The disposition of membrane systems in cardiac muscle of a lobster, Homarus americanus

TISSUF; & CELl, 1972 4 f4) 629 645 l'Mffi.shcd I0' Lon.~,nlan Group Ltd. Prinh'd it~ Great Brilail~

DAVID S. S M I T H * and M A R G A R E T E. A N D E R S O N * *

THE D I S P O S I T I O N OF M E M B R A N E S Y S T E M S IN C A R D I A C MUSCLE OF A LOBSTER, H O M A R U S A M E R I C A N U S ABSTRACT. The membrane systems of cardiac muscle o[" the lobster Homaru.s americanu.v are described. Wide radial invaginations of the plasma membrane occur at the Z level. From these arise longitudinally disposed narrower tubules leading to a collar encircling the fibril at the H level, which is associated with cisternae of the sarcoplasmic reticulum in dyadic and triadic junctions. Tiffs situation is compared with the pattern of T-system invagination previously reported in muscles of crustacea and other animals. Its physiological significance is also discussed.

(1965) describe, in crayfish, walking leg muscle, irregularly distributed cleft-like sarcolemrnal invaginations which give rise to tubules that c o n t i n u e to the A I j u n c t i o n s a n d form dyadic contacts with the SR Limulus (Sperelakis, 1971 and L e y t o n a n d S o n n e n b l i c k , 1971) and t a r a n t u l a (Sherman, personal c o m m u n i c a t i o n ) cardiac muscles also possess plasma m e m b r a n e invaginations f r o m which tubules extend to m a k e intimate c o n t a c t with the SR. In all these muscles some transverse tubules m a y originate directly frorn the cell m e m b r a n e . In n o n e are the tubules restricted to a precisely transverse orientation. An u n u s u a l feature of some crustacean muscles is the presence of two separate systems of t u b u l a r invaginations from the p l a s m a meinbrane. One t u b u l a r system enters at the level of the Z band a n d appears not to adjoin the sarcoplasmic reticulum, T h e second system enters the fiber elsewhere a l o n g the sarcomere and associates closely with the SR in dyadic contacts at the level o f the A - I j u n c t i o n in crab skeletal muscle (Peachey a n d Huxley, /964); a n d at the level of the H b a n d in s t o m a c h muscle of the spiny lobster (Atwood, 1971) a n d in cardiac muscle o f the s h r i m p (lrisawa and H a m a , 1965) and crayfish ( K o m u r o , 1969), Two separate tubular systems h a v e also been described in lobster walking leg muscle ( J a h r o m i and A t w o o d , 1971) a n d crab eyestalk muscle

Introduction

FINE structural studies on a variety of striated muscles have established the general, a l t h o u g h n o t invariable, presence of t u b u l a r invaginations of the plasma m e m b r a n e into the interior of the muscle f i b e r - - t h e transverse t u b u l a r system (TTS). Exceptions are small cylindrical or flattened tibers in which the distance between the contractile material and the plasma m e m b r a n e is minimal (e.g. Amphioxus, Peachey, 1961; Coelenterates, C h a p m a m 1962). T h e tubules are believed to provide a pathway for the i n w a r d radial conduction of excitation f r o m the fiber surface, a n d they m a k e intimate contact with the sarcoplasmic reticulum (SR) b y engaging in dyads or triads with SR cisternae. T h e tubules characteristically enter at a specific sarcomeric locus such as the Z level, or some p o i n t between this and the center of the sarcomere, pass radially into t h e fiber and c o n t a c t the S R at the level of entry. A r t h r o pod muscles exhibit considerable diversity in the o r g a n i z a t i o n o f tubular invaginations of the surface m e m b r a n e . B r a n d t et al. * Papanicolaou Cancer Research Institute and Department of Medicine, University of Miami, Miami, Florida 33136, U.S.A., and *Laboratory for Neurobiology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico 00905. ** Present address: Department of Biology, Williams College, Williamstown, Massachusetts, Manuscript received 20 JuLy 1972. 629

SMITH

630

( H o y l e a n d M c N e i l l , :1968), L e y t o n a n d S o n n e n b l i c k (1971) h a v e s u g g e s t e d t h a t I . , i m u l , s h e a r t m u s c l e a l s o p o s s e s s e s t w o sets of invaginating tubules. The myocardium of the lobster Homar.s ameHcamfs has recently been investigated by e l e c t r o p h y s i o l o g i c a l a n d e l e c t r o n m i c r o scopic techniques. Structural properties of i n t e r c e l l u l a r j u n c t i o n s (discs) h a v e b e e n d i s c u s s e d w i t h r e s p e c t to i o n t o p h o r e t i c a l l y injected dye molecules and the passive electrical p r o p e r t i e s o f t h e m u s c l e fibers, together with the structural properties of nerve muscle junctions (Anderson and S m i t h , 197.1), T h e p r e s e n t s t u d y , o n t h e s a m e material, considers the distribution of plasma membrane iuvaginations and associated c i s t e r n a / c o m p o n e n t s w i t h i n t h e fiber. I n this m u s c l e , t u b u l a r s a r c o l e m m a l i n c u r s i o n s o c c u r at t h e level o f t h e Z b a n d ; b r a n c h e s :from t h e s e t u b u l e s e x t e n d l a t e r a l l y t o w a r d

AND ANDERSON

the center of each sarcomere and dyads and t r i a d s a r e f o r m e d r e g u l a r l y at t h e level o f the H zone.

