Alteration of myofibrillar ATPase activities in hearts of cardiomyopathic hamsters (BIO 53.58)

Journal of Molecular and Cellular Cardiology (1980) 12, 445-456

Alteration

of Myofibrillar Cardiomyopathic DAVID

ATPase Activities in Hearts Hamsters (BIO 53.58)

C. PANG

AND WILLIAM

of

B. WEGLICKI

Defiartment of Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, U.S.A. (Received 6 August 1979, accepted in revised form 5 October 1979) D. C. PANG AND W. B. WEGLICKI. Alteration of Myofibrillar ATPase Activities in Hearts of Cardiomyopathic Hamsters (BIO 53.58). 3 oumal of Molecular and Cellular Cardiology (1980) 12, 445-456. Purified myofibrils were prepared from cardiac muscle of normal (RB) and cardiomyopathic (BIO 53.58) hamsters at the age of 24 to 160 days. Myofibrils from normal hamsters were sensitive to increasing concentrations of calcium buffered with EGTA. As the free calcium concentration rose from 0.1 to 10 pi, the ATPase activity increased from 0.06 to 0.35 pmol/mg/min. The free calcium concentration for half-maximal myofibrillar ATPase activity was found to be 1 x 10-s M. The myofibrillar ATPase activities from male and female normal hamsters did not vary with age. However, the fractions from cardiomyopathic hamsters behaved differently according to their sex. The maximal myofibrillar ATPase from the female cardiomyopathic hamsters (24- to 30-day-old) was less than that of the normal, but increased greatly at 36 days of age. At 44 days of age, the female myofibrillar ATPase activity was again depressed. The myofibrillar ATPase of the male cardiomyopathic hamsters was slightly increased at 36 days of age. At 44 days, the male hamsters exhibited a slight decrease in the myofibrillar ATPase activity. At 50 days of age, myofibrillar ATPase activities from both male and female cardiomyopathic hamsters were found to be depressed; this decrease was also observed at 91 days in the male and 161 days in the female animals. The free calcium concentration for the half-maximal myofibrillar ATPase activity was found to be unchanged in all the cardiomyopathic hamsters studied. KEY WORDS: Cardiomyopathic

hamsters; Myofibrils;

ATPase; Cardiac failure.

1. Introduction It has been demonstrated that cardiac contractility is depressed in the hearts of cardiomyopathic hamsters [6]. To ascertain the origin of the altered myocardial function, Dhalla et al. [5] reported a slight, but significant increase in the Mg2+ATPase of myofibrils from hearts of the cardiomyopathic hamsters (BIO 14.6). Similarly, Wada et al. [IO] also showed higher activity of myosin ATPase in the same strain BIO 14.6 than in the random bred controls. However, Bhan et al. [3] measured the actomyosin ATPases from several strains of cardiomyopathic hamsters (BIO 14.6, 50.54 and 86.62) and found that the ATPase activities were Correspondence: Dr. David C. Pang, Department of Biophysics, Box 694, MCV Richmond, Virginia 23298, U.S.A. 0022-2828/80/050445 + 12 $02.00/O 0 1980 Academic Press Inc. (London)

Station, Limited

446

D. PANG

AND

W. WEGLICKI

all lower than that in the random bred controls. In addition, activities of myosin ATPase [Z] were also found to be depressed in the hearts of the BIO 53.58 strain. The present study was initiated in order to provide clarification of the variability of the ATPase in the contractile proteins by using one particular strain of cardiomyopathic hamsters (BIO 53.58). BIO 53.58 is a relatively new strain of cardiomyopathic hamster characterized by an early myocytolysis (30 to 60 days of age), followed by some degree of heart failure [Z]. Onset of the disease process is identical to that of the BIO 14.6, from which this strain was developed. This study measured the calcium-sensitivity of the ATPase of a highly purified fraction of cardiac myofibrils, prepared by the method of Solar0 et al. [9]. Specifically, the experiments had the following goals: (1) to monitor the myofibrillar ATPase during the initial development of cardiac failure, which is synonymous to the early life-span of the cardiomyopathic hamsters [1, ,!?I; (2) to determine if the variability observed in the ATPases of the contractile proteins from other strains of animals [Z, 3, 5, 101 could also be duplicated in the BIO 53.58 strain; and (3) to ascertain whether the development of the disease is similar in the male and female cardiomyopathic hamsters. 2. Materials

