Changes in the Activities of Lysosomal Enzymes in Infarcted Canine Heart Muscle

Transcription

1 Changes in the Activities of Lysosomal Enzymes in Infarcted Canine Heart Muscle By Kurt G. Ravens, M.D., and S. Gudbjarnason, Ph.D. ABSTRACT Experimental myocardial infarction was produced in 32 mongrel dogs. The changes in activity of four lysosomal enzymes (acid phosphatase, glucuronidase, deoxyribonuclease, and y-glutamyl-transpeptidase) were examined in the soluble and the particle-bound fraction. The pattern of changes in free and particle-bound enzyme activity observed was similar for all four enzymes. During the first 48 hours after coronary occlusion, the particle-bound enzyme activity was decreased, while the free activity was moderately increased, reflecting the autolytic phase of cell and tissue destruction. Between the second and the sixth day, the soluble hydrolytic enzyme activity was maximal and the particle-bound activity was slowly increasing. During this period, the main part of tissue degradation and removal of cell debris takes place. Ten days after myocardial infarction the free hydrolytic activity had returned to control values, but the particle-bound enzyme activity was four to ten times higher in the infarcted tissue than in the control muscle. ADDITIONAL KEY WORDS tissue repair acid phosphatase y3-glucuronidase deoxyribonuclease y-glutamyl-transpeptidase Changes in energy metabolism of infarcted and ischemic myocardium and in the relative importance of various metabolic pathways in the injured heart muscle have been studied intensively in the dog (1-5). These studies have demonstrated alterations in enzyme profile and metabolite levels associated with the replacement of necrotic muscle by developing connective tissue (6-8). Further studies have illustrated changes in protein and nucleic acid synthesis in infarcted tissue (9), reflecting the anabolic phase of tissue repair. The purpose of this report is to describe changes in the activities of hydrolytic enzymes From the Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan This work was supported in part by U. S. Public Health Service Grant HE from the National Heart Institute, the American Medical Association Education and Research Foundation, the Michigan Heart Association and Detroit General Hospital Research Corporation. Dr. Gudbjarnason is recipient of U. S. Public Health Service Career Development Award No. 5-KO3-HE Received February 4, Accepted for publication April 4, which are of importance in degradation and removal of necrotic myocardial tissue. This catabolic phase of tissue repair has attracted relatively little attention, but the removal of necrotic cells and cell debris is of major importance in the proliferation of fibroblasts and stimulation of wound healing. Lysosomal enzymes play an important and well-established role in the breakdown of necrotic cells (, 11). These enzymes are mainly acid hydrolases stored in subcellular organelles (lysosomes). In this study we have examined the changes in free and particle-bound activity of certain lysosomal enzymes in normal and necrotic heart muscle after experimental myocardial infarction. Material and Methods Thirty-two adult mongrel dogs were used in these experiments. Myocardial infarction was produced surgically by ligation of several branches of the circumflex and descending coronary arteries as described previously (9). The dogs were anesthetized and killed 1, 2, 4, 6, 8, and days after the operation. The heart was removed and immediately placed on crushed ice, Circulation Reiecrch, Vol. XXIV, June

