101.
D I F F E R E N T I A T I O N OF MUSCLE F I B E R S
D U R I N G GROWTH A N D D E V E L O P H E N T *
R.
G.
CASSENS,
C.
C.
COOPER and S.
MORITA
UNIVERSITY OF WISCONSIN
F ig u re 1 c l e a r l y i l l u s t r a t e s t h a t muscle i s cornposed of more t h a n
one f i b e r t y p e . Tdo c o n s i d e r a t i o n s a r e r e l e v a n t : f i r s t , t h e fri'oer tj-pes
have d i s t i n c t l y d i f f e r e n t p r o p e r t i e s , and second, t h e g r o s s c h a r a c t e r i s t i c s
of t h e muscle a r e dependent on t h e pr opor tion of f i b e r t y p e s p r e s e n t . (Moody
and Cassens, 1968). The minimum number of f i b e r t y p e s i s two, with t h e r ? d
f i b e r being r i c h i n o x id ativ e enzymes and poor i n phosphorylase while t h e
white f i b e r i s poor i n o x id ative enzymes b u t r i c h i n phosphorylase. However,
t h e r e i s a range i n p r o p e r t i e s from r e d t o white, as t h e r e i s with most
b i o l o g i c a l phenomena, and v a r i o u s a uthor s have devised v a r l o d s schemes of
c l a s s i f i c a t i o n of f i b e r ty p in g . S u f f i c e it here t o say t h a t f i b e r s i n t e r mediate i n some property, between r e d and white, a r e well recognized. We
s h a l l confine o u rselv es t o t h e use of r e d or type I f i b e r s , white or type I1
f i b e r s and in termed iate f i b e r s . Beecher (1966) de sc r ibe d t h e complex but
d e s c r i p t i v e terminology t h a t has evolved i n regard t o snyonyms f o r f i b e r
t y p e s i n s t r i a t e d muscle. White f i b e r s have been c a l l e d a gr a nula r , b r i g h t ,
l i g h t , f a s t , l a r g e p ale, t e t a n i c , tw itc h, protoplasma r e i c h e n and F i b r i l k n s t r u k t u r whereas r e d f i b e r s have been r e f e r r e d t o as gr a nula r , dark, slow,
s m a l l , t o n i c protoplasma-armen and F e l d e r s t r u k t u r . The r e a de r i s r e f e r r e d
t o review a r t i c l e s f o r a complete account of t h e h i s t o l o g i c a l , biochemical
and p h y s i o l o g i c a l p r o p e r t i e s of red and white muscle (Needham, 1926; DenneyBrown, 1929; Cassens and Cooper, 1969).
Muscle f i b e r d i f f e r e n t i a t i o n i s t h e t o p i c of t h i s manuscript.
Denney-Brown (1929) w a s aware of t h e f a c t , some y e a r s ago, t h a t t h e prope r t i e s of muscle changed as t h e animal developed from newborn t o a d u l t . He
wrote t h a t each muscle f i b r i l , i n i t s p r i m i t i v e form, i s packed with gr a n u l e s
and t h e pr o cess of c o n t r a c t i o n i s extremely sluggish and delayed; e a r l y i n
t h e e x t r a - u t e r i n e l i f e of t h e k i t t e n , f i b e r d i f f e r e n c e s begin t o occur. The
f i r s t of t h e s e d i f f e r e n c e s observable i s t h e l o s s of gr a nula r o p a c i t y i n some
f i b e r s . T h i s p ro cess r a p i d l y progr e sse s and muscle becomes a mixture of c l e a r
and dar k f i b e r s . Today, some y e a r s a f t e r Denney-Brown spoke of such changes
i n f i b e r c h a r a c t e r i s t i c s , we a r e w e ll informed of t h e histochemical, physi o l o g i c a l and biochemical changes t h a t occur i n muscle f i b e r s from f o e t a l
s t a g e s t o t h e adult. T h is information w i l l be reviewed and t h e n a r e s m e of
our f i n d i n g s on p i g muscle w i l l be presented sinc e t h e members of t h i s
o r g a n i z a t i o n a r e i n t e r e s t e d i n a s p e c t s of muscle from domestic animals t h a t
a f f e c t s i t s use as a food.
*
T h i s c o n t r i b u t i o n w a s prepared dur ing t h e term of support by Public
HeaLth Service Grant No. UI-00266-10from t h e National Center l o r Urbcn
and I n d u s t r i a l Health.
102.
The study of d i f f e r e n t i a t i o n i s important s o t h a t nornal development i s catalogued; t h i s allows t h e r e s e a r c h e r t o e s t a b l i s h when a pathol o g i c a l p ro cess may have s t a r t e d (Dubowitz, 1965a). We have been i n t e n s e l y
i n t e r e s t e d i n t h e p ro cess of d i f f e r e n t i a t i o n i n p i g muscle because Cooper
e
a l . ( 1 9 6 9 ~ )demonstrated a p o s s i b l e a s s o c i a t i o n between f i b e r type
-t c h a r a c t e r i s t i c s and t h e development of p a l e , s o f t , exudative (PSE) muscle.
A knowledge of f i b e r ty p e d i f f e r e n t i a t i o n should t h e r e f o r e be meaningful. t o
an understanding of when, i n t h e l i f e of t h e pig, t h e sta ge i s s e t f o r t h e
PSE co n d itio n .
HISTOCHEMICAL STUDDS
Beckett and Bourne (1958) studie d suc c inic dehydrogenase i n
bic ep s b r a c h i i , gastrocnemius, t i b i a l i s a n t e r i o r and r e c t u s femoris muscles
of g o a t . Succinic dehydrogenase a c t i v i t y inc r e a se d during f o e t a l growth
bu t never reached a d u l t l e v e l s ; t h e d i f f e r e n t i a t i o n between l i g h t and dark
f i b e r s became apparent a t about 110-120 days ( t e r m i s 150 da ys) . They also
found l i t t l e s i z e d i f f e r e n c e between l i g h t and dark f i b e r s . It i s i n t e r e s t i n g i n view of some c o n s i d e r a t i o n s of i n n e r v a t i o n t o be d e a l t with l a t e r ,
t h a t a moderate c h o l i n e s t e r a s e c o n c e n t r a t i o n was found i n 48 day f o e t u s and
a d u l t l e v e l s were reached by 64 days.
Dubowitz (1963) found i n f u l l term human i n f a n t s t h a t muscle w a s
d i f f e r e n t i a t e d a t b i r t h and t h a t f i b e r s were rounded and arranged i n bundles.
F i b e r s r i c h i n phosphorylase were of s l i g h t l y g r e a t e r diameter t h a n t h o s e
r i c h i n o x id ativ e enzymes, and ATPase a c t i v i t y c o r r e l a t e d w e ll with phosphorylase a c t i v i t y i n i n d i v i d u a l f i b e r s . Muscle w a s d i f f e r e n t i a t e d a t b i r t h
i n guinea pig, r a b b i t and hamster but t h e d i f f e r e n t i a t i o n w a s most s t r i k i n g
i n guinea p i g where f i b e r s were polygonal i n shape and arranged i n bundles
t h a t resembled a d u l t muscle. I n r a b b i t and hamster muscle, t h e d i f f e r e n t i a t i o n w a s l e s s marked, t h e f i b e r s tended t o be rounded and were not so c l e a r l y
grouped i n t o bundles. F i b e r s were not d i f f e r e n t i a t e d a t b i r t h i n r a t muscle
b u t t h e d i f f e r e n t i a t i o n u s u a l l y occurred between 7 and 1 0 days post-partwn.
There were some areas of f o c a l d i f f e r e n t i a t i o n apparent at 2-3 days. The
process v a r i e d from one group of muscle t o another and w a s notic e a ble w i t h t h e
ATPase r e a c t i o n before it w a s with some of t h e o t h e r histochemical t e s t s . He
thought t h a t d i f f e r e n t i a t i o n w a s influenced by n u t r i t i o n and g e n e r a l growth
r a t e ; r a t s from smaller l i t t e r e s tended t o be l a r g e r and more mature and muscle
showed e a r l i e r maturation. Dubowitz (1963) a l s o suggested t h a t t h e d i f f e r e n c e
i n muscle maturation f o r t h e s e animals could be accounted f o r by t h e i r l e n g t h
of g e s t a t i o n and degree of g e n e r a l ma tur ity a t b i r t h . The guinea p i g ( 6 8 day
g e s t a t i o n ) has a more mature f u r a t b i r t h and i s much more mobile t h a n t h e
o t h e r animals. The r a b b i t (30-32 days g e s t a t i o n ) has b e t t e r developed f u r 2nd
i s more a c t i v e t h a n t h e r a t ( 2 1 day g e s t a t i o n ) . The hamster, although hFiving
a s h o r t e r g e s t a t i o n (16-19 days) t h a n t h e rat, also appears more a c t i v e a t
b i r t h and i t s fur develops more r a p i d l y .
Wirsen and Larsson (1964) conducted an experiment on t h e muscle of
mice ( 1 4 - 2 1 days g e s t a t i o n , new born, 1 day old and 7 days o l d ) . Succinic
dehydrogenase and l i p i d s t a i n i n g d i d not r e v e a l any d i s t i n c t d i f f e r e n c e s among
f i b e r t y p e s b efo re b i r t h and peroxidase a c t i v i t y , i n t e r p r e t e d as due t o myog lob in , w a s not demonstratable u n t i l a f t e r b i r t h . On t h e 1 6 t h g e s t a t i o n : i l d a y
some s t r o n g l y phosphorylase p o s i t i v e myotubes (primary f i b e r s ) were observed.
Also apparent were some smaller f i b e r s , i n c l o s e c o n t a c t with t h e primary f i b e r =
bu t with a weak phosphorylase r e a c t i o n (secondary f i b e r s )
The 17th and 1 8 t h
.
103.
g e s t a t i o n a l day w a s g e n e r a l l y c h a r a c t e r i z e d by r a p i d developxent of seconda r y f i b e r s which i n c e r t a i n muscle groups had become as l a r g e o r l a r g e r t h a n
primary f i b e r s . On t h e 1 9 t h g e s t a t i o n a l day, a t h i r d f i b e r type , t h e t e r t i a r y
w a s observed and it w a s p r a c t i c d l y phosphorylase ne ga tive . A t t h i s sta ge
t h e f i b e r s w e r e becoming more c l o s e l y packed and were changing from rounded
t o polygonal shape. I n newborn and 1 day old mice, a l l t h r e e f i b e r t y p e s
were seen i n s e v e r a l muscles. The a uthor s concluded t h a t dur ing f o e t a l
development t h e r e i s a gradual t r a n s i t i o n from g l y c o l y t i c t o presumably
oxidative metabolism a s r e f l e c t e d by t h e str ong phosphorylase r e a c t i o n of
primary f i b e r s compared to t h e moderate s t a i n i n g of secondary f i b e r s and t h e
very weak phosphorylase r e a c t i o n of t e r t i a r y f i b e r s . They i n t e r p r e t e d t h e i r
r e s u l t s as not supporting t h e d i r e c t c o r r e l a t i o n between g e s t a t i o n l e n g t h and
maturation of muscle as proposed by Dubowitz (1963). They thought t h a t t h e
t h r e e f o e t a l p o p u latio n s (primary, secondary and t e r t i a r y ) could be t e n t a t i v e l y c l a s s i f i e d a s white, in ter me dia te and r e d of adult musculature and
proposed t h a t t h e 3 t y p e s develop as 3 d i s t i n c t popula tions.
