Connective Tissue Cells in Healing Rat Myocardium a Study of Cell Reactions in Rhythmically Contracting Environment

Connective Tissue Cells in Healing Rat Myocardium A Study of Cell Reactions in Rhythmically Contracting Environment

Rudolf Vracko,* David Thorning,* and sible for the monophasic myocardial response have not Richard G. Fredericksont been clarified. From the Laboratory Service, VA Medical Center, the Four general explanations have been offered for the Departments ofPathology, * and Biological Structure, lack of restorative myocardial repair: 1) Cardiac myo- University ofWashington School ofMedicine, Seattle, cytes, like neurons, are terminally differentiated cells and Washington cannot divide.67 2) Myocardium, in contrast to skeletal muscle, lacks a sarcolemmal framework necessary for tissue reconstruction.4 3) Myocardium, in contrast to skele- To better understand the tendency of myocardium tal muscle, lacks myosatellite cells to serve as sources of to heal by scarring rather than regeneration, the new myocytes.7 4) Overgrowth of nonmuscle cells some- authors examined the responses of connective tis- how prevents cardiac myocyte proliferation.67 Recent sue cells (CTCs) after three types ofnecrotizing in- studies have negated the first two explanations by show- juries. Derived from myocardial interstitial cells, ing that adult mammalian left ventricular myocytes are ca- CTCs proliferated in both the connective tissue pable of DNA synthesis in vivo and in vitro and of cell space and the compartment of necrotic myocytes. division in vitro8"1' and that mammalian myocardium has They assumed various cell forms: fibrocytelike a "sarcolemmal" framework provided by a basal lamina CTCs throughout the sites ofinjury deposited extra- (BL) framework.12 The effects of myosatellite cell absence cellular scar tissue elements, established CTC- myo- is unclear at the present time, and no data are available cyte contacts, and helped anchor myocytes to scar to consider critically the effect of nonmyocyte cell over- tissue with myotendonlike specializations; CTCs growth on the process of myocardial reconstruction. with more complexforms established CTC-myocyte One of the factors unique to myocardium is its need relationships, suggesting important roles in com- to maintain forceful, rhythmic contractions throughout the munication and tissue remodeling. CTCs within healing period. It is obvious that nonlethal necrotizing scar tissue differentiated into myofibrocytes, chon- myocardial injuries interrupt myofiber continuity, that unin- drocytes, and possibly smooth muscle cells. Most jured myocytes must continue contracting, and that the scar tissue elements were disposed in the long axis forces of contraction will act on the site of injury. To our ofmyocytes. These alterations inform indicate that knowledge, the problems associated with wound healing CTCs have various roles in myocardial repair and in an environment of intermittent pulling and stretching suggest that a number of the roles are modulated have not been studied. It has been shown, however, that by contractile forces. (Am J Pathol 1989, 134: intermittent stretching can have a significant effect on the 993-1006) phenotypic expressions of nonmyocardial cells in vitro.13 15 In view of the unresolved issues, we have examined in Like other tissues, myocardium responds to necrotic inju- detail the basic reactions of adult rat left ventricular myo- ries by inflammatory exudation and connective tissue cell cardium to three different types of necrotizing injuries. (CTC) proliferation. Unlike most nonmyocardial tissues, which can heal either by restoration of structure or by scar tissue formation, myocardium seems to be limited to heal- Supported by VA Research Funds and the American Heart Association. ing by scar tissue formation. The tendency to scar and the Accepted for publication January 18, 1989. Address reprint requests to Rudolf Vracko, MD, Laboratory Service structural and some biochemical features of the process (113), VA Medical Center, 1660 South Columbian Way, Seattle, WA have been defined,1-5 however, the mechanisms respon- 98108.

