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Effects of steroid treatment on release of cardiac myosin light chain II in acute myocardial infarction in dogs
Effects of Steroid Treatment on Release of Cardiac Myosin Light Chain II in Acute Myocardial Infarctionin Dogs RYOZO NAGAI, MD, MITSUAKI ISOBE, MD, CHUNGCHENG CHIU, MD, KAZUHIDEZ YAMAOKI,
MD, YASUYOSHI
KOHJI IMATAKA,
OHUCHI, MD, SEIGO UEDA, MD,
MD, and YOSHIO YAZAKI,
MD
The effect of methylprednisolone sodium succinate (MP) on release of myosin light chain II (LCII) from the myocardium was studied in experimental myocardial infarction (MI). Acute MI was produced in conscious, closed-chest dogs by ligating the left anterior descending ooronary artery beyond the first diagonal branch. MP, 30 mg/kg, was administered intravenously just before and 24 hours after MI. After MI, LCII levels in the serum were determined serially up to 240 hours. MI slire was determined histologically 10 days after MI. In the MP group, LCII levels in the serum within 72 hours were lower than in the control, and cumulative LCII release for 3 days de-
creased from 530 f 159 to 310 f 101 ng/ml (mean f standard deviation) (p
Many studies have been performed experimentally and clinically to determine the effects of methylprednisolone sodium succinate (MP) in acute myocardial infarction (MI),l-s although the efficacy of MP is inconsistent among these studies. The inconsistency may be attributable to the differences in experimental conditions, differing subsets of subjects or methods of quantitating myocardial damage. We reported a radiloimmunoassay of a subunit of cardiac myosin molecule, myosin light chain II (LCII), and showed that the circulating LCII level can be an index of the extent of MI.7-g In particular, peak LCII in the serum or LCII release determined by a mathematical model reflects well the histologic MI size. Furthermore, since disappearance of LCII from the circulation is quite rapid, LCII levels in the serum seem to reflect the quantity of degenerating myosin filaments. In the present study wle investigated the effects of MP on the time course of LCII in the serum and LCII release
with reference to histologic MI size. We also examined the effects of MP on changes in activities of plasma creatine kinase (CK) and N-acetyl-fl-glucosaminidase (NAG), which is 1 of the lysosomal enzymes. Methods Animal preparation: Thirty mongrel dogs (7 to 12 kg) were used for the time course study of LCII and cardiac enzymes. In separate experiments, 8 dogs were used to measure the disappearance rate of LCII and another 8 were used for histologic studies. Dogs were anesthetized with pentobarbital sodium, 25 mg/kg, and intubated, and underwent left thoracotomy. To make the MI size as constant as possible, a silk snare was positioned loosely around the left anterior descending coronary artery just beyond the first diagonal branch. The ends of the silk string were attached to the skin of the chest wall. After the operation, the thorax was closed and the dogs were allowed to recover. MI was produced 7 to 10 days after the initial operation. At this time, morphine sulfate was administered intravenously in 2- to 3-mg doses intermittently over 20 minutes to provide mild sedation and a catheter for sampling blood was inserted in 1 of the peripheral veins of extremities, and the snare was closed tightly by untethering the ends of the silk string and stretching them. In the time-course study, 16 dogs were given an intravenous MP injection, 30 mg/kg, just before MI and after 24 hours, whereas 14 control dogs were injected with saline solution.
From the Third Department a4 Internal Medicine, University of Tokyo, Tokyo, Japan. Manuscript received June 28, 1983; revised manuscript received March 20,1984, accepted March 22, 1984. Address for reprints: Yoshlio Yazaki, MD, Ths Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan 113. 211
212
EFFECT OF STEROIDS ON MYOSIN LIGHT CHAIN II
TABLE I
Serial Changes in Mean Values of Serum Myosin Light Chain II
Treatment
Hours Afler Infarction 0
Control (ng/m)) Steroid @g/ml)
12
6
. . 13
16
33&23
3g*16
30
24 44
44rt12
fll
fl5 17
8
” ’
36
f5
“**14
22Flo
‘“:9
54 32
f13 *
72
96
4g*15
47
35
40
fl8
f15
30 fl0
60
48
49
2gf14
‘“:8
F8
f15 f9
120 47*21
166
144 40 f21
26
41*15
32*15
240
192 17 fl5
‘:11
‘“*lo
‘:15
20*14
14*1,
f17
216
44 fll
fl4
l
‘p <0.05; +p
drugs were not administered. No specific method of preventing ventricular fibrillation or cardiac arrest was used. The dogs were monitored by electrocardiogram continuously for 24 hours. Blood pressure was measured by the tail-cuff method periodically. Blood samples: Blood samples were obtained every 3 hours for the first 24 hours and every 6 to 12 hours up to 96 hours through the catheter, and after that every 24 hours until 10 days after MI by venipuncture. LCII was quantified by radioimmunoassay.7s CK activity was assayed at 37°C by the Rosalki method.lO NAG was assayed by the method of Woollen and Walker.” Calculations: Release of CK or LCII was calculated by the formula of Shell et a112:Et + Kd * Zb(Et - x + Et)/2 * At, where Et = serum CK (LCII) at time t, Kd = exponential disappearance rate, (Et - x + Et)/2 = average CK (LCII) value during the preceding time interval x, and At = time interval. This mathematical model can be considered as an approximate estimate of the cumulative amount of CK (LCII) that enters 1 ml of plasma (serum) from the infarcted myocardium. The disappearance rate (Kd) for CK was determined by the exponential decay method from the downslope of each CK curve. However, in the case of calculating LCII release, the Kd cannot be determined from the time course curve because LCII is assumed to be released continuously. Therefore, the Kd for LCII was determined in a separate experiment, in which 1251-labeled LCII was injected intravenously into 4 Antiarrhythmic
conscious dogs with and 4 without MP treatment at 2 days after MI, although this method is only an approximation of the true value of Kd. CK release was calculated by changes in the plasma activity of CK during 72 hours after MI, whereas LCII release was calculated by serum LCII levels for 10 days after MI. Infarct size: Dogs were killed 10 days after MI and MI size was determined macroscopically. Details of the method to determine MI size have been described? Sections stained with hematoxylin-eosin were also prepared to confirm that the macroscopically determined MI area coincided with microscopic cellular injury. After the MI margin was determined, the infarcted myocardium was excised and related to 100 g of left ventricle. Histologic features of the infarcted myocardium: In a separate experiment, 4 dogs treated with MP and 4 control dogs were killed 7 days after MI to determine light and electron microscopic changes. After the heart was removed, transverse sections were cut and stained with hematoxylineosin and Axan stains. A 2 X 2 mm piece of the central portion
CPK
*f&o.05 **p
LC II (ng/ml)
60
T
t
0
1
I
I
I
,
I
I
I
I
2
3
4
5
6
7
8
9
I
IO Days
FIGURE 1. Mean changes in serum myosin light chain II (LCII) levels in 11 control dogs (solid circles) and 11 steroid treated dogs (open circles). Bar represents standard error of the mean.
