Cardiovasc Pathol Vol. 1. No. 4 October-December 1992307-3 16
307
Microfibrillar Cardiomyopathy: An Infiltrative Heart Disease Resembling but Distinct From Cardiac Amyloidosis Stephen M. Factor, MD,*+ Mark A. Menegus, MD,+ Yvonne Kress, MS,* Sangho Cho, MD,* John D. Fisher, MD,+ Lynn Y. Sakai, PhD,* and Sidney Goldfischer, MD* From the *Departments of Pathology and ?Medicine, Albert Einstein College of Medicine, Bronx, New York; the *Departments of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon; and *the Shriners Hospital for Crippled Children, Portland, Oregon
Microfibrils-small, ubiquitous components of the extracellular matrix in many tissuesgenerally have not been recognized as causing infiltrative heart disease, except in a group of cardiac transplant patients treated with cyclosporin. Microfibrils are often associated with elastic tissue and contain the glycoprotein fibrillin, the P component of amyloid, and bound fibronectin. A genetically determined abnormality of fibrillin caused by point mutations of fibrillin genes recently was reported as the cause of Marfan’s syndrome. However, to date, no abnormalities of increased fibrillin tissue deposition have been observed. In the last two years, while examining right ventricular endomyocardial biopsies, in four patients we noted abnormal histology distinct from the usual type of congestive cardiomyopathy but with a strong resemblance to amyloidosis. The patients presented with unexplained ventricular tachycardia (N = 3) and/or congestive heart failure (N = 2). Biopsies revealed subendocardial, interstitial, and perivascular hyaline eosinophilic fibrillar material that did not stain with Congo red. Electron microscopy,revealed that this material was organized into bundles of tangled microfibrils composed of twisted and tubular structures measuring up to 17 nm wide, which did not resemble amyloid or cyclosporin-associated microfibrils. Immunoelectron microscopy of the index case, using monoclonal antibody to fibrillin, specifically identified these structures as fibrillin microfibrils; fibronectin also was bound to the interstitial microfibrils. We believe that the subendocardial and interstitial deposition of microfibrils in these four symptomatic patients may represent a new type of infiltrative cardiomyopathy, similar to but distinct from cardiac amyloidosis. We do not know yet if this disorder is genetic or acquired, or if the prognosis is better than that of cardiac amyloidosis. However, atypical cases of primary cardiac amyloidosis should be reevaluated in light of these findings.
The interstitial connective tissue matrix of the heart is composed of collagens (primarily types I, III, and IV), laminin, fibronectin, proteoglycans, elastin, and microSupported in part by grants HL-37412 from the National Institutes of Health and the Shriners Hospitals for Crippled Children. Manuscript received: December 30, 1991; accepted July 21, 1992. Address for reprints: Stephen M. Factor, MD, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. All editorial decisions concerning reviewers, revisions, and acceptability of this manuscript were handled by an Associate Editor of the Journal. 01992
by Elsewer Science Publishing
Co.. Inc
fibrils (la); the fibrillar components precisely interconnect and surround myocytes and, by so doing, play an important role in normal cardiac function (5-7). Both loss of matrix (8,9), and increases of matrix (10,ll) can have adverse affects on ventricular shape and contractility. Of the fibrous structures that constitute the matrix, microfibrils have received the least attention. They are small (range 4-25 nm, mean 11-15 nm wide) and are found in many locations throughout the body, often in association with elastic fibers (12-14). Recent studies have demonstrated that fibrillin, a newly identified 350 kd glycoprotein, is a major component of microfibrils (15,16); with immunohistochemistry, both fibronectin 1054.8807/92/$5.00
308
FACTOR ET AL. MICROFIBRILLAR
CARDIOMYOPATHY
(17) and the amyloid P glycoprotein (18) have been localized to microfibrils. With the exception of one report describing the deposition of microfibrils in the interstitium of cyclosporin-treated cardiac transplants (19) there are no descriptions of symptomatic cardiac pathology associated with idiopathic microfibril accumulation. During the course of examining right ventricular endomyocardial biopsies for the diagnosis of unexplained congestive heart failure and/or ventricular arrhythmia, we encountered an unusual histological pattern that strongly resembled amyloidosis in the endomyocardium of a 25year-old woman who presented with ventricular tachycardia. Despite a negative Congo red stain, the relatively young age of the patient, and the absence of familial history or other etiology, the diagnosis of amyloidosis was made on histological grounds. It was only when additional myocardial tissue became available to perform electron microscopy, immunohistochemistry, and immunoelectron microscopy that the correct diagnosis was made: the interstitial material was not amyloid but was composed of fibrillin and fibronectin. Over the last two years, we have seen three additional clinically symptomatic cases with virtually identical histology. Each had a characteristic pattern of amyloid-like deposits in the myocardium, with ultrastructural demonstration of microfibril accumulation in the interstitium. The histological and ultrastructural similarities of the four cases strongly suggest that this may be a new type of cardiomyopathy.
Methods Patient 1 (index case). This 25year-old, previously healthy woman presented in August 1989 with several weeks of a flu-like illness associated with mild myalgias and palpitations. An electrocardiogram (ECG) at a local hospital revealed runs of nonsustained ventricular tachycardia unresponsive to therapy, following which she was transferred to our institution. Physical examination and chest X-ray were normal. ECG revealed an incomplete right bundle branch pattern with left axis deviation and incessant runs of nonsustained VT with a left bundle-left axis configuration, at 150 beats per minute (bpm) or slower. Echocardiography showed a mildly enlarged left atrium and a normal-sized but slightly thickened left ventricle (LV), with mild redundancy of the anterior leaflet of the mitral valve. Prior to electrophysiological evaluation, the patient underwent cardiac catheterization, which revealed normal right-sided pressures and a mean wedge pressure of 18. Cardiac index was 2.4 liters per minute. Right ventriculography showed a slightly enlarged right ventricle (RV) that was mildly to moderately hyperkinetic
Cardiovasc Pathol Vol. I, No. 4 October-December 1992:307-316
and hypertrophied, giving the appearance of a twochambered RV. The levophase revealed a mildly enlarged left atrium and a normal LV with an ejection fraction (EF) of 63%. An RV endomyocardial biopsy was performed. Cardiac magnetic resonance imaging demonstrated nodularity of the RV wall secondary to hypertrophy or infiltration. The LV was hypertrophied with bright T,-weighted signals of possible infiltrative origin. Following the histopathologic diagnosis of amyloidosis on the first endomyocardial biopsy, further laboratory studies revealed negative antistreptolysin 0, negative Lyme titers, and negative C-reactive protein. Erythrocyte sedimentation rate (ESR) was 9 mm/h. Urine and serum protein electrophoresis were normal; the urine was negative for kappa and lambda light chains and Bence-Jones proteins. Quantitative immunoglobulins were normal. The patient was started on colchicine 0.6 mg three times per day in addition to her antiarrhythmic medications. The initial endomyocardial biopsy was reviewed independently by Dr. Martha Skinner at Boston University School of Medicine, and the diagnosis of cardiac amyloidosis was not supported based on a repeated negative Congo red stain. An abdominal fat pad biopsy for evaluation of systemic amyloidosis also was negative. Clinically the patient remained stable and asymptomatic on therapy. Because the diagnosis remained in question, she underwent a repeat RV endomyocardial biopsy in May 1990. Sufficient fixed and fresh frozen tissue was obtained to establish a diagnosis (see following sections). Two years after her initial symptoms, the patient continues to do well. Patient 2. This 60-year-old woman was admitted to a local hospital in February 1991 with palpitations and dizziness, and she was found to be in sustained ventricular tachycardia. The patient had a strong family history of sudden cardiac death (father age 47, sister age 35, and uncle age 28). There was a 20-year history of bilateral lower extremity weakness, and the patient had also had ptosis since childhood. Electromyography in 1982 had revealed normal conduction velocities but narrow and low action potentials, consistent with a “myopathic pattern” suggestive of a muscular dystrophy rather than polymyositis. Muscle biopsies of the left deltoid and left vastus lateralis revealed slight variability in fiber size with no fibrosis, inflammation, or vasculitis. These changes were interpreted as a mild nonspecific myopathy. In September 1990 the patient complained of palpitations and dizziness, and she was found to be in complete heart block. A permanent pacemaker was placed, and she was treated with sustained release procainamide for paroxysmal supraventricular tachycardia (SVT). From November 1990 until
Cardiovasc Path01 Vol. 1, NO. 4 October-December 1992:307-316
