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An increase of cathepsin D activity in cardiac lymph and pericardial fluid induced by experimental myocardial ischemia in the dog
Life Sciences Vol. 17, pp . 613-618 Printed in the ü .S .A .
Pergamon Press
AN INCREASE OF CATHEPSIN D ACTIVITY IN CARDIAC LYMPH AND PERICARDIAL FLUID INDUCED BY EXPERIMENTAL MYOCARDIAL ISCHEMIA IN THE DOG Haruo Araki and Fumio Takenaka Department of Pharmacology, Kumamoto University Medical School, Kumamoto Japan (Received in final form July 18, 1975) Simnary Cathepsin D activity was measured in the cardiac lymph, pericardial fluid and plasma after ligation of the left coronary artery of the dog . The activity of cathepsin D increased both in the cardiac lymph and the pericardial fluid after the coronary 1lgation, while that in the plasma did not show any increase . In sham operated group, there was practically no change in the cathepsin D activity . The increase in the cathepsin D activity in the cardiac lymph and the pericardial fluid may indicate an increase in amount of myocardial lysosomal enzymes liberated into the interstitial space during myocardial ischemia . In recent years, it has been reported that lysosomal enzymes play an undesirable role in the cause of irreversible shock (1), and disruption of cardiac lysosomes within the ischemic myocardium may prromote cardiac damage following myocardial infarction (2) . Same of these enzymes contained in the myocardial cell appear in the serum several days after the onset of myocardial infarction (3) . More recently, Spath et al (1974) have shown that, in experimental myocardial ischemia, a total amount of lysosomal enzymes in the ischemic myocardium decreases, while the ratio of "free" activity rises, and suggested that a marked de ree of lysosomal disruption occurs in the ischemic portion of the myocardium ~4) . However, this "free" activity does not necessarily mean true activity in the intracellular or interstitial fluid, since this may have been resulted from mechanical disruption of the labiled lysosamal membrane in the process of homogenation (5) . Thus, it is necessary to examine whether the myocardial lysosamal enzymes are liberated into the interstitial fluid within the ischemic region of the myocardium at the early stage of myocardial infarction . The present experiments, therefore, were designed in an attempt to quantify the release of the myocardial lysosomal enzymes following acute occlusion of a coronary artery, by measuring activity of a cardiac lysosomal enzyme (cathepsin D) in the cardiac lymph and also in the pericardial fluid. Methods The experiments were carried out in twenty mongrel dogs of either sex weighing 12-21 kg . All dogs were anesthetized with pentobarbital sodium (30 mg/kg, iv) and divided into four groups . ~Grou ~1 . Under artifitial respiration with room air, left thoracotomy was performed in the third intercostal space and the pericardium ryas incised along the anterior interventricular sulcus for about 4 cm . Small amounts of T-1824 dye (0 .1 to 0.2 ml) was injected into the subepicardial portion of the left ventricle with a tuberculin needle . After the injection of dye the 613
61 4
Cathep®in D in Myocardial iechamia
Vol . 17, No . 4
lymphatics just beneath the epicardium were stained immediately and the lymphatics were gradually stained toward the cardiac base along the coronary blood vessels . Thus, the lymphatics were clearly distinguished . The main branch of the left anterior descending coronary artery was carefully isolated for about 0 .3 cm from surrounding connective tissue and the small lymphatic branches running along to the artery . A silk thread was placed loosely round the artery . Acute ligation of the coronary artery led to ventricular fibrillation and caused a sudden death of the animal . Therefore, ligation of the artery was done for a short period and the ligature was released . This procedure was repeated for three times and then, the artery was finally ligated. Fig . 1 illustrates anatomical arrangements of cardiac lymph node and its afferent lymph vessels . Amass ligature was employed around the cardiac lymph node to interrupt the lymph flow into and out from the lymph node . A polyethylene tube, which was previously flashed with heparin solution, was cannulated into one of the afferent lymph vessels . The cardiac lymph was collected in an ice cold plastic test tube containing one drop of heparin solution (1,000 units/ml) for every one hour . Blood was sampled from the cephalic vein . Arterial blood pressure was measured from the left internal thoracic artery with an electronic manometer. The blood pressure, ECG (lead II ) and heart rate were displayed on a multipurpose polygraph-recorder (NIHON KOHDEN, Model RM-45) . Physiological saline solution was infused intravenously throughout the experiment (3 to 4 drops/min) and additional doses of pentobarbital sodium were given to maintain a proper level of anesthesia . ~GrouP~ 2. Sham operated group was exposed to the same surgical procedure except for the coronary ligation as group 1 .
