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Lysosomes and myocardial cellular injury
Lysosomes and Myocardial Cellular Injury
MICHAEL Staten
A. RICCIUTTI.
Island,
New
The stability of myocardial lysosomes in left posterior papillary muscle sites of predictable injury was investigated after 1, 2 and 4 hours of acute partial ligation of the left circumflex coronary artery of the dog heart. Lysosome stability in the papillary muscle locus of injury was significantly reduced within 1 tiour and continued to decrease as the duration of the experimental period increased. The intracellular mechanism responsible for the alteration in lysosome stability may be related to a reduction in pH of the tissue. pH of sham-operated papillary muscle was approximately 6.9, that of the 1 hour and 4 hour groups was 6.4 and 6.3, respectively. Alteration in lysosome stability is considered one of the earliest subcellular structural changes occurring during the ischemic cellular injury process in heart muscle. The extent of the lysosome involvement and the rela tively small degree of necrosis present in the papillary muscle sites by 60 minutes, when coupled with the well known hydrolytic capacity of lysosome acid hydrolysis, suggests that the intracellular release of lysosomal enzymes precedes cellular death and may initiate the cellular injury process.
PhD*
York
From the Cardiopulmonary Laboratory, U. S. Public Health Service Hospital, Staten Island. N.Y. This study was supported in part by the Federal Health Program Service, U. S. Public Health Service Project PY 72-1, and National Institutes of Health Projects HE 11829 and HE 12536. Manuscript received December 15. 1971, revised manuscript received April 11. 1972, accepted May 24, 1972. *Presented at the American College of Cardiology Meetings, Washington, D.C. February 3, 1971 as part of the manuscript honored with the Young Investigators Award for the year 1971. Address for reprints: Michael A. Ricciutti. PhD, Cardiopulmonary Laboratory, U. S. Public Health Service Hospital, Staten Island. N.Y. 10304.
490
October 1972
The American
Recent evidence has indicated that during hypoxic or ischemic conditions resulting cellular injury may be initiated through the subcellular release of hydrolytic enzymes contained within the lysosome. l-4 If lysosomes are involved in the initiation of cellular injury, alterations in lysosome stability may be expected in the early stages or prenecrotic periods of the injury process.4-10 A series of studies was designed to determine the onset of lysosome involvement in myocardial cells of the dog heart subjected to various periods of arterial hypoperfusion. 11-1s The extent of lysosome involvement and the degree of cellular injury present were simultaneously approximated in the left posterior papillary muscle, previously shown to be a predictable and relatively uniform locus of injury after acute ligation of the left circumflex coronary artery.l’-19 Methods Randomly pentobarbital
selected male and female dogs were anesthetized with sodium (30 mg/kg body weight), and respiration was maintained at a
constant rate by a Harvard respirometer. A left thoracotomy was performed, and a longitudinal incision elrposing the left atrium and ventricle was made in the pericardium. A 5 mm segment along the length of the left circumflex coronary artery was cleared of adhering fat tissue. A 3-O silk ligature was then loosely positioned around the artery. This site of ligation was always located distal to the first large ventricular branch of the circumflex artery, and at the midline of the base of the left atrium. Sham-operated dogs were surgically prepared as described and maintained in the operative state for 4 hours. The heart was then excised while still beating. In experimental dogs, the lumen of the left circumflex coronary artery was reduced approximately 60 percent according to the procedure of Lumb and Cook.20 Separate ligation experiments were allowed to
Journal of CARDIOLOGY
Volume 30
LYSOSOMES
TABLE
AND MYOCARDIAL
INJURY-RICCIUTTI
I
Subcellular
Distribution
of Acid Phosphatase
Experimental Groups Sham-operated dogs (20) Partial ligation 1 Hour series (10)
in Left Posterior
Papillary
Muscle Tissues*
Sedimentable Activity
Total Activity
Nonsedimentable Activity
Nonsedimentable/ Sedimentable
25.09 f
1.97
19.88 + 1.60
5.15 f
0.76
0.259
24.57 f
2.09
21.36 zt 1.85
3.19 f 0.66 P zto.001 2.03 f 0.54 P
0.148
2 Hour series (8)
22.35 f 1.61 P
20.26 f
1.64
4 Hour series (8)
20.02zt 1.75 P
18.77311.41
0.100 0.056
* Values are the means f the standard deviations and represent the amount of p-nitrophenol liberated per hour per gram of wet weight tissue. Figures in parentheses represent the numbers of experimental animals. P represents the probability and is based on Student’s t test of the data from the grouped control and sham-operated dogs compared to the individual series of partial ligation experiments.
continue for 60, 120 and 240 minutes, respectively. At the end of each time period the heart was excised while still beating. Since previous studies have shown that ligation of the left circumflex coronary artery at the locus described results in the production of uniform and predictable ischemit injury to the left posterior papillary muscle, this papillary muscle was sel.ected as an easily identifiable tissue sampling site in successive experiments.17-ls
The hydrolytic enzyme acid phosphatase was used as the well known marker of lysosome stability. The methods for studying subcellular distribution of acid phosphatase and the kinetics of acid phosphatase release are described in the preceding paper.zl Glycogen, potassium and pH: The glycogen and potassium contents of the left posterior papillary muscles were determined by the same operative and experimental procedures as described.21 The glycogen content of 100 mg samples was isolated by hot alkali digestion and centrifugation. The glycogen precipitate was hydrolyzed to glucose and determined calorimetrically as described by Seifter and Myrthoyler.21 All analyses were performed in duplicate and repeated jf the difference between any 2 values was greater than 5 percent.
