3oud of Molecular

Cardiac

and Cellular

Muscle

(1976)

: An Attempt

PAGE A. W. ANDERSON, Departments

Cardiology

8, 123-143

to Relate

Structure

to Function*

ANDRfiS MANRING, E. A. JOHNSON

JOACHIM

R. SOMMER

of Pathology,

Pediatrics, and Physiology, Duke University Medical Center Administration Hospital, Durham, .North Carolina 27710, U.S.A.

(Received

23 Jub

1974, accepted in revisedform

2

1 March

t AND

and Veterans

1975)

P. A. W. ANDERSON, A. MANRING, J. R. SOMMER AND E. A. JOHNSON. Cardiac Muscle: An Attempt to Relate Structure to Function. 3ouml of Molecular and Cellular Cardiology (1976) 8, 123-143). Two structure-function hypotheses were tested in this paper: Are the physiological functions-the force-frequency relationship and/or the response to low sodium media-related to the degree of differentiation of the coupling [a specialized close association of sarcolemma and sarcoplasmic reticulum] ? A comparative study of the ultrastructure of representatives of several vertebrate classes [ma-ha, aves, reptilia, amphibia, Pisces and chondrichthyes] revealed that the coupling rather than other structural differences was the best candidate for a structure-function study. A wide variation in the degree of differentiation of the coupling and the associated sarcoplasmic reticulum was found throughout the range of animal classes. Well-formed couplings occurred in all hearts except that of the frog and mudpuppy where they were sparsely distributed and poorly differentiated. Parallel comparative function studies-the force-frequency or intervalstrength relationship and the response to low-sodium media-were performed on hearts from animals from the same classes. Although all hearts developed steady tensions when exposed to low-sodium media, there were microscopic differences: In the frog the sarcomeres shortened to a steady value, whereas in hearts from other animals, groups of sarcomeres twitched repeatedly and without synchrony among groups (vermiculation). The structure-function hypothesis implied by this functional difference was disproven: the sarcomeres in the chicken embryo hearts, both with and without identifiable couplings, vermiculated; sarcomeres in hearts of the turtle and salamander, with well-formed couplings, sometimes vermiculated and sometimes shortened like those of the frog. Using the maximum rate of rise of tension in a contraction as the measure of contractility, the hearts of all animals tested fell into two classes according to the characteristics of their force-frequency relationships. In one class, which included all but amphibian hearts, contractility increased between contractions, whereas in the other class, contractility declined or did not change between contractions. Hearts without well-differentiated couplings were never found in the first class, whereas hearts with or without couplings were found in the second class. WORDS: Coupling; Structure-function relationship; Force-frequency relationship; Contracture; Vermiculation; Time to peak tension; Maximum rate of rise of tension; Sarcoplasmic reticulum; Junctional sarcoplasmic reticulum; Extended junctional sarcoplasmic reticulum; Junctional processes; Junctional granules; Sarcomere motion; Amphiuma; Bat; Cat; Dog; Salamander; Mudpuppy; Chicken; Finch; Stingray; Turtle; Goldfish; Lizard. * The results were presented, in part, at the Annual Meeting of the ASCB, 1972. t This work was supported by U. S. Public Health Service Grants HL 11307, HL 12157, HL 5372, HL 57091 and HL 12486, and HL 58305 from the National Heart and Lung Institute, Veterans Administration Grant 7905-01, and by the North Carolina Heart Association.

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1. Introduction

The physiological roles of discrete cellular structures in cardiac muscle such as the transverse tubule and the coupling [28] remain, for the most part, unknown. One conceivable approach to determining the function of a given structure is to selectively inactivate or eliminate it from the preparation and observe the ensuing changes in the mechanical, electrical or metabolic activity of the preparation. Examples of such maneuvers applied to skeletal muscle are detubulation which removes the electrical connection between the transverse tubules and the cell surface, and skinning [367 and glycerol extraction [59], both of which remove the cell membrane. In addition to excisive procedures of this kind, cardiac muscle offers a different approach to relating cell structure to function since the morphology of hearts from a relatively small range of phylogeny show marked variations in differentiation of structures which are more or lessobviously involved in excitationcontraction coupling [51, 521. T wo examples of such structures are 1. the transverse tubules, present in adult but absent in neonatal mammalian ventricular muscle [471, mammalian P fibers [28] and in hearts of other classes[25, 51, 58],2. couplings, abundant and well-developed in mammalian and chicken hearts, but so sparseand rudimentary in frog ventricular muscle that until recently they have escapeddetection [40]. The coincidence of structural differences of this kind with a physiological difference in hearts from two speciesimmediately suggestsstructurefunction relationships. Naturally, the smaller the number of structural differences between two hearts, the smaller is the list of structures potentially responsible for the physiological difference. While a physiological difference between hearts of mammals and the frog might more plausibly be attributed to the aforementioned, other differences in structure of these hearts also exist: Mammalian ventricular cells are larger than frog ventricular cells, lo-15 pm in diameter compared to 2-3 pm: mammalian cells invariably have M-lines while those of frog do not [51] ; Zlines in mammalian cardiac muscle form well-developed square lattices typical of skeletal muscle while Z-lines in frog heart apparently do not; mammalian hearts have a system of abundant sarcoplasmic reticulum with associatedwell-formed interior and peripheral couplings, whereas the sarcoplasmic reticulum in other animals, e.g. in frog hearts, is comparatively sparse and couplings are rare and poorly differentiated [40, 551. Such a plurality of structural differences leads at best to a plurality of equally plausible structure-function relationships. On the other hand, it would appear that with a single structural difference a search amongst physiological functions would yield a unique functional complement-surely the ultimate goal of all structurefunction studies.A single structural difference appears to be the only feature distinguishing frog from chicken heart fibers.* chicken heart fibers have an extensive system of sarcoplasmic reticulum with numerous and well-differentiated couplings whereas frog heart fibers do not [51]. This and the following paper are the result of

