Scanning Microscopy

Volume 2 Number 2 Article 33

2-10-1988

Structure and Function of in : Types I and III in Endomysial Struts and Pericellular Fibers

Thomas F. Robinson Albert Einstein College of Medicine

Leona Cohen-Gould Albert Einstein College of Medicine

Stephen M. Factor Albert Einstein College of Medicine

Mahboubeh Eghbali Albert Einstein College of Medicine

Olga O. Blumenfeld Albert Einstein College of Medicine

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Recommended Citation Robinson, Thomas F.; Cohen-Gould, Leona; Factor, Stephen M.; Eghbali, Mahboubeh; and Blumenfeld, Olga O. (1988) "Structure and Function of Connective Tissue in Cardiac Muscle: Collagen Types I and III in Endomysial Struts and Pericellular Fibers," Scanning Microscopy: Vol. 2 : No. 2 , Article 33. Available at: https://digitalcommons.usu.edu/microscopy/vol2/iss2/33

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STRUCTURE AND FUNCTION OF CONNECTIVE TISSUE IN CARDIAC MUSCLE: COLLAGEN TYPES I and III IN ENDOMYSIAL STRUTS AND PERICELLULAR FIBERS

Thomas F. Robinsonl,2 *, Leona Cohen-Gouldl , Stephen M. Factorl,3, Mahboubeh Eghbali4, Olga 0. Blumenfeld4

Departments of 1) Medicine, Cardiovascular Research Laboratories; 2) Physiology & Biophysics; 3) Pathology; and 4) Biochemistry Albert Einstein College of Medicine, Bronx, NY 10461

(Received for publication April 28, 1987, and in revised form February 10, 1988)

Abstract Introduction

Heart myocytes and capillaries are enmeshed in The large population of variably oriented myo­ a complex array of co nnective tissu e structures ar­ cytes of cardiac muscle are enmeshed in an elaborate ranged in several levels of organization: , and extensive array of connective tissue. Consid­ the sheath of connective tissue that surrounds mus­ erable research at the level of the light microscope cles; , which is associated with groups of was performed on myocardia l connective tissue in the cells; and , which surrounds and intercon­ early part of this century (Holmgren, 1907; nects individual cells. The present paper is a review Benninghoff, 1930), but was followed by several dec­ of work in this field with an emphasis on new, un­ ades of more modest exp loration in this field. How­ published findings, including composition of endo­ ever, within the last decade, interest in the structure mysial fibers and disposition of newly described peri­ and function of myocardial connective tissue has mysial fibers. The role of scanning electron micros­ blossomed, due largely to results achieved with the copy in the development of current understanding is scanning electron microscope and to a consequent also outlined. Biaxially arranged epimysial fibers awareness of the many possible functions of connec­ form a sheath around papillary muscles and trabe­ tive tissue in cardiac physiology and pathophysiology. culae that becomes increasingly well-oriented with (The role of the extracellu l ar matrix in development the muscle axis during stretch. Perimysial structures is a related area of active exploration, but is not are associated with groups of cells, and include covered here; the reader is referred to the mono­ weaves and septa of collagen, tendon-like fibers bet­ graph edited by Zak (1984) for extensive, recent ween weaves, ribbon-like fibers perpendicular to reviews, particularly in avian heart.) myocytes, and the newly described coiled perimysial The myocardial connective tissue, like that in fibers, which form an array in parallel with the , is generally considered to be organ­ myocytes and the epimysial net. The endomysium in­ ized on three levels. Epimysium is the sheath that cludes struts that bridge cells and pericellular fibers; surrounds the entire muscle, endomysium is the fine both contain collagen types I and III. The evidence connective tissue that surrounds and interconnects for the latter is presented in this paper and depends individual cells, and perimysium is associated with upon the use of antibody localization with fluores­ groups or bundles of cells. cent markers in light microscopy and colloidal gold The present paper contains a brief review of for scanning electron microscopy. The implications published studies that are related to the structure of the composition of collagen fibers for myocardial and function of connective tissue at all three levels function are discussed in relation to intra-cellular of organization in mammalian heart; however, the and other extra-cellular structures. emphasis is on new results related to endomysial s truts and pericellular fibers. Results from several laboratories are reported, including both unpublished results and results in press from the authors' labora­ Key Words: Cardiac muscle, connective tissue , epi­ tories. The extended discussion is aimed at putting mysi um , perimysium, endomysium, collagen, struts, the lat est findings in perspective. elastin, microfibrils, microthreads, antibody locali­ zation, scanning electron microscopy, transmission Abbreviations elec tron microscopy, light microscopy BEi backscattered electron imaging in SEM; CML collagen fibril-microthread-granule lattice; CPF co iled perimysial fibers; DIC = differential interference contrast microscopy; * Address for correspondence: FITC = fluorescein isothiocyanate; Thomas F. Robinson, LM light microscopy; Cardiovascular Research Labs., Forch. G42 PBS phosphate-buffered saline; Albert Einstein College of Medicine SEI secondary electron imaging in SEM; 1300 Morris Park Avenue SEM scanning electron microscopy; Bronx, New York 10461 TEM transmission electron microscopy. Phone No. (212) 430-2609

