The Sensitivity of Developing Cardiac Myofibrils to Cytochalasin-B (Electron Microscopy/Polarized Llght/Z-Bands/Heartbeat) FRANCIS J

Proc. Nat. Acad. Sci. USA Vol. 69, No. 2, pp. 308-312, February 1972

The Sensitivity of Developing Cardiac Myofibrils to Cytochalasin-B (electron microscopy/polarized llght/Z-bands/heartbeat) FRANCIS J. MANASEt,* BETH BURNSIDEt't, AND JOHN STROMAN* * Departments of Anatomy and Pediatrics, Harvard Medical School, Boston, Massachusetts 02115; Department of Cardiology and Pathology, The Children's Hospital Medical Center, Boston, Mass. 02115; and t Department of Anatomy, Harvard Medical School, Boston, Mass. 02115 Communicated by Keith R. Porter, November 12, 1971

ABSTRACT Developing cardiac muscle cells of 11- to (about 30-40 hr of incubation) were selected. The splanchno- 13-somite chick embryos are sensitive to cytochalasin-B. the In cultured chick embryos, ranging in development from pleure of the developing yolk sac was removed, exposing 11 to 13 somites, hearts stop beating in the presence of this heart. If care is taken to prevent damage to the anterior in- agent. Both polarized light and electron microscopic testinal portal, this surgical procedure does not interfere with examination show that cytochalasin-B disrupts existing normal development during the time periods used in this myofibrils and inhibits the formation of new ones. Dis- study. In experimental series, the culture medium was re- crete Z-bands are not present in treated heart cells and thick, presumably myosin, filaments are found in dis- placed with either 0.1 mM (50 ug/ml) or 20 ,gM (10 jsg/ml) array. These effects are reversible; after cytochalasin-B is cytochalasin-B in Tyrodes solution plus 1% dimethyl sulf- removed from the medium, heartbeat recovers and myo- oxide (Me2SO) for specified incubation times. Control em- fibrils with discrete Z-bands reappear. Fibrillar sensitivity bryos were incubated in Tyrodes solution or in 1% Me2SO appears to be a function of age since fibrils in hearts of In order to ascertain the reversibility of embryos having from 22 to 28 pairs of somites are more in Tyrodes solution. resistant. effects of treatment with cytochalasin-B, embryos were washed after incubation with cytochalasin and reincubated Cytochalasin-B has been shown to interfere with a number of with fresh Tyrodes-Me2SO medium. An older group of em- morphogenetic processes that are associated with changes of bryos having from 22 to 28 pairs of somites were also incu- cell shape (1). Examination of tissues treated with cyto- bated with 0.1 mM cytochalasin. The pericardial sac of these chalasin-B suggests that one effect of this agent is the disrup- embryos was opened to permit direct contact between the tion of a class of intracellular proteinaceous filaments (usually medium and the myocardium. After treatment, some em- called microfilaments) that measure 5-7 nm (50-70 i) in bryos were fixed with glutaraldehyde-formaldehyde fixa- diameter (1, 2). Microfilaments are found in a great variety tive (15) and prepared for electron microscopy by postfixa- of cells that are capable of cytoplasmic motility (1-8). It has tion in l% 0s04 and embedment in Araldite. Aldehyde-fixed been suggested that morphogenetic alterations induced by specimens were also prepared as unstained whole mounts for cytochalasin-B result from dispersion of these filaments and examination with polarized light. consequent prevention of normal contraction-mediated changes of cell shape (1, 2). Filaments of this size range com- RESULTS plex with heavy meromyosin (9, 10) and have chemical simi- Studies with polarized light microscopy larities to muscle actin (10-12). Surprisingly, in view of the Intact myocardia can be examined with polarized light, and sensitivity of these actin-like filaments to cytochalasin-B, re- the presence of myofibrils can be detected. This technique cent reports have indicated that striated muscle fibrils, both presents a more comprehensive picture of myofibrils than in embryonic cardiac muscle (1) and in skeletal muscle (13), does electron microscopy with its inherent sampling prob- are stable in the presence of this compound. In the present lems. Faint birefringence can be detected in the myocardium paper, we will report the effects of cytochalasin-B on cardiac of embryos at the 10-somite stage (when the first spontaneous muscle in intact chick embryos during the period of initial contractions begin) and becomes more pronounced at the fibril formation and onset of contractility. Using techniques 11-somite stage (Fig. la). Fibril formation continues at a of polarized light and electron microscopy, we -have ascer- rapid pace, both in control embryos incubated with Me2SO tained that in young embryos, cytochalasin-B reversibly and embryos grown without it. When the specimen is rotated blocks myocardial fibrillogenesis and also disrupts myofibrils by 450, sharp extinction is seen (Fig. lb). Birefringence was that have already formed. also recognized by reversal of contrast by compensation with a Brace-Kbhler X/20 mica compensator. When examined METHODS visually under higher magnification, strongly birefringent Embryos were removed from incubated fertile white leghorn periodic segments, presumed to be A bands, are clearly seen eggs and transferred to culture (14); Tyrodes solution was and are separated by narrow isotropic regions. Negative com- used as a culture medium. Embryos with 9-14 pairs of somites pensation reverses the contrast of the myofibrils. When embryos are exposed to 0.1 mM cytochalasin-B for Abbreviation: Me9SO, dimethyl sulfoxide. 45 min during the period of rapid fibril accumulation, a clearly t Address reprint requests to: Dr. M. B. Burnside, Department discernible effect can be seen with polarized light microscopy. of Anatomy, Harvard Medical School, Boston, Mass. 02115 The discrete birefringence characteristic of the long, intact 308 Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Developing Cardiac Myofibrils 309