her admission to our institution, she complained of episodic palpitations, dizziness, and chest pain. Radionuclide study revealed a dilated LV with slight hypokinesis of the septum and apex and an overall LV EF of 40%. Thyroid function studies were normal, and ESR was 8 mm/h. As left ventriculography was not performed at the initial catheterization because of arrhythmia, an LV study revealed moderate hypokinesis of the apical-lateral wall, with the remainder of the myocardium contracting normally. There was mild LV hypertrophy and an EF of 56%. End-diastolic pressures were normal. RV endomyocardial biopsy was performed (see following sections). A right quadriceps peripheral muscle biopsy revealed normal light microscopic histology and ultrastructure. She was discharged in March 1991 in stable condition, medicated with sotalol and furosemide. Currently her functional class is improved. Patient 3. The patient is a 44-year-old man without significant past medical history except for one episode of palpitations five years ago, which lasted almost six hours. He remained well until November 1990, when he was awakened from sleep with palpitations; subsequently, he was found to have wide complex tachycardia at a rate of 150 bpm. This episode was treated successfully with intravenous lidocaine. All cardiovascular studies were normal. The patient was transferred to our institution for cardiac catheterization and programmed electrical stimulation. Physical examination was remarkable only for mild pectus excavatum. Holter monitor revealed rare preventricular contractions. An exercise test showed occasional pre-excited beats at rest and none with exercise. Cardiac catheterization revealed mild to moderate enlargement of the right ventricle with akinesis of the apex and otherwise normal contraction. A levophase angiogram was normal. An RV endomyocardial biopsy was performed during catheterization. Because of limited tissue samples, all specimens were embedded in paraffin for light microscopy. Despite similar histology to the first two patients (see following sections), no tissue for electron microscopy was available for confirmation of the diagnosis. After the diagnosis was established for patients 1 and 2, we elected to study the deparaffinized biopsy by electron microscopy. Currently the patient is asymptomatic. Patient 4. The patient is a 36-year-old man with severe mental retardation who presented to a local hospital with congestive heart failure and atria1 fibrillation. He had cardiomegaly on chest X-ray and an EF of 30%. By report of his treating physician, the mental retardation could not be classified specifically. There was no family history of heart disease. An RV endomyocardial biopsy was performed at the local hospital. Sections from paraffin-embedded biopsy tissue and a
MICROFIBRILLAR
FACTOR ET AL. CARDIOMYOPATHY
309
single fragment of glutaraldehyde-fixed tissue were submitted to our Cardiovascular Pathology Laboratory for evaluation. Control Patients. Four patients were selected from a population of over 1200 individuals whose endomyocardial biopsies were studied in our laboratory. Selection criteria were (1) age comparable to the study patients; (2) c1’ ’ mica1 presentation with primary ventricular arrhythmia of unknown etiology; (3) no occlusive coronary artery disease, congenital heart disease, or significant valvular disease; and (4) biopsy tissue studied by electron microscopy. It should be noted that electron microscopy is performed relatively infrequently in our laboratory for routine endomyocardial biopsies and is reserved mainly for patients with suspected hereditary, metabolic, or inclusion body cardiomyopathies. Fewer than 50 cases have been studied in the last decade. Control patient 1 was a 27-year-old man who presented after several episodes of ventricular tachycardia. Following extensive electrophysiological testing and endomyocardial biopsy, he was discharged on propranolol, which controlled his arrhythmia. He did well for seven years, but while on vacation he died suddenly. Cardiac tissue from the autopsy performed by the medical examiner was made available for study. Control patient 2 was a 55-year-old man, patient 3 was a 54 year-old-woman, and patient 4 a 21-year-old man. These three patients all presented with ventricular tachycardia of variable duration. Patient 4 also had mitral valve prolapse. Light microscopy. Endomyocardial biopsy specimens from the first three study patients and the four control patients were processed in our laboratory; those from study patient 4 were processed elsewhere. Formaldehyde-fixed tissue was embedded in paraffin and sectioned at approximately 5 urn. Serial sections were prepared from each biopsy. Sections were stained routinely with hematoxylin and eosin, Masson’s trichrome for connective tissue, van Gieson’s stain for elastic tissue, Congo red (for amyloid), crystal violet (a nonspecific metachromatic stain for amyloid), and periodic acid Schiff (PAS) with diastase (for glycoprotein). Electron microscopy. Tissue from patients 1, 2, and 4 and the four control cases was hxed in 2.5% glutaraldehyde with Millonig’s phosphate buffer, pH 7.4, and posttixed with 1% osmium tetroxide for one hour. Tissues were dehydrated with graded alcohols and propylene oxide and embedded in 1:l araldite-epon mixture. After polymerization, 1 urn sections were prepared and stained with toluidine blue. Ultrathin sections were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. Grids were examined with a Siemens 102 electron microscope.
310
Cadiovasc Path01Vol. 1, No. 4 October-December 19!92:307-316
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
Only paraffin-embedded tissue was available from study patient 3. The most affected fragment of myocardium as determined by histology was removed from the paraffin block, deparaffinized, rehydrated, and then processed as described earlier. Antibodies. Mouse IgG monoclonal antibody (201) to fibrillin was obtained from one of the authors of this report (LYS). Polyclonal rabbit antihuman fibronectin and rabbit antilaminin (reacts with tissues derived from human, rat, mouse, and chick) were obtained commercially from Chemicon International Inc., Temecula, CA. Immunobistochemistry. Endomyocardial biopsy tissue from patient 1 (index case) was stored at -70°C for two months and processed for light microscopy with the immunoperoxidase method and for electron microscopy with the immunogold technique. Frozen biopsy sections were prepared in a cryostat at 10 urn and air-dried on gelatin-coated slides for 30 minutes. Sections were fixed in cold acetone, washed in buffer, and reacted with 3% hydrogen peroxide and 0.25% triton for 15 minutes. Following buffer washes X3, sections were blocked in 5% milk and 1% normal goat serum and then reacted with antifibrillin monoclonal antibody diluted 1:lOO for one hour. Biotynilated anti-mouse IgG diluted 1:200 was used for 30 minutes, followed by avidin-diamino benzidine. Sections were dehydrated and mounted. Immunoelectron microscopy. The frozen endomyocardial biopsy tissue from case 1 was thawed, cut into thin cubes, incubated in buffer and 0.25% triton, and washed in buffer and 5% milk. Blocking was done in 5% milk plus 1% normal goat serum at room temperature on a rotator for 1 hour. The tissue was then incubated with antifibrillin monoclonal antibody diluted 1900 overnight at 4°C on a rotator. The tissue was washed in buffer and milk and placed in goat antimouse IgG coupled to 5 nm gold particles (Janssen), diluted 1:30 in buffer and milk for 6 hours at 4°C on a rotator. Following buffer wash, the tissue was fixed in buffered 2.5% glutaraldehyde for 1 hour and postfixed in 1% osmium tetroxide. Dehydration, embedding, and staining steps were as previously described for electron microscopy. Antibodies to fibronectin and laminin were reacted with the same sections used for antifibrillin. The antifibrillin antibody was employed pre-embedding, whereas the fibronectin and laminin antibodies were used postembedding. Thin sections on nickel grids were etched with saturated sodium periodate for 1 hour. Grids were washed in buffer and 1% milk, blocked with 1% normal goat serum, and incubated in antifibronectin and antilaminin antibodies diluted 1:lOO for 1 hour, followed by antirabbit IgG coupled to 10
nm gold particles. Appropriate positive and negative controls were carried out for all antibodies.
Results Light microscopy. The histological features of all four study cases had many similarities, with deposition of PAS-positive, eosinophilic, hyaline, and generally fibrillar material immediately beneath the endocardium, focally within the interstitium, and around small blood vessels. Trichrome stain was moderately positive, but the staining intensity was less than that observed with collagen. In the first biopsy from case 1, this material surrounded, compressed, and replaced degenerated myocytes in a nodular pattern suggestive of amyloidosis (Fig. 1A). This material stained metachromatically with crystal violet, but it was negative with Congo red. Although the main site of deposition was subendocardial, interstitial and perivascular material were seen also. Vascular involvement was not observed, and myocytes were not hypertrophied. The repeat biopsy more than eight months later showed similar changes. A band of this material was present deep to the endocardium, with the fading outlines of residual myocytes still visible (Fig. 1B). Amyloid stains, including crystal violet, were all negative. The biopsy from case 2 revealed subendocardial, perivascular, and interstitial fibrillar material with focal replacement of myocytes (Fig. 1C). The deposits were somewhat less compact than those from case 1, but they still had a slightly hyaline, refractile quality more like amyloid than typical collagenous connective tissue. This material did not stain with Congo red or crystal violet. Myocytes were mildly to moderately hypertrophied. The biopsy from case 3 had the most striking appearance. There was a raised, relatively acellular, hyaline plaque in the immediate subendocardial space between endocardial cells and myocytes in one of the three biopsy fragments (Fig. 1D). Superficially the surface material resembled endocardial fibroelastosis, but the band-like deposit was more hyaline and homogeneous than that seen in that condition. Additionally, elastic tissue stain was focally positive only for a few elastic fibers, and trichrome stain was only weakly to moderately positive. Congo red and crystal violet stains were negative for amyloid. All three samples had interstitial and perivascular accumulations of similar material. Myocytes were not hypertrophied but did show focal degeneration. Several discrete interstitial collections of inflammatory cells-mainly lymphocytes and a few plasma cells-were noted without associated myocyte degeneration or necrosis (Fig. 1E). These cells
Figure 1A. The first biopsy from case 1 has a nodular interstitial mass of eosinophilic material surrounding degenerating, vacuolated myocytes (M) immediately subjacent to the endocardium (E). Note that many myocytes within the material appear to be compressed and “fading away,” a characteristic typical of cardiac amyloidosis. (Hematoxylin-eosin stain X240.)