Potassium ion content was determined according to the technique of Jennings et al.ls with omission of the fat extraction procedure. The use of a constant tissue sampling site and the extensive procedures for minimizing ionic contamination during analysis served to reduce the biological variability and experimental error normally associated with ionic analyses. The pH of sham-operated and experimental left posterior papillary muscles w(as approximated according to the method of Bittar. Tis:eue samples, 800 to 1000 mg, were rapidly minced in 2 ml of isotonic saline solution for 2 minutes. The mince was allowed to equilibrate at 4 C for 15 minutes, and the pH: of the suspension was then determined with a Beckman Expandomatic pH meter. The pH meter was standardizedi at 4 C with phosphate buffers of pH 6 and 7.
Results A comparison of the activity of acid phosphatase in left posterior papillary muscle subcellular tissue fractions from the sham-operated dogs and from
those that underwent coronary arterial ligation is presented in Table I. The total activity of acid phosphate in whole homogenate (600 X g) subcellular fractions decreased significantly (P >O.Ol) within 2 hours of coronary arterial ligation and was further reduced by the fourth hour. Sedimentable activity of the lysosome-containing subcellular fraction (20,000 X g) decreased significantly (P CO.001) within 1 . hour of coronary arterial ligation and was further reduced as the duration of in vivo ischemia increased. Sedimentable to nonsedimentable ratios from the hearts that underwent coronary arterial ligation were lower than those of the sham-operated hearts. Acid phosphatase was released from the left posterior papillary muscle tissue slices at a faster rate from the ischemic muscles than from the sham-operated group (Table II). The release of the enzyme from the sham-operated group was greatest during the first hour of in vitro incubation. The rate of release continued more slowly over the next 2 hours, with little difference between the second and third hour of incubation. A rapid release was observed from the papillary muscles of the series with 1 hour of coronary arterial ligation during the first and third hours of incubation. By the third hour of incubation, the acid phosphatase activity in the media was almost twice the 1 hour incubation level. After the 2 hour period of in vivo ischemia incubated ischemic papillary muscles released their content of acid phosphatase most rapidly during the first hour of incubation. Release of the enzyme during the second and third hour of incubation was slower. The 4 hour period of in vivo ischemia resulted in a rapid release of the enzyme during the first and second hours of in vitro incubation with a slower rate of release during the third hour. When compared to the pH values from sham-operated hearts, sodium chloride suspensions of ischemic left posterior papillary muscles showed significant reductions in the pH values of similarly prepared suspensions in the group that underwent partial liga-
October 1972
The American Journal of CARDIOLOGY
Volume 30
499
LYSOSOMES
TABLE
AND MYOCARDIAL
INJURY-RICCIUTTI
II
In Vitro Release of Acid Phosphatasefrom
Left Posterior Papillary
Muscle Tissue Slices* In Vitro Incubation Time
Experimental Groups
1 Hour
Sham-operated dogs (15) Partial ligation 1 Hour series (9) 2 Hour series (10) 4 Hour series (10)
2 Hours
At
0.12zt 0.01
...
0.18=!10.02 P
... 0.058 ... 0.095
0.15+
At
0.01
... ... 0.058 ... 0.108 ... 0.173
0.2ozk 0.02 P
... 0.153
3 Hours 0.15f
At
0.01
...
...
0.24zk 0.03 P
0.088 ... 0.140 ... 0.186
* Values are the mean optical densities f the standard deviations and represent the amount of p-nitrophenol liberated per hour per gram of wet weight tissue. t Difference between sham-operated and ischemic tissues /n soluble acid phosphatase activity. Figures in parentheses represent the numbers of experimental animals. P represents the probability and is based on Student’s t test of the data from the grouped controls and sham-operated dogs compared to corresponding times in the individual series of partial ligation experiments.
TABLE
III
Hydrogen
Ion, Glycogen and Total Pbtassium
Ion Concentration
of Left Posterior
Papillary
Muscles
In Vivo lschemia Time, Partial Ligation Series
Hydrogen
ion*
Glycogent
Potassium
ion$
2 Hours
4 Hour Sham-Operated Dogs
1 Hour
6.96 xt 0.03 (6)
6.48 f 0.14 (7) P
6.36 f
0.16 (7)
4 Hours 6.33 f
0.15 (7)
P
P
60.41 z!z 3.20 (20)
32.93 f 7.74 (10) P
16.98 f 7.46 (11) P
14.63 f ‘5.42 (12) P
38.38 f
34.87 f 1.86 (10) P <0.02
31.30 f 1.91 (8) P
27.95 f 1.89 (12) P
1.80 (7)
* Values are the means& the standard deviations and represent the pH of saline suspensions of minced papillary muscles. t The values are means =I=the standard deviations and represent the micrograms of glycogen per 100 milligrams of wet-weight tissue. $ Values are the millimoles of potassium per gram of dry-weight tissue expressed as the mean =t the standard deviation. Figures in parentheses represent the numbers of experimental animals. P represents the probability and is based on Student’s t test of the data.
tion (Table III). The pH of the 1 hour series was significantly (P >O.OOl) more acid than that of the sham-operated group (6.96). The mean pH of the 2 hour series was more acidic (6.36) than that of the 1 hour series (6.48). Little change was observed between the pH of the 2 hour series and that of the 4 hour series (6.33). After 1, 2 and 4 hours of acute ligation, glycogen content was significantly lower in left posterior papillary muscle than in sham-operated dogs (Table III). Glycogen depletion was rapid during the first 2 hours of in vivo ischemia and was almost complete by the fourth hour. This extensive hydrolysis of cardiac glycogen during the first 2 hours of hypoxic injury is well known. When compared to the sham-operated dogs, the left postetior papillary muscles from the 3 series with partial ligation demonstrated significant loss of ionic potassium (Table III) and the development of edema.
