Beating heart muscle in a skeletal muscle bed

EXPERIMENTAL

NEUROLOGY

81,

749-755

(1983)

Beating Heart Muscle in a Skeletal Muscle Bed HARALD JOCKUSCH, GERHARD MEHRKE, AND ERNST-MARTIN ~XICHTBAUER’ Developmental

Biology Unit, W7 University of Bielefeld. Federal Republic of Germany Received

April

48 Bielefeld,

4. 1983

Minced cardiac muscle from newborn rats was transplanted into a skeletal muscle bed of nude mice. The implanted tissue regenerated into autonomously beating heart muscle, the donor origin of which was shown by immunohistochemistry and isozyme analysis. Host skeletal muscle regeneration was stimulated compared with controls without implanted tissue.

INTRODUCTION Minced skeletal muscle transplanted to a muscle bed in a limb has been shown to partially regenerate (2, 3, 12) and at the same time to stimulate ingrowth and regeneration of host skeletal muscle (4). These abilities may not be restricted to skeletal muscle. It would thus be of interest to study the fate and effects of nonskeletal muscle transplanted to a skeletal muscle bed. Histologic observations on the survival and outgrowth of cardiac myoblasts at such an ectopic site have been reported (13). We transplanted minced rat cardiac muscle to a nude mouse skeletal muscle bed in order to answer the following questions: (a) Would cardiac muscle compete as successfully with host muscle as does skeletal muscle? (b) Would it stimulate the regeneration of host skeletal muscle? (c) Would the minced cardiac tissue organize itself and regain autonomous contractility? Our data showed that xenografted heart tissue regenerated in a skeletal muscle bed of the athymic host and regained contractile properties characteristic for cardiac muscle. In addition, the regeneration of host skeletal muscle was stimulated by a cardiac muscle xenograft. Abbreviations: GPI-glucose phosphate isomerase, SDH-succinic dehydrogenase. ’ We thank Heike Deschner and Monika Hepner for competent and dedicated assistance, Annette Fiichtbauer for carrying out the electron microscopy, and the Deutsche Forschungsgemeinschaft for financial support (grant Jo 84/6 and 7). 749 0014-4886/83

$3.00

Copyright 6 1983 by Academac Press. Inc. All rights of reproduction m any form reserved

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Ventricular tissue (excluding the basal part of the ventricle) from newborn Wistar rats was minced and transplanted (2) into the previously freed bed of the tibialis anterior and extensor digitorum longus muscles of noninbred nude (NMRI nu/nu) mice selected for being homozygous for the gene for glucose phosphate isomerase (GPI, EC 5.3.1.9) isozymes. After 26 to 75 days the operated animals were anesthetized with pentobarbital. The skin was opened over the transplant and action potentials were recorded extracellularly from the surface of the tissues using a twin wire or KCl-filled glass capillary and displayed on a Tektronix 5 113 storage oscilloscope. Recipient mice were perfused with 200 ml Earle’s solution to clear the transplant from host blood and the regenerated tissue was removed for biochemical, histochemical, and immunochemical analyses. Frozen sections (thickness 20 pm) were cut and stained for succinic dehydrogenase [SDH, EC 1.3.99-l (1 l)] or for acetylcholine esterase [AChE, EC 3.1.1.7 (6)]. Other sections were used to analyze the relative contributions of host and donor by immunohistochemistry using a species-directed “antirat” antiserum. This had been raised by injecting a rabbit with rat skeletal muscle homogenate ( 130 fig protein, in 200 ~1 buffer). Four immunizations were carried out during 6 weeks, the first with 200 ~1 of Freund’s complete adjuvant, the following with incomplete adjuvant. The rabbit was bled and the crude serum was absorbed by shaking 12 h at 4°C with a lo-fold volume of sonified mouse muscle homogenate. After centrifugation, the supematant was brought to 50% saturation in ammonium sulfate and centrifuged. The pellet was redissolved in calcium- and magnesium-free phosphate-buffered saline and dialyzed against the same solution. The crude gamma globulin fraction obtained was used as the first antibody for the staining of frozen sections. The second antibody was horseradish peroxidase (HRP)-conjugated swine antirabbit IgG (Dako, Boehringer Ingelheim). The HRP reaction was carried out with 0.5 mg/ml 4-chloro-I-naphtol, 0.025% H202 at pH 7.35 and room temperature for 20 min (8). Host and donor contributions to the regenerated tissue were also measured by electrophoretic separation of mouse and rat forms of glucose phosphate isomerase (GPI, EC 5.3.1.9) after extraction of tissue pieces (5 to 10 mg) or frozen sections. Electrophoresis was carried out at 4°C in 1% “Isogel” (Seakem, Johnson, Cologne) agarose, 22 mM Tris citric acid, pH 7.0, for 2 h at 300 V (5); the GPI activity was developed at pH 8.0, 37°C for 25 min by trapping the formazane reaction product in cellulose nitrate paper (10). In one instance, the recipient mouse was perfused with 1% glutaraldehyde3.7% paraformaldehyde, and the transplant was postfixed in 2% 0~0~ and embedded for electron microscopy. Ultrathin sections were stained with uranyl acetate.

