Mechanism of changes of the collagen matrix of reperfused myocardium in donor heart preservation Isolated canine hearts were preserved for 6 hours at 5° C followed by normothermic reperfusion for 2 hours. Dogs were divided into two groups: group I (group la In = 7] and group Ib In = 3] with the left ventricle unloaded during reperfusion) received a preservation solution containing potassium (20 mmol/ L), and group II (n = 9) received University of Wisconsin solution. Left ventricular diastolic function was better preserved in group II. Degradation and loss of the collagen network during reperfusion, as assessed by scanning electron microscopy, were more extensive and significantly more frequent in group la than in group II (6/7 versus 2/9; p < 0.05). Furthermore, extensive disruption of the collagen network was significantly more prevalent in hearts with a left ventricular end-diastolic pressure of more than 20 mm Hg than in hearts with a left ventricular end-diastolic pressure of less than 20 mm Hg (8/10 versus 0/6; p < 0.05), and no disruption of the collagen network occurred in group Ib, regardless of the type of preservation solution. These results suggest that the greatest disruption is caused by barotrauma resulting from an elevated left ventricular end-diastolic pressure after ventricular dysfunction caused by ischemic reperfusion injury. (J THoRAc CARDIOVASC SURG 1993;106:172-9)
Masahiro Ohnuki, MD, Makoto Sunamori, MD, Jun Amano, MD, and Akio Suzuki, MD,
Tokyo, Japan
AthOugh hypothermic cardioplegia is accepted widely as a means of myocardial preservation, it causes enzyme dysfunction,' decreased membrane stability./' 3 and calcium sequestration." Furthermore, reperfusion is associated with arrhythmias, depletion of high-energy phosphate compounds, ventricular dysfunction, and myocardial necrosis. The myocardial collagen matrix serves as an important link between force-generating myocytes and force-bearing structures.c'' Thus structural changes in the collagen matrix may decrement ventricular performance.f to Myocardial ischemia may disrupt the collagen network in considerably less time than 24 hours I 1-13 and may lead to a "stunned myocardium."!" Little is known about changes in the collagen matrix during reperfusion after hypothermic myocardial preserFrom the Department of Thoracic-Cardiovascular Surgery, Tokyo Medical and Dental University, School of Medicine, Tokyo, Japan. Received for publication Jan. 27, 1992. Accepted for publication July 9, 1992. Address for reprints: Makoto Sunamori, MD, Department of Thoracic-Cardiovascular Surgery, Tokyo Medical and Dental University, School of Medicine, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113, Japan. Copyright
1993 by Mosby-Year Book, Inc.
0022-5223/93 $1.00
172
+ .10
12/1/40862
vation, and it is an unclear argument whether degradation or loss (or both) of collagen network is a cause or result of ventricular dysfunction. We examined the hypothesis that the myocardial collagen matrix is disrupted as a result of left ventricular diastolic dysfunction during reperfusion after ischemic preservation. Materials and methods Thirty-one dogs weighing 8 to 21 kg were anesthetized with intravenous pentobarbital (30 rng/kg) and maintained by mechanical ventilation. Animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). Control group of the heart. A median sternotomy was performed, and the heart received the same procedures as those described for procurement of the heart. The great vessels of the heart and the hili of both lungs were not occluded, however, so that under well-oxygenated and stable hemodynamic conditions without cardioplegia, the left ventricular biopsy was done to analyze nonischemic myocardial biochemical and morphologic state (control, n = 12). Procurement of the heart. A median sternotomy was performed, and the superior and inferior venae cavae were controlled with 2-0 silk ligatures, both proximally and distally. The azygos vein was ligated and divided. Both common carotid