FIG . 1 Schematic representation of the experimental set-up used for collecting the cardiac lymph before and after coronary ligation . Since it was very difficult to cannulate into the cardiac lymph vessels and to obtain a constant out flow of the lymph for eight hours, we succeeded only in 10 out of 17 dogs applied . ~Grou ~3 . Left thoracotamy was perfornied in the fourth intercostal space asept c~and the pericardium was incised for one centimeter . The main branch of the left anterior descending coronary artery was isolated from sur-
Vol. 17, No . 4
Cathepsin D in Myocardial Ischemia
615
rounding connective tissue and ligated by the same way as group 1 . A polyethylene tube was placed in the pericardial space for sampling the pericardial fluid . The incised pericardium was sutured and then the chest was closed . After the operation, 6 x 10 units of penicillin 6 was given intramuscularly each day. Within twenty four hours after the operation, most of the animals recovered from anesthesia . The pericardial fluid was sampled at each time using an injection syringe and transfused into the plastic test tube containing one drop of heparin solution . ~6rou~4 . Same surgical procedure was done as group 3 except for the coronary ligation . All samples of the cardiac lymph, pericardial fluid and venous blood were centrifuged immediately after sampling at 7,000 g for 10 min at 0°C . The supernatant was used for assay of cathepsin D activity and measurement of pro tein concentration . Cathepsin D activity was measured following the method of Anson (6) ; 2 .5 % bovine hemoglobin solution (MILES LABORATORIES, INC.) was digested for 60 min at 37°C and pH 3 .5 . The split product and the protein concentration were measured by the method of Lowry et al (7) using tyrosine and bovine serum albumin for standard respectively . Results l.
Hemod amic and electrocardio ra hic data g. sows erno am c an e ectrocar iographic changes after the cor onary ligation . During the control period, the heart rate was 156 ± 9 and 146 ± 5 beats/min (mean + SE) 1n the coronary ligated and sham operated group respectively . Within seven hours after the ligation, the heart rate showed no significant changes between the two groups . The mean arterial blood pressure decreased slightly immediately after the coronary ligation and returned Thereafter, no significant changes to the control value within few minutes. At seventh hour, howevwere observed between the two groups for five hours . er, the blood pressure decreased significantly (106 ± 5 nmHg, p < 0 .05) in the ligated group, compared with that of the sham operated ones (130 ± 9) . In all cases, cardiogenic shock did not occur throughout the experiment . In ECG (lead II ), the elevation of T wave occurred immediately after the coronary ligation, followed by the depression of S-T segment . 2.
Cathe sin D activit in the cardiac 1 hand the lasma t was â~ cu t to o to n a constant ow o t e car ac lymph. The volume obtained during the control period varied from 1 .1 to 4 .1 ml/hour and the mean value was 2 .4 . After the coronary ligation the volume decreased in some cases, but increased in some others . Thus, no significant change in the volume of the cardiac lymph before and after the coronary ligation was observed . The protein concentration of the cardiac lymph and the plasma were 53 ± 5 and 76 ± 3 mg/ml during the control period and showed no significant change throughout the experiment . Fig . 3 shows the time course of cathepsin D activity in the cardiac lymph and venous plasma after the coronary ligation . Cathepsin 0 activity of the cardiac lymph was 1 .05 t 0 .04 mEq tyrosine x 10 -5 /60 min/mg protein at a control period, started to increase three hours after the ligation, and gradually increased for another several hours . At seventh hour, the cathepsin D activity markedly increased (3 .10 ± 0.44) ; approximately three times of that of the control (p < 0 .01) . On the other hand, in sham operated group there was no change in the activity for seven hours. The venous plasma showed a low value in cathepsin D activity (0 .26 f 0.03) and did not increase within seven hours . 3.
Cathe sin D activit in the ericardial fluid g. sows t e t me course o cat eps n D activity in the pericardial fluid after the coronary ligation . During the cont~ol period the activity were 1 .29 f 0.21 and 1 .10 ± 0.33 mEq tyrosine x 10 - /60 min/0 .2 ml of peri-
Cathepsin D in Myocardial Ischemia
61 6
LlOallon
3 hr
Vol . 17, No . 4
7 hr lo ».
