Prevention of hereditary cardiomyopathy in the syrian hamster with chronic verapamil therapy

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Prevention of Hereditary Cardiomyopathy in the Syrian Hamster With Chronic Verapamil Therapy STEPHEN M. FACTOR, MD, FACC, SANGHO CHO, MD, JAMES SCHEUER, MD, FACC, EDMUND H. SONNENBLICK, MD, FACC, ASHW ANI MALHOTRA, PHD Bronx, New York

The cardiomyopathic Syrian hamster develops genetically determined cardiac necrosis that invariably leads to premature death from congestive heart failure or arrhythmia. This hamster is a valuable model of human disease because it has many features in common with clinical dilated, congestive cardiomyopathy. Previous studies have shown that therapy for several weeks with the calcium channel blocking drug verapamil or the alpha-} adrenoceptor blocking drug prazosin can prevent myocardial necrosis due to microvascular spasm. Other investigations have demonstrated the positive effects of verapamil in the early stages of disease. It is not clear, however, whether continued treatment can prevent the long-term expression of the

The cardiomyopathic Syrian hamster is an inbred strain with a disease transmitted by an autosomal recessive gene associated with phenotypic expression in 100% of affected lines (1-6). The cardiomyopathy is characterized by multifocal myocardial necrosis that begins at 40 to 50 days of age and causes premature death from congestive heart failure or arrhythmia usually within 1 year (6). The pathogenesis of the disease is incompletely understood. Recent evidence (7-11) suggests that there is a generalized membrane defect that may make myocytes susceptible to the effects of transient ischemia and increased trans sarcolemmal calcium flux. Multifocal myocyte necrosis may be secondary to spasm of intramyocardial blood vessels caused by abnormal sensitivity of vascular smooth muscle cells to catecholamines (6,1214). The smooth muscle cells may have increased sensitivity From the Departments of Pathology and Medicine, Albert Einstein College of Medicine, Bronx, New York. This study was supported in part by Grants HL-18824 and HL-35882 from the National Institutes of Health, Bethesda, Maryland. Manuscript received February 19, 1988; revised manuscript received June 7, 1988, accepted July 13, 1988. Address for reprints: Stephen M. Factor, MD, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. © 1988 by the American College of Cardiology

cardiomyopathy or whether the disease is genetically predetermined. To address this question, hamsters were treated with oral verapamil for 7 to 8 months during the necrotizing, compensatory hypertrophy and early failure stages of disease. Analysis of myocardial pathologic and biochemical variables demonstrated that continuously treated animals were generally similar to unaffected control hamsters; discontinuous therapy led to partial protection. These findings demonstrate that virtually complete prevention of this hereditary disease is feasible; these results may have important implications for the treatment of human cardiomyopathy. (J Am Coli Cardiol1988;12:1599-604)

as a result of the same genetically determined membrane defect present in myocytes. In addition to these abnormalities, a number of biochemical derangements also have been described, particularly in regard to catecholamine metabolism (15-21). Regardless of the precise pathogenesis of the cardiomyopathy, the disease was prevented for a short time when these hamsters were treated with the calcium channel blocker verapamil (12) or the alpha-l adrenoceptor blocking drug prazosin (6). Both drugs protected the animals from developing myocardial necrosis for 2 to 3 weeks at the onset of disease by preventing microvascular spasm. Other investigators (22-24) also have reported beneficial effects of verapamil in this disease. In a recently published study, Kobayashi et al. (25) showed that cardiac calcium channels are increased early and that verapamil could prevent significant morphologic damage up to 90 days of age. However, because extensive myocardial damage occurs between 70 and 150 days of age and because congestive heart failure generally does not occur until after 250 days (6), these prior studies do not indicate whether drug therapy can effectively prevent cardiomyopathy. In the present report, we describe the positive effects on 0735-1097/88/$3.50

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FACTOR ET AL. PREVENTION OF SYRIAN HAMSTER CARDIOMYOPATHY

Table 1. Cardiomyopathic and Age-Matched Controls-Study Groups Group

n

Hamster

Treatment (days) Age

1 2 4

8 8 8 8

10 10 10 15 10

% Necrosis

= 150 Days

Cardiomyopathic 50 to 100 Cardiomyopathic 50 to 150 Cardiomyopathic None Control None Age

5 6 7 8 9

Killed (days)

150 150 150 150

5.42 ± 2.62 2.82 ± 1.97 7.19 ± 2.34 None

250 250 250 250 250

5.05 ± 1.90 4.61 ± 1.77 1.73 ± 1.00 12.45 ± 3.67 None

= 250 Days

Cardiomyopathic 50 to 100 Cardiomyopathic 50 to 150 Cardiomyopathic 50 to 250 Cardiomyopathic None Control None

myocardial pathology and biochemistry of long-term verapamil treatment administered for the entire period of active disease. Additional data indicate the effect on expression of the disease of treatment cessation at different time points.