993 994 Vracko, Thorning, and Frederickson AJP May 1989, Vol. 134, No. 5

Based on the need to evaluate stereologically complex closed in two layers. Rats were killed at 1 (N = 10), 4 (N interactions between the elements in necrotic, healing, = 10), 8 (N = 10), 18 (N = 10), and 42 (N = 10) days after and adjacent uninjured sites, we carefully selected tissue injury for light microscopy and at 2 (N = 2), 4 (N = 2), 8 samples oriented in the longitudinal axis of myofiber (N = 2), and 16 (N = 2) hours and 1 (N = 10), 4 (N = 10), chains and those perpendicular to that axis. In earlier re- 7 (N = 10), 14 (N = 10), 28 (N = 10), and 42 (N = 10) ports, we detailed the fate of myocardial BL framework12 days after injury for electron microscopy. and the reactions of myocytes left viable along the edge of injury.i6 In this report, we detail the reactions of connective tissue cells (CTCs). Isoproterenol Injury Our findings suggest that myocardial wound healing is a complex process that involves interactions between The method has been described.19 The animals were myocytes, CTCs, extracellular matrix elements, and phys- given a single intraperitoneal injection of isoproterenol, ical forces. CTCs appear to have multiple roles in the pro- 100 mg/kg body weight. One set was killed 2 (N = 3), 4 cess. They form and fortify scar tissue, help anchor myo- (N = 3), 8 (N = 3), and 16 (N = 3) hours after injury, fibers to scar tissue, and may play a major role in the spa- one animal used for light microscopy, and two for electron tial organization of reparative tissue elements. The microscopy from each interval. Another set was killed at formation of myotendinous junctions, unidirectional spa- 1 (N = 10), 4 (N = 10), 8 (N = 10), 16 (N = 10), 28 (N tial orientation of scar tissue elements, and fortification of = 10), and 42 (N = 10) days after injury, three animals scar tissue by smooth muscle cells and chondrocytes, were used for light and seven for electron microscopy strongly support the notion that myocardial contractions from each interval. are important determinants in the course of myocardial repair. Preparation of Tissue

Materials and Methods The excised hearts were perfused retrograde through the ascending aorta from a reservoir located 88 cm above Male and female Sprague-Dawley rats (250 to 300 g) were the heart and kept at room temperature. All hearts were anesthetized for surgical procedures with Metafane prefused first with 15 ml of 0.1% procaine in phosphate- (Methoxyfluorane). Each animal was given 800 units of buffered saline (PBS) to wash out the blood and to stop sodium heparin intravenously minutes before being killed heart contractions. For light microscopy and immunocyto- by Metafane overdose. chemistry, PBS perfusion was followed by perfusion with either 30 ml of periodate-lysine-paraformaldehyde (PLP)20 or 30 ml of 3.5% paraformaldehyde in PBS. For electron Ischemic Injury microscopy, PBS perfusion was followed by perfusion with 100 ml of an osmium mixture described by McCallis- The procedure has been described in detail.17 The left ter and Page.2' chest cavity was entered between the fourth and fifth ribs Tissues for light microscopy were postfixed overnight and the descending branch of the left coronary artery oc- in their respective perfusates. For routine examination, cluded by ligation 2 to 3 mm from its origin. The chest they were embedded in Historesin (LKB, Bromm, Swe- was closed and the animals allowed to recover. Rats were den), sectioned with glass knives at 1.5 , setting, and killed at 1 (N = 2), 2 (N = 2), 4 (N = 2), 6 (N = 2), 14 (N stained with equal volumes of 1 % azure 11 in water and 1 % = 2), and 28 (N = 2) days after injury. For each time inter- methylene blue in 1 % sodium borate. For immunochemi- val, one rat was used for light and one for electron micros- cal examination, they were embedded in low melting point copy. paraffin (55 to 58 C) and cut into 5 u thick sections. For immunoperoxidase preparations, the deparaffinized, rehydrated sections were exposed at room tempera- Freeze-Thaw Injury ture to the following: PBS (10 minutes); 0.75% H202 in PBS (30 minutes); PBS (15 minutes); 0.01% pronase in The procedure has been described in detail.'8 The abdo- PBS (20 minutes), PBS (15 minutes), normal serum (1: men was entered through a midline incision, the left lobe 100) and 2% bovine serum albumin in PBS (30 minutes); of the liver deflected, and an aluminum rod, precooled in and primary antibody (12 hours at room temperature) (1: liquid nitrogen, positioned for 10 seconds in contact with 500 HHF35, a muscle-actin-specific monoclonal antibody the diaphragm underlying the heart. The diaphragm re- obtained from Dr. Allen M. Gown, Department of Pathol- mained intact in all instances. The abdominal incision was ogy, University of Washington).22 Controls were reacted Connective Tissue Cells in Healing Myocardium 995 A/PMay 1989, Vol. 134, No. 5