FIG. 1. Polarized light micrographs of portions of intact myocardia of embryos grown under the following conditions: (a) and (b) il-somite normal embryo, showing b)irefringent, myofib)rils. In (b) 450 rotation of the specimen relative to (a) demonstrates extinction of the birefringent fibrils. (c) li-somite embryo cultured in the presence of 0.1 mi\I cytochalasin-B for 45 min. Long birefringent myofibrils cannot be demonstrated at any rotational setting of the specimien. (d) Control il-somite embryo cultured with 1 % Me2S( in Tyrodes for 45 iimiii. Note normal myofibrils. (e) After 45 mini incubaition with 0.1 inmM cytochalasin, the embryro was washed anid reincuibated with fresh medium. 10 hr later, short birefringent fibrils reappear in the nivocardiuin. All figures X468.

fibrils is lost (Fig. lo). Rotating the specimen in either direc- tic of normal hearts at this stage of development (compare tion does not reveal additional birefringent material. Very Figs. 2 and 3). Disarrayed myofibrils are plentiful (Figs. 3 faint scattered birefringence can be detected by alternating and 4) after treatment with cytochalasin. In general, these between additive and substractive compensation by use of structures consist of scattered thick filaments, which are simi- the Brace-Kohler compensator, but very rarely are these lar in appearance to myosin, surrounded by a moderately anisotropic regions aligned. Controls grown in Tyrodes plus electron-dense matrix (Figs. 3 and 4). Extensive islands of Me2SO for 45 min show normal myofibrillar development this dense material accumulate in cytochalasin-treated myo- (Fig. id). cardial cells concomitant with the loss of discrete myofibrils. l1-Somite embryos exposed to 0.1 mM cytochalasin-B for An outstanding feature of these cells is the disruption of the periods up to 45 min and subsequently washed with 1% Me2- normal association of filaments with Z-bands. Discrete SO in Tyrodes medium recover birefringent myofibrils. 5-10 Z-bands associated with myofibrils are not seen in treated hr after the embryos are washed and reincubated, discrete cells. Desmosomes appear unaffected by this concentration birefringence is again visible in the myocardium (Fig. le). of cytochalasin (Fig. 3). Of a total of nine embryos that were allowed to recover in In those specimens that are allowed to recover from the separate experiments, all regained myofibrils. effects of cytochalasin (Fig. le), the electron microscope re- The rapidity with which cytochalasin disrupts myofibrils veals myofibrils characteristic of early embryonic cardiac is dose dependent. In li-somite embryos grown in 20 ;sM myocytes (Fig. 5). cytochalasin (10 ;&g/ml), incubations ranging to 2 hr are re- quired to disrupt myofibrillar birefringence. The effects of cytochalasin-B on cardiac contractility Sensitivity to cytochalasin decreases with age. Embryos in Cytochalasin-B also causes the arrest of the embryonic heart- the 12- to 14-somite range require 2- to 3-hr exposure to 0.1 beat. When 11-somite embryos are incubated with 0.1 mM cytochalasin to disrupt their myofibrils. In older em- mM cytochalasin, visible myocardial contractions cease after bryos, having from 22 to 28 pairs of somites, there is no 30-45 min. With slightly older embryos, in the 12-14 somite noticeable decrease of birefringent myofibrils after 4 hr of range, hearts continue beating for 2-3 hr in the presence of incubation with 0.1 mM cytochalasin. the drug. In all instances, cessation of contractility is not abrupt. Contractions gradually weaken, becoming restricted Electron microscope studies to the right margin of the heart before stopping completely. In corroboration of the polarized light observations, electron In the recovery experiments, removal of the drug permits microscopic examination of cytochalasin-treated myocardial resumption of heartbeat. 5-10 hr after the embryos are washed cells reveals almost none of the intact sarcomeres characteris- and reincubated, the hearts again begin to contract spon- Downloaded by guest on September 27, 2021 310 Physiology: Manasek et al. Proc. Nat. Acad. Sci. USA 69 (1972)

