Dilated cardiomyopathy: Emerging role of endomyocardial biopsy

is currently an Associate Professor of Medicine, Section of Cardiology, at Loyola University Medical Center. He graduated from Loyola Stritch School of Medicine in 1974 and did a straight medical internship and chief residency in internal medicine and a cardiology fellowship at LUMC. He is board certified in internal medicine and cardiovascular diseases and is a Fellow of the American College of Physicians, the American College of Cardiology, American College of Chest Physicians, and the Council on Clinical Cardiology of the American Heart Association. Dr. O’Connell’s research interests over the last seven years have been in the study of dilated cardiomyopathy and myocarditis. More recently his work has been in the area of cardiac transplantation, and he serves as the medical director of Loyola’s Cardiac Transplant Program. He has authored 41 publications on this topic and edited a monograph entitled “Myocarditis: Precursor of Cardiomyopathy.”

is a graduate of the University of Bologna Medical School (Italy). She received her clinical training in internal medicine at Lutheran General Hospital in Park Ridge, Ill., and in cardiology at Loyola University Medical Center, Maywood, Ill. Dr. Costanzo-Nordin is currently an Assistant Professor of Medicine and the Assistant Medical Director of the cardiac transplant program at Loyola University Medical Center, Maywood. Her research interests include immunomodulation of myocarditis, evaluation of new immunosuppressive modalities in heart transplantation, and prognostication in dilated cardiomyopathy. 448

. obtained his medical degree from Madras University in India. His pathology residency was completed at the University of Kansas, followed by a fellowship in cardiovascular pathology at the Mayo Clinic. Presently, Dr. Subramanian is Assistant Professor of Pathology and Director of Autopsy Service at the University of Wisconsin Clinical Science Center in Mad&on, and a staff pathologist at William S. Middleton Memorial VA Hospital, Madison. His present research interests include the role of free radicals in myocardial injury during ischemia and reperfusion.

is a board-certified internist with subspecialty certification in rheumatology &liated with the Loyola University Medical Center, Maywood, Ill. A Professor of Medicine and Microbiology at Loyola, he is both Chief of the Section of Clinical Immunology and Rheumatology and Director of the Therapeutic Pheresis Unit. He serves as the immunology consultant to the cardiac transplant program. Dr. Robinson is chairman of both the biomedical research support grant committee and the research fellowship and awards committee at Loyola.

449

DILATED CARDIOMYOPATHY: EMERGING ROLE OF ENDUMYOCARDIAL BIOPSY

Congestive heart failure (CHF) remains a major worldwide cause of cardiovascular morbidity and mortality in a wide spectrum of age groups. A recent shift of prevalence of CHF etiologies has occurred in parallel with the development of modern medical technology and pharmacologic interventions. Prior to the advent of routine and safe techniques for cardiovascular surgery, valvular and coronary heart disease were commonly associated with CHF. Currently, CHF may often be palliated successfully or prevented with valve replacement. Recognition of the long-term effects of hypertension, coupled with more effective therapy, has reduced the prevalence of CHF in hypertensive patients. In direct contrast to the above conditions, however, the importance of primary heart muscle disease as a causative factor of CHF is rapidly gaining acceptance. Therefore, an emerging emphasis in cardiovascular research directed toward ascertaining the pathogenesis and treatment of heart muscle disorders has culminated in a large-scale international effort to unravel the mysteries of primary myocardial disease. We hope to underscore this rising interest by summarizing the role of endomyocardial biopsy (EMB) as a diagnostic modality that is leading to further refinement of concepts of pathogenesis and therapeutic interventions in heart muscle disease. Dilated cardiomyopathy (DC) may clinically be defined as left ventricular dilatation and systolic dysfunction in the absence of congenital, coronary, valvular, or pericardial heart disease. This condition occurs in all age groups from the pediatric through the geriatric population. The incidence ranges from five to ten new cases per 100,000 population per year.2 Patients frequently have symptoms and signs ranging from asymptomatic cardiomegaly to severe end-stage congestive heart failure. Arrhythmias or thromboembolic events may not infrequently be the first manifestation. Physical findings are variable but typically include evidence of pulmonary congestion, a displaced apex beat signifying cardiac enlargement, and an S3 gallop with or without a mitral regurgitation murmur. 450

Since etiology is not intrinsic to this definition, it is not surprising that DC represents a heterogeneous group of disorders. It is therefore also not surmising that prognostication is, at best, a problem when the patient is fk-st encountered. Although it is difficult to predict the prognosis in a given patient, the median survival is generally less than five years.’ In the Mayo Clinic study of the natural history of DC, 50% of the patients died within five years of onset of symptoms, yet 25% of the patients survived for ten years, and many of these had clinical and radiologic evidence of dramatic improvement in their clinical state.2 In our series of 109 patients followed for up to 60 months, 20% of the patients died within the first year of onset of symptoms (Fig l), and 25% showed evidence of significant improvement over the follow-up period.3 This survival rate compares favorably to that observed by Figulla et al.: who noted a similar oneyear survival rate. In the latter series, however, the spontaneous improvement or stabilization rate was greater than 50%. The large group of patients with improved survival emphasizes the inconsistency and danger in predicting prognosis for individual patients. With the advent of cardiac transplantation as an efficacious therapeutic alternative, the importance of precise prognostication in DC has to be emphasized to identify those who

la0\ 30 I

\ “i.\

80

L-...

---..

70

-.

-

---_._ .‘\ ‘..

FOLLOW-UP

(MOS)

FIG 1. Survival of dilated cardiomyopathy Medical Center. (From O’Connell Used with permission.)

over six months’ follow-up at Loyola University JB. et al: Heart Vessels 1985; l(suppl I):175 451

are likely to deteriorate rapidly and therefore should be considered as suitable candidates for transplantation early after the onset of CHF. Various prognostic indices of poor survival have been described previously (Table 11. Most authors agree that the severity of symptoms correlates closely with prognosis. Shirey and others5 noted that the five-year mortality rate of DC in the absence of CHF was only 16.4%, but if CHF was the presenting manifestation, the mortality rate was 57.4%. In our series, patients with increased severity of New York Heart Association functional class had a poor prognosis.6 In contrast, the presence of dyspnea or easy fatigability did not correlate with prognosis in the series reported by Unverferth et al.’ The severity of left ventricular dilatation is also recognized as a poor prognostic factor. When the cardiothoracic ratio was greater than 0.55 on initial examination of the chest x-ray, mortality six years later was 86%.2 Yet, left ventricular end-diastolic diameter measured echocardiographically did not correlate with prognosis in our series.6 Hemodynamics in patients with DC have been extensively studied as possible independent predictors of mortality. In general, interrelated severe decreases in cardiac output, elevation of left ventricular end-diastolic pressure, and decreases in ejection fraction predict a high risk of early mortality. Independent predictors include a left ventricular end-diastolic pressure of over 20 mm Hg that has been associated with a 50% two-year mortality; a cardiac index less than 3 L/mm/m2 has been associated with an 89% six-year mortality; and an ejection fraction less than 20% was associated with a negligible two-year survival.2z s+’ In summary, the greater the severity of derangement of left ventricular function, the greater the mortality. Mortality estimates based on ECG abnormalities, particularly ventricular arrhythmia and ambulatory monitoring, have been TABLE

1.

Poor Prognostic

Signs in Dilated

Cardiomyopathy

ACCEPTED

NYHA Class III or IV Cardiothoracic ratio >0.55 Left ventricular end-diastolic pressure Cardiac index ~3.0 L/min/m2 Ejection fraction ~20% Diffuse wall motion abnormalities CONTROVEXSIAL

Echocardiographic LV diameter Ventricular arrhythmia Histologic findings

452

>20 mm Hg

controversial. Ventricular tachycardia in DC documented by Holter monitoring is common; in our series, 40% of patients had runs of ventricular tachycardia when carefully monitored.6 Programmed electrical stimulation in the electrophysiologic laboratory has also not been helpful in prognostication based on these arrhythmias.” In some series, survival was strikingly related to the presence of these arrhythmias72 ‘I; however, in others it could not be proven.12, I3 Therefore, the role of ambulatory monitoring as a prognostic tool remains controversial. Physiologic principles suggest that a lack of appropriate compensatory hypertrophy may lead to increases in left ventricular wall stress and subsequent increase in oxygen demand per muscle unit. It is therefore logical that a greater degree of left ventricular hypertrophy may, in fact, be protective in preserving myocardial function. Early studies suggested that ECG evidence of left ventricular hypertrophy predicted a favorable prognosis.i4 This was further supported by necropsy evaluation of hearts of patients with DC. Survival was longer when the ratio between the left ventricular wall thickness and cavitary diameter was greater than 0.21 at necropsy than those with a ratio of 0.17 or less.i5 When evaluating angiographic mass-volume ratio, Feild et al.’ identified a subgroup whose high thickness/cavity volume ratios predicted improved three-year survival in a series in which the two-year overall survival was only 50%. When wall motion abnormalities on radionuclide ventriculography was clearly segmental, the survival is significantly improved over those with diffuse wall motion abnormalities.16 It is apparent that, in patients with DC and clinical, hemodynamic, and noninvasive parameters that suggest a poor prognosis, survival is accordingly quite poor. Yet, in careful analysis of individuals who improved spontaneously, we noted that 44% of these patients had a cardiac index of less than 3 Llminlm’, 15% had left ventricular filling pressures greater than 20 mm Hg, and 41% had a left ventricular ejection fraction of less than 20% (Table 2). Before precise therapeutic recommendations may be developed, more accurate prognostic factors must be defined. The treatment of DC is nonspecific (Table 3). Symptoms of congestive heart failure may now be successfully alleviated by the combination of inotropic agents, vasodilators, and diuretics. Yet, to date, no study has confirmed that this therapy improves survival. This should come as no surprise, since this therapy is directed at treating the effect (congestive heart failure) rather than the cause of the cardiomyopathic state. Recent studies suggest that beta blockade may result in dramatic symptomatic improvement.17’ l8 Retrospective, uncontrolled analysis of survival suggests chronic beta blockade may be beneficial in decreasing mortality. lg Although intriguing, firm therapeutic recommendations cannot be made until large-scale, randomized, controlled 453

TABLE 2. Comparison of Clinical and Hemodynamic Parameters of Improved and Unimproved Patients with Dilated Cardiomyopathy*t IMPROVED

No. (%o)

27 (25%) 46.6 IL 12.8$

Age, yr Sex, M/F Cl III-IV CI, L/minim’ PAW, mm Hg PA,,, mm Hg LVEDD, cm

17/10

18 2.9 14.1 32.8 6.7

(67%) r?l 0.89 t 7.611 -t 10.811 2 0.7

UNIMPROVED

82 (75%) 46.3 k 15.1 57125 63 (76%) 2.5 +- 0.7 20.8 r 10.2 44.2 5 16.6 7.0 t 8.4

*From O’Connell JB, et al: Heart Vessels 1985; l(supp1 1):175. Reproduced with permission of the American Heart Association. t Cl II-IV = NYHA Functional Class; CI = cardiac index; PAW = mean pulmonary arterial wedge pressure; PA,. = pulmonary artery systolid pressure; LVEDD = left ventricular end-diastolic dimension; EF = ejection fraction. *Mean f SD. BP-c .Ol. i/P < ,001.

