Excitation-Contraction Winifred
Coupling
T
CELL
MEMBRANE
This is a complex structure,3a4 consisting of a diffuse reticulum of collagen fibrils, a diffusemucopolysaccharide containing layer-the basement coat, and a relatively thin, enzymaticallyactive plasma membrane which is selectively permeable to certain ions and is contiguous with the cytoplasm. The possiblefunctional significance of the mucopolysaccharide-containingbasementcoat hasrecently attracted widespreadinterest and speculation,3p5,6 mainly because it contains a large number of negatively charged sites. These negatively charged sitesconfer on the basementcoat a capacity to selectively accumulate a variety of cations, including Ca’+. The possible functional significance of the basement coat’ will be discussedin detail in a later section of this review, but even at this early stageit is perhapsworth noting that it is this basementcoat which regulates the ionic environment of the milieu immediately adjacent to the plasmamembrane.That the basement coat is heterogenousis evidenced by the fact that it can be subdivided on morphological grounds From the Cardiothoracic Institute, London, England. Reprints should be addressed to Dr. W.G. Nayler, Cardiothoracic Institute, 2 Beaumont Street, London, WI, England. 0 197.5 by Grune & Stratton, Inc. Progress
in Cardiovascular
Diseases,
Vol.
XVIII,
Muscle
G. Nayler and R. Seabra-Gomes
HE DELUSIVE SIMPLICITY of the phrase’ “excitation-contraction coupling” (E-C COUpling) concealsand overshadowsthe complex sequence of events which it describes. Although many questions remain to be answeredbefore all of the events which are involved in this sequence can be accurately describedand understood at the molecular level, the broad basicprinciples are now well defined. Before discussingtheseeventsinsofar as they relate to cardiacmusclecells,it is useful to consider briefly the fine structure of the various subcellular organelleswhich are involved. Because the processof E-C coupling starts with the reversal of the transmembraneresting potential2 we will start by consideringboth the fine morphology and the distribution of the cell membraneas it occurs in cardiac musclecells. THE
in Cardiac
No.
1 (July/August),
into an inner and outer zone. For example in cat ventricular and papillary musclecells the basement coat is approximately 500 a thick and consistsof a 200 A thick inner and a 300 a thick outer zone. Similarly the 400-500 i% wide basementcoat of turkey atria1 muscle cells can be subdivided into a 300-A wide outer layer which is more denseand more osmophilic than the 150-200 A thick inner layer.8 Although cardiac muscle cells do have a well defined basementcoat this is not a peculiarity of cardiac musclecells. For example, in the giant muscle fibres of the barnacleBdanus nubihs the basement coat9 is between 1000-3000 A thick. When heavily stainedsectionsof heart musclecells are prepared it can be seen that the innermost layer of the basementcoat containsmyriads of fine filaments which are anchored to the plasmamembrane,3 and becauseof this some investigators” have questioned whether the mucopolysaccharidecontaining coat may not be an integral part of the plasma membrane, i.e., is there any justification for considering it separately from the plasma membrane?From a functional point of view there are severalreasonsfor separatingthe plasmaImembrane from the extraneous coat,” including the fact that it is the plasmamembranewhich provides the main permeability barrier and which contains the firmly bound multienzyme complexes.Neverthelessit is the mucopolysaccharide-richbasement coat which regulatesthe immediate ionic environment of the enzymatically-active, selectively-permeableplasmamembrane. THE
PLASMA
MEMBRANE
The perimeter of eachmusclecell is defined by a 75-90-A thick plasmamembrane,which provides the cell with a selectively permeablebarrier. During the past decade numerous attempts have been made to isolate plasmamembranesfrom other subcellular components. The techniques employed usually involve homogenization followed by differential centrifugation and a variety of purification steps.12-16Even so it is difficult to establish the purity of suchsubcellularpreparations,because although the relative activities of a variety of marker enzymes, including the Na+-K+ activated ATPase, the adenylate cyclase, the Cazf-activated 1975
75
76
Fi ig. 1. Longitudinal {TI. Note that the material. X 25,000
NAYLER
section sarcodasmic
of rabbit reticulum
papillary 1%)
lies
muscle, showing invagination close to the T-tubule, and
ATPase, and the 5’ nucleotidases can be readily established, this provides no guarantee of the fact that under in vivo conditions these enzymes are associated with the plasma membrane. Of course, if the isolated plasma membranes retain their mucopolysaccharide coating then this will provide a means of identification. The chemical composition of the membranes-for example, either their cholesterol-phospholipid ratioI or their sialic acid5 content can also be used to assess their purity. Determinations of the phospholipid content of plasma membrane fractions, as well as being justified in terms of providing an index of the purity of a particular preparation, can be justified on
that
AND
of the cell membrane the T-tubule is lined
SEABRA-GOMES
to form a T-tc lbule with basement coat
other grounds, including the fact that the apparent reactivities of the phospholipids with Ca’+ varies in the order phosphatidylserine > phosphatidylethanolamine S phosphatidylcholine.” Moreover, and as will be discussed later, certain phospholipids exert a regulatory effect on the activity of some of the membrane-located ATPase enzymes. The distribution of the plasma membrane is often complex, mainly because the membrane and its accompanying basement coat are not necessarily restricted to the periphery of the cell. Instead, and as is shown in the electronomicrograph reproduced as Fig. 1, the cell membrane often invaginates to form a series of slender, branching’g220 tubules
EXCITATION-CONTRACTION
Fig. 2. x 37,500
Rabbit
77
COUPLING
papillary
muscle
showing
extensive
branching
(T-tubules) which penetrate deep into the myoplasm, usually in the vicinity of the Z band. The T-tubules of cardiac muscle cells differ from those found in skeletal muscle because: (1) in cardiac but not in skeletal muscle cells the mucopolysaccharide-coating of the plasma membrane extends into and often fills the lumen of the T-tubule, (Fig. 1); (2) in cardiac cells the diameter of the lumen of the T-tubule often exceeds 1500 & whereas in skeletal muscle cells the lumen is much smaller, usually’l J.’ ranging between 200-400 a in diam-
of the
T-tubules.
