(Circulation. 1997;95:701-708.)
© 1997 American Heart Association, Inc.
Articles |
the Johannes Gutenberg-University Mainz, Second Medical Clinic (G.H., O.B., P.B., P.B., H.D., H.J.R., J.M.); the Institute for Neurosurgical Pathophysiology (A.H., O.K.); the Institute for Microbiology and Hygiene (M.L., S.B.); the Institute for Clinical Chemistry (G.H.); and Georg-August-University Gottingen, Department of Immunology (O.G.), Germany.
Correspondence to Dr Georg Horstick, Johannes Gutenberg-University Mainz, Second Medical Clinic, Langenbeckstraße 1, 55101 Mainz, Germany.
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Methods and Results Cardioprotection by C1-INH 20 IU/kg IC was examined in a pig model with 60 minutes of coronary occlusion, followed by 120 minutes of reperfusion. C1-INH was administered during the first 5 minutes of coronary reperfusion. Compared with the NaCl controls, C1-INH reduced myocardial injury (48.8±7.8% versus 73.4±4.0% necrosis of area at risk, P<.018). C1-INH treatment significantly reduced circulating C3a and slightly attenuated C5a plasma concentrations. Myocardial protection was accompanied by reduced plasma concentration of creatine kinase and troponin-T. C1-INH had no effect on global hemodynamic parameters, but local myocardial contractility was markedly improved in the ischemic zone. In the short-axis view, 137° of the anteroseptal region showed significantly improved wall motion at early and 29° at late reperfusion with C1-INH treatment.
Conclusions C1-INH significantly protects ischemic tissue from reperfusion damage, reduces myocardial necrosis, and improves local cardiac function.
Key Words: myocardial infarction ischemia
reperfusion immune system contractility
Introduction |
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Factors responsible for neutrophil influx into reperfused tissues are being investigated.8 One candidate is complement activation, which may occur when plasma gains contact with intracellular constituents (eg, mitochondria) that are released from dead cells.9 10 11 Additionally, downregulation of the complement-inhibitory molecules (eg, CD 59) on the surface of ischemic cells may play a role.12 13 Previous studies have already shown that complement is regularly activated to completion in infarcted areas of human myocardium.14 15 Moreover, there is evidence to suggest a role for complement in mediating reperfusion damage.16 Early accumulation of the terminal complement complex was observed in ischemic myocardium after reperfusion.17 Furthermore, application of C1-INH in an animal model investigated by Buerke et al18 indicated that reduction of leukocyte infiltration reduced the size of MI in ischemic and reperfused feline hearts. The present investigation was undertaken to rigorously test the contention that application of C1-INH may represent a simple and effective means to reduce reperfusion damage. For a number of reasons, we elected to use pigs as experimental animals. Schaper et al19 emphasized the very low amount of intramyocardial collateral circulation in swine. Despite the suspected similarity of collateral blood flow, MI in relation to time of coronary occlusion develops much more slowly in cats than in pigs. The results after coronary occlusion in human myocardium within the critical time limit of 6 to 12 hours are comparable to the results in the experimental system of 1 hour of coronary occlusion in the pig.20
Here we present data showing a remarkable protective effect of C1-INH application in our experimental system.
Methods |
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Coronary occlusion by a snare was applied by tunneling the LAD with a monofil suture between the proximal and medial third behind the first diagonal branch. At the same level, the vena cordis magna was cannulated for blood analysis with a small catheter. A myocardial PO2 probe was then implanted into the expected center of the area at risk. A temperature probe was positioned next to the PO2 sensor.
Experimental Protocol
Baseline values were acquired during a 1-hour preoperative period.
Coronary occlusion was achieved for 60 minutes by tightening
the snare around the LAD. The snare was then loosened, and
a 120-minute reperfusion period followed. At the beginning
of reperfusion, either 20 IU/kg body weight of C1-INH
or vehicle was infused into the LAD in a blinded fashion
under maintenance of constant flow and pressure. Hemodynamic
and PO2 measurements and
blood samples were obtained before; 5, 10, 20, 30, and 60
minutes after coronary occlusion; and after 5, 10, 20, 30,
60, 90, and 120 minutes of coronary reperfusion. Global
and regional contractility were recorded by regional
2D ultrasound before, after 15 and 60 minutes of coronary
occlusion, and after 15, 60, and 120 minutes of reperfusion.