TABLE 3. Treatment

of Dilated

CardiomvoDathv

Diuretics Vasodilators Inotropic agents Anticoagulants Beta-blockers Cardiac transplantation

trials confirm these preliminary observations. The only therapy that has to date been shown unequivocally to pro1025 survival in dilated cardiomyopathy is cardiac transplantation. In summary, all prognostic guidelines and therapeutic revelations suggest that major strides in altering the natural history of dilated cardiomyopathy will only emerge as etiologic factors are identified. It is likely that DC represents several independent disease states, all of which result in abnormalities in heart muscle function. It would be naive to think that a single therapeutic modality would be beneficial for all. Newer data-gathering techniques and development of animal models are necessary to identify etiologic factors that may be treated in a more specific way. One major step, access to human cardiac tissue during life by biopsy, is already providing major insights into etiologic subgroups of DC. 454

P.M. SHAH: A recently completed multicenter VA cooperative study on the long-term effects of vasodilator therapy on chronic heart failure randomized patients into three treatment arms: placebo group, prazosin group, and combination isordil-hydralazine group. All patients received digitalis and diuretics. The major endpoints were survival and exercise capacity. The study showed improved survival for patients on isosorbide dinitrate-hydralazine combination as compared with those on placebo or on prazosin. No concomitant improvement of exercise capacity was noted in this study; however, there seemed to be an improved ejection fraction for the effective treatment group. Further analysis of predictors of long-term outcome in this large multicenter study should be of considerable interest. (Cohn ---.. JN: N Engl J Med 1966; in press.) -

b

HISTORICAL

PERSPECTIVE

Over the past 20 years the development of angiography, echocardiography, and nuclear medicine has offered a variety of diagnostic

techniques

that have rapidly

advanced the understand-

ing of coronary, valvular, and pericardial disease. In contrast, until recently the study of primary myocardial disease has been based solely on clinical suspicion and confirmation at autopsy. These limited techniques have provided a definition of DC only in the setting of terminal disease. While several entities are known

to produce the changes noted in end-stage

DC, there has

been no reliable method available to identify the various causes of primary myocardial disease. Hence, specific and effective treatment has been impossible. These obvious limitations have emphasized the critical need for a technique that allows histologic examination of the myocardium during life. After nearly two decades of development, the modern technique of endomyocardial biopsy (EMB) fulfills this need. In its early history, EMB was a complex and risky surgical procedure. One early technique involved resection of the fourth costal cartilage under local anesthesia and direct excision of a small piece of myocardium followed by suture repair.2* Another early trans-

mural myocardial biopsy technique utilized a thin needle to remove a “core sample” of myocardium after mediastinotomy. Common complications of these procedures included ventricular arrhythmia, myocardial tear ,zTneumonia, atelectasis, and the postpericardiotomy syndrome. Direct percutaneous access later became possible with the development of two techniques, one utilizing a flexible, thin-walled Terry needle with two cutting teeth in the point, and the other, a modified Menghini needle fitted with a sharply pointed stylet. In both techniques, the biopsy needle was inserted at the left costal sternal junction or above the point of maximal impulse to approach either the right ventricle or left ventricle, advanced to the surface of the heart, quickly introduced with a rotatory motion while

suction

was applied

by a syringe,

and then rapidly 455

withdrawn. While these techniques avoided some of the risks of open chest surgery, they invariably produced ventricular and subepicardial hemorrhages, pneumothorax, and pleuritic pain. Because of limitations of the equipment used and the small sample size recoverable, the specimens so obtained were often unsatisfacto 7. In addition, only one sample per procedure could be obtained.2 -25 Later, Shirey et a1.26 obtained percutaneous left ventricular apical samples using a Silverman needle. This is a two-piece instrument consisting of a 18gauge cutting stylet inside a thinwalled 14-gauge outer cannula. The needle was advanced medially and posteriorly to the epicardial surface of the LV apex under fluoroscopic guidance, from a position on the chest wall just above and lateral to the apex beat. In rapid sequence, the inner stylet was thrust forward and cut into the myocardium, the cannula was advanced over the stylet to help shave off and grasp the sample, and both components were then withdrawn while twisting them in a clockwise direction. This technique, the best available until the early 197Os, could obtain better samples than the previous methods, but its unacceptably high morbidity limited its usefulness. Cardiac tamponade requiring thoracotomy, postpericardiotomy syndrome, or ventricular fibrillation were not infrequent occurrences.26 All the techniques had several important disadvantages, including high morbidity, inability to obtain sufficient tissue for histologic examination, and universal failure to obtain endomyocardial tissue. Meanwhile, in the early 1960s Sakakibara and Konno in Japan introduced a transvenous approach for biopsy of the heart using an instrument that remains in contemporary use. The instrument consists of an operating handle, a 100~cm shaft, and a catheter tip made of two mobile cutting jaws. These are operated on a double swivel mechanism and are controlled by a wire running within the shaft. The wire is attached to a slide mechanism on the operating handle. Wire movement operates the opening and closing of the bioptome cups during excision of the sample. Under fluoroscopic control, the jaws are advanced to the chamber to be biopsied, pulled back 1 cm, opened, pressed against the wall, closed, and forcefully withdrawn.“7-2g Myocardial biopsy was revolutionized by the Konno bioptome. Multiple samples can be obtained with this instrument, and the procedure can be repeated without significant complications. The samples recovered are much larger than those provided by the earlier biopsy techniques. The instrument can be introduced intravenously or intra-arterially, thus allowing biopsy of the right atrium, right ventricle, and left ventricle. This bioptome was to have several limitations that became apparent as its usage increased. The instrument is not durable, and its size and rigidity make intracardiac manipulation difficult. Furthermore, 456

the cutting jaws work at a poor mechanical advantage and their size requires surgical venotomy or arteriotomy for introduction. To overcome these limitations, two instruments were developed: the King’s bioptome in England and the Caves-Schultz bioptome at Stanford in the United States (Table 4). The King’s bioptome, a modified Olympus fiberoptic bronchoscope biopsy forceps, consists of an operating handle, of a 105cm long catheter shaft, and of two mobile cutting jaws measuring 1.8 mm in diameter. The operating handle is made of a slide to which is attached a rod mechanism in direct continuity with the internal wire of the biopsy forceps. A spring mechanism maintains tension on the jaws so that they are kept closed during manipulation and after excision of the sample. The jaws, finely constructed with a scissor actiun, are connected to the operating handle by an internal wire that runs inside the shaft made of coiled stainless steel and covered with [email protected]*31 The King’s bioptome offers several advantages over the Konno biopsy forceps: first, the small size of the jaws allows percutaneous introduction and avoids venotomy or arteriotomy; second, the instrument can be introduced through a sheath previously positioned in the right or lef%ventricle, which allows rapid and serial introductions without repeated intravascular manipulations; third, the high flexibility of the instrument facilitates intracardiac manipulation and sampling. The Stanford bioptome (Fig 21, first designed by Caves and Schultz32, 33 and later modified by Mason3* was developed to fulfill the need for frequent, serial biopsies of the transplanted heart-a need dictated by the striking and rapidly evolving changes that could occur during rejection. Early detection and prompt reversal of rejection are critical for patient survival and preservation of allograft function. The Stanford bioptome consists of a 9 F catheter measuring 50 cm in length. A surgical forceps at the proximal end is affixed to a movable wire within the catheter shaft, thus permitting opening and closing of the jaws at the distal end. These consist of one fixed and one mobile TABLE Comparison

4. of the Konno,

King’s,

and Stanford

Bioptomes

KONNO

KING’S

STANFORD

Sheath Operating hand Operating mechanism Catheter shaft

9 French Handle Swivel 100 cm

7 French Handle Slide 105 cm

9 French Surgical forceps Forceps opening/closing 50 cm 100 cm (for LVEMB)

Cutting jaws Mobility Internal diameter Average sample size

Both 2.5 mm 3-4 mm

Both 1.8 mm 1-3 mm

One fixed 1.5 mm 2-3 mm 457

TThTTTTTTTTT The Stanford bioptome. (inset) close-up of the cutting jaws in the open position

part, each ending in a hollow cup measuring 1.5 m m in diameter. The distal portion of the bioptome is curved in the same direction of the handle so that the operator is aware of the attitude of the cups at all times. The angle at the distal end of the bioptome can be varied to facilitate crossing of the tricuspid valve and accurate positioning in the right ventricle against the septum. Although modified bioptomes have been designed to allow left and right ventricular biopsy from the femoral artery or vein, respectively, the Stanford bioptome is usually employed for right ventricular (RV) biopsy using the right internal jugular approach (Fig 3). 34The bioptome is introduced in the right internal jugular vein through an indwelling 9 F sheath. Under fluoroscopic control, the instrument is positioned in the lower third of the right atrium with the tip pointing laterally, rotated medially 180”, and advanced across the tricuspid valve. The tip is then rotated posteriorly and positioned at the apical portion of the septum. The instrument is withdrawn 1 cm, the jaws are opened and readvanced against the myocardium, closed, and the tissue is separated *ith a gentle tug. Multiple samples are obtainable by serially reinserting the bioptome. Following removal of the sheath, the vein remains patent for subsequent cannulations. The skin puncture site heals with a small scar without suturing. The advantages provided by the Stanford bioptome are nu458

FIG 3. Right ventricular endomyocardial biopsy. Schematic drawing of the bioptome bntering the right ventricle from the right internal jugular vein.

merous. The percutaneous right internal jugular approach provides rapid and direct access to the right ventricle. The stiffness and shortness of the instrument increases the mechanical efficiency of the closing mechanism and improves maneuverability of the catheter. The fixed cup allows firm application of the forceps to the endocardial surface. Due to the sharpness of the jaws, the sample is removed by cutting and not by avulsion, which improves sample size and reduced biopsy artifacts.33, 34 In addition to the EMB techniques utilizing the King’s, Konno, and Stanford bioptomes, several other transvascular methods are available. A modified GFB gastrointestinal biopsy catheter has been used to perform right ventricular endomyocardial biopsy.35 The method proposed by Bullock combines the transvenous intracardiac approach with needle biopsy technique. A 50-cm 16-gauge sheath introduced through the right 459

internal jugular vein is positioned against the right ventricular septum. A stainless steel cutting needle is then passed through the sheath and advanced transeptally to say?le the left ventricular portion of the inter-ventricular septum. Other intravascular techniques for needle biopsy of the interventricular septum have also been described.37 Despite the variety of bioptomes available, a 1980 world survey has shown that 94% of all biopsies are performed with either the Konno, King’s, or Stanford bioptomes. This survey reported that none of the three catheters was clearly superior to the others.38 A comparative study in dogs has shown that the King’s and Konno bioptomes are equally safe and efficient, although the King’s technique appeared easier to perform and the Konno bioptome provided larger samples.39 This study also showed that the type and extent of ultrastructural artifacts did not differ significantly when percutaneously or open myocardial biopsies were compared to the transvascular technique using the King’s bioptome.40 COMPLICATIONS

The percutaneous needle biopsy method has a high incidence of serious complications, including an 8% incidence of tamponade requiring thoracotomy in half of the cases.26 In contrast, intravascular endomyocardial biopsy, when carried out by experienced operators, is a safe procedure associated with negligible discomfort for the patient. There are fewer complications associated with endomyocardial biopsy than with renal biopsy, liver biopsy, or cardiac catheterization (Table 5L41 The reported overall incidence of complications with RV EMB ranged from less than 1% to 1.7%.42,43 Only two deaths were documented in 6,739 procedures in a recent world survey.38 Perforation of the ventricular wall has been reported in between 0.16% and 0.42% of the cases.38’42 This rare complication may be managed in most cases with pericardiocentesis, and seldom requires thoracotomy. Other complications, all of which are rare, include fleeting chest pains, transient or permanent right bundle-branch block, 1” to 2” AV block, and short-lived atria1 or ventricular arrhythmias.38 Only one instance of sustained ventricular tachycardia has been reported.44 The complications related to internal jugular vein puncture (vein thrombosis, air embolism, pneumothorax, and transient nerve palsies) are almost always caused by inexperienced operators and are usually due to inaccurate identification of the anatomic landmarks.34 In over 1,000 biopsies performed in our center using the Stanford bioptome, two pneumothoraces, one episode of complete heart block, and one episode of ventricular tachycardia requiring cardioversion represent the only major complications. Cardiac perforation and 460

Biopsy __-..-~.

