F I R S T CONTRACTIONS O F THE H E A R T W I T H O I T CYTOLOGICAL DIFFERF~NTIATIOX C. M. GOSS Department of Anatomy, Universil?l of Alabama TWO FIGURES The present irivcstigatioii was uiidertakeii to determine wliether specific cytological structures appear a t the time of initiation of the beat in the heart. If such a differentiatioil is not detectable, the necessity of these structui*csf o r muscular contraction is called into question. The following comparison of heart muscle before and after the first contractions, iiidicates that visible differentiatioil is not associated with the new power of mecliariical activity. A brief description of the heart in living r a t embryos a t this stage of development has heen given in a recent publication (Goss, '38). It was recognized a t that time that these observations required confii.matioii for- two 1-casoiis. First, in t1;e intact living embryo, tlie heart cannot be observed with case because the visibility is hampered by the preseiice of overlying layers of cells which cannot be dissected away uiitil tlie embryo is somewhat older. Seconcl, the lack of cross striations in living embryonic heart muscle does not prove the absence of these structures, since they may be brought out in tixed specimens by the proper tcchiiical procedures (Lewis, '19). I n this paper, therefore, the results of a study of emhryoriic hearts in mliolc mounts pi.epai.cd according to Lewis ' nlethod and in serial sections prepared according to other cytological techniques a r e presented. MATERIAL AND METHODS A total of seventy-two embryos from eleven pregnant 1-ats were made into whole mounts in Zenker solution to whicli n 19 20 C. 31. G O S S fc\v cli.01)~of 2%) osmic acid hat1 heen added (Lewis, ’19). Of t l ~ c ~ s ctwenty-six ~, were at the age of first coiiti~ictioiis or s1iglitlF y o u i i g e ~ forty , wei’c a t ages vai*yiiigfro111 this to the 1)egiiiiiiiig of c.ir.cnlatioii and six were aft er cii.culation w a s \vc>ll cstablislied. In tlie youiigcst embryos, tlic 1ieai.t~could he f i w d fiwm theii. pei*icar(lialcovering only i)artially and Tvitli giaeatest clificulty. After the stage of a siiiglc saccnlar \*eiitriclc, the coveiiiig layers were dissected away, so tliwt tlie myocaidial cells m7ei.e bi.ouglit diiw t l y against the cover glass. Lewis’ requii.emciits of stretcliiiig tlic iriuscle slightly lo? mcaiis of pi-cssure froiii the covei’ glass wei’e ca wfullp obsei*ved. Ti1 tlic best l)rcpl-iratio~is,the embryo w a s iiiolintecl i i i ‘L’yi*ocle solution first, tlie c~oiitrwctioiisobserved thiwugli the microscope aiiil tlic pi*ogi*ess of fixation watched as osmic Zeiilicr solutioii lvas tli*awii under the covci* witli filter paper. The first sectioiied material of a desii-ed age \\-as obtained by watcliiiig f o r the hegiiiiiiiig coiiti*actioiis in cultnrccl embi-yos. ‘I’lit~ results \\-ei*cclisappoiiitiiig, liou’evei’, l~ccwusetlie tixxttmciit, whicli iiivolvccl dissectioii of the embryos from tlicii. tleciclual coats iii salt solutioii, made subsequent cyto1ogica 1 fixat ion ui i sat i sf a ct o r ~ . ‘L’lie hest fixcd niatei.ia1 was o1)taiiied in llie followiiig v-ay : The utt.i*ns was quickly i.emo\-ed from the pregnant rat to a disli of T y ~ * o dsolution e and the muscular wall dissected away from tlie cleciclua. A single cut w a s tlicii made across the latter with a pair of sharl) sciss0i.s iii such a way that the ci~ibi-yonic vesicle TWS opeiiccl near its attached eiicl. The port ioii wliicli coiitaiiictl the embryo intact witliiii its amnion, w a s tli.oppetl immccliatcly into the hottlc of fixative. This cxposed the embryo freely to the chemicals mliile its delicate structures were protc~ctedby the cleciduu froiii mecliaiiical iiijuq7 clni.ing the embedding l)i*occss. Tlic cliai.actclristic moi.phology of tlie whole ern1)i.p at tlic time of first 1icai.t coiitractioiis h a s b w n deteriiiinctl from a study of s e v c i d hunclred living ra t embryos dui*iiigtlie tcntli day of gestation. 