T-T-tubules,
E-extracellular
space.
eter; and (3) whereas in cardiac muscle cells the T-tubules are usually located at the level of the Z band, in skeletal muscle cells they are often located in the region of the A-l overlap.23 It must not be imagined, however, that T-tubules are present in all heart muscle cells. Indeed they are absent from many amphibians and reptiles. Chicken and humming-bird muscle cells,24 and some atria1 cells’ likewise lack an identifiable Tsystem. That these tubules, when they do occur, branch and ramify is clearly shown by the electron-
78
NAYLER
micrograph of part of a rabbit papillary muscle cell which is shown in Fig. 2. Although the functional significance of the Ttubules remains the subject of speculation, a careful consideration of their morphology leads directly to two conclusions. Firstly, these tubular
Fig. 3. approaches
Electronmicrograph of rabbit a T-tubule an intramembranous
papillary structure
AND
SEABRA-GOMES
invaginations provide a large increase in the surface area which is available, per unit cell volume, to facilitate both the distribution of the depolarising current,25-27 the exchange of ions between the intra and extracellular phases, and the uptake of substrates from the extracellular phase. Secondly,
muscle showing the sarcoplasmic is formed. E-extracellular space.
reticulum X75.000
(SRI.
Note
that
when
this
EXCITATION-CONTRACTION
COUPLING
it increases the area of the surface in which the various enzymes can be implanted and on which the mucopolysaccharide-containing basement coat material can be deposited. Isolated fragments of the plasma membrane coalesce, to form vesicles.12-‘6 Because the enzymatic activity which is exhibited by these vesicles will depend, to some extent at least, upon whether the vesicles have formed L‘right-side” or “insideout” it is relatively important for techniques to be developed to control this procedure. In the meantime substances such as conconavallin A can be used2’ to ensure whether ‘right-side’ or ‘insideout’ preparations have been made. THE
SARCOPLASMIC
RETICULUM
Whereas the events which are involved in E-C coupling’ start at the level of the cell membrane, the reverse phenomenon, that is the transition from the active to the resting state, starts at the level of the sarcoplasmicreticulum.s’29 This reticulum20321is a smooth reticulum, and consistsof a lace-like network of tubules which branch and ramify, crossing from sarcomere to sarcomere without apparent interruption (Fig. 3).j~~’ In marked contrast to the direct continuity which exists between the lumen of the T-tubule and the extracellular space19y2’the lumen of the sarcoplasmic reticulum is sealedoff from the extracellular spaceexcept, perhaps,in damagedcells.30 According to the morphometric studiesof Page and McCallister3r the sarcoplasmicreticulum occupies asmuch as 3.5 + 0.2% of the cell volume in myocardial cells.By contrast the transversetubular system occupies only 1.0 ? 0.1% of the total cell volume.31 This difference becomeseven more pronounced if the comparisonis madein terms of the ratio between the p sq m membranearea and the p cu m cell volume. When this is done then the ratio between the area which is occupied by the cell membrane, including the T-system, and the cell volume is 0.39 f 0.02.” The sarcotubular system provides a ratio of 1.22 f 0.0S.31 Of course morphometric data which is obtained from fixed preparations may be misleading, becauseof the changeswhich are caused by fixation, but even allowing for such inaccuracies(calculated asbeing up to 25% by Page and McCallister3’) it seems probable that the area of membranewhich is provided by the sarcoplasmicreticulum greatly exceedsthat provided by the cell membraneand its accompanying tubular invaginations. When ex-
79
pressedon a weight basisthe sarcoplasmicreticulum probably accounts for approximately 6.8 mg/g wet weight of heart muscle.3” Estimates of membrane area, volume, and mass may, however, be of little value, when applied to a morphological system such as the sarcoplasmic reticulum, becauseof the increasing amount of evidence which points towards the existence of specialization within the reticulum.3$20*23Thus whenever the facing membrances of the sarcoplasmic reticulum approach the plasmamembrane, irrespective of whether this occurs at the periphery of the cell or at the level of a T-tubule, specialized, flat, sac-like dilations form. These dilations, are part of the sarcoplasmicreticulum, and their lumen is continuous with that of the remainder of the reticulum. These specialized,sac-like dilations are commonly called cisternae,33 irrespective of whether they are located immediately below the cell membrane at the periphery of the cell (subsarcolemmalcisternae) or whether they run alongside a T-tubule (Fig. 4). The cisternae which are found in heart muscle are small when compared with those which are found in skeletal muscle cells. Nevertheless,they, like their skeletal muscle counterparts, contain intraluminal densities,often preservedas a denseline lacking the trilaminar appearance which is characteristic of membranes. This denseintraluminal structure doesnot extend uniformly throughout the whole of the sarcoplasmic reticulum. Instead, and as shown schematically in Fig. 5, it appearsto be confined to those areaswhere the reticulum either approaches or is closely apposedto the plasmamembrane. There is other evidence3’ of specializationwithin the sarcoplasmic reticulum. In some species a slender tubular profile is located at the level of the Z band.34 Undoubtedly this tubule is part of the sarcoplasmicreticulum, but the question of its functional significanceremainsunknown. There is further evidence of specialization at the level of the Z band, where those parts of the sarcoplasmic reticulum which are adjacent to the Z band olften contain 30-30 a intraluminal electron densegranules, and the walls of the reticulum may be coated with a 100-200-A thick electron densecytoplasmic coating.’ When cut in crosssection thesespecialized areasoften present the appearanceof electron densevesicles.5 The possible existence of areas of specialized function within the sarcoplasmicreticulum warrants serious consideration by investigators who
80
Fig. 4. Longitudinal drion, T-T-tubule-note tween SR and T-tubule.
