Peroxisomes (microbodies) in the myocardium of rodents and primates

Cell Tiss. Res. 175, 467-481 (1977) Cell and Tissue Research @ by Springer-Verlag 1977

Peroxisomes (Microbodies) in the Myocardium of Rodents and Primates A Comparative Ultrastructural Cytochemical Study *

Lesley Hicks and H. Dariush Fahimi

Department of Anatomy, University of Heidelberg, Heidelberg, Germany, and Department of Pathology, Harvard Medical School, Boston, Massachusetts, U.S.A.

Summary. The occurrence of peroxisomes (microbodies), their cytochemical characteristics and their ultrastructural relationship to the neighboring organelles were investigated in the ventricular myocardium of four rodent (rat, rabbit, gerbil, and guinea pig) and two primate (Macaca java and Tupaya) species. The hearts were fixed by vascular perfusion with glutaraldehyde and incubated in alkaline diaminobenzidine media for visualization of catalase. The electron-dense reaction product of catalase was found in the myocardium of all examined species and was localized in 0.2-0.5 lam oval particles, surrounded by a single limiting membrane and located usually at the junction of I and A bands. The peroxisomes in the hearts of gerbil and Macaca java were especially long and tortuous. A close spatial association was found between the myocardial peroxisomes and mitochondria, lipid droplets, and the membranes of sarcoplasmic reticulum, especially the so-called junctional sarcoplasmic reticulum. These observations demonstrate the consistent occurrence of peroxisomes in the heart of various mammalian species and suggest that peroxisomes have important metabolic and physiological functions in myocardium.

Key words: Peroxisomes - Myocardium - Rodents, primates - Ultra- structural cytochemistry.

Introduction

A peroxisome (microbody) is a cytoplasmic organdie bounded by a single limiting membrane. The finely granular matrix contains catalase and various H202-pro-

Send offprint requests to: Prof. H.D. Fahimi, Anatomisches Institut der Universit~it Heidelberg, Im Neuenheimer Feld 307, D-6900 Heidelberg, Federal Republic of Germany

* This study was supported by Grant 08533 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland and a grant from Sonderforschungs- bereich 90 (Cavas) of the Deutsche Forschungsgemeinschaft, Germany. Dr. Fahimi was the recipient of a Research Career Development Award from the National Institutes of Health, Bethesda, Maryland. The technical assistance of Ms. Gaby Kr~imer and Mr. Michel Le Hit as well as the secretarial help of Ms. Gina Folsom is gratefully acknowledged

468 L. Hicks and H.D. Fahimi

duc ing oxidases (de Duve and Baudhuin , 1966) and, in the liver and kidney, a c rys ta l l ine nucleoid , which aids in its morpho l og i c a l ident i f ica t ion ( H r u b a n and Rechcigl , 1969). Since the deve lopmen t o f the a lka l ine D A B technique for visuali- za t ion o f the pe rox ida t i c ac t iv i ty o f ca ta lase ( N o v i k o f f and Goldf ischer , 1969; F a h i m i , 1969) 0.2q3.5 Ix ca ta lase posi t ive par t ic les have been ident i f ied in m a n y tissues. In fact, it has been p r o p o s e d by H r u b a n et al. (1972) and the Nov ikof f s (1973), who co ined the t e r m , ,mic rope rox i some" for these par t ic les ( N o v i k o f f and Novikof f , 1972), tha t they are ub iqu i tous in m a m m a l i a n cells.

Recen t ly H e r z o g and F a h i m i (1974, 1976) a n d H a n d (1974) independen t ly desc r ibed the presence o f pe rox i somes in the vent r icu la r m y o c a r d i u m of the adu l t mouse and ra t respectively. These par t ic les were descr ibed as measur ing 0.2-0.5 Ix in d i a m e t e r and being in close assoc ia t ion with m i t o c h o n d r i a and the sa rcop lasmic re t icu lum. He rzog and F a h i m i (1976) also conf i rmed b iochemica l ly the presence o f ca ta lase and smal l amoun t s o f two flavin oxidases in m ic ro soma l f rac t ions o f hea r t t issue and suggested tha t the par t ic les in mouse m y o c a r d i u m resembled bo th m o r p h o l o g i c a l l y and b iochemica l ly the pe rox i somes o f o ther tissues.