1,000 Stanford bioptome

O ’CONNELL, LOYOLA. USA

4

4

0

0 0 2 0 2

a

1 6 3

Cl

i 7’<

2,337 Various instruments for transvascu1ar biopsy

RICHARDSON. WORLD SURVEY

3 1

3,097 King’s bioptome

OLSEN NATL HEART HOSP., LONDON

0

4,000 Stanford bioptome

FCIWLES-MASON. STANFORD. USA

II

6,739 Various instruments for transvascu1ar biopsy

SEKIGUCHI, WORLD SURVEY

of Endomyocardial

Overall complicat loo rate Death Cardiac tampor1ade Arrhythmias Sustained ventricular arrhythmias BBB High degree AV block Vein thrombosis Arterial injury Right recurrent laryngeal nerve paresis Homer’s syndrome Air embolism Pneumothorax Pericarditis Chest pain Catheter entrapment

No. of biopsies Bioptome

TABLE 5. Complications

tamponade has not occurred. Failure to obtain adequate tissue is rare, occurring in 2% or less of the procedures performed with the Stanford bioptome.34 LEFT

VENTRICULAR

BIOPSY

The left ventricle is the chamber predominantly involved in most cases of acquired heart disease; consequently, biopsy of this chamber can be a valuable diagnostic tool. Although left ventricular biopsy has been accomplished with the Konno, King’s, and Stanford bioptomes, these instruments have been unsatisfactory in obtaining left ventricular tissue samples because their stiffness makes it difficult to cross the aortic valve. The limitations have inspired the design of a biopsy catheter specifically intended for the LV.45 This instrument consists of a 120~cm catheter with an operating handle at the proximal end and cutting jaws at the exploring end. By rotating a knob on the operating handle, the tip can be bent 40”, allowing easy passage through the aortic arch and across the aortic valve. A straight stylet, inserted from a side-hole at the operating handle, can be advanced to the end of the catheter to obtain the stiffness necessary for excision of the tissue. Soon after the availability of these techniques greatly facilitated left ventricular biopsy, substantial controversy arose regarding the comparative safety and diagnostic usefulness of biopsies of the two ventricles (Table 6). Despite predictions that the procedure would result in grave complications such as arterial thrombosis and embolism, stroke, myocardial infarction, TABLE Right

6. vs. Left Ventricular

Biopsy catheters

Vasculaf

access

Complication Unsatisfactory sampling 462

rate

Endomyocardial

Biopsy

RVEMB

LVEMB

Konno King’s Stanford Olympus GFB gastrointestinal bioptome Right internal jugular (percutaneous) Right subclavian vein (percutaneous) Antecubital vein (percutaneous or venotomy) Femoral vein (percutaneous or venotomy) 2%

Konno King’s Modified Stanford Kawai and Kitaura

2%-13%

Brachial arteriotomy Femoral (percutaneous or venotomy) Transseptal

2% O%-2%

arrhythmias, and even death, left ventricular endomyocardial biopsy has proven to be as safe as right ventricular endomyocardial biopsy.34 There has been only one reported case of cerebral embolism attributed to left ventricular biopsy, and this involved only a transient neurologic deficit.46 In terms of tissue yield, Stanford investigators obtained inadequate samples in 2% of the right ventricular EMB and in none of the left ventricular EMB procedures. Other investigators failed to obtain a suitable sample in 13% of their right ventricular EMBs and in 2% of their left ventricular EMBs.~~ The controversy may be moot, however, since a critical analysis of several studies reveals that, in most instances, both ventricles show similar histologic changes, so that biopsy of either ventricle can be used to determine the presence and severity of the disease. In his study of pathologic findings of DC, Noda observed only minor discrepancies in the severity of myofiber hypertrophy and vacuolization between the right and the left ventricles. In contrast, other investigators found that fibrosis was significantly more severe in left ventricular than in right ventricular biopsy samples of patients with DC. The same authors reported that, in four cases of active myocarditis, inflammatory infiltrates were found in all left ventricular biopsy samples but in only half of the right ventricular biopsy samples.4R Although Brooksby and co-workers& noted that, in 60% of their patients, the morphological findings were invariably more marked in the left ventricle than in the right ventricle and recommended that right ventricular EMB be abandoned as a diagnostic technique, the validity of this observation is lessened by the fact that half of their patients had left-sided valvular disease, which logically produces morphological abnormalities more marked in the left than in the right ventricle.46 In selected conditions, however, left ventricular biopsy is superior to right ventricular biopsy. These include quantitation of hypertrophy and fibrosis in left-sided valvular disease and evaluation of hypertrophic cardiomyopathy, small vessel disease, and fibroelastosis. Left ventricular biopsy may also be useful when the right ventricular approach fails to provide diagnostic tissue (Table 7).34,42,4g TABLE

7.

Conditions in Which Left Ventricular Endomyocardial Biopsy May Be Superior to Bight Ventricular Endomyocardial Biopsy Endomyocardial fibrosis Scleroderma heart disease Left heart irradiation Cardiac fibroelastosis Small vessel disease’ Quantitation of fibrosis and hypertrophy in left-sided Evaluation of hypertrophic cardiomyopathy .-.-. -.. _ -_-I---. ---

valvular

disease

463

ENDOMYOCARDIAL

SAMPLE

PROCESSING

Accurate interpretation of myocardial histologic abnormalities requires careful handling and processing of myocardial tissue. The biopsy samples are removed from the bioptome with a fine needle and placed on filter paper (Fig 4). The tissue must not be handled with a forceps to avoid crush artifacts, which render histologic interpretation impossible. In our institution, the samples are then immediately fixed in 3% paraformaldehyde, which allows subsequent processing for both light and electron micros5oAfter two hours of fixation, the tissue to be used for elecCOPY. tron microscopy is selected with a dissecting microscope and fixed in gluteraldehyde. Tissue for light microscopy may alternatively be immersed in 10% buffered formalin, while that for electron microscopy is fixed in gluteraldehyde immediately after removal from the bioptome.42 This method may produce more ultrastructural artifacts because the tissue must be cut while still fresh. A portion of the tissue can be embedded and rapidly frozen in isopentane and dry ice to be used for immunofluorescence studies.51 After fixation, the myocardial samples are paraffin embedded and cut in 4- to 5-p sections. Five sections of all specimens are placed on each slide. Of the four necessary slides, two are stained with hematoxylin-eosin and Masson trichrome stains to evaluate fibrosis, to identify cellular infiltrates, and to analyze structural changes of the myofibers. One slide is reserved for special stains when a specific disease is suspected. Sulfated alcian blue and Congo red are used to identify amyloidosis and Prussian blue to demonstrate iron deposits in hemochromatosis.52 When acute viral myocarditis is suspected, a piece of myocardium may be placed in Earle’s medium for viral culture. Myocardial tissue can also be processed for cellular isolation and immunocytochemistry.4z

FIG 4. Typical myocardial samples obtained with the Stanford bioptome. 464

ENDOMYOCARDIAI,

BIOPSY

INTERPRETATION

Regardless of the transvascular technique employed, biopsy samples consist of small fragments of endocardium and the immediately underlying myocardium. Because biopsy samples are small and do not include myocardium deeper than 5 mm sampling error may lead to inaccurate pathologic diagnosis. 83 The likelihood of incorrect histologic interpretation resulting from sampling error is effectively reduced by obtaining at least three to five samples. At autopsy, when five or more tissue samples were examined, the diagnostic or quantitation of fibrosis errors were virtually eliminated.“4 A greater number of samples is required when the suspected pathologic entity has a focal distribution, the prime example being myocarditis. Nonrepresentative tissue samples may be obtained in patients who undergo repeated biopsies because the anatomic configuration of the right ventricle may guide the catheter to the same site repeatedly. This results in removal, not of myocardium, but of granulation tissue caused by a previous biopsy. Small segments of coronary arteries surrounded by adipose tissue can be normally seen in endomyocardial biopsy samples and do not necessarily indicate epicardial biopsy or wall perforation (Fig 51.“’ The small thrombi that frequently form on the bioptome during biopsy may be mistakenly identified as part of a mural thrombus or as myocardium. If the septum is biopsied at the base and not at the apex, a small segment of tricuspid chordae tendineae may

FIG 5. Endocardial adipose tissue (Ai. Masson‘s trichrome; original magnification

x 63. 465

be present on the sample but usua!r does not result in clinically Over- and underestimation important tricuspid regurgitation. of endocardial fibrosis is frequent because of the variable thickness of the endocardium.55 The diagnosis of myocardial hypertrophy must take into account both nuclear abnormalities and increased cell size. Stretching of myocardial cells secondary to cavity dilatation may result in a false conclusion that cells of normal diameter are present.56, 57 Myocardial disarray is a normal finding at the junction of the trabeculae carneae and ventricular myocardial and, when found in this location, must not be considered diagnostic of hypertrophic cardiomyopathy.52* 55 Densely stained contraction bands, unavoidably frequently observed in EMB samples, are artifacts caused by excessive contraction of sarcomeres induced by the biopsy procedure. These must not be interpreted as a pathologic finding (Fig 6).55358 Contraction bands and shrinkage of the tissue samples during processing may lead to the erroneous diagnosis of interstitial edema. These artifactual contraction bands, when observed by electron microscopy, reveal mitochondrial damage, sarcolemmal folding, disruption of the sarcoplasm, and displacement of glycogen and lipid vesicles. These artifacts, most likely caused by cutting fresh myocardium, can be minimized by placing the biopsy sample in fixative prior to cutting and maintaining the fixative at room temperature. Since a zone of cutting artifact ex-

FIG 6. Contraction band artifact appearing as alternate Masson’s trichrome; original magnification x 63. 466

areas of dark and light staining.

tends 0.5 to 0.6 mm from the cutting, morphological changes should not be interpreted in cells at the periphery of the specimen.5g When the diagnosis of myocarditis is entertained, one must keep in mind that many interstitial cells, when cut in cross-section, resemble lymphocytes and should not be mistaken for mononuclear infiltratesce Finally, when amyloidosis is suspected clinically, special stains should be done, because amyloid may resemble collagen on trichrome stain and may be misinterpreted as increased interstitial fibrosis.62 INDICATIONS

FOR ENDOMYOCARDIAL

BIOPSY

Endomyocardial biopsy has become an established diagnostic tool with many useful applications (Table 8). One of its most widely accepted indications is for detection and follow-up of cardiac transplant rejection (Fig 7). The successful maintenance of a cardiac allograft depends on the ability to detect rejection early and reverse it promptly with immunosuppressive therapy. The Stanford group has shown that histologic rejection can be revealed by EMB 26 to 48 hours before it can be detected by decreased ECG voltage, or any other previously used noninvasive method. Biopsy permits accurate estimation of the degree of rejection so that immunosuppressive therapy can be tailored to the severity of histologic changes. With appropriate therapy, even the more severe histologic changes of rejection may begin to resolve within 24 hours and be completely reversed within 72 hours.61 Thus, serial biopsies, which can be safely and successfully repeated as often as needed, make it possible to monitor the effectiveness of the selected therapy in controlling rejection. The histopathologic features of acute cardiac transplant rejection are graded according to the criteria developed at Stanford.42P51 Mild rejection is characterized by endocardial and interstitial edema with scanty perivascular lymphocytic infiltration. In moderate rejection, more extensive endocardial, interstitial, and perivascular infiltrates are seen. Myocyte necrosis can be seen in heavily infiltrated areas. When the myocyte injury is more extensive (severe rejection), the interstitial infiltrate may include polymorphonuclear leukocytes, and interstitial hemorrhages may be present. The infiltrating lymphocytes stain intensely with methyl green-pyronine during rejection episodes. TABLE Indications

8. for Endomyocardial

Biopsy

Detection and grading of cardial transplant rejection Detection and grading of doxorubicin cardiotoxicity Diagnosis of active myocarditis Distinction between constrictive and restrictive heart disease 467

FIG 7. Acute rejection indicated by interstitial and perivascular lymphocytic and plasma cell infiltration (arrows) and myocardial cell necrosis. Masson’s trichrome; original magnification < 100.

but lose their pyroninophilia following treatment. In patients treated with cyclosporine, sparse interstitial infiltrates of poorly staining mononuclear cells and a fine, diffuse interstitial fibrosis may be present in the absence of rejection.62 In these patients, rejection is characterized by an intensive perivascular and interstitial infiltrate of lymphocytes often associated with focal myocyte necrosis. After heart transplant recipients have undergone numerous endomyocardial biopsies, some samples that are from previous biopsy sites appear as discrete areas of necrotic myocardial cells with associated mononuclear infiltrates or granulation tissue (Fig 8).‘3 The adjacent intact myocardium cannot be used to evaluate rejection since the inflammation associated with a healingbiopsy site can extend to the surrounding myocardium, leading to an erroneous interpretation of rejection. In transplant recipients, EMB may also reveal life-threatening, opportunistic infections such as toxoplasmosis.“4 Another useful application of EMB is in the diagnosis and monitoring of doxorubicin (Adriamycin@)-induced cardiotoxicity.65 This powerful chemotherapeutic agent, although effective in man malignancies, can produce progressive congestive heart failure. 8‘a 67 Noninvasive methods of measuring left ventricular function have been used to detect the largest dose of doxorubicin that can be safely administered without producing irreversible cardiac impairment. Based on the information obtained with 468

FIG 8. Previous biopsy site appearing as a discrete area of granulation tissue (G) in the endocardium and containing small iymphomononuclear infiltrates. Masson’s trichrome: original magnification x 63.

these methods, a maximal dose of 550 mg/m’ has been commonly adopted. Morphological changes, which often precede physical signs, have been well characterized and can be reproducibly graded.68-70The histologic hallmarks of doxorubicin cardiotoxicity are myofibrillar loss, which, readily identified by EMB, causes the cells to appear shrunken, with pale homogeneous cytoplasm, and cytoplasmic coalescence of dilated sarcoplasmic reticulum.51, 71 In m ild doxorubicin myocardial damage, single myocytes show partial myofibrillar loss and swollen sarcotubular systems, while adjacent cells can be entirely normal. With moderate myocardial involvement, clusters of severely affected cells are seen. At this point, doxorubicin treatment can be cautiously continued provided that the patient has normal exercise hemodynamics. When myocardial involvement is severe, diffuse myocyte damage, including cell necrosis, is*gresent. No further doxorubicin should be given at this stage. Thus, the accurate and serial quantification of these histologic changes provided by EMB makes it possible to individualize the largest acceptable cumulative dose of doxorubicin.sg Serial EMB has provided histologic evidence that some patients tolerate larger doses than the accepted lim it of 550 mg/m’, and has identified additional risk factors such as older age, preexisting cardiac disease, hvpertension, or previous mediastinal irradiation that increase the risk .of developing doxorubicin cardiotoxicity. 41, 42. 49, 51. 72, 73 469