NAYLER
section of rabbit papillary evidence of pinocytosis,
muscle. Perfusionfixed. SR-sarcoplasmic reticulum,
use cardiac microsomal fractions prepared by homogenization and differential centrifugation. Usually attention 35336is concentrated upon the further purification of a fraction which sediments at a relatively high g, and comparatively little effort has been expended so far in trying to retrieve any heavier components of the reticulum which will
X75.000. E-extracellular G-glycogen granules,
AND
SEABRA-GOMES
space, M-mitochonJG-junctional gap
be-
have been discarded earlier in the schedule. In skeletal muscle the heavier microsomal fractions are believed to consist predominantly of the cisternal elements, and have been shown to have Ca*+ accumulating properties which are qualitatively and quantitatively different from those exhibited by the relatively lighter fractions.3’338Y39
EXCITATION-CONTRACTION
IO-2
COUPLING
BAND
Fig. 5. Schematic representation of the electron-dense feet which project from the lining membrane of the SR into the junctional gap which separates the SR from the T-system. Note the presence of basement coat material in the T-tubule, and evidence of a substructure within the sarcoplasmic reticulum in those areas where it approximates a T-tubule.
During embryological development the composition, function and structure of the sacroplasmic reticulum membranes undergo striking changes4’ the changes being more markedin the skeletal than in the cardiac muscle preparations. According to Martonosi and his colleagues4’ microsomal fractions which have been prepared from chicken embryonic skeletal muscle are largely devoid of a Ca2+ transporting system, and the appearance of such a system coincides with the appearance of a Ca2+-activated ATPase activity.40 At approximately the same time the phospholipid content of the SR membranes changes, their linoleate content increasing and the palmitate content decreasing.40 Other studies, in which freeze-etch techniques have been utilized, have shown that the age-dependant increase in the activity of the Ca2+ activated ATPase enzyme is paralleled by an increased number of spherical, intramembranous particles.41 These particles are approximately 7.5 A in diameter, and they are not restricted to the sarcoplasmic reticulum. Within the sarcoplasmic reticulum, however, they assume a density of 3000 particles/p’ membrane area.41 Whether these particles have anything at all to do with the activity of the Ca2’-activated ATPase enzyme42 is at present unknown. It is known, however, that the
81
purified enzyme contains a major polypeptide of mol w 102,000, a proteolipid of approximlately 12,000 mol w, a variable amount of high molecular weight protein, neutral lipids and phospholipids.43 The phospholipid component amounts to 450 pg/mg membrane protein.43 This phospholipid component cannot be ignored, because it dominates both the permeability characteristics pf the reticular membranes,@ and the activity of the Ca2’ sensitive ATPase enzyme. Recent studies by Martonosi and his colleague? indicate that the phospholipids may regulate the activity of the Ca2+ sensitive ATPase enzyme by positioning a histidine residue at the active site, thereby facilitating the approach of the enzyme to the phosphoprotein bond, phosphorylation of the enzyme being an intermediate step in the hydrolysis of ATP by the Ca2+-activated ATPase enzyme. The hydrolysis of ATP by sarcoplasmic reticulum membranes involves two readily distinguishable steps44 which can be summarized as follows: (1) E+ATP+E-PtADP; (2) E-P+H,O-’ E + Pi; where E is the active enzyme. On the basis of several observations Martonosi and his colleagues 44 have postulated that histidine residues are involved in the decomposition of the phosphoprotein intermediate. These same authors44 postulate that the inhibition of ATPase activity in microsomal fractions which are depleted of lipids results largely from an inhibition of the decomposition of the phosphoprotein intermediate, a decomposition pathway which involves a functional histidine residue. Several proteins4’ which exhibit relatively high affinities for Ca2+ have been isolated from the sarcoplasmic reticulum. One of these proteins, calsequestrin (mol wt 46,500) does not exhibit high affinity Ca2+-binding but binds approximately 850 nmoles Ca2’/mg in the, presence of KCI, and 900 nmoles Ca2+ in the absence of KCI. Acidic proteins with mol wts of between 20,000 and 38,000 can also be isolated from sarcoplasmic reticulum fragments, and these proteins bind from 900 to 1000 nmoles Ca’+/mg protein. Whether these proteins are distributed throughout the whole of the sarcoplasmic reticulum, or whether they are concentrated in specialized regions (possibly within the cisternae) has not yet been determined. There are many other aspects of the biochemical properties of the sarcoplasmic reticulum which warrant attention, and some of these, including the
82
NAYLER
Fig. 6. Rat ture, JG-junctional
papillary gap,
muscle. X 100,000. F-electron-dense
T-T-tubule, feet.
