3ournalof Molemlar andCellularCardiology(1977) 9, 959-966

T h e Fine S t r u c t u r e o f H e a l i n g O v e r in Mammalian Cardiac Muscle KATE M. BALDWIN

Department of Anatomy, Royal (Dick) School of Veterinary Studies, Edinburgh, Scotland and Department of Anatomy, Howard Universi~, Washington, DC 20059, U.S.A. (Received 7 December 1976, accepted in revisedforra 10 March 1977) K. M. BALDWIN.The Fine Structure of Healing Over in Mammalian Cardiac Muscle. ffournal of Molecular and Cellular Cardiology (1977) 9, 959-966. In an effort to learn the mechanism of healing over in cardiac muscle, sheep heart false tendons containing Purkinje fibers were cut, allowed to heal over in Tyrode's solution, then fixed and examined by electron microscopy. The cut cells exhibited no evidence of a new call membrane or any formed diffusion barrier at the cut surface and the dye ruthenium red passed freely into them. These results are incompatible with the hypothesis that healing over is due to new membrane formation at the cut surface and, therefore, support the hypothesis that healing over is the result of electrical uncoupling of the injured cells from the uninjured calls. The functional site of uncoupling could not be identified with certainty since there was no discernible change in intercellular junctions which were thought to be uncoupled. However, cells with apparent damage were observed 200 to 300 ~tm from the cut end, which suggests that ti,e functional site of uncoupling may be some distance from the cut surface. KEy WORDS: Cell injury; Electrical uncoupling; Healing over; Nexuses; Purkinje cells; Ruthenium red.

1. Introduction I f a sheep h e a r t false t e n d o n c o n t a i n i n g a s t r a n d of P u r k i n j e fibers is cut, there is a d r o p i n i n t r a c e U u l a r potential in cells n e a r the c u t end. W i t h i n 1 rain, however, these cells r e g a i n their n o r m a l i n t r a c e l l u l a r p o t e n t i a l a n d the s t r a n d behaves as if the cut e n d is sealed b y a n high electrical resistance [6, 30]. This f o r m a t i o n of a n high electrical resistance b a r r i e r at a cut surface is referred to as h e a l i n g over [30]. T h e ability of cardiac muscle to h e a l over is i m p o r t a n t i n t h a t the s h o r t - c i r c u i t i n g effect of a n i n j u r y is limited to a short time period, a n d thus the spread of the effects of the i n j u r y into the s u r r o u n d i n g tissue is m i n i m i z e d . T w o possible m e c h a n i s m s for the h e a l i n g over capabilities of cardiac muscle are: (1) recovery of the i n j u r e d cells as a result of the f o r m a t i o n of a n e w m e m b r a n e over the cut surface [30] a n d (2) electrical u n c o u p l i n g of the i n j u r e d cells d u e to the loss of pre-existing low resistance j u n c t i o n s [5]. I t has b e e n shown i n a m p h i bians that cardiac muscle ceils i n j u r e d b y crushing do n o t recover as i n d i c a t e d b y their i n t r a c e l l u l a r potential r e m a i n i n g n e a r zero [9]. I n this case therefore, the

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healing over was accomplished by electrical uncoupling of the injured cells from the uninjured cells. It is likely that m a m m a l i a n cardiac cells react to cutting injuries by uncoupling also, but evidence on this point is lacking. These experiments were undertaken to see which of the two proposed mechanisms would be supported b y a morphologicaJ study of healed over false tendons.

2. M a t e r i a l s a n d M e t h o d s

Adult sheep were anesthetized and the beating hearts removed and quickly placed in oxygenated Tyrode's solution. Twenty false tendons, approximately I cm long and 0.5 m m in diameter, were dissected away from the inner surface of the left ventricle or, occasionally, the right ventricle. T h e false tendons were pinned out in a bath of oxygenated Tyrode's solution and cut transversely with a sharp razor blade at 6 min, 1 rain or 5 s prior to immersion in fixative. T h e primary fixative was 1.2% glutaraldehyde in 0.067 M cacodylate buffer at p H 7.4. After a few minutes, the false tendons were placed in small vials of fresh aldehyde fixative, sometimes containing ruthenium red (see below), for further processing. Total fixation time in the aldehyde mixture was 1 h. Tissue was rinsed in 0.133 M cacodylate buffer for 1 h and post fixed for 1 h in cacodylate buffered 1.5% osmium tetroxide. All of the above procedures were carried out at 0 ~C. After fixation, the tissue was dehydrated in ethanol and embedded in either Araldite or Epon. I n one experiment, tissue was stained en bloc for 2 h in 0.5% uranyl acetate in maleate buffer before dehydration[I/]. In another experiment, false tendons were fixed with mixtures containing ruthenium red [I5]. In both these experiments the false tendons were cut either 10 rain or 5 s before beginning the fixation. Thick sections for light microscopy and thin sections for electron microscopy were cut on a Huxley microtome. T h i n sections were stained with uranyl acetate and lead citrate. T h e sections were cut parallel to the length of the false tendon; each section included the cut end and at least 1 m m of the tissue adjacent to the cut end. 3. R e s u l t s