24
48
72 Hours
FIGURE 2. Mean changes in plasma creatine kinase (CPK) activity in control (solld circles) and steroid-treated dogs (open circles). Bar represents standard error of the mean.
July 1, 1984
of the infarcted free ventricular wall was obtained for the electron microscopic study. Statistical analysis: The unpaired t test was used to compare differences between the 2 groups. Differences in plasma NAG before and after the first operation were compared by using the paired t test. The data are expressed as mean f standard deviation. A p value <0.05 was considered statistically significant. Results
A
B Size
‘LV)
NS
I
:2000
NS l
t 1500 -
0
D
C
LC II release (1 Odays) (nghl)
LC II release (3days) (ndml)
I 0
p<0.001
E
LC II release (3daysI 1 Odays) (%)
TO-
3
700
CPK release x104 VI) pco.05
p
0
t
J
f
l
0 0
I_ 1000 -
213
MP-treated dogs are shown in Figure 2. In both groups, the CK level in the plasma increased by 3 to 6 hours after MI, reached a maximum at 12 to 24 hours, and returned to almost normal by 72 hours. Peak CK activity was 4,084 f 2,246 IU/liter at 12.5 f 2.1 hours in the control dogs and 2,938 f 1,845 IU/liter at 12.7 f 2.3 hours in the treated dogs. Peak CK activity, peak CK appearance time and the time course of CK activity in the plasma were not different in the 2 groups, although serial CK activity in the MP group tended to be lower than that in the control group. Infarct size, myosin light chain II release and creatine kinase release: Histologic MI size determined at 10 days was 11.0 f 4.4% of the left ventricle (% LV) in the control group and was not reduced by MP treatment (11.8 f 4.5% LV, difference not significant [NS]) (Fig. 3A). The Kd for LCII was 0.0025 f 0.0002 min-l in the control dogs and 0.0025 f 0.0003 min-l in the treated dogs. The average value of 0.0025 min-l was used for calculation of LCII release in both groups. LCII release for 10 days in the control and MP group was 1,233 f 421 and 1,136 f 414 ng/ml, respectively (NS) (Fig. 3B). However, LCII release for 3 days, calculated by the same formula of Shell, was reduced markedly in the treated dogs (530 f 159 vs 310 f 101 ng/ml, p
In the time course study, MI was produced in 14 control dogs and in 16 dogs treated with MP. Three of the control dogs and 5 of the MP-treated dogs died of ventricular fibrillation. Heart rate and systolic blood pressure were not different between the 2 groups, and dogs that survived for 10 days after MI did not show any sign of cardiogenic shock. Serial changes in myosin light chain II in the serum: Table I shows the mean ( f standard deviation) for average LCII levels in the serum in both groups. In the control group, LCII[ began to increase by 3 to 12 hours, reached a peak level (63 f 21 ng/ml) between 12 and 120 hours (63 f 37 h.ours), and thereafter decreased gradually. In the MP group, LCII levels also increased by 3 to 12 hours, but the increase was more gradual than in the control group. That is, LCII levels between 16 and 72 hours after MI were significantly lower than those in the control group and the peak LCII level appeared later (122 f 25 hours, p tO.001). After 144 hours, LCII levels in the MP group were slightly higher than in the control, although difference is not statistically significant (Fig. 1). Serial changes in creatine kinase activity in the plasma: Changes in CK activity in the control and
Infarct
THE AMERICAN JOURNAL OF CARDIOLOGY Volume 54
%t
@ee
1.5
1 .o
00
00
0
0.5
500
i-
lo-
loo-
c
I
s
OeT
O
c
I
s
0
c
I
s
0
FIGURE 3. Infarct size (A), myosin light chain II (LCII) release for 10 days (B), LCll release for 3 days (C), the ratio of LCII release for 10 days to LCII release for 3 days (D) and creatine kinase (CPK) release for 3 days (E) in control (C)and steroid-treated dogs (S). Values are mean f standard deviation.