1005 T.F. Robinson, L. Cohen-Gould, S.M. Factor, et al.

Materials and Methods bodies to collagen type I and to collagen type III bind to different protein bands. Tissue preparation Antibody staining and localization Male W1star rats were lightly anesthetized with Samples were labelled with the indirect method. ether and then sacrificed painlessly by cervical dislo ­ Sections were equilibrated in buffer that contained cation, and the hearts quickly removed and placed in Bovine Lacto Transfer Technique Optimizer (BLOTTO) Tyrode's solution (in mM: Na+ 151.3, ca2+ 2.4 , K+ in order to minimize non-specific binding of the an­ 4.0, Mg2+ 0.5, c1- 147.3, HzPO4- 12. 0, and dex trose tibody (Johnson et al. 1984), rinsed in PBS, and then 5.5). incubated with 50 µl of the primary antibody (rabbit Ultrastructure antibody to rat collagen type I or III) at room tem­ Hearts destined for ul trastructural analvsis were perature for 30 min. in a humid chamber. cut open and fixed by immersion in buffered 6% glu­ Samples were rinsed with PBS, drained, and taraldehyde for 3 h at room temperature or overnight then labelled with the second layer in a humid cham­ in the cold. Buffer rinse was followed by dissection ber for 30 min. at room temperature. Second layer of papillary muscles and strips of ventricle wall, then for the samples destined for LM was goat-anti - rabbit post-fixation in 1 % OsO4 for 1 h, followed by buffer IgG labelled with FITC (Miles-Yeda L & D, Rehovot, rinse, and dehydration in a graded ethanol series. Israel). Samples were rinsed with PBS, drained, Samples for TEM were embedded in ERL resin mounted with a solution of 1 part 0. 3 M triethylene­ (Spurr, 1969), stained with uranium and lead salts diamine in 9 parts glycerol, and covered with glass and viewed in a JEOL 100 CX or JEOL 1200 EX. cover slips. Photomicrography was performed with a Samples for SEM were critical point dried in a Zeiss WL microscope fitted with epi- ultraviolet illu­ Samdri 790 (Tousimis Co., Rockville , MD) . Some mination and filters for FITC and rhodamine. samples were viewed after coating with Pd-Au, Kodachrome 400 or Kodak Tri-X film was used. whereas others were immersed in liquid nitrogen , Samples destined for SE M were incubated in a cracked with a pr e-co oled razor blade (Caulfield and humid chamber for 60 min. with a second layer con­ Borg, 1979) , dried by evaporation under vacuum, sisting of goat- anti-rabbit IgG labelled with col­ coated, and viewed in a JSM 25S. loidal gold particles 40 nm. in diameter (Janssen Silver staining Pharmaceutic a, Piscataway, NJ) . Samples were rinsed Hearts were prepared for silver staining by fix­ in PBS, post-fixed in 6% glutaraldehyde for 1/2 hat ation in 3. 7 % phosphat e- buffered formaldehyd e for a t room temperatur e, rinsed, post-fixed in 1% OsO4 in least 2 weeks. Frozen sections, approximately 80 µm buffer for 10 min, rinsed, dehydrated in a graded thick, were cut, floated on distilled water, placed ethanol series, and cr iti ca l point dried in CO2 in a successively in methyl alcohol, Folch' s solution, abso­ Samdri 790 (Tousimis Corp., Rockville, MD). Samples lute methyl alcohol, distilled water, 5 % potassium hy­ were photographed in a JSM 25S in our Analytical droxide, a rinse in running tap water, 1/600 potassi­ Ultrastructure Center. Paired BEI/SEI was perform ­ um permanganate, tap wat er, 5% oxalic acid for ed. Kodak 4127 film was used. bleaching, and tap water. Sections were then im­ Samples were studied in the LM by means of pregnated until they turned yellow-gray in 50% Del matched photographs of the same regions in fluores­ Rio Hortega' s lithium silver solution, washed in am­ cence and DIC or phase contrast. The expos ure moniated distilled water , reduced with 0.25 to 0.5 % times for fluorescence photographs for samples and neutral formalin, washed in distilled water, and toned controls were equal. Typical exposure times were in 1/ 500 gold chloride. Sections were then placed in 45-60 sec and controls included samples without first 5 % sodium thiosulfate, washed in distilled water, and layer (primary antibody), without second layer (FITC mounted (Robinson et al. 1983). LM was performed or colloidal gold), PBS only, and PBS and BLOTTO with a Zeiss Ultraphot with Kodachrome 25 film. only. Tissue for antibody staining The hearts for antibody staining were placed in Results and Discussion Tyrode's buffer that contained 0.1% glutaraldehyde and 4% paraformaldehyde; the apices of the hearts Epimysium were cut off and buffered fixative was circu l ated in­ Ep1mysium is the sheath of connective tissue side the chambers periodically for 3 h at room tem­ that surrounds entire muscles, for example, papillary perature. The hearts then were placed in Tyrode's muscles or trabeculae. Ultrastructural studies have buffer overnight in the refrigerator. The hearts revealed the fine structure of the endothelial cell were cut in cross section and half of the heart glued monolayer that covers collagen and elas tin fibers to the chuck of a Vibratome 1000 (Lancer, St. Louis, (Figure 1); together they compr ise most of the endo­ MO). Sections were cut at 30 or 60 µm thi ckness cardium (Lannigan and Zaki, 1966; Melax and Leeson, and picked up on gelatin-coated glass slides for LM 1967). The epimysial collagen and elastic fibers are or glass cover slips for SE M. interconnected bv and enmeshed in a CML that is Preparation of antibodies structurally and histologically similar to that of the The primary antisera were raised in rabbits to myocardial endomysium (Robinson and Winegrad, 1981; highly purified co llagen, type I from rat tail tendon Robinson et al. 1983). The overall distribution of and type III from rat skin. An IgG -e nriched fraction elastic fibers in the heart was studied by LM of his­ was precipitated with ammonium sulfate. The specif­ tological preparations by Puff and Langer ( 1965). icities of IgGs for collagen types I and III were tes­ Studies with TEM have revealed that the size of ted by ELISA (for detailed description see Eghbali et elastic fibers relative to collagen fibers is greater in al. 1987). Our antibodies to collagen types I and III the atria than in the ventricles of mammalian hearts were also tested for possible cross-reactivity with (Robinson et al. 1983) and greater in the left atria cardiac proteins. The results of immunoblot analysis than in the right atria (Klein and Bock, 1983). Elas­ on ventricular tissue extracts revealed that anti- tic fibers include the rubber-like elastin and

1006 Connective Tissue in Cardiac Muscle associated microfibrils, 10- 12 nm in diameter (Battig the tendon-like collagen "strands" between weaves and Low, 1961; Hanak and Bock, 1971), as well as (Weber et al. 1987). Ribbon-like perimysial fibers microfibrillar bundles that are not associated with also span groups of myocytes and are perpendicular elastin, even in aged hearts (Klein and Bock, 1983). to the myocyte axes (Figure 2b).

Figure 1. TEM of rat atrial trabecula cross section. Endothelium (e), EPIMYSIAL collagen (C), and elastic fibers (E) surround muscle, which includes myocytes ( M) and PERI MYSIAL collagen fibers (P). Bar = 5 µm.