_- ~.-W27>"mm-m_I-1 FIG. 2. Electron micrograph of cardiac cells from a control 11-somite embryo exposed to Tyrodes plus Me2SO for 45 min (see Fig. 1d). Note the myofibril containing parts of three sarcomeres within the section, and note the discrete Z-bands (arrows). X 15,800. taneously. The contractions are initially feeble and restricted These results are seemingly in contradiction with the find- to the right margin of the heart but they gradually increase in ings of other workers. Wessels et al. (1) report that cyto- magnitude and frequency until they involve the entire myo- chalasin does not disrupt myofibrillar structure in cultured cardium. Eight of the nine embryos used in the recovery myocytes from 8-day chick hearts, although spontaneous experiments regained spontaneous contractility. contractions are arrested. Comparison of the effects of cyto- In 11-somite embryos grown in 20 MM cytochalasin, heart- chalasin during very early stages of cardiac myogenesis (in beat continues for about 2 hr before stopping. Older embryos embryos with 11 pairs of somites) to its effects on older hearts (22-28 pairs of somites) continue beating for longer periods, (in embryos with 22-28 pairs of somites) indicates a decrease even when incubated with higher concentrations of cyto- in myofibrillar sensitivity to cytochalasin with increasing chalasin. In the presence of 0.1 mM cytochalasin, hearts of age. Perhaps the reported resistance of fibrils within 8-day these embryos continue to beat for at least 4 hr. In this age cells also represents an age-dependent decrease in sensitivity range, the effects of cytochalasin on contractility are clearly to cytochalasin. A possible explanation for decreased sensi- separated from its effects on fibril integrity. Even after cessa- tivity could be that fibrillar proteins become more refractory tion of spontaneous contractions, there is no noticeable de- as a result of addition of accessory molecules. On the other crease of birefringent myofibrils. hand, it is not possible to rule out age-dependent cytochalasin- permeability differences. It is unlikely that the suggested DISCUSSION abnormal alignment of fibrils in treated cells reported by Cytochalasin-B appears to have two effects on early em- Wessells et al. (1) is related to cytochalasin, since fibrils in bryonic myofibrils: it disrupts those already formed and in- developing heart myocytes are generally poorly aligned both hibits the production of new ones. Hearts of 11-somite em- in vivo (17-19) and in culture (20). bryos contain a readily discernible complement of birefringent Holtzer et al. (21) and Sanger et al. (13) reported that myofibrils. Treatment of these embryos with cytochalasin cytochalasin does not prevent myofibril formation or con- results in the disruption of the long, strongly birefringent tractility in cultured skeletal muscle. However, Sanger myofibrils. Thus, not only is there a failure to add myofibrils et al. (13) used a maximum concentration of 10 ug/ml (20 in the presence of cytochalasin, but the original complement ,uN) cytochalasin-B [Holtzer et al. (21) did not report the of fibrils is disrupted. concentrations used]. Since this concentration does inhibit The absence of intact myofibrils revealed by electron contractility and disrupt myofibrils in young chick hearts, but microscopic examination of the cytochalasin-treated cells only after extended incubation, it seems possible that these confirms the findings revealed by polarized light studies. authors are using near-threshold concentrations of the drug. The myofibrillar apparatus is disrupted and only loosely as- It is possible that higher concentrations, as reported here, sociated bundles of thick, presumably myosin, filaments re- which do disrupt fibrils and yet allow recovery in chick main as recognizable remnants of myofibrils. That these ef- embryo hearts, might also affect fibrillogenesis and contraction fects are not the result of agonal changes brought about by in skeletal muscle. possible cytochalasin toxicity is demonstrated by the other- The precise mechanism of fibrillar disruption is not known. wise normal ultrastructure of treated cells and more defini- It is in keeping with previous reports of disruption of actin- tively, by the ability of the cell to recover both fibrils and like filaments with cytochalasin-B (1) to suggest that dis- contractility when cytochalasin is removed. In no instances persal of fibrils results from destruction of actin filaments. did cells acquire the characteristics of dying cardiac myo- Our electron microscope observations indicate that although cytes (16). disrupted myofibrils still retain thick filaments that are Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Developing Cardiac Myofibrils 311