SR-sarcoplasmic
capacity to accumulate Ca” will be discussed in a later section of this article. Always, however, it must be recalled that there may be areas of gross biochemical as well as morphological specialization within this fine network of tubules, and that the techniques which are currently used to isolate these membranes may neither permit the preservation of these specializations nor need they provide preparations free from contamination with fragments derived from other intracellular organelles. JUNCTIONAL AREAS BETWEEN THE SARCOPLASMIC RETICULUM AND THE T-SYSTEM
Whenever the membranesof the sarcoplasmic reticulum and the T-system lie side by side, specialized “junctional areas” occur. Thesejunctional areas4’jinvolve the facing membranesof the sar-
reticulum,
showing
AND
evidence
SEABRA-GOMES
of
internal
struc-
coplasmic reticulum and the T-tubules, the intervening or ‘junctional’ gap (Fig. 4) and the periodically-arranged, election-dense profiles which project from the facing membranesof the sarcoplasmic reticulum and extend into the junctional zone as shown in the electronmicrograph (Fig. 6). In those cardiac muscle cells which have a Tsystem, junctional zones are found whenever the tubules of the SR approach a T-tubular profile, as shownin Fig. 4. This doesnot mean, however, that these specialized zones occur only in the vicinity of a T-tubule. In fact they occur whenever the sarcoplasmic reticulum approaches the plasma membrane, irrespective of whether the plasma membraneis located either at the periphery of the cell or whether it forms part of a T-tubular invagination. Hence junctional areasmay be found close to the sarcolemma,and when this happens
EXCITATION-CONTRACTION
COUPLING
the junction is usually called a “peripheral coupling.” If the muscle cell contains a T-tubular system, however, then junctional couplings will almost certainly be found alongside the T-tubule, as well as beneath the sarcolemma. Franzini Armstrong 46y47has already described the fine morphology of these specialized junctional areas in great detail, and although her observations relate primarily to smooth and skeletal muscle cells, her description holds true for cardiac muscle cells. There is, therefore, no need for another detailed description here. Several points do, however, warrant attention: (1) The electron dense, regularly spaced, feet like extensions which project from the facing membranes of the SR into the junction gap apparently do not penetrate into the opposing facing membrane. This may reflect the absence of a morphological link between the two opposing membranes. Alternatively it may only mean that the ‘invisible’ intermediate link is not electron-dense. (2) The arrangement of the ‘feet’ is always periodic4 and the width of the intervening gap remains constant4 (120-200 A) irrespective of whether the diameter of the lumen of the T-tubule is made to alter-as occurs, for example, when heart muscle is rendered hypoxic. (3) Channels linking the lumen of the SR with that of the T-tubular system have not yet been identified.47 (4) The specialization which occurs within the membranes of the SR at these junctional areas probably reflects a specialization of function different from that exhibited by the longitudinal components of the SR.47 Recognition of the existence of these specialized junctional areas has resulted in widespread speculation as to whether they could facilitate the direct transmission of an excitatory stimulus and its attendant wave of depolarisation from the level of the cell membrane and its associated T-tubules to the sarcoplasmic reticulum, including the subsarcolemmal cisternae. The membranes which line the transverse tubular system are excitable.25’48 Indeed, when normothermic conditions prevail excitability within the T-system can account for as much as 70% of the mechanical response.*’ Nevertheless, and despite the fact that a regenerative action potential can be triggered in the T-system when the surface membrane is depolarized, and despite the supporting data obtained by Peachey and Adrian,49 and Caputo and Dipola,” evidence of the direct transmission of this activity to the sarcoplasmic reticulum is lacking. But even if such a link does exist, it is still necessary to prove that
83
information which is relayed from the cell membrane to the sarcoplasmic reticulum can influence E-C coupling. The recent demonstration by Endo and Nakajima’r of the fact that depolarisaticw of the sarcoplasmic reticulum in skinned skeletal muscle fibres induces a release of Ca2+, together with the earlier findings of Constantin and Podolsky52 and of Ford et a1.53 probably establishes the proof of the latter hypothesis. Ca2+ AND
CONTRACTION
Despite our present uncertainty as to whether a depolarisingsignalcan be transferred directly from the surface membrane to the sarcoplasmicreticulum it is now generally agreedthat the transition from the resting to the active state dependsupon a sudden increasein the intracellular availability of ionized ca.56,*“>54-57 During diastole the concentration of ionized Ca which exists in the immediate vicinity of the myofibrils is probably”
Coupling
T
CELL
MEMBRANE
This is a complex structure,3a4 consisting of a diffuse reticulum of collagen fibrils, a diffusemucopolysaccharide containing layer-the basement coat, and a relatively thin, enzymaticallyactive plasma membrane which is selectively permeable to certain ions and is contiguous with the cytoplasm. The possiblefunctional significance of the mucopolysaccharide-containingbasementcoat hasrecently attracted widespreadinterest and speculation,3p5,6 mainly because it contains a large number of negatively charged sites. These negatively charged sitesconfer on the basementcoat a capacity to selectively accumulate a variety of cations, including Ca’+. The possible functional significance of the basement coat’ will be discussedin detail in a later section of this review, but even at this early stageit is perhapsworth noting that it is this basementcoat which regulates the ionic environment of the milieu immediately adjacent to the plasmamembrane.That the basement coat is heterogenousis evidenced by the fact that it can be subdivided on morphological grounds From the Cardiothoracic Institute, London, England. Reprints should be addressed to Dr. W.G. Nayler, Cardiothoracic Institute, 2 Beaumont Street, London, WI, England. 0 197.5 by Grune & Stratton, Inc. Progress
in Cardiovascular
Diseases,
Vol.