2 852 RAVENS, GUDBJARNASON and the coronary arteries were perfused for a short period with buffered 0.25M sucrose solution (ph 7.4) to remove residual blood. A sample of necrotic tissue from the center of the infarcted area of the left ventricle and a sample of muscle from the nonischemic septum were weighed (5 to 8 g), minced and homogenized in a Waring blender (for 45 seconds) in a 0.25M sucrose solution buffered with 0.1M Tris buffer, ph 7.4 (1 g tissue/ ml). The homogenate was centrifuged at l,000g in order to remove cell debris and myofibrils. The supernatant fluid was centrifuged at 20,000g for 15 minutes. The supernatant fluid was called the S-fraction, and the activity of the free or soluble enzyme was determined in this fraction. The precipitate of this centrifugation was carefully resuspended in homogenizing solution by means of a Teflon pestle. This fraction was called the P-fraction and contained the particle-bound enzyme. This fraction was thawed and frozen eight to ten times in order to release the enzymes from the lysosomes. The homogenate was prepared at 4 C, and centrifugation was carried out in a Spinco- Beckman Ultracentrifuge L2. The enzyme activities were assayed on the day of preparation. ENZYME ASSAYS Acid phosphatase activity was measured with p-nitrophenol phosphate according to a procedure described by Shibko and Tappel (12). The activity of /3-glucuronidase was estimated by an adaption of the methods of Gianetto and DeDuve (13) with phenolphthalein glucuronic acid as substrate. Deoxyribonuclease activity was measured with a purified DNA as substrate according to a method described by DeDuve et al. (14). The assay of -y-glutamyl-transpeptidase was done by a modification of the procedure described by Orlowski and Szewczuk (15), using y- glutamyl-naphthylamine as substrate. The substrates used in these assays were obtained from Sigma Chemical Company. TABLE 1 The enzyme activities were assayed spectrophotometrically according to the methods mentioned above. The assays were examined with respect to linear kinetics and optimal substrate condition for dog heart muscle (free and particlebound fraction). The enzyme reactions were carried out in a water bath at 37 C for appropriate time intervals. The protein content of the different fractions was determined using the Biuret method with bovine albumin as reference standard. The average enzyme activities in the noninfarcted septum of all experimental dogs served as a control. The enzyme activities (free and particle-bound) in the infarcted tissue at the different time intervals were expressed as percent of the control. Results Table 1 shows the average enzyme activities of the lysosomal enzymes determined in the homogenate of cardiac septal muscle. The specific activity of acid phosphatase and deoxyribonuclease (DNase) is nearly as high in the S-fraction as in the P-fraction, whereas the specific activity of acid glucuronidase and y-glutamyl-transpeptidase is twice as high in the P-fraction compared to the S-fraction, illustrating a higher storage capacity of the subcellular organelles for these enzymes. Comparison between the control values obtained from the septum and the enzyme activities in the homogenate of normal left ventricular muscle showed no significant difference. ACID PHOSPHATASE Figure 1 demonstrates the changes in free and particle-bound activity of acid phosphatase in the necrotic left ventricular wall Free and Particle-Bound Enzymatic Activities in the Nonischemic Muscle of the Septum Enzyme Acid phosphatase Deoxyribonuclease Glucuronidase 7-Glutamyl-transpeptidase No. of experiments S-fraction 1.40 ± 0.05* 0.34 ± 0.04f 0.25 ± 0.017* 0.3 ± 0.05* Fraction P-fraction 1.48 ± 0.5* 0.35 ± 0.04f ± 0.059* 0.63 ± 0.02* Means ± SE are given. * The enzymatic activity is expressed as m/imoles of product formed per minute and per mg protein, t The enzymatic activity is expressed as an increase in optical density (unit = 0.0 OD) per hour and per mg protein.

3 LYSOSOMAL ENZYMES IN MYOCARDIAL INFARCTION O300 O ac FIGURE 1 Relative changes in activity of acid phosphatase in infarcted myocardial tissue at various time intervals after surgical coronary artery occlusion compared to the control tissue (septum). Means ± SE of five experiments are given. Circles free activity (S-fraction). Triangles = particle-bound activity (P-fraction). after surgical myocardial infarction. There is a slow but continuous rise in the activity of the free, soluble enzyme, reaching a maximum after 6 days (165% above control). The free activity then declines slowly and approaches control values after days. The changes in particle-bound enzyme activity show quite a different pattern. Twenty-four hours after coronary occlusion there is a significant decrease in specific activity of the P-fraction to 51% of the control. After 2 days the enzyme activity in the P-fraction has returned to the control values, but from the second to the sixth day the enzyme activity in the P-fraction increases again at a relatively slow rate. After days the specific activity of acid phos- phatase in the P-fraction of infarcted tissue is 338% higher than in the control muscle (septum). DEOXYRIBONUCLEASE Figure 2 demonstrates a similar pattern for the free and particle-bound activity of DNase. The free activity increases gradually, reaching maximum levels after 4 days (480% of control); thereafter the activity declines slowly. At days the activity of the free enzyme is still significantly higher in infarcted tissue compared to control. The particle-bound activity is lowest after 24 hours, with only 28% of control. After 48 hours this enzyme fraction still has a significantly lower specific activity than the control tissue; from the second to the FIGURE 2 Relative changes in the activity of deoxyribonuclease in infarcted myocardial tissue at various time intervals after surgical coronary artery occlusion compared to the control tissue (septum). Means ± SE of five experiments are given. Circles = free activity (S-fraction). Triangles = particle-bound activity (P-fraction).

