oTournalof MolecularandCellularCardiology( 1977) 9, 933-943
Lysosomal Enzyme Release in the Isolated Perfused Heart o f I m m u n i z e d Rats M. FIELDS AND A. LAUFER*
Department of Pathology, The Hebrew University-Hadassah Medical School and Hadassah University Hospital, aTerusalern, Israel (Received 5 April 1976, accepted in revisedform 27 January 1977) M. FIELDSANDA, LAUFER.Lysosomal Enzyme Release in the Isolated Perfused Heart of Immunized Rats. Journal of Molecularand CellularGardiology(1977) 9, 933-943. The present study reports alterations in lysosomal enzymes in the rat myocardium and in the effluent perfusate, following immunization with homologous heart homogenates and Freund's adjuvant, and records the effect ofisoproterenol on enzyme release, employing isolated perfused rat hearts. Pathological findings in the myocardium were found in the isoproterenol treated animals wlth or without prior immunization. A selective enzyme release was observed in all groups of animals. Immunization alone decreased the activity of cathcpsin D in the myocardium but increased its activity in the effluent pcrfusatc. Although myocardial acid phosphatasc activity was increased in all experimental animals, this enzyme was not released into the effluent pcrfusatc. It is suggested that the cause of the increased enzyme release after immunization m a y bc duc to alterations in membrane permeability. Isoprotcrenol alone increased the activity of cathcpsin D and ~-glucuronidase in the myocardial tissue, but only cathcpsin D was found in the effluent pcrfusatc. Some of the findings present in the above groups of animals wcrc also encountered in animals treated with isoprotcrcnol after immunization. N o correlation was found between the increased
enzyme activity encountered in the efi]uent perfusate and the extent of myocardial damage. KEY WorDs: Immunization; Isoproterenol; Lysosomal enzymes; Isolated perfused rat heart; Acid phosphatase; ~-Glucuronidase; Cathepsin D; Enzyme release; Membrane permeability.
1. I n t r o d u c t i o n It has been shown t h a t immunization of rats with heterologous or homologous heart homogenate, reinforced with complete F r e u n d ' s a d j u v a n t (CFA), can cause myocardial necrosis a n d myocardifis. However, in these studies [1/] the titre of specific heart antibodies, measured b y the passive h a e m a g g l u t i n a t i o n technique, did not correlate with the severity of the myocardial damage. Administration o f isoproterenol to animals previously i m m u n i z e d with heterologous or homologous heart h o m o g e n a t e a n d C F A resulted in myocardial d a m a g e of greater m a g n i t u d e [11], and it was suggested that circulating anti-heart antibodies, usually non-toxic in vivo, b e c o m e cytopathic after the myofiber cell barrier * Established investigator of the Chief Scientific Bureau, Ministry of Health, Israel.
934
1K. F I E L D S A N D A. L A U F E R
is broken down by isoproterenol [11]. Furthermore, it seems that alterations in membrane structure, electrolyte shifts or changes in permeability of intracellular organelles, such as lysosomes, may result in or perpetuate myocardial damage [17]. Some clinical and experimental studies have shown that lysosomal enzyme release m a y be used as an indicator of the extent of tissue injury [21, 22]. T h e aims of the present study were (1) to determine the activity of several lysosomal enzymes in the isolated perfused hearts and in the outflow perfusate, in rats immunized with homologous heart homogenate and CFA, and in immunized rats followed by treatment with isoproterenol; (2) to attempt to correlate the degree of myocardial damage with the amount oflysosomal enzymes released.
2. M a t e r i a l s a n d M e t h o d s
Male rats of the Hebrew University strain were divided into four groups of ten animals each.