XVIII,
Muscle
G. Nayler and R. Seabra-Gomes
HE DELUSIVE SIMPLICITY of the phrase’ “excitation-contraction coupling” (E-C COUpling) concealsand overshadowsthe complex sequence of events which it describes. Although many questions remain to be answeredbefore all of the events which are involved in this sequence can be accurately describedand understood at the molecular level, the broad basicprinciples are now well defined. Before discussingtheseeventsinsofar as they relate to cardiacmusclecells,it is useful to consider briefly the fine structure of the various subcellular organelleswhich are involved. Because the processof E-C coupling starts with the reversal of the transmembraneresting potential2 we will start by consideringboth the fine morphology and the distribution of the cell membraneas it occurs in cardiac musclecells. THE
in Cardiac
No.
1 (July/August),
into an inner and outer zone. For example in cat ventricular and papillary musclecells the basement coat is approximately 500 a thick and consistsof a 200 A thick inner and a 300 a thick outer zone. Similarly the 400-500 i% wide basementcoat of turkey atria1 muscle cells can be subdivided into a 300-A wide outer layer which is more denseand more osmophilic than the 150-200 A thick inner layer.8 Although cardiac muscle cells do have a well defined basementcoat this is not a peculiarity of cardiac musclecells. For example, in the giant muscle fibres of the barnacleBdanus nubihs the basement coat9 is between 1000-3000 A thick. When heavily stainedsectionsof heart musclecells are prepared it can be seen that the innermost layer of the basementcoat containsmyriads of fine filaments which are anchored to the plasmamembrane,3 and becauseof this some investigators” have questioned whether the mucopolysaccharidecontaining coat may not be an integral part of the plasma membrane, i.e., is there any justification for considering it separately from the plasma membrane?From a functional point of view there are severalreasonsfor separatingthe plasmaImembrane from the extraneous coat,” including the fact that it is the plasmamembranewhich provides the main permeability barrier and which contains the firmly bound multienzyme complexes.Neverthelessit is the mucopolysaccharide-richbasement coat which regulatesthe immediate ionic environment of the enzymatically-active, selectively-permeableplasmamembrane. THE
PLASMA
MEMBRANE
The perimeter of eachmusclecell is defined by a 75-90-A thick plasmamembrane,which provides the cell with a selectively permeablebarrier. During the past decade numerous attempts have been made to isolate plasmamembranesfrom other subcellular components. The techniques employed usually involve homogenization followed by differential centrifugation and a variety of purification steps.12-16Even so it is difficult to establish the purity of suchsubcellularpreparations,because although the relative activities of a variety of marker enzymes, including the Na+-K+ activated ATPase, the adenylate cyclase, the Cazf-activated 1975
75
76
Fi ig. 1. Longitudinal {TI. Note that the material. X 25,000
NAYLER
section sarcodasmic
of rabbit reticulum
papillary 1%)
lies
muscle, showing invagination close to the T-tubule, and
ATPase, and the 5’ nucleotidases can be readily established, this provides no guarantee of the fact that under in vivo conditions these enzymes are associated with the plasma membrane. Of course, if the isolated plasma membranes retain their mucopolysaccharide coating then this will provide a means of identification. The chemical composition of the membranes-for example, either their cholesterol-phospholipid ratioI or their sialic acid5 content can also be used to assess their purity. Determinations of the phospholipid content of plasma membrane fractions, as well as being justified in terms of providing an index of the purity of a particular preparation, can be justified on
that
AND
of the cell membrane the T-tubule is lined
SEABRA-GOMES
to form a T-tc lbule with basement coat
other grounds, including the fact that the apparent reactivities of the phospholipids with Ca’+ varies in the order phosphatidylserine > phosphatidylethanolamine S phosphatidylcholine.” Moreover, and as will be discussed later, certain phospholipids exert a regulatory effect on the activity of some of the membrane-located ATPase enzymes. The distribution of the plasma membrane is often complex, mainly because the membrane and its accompanying basement coat are not necessarily restricted to the periphery of the cell. Instead, and as is shown in the electronomicrograph reproduced as Fig. 1, the cell membrane often invaginates to form a series of slender, branching’g220 tubules
EXCITATION-CONTRACTION
Fig. 2. x 37,500
Rabbit
77
COUPLING
papillary
muscle
showing
extensive
branching
(T-tubules) which penetrate deep into the myoplasm, usually in the vicinity of the Z band. The T-tubules of cardiac muscle cells differ from those found in skeletal muscle because: (1) in cardiac but not in skeletal muscle cells the mucopolysaccharide-coating of the plasma membrane extends into and often fills the lumen of the T-tubule, (Fig. 1); (2) in cardiac cells the diameter of the lumen of the T-tubule often exceeds 1500 & whereas in skeletal muscle cells the lumen is much smaller, usually’l J.’ ranging between 200-400 a in diam-
of the
T-tubules.