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Figure 1 A. Crossection ofniormal rat myocardiumfixed byperfusion. The capillaries are distended and the connective tissue space uwidened byperfusate. Arrowspoint to the small and inConspicuous connective tissue cells, uwhich tenid to be located near capillaries (X2600). B: Tjpical connective tissufe cell in niormal rat myocardium. The cell has two slender cell extentsionis, one in contact with a cell extension of another connective tissuie cell. The nucleus occupies most of cell volume, anid scanty perinuclear cytoplasm contains maintlyj rouigh endoplasmic reticulum (X 10,000). C: Rat mYocardium 20 hours after isoprotereniol injury. An activated connective tisste cell is located between a viable (V) and a necrotic (N) myocyte. Compared u'ith its counlterpart in (1B), its shape is less angulate, its periniuclear cytoplasm more abundant, its nucleoplasm less conidenised, anid its nucleolus more prominent (X4000).

(30 minutes) with nonimmune IgG from the same species isothiocyanite (FITC)-labeled goat anti-rabbit IgG (1:16). and at the same dilution as the primary antibody. The sec- After a final wash in PBS (10 minutes), the sections were tions were washed in PBS (10 minutes), exposed to biotin- mounted in 90% glycerol in PBS containing 5% propyl ylated IgG directed against the primary antibody and to gallate. avidin DH-biotinylated horseradish peroxidase complex Tissues for electron microscopy were selected from according to the manufacturer's instructions (Vectastain the grossly evident viable margin, healing margin, and ABC Kit, Vector Laboratories, Burlingame, CA), and re- central portion of each type of lesion. They were minced acted with 0.06% diaminobenzidine tetrachloride (5 min- with razor blades into 1 mm cubes. Twenty cubes from utes). each zone were postfixed 1 hour in chilled 2.0% OS04 in For immunofluorescence preparations, deparaffinized 0.1 M s-collidine buffer (pH 7.48), stained with 1% aque- sections were exposed at room temperature to the follow- ous uranyl acetate for 1 hour, dehydrated, and embedded ing: PBS (10 minutes); 0.01% pronase in PBS (20 min- via propylene oxide in Epon-812 or Medcast. One-half mi- utes); PBS (15 minutes); normal goat serum (1:50) and crometer-thick sections from all blocks were stained with 2% bovine serum albumin in PBS (30 minutes); and rabbit methylene blue; at least three blocks oriented longitudi- anti-fibronectin antibody (1:20 dilution; from ICN lmmu- nally with respect to myofiber bundles and three blocks nobiologicals, Lisle, IL) for 18 hours at 4 C. Controls were perpendicular to the same axis were selected for thin sec- reacted with nonimmune rabbit IgG. The sections were tioning. From the selected blocks, 60 to 90 nm-thick sec- washed with PBS (15 minutes) and treated with fluorescin tions were cut and the sections stained with saturated 996 Vracko, Thoming, and Frederickson AJPMai 1989, Vol.sw134, No. 5