4 854 RAVENS, GUDBJARNASON 400 looor 300 I g o 20 O 500 O U 0 50 I I I I I FIGURE 3 Relative changes in the activity of p-glucuronidase in infarcted myocardial tissue at various time intervals after surgical coronary artery occlusion compared to the control tissue (septum). Means ± SE of five experiments are given. Circles = free activity (S-fraction). Triangles = particle-bound activity (P-fraction). fourth day there is a rapid increase in enzyme activity of the P-fraction. Between the fourth and the sixth day the enzyme activity remains unchanged; thereafter the activity of DNase increases rapidly in the P-fraction, and 8 and days after myocardial infarction the values are to 12 times higher than the control values. 0-GLUCURONIDASE The changes in the activity of free and particle-bound /3-glucuronidase are shown in Figure 3. The pattern of changes in enzyme activity in the S- and P-fraction are similar to those described above. During the first 48 hours, there is a rapid increase in free enzyme activity which coincides with a decrease in particle-bound activity. Between the second and sixth day the free activity remains 0 0 _L I I FIGURE 4 Relative changes for the activity of y-glutamyl transpeptidase in infarcted myocardial tissue at various time intervals after surgical coronary artery occlusion compared to the control tissue (septum). Means ± SE of five experiments are given. Circles = free activity (S-fraction). Triangles = particle-bound activity (P-fraction). relatively constant and then declines gradually. The activity in the P-fraction increases slowly between the second and sixth day, but subsequently the particle-bound activity increases more rapidly and parallels the decrease in free activity. 7-GLUTAMYL-TRANSPEPTIDASE Figure 4 illustrates the changes in free and particle-bound y-glutamyl-transpeptidase activity. Following an immediate increase in soluble enzyme activity during the first 2 days, the free activity declines slowly, approaching control values after 8 days. The changes in particle-bound activity of y-glutamyl-trans-

5 LYSOSOMAL ENZYMES IN MYOCARDIAL INFARCTION 855 peptidase are similar to those described above for the other enzymes. After 24 hours there is a decrease in particle-bound activity to 53% of control values, followed by a rapid rise in activity. After 2 days, the activity of particlebound y-glutamyl-transpeptidase is fourfold higher in the infarcted area than in the control tissue. Two to six days after myocardial infarction, the particle-bound activity increases at a relatively slow rate followed by a rapid rise in activity after 6 days. Ten days after coronary artery occlusion, the activity of y-glutamyl-transpeptidase in the P-fraction has increased tenfold compared to the control value. Discussion These results demonstrate that in the homogenate of necrotic heart muscle the pattern of changes in the activity of free (soluble) as well as particle-bound enzyme is similar for all four enzymes. These observations support the view that the four enzymes examined in this study may belong to the same subcellular particles, the lysosomes. DeDuve et al. (16) have demonstrated that autolysis and tissue degradation are connected with changes in the distribution of lysosomal enzymes. In myocardial infarction, the ischemia and the subsequent necrosis render the membranes of lysosomes more permeable, leading to an escape of acid hydrolases into the cell sap (cytoplasma). The acid hydrolases digest (hydrolyze) susceptible substrates within the cell, such as proteins, nucleic acid, and polysaccharides. This release of endogenous lysosomal enzymes is only a temporary and limited pathway by which means the physiologic and pathologic cell destruction takes place. It is known from histological studies that inflamed and necrotic tissue is promptly invaded by polymorphonuclear leucocytes and other phagocytic mesenchymal cells (17). Cohn and Hirsch (18) have shown that these cells are very rich in lysosomes. The invasion of mesenchymal cell into necrotic tissue characterizes the second phase of cell and tissue degradation. These processes are called heterolytic, and the tissue destruction is chiefly mediated by an influx of phagocytic cells. Our studies on tissue homogenates cannot distinguish between autolytic and heterolytic processes, but the sequential studies reported here do reflect the dynamics of the degradation of necrotic heart muscle. During the first 24 to 48 hours after coronary occlusion, there is an inverse relationship between free and particle-bound enzyme activity (Figs. 1 to 4). These findings indicate the release of hydrolytic enzymes from endogenous lysosomes originating in heart muscle cells and local connective tissue cells. Studies by Leighty et al. (19) and by Ricciutti et al. (20) also suggest that an autolytic phase of cell destruction takes place in heart muscle during the early stage of myocardial necrosis. The second phase of tissue degradation, presumably chiefly heterolytic, can be divided into two periods. During the first period, that is from the second to the sixth day after myocardial infarction, there is a continuous rise of free enzyme activities, reaching a maximum in this time interval. The particlebound enzyme activities increase also, but at a different rate. During this period, when the highest activities of soluble hydrolytic enzymes are available, the major task of tissue degradation and removal of cell debris takes place. Histological and histochemical studies have illustrated a similar time relation of tissue destruction after myocardial infarction (1,21). Our results also indicate an increasing stability of the lysosomal membranes, resulting in an increase in particle-bound enzyme paralleling a diminution in catabolic activity. In a later stage of the second phase, the soluble hydrolytic enzyme activity returns to control levels, signaling the end of the catabolic phase. The high levels of particlebound enzyme activity observed in the infarcted tissue are no longer associated with myocardial cells, but belong to another cell population containing far more lysosomes. The lysosomal enzymes released into the necrotic tissue are also transported by lymph and blood vessels into the circulatory system.