214
EFFECT OF STEROIDS ON MYOSIN LIGHT CHAIN II
control group, MI size correlated with LCII release or CK release for 10 days. The correlation coefficients were 0.64 (p <0.05) and 0.53 (p
LC II release
Discussion We have examined the effects of MP on breakdown of myosin filaments after acute MI by determining circulating LCII levels. MP treatment at an early stage significantly reduced LCII levels in the serum and LCII release for the initial 3 days after MI. Furthermore, peak LCII levels appeared later in the MP group. However, histologic MI size determined at 10 days and LCII release for 10 days were not reduced by MP. Recently radioimmunoassays of structural proteins were developed in our laboratory and others,7-gJ3-15 and many aspects of the degradation of myofilaments in acute MI have been elucidated. In previous reports, we showed that LCII levels in the serum reach a maximum at various times between 12 and 120 hours and remain elevated for 7 to 10 days. Since the rate of disappearance of LCII from the circulation is rapid, the reason why LCII levels stay elevated for a long period can be explained by continuous liberation of LCII from the MI myocardium.7-g Similar results were obtained for circulating total myosin light chains by Khaw et all4 and for tropomyosin by Cummins et all5 Therefore, degradation of myofilaments after MI appears to be a much slower process than leakage of cardiac enzymes. In this study, the effects of MP are the most pronounced in the time-course study of LCII levels in the serum. Because the rate of disappearance of LCII was not affected by MP, the decrease in LCII levels during 72 hours could be caused by a decrease in the amount of LCII liberated during this period. However, peak LCII levels appeared later in the MP group and peak LCII levels were almost the same in both groups. These findings suggest that MP treatment delayed early dissociation of myosin filaments but failed to prevent it
(ng/ml) CPK release
2000 0
(WI)
20000 -
0
l
l 0
0
1500 -
0
.
0
0
0
0
0
0
.O
0
1
I
Infarct
0 0
0
5000 -
5
0
0
500 -
0
0
10000 -
0
0
.dJ
0 0
0
1000 -
15000 -
0
0
I
I
10
15
l
a 0
Oo
5
10
O.
0
0
0
I
20
Size (%LV)
FIGURE 4. The relation between infarct size and myosin light chain II (LCII) release for 10 days in control (solidcircles) and steroid-treated dogs (open circles). Correlation coefficients are 0.64 (p <0.05) and 0.65 (p <0.05), respectively.
0
Infarct
15
20
Size (%LV)
FIGURE 5. The relation between infarct size and creatine kinase (CPK) release in control (sofkl circles) and steroi&treated dogs (open circles). Correlation coefficients are 0.53 (p CO.1) and -0.12 (difference not significant).
July 1,1984
through the lo-day period; that is, breakdown of myosin filaments and LCII release occurred at a slower rate in the treated dogs, but finally myosin filaments were dissociated during the 10 days. Kloner et all8 examined the histologic changes induced by multiple high doses of MP in hearts of rats.. They reported that in the infarcted myocardium at ‘7days after MI, there remained large sheets of dead myocytes with preservation of striations and membranes, and that “mummified” myofilaments were more prominent in steroid treated rate. Therefore, the delayed appearance of LCII-in the serum observed in our experiment seems to be a reflection of these morphologic changes induced by MP. In our study, light and electron microscopic examination did not reveal characteristic changes in the myocardium in the MP group, although Z bands tended to be relatively well preserved in the MP group. Since our histologic study was performed in a small number of dogs, further studies are necessary to clarify the histologic interpretation of the delayed release of LCII in the MP-treated dogs. The effects of MP treatment on the time course of CK were not as pronounced as in the case of LCII, although CK release for 3 days was reduced significantly. However, the linear relation between MI size and CK release was greatly disturbed in the MP group. The reason why the relation between MI size and CK release was more strongly affected than LCII release for 10 days was not found in this study. However, as shown in previous studies,gJ7 myocardial CK can degenerate in the MI area before entering into the blood circulation. Therefore, if necrosis of the m!rocardium is delayed in the MP group, a larger fraction of myocardial CK can lose its activity in situ than in tlhe case without MP treatment. Thus, CK release does not necessarily correlate with MI size after MP treatment. The mechanism involved in the effect of steroid on ischemic myocardium is still controversial. However, recently, Blackwell and CarnucciolS reported the steroid-induced synthesis of a specific protein which in-
THE AMERICAN JOURNAL OF CARDIOLOGY Volume 54
215
hibits phospholipase A2. Phospholipase A2 is activated in ischemic myocytes and catalyzes the hydrolysis of membrane lipids, producing lysophospholipids or other cytotoxic substances.lg Therefore, a decrease in the activity of circulating lysosomal enzymes can be expected to be an index of the protective effect of steroid treatment against ischemic myocardium. Welman et a120reported that in acute MI, plasma NAG activity showed a biphasic pattern and that peak CK or NAG in patients who were treated with MP within 6 hours after the attack was lower than in control patients21 The biphasic pattern of NAG was also seen in our present study. However, the effect of MP on peak NAG activity or its peak appearance time was not obvious. One factor might be the differences in experimental
NAG(IU/I)
48
72
96 HOURS
FIGURE 6. Meanchangesin plasma N-acetyl$gl ucosaminidase (NAG) activity in control (sdkl circles) and steroi&treated dogs (open dudes). Bar represents standard error of the mean.