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-i I Figure 2 A. Rat myocardium 4 days afterfreeze-thaw injury. The connective tissue cells are most numerous in the reparative zone. Thbe interdigitate with and abut on the blunt ends ofviable myocytes. Macropbages are identifiable by their collections ofcell debris. All elements appear to be in common alignment (X25). B: Rat myocardium 4 days afterfreeze-thaw injury. The section was reacted withfluorescin isothiocyanite-labeled antibody tofibronectin. Intact myocyte stumps are visible as negative images in the lower part, unstained necrotic tissue as negative image in the upperpart, andfibronectin stained elements as the bright image in between. The stained zone containsproliferating connective tissue cells (X 100). aqueous uranyl acetate for 1 hour and with Millonig's lead scribed.12 The BL survived the acute injury intact but de- acetate for 2 minutes. The sections were examined with veloped holes about the size of inflammatory cells during either an AEI-801 or a JEOL 100-S transmission electron the exudative response to injury. Despite the holes, it con- microscope. tinued to delimit myocyte compartment from connective tissue space. Starting about 10 days after injury, myocyte BL deep within developing scar tissue was focally frag- Results mented and partially removed.12 Each method of injury initiated an exudative inflamma- Ischemia produced a transmural, left ventricular lesion in- tory reaction within 1 hour. A fluid exudate distended both volving the apical half of the anterior wall.17 Freeze-thaw- the connective tissue space and the BL-defined compart- ing produced a diaphragmatic, left ventricular lesion, 6 ments. Cellular exudates followed soon thereafter. Neu- X 8 mm in greatest diameters on its ovoid epicardial as- trophils and macrophages infiltrated sites of ischemia- pect and about 2 mm deep into heart muscle.18 Isoproter- and cold-induced necrosis, whereas only the latter cells enol produced mostly subendocardial, left ventricular le- appeared in sites of chemical-induced necrosis. The in- sions of microscopic size.19 flammatory cells entered both connective tissue space Each method of injury induced acute myocyte necro- and myocyte compartment. The necrotic debris was re- sis. The ischemic and cold injuries also destroyed most moved. Active inflammatory exudation subsided and in- of the nonmyocyte cells within lesions, although blood flammatory cells disappeared from the smaller lesions vessel cells and connective tissue cells (CTCs) were pre- over the next few days; however, macrophages were de- served in a narrow zone along the perimeter of freeze- tectable in the larger ones for another 3 weeks. thaw lesions.18 The isoproterenol injury left most blood The various reparative reactions involving myocytes at vessel cells, nerve cells, and CTCs intact throughout each the edge of injury have been described in detail.16 Briefly, lesion. necrotic myocytes detached from viable myocytes along The extracellular myocardial-supporting framework their intercalated discs. This created blunt-ended viable was not appreciably altered by the acute injuries. The fate myocyte stumps. A few of the stumps were covered by of one of its components, the myocyte BL, has been de- a new thin layer of BL, but the majority remained uncov- Connective Tissue Cells in Healing Myocardium 997 AJPMay 1989, Vol. 134, No. 5

Figure 3. Rat myocardium 3 days afterfreeze-thaw injury. Prominently elongated connective tissue cells (C) are located in connective tissue space between profiles of myocyte compartment (arrows indicate myocyte basal lamina). The myocyte compartment contains necrotic cell debris and macrophages cleaning up debris. A gap in basal lamina is noted (*). Intact capillaries (CA) and myocytes indicate the edge of necrosis; one myocyte (M) has a blunt end as the result of detachment of necrotic myocyte at its intercalated disk (X2400). ered and served as the source of reparative myocyte pro- strongly with antibody to fibronectin (Figure 2B). Most cesses. The cell processes extended into the site of injury were located within the connective tissue space (Figure within the myocyte compartment. Most developed 3), but many also were located within the myocyte com- sharply tapered, cone-shaped distal ends and became partment, where they were admixed with inflammatory el- anchored to scar tissue collagen fibers via myotendinous ements and necrotic cell debris (Figure 4). In both tissue junctions. locations, the CTCs lacked BL investments, were ob- In normal myocardium, CTCs are sparsely distributed served in mitosis, and were associated with accumulation in the connective tissue space. They have a uniform ap- of extracellular fibrillar material. These cells were decid- pearance and are the only resident cell type without a BL edly elongate and aligned in the same longitudinal axis as investment (Figure 1 A, B).23 During the first day after each the myocyte BL sheaths. method of injury, the CTCs along the edges of all lesions Fibrocytic CTCs located wholly or partially inside the and those surviving within partially necrotic lesions ap- myocyte compartment became closely apposed to newly peared to have fewer cell processes, increased perinu- developing myocyte processes, and the two cell types clear cell volumes, and more prominent cisternae of rough established multifocal cell-cell contacts (Figure 5). Asso- endoplasmic reticulum. By 48 hours, a proliferative re- ciated with these close relationships were some distinct sponse markedly increased the number of reparative and important changes: 1) The myocyte processes be- CTCs. They were by then the most numerous and most came lined with a BL, they terminated in sharply tapered prominent cells in the reparative zone. Their cytoplasms ends, and subplasmalemmal densities formed along their contained an abundance of protein synthesizing and se- tapered aspects. 2) Microfibrils, frequently arranged in creting organelles (Figure 1 C). bundles, appeared in the adjacent extracellular space The early reactive CTC forms resembled metabolically and connected the tips of myocyte processes to scar tis- active fibrocytes. They were initially concentrated along sue. 3) Sarcomeres developed out to the distal ends of the edges of lesions (Figure 2A) in a zone that stained the myocyte processes. In conjunction with these 998 Vracko, Thorning, and Frederickson AIP Mai 1989, Vol. 134, No. 5