Endomyocardial biopsy can be used to differentiate constrictive from restrictive cardiac disorders, a task often impossible using clinical and hemodynamic evaluation alone. Patients with severe heart failure, no evidence of cardiomegaly or of systolic dysfunction, lack of radiologic or echocardiographic evidence of pericardial disease, but with equalization of the diastolic pressures may be more accurately treated after histologic examination of their myocardium. At Loyola University Medical Center between 1981 and 1984, we encountered several similar cases. In ten instances, amyloid heart disease was identified by EMB. This was the sole means by which a definitive diagnosis could be made. On light microscopy with hematoxylin-eosin and sulfated Alcian blue stains, amyloid appears as a homogeneous substance surrounding individual myocytes that infiltrates the vascular wall (Fig 9). Light microscopy may fail to demonstrate amyloid early in the disease, while electron microscopy may identify the characteristic aggregates of iibrils on the basement membranes of the myocytes and vessel walls.‘4 Endomyocardial biopsy is also of diagnostic value in sarcoidosis, another infiltrative disease, in which unexplained congestive heart failure and arrhythmias are occasionally its first manifestation. Because of the dramatic response of sarcoidosis to corticosteroid therapy, the demonstration of intracardiac noncaseating granulomas has important therapeutic implications.75 It is important to realize, however, that a negative EMB result

FIG 9. Cardiac amyloid (A) appearing as a homogeneous substance that infiltrates the interstitium and the myocardial cells. Masson’s trichrome; original magnification x 100. 470

does not rule out sarcoid heart disease with absolute certainty, because sarcoid granulomas are often found in deep areas of the septum inaccessible to the bioptome.75T76 When random myocardial biopsies of seven patients with sarcoidosis were done at autopsy, 63% of right ventricular samples and only 47% of left ventricular samples were diagnostic. In contrast to cardiac sarcoidosis, hemochromatosis is almost invariably accompanied by manifest involvement of other organs. The iron overload of the myocardium produces cell damage when deposition occurs in the myocytes or stimulates fibrosis (Fig 10). Patients may demonstrate progressive congestive heart failure, massive cardiac enlargement, arrhythmias, and conduction disturbances. In this disorder, which can be primary or secondary to chronic iron overload, EMB is helpful in excluding other causes of heart failure because iron deposits in the myocytes are readily identified by the Prussian blue stain. Although EMB may underestimate myocardial iron content because of preferential deposition of iron in the subepicardial layer, serial biopsies during venisection and chelation therapy allow one to judge reversibility of heart disease by the degree of resolution of the hemosiderin deposits.7g Cardiac involvement is rare in Wilson’s disease. When CHF occurs in patients with hepatolenticular degeneration, EMB can establish whether cardiac decompensation is due to abnormal copper deposition in the myocardium or to other unrelated

FIG 10. Myocardial intracellular infiltration of dark-staining hemochromatosis. Prussian blue reaction x 250.

iron granules

in a patient with 471

causes. Quantitative copper analyses of endomyocardial biopsies in copper-induced heart disease demonstrate a copper content ten times that of normal myocardium and significantly greater than the Cu level of patients with Wilson’s disease who do not have heart failure.80 Endomyocardial biopsy can provide critical and therapeutic information in patients with endomyocardial fibrosis, a disease with clinical features indistinguishable from constrictive pericardial disease. The histologic diagnosis of endocardial fibrosis is based on the findings of a markedly thickened, fibrous endocardium without myocardial and epicardial involvement (Fig 11).81 The usefulness of EMB in this disorder is limited by the focal nature of the disease, leading to sampling error, and by the difficulty in excising the tenacious fibrous tissue with the jaws of the bioptome.4g Endomyocardial biopsy can detect endocardial fibrosis associated with carcinoid heart disease and methysergide-induced cardiotoxicity. The histologic characteristics of the two diseases may be indistinguishable because of the similarity between 5 hydroxytryptamine and the serotonin antagonist, methysergide. In both conditions severe valvular and ventricular dysfunction is attributable to a thick pannus of fibrous tissue that envelops the valve leaflets and extends to the endocardial surface. A distinguishing feature is predominant right-sided involvement with carcinoid and left-sided involvement with methysergide,41, 82

FIG 11. Endocardtal 472

fibrosis (f). Masson’s trichrome;

original magnification

x 200.

A characteristic lacework appearance of the myofibers with perinuclear vacuohzation and glycolipid-laden cytoplasmic inclusion bodies is found in Fabry’s disease, an X-linked disorder of glycosphingolipid metabolism resulting in tissue accumulation of ceramine trihexoside. The unexpected diagnosis of Fabry’s disease has been made by EMB in patients with heart disease masking as h pertrophic cardiomyopathy or restrictive cardiomyopathy.83-8 B Endomyocardial biopsy is emerging as a useful diagnostic tool in many other storage-type diseases and the skeletal myopathies.86 The increasing sensitivity of probes that detect genetic enzyme and structural protein mutations will increase its usefulness even further. Endomyocardial biopsy has added credibility to the hypothesis of a myocardial disease associated with mitral valve prolapse by demonstrating focal hypertrophy, increased endocardial and interstitial fibrosis, and mitrochondrial degyneration in many patients with this syndrome (Table 9). HISTOLOGY

OF DILATED

CARDIOMYOPATHY

The primary value of endomyocardial biopsy in DC is in confirming the diagnosis and ruling out clinically identical specific heart muscle diseases (Table 10). The histologic changes observed in DC are nonspecific; similar findings can be found in patients with congestive heart failure due to valvular and congenital causes. This implies that if a patient is suspected of having primary myocardial disease superimposed on valvular disease, it will be impossible to determine by histologic examination the relative contribution of each entity to heart failure. Thus, the nonspecific histologic changes seen on EMB become diagnostic of DC only when valvular, pericardial, or coronary artery disease have been excluded. Furthermore, in the absence TABLE 9. Specific Myocardial Diseases Diagnosed Endomyocardial Biopsy .--.-___Cardiac amyloidosis Cardiac sarcoidosis Cardiac hemochromatosib Endomyocardial fibrosis Endocardial fibroelastosis Methysergide toxicity Fabry’s disease of the heart Hepatolenticular degeneration Irradiation injury Glycogen storage disease Cardiac tumors --..-.._ -.-~- ._ - .-..- -

(Wilson’s

by

disease)

473

TABLE

10.

Possible Applications

of Endomyocardial

Biopsy

Dilated cardiomyopathy Confirmation of the clinical diagnosis Identification or exclusion of specific heart muscle disease Determination of prognosis Evaluation of response to therapy Confirmation of hypertrophic cardiomyopathy (left ventricular endomyocardial biopsy) Diagnostic aid in unexplained acute arrhythmias

of secondary causes of heart failure, endomyocardial biopsy in patients with DC cannot identify the specific underlying cause. Histologic changes found in alcoholic heart disease are indistinguishable from those found in peripartum cardiomyopathy.42 In addition to diagnostic nonspecificity, histologic changes observed in DC are of variable severity. When the clinical picture of CHF is present, EMB may be entirely normal or show severe myofiber hypertrophy, nuclear abnormalities, and fibrosis. Of 267 dilated cardiomyopathy patients studied by Olsen,57 19 (7%) had normal EMB findings. In another study of 34 patients, three (9%) exhibited no histologic changes, while 13 (38%) had m ild and 14 (41%) had severe histologic changes.55 Ths histologic characteristics of DC observed under light m icroscopy have been extensively described (Fig 12).88-g3The myofibers seen in the biopsy specimen frequently show great variability in size. When seen in cross-section, some have normal cell diameter (~20 ~1 while others appear severely hypertrophied. The striking variability commonly observed in myofiber size has been attributed to the decrease in the diameter of some but not all cells, caused by the stretching that accompanies cardiac dilatation. Abnormal branching myocytes are often seen. The nuclei of hypertrophic cells are usually enlarged, irregularly shaped, and hyperchromatic. 57 Dissociation of nuclear appearance to cell size (i.e., obvious nuclear changes of hypertrophy in cells of normal or decreased diameter) has been attributed to the aforementioned attenuation of the fibers.g4-g7 Increased interstitial collagenous tissue and areas in which fibrous tissue replaces myocytes that have undergone necrosis as a result of dilatation are frequently noted.‘* Intramyocardial vessels may be normal or show intimal thickening.g1 Focal endocardial thickening due to a prominent smooth muscle component indicative of long-standing dilatation is also seen. Interstitial lymphocytic infiltrates can be observed in DC in the absence of myocarditis and continually provoke controversy about the histologic criteria of the latter disease.” A wide variety of ultrastructural abnormalities have also been identified in dilated cardiomyopathy.100-105 The myofibrils may 474

FIG 12. Irregular myofiber hypertrophy and increased interstitial fibrosis surrounding individual myocytes typically seen in dilated cardiomyopathy. Masson’s trichrome; original magnification x 63.

be intact and normally aligned or disoriented and fragmented. Myofibrillar degeneration and loss may occur. The enlarged nuclei are bizarrely shaped owing to the convoluted nuclear membranes and intranuclear inclusions. Multinucleated myocytes can be seen. Striking m itochondrial changes, including m itochondrosis, pleomo hism, and cristolysis have been noted by most investigators? O”- lo5 Increased sarcoplasmic reticulum, prominence of the Golgi apparatus, and appearance of rough endoplasmic reticulum have been identified. Increased lysosomes, myelin figures, vacuoles, and lipofuscin granules are often evident. The intercellular spaces are widened and separated by lacuriae.*’ In similarity to the light m icroscope finding, the ultrastructural changes lack specificity. PROGNOSTICATION

BY BIOPSY

The severity of the histologic changes in DC has been studied in an effort to establish prognostic guidelines. These have produced conflicting results. When the angiographic and histologic data of 19 patients with DC were compared, no correlation was found between the degree of hemodynamic impairment and the severity of morphological alteration.106 In 12 children with DC, a rough correlation was identified between morphological abnormalities and the severity of clinical congestive heart failure, but 475

the myocardium was generally less abnormal than anticipated from the degree of hemodynamic impairment.lo7 Using histologic quantitative analysis, morphometric data of 55 patients with DC were compared with clinical and angiographic parameters. The myofiber diameter and the volume fraction of collagen tissue did not correlate with length of history, ejection fraction, and left ventricular end diastolic pressure.“’ A detailed analysis of the factors influencing the one-year mortality of dilated cardiomyopathy identified left bundle-branch block, mean right atria1 pressure, and ventricular arrhythmias as the most powerful predictors of prognosis.7 Quantitative morphological data (cell diameter, endocardial thickness, and percent fibrosis) failed to discriminate between patients at high and low risk. More recently, Schwarz and co-workers,1og using light microscopic-derived morphometry data from left ventricular biopsy samples in DC, found that myocardial fiber diameter and interstitial fibrosis had negatively correlated and volume fraction of myofibrils positively correlated to left ventricular ejection fraction. When the same authors conducted a prognostic study in their DC population, they concluded that patients with left ventricular ejection fraction lower than 35.5%, left ventricular systolic pressure greater than 120 mm Hg, 1efZ ventricular end-diastolic pressure greater than 13 mm Hg, and myofiber diameter greater than 25.5 Frn had decreased survival at one, two, and four years after EMB. However, multivariate analysis of hemodynamic and histologic data indicated that only left ventricular ejection fraction and left ventricular systolic pressure were significant independent predictors of survival. Morphometric data did not add prognostic information to that obtained from measurement of left ventricular hemodynamics.“’ In contrast to the results summarized above, other studies have demonstrated that histologic and ultrastructural changes correlate with left ventricular dysfunction and are powerful prognostic indicators. Myocardial fibrosis and hypertrophy of patients with “late stage” cardiomyopathy (defined by congestive heart failure at rest, roentgenographic cardiomegaly, ejection fraction lower than 50%, and increased end-diastolic volume) was significantly worse than in patients with “early” cardiomyopathy:“” When ultrastructural data were considered, a progressive change in the mitochondria correlated with progressive severity of myocardial disease.100 The mitochondria, initially large and increased in numbers, become small and degenerated in the late stages of the disease. Biopsy specimens in DC patients were studied by EMB using a semiquantitative morphological score based on degenerative changes (myelin figures, lipofucsin granules, lipid droplets, lysosomal changes, and lysis of myofilaments), mitochondrial abnormalities, myofibrillar changes (myofibrillar disorganization, thickened irregular Z bands), interstitial fibrosis, and myofiber 476