Materials and Methods Full

details o f t h e p r e p a r a t i o n o f t h e hear( muscle for electron microscopy have been published by Anderson and Smitln (1971). I n brief, t:he m y o c a r d i u m w a s f i x e d in ice-cold 2 ' 5 % g l u t a r a l d e h y d e in 0"05 M c a c o d y l a t e buffer at p H 7-4, c o n t a i n i n g 18% s u c r o s e . S u b s e q u e n t l y , s m a l l p i e c e s of the myocardium were prepared with a r a z o r b l a d e , w a s h e d in b u f f e r e d s u c r o s e solution; treated with cacodylate-buffered 1% o s n T i u m t e t r o x i d e ; d e h y d r a t e d in a u e t h a n o l series a n d e m b e d d e d in A r a l d i t e (CIBA, Duxford, England). Sections were cut with glass knives on an LKB Ultrotome I l l ; p i c k e d u p o n u n c o a t e d 300 m e s h grids Homarus

Fig. 1, Transverse section, el" part e r a cardiac muscle fiber of laromarll,s~aweric'anlt,s'~ The fibrils (F) are close-packed, and divided into irregular groups by clusters of mitochondria (M). Part of a nucleus (N) is included. :.t 15,000. Fig. 2, Transverse section, at higher magnification. Profiles of A and I bands are included, and the fibrils are encircled at these levels by membranes of the sarcoplasmic reticuIum (SR). The A band array of thick and thin filaments is illustrated in the inset. ::45,000. loser: :<90,000, Fig. 3. Transverse section, as Fig. 2, but passing through the H baud level of a group of fibrils, Here. two-membered (dyadic) and three-membered (triadic) associations necker at I'requent intervals around eacIr fibril (arrows). As in other muscles, these represent closely juxtaposed elements of transverse tubules and cisternae of the sarcoplasmic reticulum, farther illustrated in Figs, 4. 8.9, 10 and 12. A dyad is included, at higher nlagnification, Jn the inset. The sarcoplasmic reticulum component (sr) is identified by sligfitIy denser content than the apposed transverse (t) system profile and by the opaque b]ocks placed at intervals along the surface of the cisterna, x 45,000. Inset: >: 130,000. Fig. 4. A field illustrating the longitudinal striation pattern and disposition of interfibrillar membranes in Homaras cardiac muscle, The Z bands (in this preparation defining a sarcomere period of ca. 2"8/~) are flanked by conspicuous I bands. The A filaments are ca, 1.9f~ ilr length and the [ filaments ca. l'0t, in lengtb. The H band is here ca. 0'8t, in width and includes a narrow medial light pseudo-H zone (*). No M band is present. ]n this plane, a transversely sectioned profile e r a wide tubule (large arrows) is present at the Z band Ievet of most sarcomeres; generally the mid-sarcomere region opposite the H band is flanked by dyad or triad associations (small arrows) fin'tber illustrated in Figs. 8, 9, 10 and 12. Smaller membrane profiles o~" the sarcoplasmic reticulum occupy much of the sarcoplasm between the fibrils (SR). :: 30,000, Fig. 5 Transverse section of Homarrls cardiac muscle passing through the Z level of several peripheral fibrils (Z). Three open-mouthed Tz in-vaginations from the suri'ace plasma membrane are included, and in one instance (arrow) a narrow initial invagina~ tion leads to the wider tubule. The muscle cell is ensheathed in a basal lambm (BL) and similar material (% extends along the Tz tubules. >:40,000. Fig. 6, A longitudinal profile, illustrating the juxtaposition of a Tz tubule and a Z band. A layer of dense material is applied to the tubule surface opposite the Z band, The tubule contains opaque extracellular material (black * cf. Fig. 5), Note the abundant glycogen granules (G). >: 70,000.

i

~

~

~i

~

~

.

.~

.~ .

~

7

'
¸ ~i ¸¸ :L

.........

.....

;

,

.....

•

......

•

'

. . . .

~,,

~

, ......

,

•

,,~

.........

•

~

~ %. . . . . . . . . . . . . . . . . . . . .

~:

f ~ ; ~

;', ii~

,+

~!~7: ~, ~,

,,,:

L :'

~

,:

~,:,,~,

,

, . . . . . . . . . .

......