Dubowitz (1965a) r e s t a t e d h i s views about developing animal muscle
i n a l a t e r p u b l i c a t i o n ; t h e r e w a s a s t r i k i n g d i f f e r e n c e i n maturation of
ske1eta.l muscle i n d i f f e r e n t s p e c i e s of animals. H e thought t h e r e was a
c o r r e l a t i o n between d i f f e r e n t i a t i o n and g e n e r a l ma tur ity of muscle a t b l r t h
and a l s o w it h l e n g t h of g e s t a t i o n . He studie d guinea p i g muscle i n more
d e t a i l by i n clu d in g some f o e t a l sta ge s; muscle from newborn guinea p i g
resembled a d u l t muscle and was f u l l y d i f f e r e n t i a t e d i n t o type I and type I1
f i b e r s . I n f o e t u s of 35 t o 40 days g e s t a t i o n , most muscle showed complete
unifor mity of f i b e r s w ith NADH-diaphorase, a p a r t from some v a r i a t i o n i n
o v e r a l l s t a i n of some whole bundles as compared t o o t h e r s . I n oc c a siona l
muscles t h e r e w a s some v a r i a t i o n between i n d i v i d u a l f i b e r s w i t h i n a bundle.
With phosphorylase and ATPase, i n c o n t r a s t , most muscle showed subdivision
of f i b e r s i n t o s t r o n g l y and weakly r e a c t i n g ones, but d i d not g i v e t h e c l e a r
c u t checkerboard p a t t e r n of mature muscle. A t 50-55 days g e s t a t i o n , a l l
muscles reve aled a c l e a r c u t d i f f e r e n t i a t i o n i n t o type I and type I1 f i b e r s
with v a r i o u s enzyme r e a c t i o n s and showed t h e checkerboard p a t t e r n of mature
muscle.
Dubowitz (1965b, 1966) a l s o published f u r t h e r e xte nsive informztion
on f i b e r d i f f e r e n t i a t i o n i n developing human muscle. I n a l l i n f a n t s and
8 y ears) t h e muscle showed a checkerboard p a t t e r n with
c h i l d r e n ( 2 weeks
s u b d i v i s i o n i n t o a t l e a s t 2 f i b e r t y p e s as i n a d u l t muscle. Biopsy sampling
gave b e t t e r r e s u l t s t h a n necropsy. The muscle from newborn f u l l term i n f a n t s
had a p a t t e r n of d i f f e r e n t i a t i o n s i m i l a r t o t h a t of o l d e r i n f a n t s . Newborn
premature i n f a n t s showed a s i m i l a r p a t t e r n of d i f f e r e n t i a t i o n i n t o f i b e r t y p e s ,
b u t r e s u l t s w e r e l e s s c o n s i s t e n t t h a n i n f u l l term i n f a n t s . Most newborn
infants had an approximately eq ua l pr opor tion of type I and type I1 f i b e r s .
Foetuses were d iv id ed i n t o 2 groups on t h e b a s i s of s i z e ; l a r g e r f o e t u s e s
w e r e 20-26 weeks old and smaller f o e t u s e s were 12-20 weeks old. The l a r g e r
f o e t u s e s showed 2 groups of f i b e r s w ith t h e NADH-diaphorase reaction--one
very i n t e n s e and t h e o t h e r moderate. Phosphorylase and ATPase r e s u l t s were
not as c o n s i s t e n t b u t a number showed t h e e xa c t r e c i p r o c a l t o t h e NADHdiaphorase r e a c t i o n . The above p a t t e r n w a s not pr e se nt i n t h e smaller f o e t u s e s .
Some muscles were completely uniform and some showed v a r i a t i o n from bundle t o
bundle, but all f i b e r s w i t h i n a bundle were uniform. Some showed weakly o r
s t r o n g l y r e a c t i n g f i b e r s f o r a p a r t i c l j l a r enzyme and d i d not form t h e checkerboard p a t t e r n , b u t occurred i n c l u s t e r s . I n s e r i a l s e c t i o n s i n a number 9€
i n s t a n c e s , f i b e r s w ith a s t r o n g a c t i v i t y for one enzyme were a l s o foi,iid t o
-
104.
have a h ig h co n ten t of o t h e r enzymes so t h a t t h e y d i d not correspond t o
type I o r ty p e I1 f i b e r s of mature muscle. I n t h e s e younger f o e t u s e s t h e
f i b e r s were i n widely sep a r ate d s m a l l groups 2nd not i n compact bundles.
He d i v i d e s developing human muscle i n t o 3 phases. Phase I r e p r e s e n t s e a l y
f o e t a l l i f e t o about 20 weeks. There i s no c l e a r c.Jt d i v i s i o n i n t o f i b e r
t y p e s and c e r t a i n f i b e r s cannot be c o r r e l a t e d with type I or type I1 of
mature mtlscle. Phase I1 r e p r e s e n t s 20-26 weeks g e s t a t i o n a l age and t h e r e i s
a c l e a r c u t d i v i s i o n i n t o 2 f i b e r type s. However, type I f i b e r s comprise
only a v ery s m a l l p r o p o r t i o n of' t h e t o t a l . Phase I11 r e p r e s e n t s about 30
weeks t o f u l l term. D i f f e r e n t i a t i o n i s similar t o adult muscle with
approximately eq u al p r o p o r t i o n of type I and type I1 f i b e r s .
Developing human s k e l e t a l muscle has a l s o been examined by F e n i c h e l
(1966) d u rin g t h e g estatio nal. age 5-20 weeks. F i b e r typing could not be
accomplished d u rin g t h e p er iod 5-8 weeks: t h e mitochondrial r e a c t i o n s were
uniformly in ten se, and t h e t o t a l q u a n t i t y of a v a i l a b l e m y o f i b r i l s was i n s u f f i c i e n t t o allow conclusions t o be drawn from t h e myosin ATPase a c t i v i t y .
Light and dark myotubes could be d i s t i n g u i s h e d w ith t h e ATPase r e a c t i o n
dur in g t h e p erio d 8-10 weeks. Type I1 f l b e r s ( d a r k with ATPase and average
15-20 p i n diameter) were more numerous t h a n t h e smaller type I f i b e r s
( l i g h t ) t h a t averaged 5-10 p diameter. Mitochondrial enzyme a c t i v i t y
continu es t o be d i f f u s e l y i n t e n s e i n f i b e r s undergoing new f i l a m e n t format i o n which p reclu d es t h e i r use f o r f i b e r t y p i n g . Tye I1 f i b e r s remain l a r g e r
t h a n ty p e I f i b e r s throilgh t h e 1 5 t h g e s t a t i o n a l week and many mature niyocytes
a r e p r e s e n t which can be typed w ith mitochondrial enzyme r e a c t i o n s . The
muscle has a mature appearance by 20 weeks; t h e 2 t y p e s a r e about e qu a l i n
number but t h e i r s i z e r e l a t i o n s h i p has r e ve r se d with type I being l a r g e r t h e n
type 11. The author concluded t h a t mitochondrial enzyme r e a c t i o n s were of
uniform i n t e n s i t y i n all muscle f i b e r s dur ing t h e pe r iod of m y o f i b r i l l a r y
formation and could n o t be used f o r f i b e r t y p i n g u n t i l t h e tr a nsf or ma t i o n
from myotube t o myocyte w a s complete. The i n a b i l i t y t o use t h e o x i d a t i v e
enzyme r e a c t i o n s f o r f i b e r t y p i n g u n t i l f i b r i l l a r formation i s completed
probably r e f l e c t s t h e uniformly high r a t e of enzyme a c t i v i t y needed during
t h e e a r l y s t a g e s of muscle development i n t h e production of new m y o f i l m e n t s .
F e n i c h e l (1966) also found ATPase t o be t h e most u s e f u l r e a c t i o n , and he
concluded from h i s results, t h a t t h e 2 muscle types develop i n human embryos
as s e p a r a t e p o p u latio n s.
The phosphorylase c h a r a c t e r i s t i c s of chicken muscle have been
s t u d i e d (Cosmos, 1966; Cosmos e t al., 1965). I n &At domestic fowl,
aerobic slow f i b e r s of t h e h e w t give a r e a c t i o n s i m i l a r t o t h e r e d i o d i n e
c o l o r produced by s h o r t ch a in amylopectins. Anaerobic f a s t f i b e r s of b r e a s t
muscle gave a b lu e io d in e c o l o r resembling long c ha in amylose l i k e products
i n l e g muscle, i n ad d itio n , had f i b e r s w ith a n inte r me dia te range of p m p l e
c o l o r s . T h e i r t e s t s w ith embryo b r e a s t muscle showed an amylopectin t y p e
r e a c t i o n t h a t i n d i c a t e d t h e presence of slow a e r obic f i b e r s . D i f f e r e n t i a t i o n
t o more s p e c i a l i z e d f a s t f i b e r s began dur ing t h e f i r s t week ex ovo when t h e r e
w a s a decrease i n slow f i b e r s and a concornitant inc r e a se i n intermediLtc and
f a s t t y p e s . The adult b r e a s t muscle w a s composed mainly of f a s t f i b e r s . The
author s concluded t h a t t h e development of c e l l s of b r e a s t muscle pr og r e s s frorr
a slow f i b e r t o an in termedia te orie and f i n a l l y t o t h e highly d i f f e r e n t i a t c d
f a s t f i b e r c h a r a c t e r i z e d by polysaccharides of long unbranched c ha ic s
c -
--
.
Chinoy and George (1965) studie d t h e p e c t o r a l i s muscle of pigeon.
.
They used 15-17 day embryos and ex ovo b i r d s of i - 8 , 1 6 , 2 L and 40 d ~ ~ y s The
--
105.
d i s t i n c t i o n between t h e broad and narrow f i b e r s w a s c l e a r l y defined as
e a r l y as t h e 1 5 t h day i n ovo. The l e v e l s of f a t and suc c inic dehydrogenase appeared g r e a t e r i n narrow f i b e r s t h a n broad f i b e r s i n 2 day old
b i r d s . I n 6 day old b i r d s t h e narrow f i b e r s were more sudanophilic tha n
broad f i b e r s and contained numerous mitochondria. I n 8 day old b i r d s ,
t h e narrow f i b e r s contained considerably more f a t and suc c inic dehydrogenase t h a n broad f i b e r s i n which t h e mitochondria were fewer and smaller.
A t 16, 24, and 40 days of age, t h e narrow f i b e r s were d i s t i n c t l y more
sudanophilic and contained g r e a t e r c onc e ntr a tions of f a t , suc c inic dehydrogenase and l i p a s e t h a n d i d broad f i b e r s . The authors concluded t h a t
t h e d i f f e r e n t i a t i o n of f i b e r s i n t o broad white and narrow r e d t a k e s place
i n t h e embryo i t s e l f and not during post-embryonic l i f e , at which t i m e
t h e r e i s increased a c t i v i t y of muscle. The 2 type s of f i b e r were e a s i l y
d i s t i n g u i s h a b l e on t h e 1 5 t h day of incubation. Glycogen w a s depleted
a f t e r about t h e 3rd day ex ovo. F a t w a s t h e n incorporated i n t o t h e
narrow f i b e r s and simultaneously t h e mitochondrial, d e n s i t y and suc c inic
dehydrogenase a c t i v i t y were r a p i d l y increased i n narrow but not broad
f i b e r s . They i n t e r p r e t e d t h i s t o mean t h a t t h e narrow f i b e r s were being
prepared f o r o x id ativ e metabolism while t h e broad f i b e r s were not.