hypertrophy.11”-117 Using this scoring method, two patient groups could be delineated: the first comprised patients with a score of 4 or less. They had a 73% four-year survival. In contrast, the other group of patients with high scores (25) had an 18% two-year survival. The differential in prognosis was even more striking when patients with symptoms for less than two years were considered. The two-year mortality in this group was 100% in those with severe morphological alterations, but only 7.3% in those with mild histologic changes. Degenerative changes and alteration of the mitochondria were found almost exclusively in patients with high morphological scores, and these findings emerged as the must powerful predictors of poor outcome.ii7 Furthermore, when patients with asymptomatic left bundle-branch block, normal left ventricles, and normal coronary arteries were biopsied, the de ee of morphological change predicted the development of DC. 1*F The prognostic value of myocardial hypertrophy, fibrosis, and ultrastructural abnormalities was confirmed by Kunkel and coworkers. These investigators also demonstrated that myofiber diameter, fibrosis, and electron microscopic abnormalities increased progressively with the severity of clinical symptoms (graded according to the NYHA functional classification) and with the decline in left ventricular ejection fraction.1fg Collagen content correlated inversely with left ventricular end diastolic pressure and directly to ejection fraction.120 In our DC population, using a semiquantitative light microscopy score, we found morphological predictability at both clinical ends of the spectrum: patients dying early had high morphological scores with variable left ventricular function, while patients who spontaneously improved des ite poor initial hemodynamics had low morphological scores.’P’ Similar studies have been used recently to evaluate therapy responses. 122 Patients treated either’ with hydralazine only or hydralazine and isosorbide dinitrate demonstrated, on seral endomyocardial biopsies, regression in myocardial cellular hypertrophy.123 b P.M. SHAH: The reported observation

that hydralazine administered alone or in combination with isosorbide dinitrate has been associated with regression of myocardial cellular hypertrophy may provide an explanation for improved survival with use of these agents in the recent VA cooperative study. (N___-Engl J Med 1986; in press.) BIOCHEMICAL AND PHARMACOLOGIC ENDOMYOCARDIAL BIOPSY TISSIJE

ASSAYS

OF

The routine histologic examination of EMB in DC has not provided any specificity in differentiating this form of CHF from other etiologies. Biochemical analyses of EMB, although in their infancy,

may yield

new information

for the identification

of eti477

ologic and pathophysiologic derangements in DC. Ingwall and others124 studied left ventricular biopsy specimens from normal persons, patients with aortic stenosis, those with coronary disease without hypertrophy, and those with coronary disease with secondary hypertrophy. These authors found that the total level of creatine kinase decreases, whereas the level of MB isoenzyme increases during the hypertrophic state as well as coronary disease alone. The significance of this finding remains unclear. Other enzyme systems were studied by Peters and colleagues,125 who found a normal distribution of myofibrillar and mitochondrial enzymes in hypertrophic cardiomyopathies. However, in right ventricular EMB in DC, mitochondrial enzymes were markedly decreased, with lactic dehydrogenase increased. When the left ventricular myocardium was biopsied, mitochondrial enzymes and myofibrillar proteins were both suppressed. A decrease in myofibrillar calcium-activated ATP was similar to that found in patients with left ventricular dysfunction secondary to valvular heart disease, suggesting that defective mitochondrial function was a characteristic of DC and that perhaps the rise in LDH was compensatory. In an effort to further delineate the changes in LDH, Schultheiss et a1.126showed that the total LDH concentration was increased with a shift toward a rise in the LDH-5 isoenzyme in EMB samples. There was a strong correlation between severity of abnormalities of hemodynamics and the rise in LDH-5, suggesting that the myocardium was capable of this enzymatic shift when requiring anaerobic glycolysis due to the prolonged heart failure. This seems to be a nonspecific finding and, although interesting, will not serve as the link to correlate cellular biochemistry with function or etiology. Richardson and colleagues127 performed endomyocardial biopsies in 50 patients with DC, valvular heart disease, and alcoholic heart disease. Overall, there was no difference in total CK, LDH, MDH, alpha HBD, or SGOT values. However, when DC was compared to alcoholic heart muscle disease, elevations in CK, MDH, LHD, and alpha HBD levels were much greater in the latter. These preliminary data suggested that the ratio of alpha HBD to LDH and LDH to CK enzyme activities correlated closely with ejection fraction and represented an adaptive response in enzyme biosynthesis. More recently, the study of myocardial proteins has shifted toward the myofibrillar compartment. Yui et a1.128have demonstrated decreased myosin heavy chains in myocardial biopsies from a small number of patients with DC. Kawai et a1.12’have studied myosin heavy chains and actin in EMBs from patients with DC and showed that the myosin heavy chain to actin ratio correlated significantly with ejection fraction, and that myosin heavy chains decreased as fibrosis increased. The decrease in 478

myosin heavy chain seemed to correlate with the electron microscopic findings of lysis of myosin filaments in DC. Enzymatic analyses are difficult to perform and interpret, but the promise that technology that can identify cardiac gene expression by extraction of messenger RNA from biopsy tissue may be developed and supersede conventional biochemical approaches. Zaehringer and colleagues,13’ using surgically obtained, papillary muscle biopsy samples, studied four to five EMB-like specimens. They were able to extract enough messenger RNA content to quantitatively show its decreased synthesis in doxorubicin cardiotoxicity when compared to other forms of cardiomyopathy. Messenger RNA was also substantially increased in hearts of primary mitral regurgitation when compared to those with mitral stenosis. The potential for this technique is not in quantitation of the RNA, but in its ability to provide messenger RNA as a template to study myocyte protein synthesis in vitro. Since EMB-extractable messenger RNA is specific for cardiac myocytes, the ability to determine ratios of synthesis of contractile proteins is far greater than analysis of crude homogenates of myocardium in which fibroblast and endothelial cell proteins are also present. Future biochemical analyses may therefore be oriented toward defining the stimulus for the shift of isoenzymatic pattern and delineating the prognostic im@ation. studied ATP content in EMB Unverferth and colleagues and found that the ATP content of EMB in DC tissue was inversely correlated with right ventricular end-diastolic pressure and left ventricular end-diastolic pressure, suggesting that, as the heart muscle fails, the content of ATP concomitantly decreases. Myocardial organic acid composition has been studied by Hariguchi et a1.132These authors demonstrated that deoxyaldonic acids accumulated in large amounts in the hearts of dilated cardiomyopathy but were decreased in hypertrophic cardiomyopathy, suggesting altered organic acid metabolism in these hearts. When Yarom et a1.133studied elemental content in EMB by electron microscopic x-ray microanalysis, they detected phosphorus, sulphur, chlorine, calcium, zinc, and traces of iron and copper. Swollen, pale mitochondria had decreased levels of phosphorus, sulphur, and calcium when compared to dark mitochondria. The pathophysiologic role of these elements cannot be ascertained at present. In summary, the biochemical analyses, although promising, have been descriptive at best, and insights as to the causes of shifts in metabolic pathways and accumulation of metabolic byproducts cannot be judged based on these preliminary data. It is hoped that future interest in exploring the biochemical shifts in the failing heart, particularly those of contractile proteins, may yield more valuable information in defining a function/structure relationship in dilated cardiomyopathy. 479

RECEPTORPHARMACOLOGY

It has long been recognized that increased activity of the sympathetic nervous system is an important compensatory mechanism in CHF. The level of circulating catecholamines is elevated in heart failure in an effort to maintain the heart at its maximum inotropic capability. Cohn et a1.134have demonstrated that plasma norepinephrine levels greater than 800 pg/ml identify a CHF population with high mortality, yet myocardial catecholamine content is decreased in the failing heart. There also is decreased norepinephrine uptake at adrenergic nerve terminals. Despite this total decrease in myocardial catecholamines, there is sustained exposure of the heart to the increased plasma levels as a result of decreased clearance of norepinephrine. Evaluation of explanted human hearts has shown that beta-receptor density is decreased (down-regulation) in end-stage failing hearts secondary to high catecholamine exposure.135More recently, radioligand binding assays that measure beta-adrenergic receptors in EMB specimens have become available.136 This method confirms that down-regulation of the beta receptors is common and correlates directly with parameters of left ventricular dysfunction; that is, the more down-regulated the beta receptors, the lower the ejection fraction.137 The relevance of this observation is underscored by the recent information that chronic beta-adrenergic blockade may improve cardiac function of the myopathic heart and exercise tolerance in DC.17, l8 In uncontrolled, nonrandomized trialsi9chronic beta blockade has also appeared to improve prognosis. The mechanism by which beta blockade improves function in DC may be a result of its effect on beta-receptor density.13’ Beta blockade may result in “up-regulation” of beta receptors, and, therefore, more receptors will be occupied by the competitive antagonist. When increased catecholamines are available during periods of physical exercise, this antagonism is overcome, resulting in improvement in inotropic response and exercise tolerance due to a greater number of receptors available to respond to endogenous catecholamines. The roof of this hypothesis awaits a randomized, controlled trial.’ B’ Myocardial biopsy, therefore, may become a very useful tool in evaluating patients with end-stage DC, potentially predicting response to beta-adrenergic blockade. We can speculate that further research in receptor pharmacology of other hormone receptors may also result in improved therapies for the failing left ventricle. Currently, histologic evaluation of the myopathic heart is frequently unrewarding based on its lack of specificity, but the addition of newer biochemical studies (Table 111, particularly of contractile protein isoenzymes and receptor pharmacology, may supersede or redefine its use in the evaluation and treatment of patients with dilated cardiomyopathy. 480

TABLE

11.

Biochemical

and Pharmacologic .-

Analysis

of Biopsy Specimens

Enzymatic quantitation Total and isoenzymatic contractile protein quantitation mRNA quantitation and use as a template Organic acid studies Heavy metal quantitation Beta receptor density

BIOPSY IN ETIOLOGIC CARDIOMYOPATHY

SUBTYPES

-_

OF DILATED

MYOCARDJTIS

Historical

Perspective

It has long been postulated that infection of the heart may result in a latent form of heart disease that ultimately becomes clinically manifest as a fatal condition. The first specific reference to this hypothesis was proposed by Corvisart14’ in 1806, when he postulated that persons with carditis may become clinically improved and feel as though the disease has been cured; however, a transformation of the disease into a smoldering condition resulting in end-stage heart failure and death may result in many of these patients. Dr. Corvisart, being a pathologist, had no hard clinical or pathologic evidence supporting this data. It was only with the introduction of EMB as a clinical tool in transplantation, and the application of this technique to patients with heart disease of unknown cause, that the ability to histologically examine the human heart during life became practical. Mason and colleagues 14’in 1980 were the first to describe the findings of right ventricular EMB in patients with endrstage congestive heart failure of unknown cause who had been referred to the Standard Cardiac Transplant Program. They identified ten patients with histologic evidence of “inflammatory myocarditis” and treated them with immunosuppressive therapy. Four of these patients showed dramatic improvement, to the point that they were no longer considered transplant candidates. Four other subjects had stabilization of the previously progressive syndrome of congestive heart failure, and only two patients worsened. This important study initiated a rebirth of interest in myocarditis and cardiomyopathy. The subsequent additional experience of other investigators has changed the diagnostic approach dramatically in some patients with DC. Animal

Models

It is impossible to sequentially study the events that begin with a potentially asymptomatic viral infection of the myocardium and culminate in a DC in man. However, animal models have been developed in an effort to study the immunopathoge481

nesis of DC.‘42 When weanling male Balbic or Swiss ICR mice are infected with the known cardiotropic Coxsackie B3 virus, a self-limited, viral replication in the myocardium ensues. During this seven- to ten-day period, the animals may show no clinical signs of myocardial infection. Histologically, small areas of necrosis may be present, but there is little inflammatory infiltrate. The humoral immune system, interferon, prostaglandins, Blymphocytes, and monocytes are responsible for clearance of the virus. If these normal immunologic responses are interrupted with immunosuppressive agents during this phase, dissemination of the virus, overwhelming necrosis of the myocardium, and ultimate death results with persistence of viral replication in several organ systems. In the absence of immunomodulation, however, the virus is no longer detectable by culturing techniques after the seventh day. However, T-lymphocytes continue to be recruited to the myocardium and direct a chronic, smoldering inflammatory reaction results. These cytotoxic cells are responding either to an autoimmune cardiocyte stimulus augmented as a result of the viral infection or to the presence of a viral-initiated antigenie change of the adjacent fibroblasts. In Swiss ICR mice, the histologic inflammatory reaction persists up to six months after infection; at this point, the histologic findings of early replacement fibrosis and myocyte hypertrophy are intermediate between those of myocarditis and cardiomyopathy. If these mice are then followed for another six months, evidence of cardiac dilatation, ascites, pleural effusions, and clinical evidence of congestive heart failure becomes apparent. Histologically, inflammation is not present, but there are hypertrophied myofibers and interstitial fibrosis present. This pathologic picture is comparable to that seen in DC. Although absolute conclusions regarding the human disease cannot be drawn from this animal model, it is clear that, in some murine strains, an acute viral infection can result in persistent immunologic responses that result in a clinical and pathologic picture that is identical to DC. As further understanding of the immunologic responses in these mice occurs, comparable insights may direct research into etiologic, retrospective clues of DC in man. Human

Studies

It is obvious that extensive and similar studies in man are not possible, since infection with cardiotropic viruses are common in man and the congestive heart failure is probably an unusual manifestation of that process. Evidence supporting the infection immune theory of myocarditis is inferential at best (Table 12).143 A progressive illness initiated by a documented viral infection with clinical evidence of myocardial involvement culminating in the syndrome of idiopathic dilated cardiomyopathy has been reported. In 5% to 50% of patients with clinical acute viral myocarditis, follow-up studies document the incidence of persistent 482

TABLE

12.