At the end of reperfusion, the pigs were killed, and
their hearts were recovered for further analysis.
Hemodynamic Parameters
ECG, right atrial, pulmonary artery, and arterial pressure and
LAP were recorded on a Siemens Sirecust 404-1 at different
time points. Cardiac output was determined by thermodilution
(5 mL NaCl 0.9%, room temperature) and by continuous
measurement with a Baxter Vigilance monitor.
Myocardial PO2 Measurement
The Licox catheter probe measurement system22
was used in which the flexible Licox catheter PO2 microprobe is in direct contact
with the myocardium. The microprobe averages the local PO2 values in tissue with
a 90% response time of 60 to 90 seconds at body temperature.
Measurements were recorded on-line after in vitro calibration
and after a stabilization period of 60 minutes after implantation.
PO2 readings were obtained by
permanent compensation of the thermal PO2
probe drift.
Blood Gas and Lactate Analysis
One milliliter of heparinized venous and arterial blood was
drawn with polypropylene syringes from the femoral artery
and the vena cordis magna. Lactate and arteriovenous
O2 differences were determined before, after
60 minutes of coronary occlusion, and after 10 and
120 minutes of reperfusion. Blood gas analysis was
performed with the Radiometer Copenhagen Arterial Bloodgas
Laboratory 3. For lactate determination, samples were centrifuged
at 2000g (10 minutes at 4°C), plasma was decanted,
and lactate was measured with the Lactate Analyzer
model 23L from Yellow Springs Instrument Co Inc.
Measurements of C3a and C5a Plasma Concentrations
Blood was drawn from the vena cordis magna into polypropylene
tubes containing EDTA. Plasma samples were obtained by centrifugation
and immediately frozen in liquid nitrogen. C3a and C5a were
determined by an ELISA with monoclonal antibodies against
C3a and C5a described by Hopken et al.23
Cardiac Enzyme Analysis
Venous blood samples (3 mL) were collected in polypropylene
tubes containing citrate and were centrifuged at 2000g
for 15 minutes at 4°C. Plasma creatine kinase activity
was determined24 and
expressed as international units per milliliter. Troponin-T
was measured according to the method of Katus et al.25
Determination of Infarct Size
After 120 minutes of reperfusion, the LAD was reoccluded. Then
40 mL of Evan's blue (2% wt/vol solution) was injected into
the pulmonary artery to stain perfused myocardium. Unstained
myocardium was defined as the area at risk. After cardioplegia
with 20 mL potassium chloride IV (20%), the heart was excised.
The right ventricle, the large vessels, and fat tissue were
removed. The left ventricle was then sliced perpendicular
to the axis of the left side of the heart from the
apex to the AV groove in 4-mm slices. The unstained
part of the left ventricular myocardium was separated
from the Evan's bluestained portion and immersed
in a 0.09-mol/L sodium phosphate buffer, pH 7.4, containing
1% triphenyltetrazolium chloride (Sigma Chemie GmbH) and
8% dextran (molecular weight, 77.800) for 20 minutes at 37°C.
The tetrazolium dye forms a dark-red formazan complex in
the presence of viable myocardial cells that contain active
dehydrogenases and cofactors.26
Dead cells remained unstained.
The ischemic but nonnecrotic, red-stained tissue was separated from the unstained, infarcted tissue. The three tissue sectionsnonischemic (area not at risk), ischemic nonnecrotic (vital [V]), and ischemic necrotic tissue (MI)were weighed. The following definition was made: AR=V+MI.
Data were expressed as LVMM, AR, AR as a percent of LVMM, MI as a percent of LVMM, MI as a percent of AR, and total amount of infarcted tissue.
Myocardial Function
Global Myocardial Parameters
The arterial pressure-rate product (mean arterial pressure times
heart rate) was taken as a global parameter of myocardial
contractility. The AF was calculated as a global echocardiography
parameter in the ischemic apical and the nonischemic
basal region as the difference of end-diastolic (EDA)
and end-systolic (ESA) areas according to the formula
AF (%)=(EDA-ESA/EDA)x100.
Regional Wall Motion Analysis
A 2D cardiac ultrasound from Hewlett Packard (Sonos 500) equipped
with a 5-MHz transesophageal echo probe was used. The transesophageal
echo probe provided flexible movement with constant pressure
and contact to the myocardial surface. Markers were set
in the ischemic and nonischemic areas to constantly
achieve the same 2D view. ECG was recorded simultaneously.
