rk i n j e f i b r e s ap­pear a t a l a t e r s t age than t he more proximal pa r t s of the conduct ion system.

The development of the " t yp i c a l " Pu rk in j e f i b r e s , as well as t h a t of the Pu rk in j e f i b r e s of man, has been s t ud i ed wi th convent ional h i s t o l o g i c t e ch ­niques but not by means of h i s t ochemis t ry or e l e c t ro n microscopy. The c r i t e ­r i on fo r i d e n t i f i c a t i o n of the Pu rk in j e f i b r e s with h i s t o l o g i c t e chniques has been the appearance of c e l l s which, as compared wi th the o rd ina ry myo­cy t e s , a re more rounded and l a r g e r and have myof ib r i l s only in the pe r i ph ­e ry . Using such c r i t e r i a the Pu rk i n j e f i b r e s in human he a r t s can not be d i s ­t i ng u i s he d from o rd inary myocytes a t nine weeks of ge s t a t i o n (40) and are f i r s t d i s t i n g u i s h e d a t about 14 weeks (86 ) . At l a t e r s t ages of f e t a l deve l ­opment they 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, 86 ) . Anderson and Taylor ( 3 ) , however, de s c r i be two ce l l popu la t ions - one subendocardia l and one ou t e r l a ye r of c e l l s - which can a l r eady be recognized in the v e n t r i c l e s a t e a r l y embryonic s t age s in human h e a r t s . These autho rs propose t h a t the subendocardia l c e l l popu la t i on forms the Pu rk in j e f i b r e s and the ou t e r l aye r t he o rd inary v e n t r i c u l a r myocardium. The reason fo r the d ive rgen t d e sc r i p ­t i o n s i s probably r e l a t e d to the ba s i s f o r i d e n t i f i c a t i o n of the Pu rk in j e f i b r e s : Although Anderson and Taylor a l so desc r i bed a d i f f e r e n ce in c e l l d i ­ameter i t i s merely the observa t ion of an ex t ens ive l aye r subendocardia l l y in the v e n t r i c l e s which i s t h e i r ba s i s f o r i d e n t i f i c a t i o n . The development o f the " t yp i c a l " Pu rk in j e f i b r e s has been s t ud ied in the sheep: The Pu rk i n j e f i b r e s a re desc r i bed as d i s t i n g u i s h a b l e from o rd ina ry myocardium a t 70 mm (49) r e s p e c t i v e ly 100 mm (24) CR-length with convent i onal h i s t o l o g i c t e c h ­n iques .

For c l a r i t y i t must be po in ted out t h a t not a l l au tho rs s tudying developing he a r t s term the conduct ion c e l l s in the v e n t r i c l e s Pu rk in j e f i b r e s . I t must be remembered t h a t the 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 Pu rk in j e f i b r e s from o r ­d inary myocytes ( see above and D iscus s ion) a re va l i d f o r a d u l t h e a r t s .

The Pu rk in j e f i b r e s - h ighly d i f f e r e n t i a t e d c e l l s or embryonic remnants?

The degree of d i f f e r e n t i a t i o n of the Pu rk in j e f i b r e s i s a co n t r o ve r s i a l t op ­i c : The c e l l s a re de sc r ibed to be "embryonic", or to be highly " d i f f e r e n ­t i a t e d ^ . I t i s a l so debated whether the Pu rk in j e f i b r e s and o rd ina ry myo­cy t e s d i f f e r e n t i a t e along the same rout e but 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 along d i f f e r e n t rou te s (18 ) . Fur the r the de sc r i p t i on of the t h r ee main t ypes of Pu rk in 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 ee above) , in terms of d i f f e r e n t i a t i o n i s confus ing: The ce l l types are 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, 76) .

In old l i t e r a t u r e the Pu rk in j e f i b r e s are sa id to be c e l l s which remain un­developed and which resemble embryonic c a rd i ac muscle c e l l s ( e . g . 33, 48 ) .

8

F ie l d (24) contended t h a t the Pu rk i n j e f i b r e s were held up in t h e i r develop­ment. Also in more r ec en t s t ud i e s the Pu rk i n j e f i b r e s have been de sc r ibed as "embryonic" and " r e t a rded" in development. What a re the bases f or these sug­g es t i on s? I t i s a f a c t t h a t many of the biochemical and metabol 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 conduct i on t i s s u e from o rd ina ry myocardium in the a d u l t he a r t (amount of glycogen, r e s i s t a n c e to anoxia , a c t i v i t i e s of oxida­t i v e and g ly 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 du l t myocar­dium (18) . In a d d i t i o n , t he r e appear to be s i m i l a r i t i e s between the u l t r a ­s t r u c t u r e of the a d u l t P u rk in j e f i b r e s and t h a t of u n d i f f e r e n t i a t e d embryon­i c myocytes (6 , 47, 52 ) . This has l ead to the conc lus ion t h a t the Pu rk in j e f i b r e s have an "emb ryo nie organi za t i o n" . Virâgh and Chal l i c e (82) have f u r ­t h e r proposed t h a t the m y o f i b r i l l a r development in the Pu rk i n j e f i b r e s i s " i ncomple te " , as an exp l ana t i on of the f i lamentous components in the c e l l s . The "de f ec t s " in m yo f ib r i l l o g en es i s in the Pu rk i n j e f i b r e s ( exempl i f i ed by e . g . t he myof i lament -polyr ibosome complexes) might r ep r e s e n t c e s s a t i o n of m yo f i b r i l l o g en es i s a t an embryonic s t age (51) . I t has a l so been proposed t h a t t he conduct i ng t i s s u e r e p r e s en t s remnants of embryonic t i s s u e on the b a s i s of i t s spec i a l phys io log i ca l p r o p e r t i e s (41 ) .

A l t e r n a t i v e l y , the Pu rk i n j e f i b r e s and the o rd inary myocytes have been de­s c r i b ed as r e p r e s en t i ng d i f f e r e n t pathways of d i f f e r e n t i a t i o n , by which the Pu rk in j e f i b r e s r e t a i n the high conduct i ve p r o p e r t i e s and the spontaneous c o n t r a c t i l i t y ( 29 ) . The appearance of the m y o f i b r i l l a r mat e r i a l in the Pur ­k i n j e f i b r e s has been a t t r i b u t e d to an imbalance in s y n t he s i s and degrada­t i o n of m y o f i b r i l l a r p r o t e i n s . This imbalance may r e f l e c t a process of d i f ­f e r e n t i a t i o n in which the development of "n e r ve - l i ke " p r o p e r t i e s has taken precedence over t he development of c o n t r a c t i l i t y ( 70 ) . Fur thermore , the f a c t t h a t t he Pu rk i n j e f i b r e s change by age, e . g . i nc r e a se markedly in s i z e , would favour t h a t t he c e l l s a re not embryonic remnants (16 ) . Robb e t al (56) even proposed t h a t the conduct ion c e l l s undergo a more ex t en s iv e change than do the o rd ina ry myocytes, as the conduct ion 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 rd ina ry myocytes in human h ea r t s of l a t e f e t a l s t age s than o f e a r l y f e t a l s t a ge s . Bogusch (11) concluded in an u l t r a s t r u c t u r a l study on chicken Pu rk in j e f i b r e s dur ing development t h a t the c e l l s go through 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 roce s s . He e . g . observed t h a t t yp i ca l a d u l t f e a tu r e s such as l ep tomer ic f i b r i l s and l ep tomer i c complexes did not appear u n t i l a f ­t e r ha t ch ing and t h a t the amount of glycogen in t he Pu rk i n j e f i b r e s de­c r ea s ed dur ing development. However, u l t r a s t r u c t u r a l informa t ion on develop­ing Pu rk i n j e f i b r e s in the mammalian h e a r t i s needed, as t he r e are d i f f e r ­ences between a d u l t chicken and mammalian Pu rk i n j e f i b r e s , e . g . with r e s pec t t o amount of glycogen.