Evidence Supporting Cardiomyopathy

Myocarditis

as a Precursor

of

.Persistent immune responses result in a myopathic state in mice after enteroviral infection Clinical progression from viral myocarditis to cardiomyopathy documented 5% to 10% of patients with clinical myocarditis develop cardiomyopathy on long-term follow-up Enteroviral antibody titer elevation greater in cardiomyopathic population than controls Defects in immunoregulation documented in dilated cardiomyopathy Myocarditis identified on biopsy in clinical dilated cardiomyopathy

cardiac abnormalities and clinical evidence of DC. This far exceeds the incidence of dilated cardiomyopathy in the general populati0n.l Indirect evidence of previous cardiotropic viral infection in patients with DC is provided by analyzing neutralizing antibody titers to enteroviruses compared with control populations. A high incidence of cardiotropic viral exposure in the cardiomyopathic population has been verified. Defects in lymphocyte suppressor cell function and natural killer cell function can be also identified in a subset of patients with DC. An immunoregulatory defect could explain why only a small proportion of patients, perhaps those who immunologically “overreact,” will go on to develop a chronic myocardial lesion after exposure to appropriate viruses. Although this evidence is indirect, it further supports the hypothesis that myocarditis, at least in a subsetof patients, may serve as a precursor for dilated cardiomyopathy. Noninvasive

Studies

Before a rational therapy for myocarditis can be derived, accurate, safe, diagnostic tools must be developed. The ideal diagnostic tool is one that is easily interpretable, noninvasive, reproducible on serial studies, accurate, and safe. Currently, no such technique exists. Since most viral infections are subclinical and perhaps temporally remote from the onset of CHF, there are no clinical clues (other than heart failure of unknown cause at first onset) that arouse suspicion of myocarditis. When we compared patients with biopsy-proven myocarditis to all patients seen by us for evaluation of heart failure of unknown cause but without biopsy evidence of myocarditis, no clinical or hemodynamic characteristic differentiated the biopsy-positive and biopsy-negative groups (Table 13).144These findings are similar to those of others. Radioisotopic techniques have recently been evaluated in pa483

TABLE

13.

Characteristics Biopsy-Proved

of Patients With and Without Myocarditis*t EMB +

No. Age, yr Duration, mo Antecedent virus ESR, mm Hg CI, L/m&m’ PAW, mm Hg EF, % LVEDD, mm *From

O’Connell

5 46.4 zt 19.2 9.5 k 14.8 2 (40%) 27.4 + 13.5 2.2 2 0.4 14.4 r 5.0 23.6 +- 8.0 62.6 f 12.4

JB, et al: Circulation

EMB

47.0 13.4 25 17.1 2.5 18.5 18.4 70.3

-

63 -t 13.2 5 15.6 (40%) -c 24.2 + 0.7 +- 10.6 t 9.3 -+ 9.2

1984; 70:58. Repro-

duced with permission. tP = NS in all categories. Data expressed as mean 2 SD. EMB = endomyocardial biopsy; ESR = erythrocyte sedimentation rate; CI = cardiac index; PAW = mean pulmonary arterial wedge pressure; EF = ejection fraction; LVEDD = left ventricular’end-diastolic dimension.

tients with clinical dilated cardiomyopathy, in the search for a noninvasive diagnostic test for myocarditis. When we investigated gallium-67 ?Ga) citrate, an inflammation-avid radioisotope that is commonly used for identification of chronic inflammation in other organs, we were able to identify a subgroup of patients with DC who had significant myocardial uptake of this isotope at 72 hours after injection (Fig 13).‘45 When a series of 67Ga-positive patients were given immunosuppressive therapy, 40% of them responded with dramatic improvement in hemody-

FIG 13. Ga67 images in a patient with dilated cardiomyopathy showing dense myocardial uptake in the anterior (L) and 60” LAO (ff) projections. (From O’Connell JB, et al: J Heart Transplant 1982; 2:7. Reproduced with permission.) 484

namics, ejection fraction, and symptoms. The gallium scans in these patients all converted to negative within three months after institution of prednisone and azathioprine therapy. In the remaining 60%, persistent 67Ga uptake was coupled with continued hemodynamic instability and a very high mortality rate. As a result of this study, we postulated that 67Ga avidity to the human myocardium may also result from conditions other than active myocarditis. Subsequently, a study of parallel gallium imaging with EMB demonstrated a strong correlation between dense Ga uptake over the myocardium and biopsy-proven myocarditis (Fig 14).‘44 The incidence of myocarditis on biopsy in the overall series was 8%; however, in those patients whose gallium scan showed uptake over the myocardium, the incidence of myocarditis rose to 36%, in excess of a four-fold increase in yield of biopsy. More importantly, only one of 57 gallium scans that showed no significant uptake over the myocardium was associated with biopsyproven myocarditis; in this patient, the myocarditis was thought to be drug induced, and the 67Ga visual image was affected by dense posterior mediastinal lymph node uptake. We therefore felt that 67Ga, although only studied in a relatively small series of patients, may ultimately become a useful screening test for the presence of active myocarditis when patients are seen with heart failure of unknown cause.

FIG 14. Parallel endomyocardial biopsy specimen from patient whose gallium scan is shown in Figure 13 demonstrating active myocarditis. Hematoxylin-eosin; x 20. (From O’Connell JB, et al: J Heart Transplant 1982; 2:7. Reproduced with permission.) 485

More recently, investigators from the Massachusetts General Hospital have identified an indium-1 1 l-labeled monoclonal antibody to myosin that can detect myocyte necrosis during active myocarditis.146 Although these studies are preliminary, there is a strong suggestion that this technique is extremely sensitive for the detection of myocardial necrosis and can identify a subgroup of patients with active myocarditis. Its sensitivity is such that any condition with myocardial necrosis may be detected. It is imperative that comparison studies be done, in conditions other than active myocarditis. Ultimately, isotopic tech-

niques may be a screening endomyocardial tic interventions biopsies.

method

to select patients

biopsy and also may be used to follow to obviate the necessity for repetitive,

for

therapeufrequent

b P.M. SHAH: The authors’ use of gallium-67 uptake to identify patients with myocarditis who may respond to immunosuppressive therapy appears to be a promising approach. A noninvasive screening test is needed, so that many patients with dilated cardiomyopathy who have no evidence of active inflammation will not require biopsy studies.$$dditionally, the patients with myocarditis may be followed by such technique to assess response to therapy. Further observations in this important area are eagerly awaited.

Histologic Studies in Myocarditis Following the original report of biopsy-proven myocarditis in patients with the syndrome of DC, several others reported a wide range of biopsy-proven myocarditis (0% to 67%) in similar patient populations (Table 14).47,55S“‘, 141,147-162This highly variable incidence most likely reflects the lack of a universal definition of myocarditis and is a direct result of individualized pathologic interpretation. To confuse this issue further, a number of synonyms have been proposed, such as “inflammatory myocarditis,” 41 ”chronic active myocarditis,“154 “active lymphocytic myocarditis,“60 and “acute, chronic, and rapidly progressive myocarditis,“i4’ all of which share similar descriptive characteristics. In an effort

to create uniform

definitions,

a panel

of patholo-

gists, by mutual agreement, has defined the “Dallas criteria.“163 The definition of myocarditis is contingent upon examining a minimum

of three

(but preferably

five) biopsy

specimens.

Evi-

dence for toxic or drug-induced myocarditis as well as ischemic heart disease are exclusions. The initial biopsy may be interpreted as active myocarditis with or without fibrosis if an inflammatory infiltrate as well as damage to adjacent myocytes are present (Fig 15). There is agreement that this diagnosis should be suspected even at low power, underscoring the obvious nature of the inflammatory infiltrate. If either the cellular infiltrate or the myocyte necrosis is not readily apparent, the biopsy

TABLE

14.

Biopsy Incidence of Myocarditis in Cardiomyopathy Kunkel et al. Mason et al. Noda et al. Baandrup & Olsen Das et al. Nippoldt et al. Fenoglio et al. Hess et al. Strain et al. Unverferth et al. Parillo et al. Zee-Cheng et al. O’Connell et al. Daly et al. Bolte & Ludwig Regitz et al. Ruzyllo et al. Dee et al. Ferriere et al. Hosenpud et al. Cassling et al.

(Frankfort, 19781 (Stanford, 1980) (Osaka, 1980) (London, 1981) (India, 1981) (Mayo Clinic, 1982) (Columbia, 1983) (Virginia, 1983) (Montefiore, 1983) (Ohio State, 1983) (Boston, 1984) (St. Louis U., 1984) (Loyola, 1984) (King’s College, 1984) (Munich, 1984) (Munich, 1984) (Warsaw, 1984) (Boston, 1985) (France, 1985) iportland, 1985) (Omaha, 19851

6% 2% 1% 1% 10% 5% 25% 26% 26% 6% 25% 63% 7% 17% 20% 6% 58% 67% 5% 16% 3%

N N N N N N N N N N N N N N N N N N N N N

= = = = = = = = = = = = = = = = = = = = =

66 400 52 132 10 170 135 23 64 59 74 35 68 69 91 299 48 27 59 38 80

may be interpreted as borderline myocarditis, which should imply to the cardiologist that the biopsy should be repeated if there is strong clinical suspicion. Subsequent biopsies will then be interpreted as “ongoing or persistent,” 9esolving or healing,” or “resolved or healed” myocarditis. It is hoped that wide applicability of the “Dallas criteria” will result in improved biopsy specificity in establishing a diagnosis of myocarditis. In other words, if myocardial biopsy is to be deemed the “gold standard” against which all noninvasive techniques are compared, then uniformity of definition is mandatory. Current interpretation of the literature regarding identification and treatment of myocarditis is fraught with conflicting results largely related to inconsistent biopsy interpretation. The requirement of biopsy evidence of myocarditis before a clinical diagnosis can be affirmed is adversely affected by the potential for sampling error. Myocarditis may be patchy or focal, and the random, minute specimens obtained by biopsy may not adequately reflect the histopathologic features of the entire myocardium. If there is strong clinical suspicion in the absence of biopsy confirmation, repeated biopsy and noninvasive techniques such as isotopic imaging may be utilized to support the possibility of sampling error. Unfortunately, these noninvasive techniques have not yet been standardized or applied to large populations of patients 487

FIG 15. Active myocarditis showing lymphomononuclear sis. Hematoxylin-eosin; x 20.