T-T-tubules,
E-extracellular
space.
eter; and (3) whereas in cardiac muscle cells the T-tubules are usually located at the level of the Z band, in skeletal muscle cells they are often located in the region of the A-l overlap.23 It must not be imagined, however, that T-tubules are present in all heart muscle cells. Indeed they are absent from many amphibians and reptiles. Chicken and humming-bird muscle cells,24 and some atria1 cells’ likewise lack an identifiable Tsystem. That these tubules, when they do occur, branch and ramify is clearly shown by the electron-
78
NAYLER
micrograph of part of a rabbit papillary muscle cell which is shown in Fig. 2. Although the functional significance of the Ttubules remains the subject of speculation, a careful consideration of their morphology leads directly to two conclusions. Firstly, these tubular
Fig. 3. approaches
Electronmicrograph of rabbit a T-tubule an intramembranous
papillary structure
AND
SEABRA-GOMES
invaginations provide a large increase in the surface area which is available, per unit cell volume, to facilitate both the distribution of the depolarising current,25-27 the exchange of ions between the intra and extracellular phases, and the uptake of substrates from the extracellular phase. Secondly,
muscle showing the sarcoplasmic is formed. E-extracellular space.
reticulum X75.000
(SRI.
Note
that
when
this
EXCITATION-CONTRACTION
COUPLING
it increases the area of the surface in which the various enzymes can be implanted and on which the mucopolysaccharide-containing basement coat material can be deposited. Isolated fragments of the plasma membrane coalesce, to form vesicles.12-‘6 Because the enzymatic activity which is exhibited by these vesicles will depend, to some extent at least, upon whether the vesicles have formed L‘right-side” or “insideout” it is relatively important for techniques to be developed to control this procedure. In the meantime substances such as conconavallin A can be used2’ to ensure whether ‘right-side’ or ‘insideout’ preparations have been made. THE
SARCOPLASMIC
RETICULUM
Whereas the events which are involved in E-C coupling’ start at the level of the cell membrane, the reverse phenomenon, that is the transition from the active to the resting state, starts at the level of the sarcoplasmicreticulum.s’29 This reticulum20321is a smooth reticulum, and consistsof a lace-like network of tubules which branch and ramify, crossing from sarcomere to sarcomere without apparent interruption (Fig. 3).j~~’ In marked contrast to the direct continuity which exists between the lumen of the T-tubule and the extracellular space19y2’the lumen of the sarcoplasmic reticulum is sealedoff from the extracellular spaceexcept, perhaps,in damagedcells.30 According to the morphometric studiesof Page and McCallister3r the sarcoplasmicreticulum occupies asmuch as 3.5 + 0.2% of the cell volume in myocardial cells.By contrast the transversetubular system occupies only 1.0 ? 0.1% of the total cell volume.31 This difference becomeseven more pronounced if the comparisonis madein terms of the ratio between the p sq m membranearea and the p cu m cell volume. When this is done then the ratio between the area which is occupied by the cell membrane, including the T-system, and the cell volume is 0.39 f 0.02.” The sarcotubular system provides a ratio of 1.22 f 0.0S.31 Of course morphometric data which is obtained from fixed preparations may be misleading, becauseof the changeswhich are caused by fixation, but even allowing for such inaccuracies(calculated asbeing up to 25% by Page and McCallister3’) it seems probable that the area of membranewhich is provided by the sarcoplasmicreticulum greatly exceedsthat provided by the cell membraneand its accompanying tubular invaginations. When ex-
79
pressedon a weight basisthe sarcoplasmicreticulum probably accounts for approximately 6.8 mg/g wet weight of heart muscle.3” Estimates of membrane area, volume, and mass may, however, be of little value, when applied to a morphological system such as the sarcoplasmic reticulum, becauseof the increasing amount of evidence which points towards the existence of specialization within the reticulum.3$20*23Thus whenever the facing membrances of the sarcoplasmic reticulum approach the plasmamembrane, irrespective of whether this occurs at the periphery of the cell or at the level of a T-tubule, specialized, flat, sac-like dilations form. These dilations, are part of the sarcoplasmicreticulum, and their lumen is continuous with that of the remainder of the reticulum. These specialized,sac-like dilations are commonly called cisternae,33 irrespective of whether they are located immediately below the cell membrane at the periphery of the cell (subsarcolemmalcisternae) or whether they run alongside a T-tubule (Fig. 4). The cisternae which are found in heart muscle are small when compared with those which are found in skeletal muscle cells. Nevertheless,they, like their skeletal muscle counterparts, contain intraluminal densities,often preservedas a denseline lacking the trilaminar appearance which is characteristic of membranes. This denseintraluminal structure doesnot extend uniformly throughout the whole of the sarcoplasmic reticulum. Instead, and as shown schematically in Fig. 5, it appearsto be confined to those areaswhere the reticulum either approaches or is closely apposedto the plasmamembrane. There is other evidence3’ of specializationwithin the sarcoplasmic reticulum. In some species a slender tubular profile is located at the level of the Z band.34 Undoubtedly this tubule is part of the sarcoplasmicreticulum, but the question of its functional significanceremainsunknown. There is further evidence of specialization at the level of the Z band, where those parts of the sarcoplasmic reticulum which are adjacent to the Z band olften contain 30-30 a intraluminal electron densegranules, and the walls of the reticulum may be coated with a 100-200-A thick electron densecytoplasmic coating.’ When cut in crosssection thesespecialized areasoften present the appearanceof electron densevesicles.5 The possible existence of areas of specialized function within the sarcoplasmicreticulum warrants serious consideration by investigators who
80
Fig. 4. Longitudinal drion, T-T-tubule-note tween SR and T-tubule.