--
--
--
Germino e t al. (1965) j s e d t h e suc c inic dehydrogenase technique
t o study development of s k e l e t a l muscle i n t h e chick from t h e 3rd day of
p r e n a t a l exi sten ce t o 60 days a f t e r hatching. They found t h e development
of muscle c e l l s t o be ch ro n o lo g i c a lly p a r a l l e l up t o t h e 1 6 t h day i n
regard t o i n t e n s i t y and d i s t r i b u t i o n of t h e r e a c t i o n . D u r i n g t h e 1 7 t h
and 1 8 t h days a f e w f i b e r s appeared t h a t presented only a moderate r e a c t i o n .
These f i b e r s were c a l l e d in termedia te s as the y were thought t o a r i s e from
those having an i n t e n s e reactio n - - the only ones e x i s t i n g at t h a t time and
which were r e d f i b e r s . During t h e following days, t h e suc c inic dehydrogenase a c t i v i t y of t h e intermediate f i b e r s decreases even more and the y
become white fibers. Myofibers are a lr e a dy c o n s t i t u t e d when d i f f e r e n t i a t i o n i n red, intermediate and white f i b e r s i s seen, and it t a k e s place
between t h e 1 7 t h and 1 9 t h day of embryonic l i f e . The r e se a r c he r s made t h e
following statement about t h e i r results: t h e l a r g e amount of succinic
dehydrogenase already p resen t i n such e a r l y stages as t h e premyoblast
expresses t h e greater energy requirements of t h e muscle c e l l when one
considers su ccin ic dehydrogenase as an expression of TCA cycle a c t i v i t y .
This a c t i v i t y , which i s a t f i r s t pe r inuc le a r , would seem t o be r e l a t e d
t o t h e nucleo protoplasmic t r a n s f e r of RNA, which i s ve r y g r e a t i n t h e
e a r l y stages of growth. The energy requirements i n l a t e r stages,
e s p e c i a l l y when t h e r e a c t i o n spreads d l along t h e sarcoplasm, appears t o
be r e l a t e d t o t h e muscular co n tra c tions which are v i s i b l e from t h e 4th
and 5 t h days.
Rebollo and P i a n t e l l i (1964) have studied t h e l i p i d s i n chicken
s k e l e t a l muscle during development. I n proximal l e g muscles t h e 2 type s
of f i b e r s develop sometime a f t e r t h e 1 2 t h day. The f u t u r e s t r a i g h t l i n e
red f i b e r s have intermyof i b r i l l a r y gr a nula tions ( p o s i t i v e t o l i p i d
techniques); s i m i l a r g ran u latio ns are found i n t h e f u t u r e white f i b e r s
although i n a smaller number. From t h e 1 8 t h day on, an important inc r e a se
i n s i z e of i n t e r m y o f i b r i l l a r y granules i s observed and 3 type s of f i b e r s
are disting u ish ab le: red f i b e r s with abundant l i p i d s , white f i b e r s i n
which l i p i d s a r e scarce and f i b e r s intermediate i n t h i s a spe c t. However,
t h e f i b e r number, s i z e and d i s t r i b u t i o n a r e not y e t s i m i l a r t o t h e a d u l t
stage. I n p e c t o r a l muscle, t h e l i p i d s disappear on t h e 1 4 t h day. These
106.
f i b e r s undergo a much slower maturation from t h e histoge nic standpoint b u t
mature e a r l i e r i n regard t o d i s t r i b u t i o n of l i p i d s . Morphological f e a t u r e s
(Rebollo e t al., 1963) and l i p a s e d i s t r i b u t i o n ( P i c t n t c l l i and Rebollo, 1967)
i n developing muscle of chicken have calso bcrn r e por tc d.
-
L
Nystrom (1966) uscd t h c zdcciriic clchytir'ocf I ~ ' L J C r c n c t i o n %o study
d i f f e r e n t i a t i o n i n gastrocnenius, ;olcus m d cxtensor c.,rpi r t d i a l i s
muscles of 1, 1 0 , 15, 39 day o l d m d : d u l t c:~.ts. No d i f f e r e n t i a t i o n w a s
seen i n gsstrocnemiiis of ncwborn, a t 1 0 dayc some d i f f e r e n t i a t i o n bias v i s i b l e
and a t 15 days it w ' i s ;ccn c l e a r l y but i n t e r r i e d i a t e f i b e r s were not p r e s e n t .
All 3 f i b e r types wcre p res ent i n 39 day old c a t s but t h e muscle s t i l l d i d
not wholly resemble a d u l t muscle. No d i f f e r e n t i a t i o n was present i n s o l e u s
muscle of newborn. Between 2-7 weeks, some d i f f e r e n t i a t i o n i n t o more d a r k l y
and spa rsely stain in & f i b e r s w a s e v i d e n t . After 7 weeks, however, all f i b e r ,
stained d a r k l y and as i n t h e adult and t h e r e was no d i f f e r e n t i a t i o n i n t o
f i b e r t y p e s . The extensor c a r p i radialis i s a muscle of t h e forelimb and
d i f f e r e n t i a t i o n i s p resen t a t b i r t h ; it has a s i r i i l a r s t a i n i n g p a t t e r n t o
gastrocnemius (white muscle) of adult. Other a uthor s have shown t h a t t h e
r a d i a l nerve of t h e fo relimb i s more mature than e q u a l l y d i s t a l nerves i n
t h e hindlimb of newborn k i t t e n s as i n d i c a t e d by diameter of t h e nerve f i b e r s .
Thus, t h e au th o rs thought it probable t h a t t h e d i f f e r e n t i a t i o n mechanism
s t a r t e d at a c e r t a i n stsge of maturation i n motorneurones. Differences i n
degree of matu rity of notornewones could also e x p l a i n t h e spe c ie s d i f f e r e n c e s
found a t b i r t h i n histochemical d i f f e r e n t i a t i o n of muscle.
F i b e r d i f f e r e n t i a t i o n ha s been studie d i n r he sus monkeys by B e a t t y
Succinic dehydrogenase a c t i v i t y w a s found pr e se nt i n f e t u s e s
e
t
61.
(1967).
at 76-78 days, b u t s e c t i o n s from younger f e t u s e s r e quir e d longe r incubation
t o demonstrate a c t i v i t y . It w a s not possible t o d i f f e r e n t i a t e f i b e r s i n t o
t y p i c a l s m a l l r e d and l a r g e white types i n b r a c h i o r a d i a l i s of 90 day f e t u s .
Neither w a s it f e a s i b l e t o d i s t i n g u i s h between b r a c h i o r a d i a l i s and sol e u s on
t h e basis of f i b e r ty p e. However, by 1 2 0 days, r e d and white f i b e r s were
c l e a r l y i d e n t i f i a b l e and t h e d i s t r i b u t i o n p a t t e r n w a s s i m i l a r t o t h a t i n a d u l t
muscle. The 2 r e d f i b e r types of t h e sole us t h a t have been described f o r
t h e a d u l t c a t , r a t and rh es us monkey could be seen i n t h e 120 day f e t a l colzus.
We w i l l comment on 2 r e c e n t a dditions t o t h e l i t e r a t u r e as a conc l u s i o n t o t h i s s e c t i o n on histochemical s t u d i e s . Dubowitz (1968) has publ i s h e d an i n t e r e s t i n g book on Developing and Diseased Muscle. Much of it i s
a review or ex ten sio n of h i s e a r l i e r work b u t two observations deserve
comment. A c l e a r c u t d iv iding l i n e could not be e s t a b l i s h e d i n r a t s between
t h e phase when muscle f i b e r s were uniformly p o s i t i v e f o r a l l enzyme r e a c t i o n s
and t h e phase of complete d i f f e r e n t i a t i o n i n t o t h e a d u l t type s. It i s a
gradual process and t h e i n i t i a l occurrence i s i n random p m t s of t h e r m s c l c .
Some muscles showed e a r l y d i f f e r e n t i a t i o n with one e n z p e r e a c t i o n and comp l e t e uniformity with o t h e r s . With myosin ATPase, it w a s possible t o
recognize i s o l a t e d d a r k l y s t a i n i n g f i b e r s i n some muscles as e a r l y as t h e 1st
day of l i f e . Dubowitz t h i n k s t h a t embryologically, all muscle c o n s i s t s
i n i t i a l l y of one f i b e r type only and t h a t t h i s corresponds t o t h e t y y c I or'
r ed f i b e r ty p e. The red muscle shows no h i s t o l o g i c a l change during m a t u r a t i o n , b u t all t h e o t h e r muscles subsequently d i f f e r e n t i a t e i n t o type I, type
I1 and intermediate f i b e r s . These h i s t o l o g i c a l r e s u l t s p a r a l l e l very c l o s e l y
a number of observations by p h y s i o l o g i s t s on developing muscle t h a t T i r i l l be
described i n a subsequent s e c t i o n of t h i s manuscript.
107.
F i n a l l y , we w i l l o u t l i n e t h e r e c e nt e xte nsive histochemical
results of Nystrom (1968a) on developing c a t muscle. H e studied t h e ga str ocnemius ( f a s t white) and cru reu s (slow red, s i m i l a r t o soleus) i n k i t t e n s
1-120 days of age and i n a d u l t s . H i s A f i b e r s a r e white or l a r g e diameter
and poor i n o x id ativ e enzymes, C f i b e r s me red and r i c h i n oxida tive ensymes
and myoglobin and B f i b e r s a r e intermediate. The main type of f i b e r i n slow
red muscle w a s found not t o conform t o any of t h e t y p e s pr e se nt i n f a s t white
muscle and i s denoted as S t y p e . The f a s t white gastrocnemius had C y A and
B types. Type C stain ed d ark ly f o r suc c inic dehydrogenase, NADH-TR and
l i p i d s but f a i n t l y f o r phosphorylase, glycogen and ATPase. Type A was weak
f o r succini c dehydrogenase, NADH-TR and l i p i d s but strong f o r phosphorylase,
glycogen and ATPase. Type B w a s seen c l e a r l y with suc c inic dehydrogenase,
NADH-TR and l i p i d s and t h e i n t e n s i t y w a s c lose t o type A f o r phosphorylase,
glycogen and ATPase. The majority of f i b e r s sta ine d hemogeneously f o r
ATPase, succ in ic dehydrogenase and NADH-TR i n c r ur e us muscle. The s t a i n f o r
ATPase w a s higher t h a n f o r type C but lower t h a n f o r type B. A few a be r r a nt
f i b e r s i n t h i s muscle were v ery high f o r ATPase and suc c inic dehydrogenase
b u t not f o r NADH-TR. The g en eral type of f i b e r i n slow r e d w a s not d i r e c t l y
comparable with any of t h e 3 ty p e s i n f a s t white. During post- na ta l development i n newborn k i t t e n s t h e f i b e r s within both slow red ( sole us) and f a s t
white (gastrocnemius) appeared f a i r l y unif orm except i n ATPase s e c t i o n .