In papillary muscles the orientation of the epi­ mysial fibers changes as the muscles are stretched. A "cargo net" model has been applied to the system (Robinson, 1983; Robinson et al. 1983). Light micros ­ c opy of silver-stained sections and scanning EM of the surfaces of muscles fixed at slack length reveal­ ed that the orientations of epimysial fibers vary greatly with respect to the long axis of the muscle. As the muscle is stretched, the alignment of the fi­ bers increases with respect to the long axis of the Figure 2a. SEM of rat ventricle. Coiled PERIMYSIAL muscle. At the muscle length at which passive resis­ fiber (CPF) longitudinally associated with myocyte tance to stretch increases dramatically, fibers are ( M). Struts ( S) associated with myocyte and blood more highly oriented along the muscle axis. At this vessel (B). Bar = 10 µm. point, length is about 2.3 µm, which re­ Figure 2b. LM of silver-stained section. Myocytes are presents the upper limit of optimal cross­ seen rn association with PERICE LL ULAR fibers (p), bridge overlap with -containing filaments. On ENDOMYSIAL struts (S), and PERIMYSIAL weaves the basis of this information and the known tensile ( W). Ribbon -like perimysial fibers ( P), distinct from properties of collagen fibers, the authors concluded CPFs (double arrows), often follow cell contours (ar­ that the cargo net weave of epimysial collagen fibers row). Bar = 200 µm. are precisely arrayed to help protect the muscle from overstretch. Similar models have been proposed for Collagenous septa that partially or completely of skeletal muscle (Bairati, 1938; enmesh groups of myocytes for long distances (up to Schmalbruch, 1974; 1985), for bovine neck ligament several mm) have been described and have been cor­ (Hoeve and Flory, 1958), and for related with modification of transverse impulse pro­ (Mullins and Guntheroth, 1965). pagation among myocytes of Bachman's bundle (atri­ Perimysium um) and ventricular papillary muscles of dog and Groups of myocytes are surrounded by a weave rabbit (Dolber and Spach, 1987). The equivalence of of collagen (Figure 2; Caulfield and Borg, 1979). The collagenous septa and weaves was suggested by those weaves t hemselves are in turn interconnec t ed by authors but awaits further study. "tendon-like" collagen fibers (Caulfie l d and Borg, Ribbon-like collagenous fibers closely follow the 1979; Borg et al. 1981; Borg and Caulfield, 1981) that contours of adjacent myocytes and lie perpendicu l ar could serve to limit the displacement of groups of to the long axes of myocytes (Figure 2). myocytes during the cardiac cycle. During certain Coiled perimysial fibers (CPFs) are orien t ed types of hypertrophy, there is a thickening of the parallel or oblique to the long axes of myocytes and collagen fibers of the weave (Caulfield, 1983) and of course throughout the myocardi u m (Figure 2). The

1007 T .F. Robinson, L. Cohen-Gould, S. M. Factor, et al.

CPFs are several µmin diameter, occur (in rat papil­ cle by Mauro and Adams (1961). Many of the sur­ lary muscle) in a number ratio of coils to myocytes face fibers are associated with the splayed processes of approximately 1:3, and decrease in convolution of struts, and some of the fibers are circumferential with increase in length of the muscle. Small elastic "cuffs" that have been imaged with LM in silverim­ fibers are associated with CPFs. The structure of pregnated sections (Robinson et al. 1983). Pericellu­ the CPF array has been determined only through lar fibers often lie along the circumferential indenta­ combined use of LM with silver-stained sections for tions of the corrugated surfaces of myocytes (Figure overall patterns, SEM for determination of their 2a; Robinson et al. 1987) that lie, in turn, over the coiled nature and apposition to myocytes, and TEM Z band-planes of the contractile lattices. for elucidation of their substructure. The size, Studies with SEM also have revealed fine, lon­ distribution, and changes in configuration of the CPF gitudinally oriented cables that span portions of the array suggest that it has tensile properties important surfaces of cardiomyocytes between M band planes to myocardial physiology (CPF manuscript in prepara­ (Orenstein et al. 1980). Their composition, . their re­ tion, Robinson et al. 1987; Factor et al. 1987). The lationship with pericellular collagen, and their pos­ CPF array apparently acts in parallel with the myo­ sible function in resting tension require further in­ cytes and the epimysial cargo net in papillary mus­ vestigation. cles and trabeculae. Whether the CPF array in the Collagen-Microthread Lattice (CML) The CML ventricular wall acts, in an analogous manner, in actually exists throughout the intershtium of cardiac parallel with the perimysial weaves associated with muscle, but is described here with the endomysium, groups of myocytes under conditions of stretch, is an where it has been most extensively studied. The ex­ intriguing possibility. tracellular space is filled with individual collagen Endomysium fibrils, some of which look "spiney", that is, 30 nm - - -Struts The first known images of fine fibers granules adhere to their sides (Renteria et al. 1976), between myocytes were published in 1907 by Holm­ and a ground substance , seen in preparations fixed gren, who obtained them by the use of light micros­ with solutions that contained cationic dye ( Sommer copy of sections stained with silver. Although Holm­ and Johnson, 1979), arranged in an extensive collagen gren incorrectly concluded that the fibers were tra­ fibril- microthread-granule lattice, the "CML" (Wine­ cheal tubes, his drawings clearly indicate structures grad and Robinson, 1978; Robinson, 1980; Robinson that bridge cells. The true nature of these connec ­ and Winegrad, 1981; Robinson, 1983; Robinson et al. tions was very effectively demonstrated with SEM by 1983, 1985, 1987; Frank and Beydler, 1985; Yung and Caulfield and Borg (1979; Borg and Caulfield, 1979). Frank, 1986). The images they obtained permitted an assessment of Microthreads are 3-6 nm in diameter and inter­ the collagenous nature and disposition of struts re ­ connect collagen fibrils, interconnect cell surfaces, lative to the surfaces of myocytes and capillaries. and connect collagen fibrils to cell surfaces (Figure In addition, their SE M put images from transmission 3; Robinson and Winegrad, 1981). The microthreads EM and light microscopy into perspective, had tre ­ are probably the same fine filamentous connections mendous impact on research in the field, and mad e seen in TE M and referred to in passing by Bahr and possible a general appreciation of the extent to Jennings (1961). Robinson et al. (1983) have obser­ which myocytes and connective tissue ar e c oupl ed at ved microthreads in tissues fixed with solutions that several levels of organization. They used the word included tannic acid, alcian blue, ruthenium red, saf­ "strut" to describe the collagenous fibers that bridg e ranin-0, or cetyl pyridinium chloride. Although de­ the lateral surfaces of myocytes to each other and tails of their appearance differ in the various pre­ to capillaries. In SEM images of rodent myocardium parations, their general size and distribution do not, the struts were 120- 150 nm in diameter. and the "bottle brush" appearance is consistent with The component fibrils of struts fork and attach the idea that some of them are proteoglycan. The to the myocyte surface (Caulfield and Borg, 1979). only known antibody localization studies of micro­ The forked pattern of attachments of struts to myo ­ threads have been performed at the TEM level by cytes was actually reported earlier for skeletal mus­ Ahumada and Saffitz (1984) , who found a positive re­ cle by Khoroshkov (1976), who also used SEM. Un ­ action with antibody to fibronectin. The sparse dis­ fortunately, that paper never received much attention tribution of microthreads in the anti-fibronectin ex­ and had little impact on the field. No subsequent periments could have been due to the incomplete fix­ publications from that laboratory are known, and our ation necessary to retain antigenic reactivity, or attempts to correspond with the author have been could indicate the presence of several populations of unsuccessful. different microthreads. Robinson et al. (1987) have expanded the defini­ The most complete CML photomicrographs have tion of "strut" to include intercellular collagenous fi ­ been generated from muscles that have not been sub­ bers that are 0.1 to several µmin diameter, as visu­ jected to chemical fixation at all. Frank and Beydler alized in both SE M and in light microscopy; the lat ­ (1985) and Yung and Frank (1986) have visualized the ter serves as a complement and overview to the CML clearly in preparations that have been ultra­ higher resolution SEM images. The term "strut" has rapidly frozen in liquid helium and prepared with persisted in the literature, although in engineering deep etch and rotary shadowing techniques. Anti­ usage (McGraw-Hill Dictionary of Scientific and body localization studies, particularly if performed on Technical Terms, 1984), there is an implied role in specimens with this type of preservation, would pro­ compression, which is unlikely along the long axes of vide more of the information needed to elucidate the collagen fibers. We persist with the term "strut" in structure and function of the CML. Aside from a deference to its very widespread usage in this field. possible role in tethering, the CML could serve other Pericellular fibers. The external laminae of my­ functions. The fact that the CML is polyanionic in ocytes are closely associated with overlying collagen nature, and the additional circumstance that the car­ fibrils, as first described with TEM in skeletal mus- diac interstitium contains glycosaminoglycans