9

AIMS OF THE PRESENT INVESTIGATION

The p r i nc ipa l aims were t o answer the fo l l owing ques t i ons :

Can enzyme and immuno-his tochemist ry be employed in order to f a c i l i t a t e the i d e n t i f i c a t i o n of f e t a l Pu rk i n j e f i b r e s a t the l i g h t microscopi c l eve l ? I s i t po s s ib l e to u l t r a s t r u c t u r a l l y de f i ne the human f e t a l Pu r k in j e f i b r e s?

What a re t he morphological c h a r a c t e r i s t i c s of f e t a l Pur­k i n j e f i b r e s compared with a d u l t ones?

How do t he Pu rk 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 with t he o rd ina ry v e n t r i c u l a r and a t r i a l myocytes? Do Pu rk in j e f i b r e s show an "embryonic o rgan i za t i o n"?

10

MATERIALS AND METHODS

The o r i g in a l papers are r e f e r r e d to by t h e i r Roman numerals. In the o r i g ina l papers f u r t h e r de s c r i p t i o n s can be found.

MATERIALS

Feta l bovine h e a r t s ( I - I I I ) : Twenty-e ight f e t u se s were ob ta ined dur ing ro u t ­ine s l au g h t e r of cows. The crown-rump(CR)-lengths of the f e t u se s va r i ed be­tween 5 cm ( g e s t a t i o n a l age about 50 days) and 100 cm ( f u l l term, 260-270 days) .

The he a r t s of the f e t u se s were d i s s e c t e d out immediately a f t e r or wi th in 1-2 ti of s a c r i f i c e . From the former h e a r t s , specimens were f i xed in a s l i g h t l y s t r e t c h e d s t a t e in 2 . 5 % g l u t a r a ldehyde in Ty rode ' s b u f f e r and f u r t h e r proc­es sed fo r l i g h t microscopy ( I ) and t r an smi s s ion e l e c t r o n microscopy ( I I ) . These he a r t s were from f e t u se s with CR-lengths 7-100 cm. The specimens from h e a r t s d i s s e c t ed out with some delay a f t e r s a c r i f i c e were processed fo r en­zyme h i s t oc hem is t ry and immunofluorescence microscopy ( I , I I I ) . These hea r t s were from f e t u se s wi th CR-lengths 16-100 cm ( I ) and 5-72 cm ( I I I ) . The s m a l l e s t h ea r t s processed for c r y o - se c t i on in g (CR-lengths 5 and 8 .5 cm) were f rozen as whole p i ece s to al low sec t i o n i ng in the f r on t a l plane of the h e a r t s ( I I I ) .

Adul t bovine h e a r t s ( I I I ) : The h ea r t s of t h r ee a d u l t cows were ob t a ined dur­ing ro u t i n e s l au g h t e r of cows and were d i s s e c t e d w i th i n 1-2 h of s l au gh t e r . The specimens were processed fo r enzyme h i s t ochemis t ry and immunohistochemi- s t r y . Some specimens were f rozen t oge th e r with f e t a l specimens from c o r r e ­sponding r eg ions .

Fe ta l human he a r t s (IV, V) : The hea r t s of ten human f e tu se s ( g e s t a t i o n a l age 17-21 weeks) , ob t a ined a t l ega l i n t e r r u p t i o n s of pregnancy performed by a p ro s t a g l a n d i n i n s t i l l a t i o n procedure ( f i v e hea r t s ) or by hysterotomy ( f i v e h e a r t s ) , were i n v e s t i g a t e d . The former hea r t s were proces sed fo r enzyme and immuno-his t ochemist ry. These he a r t s were cu t i n to 3-4 b locks to al low sec­t i o n i n g in the t r a n s v e r s e and f r on t a l planes of d i f f e r e n t r egions of the h e a r t s . One h ea r t was not d ivided to a l low se r i a l s ec t i on in g in the f ron t a l p lane of the e n t i r e h ea r t .

The h ea r t s from the f e t u s e s obt a ined by hysterotomy were immediately removed from the f e t u s e s and w i th in a few minutes pe r fu s ion of the h ea r t s with an oxygenated R in ge r ' s so l u t i o n ad modum Langendorff was s t a r t e d a t room tem­p e r a t u r e . These he a r t s were processed fo r t r an smi s s ion e l ec t r o n microscopy.

Adul t human he a r t s (V) : Hear ts from two human au tops i e s were obt a ined . The specimens were processed fo r enzyme and immuno-his tochemis t ry.

Ti ssues s t ud i ed ( I -V) : The t i s s u e s s t ud i ed in the bovine h ea r t s were the Pu rk in j e f i b r e system ( I , I I , I I I ) , the o rd ina ry v e n t r i c u l a r myocardium ( I , I I , I I I ) and the o rd ina ry a t r i a l myocardium ( I I I ) . In the human hea r t s the SA- and AV-nodes, the AV-bundle and bundle branches ( IV) , the Pu rk in j e f i b r e system (IV, V), the ordinary v e n t r i c u l a r myocardium (IV, V) and the ordinary a t r i a l myocardium (IV, V) were s t ud i ed . De t a i l ed d e s c r i p t i o n s of how the specimens were cu t can be found in the o r i g in a l papers .

11

ENZYME AND IMMUNO-HISTOCHEMISTRY

General : The specimens were f rozen in freon or propane c h i l l e d with l i q u i d n i t r og en . Se r i a l s e c t i o n s were cu t in a c r y o s t a t a t -20°C and s t a i n ed fo r t he demons t ra t ion of enzyme a c t i v i t i e s , the i n t e rm ed i a t e f i l amen t subuni t s k e l e t i n or the M-l ine p r o t e i n s myomesin or MM-creatine k inase (see Di scus­s i o n ) . Other s e c t i o n s were t r e a t e d wi th con tro l se ra or s t a i n ed with rou t i ne h i s t o l o g i c a l s t a i n s . From several of the specimens a l a r g e s e r i e s of sec­t i o n s was cu t . By the s e r i a l s ec t i on in g procedure i t was pos s ib l e to prop­e r l y i d e n t i f y the d i f f e r e n t reg ions of the conduct ion system (IV).

Enzyme h i s t o c h e m i s t r y : Sec t i ons were s t a i n ed to demonstrate the 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-tetrazolium r educ t a se (NADH-TR) ( I , IV), succ i n i c dehydrogenase (SDH) ( I , I I I , IV), menadione- l inked a -g lyce rophos - phate dehydrogenase ( I , I I I , IV) or phosphorylase ( I ) . The h i s tochemica l methods were according t o Dubowitz and Brooke (19) . Sec t i ons were a l s o s t a i ne d to demons t rate the a c t i v i t y of C ho l i ne s t e r a se (IV, V): The p roce­dures were mainly according to Anderson and Taylor ( 3 ) , using Gomori ' s medi­um (26) . Other s e c t i o n s were s t a i n ed with PAS ( I ) or van Gieson ( I , IV). The s e c t i o n s were viewed in a Le i t z Dialux 20 Photomicroscope.

Immunohi stochemi s t r y : Rabbi t an t i se r a ag a i n s t bovine Pu rk i n j e f i b r e sk e l e t i n (s ee 22, 23) and a g a in s t the chicken b r e a s t muscle Mr 165,000 M-protein myomesin (s ee 21, 64, 65) were pr epared. Sheep an t i serum a g a in s t human MM- c r e a t i n e k inase was purchased from Boehr inger (FRG). Sera from normal rab­b i t s and sheep served as c o n t r o l s . Sect i ons were incubat ed wi th a n t i s k e l e t i n an t i bo d i e s ( I , I I I , IV, V) or with an t i bo d i e s ag a in s t MM-creatine kinase or myomesin ( I I I , V). The s ec t i ons were examined under a L e i t z Orthoplane Pho­tomicroscope equipped with ep i f l uo re sce nce o p t i c s .

