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Hypertrophic Cardiomyopathy in Pigs: Quantitative Pathologic Features in 55 Cases S i - k w a n g Liu, D V M , PhD, Y. T. Chiu, D V M , MS, J. J. Shyu, D V M , PhD, Stephen M . Factor, M D , R e d m a n C h u , D V M , PhD, J. H. Lin, D V M , MS, H. L. H s o u , MS, P. R. Fox, D V M , and P. C. Yang, D V M , P h D Pig Research Institute, Taiwan, Republic of China, The Animal Medical Center, New York, and Albert Einstein College of Medicine, Bronx, New York
++ Naturally occurring hypertrophic cardiomyopathy (HCM) was diagnosed in 55 purebred pigs 6 to 12 months of age. Ten (18%) of the pigs died suddenly during auction or shipment or were found dead by their keepers. The other 45 pigs failed to meet the criteria for breeding stock. Forty-six purebred and 64 hybrid pigs were studied for control. Heart weights were significantly heavier 07 < 0.001) in the pigs with HCM (473.5 + 31.8 g; heart weight [HW]/body weight [BW] ratio 4.6 + 0.7) than in the purebred (334.4 + 29.7 g; HW/BW 3.4 + 0.3) and hybrid (344.3 -I- 28.9 g; HW/BW 3.4 + 0.1) pigs without HCM. The ventricular septum (VS) in the 55 pigs with HCM was significantly thicker (26.0 + 3.1 mm; p < 0.001) than in the purebred (19.6 + 2.6 mm) and hybrid (14.1 ± 0.5 mm) pigs without HCM. The left ventricular free wall (LV) was significantly thicker 07 < 0.001) in the pigs with HCM (20.0 ± 2.7 ram) than in the purebred (18.1 + 2.1 mm) and hybrid (15.6 ± 0.3 mm) pigs without HCM. Asymmetric septal hypertrophy was evident because the ratio of VS to LV was significantly greater (p < 0.001) in the pigs with HCM (1.3 ± 0.2) than in the purebred (1.0 + 0.2) and hybrid (0.9 ± 0.01) pigs without HCM. The anterior portion of the VS appeared to bulge into and impinge upon the left ventricular outflow tract, in which a fibrotic endocardial plaque was often seen. Histologic features included marked muscle cell disorganization in the VS, LV, right ventricular free wall. Abnormal intramural coronary arteries and myocardial fibrosis were seen in most pigs with HCM. Silver impregnation stains showed that there were marked increases in perimysial coils, pericellular weaves, and cell-to-cell struts. Matrix disorientation was evident in the hearts with HCM. Quantitation revealed that the collagen protein in the hearts with HCM (23.8 ± 2.8/~g/mg protein) was significantly higher 07 < 0.001) than in the hearts of purebred (15.7 ± 1.8/~g/mg protein) and hybrid (13.9 ± 4.2/~g/mg protein) pigs without HCM. Total muscle protein in the hearts of the purebred pigs with (51.6 + 3.3 mg) and without (51.9 + 3.0 mg) HCM was not different; however, in hearts with HCM (51.6 ± 3.3 mg) it was significantly higher 07 < 0.001) than in those of hybrid pigs (47.6 + 4.4 mg) without HCM. There was 47 % to 52 % more stainable collagen in the hearts with HCM (44.7 ± 5.2 t~g collagen/mg protein) than in the purebred (30.3 ± 4.0/xg collagen/mg protein) and hybrid (28.3 ± 8.1/~g collagen/mg protein) hearts without HCM. Gross and histologic features and connective tissue abnormalities in the pigs with HCM were strikingly similar to those in humans, cats, and dogs with HCM. Thus we conclude that spontaneous porcine HCM presents a new and important model for the cardiovascular investigator.
Hypertrophic cardiomyopathy (HCM) is a primary cardiac disease in which the most characteristic morphologic features are a hypertrophied, nondilated left ventricle (1) and a myoManuscript received March 1, 1994; accepted June 30, 1994. Address for reprints: Si-kwang Liu, DVM, PhD, Pig Research Institute, P.O. Box 23, Chunan, Miaoli, Taiwan 35099, Republic of China, or The Animal Medical Center, 510 East 62nd Street, New York, NY 10021. Supported by the National Science Council, Department of Health, Council of Agriculture, Executive Yuan, Taipei, Taiwan, Republic of China. © 1994 by Elsevier Science Inc.
cardium typically showing bizarre and disorganized cellular architecture, abnormally small intramural coronary arteries, and increased amounts of matrix or replacement fibrosis (2-12). Although H C M has been a well-recognized and extensively studied clinical and pathologic entity in humans since 195 8 (2,5-9), a morphologically similar condition occurring spontaneously in cats (13-16) and dogs (15-17) has been identified recently. H C M also was described briefly in pigs 1054-8807/94/$'/.00
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(15, 18). This report describes in detail for the first time spontaneously occurring HCM in pigs that resembles HCM in humans, cats, and dogs.
Materials and Methods The study population consisted of 151 purebred pigs, of which 20 were excluded from the study because of inadequate data, and 64 hybrid pigs. The pigs came from a breeding farm, the Boar Testing Station, and the Nuclear Breeding Center of North Taiwan and were necropsied between December 1990 and December 1992. HCM was diagnosed in 55 of the 131 purebred pigs; 46 of the 76 purebred pigs and 64 hybrid pigs without HCM were studied for comparative purposes. Gross examination of the heart. After the entire heart was separated from the lung, removed from the thoracic cavity, and washed free of blood, it was fixed in 10% buffered formalin for 7 days. Ventricular slice, inflow and outflow, and four-chamber dissections were employed for gross examination of the heart (19). Ventricular septal thickness was measured in the anterior, median, and posterior septum, approximately one-third the distance between the aortic valve and apex. Thickness of the posterior left ventricular free wall was measured directly behind the midpoint of the posterior mitral valve leaflet and at the inferior extent of the mitral leaflets. The thickness of the anterior left ventricular free wall was measured approximately 2 cm lateral to the anterior descending coronary artery; that of the right ventricular free wall was measured near the tricuspid valve annulus. Care was taken to avoid including the trabeculae, papillary muscles, or the supraventricular crest in the measurement of wall thickness. Histologic examination of the heart. Transmural sections were taken perpendicular to the long axis of the left ventricle from (i) the ventricular septum, the area of maximal thickening, approximately one-third the distance between the aortic valve and left ventricular apex; (ii) the posterior left ventricular wall, approximately one-half the distance between the mitral valve annulus and left ventricular apex; and (iii) the anterior left ventricular wall, approximately 2 cm lateral to the anterior descending coronary artery. All tissue sections were embedded in paraffin, sectioned at a thickness of 6/xm, and stained with hematoxylin-eosin, Masson's trichrome, and VerhoelTs stains. The stained sections from each region were examined for muscle cell disorganization, myocardial fibrosis, and intramural coronary arterial abnormalities. Each of the lesions was graded independently, based on a three-point scale in which 0 = no pathologic abnormality, 1+ = mild pathologic changes, 2 + = moderate changes, and 3 + = severe changes.