1008 Connective Tissue in Cardiac Muscle

(Manasek, 1976), suggest that the complex could function in hydration of the interstitium and in ex­ tracellular cation binding. Elastin and microfibrils Microfibrils and small bodies of elastin are seen with TEM in the endocar­ dium (Robinson and Winegrad, 1981; Robinson et al. 1983; 1985), but have not been extensively studied.

Figure 3. (right) ENDOMYSIAL collagen fibril- micro­ thread-granule lattice in rat cardiac muscle fixed in solution that contained ruthenium red. High voltage TEM. Collagen fibril (C), myocyte (M), microthread (m), granule (g). Bar = 0.25 µm.

Figure 4. (below) LM of rat ventricle in cross section. ENDOMYSIUM labelled with first layer of antibody to collagen and FITC second layer. (a) Fluorescence photomicrograph of myocytes surrounded by type I collagen. (b) D.I.C. of same region. (c) Fluorescence photomicrograph of myocytes surrounded by type III collagen. (d) Phase contrast micrograph of same region. Bar =50 µm.

1009 T .F. Robinson, L. Cohen-Gould, S. M. Factor, et al.

Figure 5. LM of rat ventricular ENDOMYSIUM labelled with first layer of antibody to collagen and FITC second layer. Intercellular struts (arrows) and pericellular fibers (p) contain collagen types I and III. (a) Fluorescence micrograph for type I. (b) D.I.C. micrograph of same area. (c) Fluorescence micrograph for type III. (d) Phase contras t micrographof same region. Bar = 50 µ m. -

Extracellular membranous sheets "Striated magnifications and increased resolution at higher membranous sheets" (Renteria et al. 1976) have been magnifications, the colloidal gold particles and groups observed to form closed sacs in high voltage TEM of of particles, applied as a second-layer antibody, are samples preserved with ruthenium red (Robinson et visible both on readily identified struts and pericel­ al. 1983). Their nature has not been further de­ lular fibers. The staining of pericellular fibers with scribed, and their relationship to collagenous septa antibody to collagen type III is consistently stronger (Dolber and Spach, 1987) has not been explored. than that of struts in the same regions of the same Endomysial staining with an tibodi es to collagen samp les. It has not been determined whether this types I and III In the present series of experiments, difference in staining is due to different amounts of the endomysmm was labelled strongly with antibody collagen type III in the fibers or to masking of anti­ to collagen type I, as well as with antibody to col­ genic sites in the struts. SE I provides images with lagen type III (Robinson et al. 1987); these patterns good surface detail, including location of gold partic­ are seen especially well in cross sections in LM les . The BEi mode reveals considerably less surface ( Figure 4). Higher magnification LM of selected re­ detail, but shows with enhanced contrast the high gions reveals that individual struts and pericellular atomic number gold marker . In paired SEI and BEi fibers contain types I and III collagen (Figure 5). photographs (De Harven et al. 1984), the BEi images Higher resolution localization of antibodies to col­ thus serve to distinguish gold particles from extra­ lagen types I and III to inter- myocyte struts is seen cellular granules of similar size . in SE M studies (Figure 6). With overviews at low

1010 Connective Tissue in Cardiac Muscle

Figure 6. Intercellular struts and pericellular fibers labelled with antibody to co llag en type I or type III and 40 nm co lloidal gold. (a) Collagen type I. Gold particles or aggregates appear white (arrows) in BEi mode in SEM. (b) Collagen type!. SEI micrograph of same region shows less contras t for gold (arrows) and more sur­ face detail. (c) Collagen type III. BEi mode in SEM. (d) Collagen type III . SEI micrograph of same region. Bar = 5 µm . - -