False tendons consist of one or more strands of Purkinje cells surrounded by a thick layer of collagenous connective tissue and an endothelium (Plate 1). T h e observations reported here are confined to the Purkinje cells. Ceils which were located 500 ~m or more from the cut end always had the appearance of well preserved, healthy cells by standard morphological criteria and were similar in appearance to Purkinje cells previously described in the literature [2, 12, 16-18, 20, 21, 23, 25-29]. Therefore, the cells which were 500 ~m or more from the cut end were used as controls for the injured ceils at or near the cut end. Depending on the

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size of the cells and the amount of contraction resulting from the injury, 500 ~m was equivalent to 5 to 10 cell lengths.

Control cells Since normal sheep heart Purkinje cells have already been described [4, 16--18, 21, 25], their morphology will be only briefly reviewed here. The bulk of the cytoplasm was occupied by fine filaments approximately 5 to 7 n m in diameter, and granules, 12 to 20 nm in diameter, which were presumably glycogen and ribosomes. Myofibrils were located at the periphery of the cells and while they were predominantly parallel to the long axis of the cell, and thus the false tendon, they exhibited considerable irregularity in their orientation. Leptomeric fibrils (Plate 2) were frequently seen, usually in close association with the cell membrane. The sareoplasmic reticulum was inconspicuous; however, careful examination of micrographs revealed the presence of membranous profiles both in association with the myofibrils and scattered throughout the cytoplasm (Plate 2). Diadic couplings of the sarcoplasmic reticulum with the plasma membrane were seen on occasion. The nuclei, frequently two per cell, were centrally located. T h e y were uniformly pale except for one or two dense nucleoli. Associated with the nuclei were m a n y small Golgi complexes, a pair of centrioles, and varying numbers of lipofusein granules (Plate 3). Mitochondria were found near the nuclei, at the cell periphery, and scattered throughout the cytoplasm. Occasionally, mitochondria of two distinctly different morphologies were seen near the nucletqs. The more abundant type had a diameter of approximately 0.5 ~m, a pale matrix, and many cristae. The less frequent type had a diameter of approximately 0.1 ~m, very few cristae, and a dense matrix (Plate 3). Intercellular junctions were located all around the cell perimeter and were of three types. (1) Nexuses or gap junctions, characterized by a close approximation of the membranes of two adjacent ceils leaving a gap of approximately 1.5 nm between them. (2) Fasciae adherentes or insertion plaques, which were sites of attachments of the myofilaments into the cell membrane and were characterized by a membrane-associated diffuse density similar to the Z-band density. (3) Desmosomes, characterized by dense cytoplasmic plaques on either side of the junction. The desmosomes were not sites of insertion for myofilaments (Plate 6). In nonjunctional areas, the cells were closely associated with an intercellular space of approximately 10 to 15 nm.

Injured cells The appearance of the injured ceils at the cut end of the false tendon was generally similar in all experiments, regardless of time between cutting and fixing the ceils.

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T h e only obvious difference was that when false tendons were cut 6 or 10 min before fixing, there was considerable contraction and shrinkage of the injured cells away from the surrounding connective tissue (Plate 1). In the tissue fixed 1 min or less after cutting, there was no obvious shrinkage of the injured cells (Plate 11). T h e Purkinje cells which had been cut were easily recognizable in sections. Their cytoplasm ended abruptly, but in spite of the sharpness of the cellular boundary, there was no evidence of a formed diffusion barrier at the cut surface (Plates 4 and 5). While pieces of m e m b r a n e were often seen at this cut surface, there was no continuous layer similar to the plasma m e m b r a n e of intact cells. The cut cells differed from the controls in the following ways. T h e myofilaments were in a state of contracture and the cell boundaries were quite contorted. T h e cytoplasmic matrix was dense and dilated cisternae of the sarcoplasmic reticulum were prominent. T h e mitochondria were often normal in appearance, but occasionally were swollen (Plates 4, 5, 7 and 8). Intercellular junctions between cut cells and the cells adjacent to them were intact and appeared no different from those between control cells (Plates 4 to 7). Since nexuses are thought to be the sites of electrical coupling between cells, they were examined with particular care. As seen in Plates 9 and 10, the nexuses from injured tissue were similar to those from control tissue, both exhibiting a 1.5 n m gap when stained en bloc with uranyl acetate. Cells which were adjacent to cut cells showed a morphology similar to those which were actually cut. In fact, it was only when cells some distance from the cut end were examined that they appeared similar to control cells. T h e injured cell/ uninjured cell boundary (Plate 8) was always within 500 ~m, and usually within 200 to 300 ~tm, of the cut end.