D i f f e r e n t ty p es appeared i n both muscles with time, but more so i n t h e
gastrocnemius. There w a s a steady progress i n d i f f e r e n t i a t i o n t o t h e stage
of 3 adult f i b e r t y p e s i n gastrocnemius. I n soleus, however, t h e tendency
t o d i f f e r e n t i a t e i n t o v ario u s t y p e s w a s soon suppressed and subsequently only
one type w a s p resen t a p a r t from a few a be r r a nt f i b e r s . The gastrocnemius
of newborn had s i m i l a r f i b e r s with suc c inic dehydrogenase but a s l i g h t d i f f e r e n t i a t i o n i n t o d a r k l y and l i g h t l y sta ine d f i b e r s appeared by 1 0 days. The
d i f f e r e n t i a t i o n w a s more d i s t i n c t a t 15 days and B f i b e r s began t o appear a t
about 6 weeks. Soleus from newborn a l s o had uniform s t a i n i n g . There w a s a
s l i g h t d i f f e r e n t i a t i o n i n t o p ale and l i g h t f i b e r s by 2-7 weeks but t h e r e a f t e r
t h e f i b e r s s t a i n e d uniformly and darrkly except f o r a few a be r r a nt ones. The
r e s u l t s with ATPase r e a c t i o n were d i f f e r e n t . The gastrocnemius from newborn
w a s d i s t i n c t l y d i f f e r e n t i a t e d i n t o l i g h t and dark f i b e r s . The sole us showed
some d i f f e r e n t i a t i o n b u t t h e amount of l i g h t f i b e r s seemed g r e a t e r . Three
types w e r e seen i n t h e soleus a t 3 weeks of age but by 6-7 weeks, mainly 1
type w a s pr e sen t. Nystrom (1968a) f e l t t h a t 3 kinds of information are
needed t o understand t h e t r u e c h a r a c t e r i s t i c s of a f i b e r and i t s d i f f e r e n t i a t i o n . The are: (1) t h e ab so lu t e t w i t c h c o n t r a c t i o n time, ( 2 ) t h e absolute
s i z e or conductive v e l o c i t y of t h e nerve f i b e r and (3) t h e histochemical type .
PKYSIOLOGICAL STUDlES
et al. (1960a, 1960b) published 2 papers t h a t stand as
Buller c l a s s i c s i n p h y sio lo g ical s t u d i e s on r e d and white muscle. I n t h e f i r s t
paper ( B u l l e r et d.,1960a), the y point out t h a t Denney-Brown and some
o t h e r s had demonstrated some t i m e ago t h a t a l l limb muscles were slow a t
b i r t h and d i f f e r e n t i a t e d i n t o f a s t and slow type s i n m a m m a l s during t h e f i r s t
few weeks a f t e r b i r t h . The s h o r t e r a f te r - hype r pola r iz a tion of motorneurones
supplying f a s t muscles allows t h e i r f a s t frequency of f i r i n g which i s
appropriatel y r e l a t e d t o t h e c o n t r a c t i o n time of t h e i r muscles--the complimentory cond itio n s apply f o r slow muscles. B u l l e r e t al. (1960a) s e t abo-Jt
t r y i r g t o determine whether appropriate matching of motorneurones t o muscle
w a s brought about by t h e motorneurones inf lue nc ing muscle d i f f e r e n t i a t i o n
--
108.
or v i c e v e r s 3 by muscle in flue nc ing notorneurones. They recorded t h e isometr i c tw itch and t e t a n i c response of hind l i m b muscles of c a t s over :I wide
range of ages from 1 day t o ad-dt. Responses were conipared under standard
conditio n s of t e n p e r a t u r e and i n i t i a l t e n s i o n . E a r l i e r work was cor3irrned i n
t h a t all muscles were slow i n t h e newborn, and t h o s e muscles de stine d t o
be f a s t a t t a i n e d t h e i r a d u l t speed i n about 6 weeks. The slow muscles ( s o l e u s
and crureus) also quicken, b a t t o a l e s s e r e xte nt, over t h e f i r s t 5 weeks;
t h e r e a f t e r t h e r e i s a progressive slowing of t h e s c musclcs and the y apnroach
t h e adult slow muscle co n d ition i n 16-20 weei',s. The sCmcr e s u l t s were foimd
with aJ.1 3 of t h e following measurements: (1) t i m e t o surnmit of' twitc h ,
( 2 ) t i m e from s-mmit t o h a l f r e l a x a t i o n of t w i t c h and ( 3 ) i n t e r m i l between
s t i m u l i a t t h e t e t a n i c frequency b u i l d i n g LIP t o 1 / 2 t h e m a x i m a l t e t a n i c cont r a c t i o n . The d i f f e r e n t i a t i o n of f a s t muscle 1 , ~ ;v~isr t u d l y unaffected by
s p i n a l cord t r a n s e c t i o n o r by ope r a tive i s o l a t i o n of t h e lumbosacral s p i n d .
cord from all incoming impulses. However, t h e d i f f e r e n t i a t i o n of s l o w muscle
w a s g r e a t l y depressed, t h e r e being a complete f a i l u r e of t h e l a t e phase of
slowing s o t h a t i n a few weeks sole us and c r ur e us became n e a r l y as f 3 s t i n
every r e s p e c t as n o rmd f a s t muscles. The zuthors concluded t h a t t h e d i f f e r e n t i a t i o n of slow m d s c l e w a s largely e f f e c t e d by ne ur a l inf lue nc e s
exer ted from t h e s p i n a l cord. This conclLsion was i n v e s t i g a t e d i n subsequent
experiments ( B u l l e r et al., 1960b) on nerve cross-union i n c a t hind l i m b s i n
or der t o d isco v er i f motorneurones determine t h e speed of muscles or i f t h e
e f f e c t i v e in flu en ce i s i n t h e r e ve r se d i r e c t i o n ( slow-soleus and f a s t - f l e x o r
digitorum longus were t h e muscles nost o f t e n used). When a nerve from f z s t
o r phasic motorneurones has been m a d e t o innervate a slow muscle, t h e muscle
i s transformed t o a f a s t muscle--even i n t h e a d u l t . Likewise, slow o r t o n i c
rnotorneurones convert f a s t muscles t o slow. The tr a nsf or ma tion of muscle
speed w a s shown n o t o n l y by t h e t i m e course of t h e r i s i n g and f a l l i n g phases
of t h e tw itch co n tractio n , but &Is0 by t h e summation of t e t a n i c c o n t r a c t i o n s
a t vario u s freq u en cies. The slow o r f a s t muscle with d i e n inne r va tio n had
no d e t e c t a b l e in flu en ce i n t h e r e ve r se d i r e c t i o n ; i .e., on t h e motornexones.
The conduction v e l o c i t y of t h e motor axons and t h e t i m e course of t h e motorneurones a f t e r h y p erp o lariz a tion were both unchanged. These r e s e a r c h e r s also
noted t h a t t h e muscle tran sf or ma tion f e l l s h o r t of a complete change t o t h e
slow o r f a s t type. I s o l a t i o n of t h e lunbosa c r a l s p i n a l cord from all incoming impulses, with consequent s i l e n c e of motorneurones, caused a f a i l u r e of
all tr an sfo rmatio n by cross-union.
Mere t r a n s a c t i o n of t h e s p i n a l cord
g r e a t l y reduced t h e tran sfor ma tion. They concluded t h a t t h e n e u r a l influence
on muscle speed w a s n o t ex e r te d by nerve i m p d s e s a s such. It w a s p o s t u l a t e d
t h a t a substance passes down t h e axons of slow motorneurones, c r o s s e s t h e
neuromuscular ju n ctio n and t r a v e r s e s t h e muscle f i b e r s transforming them i n t o
slow c o n t r a c t i n g u n i t s and maintaining them so. Possibly t h e r e i s a l s o a
substance from f a s t motorneurones t h a t a c t s v i a a comparable pathway t o
a c c e l e r a t e muscle c o n t r a c t i o n .
Close (1364) has also studie d t h e extensor digitorum longus -tnd
soleus muscles of cat from b i r t h t o 1 4 weeks. A t b i r t h , t h e f o r n of i s o metric c o n t r a c t i o n i s s i m i l a r i n EDL a d sole us muscles. Subsequent development leads t o decreases i n t h e c o n t r a c t i o n t i m e s , t h e t i m e s f o r hrflf reldxst i o n and t h e tw itch /tetan u s r a t i o , whereas t h e r e i s an inc r e a se i n t h e
optimal frequency f o r r e p e t i t i v e s t i r n d a t i o n . The f o r c e / v e l o c i t y p r o p e r t i e s
of EDL and so leo s are v i r t u a l l y i d e n t i c a l a t b i r t h ; t h e r e a f t e r , t h e c p ~ l p dof
shortening/sarcomere, f o r any given f r h c t i o n of t h e r i R x i m u m l o t t d , incrc LSC;
by 2-1/2 t o 3 times i n EDL whereas soleus undergoes l i t t l e or no change i n
t h i s r e s p e c t . The author discussed t h e r e s u l t s i n connection with possiblr
109.
changes i n t h e time course of t h e a c t i v e state and t h e p o s s i b l e dependence
of t h e d u r a t i o n of t h e a c t i v e s t a t e upon t h e f o r c e / v e l o c i t y p r o p e r t i e s .
Nystrom (1968b, 1968c, 1968d) has studie d va r ious a s p e c t s of
inne rv atio n i n developing c a t muscle as it r e l a t e s t o d i f f e r e n t i a t i o n .
One manuscript (Nystrom, 1968b) d e a l s with t h e c h a r a c t e r i s t i c s of t h e motor
nerve t e r m i n a l s . True slow red f i b e r s ( c o l d blooded animals, some b i r d s
and mammalian e x t r a o c u l a r muscle) have a motor nerve t e r m i n a l r e f e r r e d t o
as "en grappe" while t h a t i n t r u e white f i b e r s i s "en plaque." Nystrom
(1968'13) emphasizes t h a t i n c a t muscle t h e so- c a lle d f a s t white and slow red
a r e i n f a c t both t w i t c h ty p e muscles. A d i f f e r e n c e d i d e x i s t , however,
between t h e gastrocnemius and sole us t e r m i n a l s even though both were of t h e
"en plaque" t y p e . The gastrocnemius end r a m i f i c a t i o n s were wide spreading,
long and smooth, b u t t h e so leu s end r a m i f i c a t i o n s were more t i g h t l y packed,
wrinkled and f l u t e d i n o u t l i n e . Nerve t e r m i n a l s i n 1 - 7 day old k i t t e n s were
uniform s m a l l rounded d i s c s on t h e sur f a c e , and t h e r e w a s no d i f f e r e n c e
between t h e gastrocnemius and sole us except t h a t i n t h e sole us t h e nerve
t e r m i n a l w a s sometimes l a r g e r . The t e r m i n a l r a m i f i c a t i o n s of gastrocnemius
end p l a t e s were w e l l e s t a b l i s h e d by about 2 months and t h e sole us had a t t a i n e d
a wrinkled appearance and elongated shape. The lower l e v e l s of a d u l t s i z e
were reached a t t h i s stag e and t h e r a t i o of end p l a t e diameter/muscle f i b e r
s i z e was roughly 1 / 2 i n b o th so l e us and gastrocnemius. I f t h e r a t i o of end
p l a t e s i z e t o muscle f i b e r s i z e i s i n any way r e l a t e d t o t h e t h r e s h o l d f o r
e x c i t a t i o n of muscle f i b e r s t h e n t h e above r e s u l t s cannot e x p l a i n t h e l a r g e
d i f f e r e n c e found between t h e 2 muscles with r e s p e c t t o t h e p o s t - t e t a n i c
t w i t c h p o t e n t i a t i o n . No systematic v a r i a t i o n i n s t r u c t u r e of t h e motor nerve
t e r m i n a l s was found between muscle f i b e r s of s m a l l and l a r g e size, i n g a s t r o c nemius, t h a t were presumed t o r e p r e s e n t t h e histochemical r e d and white f i b e r
t y p e s r e s p e c t i v e l y . He concluded t h a t t h e s t r u c t u r e of t h e motor nerve
t e r m i n a l s and end plate/muscle f i b e r s i z e r a t i o cannot e x p l a i n t h e d i f f e r e n c e
between s oleu s and gastrocnemius with r e s p e c t t o t h e p o s t - t e t a n i c p o t e n t i a t i o n of t w i t c h t e n s i o n .