Figure 4. Longitudinal section through rat myocardium 4 days after isoproterenol injury. All cellular and extracellular elements appear to be aligned in parallel. Metabolically active connective tissue cells are located both inlside (CI) and outside (CO) the mJyocyte compartment (arrou's indicate myocyte basal lamina). Gaps (*) in basal laminia structure are noted and macrophages are stillpresenit (X4500). changes, the intensity of staining for fibronectin de- myocardial elements. One was first observed 2 days after creased, the number of extracellular collagen fibers in- each type of injury. It resembled other metabolically active creased, and the CTCs no longer remained apposed to fibrocytes but had conspicuously elongate and tapered myocytes. Many CTCs showed degenerative changes, cell processes inserted into the branch points of normal and CTC numbers seemed to diminish significantly. appearing myocytes (Figure 7A). Fingerlike processes of The CTCs in the nearly healed environments had their distal cytoplasms interdigitated with similar pro- smaller cell bodies, markedly elongate and attenuated cell cesses of myocytes at sites devoid of myocyte BL (Figure processes, and lesser quantities of protein-synthesizing 7B). For each CTC, there appeared to be multiple points organelles. Their processes were interposed between of direct contact with a myocyte. We could not determine collagen fibers, elastic fibers, and fragments of presum- whether a single CTC made contact with more than one ably old myocyte BL (Figure 6A, B). Notable were the myocyte at this stage. common longitudinal orientation of all of these reparative The other distinct CTC form was first noted 2 weeks elements (Figure 5B, C) and the alignment of these ele- after each type of injury, was more common after isopro- ments in the longitudinal axis of uninjured myofibers. terenol injury, and was present throughout the subse- Two morphologically distinct CTC forms were ob- quent 4 weeks of observation. It was characterized by served in the borderzone between injured and uninjured complex shapes and spatial relationships, having mark- Connective Tissue Cells in Healing Myocardium 999 A/P May, 1989, Vol. 134, No. 5

Figure 5. Stages in the development ofmyotendinousjunctions. A: Rat myocardium 4 days after isoproterenol injury. The myocyte compartment outlined by basal lamina (arrows) contains two connective tissue cells which are inserted between tapered myocyte cellprocesses. The cellprocesses are associated with extracellular collagen deposits. The moreproximal ends ofthe myocyteprocesses (M) contain well orgatnized myojibrils, while the more distal ends are less uwell organized. An otherwise unidentited cellprocess is extended through a gap (*) in basal lamina. The connective tissue space contains an abundance of collagen (X5000). B: Rat myocardium 4 days after isoprotereniol injury. A connective tissue cell (C) appears to cap the distal enld ofa myocyte cellprocess (M). The myocyte basal lamina is indistinct or absent along aspects of close CTC-myocyte appositiont. Note the subplasmalemmal densities (arrowheads) along the tapered edges ofthe myocyte and the electroni-dense microfibril bunldles (open arrous) in adjacent extracellular regions (X8000). C: Rat myocardium 3 weeks afterfreeze-thau injury. Connective tissue cell (C) processes are located between myocyteprocesses (M). The subplasmalemmal myocyte denisities and the extracellular microfibril buntdles are better developed than in (B); these twlo elements appear to be continuous with an indistinct boundary. The longitudinal anidparallel alignment ofcellular and extracellular elements is obvious (X4800). 1000 Vracko, Thoming, and Frederickson AJPMay 1989, Vol. 134, No. 5

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Figure 6. Rat myocardium 5 weeks aJter isoprotereniol inlury. A: Relatively dense scar tissue consists ofpaiallel bundles ofcollagen fibers, thin and elongate cytoplasmic processes ofconnective tissue cells, elasticfibers (openi arrows), and basal lamina remnzants (closed arrow). The tip ofa myocyte (M) is presenit on the right side ofthe vieuw. Note againi the commoni longitudinal directionl of orientation ofall elemenits (X26000). B: A magnzified vieuw ofthe site outlined in A (X 10, 700).