Figure 1B. The second biopsy from case 1 has similar mate-
rial in the subendocardium surrounding degenerated, olated myocytes. (Hematoxylin-eosin stain X600.)
vacu-
biopsy from case 2 has a lightly eosinophilic, loose infiltrate of material immediately beneath the endocardium entrapping several degenerated myocytes. (Hematoxylin-eosin stain x240.)
Figure 1D. A thick, band-like infiltrate of hyaline, relatively acellular, pale eosinophilic material is present in the immediate subendocardium of this biopsy fragment from case 3. Similar material also is present around blood vessels deeper in the tissue (arrow). (Hematoxylin-eosin stain X240.)
Figure 1E. A small nodular collection of mononuclear inflammatory cells in the biopsy from case 3. This was the only inflammatory activity noted in the four cases. (Hematoxylineosin stain x600.)
Figure 1F. An interstitial and perivascutar area from case 4 has pale, hyaline, fibrillar, and slightly refractile material, similar to that seen in case 2. (Hematoxylin-eosin stain x600.)
Figure 1C. The endomyocardial
312
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
Figure 2A. A compact bundle of microfibrils (MF) is adjacent to periodically banded collagen fibrils (C) in the interstitial space of case 4. Bar = 1 urn.
Figure 2C. A highly magnified view of microfibrils from case 1 reveals they are slightly curvilinear with a suggestion of branching; they appear hollow when seen in cross-section (arrowheads), with twisting or constrictions (seen best at arrows). Bar = 100 nm.
did not appear localized to areas of increased matrix deposition. The biopsy from case 4 had an appearance virtually identical to that seen in case 2. There was a subendocardial, perivascular, and interstitial deposition of hyaline, fibrillar, and slightly refractile material (Fig. lF), without associated myocyte necrosis, degeneration, or hypertrophy. Congo red stain was negative. Stain for elastic tissue was minimally positive for a few subendocardial fibers. No inflammation was observed. Immunohistochemistry. Sections from the endomyocardial biopsy from case 1 showed focally positive antifibrillin immunoperoxidase reaction product that
CardiovascPathol Vol. 1, No. 4 October-December1992:307-316
Figure 2B. The interstitial space is diffusely infiltrated by a loose aggregation of microfibrils free of associated collagen, adjacent to a myocyte and a spindled interstitial cell (both partially seen in this field), from case 4. Bar = 0.5 pm.
Figure 2D. Immunoelectron microscopy of interstitial microfibrils from case 1, with monoclonal antifibrillin antibody linked to 5-nm gold particles (electron dense dots), shows specific binding of the antibody to the microfibrils, with no localization to the few thicker and periodically banded collagen fibrils in the field (C). Bar = 200 nm.
co-localized in the subendocardial and interstitial areas of the biopsy with amyloid-like deposits (not shown). Electron microscopy. The appearance was similar in all four cases. There were bundles and tangled masses of microfibrils morphologically distinct from collagen fibrils, with the latter showing characteristic periodic banding and wider diameter (Fig. 2A). The microfibrils were present loose in the interstitial space, often in association with the collagen; however, skeins of microfibrils were found free of collagen (Fig. 2B). The microfibrillar masses generally did not form parallel ar-
Cardiovasc Pathol Vol. I, No. 4 October-December 1992:307-316
MICROFIBRILLAR
rays, nor did they closely appose themselves to myocyte sarcolemma or basal lamina. The latter pattern is typical for amyloid microfibrils. In all four cases, the microfibrils could be found in association with elastin matrix, but this was not a prominent or consistent finding. The microfibrils were slightly curvilinear with an occasional suggestion of branching (Fig. 2C). With high magnification, twisting of individual fibrils could be appreciated, and some appeared to be hollow with questionable periodicity and beading (Fig. 2C). The width varied slightly among the four cases and ranged between 10 and 17 nm, but most were approximately 15 to 17 nm in diameter. Although the fibrils were within the same size range reported for fibrillin (15) and other microfibrils (14) the variability of the microfibrils in our four cases may reflect fixation artifacts. lmmunoelectron microscopy with monoclonal antibody to fibrillin localized the antibody-bound 5 nm gold particles specifically to the interstitial microfibrils (Fig. 2D). Ten-nanometer gold particles linked to polyclonal fibronectin antibody were bound to the same microfibrils (not shown) and to some collagen fibrils. Antilaminin antibody localized to neither microfibrils nor fibrillar collagen but was present along the myocyte basal lamina. Control patients The endomyocardial biopsies from all four control patients had moderate to serve myocellular hypertrophy, focal interstitial fibrosis, and moderate to severe intramyocardial small vessel sclerosis with perivascular fibrosis (not shown). The histopathologic changes were consistent with cardiomyopathy. No subendocardial or interstitial material similar to that observed in the study cases was identified. The postmortem cardiac tissue from control case 1 was similar to the original endomyocardial biopsy taken seven years before death. There was interstitial and focal replacement fibrosis in both the left and right ventricles, myocellular hypertrophy, and small vessel sclerosis. Electron microscopy from these cases demonstrated nonspecific and focal interstitial fibrosis, as well as changes consistent with hypertrophied myocytes. Rare and indistinct small aggregates of microfibrils could be seen in association with the interstitial collagen, but the latter was the primary intermyocellular material observed (Figs. 3A and 3B).
Discussion In the last two years we have identified four symptomatic patients who, on right ventricular endomyocardial biopsy, had very similar histological and ultrastructural features: prominent subendocardial and focal interstitial accumulations of hyaline eosinophilic material with a strong resemblance to amyloid but staining negatively with Congo red, and distinct inter-
FACTOR ET AL. CARDIOMYOPATHY
313
stitial microfibrils on electron microscopy which were uncharacteristic of amyloid. The histological similarity to amyloidosis was so strong in the index case that the diagnosis was made unequivocally and maintained until electron microscopy proved otherwise. Further study of this case revealed that the microfibrils were composed of fibrillin and fibronectin. Unfortunately, this was the only case in which fresh-frozen tissue was available for immunological studies that could not have been carried out on hxed specimens. Despite this, the striking subendocardial location and amyloid-like appearance of the deposits, with direct confirmation by electron microscopy that microfibrils are a major component of the abnormal material, strongly suggests that all four cases may represent a new type of infiltrative cardiomyopathy caused by a primary, enhanced accumulation of interstitial matrix components other than collagen alone. The pathological features of these four cases were distinct from the four controls, despite similar clinical presentations, and they were different from those of all other patients with cardiomyopathy studied in our laboratory over the last decade. A recent series of cyclosporin-treated cardiac transplant patients has been reported from the Cleveland Clinic, in which 8 of 21 individuals had interstitial microfibril accumulation as seen by electron microscopy (19). The fibrils were focal in distribution, hollow, and beaded, and they measured 8 to 10 nm in diameter. They somewhat resembled amyloid fibrils and those in our cases, but in the transplant hearts they were arranged in a more parallel pattern, and they did not have a predilection for the basal lamina, as is characteristic of amyloid. The transplant hearts appeared to have a denser accumulation and tighter packing of microfibrils than in our cases, a less tangled organization, an absence of fibril twisting, and no association with elastin matrix, which was observed in all four of our cases. The authors suggested that these microfibrils, which were otherwise unidentified, may be related to cyclosporin-induced fine interstitial fibrosis. Of interest is the fact that reduction of cyclosporin dosage has led to a disappearance of the condition in subsequent cardiac transplant patients studied by the same group (personal communication, Norman B. Ratliff, MD, 1991). Microfibrils are normal and ubiquitous components of the extracellular matrix in many organs, including the heart (12,14). They have been recognized as distinct interstitial structures for approximately 30 years (20). An association with elastic tissue has been noted by many investigators (12,13), though the fib& also occur in the absence of elastin matrix, as in the ciliary zonule of the eyes (21) which serves as the suspensory ligament of the lens. Morphological studies indicate that the fibrils play a role in structural support, Cell-Cell
314
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
Cidiovasc Path01Vol. 1, No. 4 October-December1992:307-316
Figure 3A. Representative area from control case 1 reveals dense interstitial collagen between portions of 2 myocytes (C, between 2 arrows). This was a characteristic finding in all four control cases. Bar = 200 urn.