:" L

~

~i~i~

i~ ~ : ~

~ ~ ~

~ ~i~

~ ~ ~

~

~ ~ ~

~

:,

> : .:~:~::,:<. :i:¸ /i~i ¸, >~i.::> ~?,, ,~ ~ :,~ ~, ~;~:y.,>~ ~ ~i~ . >~ii:i>~ Y ,, y~ ~

/i>, ¸ .:?!~ :z!,!ii. ~.~ ~Y

.

iii~y ¸

SMITH

636 a n d s t a i n e d in s a t u r a t e d 50% c t h a n o l i c u r a n y l a c e t a t e a n d l e a d citrate, a n d e x a m i n e d in a P h i l i p s E M 200. Results The general features of cardiac muscle of the l o b s t e r , in t r a n s v e r s e profile, a r e s u r v e y e d in Fig. I. P e r h a p s t h e m o s t s t r i k i n g f e a t u r e o f this t i s s u e at this level o f o b s e r v a t i o n is t h e irregular clustering of mitochondria interspersed among blocks of myofibrils. The o r g a n i z a t i o n o f t h e l a t t e r is seen at h i g h e r m a g n i f i c a t i o n in Fig. 2: t h e p l a n e o f t h i s s e c t i o n p a s s e s t h r o u g h A a n d I levels o f a g r o u p o f m y o f i b r i l s a n d profiles o f t h e interposed mitochondria are included. At these s a r c o m e r e levels, t h e f b r i l s a r e e n c i r c l e d b y S R profiles b u t d y a d s a n d / o r t r i a d s a r e a b s e n t . A s i l l u s t r a t e d in t h e inset (Fig. 2), t h e A band array includes thin actin filaments midway between pairs of thick myosin

AND ANDERSON

f i l a m e n t s as in m o s t insect flight m u s c l e s ( S m i t h , 1968) a n d s o m e c r u s t a c e a n f a s t fibers ( F a h r e n b a c h , 1967; R o s c n b l u t h , 1969; lahromi and Atwood, 1969), F i g u r e 3 shows that the H band array contains thick filaments alone and that the myofilaments are ringed by f r e q u e n t t w o - a n d - t h r e e membercd membrane associations respectively r e p r e s e n t i n g d y a d s o r triads. A s in o t h e r a r t h r o p o d m u s c l e s (Fig. 3) ( S m i t h , 1966a), t h e a s s o c i a t i o n b e t w e e n t h e s u r f a c e of the T-system and the membrane of the a d j o i n i n g S R c i s t e r n a is m a r k e d b y p e r i o d i c o p a q u e b l o c k s t h a t arise f r o m the S R s u r f a c e a n d a p p a r e n t l y e x t e n d all o r m o s t o f t h e w a y a c r o s s t h e ca. 100 ~ g a p s e p a r a t i n g t h e m e m b r a n e s o f t h e d y a d i c or triadic junction. S h o w n in Fig. 4 a r e t h e g e n e r a l f e a t u r e s o f l o n g i t u d i n a l l y s e c t i o n e d m u s c l e . Several d y a d a n d t r i a d profiles a r e i n c l u d e d , p l a c e d precisely at t h e m i d - s a r c o m e r e (H b a n d ) level

Fig. 7. A transverse section similar to Fig. 5, but including a more extensive array of the wide T~ tubules, encompassing a group of myofibrils at the Z level and immediately adjacent I level. Note the opaque material lining the tubule membrane bordering the Z bands (white *), (cf. Fig. 6) and the extracellular material (black *) within the Tz tubules (cf. Figs. 5, 6). >~43,000. Fig. 8. Longitudinally sectioned Homarus cardiac muscle. From the Z level, an invaginated tubule (Tz) gives rise to a longitudinally placed derivative ('T') which engages with a fiat cisterna of the sarcoplasmic reticulum in a dyad association at tbe mid-sarcomere (H band) level (arrow). A similar dyad is included (arrow) in the lower part of the field, at the middle of the next sarcomere, >' 55,000. Fig. 9. Part of the field shown in Fig. 8 at higher magnification and similarly [abelled. Note the dense material (white *) flanking the Tz tubule invagination which forms a desmosome-Iike configuration seen more extensively in transverse profiles at the Z level in Fig. 7. :. ll0,000. l-:ig. 10. A longitudinal profile similar to Fig. 6 illustrating a wide invaginated tubule (Tz) at the Z level giving rise to a longitudinal tubule ('T') flanked by sarcoplasmic reticuhlm (SR) and engaging with the latter in a mid-sarcomere dyad (arrow). • 95.000. Fig. 11. Longitudinal section passing tangentially across the surface of a fibril. A wide tubule traverses the fibril at the Z level (Tz): as described in the text, the Tz tubule is an invagination of the surface plasma membrane. This tubule extends longitudinally ('T') and subsequently encircles the center of the sarcomere as a flat belt in which narrow regions alternate with wide dilatations. These are closely flanked by cisternae and tubules of the sarcoplasmic reticulum in dyadic and triadic configurations (cf. Vigs. 3, 8-I 0), one of which is here seen in surface aspect (arrow), I 10,000. Fig. 12. A longitudinal section similar to that in Fig. 1 l, including two adjacent Z bands (Z) and, in tangential prolile, grazing sections of the intervening sarcoplasmic membranes. The mid-sarcomere level is occupied by an apposed collar of sarcoplasmic reticulum and invaginated tubular components (corresponding to the transverse tubules of other fibers): in this field, the collar includes a wide dilatation in surface aspect (long arrow) and a dyad profile in transverse section (short arrow) together with a narrow intervening tubule. The bulk of the sarcomere is ensheathed by a uniform open network of cisternae and tubules of the sarcoplasmic reticulum (SR). >: 65,C00.