Ruthenium red

W h e n ruthenium red was included in the fixatives, it was found intracellularly in cells at or near the cut surface, but was excluded from cells more than 150 to 250 ~zm from the cut end (Plate 11). As illustrated in Plate 12, the passage of ruthenium red into the strand of Purkinje ceils was abruptly stopped at an intercellular boundary. While ruthenium red penetrated the majority of cells with apparent damage, it was not seen in all such cells.

4. D i s c u s s i o n

From the results presented it appears that healing over in cardiac muscle after a cutting unjury is not due to the formation of a new diffusion barrier at the cut surface. T h e r e was no morphological evidence of such a newly formed barrier when false tendons were cut. allowed to heal over for 1 min or more. and then

P L A T E 1. Light micrograph o f a 1 ~.m Epon section stained with toludine blue. T o the right is the cut end, cut 6 m i n prior to beginning fixation. U n i n j u r e d cells are on the left. Note how the injured cells have pulled away from the cut surface of the surrounding collagen (CO), a n d t h a t darkly staining injured cells can be seen up to approximately 450 ~ m from the cut end. • 178. P L A T E 2. Control cells. T h e myofibrils are at the cell periphery a n d mitochondria are associated with them. Cisternae of the sarcoplasmic reticulum (-+) are present, b u t n o t conspicuous. A leptofibril (*) is present between a myofibril a n d the plasma m e m b r a n e . T h e s u r r o u n d i n g cytoplasm contains m a n y small granules a n d fine filaments. • 28 800. P L A T E 3. Control cells. T h e typical c o m p l e m e n t of organelles in sheep heart Purkinje fibers is represented in this micrograph. A pale nucleus is s u r r o u n d e d by mitoehondria, Golgi complexes a n d lipofuscin granules (L). T w o centrioles (C) are seen nearby. • 14 800. P L A T E S 4 a n d 5. T h e cut surfaces of ceils fixed 6 m i n (Plate 4) a n d 1 rain (Plate 5) after cutting. I n spite of the abruptness of the cellular b o u n d a r y , there is no evidence of a m e m b r a n e over the cut surface. T h e r e is contracture of the myofibrils a n d dilation of the sarcoplasmic reticulum. Note that the intercellular junctions between the injured cells are intact. Plate 4, • 27 200; Plate 5, X 14 500. P L A T E 6. Intercellular junctions between control cells. Fasciae adherentes (F) act as insertion sites for the myofilaments, while desmosomes (D) do not. At this magnification, the nexus (N) appears as a close approximation of the cell m e m b r a n e s . Note that the intercellular space in the non-junctional regions is only 10 to 15 n m wide. • 54 000. P L A T E 7. Intercellular junctions between a cut cell (above) a n d a n adjacent cell (below). Note t h a t the junctions are similar to those seen between control cells N, nexus; F, fascia adherens; D, desmosome. Tissue fixed 6 m i n after cutting. • 54 000. P L A T E 8. Intercellular b o u n d a r y located approximately 100 Exm from the cut end, which is upwards. T h e upper cell a n d all those between it a n d the cut end show morphological d a m a g e while the cell below a n d all those further away from the cut end appear normal. O n morphological grounds this cellular b o u n d a r y m i g h t be expected to be the functional site of uncoupling between injured cells and uninjured cells. Tissue fixed 1 m i n after cutting. • 15 300. P L A T E S 9 a n d 10. Comparison of nexuses between control cells (Plate 9) a n d injured cells (Plate 10) stained en bloc with uranyl acetate. T h e two junctions appear similar by this technique. Tissue fixed 6 m i n after cutting. • 175 000. P L A T E 11. Light mierograph of an unstained 2 ~zm Epon section, tissue fixed 1 m i n after cutting. R u t h e n i u m red was included in the fixatives a n d has penetrated cells up to 150 ~ m from the cut end, b u t has been excluded from the others. X 250. P L A T E 12. Electron m i c r o g r a p h of the intercellular b o u n d a r y indicated by the arrow in Plate 11. R u t h e n i u m red has penetrated the cell on the right, b u t it has not crossed into the cell on the left. x 11 300.