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a search for the functional counterpart of this structural difference. Of course, parallel comparative structural and physiological studies can never unequivocally establish a structure-function relationship for the coupling or any other cellular structure. The real power of this approach lies in the ability to disprove structurefunction relationships. In fact, the structure-function study can be considered complete only when it disproves such a relationship. We consider two aspects of excitation-contraction coupling known to differ in hearts from species to species, and hence potentially attributable to structural differences: 1. the force-frequency, or interval-strength relationship [S, 71, 2. the ability to develop contractures [31, 38, 48, 491. The force-frequency relationship describes the complex but predictable way in which the peak tension that is developed in a contraction depends on time between contractions in the past; the response to an extrasystole, to paired-pacing, or a sudden change in rate are well-known examples of this relationship. As will be seen, the force-frequency relationship of mammalian heart is markedly different from that of frog heart. A contracture (i.e., a sustained contraction), the peak tension of which was comparable to, or greater than the peak twitch tension, developed in frog ventricular strips exposed to Ringer solution containing high-potassium or low-sodium [3I]. Ventricular muscle of the mammal, like that of the frog, developed a contracture when sodium chloride of the bathing medium was replaced by potassium chloride, but, in contrast to the frog, did not develop contractures when the sodium chloride was replaced by sucrose unless the potassium concentration was also raised [49]. Can these functional differences be related to some observable structural difference? This is the question we wish to answer, unequivocably, if possible. To this end, we begin with the following argument. If the functional differences between frog and mammalian hearts are due to the abundant system of sarcoplasmic reticulum and couplings and not to other structural differences cited above, one would predict that the behavior of chicken hearts would resemble that of the mammal; if, on the other hand, the differences between frog and mammalian hearts are due to one of the other structural differences cited above, then the response of the chicken would resemble that of the frog. As will be shown, the behaviour of chicken heart resembles that of the mammal and not that of the frog so that our attention focused on the coupling and associated SR, the sole remaining candidate for our structurefunction study. This paper presents the results of a complete structure-function study in three sections : in the first section a comparative study is made of the detailed ultrastructure of the coupling and the sarcoplasmic reticulum in hearts from a wide variety of animals and a general argument for the participation of the coupling in the contraction-relaxation cycle. In the second and third sections a comparative study is made of the force-frequency relationship and of the response to low-sodium media in hearts from a similar variety of animals.

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ETAL.

2. Methods Structure

The hearts of the following animals were examined: Alligator, amphiuma, bat, chicken (adult and embryo), dog, finch (zebra and strawberry finch), frog (R. pi&ens, R. cutediana), goldfish, humming bird, lizard, mudpuppy, salamander, stingray and turtle. We have also looked, for a comparison with skeletal muscle, at the pectoral muscle of the finch and the diaphragm of the mouse. After anesthetizing the animals either by cooling on ice (frogs, alligator, amphiuma, mudpuppy, turtle, lizard, goldfish), or with ether (mammals, birds), the hearts were removed, still beating, from the animals. The hearts were quickly opened with scissorsunder glutaraldehyde (5% in 0.2 M cacodylate or phosphate buffer at pH 7.2) at room temperature and, with razor blade, several small strips (approx. 0.2 x 5 mm) of tissue were excised from the endocardial surface of the ventricles. These strips were left to fix in glutaraldehyde at room temperature for 2 to 8 h. After rinsing in distilled water, the tissuewas fixed in 2% osmium tetroxide for 1 to 2 h, contrasted in uranyl acetate (1 y0 in distilled water) for 1 h, dehydrated through alcohols and embedded in Epon 812. Fixation was judged reasonably uniform, that is to say, most cells showed no swelling of mitochondria, disruption of the sarcolemma, severe swelling of the sarcoplasmic reticulum, nor disruption of the contractile material, although some of that was seenin almost all preparations here and there. The embedded tissue was sectioned with diamond knives using either a PorterBlum MT-I, or a Reichert OM-U2 ultramicrotome. The sections were put on collodion-carbon-covered 200 mesh copper grids, contrasted with lead citrate, and viewed with a Joelco JEM IOOB electron microscope at 60 kV. Force-frequency relationshij