~: o::0~ ; i,'i !,i~ .....

i?~# ' :

<,

:!:

%

:~g %

~:~:~: i~!:2 ~

~i~ ~

,~

~:,~i ~

~ii},

~

~'i ¸¸¸

~i~ ~?~ :~!~,~ ~ ~ ~,,~,~ ~ ~ ~ ~

:

i I~¸

~i ~

~i;

~

~ ~

;. . . . . .

:~

~ ~ ~:~t~

i:'i~

-!~:~

i~< ~:~~i:~:~ :b/,~i~ ~. . . . . . . . . . ,~+~i~ ~!~ :>~:,~~ii

~ :~ < /

. . . .

~ ~

~..

,:< ~

~~,~

~,

~

7 ~ ~/~it . . . . . . . .

~ -~ ~/

~ ~,. ~i~,

~

~

/~

~~ % ;~:~~'~~<':~ <

~

~

~

S~

~ ~ ~

~

~ - j : , t ~

'~ ~ ~

~

~<~<

~ ~,

: ~-~:;~ ,~ ............

b ~'~S~ L ~' ,-

~ ~

~

~<,~i,<:i <~/:~ii ~

~ ~ . . . . . ~~~ ~ ~: ~ v ~,

~

~ ,,~:~¢,%~,,

o,~

~/~

~

,~,

~_

<' / ,~ ~

~

.... ~'<~ ~ ~<~!k~:~k~t.~ ~i'.~~i~ (i:~:~>~ ~ ~..~<<~ <

-~i ~i~ ~ .~,~,~ ~ ....

~

< ,%,.

"~L~~ ~ ~ ~ F ~: /i,'i %

:F'.

~

- <

: ~ -i,!~ ~,~~ . , ~ ÷

:; <

k~

~ili h< ¸ ~ =~

#
! c :~ ,~~:~

: ~ ~ F ~, : , ~ , ~ , ~

~

-

~i~

~

~' ~ / ~ i i , ~ F

~~

i/~'~'~ t~

~ ~!~

.......

~/i~.~,~:.~~..~!~,.~ii~/::~ ~ . ~ 9 . ~ % ~ ' ~

.

,~2 <;. :~7~,i

~-~i~;i~:~

,~b,~C ; ~(i :4,!~~

<'~ ~ . ,

¸¸<.%

640 while conspicuous tubule profiles lie in the sarcoplasm opposite most of the Z bands. In otker muscles (Franzini-Armstrong and Porter, 1964 and Smith, 1966b) transverse sections in the plane of the dyads or triads typically include the origin of the T-system invaginations passing in from the cell surface. Fields of Homarus heart muscle in the same plane as Fig. 3 and including the periphery of the cell do not show an extensive system of membrane incursions, suggesting that the plasma membrane invaginates, in this type of muscle, along the Z level tubules and via their branches to other sarcomere levels. The striation pattern of this muscle lacks an M band, but includes a pseudo H zone as in mammalian skeletal muscle (Huxley, 1965) which presumably represents a medial segment of the myosin filaments of a sarcomere devoid of cross-bridges. The tubules cccupying the Z level in crustacean muscles (here termed T,. tubules) are readily identified as invaginations of the surface plasma membrane (Fig. 5). These incursions are generally open-mouthed, but in some instances the initial invagination into the cell is a slender tubule or cleft leading into the wider T~ tubule. As shown in Fig. 5, and also in the longitudinal profile in Fig. 6 which includes a T~ tubule tightly apposed to a Z band, these tubules contain extracellular material similar in electron opacity to that of the basement membrane (basal lamina) surrounding the fiber (Fig. 5). A similar but more extensive aspect of the T , tubules is seen in Fig. 7. This micrograph illustrates a constant feature of these tubules - - t h e presence of a layer of opaque material lining the sarcoplasmic side of the tubule surface imrnediately adjoining the Z band, a feature also evident in the longitudinal section in Fig. 6. Unlike the crustacean muscles in which the Z level tubules and the dyad or triad associations at other levels have been regarded as separate systems, in heart muscle of I t o m a r u s americanus the Z level tubules appear to continue to the dyads and triads. ;Figures 8 and 9 include a transverse profile of a T~ tubule, t¥om which stems a narrow branch that courses longitudinally through the sarcoplasm to contribute to a dyad at the 14 level of the sarcomere. The tubule destined for the dyad or triad, derived from the surface membrane, corresponds to the