Strut composition The compos ition of struts 198 7). has been defined in part over the last few years. The use of antibody to collagen type III in the Fibrils of collagen are embedded in a polyanionic present study has confirmed that it is also a constit­ matrix, as discerned from samples fixed for TEM in uent of struts in rat myocardium. The co-localiza­ solutions that contained cationic dyes, such as alcian tion of collagen types I and III in struts is not blue and ruthenium red (Robinson et al. 1983, 1987). surprising in view of a similar finding in skeletal Collagen fibrils are on the order of - 50 nm in di­ muscle. Light and Champion (1984) have developed a ameter (Robinson et al. 1983; Frank and Beydler, method for the bulk separation of epimysium, peri­ 1985). Information that struts are comprised of both mysium, and endomysium for large samples of bovine collagen types I and III comes from the use of ultra­ skeletal muscle, and have found that the relative structure and LM with silver stain (Robinson et al. content of type I collagen to the total of types I 1983), reports of L M fluorescence antibody studies and III is 84% in epimysium, 72% in perimysium, and (Borg et al. 1982a, 1985), and colloidal gold antibody 38% in endomysium. No reports of such quantifica­ l ocalization (Borg et al. 1986) . Type I collagen has tion for myocardium are known. Co-formation of been localized in struts by means of indirect anti­ collagen fibrils with collagen types I and III also has body technique using FITC paired with DIC for LM been reported for human skin, cornea, amnion, aorta, and colloidal gold with both SEI and BEi SEM in and tendon by use of monoclonal IgM with TEM hearts of both rat and hamster (Robinson et al. localization (Keene et al. 1987).

1011 T .F. Robinson, L. Cohen-Gould, S. M. Factor, et al.

Strut function Caulfield and Borg (1979) hy­ comparatively high resting stiffness of cardiac myo­ pothesized that the struts played roles as tethers cytes and their profusion of intermediate filaf'lents. (tensile structures) between cells; their hypothesis was supported by the known roles of collagen in Conclusion other tissues and by the changes in convolutions of struts in hearts fixed in systole and diastole. The Cardiomyocytes are interconnected and enmes­ idea was further tested by Robinson et al. (1987) in hed in an extensive array of connective tissue struc - experiments (analogous to those of Bairati, 1938, tu res. The co- localization of collagen types I and with skeletal muscle) in which papillary muscles from III in endomysial structures suggests the possibility rat heart were pinned along one side, subjected to that the ratio of collagen types might influence the extreme lateral stretch, fixed, and structurally anal­ size of the fibers, and hence their mechanical pro­ yzed by a combination of LM, SEM, and TEM. perties. The concept that these fibers have mecha­ LM of 80 µm-thick sections stained with silver nical tethering functions, as described in the present provided overviews that showed that the large con­ paper, is supported by the work of Waldman et al. nective tissue fibers were straightened between the (1985), who showed in dynamic studies of ventricle pins, but others, away from the axis of stretch, re­ wall that the principal axes of shortening vary less mained convoluted. SEM revealed that in regions of than the myofiber directions, and have interpreted lateral stretch, the struts appeared taut and had ac - this to be evidence of interactions among neighbor­ tually pulled up patches of sarcolemma. TEM images ing myofibers. showed sarcolemma pulled away from underlying mi­ In addition to tethering by connective tissue at tochondria and along the direction of several levels of organization in normal physiologic stretch. In addition; myofibrils exhibited cusps at situations, recent studies have revealed a correlation the Z bands, which are connected by intermediate between disruption of the connective tissue frame­ filaments to the inner aspect of the sarcolemma. work and myocardial dysfunction (Robinson et al. This series of experiments provided direct evi­ 1986; Factor et al. 1987; Cohen-Gould et al. 1987). dence that the contractile lattices of adjacent myo­ For example, in a time frame of minutes to hours, cytes are tethered to each other, at least at the lev­ diminution of blood flow to the heart leads to dis­ el of some Z bands, by a series of structures that is ruption of the connective tissue framework (Sato et thus far only partially described. Struts interconnect al. 1983; Caulfield et al. 1985; Zhao et al. 1987) with the cell surfaces and often are attached lateral to a concomitant alteration in mechanical properties of the infolding over the plane of underlying Z bands the ventricle, with or without myocardial necrosis. (Caulfield and Borg, 1979, Borg et al. 1982b, 1983; Caulfield et al. (1985) have observed complete disap­ Robinson et al. 1983; 1985; 1987), or they fork and pearance of the connective tissue framework after attach on both sides of the infolding over the plane two hours of total coronary artery occlusion. Factor of underlying Z bands (Robinson et al. 1987). Fibrils et al. (1987) have found that at sites of ventricular of the struts splay and form part of the surface wall rupture in material preserved from hearts with husk of the myocyte surface (Borg et al. 1981; myocardial infarction, there is a complete absence of Robinson and Winegrad, 1981; Robinson et al. 1983; connective tissue at the rupture sites. In reactive 1985; 1987), in some cases in the form of circumfer­ hypertrophy, there is an increase in the size of con­ ential cuffs (Robinson et al. 1983; 1987). nective tissue components (Medugorac, 1980; By use of fluoresce11t - antibody, the intermediate Caulfield, 1983; Weber et al. 1987) that could be an filament protein vinculin has been localized in adaptation to increased stress. The effects of either mammalian myocardium in parallel, circumferential increased connective tissue or altered connective tis­ bands called "" that are located on both sue with diminution of blood flow on ventricular sides of the Z band plane (Pardo et al. 1983). Stu­ mechanics as well as on transverse electrical propag dies with TEM have revealed that 10 nm diameter ation (Dolber and Spach, 1987) are areas under in intermediate filaments course from Z discs to the in­ creasing investigation in a number of laboratories. ner aspect of the sarcolemma (Ferrans and Roberts, Studies of connective tissue alterations associ­ 1973; Chiesi et al. 1981; Robinson et al. 1985, 1987) . ated with disease can thus extend our knowledge ofi Severs et al. (1985), by use of freeze fracture, have certain pathologies, and should provide a wealth of found a doublet of sarcolemmal ridges that straddles additional information regarding the complementary, the Z band plane. The ultrastructural disposition of and integrative functions served by connective tissue these structures near the Z band plane and their relative to myocytes in normal myocardial function. transarcolemmal connections remain to be elucidated. The foregoing facts do suggest, however, that Z Acknowledgments bands, and hence contractile lattices, in laterally adjacent myocytes are tethered to each other in a This study was supported in part by Research tension-bearing series of structures that includes Grants HL-24336 (TFR), a Grant-in-Aid from the struts, external laminae, sarcolemmas and intermed­ American Heart Association, New York City Affiliate iate filaments. and Heart Fund (TFR), AG-05554 (SS), and HL- 18824 Price (1984) has isolated and analyzed the (EHS). The JEM 1200 EX was obtained by the Ana­ intermediate filament proteins and vimentin lytical Ultrastructure Center of AECOM with equip­ from bovine ventricular myocardium. Desmin com­ ment grants from N. S. F. and N. I. H. Biotechnology Re­ prises 2% of total protein (fivefold more than in sources. The JEM 1000 is supported by N .I.H. Bio­ skeletal muscle) and is capable of assembly into technology Resources at the H.V.E.M. Laboratory, intermediate filaments. In future studies, ultra­ University of Colorado at Boulder. We thank Drs. structural localization and determination of a possible Sam Seifter and E. H. Sonnenblick for help with many role of desmin as a major load-bearing structure are phases of the research, Ms. R. Dominitz for expert therefore of great interest, especially in view of the silver staining, Ms. J. Fant and Mr. F. Macaluso of