The end p l a t e bound a c e t y l c holine e s t e r a s e w a s a l s o studie d
(histo ch emically ) as a f u n c t i o n of p o s t n a t a l development (Nystrom, 1 9 6 8 ~ ) .
S t a i n i n g i n t e n s i t y f o r a c e t y l ch oline e s t e r a s e appeared t o be s i m i l a r i n
gastrocnemius and so leu s end p l a t e s of newborn k i t t e n s . However, it inc r e a s e d
i n gastrocnemius d u rin g p o s t n a t a l development b u t remained unchanged i n
s o l e u s . H e concluded t h a t t h e low a c e t y l c holine e s t e r a s e a c t i v i t y of soleus
end p l a t e s i n adult c a t s might p a r t l y e x p l a i n t h e well-known p o s t - t e t a n i c
r e a d i n e s s t o r e p e t i t i v e discharge found i n t h e sole us. The results obtained
with regard t o s t r u c t u r e of t h e subneural apparatus and a c t i v i t y of end p l a t e
e s t e r a s e cannot, however, f u r t h e r t h e understanding of t h e cause of d i f f e r ences i n p o s t - t e t a n i c p o t e n t i a t i o n of t w i t c h t e n s i o n following low frequency
t e t a n i z a t i o n between so leu s and gastrocnemius i n both young and a d u l t k i t t e n s .
Nystrom (1968d) also studie d t h e f i b e r diameter i n nerves t o slow
r e d and f a s t white muscles during development. The l i t e r a t u r e has shown
t h a t nerve f i b e r s t o s m a l l red a r e smaller t h a n those t o f a s t white; one n i g h t
expect t o f i n d , i n t h e newborn c a t , nerve f i b e r s of f a i r l y uniform diameter
t o f u t u r e slow r e d and f a s t white sinc e c o n t r a c t i o n time s a r e f a i r l y s i m i l a r .
Nystrom (1968d) found t h a t gastrocnemius and sole us nerve f i b e r s d i f f e r e d
inap pre cia bly d u rin g t h e f i r s t 1 0 p o s t n a t a l days. From t h e n on a d i s t i n c t
d i f f e r e n c e developed w ith t h e gastrocnemius nerve f i b e r s being l a r g e r . Results
showed c o n s i s t e n t l y smaller f i b e r s i n nerves t o slow r e d t h a n t o f z s t white
110.
muscle i n a d u l t c a t . I n k i t t e n s about 2 months old, t h e e f f e r e n t s of
gastrocnemius nerve had become d e f i n i t e l y f a s t e r t h a n t h e a f f e r e n t s and
they both conducted much f a s t e r t h a n t h e c 0 1 c . u ~fibers. A t t h i s age, as
i n t h e newborn k i t t e n , no d i f f e r e n c e w : found
~
br\t,wcx*ri t h c conduction
v e l o c i t y i n e f f e r e n t and a f f e r e n t f ibc.r-2 01' LOJ(-II: ri(,rvc . A r a pid inc r e a s e
occurred i n t h e t o t a l number of rnyc1in:itcd l ' i b c ~ r : i r i L t i c nerves during t h e
f i r s t few weeks of p o s t n a t a l l i f e *md : i d u l t V ' I I II( wc
ri':tched a t about 4-6
weeks of age. Nystrom (1968d) concluded t h . i t dil'i'c:rc r i v L observed between
soleus and gastrocnemius with regard t o post-tc:t*Lriic p o t e n t i a t i o n i n young
k i t t e n s cannot be explained by t h e s l i g h t d i f f e r e n c e i n maturation of t h e i r
nerve fib ers--alth o u g h with t h e method used, a d i f f e r e n c e i n t h e intramusc u l a r branching cannot be excluded.
I ,
Goldspink and Rowe (1968) t h i n k t h a t t h e change i n speed of muscle
during growth i s asso ciate d s o l e l y with t h e completion of "embryological"
d i f f e r e n t i a t i o n and is i n no way r e l a t a b l e t o t h e post-embryological growth
and development of t h e fibers. They also s t a t e t h a t inc r e a se i n speed of
muscle during i t s development ( b i c e p s b r a c h i i of r a t i n t h e i r case) i s due
s o l e l y t o d i f f e r e n t i a t i o n of t h e e x c i t o t o r y apparatus of t h e f i b e r and i s
not due t o any o t h e r mechanism os s t r u c t u r a l change o r a l t e r n a t i v e l y t o
change i n myosin ATPase. We should also p o i n t out t h e system Goldspink u s e s
for morphological d e s c r i p t i o n of f i b e r s . H e recognizes 3 phases: a s m a l l
phase ( 2 0 p), in terp h ase (33 )A) and l a r g e phase (60 p) which he presumes
correspond t o t h e 3 fiber types.
BIOCHEMICAT, STUDIES
It i s apparent t h a t t h e biochemical machinery of' muscle should
p a r a l l e l t h e changes t h a t have been shown t o occur with histochemical and
physio lo g ical tech n iq u es as muscle develops. We w i l l here review some of
t h e biochemical s t u d i e s on muscle development.
L a t z k o v i t s and Domonkos (1965) studie d t h e e f f e c t of p o s t n a t a l
development on carbohydrate metabolism of t o n i c and t e t a n i c muscles of t h e
r a b b i t . According t o t h e s e authors, t h e b e s t metabolic i n d i c a t o r o f t h e
t e t a n i c c h a r a c t e r of a muscle i s t h e production of aerobic l a c t a t e and
e s p e c i a l l y t h a t of aerobic pyruvate, sinc e t h e processes occur i n t e t a n i c
muscles and t h e i r f i b e r s only. No such process can be observed i n t o n i c
muscles. The l o n g i t u d i n a l i s d o r s i w a s used as a t e t a n i c muscle and abdominal.
muscles as t o n i c ; t h e y were incubated i n krebs phosphate with a i r as t h e g a s
phase. The metabolism of t h e s k e l e t a l muscles corresponds e n t i r e l y t o t h a t
of t o n i c muscles immediately following b i r t h of t h e a n i m a l s . The c h a r a c t e r i s t i c metabolism of t h e t e t a n i c muscle i s t h e r e s u l t of p o s t n a t a l d i f f e r e n t i a t i o n . The authors placed t h e d i f f e r e n t i a t i o n of metabolism p a r a l l e l
with t h e d i f f e r e n t i a t i o n of muscle f unc tion, and be f or e t h e a b i l i t y t o move
independently and locomotor f u n c t i o n have developed, t e t a n i c and t o n i c
muscles cannot be d e a l t w ith s e p a r a t e l y or cannot be d i s t i n g u i s h e d .
Cosmos (1966) h as studie d t h e development of phosphorylase i n
developing muscle ( s u p e r f i c i a l p e c t o r a l i s ) of domestic fowl and a t t h e same
t i m e h a s made t h e same o b s e r va tions i n dystrophic muscle. Adult chicken
breast muscle h a s a stro n g phosphorylase a c t i v i t y histochemically, but i n
b i r d s with muscular dystrophy t h e r e a c t i o n i s mixed. She speculated t h a t the
d i v e r s i t y of enzymatic response i n t h e va r ious adult muscles merely r e f l e c t e d
111.
s tages of development of one common f i b e r type and t h e r e should be a period
i n development when a l l f i b e r s give a s i m i l a r phosphorylase response. With
f u r t h e r maturation, some f i b e r s should continue t o d i f f e r e n t i a t e t o those
s pecialized f o r s p e c i f i c metabolic f unc tion while o t h e r s continue t o grow
with no a l t e r a t i o n i n s p e c i f i c enzyme response. Thus a t some period of
development, t h e most h ig h ly d i f f e r e n t i a t e d f i b e r s must give a phosphorylase
r e a c t i o n s i m i l a r t o t h a t of a d u l t f i b e r s showing minimal enzyme a l t e r a t i o n .
Cosmos (1966) found t h a t t o t a l phosphorylase a c t i v i t y i n t h e embryo was
s i m i l a r i n both normal and abnormal muscle. Dming t h e e a r l y period ex ovo
t h e r e w a s a r a p i d in crease of a c t i v i t y t h a t r e f l e c t e d white f i b e r developrient.
M a x i m u m a c t i v i t y w a s a t t a i n e d i n 6 - 8 weeks. I n dystrophic muscle, t h e slower
r a t e of development during t h e f i r s t week w a s followed by a r a p i d inc r e a se
during t h e second week; a change i n rate of enzyme development a f t e r t h i s
period l e d t o a decrease i n phosphorylase a c t i v i t y and low l e v e l s of t h e
enzyme were maintained throughout rnzturity . She equated percent a c t i v e
phosphorylase as being phosphorylase a l t o t a l phosphorylase. All va lue s were
g r e a t e r t h a n 65-70$ from 4-32 days ex ovo which i n d i c a t e d t h a t phosphorylase n
w a s t h e predominant enzyme i n t h e homogenate. I n t h e embryo? however, a l l
values were low and w e r e taken t o mean t h e r e l a t i v e absence of phosphorylase a
a t t h a t perio d . Other evidence on t h e c o l o r of iodine r e a c t i o n i n homogenates
i s c i t e d t o show a stro n g er r e a c t i o n as t h e muscle develops, p a r t i c u l a r l y
1 4 - 7 4 days. To summarize, t h e r e w a s a r a p i d enzyme inc r e a se i n b r e s s t f i b e r ;
during e a r l y ex ovo l i f e . A 50 f o l d inc r e a se i n enzyme a c t i v i t y i s seen from
hatching t o 6 weeks and probably r e f l e c t s r a t e of white f i b e r d i f f e r e n t i a t i o n .
The white f i b e r s we h ig h ly d i f f e r e n t i a t e d c e l l s adapted t o anaerobic
functions.
Hartshorne and P erry (1962) studie d sarcoplasmic p r o t e i n s f r o a
f o e t a l and a d u l t r a b b i t muscle. They found g e n e r a l l y t h a t t h e more p o s i t i v e l y
charged p r o t e i n components, which are r e a d i l y e l u t e d from diethylaminoethylc e l l u l o s e , are r e l a t i v e l y much decreased i n sarcoplasm i s o l a t e d from f oe tFJr a b b i t s k e l e t a l muscle and from a d u l t h e a r t muscle compared with t h e amolints
present i n t h e corresponding f r a c t i o n s from a d u l t s k e l e t a l muscle. More
s p e c i f i c a l l y , ald o lase a c t i v i t y i n foetal s k e l e t a l muscle sarcoplasrn i s comparable with t h a t i n a d u l t h e a r t muscle sarcoplasm, but much lower t h a n t h a t
i n a d u l t r a b b i t s k e l e t a l muscle sarcoplasm. Enzymic a c t i v i t y r i s e s as t h e
f o e t u s develops, and t h e g en eral conclusion of t h i s comparison i s t h a t , art
l e a s t f o r t h e components moving toward t h e cathode, f o e t a l muscle sarcoplasm
i s more comparable with t h a t of a d u l t h e a r t muscle t h a n t h a t of a d u l t white
s k e l e t a l muscle. The sarcoplasm of r e d s k e l e t a l muscle appears t o be i n t e r mediate between t h e s e 2 types. The a uthor s thought t h a t t h e high concentrat i o n of aldo lase i n a d u l t s k e l e t a l muscle sarcoplasm might be expected i n view
of t h e w e l l developed a b i l i t y of t h a t t i s s u e t o f u n c t i o n a na e r obic a lly and
furthermore suggested t h a t after b i r t h , a ldola se and othe r p r o t e i n s or' high
i s o e l e c t r i c p o in t in crease r a p i d l y i n amount i n response t o t h e increased
a c t i v i t y of t h e s k e l e t a l muscle.