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PLATES 1 and 2

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P L A T E S 4 and 5

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processed for examination by electron microscopy. Furthermore, the dye ruthenium red freely passed into the cut ceils of healed over false tendons and was observed in cells at a distance of 150 to 250 ~m from the cut surface. Since the data presented here are inconsistent with the new membrane formation hypothesis of healing over, they support the hypothesis of electrical uncoupling as the mechanism of healing over. Additional support for the uncoupling hypothesis comes from the work of D616ze [7] on injury in rat heart using the tracer Procion Yellow. He also concluded that the injured cells uncoupled from the uninjured ceils. Electrical uncoupling of injured cells from uninjured cells has been reported in a variety of tissues [9, 14, 19, 22, 24], therefore, it is no surprise that uncoupling also occurs after cutting injuries in cardiac muscle. T h e lack of morphological evidence of uncoupling, while disappointing, was not unexpected. Bullivant and Loewenstein [3] found no anatomical change when insect salivary gland cells were uncoupled, and this author [1] found no morphological correlation to uncoupling after crushing injuries in frog heart. However, Pappas et al. [22] did see separation of axon segments at an electrotonic synapse after uncoupling resulting from mechanical trauma, so morphological correlates to uncoupling can occur. Loewenstein [13] has proposed that uncoupling can be brought about by increasing the intracellular calcium concentration above normal levels. This is supported by DeMello [8] who uncoupled cardiac muscle ceils by injecting calcium intracellularly. D616ze's work [6] showing that calcium is required for healing over to occur supports the proposals that uncoupling is the mechanism of healing over and that increased intraeellular calcium causes the uncoupling. Furthermore, the experimental findings of contracture of the myofilaments and dilated cisternae of the sarcoplasmic reticulum in the injured cells are consistent with the proposal of increased intracellular calcium in these cells. If, then, the mechanism of healing over is the uncoupling of injured from uninjured cells, it would be of interest to know at what level this occurs. Rose [24] injured electrically coupled ceils in Chironomus salivary gland and found that not only the directly injured cells, but also those immediately adjacent became uncoupled. The results presented here are suggestive that functional uncouPling might occur some distance from the cut end. In the healed over false tendons, morphological damage was often seen in cells up to 300 ~m from the cut surface. Furthermore, ruthenium red penetrated all cells up to 150 to 250 ~m from the cut end of the false tendons. Since ruthenium red will not cross the cell membrane of an intact, viable cell [15], the Purkinje cells which were penetrated by this dye were certainly injured. Unfortunately, however, ruthenium red does not enter all cells with apparent damage as reported here and in an earlier study[/], and therefore the extent of ruthenium red penetration cannot with any confidence be identified as the extent of injury. Thus there are two apparent boundaries: the injured cell/uninjured cell boundary, usually seen 200 to 300 tLm away from the cut end, and the boundary of the

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extent of ruthenium red penetration, usually 150 to 250 ~zm from the cut end. At this time, neither of these boundaries can be identified as the functional site of uncoupling, but they both suggest that this site is several cell diameters from the

cut surface. W h c n thcsc cxpcrimcnts wcrc undertaken, it was cxpccted that falsc tcndons fixcd l mln or morc aftcr cutting would havc hcalcd ovcr and that thosc fixcd only a fcw scconds aftcr cutting would not have. In vicw of thc similarity of thc rcsults obtained, irrcspcctivc of timc bctwccn cutting and immcrsion in fixativc,and particularly thosc cxpcrimcnts using ruthenium red, it sccms likely that healing ovcr occurrcd in all thcsc falsc tcndons. Ddl~zc [6] has shown healing over to take placc in this tissuc within I min and it m a y prcccdc fixation since glutaraldchydc pcnctratcs biological tissuesrclativclyslowly ] 10]. In summary, this study has shown that ncw mcmbranc formation cannot bc thc mechanism of hcaling ovcr aftcr cutting injurics in shccp heart false tendons and, thcrcforc, supports thc hypothcsis that clcctrical uncoupling of injured cclls from uninjurcd ccUs is thc mcchanism of healing ovcr. While no conclusion can bc made as to the functional siteof uncoupling, the data suggest that it is several ccll Icngths from thc cut surface.

