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American Journal of Pathology, Vol. 130, No. 2, February 1988 Copyright © American Association of Pathologists

Selective Morphologic Alterations of the Cardiac Conduction System in Calves Deficient in Vitamin E and Selenium

SEAMUS KENNEDY, MVB, PhD, and DESMOND A. RICE, MVB, PhD

From the Departments of Pathology and Biochemistry, Veterinary Research Laboratories, Belfast, Northern Ireland

Vitamin E and selenium (E-Se) deficiency causes necrosis of the contractile myocardium in many species but does not usually affect the cells of the conduction system. In the present study, experimental E-Se deficiency in cattle produced preferential degeneration and necrosis of Purkinje cardiocytes. Calves fed deficient diets for 127-137 days had sublethal damage characterized histologically by sarcoplasmic accumulation of lipopigment granules; ultrastructurally, these granules corresponded to cytolysosomes that had a heterogeneous ultrastructure. Alterations in necrotic cells included mitochondrial mineralization, sarcoplasmic condensation, and plasmalemmal fragmentation. Necrosis of Purkinje cells was followed by macrophagic penetration ofthe external lamina, phagocytosis of ne-

crotic sarcoplasm, and repair by fibrosis. Furthermore, E-Se depletion of calves resulted in only minimal alterations in the contractile myocardium. In contrast, feeding supplements of polyunsaturated fatty acids to E-Se-deficient calves intensified the Purkinje cell damage but also resulted in widespread degeneration and necrosis ofthe contractile myocardium. Accumulation of lipopigment supports a pathogenetic role for lipoperoxidation in development of the cardiac lesions of E-Se deficiency. These lesions constitute a unique example of preferential damage to Purkinje cardiocytes. This model offers an attractive method of studying damage and repair to the cardiac conduction system. (Am J Pathol 1988, 130:315 - 325)

VITAMIN E AND SELENIUM (E-Se) deficiency diseases of farm animals are of major economic importance in many countries of the world. They are characterized by cardiac and skeletal myonecrosis and a variety of other lesions depending on species. There have been numerous reports of cardiac lesions in many species fed either natural or experimental E-Se-deficient diets. '9 Most of these reports, document degeneration and necrosis of the contractile myocardium but, with few exceptions,'s'3 indicate that Purkinje cardiocytes are unaffected by E-Se deficiency. Nutritional degenerative myopathy commonly develops in E-Se-deficient cattle after access to pasture in the spring. It is characterized by skeletal and cardiac myonecrosis. We have developed an experimental model to reproduce this disease.'4 It is based on feeding an E-Se-deficient diet for several months, followed by feeding polyunsaturated fatty acids (PUFA) to simulate ingestion of large quantities of linolenic acid as found in lush spring grass. Degeneration and

necrosis of Purkinje cardiocytes are consistent findings in this model.'S,16 This paper describes histopathologic and ultrastructural features of the induced alterations in these cells.

Materials and Methods Twenty 14-week-old male Friesian calves were divided into a principal group of 14 calves and a control group of 6 calves. The principal group was fed a low E-Se diet containing 1.0 mg/kg a-tocopherol and 0.03 mg/kg selenium; the control calves received a similar diet supplemented with 80 mg/kg a-tocopherol and 0.24 mg/kg selenium. Details of diet preparation, and clinical, biochemical, and electrocardiographic changes in the principal calves have been reAccepted for publication September 15, 1987. Address reprint requests to Seamus Kennedy, MVB, PhD, Veterinary Research Laboratories, Stoney Road, Stormont, Belfast BT4 3SD, N. Ireland.