In this s tudy we have inves t iga ted the occurrence o f mic robod ie s in the ven- t r i cu la r m y o c a r d i u m of several o the r rodents and two p r ima te species, using ca ta- lase as a m a r k e r o f this organelle. The enzyme was de mons t r a t e d cy tochemica l ly in all species, and in the rodents its presence was conf i rmed biochemical ly . The resul ts indica te tha t m ic robod ie s occur in the m y o c a r d i u m of all species examined . A de ta i l ed desc r ip t ion o f their u l t ras t ruc ture wi th special cons ide ra t ion o f their r e l a t ionsh ips to ne ighbor ing organel les is p resen ted here.

Materials and Methods

Animals

Myocardial tissue of adult males from four rodent species were examined both cytochemically and biochemically. Albino Charles River Strain rats weighing 200-300 g, albino rabbits weighing approximately 1,000 g, Guinea pigs weighing 300-400 g, and Mongolian gerbils (Meriones unguicutatus) weighing 100 150g were kept on normal diets and fasted overnight before sacrifice. The Tupaya, or tree shrew, studied was a 160g adult female from the stock of the Batelle Institute in Frankfurt/ Main, Germany, and the Macacajava was a 1,600 g male obtained through the courtesy of the Pathology Department of the University of Heidelberg.

Cytochemical Procedure

The rodents were perfused in situ through the left ventricle with physiological saline for 1-3 min followed by 3~ glutaraldehyde in 0.1 M Na-cacodylate buffer at pH 7.4 containing 0.05~o CaC12 for 10rain at room temperature. The left ventricle was cut into thin strips and immersion-fixed in the same solution at 4~ for an additional hour. After a brief rinse in 0.15 M Na-cacodylate, the tissue was chopped into 30-50 ~t sections directly into the preincubation media by means of a TC-2 Smith- Farquhar (1965) tissue chopper. After 2 h of preincubation the reaction was started by the addition of H202 and carried out for 2 h with renewal of media after the first hour. The incubation was performed in the dark at room temperature under continuous agitation. The incubation media contained 1, 2.5, or 4rag DAB/ml prepared in Teorell-Stenhagen (1938) buffer at pH 10.5 with 0.02, 0.06, or 0.15~ H20 2 respectively. A modified Novikoff medium (1972) containing 2mg DAB per ml with 10 -4 M KCN and 0.05~ H202 at pH 10.5 was also employed. As a control the reaction was inhibited by 0.02 M 1,2,4-triaminotrizole (Fahimi, 1969).

Peroxisomes in the Mammalian Heart 469

The reaction was stopped by transferring the sections to 0.15 M Na-cacodylate, pH 7.4. The material was then postfixed in 2 ~ aqueous osmium tetroxide for 90 min, dehydrated in ethanol, and embedded in Epon 812.

The primates were anesthetized with Nembutal and perfused through the abdominal aorta according to the method of Forssmann et al. (1967), using 3 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, containing 2.5~ PVP, at 4 ~ C for 10 rain. The left ventricle was cut into thin strips and immersion-fixed for an additional hour in 3 ~ glutaraldehyde in 0.1 M Na-cacodylate buffer at 4~ The tissues were chopped, incubated, and processed for electron microscopy in the same manner as described above for the rodent species.

One micron-thick sections were examined unstained under a light microscope. Ultra-thin sections, stained with lead or lead plus uranyl acetate, were studied in a Philips EM 301 electron microscope.

Biochemical Determinations

For the measurement of catalase the animals were perfused through the left ventricle for about 5 rain with saline to wash out the blood ceils. The left ventricles were minced and homogenized in ice-cold 0.25 M sucrose (1:10 w/v). The homogenate was filtered through a double layer of cheese-cloth and centrifuged at 4~ at 3,500g/min to remove unbroken cells, nuclei, and cell debris. Triton-X-100 was added to a final concentration of 0.1 ~ , and the samples were sonicated for 5 min in an ice-cold bath. Catalase was determined immediately according to Lfick (1965) using a Philips-Unicam recording spectrophotometer. Protein was determined according to the Lowry technique (1951) with bovine serum albumin as standard. Catalase activity is expressed as International Units per mg protein.