Figure 3B. A deposit of microfibrils (arrow) in the interstitial space from control case 1, amidst isolated collagen fibrils and amorphous ground substance. In contrast to the masses of microfibrils in the four study cases, microfibrils were observed
rarely in the four controls. Bar = 500 urn.
interconnection, and tissue elasticity. Although most studies of these fibrils have been descriptive, recent work by several investigators has led to a better understanding of the composition of these structures. Goldfischer et al. (17) demonstrated that the microfibrils were associated or coated with fibronectin. InouC and associates (18) showed that amyloid P could be localized by immunohistochemistry, and they proposed a structure with a hollow core and repetitive amyloid P elements (14). Recently, Sakai and colleagues (1.5) showed that a major component of both elastin and nonelastin associated microfibrils is a newly identified 350 Kd glycoprotein named fibrillin. These investigators have identified abnormalities of fibrillin in Marfan’s syndrome and have demonstrated linkage of
Mar-fan’s syndrome to defective fibrillin genes (22-29, thus providing a reasonable explanation for the cardiovascular and ocular phenotypic manifestations of the disease (e.g., microfibril abnormalities of aortic elastic tissue and the ciliary zonule). The tinctorial properties and deposition pattern of the eosinophilic, hyaline material in our four cases strongly suggested a diagnosis of amyloidosis with hematoxylin and eosin stains. The amyloid-like appearance may be accounted for by the presence of amyloid P containing microfibrils in the interstitium with a size similar to amyloid microfibrils (26). In our case 1, from whom fresh tissue was avaiiable, analysis of one biopsy fragment for amyloid P was unsuccessful (personal communication, Peter Gorevic, MD, State University
Pathol Vol. I, No. 4 October-December1992:307-316 Cardiovasc
of New York at Stony Brook, 1990); however, it is possible that this specimen, which was not examined histologically, did not have significant microfibrillar deposition. None of our cases had apple-green birefringence with Congo red stain when viewed by polarized light, a relatively specific diagnostic feature of amyloid of all types related to its organization into beta-pleated sheets (26). A consistent feature of all four cases was the presence of prominent subendocardial localization of the microfibrils, in addition to its focal deposition in the interstitium. This subendocardial pattern, particularly that seen in case 3, was suggestive of endocardial fibroelastosis; however, this case, as well as the other three, had only rare elastic fibers with histological staining. However, the known association of microfibrils with elastic tissue (12,13), and identification of microfibrils in the normal subendocardial space (12), may account for its presence in the subendocardium in our cases, albeit in increased amounts. The increased prevalence of subendocardial localization may be partially a result of tissue selection; however, this appears unlikely. Case 1 hat magnetic resonance imaging suggestive of diffuse infiltration, cases 2 and 3 had focal areas of ventricular dyskinesis or akinesis, and case 4 had a moderately severe reduction in ejection fraction. Thus it is probable that all four cases have relatively generalized disease. We have no information concerning a possible familial occurrence of this condition; only case 2 has a strong family history of sudden death without known cause, as well as myopathy. Case 3 has mild pectus excavatum, a condition seen in connective tissue disorders such as Marfan’s syndrome. Case 4 is severely retarded, but the etiology is uncertain. Prognosis cannot be determined with this small series followed for only a short time period. Only the index case has been followed for two years, and she is doing well. Parenthetically, if she actually had cardiac amyloidosis, we would have anticipated that she would be severely symptomatic or dead at two-year follow-up (27-29). In summary, we have identified four patients with cardiac arrhythmia and/or ventricular dysfunction who have an amyloid-like subendocardial and interstitial matrix material on endomyocardial biopsy, which by ultrastructure shows increased accumulation of microfibrils. In one case we were able to show that the microfibrils were composed of fibrillin and fibronectin; in the three other cases the morphology and staining characteristics of the microfibrils was similar, but their composition is unproven. None of the cases is consistent with amyloidosis by Congo red staining or electron microscopy. The etiology of the microfibrillar deposition is unknown. Determination of whether it represents a precursor to amyloidosis, a disproportional
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
315
increase of a matrix component occurring concurrently with collagen fibrosis in the usual type of congestive cardiomyopathy, or a specific synthetic abnormality of fibrillin or other microfibrils will have to await study of additional cases. Regardless, patients with a diagnosis of. cardiac amyloidosis on endomyocardial biopsy, who do not have a positive Congo red stain or associated clinical disease, should have the diagnosis confirmed by electron microscopy to preclude the presence of nonamyloid microfibrils. We sincerely appreciate the contribution of Dr. Celi Reyes, who assisted with the electron microscopy of case 1. We thank Drs. Thomas Pappas and J.C. Lee, who allowed us to study case 4. Portions of this paper were presented at the 1991 U.S. and Canadian International Academy of Pathology, Chicago, Illinois, and were published in abstract (Lab Invest 1991;64374A).
References 1. Eghbali M, Blumenfeld 00, Seifter S, et al. Localization of types I, III, and IV collagen mRNA in rat heart cells by in situ hybridization. J Mol Cell Cardiol 1988;20:267-276. 2. Robinson TF, Cohen-Gould L, Factor SM. Skeletal framework of mammalian heart muscle: arrangement of inter- and pericellular connective tissue structures. Lab Invest 1983;49:482-498. 3. Robinson TF, Cohen-Gould L, Factor SM, Eghbali M, Blumenfeld 00. Structure and function of connective tissue in cardiac muscle: collagen type III in endomysial struts and pericellular fibers. Scan Microsc 1988;2:1005-1015. 4. Robinson TF, Factor SM. Capasso JM, Wittenberg BA, Blumenfeld 00, Seifter S. Morphology, composition, and function of struts between cardiac myocytes of rat and hamster. Cell Tissue Res 1987;249:247-25.5. ,5. Borg TK, Caulfield JB. The collagen matrix of the heart. Fed Proc 1981;40:2037-2041. 6. Factor SM, Robinson TF. Comparative connective tissue structure-function relationshins in biologic _. Dumps. - Lab Invest 1988; 58:15&156. 7. Factor SM. Flomenbaum M, Zhao MJ, Eng C, Robinson TF. The effects of acutely increased ventricular cavity pressure on intrinsic myocardial connective tissue. J Am Co11 Cardiol 1988;12: 1582-1589. 8. Factor SM, Robinson TF, Dominitz R, Cho S. Alterations of the myocardial skeletal framework in acute myocardial infarction with and without ventricular rupture: a preliminary report. Am J Cardiovasc Path01 1987;1:91-97. 9. Zhao MJ, Zhang H, Robinson TF, Factor SM, Sonnenblick EH, Eng C. Profound structural alterations of the extracellular collagen matrix in postischemic dysfunctional (“stunned”) but viable myocardium. J Am Co11Cardiol 1987;10:1322-1334. 10. Abrahams C, Janicki JS, Weber KT. Myocardial hypertrophy in Mucacu fascicularis: structural remodeling of the collagen matrix. Lab Invest 1987;56:676-683. 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 Co11Cardiol 1991;17:1343-1351. 12. Haust MD. Fine fibrils of extracellular space (microfibrils): their structure and role in connective tissue organization. Am J Path01 1965;47:1113-1137.
316
FACTOR ET AL. MlCROLlBRILLAR
CARDIOMYOPATHY
13. Goldfischer S, Coltoff-Schiller B, Schwartz E, Blumenfeld 00. Ultrastructure and staining properties of aortic microfibrils. J Histochem Cytochem 1983;31:382-390. 14. Inoue S, Leblond CP. The microfibrils of connective tissue: I. Ultrastructure. Am J Anat 1986;176:121-138. 15. Sakai LY, Keene DR, Engvall E. Fibrillin, a new 3%kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol 1986;103:2499-2509. 16. Keene Dr, Maddox BK, Kuo H-J, Sakai LY, Glanville RW. Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils. J Histothem Cytochem 1991;39:441449. 17. Goldfischer S, Coltoff-Schiller 8, Schwartz E, Blumenfeld 00. Microfibrils, elastic anchoring components of the extracellular matrix, are associated with fibronectin in the zonule of Zinn and aorta. Tissue Cell 1985;17:441450. 18. Inoue S, Leblond CP, Grant DS, Rico P. The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component. Am J Anat 1986;176:139-152. 19. Myles JL, Ratliff NB, McMahon JT, et al. Cyclosporin-associated microfibrils in cardiac transplant patients. Am J Cardiovasc Path01 1988;2:127-132. 20. Low FN. Microfibrils, fine filamentous components of the tissue space. Anat Ret 1962;142:131-137. 21. Raviola G. The fine structure of the ciliary zonule and ciliary
Cardiovasc Pathol Vol. I, No. 4 October-December 1992:307-3 16
epithelium: with special regard to the organization and insertion of the zonular fibrils. Invest Ophthalmol 1971;10:851-869. 22. Hollister DW, Godfrey M, Sakai LY, Pyeritz RE. Immunohistologic abnormalities of the microfibrillar-fiber system in the Marfan syndrome. N Engl J Med 1990;323:152-159. 23. Lee B, Godfrey M, Vitale E, et al. Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes. Nature 1991;352:33&334. 24. Maslen CL, Corson GM, Maddox BK, Glanville RW, Sakai LY. Partial sequence of a candidate gene for the Marfan syndrome. Nature 1991;352:334-337. 25. Dietz HC, Cutting GR, Pyeritz RE, et al. Marfan syndrome caused by a recurrent de nova missense mutation in the fibrillin gene. Nature 1991;352:337-339. 26. Glenner GG. Amyloid deposits and amyloidosis. The betafibrilloses [first of two parts]. N Engl J Med 1980;302:1283-1292. 27. Kyle RA, Greipp PR. Amyloidosis (AL): clinical and laboratory features in 229 cases. Mayo Clin Proc 1983;58:665-683. 28. Roberts WC, Waller BF. Cardiac amyloidosis causing cardiac dysfunction: analysis of 54 necropsy patients. Am J Cardiol1983; 52:137-146. 29. Pellikka PA, Holmes DR Jr, Edwards WD, Nishimura RA, Tajik J, Kyle RA. Endomyocardial biopsy in 30 patients with primary amyloidosis and suspected cardiac involvement. Arch Intern Med 1988;148:662-666.