Quantitative collagen and total protein determinations. A previously described method (20) that provides a quantitative and sensitive measure of collagen and total protein content in formalin-fixed and paraffin-embedded tissue sections was used. This method utilizes the selective binding of Sirius red F3BA to collagen and fast green FCF to noncol-
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lagenous protein when both are dissolved in aqueous saturated picric acid. When the dyes are eluated from the tissue sections with sodium hydroxide-methanol, the absorbances of both are obtained at 540 and 605 nm, respectively. When compared with color equivalences determined and verified previously with multiple tissue samples (20), the absorbances provide a relative measure of the collagen/protein ratio in given section. Because determination of collagen is relative to the concentration of protein per milligram for each tissue section analyzed, the thickness or area of the histologic preparation is not a significant variable. Matrix silver impregnation and histology. The myocardial interstitial matrix connective tissue is relatively invisible with routine histologic staining procedures, even with stains such as Masson's trichrome, which binds collagen fibers. We used a modification of a silver impregnation method originally described by del Rio Hortega and others for central nervous system histology (21,22). This technique employs thick (25-50/xm) frozen sections of formalin-fixed myocardium that are impregnated with silver. The silver binds selectively to several different components of the connective tissue matrix, as previously described by Factor and colleagues (11,23), among others. These components include struts that interconnect the lateral surfaces of myocytes, weaves that encircle the sarcolemma of individual myocytes as a mesh or grid, and perimysial coils that course as variably thick, spring-like structures in the interstitium between myocytes and are connected to them and to each other by struts. Thus cardiac muscle has a complex connective tissue matrix between and around individual myocytes; in addition, similar matrix components are iterated in ever-increasing complexity around larger groups of myocytes (fascicles) and myocardial layers. Statistics. All quantitative data are expressed as mean + standard deviation (SD). Differences between groups were evaluated with Student's t test. P values less than 0.05 were considered significant.
Results Clinical findings. Ten (18%) of the 55 purebred pigs with HCM died suddenly during auction of shipment or were found dead by their keepers. Six of the 10 pigs that died were male. The other 121 pigs failed to meet the criteria for breeding stock. Thirty-one (56%) of the 55 pigs with HCM were male, Table 1. Heart Weights, Body Weights, and Heart Weight to Body Weight Ratios in 165 Pigs Heart Weight (gm) Heart Weight BodyWeight to Body Weight (kg) (gin) (kg) Ratio HCM (55) 473.5 + 31.8" 104.2 + 7.1 4.6 + 0.7* Purebred (46) 334.4+ 29.7 98.8 5:8.2 3.4 5:0.3 Hybrid (64) 344.3 + 28.9 101.65:9.3 3.4 + 0.1 HCM = hypertrophiccardiomyopathy. * Significant differenceofp < 0.001.
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Table 2. Left Ventricular Wall Thickness in Pigs with and without Hypertrophic Cardiomyopathy HCM Purebred Hybrid
n
VS
LV
RV
VS/LV
55 46 64
26.4 + 3.1" 19.6 + 2.6 14.1 + 0.5
20.0 + 2.7* 18.1 + 2.1 15.6 + 0.3
8.9 + 2.3* 7.5 + 1.8 5.4 + 1.7
1.3 + 0.2* h 0 + 0.2 0.9 + 0.1
* Significant differenceofp < 0 . 0 0 1 . HCM = hypertrophiccardiomyopathy;VS = ventricularseptum; LV = left ventricular free wall; RV = right ventricular free wail.
16 (35%) of the 46 purebred pigs without HCM were male, and 31 (48%) of the 64 hybrid pigs without HCM were male. There was a higher incidence of HCM in the Landrace pig than in the other breeds. Of the 55 pigs with HCM, 44 (80%) were Landrace, 9 (16%) were Yorkshire, and 2 (4%) were Duroc. Of the 46 purebred pigs without HCM, 20 (43 %) were Landrace, 15 (33 %) were Yorkshire, and 11 (24 % ) were Duroc.
Gross anatomic findings. The hearts were significantly heavier (p < 0.001) in the 55 pigs with HCM (473.5 5:31.7 g; range 396-619 g; heart weight/body weight ratio [HW/BW] 4.6 + 0.7) than in the purebred (334.4 + 29.7 g; HW/BW, 3.4) and hybrid (344.3 + 28.9 g; HW/BW 3.4 -t- 0.1) pigs without HCM (Table 1). The ventricular septum (VS) in the pigs with HCM (26.4 + 3.1 mm) was significantly thicker (p < 0.001) than in the purebred (19.6 + 2.6 mm) and hybrid
Figure 1. Gross morphologic features of hearts from pigs with hypertrophic cardiomyopathy. (/%) Seven-month-old Duroc boar. Notice hypertrophy of anterior septum (arrow)and left ventricular free wall, bulging of anterior septum, and subendocardial fibrosis on the outflow tract (0). 01l) Six-month-old Yorkshire boar. Notice hypertrophy of ventricular septum (S) 26 mm and left ventricular free wall (L) 19 mm, thickening of anterior mitral leaflet, and fibrotic endocardial plaque on the outflow tract (arrow). Right ventricular free wall (R). (C) Five-month-old Landrace boar. Notice uniform hypertrophy of the anterior septum (S) and left ventricular free wall (L), and hypertrophy of supraventricular crest (arrow)bulging into the right ventricular (R) outflow tract.
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Figure 2. Distorted architecture of ventricular septum (A, B, and D) and left ventricular free wall (C) with hypertrophic cardiomyopathy. (A) Markedly disordered arrangement of small groups of muscle cells with adjacent cells arranged at acute angles to large groups of cells. There is intercellular fibrosis. (B) Cellular disorganizationin which broad bundles of muscle cells are arranged at oblique or perpendicular angles. Cells within these bundles are normally arranged. (C) Relatively narrow longitudinally cut bundles of cardiac muscle cells are interlaced among larger groups of transversally cut cells. (D) Cardiac muscle is embedded in a framework of fibrous connective tissue. (A-D: Hematoxylin-eosin stain, x 100.)
(14.1 5:0.5 mm) pigs without HCM. The left ventricular free wall (LV) was significantly thicker (p < 0.001) in the pigs with HCM (20.0 5:2.7 mm) than in the purebred (18.1 + 2.1 mm) and hybrid (15.6 5: 0.3) pigs without HCM. Asymmetrical septal hypertrophy was evident because the VS/LV ratio was significantly greater (p < 0.001) in the pigs with HCM (1.3 5: 0.2) than in the purebred (1.0 + 0.2) and hybrid (0.9 ___0.01)
pigs without HCM. The right ventricular free wall in the pigs with HCM (8.9 + 2.3 mm) was significantly thicker (p < 0.001) than in the purebred (7.5 5:1.8 mm) and hybrid (5.4 5: 1.7 ram) pigs without HCM (Table 2). The anterior portion of the ventricular septum appeared to bulge into and impinge upon the left ventricular outflow tract, in which a fibrotic endocardial plaque was often seen
Table 3. Histologic Findings in Pigs with and without Hypertrophic Cardiomyopathy Cellular Disorganization
With H C M Without H C M
Abnormal ICA
Abnormal Matrix Collagen
n
%
Grade
%
Grade
%
Grade
55 46
45 15
1.2 -i- 0.1" 0.5 + 0.1
84 4
1.6 + 0.1" 0.5 + 0.1
91 4
2.8 + 0.5* 0.4 + 0.1
ICA = Intramural coronary artery. Grades from 1 to 3. * Significant difference o f p < 0,001.
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Figure 3. Histologic abnormalities of intramural coronary arteries of left ventricular free wall (A) and ventricular septum at left ventricular outflow tract 011)with hypertrophic cardiomyopathy. (A) Thickening of intramural coronary arterial wall due to proliferation of smooth muscle ceils and fibrous tissue in the media, hyperplasia of endothelial ceils, and extremely narrow lumen. (Hematoxylineosin stain, x 200). fiB)Extreme endocardial thickening consistent with a septal contact lesion or fibrotic plaque. The adjacent' myocardium muscle cells are disorganized, and there are several abnormally thickened coronary arteries with narrowed or obliterated lumen and markedlythickenedwalls, which appeared to include intimal and medial proferation of smooth muscle cells. (Trichrome stain, x60).