NAYLER
section of rabbit papillary evidence of pinocytosis,
muscle. Perfusionfixed. SR-sarcoplasmic reticulum,
use cardiac microsomal fractions prepared by homogenization and differential centrifugation. Usually attention 35336is concentrated upon the further purification of a fraction which sediments at a relatively high g, and comparatively little effort has been expended so far in trying to retrieve any heavier components of the reticulum which will
X75.000. E-extracellular G-glycogen granules,
AND
SEABRA-GOMES
space, M-mitochonJG-junctional gap
be-
have been discarded earlier in the schedule. In skeletal muscle the heavier microsomal fractions are believed to consist predominantly of the cisternal elements, and have been shown to have Ca*+ accumulating properties which are qualitatively and quantitatively different from those exhibited by the relatively lighter fractions.3’338Y39
EXCITATION-CONTRACTION
IO-2
COUPLING
BAND
Fig. 5. Schematic representation of the electron-dense feet which project from the lining membrane of the SR into the junctional gap which separates the SR from the T-system. Note the presence of basement coat material in the T-tubule, and evidence of a substructure within the sarcoplasmic reticulum in those areas where it approximates a T-tubule.
During embryological development the composition, function and structure of the sacroplasmic reticulum membranes undergo striking changes4’ the changes being more markedin the skeletal than in the cardiac muscle preparations. According to Martonosi and his colleagues4’ microsomal fractions which have been prepared from chicken embryonic skeletal muscle are largely devoid of a Ca2+ transporting system, and the appearance of such a system coincides with the appearance of a Ca2+-activated ATPase activity.40 At approximately the same time the phospholipid content of the SR membranes changes, their linoleate content increasing and the palmitate content decreasing.40 Other studies, in which freeze-etch techniques have been utilized, have shown that the age-dependant increase in the activity of the Ca2+ activated ATPase enzyme is paralleled by an increased number of spherical, intramembranous particles.41 These particles are approximately 7.5 A in diameter, and they are not restricted to the sarcoplasmic reticulum. Within the sarcoplasmic reticulum, however, they assume a density of 3000 particles/p’ membrane area.41 Whether these particles have anything at all to do with the activity of the Ca2’-activated ATPase enzyme42 is at present unknown. It is known, however, that the
81
purified enzyme contains a major polypeptide of mol w 102,000, a proteolipid of approximlately 12,000 mol w, a variable amount of high molecular weight protein, neutral lipids and phospholipids.43 The phospholipid component amounts to 450 pg/mg membrane protein.43 This phospholipid component cannot be ignored, because it dominates both the permeability characteristics pf the reticular membranes,@ and the activity of the Ca2’ sensitive ATPase enzyme. Recent studies by Martonosi and his colleague? indicate that the phospholipids may regulate the activity of the Ca2+ sensitive ATPase enzyme by positioning a histidine residue at the active site, thereby facilitating the approach of the enzyme to the phosphoprotein bond, phosphorylation of the enzyme being an intermediate step in the hydrolysis of ATP by the Ca2+-activated ATPase enzyme. The hydrolysis of ATP by sarcoplasmic reticulum membranes involves two readily distinguishable steps44 which can be summarized as follows: (1) E+ATP+E-PtADP; (2) E-P+H,O-’ E + Pi; where E is the active enzyme. On the basis of several observations Martonosi and his colleagues 44 have postulated that histidine residues are involved in the decomposition of the phosphoprotein intermediate. These same authors44 postulate that the inhibition of ATPase activity in microsomal fractions which are depleted of lipids results largely from an inhibition of the decomposition of the phosphoprotein intermediate, a decomposition pathway which involves a functional histidine residue. Several proteins4’ which exhibit relatively high affinities for Ca2+ have been isolated from the sarcoplasmic reticulum. One of these proteins, calsequestrin (mol wt 46,500) does not exhibit high affinity Ca2+-binding but binds approximately 850 nmoles Ca2’/mg in the, presence of KCI, and 900 nmoles Ca2+ in the absence of KCI. Acidic proteins with mol wts of between 20,000 and 38,000 can also be isolated from sarcoplasmic reticulum fragments, and these proteins bind from 900 to 1000 nmoles Ca’+/mg protein. Whether these proteins are distributed throughout the whole of the sarcoplasmic reticulum, or whether they are concentrated in specialized regions (possibly within the cisternae) has not yet been determined. There are many other aspects of the biochemical properties of the sarcoplasmic reticulum which warrant attention, and some of these, including the
82
NAYLER
Fig. 6. Rat ture, JG-junctional
papillary gap,
muscle. X 100,000. F-electron-dense
T-T-tubule, feet.