CONVENTIONAL LIGHT MICROSCOPY AND TRANSMISSION ELECTRON MICROSCOPY

Feta l bovine h ea r t s ( I I ) : The g lu t ar a ldehyde f i xed specimens were cu t i n to small b locks , pos t f i x ed in 1% OSO4 , r i n sed in b u f f e r and dehydra t ed . Em­bedding was c a r r i e d out in Vestopal W. Survey semi t h i n and f i ne s e c t i o n s were cu t in a LKB Ultrotome I or I I I . The survey s e c t i on s were s t a i n ed with t o i u id i ne blue and the f i ne s e c t i o ns with uranyl a c e t a t e and l ead c i t r a t e . Some of the f i ne s e c t i on s were c o l l e c t e d on gold g r i d s and s t a i n ed with pe­r i o d i c a c id - 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) (see 6 6 , 69) . L igh t microscopi c examinat ion was c a r r i e d out in a L e i t z Dialux 20 Photomic­roscope and e l e c t r o n microscopi c examinat ion in a P h i l i p s EM 300 o r in a Siemens Elmiskop 1A.

Feta l human h ea r t s (V) : The h ea r t s were i n i t i a l l y f i xed by add i t i on of 2.5% g lu t a r a ldehyde to the per fus ion f l u i d . Af te r the pe r f u s i on , the h ea r t s were placed in g lu t a r a ldehyde for a f u r t h e r 2 h. The he a r t s were then r i n sed in Ty rode ' s bu f f e r and s e l e c t ed a r ea s were c u t out . P o s t f i x a t i o n and f u r t h e r procedures were as de sc r ibed fo r the bovine h e a r t s .

12

MAIN OBSERVATIONS

The obse r va t i o ns on the Pu rk i n j e f i b r e s a r e descr i bed in r e l a t i o n to the spe c i e s s t ud i ed and the t echn iques used. The t i s s u e s s t ud i ed in p a r a l l e l a r e a l so 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 the r e s u l t s a re found in the o r i g in a l papers .

COW

Semi t h i n p l a s t i c s e c t i on s ( I ) : Even a t the e a r l i e s t s t age s t ud i ed (CR-length 7 cm) t he Pu rk in 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 rd ina ry v e n t r i c u l a r myocytes. The d i s t i n g u i s h i n g f ea t u r e s f o r the Pu rk i n j e f i b r e s were t h e i r ag­g r e ga t i on i n to bundles , the connec t i ve t i s s u e sheath and the c l e a r cytoplasm o f the c e l l s . However, in the l a t e f e t a l s t age the Pu r k i n j e f i b r e s were moree a s i l y d i s t i n g u i s h e d , due to a d i f f e r e n ce in c e l l diameter : They had l a r g e rd iame te r s than t he o rd inary v e n t r i c u l a r myocytes a t t h i s s t ag e , whi le in e a r l y f e t a l s t ages the two c e l l types had approximately the same d iameter s . Fur thermore, in f e t u s e s with CR-lengths 20 cm and more the 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 ed from o rd ina ry v e n t r i c u l a r myocytes by a s t r ong e r PAS-re- a c t i o n , whi le in smal le r f e t u s e s the t i s s u e s showed a comparable PAS-reac- t i o n . The Pu rk in j e f i b r e s seemed to become b in uc l e a t e more f r e qu en t l y as age i n c r ea s ed . Occasional m i t o t i c f i gu re s were observed in the Pu rk in j e f i b r e s , wh ile they were more f r equen t in the ord inary v e n t r i c u l a r myocytes.

Immunohistochemistry ( I , I I I ) : In a l l the f e t a l s t age s s t ud ied the Pu rk in j e f i b r e s were d i s t i n g u i s h ed from the o rd inary v e n t r i c u l a r myocytes by an i n t e n se f l uo re scence in s e c t i o ns incuba t ed with an t i bo d i e s ag a i n s t the i n t e rm ed ia t e f i l amen t subun i t s k e l e t i n . The s t r ong f l uo re scence in the Pu rk i n j e f i b r e s emanated from the cen t r a l p a r t of the cytoplasm of the c e l l s . ’ The nucle i were devoid of f l uore scence and the f l uore scence was weaka t c e l l borders f ac ing o the r c e l l s in the bundles .

The M-l ine p ro t e in MM-creatine k inase was de t ec t ed a t an e a r l i e r s t age in Pu rk i n j e f i b r e s and a t r i a l myocytes than in o rd ina ry v e n t r i c u l a r myocytes. The o t h e r M-line p r o t e in , myomesin, could be de t ec t ed in a l l t h r ee ce l l t ypes even a t the e a r l i e s t s t age s t ud ied (CR-length 5 cm). At t h i s s t age , however, d e t e c t a b i l i t y of myomesin was more obvious in the Pu rk i n j e f i b r e s than in t he o rd ina ry v e n t r i c u l a r myocytes.

Enzyme h i s t och em is t ry ( I , I I I ) : The Pu rk in j e f i b r e s showed a higher a c t i v i t y o f SDH than t he o rd ina ry v e n t r i c u l a r myocytes in f e t u se s with CR-length 32 cm or l e s s . In l a r g e r f e t u se s the a c t i v i t y of SDH seemed to be equal in the two ce l l pop u l a t i ons , while in a d u l t he a r t s the Pu rk i n j e f i b r e s showed a lower a c t i v i t y . The Purk in j e f i b r e s showed a h igher a c t i v i t y o f « - g ly c e ro ­phosphate dehydrogenase (a high a c t i v i t y c o r r e l a t e s with g l y c o l y t i c a c t i v i ­t i e s ) than the ord inary v e n t r i c u l a r myocytes, with the except ion t h a t , in h e a r t s of s t age s 11-32 cm, the ord inary v e n t r i c u l a r myocytes eve n tua l l y ex­h 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 of SDH and a -g l ycerophospha t e de­hydrogenase of Pu rk i n j e f i b r e s seemed to remain unchanged from ea r l y to l a t e f e t a l s t age s . The a c t i v i t y of SDH g radua l l y i nc r ea sed for both ord ina ry ven­t r i c u l a r and a t r i a l myocytes. The a c t i v i t y of a -g lyc e ro pho sph a t e dehydro­genase was h igher in the a t r i a than in the v e n t r i c l e s of the sm a l l e s t f e ­t u s e s (CR-length 5 and 8 .5 cm), lower in the a t r i a of l a r g e r f e t u se s and low in both t i s s u e s of a d u l t he a r t s .

13

Elec t ron microscopy ( I I ) : In the e a r l i e s t s t age s (CR-length 7 and 10 cm) t he appearance of the i n t e r c a l a t e d d i sks and the m y of ib r i l s was qu i t e s i m i l a r in Pu rk in j e f i b r e s and o rd ina ry v e n t r i c u l a r myocytes. With i nc r e a s i ng g e s t a ­t i o na l age the l a t t e r c e l l s became more packed with m yo f ib r i l s and a t t a i n e d more s t ep -w i se formed i n t e r c a l a t e d d i sk s . The m y o f i b r i l l a r M-band became more ev iden t with age, the Pu rk in j e f i b r e s c l e a r l y pr eceeding the o rd ina ry v e n t r i c u l a r myocytes in t h i s r e s pe c t . L e p t o f i b r i l s were observed in both ce l l types only in old f e t u s e s . I n t e rmedia t e f i lamens were more abundant in t he Pu rk i n j e f i b r e s . Myof i lament-polyribosome complexes t yp i ca l of a d u l t cow Pu rk in j e f i b r e s were not observed. The mi tochondr ia were few and s c a t t e r e d i n the cytoplasm in both ce l l types in e a r l y s t ag e s . In the o l d e s t f e t u se s the mi tochondr ia formed l ong i t ud in a l rows between the m y of ib r i l s and pe r i nu ­c l e a r masses around the nuclei in the ord inary v e n t r i c u l a r myocytes, whi le they were s t i l l s c a t t e r e d in the cytoplasm in the Pu rk i n j e f i b r e s . In the e a r l i e s t s t age s both ce l l types had abundant glycogen g r anu l e s , whi le in l a t e r s t age s the amount was h igher in the Pu rk i n j e f i b r e s . T - tubul e s were seen only in the ord inary v e n t r i c u l a r myocytes of the l a r g e s t f e t u s .