(Figs. 1A and 1B). Hypertrophy of the anterior papillary muscle and thickening of the anterior mitral leaflet were also evident in the pigs with HCM (Figs. IA and 1B). Hypertrophy of the supraventricular crest, which appeared to bulge into and impinge on the right ventricular outflow tract (Fig. 1C), was observed in 13 (24%) of the 55 pigs with HCM.
LIU ET AL. HYPERTROPHIC CARDIOMYOPATHY IN PIGS
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I-listoiogic features. Normal parallel alignment of cardiac muscle cells was observed in 30 (55 %) of the 55 pigs with HCM and 38 (85%) of the 46 purebred pigs without HCM. The foci of broad bundles of cardiac muscle cells were arranged perpendicularly or obliquely to one another in 25 (45%) of the 55 pigs with and in 8 (15%) of the 46 pigs without HCM (Figs. 2A-2C). The area of cardiac muscle cell disorganization in the ventricular septum was significantly greater (p < 0.001; mean grades 1.2 + 0.1; range 0-3+) in the pigs with HCM than in the pigs without HCM (0.5 + 0.1; range 0 - 2 + ; Table 3). Abnormal intramural coronary arteries were characterized by both large size and thickened walls. The lumen appeared narrow and was occasionally obliterated. Thickening of the arterial wall was caused mainly by the proliferation of collagen and smooth muscle cells in the intimal and medial components (Figs. 3A and 3B). In a few obliterated arteries, there was extreme proliferation of elastic fibers, collagen, and smooth muscle cells in the intimal and medial layers of the arterial walls and irregular elastic membranes and endothelial hyperplasia in the obstructed lumens. Segmental, intimal proliferation of loose fibrous tissue was identified in some of the coronary small arteries. Abnormal intramural coronary arteries were identified in tissue sections from either the ventricular septum or the left ventricular free wall in 46 (84%) of the 55 pigs with and 2 (4%) of the 46 pigs without HCM. The arterial lesions were significantly more severe (p < 0.001; mean 1.6 + 0.1; range 0-3+) in the pigs with HCM than in the pigs without HCM (mean 0.5 + 0.1; range 0-2+). Subendocardial fibrosis was found in the ventricular septurn on the surface of the outflow tract in the pigs with HCM (Figure 3B). Subendocardial lesions were significantly more severe in 50 (91%) of 55 pigs with and in 4 (9%) of the pigs without HCM. Subendocardial lesions were significantly more severe in the pigs with HCM (p < 0.001; mean 1.4 + 0.1; range 0-3+) than in the purebred pigs without HCM (mean 0.3 + 0.1; range 0-2+). Myocardial fibrosis in the pigs with HCM was significantly more severe (p < 0.001; mean 1.0 + 0.1; range 0-3+) than in the pigs without HCM (mean 0.3 + 0.1; range 0-2+; Fig. 2D). As for collagen content, the data from the Sirius red and fast green elution studies are summarized in Table 4. The col-
Table 4. Subendocardial and Myocardial Fibrosis in Pigs with and without Hypertrophic Cardiomyopathy Subendocardial Fibrosis
With HCM Without HCM
Myocardial Fibrosis
n
%
Grade
%
Grade
55 46
91 9
1.4 5: 0.1" 0.3 5:0.1
47 3
1.0 + 0.1" 0.3 + 0.1
Grades from 1 to 3. * Significant difference of p < 0.001.
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lagen protein in the hearts of pigs with HCM (23.8 4- 2.8/~g/mg protein) was significantly higher (p < 0.001) than in the hearts of purebred pigs (15.7 4- 1.8 #g/rag protein) and hybrid pigs (13.9 4- 4.2/zg/mg protein) without HCM. Total muscle protein in purebred pigs with (51.6 4- 4.2 mg) and without HCM (51.9 4- 3.0 rag) was not different; however, total muscle protein in purebred pigs was greater (t7 < 0.001) than that in hybrid pigs (47.6 + 4.4 rag). There was 47% to 57% more stainable collagen in the hearts with HCM (44.7 4- 5.2/xg collagen/mg protein) than in the purebred (30.3 4- 4.0 #g/mg) and hybrid (28.3 4- 8.1/xg/mg) pigs without HCM (Table 3). The silver-impregnated HCM specimens showed marked increases in all components (coils, struts, and weaves) of the matrix, as well as evidence of matrix disorganization (Figs. 4A-4C). Matrix condensation was appreciated in areas of interstitial, replacement, and perivascular scarring that stained with trichrome. Other areas without pathologic fibrosis could still be recognized as fibrotic in the silver-impregnated sections, because scars were counterstained with the gold toning used in the del Rio Hortega method (Fig. 2B). In 50 (91%) of the 55 pigs with and 2 (4%) of the 46 pigs without HCM, there were marked increases in matrix connective tissue. Abnormal connective tissue matrix in the pigs with HCM (mean 2.8 + 0.5; range 0-3+) was significantly more severe (p >
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0.001) than in the pigs without HCM (mean 0.4 4- 0.1; range, 0-2+; Tables 4 and 5).
Discussion The present report describes the spontaneous occurrence of hypertrophic cardiomyopathy in pigs. Pathologic findings in these pigs are similar in many respects to the manifestations of hypertrophic cardiomyopathy in humans, cats, and dogs. First, each of pig with HCM showed marked cardiac hypertrophy- i.e., cardiac mass and mean ratio of heart weight to body weight in the pigs with HCM was significantly greater than in the pigs without HCM. Second, the mean ratios of septal to free wall thickness in the pigs judged to have hypertrophic cardiomyopathy were significantly greater than in pigs with normal hearts. It should be emphasized that the mean septal to free wall thickness ratio was 1.3 + 0.2 (60% > 1.3) in the 55 pigs with hypertrophic cardiomyopathy, thereby meeting the diagnostic criterion generally used to identify disproportionate septal thickening in humans (mean septal to free wall ratio, 1.3) (8) and in dogs and cats (i>1.1) (13,15-17). The uniform thickness of the anterior ventricular septum and left ventricular free wall seen in the pigs with HCM is not as commonly seen in cats, dogs, and human beings
Figure 4. Histologic abnormalities of matrix connective tissue in myocardiumwith hypertrophic cardiomyopathy.(A) Dense matrix connective tissue, representing increased perimysial coils running in different directions and associated with disarray of myocytes (Hortega silverstain, x200). (B) Increasedperimysialcoil and weave fibers of matrix connectivetissue spacesbetweenmyocytes(Hortega silver stain, x200). (C) Connective tissue whorl composed of stretched and coiled perimysial coils running in differentdirections. The straightenedcoils most likely reflectabnormal contractileforces in areas of myocyte disarray (Hortega silver stain, x300).
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LIU ET AU HYPERTROPHIC CARDIOMYOPATHYIN PIGS
Table 5. Collagen Determination in the Hearts of Pigs with and without Hypertrophic Cardiomyopathy Collagen Total Protein Protein n (~g) (mg) HCM 29 23.8 + 2.8* 51.6 + 3.3* Purebred 15 15.7 + 1.8 51.9 5:3.0 Hybrid 20 13.9 + 4.1 47.6 5:4.4 * Significantdifferenceof p < 0.001.