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RESULTS Six heart tissue transplantations and two control operations without implanted minced muscle were carried out. Data from a series of skeletal muscle transplantations served comparative purposes. In heart tissue transplants the total regenerated tissue weighed 15 to 40 mg or 30 to 60% of the unoperated control muscle whereas no regenerated muscle tissue was found in controls without implanted tissue. Histochemical staining for SDH revealed three main zones in the cross section of the regenerated tissue (Fig. la, d): skeletal muscle with heterogenous fiber pattern staining, connective and adipose tissue with no or weak staining, and heart muscle tissue with intense homogenous staining. The latter identification was confirmed by transmission electron microscopy of ultrathin sections (Fig. 1b, c). Staining of parallel sections for AChE revealed typical end-plates, confined to the zone of skeletal muscle fibers. No other AChE-positive structures were observed under the staining conditions used (15 min at 37°C pH 6.7). The donor origin of regenerated tissue was identified by two methods. A rabbit antiserum against rat skeletal muscle, preabsorbed with mouse skeletal muscle homogenate was used in conjunction with HRP-conjugated second antibody to identify rat tissue in frozen sections. This antibody reacted with species-specific intracellular antigens which were also present in cardiac muscle and connective tissue. The specificity of the reaction was checked with control sections of rat and mouse cardiac and skeletal muscle and with a weakly reactive antimouse serum which was preabsorbed with rat muscle homogenate (not shown). Figure 1e shows the identification of areas containing rat antigen in the cross section of a regenerated transplant. It is seen that the skeletal muscle was unstained and must therefore be derived from the mouse host, whereas the cardiac tissue was strongly positive for rat antigen. In a case of optimal heart muscle regeneration the following relative crosssectional areas were calculated: 65% mouse skeletal muscle; 17% rat cardiac muscle; 18% connective and adipose tissue containing rat antigen (corresponding to regions S, H, and C, respectively, in Fig. 1a). The electrophoretic separation of mouse and rat forms of GPI yielded donor contributions ranging from 10 to 25% of the total tissue in the muscle bed (Fig. 2). In four of six cases, upon opening the skin the regenerated tissue was seen pulsating at a frequency of about 0.2 Hz. In the remaining two cases rhythmic contractions were initiated by the tactile stimulus when the electrode was placed on the regenerate. The electrical activity of the cardiac transplant was recorded extracellularly. Measured spiking patterns were always paralleled by visible contractions. In addition to regular low-frequency pulsations (Fig. 3a) we observed spontaneous higher-frequency “runs” (Fig. 3c) during which the amplitude increased gradually, followed by periods of quiescence. Me-

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FIG. 1. Histology and histochemistry of cardiac transplants. a, d-frozen section (20 pm) of regenerated tissue, stained for SDH activity (5 min. pH 7.2, 20°C). Sample taken 36 days after transplantation. a, Overview: region C, connective and some adipose tissue; H, heart muscle; S, skeletal muscle. X60. d, Border between regions H and S, heart muscle to the left, skeletal muscle to the right. X240. b, c-electron micrographs of heart muscle cells, 42 days after transplantation. Cells shown were from a region corresponding to H in Fig. 1a. b, Two heart muscle cells connected by an intercalated disc (arrow), one showing a nucleus (N). c, A bundle of cardiac myofibrils. X3600. e-immunoperoxidase staining with antirat muscle serum. Section from the same series as Id. X240.

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123 FIG. 2. Electrophoretic analysis of glucose phosphate isomerase (GPI) variants in regenerated tissues, 71 days after transplantation of cardiac cells. Photograph of a cellulose nitrate sheet containing the tetrazolium reaction product of the GPI test. More anodic component, rat GPI, less anodic component, mouse GPI-B/B. Track l-mixture of rat and mouse red blood cell lysates; track 2-extract from 5 mg regenerated tissue; track 3-extract from the host tibialis contralateral to the operated leg. Scanning of track 2 with a densitometer yielded peak areas corresponding to 78% host (mouse) GPI and 22% donor (rat) GPI.