MAN

Immunohistochemistry ( IV, V) : In s e c t i on s i ncubat ed wi th an t i bo d i e s ag a i n s t the i n t e rmed ia t e f i l amen t subuni t s k e l e t i n a subendocardia l c e l l popul at i on was d i s t i n g u i s h e d from the o rd inary v e n t r i c u l a r myocardium in the human f e ­t a l he a r t s a t m idgest a t i on by an i n t ense f l uo re scence . By s e r i a l s ec t i o n i ng i t was observed t h a t the bundle branches took con t a c t with t h i s c e l l popu­l a t i o n (see below). In f a c t , p a r t s of the bundle branches were a l so composed o f i n t e n se ly f l u o r e s c i n g c e l l s . A s h i f t from moderate f l uo re scence (o f com­pa rab l e magni tude to t h a t of the ord inary myocardium) to i n t ense f l u o r e s ­cence occur r ed in the i n i t i a l p a r t of the l e f t bundle branch and in the d i s ­t a l i n t r a s e p t a l p a r t of the r i g h t bundle branch. The i n t en se l y f l u o r e s c i ng c e l l s a re i n t e r p r e t e d as the Pu rk i n j e f i b r e s .

Also the o rd ina ry v e n t r i c u l a r and a t r i a l myocytes showed f l u o re sce nc e , which emanated from the Z d isk s and the i n t e r c a l a t e d disk r eg i on s , in s e c t i on s i n ­cubated wi th a n t i s k e l e t i n an t i b o d i e s . The f l uo re scence in the more i n t en se l y f l u o re sc i n g Pu rk in j e f i b r e s was more uniformly spread in the cytoplasm.

The Pu rk in j e f i b r e s and the o rd ina ry v e n t r i c u l a r and a t r i a l myocytes of the f e t a l he a r t s a t midges t a t i on 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 on s incubat ed wi th an t i b od i e s ag a i n s t the M-line p ro t e in myomesin. The Pu rk 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 to t h e i r high s k e l e t i n r e ac ­t i v i t y and high Cho l i ne s t e r a se a c t i v i t y ( see below). In s ec t i on s incubat ed wi th a n t i b od i e s a g a in s t the M-l ine p ro t e in MM-creatine k ina se the Pu rk in j e f i b r e s and some a t r i a l myocytes showed a c r o s s - s t r i a t e d p a t t e r n , whi le the o rd ina ry v e n t r i c u l a r myocytes did not . In ad u l t hea r t s s t r i a t i o n s were e v i ­dent in the myocytes of a l l t h r ee t i s s u e s in s e c t i o ns t r e a t e d to demonstrate presence of MM-creatine k ina se .

Enzyme h i s t o che mi s t ry (IV, V) : Sec t i ons s e r i a l to those i ncubat ed with a n t i - bodies were prepared for enzyme h i s t o che m is t ry . This enabled the i d e n t i f i c a ­t i o n of the d i f f e r e n t p a r t s of the conduct ion system (IV). All pa r t s of the conduct i on system showed a high a c t i v i t y of C ho l i n e s t e r a se .

E lec t ron microscopy (V) : Ult r a s t r u c t u r a l l y two ce l l popu la t ions were d i s t i n - guished in the v e n t r i c l e s in the human f e t a l h ea r t s : A subendocardial c e l l popu la t ion in which the ma j o r i t y of the my o f i b r i l s showed dense M-bands and t he r e s t of the myocardium in which the my o f i b r i l s showed no dense M-bands.

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The subendocardia l c e l l popul a t ion i s i n t e r p r e t e d to r e p r e s en t the Pu rk in j e f i b r e s . Also in some reg ions in the a t r i a the m y of ib r i l s showed dense M-bands.

Pu rk in j e f i b r e s formed bundles or l oose ly a rranged ce l l format ions su r ­rounded by abundant connec t i ve t i s s u e and few c a p i l l a r i e s . Some of them were l y in g in c on t ac t wi th o rd ina ry myocardial t i s s u e . In general the Pu rk in j e f i b r e s had a more rounded c e l l u l a r and nuc lea r shape and l a r g e r diameters than the ord ina ry v e n t r i c u l a r and a t r i a l myocytes. The number of sp ec i a l i z e d c e l l con t a c t s seemed to be h ig hes t between the Pu rk in j e f i b r e s . I n t e rmed ia te f i l am en t s were most f r equen t in the Pu rk in j e f i b r e s . The my of ib r i l s occupied only pa r t s of the cytoplasm and had a varying appearance in a l l t h r ee ce l l types s t ud i ed ; howewer they va r i ed to the g r e a t e s t ex t e n t in the Pu rk in j e f i b r e s . Several f e a t u r e s were common to the t h r ee ce l l t ypes : eg. a s i ng l e nucleus in a lmost a l l c e l l s , no wel l - formed s tepwi se i n t e r c a l a t e d d i sk s , presence of e l e c t r o n dense masses (mainly composed of f i l amen t s and r i bo ­somes), few and s c a t t e r e d mi tochondr i a , abundant glycogen granu les and no T- t ub u l e s . In the a t r i a l myocytes t he 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 anul e s .

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DISCUSSION

THE PURKINJE FIBRES IN THE DEVELOPING HEART

The development of "highly d i f f e r e n t i a t e d " ( " t y p i c a l " ) (bovine) and " l e s s d i f f e r e n t i a t e d " (human) Pu rk in j e f i b r e s ( c f . 50, 76) has been s t ud ied in the p r e s en t i n v e s t i g a t i o n . A subendocardial c e l l popul a t ion in the v e n t r i c l e s , i n t e r p r e t e d t o r ep r e s e n t the Pu r k in j e f i b r e s , could be d i s t i ng u i sh ed in both the bovine and human f e t a l h e a r t s . The accuracy 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 cussed in r e l a t i o n t o t he i d e n t i f i c a t i o n , the terminology and the func t i on of the c e l l s .

I d e n t i f i c a t i o n

One way to i d e n t i f y the Pu rk in j e f i b r e s in the v e n t r i c l e s i s to t r a c e the d i s t r i b u t i o n of t he bundle branches p e r i p h e r a l l y . This was enabled by s e r i a l s ec t i o n i ng and by the f a c t t h a t the bundle branches were c l e a r l y d i s t i n ­guished from o rd ina ry myocardium by enzyme h i s t oc he m is t ry . For both the bo­v ine and the human f e t a l h ea r t s i t was c l e a r l y demons trated t h a t the bundle branches became d i s t r i b u t e d i n to the subendocardia l c e l l pop u l a t i ons . Thus t he s e c e l l popu la t ions r e p r e s en t the conduct ion c e l l s in the v e n t r i c l e s .

Another way to " i d en t i f y " Pu rk in j e f i b r ë s in the v e n t r i c l e s in t i s s u e sec­t i o n s would be to search fo r c e l l s wi th "Purk inj e f i b r e - l i k e " morphological c h a r a c t e r i s t i c s . That was performed in specimens of subendocardia l r egions o f the v e n t r i c l e s and in t r an sv e r s e s e c t i on s through the 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 looked fo r were the ones de scr i bed to be t yp i ca l of ad u l t Pu rk in j e f i b r e s , such as t h e i r gross morphological f e a t u r e s , a high con ten t o f s k e l e t i n , a high con t en t of glycogen and t yp i ca l enzyme h is tochemical c h a r a c t e r i s t i c s wi th r e s pe c t to ox ida t i ve and g ly c o l y t i c a c t i v i t i e s ( c f . 1 , 17, 23, 46, 50, 60, 75) .

In the bovine f e t a l h e a r t s , g ros s morphological 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 the Pu rk in j e f i b r e s were a p p l i c a b l e a t the e a r l i e s t s t age i n v e s t i g a t e d (CR-length 7 cm). The d i s t i n c t i o n was, however, c l e a r e r in l a t e r s t a ge 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 rd ina ry myocardium by a h igher con t en t o f s k e l e t i n , as seen immunohistochemical ly. The metabol i c c r i t e r i a f o r the i d e n t i f i c a t i o n of bovine Pu rk i n j e f i b r e s changed dur ing development.