1012 Connective Tissue in Cardiac Muscle the AECOM AUC for excellent ultrastructural muscle of cardiomyopathic Syrian hamsters. Am. J. resources, Mr. G. Wray and Mr. G. Charlie for help Pathol. 127, 327-334. with HVEM, Mrs. M. Abercrombie and Ms. L. DiDia De Harven E, Leung R, Christensen H (1984). A for translation of papers in Italian, and Mrs. K. novel approach for scanning electron microscopy of Cohen for translation of papers in German. colloidal gold-labelled cell surfaces. J. Cell Biol. 99, 53-57. - References Dolber PC, Spach MS (1987). Thin collagenous septa in cardiac muscle. Anat. Rec. 218, 45-55. Ahumada GG, Saffitz JE (1984). Fibronectin in Eghbali M, Robinson TF, Seifter S, Blumenfeld rat heart: a link between cardiac myocytes and coll - 00 (1987). Enzyme-antibody histochemistry: a method agen. J. Histochem. Cytochem. 32, 383-388. for detection of collectively. Histochem. Bahr GF, Jennings RB (1961). Ultrastructure of 87, 257-262. normal and asphyxic myocardium of the dog. Lab. - Factor SM, Robinson TF, Dominitz R, Cho SH Invest. 10, 548-571. (1987). Alterations of the myocardial skeletal frame­ Bairati A (1938). Struttura e proprieta fisiche work in acute myocardial infarction with and without del sarcolemma dell a fibra muscolare stria ta. Z. ventricular rupture: a preliminary report. Amer. J. Zellforsch. 27, 100-124. Cardiovasc. Pathol. 1, 91-97. Battig CG, Low FN (1961). The ultrastructure of Ferrans VJ, Roberts WC (1973). Intermyofibrillar human cardiac muscle and its associated tissue space. and nuclear-myo-fibrillar connections in human and Am. J. Anat. 108, 199-252. canine myocardium: an ultrastructural study. J. Mol. Benninghill A (1930). Das perimysium internum. Cell. Cardiol. 5, 247- 257. Handbuch der mikrosk. Anatomie von v. Mollendorf 6, Frank J, Beydler S (1985). Intercellular connec­ 192-196. - tions in rabbit heart as revealed by quick-frozen , Borg TK, Caulfield JB ( 1979). Collagen in the deep- etched, and rotary-replicated papillary muscle. heart. Texas Rep. Biol. Med . 39, 321-333. J. Ultrastruc. Res. 90, 183-193. Borg TK, Caulfield JB (198T). The collagen mat­ Hanak H, Bock P (1971). Die Feinstruktur der rix of the heart. Fed. Proc. Fed . Am. Soc. Exp. Biol. Muskel-Se hnenverbindung von Skelettund Herzmuskel. 40, 2037-2041. J. Ultrastrct. Res. 36, 68-85. - Borg TK, Ranson WF, Moslehy FA, Caulfield JB Hoeve CAJ, Flory PJ (1958). The elastic proper­ (1981). Structural basis of ventricular stiffness. Lab. ties of elastin. J. Amer. Chem. Soc. 80, 6523-6526. Invest. 44, 49- 54. Holmgren E (1907). Uber die Trophospongien der Borg TK, Gay RE, Johnson LD (1982a). Changes quergestreiften Muskelfasern, nebst Bemerkungen in the distribution of fibronectin and collagen during uber den allgemeinen Bau dieser Fasern. Arch. development of the neonatal rat heart. Collagen Rel. Mikrosk. Anat. 71, 165-247. Re s. 2, 211-218. Johnson DA-;- Gautsch JW, Sportsman JR, Elder Borg TK, Johnson LD, Gay R (1982b). Specific JH (1984). Improved technique utilizing nonfat dry attachment of collagen to cardiac myocytes in vivo milk for analysis of protein and nucleic acids trans­ and in vitro. In : Extracellular Matrix : Symp~ ferred to nitrocellulose. Gene Anal. Tech. 1, 3- 8. Hawkes S, Wang JL (e ds.). Academic Press, New Keen e DR, Sakai LY, Bae hinger H- P, Burgeson York, pp. 253-257. RE (1987). Type III collagen can be present on band­ Borg TK, Johnson LD, Lill PH (1983). Specific ed collagen fibrils regardless of fibril diameter. J. attachment of collag en to cardiac myocytes in vivo Cell Biol. 105, 2393 - 2402. and in vitro. Devel. Biol. 97, 417-423. --- Khoroshkov YA (1976). (Title in Russian) The Borg TK, Klevay LM, Gay RE, Siegel R, Bergin structure of the collagenous framework of skeletal ME (1985). Alteration of the connective tissue net­ muscle. Arkhiv. Anatomii Gistologii 71, 97-100. work of striated muscle in copper deficient rats. J. Klein W, Bock P (1983). Elastica=-Positive mate­ Mol. Cell. Cardiol. 17, 1173-1183. rial in the atrial endocardium: Light and electron Borg TK, Buggy - J , Sullivan T, Laks J, Teraccio microscopic identification. Acta Anat. 116, 106-113. L (1986). Morphological and biochemical characteris­ Lannigan RA, Zaki SA (1966). Ultrastructure of tics of the connective tissue network during normal the normal atrial myocardium. Br. Heart J. 28, 785- development and hypertrophy. J. Molec. Cell. Cardiol. 795. - 18 (Suppl. 1), 247 (Abstract) . Light ND, Champion AE (1984). Characterization - Caulfield JB, Borg TK (1979). The collagen net­ of muscle epimysium, perimysium, and endomysium work of the heart. Lab. Invest. 40, 364 - 372. collagens. Biochem. J. 219, 1017-1026. Caulfield JB (1983). Alterations in cardiac Manasek FJ. (1976)--:-Macromolecules of the ex­ collagen with hypertrophy. In : Perspectives in Cardi­ tracellular compartment of embryonic and mature ovascular Research, Vol. 8, Tarazi RC, Dunbar JB hearts. Circ. Res. 38, 331-337. (eds . ). Raven Press, New York, pp. 49-57. Mauro A, Adams WR (1961). The structure of Caulfield JB, Xuan JC, Ranson W (1985). Mor­ the sarcolemma of the frog skeletal muscle fiber. J. phology and ventricular strain rates with infarction. Biophys. Biochem. Cytol. 10, 177-186. Proc. 38th Annual Conf. Engineering in Med. & Biol., McGraw-Hill Dictionary of Scientific and Alliance for Engineering in Medicine and Biology, Technical Terms (1984). 3rd Ed., Parker SP (ed.). Washington DC, 198 (Abstract). New York, p.1571. Chiesi M, Inesi G, Somlyo AV, Somlyo AP Medugorac I (1980). Collagen content in differ­ (1981). Primary role of in ent areas of normal and hypertrophied rat myocard­ phasic contractile activation of cardiac myocytes ium. Cardiovasc. Res. 14, 551-554. with shunted myolemma. J. Cell Biol. 91, 728-742. Melax H, Leeson fs (1967). Fine structure of Cohen-Gould L, Robinson TF, Factor SM (1987). the endocardium in adult rats. Cardiovasc. Res 1, Intrinsic connective tissue abnormalities in the heart 349-355. -