307
Microfibrillar Cardiomyopathy: An Infiltrative Heart Disease Resembling but Distinct From Cardiac Amyloidosis Stephen M. Factor, MD,*+ Mark A. Menegus, MD,+ Yvonne Kress, MS,* Sangho Cho, MD,* John D. Fisher, MD,+ Lynn Y. Sakai, PhD,* and Sidney Goldfischer, MD* From the *Departments of Pathology and ?Medicine, Albert Einstein College of Medicine, Bronx, New York; the *Departments of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon; and *the Shriners Hospital for Crippled Children, Portland, Oregon
Microfibrils-small, ubiquitous components of the extracellular matrix in many tissuesgenerally have not been recognized as causing infiltrative heart disease, except in a group of cardiac transplant patients treated with cyclosporin. Microfibrils are often associated with elastic tissue and contain the glycoprotein fibrillin, the P component of amyloid, and bound fibronectin. A genetically determined abnormality of fibrillin caused by point mutations of fibrillin genes recently was reported as the cause of Marfan’s syndrome. However, to date, no abnormalities of increased fibrillin tissue deposition have been observed. In the last two years, while examining right ventricular endomyocardial biopsies, in four patients we noted abnormal histology distinct from the usual type of congestive cardiomyopathy but with a strong resemblance to amyloidosis. The patients presented with unexplained ventricular tachycardia (N = 3) and/or congestive heart failure (N = 2). Biopsies revealed subendocardial, interstitial, and perivascular hyaline eosinophilic fibrillar material that did not stain with Congo red. Electron microscopy,revealed that this material was organized into bundles of tangled microfibrils composed of twisted and tubular structures measuring up to 17 nm wide, which did not resemble amyloid or cyclosporin-associated microfibrils. Immunoelectron microscopy of the index case, using monoclonal antibody to fibrillin, specifically identified these structures as fibrillin microfibrils; fibronectin also was bound to the interstitial microfibrils. We believe that the subendocardial and interstitial deposition of microfibrils in these four symptomatic patients may represent a new type of infiltrative cardiomyopathy, similar to but distinct from cardiac amyloidosis. We do not know yet if this disorder is genetic or acquired, or if the prognosis is better than that of cardiac amyloidosis. However, atypical cases of primary cardiac amyloidosis should be reevaluated in light of these findings.
The interstitial connective tissue matrix of the heart is composed of collagens (primarily types I, III, and IV), laminin, fibronectin, proteoglycans, elastin, and microSupported in part by grants HL-37412 from the National Institutes of Health and the Shriners Hospitals for Crippled Children. Manuscript received: December 30, 1991; accepted July 21, 1992. Address for reprints: Stephen M. Factor, MD, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. All editorial decisions concerning reviewers, revisions, and acceptability of this manuscript were handled by an Associate Editor of the Journal. 01992
by Elsewer Science Publishing
Co.. Inc
fibrils (la); the fibrillar components precisely interconnect and surround myocytes and, by so doing, play an important role in normal cardiac function (5-7). Both loss of matrix (8,9), and increases of matrix (10,ll) can have adverse affects on ventricular shape and contractility. Of the fibrous structures that constitute the matrix, microfibrils have received the least attention. They are small (range 4-25 nm, mean 11-15 nm wide) and are found in many locations throughout the body, often in association with elastic fibers (12-14). Recent studies have demonstrated that fibrillin, a newly identified 350 kd glycoprotein, is a major component of microfibrils (15,16); with immunohistochemistry, both fibronectin 1054.8807/92/$5.00
308
FACTOR ET AL. MICROFIBRILLAR
CARDIOMYOPATHY
(17) and the amyloid P glycoprotein (18) have been localized to microfibrils. With the exception of one report describing the deposition of microfibrils in the interstitium of cyclosporin-treated cardiac transplants (19) there are no descriptions of symptomatic cardiac pathology associated with idiopathic microfibril accumulation. During the course of examining right ventricular endomyocardial biopsies for the diagnosis of unexplained congestive heart failure and/or ventricular arrhythmia, we encountered an unusual histological pattern that strongly resembled amyloidosis in the endomyocardium of a 25year-old woman who presented with ventricular tachycardia. Despite a negative Congo red stain, the relatively young age of the patient, and the absence of familial history or other etiology, the diagnosis of amyloidosis was made on histological grounds. It was only when additional myocardial tissue became available to perform electron microscopy, immunohistochemistry, and immunoelectron microscopy that the correct diagnosis was made: the interstitial material was not amyloid but was composed of fibrillin and fibronectin. Over the last two years, we have seen three additional clinically symptomatic cases with virtually identical histology. Each had a characteristic pattern of amyloid-like deposits in the myocardium, with ultrastructural demonstration of microfibril accumulation in the interstitium. The histological and ultrastructural similarities of the four cases strongly suggest that this may be a new type of cardiomyopathy.
Methods Patient 1 (index case). This 25year-old, previously healthy woman presented in August 1989 with several weeks of a flu-like illness associated with mild myalgias and palpitations. An electrocardiogram (ECG) at a local hospital revealed runs of nonsustained ventricular tachycardia unresponsive to therapy, following which she was transferred to our institution. Physical examination and chest X-ray were normal. ECG revealed an incomplete right bundle branch pattern with left axis deviation and incessant runs of nonsustained VT with a left bundle-left axis configuration, at 150 beats per minute (bpm) or slower. Echocardiography showed a mildly enlarged left atrium and a normal-sized but slightly thickened left ventricle (LV), with mild redundancy of the anterior leaflet of the mitral valve. Prior to electrophysiological evaluation, the patient underwent cardiac catheterization, which revealed normal right-sided pressures and a mean wedge pressure of 18. Cardiac index was 2.4 liters per minute. Right ventriculography showed a slightly enlarged right ventricle (RV) that was mildly to moderately hyperkinetic
Cardiovasc Pathol Vol. I, No. 4 October-December 1992:307-316
and hypertrophied, giving the appearance of a twochambered RV. The levophase revealed a mildly enlarged left atrium and a normal LV with an ejection fraction (EF) of 63%. An RV endomyocardial biopsy was performed. Cardiac magnetic resonance imaging demonstrated nodularity of the RV wall secondary to hypertrophy or infiltration. The LV was hypertrophied with bright T,-weighted signals of possible infiltrative origin. Following the histopathologic diagnosis of amyloidosis on the first endomyocardial biopsy, further laboratory studies revealed negative antistreptolysin 0, negative Lyme titers, and negative C-reactive protein. Erythrocyte sedimentation rate (ESR) was 9 mm/h. Urine and serum protein electrophoresis were normal; the urine was negative for kappa and lambda light chains and Bence-Jones proteins. Quantitative immunoglobulins were normal. The patient was started on colchicine 0.6 mg three times per day in addition to her antiarrhythmic medications. The initial endomyocardial biopsy was reviewed independently by Dr. Martha Skinner at Boston University School of Medicine, and the diagnosis of cardiac amyloidosis was not supported based on a repeated negative Congo red stain. An abdominal fat pad biopsy for evaluation of systemic amyloidosis also was negative. Clinically the patient remained stable and asymptomatic on therapy. Because the diagnosis remained in question, she underwent a repeat RV endomyocardial biopsy in May 1990. Sufficient fixed and fresh frozen tissue was obtained to establish a diagnosis (see following sections). Two years after her initial symptoms, the patient continues to do well. Patient 2. This 60-year-old woman was admitted to a local hospital in February 1991 with palpitations and dizziness, and she was found to be in sustained ventricular tachycardia. The patient had a strong family history of sudden cardiac death (father age 47, sister age 35, and uncle age 28). There was a 20-year history of bilateral lower extremity weakness, and the patient had also had ptosis since childhood. Electromyography in 1982 had revealed normal conduction velocities but narrow and low action potentials, consistent with a “myopathic pattern” suggestive of a muscular dystrophy rather than polymyositis. Muscle biopsies of the left deltoid and left vastus lateralis revealed slight variability in fiber size with no fibrosis, inflammation, or vasculitis. These changes were interpreted as a mild nonspecific myopathy. In September 1990 the patient complained of palpitations and dizziness, and she was found to be in complete heart block. A permanent pacemaker was placed, and she was treated with sustained release procainamide for paroxysmal supraventricular tachycardia (SVT). From November 1990 until
Cardiovasc Path01 Vol. 1, NO. 4 October-December 1992:307-316