Results

T h e p e r o x i s o m e s o f t h e v e n t r i c u l a r m y o c a r d i u m o f t he f o u r r o d e n t a n d t w o

p r i m a t e spec ies e x a m i n e d c o u l d be v i sua l i zed u n d e r t h e l ight m i c r o s c o p e o n l y

w i t h a n oil i m m e r s i o n lens. By e l e c t r o n m i c r o s c o p y , h o w e v e r , m a n y d i s t i n c t

All figures are from the left ventricular myocardium fixed by vascular perfusion with glutaraldehyde, incubated for cytochemical localization of catalase, and processed for electron microscopy. All sections were counterstained with lead citrate

Fig. 1. Low power electron micrograph of rat ventricular myocardium in cross section. Incubation of tissue resulted in a dark even reaction product, filling the matrix of the peroxisomes. These appear in close association with the sarcoplasmic reticulum and mitochondria, x 21,600

Fig. 2. Longitudinal section of ventricular myocardium of Macaca java incubated in 2 mg/ml DAB with 10-4 M KCN (Novikoff et al., 1972). Note the irregular shape of two peroxisomes on the left, which is typical for this species. • 21,150

Figs. 3 and 4. Association of peroxisomes (P) with the sarcolemmal membrane in the heart of rat Fig. 3 and Macacajava Fig. 4. In Figure 3a segment of junctional SR (JSR) is found between the microbody and the adjacent cell membrane. Note also the extreme length of the peroxisome in the heart of Macacajava (Fig. 4). Figure 3: x 39,600; Figure 4: • 58,500

Figs. 5 and 6. Association of peroxisomes with T-tubules. Usually a portion of junctional SR (JSR) is present between the peroxisome (P) and the T-tubule (73, analogous to the arrangement at the cell membrane. Figure 5: Rat, x 45,900; Figure 6: Gerbil, • 45,900

Figs. 7 and 8. Peroxisomes (P) in close association with lipid droplets. Both figures are from Macaca java and illustrate the irregular shape of the microbodies in this species. Note the low electron density of lipids in comparison to positively reacted peroxisomes. Figure 7: • 60,750; Figure 8: x 59,850


Captions see p. 469


Captions see p. 469

Figs. 9-12. These figures illustrate the close association of peroxisomes (P) with the sarcoplasmic reticulum (SR). Some of these profiles suggest continuity between the limiting membrane ofperoxisomes and SR (arrows). Rarely, as seen in Figure 12, reaction product was observed extending into a membranous tail of a microbody. Figure 9: Rat, • 54,000; Figure 10: Rat, x 49,500; Figure 11: Tupaya, • 76,500; Figure 12: Gerbil, x 53,100


Table 1. The size of myocardial peroxisomes in different species. For each species 75-100 particles were measured, and the ratio of length to width was determined

Animal Size of peroxisomes in p Ratio of length to width

length width

Rat 0.29 +0.07 0.20 +0.05 1.5 Rabbit 0.41 +0.12 0.28 +0.10 1.5 Guinea pig 0.36 -0.12 0.26 +0.09 1.4 Gerbil 0.36+0.17 0.17+0.06 2.1 Tupaya 0.30+0.11 0.17+0.05 1.8 M.java 0.33 +0.20 0.14 +0.05 2.4

particles were observed (Figs. 1, 2) bounded by a single membrane and containing a granular electron-dense reaction product distributed evenly over the matrix (Figs. 3-6). In longitudinal sections the microbodies were frequently found at the junction of the A and I bands (Fig. 2). Many particles were located near the sarcolemmal membrane (Figs. 3 and 4) as well as the intracellular extensions of the latter, the T-tubules (Figs. 5, 6). Almost invariably a small cisterna of sarcoplasmic reticulum (SR), the junctional SR (Sommer and Waugh, 1976), was found sandwiched between the microbody and the cell membrane or T-tubule (Figs. 3, 5, 6). The particles often appeared in close association with lipid droplets (Figs. 7, 8), the sarcoplasmic reticulum (SR) (Figs. 9-12), and the mitochondria (Figs. 13