1013 T .F. Robinson, L. Cohen-Gould, S. M. Factor, et al.

Mullins GL, Guntheroth WG (1965). A collagen ventricle. Circ . Res. 57, 152-163. net hypothesis for force transference of smooth mus­ Weber KT, Janicki JS, Pick R, Abrahams C, cle. Nature 20, 592-594. Schroff SG, Bashey RI , Chien RM (1987). Collagen Orenstem. J, Hogan D, Bloom S ( 1980). Surface in the hypertrophied, pressure-overloaded myocar­ cables of cardiac myocytes. J. Molec. Cell. Cardiol. dium. Circulation 75 (Suppl I) 1-40. 12, 771-780. Winegrad S, Robinson TF (1978) . Force genera­ - Pardo JV, Siliciano JD, Craig SW (1983). Vin­ tion among cells in the relaxing heart. Eur.J .Cardiol. culin is a component of an e:x:tensive network of 7(Suppl.), 63-70. -sarcolemma attachment regions in cardiac - Yung R, Frank JS (1986). Extracellular matrix­ muscle fibers. J. Cell Biol. 97, 1081-1088. sarcolemmal surface interconnections: a quick-freeze Price M. (1984). Molecular analysis of intermed­ deep- etch study. J. Ultrastruct. Molec. Res. 96, iate filament cytoskeleton: a putative load-bearing 160-171. - structure. Am. J. Physiol. 246, H566 - H572. Zak R. (1984) . Growth of the Heart in Health Puff A, Langer H (1965)--:-Ilas Problem der dia ­ and Disease. Raven Press, New York, pp. 1-185. stolischen Entfaltung der herzkammer (Eine Unter­ Zhao M, Zhang H, Robinson TF, Factor SM, suchung uber das elastische Gewebe im Myocard). Sonnenblick EH, Eng C (1987). Profound structural Gegenhaurs Morphol. J. 7, 184-212. alterations of the extracellular matrix in post­ Renteria VG, Ferrans VJ, Jones M (1976). Stri ­ ischemic dysfunctional "stunned" but viable myocar­ ated membranous structures in human hearts. Amer. dium. J. Amer . Coll. Cardiol. !_()_,1322-1334. J. Pathol. 85, 85-94. Robinson TF (1980). Lateral connections bet­ Discussion with Reviewers ween heart muscle cells as revealed by conventional and high voltage transmission electron microscopy. T. K. Borg: What is your evidence that the silver Cell Tissu e Res. 211, 353-359. stam actually stains only collagen and not proteo­ Robinson TF-;-Winegrad S (1981). A variety of glycans or glycoproteins that may mask the actual intercellular connections in heart muscle. different types of collagen fibrils? J. Mol.Cell.Cardiol. 13, 185-195. Authors : The large collagen fibers of the annular Robinson TF, Cohen-Gould L, Factor SM ( 1983). valve rmgs and the cortex of the papillary muscle­ The skeletal framework of mammalian heart muscle: tendon junction are presumably mostly collagen type Arrangement of inter-and pericellular connective tis­ I and do not stain with our silver impregnation tech­ sue structures. Lab.Invest. 49, 482-498. nique. Collagen -c ontaining fibers of the myocardial Robinson TF (1983). Thephysiological relation­ endomysium and perimysium do stain with our tech­ ship between connective tissue and contractile fila­ nique, as determined from examination of sections ments in heart muscle. Einstein Q. 1, 121-127. from re-embedded silver-stained sections with high Robinson TF, Cohen-Gould 1-;-Remily, RM, voltage transmission EM, as well as from comparative Capasso JM, Factor, SM (1985). Extracellular Struc ­ morphology of the relevant structures with other tures in Heart Muscle. In: Advances in Myocardiol ­ techniques, such as light microscopy with trichrome­ ogy, Vol. 5, Harris P, Poole-Wilson PA (eds.). stained sections, differential interference contrast of Plenum, New York, pp. 243-255. unfixed, unstained tissue, and antibody-labelled sec­ Robinson TF, Factor SM, Sonnenblick EH (1986). tions, as well as conventional and antibody-labelled The heart as a suction pump. Sci. Amer. 254, 84- 91. scanning EM, and transmission EM. Robinson TF, Factor SM, Capasso JM, Witten­ The silver, however, probably does not stain the berg BA, Blumenfeld 00 , Seifter S (1987). Morpholo­ collagen itself. In myocardium treated with pronase, gy, composition, and function of struts between car­ in which all protein except collagen was digested, we diac myocytes of rat and hamster. Cell Tissue Res. found that the treated collagenous fibers did not 249, 247-255. stain with silver, although their familiar morphology - Sato S, Ashraf M, Millard RW, Fujiwara H, was maintained, as seen in differential interference Schwartz A (1983). Connective tissue changes in ear­ contrast L M and in scanning EM (unpublished results, ly ischemia of porcine myocardium: an ultrastructural 1987). The stain might react with proteoglycans study. J. Molec. Cell. Cardiol. 15, 261-275. and/or glycoproteins that are normally bound to the Schmalbruch H (1974). The sarcolemma of ske l e­ constituen t collagen fibrils. tal muscle fibres as demonstrated by a replica tech­ nique. Cell Tiss. Res. 150, 377-387. T .K. Borg: What is the evidence that th e "collagen Schmalbruch H (1985}": Skeletal Muscle. Springer threads" are actually co llag en? Are they digestible Verlag, Berlin, pp. 16-20 . with purified collagenase? Severs NJ, Slade AM, Powell T, Twist VW, Authors: The term "collagen threads" is not used in Jones GE (1985). Morphometric analysis of the iso­ this manuscript. Fibers containing collagen fibrils lated calcium-tolerant cardiac myocyte : organelle are recognized by their morphology, labelling witli volumes, sarcomere length, plasma membrane surface ant ibody , resistance to pronase digestion, and sus­ folds, and intramembrane particle density and cep tibility to digestion with crude collagenase. distribution . Cell Tissue Res. 240, 159 - 168. Sommer J, Johnson T (197~ Ultrastru cture of T .K. Borg: What is the significance in changes in Cardiac Muscle. In: Handbook of Physiology: The the amount of type I and type III collagen in the Cardiovascular System I, 113-186. heart? Spurr AR (1969). A low-viscosity epoxy resin Authors: Our interpretation of the present data is embedding medium for electron microscopy. J. Ultra­ that collagen types I and III are co-localized in the struct. Res. 26, 31-43. endomysium. We do not have quantitative data on Waldman LK, Fung YC, Covell JW (1985). Trans­ the amounts of collagen types present, or the differ­ mural myocardial deformation in the canine left ent ratios of types I and III, as determined, for ex-