Rabbits, guinea pigs and rats were killed by a sharp blow to the neck or head. Chickens were killed by neck-wringing. Dogs and cats were anaesthetized with an intraperitoneal injection of pentobarbital, 25 mg/kg. The frog, goldfish, salamander, mudpuppy, turtle and amphiuma were decapitated. Hearts were excised from all animals within 30 s. Mammalian, chicken and goldfish hearts were placed immediately into Krebs-Henseleit solution [30], aerated with a gas mixture of 95% 02 and 5% COa, at 38°C for mammalian and chicken hearts and at room temperature (20 to 25°C) for the goldfish hearts. Hearts from all other animals were placed immediately into Ringer solution (NaCl, 118 mM; KCl, 1.6 mu; NasHPOd, 1.06 mu; NaHaPO+ 0.36 mM; CaCla, 1.25 mM; dextrose, 5.56 mM) at room temperature. Papillary muscles from rabbit and cat right ventricle and ventricular strips or trabeculae from other animals were mounted in a thermostatically controlled tissue chamber at 25°C for the poikileotherms and at 38°C for homeotherms. The

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tissue bath was a channel, 1 ml in volume, milled into one face (2 cm x 4.5 cm) of a rectangular block of silver (2 cm x 4.5 cm x 1.5 cm). The channel was closed with a Lucite lid in which a strip of silver (1 cm x .3 cm) was embedded. This silver strip together with the walls and floor of the bath formed the two stimulating electrodes. The bathing solution passed (at 5 ml/min) through a heat exchanger, formed by a series of interconnecting tunnels, 0.15 cm in diameter and 25 cm in length drilled into the walls of the bath, before it entered through an opening in the floor at one end of the bath and spilled out through a hole at the opposite end. The temperature of the silver block was thermostatically controlled to within O.Ol”C at any desired temperature from 20°C to 40°C through a thin nichrome ribbon heating element wrapped around and insulated electrically, but not thermally, from the silver. Stainless steel hooks pierced each end of the ventricular strips and were linked to stainless steel loops, one of which was fixed to a post at one end of the chamber while the other was attached to a semiconductor strain gauge force transducer (resonant frequency approximately 250 Hz ; compliance 10-s cm/dyne). The papillary muscle was tied using 4-O silk with a sheet bend knot and a loop was provided for a stainless steel hook to link the muscle to the force transducer. The ventricular base of the papillary muscle was firmly tied to the silver post at the other end of the bath by slipping the button of ventricular muscle through a 4-O silk loop and garotting the papillary muscle at its junction with the ventricular muscle to the post. The two strain gauges of the transducer formed two arms of the bridge of a Tektronix Carrier Amplifier Unit, the output of which was differentiated using an operational amplifier as a differentiator (high frequency cut-off, 200 Hz; gain constant, 300 ms ; rise time, 4 ms). Both the force signal and Its derivative were displayed and photographed on a Tektronix Type 564 storage oscilloscope. When the preceeding regular contraction and the test contraction were fused, the peak tension for the test contraction was obtained by subtracting the waveform of the previous regular contraction from that of the fused contractions, and the maximum rate of rise of tension for the test contraction was obtained by subtracting the waveform of the first derivative of the previous regular contraction from that of the fused contractions. Extra contractions for which the peak tension occurred earlier than the maximum rate of rise of tension of the normal regular contraction were rejected. Although this criterion for rejection was arbitrary, it was based on the observation that the extracted waveforms of the extra contractions at such short stimulus intervals were abnormal in that the rising phase of tension was shortened and the relaxation phase accelerated. Response to low-sodium

media

Preparations of cardiac muscle that were used for studying the response to lowsodium media were obtained in the same way as those used for the force-frequency experiments.

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Muscles were stimulated at a low constant rate (usually l/4 s) for 1 h prior to and during the contracture. Contractures in both microscopic studies and tension studies were initiated by switching the flow of bathing solution from normal (normal solution AN or BN for mammal, chicken and goldfish hearts, and normal solution CN for other hearts) to the appropriate low-sodium solution. Normal solution AN was Krebs-Henseleit solution. Normal solution BN contained 140 mM NaCl, 5.56 mM dextrose, 2.5 mM CaCl s, and 3 mM HEPES (N-2-hydroxyethylpiperazine-./V-2ethanesulfonic acid); it was buffered to a pH of 7.2 by titrating with KOH, and enough KC1 was added to raise the potassium concentration to 5.0 mM. Normal solution CN was Ringer’s solution (see above). Low-sodium/high-potassium solutions AK, BK, and CK were made by replacing the sodium salts in the normal solutions (except the 1.06 mM NasHPOd in normal solution CN) with the corresponding potassium salt. Low-sodium/sucrose solutions BS and CS were made by replacing the NaCl in normal soutions BN and CN with an equiosmolar concentration of sucrose; in CS, the NaHsP04 was replaced by KHsPOd and the KC1 concentration was reduced to 0.54 rnM to make the total potassium concentration the same in the normal and low-sodium solutions. Other low-sodium solutions were made like solutions BS and CS, above, except that the NaCl was replaced on an equiosmolar basis by one of the following: LiC 1, MgS04, dextrose, or (using solution B) CaCl2. A detailed description of the apparatus used for the microscope studies of living cardiac muscle has been made elsewhere [18]. Trabeculae used for this study were removed from atria or ventricles. In a few instances, when no trabeculae could be found, bundles of fibers attached along their length to the thin, translucent wall of the atrium were used. Hearts from chicken embryos were snared with a loop made by passing both ends of a nylon filament through a blunt, hollow rod made from a hypodermic needle. 3. Results Comjarative