Group no. 1 This group consisted of untreated rats, weighing 300 g. The animals were anaesthetized with ether, their hearts were immediately removed and perfused for 60 rain in a modified Langendorff apparatus at constant flow (4 ml.min -1) with Krebs bicarbonate solution (pH 7.4) containing 5 m ~ glucose. The solution was gassed with 95% O3 and 5% COs, and the temperature was kept at 37~ The effluent perfusate was collected at 15 min intervals, but the first collection after 15 min was discarded. The perfusion fluid was analyzed for acid phosphatase, according to Andersch and Szczypinski [2], ~-glucuronidase according to Fishman [9], and cathepsin D according to Press et aL [20]. At the end of the perfusion, the hearts were removed, placed in cold saline, blotted dry, and weighed. Portions of myocardium were taken for histological examination. The hearts were then cut into small pieces and homogenized in 10 vol. ice cold 0.25 M sucrose containing 0.002 E D T A Na and 0.02 M Tris (pH 7.4) using the ultraturrax homogenizer. The homogenates were subjected to a three-step fractionation procedure to provide three subcellular fractions as described by Ravens and Gudbjarnason [22]. The activity of acid phosphatase, ~-glucuronidase and cathepsin D was determined on the three subcellular fractions obtained. Protein was determined on each subcellular fraction according to Lowry et al. [18].
Group no. 2 Rats weighing 100 to 150 g were immunized with 5.0 mg of rat heart homogenate reinforced with CFA and dried mycobacteriurn tuberculosis in 0.5 ml of saline once
LYSOSOMAL ENZYME R E L E A S E F O L L O W I N G IMMUNIZATION"
935
a week for 6 weeks [12]. One week after the cessation of the injections the animals were killed and their hearts were perfused as described above. Lysosomal enzymatic activity was determined on the subcellular fractions as well as on the perfusion fluid as described above. At the time of death the rats weighed 300 g.
Group no. 3 Rats weighing 300 g were injected with a single dose of 0.5 ml isoproterenol sulfate in saline (100 mg.kg-1). Twenty-four hours thereafter, the animals were anaesthetized with ether, their hearts were removed immediately, and perfused as described before. T h e biochemical estimations on the perfusion fluid and on the subcellular fractions whieh were prepared from the heart muscle at the end of the perfusion period, were done as described above.
Group no. 4 Rats were immunized as in group no. 2. O n e week after the cessation of the injections, they were injected with a single injection of 0.5 ml isoproterenol sulfate in saline (100 mg.kg-1). Twenty-four hours after the injection of isoproterenol, the animals were killed (weighing 300 g), their hearts were removed and perfused as described before. T h e biochemical procedures were carried out as above. Tissue activities of the same lysosomal enzymes in non-perfused hearts that were removed from another set of animals, four groups of ten rats each (control, isoproterenol, immunized and immunization q- isoproterenol) were assayed. T h e effect of different flow rate on the mechanical activity of the isolated perfused hearts (left ventricular pressure, ECG, heart rate and force), as well as the effect on pH, O~ and CO2 of the effluent perfusate was studied. Stable levels were obtained after 15 rain of perfusion. T h e flow rate of 4 ml.min -1 was selected as the best rate for the stability of the preparation.
Histological examination Portions of myocardium were taken for histological examination from non-perfused and perfused hearts before homogenization.
3. R e s u l t s
T h e perfused hearts remained in good active condition throughout the perfusion procedure, and no physiological and mechanical abnormalities were seen after I h ofperfusion.
936
M. F I E L D S A N D A. L A U F E R
Histologicalfindings Multiple sections stained with haematoxylin and eosin were examined. Only the sections from groups 3 and 4 showed histological d a m a g e (Plates 1 and 2). Twentyfour hours following isoproterenol treatment, foei of myocardial necrosis were found. These areas of necrosis were either single, multiple or confluent. Inflammatory lesions showed a mixture of lymphoid cells, histiocytes and occasional fibroblasts. T h e group of immunized animals followed by a single injection of isoproterenol showed essentially similar histological findings, which were, however, more prominent. The immunized animals did not reveal any histological findings. No histological differences were found between perfused and non-perfused hearts.