500
October 1972
The American Journal of CARDIOLOGY
Ionic potassium loss was greatest in the series that underwent 4 hours of ligation. The series with 1 hour of ligation also showed a significant (P CO.02) potassium loss.
Discussion Myocardial lysosome instability and developing cellular injury were simultaneously approximated in sham-operated and experimental left posterior papillary muscle sites after acute ligation of the left circumflex coronary artery of the dog heart. The experimental model utilized is well known and has been in use for at least 12 years.17-ls Its value lies in the predictability and uniformity of the cellular injury to the left posterior papillary muscle of the dog heart after ligation of the left circumflex coronary artery. The biochemical analyses of subcellular alterations in structure and function associated with the cellular injury process require the site of injury to be easily
Volume 30
LYSOSOMES
identifiable and the extent of injury to be hdmogenous in successive experiments so as to minimize dilution of the experim.ental findings. These conditions are known to be satisfied in the model utilized which also permitted simultaneous monitoring, at the same site of injury, of the extent of lysosome involvement relative to the degree of developing cellular injury. Since the transition from reversible to irreversible injury requires a continuum of subcellular alterations in structure and function which is poorly understood, no attempt was made to characterize the injury at the time of lysosome analysis as reversible. Rather, the degree of cellular injury was approximated on the basis of potassium. ion depletion and glycogen loss. Potassium ion loss and glycogen depletion from myocardial cells subjected to arterial hypoperfusion are among the earliest indications of cellular injury. Comparison of potassium ion depletion in control and experimental tissule is an acceptable method for approximating the degree of cellular injury present in injured tissue.18,1g The limitations of this technique are realized, and no attempt was made to characterize the state of injury as reversible or irreversible. The measurements of potassium ion depletion and glycogen loss were therefore of value in defining the state of the papillary muscles after each experimental period. The onset and extent of glycogen depletion further indicated the high degree of anae:obiosis to which the papillary muscles were subjected. Using dogs .with permanently rather than partially occluded circumflex arteries, Jennings et al.ls,lg have defined t,he state of the left posterior papillary muscle in the dog heart after 1, 2 and 4 hours of acute ligation of the left circumflex coronary artery. Our data approximate their findings. ?‘he results of this study demonstrate that within 1, 2 and 4 hours of acute reduction in blood flow through the left circumflex coronary artery of the dog heart, lysosome stability was significantly decreased in left posterior papillary muscle sites of injury. When compared to the findings from the sham-operated dogs, this reduction in lysosome stability was supported by the following observations. (1) There were significant reductions in the total activity of acid phosphatase in the whole homogenate subcellular fractions. (2) The acid phosphatase content of the lysosome containing sedimentable fractions was significant1.y reduced. (3) The ratios of nonsedimentable to sedimentable activity progressively decreased with time. (4) After each in vivo period of ischemia, left posterior papillary muscle tissue slices released acid phosphatase at significantly faster rates during ,the 3 hours of in vitro incubation than did similar1.y prepared preparations from sham-operated dogs. The extent of lysosome instability appeared to increase as the duration of the experimental period increased. This finding was based on the comparison of the extent of reduction in whole homogenate activi-
AND MYOCARDIAL
INJURY-RICCIUTTI
ty, the progressive fall in nonsedimentable ratios, the continued decrease in acid phosphatase activity of the lysosome-containing fractions, and the greater rates of in vitro release of acid phosphatase from the 4 hour series, when compared to the 1 hour series.
Correlation of decreased lysosome stability and myocardial necrosis: Decreased lysosome stability
may be related to the degree of developing injury within the cells of the papillary muscles. Glycogen depletion was almost complete by the second hour, thus indicating a high level of anaerobic activity during the first 2 hours after ligation. Analysis of the potassium ion content of these sites demonstrated significant reductions in the 1 hour series which continued to decrease with time. Jennings et al.19 reported that within 60 minutes of temporary ligation of the left circumflex coronary artery of the dog, the left posterior papillary muscle was irreversibly injured and had lost significant amounts of potassium. Their estimates of the percent of necrotic cells in these papillary muscles were based on a comparison of the potassium content of the ischemic site and the content of the nonischemic ventricular loci in the same heart. Values of 6.6, 8.6 and 21.7 percent necrosis were reported for left posterior papillary muscles subjected to 60, 120 and 240 minutes, respectively, of permanent ischemia in different experiments. In the present investigation similar estimates were made of the percent necrosis present in the papillary muscles of the 1, 2 and 4 hour experimental series. For the 1, 2 and 4 hour series of acute ligation experiments, respective estimates of 8.9, 18.28 and 28.2 percent necrosis were obtained. An inverse relation was apparent between the percent of necrosis and the reduction in nonsedimentable/sedimentable acid phosphatase ratios.
Lysosome instability and initiation of the injury process: Lysosome instability in injured left poste-
rior papillary muscle sites during the first 4 hours of coronary arterial ligation indicates myocardial lysosomes are involved in the early stages of ischemic injury to the heart. Significant lysosome instability ‘was demonstrated within 1 hour in left posterior ‘papillary muscle sites that were estimated to be only 8.9 percent necrotic. When coupled with the well known ability of lysosome enzymes to hydrolyze intracellular constituents, these data suggest that the development of lysosome instability occurs before cellular death and necrosis and may therefore initiate the injury process. The indication that the lysosomes may initiate the injury process is further supported by the studies21 which demonstrated lysosome instability in right .ventricular tissue and left anterior papillary muscles which had lost glycogen but not potassium at the end of a four hour period of acute total ligation of the left circumflex coronary artery of the dog heart. The mechanism involved in the induction of lysosome instability observed may be related to an in-
October 1972
The American Journal of CARDIOLOGY
Volume 30
501
LYSOSOMES
AND MYOCARDIAL
INJURY-RICCIUTTI
crease in the acidity of the cells. The ability of an acid pH to induce a graded release of lysosomal enzymes has been reported. 2,s~~ In this investigation the pH of saline suspension of minced papillary muscles fell to levels of approximately 6.48 by the end of the first hour and to 6.33 by the fourth hour of ischemia, when compared to sham-operated controls which evidenced a mean value of 6.96.