011 tlic I)asis of this study aiitl by means of c.ompai.isoiis witli fixed c u l t u i d embryos, the ilesiiwl stages of clevc~lopmeiitliavc lieen selected f i v m a number of carefully timed series. Tlic Cyltampy-Knll tecliiiique a s giveii in JlcCluiig ( ’3T) was used on twenty-seven embryos. Ten cnibrj-os fixed in Rcgaud’s niixtui.e, tliit*tecii in Hclly ’s arid forty-eight in Bouin’s wet^> vaiiously staiiied with Heideiihaiii’s iron liaematoxylin, ;izureosiii aiicl ,\lasson’s staiii. A few l)rcqiai*atioiissupt*uvitally staiiictl with Jaiius greeii \ z ~ c made. i~ They sliowed the size, sliapc ant1 distribution of the mitoclioiidi*ia I)nt otiiei*wiseatldetl nothing to tlic oIisei*vittions with liaiigiiig cli*op cnltuiw of ~ l 1 0 l eenihiyos nlt*eacl?tlesciibecl. RESULTS Tlici i-eadei. is refei*t*cdto tlie description of tlie rat cmbrj-o a t tlic time of first contractioiis in the cai.lier papei’ (Goss, ’ 3 8 ) foi. details of moi*phology aiitl deve1ol)mcnt. Tlic p i + moixliuni of tlic heal4 is coiitaiiictt in tlie mcsoclei*m 011 eithci. side of the foregut invagiiiatioii just hefore the first coiit i*actioiis arc visible. Each lateral heart is composed of two pii3s, ( a ) a core of endocardium aiicl ( h ) an iiifoltliiig or groove in tlie sl)laiicliiiic mesodci*m wliicli pai.tiaily surrouiids tlic c ~ i c l o ca idium, aiid is usunlly called tlic iiiyoepicaidial mantle. The ciiclocardium is formed I)p a cliimpiiig of the scattercd cells of tlic vascular layclr. It is composed of fusiform aiitl stellatc cells which are separated from tlic spluncliiiic mesot1ei.m by a i i a i ~ i - o wspace wliicli is occwioiially Iwidgetl 11y pt*ocesses of tlie stclllate cells. X lumcii is frequeiitlp pt*eseiit a t tlie point of first contractioii, but is more in tlic forni of irregular vesicles than ;t continuous tube. The iiicli\ricliial ciitlocaidial cells are not distinguisliable f rom tlic eiidotlielium clcvelopiiig in the aortae or tlie yolk sac vessels. Before contractioii, the myoepicardial niantlc is com1)osed of low columiiai* cells (fig. 1). No distirictioii can be made hctmceii myocardium aiid epicardium. Tlicre is a single row of nuclei aiitl tlie cell boundaries can be distinguislied iii most cases. Observations of later stages indicate that furictioiially this mantle is almost entirely myocardium. The cells linvc 110 22 C. 31. GOSS cytological chai~ictci.isticswliicli clistiiiguisli them from other enihi*yoiiic cells. They liavc relatively large vesicular iiuclei wi tli 1)i’ominciit iiucleoli. The c-jtoplasm al-years homogeneous in the living cells exccllt f o r rather sparse mitoclioiidria iii the shape of short rods and gi*anules. Occasional f a t globules may he seen, but there ai’c n o fibrillac 1101- cross m a r k i n ~ s . Tlic myocardial cells in osmic Zenker wliolc iiioiiiits closely i*cseniblc tlic living cells ; all s t i w t u i w arc moi*c refractive becauw of tlic fixation but no cross striatioils o r fibrillac, ai*e l)i*ouglit out. The (&ellsin scctioiied matei*ial va1.y somewhat according to the fixative, but they retain their i*eseiiiblanice to thc otliei. cells of tlic embiaj-o. After good cytological fisalives SLICII a s Cliampy ’s 01’ Hcllly ’s, t h e w is a homogeneous gi.omicl substwiice. Tlie mitoclioiicliia do not stain rcacIilj7 i n tlicse j-o~iiigcnibi.yos. C’liampy- Kull \\-as the o i i l ~mcltliod which houglit thclm out tlcl)eiitlably aiicl Iic~rotlicy 1ve1.c not stained a s licavily a s in oltler ciiil)iyos. Tliere a1.c’ no fi1)rillae or striwtioiis. Aftcia poor cytologicxl fixatives such a s Rouiii ’s, tlic cyto1)lasrn 1xeseiits a fiiic feltwork, but is 110 iiioi’e fibrillarj- tliaii iii all the otliei. cells of tlie eiiibryo. FI1;ST CONTIL%CTIONS O F T H E H E A R T 23 The myocardial ceIls seen in tlie liviiig embryo shortly after their first contractions, h a r e the same embryonic cliaracteristics, iiamcly, vesicular nuclei arid homogeneous cpto1)lasm with scattered mitochoiicli*ia. This is true of tlic osmic Zeiiker whole niouiits also. Neither striations nor fibiaillae a r c seen. I n the Champy fixed material, tlic myocardial cells are essentially the same as before coiitiwtioii. They are more columiiai-, larger and have more cytoplasm, but s1)ecific structures such a s fibriilae 01’ striations are still lacking (fig. 2 ) . Fig. 2 Swtioii througli primitire 1ater:rl heart : i p p ~ o x i ~ i i : i t 3~ ~hours l~ after the initiation of contraction. R a t ein1)ryo. (‘halupp-Kull. X 1500. Tlic coiitractiiig cells w e i ~identified in tlic liviiig embryo. A t this stage they occupy the fold of spla~icliiiiciiiesodc1i.m which iriimecliately sui*rouiids the ciidocai.clia1 tube of tlic lateral heal$. In