ultrastructure

of the coupling

A coupling [28] is a close association of sarcolemma with junctional sarcoplasmic reticulum (JSR, syn. terminal cisterna, 42) (Plates l-10). The latter is defined as a subsarcolemmal tubule of SR filled with junctional granules (JG), and from which project junctional processes (JP) or feet that seem to attach to sarcolemma [5, 13, 14, 29, 43, Sr]. The term junctional granules is a generic and purely descriptive and often quasi-memterm for the usually granular [16,41], sometimes cribriform, branous electron-dense material seen in various amounts in the JSR of both skeletal (Plates 5-7) and cardiac (Plates l-4, 8-10) muscle [25, 26, 28, 39, 45, 51, 52, 611. When couplings involve sarcolemma of the fiber surface, they are called peripheral couplings, and when they involve the membrane of transverse tubules they are

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called interior couplings [52, 571. When JSR occurs separate from sarcolemma it is called extended junctional SR (EJSR) [25l. The remainder of the SR which is continuous with the JSR/EJSR is named free-SR. In practice, the question of whether couplings exist or not is always a matter of clearly identifying JSR with its JG’s and, especially JP’s. Free-SR was present in all preparations of cardiac muscle studied and also, occasionally, subsarcolemmal-SR, i.e., segments of SR lying in close apposition to sarcolemma but in which JG’s and JP’s were uncertain [51]. Only when subsarcolemmal-SR contained JG’s and two or more JP’s about 250 A apart was it classified as JSR. In mammalian (Plates 1-4) and avian cardiac muscle, well-formed couplings with many JP’s in register were common. In the hearts of birds, especially those with fast heart rates (finch, sparrow, humming bird, canary, etc., as opposed to the chicken), SR having the characteristics of JSR was found not only in association with the sarcolemma in the form of couplings but also throughout the cell, as EJSR [25]. In contrast, the SR in the frog and mudpuppy (Plate 9) hearts was sparse and the couplings were rudimentary in that the JG’s and JP’s were poorly differentiated and, thus, much more difficult to visualize. As a result, the couplings in frog cardiac muscle have long escaped detection [51, 58 but cf. 24, 40, 531, a story similar to the belated discovery in chicken cardiac muscle [26, cf. 501, of EJSR which is so prominent in other birds [25]. In all animals, couplings, whether interior or peripheral and including the EJSR of birds, almost always are associated with the Z-I region of the sarcomere.* JSR in some animals, and EJSR in bird cardiac muscle, is remarkably constant in width, at least in one plane of sectioning, in contrast to the relatively great variability in width of free-SR. In other animals, such as the alligator, amphiuma (Plate 8), fish, turtle, salamander, lizard [53] and chicken, the JSR appears often quite distended (apparently in several planes), as compared with the tubules of the freeSR in these animals. Correspondingly, the JG’s are most prominent in the latter group of animals, in which they often form a cribriform pattern (e.g. Plate 8) within the JSR. In contrast, the JG’s in the former group of animals are sparse and tend to form central quasi-membranes bisecting the JSR (e.g. Plates 2, 4 and 10). Consonant with its intercalated position within the SR network, the JSR/EJSR shares some features with the free-SR and is different in others. In isolated preparations it, so far, has not been possible to separate free-SR from JSR or EJSR. Thus, while at least the ability of a total preparation of SR vesicles to pump calcium, in vitro, from the outside to the inside of the vesicles is well established [22,23], direct evidence that might elucidate the function of the JSR/EJSR as opposed to the freeSR, and consequently that of the coupling, is still lacking. Significant cytochemical differences between the JSR and free-SR have not yet been convincingly demonstrated [17,46,54, 601 except, perhaps, for the evidence concerning the presence of * Page and Niedergerke 2nd paragraph].

have since established

that relationship

also for the frog [35, cf. 5 1, p. 449,

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ETAL.