and Methods

In this study, male and female cardiomyopathic hamsters of the strain BIO 53.58 (approximately 24- to 160-days-old) were used along with random bred control (RB) animals of the same age. Hamster hearts, removed from animals anesthetized with ether, were immediately washed with saline at 4°C. Fat and connective tissues were trimmed away and the muscle was cut into small pieces for homogenation in a Sorvall Omnimixer (DuPont Co., Newton, CT). Five hearts were pooled in each group. Myofibrils were isolated and purified from the homogenate using Triton X-100

lI91. Myofibrillar ATPase activities were determined from the rate of release of inorganic phosphate in an incubation medium containing myofibrils (0.1 mg/ml), 2 mM ATP, 3 mM McCl,, 50 mM KCl, 20 mM imidazole, pH 7.0. 1 mM EGTA and various concentrations of CaCl, at 37°C. Free calcium ion concentration was calculated using an apparent formation constant of 1.1 x lo6 M-l for the calcium/ EGTA reaction at pH 7.0 [4]. Inorganic phosphate was measured by the method of Wahler and Wollenberger [II]. Results are given as mean & S.E. 3. Results Figure 10 PM, greatly

1 shows that, on increasing the concentration of free calcium from 0.1 to the myofibrillar ATPase from normal 24-day-old female hamster was stimulated. The maximal ATPase activity was 0.35 pmol/mg/min and

MYOFIBRILLAR

ATPASE

IN CARDIOMYOPATHIC

(Free calcium

concentration

HAMSTERS

447

)M

FIGURE 1. Effect of calcium on the myofibrillar ATPase activity in a 24-day-old female normal hamster. The enzyme activities were determined in the presence of 0.1 mg/ml myofibrils, 2 rnM ATP, 3 mM M&l, 50 rnM KCl, 20 mm imidazole, pH 7.0, 1 rnM EGTA and CaCl, at 37°C.

the free calcium concentration for half-maximal ATPase activity was around 1 FM. The myofibrillar ATPase was inhibited 93% in the presence of 2 mM EGTA and no added CaCl,, an inhibition comparable to that observed in myofibrils from canine cardiac muscle [9]. The basal ATPase of the myofibrils from normal and cardiomyopathic hamsters did not vary and fell within the range of 0.02 to 0.04 pmol/mg/min. Consistent with the finding that canine cardiac myofibrils, isolated by the treatment of Triton X-100, were virtually free of contamination by mitochondrial, sarcolemmal and sarcoplasmic reticulum membranes [9], the myofibrils from hamsters (normal and cardiomyopathic) did not contain any detectable (Naf + K+) ATPase and cytochrome oxidase activities. In addition, the myofibrillar ATPase was not affected by the addition of 10 mM azide (for mitochondrial ATPase) and 1 mM quinidine (for sarcotubular ATPase) [9]. Furthermore, addition of oxalate did not alter the calcium binding to the myofibrils. Figure 2 shows the activities of the myofibrillar ATPase from both the normal and cardiomyopathic hamsters at 24 days old. The data represent averages from four sets of experiments for each hamster group and are expressed as a percentage of myofibrillar ATPase activity of the normal male and female hamster.