Acknowledgements This work was supported by Science Research Council of Great Britain Grant B/SR/6997 and an Institutional Grant from Howard University. The author is indebted to the late Professor Alan R. Muir for the opportunity to work in his laboratory and for much helpful advice and criticism. The author would also like to thank Dr Gayle M. Crosby for suggestions regarding this manuscript.

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the Heart. B. Taccardi and G. Marchetti, Eds. pp. 147-148. Oxford : Pergamon ( 1965 ). 6. 7. 8. 9. 10. 11. 12. 13. 14. I5. 16. 17. 18. 19.

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D~L~ZE, J. The recovery of resting potential and input resistance in sheep heart injured by knife or laser. Journal of Physiology208, 547-562 (1970). D~Li~ZE,J. The site of healing over after a loeal injury in the heart. In Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 5. N. S. Dhalla, Ed: pp. 223-225. Baltimore: University Park Press (I 975). DEMEL~.O, W. C. Effect of intracellular injection of calcium and strontium on cell communication in heart. Journal of Physiology250, 231-245 (1975). HENRY, M., IVIATsUMOTO,C. & SALAFSKY,B. Injury potentials of frog cardiac muscle. Project Reports, Vol. 12. Seattle: Department of Physiology and Biophysics, University o f Washington (1961). Data quoted and discussed in Reference [1] above. HoPwooD, D. Some aspects of fixation with glutaraldehyde. ~7ournalof Anatom2 101, 83-92 (1967). KAP~OVSKY, M. J. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer, jTournalof Cell Biology35, 213-236 (1967). KAWAMURA,K. & JAMES,W. N. Comparative ultrastructure of cellular junctions in working myocardium and the conduction system under normal and pathologic conditions. ~7ournalof Molecular and CellularCardiology3, 31-60 ( 1971). LOEW~NSTEIN,W. R. Permeability of membrane junctions. Annals of the New York Academyof Sciences 137, 441-472 (1966). LOEWENSTErN,W. R., NA~S, M. & SOCOLAX,S. J. Junctional membrane uncoupling. Journal of GeneralPhysiology50, 1865-1891 (1967). LUST,J. H. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. AnatomicalRecord 171, 347-368 (1971). MOBLEY, B. A. & PAOE, E. The surface area of sheep eardiac Purkinje fibers, oTournal of Physiology220, 547-563 (1972). MUIR, A. R. Observations on the fine structure of the Purkinje fibers in the ventricles of the sheep's heart. ~7ournalof Anatomy 91, 251-258 (I 957). MUIR, A. R. Further observations on the cellular structure of cardiac muscle, oTournal of Anatomy 99, 27-46 (1965). O'LAGUE, P. & DALEN, H. Low resistance junctions between normal and between virus transformed fibroblasts in tissue culture. ExperimentalCell Research86, 374-382 (1974). OSCULATI,F. & GARIBALDI,E. Fine structural aspects of the Pm'kinje fibers of the dog's heart, journal of SubmicroscopicCytology6, 39-53 (1974). PAOE,E., POWER, B., FOZZARD,H. H. & MEDDOFF, D. A. Sarcolemmal invaginations with knob-like or stalked projections in Purkinje fibers of the sheep's heart, oTournal of UltrastructureResearch28, 288-300 (1969). PAPPAS, G. D., ASADA,Y. & BENNETT, i . V. L. Morphological correlates of increased coupling resistance at an electrotonic synapse. Journal of Cell Biology49, 173-188 ( 1971). RHOOIN, J. A. G., DEL MISSIER, P. & REID, L. C. The structure of the specialized impulse conducting system of the steer heart. Circulation24, 349-367 (1961). RosE, B. Intercellular communication and some structural aspects of membrane junctions in a simple cell system, oTot.rnalof Membrane Biology5, I- 19 ( 1971). SOMMER,J. R. & JOHNSON, E. A. Cardiac muscle. A comparative study of Purkinje fibers and ventricular fibers. Journal of Cell Biology36, 497-526 (1968). TI~ORNELL,L. E. Myofilament polyribosome complexes in the conducting system of hearts from cow, rabbit, and cat. jTournalof Ultrastructure Research41,579-596 (1972). THORNELL,L. E. Distinction of glycogen and ribosome particles in cow Purkinje fibers by enzymatic digestion en bloc and in sections. Journal of Ultrastructure Research 47, 153-168 (1974).

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The fine structure of healing over in mammalian cardiac muscle.

3ournalof Molemlar andCellularCardiology(1977) 9, 959-966 T h e Fine S t r u c t u r e o f H e a l i n g O v e r in Mammalian Cardiac Muscle KATE M...
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