End diastole was defined according to the recommendations
for quantification of the left ventricle by 2D echocardiography
published by the American Society of Echocardiography.27 A short-axis view from the lateral
wall of the left ventricle was used. The quantitative
analysis was performed from digitized frames of recorded
videotapes at different times and analyzed with a semiautomatic
computer system (Cardio 500, Kontron). Extrasystolic
and postextrasystolic cycles were excluded from analysis.
For quantitative regional wall motion analysis, the fixed centerline method, which is based on a system of automatic diastolic center point determinations28 (Cardio 500, Kontron), was used. Motion was measured along 100 chords drawn perpendicularly to a centerline constructed midway between the end-diastolic and end-systolic contours. The measured motion of each chord was normalized for heart size by dividing through the length of the end-diastolic perimeter. This results in a dimensionless "shortening fraction". The starting and end points of the diastolic and systolic circumference were the transition between the posterior septum and the posterior free wall.
Values before coronary occlusion in each group served as controls. Radiant shortening fraction before coronary occlusion conformed to a normal distribution. Therefore, each chord of either group was compared for significant wall motion differences between the C1-INH and vehicle-treated groups.
Statistical Analysis
Differences between the two experimental groups at baseline
and after ischemia and reperfusion were determined with
unpaired Student's t test. Paired Student's
t test was applied on effects before and after
120 minutes of reperfusion. If values did not show
a normal distribution, the Wilcoxon or Mann-Whitney test
was performed. For repeated measures (C3a, C5a, troponin-T,
creatine kinase, and cardiac output), ANOVA with the multiple
comparison method (Student-Newman-Keuls test) was used.
Differences of wall motion analysis were determined with unpaired Student's t test. Data of corresponding chords of the C1-INH and vehicle-treated groups were compared. Statistical significance was accepted at a value of P<.05 between groups.
Average values in text and figures are mean±SEM.
Results |
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Cardiac output was not significantly different between the vehicle and C1-INH groups at any time of the experiment. Fig 1A gives the measured values for cardiac output.
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Pressure-rate product, expressed as mean arterial blood pressure times heart beats per minute, was 5502±532 mm Hgxbpm in the C1-INH group and 7234±869 mm Hgxbpm in the NaCl group at baseline. No significant differences of pressure-rate product were observed at any time point between groups.
Myocardial Oxygen Pressure
Intramyocardial PO2 served as
a sensitive and rapid indicator of myocardial ischemia
and successful reperfusion (Fig 1B).
The mean PO2 of the myocardium
of all 12 pigs before coronary occlusion was 30.3±4.7
mm Hg. During the first 5 minutes of coronary occlusion,
the PO2 dropped dramatically.
After 60 minutes of coronary occlusion, PO2
had decreased to 1.5±1.3 mm Hg. Myocardial PO2 started to increase within the
first minutes of reperfusion. Five minutes after reperfusion,
PO2 was 2.4±0.9
mm Hg; 10 minutes after reperfusion, it was 3.8±1.4
mm Hg. Compared with the PO2
after 60 minutes of myocardial ischemia, the recovery
at 20 minutes after reperfusion was significant (18.9±6.1
mm Hg, P<.05). At the end of reperfusion, PO2
had increased to 80.1±6.4 mm Hg. Compared with data
under control conditions, there was a significant increase
in PO2 60 minutes
after reperfusion (65.9±7.7 mm Hg, P<.05) that
was still present at the end of the experiment in all 12
pigs.
Blood Gas and Lactate Analysis
There was a significant drop in AV DO2
in the C1-INH and NaCl-treated groups 10 minutes
after reperfusion (P<.05). At the end of
reperfusion, the decrease in AV DO2
was still significant (P<.05), however, without
any difference between the groups.
AV lactate difference decreased during ischemia in both groups. At early reperfusion (10 minutes after coronary occlusion) AV lactate difference was significantly different between the vehicle- (-1.15±0.5 mmol/L) and the C1-INH treated group (0.58±0.4 mmol/L, P<.05) with a higher lactate production in the NaCl group. Values at the end of reperfusion time remained significantly different from baseline values (Fig 1C).