448

D. PANG

120

AND

W. WEGLICKI

120

female

Male 0 Control ( RB 1

A 810 53.58 100 -

z 800 E a =b 60C e 5 % 408

-40

-20

200' 10-T

10-e

I o-6

I o-5 10-T

10-S

Free calctum ( M )

FIGURE 2. Comparison of the myofibrillar ATPase activities from 24-day-old cardiomyopathic hamsters. Conditions the same as that in Figure 1.

r

and

Female

Male 0 Control-- (RB) _^ A

normal

8Ic

30 $0

I o-7

10-e

10-S

I o-6

10-T

Free calcium(

I o-5

M1

FIGURE 3. Comparison of the myofibrillar ATPase activities from 30-day-old cardiomyopathic hamsters. Conditions the same as that in Figure 1.

normal

and

MYOFIBRILLAR

ATPASE

IN CARDIOMYOPATHIC

Mole

449

HAMSTERS

Female

II ( RB) 3.58

1’

1 36 Days 1

6

Y

Freecalcium FIGURE 4. Comparison of the myofibrillar ATPase activities from 36-day-old cardiomyopathic hamsters. Conditions the same as that in Figure 1.

normal

and

The maximal levels of myofibrillar ATPase activities from the normal male and female hamsters were 0.34 f 0.06 pmol/mg/min and 0.35 f 0.02 pmol./mg/min respectively. There was no difference in activities of the myofibrillar ATPase towards free calcium concentrations between the 24-day-old normal and cardiomyopathic male hamsters (Figure 2). However, the myofibrillar ATPase from the female cardiomyopathic hamsters was depressed to 84% of the control at a free calcium concentration of 1O-5 M. Figure 3 shows that the myofibrillar ATPases of the 30-day-old cardiomyopathic hamsters exhibited a pattern similar to that of the 24-day-old hamsters. The myofibrillar ATPase activities of the male cardiomyopathic hamsters were unchanged, whereas the female enzyme activities were depressed. However, a significantly different pattern in the myofibrillar ATPase was observed in the 36-day-old male and female cardiomyopathic hamsters (Figure 4). There was a slight decrease in the ATPase activity in the male cardiomyopathic hamsters. Conversely, there was an increase in the maximal ATPase activity to 120% for the female cardiomyopathic hamsters. Figure 5 shows that the myofibrillar ATPase activity of the male cardiomyopathic hamsters was increased to 110% at 44 days of age in the presence of 1O-5 M free calcium, whereas the female cardiomyopathic hamsters exhibited a decrease in the ATPase activity. Figure 6 shows that both the female and male cardiomyopathic hamsters exhibited depressed myofibrillar ATPase activities at 50 days of age. This

450

D. PANG l20-

AND

W. WEGLICKI

Mole 0 Control ( RB) A 610 53.58

Female

120

100 -

10-S

10-T

Free calcium(

M)

FIGURE 5. Comparison of the myofibrillar ATPase activities from 44-day-old cardiomyopathic hamsters. Conditions the same as that in Figure 1.

Female

F a k G h

normal

and

normal

and

120

I 60

t I

‘,[&& Io-7

, Io-6

p 10-5

, 10-7

I o-6

lo 10-5

Free calcium (M )

FIGURE 6. Comparison of the myofibrillar ATPase activities from 50-day-old cardiomyopathic hamsters. Conditions the same as that in Figure 1.

MYOFIBRILLAR

ATPASE

IN CARDIOMYOPATHIC

451

HAMSTERS

depression of ATPase activities extend to 72 days in the female and 66 days in the male (Figure 7). Table 1 summarizes the alterations in the myofibrillar ATPase activities of the cardiomyopathic hamsters during the ages of 24 to 161 days. Both the male and female group exhibited a definite pattern of alterations with age: the myofibrillar ATPase activity decreased initially, then increased, and finally decreased with respect to that of the normal hamsters. TABLE

1. Maximal

myofibrillar ATPase activities of cardiomyopathic o/0 Maximal myofibrillar ATPase

Age (days) Male Female

hamsters with age

24

30

36

44

50

66

72

91

161

100

100

94

110

79

82

-

85

-

84

83

120

76

85

-

83

-

84

o/0 Maximal

myofibrillar

ATPase =

cardiomyopathic normal

x 100.