SMITH AND ANDERSON 'transverse tubule' of other muscle cells: it recovers a transverse orientation as it encircles the fibril at the H level, but the longitudinal connecting links with the Tz iuvaginations are, in Homarus muscle, morphologically an integral part of the complex system of cell membrane incursions. A further example of this relationship between the dyad and the initial Z level invagination is illustrated in Fig. 10. Sections passing tangentially over the fibril surface reveal further details of the topography of internal membrane systems in Hon~arus cardiac muscle. The plane of section in Fig. 11 is approximately normal to that in Figs. 8-10. Here, a T~ tubule and a membrane collar at the mid-sarcomere level are seen in surface aspect as they run transversely around the fibril, and the longitudinally directed tubule linking the two is included in the plane of section. Transverse sections of the fiber (Fig. 3) indicate that the dyads and triads vary considerably in length (ca. 0'15-0"4~)~ this is reflected in the periodic dilatation and constriction of the flat tubule encircling the central zone of the sarcomere and of the flanking sarcoplasmie reticulum cisternae. This variation in width is seen in Fig. l l , and further in Fig. [2. The latter field includes, in addition to a surface view of the irregularly anastomosing tubular or cisternal elements of the general SR, a dyad in transverse aspect leading via a narrow tubular segment to a dilatation ca. 0-3tL in diameter, which as a consequence of the curvature of the underlying fibril surface, is seen in surface aspect. This dilatation represents a surface view of a dyad or triad; its enhanced opacity compared with the neighboring SR cisternae is attributed to the stacking of 4 or 6 membranes depending on whether the association is dyadic or triadic. Figure 13 represents a three-dimensional reconstruction of the membranes of Homarus heart muscle. This diagram is based on the accompanying electron micrographs and incorporates the present finding of the dyads and triads in this muscle, though occupying the central sarcomere zone, nevertheless receive their plasma membrane component (the 'transverse tubule' of other muscles) primarily via branches stemming from invaginations originating at the level of the Z band.

MEMBRANE

SYSTEMS

IN CARI)IAC

MUSCLE

64

H

@ Fig. 13. Three-dimensional reconstruction o[" the disposition of membrane systems oF the Hotllarizs myocardium based on the accompanying electron micrographs. The origin o[" the Tz tubules as open-mouthed invaginations of the plasma membrane is indicated by arrowheads. These traverse the fiber at the Z band level, and at intervals give rise to longitudinally oriented branches that continue into a periodically dilated collar around the center (H level) of tlne sarcomere, The general librillar surface is ensheathed in a fenestrated cisternal and tubular sarcoplasmic reticulum; at the It level the reticulum and invaginated membrane collar become closely associated in dyad and triad configurations (arrow).

SMITH AND ANDERSON

642 Discussion Arthropod muscles are unusually diverse with respect to sarcomere length, shape of the Z band, actin-myosin ratio and filament array, topography of plasma membrane in-. vaginations and the location between these invaginations and cisternae of the SR. Unfortuz?atcly, precise comparison between fiber types is rendered difficult by the uneven emphases of tine structural accounts. iln general, however, as Atwood (1971) summarizes the situation, in most crustacean [ibers there have been described two parallel systems of tubules open to the external environment which enter' the fiber as invaginations> These have been distinguished in the past by their position with respect to the sarcomerc----one series entering at the Z level, the other at a level characteristic of a particular tibet" and generally near the A--I junction or the H level. The only correlated structural and physiological study on these membranes is that of Peachey and Huxley (11964.) who in micropolarization experiments employing an external current-passing electrode on crab muscle failed to elicit a response when the electrode was placed at the Z level, but observed local contractile responses when the electrode was at the A - I level where dyad associations are located. These results convincingly implicated an invaginated component at the A--I junction that was in contact with the dyad through which excitation-contraction coupling is accomplished, and, in crab muscle, appeared to relegate the Z tubules to some other unidentified function. Subsequently, tubules at the Z level have been described in other crustacean muscles and assumed to be separate from the tubules that contact the SR (Irisawa and Hams, 1965: Reger, 1967; Hoyle and McNeill, 1968; Komuro, 1969: Atwoo& 1971, and Jahromi and Atwood, 197[). l,eyton and Sonnenblick (1971) also report two systems of tubules in LimHlHs heart inusde. Dyads (and triads) occur in cardiac fibers and in pyloric muscles at the center of the sarcomere, and in other instances at or close to the ends of the A band. All accounts except those of lrisawa and Hama (1965), Komuro (/969) and Howse et af. (1971 ) that consider the question agree that dyads or triads are never observed to involve apposition of SR cistcrnae and the tubules that