Figs. 13 and 14. Peroxisomes (P) in close association with mitochondria. Figure 13: Rabbit, x 51,300; Figure 14: Macacajava, x 47,250

Fig. 15. A tortuous and segmented peroxisome (P) from the myocardium of Macacajava. x 88,740

Fig. 16. A group of four peroxisomes in close association with each other from the rat myocardium. x 85,050

Figs. 17 and 18. Although lipofuscin-bodies (LF) have an electron-dense matrix somewhat similar to that of the reacted microbodies (P), the two organelles can be easily distinguished from one another

Fig. 17. Macaca java heart incubated for catalase. Both the peroxisome and lipofuscin-body are electron-dense; the latter, however, is more granular and is surrounded by a double membrane, while the peroxisome has a single membrane. • 51,750

Fig. 18, Gerbil myocardium incubated in the presence of 0.02 M 1,2,4-triaminotriazole. Here the lipofuscin-body retains its electron density, whiletheperoxisomeexhibits no reaction product, x 36,180

Fig. 19. Aminotriazole control preparation of rat myocardium showing 2 microbodies (MB) identifiable by their single limiting membrane and finely granular matrix. Note the membranous tail attached to one microbody (arrows). x 54,900

Fig. 20. A unique example of a crystalline inclusion in the matrix of a myocardial microbody. The 80 A periodicity and positive DAB reaction suggest that this is a catalase crystal. Rabbit left ventricle. • 78,300


Captions see p. 473


Captions see p. 473

476

Table 2. Catalase activity in homogenates of left ventricles of various rodents

L. Hicks and H.D. Fahimi

Animal Catalase IU/mg protein

Rat 25 -+ 2 Rabbit 79 -+ 2 Guinea pig 57 -+ 5 Gerbil 78 -+ 12

14). Membranous tails at the periphery of some particles were observed, which suggested possible direct continuity with the SR (Figs. 9-12). Although the extensions were usually void of reaction product, in rare instances a trace of electron- dense material was found in them (Fig. 12).

Measurement of 75-100 particles per species indicated that although there is a slight difference in size, the microbodies are generally oval-shaped with dimen- sions in the 0.2-0.5 ~t range (Table 1). The ratio of length to width varied from 1.5 in the rat, rabbit, and Guinea pig to 2.4 in the Macaca java. The shape of the Macaca java microbodies was particularly interesting in that they were often elongated with one or more constrictions (Figs. 2, 4, 7, 8, 15). In other species occasionally two or more microbodies were observed close to each other, suggesting possible interconnections (Fig. 16).

Of the various cytoplasmic inclusions in the heart, the lipofuscin bodies containing an electron-dense matrix somewhat resembled the positively reacted microbodies (Fig. 17). In control preparations, however, the reaction in microbodies was abolished, but the lipofuscin bodies remained electron-dense (Fig. 18). In such aminotriazole controls the microbodies could be recognized by their location at the junction of A and I bands and by their typical appearance with a finely granular matrix bounded by a single membrane (Figs. 18, 19). No evidence of crystalline nucleoids was observed in the myocardial microbodies, except for a single instance in the rabbit heart,.where a DAB-positive crystal with a periodicity of approximately 80 A partially filling the matrix was found (Fig. 20).

The results of biochemical determination of catalase activity in rodent hearts is presented in Table 2, which confirms the presence of this enzyme in myocardial tissue of all the examined species.