SR-sarcoplasmic
capacity to accumulate Ca” will be discussed in a later section of this article. Always, however, it must be recalled that there may be areas of gross biochemical as well as morphological specialization within this fine network of tubules, and that the techniques which are currently used to isolate these membranes may neither permit the preservation of these specializations nor need they provide preparations free from contamination with fragments derived from other intracellular organelles. JUNCTIONAL AREAS BETWEEN THE SARCOPLASMIC RETICULUM AND THE T-SYSTEM
Whenever the membranesof the sarcoplasmic reticulum and the T-system lie side by side, specialized “junctional areas” occur. Thesejunctional areas4’jinvolve the facing membranesof the sar-
reticulum,
showing
AND
evidence
SEABRA-GOMES
of
internal
struc-
coplasmic reticulum and the T-tubules, the intervening or ‘junctional’ gap (Fig. 4) and the periodically-arranged, election-dense profiles which project from the facing membranesof the sarcoplasmic reticulum and extend into the junctional zone as shown in the electronmicrograph (Fig. 6). In those cardiac muscle cells which have a Tsystem, junctional zones are found whenever the tubules of the SR approach a T-tubular profile, as shownin Fig. 4. This doesnot mean, however, that these specialized zones occur only in the vicinity of a T-tubule. In fact they occur whenever the sarcoplasmic reticulum approaches the plasma membrane, irrespective of whether the plasma membraneis located either at the periphery of the cell or whether it forms part of a T-tubular invagination. Hence junctional areasmay be found close to the sarcolemma,and when this happens
EXCITATION-CONTRACTION
COUPLING
the junction is usually called a “peripheral coupling.” If the muscle cell contains a T-tubular system, however, then junctional couplings will almost certainly be found alongside the T-tubule, as well as beneath the sarcolemma. Franzini Armstrong 46y47has already described the fine morphology of these specialized junctional areas in great detail, and although her observations relate primarily to smooth and skeletal muscle cells, her description holds true for cardiac muscle cells. There is, therefore, no need for another detailed description here. Several points do, however, warrant attention: (1) The electron dense, regularly spaced, feet like extensions which project from the facing membranes of the SR into the junction gap apparently do not penetrate into the opposing facing membrane. This may reflect the absence of a morphological link between the two opposing membranes. Alternatively it may only mean that the ‘invisible’ intermediate link is not electron-dense. (2) The arrangement of the ‘feet’ is always periodic4 and the width of the intervening gap remains constant4 (120-200 A) irrespective of whether the diameter of the lumen of the T-tubule is made to alter-as occurs, for example, when heart muscle is rendered hypoxic. (3) Channels linking the lumen of the SR with that of the T-tubular system have not yet been identified.47 (4) The specialization which occurs within the membranes of the SR at these junctional areas probably reflects a specialization of function different from that exhibited by the longitudinal components of the SR.47 Recognition of the existence of these specialized junctional areas has resulted in widespread speculation as to whether they could facilitate the direct transmission of an excitatory stimulus and its attendant wave of depolarisation from the level of the cell membrane and its associated T-tubules to the sarcoplasmic reticulum, including the subsarcolemmal cisternae. The membranes which line the transverse tubular system are excitable.25’48 Indeed, when normothermic conditions prevail excitability within the T-system can account for as much as 70% of the mechanical response.*’ Nevertheless, and despite the fact that a regenerative action potential can be triggered in the T-system when the surface membrane is depolarized, and despite the supporting data obtained by Peachey and Adrian,49 and Caputo and Dipola,” evidence of the direct transmission of this activity to the sarcoplasmic reticulum is lacking. But even if such a link does exist, it is still necessary to prove that
83
information which is relayed from the cell membrane to the sarcoplasmic reticulum can influence E-C coupling. The recent demonstration by Endo and Nakajima’r of the fact that depolarisaticw of the sarcoplasmic reticulum in skinned skeletal muscle fibres induces a release of Ca2+, together with the earlier findings of Constantin and Podolsky52 and of Ford et a1.53 probably establishes the proof of the latter hypothesis. Ca2+ AND
CONTRACTION
Despite our present uncertainty as to whether a depolarisingsignalcan be transferred directly from the surface membrane to the sarcoplasmicreticulum it is now generally agreedthat the transition from the resting to the active state dependsupon a sudden increasein the intracellular availability of ionized ca.56,*“>54-57 During diastole the concentration of ionized Ca which exists in the immediate vicinity of the myofibrils is probably”