6 856 RAVENS, GUDBJARNASON Significant changes in the serum activity of lysosomal enzymes in patients with myocardial infarction have been observed (22). Because of the relatively late increase in serum activity of these enzymes following coronary occlusion, the determination of the activity of these enzymes may be of some interest in the follow-up of myocardial infarction in patients, at a time when the activity of other enzymes has returned to normal levels. References 1. BING, R. J., CASTEIXANOS, A., GRADEL, E., LUPTON, C, AND SIECEL, A.: Experimental myocardial infarction: circulatory, biochemical and pathologic changes. Am. J. Med. Sci. 232: 533, SHNITKA, T. K., AND NACHLAS, M. M.: Histochemical alterations in ischemic heart and early myocardial infarction. Am. J. Pathol. 27: 507, BRAASCH, W., GUDBJABNASON, S., PURI, P. S., RAVENS, K. G., AND BING, R. J.: Early changes in energy metabolism in the myocardium following acute coronary artery occlusion in anesthetized dogs. Circulation Res. 28: 429, WOLLENBERCER, A., AND KRAUSE, E. G.: Metabolic control characteristics of the acutely ischemic myocardium. Am. J. Cardiol. 22: 349, FLECKENSTEIN, A.: Herzstoffwechsel bei Koronarverschluss und Herzstillstand. In: Herzinsuffizienz, Haemodynamik und Stoffwechsel, edited by E. Wollheim and K. W. Schneider. Stuttgart, Thieme, 1964, p GUDBJARNASON, S., COWAN, C., BRAASCH, W., AND BINC, R. J.: Changes in enzyme pattern of infarcted heart muscle during tissue repair. Cardiologia 51: 148, GUDBJARNASON, S., FENTON, J., WOLF, P. L., AND BING, R. J.: Stimulation of reparative processes following experimental myocardial infarction. Arch. Internal Med. 118: 33, GUDBJARNASON, S., BRAASCH, W., COWAN, C, AND BING, R. J.: Metabolism of infarcted heart muscle during tissue repair. Am. J. Cardiol. 22: 360, GUDBJARNASON, S., DESCHBYVER, F., CHIBA, C., YAMANAKA, F., AND BING, R. J.: Protein and nucleic acid synthesis during the reparative process following myocardial infarction. Circulation Res. 15: 328, DEDUVE, C: The lysosome concept. In: Lysosomes, Ciba Foundation Symposium, edited by A. V. S. de Rendt. Boston, Little, Brown and Company, 1963, p WEISSMANN, G.: Lysosomes. New Engl. J. Med. 273: 84, SHIBKO, S., AND TAPPEL, A. L.: Acid phosphatase of lysosomal and soluble fraction of rat liver. Biochem. Biophys. Acta 73: 76, GIANETTO, R., AND DEDUVE, C: Tissue fractionation studies: 4. Comparative study of the binding of acid phosphatase, /3-glucuronidase and cathepsin by rat-liver particles. Biochem. J. 59: 433, DEDUVE, C, PRESSMANN, B. C, GIANETTO, R., WATTIAUX, R., AND APPELMANS, F.: Tissue fractionation studies: 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem. J. 60: 604, ORLOWSKI, M., AND SZEWCZUK, A.: Determination of 7-glutamyl transpeptidase activity in human serum and urine. Clin. Chem. Acta 7: 755, DEDUVE, C: Lysosomes and cell injury. In: International Symposium on Injury, Inflammation and Immunity, England 1962, edited by L. Thomas, F. W. Uhr, and L. Grant. Baltimore, Williams and Wilkins, 1962, p JANOFF, A., AND ZWERFACH, B. W.: Production of inflammatory changes in microcirculation by cationic proteins extracted from lysosomes. J. Exptl. Med. 120: 747, COHN, Z. A., AND HIRSCH, J. G.: Influence of phagocytosis on the intracellular distribution of granule-associated components of polymorphonuclear leucocytes. J. Exptl. Med. 112: 15, LEICHTY, E. G., STONER, C. D., RESSALLAT, M. M., TASANANTI, G. T., AND SIHAK, H. D.: Effects of acute asphyxia and deep hypothermia on the state of binding of lysosomal acid hydrolases in canine cardiac muscle. Circulation Res. 21: 59, RICCIUTTI, M., SCHERLAG, B., STEIN, E., AND DAMATO, A.: Lysosome stability and coronary artery occlusion. Clin. Res. 26: 245, MALLORY, G. K., WHITE, P. D., AND SALCEDO SALGAR, J.: Speed of healing of myocardial infarction. Am. Heart J. 18: 647, RAVENS, K. G., GUDBJARNASON, S., COWAN, C, AND BING, R. J.: 7-glutamyl transpeptidase in myocardial infarction. Circulation, in press.