cell infiltration with myocyte necro-

VIROLOGIC AND IMMUNOLOGIC STUDIES IN DILATED CARDIOMYOPATHY AND MYOCARDITIS Easy and safe access to myocardium during life to obtain tissue for viral culture and high-level architectural studies was not available until the advent of EMB. In the autopsy studies and scattered instances of transthoracic myocardial biopsies, unusual cytoarchitecture, m itochondrial disarray, and suspect viral particles have been described.164-166A fatal instance of viral myocarditis has provided direct evidence that Coxsackie viruses could cause clinical myocarditis when grown from the postmortem heart.167 This observation, by definition, could not support the contention that viral infection m ight subsequently lead to DC. When a heart from a patient dying of dilated cardiomyopathy was studied, abnormal cell structures and virus-like particles were also described, but no specific m icrobiologic diagnosis could be rnade.ia One patient with DC underwent thoracotomy for biopsy, but only nonspecific, viral-like particles were found.‘66 The availability of EMB has now provided a means to test the etiologic hypothesis that an initiating agent sets up protracted abnormal immunologic responses that culminate in DC. A patient with varicella infection developed clinical myocarditis; 100 nm, hexagonal, dense-core herpes particles were seen in the myocytes at biopsy.16”His acute illness progressed to DC and he ultimately underwent cardiac transplantation. The explanted 488

heart had histologic features characteristic of DC, but no detectable varicella virus. Documentation of virus-initiated cardiomyopathy will continue to appear as EMB tissue is now studied with new techniques, especially recombinant DNA technology. Recombinant DNA methods are revolutionizing medicine. They have provided a wide range of advances, from the means for industrial production of interferon to constructing a vaccinia virus vaccine vector with multiple antigenic viral proteins to preparation of the first possibly effective malarial vaccine. Recombinant DNA technology is also redefining the limits of clinical laboratory testing, especially in the area of microbiology. The absolute complemeatarity and specificity of nucleic acid bases for identical copies of each other and the availability of nucleic acid fragmenting enzymes make the method possible. The restrictive endonucleases are used to fragment large DNA or RNA molecules into shorter base sequences. These sequences can be catalogued, synthesized, and reinserted into the genomes of appropriate producers (usually bacterial) or into vectors (usually viral). The recombinant gene products are then analyzed in a search for the desired one. Once a nucleic acid base fragment is identified as that carrying the appropriate genetic information for gene expression, it can be utilized for production or as a probe. The radio-labeled probe usually has absolute specificity for its complementary genetic area and can be used as a highly sensitive tracer for the presence of this genetic sequence in cells, tissue, fluids, etc. An illustrative example would be the following: a known genetic base fragment specific for the Coxsackie viral genome can be cloned, labeled, and used as the tracer for detecting Coxsackie genomes in infected tissue, e.g., myocardial biopsies. Since Southern blot electrophoretic analysis is capable of detecting a specific gene sequence in tissue at a sensitivity of 1: 10” DNA molecules, the sensitivity of this technique is manyfold higher than any current viral detection method. Of special interest will be the use of this exquisitely sensitive technique of in situ hybridization of viral nucleic acid probes with their mirror images in the EMB of patients with myocarditis or DC. This methodology may provide a quantum jump forward in the understanding of viral pathogenesis of primary myocardial disease. IMMUNOLOGIC CARDIOMYOPATHY

STUDIES

OF DILATED

AUTOIMMUNE PATHOGENENS

Rationale(s) for implicating antibodies, a major effector arm of the immune response, as a cause of DC have progressed through a predictable sequence. Three decades ago, antibody-mediated 489

diseases held etiologic sway, and the anticipation that DC may be antibody mediated was no exception.i6” 7o The main reason for this enthusiasm was not theoretical but mainly technical, for there had been a disproportionate, rapid increase in understanding in structure and function of antibodies in contrast to cellular functions. Newly described methodology that detected antibodies in serum and tissue were rapidly applied to the study of DC. Circulating heart antibodies have no diagnostic or prognostic specificity and most likely represent an epiphenomenon of myocardial damage, past or present.17i The direct immunofluorescent technique, that is, detection of in vivo bound antibody to myocardium, was used in the study of postmortem specimens or tissue samples obtained at thoracotomy. The latter approach led to the exciting finding that there was intense binding of immunoglobulins to atrial appendages that had been removed at the time of valve replacement from patients with a history of rheumatic fever.171 This report was the signal impetus to develop new perspectives and immunologic hypotheses for several heart diseases. The detection of myocardial antibodies in Chagas’ disease, a cardiomyopathy with an infectious-immunologic etiology, provided support for a similar etiology of DC.172 There is a high incidence of heart in vivo bound antibodies found in all the postmyocardial trauma syndromes. Immunoglobulins bound to myocardial tissue have been described in varying frequencies in the cardiomrogathies, but have not provided any diagnostic clue.173P7 They, like circulating heart antibodies, bind to sarcolemmal membranes, various intramyocyte proteins, and adjacent cytostructures. Clinical associations that might be drawn from the numerous direct and indirect antibody studies are that DC, in contrast to hypertrophic or restrictive cardiomyopathy, seems to have a higher incidence (1OYo to 55%) of myocardial bound immunoglobulins. As more and more patients with cardiomyopathies are studied with EMB, however, it has been evident that only a small proportion of patients will have myocardial bound immunoglobulin heavy chains and complement components. In one study, ten (14%) of 70 EMBs had either antibody or C3 present in EMB: ‘When these ten biopsies were compared to 60 biopsies without abnormal immunoglobulin deposition, there was no correlation with clinical presentation, hemodynamics, or outcome.175 In another biopsy analysis of 24 patients with DC, there seemed to be an inverse relationship between immunoglobulin myocardial deposition and ejection fraction.“* This study also suggested that there was a negative correlation between the presence of myocardial bound antibody and alcoholic heart disease. This is in contrast to others who have described circulating, organ-specific heart antibody in 11% of patients with alcoholic cardiomyopathy,176 Overall, a consensus would be that 490

antibody bound to myocardium is more likely to be found in patients with DC than in normal persons; that there is only a very loose correlation between the presence of antibody and the severity of involvement that may simply relate to the temporal course of the illness; and that heart bound antibody, similar to circulating myocardial specific antibody, simply represents an epiphenomenon of myocardial damage. Our initial protocol for immunologic testing of EMB included routine direct immunofluorescence methods that detected all immunoglobulin heavy chains, C3, and Clq. There was frequent, unexplainable low-intensity binding of fluoresceinated immunoglobulin antiserum to subsarcolemmal regions, but, in over 300 EMBs, we could not correlate the deposition with a specific disease process. We have rarely noted that in certain syndromes associated with circulating immune complexes (i.e., SLE, drug hypersensitivity) there will be an intense antibody deposition in the intramyofiber areas. Myocardial amyloid deposits have also been incidentally detected during fluorescent anti-immunoglobulin biopsy screening. The detection of myocardial antibodies has also been reported in patients with acute myocarditis.f77 Myocardial antibody was found in seven of 44 patients and occurred randomly throughout the various histologic subsets that varied from the presence of mononuclear inflammatory Siltrates to normal histologic find, ings. Direct immunofluorescence is a technique that can detect any antigen if a suitable fluoresceinated tracer is available. The restricted current perspective that attempts to implicate antibody involvement has been nonrevealing but may shift to the detection of other cell surface markers. The detection and distribution of various types of collagen has already been reported in patients with cardiomyopathy.178 New methods such as the’ immunoperoxidase stain and exquisitely specific and wide-ranging monoclonal antibody probes may supersede fluorescent techniques and will be applied to biopsy analysis, especially in the area of structural proteins and nuclear abnormalities. EFFECTORCELL STUDIESIN DILATED CARDIOMYOPATHY As noted previously, a strong parallel exists between the available immunologic methods and rationales generated to explain DC. Prior to access to myocardial tissue during life, detection of abnormal cellular immune functions in DC have been limited to measurement of quantity or function of circulating effector cells. A straightforward study that, surprisingly, was not done until recently was the assessment of cellular immunity in DC by in vivo skin testing to common antigens supplanted with standard in vitro assays that measured delayed hypersensitivity and cellular mitogenic responses. Twenty-seven patients 491

with DC were studied in such a way and found to be normal in all respects.17’ Another 19 patients with DC were assessed for their lymphocytes’ ability to transform to mitogens. There was no significant difference between normal persons and those with DC in terms of response to phytohemagglutinin; however, some patients had an attenuation of responses to a T-lymphocyte mitogen, Concanavalin A, but an increased response to a T-celldependent B-cell activator, pokeweed mitogen.18’ The significance of these findings, if any, remains unknown. Access to peripheral mononuclear cells has provided a number of studies attempting to detect significant alterations in lymphocyte subsets in DC. In general, there is no apparent difference in the percentage of circulating T-lymphocytes when measured by a general T-cell antigen (OKT3); however, two studies have shown an increase in the ratio between he1 er/inducer cells (OKT4) and suppressor/cytotoxic cells (OKT8J.l r:‘, 182These have been offered as circumstantial support for a postulated suppressor cell defect in immunoregulation in DC.ls3 The recent realization that chronic illness, protein calorie depletion, intercurrent infection, and multiple medications can alter T4/T8 ratios make conclusions drawn from study of peripheral 1 mphocyte subsets in DC highly suspect. In 1978, Fowles et al.” 1 reported that mononuclear cells of patients with DC were defective in suppressing lymphocyte alloreactivity. It was postulated that an in vivo suppressor cell defect could not properly regulate the host immune system after an initial viral infection initiated an autoreactive myocardial destructive process. Shortly thereafter, a similar report described significant reduction in suppressor cell activity in DC.lM We, and others, have studied 26 patients with DC with a similar Con A suppressor assay. We have been unable to show any suppressor defect in our group of patients.ls5 Another mechanism by which immunoregulation may prove to be faulty in DC could be a defect in natural killer (NK) cell function. Natural killer cells are a subpopulation of circulating lymphocytes that are capable of immediate immune surveillance of altered surface structures, that is, they do not require prior exposure to antigen. Initial studies by Anderson et a1.1a6suggested that NK activity was deficient in patients with DC. Normal NK activity in DC was reported shortly thereafter.18’ The same deficiencies that haunted the search for a definitive circulating organ-specific antibody in DC are present in the function studies of peripheral effector cells. They do not reveal what is actually occurring within the heart itself. Precedence for this concept is the lack of clear linkage between peripheral lymphocyte subsets and the pulmonary infiltrates of sarcoidosis and synovial cell populations of rheumatoid arthritis. This provides the basis for continuing skepticism concerning diagnostic and etiologic elaims based on peripheral immune cell assays in DC. It would seem reasonable to study antigen-specific T-cell-me492

diated cytotoxicity directed against cardiac myocytes, Ample evidence exists in the murine model that this occurs. Are they present in DC? Jacobs et al.“* studied a heterogeneous group of patients with primary myocardial disease and showed that 30% of these patients had mononuclear cells that were cytotoxic toward a human myocardial cell line. The degree of cytotoxicity was no different, however, than that found in patients with other cardiac diseases. The authors concluded that this lymphocyte activity was probably a nonspecific end result of myocardial damage and certainly not specific for myocardial disease, a contention similar to the epiphenomenon of myocardial antibodies arising secondary to any type of myocardial damage. Lowry et al. lag studied delayed (T-cell) hypersensitivity responses in DC using two in vitro techniques of leukocyte migration and inhibition of leukocyte transformation by a crude cardiac muscle antigen, There was no significant difference between patients with DC and normal persons in leukocyte migration or lymphocyte transformation (Table 15). IN SITU CELL ANALYSIS

Cells obtained from EM3 tissue may provide the single most important source for definitive immunologic studies of DC (Table 16). The ability to analyze the cellular constituency of the myocardium obtained in vivo provides direct evidence of the actual participants at the site of damage. Sensitive monoclonal immunofluorescent and immunoperoxidase antibody techniques that can identify lineage of lymphocytes, monocytes, and granulocytes can be performed on either fresh/frozen or even paraffin-embedded, formalin-fixed tissue. The light microscopic histologic criteria for the normal distribution of lymphocytes in myocardium has been a thorny issue. The timely study of Linder and colleagues1g0 is a classic example TABLE 15. Mononuclear Cells and Functions Cardiomyopathy LYMPHOCYTES

in Dilated

.---._ (LY)

SUBSETS

Peripheral blood (8) Total T-LY: normal’“‘~ lrJ2 Helper T-LY: normal,‘“’ increased”’ Suppressor T-LY: decreased.lR2 normall”

Biopsy: not available LYMPHOCYTE

FUNCTIONS

In vivo: cellular immunity: normal’79 In vitro: suppressor cell function: decreasedla3* lx4 normal’85 In vitro: natural killer cell function: reduced’% normalla