Also in the human f e t a l h e a r t s , subendocardial c e l l s surrounded by connec­t i v e t i s s u e were d i s t i n g u i s h e d in the v e n t r i c l e s in semi t h i n p l a s t i c sec ­t i o n s (unpubl ished observa t ions ) . . However, i t was not po s s ib l e to unequivo­c a l l y i d e n t i f y t he se c e l l s as Pu rk i n j e f i b r e s ( c f . 40, 56, 86 ) . On the o the r hand, the Pu rk in j e f i b r e s were i d e n t i f i e d due to t h e i r high con t en t of sk e l ­e t i n , as seen immunohistochemical ly. Also with o the r t echn iques a c l e a r sep­a r a t i o n between two c e l l types in the v e n t r i c l e s was p o s s ib l e . The Pu rk in j e f i b r e s , in comparison with the c e l l s in the r e s t of the v e n t r i c u l a r myocar­dium, showed a h igher a c t i v i t y of Cho l i ne s t e r a se , were l a b e l l e d by an t ibod­i e s ag a in s t MM-creatine k inase and showed M-bands u l t r a s t r u c t u r a l l y . The use of immuno- and enzyme h i s t oc hem is t ry had thus enhanced the p o s s i b i l i t y to i d e n t i f y Pu rk in j e f i b r e s in the human f e t a l he a r t a t the l i g h t microscopic l e v e l . In f a c t , a l r e ady a t ten weeks ge s t a t i on a l age an ex t ens ive ce l l popu­l a t i o n i s d i s t i n g u i s h ed subendocardi a l l y in the v e n t r i c l e s , due to a high con t en t of s k e l e t i n and a high a c t i v i t y of Cho l i ne s t e r a se (unpubl ished ob­s e r v a t i o n s ) . Fur thermore, i t was pos s ib l e to 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 the

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human f e t a l Pu rk in j e f i b r e s a t midges t a t i on as these c e l l s but not the o r d i ­nary myocytes in the v e n t r i c l e s had a t t a i n e d e l ec t r o n dense M-bands and con­t a i ne d MM-creat ine k ina se . Immunohistochemical de t ec t i o n of MM-creatine k i ­nase c o r r e l a t e s with the presence of an e l e c t r o n dense M-band (64, 65, 85) . The func t i on of the M-l ine bound MM-creat ine k inase i s proposed to be a par ­t i c i p a t i o n in the r e gu l a t i o n of the ATP metabolism r e l a t e d to muscular con­t r a c t i o n (65, 85 ) .

Terminology

I f we conclude t h a t the c e l l s in the subendocardial c e l l popu la t ions are conduct 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 ng u i sh ed from ord inary myo­cardium on the same ba s i s as a d u l t Pu rk in j e f i b r e s , i s i t a ccu ra t e to c a l l t he c e l l s Pu rk in j e f i b r e s ? C lea r l y the c e l l s in the bovine f e t a l h e a r t s , from an ea r l y f e t a l s t ag e , can be c a l l e d Pu rk in j e f i b r e s , mainly as they could be d i s t i ng u i sh ed by gros s morphological f e a t u r e s and a high con t en t of s k e l e t i n . These f e a t u r e s are two main c r i t e r i a of a d u l t Pu rk in j e f i b r e s (e . g. 23, 76) .

A high con t en t of s k e l e t i n , as demonstrated immunohistochemical ly ( IV) , can a l s o be used as a c r i t e r i o n to def i ne human Pu rk in j e f i b r e s ( c f . 23) . Thus from a t l e a s t ten weeks ge s t a t i o n a l age, which i s the e a r l i e s t s t age we have s t u d i e d h i t h e r t o , and onwards, conduct ion c e l l s which mer i t the name Pur­k in j e f i b r e s are p r e s en t in human f e t a l h e a r t s . Fur thermore, we observed t h a t t he 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 occur r ed a t spec i a l l e v e l s : In the i n i t i a l p a r t of the l e f t bundle branch and in the d i s t a l p a r t of the i n t r a ­mural r i g h t bundle branch. Thus from these l e v e l s and f u r t h e r p e r i p h e ra l l y t he conduct i on c e l l s might be ca l l ed Pu rk in j e f i b r e s . This i s of s i g n i f i ­cance fo r the human conduct ion system as the border between bundle branch and pe r i phe ra l Pu rk i n j e f i b r e c e l l types f o r a long t ime has been d i scussedand not been determined ( c f . 28, 62 ) .

Funct i onal a spec t s

What do the obse rva t i ons in the p r e sen t s t u d i e s imply wi th r e sp ec t to the func t i on of f e t a l Pu rk in j e f i b r e s , as compared with a d u l t ones? E lec t rophys- i o l o g i c a l l y the behaviour of the human f e t a l h e a r t i s comparable to t h a t of t he a d u l t he a r t (25, 35, 79) . J ans e e t al (35) argued t h a t the e l e c t r o p h y s i ­ol ogica l ma tur i ty of the human f e t a l h ea r t was produced d e sp i t e a "morpholo­g ica l immaturi ty" of the conduct ion system. However, the p r e s en t s t ud i e s i n ­d i c a t e t h a t the f e t a l Pu rk in j e f i b r e s are not "immature". Morphologica l ly i t appears t h a t the c e l l s i n s t e ad have a sp e c i a l i z e d func t i on a l r eady a t e a r l y f e t a l s t age s .

The format ion a f bundles a l r eady a t the e a r l y f e t a l s t age of bovine hea r t s may favour t h a t the Pu rk in j e f i b r e s e a r l y have f a s t conduct ion p r o p e r t i e s . Whether nexuses are more abundant between Pu rk in j e f i b r e s than ord inary myo­cy t e s a l r eady in f e t a l h e a r t s , which i s our p r e l im inary opinion (V), mustawa it 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 ly ce r op hos ph a t e de­hydrogenase and t he presence of dense M-bands in the bovine Pu rk in j e f i b r e s a t e a r l y f e t a l s t age s shows t h a t the c e l l s a t the se s t ages are more d i f f e r ­e n t i a t e d than the o rd inary myocytes ( see below). The high a c t i v i t y of the two enzymes favours t h a t the energy demand i s h igher in the Pu rk in j e f i b r e s as compared wi th the o rd ina ry myocytes a t e a r l y f e t a l s t age s . The f a c t t h a t s k e l e t i n i s p r e s en t in l a rge amounts in f e t a l Pu rk in j e f i b r e s , pos s ib ly i n ­s t e ad of m y o f i b r i l l a r p r o t e in s , sugges ts t h a t the c e l l s e a r l y are not d i ­r ec t ed to c o n t r a c t i l i t y .

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The high a c t i v i t y of Cho l i n e s t e r a se in the human f e t a l Pu rk i n j e f i b r e s i s l i k e l y to r e f l e c t high a c t i v i t y of the Pu rk i n j e f i b r e s t hemselves , as no nerves were seen with c e r t a i n t y . In the ad u l t human h e a r t , on the o the r hand, c h o l i n e s t e r a s e - p o s i t i v e nerves are f r equen t in a s s o c i a t i o n with the Pu rk i n j e f i b r e s (37) . One i n t e r p r e t a t i o n i s t h a t the high a c t i v i t y observed in the p r e se n t study i s r e l a t e d to a process of d i f f e r e n t i a t i o n of the Pur­k in j e f i b r e s ( c f . 54) . Another i n t e r p r e t a t i o n i s t h a t i t r e f l e c t s an ea r l y s t ep in the development of nerves , as a high C ho l in e s t e r a se a c t i v i t y i s de­t e c t e d in a t i s s u e p r i o r to neurogenes is occurs (36) . Species d i f f e r e nc es wi th r e s pe c t to C ho l i ne s t e r a se e x i s t : The bovine f e t a l Pu rk in j e f i b r e s , in c o n t r a s t to the human ones, do not show a c l e a r l y h igher a c t i v i t y than o r d i ­nary myocytes (unpub l i shed ob se r va t i o ns ) .