tlie fixed matci*ial, tlmefore, the contractile cells were identified by their position in the fold adjacent to the endocardial tulle. Contraction is well estabiislied in the lateral liearts for 3 o r 4 1iolu.s before tlie single sacculwr ventricle develops iiito an active oi*gaii. Although the resemblance between tlie contracting cells arid embryonic cells in general illusti~atcsthe lack of cytological diffeiwitiation, tlic most sti.ikiiig example of similarity be- 24 C. &I. GOSS tlveeli active aiicl iiiactire cells is fouiid w i t h a single layer, tlic splaiicliiiic mesoderm. The cells of t h at entire layer resemble each other vei‘y closely in size, sliape, ari*aiigeriieiit, aiicl cytological detail. Tlie layer can be divided into three portions, however, oii tlic basis of tlieir activity. F i rs t , tlie myocardiuiii of tlic lateral 1icai.ts is coiitractile. Seco11cl, tlie iiiidtllc caidiac platc, remains iiiactivc foi* 3 o r 4 1ioiii.s before it is iiicoi*l)oratctlin the siiigle sacenlar veiitriclc. Tliir(l, that portioii of tlic splaiicluiic layer which exteiids medially anel late~-allyfrom t h e iiifoldiiig \vliich marks tlie lateral liearts joins t l i ( 1 somatic mesoclcim in the forniatioii of tlie primitim 1)arietal 1)ei.icardiuin. Iii othei. \voids, cells in the same embi*yoiiic layer arc iiitlistiiiguisli~blefroiii cwcli other cj-tologically, but some ai’e contractile, some liwve iiot yet begun t o contract aiid others will iicver contract. -1 differciice 1)ctweeii myocaidial cells aiicl other cells of the embryo becoiiics noticeable after the single sacculnr vcnti.icle lias foimecl. Jlitoclioiidi.ia t i r e lai.ger, m o m iiumei’ous and staiii mow clecply. h i certain areas tlic cell sui+’aces a r e so (121 1.1wii cd that t 1i e p 1111(lonlr t cv 11y ma i . 1 ~ t 11c f o wi7i I i 11c’ 1’s of fibi*ill:icl. Sonicwliat Intci. the fibrillae can I)e tlistinguisliccl n-it11 cei-taiiity, a t first at the bortlcix of the cells aiicl theii Ti-i t h iii t 1iem. IT omog:.cixou s fibr illa e a 1111e a r e a 1.1i c r i ii o smi Zeiiltei. prcparatioiis tliwii iii a n y other kind of matei*ial,but they (lo iiot sliow uiitil 2 01’ 3 Iiours a f t e r the single saccular ciiwlatioii bcgiiis, veiiti.icle lias forniecl. At tlie time ~~71ieii the staiiiecl fibrillae make tlie myocartliuiii stand ont fiwm all other pai.ts of tlic cmhi.yo, crcii iiiider low iiingiiificatioii. The y m i g e s t embryo iii \vliic~lici’oss sti.iatioiis lia\-e 1)ecii s e ~ i iii i osmic Zciiker \vliole iiionnts w a s fised aftel. tlie cstal)lislimeiii of ciiwlatioii. Approsimately It; 1ioui.s elapse hch7eeii tlic first coiitractions a i d tlic lqiiiiiiiig of cii.culatioii. t b DTH(’I’SSJ 0s Tliesc obsei-vatioiis co1ifii.m the findiiigs of (’o11eiiliavcr ( ’39) with Ambl~-stoniatliat cross striations a w iiot seeii a t tlie time of first coiitrwctioiis. After calling atteiitioii t o Patten FIRST CONTRACTIOhTS O F THE HEAHT 25 and Kramer ’s ( ’ 3 3 ) statement that initiation of contraction in the chick heart occurs at the same stage as that in which Lewis (’19) found tlie first evidence of cross striations, Copenhaver gives his results with Lewis’ fixative as well as another osmic Zenker solution of his own. Concerning the latter he writes: “Serial sections of material fixed in this manner showed well-stained cardiac striations in older embryos but, like all otlicr methods used, gave no indication of striations at the time of beat initiatioii.” Our observatioiis also confirm those of Lewis (’19) that the osmic Zenker whole mounts bring out the striatioils a t an earlier age than any other teclinique. After circulation was established, tlie striations showed in a manner exactly like that described by Lewis. The lack of agreement between results with the chick and with Amblystoma or tlie r a t is more apparent than real. Lewis does not mention the initiation of contraction. Instead she makes tlie statemciit, “ K O preparation of a living- heart was obtained in which contraction did not occur.” The youngest embryos slie stndicd were ‘about 10 mpotomes.’ Patten and Iiramer, on the other hand, described the initiation of contraction in 9-somite embryos. The stages of development based on somites were only approximately the same in the two investigations. Observations with the rat have shown that tlie number of somites does not correspond closely to the development of tlie heart, and that there is a relatively great unfolding of the lieart during the formation of two or three somites at this early stage. The worli of Copenliaver, tlie present observations with fixed material, aiid the earlier studies with living embryos ( GOSS,’38) all point to the development of contractility before specialized cytological structure. If tlie contractions were found only in muscle with cross striations, it would be strong evidence tliat striations are a necessary part of the mechanism. Since contraction occurs before striations develop, the latter must not lie necessary for simple muscular motion. The development of such a physiological power, before differentiation, brings forward an important concept in physio- 26 C. N. GOSS logical embryology. It is particularly sigiiificant ill tlic c a w of niusclc because there is eviclctice that full differentiation clocs iiot take place unless there is functioiial adaptation of this power. In tissue cultures of cardiac muscle, cross striations form in portions of muscle wliich exert a pull against meclianiwillereas groups of cells encapsulated cal tension (Cross, '3), Iry coiiriective tissue coiitiiiuc t o coiltract for tiioiitlis but show 110 signs of diiTci+ciitiatioii. A distiiict ion may be matlc, tlicrefoi-e, iii tlic cliscnssioii of iriusclc, between sini1)le contractile 1)ower and iiiuwiilar fuiictioii. The latter involves a liaimssiiig of the coiitractioii s o tliat it perforiiis acts wliich are of sigiiificaiice to tllc oi-gaiiisiii. It seeiris to be this performance of w o ~ l rwhicli is associated with clif'ferentiation rather than tlie mere motion of the cell. Tlie contraction of undifferentiated muscle cells iii tissue cultures has been described by many investigators. Tlie valiclity of tlic observation has been questioned because the coiidtioiis in culture do riot exactly reproduce those iii tlic intact animal and because cells in culture have a teiideiicy to go through a morphological change wliich has been called dediffewentiation. Smooth muscle provides us with an maniple of contraction without benefit of cross striations. Its importance has been ignored or brushed aside with the cominent that the type of contraction is different from that of striated muscle. Tlie type of contraction, disregarding strength, is the same i r i the early embryonic and adult hearts. In both, a rapid contraction is followed immediately by relaxation in regular 1.1iytliin. The primitive lieart tube is occasionally i+eclnccclto liwlf its clianictcr by the coiiti.action. The structurcbs of tlich embryo are cxtrcmcly delicate in texture, liowcvei*,aiid p w b iiljly 1 1 0 great amomit of tension is pi*oduced. After the circulatioii of blood begins, glcttei. f o i m is called into play m d it is tlieii that difTcrentiatiori makes its appearatice. SUMMARY 'l'lie cells of primorclia of tlie heart bcfom the first coiitractioiis and tlic primitive heart lubes a few hours after the FIRST CONTIiACTIOSS 01.’ THE: HEAItT 27 initiation of contraction were studied in r a t embryos. The observations with fixed material i n wliole mounts and ill stained sections confirm those with living embryos that the specialized cytological structures, fibrillae and cross striations, a r e not elaborated until some hours after contractile activity is well established. Cytological differentiation is presented a s less associated with contraction than with a harnessing of the contractile force in order to exert meclianical pull outside the cell. LITERATUIIE CITED COPENITAVER, MT. Ill. 1939 Initiation of beat and intrinsic eontraction ratcs in different parts of the Amblystoma heart. J. Esp. Zool., vol. 80, pp. 193-224. Goss, C. B1. 1933 Further observations on the differentiation of cardiac muscle in tissue cultures. Arch. esp. Zellforsch., vol. 14, pp. 175-201. 1938 The first contractions of the heart in rat embryos. Anat. Rec., vol. 70, pp. 505-524. LEKIS, RI. R. 1919 The development of cross-striations i n the heart muscle of the chick embryo. Johns Hopkins Hosp. Bull., vol. 30, pp. 176-181. NCCLUNG,C. E. 1937 Handbook of Rlieroscopical Technique. 2nd ed. Hoeber, Xew York. P a T T E X , B. M., AND T. c. KRAXER1933 The initiation of contraction i n the embryonic chiek heart. h i . J. Anat., vol. 53, pp. 349-375.