Myocardial ultrastructure in systole and diastole using ballistic cryofixation

© 1968 by Academic Press lr.c.

22

J. ULTRASTRUCTURE RESEARCH

22, 22-36 (1968)

Myocardial U ltrastructure in Systole and Diastole Using Ballistic Cryofixation 1 R. GRIER MONROE,2 W. J. GAMBLE, C. G. LA FARGE, R. GAMBOA, C. L. MORGAN, A. ROSENTHAL, AND S. BULLIVANT Department of Pediatrics, Harvard Medical School and the Cardiology Division of the Medical Service, Children's Hospital Medical Center, Boston, and the Mixter Laboratory for Electron Microscopy, Neurosurgical Service, Massachusetts General Hospital, Boston Massachusetts Received August 18, 1967, and in revised form November 9, 1967 A method is described whereby sections of myocardium were obtained for electron microscopy from an actively beating heart during specific phases of its contraction cycle. Small section were rapidly removed by hollow projectiles accelerated to supersonic velocities by an explosive. Cryofixation was achieved using a mixture of liquid and frozen propane as the coolant. The method employed is discussed in terms of its application, results, and limitations. Recent studies of the length-tension characteristics of both striated and cardiac muscle, carried out in conjunction with electron microscopic examination of the sarcomere during relaxation, have shed new light on the ultrastructural basis of the muscle function. There is now considerable evidence that disengagement of the thick from the thin filaments of the sarcomere, in diastole or relaxation, is causally related to the descending limb of the Frank-Starling mechanism (4, 5, 9). Implicit in this observation is the possibility of a precise, anatomical definition of normal myocardial contractility as opposed to the abnormal pattern in cardiac failure where overdistention of the ventricle is a prominent feature. Less ultrastructural information has been obtained about cardiac muscle in a mechanically active state from a spontaneous or electrically stimulated beat. Phase and interference microscopy of striated muscle have yielded considerable information within the limits of their resolution (3, 6). Furthermore, it is well known that a state of supercontraction can be induced by fixation of muscle in 10% formalin prior to preparation of the specimen for electron microscopy. Under the latter circumstances, of course, the contraction is artifactually induced. z Supported in part by N.I.H. Grants H-6144, HE-10436, and CA-07368. 2 This work was performed in part under the tenureship of an Advanced Research Fellowship of the American Heart Association, and in part under the tenureship of a Career Development Award from the N.I.H.

23

MYOCARDIAL ULTRASTRUCTURE IN SYSTOLE AND DIASTOLE

SOLENOID TRIGGER

t

"~:J,~V~::",

.':. "

~X,~PLOSION

f CHAMBER

I

1

I

S,,b

[]

COLLECTION CHAMBER CONTAINING TISSUE FRAGMENTS IN LIQUID PROPANE AT -188° C

SWITCH

DELAY CIRCUIT

STIMULATOR

FIG. 1. Schematic diagram of technique. Stimulus inducing contraction in isolated heart may be variably delayed to trigger explosion, driving biopsy projectile into ventricle at any time in contraction cycle. Projectile deposits contents into propane slurry ( - 188°C). Attempts were made, therefore, to develop a technique for obtaining biopsies rapidly, at specific points in the contraction cycle of an active left ventricle using a "quick freeze" technique. Inasmuch as the period of systole during which the ventricle remains contracted is a short one, approximately 0.2 second, efforts were made to obtain the biopsy in the shortest time possible. To this end the authors took advantage of knowledge gained in one of man's long-standing, albeit often misguided, preoccupations, and designed a system whereby the biopsy could be obtained by a hollow projectile which had been accelerated with gunpowder. The collecting projectile, while still in flight, deposited its contents in an appropriate coolant contained in a collection chamber so that the specimen would be frozen in as short a time as possible. While the biopsy specimen was being cooled one could gain advantage of the fact that such cooling, of itself, prolongs the active state (8).

MATERIALS AND METHODS A schematic diagram of the technique used is seen in Fig. 1. Tubular stainless steel projectiles made from 6 cm segments of No. 22 gauge hypodermic needle tubing (o.d., 0.71 mm; i. d, 0.39 mm) were accelerated to supersonic velocities (400 meters per second) by the explosion of 0.144 g of gunpowder (dupont 3031), directed into the left ventricle of an isolated heart preparation, and decelerated in a collection chamber containing a mixture of liquid and frozen propane (temperature, -188°C). Upon entering the collection chamber the biopsy specimen collected by the projectile was extruded into the propane mixture.

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R. GRIER MONROE ET AL.