(2-8,16). Similarly, although anterior septal hypertrophy that encroached upon the left ventficular outflow tract, hypertrophy of the anterior papillary muscles, thickening of the anterior mitral leaflet, and fibrous endocardial plaque were commonly observed in the pigs with HCM, as they are in human beings with the obstructive form of HCM, they are uncommon features in cats and dogs (16,17) with the disease. The phenomenon of the hypertrophic ventricular septum-particularly in the supraventricular crest-bulging into and impinging upon the right ventricular outflow tract that was seen in the pigs with HCM has not been described in humans, cats, or dogs with the disease (3,4,7,8,16). Only 25 (45%) of the 55 pigs with hypertrophic cardiomyopathy showed marked disorganization of cardiac muscle cells in the ventricular septum and left ventricular free wall. This type of disorganization is typical of humans (2,16,24,25), cats (13,15,16), and dogs (15-17) with hypertrophic cardiomyopathy. However, the relative infrequency of this phenomenon in cats and dogs, and the higher frequency in human beings, underlies the fact that HCM in pigs is similar but not identical in all anatomic respects to hypertrophic cardiomyopathy in humans, cats, and dogs. In each pig with HCM, disorganization was predominantly of the type 1B pattern, in which broad bundles of cells were orientated at oblique and perpendicular angles; in humans, cats, and dogs with HCM, disorganization is predominantly of the type IA pattern, which involves multiple small foci of cell-to-cell disarray (5-7,13-17). Abnormal intramural coronary arteries (that is, larger and with thickened walls) were identified in tissue sections taken from either the septum or left ventricular free wall in 45 (84 %) of the 55 pigs with HCM. This vascular abnormality is frequently observed in humans, cats, and dogs with HCM but is uncommon in these species with other forms of heart disease. (12,16). Subendocardial fibrosis was found in the ventricular septurn on the surface of the outflow tract in 50 (91%) of the 55 pigs with HCM; this degree of involvement is more severe than that in humans (9), cats (16), and dogs (16) with the disease. The elution technique showed a 47 % to 57 % increase of measurable collagen in the hypertrophic cardiomyopathy specimens compared with hearts of either the purebred or hybrid pigs without HCM. This increase of stainable collagen in the
267
Collagen to Total Protein Ratio (/tg:mg) 44.7 + 5.2* 30.3 + 4.0 28.3 5:8.1
porcine hearts with HCM is similar to that in human and feline hearts with the disease (11,25). The silver-impregnated porcine specimens with HCM had increases in all the components of matrix (perimysial coils, cell-to-cell struts, and pericellular weaves) and also showed matrix disorganization, characteristic of human and feline hearts with the disease (11,25). Certain clinical features of HCM in the pigs described in this report are similar to those of humans, cats, and dogs with the disease (9-10,13-17). One point of striking similarity is the occurrence of sudden and unexpected death even in humans, cats, and dogs without previous symptomatic manifestations of cardiac disease (9-10,13-17). Further clinical, functional, and genetic evaluations are presently being carried out in order to characterize the porcine disease more completely. In conclusion, it is evident from the data presented in this report that a primary myocardial disease, with certain pathologic features similar to those of hypertrophic cardiomyopathy in humans, cats, and dogs, also occurs spontaneously in pigs. The existence of this porcine disease may prove to be a new important model in the investigation of human cardiomyopathies.
References 1. MaronBJ, Epstein SE. Hypertrophic cardiomyopathy: a discussion of nomenclature. Am J Cardiol 1979;43:1242-1244. 2. Teare D. Asymmetric hypertrophy of the heart in young adults. Br Heart J 1958;20:1-8. 3. Olsen EG. Anatomic and light microscopic characterization of hypertrophic obstructive and nonobstructive cardiomyopathy. Eur Heart J 1983;4(Suppl F): 1-8. 4. Ferrans VJ, Morrow AG, Roberts WC. Myocardial ultrastructure in idiopathic hypertrophic subaortic stenosis: a study of operatively excised left ventricular outflow tract muscle in 14 patients. Circulation 1972;45:769-792. 5. Maron BJ, Roberts WC. Hypertrophic cardiomyopathyand cardiac muscle cell disorganization revisited: relation between the two and significance. Am Heart J 1981;102:95-110. 6. Maron BJ, Anron TJ, Robert WC. Quantitative analysis of the distribution of cardiac muscle cell disorganization in the left ventricular wall of patients with hypertrophic cardiomyopathy. Circulation 1981;63: 882-894. 7. Maron BJ, Roberts WC. Quantitative analysis of cardiac muscle cell disorganization in the ventricular septum of patients with hypertrophic cardiomyopathy. Circulation 1979;59:689-706. 8. MaronBJ, Epstein SE. Hypertrophic cardiomyopathy: recent observa-
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tions regarding the specificity of three hallmarks of the disease: asymmetric hypertrophy, septal disorganization, and systolic anterior motion of anterior mitral leaflet. Am J Cardiol 1980;45:141-145. 9. Maron BJ, Bonow RO, Canon RD, Leon MB, Epstein SE. Hypertrophic cardiomyopathy: interrelation of clinical manifestations, pathophysiology and therapy. N Engl J Med 1987;316:780-789, 844-852. 10. McKenna WJ, Camm AJ. Sudden death in hypertrophic cardiomyopathy: assessment of patients at high risk. Circulation 1989;80:1489-1492. 11. Factor SM, Butany J, Sole MJ, Wigle ED, Williams WC, Rojkind M. Pathologic fibrosis and matrix connective tissue in the subaortic myocardium of patients with hypertrophic cardiomyopathy. J Am Coil Cardiol 1991;17:1343-1351. 12. Maron BJ, Wolfson JK, Epstein SE, Roberts WC. Intramural ("small vessel") coronary artery disease in hypertrophic cardiomyopathy. J Am Coil Cardiol 1986;8:545-557. 13. Liu SK, Maron BJ, Tilley LP. Feline hypertrophic cardiomyopathy: gross anatomic and quantitative histologic features. Am J Patho11981; 102:388-395. 14. Tilley LP, Liu SK, Gilbertson SR, Wagner BM, Ford PF. Primary myocardial disease in the cat. Am J Pathol 1977;87:493-522. 15. Liu SK, Hsu FS, Lee RCT. An Arias of Cardiovascular Pathology. Taiwan, ROC: Pig Research Institute, 1989. 16. Liu SK, Roberts WC, Maron BJ. Comparison ofmorphologic findings in spontaneously occurring hypertrophic cardiomyopathy in humans, cats and dogs. Am J Cardiol 1993;72:944-951.
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17. Liu SK, Maron BJ, Tilley LP. Hypertrophic cardiomyopathy in the dog. Am J Pathol 1979;94:497-508. 18. Liu SK, Chiu YT, Tsau HL, Shyu JJ, Lin JH. Morphologic features of hypertrophic cardiomyopathy in pigs. In: Third International Symposium on Pig Model for Biomedical Research. Talwan, ROC: Pig Research Institute, 1992:9-22. 19. Liu SK. Postmortem examination of the heart. Vet Clin North Am 1983;13:379-394. 20. Lopez DeLeon A, Rojkind A. A simple micromethod for collagen and total protein determination hi formalin-fixed paraffin-embedded sections. J Histochem Cytochem 1985;33:737-743. 21. delRioHortegaP. El metododel carbonatoargentico: revision general de sus tecnicas y aplicaciones en histologia normal y pathologia. Arch Histo Normo Patol Buenos Aires 1943;2:231-243. 22. Hasegawa T, Raven JK. A metallic impregnation method for the demonstration of cerebral vascular patterns. Acta Neuropathol (Bed) 1968; 10:183-188. 23. Robinson TF, Cohen-Gould L, Factor SM. Skeletal framework of mammalian heart muscle: arrangements of inter- and peri-cellular connective tissue. Lab Invest 1983;49:482--498. 24. Maron BJ. Asymmetry in hypertrophic cardiomyopathy: the septal to free wall thickness ratio revisited. Am J Cardiol 1985;55:835-838. 25. Liu SK, Chiu YT, Fox PR, Factor SM, Shyu JJ, Chu RM. Interstitial connective tissue abnormalities in feline cardiomyopathies. Lab Invest 1994;70:20A.