Methods Animals and treatment protocol. Seven groups of cardiomyopathic Syrian hamsters (strain 53.58) were entered into the study with five cardiomyopathic groups begun on oral verapamil therapy (1 g verapamil in I liter of drinking water) at 50 days of age; average intake was 20 ml/day. The disease is manifested 15 to 20 days later in the 53.58 strain than in the 14.6 strain of animals (50 to 60 days versus 35 to 40 days). Two groups of age-matched noncardiomyopathic golden hamsters were used as normal controls. Treatment duration and study times (Table 1) were chosen to correspond to observed features of disease in these animals. Active myocardial necrosis occurs up to 150 days of age, followed by a stage of compensatory hypertrophy with a diminution of necrosis that extends up to 250 days. Subsequent congestive heart failure or arrhythmic sudden death ensues before the animals are 1 year old. The time frames for treatment were chosen to test whether relatively short courses of verapamil during the active necrotic phase can abort the disease entirely or delay its onset, or whether treatment is required for the lifetime of the animal. Pathology and morphometry. At the time of study, animals were anesthetized with ether and the heart was removed. For those hearts undergoing myocardial pathologic study, two circumferential left ventricular rings were cut and fixed in 3.7% buffered formaldehyde. The remaining ventricular muscle was frozen at -80°C in buffered 50% glycerol (pH 7.0) for subsequent biochemical analysis. Morphometric determination of myocardialnecrosis was performed with an interactive image analyzing system (Zeiss

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Videoplan II) and a Zeiss Photomicroscope III. The areas of left ventricular rings and summed areas of myocardial necrotic lesions were determined for each animal, by group, and expressed as percent necrosis per ventricle. Ventricular areas and necrotic lesions were assessed with a 1x or 2.5 x microscope objective. Questionable lesions or lesions with uncertain margins were checked at higher magnifications. Sections were stained with hematoxylin-eosin and Masson's trichrome stains. The predominant lesion of the heart of affected animals is heavily calcified with sharp, relatively discrete borders (26). This demarcation facilitated tracing the lesion edge; however, all lesions were quantitated, including those with recent necrosis, fibrosis, or calcification. The viewing system was integrated through a drawing tube with a cursor, a digitizing tablet and a computer. The cursor projected a point source of light back through the drawing tube onto the tissue section within the viewing plane. Direct tracing of ventricular area and lesions could therefore be performed on the microscopic image. Ventricular area was calculated by tracing the epicardial surface and subtracting the area encompassed by the ventricular cavity. Lesion area was sequentially added and percent involvement was determined for the combined ring areas. Biochemistry. Biochemical studies were carried out on the previously frozen heart in those animals killed at 250 days. Myofibrils were isolated and purified with Triton X-IOO by a previously described method (27). Magnesium-calcium (Mg 2+-Ca2+) myofibrillar adenosine triphosphatase activity, defined as Ca2+ -stimulated adenosine triphosphatase measured in the presence of Mg2+ (3 mM) and Ca2+ (0.1 mM) concentrations, and ethylene glycol doubled (B-aminoethyl ether)-N,N,N' ,N'-tetraacetate (EGTA) adenosine triphosphatase activity, defined as basal adenosine triphosphatase (Mg2+ adenosine triphosphatase) measured in the absence of free Ca2+ but in the presence of Mg2+ and 2 mM EGTA, were determined (28). Myosin isoenzymes in myofibrillar preparations were analyzed by polyacrylamide gel electrophoresis under nondissociating conditions at 2°C (29,30) in the Pharmacia Apparatus (GE 2/4). Densitometric scans of gels were recorded at 605 nm on an E-C apparatus densitometer attached to a Hewlett-Packard integrator (3390A). Statistics. Statistical comparisons between groups were carried out by analysis of variance and the Scheffe multiple comparison test. A p value < 0.05 was considered statistically significant.