The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 1
arteries, the left subclavian artery, and the descending aorta also were encircled proximally and distally with 2-0 silk ligatures, as were the hili of both lungs. A IOF arterial cannula was inserted from the proximal right subclavian artery, and a 24F venous cannula was placed in the right ventricle through the right atrial appendage. Approximately 500 ml of blood was withdrawn from the venous cannula, heparinized, and saved for transfusion during reperfusion. The previously encircled arteries were ligated, and the pulmonary hili were ligated after ventilation was stopped. Immediately after aortic occlusion, cardioplegia was induced by the infusion of cold (50 C) cardioplegic solution via the arterial cannula. The initial volume infused was 10 ml/kg in group I and 30 ml/kg in group II, with perfusion pressure of 70 mm Hg for 2 minutes. The superior and inferior venae cavae were ligated and divided, and the heart was removed. Preservation of the heart. The heart was immersed for 6 hours in cold (50 C) saline solution in group I (n = 10) and cold University of Wisconsin (UW) solution (ViaSpan, E. I. duPont de Nemours & Co., Inc., Wilmington, Del.) in group II (n = 9). Cardioplegic solution used in group I consisted of the following: K+, 20 mmol/L; Na", 10 mmol/L; Mg2+, 16 mmol/L; Ca2+, I mrnol/L; mannitol, 100 mrnol/L; and glucose 245 mmol/L; with an osmolarity of 450 mOsm and a pH of 7.50, adjusted by sodium bicarbona te 10 mmol/L. UW solution consists of these components: Na+, 20 mrnol/L; Ca2+, 0 mmol/L; KCl, 15 mmol/L; KH 2P04 , 25 rnrnol/L; K-lactobionate, 100 mmol/L (final K+ concentration 140 mrnol/L); Mg2+, 10 mrnol/L; adenosine, 5 mmol/L; glutathione, 3 mmol/L; raffinose, 30 mrnol/L; allopurinol, 1.0 rng/L; and pentastarch, 5%/L; with an osmolarity of 320 mOsm/L and a pH of?.4. One hour before reperfusion, a latex balloon was placed in the left ventricle and secured with a holding apparatus sutured in the mitral position. The balloon was connected to a transducer (Statham P23DB, Viggo-Spectramed Inc., Critical Care Division, Oxnard, Calif.), and left ventricular pressure during reperfusion was measured using a polygraph (Nihon Kohden Co., Tokyo, Japan). Special care was taken to avoid mechanically induced aortic regurgitation. Thirty minutes before reperfusion, the heart was exposed to room temperature without immersion in either cold saline or UW solution. Reperfusion. A second dog was anesthetized, lungs ventilated, and maintained hemodynamically by the infusion of Ringer's lactate solution. Both carotid arteries were cannulated with IOFcatheters and connected to the arterial cannula placed in the preserved heart. A pressure transducer and a magnetic flowmeter (N ihon) were connected to the circuit to measure perfusion pressure and flow. Coronary sinus blood flow was measured by a magnetic flowmeter as an estimate of coronary blood flow. Blood from the cannula in the right ventricle and vented from the left ventricle was collected in a reservoir and rein fused into the supporting dog by a pump. A heat exchanger maintained normothermia. Reperfusion was continued for 2 hours, and defibrillation was performed when the heart developed ventricular fibrillation during the early phase of reperfusion. After 5 minutes of reperfusion, all dogs were paced at 130 beats/min. No cardiotonic drugs were administered. At the end of the period of arrest and during reperfusion while the heart was beating, a tissue sample was excised from the subendocardium of the left ventricle. Tissue concentrations of adenosine triphosphate (A TP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and creatine phosphate were measured using previously described methods.'> The tissue calcium concentration was measured by atomic absorp-
Ohnuki et al.