1014 Connective Tissue in Cardiac Muscle ample, in bovine skeletal muscle epimysium, perimy­ that the size and disposition of microthreads with sium, and endomysium [ Light N, Champion AE. ( 1984). respect to myocytes and collagen fibrils did not Characterization of muscle epimysium, perimysium, differ significantly from samples fixed in solutions and endomysium collagens. Biochem. J. 219, that contained cationic dye. 1017-1026.J -

J.B. Caulfield: There are data [Cardiac Mechanics, ed ited by Mirsky I, Ghista DN, Sandler H, John Wiley & Sons, 1979, pp. 344-345], that indicated the elastic modulus of papillary muscle is greater than that of the ventricle. Have you any anatomic data consist­ ent with this? Authors: On the basis of our thus-far qualitative data, we cannot yet address this interesting point.

J .S . Frank: Are the "pericellular fibers" or "circum­ ferential cuffs" extensions of the struts? Authors: Many of them are (e.g., text references: Caulfield and Borg, 1979; Robinson et al, 1987). Our experiments involving lateral stretch of papillary muscles, reported in the latter reference, indicate that struts are functionally continuous with the cy­ toskeleton, as well as the external laminae of th e myocytes that they interconnect.

M. Ashraf: There is an extensive network of colla­ gen and other connective tissue fibrils surrounding myocytes. They are not as prominent in the fixed tissue. I wonder if the fixation has any adverse ef­ fect on their structure. It would be extremely useful to examine the quickly frozen tissue with helium after preparing with freeze-fracture t echnique. Have the authors done work utilizating this preparation? Authors: We have found that co llagen fibers, colla­ gen fibrils, and elastic fibers are well - preserved in all of the fixation procedures used in the present study . Extensive, reliable preservation of micro­ threads [Robinson TF, Winegrad S. (197 8). Force generation among cells in the relaxing heart. Europ. J. Cardiol. 7 (Suppl.), 63-70], 3-6nmindiameter, require fixafion procedures other than just glutar­ alde hyd e and osmium tetroxide. We have used addi­ tional componen ts in the fixation solution, such as tannic acid or the cationic dyes alcian blue, ruthe­ nium red, or safranin-0 in conventional and stereo high voltage transmission electron microscopy. Good preservation ensued, and studies revealed a three­ dimensional lattice of microthreads that connected myocytes to other myocytes and to collagen fibrils, interconnected collagen fibrils, and filled the extra­ celluar space [Robinson TF, Winegrad S. (1981). A variety of intercellular connections in heart muscle. J. Molec. Cell. Cardiol. 13, 185-195; Robinson TF, Cohen-Gould L, Factor SM-:-(1983). Skeletal frame­ work of mammalian heart muscle : arrangement of in­ ter- and pericellular connective tissue structures. Lab. Invest. 49, 482- 498]. Perhaps the best views of this extracellular matrix are seen in rabbit cardiac papillary muscles that were ultrarapidly frozen at the temperature of liquid helium, deep etched, and rotary shadowed [ Frank J, Beydler S. (1985) . In tercellular connections in rabbit heart as revealed by quick-frozen, deep­ etched, and rotary-replicated papillary muscle. J. Ultrastruc. Res. 90, 183-193; Yung R, Frank JS (1986). Extracellu!ar matrix-sarcolemmal surface interconnections: a quick-freeze deep-etch study. J. Ultrastruct. Molec. Res. 96, 160-171]. Based on micrographs of such samples, the authors concluded

1015