Results Pathology and morphometry (Table 1). For those hamsters killed at 150 days of age, continuous treatment had a significant effect on the extent and frequency of myocardial necrosis (Group 2 versus Group 3 [untreated]: 2.82 ± 1.97 versus 7.19 ± 2.34%, p < 0.01), whereas discontinuous therapy led to intermediate protection (Group 1 versus

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FACTOR ET AL. PREVENTION OF SYRIAN HAMSTER CARDIOMYOPATHY

Table 2. Contractile Protein Biochemistry at 250 Days of Age Myofibrils % Necrosis Mg2+ Ca2+ ATPase p Value Preparations EGTA ATPase p Value Preparations Myosin isoenzyme %V I %V 3 p Value Preparations Mg2+ Ca2+ ATPase/EGTA ATPase ratio ATPase

Control

Group 8

Group 6

Group 7

None 0.095 ± 0.004

12.45 0.139 ± 0.008

4.81 0.100 ± 0.005

1.73 0.102 ± 0.008

Control vs. 8 <0.001

8 vs. 6 <0.01

(N) (5) 0.025 ± 0.002

(8) 0.073 ± .006 Control vs. 8 <0.001

(10) 0.030 ± 0.003 8 vs. 6 <0.001

8 vs. 7 <0.05

(6) 0.038 ± 0.002

8 vs. 7 <0.01

(N) (5)

(8)

(9)

(6)

83.2 ± 5 16.8 ± 5

60.2 ± 2 39.8 ± 2

70.9 ± 1.6 29.1 ± 1.6

76.7 ± 2.3 23.3 ± 2.3

Control vs. 8 <0.001

8 vs. 6 <0.05

(N) (5) 3.80

8 vs. 7 <0.01

(II)

(13)

1.90

3.33

(8) 2.70

= adenosine triphosphatase; VI and V3 are myosin isoenzyme bands.

Group 3: 5.42 ± 2.62 versus 7.19 ± 2.34%, P < 0.01). Analysis of hearts at 250 days of age demonstrated marked protection by continuous verapamil therapy (Group 7 versus Group 8 [untreated]: 1.73 ± 1.00 versus 12.45 ± 3.67%, p < 0.01). Discontinuous therapy, whether it was stopped at 100 or 150 days of age, was only partially protective (Group 5 versus Group 8: 5.05 ± 1.90 versus 12.45 ± 3.67%, P < 0.01). The number of individual lesions in each heart was not quantitated nor were their size variations determined as a function of treatment; however, subjective analysis revealed that, with long-term treatment, the lesions were small, discrete and scattered throughout the myocardium, predom-

inantly in the midwall and epicardial layers. Overall, as demonstrated by the area measurements, there were significantly fewer foci of necrosis in the continuously treated hamsters. Several hamsters with long-term treatment were virtually free of lesions; this observation was never seen in untreated or partially treated cardiomyopathic animals. Biochemistry (Table 2, Fig. 1 and 2). The contractile protein and myosin isoenzymes were analyzed in four groups of hamsters: control (Group 9), untreated (Group 8), discontinuously treated (Group 6) and continuously treated (Group 7). The basal Mg2+ adenosine triphosphatase (EGTA adenosine triphosphatase) and Ca2+ -stimulated Mg2+ adenosine triphosphatase activity of myofibrils was significantly

.16

c

E ..... C>

E .....

cL .12 ~

Figure 1. Myofibrillar adenosine triphosphatase (ATPase) activity for verapamil-treated (Groups 6 and 7) and untreated hamsters (Group 8) at 250 days of age. *Significantiy different (p ::5 0.05) from Groups 6, 7 and 9. No other significant differences were observed. EGTA ATPase = basal Mg2+ ATPase activity (left); Ca2+ ATPase = Ca2+-stimulated Mg2+ ATPase activity (right).

~

>-

l-

S;

;::: () « .08 Q)

Ul

'"

c..

I-

« « a:

-'

-' .04

cr III Li:

o>~

2mM EGTA

O.1mM Ca 2 +

1602

FACTOR ET AL. PREVENTION OF SYRIAN HAMSTER CARDIOMYOPATHY

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100 w

:E 80

>N

Figure 2. Myosin isoenzyme distribution in the heart of verapamil-treated (Groups 6 and 7) and untreated (Group 8) hamsters at 250 days of age. *Significantiy different (p :s 0.05) from Groups 6, 7 and 9. **Significantiy different from Group 6. No other significant differences were observed. V I and V3 = bands of myosin enzymes.

Z

w 60 5!?