Plasma Anaphylatoxin Levels
Levels of C3a and C5a in the two groups did not differ significantly
before ischemia and after 60 minutes of coronary occlusion
(Fig 2).
C3a levels increased in the vehicle-treated animals significantly
after 20 minutes and remained elevated throughout the experiment
(Fig 2A).
C1-INH treatment reduced the increase in C3a; at the end
of the reperfusion period, C3a plasma levels in the C1-INH
group remained significantly lower than in the vehicle group
(P<.05; Fig 2A).
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C5a determinations revealed a significant increase in C5a at 90 and 120 minutes of reperfusion compared with baseline and ischemic values (60 minutes of coronary occlusion, P<.05) in the vehicle-treated group. However, these increases in C5a levels were much smaller than those found for C3a, and differences between the two groups were not significant (Fig 2B).
Cardiac Creatine Kinase and Troponin-T
Plasma creatine kinase activity remained constant in both groups
during the whole ischemic period. The washout of creatine
kinase into the circulating blood occurred during the
first minutes of reperfusion and was significantly
higher in the group receiving vehicle, with maximum
reached 10 minutes after reperfusion (P<.05). Fig
3A gives the measured
values for creatine kinase.
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Troponin-T was also released into the circulation at the beginning of reperfusion. C1-INHtreated pigs had significantly lower values compared with the vehicle-treated group. This effect remained until the end of the observation period and was statistically significant 90 and 120 minutes after ischemia (P<.05) compared with untreated pigs (Fig 3B).
Myocardial Necrosis
Total LVMM and total wet weight of the AR were determined at
the end of the experiment. Wet weight of LVMM in the C1-INH
group (67.4±2.5 g) was not significantly different
from that of the NaCl group (70.8±4.2 g; Fig
4A). Neither
AR (C1-INH, 21.4±2.8 g; NaCl, 25.0±1.8 g;
Fig 4B)
nor the wet weight of the AR expressed as a percentage of
LVMM (C1-INH, 31.4±3.2%; NaCl, 35.9±2.9%;
Fig 4C)
showed a significant difference. This showed that the region
of myocardial ischemia was comparable in size in both groups.
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In the NaCl-treated group, 73.4±4.0% of the ischemic AR became necrotic (Fig 4D). In contrast, C1-INH treatment reduced the necrotic area expressed in relation to AR (48.8±7.8%, P<.018; Fig 4D) or LVMM (14.5±2.1%; NaCl, 26.4±2.6%; P<.005; Fig 4E) or as total necrotic tissue (9.9±1.7 g wet weight; NaCl, 18.2±1.1 g wet weight; P<.002; Fig 4F). Thus, C1-INH markedly reduced the size of infarctions.
Area Ejection Fraction
The AF was measured as a global parameter of contractility.
In the apical infarcted area, the C1-INHtreated group had an AF of 74.6±1.9% and the vehicle-treated group had an AF of 68.9±3.8% before coronary occlusion (Fig 5A). There was no significant difference between groups. During ischemia, there was a significant decrease in AF (C1-INH, 32.3±4.5%, P<.05; NaCl, 25.8±5.4%, P<.05) with no statistical difference between C1-INH and vehicle. With reperfusion and drug treatment, AF increased significantly in both groups until the end of the experiment. After 120 minutes of reperfusion, AF was 53.4±2.6% in the C1-INHtreated group and 39.2±4.7% in the vehicle-treated group. In both groups, a significant reduction persisted after 120 minutes of reperfusion (C1-INH and NaCl, P<.05). The recovery of AF was statistically better with C1-INH treatment 15, 60, and 120 minutes after coronary occlusion than with vehicle treatment (Fig 5A).
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The basal AF in the nonischemic area was not different in both groups (Fig 5B). During ischemia, there was a significant drop in the baseline AF in both groups (C1-INH and NaCl, P<.05). After reperfusion, AF increased in both groups, and no statistical difference was observed between the C1-INH and vehicle treatment at any time point (Fig 5B).
Regional Wall Motion Analysis
Regional wall motion before coronary occlusion showed normal
distribution with no significant difference in both groups
(Fig 6A).
After 15 minutes of coronary occlusion, wall motion dropped
significantly in the anteroseptal region in both groups
(Fig 6B).