Table 2 shows the free calcium concentrations for half-maximal myofibrillar ATPase activities from the normal and cardiomyopathic hamsters. There were no significant changes in the calcium requirement during the development of the cardiomyopathic hamsters. TABLE

2. Free calcium concentration for half-maximal myopathic and normal hamsters Free calcium

myofibrillar

concentration ATPase Activity

ATPase

in cardio-

for half-maximal (FM)

Age (days)

24

30

36

44

50

Male Normal Cardiomyopathic

0.9 0.9

1.1 1.1

1.8 1.1

1.8 1.4

1.0 0.9

Female Normal Cardiomyopathic

1.0 0.9

1.1 0.9

1.3 1.2

1.2 1.0

0.9 0.7

Since it has been demonstrated that the myocardial calcium content increases progressively during the stage of early myocytolysis in the cardiomyopathic hamsters [8], calcium may be responsible for the elevation and/or depression of the myofibrillar ATPase activities observed in this study. Thus, cardiac myofibrils (0.1 mg/ml) from normal hamsters (30- to 60-day-old, male and female) were

452

D. PANG

AND

W. WEGLICKI

Free colcium(u

)

100 -

FIGURE 7. Comparison normal and cardiomyopathic

of the myofibrillar ATPase activities from 66-day-old hamsters. Conditions the same as that in Figure 1.

0 Preincubatlon

and 72-day-old

with calcium

Free calcium ( M )

FIGURE 8. Preincubation of myofibrils from normal hamsters with calcium. Myofibrils (0.1 mg/ml) were preincubated for 30 min with CaCl, and 1 rnM EGTA in the presence of 50 mM KC1 and 20 rnM imidazole, pH 7.0, at 37°C. 2 mM ATP and 3 rnM MgCl, were then added to start the assay of the ATPase activities. 0, Control-no preincubation; 0, 30-min preincubation.

MYOFIBRILLAR

ATPASE

IN CARDIOMYOPATHIC

HAMSTERS

453

incubated in a medium containing CaCl, and 1 mM EGTA, together with 50 mM KC1 and 20 mM imidazole, pH 7.0, for 30 min at 37°C prior to the addition of 2 mM ATP and 3 mM MgCl, for the assay of the myofibrillar ATPase activities. Figure 8 shows that the myofibrillar ATPase activity was greatly stimulated at low concentration of free calcium (l-7 x 10-v M) after the pre-incubation. However, on increasing the free calcium concentration beyond 1 x 1O-s M, the

6$,:::;:f..., , 0

0’

30'

60'

, 90'

,1 120'

Time of preincubutlon

FIGURE 9. Time course of myofibrillar pre-incubation. Myofibrils (0.1 mg/ml) were incubated in the presence of 50 mM KC1 and 20 mM imidazole, pH 7.0, with (0) and without (0) CaCl, and 1 rnM EGTA at 37”C, before the addition of 2 rnM ATP and 3 mu MgCl, for the assay of the ATPase activities.

myofibrillar ATPase activity was depressed. The enzyme activity was inhibited down to 85% of the control at free calcium concentration of 1 x 1O-5 M. Figure 9 shows that the pre-incubation of the cardiac myofibrils with only 50 mM KC1 and 20 mM imidazole, pH 7.0, did not alter the myofibrillar ATPase activity. However, addition of 5 x lo-’ M and 1 x 1O-5 M free calcium resulted in stimulation and inhibition of the enzyme activities respectively.

454

D.

PANG

AND

W.