THE DIFFERENTIATION PATTERN OF THE PURKINJE FIBRES

Pu rk in j e f i b r e s , forming an ex t ens ive subendocardial popul at i on of c e l l s , were a l r e ady d i s t i n g u i s h ed a t e a r l y f e t a l s t ages of both human (unpubl ished obse r va t i ons ) and bovine h e a r t s . This observa t ion suppo r t s the proposal t h a t t he Pu r k in j e f i b r e s d i f f e r e n t i a t e in s i t u in the v e n t r i c l e s ( c f . 3 , 34, 84 ) . The d i f f e r e n t i a t i o n pa t t e r n of the c e l l s can be d i s cussed in r e l a t i o n to t h a t of o rd inary v e n t r i c u l a r and a t r i a l myocytes.

Comparison wi th o rd ina ry v e n t r i c u l a r myocytes

The mode of development of the o rd ina ry v e n t r i c u l a r myocytes in the bovine he a r t s conformed to previous d e s c r i p t i o n s fo r o the r sp ec i e s . This i nc ludes a gradual ma tur a t i on of the m y o f i b r i l l a r apparatus and the i n t e r c a l a t e d d i s k s , a g r adua l l y i nc r e a s i ng aerobi c and decr eas ing anae robi c metabolism and a compa ra t i ve ly l a t e development of T - tub u l e s . However, t he r e are i n t e r s p e c i e s d i f f e r e n c e s with r e s pe c t to myocardial matur a t ion a t b i r t h . I t seems as i f t he e x t e n t to which the o rd ina ry myocytes in neonates are d i f f e r e n t i a t e d cor r esponds to the degree of general development of the animal . Thus, the newborn c a l f i s born in a qu i t e mature s t a t e and the o rd ina ry myocytes are we l l - deve loped ( e . g . have T - tu b u l e s ) , whi le e . g . the r a t and the mouse are born in a more immature s t a t e and the ord inary myocytes a re l e s s d i f f e r e n ­t i a t e d ( c f . 30, 31, 39) .

Both the o rd ina ry v e n t r i c u l a r myocytes and, in c o n t r a s t to previous d e s c r i p ­t i o n s (24, 29 ) , the Pu rk in j e f i b r e s , e xh i b i t ed mi to t i c f i g u r e s . Both ce l l t ypes a l so d iv ide m i t o t i c a l l y in the human f e t a l he a r t a t m idgest a t i on (un­pub l i shed ob se r v a t i o n s ) . In the r a t m i t o t i c d i v i s i on of o rd inary myocytes i s s t i l l appa rent a t b i r t h but has almost completely ceased wi th in the f i r s t few weeks of neonatal l i f e (89) . He re a f t e r the o rd ina ry myocytes en l a rge but do not p r o l i f e r a t e .

The Pu rk in j e f i b r e s in the bovine f e t a l h ea r t s develop along a qu i t e d i f f e r ­en t pathway from the o rd ina ry v e n t r i c u l a r myocytes. Some f e a t u r e s were d i s ­t i n g u i s h in g through a l l f e t a l s t age s : The bundle forma tion , the more rounded c e l l u l a r and nuc lea r shape and the h igher s k e l e t i n co n t en t of the Pu rk in j e f i b r e s . Other d i f f e r e n c e s between the ce l l types were man i f e s t only in the l a t e f e t a l s t age: The amount and the o r i e n t a t i o n of the m yo f ib r i l s , the a r ­rangement of the i n t e r c a l a t e d d i sks and the format ion of T - tub u l e s . The two ce l l types showed qu i t e d i f f e r e n t d i f f e r e n t i a t i o n p a t t e r n s with r e s p ec t to c e l l metabolism and development of the m y o f i b r i l l a r M-band.

As evidenced from both enzyme a c t i v i t i e s and u l t r a s t r u c t u r a l obse rva t i ons t he metabol i c d i f f e r e n c e s between Pu rk i n j e f i b r e s and o rd ina ry v e n t r i c u l a r

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myocytes in bovine f e t a l he a r t s are qu i t e d i f f e r e n t a t var i ous s t age s of de­velopment. I t seems as i f t h i s i s mainly due to the matur a t ion of the o r d i ­nary v e n t r i c u l a r myocytes. The a c t i v i t i e s of SDH and a -g lyce rop hos pha t e de­hydrogenase in t he se c e l l s , but not in the Pu rk i n j e f i b r e s , changed cons id­e r ab l y dur ing f e t a l development. Also u l t r a s t r u c t u r a l l y (amount of glycogen- g r anu l e s , arrangement of mi tochondr ia) t he changes in the Pu rk i n j e f i b r e s were minimal as compared with the o rd ina ry v e n t r i c u l a r myocytes. These ob­s e r v a t i o n s may r e f l e c t an ea r l y s p e c i a l i z a t i o n of the Pu rk i n j e f i b r e s (see above) .

The proces s of d i f f e r e n t i a t i o n of c a rd i ac myocytes i nvolves the o rgan i za t i on o f c o n t r a c t i l e p r o t e in s i n to c h a r a c t e r i s t i c c r o s s - s t r i a t e d m yo f ib r i l s . V i s i ­b l e M-bands in the sarcomeres i s a comparat i ve ly l a t e phenomenon and r ep re ­s e n t s a s ign of te rminal m y o f i b r i l l a r matur at i on (eg. 2 , 5 ) . The presence of dense M-bands and the d e t e c t i o n of MM-creatine kinase a t an e a r l i e r s t age in Pu rk in j e f i b r e s than in o rd ina ry v e n t r i c u l a r myocytes shows t h a t t h i s p a r t o f f i n a l m y o f i b r i l l a r ma tu ra t ion proceeds independent ly and t imely sepa ra t ed in the two t i s s u e s . The second M-line p ro t e in known a t p r e s en t , myomesin, was de t ec t ed in both Pu rk i n j e f i b r e s and o rd ina ry v e n t r i c u l a r myocytes a t e a r l y s t ages of bovine f e t u s e s . However, the s t r i a t i o n s were more c l e a r l y seen in the Pu rk i n j e f i b r e s a t t he se s t ag e s . This p ro t e in has been repo r ted t o be an i n t eg ra l component of the m y o f i b r i l l a r s t r u c t u r e and to be p r e sen t a l r e ady a t the e a r l i e s t s t age s of my of ib r i l l o ge n es i s in chicken sk e l e t a l muscle c e l l s (21, 65) .

Comparison wi th a t r i a l myocytes

The a t r i a l myocytes in the human f e t a l h ea r t s a t m idgest a t i on mainly d i f ­f e r ed from the o rd inary v e n t r i c u l a r myocytes by presence of dense M-bands and so c a l l e d a t r i a l s p e c i f i c g r anul e s . Other d i f f e r e n c e s wi l l develop a t l a t e r s t age s of development (s ee I I I ) . In the bovine f e t a l he a r t s we ob­served t h a t the a t r i a l myocytes e a r l i e r a t t a i n e d m y o f i b r i l l a r M-bands and showed a d i f f e r e n t pathway of g ly c o l y t i c a c t i v i t i e s than the o rd ina ry ven­t r i c u l a r myocytes.

With r e s p e c t to t he development of the m y o f i b r i l l a r M-band, the Pu rk in j e f i b r e s ' a r e more “a t r i a l - l i k e " than " v e n t r i c u l a r - ! i k e " . In f a c t , we have ob­served a l so o the r s i m i l a r i t i e s between P u rk in j e f i b r e s and a t r i a l myocytes i n f e t a l h ea r t s : Darkly s t a i n ed dense bodi es , which appear s i m i l a r to the a t r i a l sp e c i f i c g ranu l es t yp i ca l of a t r i a l myocytes, a re p r e s en t in Pu rk i n j e f i b r e s but not in o rd inary v e n t r i c u l a r myocytes in human f e t a l h e a r t s . Fur­t hermore , in e a r l y f e t a l s t age s of the bovine he a r t the Pu rk i n j e f i b r e s as well as the a t r i a l myocytes show an i n t ense f l uo re scence in s ec t i on s incu­ba t ed wi th ah t i bod i e s ag a in s t a t r i a l myosin (unpubl ished ob se r v a t i o n s ) .