17 3
tion.!" Transmission electron microscopy was used to evaluate mitochondrial ultrastructural changes by semiquantitative morphometry.!? Regarding morphometry, six electron micrographs were made for each dog. In each micrograph, ten mitochondria were selected at random. Intact mitochondrial membranes were scored as 10, and completely destroyed membranes were scored as O. Pathologic changes in membranes were graded with scores between 0 and 10. Therefore total score was scaled between 0 (complete destruction) and 100 (intact) in each electron micrograph. The average score of six electron micrographs was calculated. Mitochondrial cristae were evaluated by the same method as described for membranes. Scanning electron microscopy (SEM) was used to evaluate changes in the myocardial collagen matrix. Disruption of the collagen matrix was defined as disruption of the collagen weaves." The heart showing this finding in all 10 different sections was defined to have the disruption of collagen matrix. Left ventricular end-systolic and end-diastolic pressures were measured by means of a balloon inflated with saline volumes of 5, 10, 15, and 20 ml in both subgroup la (n = 7) and group II (n = 9). Reperfusion was observed without loading a balloon with saline volumes of 0 ml in subgroup Ib (n = 3). The left ventricular end-systolic pressure-volume relationship and enddiastolic pressure-volume relationship were analyzed to evaluate functional recovery of the left ventricle. Data within each group were analyzed by Student's paired t test, and between groups by the Mann- Whitney test or X2 test. p Values less than 0.05 were considered statistically significant.
Results Perfusion pressure, flow during reperfusion, coronary flow, hematocrit level, temperature, and left ventricular weight were matched between the two groups. Defibrillation was performed in all dogs in both groups. All dogs in both groups survived the 2 hours of reperfusion. At the end of both preservation and reperfusion, the myocardial ATP concentration in group I was significantly lower than in group II (p < 0.05 and p < 0.01). At the end of reperfusion, myocardial ADP in group I was significantly lower than in group II. The myocardial ADP concentration decreased significantly during reperfusion in group I (p < 0.001) but was unchanged in group II. The myocardial AMP concentration decreased during reperfusion in both groups, but at the end of reperfusion, myocardial AMP in group I was significantly lower than in group II (p < 0.05). At the end of both preservation and reperfusion, myocardial total adenine nucleotide and creatine phosphate in group I were significantly lower than in group II (p < 0.05 and p < 0.01). Tissue calcium concentration increased significantly during reperfusion in both groups, but the difference between groups was not significant at the end of either preservation or reperfusion (Table I).
Left ventricular functional recovery. The end-systolic pressure during reperfusion was satisfactory in both groups though, in general, and the end-systolic pressure in group I was better than in group II (Fig. 1). The end-
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Table I. Comparison of myocardial concentration of high-energy phosphate compounds and calcium in canine hearts preserved with two types of cardioplegic solution Control (n = 12)
ATP
C
ADP
I II C I II
4.87 ± 0.39
C
0.70 ± 0.14
AMP
C
C
13.70 ± 1.18*t 13.45 ± 2.67*:j:
2.89 ± 0.34§ 1.40 ± 0.30*
3.53 ± 0.32§ 3.45 ± 0.17:j:§
1.91 ± 0.84 2.60 ± 0.06§
0.72 ± 0.16 0.50 ± 0.08t
12.24 ± 2.45* 5.11 ± 3.09*
17.90 ± 1.42*t 17.40 ± 2.85*:j:
3.07 ± 1.38* 3.42 ± 2.39*
6.21 ± 1.44*t 18.78 ± 4.96:j:
34.1 ± 2.9 121.0 ± 18.3*
38.8 ± 3.8 95.1 ± 17.8*
18.61 ± 1.69
I II
Ca
7.14 ± 2.60* 3.49 ± 2.80*
26.77 ± 1.03
I II
CP
Group II (n = 9)
20.20 ± 0.82
I II
TAN
Group la (n = 7)
C
42.88 ± 3.1
I II
Data are reported as mean ± standard error of the mean, micrograms per milligram of protein, except for calcium, which is reported in micrograms per gram of tissue. Group [a received cardioplegic solution containing K+ 20 rnrnol/L: group [I received UW solution. ATP. Adenosine triphosphate: ADP. adenosine diphosphate: AMP. adenosine monophosphate: TAN. total adenine nucleotides; CPo creatine phosphate; Ca. calcium: I. end of preservation: II. end of reperfusion: C. before preservation. * p < 0.0 I. significance versus control.
s» < 0.05. significance between groups [a and II. :j:p < 0.01. significance between groups la and II. §p < 0.05. significance versus control.