Figure 7. Rat myocardium 3 days afterfreeze-thaw injury. A: A metabolically active appearing connective tissue cell occupies the connective tissue space between intact myocytesandinsertsa taperedprocess (*) inito a myocyte branchbpoint (X4500). B: Enlarged view ofA. The myocyte basal lamina (arrows) is absent at the site ofconnective tissue cell (*) initerdigitationi and contact (arrowhead) with the myocyte (X 10,300). Connective Tissue Cells in Healing Myocardium 1001 AJPMay 1989, Vol. 134, No. 5

Figure 8. Rat myocardium 4 weeks after isoproterenol injury. A: A connective tissue cell has ramifiedprocesses thatsurroundportions of myocytes, insert deeply into the bifurcation of one cell, and passfingerlike terminals through gaps in basal lamina to contact myocytes directly (arrowbeads) (X6400). B: Enlarged view ofcontact site. The terminalprocesses ofone or two ramified connective tissue cellspass tbrough basal lamina (arrows) to contact myocyte (arrowheads) (X 16,000). edly elongate, attenuated, and irregularly ramified cell pro- served in the subepicardial aspects of healed ischemia- cesses, which followed the contours of nearby myocytes, and cold-induced lesions. capillaries, or extracellular matrix elements (Figures 8 and 9). In certain planes of section, the cell embraced all or a large portion of one or several myocyte processes (Fig- ures 8 and 10) and established either broad or fingerlike Discussion CTC-myocyte contacts at sites devoid of myocyte BL (Figures 8, 9, and 10). Our experimental approach involved injuring left ventricu- Three additional, well differentiated cell forms ap- lar myocardium of adult rats and examining tissue sam- peared within the maturing scar tissue (Figure 1 1). Typical ples from the edge of viable myocardium with light and myofibrocytes became apparent by 4 days after injury electron microscopy at intervals up to 6 weeks after injury. (Figure 1 1 A) and typical chondrocytes by 2 weeks (Figure To avoid changes that could be unique for a specific form 11D, E). The myofibrocytes were distributed throughout of injury, we used three different methods of injury; to developing scar tissue, whereas the chondrocytes were avoid tissue distortion by myocyte contractions, we localized to subendocardial aspects of markedly thin stopped the heart action with procaine and fixed each transmural scars. Both of these were observed in forms heart by perfusion; and to better envision the stereologi- transitional between theirs and typical fibrocytic CTCs. cally complex interactions between the various myocar- Typical smooth muscle cells were first noticed 8 days af- dial elements, we used tissue sections oriented longitudi- ter injury (Figure 1 1 B, C). They were most commonly ob- nally and perpendicularly to the long axis of myocytes. 1002 Vracko, Thoming, and Fredenckson AJPMay 1989, Vol. 134, No. 5