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ported.'7-'9 Control calves were killed by intravenous pentobarbitone overdosage and exsanguination on Days 29, 65, 71, 117, 140, and 179. Two principal calves were killed on each of Days 127, 136, and 137. Linseed oil (54% linolenic acid) was "protected" against ruminal hydrogenation by coating droplets of the oil with formaldehyde-treated casein, and then introduced at a rate of 250 g daily as a source of PUFA to the low E-Se diet of the 8 remaining calves. Six of these calves were killed after 6-1 1 days of PUFA feeding; the other 2 calves died after 6 and 8 days of PUFA feeding, respectively. At necropsy, hearts were rapidly removed from all euthanatized calves, and small blocks were collected from the left and right ventricular and septal myocardium. Regions sampled included left and right branches ofthe bundle of His and peripheral Purkinje network from several widely spaced regions of the ventricular free walls. The blocks were trimmed into 1-mm cubes, fixed in cacodylate-buffered 4% glutaraldehyde, postfixed in osmium tetroxide in phosphate buffer, and embedded in Durcupan resin (EM Scope Lab plc, Kent, England). Semithin (1 -,u) sections were cut and stained with alkaline toluidine blue for light-microscopic examination. Ultrathin sections of selected blocks containing Purkinje cardiocytes were cut and stained with lead citrate and uranyl acetate and examined by electron microscopy. Portions of myocardium, including bundle branches and peripheral Purkinje network, were either quench-frozen or fixed in 10% neutral formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for histopathologic examination. In order to characterize sarcoplasmic granules in Purkinje cardiocytes of the principal calves, we performed the following tests. Selected unstained paraffin and cryostat sections were examined in plane-polarized and ultraviolet light or stained with periodic acid-Schiff (PAS) after diastase digestion, long Ziehl-Neelsen, Schmorl's ferric-ferricyanide, Masson-Fontana, Sudan black B, oil red 0, alizarin red S, Feulgen reaction, and Perl's Prussian blue. Unstained, deparaffinized (rehydrated) sections were checked for resistance to alkali (5% NaOH) and a chloroform-methanol mixture (2: 1 vol/vol) by immersion in the respective solutions for 1 hour at 37 C, followed by staining with H&E or examination in ultraviolet light.

Results

Histopathologic Changes E-Se-Deficient Calves-No PUFA Supplementation Many Purkinje cardiocytes in the hearts of the 6 E-Se-depleted calves killed after 126-137 days, con-

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tained multiple sarcoplasmic granules that appeared yellow-brown in hematoxylin and eosin-stained sections. These granules were acid-fast, PAS positive (diastase-resistant) (Figures 1 and 2) but did not stain with oil red 0 in paraffin-embedded or frozen sections. They were identified as lipopigment on the basis of their histologic and histochemical staining properties (Table 1). The granules were approximately spherical, with diameters ranging from 2 to 8 i. Several intracellular distribution patterns were seen, the most common of which was aggregation in the central region of the cell. A definite perinuclear distribution was seen when relatively small numbers of granules were present. When aggregations of large granules were present, the surrounding sarcoplasm was frequently vacuolated. Conduction cells of the bundle branches and peripheral Purkinje network were equally affected. Sarcoplasmic lipopigment granule accumulation and vacuolation were the only changes seen in the Purkinje cardiocytes of 1 E-Se-depleted calf. However, many Purkinje cardiocytes of the other 5 depleted calves had coagulative necrosis (Figure 3). Necrotic cells stained deeply with eosin and were shrunken, which resulted in prominent separation between the necrotic cells and apposed normal conduction cells or the surrounding peri-Purkinje connective tissue sheath. Necrotic cells were invaded by macrophages, and increased numbers of mononuclear cells were seen in the adjacent connective tissue sheath. Lipopigment-laden macrophages were frequently seen within necrotic cells and in the adjacent connective tissue sheath. Mild loss of conducting cardiocytes from Purkinje fiber tracts resulted in the tracts having a vacuolated appearance with little or no fibroblastic reaction, but severe loss was associated with replacement fibrosis. Surviving cardiocytes in fibrosed tracts were frequently atrophic. Lesions were widely distributed throughout all myocardial sections examined. There was no evidence of regeneration of surviving

cardiocytes. Degeneration and necrosis of a few contractile cardiocytes were also seen throughout the myocardia of the E-Se-depleted calves, but these changes were relatively mild and infrequent.

E-Se-Deficient Calves-PUFA Supplementation Widespread degeneration and necrosis of Purkinje and contractile cardiocytes were seen in the hearts of the 8 calves fed PUFA. Purkinje cell lesions in these calves were qualitatively similar to those in the 6 calves that did not receive PUFA but were much more severe, as indicated by a higher proportion of necrotic

PURKINJE CARDIOCYTE DAMAGE

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