Table II. Comparison of changes in mitochondrial ultrastructure as measured by semiquantitative morphometry in canine hearts preserved with two types of cardioplegic solution Group fa (n = 7) Mitochondrial membrane I 64.0 ± 2.7 II 64.4 ± 4.0 Mitochondrial cristae I 42.3 ± 3.5 II 44.9 ± 2.9
Group II (n
= 9)
Significance
58.3 ± 1.1 64.0 ± 2.1
p
< 0.05
34.8 ± 1.5 49.6 ± 4.0
p
< 0.05
Score indicates intact structure (100) and complete destruction (0). I. End of preservation; II, cnd of reperfusion. Group Ia received cardioplegic solution containing K+ (20 mrnol/L); group [[ received University of Wisconsin solution.
The myocardial collagen network was fairly well preserved at the end of preservation, but at the end of reperfusion collagen weaves? were disrupted, and the collagen fine network was destroyed (Fig. 3). The changes in the collagen network were more extensive and significantly more frequently seen in hearts with left ventricular end-diastolic pressure of more than 20 mm Hg than in hearts with left ventricular end-diastolic pressure of less than 20 mm Hg (8/ 10 hearts versus 0/6 hearts, respectively; p < 0.05, Fig. 4). Disruption of the collagen matrix occurred in six of seven hearts in group Ia and in two of nine hearts in group II (p < 0.05). Furthermore, no disruption of the collagen matrix occurred in group lb. Discussion
diastolic pressure was significantly more impaired at left ventricular volume of 15 and 20 ml during reperfusion in group I than in group II (Fig. 2). At 120 minutes of reperfusion, the left ventricular end-diastolic pressure in group I was significantly higher than in group II (p < 0.05). Ultrastructural changes. At the end of preservation, but not reperfusion, mitochondrial cristae and membranes were slightly better preserved in group I than in group II (p < 0.05) (Table II).
The current method of cardioplegia allows depolarization of the myocytic membrane. Depolarization increases permeability and subsequently leads to the accumulation of sodium and calcium, cellular swelling, calcium overload, damage to subcellular organelles, and ventricular fibrillation during reperfusion after ischemia. At present, calcium overload is thought to be a major cause of reperfusion. 18 The method of myocardial protection utilized in this investigation differs from the standard clinical practice in
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_
Ohnuki et al.
* **
K+20 Solution, n=7 UW Solution, n=9
CJ
I 75
p<0.05 p<0.01
200
200
150
150
100
100
50
50
0
LV Volume, mL 120 min Fig. 1. Comparison of sequential changes in the left ventricular end-systolicpressure (LVESPj during reperfusion after a 6-hour preservationof canine hearts in cardioplegicsolution containing K+ 20 mmol/L (group I) or University of Wisconsin (UW) solution (group 11). Left ventricular (LV) volume was varied using an inflatable balloon sutured in the mitral position. Data are presented as the mean. *p < 0.05; **p < 0.01.