O
u W
O 2
160
E E
C
E é 100
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0. 0
0
0
00
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FIG. 2 Upper tracing; Effects of coronary li ation on arterial blood Lower panel ; Changes pressure, heart rate and ECG (lead II ~ . in mean arterial blood pressure and heart rate before and after coronary ligation and in sham operation. All values are * p < 0 .05; compared with the sham operated group. mean ± SE . cardial fluid, in both groups of coronary ligated and sham operated ones respectively . Three hours after the ligation the activity increased significantly (p < 0.05) and progressively increased over the next several hours . The peak value was attained 24 hours after the ligation . The mean value obtained at 24 hour was approximately four-folds of that of the control (4 .41 ± 1 .Olx p < 0.05) . Thereafter, the activity showed a tendency to decrease at 48 hour . In one out of six cases examined, however, the cathepsin D activity returned to the control value at 24 hour . Discussion It has been generally accepted that the cardiac lymph reflects the changes in the interstitial fluid of the myocardium and large molecules such as enzymes are carried away from the interstitial space via the lymphatics rather than the blood vessels . Recently, Szabó et al (1974) have reported that in experimental myocardial ischemia, cardiac enzymes of creatine phosphokinase, malate dehydrogenase, glutamic oxaloacetic transaminase and lactic dehydrogenase increased in the cardiac lymph in lar er amounts and In the present earlier than those measured in the coronary sinus blood (8~ . experiments, it has been demonstrated that activity of cathepsin D increased gradually in the cardiac lymph after the coronary ligation, while no change
Vol . 17, No . 4
Cathepsin D in Myocardial Iachemia
617
Q W E 0
O
1
2
3
4
Tima (hour )
e
6
7
FIG. 3 Cathepsin D Activity in Cardiac Lymph and Venous Plasma . Abscissa indicates time after coronary liga~ion (hour) . Ordinate, activity of Cathepsin D (mEq tyrosine x 10 - /60 min/mg protein) . (O) Activity in cardiac lymph in coronary ligated group, n = 6; ( " ) in sham operation, n s 4 . (O) Activity in venous plasma in coronary l igated group, n = 6. All values are mean ± SE . +r+t p < 0 .01, p < 0.001 ; compared with the sham operated group.
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4.0 3 .0
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Time
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24
49
FIG . 4 Cathepsin D Activity in the Pericardial Fluid . Abscissa indicates time after coronary ligati n (hour) . Ordinate, activity of Cathepsin D (mEq tyrosine x 10 -~/60 min/0.2 ml of the pericardial fluid) . Ligation of coronary artery (O), .n=6 ; and sham operation ( " ), n = 4. All values are mean f SE . * p < 0 .05, ~ p< 0 .01 ; compared with the sham operated group .
61 8
Cathepein D in Myocardial Ischemia
Vol . 17, No . 4
was observed in the venous plasma activity of cathepsin D . The Increase in the cathepsin D activity in the cardiac lymph indicates the Increase of the lysosomal enzymes liberated into the interstitial fluid of the myocardiun exposed to ischemia . In this experiment, however, the lymph draining the nonischemic area was also collected . Therefore, it is most likely that the ischanic area itself might have higher activity of cathepsin D . The normal pericardial fluid is probably representative of the myocardial interstitial fluid (9) : in hypoxic state, Rubio et al (1969) demonstrated the increase of thé concentration of adenosine in pe~cârdial perfusates (10) . In the present experiments, cathepsin D activity also increased 1n the pericardial fluid after the coronary ligation as observed in the cardiac lymph, indicating an increase in cathepsin D activity in the interstitial fluid of the myocardium exposed to ischemia . At present, it is not clear as to whether this enzymatic increase in the myocardial interstitial fluid originates from the ischemic myocardium itself or infiltrated leukocytes . It is most likely, however, that this originates from the ischemic myocardial cells, since the content of cathepsin D decreases in~the ischemic myocardium five hours after the coronary ligation in the cat (11) and during the first 24 hours of the myocardial infarction, the leukocytec infiltration is slight (12) . Whether the myocardial lysosomal enzymes in the interstitial fluid have some deleterious influences on the course of myocardial infarction is yet to be explored . However, it is possible to speculate that the enzymes may have some depressant effects on cardiac function, since it has been shown that during shock, the pancreatic lysosomal enzymes are activated, which produce a "myocardial depressant factor" (1) . An infusion of splanchnic lysosomal enzymes into the isolated cat heart preparation showed a negative inotropic action and a decrease of coroner flow (13) . Furthernare, some drugs which stabilize lysosamal membrane (14~ or Inhibit proteases (11), have some beneficial effects on the treatment of myocardial infarction . The role of the myocardial lysosomal enzymes in the myocardial infarction must be studied further on . Acknowledgements The authors wish to express to gratitude to Dr . K . Nishi, Department of Pharniacology, Kumamoto University Medical School, for reading the manuscript and comments on it . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