WEGLICKI

4. Discussion The major new observation in the present study is that there is a definite pattern of alterations in the myofibrillar ATPase of the young cardiomyopathic hamsters, BIO 53.58 (Table 1 and Figures 2 to 7). The pattern of first depression, then elevation, and finally depression in the enzyme activities is apparent in both the male and female hamsters. It is especially interesting that there is a transitional stage between the elevation and depression in the enzyme activities in both animals. In the 50-day-old male (Figure 6) and 44-day-old female (Figure 5) cardiomyopathic hamsters, the myofibrillar ATPase is stimulated at low free calcium concentration (l-7 x IO-7 M) and depressed at high calcium concentration (2 x 10-6-l x 1O-5 M). The depression in the myofibrillar ATPase activity from 50 days on is significant, because it has been shown that the rate of force development and the developed force of the hearts of 60-day-old cardiomyopathic hamsters (BIO 14.6) are lowered to 50% and 60% of the control respectively [6]. The sequence of initial elevation followed by reduction in the ATPase of the contractile proteins has also been shown in a canine model of aortic stenosisinduced heart failure. An elevation of 15% in the left ventricular myosin ATPase activity is associated with animals of mild aortic stenosis where a pressure gradient of 25 mmHg is induced [12]. On increasing the pressure gradient to 50 mmHg, the myosin ATPase activity is shown to be 15% depressed [12]. The cardiomyopathic hamsters have been bred with an autosomal recessive trait that produces spontaneous cardiomyopathy [I]; thus, the ages of the hamsters can be correlated with the different stages of cardiac failure. Hence, the alteration in the enzyme activity of the myofibrils of the cardiomyopathic hamsters may be viewed as an early indication of subsequent development of heart failure. In addition, the present study also shows that the female cardiomyopathic hamsters exhibit signs of alteration in the myofibrils much earlier than that of the male animals (Table 1). The finding in the present study may also be used to explain the inconsistencies in the observations of either elevated [.5, 101 or depressed 12, 31 ATPase activities of the contractile proteins measured in other laboratories. The differing results of these experiments [2, 3, 5, 101 could not have been due to the differences in the strains of cardiomyopathic hamsters and the methods of preparation of the contractile proteins, since similar results have been obtained in the present study using one strain of cardiomyopathic hamsters (BIO 53.58) and one method of preparation. The data presented in this study indicate that the disagreement among the other laboratories could possibly be due to the lack of recognition of the importance of the age of the animals and the different time of onset of the early myocytolysis in the male and female cardiomyopathic hamsters during the development of cardiac failure. Any combination of animals with different ages

MYOFBRILLAR

ATPASE

IN CARDIOMYOPATHIC

HAMSTERS

455

and sex could result in the observation of either elevation or depression in the myofibrillar ATPase activity (Table 1, Figures 2 to 7). Clear evidence for the formulation of a possible mechanism responsible for the alteration in the myofibrillar ATPase has not yet been presented. It has been proposed that the light chains in the myosin could be the cause in the depression of the myosin ATPase, because the myosin from the cardiomyopathic hamsters (BIO 53.58) shows a loss of the light chain LC, [Z]. However, close analyses ofthe myofibrils and actomyosin reveal that the light chain LC, is present in these preparations from the cardiomyopathic hamsters, and the LC, is lost during the purification of the myosin [Z]. Despite the presence of LC, in the myofibrils and actomyosin, the corresponding ATPase activities are depressed [2]. Thus, at least in the cardiomyopathic hamsters, the light chain LC, is not responsible for the alterations in the myofibrillar ATPase activity. However, it is curious that the calcium content of the cardiac muscle is immensely increased in the 56- to 7 1-dayold cardiomyopathic hamsters, BIO 14.6 [8], the same age for early myocytolysis in this strain [I]. It has been suggested that the increase in the calcium content is due to influx of calcium into the cardiac cell [8]. The direct relationship between the calcium influx and early myocytolysis is strengthened by the fact that injection of verapamil, a calcium antagonist, prevents the development of heart lesions in the cardiomyopathic hamsters during early myocytolysis [7]. If there is also calcium influx into the cardiac cell in the BIO 53.58 strain, then the alteration in the myofibrillar ATPase would simply be due to the progressive increase in the calcium content in the myoplasm, as suggested by the results of incubation of myofibrils from normal hamsters with calcium (Figures 8 and 9). It may be coincidental that the pattern of changes after preincubation (Figure 8) is identical to that in the 44-day-old female (Figure 5) and 50-day-old male cardiomyopathic hamsters, However, the similarity does lend credence to the role played by calcium in the induction of myofibrillar dysfunction. Understandably, the mechanism for the alteration in the myofibrils may not be that simple and straight forward. Nonetheless, the collapse of calcium homeostasis in the cardiac cell may be one of the factors leading to the depression of contractility in the cardiomyopathic hamsters. In conclusion, the present study incidates that the myofibrillar ATPase may be a useful indicator of pending development of heart failure in the cardiomyopathic hamsters. Acknowledgements