FIGURE 7. Electron micrographs of the center of a 7-day-old infarct in a steroid-treated dog (A) and in a control dog (B). Myofilaments are disrupted and wide intermyofibrillar spaces are observed in both dogs. However, Z bands can be recognized better in the MP-treated dog. Bar represents 3 pm.
216
EFFECT OF STEROIDS ON MYOSIN LIGHT CHAIN II
conditions, since plasma NAG activity is affected by many factors, such as species of the animals, age of the subjects or previous history of illness.22 Hence, our results do not necessarily contradict those of Welman et a1.20 The clinical usefulness of steroid treatment in acute MI remains to be settled because the results obtained by different investigators are inconsistent. Our results suggest that MP treatment early after attack only retards complete necrosis of the myocardium and that protection against MI myocardium through pharmacologic intervention cannot be assessed at a single time point when myocardial necrosis is not complete. Although further studies are necessary to clarify the importance of degradation of myosin filaments in the progress of reversible to irreversible myocyte injury, delay of necrosis may be desirable if further intervention such as revascularization of the coronary artery can be undertaken within several hours after the attack. In contrast, long-term steroid treatment may be harmful because delay in repair of the MI myocardium results in formation of a ventricular aneurysm as shown clinically.23 References 1. Libby P, Msroko PR, Bloor CM, Bobol BE. Reduction of experimental myocardial infarct size by corticosteroid administration. J Clin Invest 1973;52:599-607. 2. Spath JA, Lane DL, Lefer AM. Protective action of methylprednlsolone on the myocardium during experlmental myocardial ischemla in cat. Circ Res 1974;35:44-51. 3. MorrIson J, Reduto L, Plzzarello R, Geller K, Maley T, Gulotta B. Modification of myocardial injury in man by corticosteroid administration. Circulatlon 1976;53:suppl l:l-200-l-203. 4. Vogel WY, Zannonl VG, Abram8 GD, Luchessl BR. Inability of methylprednisolcne sodturn succinate to decrease infarct size or preserve enzyme activity measured 24 hours after coronary occlusion in the dog. Circulation
1977;55:566-595. 5. Bulkfey BH, Roberts WC. Steroid therapy during acute myocardial infarction: a cause of delayed healing and of ventricular aneurysm. Am J Med 1974;
__ _
!iR~3AA-3!ill
___
6. Madlas JE, Hood WB Jr. Effects of methylprednisolone on the ischemic damage in patients with acute myocardial infarction. Circulation 1962; 65:1106-1113. 7. Nagal R, Ueda S, Yazakl Y. Radioimmunoassay of cardiac myosin light chain II in the serum following experimental myocardiil infarction. Biochem Biophys Res Commun 1979;86:683-688. 6. Nagal R, Yazakl Y. As.s+smsnt of myocardial infarct sife by serial changes ;aerum cardrac myosln light chain II In dogs. Jpn Crrc J 1981;45:661-
___.
9. Nagal R, Chlu CC, Yamaokl K, Ohuchl Y, Ueda S, lmataka K, Yarakl Y.
Evaluationof methodsfor estimatinainfarct size bv mvosinliaht chain 2: comparison with cardiac enzymes. Am J Physiol 198$245:H213-H419. 10. Rosalkl SB. An improved procedure for serum creatine phosphokinase determination. J Lab Clin Med 1967;69:896-705. 11. Woollen JW, Walker PG. The fluorimetric estimation of Nacetyl-&glucosaminidase and ,#-galactosidase in blood plasma. Clin Chimics Acta 1965: 12:647-658. 12. Shell.WE, KjekshusJK, Sobel BE. Quantitative assessment of the extent of myocardial infarction in the conscious dog by means of analysis of serial chanaes in serum creatine .ohosohokinase activitv. J Clin Invest 1971: . 50:2614-2625. 13. Trahern CA, Gere JB, Krauth GH, Blgham DA. Clinical assessment of serum myosln light chains in the diagnosis of acute myocardial infarction. Am J Cardiol 1978;41:841-645. 14. Khaw BA, Gokf HK, Fallon JT, Haber E. Detecticn of serum cardiac myosln lioht chains in acute exoerimsntal mvocardial Infarction: radloimmunoassav 07 cardiac myosin light chains. Circulation 1978;58:1130-1138. . 16. Cummlns P, YcGurk B, Llttter WA. Radioimmunoassay of human cardiac tropomyosin in acute myocardial infarction. Clin Sci 1981;60:251-259. 16. Kloner RA, Flshbeln MC, Lew H, Maroko PR, Braunwakl E. Mummification of the infarcted myocardium by high dose corticosterolds. Circulation 1978;57:56-63. 17. Clark GL, Roblnson AK, Gnepp DR, Roberts R, Sobel BE. Effects of lymphatic transport of enzyme on plasma creatine kinase time-activity curves after myocardial infarction in dogs. Circ Res 1978;43:162-169. 16. Blackwell GJ, Carnucclo R. Macrocortin: a polypeptide causing the antiphospholi ase effect of glucocorticoids. Nature 1980;287:147-149. 19. Franson R C, Waite BM, Wegllckl WB. Phospholipase A activity of lysosomes of rat myocardial tissue. Biochemistry 1972;11:472-478. 20. Welman E, Selwyn AP, Peters TJ,Co!beck JF, Fox K,M. Plasma lysosomal in&?; activrty In acute myocardral Infarction. Cardrovasc Res 1978; 12: 21. Welman E, Selwyn AP, Fox KM. Lysosomal and cytosolic enzyme release in acute myocardial infarction: effects of methylprednisolone. Circulation 1979;59:730-733. 22. Woollen JW, Turner P. Plasma N-acetyl-/‘?-glucosaminidase and &glucuronidase in health and disease. Clin Chimica Acta 1965;12:671-883. 23. Roberts R, DeMetto V, S&et BE. Deleterious effects of methylprednisolone in patients with myocardial infarction. Circulation 1976;53:1-204-I-206.