T . M . Glenn and A . M . Lefer, Circ . Res . 27, 783-797 (1970) M . A . Ricciutti, Am . J . CardioT30,~98~02 (1972) . K . G . Ravens, S . ~db~arnason, C .~ . Cowan and R . J . Bing, Circulation 39, 693-700 (1969) J. A . Spath, Jr ., D . L . Lane and A . M . Lefer, Circ . Res . 35, 44-51 (1974) C . C . Griffin, V . S . Waravdekar, B . F . Trump, >r7 . ~ófdbTätt and R . E . Stowell, Am . J . Pathol . 47, 833-850 1965 M . L . Anson, J .~en. ~PFY T. ~6, 565-574 19363 0 . H . Lowry, N. ~Rosebrough,~C. L . Farr and R . J . Randoll, J . Biol . Chem . 193, 265-275 (1951) G . Szabb, Z . Magyar and A . Réffy, L holo 7, 37-44 (1974) F . M . Maurer, M . F . Warren and C . K . r n er, _Äm . _J . P siol . _129, 635-644 (1940) R . Rubio and R . M . Berne, Circ . Res . 25, 407-415 (1969) A . M . Lefer and J . A . Spat íß,3r., Arch .~nt . Pharmacod 211, 225-236 (1974) R . Tennant and D . M . Grayzel, Am . Heart J . 21 , 168-173~(T~6) T . M . Glenn and A . M . Lefer, New As ectsof~ras lol Therapy , 5 ; p 53) F . K . Schattauer Verlag, Stuttgart~orTc P . Libby and P . R . Maroko, J . Clin . Invest . 52, 599-607 (1973)
~s
Pergamon Press
AN INCREASE OF CATHEPSIN D ACTIVITY IN CARDIAC LYMPH AND PERICARDIAL FLUID INDUCED BY EXPERIMENTAL MYOCARDIAL ISCHEMIA IN THE DOG Haruo Araki and Fumio Takenaka Department of Pharmacology, Kumamoto University Medical School, Kumamoto Japan (Received in final form July 18, 1975) Simnary Cathepsin D activity was measured in the cardiac lymph, pericardial fluid and plasma after ligation of the left coronary artery of the dog . The activity of cathepsin D increased both in the cardiac lymph and the pericardial fluid after the coronary 1lgation, while that in the plasma did not show any increase . In sham operated group, there was practically no change in the cathepsin D activity . The increase in the cathepsin D activity in the cardiac lymph and the pericardial fluid may indicate an increase in amount of myocardial lysosomal enzymes liberated into the interstitial space during myocardial ischemia . In recent years, it has been reported that lysosomal enzymes play an undesirable role in the cause of irreversible shock (1), and disruption of cardiac lysosomes within the ischemic myocardium may prromote cardiac damage following myocardial infarction (2) . Same of these enzymes contained in the myocardial cell appear in the serum several days after the onset of myocardial infarction (3) . More recently, Spath et al (1974) have shown that, in experimental myocardial ischemia, a total amount of lysosomal enzymes in the ischemic myocardium decreases, while the ratio of "free" activity rises, and suggested that a marked de ree of lysosomal disruption occurs in the ischemic portion of the myocardium ~4) . However, this "free" activity does not necessarily mean true activity in the intracellular or interstitial fluid, since this may have been resulted from mechanical disruption of the labiled lysosamal membrane in the process of homogenation (5) . Thus, it is necessary to examine whether the myocardial lysosamal enzymes are liberated into the interstitial fluid within the ischemic region of the myocardium at the early stage of myocardial infarction . The present experiments, therefore, were designed in an attempt to quantify the release of the myocardial lysosomal enzymes following acute occlusion of a coronary artery, by measuring activity of a cardiac lysosomal enzyme (cathepsin D) in the cardiac lymph and also in the pericardial fluid. Methods The experiments were carried out in twenty mongrel dogs of either sex weighing 12-21 kg . All dogs were anesthetized with pentobarbital sodium (30 mg/kg, iv) and divided into four groups . ~Grou ~1 . Under artifitial respiration with room air, left thoracotomy was performed in the third intercostal space and the pericardium ryas incised along the anterior interventricular sulcus for about 4 cm . Small amounts of T-1824 dye (0 .1 to 0.2 ml) was injected into the subepicardial portion of the left ventricle with a tuberculin needle . After the injection of dye the 613