her admission to our institution, she complained of episodic palpitations, dizziness, and chest pain. Radionuclide study revealed a dilated LV with slight hypokinesis of the septum and apex and an overall LV EF of 40%. Thyroid function studies were normal, and ESR was 8 mm/h. As left ventriculography was not performed at the initial catheterization because of arrhythmia, an LV study revealed moderate hypokinesis of the apical-lateral wall, with the remainder of the myocardium contracting normally. There was mild LV hypertrophy and an EF of 56%. End-diastolic pressures were normal. RV endomyocardial biopsy was performed (see following sections). A right quadriceps peripheral muscle biopsy revealed normal light microscopic histology and ultrastructure. She was discharged in March 1991 in stable condition, medicated with sotalol and furosemide. Currently her functional class is improved. Patient 3. The patient is a 44-year-old man without significant past medical history except for one episode of palpitations five years ago, which lasted almost six hours. He remained well until November 1990, when he was awakened from sleep with palpitations; subsequently, he was found to have wide complex tachycardia at a rate of 150 bpm. This episode was treated successfully with intravenous lidocaine. All cardiovascular studies were normal. The patient was transferred to our institution for cardiac catheterization and programmed electrical stimulation. Physical examination was remarkable only for mild pectus excavatum. Holter monitor revealed rare preventricular contractions. An exercise test showed occasional pre-excited beats at rest and none with exercise. Cardiac catheterization revealed mild to moderate enlargement of the right ventricle with akinesis of the apex and otherwise normal contraction. A levophase angiogram was normal. An RV endomyocardial biopsy was performed during catheterization. Because of limited tissue samples, all specimens were embedded in paraffin for light microscopy. Despite similar histology to the first two patients (see following sections), no tissue for electron microscopy was available for confirmation of the diagnosis. After the diagnosis was established for patients 1 and 2, we elected to study the deparaffinized biopsy by electron microscopy. Currently the patient is asymptomatic. Patient 4. The patient is a 36-year-old man with severe mental retardation who presented to a local hospital with congestive heart failure and atria1 fibrillation. He had cardiomegaly on chest X-ray and an EF of 30%. By report of his treating physician, the mental retardation could not be classified specifically. There was no family history of heart disease. An RV endomyocardial biopsy was performed at the local hospital. Sections from paraffin-embedded biopsy tissue and a
MICROFIBRILLAR
FACTOR ET AL. CARDIOMYOPATHY
309
single fragment of glutaraldehyde-fixed tissue were submitted to our Cardiovascular Pathology Laboratory for evaluation. Control Patients. Four patients were selected from a population of over 1200 individuals whose endomyocardial biopsies were studied in our laboratory. Selection criteria were (1) age comparable to the study patients; (2) c1’ ’ mica1 presentation with primary ventricular arrhythmia of unknown etiology; (3) no occlusive coronary artery disease, congenital heart disease, or significant valvular disease; and (4) biopsy tissue studied by electron microscopy. It should be noted that electron microscopy is performed relatively infrequently in our laboratory for routine endomyocardial biopsies and is reserved mainly for patients with suspected hereditary, metabolic, or inclusion body cardiomyopathies. Fewer than 50 cases have been studied in the last decade. Control patient 1 was a 27-year-old man who presented after several episodes of ventricular tachycardia. Following extensive electrophysiological testing and endomyocardial biopsy, he was discharged on propranolol, which controlled his arrhythmia. He did well for seven years, but while on vacation he died suddenly. Cardiac tissue from the autopsy performed by the medical examiner was made available for study. Control patient 2 was a 55-year-old man, patient 3 was a 54 year-old-woman, and patient 4 a 21-year-old man. These three patients all presented with ventricular tachycardia of variable duration. Patient 4 also had mitral valve prolapse. Light microscopy. Endomyocardial biopsy specimens from the first three study patients and the four control patients were processed in our laboratory; those from study patient 4 were processed elsewhere. Formaldehyde-fixed tissue was embedded in paraffin and sectioned at approximately 5 urn. Serial sections were prepared from each biopsy. Sections were stained routinely with hematoxylin and eosin, Masson’s trichrome for connective tissue, van Gieson’s stain for elastic tissue, Congo red (for amyloid), crystal violet (a nonspecific metachromatic stain for amyloid), and periodic acid Schiff (PAS) with diastase (for glycoprotein). Electron microscopy. Tissue from patients 1, 2, and 4 and the four control cases was hxed in 2.5% glutaraldehyde with Millonig’s phosphate buffer, pH 7.4, and posttixed with 1% osmium tetroxide for one hour. Tissues were dehydrated with graded alcohols and propylene oxide and embedded in 1:l araldite-epon mixture. After polymerization, 1 urn sections were prepared and stained with toluidine blue. Ultrathin sections were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. Grids were examined with a Siemens 102 electron microscope.
310
Cadiovasc Path01Vol. 1, No. 4 October-December 19!92:307-316
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
Only paraffin-embedded tissue was available from study patient 3. The most affected fragment of myocardium as determined by histology was removed from the paraffin block, deparaffinized, rehydrated, and then processed as described earlier. Antibodies. Mouse IgG monoclonal antibody (201) to fibrillin was obtained from one of the authors of this report (LYS). Polyclonal rabbit antihuman fibronectin and rabbit antilaminin (reacts with tissues derived from human, rat, mouse, and chick) were obtained commercially from Chemicon International Inc., Temecula, CA. Immunobistochemistry. Endomyocardial biopsy tissue from patient 1 (index case) was stored at -70°C for two months and processed for light microscopy with the immunoperoxidase method and for electron microscopy with the immunogold technique. Frozen biopsy sections were prepared in a cryostat at 10 urn and air-dried on gelatin-coated slides for 30 minutes. Sections were fixed in cold acetone, washed in buffer, and reacted with 3% hydrogen peroxide and 0.25% triton for 15 minutes. Following buffer washes X3, sections were blocked in 5% milk and 1% normal goat serum and then reacted with antifibrillin monoclonal antibody diluted 1:lOO for one hour. Biotynilated anti-mouse IgG diluted 1:200 was used for 30 minutes, followed by avidin-diamino benzidine. Sections were dehydrated and mounted. Immunoelectron microscopy. The frozen endomyocardial biopsy tissue from case 1 was thawed, cut into thin cubes, incubated in buffer and 0.25% triton, and washed in buffer and 5% milk. Blocking was done in 5% milk plus 1% normal goat serum at room temperature on a rotator for 1 hour. The tissue was then incubated with antifibrillin monoclonal antibody diluted 1900 overnight at 4°C on a rotator. The tissue was washed in buffer and milk and placed in goat antimouse IgG coupled to 5 nm gold particles (Janssen), diluted 1:30 in buffer and milk for 6 hours at 4°C on a rotator. Following buffer wash, the tissue was fixed in buffered 2.5% glutaraldehyde for 1 hour and postfixed in 1% osmium tetroxide. Dehydration, embedding, and staining steps were as previously described for electron microscopy. Antibodies to fibronectin and laminin were reacted with the same sections used for antifibrillin. The antifibrillin antibody was employed pre-embedding, whereas the fibronectin and laminin antibodies were used postembedding. Thin sections on nickel grids were etched with saturated sodium periodate for 1 hour. Grids were washed in buffer and 1% milk, blocked with 1% normal goat serum, and incubated in antifibronectin and antilaminin antibodies diluted 1:lOO for 1 hour, followed by antirabbit IgG coupled to 10
nm gold particles. Appropriate positive and negative controls were carried out for all antibodies.
Results Light microscopy. The histological features of all four study cases had many similarities, with deposition of PAS-positive, eosinophilic, hyaline, and generally fibrillar material immediately beneath the endocardium, focally within the interstitium, and around small blood vessels. Trichrome stain was moderately positive, but the staining intensity was less than that observed with collagen. In the first biopsy from case 1, this material surrounded, compressed, and replaced degenerated myocytes in a nodular pattern suggestive of amyloidosis (Fig. 1A). This material stained metachromatically with crystal violet, but it was negative with Congo red. Although the main site of deposition was subendocardial, interstitial and perivascular material were seen also. Vascular involvement was not observed, and myocytes were not hypertrophied. The repeat biopsy more than eight months later showed similar changes. A band of this material was present deep to the endocardium, with the fading outlines of residual myocytes still visible (Fig. 1B). Amyloid stains, including crystal violet, were all negative. The biopsy from case 2 revealed subendocardial, perivascular, and interstitial fibrillar material with focal replacement of myocytes (Fig. 1C). The deposits were somewhat less compact than those from case 1, but they still had a slightly hyaline, refractile quality more like amyloid than typical collagenous connective tissue. This material did not stain with Congo red or crystal violet. Myocytes were mildly to moderately hypertrophied. The biopsy from case 3 had the most striking appearance. There was a raised, relatively acellular, hyaline plaque in the immediate subendocardial space between endocardial cells and myocytes in one of the three biopsy fragments (Fig. 1D). Superficially the surface material resembled endocardial fibroelastosis, but the band-like deposit was more hyaline and homogeneous than that seen in that condition. Additionally, elastic tissue stain was focally positive only for a few elastic fibers, and trichrome stain was only weakly to moderately positive. Congo red and crystal violet stains were negative for amyloid. All three samples had interstitial and perivascular accumulations of similar material. Myocytes were not hypertrophied but did show focal degeneration. Several discrete interstitial collections of inflammatory cells-mainly lymphocytes and a few plasma cells-were noted without associated myocyte degeneration or necrosis (Fig. 1E). These cells
Figure 1A. The first biopsy from case 1 has a nodular interstitial mass of eosinophilic material surrounding degenerating, vacuolated myocytes (M) immediately subjacent to the endocardium (E). Note that many myocytes within the material appear to be compressed and “fading away,” a characteristic typical of cardiac amyloidosis. (Hematoxylin-eosin stain X240.)