100 ~ linear liter ~J spirometer r~E differential air chamber[[ ~ - [ i ~:: transformer maW2t2rter~

I, ~ ; ~

~I

loading valve

~pressure perfusionvia aorta . - - ~ ~ t r a n s d u c e r returnvia P A ~ ( LV "~

~

stimulator

FIG. 2. Schematic of isolated heart preparation. End-diastolic pressure is determined by pressure surrounding spirometer and transmitted to ventricle via J valve. When loading valve is open, ventricle can contract isobarically while absolute ventricular volume is monitored by spirometer, and intraventricular pressure by transducer. Contraction of the ventricle was induced by an electrical stimulus, after ligation of the bundle of His. The stimulus, having been delayed by a multivibrator circuit, also served to initiate the explosion by the mechanical firing of a primer (Winchester 120M) through a solenoid release (Fig. 1). The delay circuit was so programmed that the biopsy could be obtained at any specific time following the stimulus and therefore, in any desired phase of the heart's contraction cycle. Collection and release of the biopsy specimen occurred in less than a millisecond. Cooling rates were estimated to be in excess of 3 degrees per millisecond from studies in which the same projectiles containing thermocouples were suddenly immersed in the - 188°C propane mixture. The entire contents of the collection chamber were then passed through a 37-ff sieve at low temperature. The tissue fragments retained by the sieve were subsequently substituted for 2 weeks in absolute ethanol at - 7 5 ° C to which no osmium or other fixatives were added. At no time in their handling did the temperature of the tissue fragments rise above - 7 5 ° C until this 2-week period had elapsed. After freeze substitution in ethanol the specimens were immersed in a mixture of 50 % absolute ethanol and 50 % Epon resin and remained there for 24 hours at - 2 5 ° C (1). After this period the specimens were allowed to rise to room temperature and were embedded in Epon blocks. The blocks were hardened at 60°C for 24 hours or more. Sections were subsequently cut for either phase or electron microscopy, and measurements of sarcomere length were carried out on suitably calibrated phase photomicrographs or electron micrographs. For electron microscopy the thin sections were stained with uranyl acetate and lead hydroxide.

MYOCARDIAL ULTRASTRUCTURE IN SYSTOLE AND DIASTOLE

25

ECG

IMPACT MARKER

INTRAVENTRICULAR

I0 30 50

VOLUME

70

ml

90

INTRAVENTRICULAR PRESSURE mm Hg

60 40

2O 0

F~6.3. Recording of physiological data during isobaric contraction of ventricle. Upper panel, electrocardiogram. Second panel, moment of impact. Third panel, intraventricular volume. Bottom panel, intraventricular pressure.

The isolated heart preparation has been previously described in detail and will be outlined only briefly herein (7, 8). Hearts from healthy mongrel dogs were rapidly excised under chloralose (60 mg/kg) and urethane (900 rng/kg) anesthesia. A Lucite button was ligated in the root of the ascending aorta just below the ostia of the coronary vessels, thereby separating the left ventricular cavity from the coronary circulation. The bundle of His was ligated, and the orifices leading to the right atrium were closed. Cannulae were ligated in the aorta, the pulmonary artery, and the mitral orifice. The heart was then perfused via the a o r t a with blood from a healthy anesthetized donor, the blood being returned from the coronary sinus through the pulmonary artery and pumped back to the donor. The heart was next attached to a Krogh spirometer by the mitral cannula, as illustrated in Fig. 2. During most of the reported studies the centriele contracted isobarically by compressing air into a relatively large chamber (100 liters). Under these circumstances the loading valve in Fig. 2 remained open, and the pressure distending the ventricle was equal

26

R. GRIER MONROE ET AL. INTRAVENTRICULAR 120'

PRESSURE

(2)

mm Hg I00' (t) 80"

60.

40.

(6),, .(4)

(5) (7)

20"