In the a d u l t h e a r t t he r e are d i f f e r e n c es between a t r i a l and v e n t r i c u l a r myo­cy t e s which, in p r i n c i p a l , a re comparable to d i f f e r e n c es between Pu rk in j e f i b r e s and v e n t r i c u l a r myocytes: These inc lude l e s s s tepwi se formed i n t e r c a ­l a t e d d i sk s , fewer mi tochond r ia , l e s s developed T - tubu lu s system, more g ly­cogen and somewhat lower a c t i v i t y of ox id a t i ve enzymes in a t r i a l vs v e n t r i ­c u l a r myocytes (12, 15, 31, 38, 44, 74) . The d i f f e r e n c e s a r e , however, prob­ably more pronounced beween Pu rk in j e f i b r e s and v e n t r i c u l a r myocytes. A problem i s t h a t comparisons between a t r i a l myocytes and Pu rk i n j e f i b r e s are very few in the l i t e r a t u r e . However, i t i s c l e a r t h a t the P u rk in j e f i b r e s in seve ra l r e s pe c t s are more " a t r i a l - l i k e " than " v e n t r i c u l a r - l i k e " both during d i f f e r e n t i a t i o n and in the a d u l t s t age . Ne ve r the l e s s , t he r e are c l e a r d i f ­f e r e nce s between Pu rk i n j e f i b r e s and a t r i a l myocytes, eg. with r e s pe c t to

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ce l l shape and volume ( c f . 38, 77) .

Comment on t he proposed "embryonic o rgan i za t i on"

The ques t i on of whether Pu rk in j e f i b r e s show an "embryonic o rgan i za t i o n" or no t i s i n t i m a t e l y r e l a t e d to the d e f i n i t i o n of t h i s "embryonic o rgan i za ­t i o n " . I t i s a f a c t t h a t the m y of ib r i l s a re l e s s p a r a l l e l l y a r ranged, the i n t e r c a l a t e d d i sk s have another c on f i gu r a t i on ( l e s s s t epwi se formed) and the g l y c o l y t i c a c t i v i t i e s are h igher in Pu rk i n j e f i b r e s than in ord ina ry ven­t r i c u l a r myocytes in the ad u l t h ea r t . These are t yp i ca l examples of f e a t u r e s by which t he Pu rk i n j e f i b r e s are a s c r i bed to show s i m i l a r i t i e s to u n d i f f e r ­e n t i a t e d o rd ina ry myocytes (s ee 18, 47 ) . However, what i s most impor t ant i s t h a t the Pu rk i n j e f i b r e s do not show an "embryonic o rgan i za t i on " in the sense t h a t the c e l l s a t t a i n t h e i r t yp i ca l f e a t u r e s by a " r e t a rd a t i o n " of f e a t u r e s of u n d i f f e r e n t i a t e d o rd ina ry myocytes. I n s t ead the p r e sen t s t ud i e s show t h a t t he two c e l l types in s everal r e s p e c t s are d i f f e r e n t a l r eady a t e a r l y s t ag e s , develop along d i f f e r e n t pathways and t h a t in f a c t the Pu rk i n j e f i b r e s in some r e s p ec t s e a r l i e r show f e a t u r e s t yp i ca l of a mature , d i f f e r e n ­t i a t e d s t a t e than the ordina ry myocytes. The presence of a m y o f i b r i l l a r M- band and a high a c t i v i t y of ox ida t i ve enzymes are such f e a t u r e s .

With r e s p ec t to t he proposed "embryonal i ty" of Pu rk i n j e f i b r e s comments can a l s o be made on s p e c i f i c o r g a n e l l e s . The myof i lament -polyr ibosome complexes t yp i ca l o f a d u l t Pu rk in j e f i b r e s do not r e p r e s e n t c e s s a t i o n of m y of ib r i l l o - genes i s a t an embryonic s t age ( c f . 51 ) , as these complexes were not observed in the f e t a l h e a r t s . L e p t o f i b r i l s a re not embryonic remnants , as p r ev ious ly proposed (13 ) , but develop with age ( c f . 11) . Recent ly i t was a l s o shown t h a t l e p t o f i b r i l s appear in o rd ina ry myocytes as a r e s u l t of mechanical un­load ing (73) . The high con t en t of s k e l e t i n as seen immunohistochemical ly and t he high amount of i n t e rmed ia t e f i l amen t s as seen u l t r a s t r u c t u r a l l y in the Pu rk in j e f i b r e s i s in favour of d i f f e r e n t i a t i o n and s p e c i a l i z a t i o n . Sk e l e t i n seems to r ep r e s en t a sp ec i a l i z e d form of i n t e rmed ia t e f i l am en t p r o t e in : Dur­ing sk e l e t a l muscle d i f f e r e n t i a t i o n t he r e i s a s h i f t of syn t he s i s from one type of i n t e rm ed ia t e f i l amen t subun i t (viment in) t o the muscle s p e c i f i c type (desmin, sk e l e t i n ) (10, 8 8 ) . The abundance of i n t e rmed ia t e (sometimes e r r o ­neously de scr i bed as " f i ne" ) f i l am en t s in a d u l t Pu rk i n j e f i b r e s has been one o f t he f e a t u r e s which have been r e l a t e d to an "embryonic o rgan i za t i o n" (6 ) .

F u r t he r s t ud i e s on the d i f f e r e n t i a t i o n p a t t e r n s o f the Pu rk i n j e f i b r e s , the c e l l s of the proximal p a r t s of the conduct ion system and the a t r i a l and v e n t r i c u l a r myocytes are in p rogre ss in our l a b o r a t o r y . One obse rva t i on i s t h a t t he m y o f i b r i l l a r M-band, as evidenced by de t e c t i o n of MM-creatine k ina s e , develops a t a l a t e r s t age in the AV node c e l l s than in the Pu rk i n j e f i b r e s and a t r i a l myocytes in t he bovine f e t a l h e a r t . This shows t h a t m y o f i b r i l l a r ma tu ra t ion proceeds d i f f e r e n t l y not only between Pu rk in j e f i b r e s and o rd ina ry myocytes but a l so between Pu rk i n j e f i b r e s and AY-node c e l l s . Another observa t ion i s t h a t the bovine Pu rk i n j e f i b r e s from being homogeneously s t a i n e d in e a r l y f e t a l s t age s become he te rogeneous ly s t a i n e d in l a t e f e t a l s t ag e s , in s e c t i o n s incubat ed wi th an t i b od i e s ag a in s t a t r i a l myosin. Thus, i t seems as i f the he t e ro gen e i t y of a d u l t Pu rk in j e f i b r e s wi th r e s p e c t to myosin composi t ion ( c f . 58, 72) i s a r e s u l t of a d i f f e r e n t i a t i o n p roce s s .

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GENERAL SUMMARY AND CONCLUSIONS

The development of Pu rk in j e f i b r e s and o rd ina ry v e n t r i c u l a r and a t r i a l myo­cy t e s in bovine and human f e t a l h ea r t s was s t ud ied :

1) By s e r i a l s e c t i o n in g and t r a c in g of the bundle branches p e r i p h e ra l l y the Pu rk in j e f i b r e s were i d e n t i f i e d . In both bovine and human f e t a l h ea r t s they could f u r t h e r be i d e n t i f i e d in s e c t i on s incubat ed wi th an t i bod i e s ag a in s t t he i n t e rmed ia t e f i l am en t subun i t s k e l e t i n , due to a s t r o nge r immunofluore­scence r e ac t i on than ord inary v e n t r i c u l a r myocytes. From ea r l y f e t a l s t ages and onwards the bovine Pu rk i n j e f i b r e s could a l so be d i s t i ng u i s he d in semi- t h i n p l a s t i c s e c t i o n s and u l t r a s t r u c t u r a l l y due to gross morphological f e a ­t u r e s ( aggrega t i on i n to bundles , a connect ive t i s s u e shea th , a c l e a r cy to­plasm). The Pu rk in j e f i b r e s in the human f e t a l hea r t s were not c l e a r l y iden­t i f i e d by such c r i t e r i a but could , on the o the r hand, be d i s c r im ina t ed from o rd ina ry myocytes due to a high a c t i v i t y of C ho l i ne s t e r a se .