Figure 1B. The second biopsy from case 1 has similar mate-
rial in the subendocardium surrounding degenerated, olated myocytes. (Hematoxylin-eosin stain X600.)
vacu-
biopsy from case 2 has a lightly eosinophilic, loose infiltrate of material immediately beneath the endocardium entrapping several degenerated myocytes. (Hematoxylin-eosin stain x240.)
Figure 1D. A thick, band-like infiltrate of hyaline, relatively acellular, pale eosinophilic material is present in the immediate subendocardium of this biopsy fragment from case 3. Similar material also is present around blood vessels deeper in the tissue (arrow). (Hematoxylin-eosin stain X240.)
Figure 1E. A small nodular collection of mononuclear inflammatory cells in the biopsy from case 3. This was the only inflammatory activity noted in the four cases. (Hematoxylineosin stain x600.)
Figure 1F. An interstitial and perivascutar area from case 4 has pale, hyaline, fibrillar, and slightly refractile material, similar to that seen in case 2. (Hematoxylin-eosin stain x600.)
Figure 1C. The endomyocardial
312
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
Figure 2A. A compact bundle of microfibrils (MF) is adjacent to periodically banded collagen fibrils (C) in the interstitial space of case 4. Bar = 1 urn.
Figure 2C. A highly magnified view of microfibrils from case 1 reveals they are slightly curvilinear with a suggestion of branching; they appear hollow when seen in cross-section (arrowheads), with twisting or constrictions (seen best at arrows). Bar = 100 nm.
did not appear localized to areas of increased matrix deposition. The biopsy from case 4 had an appearance virtually identical to that seen in case 2. There was a subendocardial, perivascular, and interstitial deposition of hyaline, fibrillar, and slightly refractile material (Fig. lF), without associated myocyte necrosis, degeneration, or hypertrophy. Congo red stain was negative. Stain for elastic tissue was minimally positive for a few subendocardial fibers. No inflammation was observed. Immunohistochemistry. Sections from the endomyocardial biopsy from case 1 showed focally positive antifibrillin immunoperoxidase reaction product that
CardiovascPathol Vol. 1, No. 4 October-December1992:307-316
Figure 2B. The interstitial space is diffusely infiltrated by a loose aggregation of microfibrils free of associated collagen, adjacent to a myocyte and a spindled interstitial cell (both partially seen in this field), from case 4. Bar = 0.5 pm.
Figure 2D. Immunoelectron microscopy of interstitial microfibrils from case 1, with monoclonal antifibrillin antibody linked to 5-nm gold particles (electron dense dots), shows specific binding of the antibody to the microfibrils, with no localization to the few thicker and periodically banded collagen fibrils in the field (C). Bar = 200 nm.
co-localized in the subendocardial and interstitial areas of the biopsy with amyloid-like deposits (not shown). Electron microscopy. The appearance was similar in all four cases. There were bundles and tangled masses of microfibrils morphologically distinct from collagen fibrils, with the latter showing characteristic periodic banding and wider diameter (Fig. 2A). The microfibrils were present loose in the interstitial space, often in association with the collagen; however, skeins of microfibrils were found free of collagen (Fig. 2B). The microfibrillar masses generally did not form parallel ar-
Cardiovasc Pathol Vol. I, No. 4 October-December 1992:307-316
MICROFIBRILLAR
rays, nor did they closely appose themselves to myocyte sarcolemma or basal lamina. The latter pattern is typical for amyloid microfibrils. In all four cases, the microfibrils could be found in association with elastin matrix, but this was not a prominent or consistent finding. The microfibrils were slightly curvilinear with an occasional suggestion of branching (Fig. 2C). With high magnification, twisting of individual fibrils could be appreciated, and some appeared to be hollow with questionable periodicity and beading (Fig. 2C). The width varied slightly among the four cases and ranged between 10 and 17 nm, but most were approximately 15 to 17 nm in diameter. Although the fibrils were within the same size range reported for fibrillin (15) and other microfibrils (14) the variability of the microfibrils in our four cases may reflect fixation artifacts. lmmunoelectron microscopy with monoclonal antibody to fibrillin localized the antibody-bound 5 nm gold particles specifically to the interstitial microfibrils (Fig. 2D). Ten-nanometer gold particles linked to polyclonal fibronectin antibody were bound to the same microfibrils (not shown) and to some collagen fibrils. Antilaminin antibody localized to neither microfibrils nor fibrillar collagen but was present along the myocyte basal lamina. Control patients The endomyocardial biopsies from all four control patients had moderate to serve myocellular hypertrophy, focal interstitial fibrosis, and moderate to severe intramyocardial small vessel sclerosis with perivascular fibrosis (not shown). The histopathologic changes were consistent with cardiomyopathy. No subendocardial or interstitial material similar to that observed in the study cases was identified. The postmortem cardiac tissue from control case 1 was similar to the original endomyocardial biopsy taken seven years before death. There was interstitial and focal replacement fibrosis in both the left and right ventricles, myocellular hypertrophy, and small vessel sclerosis. Electron microscopy from these cases demonstrated nonspecific and focal interstitial fibrosis, as well as changes consistent with hypertrophied myocytes. Rare and indistinct small aggregates of microfibrils could be seen in association with the interstitial collagen, but the latter was the primary intermyocellular material observed (Figs. 3A and 3B).
Discussion In the last two years we have identified four symptomatic patients who, on right ventricular endomyocardial biopsy, had very similar histological and ultrastructural features: prominent subendocardial and focal interstitial accumulations of hyaline eosinophilic material with a strong resemblance to amyloid but staining negatively with Congo red, and distinct inter-
FACTOR ET AL. CARDIOMYOPATHY
313
stitial microfibrils on electron microscopy which were uncharacteristic of amyloid. The histological similarity to amyloidosis was so strong in the index case that the diagnosis was made unequivocally and maintained until electron microscopy proved otherwise. Further study of this case revealed that the microfibrils were composed of fibrillin and fibronectin. Unfortunately, this was the only case in which fresh-frozen tissue was available for immunological studies that could not have been carried out on hxed specimens. Despite this, the striking subendocardial location and amyloid-like appearance of the deposits, with direct confirmation by electron microscopy that microfibrils are a major component of the abnormal material, strongly suggests that all four cases may represent a new type of infiltrative cardiomyopathy caused by a primary, enhanced accumulation of interstitial matrix components other than collagen alone. The pathological features of these four cases were distinct from the four controls, despite similar clinical presentations, and they were different from those of all other patients with cardiomyopathy studied in our laboratory over the last decade. A recent series of cyclosporin-treated cardiac transplant patients has been reported from the Cleveland Clinic, in which 8 of 21 individuals had interstitial microfibril accumulation as seen by electron microscopy (19). The fibrils were focal in distribution, hollow, and beaded, and they measured 8 to 10 nm in diameter. They somewhat resembled amyloid fibrils and those in our cases, but in the transplant hearts they were arranged in a more parallel pattern, and they did not have a predilection for the basal lamina, as is characteristic of amyloid. The transplant hearts appeared to have a denser accumulation and tighter packing of microfibrils than in our cases, a less tangled organization, an absence of fibril twisting, and no association with elastin matrix, which was observed in all four of our cases. The authors suggested that these microfibrils, which were otherwise unidentified, may be related to cyclosporin-induced fine interstitial fibrosis. Of interest is the fact that reduction of cyclosporin dosage has led to a disappearance of the condition in subsequent cardiac transplant patients studied by the same group (personal communication, Norman B. Ratliff, MD, 1991). Microfibrils are normal and ubiquitous components of the extracellular matrix in many organs, including the heart (12,14). They have been recognized as distinct interstitial structures for approximately 30 years (20). An association with elastic tissue has been noted by many investigators (12,13), though the fib& also occur in the absence of elastin matrix, as in the ciliary zonule of the eyes (21) which serves as the suspensory ligament of the lens. Morphological studies indicate that the fibrils play a role in structural support, Cell-Cell
314
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
Cidiovasc Path01Vol. 1, No. 4 October-December1992:307-316
Figure 3A. Representative area from control case 1 reveals dense interstitial collagen between portions of 2 myocytes (C, between 2 arrows). This was a characteristic finding in all four control cases. Bar = 200 urn.