0

z

Ci5 40

0

>-

:E i?- 20

higher in myocardium from untreated animals, whereas treatment with verapamil normalized the differences in net adenosine triphosphatase activation (Ca2+ _Mg2+ activity minus basal EGTA adenosine triphosphatase) in control and myopathic groups at 0.1 mM Ca2+ concentration (28). The distribution of myosin isoenzymes demonstrated a predominance of the VI band in the control group, with a decrease of 28% in untreated hamsters. The inverse pattern was seen with the V3 isoenzyme. A partial correction was attained with discontinuous verapamil therapy, and a complete reversal of the control pattern was attained with continuous treatment. Thus, the biochemical indexes of cardiomyopathy in this genetic disease paralleled the morphologic indexes in their sensitivity to and normalization by long-term verapamil therapy.

Discussion Verapamil protection. These studies indicate that institution of oral verapamil therapy when active myocardial necrosis begins, with continuation of treatment until the hamsters would ordinarily begin to develop congestive heart failure or manifest sudden death, can prevent the significant manifestations of cardiomyopathy in this model. With the exception of the effect of discontinuous treatment on myofibrillar adenosine triphosphatase activity, interrupted therapy generally led to partial protection with intermediate indexes of necrosis and myosin isoenzyme distribution. These latter observations suggest that verapamil therapy either delays the onset of disease until such therapy is stopped, or that disease progresses through the 150 to 250 day hypertrophic stage when necrosis is generally thought to be quiescent. In fact, the comparison between Group 3 (untreated) with 7.19 ± 2.34% necrosis at 150 days and Group 8 (untreated) with 12.45 ± 3.67% necrosis at 250 days supports the view that necrosis is an ongoing process that probably continues for the life of the animal. Continuous treatment prevented the development of myocardial necrosis and maintained biochemical variables

within the normal range. This finding indicates that the hallmarks of this hereditary congestive cardiomyopathy (for example, focal myocardial necrosis and ventricular mural scarring with concomitant biochemical changes) are not genetically predetermined but are inducible abnormalities that can be ameliorated by appropriate therapy. Recently, Kobayashi et al. (25) demonstrated protective effects of verapamil therapy in a small group of cardiomyopathic hamsters treated during the early phase of myocyte necrosis (up to 90 days). These investigators also demonstrated increased alpha-l and beta-adrenoceptor binding sites in cardiomyopathic hearts during the active phase of disease (70 days); they also showed increased [H3] nitrendipine-binding sites in young hamsters before necrosis. Additionally, they were able to measure significantly increased lipid peroxides in cardiomyopathic hearts. These studies did not continue into the more chronic stages of disease and, thus, could not determine whether cardiomyopathy was ultimately prevented by verapamil therapy. Pathogenesis of Syrian hamster cardiomyopathy. Our group has been investigating the pathogenesis of cardiomyopathy in the hamster model for > 15 years. We have identified a number of abnormalities including increases of catecholamine activity (15,16), an increased sensitivity of in vivo blood vessels to catecholamines (14), the presence of microvascular spasm in the myocardial microcirculation (12) and the short-term prevention by either verapamil or prazosin therapy of reperfusion necrosis characteristic of microvascular spasm (6,12). Studies from other laboratories also have confirmed the acute positive effects of verapamil or prazosin (22-24) and have shown that treatment with the beta-adrenoceptor antagonist propranolol is incompletely protective in this model (31). A number of biochemical abnormalities also have been described (15-21), but many of these were observed in the latter stages of disease when they may be secondary to congestive heart failure. Although the precise genetic defect in the Syrian hamster has not been determined, evidence to date suggests that there is an abnormality of ceU membranes and surface