Kendrick-Jones and P err y (1967) state t h a t biochemical development i n muscle involves t h e ev o lu tion and growth of a c o n t r a c t i l e system and
t h e development of enzyme systems t o convert metabolic f u e l s t o ATP. The
c o n t r a c t i l e system of m a m m a l i a n s t r i a t e d muscle i s very s i m i l a among muscle
t y p e s as t o s t r u c t u r e and biochemical composition, but t h e major diffcrcrices
among types are r e f l e c t e d mainly i n t h e non-myofibrillar f r a c t l o n . Thcsc
r e s e a r c h e r s stu d ied AMP-deaminase and c r e a t i n e phosphokinase. Leg, c z r dia c
112.
and diaphragm muscles were studie d i n r a b b i t . All muscles had low a c t i v i t y
i n 20-24 day f o e t u s . Cardiac muscle a c t i v i t y remained low throughout t o t h e
a d u l t stage. Diaphragm muscle a c t i v i t y inc r e a se d r a p i d l y 4-5 days before
p a r t u r i t i o n , reaching a m a x i n u m be f or e b i r t h , and t h e n f e l l slowly t o t h e
adult l e v e l . Leg muscle w a s c onsta nt u n t i l 8-9 days a f t e r b i r t h and t h e n
r o s e s t e a d i l y t o t h e adult l e v e l by about 1 4 days ( 7- 8 t i m e s i n c r e a s e ) . T h i s
i n c r e a s e i n l e g muscle AMP-deaminase occurred during t h e period when young
animdls began t o leav e t h e n e s t md move about independently. AMI?-deminase
increased r a p i d l y i n rat l e g muscle as t h e newborn began t o move about b u t i n
chick and guinea p i g l e g muscle it r o s e r a p i d l y be f or e b i r t h and reached t h e
adult l e v e l soon afterw ard . Adenylate kinase c l o s e l y p a r a l l e l e d t h e change
i n AMP-deaminase i n a l l s p e c i e s . Likewise, c r e a t i n e phosphokinase a c t i v i t y
was low i n foetal. t i s s u e b u t inc r e a se d a t t h a t sta ge of development a t which
a c t i v i t y r o s e depending on t h e s p e c i e s and muscle. These r e s e a r c h e r s thought,
from i n t e r p r e t a t i o n of t h e da ta , t h a t use of t h e muscle might be a stimulus
for r i s e i n s p e c i f i c a c t i v i t y of t h e enzTymes. Therefore, young r a b b i t s were
encouraged t o move about e a r l i e r t h a n usua l and i n t h e s e d i s t u r b e d l i t t e r s
t h e c r e a t i n e phosphokinase a c t i v i t y increased more r a p i d l y and a d u l t va l u e s
were approached e a r l i e r t h a n i n c o n t r o l animals. The r e s u l t s a r e most
i n t e r e s t i n g i n view of t h e f a c t t h a t AMP-deaminase i s most s p e c i f i c a l l y
a s s o c i a t e d w ith muscle. I n r a b b i t , t h e muscle a c t i v i t y i s 60-100 tim e s
g r e a t e r t h a n i n any o t h e r t i s s u e . I t ' s s i g n i f i c a n t t h a t t h e r e w a s a good
c o r r e l a t i o n between r a p i d i n c r e a s e i n enzyme a c t i v i t y and time a t which
c o n t r a c t i l e a c t i v i t y began t o r i s e r a p i d l y . The results s t r o n g l y suggested
t h a t change i n a c t i v i t y w a s not i n response t o a systemic stimulus but
endogenous t o muscle i t s e l f . It could be i n response t o developnent of
nerve system of muscle o r frequency of s t i m u l i r e c e ive d; i . e . , a c t i v i t y
p a t t e r n . The f a c t t h a t animals born at a more advanced sta ge of development ( l i k e guinea pig) haxe a l r e a d y high enzyme a c t i v i t y f i t s w e l l w i t h
previous histochemical. o b se r va tions (see Dubowitz, 1968). The a uthor s p o i n t
out t h a t t h e p o s s i b i l i t y of a c t i v a t i o n of enzyme pr e c ur sor s or change i n
isoenzyme complement t o more a c t i v e enzymic components cannot be r u l e d o u t .
The m y o f i b r i l l a r p r o t e i n s have been considered dur ing development
of r a t s k e l e t a l muscle by Ermine and Schaub (1968). They used m y o f i b r i l s
f o r assay and found t h e major changes dur ing t h e f i r s t 3 weeks of f o e t a l l i f e .
++ ATPase a c t i v i t y
The Mg"
ATPase a c t i v i t y in c r e a se d about 4 times, whereas C a
at low i o n i c s t r e n g t h only doubled. Ca"
ATPase a t high i o n i c s t r e n g t h and
pH 9 . 0 increased about 2 t i m e s . The low a c t i v i t y of Ca? ATPase of n a t u r a l
actomyosin a t low i o n i c s t r e n g t h i s due t o tropomyosin bound t o t h e complex
under t h e s e co n d itio n s. When tropomyosin i s removed, t h e ATPase a c t i v i t y
becomes as h ig h as i n t h e presence of Mg++ and a t t h e same time t h e actonyosin
and Cz++ a c t i loses i t s EGTA s e n s i t i v i t y . The d i f f e r e n c e between t h e Mg*
vated ATPase of my o fib rils at low i o n i c s t r e n g t h i n c r e a s e s with f u r t h e r
development. T h i s might r e f l e c t a continuous r i s e of tropomyosin c ont e n t of
t h e m y o f i b r i l . The EGTA s e n s i t i v i t y also i n c r e a s e s s i g n i f i c a n t l y during the
l a t e r s t a g e s of development; t h e EGTA s e n s i t i v i t y i s r e a l i z e d b y t h e j o i n t
a c t i o n of t r o p o n i n and tropomyosin. Thus, t h e s e r e g u l a t o r y p r o t e i n s are
s t i l l developing a t a t i m e when t h e enzymatic a c t i v i t i e s of t h e c o n t r ? c t i l e
p r o t e i n s have reached t h e a d u l t l e v e l . The a uthor s also point out 'chat t h e
ATPase a c t i v i t y of r e d muscle i s as high as it i s f o r white muscle i n t h e
r z t . T h is i s i n c o n t r a s t t o o t h e r work with r a b b i t where r e d nosel,? ATPacc
w a s much lower t h a n t h a t from white muscle. I n r a t , t h e r e s i s t a n c ? t o i n a c t i v a t i o n of m y o f i b r i l l a r ATPase from r e d compared t o white muscle w a s t h e
o n l y apparent d i f f e r e n c e between t h e 2 t y p e s .
113.
The enzyme probably most studie d dur ing t h e developmental changes
i s l a c t i c dehydrogenase. F iv e e l e c t r o p h o r e t i c forms of l a c t i c dehydrogenase
are found i n t h e muscle of animals; t h e y r e p r e s e n t t w o d i s t i n c t t y p e s of
enzyme ( M and H) with 3 in termedia te hybr ids ( Fine e t al., 1963) . When
p a t t e r n s do not follow a binomial d i s t r i b u t i o n , more t h a n one c e l l type i s
involved. The above r e search ers followed development a3 changes i n hwnsn
s k e l e t a l muscle. The r e s u l t s were expressed a s percent E-I s u b u n i t s and t h e y
found 99% i n muscle from 6 week old f o e t u s e s . Eighty- f ive pe r c e nt i n 3
month old, 60% i n 7 month o ld and 1 2 , 2 2 and 26% i n 3 d i f f e r e n t adults.
--
Clausen and H u s t r u l i d (1969) studie d l a c t i c dehydrogenase,
glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase i n
human f o e t u s e s ranging i n age from 13-25 weeks g e s t a t i o n . They used locomotive (gastrocnemius, psoas n a j o r , s a r t o r i u s ) and postur e ( g l u t e u s maximus,
quadriceps femoris, soleus) muscles. During f o e t a l development a l i n e a r
steady i n c r e a s e i n t o t a l l a c t a t e dehydrogenase a c t i v i t y a s w e l l as a l i n e a r
decrease i n t h e H/M subunit r a t i o of t h e isoenzymes was found. No s i g n i f i c a n t changes were found i n t h e a c t i v i t i e s of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The changes found suggest a
steady incre ased s y n t h e s i s of l a c t a t e dehydrogenase M-subunits i n human
s k e l e t a l muscles during f o e t a l development. The weekly changes i n t h e totc&
l a c t i c dehydrogenase a c t i v i t y and i n l a c t i c dehydrogenase isoenzymes a r c
lower i n muscles involved i n support t h a n i n those involved i n p e r i o d i c cont r a c t i l i t y . These f i n d i n g s , t o g e t h e r with t h e l i t e r a t u r e a v a i l a b l e , z r e
c o n s i s t e n t w ith t h e morphological f a c t t h a t f o e t a l development of s k e l e t a l
muscle mostly concerns t h e white muscle f i b e r s and not t h e r e d muscle
fibers.
Singh and Kanungo (1968) hzve a l s o published r e s u l t s on l a c t i c
dehydrogenase isoenzymes i n developing r a t muscle.
Syromy and Gutmann (1967) studie d t h e f a s t p o s t e r i o r l a t i s s i m u s dorsli
and slow a n t e r i o r l a t i s s i m u s d o r s i i n 20 day embryo, 1 day, 8 day and 30 day
chicks. T he ir d a t a suggested t h a t d i f f e r e n t i a t i o n may involve 2 d i f f e r e n t
mechanisms, one e s s e n t i a l f o r t h e f a s t muscle and r e f l e c t i n g a predominance
of g l y c o l y t i c processes, t h e o t h e r e s s e n t i a l f o r slow muscle and r e f l e c t i n g a
f a s t e r tur n o v er of p r o t e i n s .
CROSS-INNERVATION STUDIES
W
e have, up t o t h i s p oint, considered t h e histochemical, physiologi c a l and biochemical changes t h a t a r e c h a r a c t e r i s t i c of t h e process of muscle
f i b e r d i f f e r e n t i a t i o n ; however, we have l e f t unanswered t h e que s t i o n of what
c o n t r o l s and r e g u l a t e s t h i s p ro ce ss of d i f f e r e n t i a t i o n . The b e a u t i f u l e x p e r i ments on cr o ss-in n erv atio n shed some l i g h t on t h i s que stion.
_-
The i n i t i a l experiment of B u l l e r e t a l . (1960b) on nerve c r o s s union w a s considered e a r l i e r i n t h e physiology s e c t i o n . You w i l l r e c a l l t h a t
t h e i r p o s t u l a t e involved t h e a c t u a l passage of a substance down t h e axons.