MD, YASUYOSHI
KOHJI IMATAKA,
OHUCHI, MD, SEIGO UEDA, MD,
MD, and YOSHIO YAZAKI,
MD
The effect of methylprednisolone sodium succinate (MP) on release of myosin light chain II (LCII) from the myocardium was studied in experimental myocardial infarction (MI). Acute MI was produced in conscious, closed-chest dogs by ligating the left anterior descending ooronary artery beyond the first diagonal branch. MP, 30 mg/kg, was administered intravenously just before and 24 hours after MI. After MI, LCII levels in the serum were determined serially up to 240 hours. MI slire was determined histologically 10 days after MI. In the MP group, LCII levels in the serum within 72 hours were lower than in the control, and cumulative LCII release for 3 days de-
creased from 530 f 159 to 310 f 101 ng/ml (mean f standard deviation) (p
Many studies have been performed experimentally and clinically to determine the effects of methylprednisolone sodium succinate (MP) in acute myocardial infarction (MI),l-s although the efficacy of MP is inconsistent among these studies. The inconsistency may be attributable to the differences in experimental conditions, differing subsets of subjects or methods of quantitating myocardial damage. We reported a radiloimmunoassay of a subunit of cardiac myosin molecule, myosin light chain II (LCII), and showed that the circulating LCII level can be an index of the extent of MI.7-g In particular, peak LCII in the serum or LCII release determined by a mathematical model reflects well the histologic MI size. Furthermore, since disappearance of LCII from the circulation is quite rapid, LCII levels in the serum seem to reflect the quantity of degenerating myosin filaments. In the present study wle investigated the effects of MP on the time course of LCII in the serum and LCII release
with reference to histologic MI size. We also examined the effects of MP on changes in activities of plasma creatine kinase (CK) and N-acetyl-fl-glucosaminidase (NAG), which is 1 of the lysosomal enzymes. Methods Animal preparation: Thirty mongrel dogs (7 to 12 kg) were used for the time course study of LCII and cardiac enzymes. In separate experiments, 8 dogs were used to measure the disappearance rate of LCII and another 8 were used for histologic studies. Dogs were anesthetized with pentobarbital sodium, 25 mg/kg, and intubated, and underwent left thoracotomy. To make the MI size as constant as possible, a silk snare was positioned loosely around the left anterior descending coronary artery just beyond the first diagonal branch. The ends of the silk string were attached to the skin of the chest wall. After the operation, the thorax was closed and the dogs were allowed to recover. MI was produced 7 to 10 days after the initial operation. At this time, morphine sulfate was administered intravenously in 2- to 3-mg doses intermittently over 20 minutes to provide mild sedation and a catheter for sampling blood was inserted in 1 of the peripheral veins of extremities, and the snare was closed tightly by untethering the ends of the silk string and stretching them. In the time-course study, 16 dogs were given an intravenous MP injection, 30 mg/kg, just before MI and after 24 hours, whereas 14 control dogs were injected with saline solution.
From the Third Department a4 Internal Medicine, University of Tokyo, Tokyo, Japan. Manuscript received June 28, 1983; revised manuscript received March 20,1984, accepted March 22, 1984. Address for reprints: Yoshlio Yazaki, MD, Ths Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan 113. 211
212
EFFECT OF STEROIDS ON MYOSIN LIGHT CHAIN II
TABLE I
Serial Changes in Mean Values of Serum Myosin Light Chain II
Treatment
Hours Afler Infarction 0
Control (ng/m)) Steroid @g/ml)
12
6
. . 13
16
33&23
3g*16
30
24 44
44rt12
fll
fl5 17
8
” ’
36
f5
“**14
22Flo
‘“:9
54 32
f13 *
72
96
4g*15
47
35
40
fl8
f15
30 fl0
60
48
49
2gf14
‘“:8
F8
f15 f9
120 47*21
166
144 40 f21
26
41*15
32*15
240
192 17 fl5
‘:11
‘“*lo
‘:15
20*14
14*1,
f17
216
44 fll
fl4
l
‘p <0.05; +p
drugs were not administered. No specific method of preventing ventricular fibrillation or cardiac arrest was used. The dogs were monitored by electrocardiogram continuously for 24 hours. Blood pressure was measured by the tail-cuff method periodically. Blood samples: Blood samples were obtained every 3 hours for the first 24 hours and every 6 to 12 hours up to 96 hours through the catheter, and after that every 24 hours until 10 days after MI by venipuncture. LCII was quantified by radioimmunoassay.7s CK activity was assayed at 37°C by the Rosalki method.lO NAG was assayed by the method of Woollen and Walker.” Calculations: Release of CK or LCII was calculated by the formula of Shell et a112:Et + Kd * Zb(Et - x + Et)/2 * At, where Et = serum CK (LCII) at time t, Kd = exponential disappearance rate, (Et - x + Et)/2 = average CK (LCII) value during the preceding time interval x, and At = time interval. This mathematical model can be considered as an approximate estimate of the cumulative amount of CK (LCII) that enters 1 ml of plasma (serum) from the infarcted myocardium. The disappearance rate (Kd) for CK was determined by the exponential decay method from the downslope of each CK curve. However, in the case of calculating LCII release, the Kd cannot be determined from the time course curve because LCII is assumed to be released continuously. Therefore, the Kd for LCII was determined in a separate experiment, in which 1251-labeled LCII was injected intravenously into 4 Antiarrhythmic