Hypothesis of homeostatic lysosomal nism: The presence of lysosome instability
mecha-
in right ventricular sites which had lost glycogen and not potassium and the demonstration in this study of significant lysosome involvement in left posterior papillary muscle sites within one hour of coronary artery ligation was of further interest. Lysosomes are known to contain glycosidases which hydrolyze glycogen. At acid pH ranges, acid hydrolases can be released from lysosomes in a graded manner. In the absence of an adequate arterial blood supply, the myocardial cell rapidly metabolizes its intracellular stores of glycogen in an attempt to maintain its function integrity. A consideration worthy of further investigation therefore is the possibility that myocardial lysosomes may initially function to facilitate the hydrolysis of
intracellular energy stores such as glycogen in response to the needs of the cell, subsequent to inadequate oxygenation and prior to ischemic disruption of these biochemical and biophysical mechanisms which maintain membrane integrity and regulate ion transport. If the injurious conditions persist, continued intracellular hydrolysis occurs and cellular injury is initiated. Proof for this hypothesized homeostatic lysosomal mechanism is presently being gathered. Recknt evidence has indicated the ability of hepatic lysosomes to hydrolyze glycogen when normal glycolytic pathways are inhibited through the action of insulin or glucose infusion.25 The activity of glycolytic enzymes is suppressed as pH is reduced and at a level of approximately 6.3 inhibition ensues. In contrast, lysosomal acid hydrolases are released in a graded manner as pH is reduced and are considered to have a pH optima in the range of 6.5 to 4.5.
Acknowledgment Sincere appreciation is extended to Dr. Anthony N. Damato for creating and sustaining the scientific environment in which this project was completed. Without his unending patience, good will and financial support this 4 year study would not have been possible.
References 1. Holtzer BS, Van Lancker J: The release of acid phosphatase and beta glucuronidase from cytoplasmic granules in the early course of autolysis. Amer J Path 35563-573, i 958 of ischemia on the 2. De Duve C, Beaufoy H: Influences state of some bound enzymes in rat liver. Biochem J 73:610-61631959 3. De Duve C, Wattiaux R: Functions of lysosomes. Ann Rev Physiol 28:435-492, 1965 4. Nelson D: Hepatic lysosome and serum enzyme alterations in rats exposed to high altitude. Amer J Physiol 211:651-655, 1966 5. Tappel AL, Shibko S: Increased lysosomal enzymes in geArch Biochem Biophysic muscular dystrophy. netic 96:340-34631962 in animals, 6 Tappel AL, Shibko S: Lysosomes: distribution hydrolytic capacity and other properties. In, Lysosomes. Ciba Foundation Symposium 1963, Boston, Little Brown, p 78-113 Lysosomes and pathological cell 7 Slater TF, Wang DY: damage. In, Ref. 6, p 311-334 a. Slater F, Greenbaum AL: Changes in lysosomes in acute experimental liver injury. Biochem J 96:464-491, 1965 9. Bitensky L: The reversible activation of lysosomes in normal cells and the effects of pathological conditions. In, Ref. 6, p 363-363 10. Bertini F, Brandes D: Role of lysosomes in cellular lytic processes. Exper Molec Path 4:245-265, 1965 Lysosome stability and coronary 11. Ricciutti MA, Damato AN: artery occlusion (abstr). Clin Res 16:245. 1966 MA, ~Damato -AN: Myocardial lysosomes and 12. Riccuitti ischemic injury (abstr). Circulation 60:169, 1969 Ultrastructure autoradiography and lysosome 13. Wheat MW: studies in myocardium. J Mount Sinai Hosp NY 32:107-120,
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October 1972
The American Journal of CARDIOLOGY
1965 14. Kottimeier CA, Wheat MW: Myocardial lysosomes in atrial septal defects. Circ Res 21: 17-27, 1967 15. Leighty EG, Sirak HD: Effects of acute asphyxia and deep hypothermia on the state of binding of lysosomal acid hydrolases in canine cardiac muscle. Circ Res 21:59-67, 1967 S, Raven G: Changes in the activities of ly16. Gudbjornasow sosomes in infarcted canine heart muscle. Circ Res 24:85i-857,1969 17. Jennings RB, Wartman RB: Production of an area of homogeneous myocardial infarction in the dog. Arch Path (Chicago) 63:580-585,1957 ia. Jennings RB, Crout JR, Smelters GW: Studies in the distribution and localization of potassium in early myocardial ischemic injury. Arch Path (Chicago) 63:566-592, 1957 19. Jennings RB, Sommers HM, Kaltenbach JP, et al: Electrolyte alterations in acute myocardial ischemic injury. Circ Res 14:260-269, 1964 The results of varying degrees of nar20. Lumb G, Cook JB: rowing on the left circumflex coronary artery in dogs. Amer J Path 36:113-123, 1960 Myocardial lysosome stability in the early 21. Ricciutte MA: StageS of acute ischemic injury. Amer J Cardiol 30:4g2497,1972 22. Seifter S, Myrthoyler E: The estimation of glycogen with the anthrone reagent. Arch Biochem 25:191-l 99, 1949 23. Blttar E: pH in relation to the cell. In, Cell pH (Bittar E, ed). Butterwprth, Washingtqn DC, 1964, p 16-23 HM: Lysosomes in human cell cul24. Gordis L, Nitowsky tures. Exp Cell Res 36556-569, 1965 25. Kotoulas 0, Adochi F, Phillips M: Fine structural aspects of the mobilization of hepatic glycogen. Inhibition of glycogen breakdown. Amer J Path 62:23-34, 1971