2) U l t r a s t r u c t u r a l l y , the m y o f i b r i l l a r M-band became more ev iden t with age in the bovine f e t a l h e a r t s , the Pu rk in j e f i b r e s c l e a r l y preceeding the o r d i ­nary v e n t r i c u l a r myocytes in t h i s r e s p ec t . This was confi rmed by presence of MM-creat ine k ina s e , as demonstrated immunohistochemical ly, a t an e a r l i e r s t ag e in the former c e l l s . MM-creatine k inase i s known to make an e s s e n t i a l c o n t r i b u t i o n to t he e l e c t r on dens i t y of the M-band. In human f e t a l h ea r t s a t m idges t a t i on t he Pu rk i n j e f i b r e s , but not the ord inary v e n t r i c u l a r myocytes, were s t a i n ed in s e c t i o n s incuba ted with an t i b od i e s a g a in s t MM-creatine k ina ­se and showed M-bands u l t r a s t r u c t u r a l l y .

3) In the human f e t a l he a r t a t midges t a t i on and in the h ea r t s of small bovi­ne f e t u se s t he r e were few d i s t i n c t u l t r a s t r u c t u r a l d i f f e r e n c e s between Pur­kinje* f i b r e s and o rd inary v e n t r i c u l a r myocytes: Apart from the d i f f e r e n ce in appearance of the m y o f i b r i l l a r M-band the Pu rk i n j e f i b r e s had a g r e a t e r amount of i n t e rm ed i a t e f i l a m e n t s . In the l a t e f e t a l s t age of the bovine he a r t s t he r e were c l e a r d i f f e r e n ce s between the two ce l l t ypes . These i n ­cluded t he amount and o r i e n t a t i o n of the m y o f ib r i l s , the format ion of the i n t e r c a l a t e d d i s k s , the arrangement of the mi tochond r i a , the amount of g ly ­cogen and the format ion of T - tub u l e s .

4) The a c t i v i t i e s of succ in i c dehydrogenase (SDH) and a -g lyce rophospha t e de­hydrogenase seemed to remain unchanged in the Pu rk i n j e f i b r e s from ea r l y to l a t e f e t a l s t age s of the bovine h e a r t s . The a c t i v i t y of SDH g radua l l y i n ­c r ea sed for both o rd ina ry v e n t r i c u l a r and a t r i a l myocytes, whi le the a c t i v i ­t y of a -g lycerophospha t e dehydrogenase was high a t d i f f e r e n t s t ages of e a r l y f e t a l development in the two t i s s u e s to become low in the a d u l t s t age . The a c t i v i t y of SDrf o f the Pu rk in j e f i b r e s was h igher in small f e t u s e s , equal in l a r g e f e t u se s and lower in ad u l t h e a r t s , as compared with t h a t of o rd inary v e n t r i c u l a r myocytes. In a l l s t ages of the bovine h ea r t s the Pu rk i n j e f i b r e s showed a high a c t i v i t y of a-glycerophospha te dehydrogenase.

5) In a t r i a l myocytes of both bovine and human hea r t s e l e c t r o n dense M-bands and de t ec t i o n o f *MM-creatine kinase was ev iden t a t an e a r l i e r s t age than in o rd ina ry v e n t r i c u l a r myocytes. In the human f e t a l he a r t s a t midgest a t i on so c a l l e d a t r i a l s p e c i f i c g ranul es were numerous in the a t r i a l myocytes.

The obse rva t i ons show th a t :

21

1) Pu rk i n j e f i b r e s can be i d e n t i f i e d a l r eady a t an ea r l y f e t a l s t age .

2) The use of enzyme and immuno-histochemistry has enhanced the p o s s i b i l i t y t o i d e n t i f y the f e t a l Pu rk in j e f i b r e s . A high con t en t of s k e l e t i n , as demon­s t r a t e d immunohis tochemical ly , seems to be a general c r i t e r i o n to i d e n t i f y not only a d u l t , bu t a l so f e t a l , Pu rk in j e f i b r e s . By comparisons of immuno- h is tochemica l and u l t r a s t r u c t u r a l obse rva t i ons i t was p os s i b l e to d i s t i n ­guish the human f e t a l Pu rk in j e f i b r e s a t midges t a t i on a t the ul t r a s t r u c t u r a l 1 e v e l .

3) The Pu rk in j e f i b r e s show f ea t u r e s of a d i f f e r e n t i a t e d s t a t e , as format ion o f bund les , presence of dense M-bands and high a c t i v i t y of ox id a t i v e en­zymes, a t e a r l y f e t a l s t ag e s . This may sugges t t h a t the c e l l s e a r l y have a sp e c i a l i z e d func t i on .

4) The Pu rk in j e f i b r e s d i f f e r e n t i a t e along a l i n e s epa ra t e from o rd ina ry v e n t r i c u l a r myocytes.

5) The Pu rk in j e f i b r e s do not a t t a i n t h e i r t yp i ca l f e a t u r e s by a " r e t a r d a ­t i on " of f e a t u r e s of u n d i f f e r e n t i a t e d o rd ina ry myocytes. The d e s c r i p t i o n of an "embryonic o rg an i za t i o n" of a d u l t Pu rk i n j e f i b r e s i s thus mi s lead ing .

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ACKNOWLEDGEMENTS

I would l i k e to exp re ss my s i n ce r e ap p rec i a t i on and g r a t i t u d e to:

Doc La r s -E r i c Tho rn e l l , my su pe r v i s o r , who in t roduced me i n to the f i e l d of the h ea r t conduct ion system. His ex t ens ive knowledge in the r e s ea rch f i e l d of the h ea r t and the methodological procedures used i s g r e a t f u l l y acknowl­edged. His pa t i e n ce , encouraging and c o n s t ru c t i v e a t t i t u d e and never f a i l i n g personal suppor t have been p r e r e q u i s i t e s f o r the r e a l i z a t i o n of t h i s i nves­t i g a t i o n .

Doc Anders E r iks son , f o r p l e a s a n t c o l l a b o ra t i o n with immunohistochemical p a r t s , f o r h is knowledge in wordings , and fo r va luab l e c r i t i c i s m .

P ro f He rber t Helander , f or h i s p o s i t i v e a t t i t u d e when I i n i t i a l l y s e t f o o t upon the Research d i v i s i o n of the I n s t i t u t e .

Emanuel S t r e h l e r , PhD, fo r h i s knowledge in the mys te r i e s of M-band p r o t e ­i n s .

Eva Ca r l s son , MB, and Uno K j ö r e l l , DDS, f o r p l e a s an t c o l l a b o r a t i o n .

J e s p e r Barkman, Lena Ca r l s son , Margaretha E ne r s t e d t , B r i t t a Gy l l eb r ing , B i r g i t t a Holmbom, Inga Johans son, Marlene Lundström, and E l i s a b e t h Rubing f o r s k i l f u l t e chn i ca l a s s i s t a n c e .

Bror Berggren and B i r g i t Be rgva l l , f or the photographic work, and Bror a l so fo r h is knowledge in a l l photot echni ca l problems.

Grethel Jangvad, I ng r id N i l s son , Anna-Lena Sandström and Lolomai Örnehul t f o r typing my manuscr ip ts .

Bengt Andersson, Tors t en Edström, Yngve Lindst röm and Gösta Lundberg for va luab l e t e chn i ca l a s s i s t a n c e .

All o t he r members of the s t a f f a t the I n s t i t u t e of Anatomy, U n ive r s i t y of Umeå, who d i r e c t l y or i n d i r e c t l y have been involved in t h i s work.

Kar in , f o r her suppor t , and fo r being i ndu lgen t towards me dur ing these y e a r s .

This work was Supported; by the Swedish Medical Research Council (12X-3934), t he Muscular Dystrophy Assoc i a t i on , USA, the Expressen P rena t a l Fund and the Facu l t y of Medicine, U n ive r s i t y of Umeå.

23

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