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Hypertrophic Cardiomyopathy in Pigs: Quantitative Pathologic Features in 55 Cases S i - k w a n g Liu, D V M , PhD, Y. T. Chiu, D V M , MS, J. J. Shyu, D V M , PhD, Stephen M . Factor, M D , R e d m a n C h u , D V M , PhD, J. H. Lin, D V M , MS, H. L. H s o u , MS, P. R. Fox, D V M , and P. C. Yang, D V M , P h D Pig Research Institute, Taiwan, Republic of China, The Animal Medical Center, New York, and Albert Einstein College of Medicine, Bronx, New York
++ Naturally occurring hypertrophic cardiomyopathy (HCM) was diagnosed in 55 purebred pigs 6 to 12 months of age. Ten (18%) of the pigs died suddenly during auction or shipment or were found dead by their keepers. The other 45 pigs failed to meet the criteria for breeding stock. Forty-six purebred and 64 hybrid pigs were studied for control. Heart weights were significantly heavier 07 < 0.001) in the pigs with HCM (473.5 + 31.8 g; heart weight [HW]/body weight [BW] ratio 4.6 + 0.7) than in the purebred (334.4 + 29.7 g; HW/BW 3.4 + 0.3) and hybrid (344.3 -I- 28.9 g; HW/BW 3.4 + 0.1) pigs without HCM. The ventricular septum (VS) in the 55 pigs with HCM was significantly thicker (26.0 + 3.1 mm; p < 0.001) than in the purebred (19.6 + 2.6 mm) and hybrid (14.1 ± 0.5 mm) pigs without HCM. The left ventricular free wall (LV) was significantly thicker 07 < 0.001) in the pigs with HCM (20.0 ± 2.7 ram) than in the purebred (18.1 + 2.1 mm) and hybrid (15.6 ± 0.3 mm) pigs without HCM. Asymmetric septal hypertrophy was evident because the ratio of VS to LV was significantly greater (p < 0.001) in the pigs with HCM (1.3 ± 0.2) than in the purebred (1.0 + 0.2) and hybrid (0.9 ± 0.01) pigs without HCM. The anterior portion of the VS appeared to bulge into and impinge upon the left ventricular outflow tract, in which a fibrotic endocardial plaque was often seen. Histologic features included marked muscle cell disorganization in the VS, LV, right ventricular free wall. Abnormal intramural coronary arteries and myocardial fibrosis were seen in most pigs with HCM. Silver impregnation stains showed that there were marked increases in perimysial coils, pericellular weaves, and cell-to-cell struts. Matrix disorientation was evident in the hearts with HCM. Quantitation revealed that the collagen protein in the hearts with HCM (23.8 ± 2.8/~g/mg protein) was significantly higher 07 < 0.001) than in the hearts of purebred (15.7 ± 1.8/~g/mg protein) and hybrid (13.9 ± 4.2/~g/mg protein) pigs without HCM. Total muscle protein in the hearts of the purebred pigs with (51.6 + 3.3 mg) and without (51.9 + 3.0 mg) HCM was not different; however, in hearts with HCM (51.6 ± 3.3 mg) it was significantly higher 07 < 0.001) than in those of hybrid pigs (47.6 + 4.4 mg) without HCM. There was 47 % to 52 % more stainable collagen in the hearts with HCM (44.7 ± 5.2 t~g collagen/mg protein) than in the purebred (30.3 ± 4.0/xg collagen/mg protein) and hybrid (28.3 ± 8.1/~g collagen/mg protein) hearts without HCM. Gross and histologic features and connective tissue abnormalities in the pigs with HCM were strikingly similar to those in humans, cats, and dogs with HCM. Thus we conclude that spontaneous porcine HCM presents a new and important model for the cardiovascular investigator.
Hypertrophic cardiomyopathy (HCM) is a primary cardiac disease in which the most characteristic morphologic features are a hypertrophied, nondilated left ventricle (1) and a myoManuscript received March 1, 1994; accepted June 30, 1994. Address for reprints: Si-kwang Liu, DVM, PhD, Pig Research Institute, P.O. Box 23, Chunan, Miaoli, Taiwan 35099, Republic of China, or The Animal Medical Center, 510 East 62nd Street, New York, NY 10021. Supported by the National Science Council, Department of Health, Council of Agriculture, Executive Yuan, Taipei, Taiwan, Republic of China. © 1994 by Elsevier Science Inc.
cardium typically showing bizarre and disorganized cellular architecture, abnormally small intramural coronary arteries, and increased amounts of matrix or replacement fibrosis (2-12). Although H C M has been a well-recognized and extensively studied clinical and pathologic entity in humans since 195 8 (2,5-9), a morphologically similar condition occurring spontaneously in cats (13-16) and dogs (15-17) has been identified recently. H C M also was described briefly in pigs 1054-8807/94/$'/.00
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(15, 18). This report describes in detail for the first time spontaneously occurring HCM in pigs that resembles HCM in humans, cats, and dogs.
Materials and Methods The study population consisted of 151 purebred pigs, of which 20 were excluded from the study because of inadequate data, and 64 hybrid pigs. The pigs came from a breeding farm, the Boar Testing Station, and the Nuclear Breeding Center of North Taiwan and were necropsied between December 1990 and December 1992. HCM was diagnosed in 55 of the 131 purebred pigs; 46 of the 76 purebred pigs and 64 hybrid pigs without HCM were studied for comparative purposes. Gross examination of the heart. After the entire heart was separated from the lung, removed from the thoracic cavity, and washed free of blood, it was fixed in 10% buffered formalin for 7 days. Ventricular slice, inflow and outflow, and four-chamber dissections were employed for gross examination of the heart (19). Ventricular septal thickness was measured in the anterior, median, and posterior septum, approximately one-third the distance between the aortic valve and apex. Thickness of the posterior left ventricular free wall was measured directly behind the midpoint of the posterior mitral valve leaflet and at the inferior extent of the mitral leaflets. The thickness of the anterior left ventricular free wall was measured approximately 2 cm lateral to the anterior descending coronary artery; that of the right ventricular free wall was measured near the tricuspid valve annulus. Care was taken to avoid including the trabeculae, papillary muscles, or the supraventricular crest in the measurement of wall thickness. Histologic examination of the heart. Transmural sections were taken perpendicular to the long axis of the left ventricle from (i) the ventricular septum, the area of maximal thickening, approximately one-third the distance between the aortic valve and left ventricular apex; (ii) the posterior left ventricular wall, approximately one-half the distance between the mitral valve annulus and left ventricular apex; and (iii) the anterior left ventricular wall, approximately 2 cm lateral to the anterior descending coronary artery. All tissue sections were embedded in paraffin, sectioned at a thickness of 6/xm, and stained with hematoxylin-eosin, Masson's trichrome, and VerhoelTs stains. The stained sections from each region were examined for muscle cell disorganization, myocardial fibrosis, and intramural coronary arterial abnormalities. Each of the lesions was graded independently, based on a three-point scale in which 0 = no pathologic abnormality, 1+ = mild pathologic changes, 2 + = moderate changes, and 3 + = severe changes.