61 4
Cathep®in D in Myocardial iechamia
Vol . 17, No . 4
lymphatics just beneath the epicardium were stained immediately and the lymphatics were gradually stained toward the cardiac base along the coronary blood vessels . Thus, the lymphatics were clearly distinguished . The main branch of the left anterior descending coronary artery was carefully isolated for about 0 .3 cm from surrounding connective tissue and the small lymphatic branches running along to the artery . A silk thread was placed loosely round the artery . Acute ligation of the coronary artery led to ventricular fibrillation and caused a sudden death of the animal . Therefore, ligation of the artery was done for a short period and the ligature was released . This procedure was repeated for three times and then, the artery was finally ligated. Fig . 1 illustrates anatomical arrangements of cardiac lymph node and its afferent lymph vessels . Amass ligature was employed around the cardiac lymph node to interrupt the lymph flow into and out from the lymph node . A polyethylene tube, which was previously flashed with heparin solution, was cannulated into one of the afferent lymph vessels . The cardiac lymph was collected in an ice cold plastic test tube containing one drop of heparin solution (1,000 units/ml) for every one hour . Blood was sampled from the cephalic vein . Arterial blood pressure was measured from the left internal thoracic artery with an electronic manometer. The blood pressure, ECG (lead II ) and heart rate were displayed on a multipurpose polygraph-recorder (NIHON KOHDEN, Model RM-45) . Physiological saline solution was infused intravenously throughout the experiment (3 to 4 drops/min) and additional doses of pentobarbital sodium were given to maintain a proper level of anesthesia . ~GrouP~ 2. Sham operated group was exposed to the same surgical procedure except for the coronary ligation as group 1 .
FIG . 1 Schematic representation of the experimental set-up used for collecting the cardiac lymph before and after coronary ligation . Since it was very difficult to cannulate into the cardiac lymph vessels and to obtain a constant out flow of the lymph for eight hours, we succeeded only in 10 out of 17 dogs applied . ~Grou ~3 . Left thoracotamy was perfornied in the fourth intercostal space asept c~and the pericardium was incised for one centimeter . The main branch of the left anterior descending coronary artery was isolated from sur-
Vol. 17, No . 4
Cathepsin D in Myocardial Ischemia
615
rounding connective tissue and ligated by the same way as group 1 . A polyethylene tube was placed in the pericardial space for sampling the pericardial fluid . The incised pericardium was sutured and then the chest was closed . After the operation, 6 x 10 units of penicillin 6 was given intramuscularly each day. Within twenty four hours after the operation, most of the animals recovered from anesthesia . The pericardial fluid was sampled at each time using an injection syringe and transfused into the plastic test tube containing one drop of heparin solution . ~6rou~4 . Same surgical procedure was done as group 3 except for the coronary ligation . All samples of the cardiac lymph, pericardial fluid and venous blood were centrifuged immediately after sampling at 7,000 g for 10 min at 0°C . The supernatant was used for assay of cathepsin D activity and measurement of pro tein concentration . Cathepsin D activity was measured following the method of Anson (6) ; 2 .5 % bovine hemoglobin solution (MILES LABORATORIES, INC.) was digested for 60 min at 37°C and pH 3 .5 . The split product and the protein concentration were measured by the method of Lowry et al (7) using tyrosine and bovine serum albumin for standard respectively . Results l.
Hemod amic and electrocardio ra hic data g. sows erno am c an e ectrocar iographic changes after the cor onary ligation . During the control period, the heart rate was 156 ± 9 and 146 ± 5 beats/min (mean + SE) 1n the coronary ligated and sham operated group respectively . Within seven hours after the ligation, the heart rate showed no significant changes between the two groups . The mean arterial blood pressure decreased slightly immediately after the coronary ligation and returned Thereafter, no significant changes to the control value within few minutes. At seventh hour, howevwere observed between the two groups for five hours . er, the blood pressure decreased significantly (106 ± 5 nmHg, p < 0 .05) in the ligated group, compared with that of the sham operated ones (130 ± 9) . In all cases, cardiogenic shock did not occur throughout the experiment . In ECG (lead II ), the elevation of T wave occurred immediately after the coronary ligation, followed by the depression of S-T segment . 2.