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receptors. Several studies (32,33) have shown that calcium channel receptors are increased in both smooth and cardiac muscle, although this could not be confirmed in a recent study (34). Adrenoceptors also have been found to be abnormal in this model (35). Morphologic studies (36,37) of hamster cell membranes using freeze-fracture techniques have shown differences in the fracture faces between control and myopathic animals, possibly related to changes in surface receptor sites. Additionally, membrane biochemistry has revealed differences in lipid composition compared to normal (38). These data strongly implicate a genetic defect expressed at the cell surface of smooth and striated muscle. Rate of vasospasm. As noted previously, we recently demonstrated (14) increased vessel reactivity in cardiomyopathic hamster cremaster muscle preparations with topical application of catecholamines. Hunter and Elbrink (39) also showed increased contractility of aortic rings from cardiomyopathic animals. These abnormalities of vascular smooth muscle support the view that microvascular spasm induced by hypersensitivity of blood vessels to catecholamines may lead to ischemic damage of susceptible cardiac myocytes. This sequence of events would explain why disease in cardiomyopathic hamster hearts is not diffuse (as might be anticipated with a generalized degenerative biochemical defect) but rather is focal and discrete. The genetically determined diffuse biochemical abnormality (of cell membranes?) simply causes myocytes to be susceptible to ischemia induced by hypersensitive vascular smooth muscle cells undergoing spasm. Interruption of this pathway-by inhibiting vasospasm with verapamil or prazosin-Ieads to myocyte protection without affecting the fundamental membrane defect. Similarly, prevention of necrosis maintains the biochemical variables of cardiomyopathy associated with ventricular hypertrophy or failure within the normal range. Discontinuous verapamil therapy. The partial protection afforded by discontinuous verapamil therapy suggests that the susceptibility of the myocytes to necrosis continues for the lifetime of the hamster. Amelioration of disease with discontinuous therapy is feasible (as in Groups 1, 5 and 6), but the more extended benefits observed with continuous treatment were not obtained. We recognize that prevention of necrosis and maintenance of normal contractile protein biochemistry does not prove unequivocally that these animals were cured of their disease. Only demonstration of a normal lifespan could conclusively show the benefits of long-term verapamil therapy in this model. This demonstration would have required treatment protocols for up to 2 years at prohibitive expense. Nevertheless, virtually complete preservation of morphology and cardiac biochemical variables of disease for 7 to 8 months suggests that significant cardiomyopathy was prevented. Conclusions. Our findings clearly suggest that long-term therapy with verapamil can effectively prevent the genetically determined manifestations of this cardiomyopathy. The

FACTOR ET AL. PREVENTION OF SYRIAN HAMSTER CARDIOMYOPATHY

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observations suggest that the genetically determined abnormality in the Syrian hamster, which may affect cell membranes or calcium channel receptors, acts as a substrate for disease induction but may not independently lead to significant disease. This finding suggests that, in patients with certain hereditary and possibly acquired human cardiomyopathies, it may be possible to abort the disease if appropriate therapy is instituted early enough.

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29. Hoh JFY, McGrath PA, Hale PT. Electrophoretic analysis of multiple forms of rat cardiac myosin: effects of hypophysectomy and thyroxine replacement. J Mol Cell Cardiol 1977;10:1053-76. 30. D' Albis A, Pantaloni C, Bechet JJ. An electrophoretic study of native myosin isozymes and of their subunit content. Eur J Biochem 1979;99: 261-72. 31. Jasmin G, Solymoss B, Proschek L. Therapeutic trials in hamster dystrophy. Ann NY Acad Sci 1979;317:338-48. 32. Wagner JA, Reynolds II, Weisman HF, Dudeck P, Weisfeldt ML, Snyder SH. Calcium antagonist receptors in cardiomyopathic hamster: selective increases in heart, muscle, brain. Science 1986;232:515.:..S. 33. Finkel MS, Marks ES, Patterson RE, Speir EH, Steadman K, Keiser HR. Increased cardiac calcium channels in hamster cardiomyopathy. Am J Cardiol 1986;57: 1205~. 34. Bazan E, Schwartz A, Gardner S, Wells JW, Sole MJ, Johnson CL. Receptors for calcium channel antagonists in cardiomyopathy (abstr). Fed Proc 1987;46:852. 35. Karliner JS, Alabaster C, Stephens H, Barnes P, Dollery C. Enhanced noradrenaline response in cardiomyopathic hamsters: possible relation to changes in adrenoceptors studied by radioligand binding. Cardiovasc Res 1981 ;15:296-304. 36. Berry B, Poulsen R, Yunge L, et al. Numerical densities of intramembrane particles in the cardiac sarcolemma of normal and myopathic Syrian hamsters. J Mol Cell Cardiol 1983;15:503-13. 37. Graham KA, Shivers RR, Atkinson BG. A freeze-fracture analysis of intramembrane particle densities on dystrophic hamster "beart sarcolemma. Muscle Nerve 1984;7:513-23. 38. Slack BE, Boegman RJ, Downie JW, Jasmin G. Cardiac membrane cholesterol in dystrophic and verapamil-treated hamsters. J Mol Cell CardioI1980;12:179.:..S5. 39. Hunter EG, Elbrink J. Increased contractility in vascular smooth muscle of dystrophic hamsters. Can J Physiol Pharmacol 1983;61: 182-5.