At 60 minutes of coronary occlusion, the decrease was still
significant without differences between groups (Fig 6C). Fifteen
minutes after reperfusion, a significant difference in wall
motion was detectable in the anteroseptal region and the
posterior sector. Wall motion in the NaCl-treated group
was significantly lower from chord 36 to 74, including
a region of profound dyskinesia (136.8° of the
anteroseptal area of 360° of the short-axis view;
Fig 6D).
In the posterior region, wall motion was significantly increased
from chord 7 to 13 as a sign of compensatory hyperkinesia
in the NaCl-treated group.
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Sixty minutes after reperfusion, the region with significantly lower wall motion in the vehicle-treated group went from chord 42 to 75, representing 118.8° of the anteroseptal area of the short-axis view (Fig 6E). The area of dyskinesia became smaller. Posterior wall motion was significantly better from chord 10 to 13 and from chord 19 to 20 compared with the C1-INH group.
At the end of reperfusion (120 minutes), wall motion was significantly higher in the C1-INHtreated group from chord 65 to 72. This represents 28.8° of the total left ventricular circumference (Fig 6F). There was no differentiation between groups in posterior wall motion at that time point.
Control pigs without ischemia showed no statistical differences in global cardiac function and regional wall motion at different time points throughout the 3-hour observation period.
Discussion |
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Complement activation occurs to completion in ischemic and infarcted tissues,9 10 14 16 and several studies indicate that classic pathway activation through C1 fixation is one indicating mechanism.29 Thus, it has been shown that myocardial ischemia causes release of subcellular constituents that bind C1 in vivo.9 10 An accumulation of C1 in ischemic canine muscle has been demonstrated immunohistochemically.29 Furthermore, application of C1-INH reduced C1 deposition and leukocyte infiltration in reperfused feline myocardium.18
The potential importance of neutrophil infiltration and activation in the pathogenesis of reperfusion damage is undisputed.30 In reperfused myocardium, neutrophil accumulation was observed preferentially in the subendocardial region.3 Neutrophils may contribute to reperfusion damage by releasing reactive oxygen metabolites,31 lipid mediators, and proteases.32 33
A correlation exists between reperfusion-dependent neutrophil infiltration and infarct size,6 and neutrophil activation in reperfusion areas appears to have detrimental consequences on cardiac function.34 Complement anaphylatoxins now represent logical candidates as initiators of the detrimental processes.
We used pigs as experimental animals. This system provided the following advantages over previously used models. First, there is little collateral blood flow from other coronary arteries to the vena cordis magna, which drains the blood of the LAD.19 Second, hemodynamic parameters are obtainable in exactly the same fashion as in humans. Third, myocardial and coronary anatomies are very similar in pigs and humans. In these first experiments, we applied C1-INH intracoronarily to guarantee rapid access of the inhibitor in high concentration to the AR. The dose of 20 IU/kg body weight was selected on the basis of pilot experiments that had indicated this dose to be effective. Possible dose-related toxic effects of C1-INH have not been studied to date. Such studies would have to be performed before therapeutic trials could be conducted in humans. Of note, the applied dose is not particularly high. The physiological concentration of C1-INH in serum is appr. 1 IU (0.2 mg)/mL; hence, application of 20 IU/kg will lead to a maximal systemic increase of <50% of C1-INH plasma levels.
C1-INH inhibits C1s esterase at the site of its formation. Increasing C1-INH plasma levels will not be expected to affect overall CH50 complement titers; indeed, no such systemic effects were observed (not shown). However, at the sites of classic pathway complement activation, C1-INH would be expected to suppress formation of C3 convertase and thus attenuate C3a generation. We anticipate that the marked protective effects of C1-INH observed in this study were due to very high concentrations attained locally at the site of application. Because C3a would be liberated only locally, plasma samples were collected directly at the sites of blood efflux from the reperfused zones. The C3a determinations were significant for two important reasons. First, they provided unequivocal evidence that C3a is indeed generated in reperfused tissue; such direct evidence for anaphylatoxin generation during reperfusion had not been obtained before. Second, they showed that C3a generation was markedly suppressed when C1-INH was added to the reperfusion fluid; this supported the previous contention that C1 fixation is indeed a relevant pathway of complement activation during reperfusion. A similarly marked suppression of C5a generation by C1-INH could not be documented, however; because the increases in C5a levels during complement activation are much smaller compared with C3a, detection of significant differences will in general probably be difficult.