Volume 30
MICHAEL Staten
A. RICCIUTTI.
Island,
New
The stability of myocardial lysosomes in left posterior papillary muscle sites of predictable injury was investigated after 1, 2 and 4 hours of acute partial ligation of the left circumflex coronary artery of the dog heart. Lysosome stability in the papillary muscle locus of injury was significantly reduced within 1 tiour and continued to decrease as the duration of the experimental period increased. The intracellular mechanism responsible for the alteration in lysosome stability may be related to a reduction in pH of the tissue. pH of sham-operated papillary muscle was approximately 6.9, that of the 1 hour and 4 hour groups was 6.4 and 6.3, respectively. Alteration in lysosome stability is considered one of the earliest subcellular structural changes occurring during the ischemic cellular injury process in heart muscle. The extent of the lysosome involvement and the rela tively small degree of necrosis present in the papillary muscle sites by 60 minutes, when coupled with the well known hydrolytic capacity of lysosome acid hydrolysis, suggests that the intracellular release of lysosomal enzymes precedes cellular death and may initiate the cellular injury process.
PhD*
York
From the Cardiopulmonary Laboratory, U. S. Public Health Service Hospital, Staten Island. N.Y. This study was supported in part by the Federal Health Program Service, U. S. Public Health Service Project PY 72-1, and National Institutes of Health Projects HE 11829 and HE 12536. Manuscript received December 15. 1971, revised manuscript received April 11. 1972, accepted May 24, 1972. *Presented at the American College of Cardiology Meetings, Washington, D.C. February 3, 1971 as part of the manuscript honored with the Young Investigators Award for the year 1971. Address for reprints: Michael A. Ricciutti. PhD, Cardiopulmonary Laboratory, U. S. Public Health Service Hospital, Staten Island. N.Y. 10304.
490
October 1972
The American
Recent evidence has indicated that during hypoxic or ischemic conditions resulting cellular injury may be initiated through the subcellular release of hydrolytic enzymes contained within the lysosome. l-4 If lysosomes are involved in the initiation of cellular injury, alterations in lysosome stability may be expected in the early stages or prenecrotic periods of the injury process.4-10 A series of studies was designed to determine the onset of lysosome involvement in myocardial cells of the dog heart subjected to various periods of arterial hypoperfusion. 11-1s The extent of lysosome involvement and the degree of cellular injury present were simultaneously approximated in the left posterior papillary muscle, previously shown to be a predictable and relatively uniform locus of injury after acute ligation of the left circumflex coronary artery.l’-19 Methods Randomly pentobarbital
selected male and female dogs were anesthetized with sodium (30 mg/kg body weight), and respiration was maintained at a
constant rate by a Harvard respirometer. A left thoracotomy was performed, and a longitudinal incision elrposing the left atrium and ventricle was made in the pericardium. A 5 mm segment along the length of the left circumflex coronary artery was cleared of adhering fat tissue. A 3-O silk ligature was then loosely positioned around the artery. This site of ligation was always located distal to the first large ventricular branch of the circumflex artery, and at the midline of the base of the left atrium. Sham-operated dogs were surgically prepared as described and maintained in the operative state for 4 hours. The heart was then excised while still beating. In experimental dogs, the lumen of the left circumflex coronary artery was reduced approximately 60 percent according to the procedure of Lumb and Cook.20 Separate ligation experiments were allowed to
Journal of CARDIOLOGY
Volume 30
LYSOSOMES
TABLE
AND MYOCARDIAL
INJURY-RICCIUTTI
I
Subcellular
Distribution
of Acid Phosphatase
Experimental Groups Sham-operated dogs (20) Partial ligation 1 Hour series (10)
in Left Posterior
Papillary
Muscle Tissues*
Sedimentable Activity
Total Activity
Nonsedimentable Activity
Nonsedimentable/ Sedimentable
25.09 f
1.97
19.88 + 1.60
5.15 f
0.76
0.259
24.57 f
2.09
21.36 zt 1.85
3.19 f 0.66 P zto.001 2.03 f 0.54 P
0.148
2 Hour series (8)
22.35 f 1.61 P
20.26 f
1.64
4 Hour series (8)
20.02zt 1.75 P
18.77311.41
0.100 0.056
* Values are the means f the standard deviations and represent the amount of p-nitrophenol liberated per hour per gram of wet weight tissue. Figures in parentheses represent the numbers of experimental animals. P represents the probability and is based on Student’s t test of the data from the grouped control and sham-operated dogs compared to the individual series of partial ligation experiments.
continue for 60, 120 and 240 minutes, respectively. At the end of each time period the heart was excised while still beating. Since previous studies have shown that ligation of the left circumflex coronary artery at the locus described results in the production of uniform and predictable ischemit injury to the left posterior papillary muscle, this papillary muscle was sel.ected as an easily identifiable tissue sampling site in successive experiments.17-ls
The hydrolytic enzyme acid phosphatase was used as the well known marker of lysosome stability. The methods for studying subcellular distribution of acid phosphatase and the kinetics of acid phosphatase release are described in the preceding paper.zl Glycogen, potassium and pH: The glycogen and potassium contents of the left posterior papillary muscles were determined by the same operative and experimental procedures as described.21 The glycogen content of 100 mg samples was isolated by hot alkali digestion and centrifugation. The glycogen precipitate was hydrolyzed to glucose and determined calorimetrically as described by Seifter and Myrthoyler.21 All analyses were performed in duplicate and repeated jf the difference between any 2 values was greater than 5 percent.