Biochemicalfindings T h e non-treated animals served as controls. Enzyme activities in the myocardial tissue were expressed as the amount of product formed per mg protein per h. T h e enzymatic activity of acid phosphatase was expressed as m ~ of p-nitrophenol formed.mg protein -1. h -1. T h e enzymatic activity of ~-glucuronidase was expressed as ~tg glucuronic acid formed.mg protein -1. h -1. T h e activity of cathepsin D was expressed as ~tg tyrosine.mg protein-1, h-1. T h e enzymatic activity from the effluent perfusate was expressed as the amount of product formed.ml-l.g heart wet weight-X, h - l . All results were expressed as means 4- standard deviation (S.D.). T h e statistical significance of differences between means of control and experimental animals was calculated with the Student t-test. H e a r t weight and total protein content are summarized in Table I. Myocardial lysosomal activities of perfused and nonperfused hearts are summarized in Tables 2 (a) and 2 (b). Tissue lysosomal enzymatic activities without perfusion were similar to those of perfused hearts [Table 2(b)]. TABLE I. Heart wet weight and total protein content
Control Immunization Isoproterenol Immunization + isoproterenol
Wet heart weight (g)
Total protein content (mg)
0.8 4- 0.01 0.6 4- 0.02* P < 0.001 1.1 4- 0.01t P < 0.001
86.9 -4- 13.4 72.1 4-4.6* P < 0.01 141.1 4- 22.4t P < 0.001
0.9 4- 0.01++ P < 0.001
80.5 4- 6.7
Results are expressed as mean often observations 4- (S.D.) Tests of significance: * immunization vs control; t isoproterenol vs control; $ immunization -? isoproterenol vs control.
937
LYSOSOMAL E N Z Y M E R E L E A S E F O L L O W I N G I M M U N I Z A T I O N
TABLE 2(a). Lysosomal enzymatic activities in subcellulax fractions of rat myocardium following perfusion Nuclear fraction Acid phosphatase Control Immunization Isoproterenol Immunization + isoproterenol Cathepsin D Control Immunization Isoproterenol Immunization + isoproterenol [3-Glucuronidase Control Immunization Isoproterenol Immunization + isoproterenol
5.9 -4- 1.7 4.7 4- 0.3
Soluble (free) fraction
3.4 4- 0.3 t P < 0.01
8.0 414.4 -t*P < 12.5 4J'P
Lysosomal Enzyme Release in the Isolated Perfused Heart o f I m m u n i z e d Rats M. FIELDS AND A. LAUFER*
Department of Pathology, The Hebrew University-Hadassah Medical School and Hadassah University Hospital, aTerusalern, Israel (Received 5 April 1976, accepted in revisedform 27 January 1977) M. FIELDSANDA, LAUFER.Lysosomal Enzyme Release in the Isolated Perfused Heart of Immunized Rats. Journal of Molecularand CellularGardiology(1977) 9, 933-943. The present study reports alterations in lysosomal enzymes in the rat myocardium and in the effluent perfusate, following immunization with homologous heart homogenates and Freund's adjuvant, and records the effect ofisoproterenol on enzyme release, employing isolated perfused rat hearts. Pathological findings in the myocardium were found in the isoproterenol treated animals wlth or without prior immunization. A selective enzyme release was observed in all groups of animals. Immunization alone decreased the activity of cathcpsin D in the myocardium but increased its activity in the effluent pcrfusatc. Although myocardial acid phosphatasc activity was increased in all experimental animals, this enzyme was not released into the effluent pcrfusatc. It is suggested that the cause of the increased enzyme release after immunization m a y bc duc to alterations in membrane permeability. Isoprotcrenol alone increased the activity of cathcpsin D and ~-glucuronidase in the myocardial tissue, but only cathcpsin D was found in the effluent pcrfusatc. Some of the findings present in the above groups of animals wcrc also encountered in animals treated with isoprotcrcnol after immunization. N o correlation was found between the increased
enzyme activity encountered in the efi]uent perfusate and the extent of myocardial damage. KEY WorDs: Immunization; Isoproterenol; Lysosomal enzymes; Isolated perfused rat heart; Acid phosphatase; ~-Glucuronidase; Cathepsin D; Enzyme release; Membrane permeability.