Myocardial damage was quantified by use of a double-staining technique. These experiments revealed a dramatic protective effect of C1-INH. Regardless of whether the infarcted areas were compared directly with each other or whether ratios were formed, eg, with the AR, highly significant differences between the C1-INH and the vehicle-treated groups were observed. Parallel to these morphological criteria, plasma creatine kinase and troponin-T levels, two biochemical markers for myocardial necrosis, were measured. Again, clear attenuation of plasma levels of both markers was found in the C1-INH group.
Our observation that C1-INH reduces infarct size in reperfusion experiments can be compared with two related publications. A 44% reduction of myocardial necrosis was reported in a rat model of reperfusion injury after application of sCR1, which inhibits complement at the level of C3.16 A second study with a feline model recently reported 65% reduction in myocardial necrosis after application of C1-INH.18 Our results in the pig model are in line with these previous findings. Of note, the extent of infarction in the vehicle group varies considerably, depending on the animal species. Thus, only 28% of the ischemic myocardium became necrotic in the feline model despite longer periods of occlusion (90 minutes) and reperfusion (4.5 hours),18 whereas we observed 73% necrosis of the AR after only 60 minutes of occlusion. The differences in protection afforded by C1-INH should be considered in the light of these major initial differences. The reasons for such differing primary sensitivity toward reperfusion damage between cat and pig are not known in detail but probably derive from differences in capillarization and collateralization.19 20 35 36 37 In the feline model, C1-INH was administered intravenously before reperfusion. Experiments to determine the optimal time and mode of C1-INH administration in the pig model are currently underway.
Although documentation of reduced infarction area is in itself a relevant issue, functional aspects of any given therapeutic measure also need to be addressed. Selection of appropriate parameters is thereby of essence. For example, many studies use measurements of global parameters such as dP/dtmax and pressure-rate product. Improved dP/dtmax has been reported after inflammatory intervention in some reperfusion studies.18 38 Our data diverge from those findings in that no differences in any of the measured global cardiac parameters were discerned. However, the use of more sensitive assays revealed that C1-INH application had dramatic effects, specifically on the regional function of the ischemic myocardium.
The parameter used in our study was the assessment of wall motion combined with calculations of the AF, which are the criteria used in clinical ventriculography. Some studies compared wall thickening with wall motion analysis. We did not use the epicardial-endocardial Doppler crystal technique, which detects changes in regional thickening rather than net endocardial motion and does not measure the contribution of epicardial motion to endocardial excursion.39 This technique is also very vulnerable to disturbances such as those that occur during defibrillation. Instead, we elected to monitor the short-axis view; the accuracy of this approach has been validated for severe ischemic regional dysfunction and discussed by many investigators.40 41 42
With this technique, clear findings emerged. During coronary occlusion of the LAD, the left ventricle developed anteroseptal hypokinesia with a small area of dyskinesia. In the vehicle-treated group, there was a depression of anteroseptal cardiac function at early reperfusion with compensatory hyperkinesia in the posterior area. Anteroseptal wall motion improved slightly up to the end of reperfusion. This improvement was markedly enhanced by application of C1-INH.
In accordance with the wall motion data, the AF decreased in the ischemic part of the left ventricle. Application of C1-INH markedly improved the AF in the ischemic zone. The basal nonischemic area that also exhibited some wall thickening showed a reduction in AF during ischemia that was not affected by C1-INH application. Overall, we thus detected an improved local contractility of the ischemic myocardium that was dependent on the presence of C1-INH. In contrast, C1-INH had no effect on the function of nonischemic myocardium.
C1 inhibitor exerts multiple functions. It not only inhibits C1s activity but also is effective against several other physiological serine proteases. Therefore, it influences the bradykinin-kinine system, the clotting cascade, and fibrinolysis. Our present study has addressed only its role in suppressing complement activation, and the results underline the important role of the classic complement pathway activation. They are in perfect agreement with recent data of Weiser et al,43 who showed that depletion of plasma immunoglobins or C4 also led to marked protection against reperfusion injury.43 Protection of C1-INH may emerge as a generally effective measure to protect against reperfusion injury in clinical settings.
Selected Abbreviations and Acronyms |
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Acknowledgments |
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Received April 25, 1996; revision received August 29, 1996; accepted September 9, 1996.
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