Quantitative collagen and total protein determinations. A previously described method (20) that provides a quantitative and sensitive measure of collagen and total protein content in formalin-fixed and paraffin-embedded tissue sections was used. This method utilizes the selective binding of Sirius red F3BA to collagen and fast green FCF to noncol-
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lagenous protein when both are dissolved in aqueous saturated picric acid. When the dyes are eluated from the tissue sections with sodium hydroxide-methanol, the absorbances of both are obtained at 540 and 605 nm, respectively. When compared with color equivalences determined and verified previously with multiple tissue samples (20), the absorbances provide a relative measure of the collagen/protein ratio in given section. Because determination of collagen is relative to the concentration of protein per milligram for each tissue section analyzed, the thickness or area of the histologic preparation is not a significant variable. Matrix silver impregnation and histology. The myocardial interstitial matrix connective tissue is relatively invisible with routine histologic staining procedures, even with stains such as Masson's trichrome, which binds collagen fibers. We used a modification of a silver impregnation method originally described by del Rio Hortega and others for central nervous system histology (21,22). This technique employs thick (25-50/xm) frozen sections of formalin-fixed myocardium that are impregnated with silver. The silver binds selectively to several different components of the connective tissue matrix, as previously described by Factor and colleagues (11,23), among others. These components include struts that interconnect the lateral surfaces of myocytes, weaves that encircle the sarcolemma of individual myocytes as a mesh or grid, and perimysial coils that course as variably thick, spring-like structures in the interstitium between myocytes and are connected to them and to each other by struts. Thus cardiac muscle has a complex connective tissue matrix between and around individual myocytes; in addition, similar matrix components are iterated in ever-increasing complexity around larger groups of myocytes (fascicles) and myocardial layers. Statistics. All quantitative data are expressed as mean + standard deviation (SD). Differences between groups were evaluated with Student's t test. P values less than 0.05 were considered significant.
Results Clinical findings. Ten (18%) of the 55 purebred pigs with HCM died suddenly during auction of shipment or were found dead by their keepers. Six of the 10 pigs that died were male. The other 121 pigs failed to meet the criteria for breeding stock. Thirty-one (56%) of the 55 pigs with HCM were male, Table 1. Heart Weights, Body Weights, and Heart Weight to Body Weight Ratios in 165 Pigs Heart Weight (gm) Heart Weight BodyWeight to Body Weight (kg) (gin) (kg) Ratio HCM (55) 473.5 + 31.8" 104.2 + 7.1 4.6 + 0.7* Purebred (46) 334.4+ 29.7 98.8 5:8.2 3.4 5:0.3 Hybrid (64) 344.3 + 28.9 101.65:9.3 3.4 + 0.1 HCM = hypertrophiccardiomyopathy. * Significant differenceofp < 0.001.
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Table 2. Left Ventricular Wall Thickness in Pigs with and without Hypertrophic Cardiomyopathy HCM Purebred Hybrid
n
VS
LV
RV
VS/LV
55 46 64
26.4 + 3.1" 19.6 + 2.6 14.1 + 0.5
20.0 + 2.7* 18.1 + 2.1 15.6 + 0.3
8.9 + 2.3* 7.5 + 1.8 5.4 + 1.7
1.3 + 0.2* h 0 + 0.2 0.9 + 0.1
* Significant differenceofp < 0 . 0 0 1 . HCM = hypertrophiccardiomyopathy;VS = ventricularseptum; LV = left ventricular free wall; RV = right ventricular free wail.
16 (35%) of the 46 purebred pigs without HCM were male, and 31 (48%) of the 64 hybrid pigs without HCM were male. There was a higher incidence of HCM in the Landrace pig than in the other breeds. Of the 55 pigs with HCM, 44 (80%) were Landrace, 9 (16%) were Yorkshire, and 2 (4%) were Duroc. Of the 46 purebred pigs without HCM, 20 (43 %) were Landrace, 15 (33 %) were Yorkshire, and 11 (24 % ) were Duroc.
Gross anatomic findings. The hearts were significantly heavier (p < 0.001) in the 55 pigs with HCM (473.5 5:31.7 g; range 396-619 g; heart weight/body weight ratio [HW/BW] 4.6 + 0.7) than in the purebred (334.4 + 29.7 g; HW/BW, 3.4) and hybrid (344.3 + 28.9 g; HW/BW 3.4 -t- 0.1) pigs without HCM (Table 1). The ventricular septum (VS) in the pigs with HCM (26.4 + 3.1 mm) was significantly thicker (p < 0.001) than in the purebred (19.6 + 2.6 mm) and hybrid
Figure 1. Gross morphologic features of hearts from pigs with hypertrophic cardiomyopathy. (/%) Seven-month-old Duroc boar. Notice hypertrophy of anterior septum (arrow)and left ventricular free wall, bulging of anterior septum, and subendocardial fibrosis on the outflow tract (0). 01l) Six-month-old Yorkshire boar. Notice hypertrophy of ventricular septum (S) 26 mm and left ventricular free wall (L) 19 mm, thickening of anterior mitral leaflet, and fibrotic endocardial plaque on the outflow tract (arrow). Right ventricular free wall (R). (C) Five-month-old Landrace boar. Notice uniform hypertrophy of the anterior septum (S) and left ventricular free wall (L), and hypertrophy of supraventricular crest (arrow)bulging into the right ventricular (R) outflow tract.
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Figure 2. Distorted architecture of ventricular septum (A, B, and D) and left ventricular free wall (C) with hypertrophic cardiomyopathy. (A) Markedly disordered arrangement of small groups of muscle cells with adjacent cells arranged at acute angles to large groups of cells. There is intercellular fibrosis. (B) Cellular disorganizationin which broad bundles of muscle cells are arranged at oblique or perpendicular angles. Cells within these bundles are normally arranged. (C) Relatively narrow longitudinally cut bundles of cardiac muscle cells are interlaced among larger groups of transversally cut cells. (D) Cardiac muscle is embedded in a framework of fibrous connective tissue. (A-D: Hematoxylin-eosin stain, x 100.)
(14.1 5:0.5 mm) pigs without HCM. The left ventricular free wall (LV) was significantly thicker (p < 0.001) in the pigs with HCM (20.0 5:2.7 mm) than in the purebred (18.1 + 2.1 mm) and hybrid (15.6 5: 0.3) pigs without HCM. Asymmetrical septal hypertrophy was evident because the VS/LV ratio was significantly greater (p < 0.001) in the pigs with HCM (1.3 5: 0.2) than in the purebred (1.0 + 0.2) and hybrid (0.9 ___0.01)
pigs without HCM. The right ventricular free wall in the pigs with HCM (8.9 + 2.3 mm) was significantly thicker (p < 0.001) than in the purebred (7.5 5:1.8 mm) and hybrid (5.4 5: 1.7 ram) pigs without HCM (Table 2). The anterior portion of the ventricular septum appeared to bulge into and impinge upon the left ventricular outflow tract, in which a fibrotic endocardial plaque was often seen
Table 3. Histologic Findings in Pigs with and without Hypertrophic Cardiomyopathy Cellular Disorganization
With H C M Without H C M
Abnormal ICA
Abnormal Matrix Collagen
n
%
Grade
%
Grade
%
Grade
55 46
45 15
1.2 -i- 0.1" 0.5 + 0.1
84 4
1.6 + 0.1" 0.5 + 0.1
91 4
2.8 + 0.5* 0.4 + 0.1
ICA = Intramural coronary artery. Grades from 1 to 3. * Significant difference o f p < 0,001.