some glycoprotein that is present in JSR but absent in free-SR [20, 211. Recent insitu negative staining of the SR of skeletal muscle (Plates 5 and 6) indicate that there is no significant difference in luminal anatomy between JSR and free-SR [64, Plate 61. This is very surprising, indeed, because one would have expected negative staining to reveal to good advantage the JG’s so prominent in conventional sections of the JSR (cf. Plate 7 with 5 and 6). This failure to do so raises new questions regarding the structure of the JG’s [cf. 21, 45, 611, although with yet another method which may represent evidence for an endogenous peroxidase in both freeSR and JSR [62], we have recently been able to demonstrate JG’s in negative contrast in cardiac muscle. The in-situ negative staining of the SR has also confirmed that the lumen of the free-SR is continuous with that of the JSR, and that fenestrations occur in both JSR and free-SR of at least skeletal muscle (Plate 6). Freeze-fracture studies are beginning to make some contribution to the differential anatomy of the JSR/EJSR vs. free-SR [4, 50, 551. As in skeletal muscle [15], in cardiac muscle, too, only the free-SR seems to have a very dense population of particles [.55], JSR being relatively smooth. Further evidence that the JSR/EJSR is a discrete compartment within the free-SR network, rather than a segment of SR which merely has points of attachment to other structures, is the discovery in a ventricular cell of an adult opossum of what appears to be a hyperplasia of sheets of EJSR membranes stacked on top of one another [SS, 631. This structure suggests that EJSR is capable of replicating independently from the free-SR network, thus asserting its independence from it, while at the same time not losing its intergal position within it. The JP’s of adjacent membranes in those stacks of EJSR membranes apparently touch each other? often in register, providing yet another instance in which JP’s make contact with cellular elements other than the sarcolemma. On balance, it is clear from the foregoing that both JSR and EJSR are, indeed, a discrete, and probably not trivial, morphologic differentiation within the SR. AS a result, it is reasonable to posit a function, sui generis, for the JSR/EJSR, which is different, at least in some respects, from that of the free-SR. Because of the close association of the JSR with sarcolemma which carries the action potential, and with the free-SR which is believed to harbor a calcium pump, it is reasonable to imagine that the JSR is involved in some aspect of excitationobjections contraction coupling. Franzini-Armstrong [13, 141 h as raised theoretical based on anatomical considerations to the notion that the coupling represents a site of electrical couplings, be it capacative or resistive between the transverse tubular system and the SR in skeletal muscle. The surface area of the SR is some 90 times that of the associated sarcolemma [41] and, for this reason alone, the two membrane systems must be essentially decoupled, electrically, at all times, since the membrane capacitance per unit length would far exceed that observed in impedance studies or that which would be compatible with observed propagation velocities were the coupling transient during all or part of the action potential. AS

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a result, excitation-contraction coupling is seen either as a regenerative or autocatalytic release of activator: a tiny transmembrane flux of calcium ions during the action potential causes the release of a large quantity of calcium ions from the SR. Or it is seen, perhaps more realistically, as some form of chemical transmission between sarcolemma and SR analogous to the coupling between nerve and endplate membrane in skeletal muscle. Be that as it may, when considering the process of intracellular calcium movements during excitation-contraction coupling, at least three important anatomical realities must be kept in mind. 1. The SR in both skeletal and cardiac muscle is differentiated into at least two anatomically discrete portions: the free-SR on the one hand and the JSR on the other. The latter is located almost exclusively at the Z-I region of the sarcomere and is intercalated into the tubules of the free-SR. 2. The free-SR tightly enmeshes all contractile material without being interposed, at any point, between the JSR/EJSR and the contractile material. 3. EJSR, the structural homologue of the JSR of the coupling, is located deep inside cardiac cells and lacks any sarcolemmal contact except through being continuous with free-SR that eventually forms peripheral couplings when it turns into JSR that in turn (by definition) make contact with the peripheral sarcolemma via JP’s. This anatomical arrangement does not favor the notion that calcium released for contraction comes from near the cell surface since before reaching the myofibrils that calcium would have to pass through the meshes of the SR which contains a very efficient calcium pump. On the other hand, this anatomical arrangement favors the JSR as the site. Response to low-sodium Macroscopic

media

0bservation.s

Scholz [48, 491 found that atria1 and ventricular muscle of the guinea pig and calf, like frog ventricular muscle, developed contractures when exposed to sodium-free media in which the NaC 1 was replaced in whole or in part by KC 1. He also found that whereas mammalian atria1 muscle, like frog ventricular muscle, developed contractures when the sodium in the bathing medium was replaced entirely by sucrose, mammalian ventricular muscle did not develop contractures unless part of the sodium was replaced by potassium. He speculated that the transverse tubular system, present in mammalian but absent in frog ventricular muscle and rudimentary in mammalian atria1 muscle, was responsible for the physiological difference. Unlike Scholz, we found that mammalian ventricular muscle developed strong contractures when exposed to sodium-free media, whether or not the potassium concentration was raised. In fact, contractures developed in all sodium-free media tested, whether sodium was replaced by sucrose [Figure 1 (b)], dextrose, potassium [Figure 1 (a)], lithium, magnesium, or calcium (see Methods). Although a quantitative analysis of the complex and sometimes variable features of contractures is of

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IO)

4 Hi K Normal

FIGURE 1. Response to low-sodium media. (a) Puppy ventricular muscle on exposure to HiK solution (AK) stopped contracting (peak tension, A) while it develops a sudden increase in resting tension, a contracture. (b) In response to a high-sucrose, low-sodium media (BS), the contracture developed more slowly (a) while the peak tension of contractions (A) increased before disappearing.

no interest from a structure-function point of view, we do present a comparison between the time-course of two contractures induced by sodium-free media. The comparison suggests an explanation for the conflict between our results and those of Scholz. When ventricular muscle was transferred from either normal solution AN or BN into one of the potassium-rich solutions, AK or BK, the contractions were extinguished within a few seconds and the muscle went into a contracture; the tension rose rapidly and almost immediately to a fairly stable value [Figure 1 (a)]. When the muscle was returned to the normal solution, it relaxed fully and contracted when stimulated. On the other hand, when ventricular muscle was transferred from normal solution BN to one in which the sodium was replaced by sucrose, the resulting contracture took many minutes to develop [Figure 1 (b)]. When the muscle was returned to the normal solution, it relaxed fully and would again contract

when

stimulated.