1. I n t r o d u c t i o n It has been shown t h a t immunization of rats with heterologous or homologous heart homogenate, reinforced with complete F r e u n d ' s a d j u v a n t (CFA), can cause myocardial necrosis a n d myocardifis. However, in these studies [1/] the titre of specific heart antibodies, measured b y the passive h a e m a g g l u t i n a t i o n technique, did not correlate with the severity of the myocardial damage. Administration o f isoproterenol to animals previously i m m u n i z e d with heterologous or homologous heart h o m o g e n a t e a n d C F A resulted in myocardial d a m a g e of greater m a g n i t u d e [11], and it was suggested that circulating anti-heart antibodies, usually non-toxic in vivo, b e c o m e cytopathic after the myofiber cell barrier * Established investigator of the Chief Scientific Bureau, Ministry of Health, Israel.
934
1K. F I E L D S A N D A. L A U F E R
is broken down by isoproterenol [11]. Furthermore, it seems that alterations in membrane structure, electrolyte shifts or changes in permeability of intracellular organelles, such as lysosomes, may result in or perpetuate myocardial damage [17]. Some clinical and experimental studies have shown that lysosomal enzyme release m a y be used as an indicator of the extent of tissue injury [21, 22]. T h e aims of the present study were (1) to determine the activity of several lysosomal enzymes in the isolated perfused hearts and in the outflow perfusate, in rats immunized with homologous heart homogenate and CFA, and in immunized rats followed by treatment with isoproterenol; (2) to attempt to correlate the degree of myocardial damage with the amount oflysosomal enzymes released.
2. M a t e r i a l s a n d M e t h o d s
Male rats of the Hebrew University strain were divided into four groups of ten animals each.
Group no. 1 This group consisted of untreated rats, weighing 300 g. The animals were anaesthetized with ether, their hearts were immediately removed and perfused for 60 rain in a modified Langendorff apparatus at constant flow (4 ml.min -1) with Krebs bicarbonate solution (pH 7.4) containing 5 m ~ glucose. The solution was gassed with 95% O3 and 5% COs, and the temperature was kept at 37~ The effluent perfusate was collected at 15 min intervals, but the first collection after 15 min was discarded. The perfusion fluid was analyzed for acid phosphatase, according to Andersch and Szczypinski [2], ~-glucuronidase according to Fishman [9], and cathepsin D according to Press et aL [20]. At the end of the perfusion, the hearts were removed, placed in cold saline, blotted dry, and weighed. Portions of myocardium were taken for histological examination. The hearts were then cut into small pieces and homogenized in 10 vol. ice cold 0.25 M sucrose containing 0.002 E D T A Na and 0.02 M Tris (pH 7.4) using the ultraturrax homogenizer. The homogenates were subjected to a three-step fractionation procedure to provide three subcellular fractions as described by Ravens and Gudbjarnason [22]. The activity of acid phosphatase, ~-glucuronidase and cathepsin D was determined on the three subcellular fractions obtained. Protein was determined on each subcellular fraction according to Lowry et al. [18].
Group no. 2 Rats weighing 100 to 150 g were immunized with 5.0 mg of rat heart homogenate reinforced with CFA and dried mycobacteriurn tuberculosis in 0.5 ml of saline once
LYSOSOMAL ENZYME R E L E A S E F O L L O W I N G IMMUNIZATION"
935
a week for 6 weeks [12]. One week after the cessation of the injections the animals were killed and their hearts were perfused as described above. Lysosomal enzymatic activity was determined on the subcellular fractions as well as on the perfusion fluid as described above. At the time of death the rats weighed 300 g.
Group no. 3 Rats weighing 300 g were injected with a single dose of 0.5 ml isoproterenol sulfate in saline (100 mg.kg-1). Twenty-four hours thereafter, the animals were anaesthetized with ether, their hearts were removed immediately, and perfused as described before. T h e biochemical estimations on the perfusion fluid and on the subcellular fractions whieh were prepared from the heart muscle at the end of the perfusion period, were done as described above.