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Figure 3. Histologic abnormalities of intramural coronary arteries of left ventricular free wall (A) and ventricular septum at left ventricular outflow tract 011)with hypertrophic cardiomyopathy. (A) Thickening of intramural coronary arterial wall due to proliferation of smooth muscle ceils and fibrous tissue in the media, hyperplasia of endothelial ceils, and extremely narrow lumen. (Hematoxylineosin stain, x 200). fiB)Extreme endocardial thickening consistent with a septal contact lesion or fibrotic plaque. The adjacent' myocardium muscle cells are disorganized, and there are several abnormally thickened coronary arteries with narrowed or obliterated lumen and markedlythickenedwalls, which appeared to include intimal and medial proferation of smooth muscle cells. (Trichrome stain, x60).
(Figs. 1A and 1B). Hypertrophy of the anterior papillary muscle and thickening of the anterior mitral leaflet were also evident in the pigs with HCM (Figs. IA and 1B). Hypertrophy of the supraventricular crest, which appeared to bulge into and impinge on the right ventricular outflow tract (Fig. 1C), was observed in 13 (24%) of the 55 pigs with HCM.
LIU ET AL. HYPERTROPHIC CARDIOMYOPATHY IN PIGS
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I-listoiogic features. Normal parallel alignment of cardiac muscle cells was observed in 30 (55 %) of the 55 pigs with HCM and 38 (85%) of the 46 purebred pigs without HCM. The foci of broad bundles of cardiac muscle cells were arranged perpendicularly or obliquely to one another in 25 (45%) of the 55 pigs with and in 8 (15%) of the 46 pigs without HCM (Figs. 2A-2C). The area of cardiac muscle cell disorganization in the ventricular septum was significantly greater (p < 0.001; mean grades 1.2 + 0.1; range 0-3+) in the pigs with HCM than in the pigs without HCM (0.5 + 0.1; range 0 - 2 + ; Table 3). Abnormal intramural coronary arteries were characterized by both large size and thickened walls. The lumen appeared narrow and was occasionally obliterated. Thickening of the arterial wall was caused mainly by the proliferation of collagen and smooth muscle cells in the intimal and medial components (Figs. 3A and 3B). In a few obliterated arteries, there was extreme proliferation of elastic fibers, collagen, and smooth muscle cells in the intimal and medial layers of the arterial walls and irregular elastic membranes and endothelial hyperplasia in the obstructed lumens. Segmental, intimal proliferation of loose fibrous tissue was identified in some of the coronary small arteries. Abnormal intramural coronary arteries were identified in tissue sections from either the ventricular septum or the left ventricular free wall in 46 (84%) of the 55 pigs with and 2 (4%) of the 46 pigs without HCM. The arterial lesions were significantly more severe (p < 0.001; mean 1.6 + 0.1; range 0-3+) in the pigs with HCM than in the pigs without HCM (mean 0.5 + 0.1; range 0-2+). Subendocardial fibrosis was found in the ventricular septurn on the surface of the outflow tract in the pigs with HCM (Figure 3B). Subendocardial lesions were significantly more severe in 50 (91%) of 55 pigs with and in 4 (9%) of the pigs without HCM. Subendocardial lesions were significantly more severe in the pigs with HCM (p < 0.001; mean 1.4 + 0.1; range 0-3+) than in the purebred pigs without HCM (mean 0.3 + 0.1; range 0-2+). Myocardial fibrosis in the pigs with HCM was significantly more severe (p < 0.001; mean 1.0 + 0.1; range 0-3+) than in the pigs without HCM (mean 0.3 + 0.1; range 0-2+; Fig. 2D). As for collagen content, the data from the Sirius red and fast green elution studies are summarized in Table 4. The col-
Table 4. Subendocardial and Myocardial Fibrosis in Pigs with and without Hypertrophic Cardiomyopathy Subendocardial Fibrosis
With HCM Without HCM
Myocardial Fibrosis
n
%
Grade
%
Grade
55 46
91 9
1.4 5: 0.1" 0.3 5:0.1
47 3
1.0 + 0.1" 0.3 + 0.1
Grades from 1 to 3. * Significant difference of p < 0.001.
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lagen protein in the hearts of pigs with HCM (23.8 4- 2.8/~g/mg protein) was significantly higher (p < 0.001) than in the hearts of purebred pigs (15.7 4- 1.8 #g/rag protein) and hybrid pigs (13.9 4- 4.2/zg/mg protein) without HCM. Total muscle protein in purebred pigs with (51.6 4- 4.2 mg) and without HCM (51.9 4- 3.0 rag) was not different; however, total muscle protein in purebred pigs was greater (t7 < 0.001) than that in hybrid pigs (47.6 + 4.4 rag). There was 47% to 57% more stainable collagen in the hearts with HCM (44.7 4- 5.2/xg collagen/mg protein) than in the purebred (30.3 4- 4.0 #g/mg) and hybrid (28.3 4- 8.1/xg/mg) pigs without HCM (Table 3). The silver-impregnated HCM specimens showed marked increases in all components (coils, struts, and weaves) of the matrix, as well as evidence of matrix disorganization (Figs. 4A-4C). Matrix condensation was appreciated in areas of interstitial, replacement, and perivascular scarring that stained with trichrome. Other areas without pathologic fibrosis could still be recognized as fibrotic in the silver-impregnated sections, because scars were counterstained with the gold toning used in the del Rio Hortega method (Fig. 2B). In 50 (91%) of the 55 pigs with and 2 (4%) of the 46 pigs without HCM, there were marked increases in matrix connective tissue. Abnormal connective tissue matrix in the pigs with HCM (mean 2.8 + 0.5; range 0-3+) was significantly more severe (p >
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0.001) than in the pigs without HCM (mean 0.4 4- 0.1; range, 0-2+; Tables 4 and 5).
Discussion The present report describes the spontaneous occurrence of hypertrophic cardiomyopathy in pigs. Pathologic findings in these pigs are similar in many respects to the manifestations of hypertrophic cardiomyopathy in humans, cats, and dogs. First, each of pig with HCM showed marked cardiac hypertrophy- i.e., cardiac mass and mean ratio of heart weight to body weight in the pigs with HCM was significantly greater than in the pigs without HCM. Second, the mean ratios of septal to free wall thickness in the pigs judged to have hypertrophic cardiomyopathy were significantly greater than in pigs with normal hearts. It should be emphasized that the mean septal to free wall thickness ratio was 1.3 + 0.2 (60% > 1.3) in the 55 pigs with hypertrophic cardiomyopathy, thereby meeting the diagnostic criterion generally used to identify disproportionate septal thickening in humans (mean septal to free wall ratio, 1.3) (8) and in dogs and cats (i>1.1) (13,15-17). The uniform thickness of the anterior ventricular septum and left ventricular free wall seen in the pigs with HCM is not as commonly seen in cats, dogs, and human beings
Figure 4. Histologic abnormalities of matrix connective tissue in myocardiumwith hypertrophic cardiomyopathy.(A) Dense matrix connective tissue, representing increased perimysial coils running in different directions and associated with disarray of myocytes (Hortega silverstain, x200). (B) Increasedperimysialcoil and weave fibers of matrix connectivetissue spacesbetweenmyocytes(Hortega silver stain, x200). (C) Connective tissue whorl composed of stretched and coiled perimysial coils running in differentdirections. The straightenedcoils most likely reflectabnormal contractileforces in areas of myocyte disarray (Hortega silver stain, x300).