•
*
K+20 Solution, n=7 UW Solution, n=9
o
**
p<0.05 p<0.01 LVEDP, mmHg
LVEDP , mmHg
100
100
80
80
60
60
40
40
20
20
o
o
30
min
LV Volume, mL 120 min Fig. 2. Comparison of sequential changes in the left ventricular end-diastolic pressure (LVEDPj during reperfusion after a 6-hour preservation of canine hearts in cardioplegic solution containing K+ 20 mmol/L (group Ia) or UW solution (group II). Left ventricular (LV) volume was varied using an inflatable balloon sutured in the mitral position. Data are presented as the mean. *p < 0.05; **p < 0.01. the composition and volume of cardioplegic solution administered and in the solutions for storage. These inconsistent variables between two groups likely have affected myocardial viability. However, we needed these
two different conditions to examine what changes in the myocardial collagen network occur. It is not our purpose to elucidate the quality of myocardial preservation comparing the two solutions. Furthermore, it is clear that UW
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The Journal of Thoracic and Cardiovascular Surgery July 1993
Fig. 3. Representative scanning electron photomicrograph of the subendocardium of a canine heart after a 6-hour preservation (top) and 2 hours of reperfusion (bottom). Hearts with a left ventricular end-diastolic pressure less than 20 mm Hg (top left) and more than 20 mm Hg (top right); heart with a left ventricular balloon deflated (bottom left); hearts with a left ventricular end-diastolic pressure less than 20 mm Hg (bottom middle) and more than 20 mm Hg (bottom right).
solution gives an excellent cardiac preservation up to 10 to 15 hours. 19-21 High-energy phosphate compounds are catabolized during ischemia, and the depletion of high-energy phosphate compounds and their intermediates leads to irreversible myocyte damage during reperfusion. Our data show that myocardial high-energy phosphate compounds were better preserved in group II than in group I at the end of both preservation and reperfusion. This clearly shows that the extent of injury during reperfusion after hypothermic cardioplegia is less in hearts preserved with
UW solution than in hearts preserved with a depolarizing K+ 20 mmol/L solution. Our data are consistent with recent reports. I9-21 It is interesting that left ventricular diastolic function was less compromised with UW solution than with K + 20 mmol/L solution. UW solution was developed to prevent inflammation, particularly free radical production, in the myocardium during ischemic reperfusion. UW solution contains allopurinol and glutathione, while the K+ 20 mmol/L solution used in group I did not. Bolli22 has reported that free radicals are responsible for
The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 1
ventricular dysfunction during early reperfusion. This study confirmed a salutary effect of UW solution on either inhibition of cellular damage or ventricular function. A number of factors, alone or in combination, can elevate left ventricular diastolic pressure through two mechanisms: an increase in passive chamber stiffness and a decrease in relaxation.P In acute experimental models like ours, both ischemia and abnormal cytosolic Ca 2+ flux decrease ventricular diastolic relaxation. Elevation of left ventricular end-diastolic pressure was more pronounced in group I, which also had a higher tissue calcium concentration than group II. Degradation of the collagen matrix occurs by three mechanisms: 1. Collagenases are activated by sulfhydryl-oxidizing agents. Reduced glutathione added to fresh UW solution is oxidized during storage to form oxidized glutathione.i" and the concentration of oxidized glutathione increases with time. Oxidized glutathione has been shown to activate collagenase, which results in the digestion of the myocardial collagen matrix. 25, 26 This factor was likely responsible for disruption of the collagen matrix, although we used only UW solution that was less than 6 months 01d. 25 However, the 2-hour reperfusion after the hypothermic preservation is inadequate to have any activated collagenases damage the collagen matrix to a point that would be visible by SEM. 24, 25 2. Proteases also can degradate the collagen matrix. Since proteases are temperature sensitive and the hearts in both groups were preserved at 5° C, proteases are unlikely to have had a major effect in this study. That the collagen matrix appeared intact by SEM at the end of preservation provides some supporting evidence for this assumption. Disruption of the collagen matrix was more extensive at the end of reperfusion than at the end of preservation in both groups. In this study, however, group Ib, in which the left ventricle was unloaded, showed no change in the myocardial collagen matrix; therefore ·it is suggested that proteases are not involved in degradation of the collagen matrix during 2-hour reperfusion. 3. Cannon and colleagues 11 have reported that degradation of collagen occurs soon after acute myocardial infarction in the rat and observed that within the 24-hour infarction zone, the myocardial concentration of hydroxyproline decreased significantly in control rats while remaining unchanged in leukopenic rats. Cannon and colleagues interpreted this to mean that collagen degradation is mediated by inflammatory cellular proteases during the first 24 hours after acute myocardial infarction in the rat. On the other hand, collagen breakdown in ischemic areas occurs in considerably less time than 24
Ohnuki et al.
