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

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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 cyto- chalasin-B for 45 min. Long birefringent myofibrils cannot be demon- strated 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.
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