Discussion

The results of this study have established the occurrence of catalase-positive particles in the ventricular myocardium of two primate and several rodent species. These particles appear identical to the microbodies or peroxisomes described recently in mouse (Herzog and Fahimi, 1974, 1976) and rat (Hand, 1975) hearts, and it seems likely that they are a constituent organelle of all mammalian cardiac muscle cells. In control preparations they exhibit a finely granular matrix surrounded by a single membrane but lack the crystalline nucleoids (for Fig. 20 see discussion below) typical for liver and kidney microbodies (Hruban and Rechcigl, 1969). The Novikoffs (1972) introduced the term "microperoxisome" for such catalase-positive particles that lack crystalline nucleoids, but de Duve (1973)

Peroxisomes in the Mammal ian Heart 477

has cautioned that ifcatalase as well as oxidases were localized in the same particle, it should essentially be considered a true peroxisome. Thus the particles in small intestine for which the designation "microperoxisome" was originally introduced (Novikoff and Novikoff, 1972) were shown by Connock et al. (1974) to contain catalase and D-amino-acid oxidase. Similarly, in the mouse heart Herzog and Fahimi (1976) found the presence of catalase and D-amino-acid- and L-a-hydroxy- acid-oxidases in the microsomal fraction and showed that this fraction contained large numbers of catalase-positive particles, thereby indicating the presence of true peroxisomes in this tissue.

In the present study biochemical evidence of catalase activity was found in the mouse heart of all rodent species (Table 2), thus confirming the results of cytochemical observations and the specificity of the DAB-method used. The oxidases, however, were not measured because of their low levels of activity, as known from the mouse heart, and due to the lack of a sufficiently sensitive method (Herzog and Fahimi, 1976). The close ultrastructural and cytochemical resem- blance of these particles to those in the mouse heart nevertheless justifies the assumption that they also represent true peroxisomes. Further studies on the localization of oxidases in myocardial peroxisomes and the assessment of their substrate preference and specificity could clarify the exact biological function of these particles in the myocardium. A recent improvement by Hand (1975) of the cytochemical method for oxidases of Shnitka and Talibi (1971) should be use- ful in such future studies.

The failure of previous investigators to recognize the existence of peroxisomes in the myocardium is probably due to their small size and similarity of their contents with the profiles of sarcoplasmic reticulum. Indeed, Hand (1974) pointed out that in unreacted preparations of striated muscle it was virtually impossible to differentiate peroxisomes from dilated cisternae of SR. Our control preparations show the indistinctive appearance ofperoxisomes in the absence of a positive DAB-reaction; nevertheless, their finely granular matrix surrounded by a distinct limiting membrane (Figs. 18, 19) and their location at the junction of A and I bands help to distinguish them from other cytoplasmic organelles. As noted, the myocardial peroxisomes lack crystalline nucleoids, but in one instance (Fig. 20) a DAB-positive crystal with a periodicity of approximately 80 A was found in the heart of a rabbit. The periodicity and the positive DAB reaction strongly suggested that this was a catalase crystal. Similar crystalline inclusions of catalase have been described in plant microbodies (Vigil, 1973) but are unusual for mammalian peroxisomes.

Association with Sarcoplasmic Reticulum

One of the well-known morphological features of microbodies has been their close association with the endoplasmic reticulum (ER). Especially in regenerating (Novikoff and Shin, 1964) and fetal (Essner, 1967) livers and after treatment with various hypolipidemic drugs (Reddy and Svoboda, 1971) direct continuities between the limiting membranes of microbodies and the ER have been described. Recently Gulyas and Yuan (1975) emphasized that in corpus luteum peroxisomes


were continuous with membranes of smooth but not rough ER. In a recent study from our laboratory (Fahimi et al., 1976) it was also shown that in a cell-type which contains only rough ER but lacks smooth ER, such as the hepatic Kupffer cells (Wisse, 1974), continuities between ER and microbodies are absent. Since the SR of myocardial cells is for the most part of the smooth variety, one might expect to find direct continuities between the two organelles in this tissue. We indeed found close associations between SR and peroxisomes in addition to peroxisomes with tail-like extensions void ofcatalase reaction product (Figs. 9-12, 19). The demonstration of definite continuity between the two organelles, however, requires specialized techniques such as serial sectioning and tilting of sections on a goniometer stage (Novikoff and Novikoff, 1972). Such studies are now in progress to clarify the question of direct continuities between the myocardial peroxisomes and the membranes of the neighboring organelles.