The

extreme

difference

in the

time

scales

for

these

contrac-

tures suggests an explanation for the conflict between our results and those of Scholz; perhaps the ventricular muscle preparations of Scholz were exposed for too short a time to the sucrose-rich solution for the contracture to develop. Whatever the reason for the discrepancy, our finding that both mammalian and frog ventricular muscle developed contractures in sodium-free media vitiated any further con-

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sideration of a contractme-structure relationship. However, as we will see in the next section, this indistinguishability between the mechanical responses of frog and mammalian hearts to sodium-free media was more apparent than real. Microscopic

observations

When frog ventricular or atria1 trabeculae were exposed to a low-sodium solution, the sarcomeresthroughout the muscle shortened slowly and continuously for several secondsuntil motion stopped. Except for the slowness,the processresembled the shortening phase of a normal twitch. In one frog ventricular trabecula (18 pm in diameter, 2.5 m initial sarcomere length) at room temperature, the shortening phasein a normal twitch lasted for about 440 ms, whereasthe shortening phase of a contracture produced by exposing the muscle to a low-sodium Ringer’s solution, CS (sucrose replacing sodium), lasted about 25 s. The sarcomeres remained shortened as long as the muscle was exposed to the low-sodium solution (minutes) and relaxed fully when returned to Ringer’s solution. In mammalian heart muscle, the microscopic activity following exposure to sodium-free media was considerably more complicated than that of frog heart muscle. Almost immediately after atrial muscle and ventricular trabecula were exposed to a sodium-free medium, small groups of one to about five adjacent sarcomeres in surface fibers began to twitch repeatedly, the contraction-relaxation cycle lasting about 50 ms. There appeared to be no synchrony among the contracting groups. These focal twitches had two visible effects. The first appeared to be purely passive: sarcomeresalong the fibers being stretched whilst adjacent fibers were pulled. The second effect was more intriguing: the focal twitching seemedto initiate a slow propagating contractile wave (by analogy with similar waves observed and named by Matthaei and Tiegs [35] in arthropod leg muscle fibers) traveling at about l-3 mm/s which could not have been passive[3.?, 331. The waves closely resembled those observed by Marco and Nastuk [34] and Coleman et al. [S] in skeletal muscle exposed to low dosesof caffeine and by Fabiato and Fabiato [II] in disrupted cardiac muscle fibers. The wave front consisted of a bulge in the fiber approximately 10 pm long and about 1.2 fiber diameter wide. Colliding waves annihilated one another. Within a minute or so, the focal twitching spread rapidly throughout the preparation reaching a crescendo of seething, chaotic motion: with rapid, asynchronous twitching in every fiber; with fibers vigorously tugging one another, now sideways, now out of the plane of focus; with waves traveling and colliding throughout the field of view. Becausethis activity was reminiscent of a massof writhing worms, we named this phenomenon vermiculation. Vermiculation persisteduntil the low-sodium solution wasreplaced by the normal solution, at which time the focal twitching became lessand lessfrequent, stopping altogether within a few minutes. Even with prolonged or repeated exposure, mammalian heart muscle was never observed to develop frog-like contractures. The

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question immediately arose: Does chicken heart muscle, which structurally in many ways resembles frog heart much more closely than mammalian, respond to lowsodium media with a frog-like contracture, or does it vermiculate? It vermiculated. Two possible (not mutually exclusive) structure-function relationships for the coupling therefore arose: 1. The steady contracture-like response of frog hearts results from the scarcity and rudimentary development of couplings in frog hearts. 2. Vermiculation in mammalian and chicken hearts is in some way the result of the couplings in these two hearts. One might imagine, for example, that the coupling was the storage site for the activator of contraction and that the exposure to lowsodium media caused the spontaneous, repetitive, release of activator, rather like the spontaneous vesicular release of acetylcholine from motor nerve terminals in media low in calcium or high in magnesium. We tested hypotheses 1. and 2. by determining the response of hearts of other animals to low-sodium media. The second hypothesis was supported by the finding that hearts of the goldfish, turtle, amphiuma, and salamander-all of which have well-formed couplings-vermiculated. Both hypotheses were disproved by the subsequent finding that chicken embryo hearts vermiculated at a stage of development not only when couplings were first found (17-20 somites), but also at an earlier stage (14-15 somites) when couplings were not found. Furthermore, the hopelessness of finding a clear-cut relationship between the response to low-sodium media and any other structure was underscored by the finding that hearts of the turtle and salamander either developed frog-like contractures or vermiculated, depending on the experimental conditions: the turtle heart developed a frog-like contracture in response to its first exposure to low-sodium Ringer’s, but the second and subsequent exposures caused it to vermiculate. The salamander heart developed a frog-like contracture in the presence of low-sodium Ringer’s, but when this solution was replaced in part by the normal Ringer’s, the contracture relaxed, and the muscle vermiculated. Although the possibility still remains that the response to low-sodium media is determined by some quantitative, rather than qualitative, structural feature (e.g. the relative amount of SR), this possibility cannot be decided, since it would require an EM stereological study, which is beyond the scope of this investigation.