conscious dogs with and 4 without MP treatment at 2 days after MI, although this method is only an approximation of the true value of Kd. CK release was calculated by changes in the plasma activity of CK during 72 hours after MI, whereas LCII release was calculated by serum LCII levels for 10 days after MI. Infarct size: Dogs were killed 10 days after MI and MI size was determined macroscopically. Details of the method to determine MI size have been described? Sections stained with hematoxylin-eosin were also prepared to confirm that the macroscopically determined MI area coincided with microscopic cellular injury. After the MI margin was determined, the infarcted myocardium was excised and related to 100 g of left ventricle. Histologic features of the infarcted myocardium: In a separate experiment, 4 dogs treated with MP and 4 control dogs were killed 7 days after MI to determine light and electron microscopic changes. After the heart was removed, transverse sections were cut and stained with hematoxylineosin and Axan stains. A 2 X 2 mm piece of the central portion
CPK
*f&o.05 **p
LC II (ng/ml)
60
T
t
0
1
I
I
I
,
I
I
I
I
2
3
4
5
6
7
8
9
I
IO Days
FIGURE 1. Mean changes in serum myosin light chain II (LCII) levels in 11 control dogs (solid circles) and 11 steroid treated dogs (open circles). Bar represents standard error of the mean.
24
48
72 Hours
FIGURE 2. Mean changes in plasma creatine kinase (CPK) activity in control (solld circles) and steroid-treated dogs (open circles). Bar represents standard error of the mean.
July 1, 1984
of the infarcted free ventricular wall was obtained for the electron microscopic study. Statistical analysis: The unpaired t test was used to compare differences between the 2 groups. Differences in plasma NAG before and after the first operation were compared by using the paired t test. The data are expressed as mean f standard deviation. A p value <0.05 was considered statistically significant. Results
A
B Size
‘LV)
NS
I
:2000
NS l
t 1500 -
0
D
C
LC II release (1 Odays) (nghl)
LC II release (3days) (ndml)
I 0
p<0.001
E
LC II release (3daysI 1 Odays) (%)
TO-
3
700
CPK release x104 VI) pco.05
p
0
t
J
f
l
0 0
I_ 1000 -
213
MP-treated dogs are shown in Figure 2. In both groups, the CK level in the plasma increased by 3 to 6 hours after MI, reached a maximum at 12 to 24 hours, and returned to almost normal by 72 hours. Peak CK activity was 4,084 f 2,246 IU/liter at 12.5 f 2.1 hours in the control dogs and 2,938 f 1,845 IU/liter at 12.7 f 2.3 hours in the treated dogs. Peak CK activity, peak CK appearance time and the time course of CK activity in the plasma were not different in the 2 groups, although serial CK activity in the MP group tended to be lower than that in the control group. Infarct size, myosin light chain II release and creatine kinase release: Histologic MI size determined at 10 days was 11.0 f 4.4% of the left ventricle (% LV) in the control group and was not reduced by MP treatment (11.8 f 4.5% LV, difference not significant [NS]) (Fig. 3A). The Kd for LCII was 0.0025 f 0.0002 min-l in the control dogs and 0.0025 f 0.0003 min-l in the treated dogs. The average value of 0.0025 min-l was used for calculation of LCII release in both groups. LCII release for 10 days in the control and MP group was 1,233 f 421 and 1,136 f 414 ng/ml, respectively (NS) (Fig. 3B). However, LCII release for 3 days, calculated by the same formula of Shell, was reduced markedly in the treated dogs (530 f 159 vs 310 f 101 ng/ml, p
In the time course study, MI was produced in 14 control dogs and in 16 dogs treated with MP. Three of the control dogs and 5 of the MP-treated dogs died of ventricular fibrillation. Heart rate and systolic blood pressure were not different between the 2 groups, and dogs that survived for 10 days after MI did not show any sign of cardiogenic shock. Serial changes in myosin light chain II in the serum: Table I shows the mean ( f standard deviation) for average LCII levels in the serum in both groups. In the control group, LCII[ began to increase by 3 to 12 hours, reached a peak level (63 f 21 ng/ml) between 12 and 120 hours (63 f 37 h.ours), and thereafter decreased gradually. In the MP group, LCII levels also increased by 3 to 12 hours, but the increase was more gradual than in the control group. That is, LCII levels between 16 and 72 hours after MI were significantly lower than those in the control group and the peak LCII level appeared later (122 f 25 hours, p tO.001). After 144 hours, LCII levels in the MP group were slightly higher than in the control, although difference is not statistically significant (Fig. 1). Serial changes in creatine kinase activity in the plasma: Changes in CK activity in the control and
Infarct
THE AMERICAN JOURNAL OF CARDIOLOGY Volume 54
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FIGURE 3. Infarct size (A), myosin light chain II (LCII) release for 10 days (B), LCll release for 3 days (C), the ratio of LCII release for 10 days to LCII release for 3 days (D) and creatine kinase (CPK) release for 3 days (E) in control (C)and steroid-treated dogs (S). Values are mean f standard deviation.