Cathe sin D activit in the cardiac 1 hand the lasma t was â~ cu t to o to n a constant ow o t e car ac lymph. The volume obtained during the control period varied from 1 .1 to 4 .1 ml/hour and the mean value was 2 .4 . After the coronary ligation the volume decreased in some cases, but increased in some others . Thus, no significant change in the volume of the cardiac lymph before and after the coronary ligation was observed . The protein concentration of the cardiac lymph and the plasma were 53 ± 5 and 76 ± 3 mg/ml during the control period and showed no significant change throughout the experiment . Fig . 3 shows the time course of cathepsin D activity in the cardiac lymph and venous plasma after the coronary ligation . Cathepsin 0 activity of the cardiac lymph was 1 .05 t 0 .04 mEq tyrosine x 10 -5 /60 min/mg protein at a control period, started to increase three hours after the ligation, and gradually increased for another several hours . At seventh hour, the cathepsin D activity markedly increased (3 .10 ± 0.44) ; approximately three times of that of the control (p < 0 .01) . On the other hand, in sham operated group there was no change in the activity for seven hours. The venous plasma showed a low value in cathepsin D activity (0 .26 f 0.03) and did not increase within seven hours . 3.
Cathe sin D activit in the ericardial fluid g. sows t e t me course o cat eps n D activity in the pericardial fluid after the coronary ligation . During the cont~ol period the activity were 1 .29 f 0.21 and 1 .10 ± 0.33 mEq tyrosine x 10 - /60 min/0 .2 ml of peri-
Cathepsin D in Myocardial Ischemia
61 6
LlOallon
3 hr
Vol . 17, No . 4
7 hr lo ».
O
u W
O 2
160
E E
C
E é 100
C
0. 0
0
0
00
m 0
"
0
1
7
Tlm~
Ihourl
7
7
-
S
0
FIG. 2 Upper tracing; Effects of coronary li ation on arterial blood Lower panel ; Changes pressure, heart rate and ECG (lead II ~ . in mean arterial blood pressure and heart rate before and after coronary ligation and in sham operation. All values are * p < 0 .05; compared with the sham operated group. mean ± SE . cardial fluid, in both groups of coronary ligated and sham operated ones respectively . Three hours after the ligation the activity increased significantly (p < 0.05) and progressively increased over the next several hours . The peak value was attained 24 hours after the ligation . The mean value obtained at 24 hour was approximately four-folds of that of the control (4 .41 ± 1 .Olx p < 0.05) . Thereafter, the activity showed a tendency to decrease at 48 hour . In one out of six cases examined, however, the cathepsin D activity returned to the control value at 24 hour . Discussion It has been generally accepted that the cardiac lymph reflects the changes in the interstitial fluid of the myocardium and large molecules such as enzymes are carried away from the interstitial space via the lymphatics rather than the blood vessels . Recently, Szabó et al (1974) have reported that in experimental myocardial ischemia, cardiac enzymes of creatine phosphokinase, malate dehydrogenase, glutamic oxaloacetic transaminase and lactic dehydrogenase increased in the cardiac lymph in lar er amounts and In the present earlier than those measured in the coronary sinus blood (8~ . experiments, it has been demonstrated that activity of cathepsin D increased gradually in the cardiac lymph after the coronary ligation, while no change
Vol . 17, No . 4
Cathepsin D in Myocardial Iachemia
617
Q W E 0
O
1
2
3
4
Tima (hour )
e
6
7
FIG. 3 Cathepsin D Activity in Cardiac Lymph and Venous Plasma . Abscissa indicates time after coronary liga~ion (hour) . Ordinate, activity of Cathepsin D (mEq tyrosine x 10 - /60 min/mg protein) . (O) Activity in cardiac lymph in coronary ligated group, n = 6; ( " ) in sham operation, n s 4 . (O) Activity in venous plasma in coronary l igated group, n = 6. All values are mean ± SE . +r+t p < 0 .01, p < 0.001 ; compared with the sham operated group.