An interesting aspect of the association of peroxisomes with SR was their consistent presence near a specialized region of SR, which is known as the junctional SR (Waugh and Sommer, 1974; Sommer and Waugh, 1976), or the terminal cisterna (McNutt and Fawcett, 1974). These are segments of SR which are located beneath the sarcolemma or its intracellular extensions, the T-tubules, and which are periodically attached to the latter by poorly defined globular densities re- ferred to as junctional processes (Waugh and Sommer, 1974; Sommer and Waugh, 1976) or simply "SR feet" (Franzini-Armstrong, 1970). It is generally believed that the coupling of excitation to contraction takes place at the level of these junc- tions, but elaborate studies of Franzini-Armstrong (1975) have ruled out the existence of specialized junctional complexes similar to the low capacitance junc- tions of epithelial cells at these sites.

The important role of sarcoplasmic reticulum in myocardial lipid metabolism was revealed by Stein and Stein (1968), who also suggested that the junctional SR may be the site of esterification of fatty acids. The close association of peroxisomes with such junctional complexes points to the possible participation of these particles in transport and metabolism of lipids in the heart.

Participation in Lipid Metabolism

Fatty acids are the principal metabolic fuel of the adult mammalian heart (Opie, 1968). Because there is some circumstantial evidence suggesting involvement of peroxisomes in lipid metabolism, these particles could have an important metabolic function in the heart.

Of the various enzymes known to occur in peroxisomes, two deserve special attention: carnitine acetyl transferase (CAT) and NAD-e-glycerophosphate dehydrogenase. Markwell et al. (1973) have shown that in rat liver 10-15~ of CAT is localized in peroxisomes. In mitochondria this enzyme transfers acetyl moieties bound to Co-A across the inner mitochondrial membrane, but its function in liver peroxisomes is not known. It has been suggested that it may serve as a reservoir for acetyl groups or for Co-A (Markwell et al., 1973). The presence of high levels of CAT in the mammalian heart has been demonstrated (Neely and Morgan, 1974), and it has been shown that addition of carnitine to tissue culture preparations of


chicken heart cells stimulates markedly (30-70~) the rate of fatty acid oxidation (Rosenthal and Warshaw, 1973). Although it is not known whether any of the CAT activity in the heart is associated with the peroxisomes, its presence in this organelle could help to explain the close morphological association seen between peroxisomes and lipid droplets (Figs. 7, 8) on the one hand and between the peroxisomes and mitochondria (Figs. 13, 14) on the other.

Another enzyme localized in peroxisomes is the NAD-linked e-glycerophosphate dehydrogenase (Gee et al., 1974). This enzyme, most probably involved in lipid degradation rather than synthesis, converts glycerophosphate to dehydroxy- acetone phosphate. The possible participation of peroxisomes in degradation of stored lipids was suggested recently by Gulyas and Yuan (1975) on the basis of electron micrographs showing the partial indentation of lipid droplets in areas adjacent to peroxisomes. Similar observations were made in the present study (Figs. 7, 8).

Other evidence suggesting involvement of peroxisomes in lipid metabolism are the observations that clofibrate and other hypolipidemic drugs cause marked increase in the microbody profiles of liver parenchymal cells (Reddy et al., 1974; Reddy and Krishnakantha, 1975), and the occurrence of large numbers of microbodies in cells actively metabolizing lipids, such as steroid-secreting cells (Reddy and Svoboda, 1972; Black and Bogart, 1973) and the cells of brown and white adipose tissue (Ahlabo and Barnard, 1971).

De Duve and Baudhuin (1966) suggested that peroxisomes may be involved in gluconeogenesis. Indeed, in fatty seedlings peroxisomes are involved in the conversion of lipids to carbohydrates (Vigil, 1973). Although in higher verte- brates it is generally reported that such a conversion does not occur, Hand (1973) recently mentioned that acinar cells of rat parotid are capable of converting cytoplasmic fat droplets into glycogen. In this respect the c~-Keto acids produced by the oxidases of peroxisomes could be implicated as the source for the formation of glycogen. Since cardiac glycogen is mobilized mainly under extreme conditions of anoxia (Opie, 1968), further investigation of the function of peroxisomes and their role in the metabolism of glycogen in the heart should be of great interest in the elucidation of the pathophysiology of ischemic heart disease.

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Accepted August 22, 1976

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Peroxisomes (microbodies) in the myocardium of rodents and primates