This investigation was supported by research grants from the USPHS, HL-18824, HL-19148, HL-21493 and from the American Heart Association, Virginia Affiliate and the Northern Virginia Chapter. David C. Pang is a recipient of a Research Career Development Award (5 K04 HL-00488) from the National Heart, Lung, and Blood Institute.

456

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2.

7. a. 9. 10. 11. 12.

w$h special reference to involvement of the cardiovascular system in the myopathy of the hamster. Annals of the New York Academy of Sciences 138, 213-231 (1966). BHAN, A., MALHOTRA, A., HATCHER, V. B., SONNENBLICK, E. H. & SCHEUER, J. Depressed myosin ATPase activity in hearts of myopathic hamsters: dissociation from neutral protease activity. Journal of Molecular and Cellular Cardiology 10, 769-777 (1978). BHAN, A., MALHOTRA, A., SONNENBLICK, E. H. & SCHEUER, J. Decreased actomyosin ATPase activity in hamster myopathy. Circulation 51,52, II-161 (Abstract) (1975). BRIGGS, F. N. & FLEISHMAN, M. Calcium binding by particle-free supernatants of homogenates of skeletal muscle. Journal of General Physiology 49, 13 1-149 (1965). DHALLA, N. S., SULAKHE, P. F., FEDELSOVA, M. & YATES, J, C. Molecular abnormalities in cardiomyopathy. Advances in Cardiology 13, 283-300 (1974). FORMAN, R., PARMLEY, W. W. & SONNENBLICK, E. H. Myocardial contractility in relation to hypertrophy and failure in myopathic Syrian hamsters. Journal of Molecular and Cellular Cardiology 4, 203-2 11 (1972). JASMIN, G., SOLYMOSS, B. & PROSCHEK, L. Therapeutic trials in hamster dystrophy. Annals of the .New 2Tork Academy of Sciences 317, 338-348 (1979). LOSSNITZER, K. & BAJUSZ, E. Water and electrolyte alterations during the life course of the BlO 14.6 Syrian Golden Hamster. A disease model of a hereditary cardiomyopathy. Journal of Molecular and CeUular Cardiology 6, 163-I 77 (1974). SOLARO, R. J., PANG, D. C. 8r BRIGGS, F. N. The purification of cardiac myofibrils with Triton X-100. Biochimica et biophysics acta 245, 259-262 (1971). WADA, A., YONEDA, H., SHIBATA, N., INUI, Y. & ONISHI, S. Morphological and biochemical studies on the heart of the Cardiomyopathic Syrian Hamster. Recent Advances in Studies on Cardiac Structure and Metabolism 6,275-282 (1975). WAHLER, B. F. & WOLLENBERGER, A. Measurement of orthophosphate using molybdate salt. Biochemische zeitschrzft 329, 508-520 (1958). WILKMAN-COPFELT, J., FENNER, C., WALSH, R., SALEL, A., KAMIYAMA, T. & MASON, D. T. Comparison of mild vs severe pressure overload on the enzymatic activity of myosin in the canine ventricles. Biochemical Medicine 14, 139-146 (1975).