É N Ó
s.o
C ~ 'S
b ó
4.0 3 .0
x~
r E
o 1.0 0
3
Time
~
(hour )
24
49
FIG . 4 Cathepsin D Activity in the Pericardial Fluid . Abscissa indicates time after coronary ligati n (hour) . Ordinate, activity of Cathepsin D (mEq tyrosine x 10 -~/60 min/0.2 ml of the pericardial fluid) . Ligation of coronary artery (O), .n=6 ; and sham operation ( " ), n = 4. All values are mean f SE . * p < 0 .05, ~ p< 0 .01 ; compared with the sham operated group .
61 8
Cathepein D in Myocardial Ischemia
Vol . 17, No . 4
was observed in the venous plasma activity of cathepsin D . The Increase in the cathepsin D activity in the cardiac lymph indicates the Increase of the lysosomal enzymes liberated into the interstitial fluid of the myocardiun exposed to ischemia . In this experiment, however, the lymph draining the nonischemic area was also collected . Therefore, it is most likely that the ischanic area itself might have higher activity of cathepsin D . The normal pericardial fluid is probably representative of the myocardial interstitial fluid (9) : in hypoxic state, Rubio et al (1969) demonstrated the increase of thé concentration of adenosine in pe~cârdial perfusates (10) . In the present experiments, cathepsin D activity also increased 1n the pericardial fluid after the coronary ligation as observed in the cardiac lymph, indicating an increase in cathepsin D activity in the interstitial fluid of the myocardium exposed to ischemia . At present, it is not clear as to whether this enzymatic increase in the myocardial interstitial fluid originates from the ischemic myocardium itself or infiltrated leukocytes . It is most likely, however, that this originates from the ischemic myocardial cells, since the content of cathepsin D decreases in~the ischemic myocardium five hours after the coronary ligation in the cat (11) and during the first 24 hours of the myocardial infarction, the leukocytec infiltration is slight (12) . Whether the myocardial lysosomal enzymes in the interstitial fluid have some deleterious influences on the course of myocardial infarction is yet to be explored . However, it is possible to speculate that the enzymes may have some depressant effects on cardiac function, since it has been shown that during shock, the pancreatic lysosomal enzymes are activated, which produce a "myocardial depressant factor" (1) . An infusion of splanchnic lysosomal enzymes into the isolated cat heart preparation showed a negative inotropic action and a decrease of coroner flow (13) . Furthernare, some drugs which stabilize lysosamal membrane (14~ or Inhibit proteases (11), have some beneficial effects on the treatment of myocardial infarction . The role of the myocardial lysosomal enzymes in the myocardial infarction must be studied further on . Acknowledgements The authors wish to express to gratitude to Dr . K . Nishi, Department of Pharniacology, Kumamoto University Medical School, for reading the manuscript and comments on it . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
T . M . Glenn and A . M . Lefer, Circ . Res . 27, 783-797 (1970) M . A . Ricciutti, Am . J . CardioT30,~98~02 (1972) . K . G . Ravens, S . ~db~arnason, C .~ . Cowan and R . J . Bing, Circulation 39, 693-700 (1969) J. A . Spath, Jr ., D . L . Lane and A . M . Lefer, Circ . Res . 35, 44-51 (1974) C . C . Griffin, V . S . Waravdekar, B . F . Trump, >r7 . ~ófdbTätt and R . E . Stowell, Am . J . Pathol . 47, 833-850 1965 M . L . Anson, J .~en. ~PFY T. ~6, 565-574 19363 0 . H . Lowry, N. ~Rosebrough,~C. L . Farr and R . J . Randoll, J . Biol . Chem . 193, 265-275 (1951) G . Szabb, Z . Magyar and A . Réffy, L holo 7, 37-44 (1974) F . M . Maurer, M . F . Warren and C . K . r n er, _Äm . _J . P siol . _129, 635-644 (1940) R . Rubio and R . M . Berne, Circ . Res . 25, 407-415 (1969) A . M . Lefer and J . A . Spat íß,3r., Arch .~nt . Pharmacod 211, 225-236 (1974) R . Tennant and D . M . Grayzel, Am . Heart J . 21 , 168-173~(T~6) T . M . Glenn and A . M . Lefer, New As ectsof~ras lol Therapy , 5 ; p 53) F . K . Schattauer Verlag, Stuttgart~orTc P . Libby and P . R . Maroko, J . Clin . Invest . 52, 599-607 (1973)
~s