17 7
hours and occurs before leukocytes are present in any number. 12, 13 At present, little is known about collagen degradation by proteases during ischemic reperfusion. On the other hand, ischemia results in changes in electrolyte concentrations and pH, both of which affect the chemical structure of collagen fibers.F' Together, these factors certainly could disrupt the collagen weave, but hearts in group Ib showed no disruption at the end of both preservation and reperfusion; therefore it is difficult to explain our findings based on these factors alone. We observed disruption of the collagen network when the left ventricular end-diastolic pressure was elevated more than 20 mm Hg at more than one point of left ventricular balloon volume from 10 to 20 ml, but not when it was less than 20 mm Hg. Also, there was a significant difference in the prevalence of disruption of the collagen matrix between the two ranges of end-diastolic pressure with a 20 mm Hg borderline. Furthermore, no disruption of the collagen matrix occurred after 2 hours of reperfusion in the heart without loading the left ventricle (group Ib). These findings suggest that disruption of the collagen network occurs during reperfusion and that elevation of the left ventricular end-diastolic pressure is the primary determinant in the loss of the collagen network. Zhao and colleagues'" have discussed in some detail the potential effect on cardiac mechanical performance of structural changes in the collagen network. They established that ventricular stiffness increases and diastolic relaxation is impaired as a result of biochemical and morphologic abnormalities that occur because of ischemia. The structural degradation of the extracellular collagen matrix may be one contributing factor in the development of the reversible postischemic dysfunction ("stunned" myocardium). Zhao and colleagues suggested that degradation of the collagen network, particularly the heterogeneous loss of collagenous force-bearing structures, may compromise optimal myocardial performance during systole by uncoupling cells and muscle fibers. Systolic performance was fairly well preserved in this study, but diastolic function was markedly disturbed in both groups, although to a greater extent in group I than in group II. In group I a significant reduction in the concentrations of myocardial A TP and total adenine nucleotides was associated with accumulation of tissue calcium. These factors are known to cause left ventricular dysfunction, especially diastolic relaxation. It is important to note that, although the collagen network was fairly well preserved in both groups at the end of preservation, disruption was more prevalent in group Ia than in group II. Also of importance, the collagen network was disrupted in eight of ten hearts with end-diastolic pressure more
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The Journal of Thoracic and Cardiovascular Surgery July 1993
Ohnuki et al.
l VEDP (mmHg)
80
o A - G (Group la) o H - P (Group II)
60
*
40
F
20
o I
10
I
I
20
15
lV balloon volume (ml)
Fig. 4. Relationship between the left ventricular end-diastolic pressure (LVEDP) and left ventricular (LV) balloon volume at 2 hours of reperfusion after 6 hours of hypothermic cardioplegia. The heart with disruption ofthe collagen matrix (*): six of seven hearts (group la) versus two of nine hearts (group II), p < 0.05; none of six hearts (LVEDP <20 mm Hg) versus eight of ten hearts (LVEDP :0::20 mm Hg), p < 0.05.
than 20 mm Hg. Conversely, little change occurred when the end-diastolic pressure was less than 20 mm Hg. This finding was independent of the type of cardioplegic solution administered to studied groups. These data suggest that the bulk of the disruption results from barotrauma brought about by an increase in the left ventricular enddiastolic pressure, since the increase in the end-diastolic pressure is the direct result of ventricular dysfunction caused by the injuries associated with prolonged ischemia. We express our gratitude to Mr. T. Nakamura for his excellent technical assistance. I.
2.
3.
4.
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