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*AW FIG. 3. Electron micrograph of cell from myocardium of an ll-somite embryo grown in 0.1 mM cytochalasin-B for 45 min. Note the disrupted myofibril (inset). Discrete Z-bands are lacking, but numerous dense bodies (inset and small arrows) appear in cytochalasin-B- treated heart cells. Desmosomes do not seem to be altered by cytochalasin-B. X26,070. Inset X48,190. FIG. 4. Disrupted myofibril from the same heart shown in Fig. 3. Thick filaments are visible (arrows) and an extensive moderately electron-dense matrix (M) that is not found in normal hearts is found in the vicinity of disrupted fibrils. X73,470.

probably myosin, few actin filaments remain recognizable. Cytochalasin-B also inhibits spontaneous contractions of If polymerized actin filaments are indeed absent, then the embryonic cardiac muscle. Wessells et al. (1) described an islands of amorphous material that accumulate in cyto- irreversible inhibitory effect on cultured 8-day embryonic chalasin-treated cells might represent unpolymerized precursor myocytes, even though these cells retain their myofibrils. material. This interpretation is complicated, however, by the Our experiments with whole embryos show that cytochalasin observation that Z-bands appear to be attacked by cyto- also interferes with contractility in intact hearts. Although chalasin. Although loose bundles of filaments are seen in in the younger embryos used in this study the time-course of cytochalasin-treated cells, they are almost never associated contraction inhibition and recovery approximates that of with discrete Z-bands. It is possible that disruption of Z- fibril disruption and reappearance, the full complement of bands may allow such disarrangement of other myofibrillar birefringent myofibrils is still present in older hearts that have elements that actin is difficult to discern in sections. On the stopped beating in the presence of cytochalasin. This observa- other hand, if the primary site of cytochalasin action is the tion suggests that under these conditions cytochalasin- actin component, then similar myofibrillar breakdown would mediated inhibition of contractility is not the result of fibril be expected. It is not possible on the basis of our observations disruption, but rather that cytochalasin has some other effect to decide between these alternatives. such as interfering with contraction of the fibrils, altering Downloaded by guest on September 27, 2021 312 Physiology: Manasek et al. Proc. Nat. Acad. Sci. USA 69 (1972)