~PRESSURE-VOLUME o

o

Ib

2'o

3b

do

5'o

CURVE

go

VOLUME ml

FIG. 4. Left ventricular pressure-volume plot with pressure in millimeters Hg (vertical axis) and volume in milliliters (horizontal axis). Numbers in parentheses refer to specific biopsies: (6) and (4) obtained during diastole; (1), (2), (5), and (7) obtained during systole. to that in the chamber surrounding the spirometer. Isovolumic contraction of the ventricle was achieved by filling the ventricle with saline and closing the loading valve. Intraventricular pressure was monitored with a pressure transducer (Sanborn 267B) and the absolute intraventricular volume monitored with a linear differential transducer (Sanborn 575 DT 250) attached to the spirometer. The exact moment of impact of the projectile was marked electronically as it passed through an aluminum foil screen placed next to the ventricle. The pressure, volume, and impact signals together with an electrocardiogram, were continuously recorded on a Sanborn polyviso recorder (Sanborn 964) as illustrated in Fig. 3. Since at the moment of impact, the absolute ventricular volume and intraventricular pressure were simultaneously recorded, it was possible to construct pressure-volume diagrams of ventricular function and label the points at which the biopsies were obatined (Fig. 4). The explosion chambers were machined to the dimensions of a 30-06 rifle cartridge thereby allowing them, for safety, to be housed in the breech of a 30-06 military rifle. Fig. 5 is a photograph of the explosion chambers and projectiles. Fig. 6 is a photograph of the assembled biopsy equipment. In addition, for reasons discussed below, attempts were made to obtain biopsies of skeletal muscle during rest, passive stretch, and active contraction. The sartorius muscle of an anesthetized dog was used for these studies. Stretch was imposed by a 500 g weight attached

MYOCARDIAL ULTRASTRUCTURE IN SYSTOLE AND DIASTLOE

27

Fro. 5. Photograph of assembled (top) and disassembled (middle) explosion chambers. Three projectiles are shown: one protruding from barrel adapter (middle), and two below.

to a pulley. Contraction was then induced with a tetanic stimulus (12 V, 60 ~ AC) applied to the nerve ennervating the muscle. RESULTS

Rapidfreezing of cardiac muscle Despite the fact that formation of ice crystals frequently disrupted most areas of the biopsy specimens examined, good electron micrographs could nevertheless be obtained with this technique, as shown in Fig. 7. In the section illustrated, which was obtained in diastole at an end-diastolic pressure of 10 m m Hg, some ice crystal damage is evident, particularly in the region of the A band. However, the boundaries of the A and I bands can clearly be discerned and the Z bands readily identified. The mitochondria were, in general, less subject to ice damage. Infoldings of the plasma membrane can be seen at the Z band level. Scattered opaque deposits of glycogen are also seen, primarily in the vicinity of the mitochondria. In the center of the sarcomere the M and L bands are quite evident. In some areas of the section shown in Fig. 7, H bands can clearly be discerned despite the fact that the ventricle was distended in this case with only a pressure of

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R. GRIER MONROE ET AL.

FIG. 6. Photograph, from left to right, of 30-60 military rifle housing explosion chamber, aluminum foil screen to mark moment of biopsy, and collection chamber containing liquid propane mixture. Heart or skeletal muscle is positioned between marker and collection chamber.

10 m m Hg. In the opinion of the authors this is an unfortunate artifact of the method, as subsequently discussed. Contraction bands were not found in those sections that were in a marked state of contraction. Although some excellent micrographs could be obtained with this technique as shown in Fig. 7, correlation between ventricular volume and sarcomere length was disappointing. All biopsies were obtained either at peak systole or end-diastole, and as intraventricular volume was constantly monitored it was possible to measure the volume at the precise moment of biopsy. Fig. 8 is a plot of sarcomere length against the cube root of the volume at which the biopsy was obtained, divided for normalization by the cube root of the volume at an end-diastolic pressure of 20 mm Hg. The graph shows the cumalutive results from six hearts, points obtained in diastole being marked with X and those obtained in systole being marked with a dot. Only those sarcomeres were counted in which the width of the A band was 1.5/~. With this provision one could be reasonably confident that the plane of section was similar in the sections compared. Three things should be noted from the results shown in Fig. 8. First, one cannot demonstrate with any certainty a correlation between sarcomere length and ventricular volume. Second, there is a wide scatter in sarcomere length at any given ventricular volume. Third, in general, the sarcomeres are more extended or stretched than they should be regardless of whether they were obtained in systole or diastole. In this connection it should be pointed out that the end-diastolic pressures did not exceed 20 m m Hg.

FIG. 7. Electron micrograph of heart muscle obtained in diastole at 10 mm Hg end-diastolic pressure. Note fair preservation of ultrastructure in center of electron micrograph despite ice crystal damage at periphery. Further description in text. × 15,000.

MYOCARDIAL ULTRASTRUCTURE IN SYSTOLE AND DIASTOLE

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30

R. GRIER MONROE ET AL.

~X

ZtoZ length P

.4

.6

.8

1.0

,~ vol.

v6r20 FIG. S. Plot of sarcomere length in microns (vertical axis) against 3~ / v ~ ~vo~20 (horizontal axis). 31/~ol = ventricular volume at moment of biopsy; 3~/v~0 = ventricular volume at 20 m m Hg enddiastolic pressure. Points marked x were obtained in diastole; points marked with dots, in systole. No correlation could be demonstrated between sarcomere length and ventricular volume. Further description in text.