Figure 3B. A deposit of microfibrils (arrow) in the interstitial space from control case 1, amidst isolated collagen fibrils and amorphous ground substance. In contrast to the masses of microfibrils in the four study cases, microfibrils were observed
rarely in the four controls. Bar = 500 urn.
interconnection, and tissue elasticity. Although most studies of these fibrils have been descriptive, recent work by several investigators has led to a better understanding of the composition of these structures. Goldfischer et al. (17) demonstrated that the microfibrils were associated or coated with fibronectin. InouC and associates (18) showed that amyloid P could be localized by immunohistochemistry, and they proposed a structure with a hollow core and repetitive amyloid P elements (14). Recently, Sakai and colleagues (1.5) showed that a major component of both elastin and nonelastin associated microfibrils is a newly identified 350 Kd glycoprotein named fibrillin. These investigators have identified abnormalities of fibrillin in Marfan’s syndrome and have demonstrated linkage of
Mar-fan’s syndrome to defective fibrillin genes (22-29, thus providing a reasonable explanation for the cardiovascular and ocular phenotypic manifestations of the disease (e.g., microfibril abnormalities of aortic elastic tissue and the ciliary zonule). The tinctorial properties and deposition pattern of the eosinophilic, hyaline material in our four cases strongly suggested a diagnosis of amyloidosis with hematoxylin and eosin stains. The amyloid-like appearance may be accounted for by the presence of amyloid P containing microfibrils in the interstitium with a size similar to amyloid microfibrils (26). In our case 1, from whom fresh tissue was avaiiable, analysis of one biopsy fragment for amyloid P was unsuccessful (personal communication, Peter Gorevic, MD, State University
Pathol Vol. I, No. 4 October-December1992:307-316 Cardiovasc
of New York at Stony Brook, 1990); however, it is possible that this specimen, which was not examined histologically, did not have significant microfibrillar deposition. None of our cases had apple-green birefringence with Congo red stain when viewed by polarized light, a relatively specific diagnostic feature of amyloid of all types related to its organization into beta-pleated sheets (26). A consistent feature of all four cases was the presence of prominent subendocardial localization of the microfibrils, in addition to its focal deposition in the interstitium. This subendocardial pattern, particularly that seen in case 3, was suggestive of endocardial fibroelastosis; however, this case, as well as the other three, had only rare elastic fibers with histological staining. However, the known association of microfibrils with elastic tissue (12,13), and identification of microfibrils in the normal subendocardial space (12), may account for its presence in the subendocardium in our cases, albeit in increased amounts. The increased prevalence of subendocardial localization may be partially a result of tissue selection; however, this appears unlikely. Case 1 hat magnetic resonance imaging suggestive of diffuse infiltration, cases 2 and 3 had focal areas of ventricular dyskinesis or akinesis, and case 4 had a moderately severe reduction in ejection fraction. Thus it is probable that all four cases have relatively generalized disease. We have no information concerning a possible familial occurrence of this condition; only case 2 has a strong family history of sudden death without known cause, as well as myopathy. Case 3 has mild pectus excavatum, a condition seen in connective tissue disorders such as Marfan’s syndrome. Case 4 is severely retarded, but the etiology is uncertain. Prognosis cannot be determined with this small series followed for only a short time period. Only the index case has been followed for two years, and she is doing well. Parenthetically, if she actually had cardiac amyloidosis, we would have anticipated that she would be severely symptomatic or dead at two-year follow-up (27-29). In summary, we have identified four patients with cardiac arrhythmia and/or ventricular dysfunction who have an amyloid-like subendocardial and interstitial matrix material on endomyocardial biopsy, which by ultrastructure shows increased accumulation of microfibrils. In one case we were able to show that the microfibrils were composed of fibrillin and fibronectin; in the three other cases the morphology and staining characteristics of the microfibrils was similar, but their composition is unproven. None of the cases is consistent with amyloidosis by Congo red staining or electron microscopy. The etiology of the microfibrillar deposition is unknown. Determination of whether it represents a precursor to amyloidosis, a disproportional
FACTOR ET AL. MICROFIBRILLAR CARDIOMYOPATHY
315
increase of a matrix component occurring concurrently with collagen fibrosis in the usual type of congestive cardiomyopathy, or a specific synthetic abnormality of fibrillin or other microfibrils will have to await study of additional cases. Regardless, patients with a diagnosis of. cardiac amyloidosis on endomyocardial biopsy, who do not have a positive Congo red stain or associated clinical disease, should have the diagnosis confirmed by electron microscopy to preclude the presence of nonamyloid microfibrils. We sincerely appreciate the contribution of Dr. Celi Reyes, who assisted with the electron microscopy of case 1. We thank Drs. Thomas Pappas and J.C. Lee, who allowed us to study case 4. Portions of this paper were presented at the 1991 U.S. and Canadian International Academy of Pathology, Chicago, Illinois, and were published in abstract (Lab Invest 1991;64374A).
References 1. Eghbali M, Blumenfeld 00, Seifter S, et al. Localization of types I, III, and IV collagen mRNA in rat heart cells by in situ hybridization. J Mol Cell Cardiol 1988;20:267-276. 2. Robinson TF, Cohen-Gould L, Factor SM. Skeletal framework of mammalian heart muscle: arrangement of inter- and pericellular connective tissue structures. Lab Invest 1983;49:482-498. 3. Robinson TF, Cohen-Gould L, Factor SM, Eghbali M, Blumenfeld 00. Structure and function of connective tissue in cardiac muscle: collagen type III in endomysial struts and pericellular fibers. Scan Microsc 1988;2:1005-1015. 4. Robinson TF, Factor SM. Capasso JM, Wittenberg BA, Blumenfeld 00, Seifter S. Morphology, composition, and function of struts between cardiac myocytes of rat and hamster. Cell Tissue Res 1987;249:247-25.5. ,5. Borg TK, Caulfield JB. The collagen matrix of the heart. Fed Proc 1981;40:2037-2041. 6. Factor SM, Robinson TF. Comparative connective tissue structure-function relationshins in biologic _. Dumps. - Lab Invest 1988; 58:15&156. 7. Factor SM. Flomenbaum M, Zhao MJ, Eng C, Robinson TF. The effects of acutely increased ventricular cavity pressure on intrinsic myocardial connective tissue. J Am Co11 Cardiol 1988;12: 1582-1589. 8. Factor SM, Robinson TF, Dominitz R, Cho S. Alterations of the myocardial skeletal framework in acute myocardial infarction with and without ventricular rupture: a preliminary report. Am J Cardiovasc Path01 1987;1:91-97. 9. Zhao MJ, Zhang H, Robinson TF, Factor SM, Sonnenblick EH, Eng C. Profound structural alterations of the extracellular collagen matrix in postischemic dysfunctional (“stunned”) but viable myocardium. J Am Co11Cardiol 1987;10:1322-1334. 10. Abrahams C, Janicki JS, Weber KT. Myocardial hypertrophy in Mucacu fascicularis: structural remodeling of the collagen matrix. Lab Invest 1987;56:676-683. 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 Co11Cardiol 1991;17:1343-1351. 12. Haust MD. Fine fibrils of extracellular space (microfibrils): their structure and role in connective tissue organization. Am J Path01 1965;47:1113-1137.
316
FACTOR ET AL. MlCROLlBRILLAR
CARDIOMYOPATHY
13. Goldfischer S, Coltoff-Schiller B, Schwartz E, Blumenfeld 00. Ultrastructure and staining properties of aortic microfibrils. J Histochem Cytochem 1983;31:382-390. 14. Inoue S, Leblond CP. The microfibrils of connective tissue: I. Ultrastructure. Am J Anat 1986;176:121-138. 15. Sakai LY, Keene DR, Engvall E. Fibrillin, a new 3%kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol 1986;103:2499-2509. 16. Keene Dr, Maddox BK, Kuo H-J, Sakai LY, Glanville RW. Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils. J Histothem Cytochem 1991;39:441449. 17. Goldfischer S, Coltoff-Schiller 8, Schwartz E, Blumenfeld 00. Microfibrils, elastic anchoring components of the extracellular matrix, are associated with fibronectin in the zonule of Zinn and aorta. Tissue Cell 1985;17:441450. 18. Inoue S, Leblond CP, Grant DS, Rico P. The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component. Am J Anat 1986;176:139-152. 19. Myles JL, Ratliff NB, McMahon JT, et al. Cyclosporin-associated microfibrils in cardiac transplant patients. Am J Cardiovasc Path01 1988;2:127-132. 20. Low FN. Microfibrils, fine filamentous components of the tissue space. Anat Ret 1962;142:131-137. 21. Raviola G. The fine structure of the ciliary zonule and ciliary
Cardiovasc Pathol Vol. I, No. 4 October-December 1992:307-3 16
epithelium: with special regard to the organization and insertion of the zonular fibrils. Invest Ophthalmol 1971;10:851-869. 22. Hollister DW, Godfrey M, Sakai LY, Pyeritz RE. Immunohistologic abnormalities of the microfibrillar-fiber system in the Marfan syndrome. N Engl J Med 1990;323:152-159. 23. Lee B, Godfrey M, Vitale E, et al. Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes. Nature 1991;352:33&334. 24. Maslen CL, Corson GM, Maddox BK, Glanville RW, Sakai LY. Partial sequence of a candidate gene for the Marfan syndrome. Nature 1991;352:334-337. 25. Dietz HC, Cutting GR, Pyeritz RE, et al. Marfan syndrome caused by a recurrent de nova missense mutation in the fibrillin gene. Nature 1991;352:337-339. 26. Glenner GG. Amyloid deposits and amyloidosis. The betafibrilloses [first of two parts]. N Engl J Med 1980;302:1283-1292. 27. Kyle RA, Greipp PR. Amyloidosis (AL): clinical and laboratory features in 229 cases. Mayo Clin Proc 1983;58:665-683. 28. Roberts WC, Waller BF. Cardiac amyloidosis causing cardiac dysfunction: analysis of 54 necropsy patients. Am J Cardiol1983; 52:137-146. 29. Pellikka PA, Holmes DR Jr, Edwards WD, Nishimura RA, Tajik J, Kyle RA. Endomyocardial biopsy in 30 patients with primary amyloidosis and suspected cardiac involvement. Arch Intern Med 1988;148:662-666.