493

TABLE 16. Novel Immunologic Opportunities Provided by Endomyocardial Biopsy In situ lymphocyte, monocyte, and endothelial cell immunohistopathology Interleukin-expansion and functional analysis of myocardial lymphocytes In situ hybridization technology for viral and other antigen detection Testing ‘neoantigen’ and autocytotoxic hypotheses of DC

of new technology utilization; using immunohistochemical techniques, they could differentiate lymphocytes from other mononuclear, nonmyocyte cells, such as interstitial macrophages, fibroblasts, endothelial cells, and pericytes and provide quantitation and normal distributions for reference. In the uninflamed heart there were 3.6 lymphocytes, mostly of T lineage, in a relatively normal ratio of subsets per square m illimeter of tissue. A monoclonal NK specific probe detected 2.7 NK positive phenotypes per square m illimeter of tissue. These normal distributions can now be used as controls to better test the hypotheses of impaired NK activity or suppressor regulation in DC. With regard to the latter, the emergence of monoclonal probes that can separate suppressor T cells from cytotoxic T cells will be of specific interest; previously, both were detected as one subset without discrimination by the monoclonal OKT8. The prevalence of lymphocytes, helper/inducer, and cytotoxic/suppressor subsets has been reported in EM3 from patients with myocarditis.igl The diagnostic and prognostic importance of this type of analysis is unknown at present. There is growing awareness of the importance of endothelial cells as intraorgan antigen presenters, displayers, and stimulators of rejection. Cells that are capable of presenting antigens to lymphocytes are of paramount importance for an effective immune response. Macrophages and monocytes were previously thought to be the sole arbiters of effective antigen presentation to the immune system, but it is now apparent that other cells such as dendritic, Langhans, and endothelial cells are also effective antigen presenting cells. This was realized during studies using HLA-DR directed antiserum probes that detect cell surface products associated with immune response genes. DR products are extremely strong stimulators of responses that reflect histoincompatibility, and they increase in density on cell surfaces during immunologic reactions, especially ones characterized by T-cell activation such as transplant rejection.lg2 They are also displayed .in situations where an autoimmune time reaction is occurring; for example, normally they are not present in normal thyroid or colon, but in patients with Hashimoto’s thyroiditis or ulcerative colitis, they are easily detectable on colonic ep494

ithelium or thyroid acinar cells. Their novel appearance suggests they are acting as some type of antigen presenting cells that stimulate an autodirected response. It is conceivable that viral infections of the heart may also stimulate the appearance of DR on myocardial endothelium, similar to that already documented during cardiac rejection, and be a driving force for attraction and protracted intramyocardial recruitment of cytotoxic cells. The most exciting area of speculation, however, stems from the reality that EMB provides a source of viable myocardial and immunologic effector cell populations for further study. With regard to the former, 3 x lo5 myocardial cells can be isolated from EMB and reacted with autologous lymphocytes to assess autoimmune responses. lg3 EMB also provides a source for isolating the actual effector cells present at the site of injury. Until recently, the cell yield was too small for definitive quantitative analysis or functional study. This can now be obviated, however, by incubation of biopsy fragments with interleukin-2 (IL-21, a potent propagator of activated lymphocytes and now available in abundance by recombinant cell production. The basic prerequisite immunologic tenet that exploits the use of IL-2 in this fashion is that lymphocytes that have been activated, usually by an antigen-specific stimulus, proliferate vigorously in its presence. Cell populations amplified by this approach may reflect the character of original mediator cells. Analysis of in vivo activated lymphoblasts grown from renal allograft biopsy specimens provide support for this hypothesis.lg4 Lymphoblasts generated by IL-2-stimulated growth from needle renal biopsy specimens had mature T-cell phenotypes with a preponderance of the cytotoxic/suppressor (OKT8) subset, proliferated in response to donor allograft antigens, and, exhibited donor specific cytotoxicity. The ability to expand and isolate antigen-activated lymphocytes from EMB will provide a means for phenotypic characterization, cytotoxic and suppressor functional analyses of cells present during myocarditis. More importantly, it will provide a source of cells by which to test the autoimmune and neoantigen theories of myocarditis and DC. TREATMENTOF

MYOCARDITIS

The importance of establishing an accurate diagnosis of myocarditis is directly linked to the hypothesis that inflammation of the heart is reversible. Early experience with immunosuppressive therapy of myocarditis in man has been very encouraging. In fact, preliminary experience with prednisone and azathioprine has shown substantial clinical and hemodynamic improvement in from 40% to 100% of the cases reported.‘41* 14’, 5oX15’ 495

However, each of these studies was uncontrolled and consisted of only small numbers of patients (Table 17). The experience of Sekiguchi and colleagueslg5 suggests that myocarditis may spontaneously remit in many patients. This has given rise to a legitimate concern as to whether patients should be subjected to the morbidity of immunosuppressive therapy if their disease is likely to improve spontaneously. Dee et al.’ 6 have shown that histologic evidence of myocarditis may improve in the face of clinical deterioration or vice versa, and clinical improvement may occur while histologic findings do not. More recently, patients with biopsy-proven myocarditis and who were treated with immunosuppressive therapy have shown little to no clinical response. 16’Based on all the above, a recommendation for immunosuppression in all patients with active myocarditis cannot be made at present. However, if a patient experiences severe heart failure and has histologic evidence of active myocarditis, immunosuppressive therapy may be instituted if the clinical course is clearly deteriorating. Many of the problems posed in the above discussion may be resolved by the institution of a multicentered trial of immunosuppressive therapy for active myocarditis. This trial is designed to enter patients with biopsy-proven myocarditis determined by the “Dallas criteria” and to randomize therapy to either conventional medical therapy for the heart failure or conventional therapy plus the addition of one of two immunosuppressive arms-either prednisone and azathioprine or low-dose prednisone and cyclosporine, the potent immunosuppressive drug used in suppression of transplant rejection. Serial myocardial biopsies will be performed during this trial so that a close correlation between clinical, hemodynamic, and histologic sequelae can be obtained. Two vital questions will be answered by this study: Will immunosuppressive therapy improve the natural history of biopsy-proven myocarditis? What is the natural history of myocarditis in those patients with a picture of congestive heart failure of unknown cause? Finally, stratification of severity of myocarditis and severity of hemodynamic abnormalities may result in therapeutic recommendations based on strong supportive scientific evidence.

TABLE 17. Controversies Regarding Immunosuppression Proved Myocarditis

in Biopsy-

Myocarditis may remit spontaneously Histology may improve without improvement in function Clinical improvement may occur in the absence of histologic resolution 496

OTHER ETIOLOGIC CARDIOMYOPATHY

SUBTYPES

OF DILATED

FAMILIAL CARDIOMYOPATHY

The propensity for DC to occur in familial clusters has been well described. By inference, this clustering suggests a genetic predisposition for development of DC. Although no unifying inherited defect has been described, independent ultrastructural, metabolic, and immunoregnlatory abnormalities have been found in isolated families. In one reported familial study, an association of the cardiomyopathy with abnormalities in mitochondrial ultrastructure and the development of ring-shaped organelles have been found as a marker of the clinical cardiomy0pathy.l” These ring-mitochondria, although not unique to this family, occurred at a much greater frequency than other disease states and suggested abnormalities of mitochondrial function as the cause of heart failure. Carnitine deficiency has been described as a cause of DC in the pediatric population.ig8 When these young patients were given replacement carnitine, substantial clinical improvement occurred suggesting the presence of an inherited reversible, defect in carnitine synthesis. Recently, we described two families in which multiple family members developed congestive heart failure over a period of 15 years.1gg Endomyocardial biopsy of the index case revealed active myocarditis, and when autopsy slides from the myocardium of previously affected family members were studied, active myocarditis was present in each. Lymphocyte suppressor studies in the index case showed a defect in suppressor cell function and we suggested that the inherited trait was not an intrinsic cardiac defect, but a defect in immunoregulation that predisposed family members affected to a chronic inflammatory reaction culminating in dilated cardiomyopathy after infection with a cardiotropic virus. It is of interest that substantial histologic improvement resulted when the proband was given immunosuppressive therapy. Other surviving family members were studied with noninvasive cardiac testing and showed no evidence of LV and suppressor cell dysfunction. It is conceivable that immunoregulation is perfectly normal in the absence of active viral infection, but that a defect in immunoregulation is unmasked when exposed to the appropriate virus and the appropriate environmental conditions. It seems reasonable that, if a familial form of cardiomyopathy is recognized, an aggressive approach to document the inherited trait of additional family members be adopted in the hope that preventive measures may delay the onset of clinical heart failure.

497

PERIPARTUMCARDIOMYOPATHY When dilated cardiomyopathy first presents in the third trimester of pregnancy or the first six months post par-turn, it is frequently referred to as peripartum cardiomyopathy. This syndrome was initially described in lower socioeconomic populations, and nutritional factors and hypertension were thought to be important in the development of this state. More recently, EMB studies have demonstrated that peripartum cardiomyopathy may be associated with histologic evidence of myocarditis when the biopsy is performed early after onset of symptoms.200 The natural history of this state, which shows a very high incidence of spontaneous improvement, may lend supportive evidence to the myocarditis theory. Furthermore, it is now recognized that a physiologic augmentation of suppressor cell function occurs toward the end of the third trimester of pregnancy. This apparent immunoregulatory defect theoretically serves to prevent maternal immunologic recognition of the fetal antigens that would initiate fetal destruction and also proscribe future pregnancies. Viral myocarditis may therefore be somewhat analogous to the rather vehement form of influenza pneumonia that may develop in some women during the third trimester. Both may result from augmented suppressor cell function. If the woman is exposed to a cardiotropic virus during this period of immunoregulatory change, the likelihood of development of clinical evidence of myocarditis is increased. A prospective trial of immunosuppressive therapy has not been attempted in these patients. However, Melvin and colleagues201 have described dramatic improvement in three cases of peripartum myocarditis when immunosuppressive therapy was instituted. A large trial evaluating immunosuppressive therapy in biopsy-proven peripar-turn “myocarditis” should be performed in an effort to clarify its role, if any, in the treatment of this condition. SUMMARY

AND

CONCLUSION

The development of safe techniques of endomyocardial biopsy has led to a significant increase in our understanding of the etiology ‘and pathogenesis of dilated cardiomyopathy. The scope of patients for whom this technique is absolutely clinically indicated, however, remains quite narrow and should be restricted to those centers with active cardiac transplant programs, large oncology practices, or those involved with active research into the etiology and treatment of patients with heart muscle disease. The most exciting concept to emerge from the use of EMB is the role of myocarditis in the development of dilated cardiomyopathy. It can accurately be stated that a subset of patients with the clinical presentation of dilated cardiomyopathy may in fact have histologic evidence of myocarditis. Although there is a

suspicion that immunosuppressive therapy may be helpful, a randomized trial of large numbers of patients is necessary before definitive conclusions may be drawn. If immunosuppressive therapy proves to be efficacious in active myocarditis, then one could argue that all patients with heart failure of unknown cause with no evidence of valvular or coronary artery disease should undergo endomyocardial biopsy as part of the routine diagnostic workup. However, this recommendation must be considered premature. Since routine histologic findings are nonspecific in dilated cardiomyopathy, biochemical, pharmacologic, and cell culture techniques may provide more definitive information regarding the functional state of the heart muscle. In conclusion, endomyocardial biopsy is rapidly emerging as a useful diagnostic tool in the evaluation of patients with heart failure of unknown cause. REFERENCES 1. O’Connell JB, Gunnar RM: Dilated-congestive cardiomyopathy: Prognostic features and therapy. J. Heart Transplant 1982; 27. 2. Fuster V, et al: The natural history of idiopathic dilated cardiomyopathy. Am J Cardiol 1981; 47~525. 3. O’Connell JB, et al: Prognosis and treatment of cardiomyopathy and myocarditis. Heart Vessels 1985; lkmppl 1):175. 4. Figulla HR, et al: Spontaneous hemodynamic improvement or stabilization and associated biopsy findings in patients with congestive cardiomyopathy. Circulation 1985; 71:1095. 5. Shirey EK, et al: Primary myocardial disease: Correlation with clinical findings, angiographic and biopsy diagnosis. Am Heart J 1980; 99:198. 6. Costanzo-Nordin MR, et al: Dilated cardiomyopathy: Functional status, hemodynamics, arrhythmias, and prognosis. Cathet Cardiovasc Diugn 1985; 11:445. 7. Unverferth DV, et al: Factors influencing the one-year mortality of dilated cardiomyopathy. Am J Cardiol 1984; 54:147. 8. Convert P, et al: Etude prognostique des myocardiopathies primitives non obstructives. Arch Ma1 Coeur 1979; 73:227. 9. Feild B, et al: Left ventricular function and hypertrophy in cardiomyopathy with depressed ejection fraction. Circulation 1973; 47:1022. 10. Stamato NJ, et al: The response of patients with dilated cardiomyopathy to programmed electrical stimulation. Am Heart J, in press. 11. Meinertz T, et al: Significance of ventricular arrhythmias in idiopathic dilated cardiomyopathy. Am J Cardiol 1984; 53:902: 12. Von Olschausen K, et al: Ventricular arrhythmias in idiopathic dilated cardiomyopathy. Br Heart J 1984; 51:195. 13. Huang SK, et al: Significance of ventricular tachycardia in idiopathic dilated cardiomyopathy: Observations in 35 patients. Am J Cardiol 1983; 51:507. 14. Hatle L, et al: Chronic myocardial disease: II. Clinical picture related to lone-term nroanosis. Acta Med Stand 1976: 199399. 15. Benjamin i, ei al: Cardiac hypertrophy in idiopathic dilated cardiomyopathy: A clinicopathologic study. Circulation 1984; 64:442. 16. Wallis DE, et al: Segmental wall motion abnormalities in dilated cardiomyopathy: A common finding and good prognostic sign. J Am Co11 Cardiol 1984; 4:674. 17. Engelmeier RS, et al: Improvement in symptoms and exercise tolerance by metoprolol in patients with dilated cardiomyopathy: A double-blind, randomized, placebo-controlled trial. Circulation 1985; 72:536. 499

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