FIG. 3. Electrical activities in cardiac transplants. a-spontaneous slow rhythmic spiking recorded 42 days after transplantation. b-control recording from the tibialis anterior of the contralateral leg of the same animal as in a. The muscle spikes observed were always correlated with visible leg movements. c-spontaneous change in spiking frequency and amplitude. Recording taken 7 I days after transplantation (the very small spikes are artifacts due to breathing movements).

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chanical or electrical stimulation led to similar bursts of activity (not shown). None of these spiking activities were ever observed in contralateral unoperated tibialis anterior muscles (Fig. 3b) or in about 20 skeletal muscle transplants. DISCUSSION Cardiac myoblasts differentiate autonomously under a variety of experimental conditions. In tissue culture, explanted avian heart cells develop the ability to contract rhythmically (1). Electrical activity has been reported of cardiac cells transplanted to a subcutaneous site (14). Our work showed the reorganization of minced heart into a synchronously beating structure in a skeletal muscle bed. Donor cardiac cells competed successfully with ingrowing host skeletal muscle fibers, yet to a lesser degree than skeletal muscle transplants (5). In a freed skeletal muscle bed, regeneration only occurs if viable (2) skeletal muscle is implanted yet as much as 100% of the regenerate can be of host origin (4, 5). Therefore viable donor muscle cells exert a stimulatory influence on the regeneration of host skeletal muscles. Our results on cardiac muscle xenografis show this influence to be neither organ- nor species-specific. The possibility of foreign innervation of ectopically transplanted cardiac muscle is of particular interest because of its bearing on the problem of musclespecific innervation. Intact embryonic heart grafted to the anterior chamber of the eye has been shown to receive sympathetic and parasympathetic innervation (9). Whether or not cardiac muscle in a skeletal muscle bed would accept spinal motor nerves remains to be investigated. The converse experiment has been done successfully in the frog, where ectopically transplanted skeletal muscle was innervated by the vagus nerve (7). REFERENCES 1. BURROWS,M. T. 1912. Rhytmische Kontraktionen bei isolierten Herzmuskelzellen auBerhalb des Organismus. Miinch. Med. Wochenschr. 59: 1473. 2. CARLSON, B. M. 1972. The Regeneration ofMinced Muscles. Karger, New York. 3. ELSON, J. 1929. Auto- and home-transplantation of cross-striated muscle tissue in the rat. Am. J. Pathol.

5: 425.

4. GROUNDS, M., T. A. PARTRIDGE, AND J. C. SLOPER. 1980. The contribution of exogenous cells to regenerating skeletal muscle: an isoenzyme study of muscle allogratts in mice. J. Pathol. 132: 325-34 1. 5. JOCKUSCH,H. 1982. Muscle transplantation in mammals: a tool to study neuromuscular mutations and specificity of innervation. Pages 195-198 in L. JAENICKE, Ed., 33rd Colloquium-Mosbach, 1982: Biochemistry of Dlrerentiation and Morphogenesis. SpringerVerlag, Berlin/Heidelberg. 6. KARNOVSKY, M. J., AND L. ROOTS. 1964. A “direct-coloring” thiocholine method for cholinesterases. J. Histochem. Cytochem. 12: 2 19-22 1. 7. LANDMESSER,L. 1972. Pharmacological properties, cholinesterase activity and anatomy of nerve-muscle junctions in vagus-innervated frog sartorius. J. Physiol. (London) 220: 243256.

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8. NAKANE, P. K. 1968. Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study of pituitary glands of the rat. J. Histochem. Cytochem.

16: 557-560.

9. OLSON, L., AND A. SEIGER.1976. Beating intraocular hearts: light-controlled rate by autonomic innervation from host iris. J. Neurobiol. 7: 193-203. 10. PETERSON, A. C., P. M. FRAIR, AND G. G. WONG. 1978. A technique for detection and relative quantitative analysis of glucosephosphate isomerase isozymes from nanogram tissue samples. Biochem. Genet. 16: 68 l-690. 11. SPAMER, C., AND D. PETTE. 1977. Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle. Histochemistry 52: 207-216. 12. STUDITSKY, A. N. 1952. The restoration of muscle by means of transplantation of minced muscle tissue. Dokl. Akad. Nauk SSSR 84: 389-392. [in Russian]. 13. STUDITSKY, A. N. 1954. The development of myoblasts after the transplantation of cardiac muscle tissue in the place of a removed skeletal muscle. Dokl. Akad. Nauk SSR 95: 13551358. [in Russian]. 14. VORNOVITSKI~, E. G. 1977. Changes in the electrical activity of myocardial fibers in the heart transplanted subcutaneously into the mouse ear. Byull. Eksp. Biol. Med. 84: 396400. [in Russian].