214
EFFECT OF STEROIDS ON MYOSIN LIGHT CHAIN II
control group, MI size correlated with LCII release or CK release for 10 days. The correlation coefficients were 0.64 (p <0.05) and 0.53 (p
LC II release
Discussion We have examined the effects of MP on breakdown of myosin filaments after acute MI by determining circulating LCII levels. MP treatment at an early stage significantly reduced LCII levels in the serum and LCII release for the initial 3 days after MI. Furthermore, peak LCII levels appeared later in the MP group. However, histologic MI size determined at 10 days and LCII release for 10 days were not reduced by MP. Recently radioimmunoassays of structural proteins were developed in our laboratory and others,7-gJ3-15 and many aspects of the degradation of myofilaments in acute MI have been elucidated. In previous reports, we showed that LCII levels in the serum reach a maximum at various times between 12 and 120 hours and remain elevated for 7 to 10 days. Since the rate of disappearance of LCII from the circulation is rapid, the reason why LCII levels stay elevated for a long period can be explained by continuous liberation of LCII from the MI myocardium.7-g Similar results were obtained for circulating total myosin light chains by Khaw et all4 and for tropomyosin by Cummins et all5 Therefore, degradation of myofilaments after MI appears to be a much slower process than leakage of cardiac enzymes. In this study, the effects of MP are the most pronounced in the time-course study of LCII levels in the serum. Because the rate of disappearance of LCII was not affected by MP, the decrease in LCII levels during 72 hours could be caused by a decrease in the amount of LCII liberated during this period. However, peak LCII levels appeared later in the MP group and peak LCII levels were almost the same in both groups. These findings suggest that MP treatment delayed early dissociation of myosin filaments but failed to prevent it
(ng/ml) CPK release
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FIGURE 4. The relation between infarct size and myosin light chain II (LCII) release for 10 days in control (solidcircles) and steroid-treated dogs (open circles). Correlation coefficients are 0.64 (p <0.05) and 0.65 (p <0.05), respectively.
0
Infarct
15
20
Size (%LV)
FIGURE 5. The relation between infarct size and creatine kinase (CPK) release in control (sofkl circles) and steroi&treated dogs (open circles). Correlation coefficients are 0.53 (p CO.1) and -0.12 (difference not significant).
July 1,1984
through the lo-day period; that is, breakdown of myosin filaments and LCII release occurred at a slower rate in the treated dogs, but finally myosin filaments were dissociated during the 10 days. Kloner et all8 examined the histologic changes induced by multiple high doses of MP in hearts of rats.. They reported that in the infarcted myocardium at ‘7days after MI, there remained large sheets of dead myocytes with preservation of striations and membranes, and that “mummified” myofilaments were more prominent in steroid treated rate. Therefore, the delayed appearance of LCII-in the serum observed in our experiment seems to be a reflection of these morphologic changes induced by MP. In our study, light and electron microscopic examination did not reveal characteristic changes in the myocardium in the MP group, although Z bands tended to be relatively well preserved in the MP group. Since our histologic study was performed in a small number of dogs, further studies are necessary to clarify the histologic interpretation of the delayed release of LCII in the MP-treated dogs. The effects of MP treatment on the time course of CK were not as pronounced as in the case of LCII, although CK release for 3 days was reduced significantly. However, the linear relation between MI size and CK release was greatly disturbed in the MP group. The reason why the relation between MI size and CK release was more strongly affected than LCII release for 10 days was not found in this study. However, as shown in previous studies,gJ7 myocardial CK can degenerate in the MI area before entering into the blood circulation. Therefore, if necrosis of the m!rocardium is delayed in the MP group, a larger fraction of myocardial CK can lose its activity in situ than in tlhe case without MP treatment. Thus, CK release does not necessarily correlate with MI size after MP treatment. The mechanism involved in the effect of steroid on ischemic myocardium is still controversial. However, recently, Blackwell and CarnucciolS reported the steroid-induced synthesis of a specific protein which in-
THE AMERICAN JOURNAL OF CARDIOLOGY Volume 54
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hibits phospholipase A2. Phospholipase A2 is activated in ischemic myocytes and catalyzes the hydrolysis of membrane lipids, producing lysophospholipids or other cytotoxic substances.lg Therefore, a decrease in the activity of circulating lysosomal enzymes can be expected to be an index of the protective effect of steroid treatment against ischemic myocardium. Welman et a120reported that in acute MI, plasma NAG activity showed a biphasic pattern and that peak CK or NAG in patients who were treated with MP within 6 hours after the attack was lower than in control patients21 The biphasic pattern of NAG was also seen in our present study. However, the effect of MP on peak NAG activity or its peak appearance time was not obvious. One factor might be the differences in experimental
NAG(IU/I)
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72
96 HOURS
FIGURE 6. Meanchangesin plasma N-acetyl$gl ucosaminidase (NAG) activity in control (sdkl circles) and steroi&treated dogs (open dudes). Bar represents standard error of the mean.
FIGURE 7. Electron micrographs of the center of a 7-day-old infarct in a steroid-treated dog (A) and in a control dog (B). Myofilaments are disrupted and wide intermyofibrillar spaces are observed in both dogs. However, Z bands can be recognized better in the MP-treated dog. Bar represents 3 pm.