We s h a l l con sid er now 4 a d d i t i o n a l manuscripts on c r o s s - i n n e r v a t i o n t h a t a r e
p e r t i n e n t t o t h e a i m s of t h i s review.
Dubowitz (1967) employed t h e slow sole us and f a s t f l e x o r h a l u c i s
longus (FHL) o r f a s t f l e x o r digitorum longus (FDL) of newborn k i t t e n c ?id
114.
r a b b i t s and a d u l t c a t s i n h i s experiments. Cross-innervation produced
dramatic changes i n t h e histochemical p a t t e r n of f a s t miiscle with t h e
development of a r e a s i n d i s t i n g u i s h a b l e from normal s o l e u s . The c r o s s i n n e r v a t i o n FHL showed a s t r i k i n g appearance on enzyme histochemical
assessment. Areas of t h e muscle had a normal FHL p a t t e r n with a checkerboard of ty p e I and I1 f i b e r s , although w ith an apparent absence of i n t e r mediate f i b e r s . I n ad d itio n, t h e r e were a r e a s of muscle composed e n t i r e l y
of type I f i b e r s and i n d i s t i n g u i s h a b l e from normal s3le us. These l a t t e r
zones cornprised e i t h e r s i n g l e or r i u l t i p l e b d d l e s of f Lbe r s o r a t t i m e s o d y
p a r t of a bundle. The converse change from t h e h i s t o c h e a i c a l p a t t e r n of slow
soleus t o t h a t of f a s t muscle also o c c n r e d b u t was l e s s c o n s i s t e n t . When
f a s t muscle i s r e i n n e r v a t e d by sole us motornetxones, it produces EL slower
t w i t c h and a low m a x i m u m r a t e of t e n s i o n developnent dur ing isome tr ic
tetanus--b o th a r e c h a r a c t e r i s t i c of normal slow muscle. Solnus inncrvztcd
by f a s t FHL motorneurones produces a f a s t e r t w i t c h , b u t r e t a i n s a 1 o ~ m
i aximum r a t e of t e n s i o n development dur ing isome tr ic t e n s i o n . Thus, it f a l l s
s h o r t of complete conversion and i s an inte r me dia te hybrid. H istoc hen i c a l l y ,
all t h e transformed cro ss-i nne r va te d nuscle s z r e hybr ids. The incomplete
conversion may be due t o some i n e v i t a b l e s e l f - i n n e r v a t i o n , even when t h e
crossed nerves a r e w e l l sepa r a te d from each o t h e r . Ditbowitz (1967) concluded
t h a t n eu ral i n f l u e n c e determining t h e c o n t r a c t i l e p r o p e r t i e s of f a s t and
slow muscle h as a profound c o n t r o l l i n g inf lue nc e on t h e s t r u c t u r e and metab o l i c a c t i v i t y of t h e muscle f i b e r s . It i s i n t e r e s t i n g t h a t he could not
demonstrate biochemically a s i g n i f i c a n t change i n t h e ATPase a c t i v i t i e s o r
t h e f a s t and slow muscles following c r oss- inne r va tion.
Romanul and Van Der Meulen (1967) studie d c r o s s union o r reunion
of nerves t o so leu s and FDL o r FHL i n young and a d u l t c a t s and r a t s . Crossinnervated muscles rev ersed t h e i r speed of c o n t r a c t i o n and t h e enzymatic
c h a r a c t e r i s t i c s of t h e i r f i b e r s . Thus t h e high oxida tive and low g l y c o l y t i c
enzymatic p r o f i l e of t h e s o l e u s muscle f i b e r s was changed t o a low oxl d a t i v e
and high g l y c o l y t i c p a t t e r n of t h e normal FDL and FHL f i b e r s . Converse
changes occurred i n t h e FDL o r FHL. Reinnervated muscles showed no change
i n t h e speed of c o n t r a c t i o n o r pr opor tion of f i b e r s of a ppr opr iz te enzynatie
t y p e s . The results prove t h a t t h e energy metabolism of t h e muscle f i b e r s i s
determined by t h e nerve supply, as i n t e r p r e t e d by t h e s e a u t h o r s . An a d d i t i o n a l o b serv atio n made was t h a t i n normal muscle t h e f i b e r s of d i f f e r e n t
histochemical types were s c a t t e r e d among each o t h e r . I n c r oss- inne r v a t e d and
reinn erv ated m s c l e , f i b e r s of t h e same histoc he mic a l type were arranged i n
s m a l l groups. Some groups of f i b e r s of a s i n g l e histochemical type and equal
s i z e had a diameter which d i f f e r e d markedly from t h a t of a l l o t h e r fiber::
r e g a r d l e s s of t y p e . Romanul and Ven Der Meulen (1967) thought t h i s suggested
s t r o n g l y t h a t t h e motor units were histoc he mic a lly homogeneous. Gross observ a t i o n rev ealed t h a t cro ss-inne r va te d muscles were changed i n c olor ; i . e . ,
soleus p a l e r and FDL d ark er r e d .
K a r p a t i and E n g e l (1967) studie d t h e sequence of histochemic -1
changes from a mixed to uniform p a t t e r n i n guinea p i g soleus e x t r a f u s n l ni x l e
f i b e r s from 50th day g e s t a t i o n t o 6 weeks p o s t n a t a l and also t h e prevention
of t h a t change by n eo n atal denervation. The sole us, i n a d u l t guinea p i c , i r
uniform w ith only ty p e I f i b e r s t h a t a r e l l g h t with ATPase and phosphor)l?s?
and dark w ith most o t h e r dehydrogenases. The sole us, i n newborn guine.1 pic,
i s mixed w ith 55-655 ty p e 11 f i b e r s . Type I1 f i b e r s diminish up t o 6 b d ~ ~ ~ ~ k c
age when t h e y a r e no lo n g e r p r e s e n t . I n newborn k i t t e n t h e r e rrc’ ,ip’roxirnti,ly
115.
equal numbers of type I and type I1 f i b e r s , and i n a d u l t c a t t h e r e a r e
occasional type I1 ( ( 3 % ) f i b e r s . I n newborn r a t t h e r e i s l e s s d i f f e r e n t i a t i o n and some myotubes are even pr e se nt; about equal numbers of type I and
type I1 f i b e r s a r e apparent by 1 0 days p o s t n a t a l and i n a d u l t t h e r e i s
g r e a t e r t h a n 90% type I. The l o s s of type I1 f i b e r s i n guinea p i g soleus
during maturation i s prevented by neonatal s c i a t i c denervation, but nct by
neonatal tenotomy. The mixed f i b e r population p e r s i s t s i n denervated b L t
t h e c o n t r a l a t e r a l c o n t r o l muscle develops t h e a d u l t p a t t e r n . The "loss"
of type I1 f i b e r s i s delayed by nerve crush followed by r e inne r va tion
but does occur a t a l a t e r t i m e . The l l l o s s l tcould be atrophy or t r a n s formation, but t h e authors fav o r transformation because: (1) a bsolute number
of f i b e r s in c reases, ( 2 ) diameter of type I1 f i b e r s inc r e a se s u n t i l the y
gradually disappear and (3) n ecrotic f i b e r s are not seen. Two conclusions
w e r e reached. F i r s t , t h e maturing motor nerve has a d e c i s i v e r o l e i n
determining t h e normal histochemical p r o f i l e of developing muscle c e l l s .
It i s possibl e t h a t t h i s fu n ctio n of t h e lower motorneurone i s dependent
upon suprasegmental in n erv atio n and would be absent i n de a f f e r e nta te d l o v e r
motorneurones. Second, t h e authors s t a t e t h a t t h e histochemical p a t t e r n
of e x t r a f u s a l muscle f i b e r s i n soleus of newborn animals i s not w e l l
c o r r e l a t e d with speed of co n tract ion. They quote s t u d i e s with c a t ( B u l l e r
et al., 1960a) t h a t show a t b i r t h all muscles including soleus a r e slow
t w i t c h and w ith in a f e w weeks c e r t a i n (EDL and gastrocnemius) become f a s t
twitch, whereas soleus remains slow tw itc h. The r e se a r c he r s ( Ka r pa ti and
Engel, 1967) t h i n k t h a t t h e profound l o s s of type I1 f i b e r s from s o l e s of
newborn r a t and guinea pig, as the y develop t o maturity, should be accompanied
by considerable physiologic slowing t h a t has not a c t u a l l y been shown with c a t .
Therefore, t h e y suggest t h a t all type I1 f i b e r s a r e not f a s t tw itc h physiol o g i c a l l y , a t l e a s t i n developing muscle.
F u r t h e r thoughts have been gleaned from c r oss- inne r va tion e xpe r iments by Robbins, Karpati and Engel (1969) who studied t h e isometric cont r a c t i l e p r o p e r t i e s of guinea p i g soleus muscles i n v i t r o . Although s o l e i
are normally slow t w i t c h muscles, s o l e i cross-innervated by peroneal or
t i b i a l nerves (which normally innervate f a s t muscle) show speeding of isometric c o n t r a c t i l e p r o p e r t i e s :
shortening of t w i t c h t i m e t o peak, s l i g h t
increase i n m a x i m u m rate of isometric shortening and diminished build-up
of t e n s i o n during a low frequency t e t a n u s . Normal guinea pig soleus a r e a l l
histochemically type I, b u t a f t e r c r oss- inne r va tion a v a r i a b l e percent of
type I1 f i b e r s appeared as defined by m y o f i b r i l l a r ATPase r e a c t i o n . The
physiologic d a t a were c o n s i s t e n t with t h e hypothesis t h a t a l l f i b e r s w ithin
t h e cross-innervated so leu s a r e p a r t i a l l y "speeded". The d a t a were incomp a t i b l e with t h e hypothesis t h a t some f i b e r s i n X-innervated muscle a r e
completely ''Speeded'' becoming l i k e normal f a s t f i b e r s , t h e r e s t remaining
completely slow. The c o r r e l a t i o n s of percent of type I1 f i b e r pe r crozss e c t i o n a l a r e a with t w i t c h time-to-peak or 5/sec tetan-0s:twitch r a t i o l e d t o
t h e i n t e r p r e t a t i o n t h a t with t h e ATPase r e a c tion, change i n histochemical
f i b e r type occurs when physiologic "speeding" exceeds a c e r t a i n thr e shold.
Figur es are given t o i n d i c a t e t h e e xte nt of type I1 f i b e r s p r e s e n t . I n a l l
cross-innervated s o l e i a v a r i a b l e number of t y p i c a l type I1 e x t r a f u s a l f i b c r s
of normal diameter and a r c h i t e c t u r e appeared, e i t h e r s i n g l y or i n groups. The
p ropor tion of type I1 f i b e r s , expressed as percent of t o t a l c r o s s - s e c t i o n a l
area of muscle a t t h e midpoint, ranged from 5 t o 37 percent with a mean of 18.3
percent; i n s e l f re-innervated and c o n t r a l a t e r a l s o l e i , no type I1 fibers were
seen and i n EDL t h e c r o s s - s e c t i o n a l a r e a f o r type I1 w a s about 88 pe r c e nt.
--
116.