Potassium ion content was determined according to the technique of Jennings et al.ls with omission of the fat extraction procedure. The use of a constant tissue sampling site and the extensive procedures for minimizing ionic contamination during analysis served to reduce the biological variability and experimental error normally associated with ionic analyses. The pH of sham-operated and experimental left posterior papillary muscles w(as approximated according to the method of Bittar. Tis:eue samples, 800 to 1000 mg, were rapidly minced in 2 ml of isotonic saline solution for 2 minutes. The mince was allowed to equilibrate at 4 C for 15 minutes, and the pH: of the suspension was then determined with a Beckman Expandomatic pH meter. The pH meter was standardizedi at 4 C with phosphate buffers of pH 6 and 7.
Results A comparison of the activity of acid phosphatase in left posterior papillary muscle subcellular tissue fractions from the sham-operated dogs and from
those that underwent coronary arterial ligation is presented in Table I. The total activity of acid phosphate in whole homogenate (600 X g) subcellular fractions decreased significantly (P >O.Ol) within 2 hours of coronary arterial ligation and was further reduced by the fourth hour. Sedimentable activity of the lysosome-containing subcellular fraction (20,000 X g) decreased significantly (P CO.001) within 1 . hour of coronary arterial ligation and was further reduced as the duration of in vivo ischemia increased. Sedimentable to nonsedimentable ratios from the hearts that underwent coronary arterial ligation were lower than those of the sham-operated hearts. Acid phosphatase was released from the left posterior papillary muscle tissue slices at a faster rate from the ischemic muscles than from the sham-operated group (Table II). The release of the enzyme from the sham-operated group was greatest during the first hour of in vitro incubation. The rate of release continued more slowly over the next 2 hours, with little difference between the second and third hour of incubation. A rapid release was observed from the papillary muscles of the series with 1 hour of coronary arterial ligation during the first and third hours of incubation. By the third hour of incubation, the acid phosphatase activity in the media was almost twice the 1 hour incubation level. After the 2 hour period of in vivo ischemia incubated ischemic papillary muscles released their content of acid phosphatase most rapidly during the first hour of incubation. Release of the enzyme during the second and third hour of incubation was slower. The 4 hour period of in vivo ischemia resulted in a rapid release of the enzyme during the first and second hours of in vitro incubation with a slower rate of release during the third hour. When compared to the pH values from sham-operated hearts, sodium chloride suspensions of ischemic left posterior papillary muscles showed significant reductions in the pH values of similarly prepared suspensions in the group that underwent partial liga-
October 1972
The American Journal of CARDIOLOGY
Volume 30
499
LYSOSOMES
TABLE
AND MYOCARDIAL
INJURY-RICCIUTTI
II
In Vitro Release of Acid Phosphatasefrom
Left Posterior Papillary
Muscle Tissue Slices* In Vitro Incubation Time
Experimental Groups
1 Hour
Sham-operated dogs (15) Partial ligation 1 Hour series (9) 2 Hour series (10) 4 Hour series (10)
2 Hours
At
0.12zt 0.01
...
0.18=!10.02 P
... 0.058 ... 0.095
0.15+
At
0.01
... ... 0.058 ... 0.108 ... 0.173
0.2ozk 0.02 P
... 0.153
3 Hours 0.15f
At
0.01
...
...
0.24zk 0.03 P
0.088 ... 0.140 ... 0.186
* Values are the mean optical densities f the standard deviations and represent the amount of p-nitrophenol liberated per hour per gram of wet weight tissue. t Difference between sham-operated and ischemic tissues /n soluble acid phosphatase activity. Figures in parentheses represent the numbers of experimental animals. P represents the probability and is based on Student’s t test of the data from the grouped controls and sham-operated dogs compared to corresponding times in the individual series of partial ligation experiments.
TABLE
III
Hydrogen
Ion, Glycogen and Total Pbtassium
Ion Concentration
of Left Posterior
Papillary
Muscles
In Vivo lschemia Time, Partial Ligation Series
Hydrogen
ion*
Glycogent
Potassium
ion$
2 Hours
4 Hour Sham-Operated Dogs
1 Hour
6.96 xt 0.03 (6)
6.48 f 0.14 (7) P
6.36 f
0.16 (7)
4 Hours 6.33 f
0.15 (7)
P
P
60.41 z!z 3.20 (20)
32.93 f 7.74 (10) P
16.98 f 7.46 (11) P
14.63 f ‘5.42 (12) P
38.38 f
34.87 f 1.86 (10) P <0.02
31.30 f 1.91 (8) P
27.95 f 1.89 (12) P
1.80 (7)
* Values are the means& the standard deviations and represent the pH of saline suspensions of minced papillary muscles. t The values are means =I=the standard deviations and represent the micrograms of glycogen per 100 milligrams of wet-weight tissue. $ Values are the millimoles of potassium per gram of dry-weight tissue expressed as the mean =t the standard deviation. Figures in parentheses represent the numbers of experimental animals. P represents the probability and is based on Student’s t test of the data.
tion (Table III). The pH of the 1 hour series was significantly (P >O.OOl) more acid than that of the sham-operated group (6.96). The mean pH of the 2 hour series was more acidic (6.36) than that of the 1 hour series (6.48). Little change was observed between the pH of the 2 hour series and that of the 4 hour series (6.33). After 1, 2 and 4 hours of acute ligation, glycogen content was significantly lower in left posterior papillary muscle than in sham-operated dogs (Table III). Glycogen depletion was rapid during the first 2 hours of in vivo ischemia and was almost complete by the fourth hour. This extensive hydrolysis of cardiac glycogen during the first 2 hours of hypoxic injury is well known. When compared to the sham-operated dogs, the left postetior papillary muscles from the 3 series with partial ligation demonstrated significant loss of ionic potassium (Table III) and the development of edema.