216
EFFECT OF STEROIDS ON MYOSIN LIGHT CHAIN II
conditions, since plasma NAG activity is affected by many factors, such as species of the animals, age of the subjects or previous history of illness.22 Hence, our results do not necessarily contradict those of Welman et a1.20 The clinical usefulness of steroid treatment in acute MI remains to be settled because the results obtained by different investigators are inconsistent. Our results suggest that MP treatment early after attack only retards complete necrosis of the myocardium and that protection against MI myocardium through pharmacologic intervention cannot be assessed at a single time point when myocardial necrosis is not complete. Although further studies are necessary to clarify the importance of degradation of myosin filaments in the progress of reversible to irreversible myocyte injury, delay of necrosis may be desirable if further intervention such as revascularization of the coronary artery can be undertaken within several hours after the attack. In contrast, long-term steroid treatment may be harmful because delay in repair of the MI myocardium results in formation of a ventricular aneurysm as shown clinically.23 References 1. Libby P, Msroko PR, Bloor CM, Bobol BE. Reduction of experimental myocardial infarct size by corticosteroid administration. J Clin Invest 1973;52:599-607. 2. Spath JA, Lane DL, Lefer AM. Protective action of methylprednlsolone on the myocardium during experlmental myocardial ischemla in cat. Circ Res 1974;35:44-51. 3. MorrIson J, Reduto L, Plzzarello R, Geller K, Maley T, Gulotta B. Modification of myocardial injury in man by corticosteroid administration. Circulatlon 1976;53:suppl l:l-200-l-203. 4. Vogel WY, Zannonl VG, Abram8 GD, Luchessl BR. Inability of methylprednisolcne sodturn succinate to decrease infarct size or preserve enzyme activity measured 24 hours after coronary occlusion in the dog. Circulation
1977;55:566-595. 5. Bulkfey BH, Roberts WC. Steroid therapy during acute myocardial infarction: a cause of delayed healing and of ventricular aneurysm. Am J Med 1974;
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!iR~3AA-3!ill
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6. Madlas JE, Hood WB Jr. Effects of methylprednisolone on the ischemic damage in patients with acute myocardial infarction. Circulation 1962; 65:1106-1113. 7. Nagal R, Ueda S, Yazakl Y. Radioimmunoassay of cardiac myosin light chain II in the serum following experimental myocardiil infarction. Biochem Biophys Res Commun 1979;86:683-688. 6. Nagal R, Yazakl Y. As.s+smsnt of myocardial infarct sife by serial changes ;aerum cardrac myosln light chain II In dogs. Jpn Crrc J 1981;45:661-
___.
9. Nagal R, Chlu CC, Yamaokl K, Ohuchl Y, Ueda S, lmataka K, Yarakl Y.
Evaluationof methodsfor estimatinainfarct size bv mvosinliaht chain 2: comparison with cardiac enzymes. Am J Physiol 198$245:H213-H419. 10. Rosalkl SB. An improved procedure for serum creatine phosphokinase determination. J Lab Clin Med 1967;69:896-705. 11. Woollen JW, Walker PG. The fluorimetric estimation of Nacetyl-&glucosaminidase and ,#-galactosidase in blood plasma. Clin Chimics Acta 1965: 12:647-658. 12. Shell.WE, KjekshusJK, Sobel BE. Quantitative assessment of the extent of myocardial infarction in the conscious dog by means of analysis of serial chanaes in serum creatine .ohosohokinase activitv. J Clin Invest 1971: . 50:2614-2625. 13. Trahern CA, Gere JB, Krauth GH, Blgham DA. Clinical assessment of serum myosln light chains in the diagnosis of acute myocardial infarction. Am J Cardiol 1978;41:841-645. 14. Khaw BA, Gokf HK, Fallon JT, Haber E. Detecticn of serum cardiac myosln lioht chains in acute exoerimsntal mvocardial Infarction: radloimmunoassav 07 cardiac myosin light chains. Circulation 1978;58:1130-1138. . 16. Cummlns P, YcGurk B, Llttter WA. Radioimmunoassay of human cardiac tropomyosin in acute myocardial infarction. Clin Sci 1981;60:251-259. 16. Kloner RA, Flshbeln MC, Lew H, Maroko PR, Braunwakl E. Mummification of the infarcted myocardium by high dose corticosterolds. Circulation 1978;57:56-63. 17. Clark GL, Roblnson AK, Gnepp DR, Roberts R, Sobel BE. Effects of lymphatic transport of enzyme on plasma creatine kinase time-activity curves after myocardial infarction in dogs. Circ Res 1978;43:162-169. 16. Blackwell GJ, Carnucclo R. Macrocortin: a polypeptide causing the antiphospholi ase effect of glucocorticoids. Nature 1980;287:147-149. 19. Franson R C, Waite BM, Wegllckl WB. Phospholipase A activity of lysosomes of rat myocardial tissue. Biochemistry 1972;11:472-478. 20. Welman E, Selwyn AP, Peters TJ,Co!beck JF, Fox K,M. Plasma lysosomal in&?; activrty In acute myocardral Infarction. Cardrovasc Res 1978; 12: 21. Welman E, Selwyn AP, Fox KM. Lysosomal and cytosolic enzyme release in acute myocardial infarction: effects of methylprednisolone. Circulation 1979;59:730-733. 22. Woollen JW, Turner P. Plasma N-acetyl-/‘?-glucosaminidase and &glucuronidase in health and disease. Clin Chimica Acta 1965;12:671-883. 23. Roberts R, DeMetto V, S&et BE. Deleterious effects of methylprednisolone in patients with myocardial infarction. Circulation 1976;53:1-204-I-206.