invaginate at the Z level. Irisawa and Hama (1965) describe dyads at the M Ievel in &juilla heart muscle but do not convincingly document a supposed triadic association of the SR with the Z level invaginations. Komuro (11969) states that dyads and triads occur at both the M and Z bands in the crayfish heart; however, the tubular associations at the Z level do not resemble the obviously dyadic structures of the M level. It should be noted that none of the above accounts includes a micrograph showing membrane invaginations at the dyad level by revealing open-mouthed iuvaginations in transverse sections in the plane of the dyad, such as have been documented in skeletal muscles of other animals (Franzini-Armstrong and Porter, 1964 and Snfith, 1966b). The only description providing any evidence for a physical link between the two tubular arrays is that of Reger (1967) in which a micrograph of crab abductor claw muscle shows a lateral extension :from the Z tubule to the dyad at the center of the sarcomere: the account implies that other tubules enter the fiber at the level of the dyads, but this is not shown. Atwood (1971) observed no longitudinally oriented tubules and suggests that these, if present at all, in lobster pyloric muscles, are sparse. Leyton and Sonnenblick (197l, Fig. 9) described, in LimHiu,s
MEMBRANF,

SYSTEMS

tN C A R D I A C

MLJSC]~E

noted that this tissue shows a regular pattern of in tern al mem brahe d ist ri buti o n which p uts into new perspective the lubular arrays in this tiber type. As documented in the electron micrograplns, this muscle (a) exhibits conspicuous radial invaginations at the Z level; (b) exhibits dyads and triads at the center of the H zone; (c) does not exhibit a system of open-mouthed invaginations at the level occupied by the dyads and triads and (d) exhibits convincing evidence of the frequent presence of longitudinal tubules cotmecting the invaginations at the Z level with the circumtibrillar 1[ level collar participating with SR cisternae in the dyads and triads, It should be stressed that while confluence between the two series of tubules is well established in HomarHs cardiac muscle, this by no means indicates that the same is true of other fibers that ihave been studied in detail. For example, the micropolarization studies of Peachey and Huxley provide highly persuasive evidence for the existence of two different systems of tubules in crab muscle. Further micropolarization studies on other types of muscles would be useful in clarifying this issue. The responses of lobster cardiac muscles, w,hich appear to possess only one system of sarcolemmal incursious~ could be compared with the responses of muscles that are thougl:~t to possess two such systems. In the ttomarlls myocardium the principal series of membrane invaginations occur at the Z level and contribute, via connecting tubules, to the dyads and triads and thus is the :morphological counterpart of the transverse (T-system) tubule of other fibers, We have termed these iavaginations T~ tubules to indicate their position and our interpretation, in fine absence of a system of transverse invaginations from the peripheral plasma membrane elsewhere along the sarcomere, of their functional significance in this muscle. Similarly, the longitudinal connecting links, despite their contrary orientation, form part of the membrane pattern corresponding to the 'transverse tubular system' of other fibers. The TTS of lobster cardiac muscle is therefore similar to that of crayfish skeletal muscle (Braudt el al., 1965) and Limuhls (Leyton and Sonnenblick, 1971 and Sperelakiso 1971) and tarantula (Sherman, personal communication) and crayfish (Howse et al., 1971) heart muscles in that it departs from

643

a transverse orientation, It appears to differ from these muscles by its organization in a precisely organized array, Further, dyads in lobster and crayfish cardiac muscle libe]s are formed at tile H level whereas they are formed at the A-o-Ijunction in LimH/te.v heart (Leyton and Sonnenblick, 1971) and craylish walking leg muscle (Brandt c t a / . , 1965) and usually near the A band in tarantula heart muscle (Sherman, persona[ communication), Dyads in crayl~sh walking leg muscles (Brandt eg al., 1965) are formed between the SIR and T-tubules, the SR and the sarcolemma and the SR and the sarco., lemmal invaginations; in lobster heart muscle dyads are formed only between cisternae of the SR and the T-tubules. The Tz tubules of HoynarHs heart muscle may be analagous to the regular sarcolemmal invaginations at the Z level described for Litn~dus (Sperelakis, 1971) and tarantula (Sherman, personal communication) heart muscles. (Leytou. and Sonnenblick, ]971, observed invaghmfions at the Z ]ine only occasionally in LimHhts; they state that most tubules, presumably of a differe~t system from those that enter at the Z lines, invaginate at the A. I junction.) The regularly occurring incursions possess extl'acellulaJ lining and give off lateral, unlined T-tubules which run paralie] to the long axis of the fibers and form dyads and triads with tlne SR. The sarcolemmal in.vaginations in crayfish walking leg muscles are similar in all of these respects to the invaginations of other fiber types except that they occur irregularly with respect to the sarcomere pattern. Unlined T-tubules do not originate directly from the sm'face membrane in lobster cardiac muscle as they do iu crayfish walking leg muscle (Brandt et al., 1965) and LimldHs (Sperelak]s, 197/) and tarantula (Sherman, personal communication) heart muscles, Perhaps the closest correspondence to the present account is :found in the description of cardiac muscle fiber of the crayfish by Howse e t a / . (1971) and in Reger's (1967) description of the abductor claw muscle of the crab Pi,mi,via in which certain fibers exhibit plasmaqemmal invagiuations at the level of the Z band which are linked by lateral tubules to the dyads at the center of the sarcomere. It is of interest to note that: the TTS of frog slow muscle fibers, although forming dyads and triads with the SR at the