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Ionic potassium loss was greatest in the series that underwent 4 hours of ligation. The series with 1 hour of ligation also showed a significant (P CO.02) potassium loss.
Discussion Myocardial lysosome instability and developing cellular injury were simultaneously approximated in sham-operated and experimental left posterior papillary muscle sites after acute ligation of the left circumflex coronary artery of the dog heart. The experimental model utilized is well known and has been in use for at least 12 years.17-ls Its value lies in the predictability and uniformity of the cellular injury to the left posterior papillary muscle of the dog heart after ligation of the left circumflex coronary artery. The biochemical analyses of subcellular alterations in structure and function associated with the cellular injury process require the site of injury to be easily
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identifiable and the extent of injury to be hdmogenous in successive experiments so as to minimize dilution of the experim.ental findings. These conditions are known to be satisfied in the model utilized which also permitted simultaneous monitoring, at the same site of injury, of the extent of lysosome involvement relative to the degree of developing cellular injury. Since the transition from reversible to irreversible injury requires a continuum of subcellular alterations in structure and function which is poorly understood, no attempt was made to characterize the injury at the time of lysosome analysis as reversible. Rather, the degree of cellular injury was approximated on the basis of potassium. ion depletion and glycogen loss. Potassium ion loss and glycogen depletion from myocardial cells subjected to arterial hypoperfusion are among the earliest indications of cellular injury. Comparison of potassium ion depletion in control and experimental tissule is an acceptable method for approximating the degree of cellular injury present in injured tissue.18,1g The limitations of this technique are realized, and no attempt was made to characterize the state of injury as reversible or irreversible. The measurements of potassium ion depletion and glycogen loss were therefore of value in defining the state of the papillary muscles after each experimental period. The onset and extent of glycogen depletion further indicated the high degree of anae:obiosis to which the papillary muscles were subjected. Using dogs .with permanently rather than partially occluded circumflex arteries, Jennings et al.ls,lg have defined t,he state of the left posterior papillary muscle in the dog heart after 1, 2 and 4 hours of acute ligation of the left circumflex coronary artery. Our data approximate their findings. ?‘he results of this study demonstrate that within 1, 2 and 4 hours of acute reduction in blood flow through the left circumflex coronary artery of the dog heart, lysosome stability was significantly decreased in left posterior papillary muscle sites of injury. When compared to the findings from the sham-operated dogs, this reduction in lysosome stability was supported by the following observations. (1) There were significant reductions in the total activity of acid phosphatase in the whole homogenate subcellular fractions. (2) The acid phosphatase content of the lysosome containing sedimentable fractions was significant1.y reduced. (3) The ratios of nonsedimentable to sedimentable activity progressively decreased with time. (4) After each in vivo period of ischemia, left posterior papillary muscle tissue slices released acid phosphatase at significantly faster rates during ,the 3 hours of in vitro incubation than did similar1.y prepared preparations from sham-operated dogs. The extent of lysosome instability appeared to increase as the duration of the experimental period increased. This finding was based on the comparison of the extent of reduction in whole homogenate activi-
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ty, the progressive fall in nonsedimentable ratios, the continued decrease in acid phosphatase activity of the lysosome-containing fractions, and the greater rates of in vitro release of acid phosphatase from the 4 hour series, when compared to the 1 hour series.
Correlation of decreased lysosome stability and myocardial necrosis: Decreased lysosome stability
may be related to the degree of developing injury within the cells of the papillary muscles. Glycogen depletion was almost complete by the second hour, thus indicating a high level of anaerobic activity during the first 2 hours after ligation. Analysis of the potassium ion content of these sites demonstrated significant reductions in the 1 hour series which continued to decrease with time. Jennings et al.19 reported that within 60 minutes of temporary ligation of the left circumflex coronary artery of the dog, the left posterior papillary muscle was irreversibly injured and had lost significant amounts of potassium. Their estimates of the percent of necrotic cells in these papillary muscles were based on a comparison of the potassium content of the ischemic site and the content of the nonischemic ventricular loci in the same heart. Values of 6.6, 8.6 and 21.7 percent necrosis were reported for left posterior papillary muscles subjected to 60, 120 and 240 minutes, respectively, of permanent ischemia in different experiments. In the present investigation similar estimates were made of the percent necrosis present in the papillary muscles of the 1, 2 and 4 hour experimental series. For the 1, 2 and 4 hour series of acute ligation experiments, respective estimates of 8.9, 18.28 and 28.2 percent necrosis were obtained. An inverse relation was apparent between the percent of necrosis and the reduction in nonsedimentable/sedimentable acid phosphatase ratios.
Lysosome instability and initiation of the injury process: Lysosome instability in injured left poste-
rior papillary muscle sites during the first 4 hours of coronary arterial ligation indicates myocardial lysosomes are involved in the early stages of ischemic injury to the heart. Significant lysosome instability ‘was demonstrated within 1 hour in left posterior ‘papillary muscle sites that were estimated to be only 8.9 percent necrotic. When coupled with the well known ability of lysosome enzymes to hydrolyze intracellular constituents, these data suggest that the development of lysosome instability occurs before cellular death and necrosis and may therefore initiate the injury process. The indication that the lysosomes may initiate the injury process is further supported by the studies21 which demonstrated lysosome instability in right .ventricular tissue and left anterior papillary muscles which had lost glycogen but not potassium at the end of a four hour period of acute total ligation of the left circumflex coronary artery of the dog heart. The mechanism involved in the induction of lysosome instability observed may be related to an in-
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crease in the acidity of the cells. The ability of an acid pH to induce a graded release of lysosomal enzymes has been reported. 2,s~~ In this investigation the pH of saline suspension of minced papillary muscles fell to levels of approximately 6.48 by the end of the first hour and to 6.33 by the fourth hour of ischemia, when compared to sham-operated controls which evidenced a mean value of 6.96.