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LIU ET AU HYPERTROPHIC CARDIOMYOPATHYIN PIGS
Table 5. Collagen Determination in the Hearts of Pigs with and without Hypertrophic Cardiomyopathy Collagen Total Protein Protein n (~g) (mg) HCM 29 23.8 + 2.8* 51.6 + 3.3* Purebred 15 15.7 + 1.8 51.9 5:3.0 Hybrid 20 13.9 + 4.1 47.6 5:4.4 * Significantdifferenceof p < 0.001.
(2-8,16). Similarly, although anterior septal hypertrophy that encroached upon the left ventficular outflow tract, hypertrophy of the anterior papillary muscles, thickening of the anterior mitral leaflet, and fibrous endocardial plaque were commonly observed in the pigs with HCM, as they are in human beings with the obstructive form of HCM, they are uncommon features in cats and dogs (16,17) with the disease. The phenomenon of the hypertrophic ventricular septum-particularly in the supraventricular crest-bulging into and impinging upon the right ventricular outflow tract that was seen in the pigs with HCM has not been described in humans, cats, or dogs with the disease (3,4,7,8,16). Only 25 (45%) of the 55 pigs with hypertrophic cardiomyopathy showed marked disorganization of cardiac muscle cells in the ventricular septum and left ventricular free wall. This type of disorganization is typical of humans (2,16,24,25), cats (13,15,16), and dogs (15-17) with hypertrophic cardiomyopathy. However, the relative infrequency of this phenomenon in cats and dogs, and the higher frequency in human beings, underlies the fact that HCM in pigs is similar but not identical in all anatomic respects to hypertrophic cardiomyopathy in humans, cats, and dogs. In each pig with HCM, disorganization was predominantly of the type 1B pattern, in which broad bundles of cells were orientated at oblique and perpendicular angles; in humans, cats, and dogs with HCM, disorganization is predominantly of the type IA pattern, which involves multiple small foci of cell-to-cell disarray (5-7,13-17). Abnormal intramural coronary arteries (that is, larger and with thickened walls) were identified in tissue sections taken from either the septum or left ventricular free wall in 45 (84 %) of the 55 pigs with HCM. This vascular abnormality is frequently observed in humans, cats, and dogs with HCM but is uncommon in these species with other forms of heart disease. (12,16). Subendocardial fibrosis was found in the ventricular septurn on the surface of the outflow tract in 50 (91%) of the 55 pigs with HCM; this degree of involvement is more severe than that in humans (9), cats (16), and dogs (16) with the disease. The elution technique showed a 47 % to 57 % increase of measurable collagen in the hypertrophic cardiomyopathy specimens compared with hearts of either the purebred or hybrid pigs without HCM. This increase of stainable collagen in the
267
Collagen to Total Protein Ratio (/tg:mg) 44.7 + 5.2* 30.3 + 4.0 28.3 5:8.1
porcine hearts with HCM is similar to that in human and feline hearts with the disease (11,25). The silver-impregnated porcine specimens with HCM had increases in all the components of matrix (perimysial coils, cell-to-cell struts, and pericellular weaves) and also showed matrix disorganization, characteristic of human and feline hearts with the disease (11,25). Certain clinical features of HCM in the pigs described in this report are similar to those of humans, cats, and dogs with the disease (9-10,13-17). One point of striking similarity is the occurrence of sudden and unexpected death even in humans, cats, and dogs without previous symptomatic manifestations of cardiac disease (9-10,13-17). Further clinical, functional, and genetic evaluations are presently being carried out in order to characterize the porcine disease more completely. In conclusion, it is evident from the data presented in this report that a primary myocardial disease, with certain pathologic features similar to those of hypertrophic cardiomyopathy in humans, cats, and dogs, also occurs spontaneously in pigs. The existence of this porcine disease may prove to be a new important model in the investigation of human cardiomyopathies.
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tions regarding the specificity of three hallmarks of the disease: asymmetric hypertrophy, septal disorganization, and systolic anterior motion of anterior mitral leaflet. Am J Cardiol 1980;45:141-145. 9. Maron BJ, Bonow RO, Canon RD, Leon MB, Epstein SE. Hypertrophic cardiomyopathy: interrelation of clinical manifestations, pathophysiology and therapy. N Engl J Med 1987;316:780-789, 844-852. 10. McKenna WJ, Camm AJ. Sudden death in hypertrophic cardiomyopathy: assessment of patients at high risk. Circulation 1989;80:1489-1492. 11. Factor SM, Butany J, Sole MJ, Wigle ED, Williams WC, Rojkind M. Pathologic fibrosis and matrix connective tissue in the subaortic myocardium of patients with hypertrophic cardiomyopathy. J Am Coil Cardiol 1991;17:1343-1351. 12. Maron BJ, Wolfson JK, Epstein SE, Roberts WC. Intramural ("small vessel") coronary artery disease in hypertrophic cardiomyopathy. J Am Coil Cardiol 1986;8:545-557. 13. Liu SK, Maron BJ, Tilley LP. Feline hypertrophic cardiomyopathy: gross anatomic and quantitative histologic features. Am J Patho11981; 102:388-395. 14. Tilley LP, Liu SK, Gilbertson SR, Wagner BM, Ford PF. Primary myocardial disease in the cat. Am J Pathol 1977;87:493-522. 15. Liu SK, Hsu FS, Lee RCT. An Arias of Cardiovascular Pathology. Taiwan, ROC: Pig Research Institute, 1989. 16. Liu SK, Roberts WC, Maron BJ. Comparison ofmorphologic findings in spontaneously occurring hypertrophic cardiomyopathy in humans, cats and dogs. Am J Cardiol 1993;72:944-951.
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17. Liu SK, Maron BJ, Tilley LP. Hypertrophic cardiomyopathy in the dog. Am J Pathol 1979;94:497-508. 18. Liu SK, Chiu YT, Tsau HL, Shyu JJ, Lin JH. Morphologic features of hypertrophic cardiomyopathy in pigs. In: Third International Symposium on Pig Model for Biomedical Research. Talwan, ROC: Pig Research Institute, 1992:9-22. 19. Liu SK. Postmortem examination of the heart. Vet Clin North Am 1983;13:379-394. 20. Lopez DeLeon A, Rojkind A. A simple micromethod for collagen and total protein determination hi formalin-fixed paraffin-embedded sections. J Histochem Cytochem 1985;33:737-743. 21. delRioHortegaP. El metododel carbonatoargentico: revision general de sus tecnicas y aplicaciones en histologia normal y pathologia. Arch Histo Normo Patol Buenos Aires 1943;2:231-243. 22. Hasegawa T, Raven JK. A metallic impregnation method for the demonstration of cerebral vascular patterns. Acta Neuropathol (Bed) 1968; 10:183-188. 23. Robinson TF, Cohen-Gould L, Factor SM. Skeletal framework of mammalian heart muscle: arrangements of inter- and peri-cellular connective tissue. Lab Invest 1983;49:482--498. 24. Maron BJ. Asymmetry in hypertrophic cardiomyopathy: the septal to free wall thickness ratio revisited. Am J Cardiol 1985;55:835-838. 25. Liu SK, Chiu YT, Fox PR, Factor SM, Shyu JJ, Chu RM. Interstitial connective tissue abnormalities in feline cardiomyopathies. Lab Invest 1994;70:20A.