The auth o rs g iv e c a l c u l a t i o n s and reasoning t h a t change i n c o n t r a c t i l c
p r o p e r t i e s of slow muscle, cross-innervated by a nerve normhlly inne rv z t i n g
f a s t muscle, i s p resen t i n most or a l l of i t s component f i b e r s . The ir r e s u l t s
i n d i c a t e t h a t t h e percent of type I1 f i b c r c a c t s only as an indic:Ltor of t h e
degree of speeding of t h e whole f i b e r population. For insta nc e , t h e p o s t n a t a l
developing r a t so leu s shows a progressive c h o r t c n i w of c o n t r a c t i o n time ( i. e . ,
"speeding") w h i l e t h e percent of type TI i'ib~r.:;i:; .ictu;Llly decreasing. It
i s possib le t h a t while t h e mean c o n t r a c t i o n t i n c i:, slowly moving i n t h e
d i r e c t i o n of increased percent speeding, t h e histochemical "turnover p o i n t "
i s moving t o a h ig h er percent speedin€ a t ~1 s t i l l f z s t e r r a t e .
SIGNIFICANCE TO MEAT mSEARCH
W e have examined i n d e t a i l t h e process of f i b e r d i f f e r e n t i a t i o n .
We have done so because t h e r e i s an important a r e a of a p p l i c a t i o n t o meat
science; t h e f i b e r type composition of meat animals can markedly influence t h e
post-mortem change and u l t i m a t e value f o r use as food (Cooper et al., 1 9 6 9 ~ ) .
I n order t o understand and solve t h e problem, w e must know how t h e f i b e r
populatio n develops and what f a c t o r s inf lue nc e t h e development of normal or
abnormal fiber populations. Dubowitz (1963) s t a t e d i n one of h i s e a r l y papers
t h a t l a c k of d i f f e r e n t i a t i o n i n muscle f i b e r s i n some d i s e a s e s may r e p r e s e n t
a f a i l u r e of maturation of t h e muscle and the r e by a study of d i f f e r e n t i a t i o n
might y i e l d valuable i n s i g h t t o understanding t h e d i s e a s e . W e thought l i k e w i s e t h a t ab n o rmalities i n t h e muscle of meat animals (such a s Giant F i b e r s ,
Cassens et a l . , 196973) might be b e t t e r understood by having a v a i l a b l e a
complete histochemical d e s c r i p t i o n of f i b e r d i f f e r e n t i a t i o n .
--
Cassens e t al. (1969a) have used t h e Sudan black B technique t o
v i s u a l i z e red and white f i b e r s i n porcine longissimus muscle a t t h e following
developmental stages: 8-9 weeks g e s t a t i o n , 9-1/2-11 weeks g e s t a t i o n , 1 2 - 1 3
weeks g e s t a t i o n , 1 day p o s t n a t d , 13 day old, 180 day o l d and 24 month o l d .
There w a s no d i f f e r e n t i a t i o n of f i b e r t y p e s i n any of t h e f o e t a l s t a g e s or at
1 day of age. Much lo o se connective t i s s u e w a s pr e se nt i n t h e f o e t a l s t a g e s ,
t h e f i b e r s were round r a t h e r t h a n polygonal and t h e f i b e r s were arranged i n
c l u s t e r s t h a t resembled primary bundles. The f i b e r s from t h e foregoing
s t a g e s were Sudan b lack B p o s i t i v e and t h e r e f o r e t a k e n as type I. The d i f f e r e n t i a t i o n of fiber t y p e s w a s c l e a r by t h e t i m e t h e animal had reached 13 days
of age and about 60 percent of t h e f i b e r s were Sudan black B negative o r
type 11. Type I f i b e r s composed only a small p o r t i o n of t h e t o t a l f i b e r s
(about 15%) by t h e time t h e animal had reached 180 days of age and t h e r e
appeared t o be l i t t l e change as t h e p i g matured t o 24 months of age. T h i s
work e s t a b l i s h e d t h e g e n e r a l a s p e c t s of f i b e r d i f f e r e n t i a t i o n i n p i g l o n g i s simus muscle.
Cooper e t al. (1969a) have completed an e xte nsive histochemical study
of f i b e r d i f f e r e n t i a t i o n i n p i g muscle (longissimus) by using DPNH-TR, amyloet a l . , 1969b)
phosphorylase and ATPase techniques. I n a ddition, t h e y (Cooper have run a complementary study with biochemical techniques (phosphorylase,
l a c t i c dehydrogenase, glutamate-oxaloacetate transaminase and l a c t i c dehydrogenase, glutamate-oxaloacetate transaminase and l a c t i c dehydrogenase isoenzymes)
The p i g s were Poland China, Yorkshire and t h e r e c i p r o c a l c r o s s e s between t h e
ages of 1 day and about 6 months. Figur e s 2, 3, 4 and 5 show t h e process of
d i f f e r e n t i a t i o n as revealed with histoc he mistr y. F i b e r d i f f e r e n t i a t i o n had
not t a k e n p lace a t 1 day of age according t o histochemical observations a i though t h e myosin-ATPase appeared t o show some s l i g h t d i f f e r e n t i a t i o n . At l
-
L
.
117.
week of age t h e ty p e I f i b e r s were d i s t i n g u i s h a b l e from t h e othe r f i b e r s .
Type I1 and in termed iate f i b e r s could be s e p a r a t e l y c l a s s i f i e d a t 4 wceks of
age. Percentage of a p a r t i c u l a r f i b e r type , c a l c u l a t e d on n. c r oss- se c tion3 1
area b a s i s , i n d i c a t e d t h a t type I1 f i b c r ; increased ovcr t h e period of 4
weeks t o 6 months a t t h e expense of intcrrricdi.&r’ C i h c r s , bJherrrJs t h e type I
f i b e r s decreased only s l i g h t l y . Number ol’ t’ibcr:, vcr:;u, ‘ ~ r c of
‘ ~ f i b e r s gdve
a bimodal d i s t r i b u t i o n f o r t h e 1 day o l d pi(. .itid ‘ I unirqod’i L onc Lor t h e 6
month old a n i q a l . Over t h e period studic d t , h > r C , w:~.: ‘ 1 r r ~ l * i t i o n s h i between
p
f i b e r s i z e and age. Biochemical dctcrrriin.il 1 onL: of’ phO;phorylrJSc :And lr,ctic
dehydrogenase a c t i v i t y increased ovcr the : L ~ c ‘ periods studie d ‘Ind %reed with
t h e increase of histochemically c l a s s i f i e d type I1 f i b e r s . The glutarinteoxaloacetate transaminase l e v e l s increased over t h e f i r s t f e w weeks and t h e n
f e l l as t h e animal grew o l d e r .
Morita et a l . , (1969) have employed t h e histochemical myoglobin
technique t o t h e same group of p i g s described i n t h e preceding paragraph.
They found e s s e n t i a l l y a negative myoglobin r e a c t i o n i n longissimus rnuscle
of 1 day old p i g s . By 3 weeks t h e t y p i c a l a d u l t p a t t e r n w a s e vide nt and t h e
c a p i l l a r y d i s t r i b u t i o n w a s c l e a r and s i m i l a r t o t h a t i n adult muscle.
An a d d i t i o n a l observation, t h a t might be q u i t e s i g n i f i c a n t , should
be mentioned a t t h i s p o i n t . I n all t h e f i g u r e s shown ( Figur e s I, 2, 3 ,
4, 5), t h e arrangement of type I f i b e r s i s i n t h e d i s t i n c t clumps o r groups-t h i s i s ver y u n lik e t h e checkerboard o r s c a t t e r e d arrangement t h a t i s known
f o r o t h e r mammalian muscle. T h is p a t t e r n i s reminiscent of t h e s m a l l g r o ~ p s
of one f i b e r ty p e seen i n cross-innervated o r reinnervated muscle (Romanul
and Van Der Meulen, 1967). The r e a l importance of t h i s observation i n pig
muscle i s unknown but c e r t a i n l y merits f u r t h e r i n v e s t i g a t i o n .
D i f f e r e n t i a t i o n h as a l s o been studie d i n beef muscle (Waldman, 1 9 6 7 ) .
H e studied longissimus muscle from b i r t h t o about 1300 l b s animal weight and
g e n e r a l l y found a decrease i n red fibers as t h e animal matured.
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( I n prepara-
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Figure 1. Red and white f i b e r d i s t r i b u t i o n i n porcine longissimus muscle.
The red f i b e r s a r e grouped i n clumps i n c o n t r a s t t o t h e
checkerboard arrangement t h a t has been r e por te d f o r o t h e r
mammalian muscle. Secion r e a c te d f o r DPNH-TR; A i s 20 X and B
i s 12%.
122.
Figur e 2.
S ectio n s (p o rcin e longissimus muscle) r e a c te d f o r DPNH-TR t h a t
show t h e d i f f e r e n t i a t i o n of f i b e r type s. A (1day old; 496 X)
shows a uniform s tr ong r e a c t i o n , but does r e v e a l some l a r g e
f i b e r s and some small f i b e r s . B (1week old; 3 1 2 X) shows a
d i f f e r e n t i a t i o n of s t r o n g l y and weakly r e a c t i n g f i b e r s , but
t h e weakly r e a c t i n g s t i l l d i s p l a y a gr a nula r p a t t e r n . C
( 2 week old; 312 X) shows a more d i s t i n c t d i f f e r e n t i a t i o n .
123.
Figure 3.
Sections (p o rc ine longissimus muscle) t h a t show t h e d i f f e r e n t i a t i o n of f i b e r type s. A ( 3 weeks old; 3 1 2 X) s t i l l shows
t h a t l i g h t l y s t a i n i n g f i b e r s have a gr a nula r r e a c t i o n p a t t e r n .
B ( 4 weeks; 312 X) and C ( 5 weeks; 312 X) i l l u s t r a t e t h e time
when white f i b e r s begin t o lose g r a n u l a r i t y and intermediate
f i b e r s can be s e p a r a t e l y c l a s s i f i e d . D Shows a sectTon from
8 week o l d p i g a t 1 2 4 X .
124.
Figure 4.
S ectio n s (p o rcine longissimus muscle) t h a t show t h e d i f f e r e n t i a t i o n of f i b e r t y p e s . A i s from 1 6 week old p i g at 1 2 4 X
and B and C are from 20 and 24 week old p i g s r e s p e c t i v e l y at
50 X. From t h e period 4 weeks t o 6 months, type I1 f i b e r s
i n c r e a s e a t t h e expense of inte r me dia te f i b e r s whereas type I
fibers decrease s l i g h t l y .
125.
Figure 5.
S e r i a l s e c t i o n s r e a c t e d f o r diphosphopyridine nuc le otide
t e t r a z o l i u m reductase ( A ) , mylophosphorylase (B) and
ATPase ( C ) . The g r e a t e r s e l e c t i v i t y of t h e ATPase i s
i l l u s t r a t e d . All at 1 2 4 X.
126.
BOB CASSENS:
Paper I n .
JIM KEMP: Thank you ve r y much Bob and i f we have t i m e when we
g e t through, w e ' l l l e t him show t h e s e . Now t h e next speaker w i l l be using
t h e other p r o j e c t o r , so we w i l l go ahead.
BOB CASSENS:
P r o j e c t o r ' s f i x e d so on with t h e s l i d e s .
JIM KEMP: Thank's Bob for a ve r y e x c e l l e n t paper d e s p i t e th e
inconvenience. Our second speaker i s from t h a t "show me" s t a t e of Missouri.
He showed me t h a t he knows q u i t e a b i t about h i s subje c t when I read his
r ecent paper t h a t came from t h e Symposium on Body Composition i n Animals
and Man. Milt B a i l e y w i l l now speak t o us on "Changes i n P r o t e i n s and
Related Substances i n Muscle During Growth and Development. " M i l t .
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