(~44

same sarcomere level at which it originates from the fiber surface, resembles the TTS of lobster cardiac and other muscles in spread~ ing longitudinally; the tubules that are parallel to the myofilaments are estimated to account for as much as 50% of the volume of the total T-system (Flitney, 1971). The capacitance of lobster cardiac muscle fibers (mean, 8"37 /,F/cm e, Anderson and Smith, J 971 ) is somewhat lower than that of other crustacean peripheral muscle fibers (30~55"0 /~F/cm e) (Fatt and Katz, 1953 and Atwood, 7963). It is generally agreed that the capacitance of the walls of the transverse tubular channels contributes substantially to the capacitance of the fiber (Falk and k'att, 1964 and Gage and Eisenberg, 1969); thus assuming a given value of capacitance for the surface membrane itself, the rnore extensive the interior tubular invaginations along which current spreads toward the center of the fiber, the greater will be the apparent capacitance per unit area of surface (Falk and Fatt, 1964). Some studies correlating electrical properties and morphological features have been made on vertebrate muscle fibers. Luff and Atwood (1972) observed greater values of membrane capacitance iT7 mouse fast extensor digitorum Iongus muscle fibers than in slow soleus fibers. Ultrastructural studies of the same tissues revealed a greater surface area and volume of the TTS and more frequent occurrence of triads in the fast than in the slow fibers, although the arrangement of the TTS was similar in both fiber types. Adrian and Peachey (1965) found the capacitance per unit area of surface to be about three times as great in the fast fibers as in the slow fibers of the tonus bundle of iliofibularis it/ frog° The slow fibers were shown by electron microscopy to possess a much less developed TTS than the fast. Thus, it could be argued that the regular pattern of Z tubules and lateral cc>nnecting links in lobster cardiac muscle is a less extensive system of invaginations than that of muscle fibers that

SMITH AND ANDERSON exhibit greater values of apparent membrane capacitance. Support of this argument would require comparative analyses, similar to the studies done on vertebrate muscle fibers, of the tubular systems and the passive electrical properties of different muscle fibers. As in other muscles, the dyads are regularly placed at one preferred sarcomere level in lobster cardiac muscle (as, for example, in rat skeletal muscle: Porter and Palade, 1957) instead of at two levels per sarcomere (e.g., in amphibian skeletal muscle, Porter and PaJade, 1957). Assuming that, by analogy with other fibers, excitationcontraction coupling involves the release of calcium from the SR component of the dyad the diffusion path for the ion is of the same length whether the dyad lies at the Z or the H level. For all but the most peripheral fibrils, the addition to the length of tubule that links the Z tubules with the dyads is probably negligible. At the present time, it appears that the positioning of dyads and triads with respect to the sarcomere divisim~s offers no obvious correlation with physiological activity.

Acknowledgements It is a pleasure to thank Miss Elisabeth Lindgren for technical assistar~ce. This work was supported by grant GB-12117X (DSS) from the National Science Foundation, and by National Institutes of Health postdoctoral fellowship 1-F2-HE-35, 383-02 (MEAL

Note added in proof: Forbes et. al. (1972) describes a single system of invaginating tubules for the L i n m l i u s heart which is sirnilar to that of the lobster heart. Sarcolemmal invaginations enter the fiber at the Z level and T-tubercles, oriented mainly longitudinally, arise from the invaginations. Dyadic contact with the SR appears to occur at the A-I level.