connective tissue space through gaps in BL, and most formed myotendinous junctions with scar tissue collagen fibers. CTCs played major roles in these events. They origi- nated from relatively inconspicuous interstitial cells.23 Im- mediately after injury, the progenitor cells increased in size, multiplied, and provided a distinct zone of CTC hy- percellularity along the edge of each lesion. This zone was associated with fibronectin-rich deposits similar to those in nonmyocardial healing wounds.25 The reactive CTCs were located in the connective tissue space and within the compartment of necrotic myocytes. Although this is unproven, they probably migrated from interstitium into the compartment through the afore- mentioned holes in myocyte BL. The incursion of CTCs into the myocyte compartment could represent a major factor in the outcome of the healing process. In noncardiac tissues with intact BL, proliferating CTCs are generally contained within the connective tissue space and do not enter BL-defined parenchymal tissue compartments; under these conditions, these tissues generally heal by restoration of normal tissue structure. In contrast, when noncardiac necrotic tissue injuries are also associated with disruption or removal of parenchymal BLs, healing is Figure 9. Rat myocardium 3 weeks afterfreeze-thaw injury. with of ele- Slender connective tissue cellprocessesfollow the contours of associated overgrowth connective tissue a capillary (CA) and several myocytes. They establish cell-cell ments in interstitium and within BL-disrupted parenchymal contacts with myocytes (arrowheads) (X 6000). compartments, and this overgrowth is associated with formation of scar tissue.242631 We have examined the reparative interactions be- Myocardial CTCs assumed a variety of morphologic tween CTCs, the BL framework of necrotic myocytes, forms and spatial relationships during repair. The predom- and the viable myocytes at the edge of the injury and have inant CTC form had the morphologic features of typical, not yet evaluated the roles of the fibrous extracellular ma- metabolically active fibrocytes.32 These CTCs appeared trix, the vessels, and the nerves. The fate of myocyte BL to be responsible for deposition of extracellular scar tis- has been reported.12 Briefly, myocardium, like most other sue matrix located both in interstitium and within dam- tissues,24 possesses a BL framework.12 This framework aged myocyte compartments. CTCs located in the myo- is normally sheetlike without disruptions; however, during cyte compartment were closely approximated to repara- the first day after myocyte necrosis, it develops numerous tive myocyte processes, and the two cell types seemed holes. We do not know whether the holes were made by to collaborate in the anchorage of myocytes to scar tissue migrating inflammatory cells, by the pull of contracting collagen fibers. The completed myotendonlike anchor- myocardium, by both of these mechanisms, or by some ages resembled myotendineous junctions at the tips of other undefined mechanism. Except for the holes, the papillary muscles.33'34 acellular BL sheath of necrotic cardiac myocytes re- Two other CTC forms developed spatially intimate rela- mained intact for at least 10 days after each type of injury; tionships with myocytes by establishing close cell-cell it was focally removed later in the course of repair. contacts suggestive of some form of direct communica- The reactions of myocytes at the edge of injury also tion. One of these was observed between the second and have been reported.'6 Briefly, the stumps of myocytes ex- the seventh day after all forms of injury, resembled the tended cell processes into the site of injury and the pro- typical reactive fibrocyte, but differed in its relationships cesses seemed to advance, at least initially, along the re- to normal appearing myocytes located at the initial edge sidual BL framework. This process is similar to the growth of injury. It inserted elongate, tapered, cell processes into of regenerating cells in other tissues,24 however, in con- myofiber branch points, inserted fingerlike cell projections trast to other tissues, the myocyte processes did not ex- through myocyte BL, and established direct contacts with tend far into the injured site and did not appear to be asso- the myocytes. The second CTC form was observed from ciated with cell replication. Some myocyte processes ex- the second week after injury throughout the remaining 4 tended out of the myocyte compartment into the weeks of observation, had an usual ramified structure, Connective Tissue Cells in Healing Myocardium 1003 AJPMay 1989, Vol. 134, No. 5