FIG. 5. Electron micrograph of myocardial cells from a chick embryo that was treated for 45 mm with 0.1 mM cytochalain-B at the li-somite stage, washed and allowed to recover for 10 hr in Tyrodes plus Me2SO. Recovery was indicated by return of normal heartbeat and reappearance of birefringent fibrils (see Fig. le). Ultrastructurally, one finds long fibrils showing several sarcomeres demarcated by discrete Z-bands (arrows) within a single section. X 16,960.

cation permeability of the membranes, or uncoupling excita- 6. Nagai, R. & Rebhun, L. I., (1966) J. Ultrastruct. Res. 14, 571-589. tion-contraction. 7. Pollard, T. D. & Ito, S. (1968) J. Cell Biol. 39, 106A. In summary, cytochalasin-B reversibly disrupts the de- 8. Wolfarth-Botterman, K. E. (1964) Int. Rev. Cytol. 16, veloping fibrils of early embryonic chick hearts and also 61-131. causes the reversible arrest of the embryonic heartbeat. This 9. Ishakawa, H., Bischoff, R. & Holtzer, H. (1969) J. Cell cytochalasin sensitivity appears to be age-dependent and de- Biol. 43, 312-328. 10. Pollard, T. E., Shelton, E., Weihing, R. R. & Korn, E. V. creases in older embryos. (1970) J. Mol. Biol. 50, 91-97. 11. Adelman, M. R., Borisy, G. G., Shelanski, M. L., Weisen- This research was supported in part by grants from the Medical berg, R. C. & Taylor, E. W. (1968) Fed. Proc. 27, 1186-1193. Foundation of Boston, Inc. and from the National Heart In- 12. Nachmias, V. T., Huxley, H. E. & Kessler, D. (1970) J. stitute (HE 10436), and in part by the National Institutes of Mol. Biol. 50, 83-90. Health Postdoctoral Fellowship GM-30,828-02. FJM is a Fellow 13. Sanger, J. W., Holtzer, S. & Holtzer, H. (1971) Nature 229, of the Medical Foundation of Boston, Inc. 121-123. 14. New, D. A. T. (1955) J. Embryol. Exp. Morphol. 3, 320-331. 15. Karnovsky, J. J. (1965) J, Cell Biol. 27, 137A. 1. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. 0., 16. Manasek, F. J. (1969) J. Embryol. Exp. Morphol. 21, 271- Luduena, M. A., Taylor, E. L., Wrenn, J. T., & Yamada, 284. K. M. (1971) Science 171, 135-143. 17. Wainrach, S. & Sotello, J. R. (1961) Z. Zellforsch. Mikrosk. 2. Schroeder, T. E. (1970) Z. Zellforsch. Mikrosk. Anat. 109, Anat. 55, 622-634. 431-449. 18. Manasek, F. J. (1968) J. Morphol. 125, 329-366. 3. Baker, P. & Schroeder, T. E. (1967) Develop. Biol. 15, 432- 19. Manasek, F. J. (1970) Amer. J. Cardiol. 25, 149-168. 450. 20. Muscatello, U., Pasquali-Ranchetti, I. & Barasa, A. (1968) 4. Buckley, I. K. & Porter, K. R. (1967) Protoplaama 64, 349- J. Ultrastruct. Re8. 23, 44-59. 380. 21. Holtzer, H., Sanger, J. & Ishikawa, H. (1971) J. Gen. 5. Cloney, R. A. (1966) J. Ultrastruct. Res. 14, 300-328. Physiol. 57, 245A. Downloaded by guest on September 27, 2021