Force-frequency relationship General remarks The relationship between the steady-state value of the peak tension that is developed in contractions of heart muscle and the rate of stimulation (the steady-state forcefrequency relationship) has been shown to differ markedly from species to species [S, 71. Unfortunately, these differences are not obviously correlated with any cell

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structure, since there are no clear and consistent similarities in the steady-state force-frequency relationship among mammalian hearts which have essentially identical morphology, viz. rat and cat ventricles, let alone differences in the relationship between hearts with markedly different morphology, e.g. cat and frog ventricles. It must be noted, however, that before a steady-state is reached after a change in the rate of stimulation, the peak tension developed in successive contractions goes through two sequences of changes. In the first sequence, the so-called positive or negative staircase, the change from beat to beat is large but diminishes rapidly so that the peak tension reaches a quasi steady-state value within IO-20 beats. These and other large beat to beat changes, such as those produced by an extra systole (post-extrasystolic potentiation) and paired-pacing, are all of the same genre, and all exemplify the so-called short-term force-frequency relationship 13, 271. In the second sequence, the change from beat to beat is small : the peak tension of successive contractions slowly changes from the initial quasi steady-state value to a true steady-state value over the next hundred or so contractions. These two kinds of changes, particularly if they were independent of one another, could account for the lack of any consistent difference or similarity in the relationships from animal to animal, obscuring a correlation between similarities in some aspect of either the rapid or slow phases. In order to separate these slow and fast changes we used the procedure of Johnson et al. [27] in which the short-term force-frequency relationship is analysed using data from a two-part experiment, the details and typical results or which are described in Figure 2. Essentially, the first part of the experiment examines how contractility changes between contractions at a constant rate, and the second part, through a pertubation in rate, determines how this time course differs from one rate to another. The choice of the measure of contractility in these experiments was found to be crucial to their success. In preliminary studies, when using the peak tension developed in a contraction as a measure, not only was no consistent scheme found that would classifjr animal species according to the force-frequency relationship, but also the relationships sometimes differed qualitatively from animal to animal in the same species and even with time in the same heart ! The reason for these inconsistensies was that changes in peak tension were the result of two changes in the waveform of the contraction-the rate of rise of tension and the time to peak tensionboth of which depend on the rate and pattern of stimulation. Evidence from the literature [I, 2,661 and from the accompanying paper support the idea that changes in the time to peak tension result from changes in configuration of the concomitant action potential, the latter being, in itself, a complex function of the rate and pattern of stimulation [19]. The area of action potentials as a function of the rate and pattern of stimulation correlates well with the corresponding short-term changes in time to peak tension: the larger the area, the longer the time to peak.

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ET AL.

T

VA

T

T

VI0

v

o, 0

2 s

FIGURE 2. The results of the two-part experiment were obtained from rabbit papillary muscle in the following way. The muscle was stimulated to contract at a low constant rate and in the lirst part of the experiment an extra stimulus was interpolated sufficiently infrequently (in this case between every 5th and 6th regular stimulus), so that its effects on subsequent contractions had time to die away before the next extra stimulus was applied. The maximum rate of rise of tension developed in the contraction (Pm& in response to this extra stimulus was plotted (A) as a function of the time interval between the extra stimulus and the previous regular stimulus (the extra stimulus interval), with the previous regular contraction ( a) serving as the controls. In the second part of the experiment the extra stimulus was positioned at a fixed extra stimulus interval (250 ms for this example) and the change in the time course of contractility after this stimulus was measured by introducing a second extra stimulus after the first and plotting the maximum rate of rise of tension in the contraction in response to the second extra stimulus (v) as a function of the interval between the test stimulus and the preceding stimulus at the regular rate. The previous regular contractions were the controls ( V) In a complete experimental analysis, the second part of the experiment is repeated for several different (fixed) values of the lirst extra stimulus interval. However, in the present paper, one value was used since it was found that it provided sufficient data to permit the force-frequency relationships of the different animal hearts to be distinguished from one another. Regular rate was 0.3 Hz.

However, when the maximum tension was used as the measure to above vanished.*

rate of rise of tension (imax) of contractility, inconsistencies

rather than the peak of the kind referred

* Care must be taken to avoid using very short extra stimulus intervals, especially in the amphibia where changes in time to peak tension are large and predominantly determine the peak tension developed in a contraction. In these circumstances, the time to peak tension can be so severely curtailed that the rate of rise of tension cannot reach its maximum before relaxation commences. When such contractions were deleted, all the inconsistencies in the force-frequency relationships disappeared.