As shown in Fig. 9 one could, by selection, find areas in which the sarcomere lengths obtained from the same heart in both systole and diastole correlated well with the volume change. This, it should be emphasized, could be done only by selection and does not represent the overall picture of the data obtained.

Rapidfreezing of skeletal muscle In view of the overall poor correlation between sarcomere length and ventricular volume noted in Fig. 8, an attempt was also made to obtain biopsies from the sartorius muscle of a dog using the same technique during passive stretch, relaxation, and active contraction induced by electrical stimulation of the nerve while the muscle was under a light load. Efforts were then made to correlate these with the physical state of the muscle by marking a length of the muscle from which the biopsy was to be obtained with two silk sutures, photographing the muscle on or about the moment of biopsy, and measuring the distance between the sutures. The results of one of these studies are shown in Fig. 10 where the sarcomere lengths obtained from the biopsies are plotted against the muscle lengths obtained from the photographs. Here, at first glance, there may appear to be a better correlation than that shown between sarcomere length and ventricular volume on Fig. 8. But on closer scrutiny the correlation between sarcomere length and muscle length on Fig. 10 is highly dubious, despite the fact that in contraction the muscle shortened to one-third of its stretched length. As in the case of cardiac muscle there is considerable scatter in the sarcomere length at a given muscle length. Further, the sections obtained show

MYOCARDIAL ULTRASTRUCTURE IN SYSTOLE AND DIASTOLE

31

FIG. 9. Comparison of electron micrographs obtained from same heart at peak systole (top) and end-diastole (bottom). In this comparison changes in sarcomere length corresponded with volume changes. End-diastolic pressure, 12 m m Hg; stroke volume, 23 ml. Top electron micrograph corresponds to point (5) and bottom electron micrograph corresponds to point (4) of Fig. 4. Scale marker (lower left) = 1/~. Both pictures at identical magnification: × 21,000.

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R. GRIER MONROE ET AL.

.Z"7'

Zto z length

muscle length (cm) Fro. 10. Plot of sarcomere length in microns (vertical axis) against muscle length in centimeters (horizontal axis). Biopsies obtained from dog sartorius during active contraction (6.5 cm), rest (16 cm), and passive stretch (19,5 cm). Further description in text. the sarcomere to be stretched more than one would anticipate in all three states of passive stretch, relaxation, and active contraction.

Formalin fixation, using the rapid biopsy technique In the studies described, sections of both cardiac and striated muscle were also obtained using the same biopsy technique but collected in 10% formalin at room temperature instead of liquid propane at -188°C. Upon removal from the formalin the tissue fragments were fixed in osmium, embedded, sectioned, and stained as described in the methods. All sections so obtained were markedly contracted as seen in Fig. 11 whether they were obtained in systole or diastole. Using this technique the sarcomere lengths averaged 1.73 #. It should be noted, however, that one set of sections obtained from striated muscle and fixed in glutaraldehyde instead of formalin were stretched when obtained during passive stretch and contracted when obtained during active contraction. DISCUSSION From the figures shown it is evident that electron micrographs with reasonable structural detail can be obtained from both cardiac and striated muscle using the described technique. This does not imply that all parts of all sections were iq the state of preservation illustrated. Frequently many cuts of a biopsy specimen had to be made before encountering an area where the ultrastructure was not completely disrupted by ice crystal formation. The primary disadvantage of the technique, therefore, lay in the formation of ice crystals within the specimen with their attending distortion of the relation of the thick

MYOCARDIAL ULTRASTRUCTURE IN SYSTOLE AND DIASTOLE

33

Fro. 11. Electron micrograph of section obtained with rapid biopsy technique but collected in 10% formalin at room temperature. Biopsy obtained during diastole. Note extreme state of contraction with narrowing of I bands, x 30,000. 3 - 681834 J . Ultrastrueture Research

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R. GRIER MONROE ET AL.