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Figure 10. Rat- myocardium 3 weeksI after isoproterenol injury. A: Crossections oftwo myocyte cellprocesses arepartially surrounded by connective tissue cellprocesses. The myocytes have subplasmalemmal densities and are lined by basal lamina. One interstitial cellprocess (*) broaches myocyte basal lamina (arrows) and establishes contact with the myocyte (arrowhead). This constellation is surrounded by collagen and elastic fibers (X 15,800). B: Oblique sections through two myocyte processes (M), which are sur- routnded, int part, by complex connective tissue cellprocesses that pass through gaps in myocyte basal lamina to establish cell-cell contacts (arrowheads). Portions ofredundant basal lamina (arrows) arepresent (X 10,100). and established complex spatial relationships with other within the reparative zone. These suggest the possibility reparative tissue elements. Its numerous slender and long of an instructive role integrating reparative tissue struc- cell processes tended to surround portions of myocytes, tures in accordance with existing normal tissue structures capillaries, and connective tissue fibers and to establish and mechanical forces. Cell-cell contacts, similar to intimate CTC-myocyte contacts through gaps in myocyte those we observed, have been observed between mes- BL. Some of these ramified cells developed attenuated enchymal and epithelial cells during embryogenesis35-38 processes that virtually enveloped slender myocyte pro- and postnatal growth39 and were interpreted as indicators cesses. of inductive tissue interactions40 necessary for continuing We do not know the exact roles of these two reparative development and growth. CTCs with spatially distinct relationships nor do we know Three additional differentiated CTC cell forms were ob- whether the form observed earlier in repair is related to served within relatively mature scar tissue. Typical myofi- that observed later. Although their topographic relation- brocytes41 were distributed throughout scar tissue and ships suggested a communicative role between cells, presumably contracted to diminish the final size of the they also suggested a broader role in reparative structural scar. Typical chondrocytes appeared mainly in the suben- remodelling. The one form extended from the reparative docardial regions of transmural scars and presumably zone to normal structures at the edge of injury, and the provided relatively inflexible resistance to stretching other form interacted with cells and extracellular elements forces. Typical smooth muscle cells appeared in orderly 1004 Vracko, Thorning, and Frederickson AJPMa)' 1989, Vol. 134, No. 5

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Figure 11 A. Rat myocardium 4 days after ischemic injury. Connective tissue cells are surrounded by collagenfibrils. One cell is in mitosis; anzother containssmooth musclefilamenits (opent arrow) along itsperipheral aspect. Neithercell has basal lamina investment (X3000). B: Rat myocardium 3 weeks afterfreeze-thaw injury. This immunoperoxidase prepared section was reacted with antibody to mnuscle specific actin. Longitudinally sectionied mYocytes are located adjacen t to scar tissue in the lowerportion ofview. Between the myocytes and the epicardiuim, in the upperportion ofview, are numerous smooth muscle cells ald myofibrocytes (X60). C: Rat myocardium 3 weeks afterfreeze-thaw inijury,. These tjpical appearinigsmooth muscle cells were located within densely collagenous scar (X 15,800). D: Rat myocardium 2 weeks after ischemic injury. The cross-sectioned, scarred, papillary, muscle is almost etntirely replaced by, chondrocytes. Similar cells were presenit at other sites in scarred suibendocardium (X25). E: Rat myocardium 3 weeks afterfreeze-thaw injury. The chondrocyte has a scalloped outlinie, abunidanzt rough endoplasmic reticulum, and no basal lamina. It is surrounded bJy an abundanice ofinterwoven collageni fibrils (X9300). Connective Tissue Cells in Healing Myocardium 1005 AJPMay 1989, Vol 134, No. 5 layers located predominantly in the subepicardial aspects 7. Rumyantsev PP: Interrelations of the proliferation and differ- of large scars; their spatial arrangements were reminis- entiation processes during cardiac myogenesis and regen- cent of arterial media, and they presumably could con- eration. Int Rev Cytol 1977, 51:186-273 tract or relax as a unit to provide variable resistance to 8. Cantin M, Ballak M, Beuzeron J, Anand MB, Tautu C: DNA stretching forces. At no time during myocardial repair synthesis in cultures adult cardiocytes. Science 1981, 214: were we able to detect cell forms suggesting interconver- 569-570 9. Claycomb WC: Long-term culture and characterization of in nu- sion of CTCs and myocytes, a process suggested the adult ventricular and atrial cardiac muscle cell. Basic Res merous earlier studies.6'7'42'43 Cardiol 1985, 80(suppl 2):171-174 During the course of healing, all reparative tissue ele- 10. Nag AC, Cheng M: Adult mammalian cardiac muscle cells ments became oriented in alignment with the longitudinal in culture. Tissue Cell 1981,13:515-523 direction of the original myofiber bundles, and they re- 11. Nag AC, Cheng M: DNA synthesis of adult mammalian car- mained this way in the final scar product. This distinct ori- diac muscle cells in long-term culture. Tissue Cell 1986, 18: entation, the anchorages of myocytes to scar tissue, and 491-497 the fortification of scar tissue by smooth muscle cells and 12. 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