Group no. 4 Rats were immunized as in group no. 2. O n e week after the cessation of the injections, they were injected with a single injection of 0.5 ml isoproterenol sulfate in saline (100 mg.kg-1). Twenty-four hours after the injection of isoproterenol, the animals were killed (weighing 300 g), their hearts were removed and perfused as described before. T h e biochemical procedures were carried out as above. Tissue activities of the same lysosomal enzymes in non-perfused hearts that were removed from another set of animals, four groups of ten rats each (control, isoproterenol, immunized and immunization q- isoproterenol) were assayed. T h e effect of different flow rate on the mechanical activity of the isolated perfused hearts (left ventricular pressure, ECG, heart rate and force), as well as the effect on pH, O~ and CO2 of the effluent perfusate was studied. Stable levels were obtained after 15 rain of perfusion. T h e flow rate of 4 ml.min -1 was selected as the best rate for the stability of the preparation.
Histological examination Portions of myocardium were taken for histological examination from non-perfused and perfused hearts before homogenization.
3. R e s u l t s
T h e perfused hearts remained in good active condition throughout the perfusion procedure, and no physiological and mechanical abnormalities were seen after I h ofperfusion.
936
M. F I E L D S A N D A. L A U F E R
Histologicalfindings Multiple sections stained with haematoxylin and eosin were examined. Only the sections from groups 3 and 4 showed histological d a m a g e (Plates 1 and 2). Twentyfour hours following isoproterenol treatment, foei of myocardial necrosis were found. These areas of necrosis were either single, multiple or confluent. Inflammatory lesions showed a mixture of lymphoid cells, histiocytes and occasional fibroblasts. T h e group of immunized animals followed by a single injection of isoproterenol showed essentially similar histological findings, which were, however, more prominent. The immunized animals did not reveal any histological findings. No histological differences were found between perfused and non-perfused hearts.
Biochemicalfindings T h e non-treated animals served as controls. Enzyme activities in the myocardial tissue were expressed as the amount of product formed per mg protein per h. T h e enzymatic activity of acid phosphatase was expressed as m ~ of p-nitrophenol formed.mg protein -1. h -1. T h e enzymatic activity of ~-glucuronidase was expressed as ~tg glucuronic acid formed.mg protein -1. h -1. T h e activity of cathepsin D was expressed as ~tg tyrosine.mg protein-1, h-1. T h e enzymatic activity from the effluent perfusate was expressed as the amount of product formed.ml-l.g heart wet weight-X, h - l . All results were expressed as means 4- standard deviation (S.D.). T h e statistical significance of differences between means of control and experimental animals was calculated with the Student t-test. H e a r t weight and total protein content are summarized in Table I. Myocardial lysosomal activities of perfused and nonperfused hearts are summarized in Tables 2 (a) and 2 (b). Tissue lysosomal enzymatic activities without perfusion were similar to those of perfused hearts [Table 2(b)]. TABLE I. Heart wet weight and total protein content
Control Immunization Isoproterenol Immunization + isoproterenol
Wet heart weight (g)
Total protein content (mg)
0.8 4- 0.01 0.6 4- 0.02* P < 0.001 1.1 4- 0.01t P < 0.001
86.9 -4- 13.4 72.1 4-4.6* P < 0.01 141.1 4- 22.4t P < 0.001
0.9 4- 0.01++ P < 0.001
80.5 4- 6.7
Results are expressed as mean often observations 4- (S.D.) Tests of significance: * immunization vs control; t isoproterenol vs control; $ immunization -? isoproterenol vs control.
937
LYSOSOMAL E N Z Y M E R E L E A S E F O L L O W I N G I M M U N I Z A T I O N
TABLE 2(a). Lysosomal enzymatic activities in subcellulax fractions of rat myocardium following perfusion Nuclear fraction Acid phosphatase Control Immunization Isoproterenol Immunization + isoproterenol Cathepsin D Control Immunization Isoproterenol Immunization + isoproterenol [3-Glucuronidase Control Immunization Isoproterenol Immunization + isoproterenol
5.9 -4- 1.7 4.7 4- 0.3
Soluble (free) fraction
3.4 4- 0.3 t P < 0.01
8.0 414.4 -t*P < 12.5 4J'P