and thin filaments. It is known that the formation of these crystals can be avoided only by very rapid freezing (11, 12). For this reason liquid propane was selected as the best coolant, having a reasonably low freezing point and a relatively high boiling point. As has been shown by Bullivant, these properties give liquid propane an advantage over the colder liquid helium as a coolant for rapidly freezing tissue wlm minimal ice crystal disruption of the ultrastructure (1). In this connection it was also desirable to ensure that the biopsy specimen had the smallest mass possible, within the limits of practical handling, thereby also increasing the rate of freezing (11). Doubtless the velocity of the projectile and its tissue fragments ensured good mixing of the coolant, an additional factor which would tend to increase this rate. As stated in the results, there was a poor correlation between the physical state of contraction of the muscle and sarcomere length. Although, as shown in Fig. 10, the sarcomere lengths of striated muscle biopsied during contraction averaged 2.37 # and were in general shorter than those biopsied during relaxation and stretch, they were not in a state of contraction consistent with the observed change in muscle length. Likewise, the sarcomere lengths of muscle biopsied during rest averaged 2.71/~, a figure certainly higher than the previously established sarcomere length of resting striated muscle (4, 5). Two reasons come to mind to explain the observed discrepancy. First, one might postulate that the sarcomere length is altered after freezing during the freeze substitution process. This would appear an unlikely possibility as the elastic elements of striated muscle would tend to draw it to a shorter length than that observed in sections obtained during relaxation. Second, one might postulate that the section of muscle is stretched during its acceleration when captured by the projectile and possibly stretched again during its release from the projectile. Visualizing the section of muscle which is captured and released as a cylinder, it should be pointed out that, in general, the long axis of this cylinder is at right angles to the myofibrils. Furthermore, this cylinder may be regarded as a stack of thin discs, the diameter of each being the internal diameter of the projectile, with the myofibrils extending in the plane of the discs. Postulating that the forces of acceleration are frictional forces directed at the edges of the discs while the opposing inertial forces are directed at their centers, one can readily see how the myofibrils might be stretched during the capture of the specimen. Conversely, during deceleration, the decelerative forces would be directed at the center of the disc, by the liquid propane entering the projectile, while the frictional forces would again be directed at the edges, and the myofibrils could be stretched further. To the authors the possible stretching of the sarcomere with the described technique seems the most likely explanation for the observed discrepancy between the gross and ultrastructural state of the muscle. In this connection when one considers the accelerative and decelerative forces in-

MYOCARDIAL ULTRASTRUCTURE IN SYSTOLE AND DIASTOLE

35

volved in raising the velocity of the tissue obtained to the speed of sound and decelerating it in less than a millisecond one might indeed wonder how the tissues bear any resemblance at all to its natural appearance. In the face of the data one can appreciate that the ultrastructure of the sarcomere at the molecular level must have an extraordinary cohesiveness to withstand such an insult with only the small distortion shown. Those sections collected with the ballistic technique described and deposited in formalin at room temperature, instead of frozen, were consistently found to be in a supercontracted state. Here, it is safe to assume that the contraction was induced by the formalin, after immersion, and was evidently sufficient to overcome the stretch induced by the accelerative and decelerative forces. Although this illustrates a wellestablished effect of formalin as a fixative it must be remembered that the sections so fixed in this study were free and not restricted from contracting. Glutaraldehyde did not appear to induce supercontraction in the one instance in which it was used as a fixative and to this extent may be more useful than formalin, as has been implied by others (10). In spite of its evident drawbacks the authors are hopeful that the technique described may provide a way of collecting tissue for the chemical analysis of muscle at various points in the contraction cycle. This method could be used, then, to explore the migration of sodium, potassium, and calcium ions during muscular contraction and relaxation, using suitable techniques for the analysis of these ions at the ultrastructural level (2, 13, 14). The method has the obvious advantage of rapidly fixing muscle without the addition of foreign elements and to this extent is less artifactual and probably faster than chemical methods. To date the authors know of no electron micrographs, including their own, which inarguably demonstrate the unique molecular architecture of the mechanically active state. Hopefully this or similar techniques may provide evidence to this end. Further, the method may serve as a means of obtaining specimens from other organs under circumstances in which tissue obtained at a precise point in time will provide useful information. Note added in proof." Since this report was accepted for publication a new paper by Sonnenblick et aI. was published [Circulation Res. 21,423 (1967)] which describes an original method of obtaining electron micrographs of heart muscle in systole using glutaraldehyde fixation. The authors are indebted for the technical assistance of Sheldon Rich, who designed and constructed the biopsy equipment, and for the technical assistance of Philip E. Waithe in the surgery performed. The authors are also indebted to Dr. Betty Uzman for the phase microscopy and her stimulation and advice in the preparation of the manuscript, and to Dr. Don Fawcett for his advice and direction.

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BULLIVANT,S., Lab. Invest. 14, 1178 (1965).

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