Hypothesis of homeostatic lysosomal nism: The presence of lysosome instability
mecha-
in right ventricular sites which had lost glycogen and not potassium and the demonstration in this study of significant lysosome involvement in left posterior papillary muscle sites within one hour of coronary artery ligation was of further interest. Lysosomes are known to contain glycosidases which hydrolyze glycogen. At acid pH ranges, acid hydrolases can be released from lysosomes in a graded manner. In the absence of an adequate arterial blood supply, the myocardial cell rapidly metabolizes its intracellular stores of glycogen in an attempt to maintain its function integrity. A consideration worthy of further investigation therefore is the possibility that myocardial lysosomes may initially function to facilitate the hydrolysis of
intracellular energy stores such as glycogen in response to the needs of the cell, subsequent to inadequate oxygenation and prior to ischemic disruption of these biochemical and biophysical mechanisms which maintain membrane integrity and regulate ion transport. If the injurious conditions persist, continued intracellular hydrolysis occurs and cellular injury is initiated. Proof for this hypothesized homeostatic lysosomal mechanism is presently being gathered. Recknt evidence has indicated the ability of hepatic lysosomes to hydrolyze glycogen when normal glycolytic pathways are inhibited through the action of insulin or glucose infusion.25 The activity of glycolytic enzymes is suppressed as pH is reduced and at a level of approximately 6.3 inhibition ensues. In contrast, lysosomal acid hydrolases are released in a graded manner as pH is reduced and are considered to have a pH optima in the range of 6.5 to 4.5.
Acknowledgment Sincere appreciation is extended to Dr. Anthony N. Damato for creating and sustaining the scientific environment in which this project was completed. Without his unending patience, good will and financial support this 4 year study would not have been possible.
References 1. Holtzer BS, Van Lancker J: The release of acid phosphatase and beta glucuronidase from cytoplasmic granules in the early course of autolysis. Amer J Path 35563-573, i 958 of ischemia on the 2. De Duve C, Beaufoy H: Influences state of some bound enzymes in rat liver. Biochem J 73:610-61631959 3. De Duve C, Wattiaux R: Functions of lysosomes. Ann Rev Physiol 28:435-492, 1965 4. Nelson D: Hepatic lysosome and serum enzyme alterations in rats exposed to high altitude. Amer J Physiol 211:651-655, 1966 5. Tappel AL, Shibko S: Increased lysosomal enzymes in geArch Biochem Biophysic muscular dystrophy. netic 96:340-34631962 in animals, 6 Tappel AL, Shibko S: Lysosomes: distribution hydrolytic capacity and other properties. In, Lysosomes. Ciba Foundation Symposium 1963, Boston, Little Brown, p 78-113 Lysosomes and pathological cell 7 Slater TF, Wang DY: damage. In, Ref. 6, p 311-334 a. Slater F, Greenbaum AL: Changes in lysosomes in acute experimental liver injury. Biochem J 96:464-491, 1965 9. Bitensky L: The reversible activation of lysosomes in normal cells and the effects of pathological conditions. In, Ref. 6, p 363-363 10. Bertini F, Brandes D: Role of lysosomes in cellular lytic processes. Exper Molec Path 4:245-265, 1965 Lysosome stability and coronary 11. Ricciutti MA, Damato AN: artery occlusion (abstr). Clin Res 16:245. 1966 MA, ~Damato -AN: Myocardial lysosomes and 12. Riccuitti ischemic injury (abstr). Circulation 60:169, 1969 Ultrastructure autoradiography and lysosome 13. Wheat MW: studies in myocardium. J Mount Sinai Hosp NY 32:107-120,
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1965 14. Kottimeier CA, Wheat MW: Myocardial lysosomes in atrial septal defects. Circ Res 21: 17-27, 1967 15. Leighty EG, Sirak HD: Effects of acute asphyxia and deep hypothermia on the state of binding of lysosomal acid hydrolases in canine cardiac muscle. Circ Res 21:59-67, 1967 S, Raven G: Changes in the activities of ly16. Gudbjornasow sosomes in infarcted canine heart muscle. Circ Res 24:85i-857,1969 17. Jennings RB, Wartman RB: Production of an area of homogeneous myocardial infarction in the dog. Arch Path (Chicago) 63:580-585,1957 ia. Jennings RB, Crout JR, Smelters GW: Studies in the distribution and localization of potassium in early myocardial ischemic injury. Arch Path (Chicago) 63:566-592, 1957 19. Jennings RB, Sommers HM, Kaltenbach JP, et al: Electrolyte alterations in acute myocardial ischemic injury. Circ Res 14:260-269, 1964 The results of varying degrees of nar20. Lumb G, Cook JB: rowing on the left circumflex coronary artery in dogs. Amer J Path 36:113-123, 1960 Myocardial lysosome stability in the early 21. Ricciutte MA: StageS of acute ischemic injury. Amer J Cardiol 30:4g2497,1972 22. Seifter S, Myrthoyler E: The estimation of glycogen with the anthrone reagent. Arch Biochem 25:191-l 99, 1949 23. Blttar E: pH in relation to the cell. In, Cell pH (Bittar E, ed). Butterwprth, Washingtqn DC, 1964, p 16-23 HM: Lysosomes in human cell cul24. Gordis L, Nitowsky tures. Exp Cell Res 36556-569, 1965 25. Kotoulas 0, Adochi F, Phillips M: Fine structural aspects of the mobilization of hepatic glycogen. Inhibition of glycogen breakdown. Amer J Path 62:23-34, 1971
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