MEMBRANE

SYSTEMS

IN CARDIAC

MUSCLE

645

References ADRIAN, R. H, and PIiACHEY~ L. D. 1965, The membrane capacity of frog twitch and slow muscle fibres, J. Phvsiol,, 181, 324~436, AND~:RSON, M. E, and SMrrH, D. S. 1971. Electrophysiological and structural studies oil tile heart muscle of tile lobster l[omarus americ'anus~ Tissue & Cell, 3, 191-205. A'lwooD, H. L. 1963. Differences in muscle fiber properties as a factor ill 'fast' and 'slow' contraction in Careinus. Comp. Bioehem. P/lysiol., I11, 17-32. A'rwoon, H. L. 1971. Z and T tubules in stomach muscles of tile spiny lobster. J. Cell Biol., 50, 264 -268. Fh~,ANDI, P. W.. RISUBF;N, J. P., GIRARDIER, L. and GRUND~:Esr, H, 1965. Correlated nnorphological and physiological studies on isolated single muscle fibers. I. Vine structure of tbe crayfish lUuscle fber, J. Cell Biol., 25, 233-260. CIIAPMAN, D. M. 1962. Muscle in coelenterates, Rev. can, Biol., 21, 2 6 7 2 7 8 . FAHP,ENI~ACIt~ W. H. 1967. The line structure of fast and slow crustacean muscles. ,1. Cell Biol., 35, 6 9 7 9 . FALK, G. and FA r'r, P. 1964, Linear electrical properties of striated muscle fibres observed with intracellular electrodes. Proc'. R. Soc. B, 160, 69-123. FA rl~ P. and KA'IZ, B. 1953. The electrical properties of crustacean muscle fibres~ .L Physiol., 120, 17D 204° FI,ITNEY, F. W, 1971. The volume of the T-system and its association with the sarcoplasmic reticulum in slow muscle fibres of the frog. J. Physiol., 217, 243- 257. FaANZJN1-ARMS'rRONO, C. and PORTER, K. R. 1964. Sarcolemmal invaginations constituting the 3' system in fish nmscle fbers. J. Cell Biol., 22, 675 696, GAGE, P. W. and ~ISENBER6, R. S, 1969, Capacitance of line surface and lransverse tubular m e m b r a n e of frog sartorius muscle fibers. J. gem Physiol., 53, 265-278. FIOYLE, 11, and McN~l~t,, P. A. 1968o Correlated physiological and ultraslructural studies on specialised muscles, lb. Ultrastructure of white and p i n k fibers of the levator of the eyestalk of Podophthahnua' vigil (Weber)..I. £X'p. Zool., 167, 487-522. H u x l , E¥, H. E. 1965. Tile mechanism of muscular contraction. Sci. Amer., 213, 18--27. IRISA\¥A, A. and HAMA, K. 1965. Some observations on tile fine structure of the mantis shrimp hearL Z. Zell/bJwch. mikroslc Anat., 68, 674-688. JAHROMI, S~ S. and ATWOOD, H. L, 1969. Correlation of structure, speed of contraction, and total tension in I)tst and slow a b d o m i n a l nmscle fibers of" the lobster (Homarus amerieaHHs). J. exp. Zool., 171, 2 5 3 8 . JaHRo~al, S, S. and A rWOOD, H. l,. 1971. Structural and contractile properties of lobster leg-muscle fibers. J. Exp. Zool., 176, 475-486. KOMUI~Q T. 1969. The fine structure of the crayfish cardiac muscle. J. Electron Microsc,. 18, 291 297. LI2YTON, R. A. and SONNENBL1CK~ E. H. 1971. Cardiac muscle of tile horseshoe crab, Limit/us po@phenlu,s'. J. Cell Biol., 48, 10I--119. L,UFF~ A. R. and ArwOoD, H, L. 1972, M e m b r a n e properties and contraction of single muscle fibers in the mouse. Am. J. Physiol., 222, 1435 1440, PI~ACIII-;Y, L, D. 1961. Structure of the longitudinal body muscles of Amphio.vus. J. hiophys, hiochenz. CytoL !0 suppl., I59 176, PEACHEV, L. D. and HuxL~;V, A. F. 1964. Transverse tubules in heart muscle, J. Cell Biol., 23, 70A. PoR'r~l< K. R. and PALADin, G. E. 1957. Studies on tile endoplasmic reticulum. 111. Its form and distribution in striated muscle cells. J. biophys, bioehem. Qytol,, 3, 269-300. REGH~, J. F. 1967. A comparative study on striated muscle fibers of the first antenna and the claw muscle of the crab Pinnixia sp. J. Ultrastruct. Res., 7,0, 72--82. ROS~NBLITr~I, J. 1969. Sarcop/asmic reticulum of an unusually fast-acting crustacean muscle. J. (>ll Biol, 42, 534-547. SHH~MAN, R. G. (in preparation). Ultrastructural features of cardiac muscle celIs in a t a r a n t u l a spider. SMITh, D. S. 1966a, The organization and function of fine sarcoplasmic reticulum and T-system of muscle cells. Prog. Biophys. Alolec. Biol., 16, 107 142. S~J~TH, D. S. /966b. The organization of flight muscle fibers in the Odonata. J. Cell Biol., 28, 109 126. SMITH, D. S. [968, lnsect Cells: Their Strztcture and Ftmction. Oliver & Boyd, Edinburgh. SVER~:LAK1S, N. 1971. Ultrastructure of the neurogenic heart ofLimulus po!vphemus. Z. Zell/brsch. mikrosk, Anat., 1116, 443-463.