THE

Classijication

of the force-frequency

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relationship

The comparative analysis of the short-term force-frequency relationships showed that all the animals studied could be grouped into two classes: in one class contractility always increased between contractions, in the other, contractility declined or did not change between contractions. The force-frequency relationship of ventricular muscle of goldfish, turtle, chicken, rat, cat, dog, rabbit and guinea pig fell into the first class; in this group p,, of the first extra contraction increased from a minimum, as the first extra stimulus interval was increased, to approach the value of p max for the previous regular contraction (Figure 3). Except in the goldfish heart, P maX of the second extra contraction rose IO A

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FIGURE 3. The maximum rate of rise of tension developed in contractions of a dog trabeculum; Results of the first (a) and second parts (A) of th e experiment. Basic rate was 0.33 Hz. The fixed first extra stimulus interval was 200 ms.

monotonically from a minimum to approach the steady-state value of p,, which was greater than the steady-state of P max of the first extra contraction. In the case of the goldfish, the steady-state value of the second extra contraction always rose to a value equal to and never exceeded that of the steady-state value of Jj,,,, of the first extra contraction. The force-frequency relationship of ventricular muscle of all the amphibia tested (amphiuma, mudpuppy, salamander and frog [R. @piens and R. catesbiana]) fell into

138

P. A.

W.

ANDERSON

ETAL.

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.

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s FIGURE 4. The maximum rate of rise of tension developed in contractions ventricular strip; results of the first (@) and second parts (A) of the experiment. Pmsx of the previous regular contractions. The basic rate was 0.25 Hz. The fixed interval was 1200 ms.

of an amphiuma (0, A) represent first extra stimulus

the second class : 1. pmax of the first extra contraction had a value equal to that of the previous regular contraction and remained constant for all values of the extra stimulus interval, or 2. p,, of the extra contraction declined from a maximum greater than to a plateau value equal to that of the previous regular contraction as the extra stimulus interval was increased (Figure 4). The ventricular muscle of both the frog and the amphiuma always behaved in the second way, whereas, in the mudpuppy and the salamander, P mex sometimes varied in one and sometimes in the other way, from animal to animal in the mudpuppy and from hour to hour in the same piece of muscle in the salamander. Thus, hearts without well-differentiated couplings were never found in the first class, whereas hearts with or without couplings were found in the second class. Unfortunately, the only decisive case, which would have permitted a structurefunction relationship to be disproven, namely the case of a force-frequency relationship of the first (i.e., mammalian-type) class in a heart without well-differentiated couplings, was never encountered. 4. Discussion

The reader might be disappointed that our searchfor a structure-function relationship did not begin with a detailed formulation followed by testsof hypothesesabout

THE

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the cellular mechanisms underlying the functional differences we have described. Our reason was not that hypotheses did not come to mind, but rather that we considered it best to first establish the existence of a relationship. Clearly, differences in the force-frequency relationship, for example, do invite speculation about mechanisms. As pointed out, we might have imagined, that the coupling acted as a storage site for the activator in excitation-contraction coupling, the amount of activator stored being a function of the interval between beats in the past. Indeed, such a speculation would also be in keeping with the findings of Antoni and others [2, 661: in mammalian heart distorting the waveform of the action potential with electrical current had little effect on the waveform of the contraction in response to the distorted action potential but markedly altered the peak tension developed in the contraction in response to the next normal action potential. Moreover, this “delayed potentiation” appears to be shared by all hearts that have a forcefrequency relationship of the mammalian kind, i.e., one characterized by such phenomana as post-extrasystollic potentiation and so-called ‘Lmemory” [3, 271. In frog heart, on the other hand, where couplings are sparse and poorly differentiated, the affects of the applied current were confined to the contraction in response to the distorted action potential-no delayed potentiation was seen [2]. Such speculations involve two kinds of hypotheses, one as to mechanism, of which there would seem to be countless and equally plausible alternatives, and the other, a more basic hypothesis which is shared by all: that in fact the given structure is related to the given function regardless of the mechanism involved. The coincidence in a heart of a cell structure, such as the coupling, with a given physiological activity and the absence in another heart of both the structure and the activity suggest, but can never prove, a structure-function relationship. Physiological and morphological studies of hearts from animals of other species might support the proposed relationship, but the results of such comparisons are conclusive only when they disprove the relationship. It has been our experience that an ultrastructural difference between hearts is more likely to be one of relative abundance and degree of differentiation of a structure rather than the total absence of the structure from one of the hearts. Even when no contradictory evidence exists, the coincidence of a physiological difference with a given structural difference could be fortuitous. The real reason for the physiological difference might, with equal likelihood, be some subtle (possibly chemical) difference undetectable by morphological techniques or destroyed during the preparative process.

Acknowledgement The superb technical acknowledged.

assistance

by Mr

I. Taylor

and

Pat Worthy

is gratefully

p. A. W. ANDERSON

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ETAL.

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